Vitiligo-Specific Soluble Biomarkers as Early Indicators of Response to Immune Checkpoint Inhibitors in Metastatic Melanoma Patients

Vitiligo-Specific Soluble Biomarkers as Early Indicators of Response to Immune Checkpoint Inhibitors in Metastatic Melanoma Patients | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Vitiligo-Specific Soluble Biomarkers as Early Indicators of Response to Immune Checkpoint Inhibitors in Metastatic Melanoma Patients Maria Luigia Carbone, Gabriele Madonna, Marianna Bove, Simona Mastroeni, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-753314/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Mar, 2022 Read the published version in Scientific Reports → Version 1 posted You are reading this latest preprint version Abstract Background: Immunotherapy with checkpoint inhibitors strongly improved the outcome of metastatic melanoma patients. However, not all the patients respond to treatment and identification of prognostic biomarkers able to select patients who would respond to this therapy is of outmost importance. Considering that development of vitiligo-like depigmentation in melanoma patients represents both an adverse event of immunotherapy and a favorable prognostic factor for overall survival, we analyzed known soluble biomarkers of vitiligo to validate them as early indicators of response to checkpoint inhibitors in metastatic melanoma. Methods: Fifty-seven patients with metastatic melanoma receiving anti-PD-1 checkpoint inhibitor immunotherapy were enrolled. Patient sera and plasma were evaluated for vitiligo biomarkers at pre-treatment and after 1 and 3 months of therapy. Patients were divided and analyzed according to the best overall response to treatment. Characteristic vitiligo proteins were analyzed by ELISA, while expression of circulating microRNAs, distinctive of vitiligo, was determined using real-time RT-PCR. Results: Basal serum CD25 levels were higher in stable and responder melanoma patients and remained higher during the first 3 months of anti-PD-1 therapy compared to non-responder patients. The chemokine CXCL9 was absent in non-responder patients before therapy beginning. Moreover, an increase of CXCL9 levels was observed at 1 and 3 months of therapy for all patients, although higher CXCL9 amounts were present in stable and responder compared to non-responder patients. Finally, higher levels of miR-19b, miR-25 and miR-16 were observed after 1 month of therapy in plasma of stable and responder compared to non-responder patients. Conclusions: Serum levels of CD25 and CXCL9 before and during the first months of treatment could represent biomarkers of response to anti-PD-1 immunotherapy in metastatic melanoma patients. Plasmatic miR-19b, miR-25 and miR-16 could also represent possible early biomarkers of response to anti-PD-1 treatment, but they must be validated in a higher number of patients. Biomedical Engineering Biomaterials melanoma vitiligo immunotherapy checkpoint inhibitor anti-PD-1 pembrolizumab nivolumab chemokines microRNA. Figures Figure 1 Figure 2 Figure 3 Background Identification of biomarkers for predicting response to therapies and monitoring disease progression is a rapidly growing field in cancer research. In the last decade, development of checkpoint inhibitors (CPIs) has represented the most significant breakthrough in cancer therapy, generating major clinical benefit in patients diagnosed with advanced metastatic cancer [ 1 , 2 ]. Despite the positive results obtained in around 40% of patients [ 3 – 5 ], therapy with anti-programmed death (PD)-1 antibodies is hampered by a substantial number of unresponsive individuals. Therefore, it is necessary to study prognostic biomarkers able to identify patients who would respond to therapy, so to avoid exposure of non-responders to potentially harmful side-effects, delaying introduction for them of alternative therapies [ 6 – 8 ]. Biomarkers reflecting tumor immune microenvironment and tumor cell-intrinsic features, directly obtained from tumor samples, have been already studied as potential biomarkers of response to CPIs in metastatic melanoma, i.e. intratumor programmed death-ligand (PD-L)1 expression, tumor-infiltrating lymphocytes, tumor mutational burden [ 9 – 12 ]. However, due to intratumor heterogeneity and dynamic changes in protein expression associated with the tumor microenvironment, these tissue biomarkers are not appropriate for monitoring tumor response during treatment. Additional biomarkers from peripheral blood samples have been studied in melanoma, e.g., neutrophil-to-lymphocyte ratio (NLR) [ 13 , 14 ], serum lactate dehydrogenase (LDH) levels [ 15 , 16 ], cytokines and lymphocyte populations [interleukin (IL)-6, cluster of differentiation (CD)73, CD8 + CD73 + T cells] [ 17 – 19 ]. Data about these biomarkers could be easier obtainable and some of them have been already proposed for prognostic and diagnostic purposes. During immunotherapy with CPIs, several immune-related adverse event (irAE) can be developed, in particular dermatologic toxicity associated with inflammatory and autoimmune responses [ 20 , 21 ]. These toxicities include vitiligo-like depigmentation or leukoderma and, to a lesser extent, psoriasis, bullous pemphigoid, and lichenoid reactions [ 22 – 24 ]. Leukoderma development is the only irAE associated with a favorable outcome in melanoma patients receiving immunotherapy with CPIs [ 25 – 30 ]. Vitiligo biomarkers, characterizing active vitiligo status early on and in the absence of clinical signs of the pathology, have been previously identified [ 31 ]. They include an augmented presence in patient circulation of inflammatory molecules such as the chemokine (C-X-C motif) ligand (CXCL)9/MIG [ 32 – 34 ], CXCL10/IP-10 [ 32 , 35 , 36 ], and CXCL11/I-TAC [ 36 ], S100B [ 37 , 38 ], IL-17A [ 39 , 40 ], soluble forms of CD25/IL-2 receptor alpha (IL-2 Rα) [ 41 – 43 ] and CD27/TNFRSF7 [ 43 , 44 ]. Moreover, some microRNAs (miRNAs), such as miR-16, miR-19b, and miR-25, were highly expressed in vitiligo patients, while miR-574 was downregulated, compared with healthy controls [ 45 – 47 ]. Considering that leukoderma development has been described in melanoma patients receiving CPI immunotherapy and it has been associated with a positive response to therapy [ 25 – 29 ], there is the possibility that immunological mechanisms like those activated in vitiligo pathogenesis could be triggered also in melanoma patients responding to CPIs. If this would be the case, the same biomarkers that identify vitiligo active phase could also represent biomarkers of melanoma patient response to CPI immunotherapy. In this prospective, we analyzed several biomarkers of active vitiligo in serum and plasma samples derived from metastatic melanoma patients treated with anti-PD-1 inhibitors and correlated their expression levels with patient response to therapy. Methods Patients, treatment, and clinical assessment. This study was conducted according to the Good Clinical Practice Guidelines and the Declaration of Helsinki. The study was approved by the Institutional Review Boards of Istituto Dermopatico dell’Immacolata (IDI)-IRCCS (510/3, April 2018), and Istituto Nazionale Tumori-IRCCS, Fondazione “G. Pascale” (20/14 oss, July 2014). All patients enrolled in the study provided written informed consent. This study included fifty-seven patients with unresectable metastatic melanoma, stage IIIc or IV based on American Joint Committee on Cancer (AJCC, 8th edition) staging [ 48 ], enrolled for treatment with CPIs (anti-PD-1) at IDI-IRCCS or Istituto Nazionale Tumori - Fondazione “G. Pascale” since June 2014. Patient characteristics, blood analysis, clinicopathologic and demographic information were collected. Peripheral blood samples have been sequentially collected before therapy onset and up to one-year therapy, disease progression or leukoderma appearance. Baseline evaluation included medical history, physical examination, and radiologic tumor assessment with computed tomography scans. Nivolumab (Opdivo®) was given at the dose of 240 mg every 2 weeks or 480 mg every 4 weeks, pembrolizumab (Keytruda®) at the dose of 200 mg every 3 weeks. Patients underwent physical examination and assessment of biochemical parameters monthly, whereas investigator-determined objective response was assessed radiologically with computed tomography scans approximately every 12 weeks after treatment initiation. Tumor response was classified as immune complete response (iCR), partial response (iPR), stable disease (iSD), or progressive disease (iPD), according to the immune response evaluation criteria in solid tumors (iRECIST) [ 49 , 50 ]. iPD includes both the unconfirmed progressive disease, a pseudo progression response to be confirmed in a re-evaluation follow-up visit after 4–8 weeks, and confirmed progressive disease [ 50 ]. Therapy efficacy evaluation was based on best overall response (iBOR) and progression-free survival (PFS). iBOR was determined as best time point response according to iRECIST. PFS was defined as time from the anti-PD-1 treatment start until diseases progression or death; if no such an event occurred, the closing date was November 11th, 2020. Serum preparation and molecules quantification by ELISA. Total blood samples were collected into vacutainer tubes (cat. no. 366881, BD Biosciences, Plymouth, UK), allowed to clot for 1 hour at 37°C, and centrifuged for 15 min at 1700 rcf at 4°C. Then, serum was aliquoted and stored at -80°C until use. Concentrations of human CD25/IL-2 Rα (cat. no. DY223, R&D Systems, Minneapolis, USA), CD27/TNFRSF7 (cat. no. DY382-05, R&D Systems, Minneapolis, USA), IL-17A (cat. no. DY317, R&D Systems, Minneapolis, USA), S100B (cat. no. DY1820-05, R&D Systems, Minneapolis, USA), CXCL9/MIG (cat. no. DY392, R&D Systems, Minneapolis, USA) and CXCL11/I-TAC (cat. no. DY672, R&D Systems, Minneapolis, USA) were measured using DuoSet ELISA kits (R&D Systems, Minneapolis, USA). CXCL10/IP-10 levels were evaluated with a BD Pharmingen kit (cat. no. 550926, San Diego, USA). The plates were analyzed in an ELISA iMark Microplate Reader (Bio-Rad, California, USA). Circulating total RNA extraction and quantitative miRNA real-time RT‑PCR. Total blood samples were collected into vacutainer sodium citrate tubes (cat. no. 367704, BD Biosciences, Plymouth, UK), centrifuged twice for 10 min at 1260 rcf at 4°C, and the plasma was aliquoted and stored at -80°C. Total RNA was extracted and purified from 200 µl of plasma using a miRNeasy Serum/Plasma kit (QIAGEN, Hilden, Germany), according to manufacturer’s instructions. A fixed volume of 2 µl of total RNA was used as input for consecutive reactions including poly(A) tailing, ligation, reverse transcription, and miR-Amp reaction with a TaqMan Advanced miRNA cDNA Synthesis kit (Applied Biosystems, CA, USA). The miRNA levels were then assessed by TaqMan Advanced miRNA Assay with TaqMan Fast Advanced miRNA master mix (Applied Biosystems, CA, USA). Each PCR plate was run in the QuantStudio 5 Real-Time PCR System (Applied Biosystems, CA, USA) according to manufacturer’s recommendations. The TaqMan Advanced miRNA probes (5'-phosphorylated) were as follows: hsa-miR-19b-3p (ID 478264_mir), hsa-miR-25-3p (ID 477994_mir), hsa-miR-16-5p (ID 477860_mir), hsa-miR-574-3p (ID 478163_mir). For quality control and analyses of miRNA expression, cel-miR-39-3p (ID 478293_mir, cat. no. A25576, Applied Biosystems, CA, USA) was used as an exogenous spike-in. Expression of miRNAs relative to cel-miR-39-3p was determined using the formula 2 −ΔCt , where ΔC T = C TmiRNA - C TmiR−39 , and C T (i.e., threshold cycle) indicates the fractional cycle number at which the amount of amplified target reaches a fixed threshold. To simplify data presentation, the relative expression values were multiplied by 10 4 . All determinations were performed at least three times, each in duplicate. Statistical analysis. Groups based on treatment-response were compared using the Fisher's exact test for categorical variables, the non-parametric Mann-Whitney U test or the Kruskal-Wallis test for continuous variables. The Spearman’s coefficient was used to test correlation between levels of analyzed molecules at baseline. To evaluate before-after treatment differences, the Wilcoxon matched-pairs signed-rank test was used. For data analysis, the value of 0.01 pg/ml was assigned to samples with values under the lower limit of ELISA detection. Statistical significance was set at p < 0.05. All statistical analyses were conducted using GraphPad prism Software (La Jolla, CA, USA). Results Patients’ characteristics and responses. For this study, fifty-seven melanoma patients were analyzed. Considering the iBOR, 28 patients responded positively to CPI therapy with an iCR (n = 11) or an iPR (n = 17), 16 patients had no objective response (iSD), and 13 patients developed an iPD. The median follow-up time for PFS was 12.2 months (mean 17.1 month, ranging from 1.1 to 61.7 months). At 1 year, PFS was 57.7%, with a median PFS of 33.4 months for iCR, 20.7 for iPR, 9.7 for iSD, and 2.8 for iPD patients. There were no patients lost to follow-up. Fifteen patients developed leukoderma at a mean time of 6.8 months after CPI therapy starting (Table 1 ). Table 1 Characteristics and treatment outcomes of enrolled patients. Patient Stage a Checkpoint inhibitor a iBOR b PFS c Progression c Leukoderma d 1 IIIc nivo iCR 10.8 no 8.7 2 M1a nivo iCR 40.7 no 2.1 3 M1a nivo iCR 13.5 no - 4 M1a nivo iCR 12.2 no - 5 M1b pembro iCR 21.9 no 7.6 6 M1c nivo iCR 33.4 no 4.7 7 M1c pembro iCR 37.3 no 11.1 8 M1c pembro iCR 37.3 no 9.7 9 M1c pembro iCR 48.1 no - 10 M1c nivo iCR 13.3 no - 11 M1d pembro iCR 62.6 no - 12 M1a nivo iPR 40.5 no 13.1 13 M1a pembro iPR 26.2 no - 14 M1a pembro iPR 20.7 no 4.3 15 M1a pembro iPR 19.1 no - 16 M1a pembro iPR 7.1 yes - 17 M1a nivo iPR 11.5 no - 18 M1a nivo iPR 10.8 no - 19 M1b pembro iPR 33.6 no 2.1 20 M1b pembro iPR 9.2 no 6.7 21 M1c nivo iPR 8.9 yes - 22 M1c nivo iPR 42.3 no - 23 M1c nivo iPR 36.8 no 9.7 24 M1c pembro iPR 39.1 no - 25 M1c pembro iPR 38.0 no 7.7 26 M1c pembro iPR 36.8 no 6.9 27 M1c nivo iPR 13.9 yes - 28 M1c nivo iPR 12.9 no - 29 M1a pembro iSD 7.7 yes - 30 M1a nivo iSD 12.9 no - 31 M1b nivo iSD 18.2 yes - 32 M1b nivo iSD 11.7 yes - 33 M1c nivo iSD 5.9 yes - 34 M1c nivo iSD 5.3 yes - 35 M1c nivo iSD 36.4 no 4.3 36 M1c nivo iSD 6.8 yes - 37 M1c pembro iSD 5.2 yes - 38 M1c pembro iSD 4.1 yes - 39 M1c pembro iSD 32.5 yes - 40 M1c pembro iSD 6.3 yes - 41 M1c pembro iSD 26.4 yes - 42 M1c pembro iSD 15.8 no - 43 M1d pembro iSD 4.8 no 2.8 44 M1d pembro iSD 17.3 no - 45 IIIc pembro iPD 2.8 yes - 46 M1a pembro iPD 2.7 yes - 47 M1a pembro iPD 2.7 yes - 48 M1c nivo iPD 3.6 yes - 49 M1c nivo iPD 3.7 yes - 50 M1c nivo iPD 4.1 yes - 51 M1c nivo iPD 1.8 yes - 52 M1c pembro iPD 2.0 yes - 53 M1c pembro iPD 1.1 yes - 54 M1c pembro iPD 4.0 yes - 55 M1c pembro iPD 3.1 yes - 56 M1c pembro iPD 1.4 yes - 57 M1c nivo iPD 3.9 yes - a Staging before starting therapy: nivo, nivolumab; pembro, pembrolizumab. b iBOR, best overall response according to the iRECIST criteria: iCR, complete response; iPR, partial response; iSD, stable disease; iPD, progressive disease. c Data cut off on 11th November 2020. PFS, progression-free survival (months). d Patients who had developed (months) or not (-) leukoderma at the cut off data. We analyzed patient characteristics and tumor-associated factors at the beginning of the treatment with anti-PD-1 antibodies. As shown in Table 2 , 37 patients were males (64.9%) and 20 females (35.1%). The mean age at treatment start was 67.3 years. Levels of circulating LDH were found high in 13 patients (26.5%) and were not assessed for 8 patients. The M staging of the extent of metastatization was stage IIIc-M1b for 21 patients (36.8%), and M1c-M1d for 36 patients (63.2%). In particular, stage IIIC for 2 patients, stage M1a for 12 patients (M1a(0) for 8, M1a(1) for 3, LDH not assessed for 1), stage M1b for 7 patients (M1b(0) for 5, M1b(1) for 2), stage M1c for 33 patients (M1c(0) for 20, M1c(1) for 8, LDH not assessed for 5), stage M1d for 3 patients (M1d(0) for 2, LDH not assessed for 1). BRAF gene mutation was detected in 15 patients (26.8%). Thirty-one patients (54.4%) were treated with pembrolizumab and 26 patients (45.6%) with nivolumab. First-line treatment was performed by 32 patients (56.1%), and second-to-fourth line by 25 patients (43.9%). Leukoderma was developed by 15 patients (26.3%). Of those, n = 6 iCR patients (40.0%), n = 7 iPR patients (46.7%), n = 2 iSD patients (13.3%), none iPD patient. Clinical characteristics of the response groups were similar for sex, age, metastasis stage, serum LDH amount, BRAF mutation, drug administrated, and line of treatment. Patients who developed leukoderma were associated with a good response to CPI therapy ( p = 0.003). Out of 15 patients with leukoderma, 12 (80%) developed it during a first-line treatment. Table 2 Patient characteristics, treatments, and leukoderma development. Characteristics All (n = 57) iPD (n = 13) iSD (n = 16) iPR (n = 17) iCR (n = 11) p value a Sex Male 37 (64.9) 9 (69.2) 9 (56.3) 12 (70.6) 7 (63.6) Female 20 (35.1) 4 (30.8) 7 (43.7) 5 (29.4) 4 (36.4) 0.866 Age, years mean (SD) 67.3 (12.8) 62.0 (12.0) 67.1 (13.5) 73.2 (12.2) 64.7 (11.3) median (IQR) 67 (59–78) 65 (54–68) 67 (60–79) 76 (67–81) 65 (56–72) 0.084 b ≤ 65 22 (38.6) 6 (46.1) 7 (43.7) 4 (23.5) 5 (45.4) > 65 35 (61.4) 7 (53.9) 9 (56.3) 13 (76.5) 6 (54.6) 0.509 Serum LDH Normal 36 (73.5) 8 (80.0) 10 (71.4) 11 (68.8) 7 (77.8) Elevated 13 (26.5) 2 (20.0) 4 (28.6) 5 (31.2) 2 (22.2) 0.970 Stage IIIc-M1b 21 (36.8) 3 (23.1) 4 (25.0) 9 (52.9) 5 (45.4) M1c-M1d 36 (63.2) 10 (76.9) 12 (75.0) 8 (47.1) 6 (54.6) 0.263 BRAF status Mutation 15 (26.8) 5 (38.5) 4 (25.0) 3 (18.7) 3 (27.3) No mutation 41 (73.2) 8 (61.5) 12 (75.0) 13 (81.3) 8 (72.7) 0.702 Drug Pembrolizumab 31 (54.4) 5 (38.5) 7 (43.7) 8 (47.1) 6 (54.6) Nivolumab 26 (45.6) 8 (61.5) 9 (56.3) 9 (52.9) 5 (45.4) 0.907 Line of treatment 1 32 (56.1) 5 (38.5) 7 (43.7) 11 (64.7) 9 (81.8) 2–4 25 (43.9) 8 (61.5) 9 (56.3) 6 (35.3) 2 (18.2) 0.117 Leukoderma No 42 (73.7) 13 (100.0) 14 (87.5) 10 (58.8) 5 (45.4) Yes 15 (26.3) - 2 (12.5) 7 (41.2) 6 (54.6) 0.003 Totals may vary because missing values. Data are n (%) unless otherwise stated. Abbreviation: iPD, progressive disease; iSD, stable disease; iPR, partial response; iCR, complete response. SD, Standard Deviation; IQR, Interquartile Range; LDH, lactate dehydrogenase. p value was calculated using the Fisher's exact test ( a ) or Kruskal-Wallis test ( b ). In a sub-group of 44 patients for whom blood analysis data were available (patients no. 1–5, 7, 10, 14–16, 18–28, 30–39, 42–44, 46, 47, 49–55, 57 of Table 1 ), we observed higher neutrophil count at the beginning of the therapeutic plan in iPD patients in respect with iCR patients (p = 0.043; Table 3 ). Moreover, our data indicated higher baseline values of NLR and derived NLR (dNLR) in iPD patients rather than in iCR patients, suggesting that these higher basal levels could associate with a tendency towards progression disease after treatment with CPIs (Table 3 ). Table 3. Analysis of the relationship between neutrophils and lymphocytes before the start of immunotherapy with CPIs. Neutrophil count (µL); NLR, neutrophil-to-lymphocyte ratio (neutrophils / lymphocytes); dNLR, derived neutrophil-to-lymphocyte ratio [neutrophils / (leukocytes-neutrophils)]. Best overall response according to iRECIST criteria: iPD, progressive disease (n. 10 patients); iSD, stable disease (n. 13 patients); iPR, partial response (n. 14 patients); complete response, iCR (n. 7 patients). Data are expressed as mean value ± standard error of the mean (SEM). (*) p = 0.043 iCR versus iPD, assessed by Mann-Whitney U test. CD25 and CXCL9 as potential early biomarkers of response to CPI therapy. To search for potential biomarkers that could predict patient clinical response or that could represent early indicators of response to CPIs, selected biomarkers of the vitiligo active phase were analyzed in serum samples from patients before (T0) and after 1 month (T1) and 3 months (T2) of treatment. We analyzed circulating levels of CD25, CD27, CXCL9, CXCL10, CXCL11, S100B, and IL-17A. For the patients examined, the circulating amount of either S100B or IL-17A was below the ELISA detection level (data not shown). Regarding CD25, iSD and iPR patients had significantly higher levels of CD25 at baseline than iPD patients (Fig. 1 a, b). A tendency to have higher serum amounts of CD25 was observed also for iCR patients, but it did not reach statistical significance (Fig. 1 a, b). Basal CD25 levels (T0) significantly increased at T1 and T2 for iPD patients, but values remained lower than the other patient groups at the same time point. As far as CD27, iCR patients tended to have less circulating protein amount than other groups at all time-point analyzed. Statistical significance was observed only at T1 between iCR and iPD patients (Fig. 1 c, d). CXCL9 was undetectable at T0 in the sera of iPD compared to other patients who had baseline higher chemokine circulating levels (significance was achieved at T0 for iSD and iCR versus iPD patients; Fig. 2 a, b). Moreover, the trend of lower circulating amount of CXCL9 in iPD patients compared to the other groups was maintained after 1 and 3 months of therapy (significance was achieved at T1 and T2 for iPR versus iPD patients). All three groups of patients showed CXCL9 serum basal levels (T0) that significantly increased after 1 month of therapy (T1). Interestingly, while for iPR and iCR patients CXCL9 levels continued to significantly growth at T2, for iPD and iSD patients the levels of CXCL9 decreased at T2. CXCL10 amounts significantly increased at T1 compared to T0 for iSD, iPR and iCR patients, and a significative increment was also present for iCR patients at T2 (Fig. 2 c, d). No significant differences in serum concentration among the three response groups of patients was observed. Similarly, CXCL11 concentrations among the patient groups was significantly different only at T1 for iCR versus iPD patients (Fig. 2 e, f). CXCL11 levels increased between T0 and T1 for iPD and iPR patients, and between T0 and T2 for iSD patients (Fig. 2 e, f). In iPD patients, the increment observed at T1 was followed by a reduction in CXCL11 levels at T2 (Fig. 2 e, f). Since both CD25 and CXCL9 levels could represent early biomarkers of response to CPI therapy, we also observed that no correlation was found between the two proteins levels (Spearman’s ρ = 0.03, p = 0.821). miR-19b, miR-25 and miR-16 as promising indicators of response to CPIs therapy. For a subset of 29 patients, for whom plasma samples were available, circulating miRNAs, previously identified as biomarkers of vitiligo, were analyzed. Patients no. 1, 3–5, 9–11, 13–18, 20, 27–30, 32, 40–47, 56, 57 of Table 1 were examined (iPD, 5 patients; iSD, 8 patients; and iPR/iCR, 16 patients; iPR and iCR were assembled due to the small number of patients analyzed). As shown in Fig. 3 and in Additional Table 1 , although statistical significance was not achieved in the comparisons between patient groups or times of drugs administration, some trends towards significance were observed. Higher levels of miR-19b and miR-25 were present at all time points of treatment in the plasma of iSD and iPR/iCR patients in respect to iPD individuals. For miR-16, a higher amount was present in iSD patients at T1 ant T2 and in iPR/iCR patients at T2 compared to iPD patients at the corresponding time points. Furthermore, miR-19b, miR-25, and miR-16 showed an increment at T1 versus T0 in iSD and iPR/iCR patients, but not in iPD patients, whose values remained low. No major differences in the amount of miR-574 among the three patient groups and at any time of treatment were observed. Interestingly, regarding miR-19b, miR-25 and miR-16, most of the iPR/iCR outliers that fall outside the distribution of miRNA levels, were the same patients who developed leukoderma during CPI therapy (stars in Fig. 3 ). Discussion To date, it is urgent to develop non-invasive methods predicting melanoma patient response or resistance to immunotherapy to better address them towards the most efficacious therapies. In this regard, the intent of our work was the identification of predictive and/or early biomarkers of response to anti-PD-1 antibodies, taking advantages of previously identified biomarkers of the autoimmune vitiligo pathology, since leukoderma represents an irAE that often occurs during CPI therapy in melanoma patients and correlates with a positive outcome. We confirmed in our patient subset the association between development of leukoderma and a favorable clinical response in melanoma patients [ 30 ]. Then, we analyzed specific vitiligo biomarkers as possible indicators of response to anti-PD-1 treatment in metastatic melanoma patients. Our data suggest that, among the molecules that characterize the active phase of vitiligo, CD25 and CXCL9 represent independent predictive biomarkers of response to CPI treatment. In fact, higher levels of these proteins were observed at baseline (T0) in the serum of patients who respond positively or stably to CPI therapy compared to patients who advanced towards progression disease. In addition, a constant increment of CXCL9 circulating amounts during the therapeutic period could represent an early biomarker of response to CPIs. CD25, the membrane IL-2 Rα that can be shed into a soluble molecule upon immune cell activation, plays a role in the regulation of T cell function and in the maintenance of immune tolerance [ 51 , 52 ]. Blockage of the PD-1/PD-L1 interaction leads to an early activation of T cells, inducing several distinct signal transduction pathways also involved in IL-2 expression [ 53 ]. When high levels of IL-2 are available, cells that express CD25, i.e. natural killer cells and T lymphocytes, will be activated, leading to an important immune response towards the tumor. Indeed, high dose IL-2 treatment was the first immunotherapy approach used in advanced melanoma. However, IL-2 benefits were associated with substantial toxicity [ 54 – 56 ]. In the present investigation, we observed that stable or responding patients had higher levels of soluble CD25 at baseline and in the first months of anti-PD-1 therapy compared with patients who did not respond to treatment. Thus, we hypothesize that the increment of serum CD25 in melanoma patients who respond to anti-PD-1 therapy could be an indicator of an IL-2-mediated T cell activation, predictive of an effective anti-tumor immune response [ 53 , 57 ]. In future studies, it would be interesting to deeply analyze the mechanism of action of the CD25/IL-2 pathway and its time schedule in the response to anti-PD-1 treatment, and to evaluate the possible beneficial effects of a combined therapy of low doses of IL-2 together with the anti-PD-1 treatment. Chemokines play key roles in the recruitment of effector immune cells into the tumor [ 58 ]. In particular, CXCR3, the receptor for the interferon-inducible chemokines CXCL9, CXCL10, and CXCL11, is highly expressed on activated T cells and plays an essential role in T cell migration into lymphoid and peripheral tissues [ 59 – 61 ]. A few studies have been already conducted on the circulating expression of CXCR3 ligands after CPI therapies [ 6 , 8 , 62 ]. An increase of circulating CXCL9 and CXCL10 in melanoma patients was observed during anti-PD-1 treatment [ 63 ], and correlated with treatment outcome [ 64 ]. In our study, we observed that, during anti-PD-1 immunotherapy, CXCL9 increased in the serum of responder and stable patients compared to non-responder, suggesting that this chemokine could be an early indicator of a positive therapeutic response. Moreover, circulating CXCL9 was absent in non-responder patients before the beginning of the treatment and increased after anti-PD-1 treatment in the sera of responder and stable patients, suggesting that a high amount of CXCL9 is important to mount an effective T cells-mediated antitumor immune response. It would be interesting to study if patient pretreatment with a compound able to specifically induce CXCL9 expression could be helpful for obtaining a better response to anti-PD-1 antibodies as shown in the mouse model [ 64 ]. Our data also indicate that CXCL10 significantly increases in stable and responder patients after the first month of therapy, suggesting an initial activation of the immune system fundamental for effective treatment response and tumor clearance. Conversely, elevated CXCL11 levels in blood have been previously linked to poorer outcome for CPI-treated melanoma [ 66 ]. CXCL11 has distinct immunoregulatory functions from those of CXCL9 and CXCL10 [ 67 ]. CXCL11 limits T cell effector functions through induction and/or recruitment of regulatory T cells, and it is capable of binding to CXCR7, commonly associated with tumor cells growth and invasiveness [ 67 , 68 ]. Indeed, we also observed that CXCL11 significantly increased in non-responder compared to complete responder patients after one month of CPI therapy. Other potential biomarkers of response to CPIs could be represented by miRNAs. Expression profiles of miRNAs are frequently dysregulated during cancer progression, and they are also involved in the modulation of the immune microenvironment [ 69 , 70 ]. Altered expression of miRNAs frequently correlates with poor prognosis and/or inadequate response to treatments in all stages of malignant melanoma [ 71 – 73 ]. Several circulating miRNAs have already been described as potential biomarkers of progression and prognosis in melanoma [ 74 – 76 ]. In this work, we decided to analyze the miRNAs that characterize the vitiligo disease. We showed that miR-16, miR-19b, miR-25 levels have the tendency to increase in stable and responder melanoma patients, but not in non-responder ones. Moreover, in agreement to published data [ 77 ], we observed that circulating miR-16 levels tended to decrease in non-responder melanoma patients after CPI therapy. The lack of statistical association in our study may be due to the low number of samples analyzed that will be increased in future investigations. Despite the presence of literature data on the role of miR-19b and miR-25 in melanoma cells [ 78 – 80 ], their expression levels have not been previously analyzed in patient plasma. Therefore, for the first time, our data indicate that in metastatic melanoma patients the levels of miR-19b and miR-25 increase in stable and responder patients after one month of CPI therapy. Most responder patients with high plasma levels of miR-19b, miR-25 and miR-16 were the same patients who developed leukoderma, further implying existence of a connection between the molecular mechanisms that drive the autoimmune response in vitiligo and those that induce response to CPIs. Finally, among the patient characteristics already described as potential biomarkers of response to immunotherapy with CPIs, we confirmed in our patient subset that basal higher neutrophil count, NLR, and dNLR could be indicators of progression disease during CPI treatment [ 13 , 14 ]. Conclusions Our data confirm that leukoderma is associated with a favorable outcome in metastatic melanoma patients treated with CPIs and indicate that biomarkers of the active phase of vitiligo could represent promising indicators of response to CPIs. When validated in a larger cohort, dosage of CD25 and CXCL9 circulating levels before and in the first months of therapy could represent a tool for predicting patient response to anti-PD-1 antibodies. Finally, both CD25 and CXCL9 should be further investigated as therapeutic targets to overcome resistance to CPI treatment in metastatic melanoma. Abbreviations CPIs checkpoint inhibitors; PD-1:programmed death-1; PD-L1:programmed death-ligand 1; NLR:neutrophil-to-lymphocyte ratio; LDH:serum lactate dehydrogenase; irAE:immune-related adverse event; CXCL:(C-X-C motif) ligand; IL:interleukin; CD:cluster of differentiation; IL-2 Rα:IL-2 receptor alpha; miR:microRNA; AJCC:American Joint Committee on Cancer; iCR:complete response; iPR:partial response; iSD:stable disease; iPD:progressive disease; iRECIST:immune response evaluation criteria in solid tumors; iBOR:best overall response; PFS:progression-free survival; dNLR:derived NLR. Declarations Ethics approval and consent to participate. Informed consent was obtained from all patients under approved human research ethics committee protocols from the Istituto Dermopatico dell’Immacolata IDI-IRCCS (510/3, April 2018) and the Istituto Nazionale Tumori-IRCCS - Fondazione “G. Pascale” (20/14 oss, July 2014 ). Consent for publication. Not applicable. Availability of data and materials. All data generated or analyzed during this study are available from the corresponding author on reasonable request. Competing interests. Paolo A. Ascierto has/had a consultant/advisory role for Bristol Myers Squibb, Roche-Genentech, Merck Sharp & Dohme, Novartis, Array, Merck Serono, Pierre-Fabre, Incyte, Medimmune, AstraZeneca, Syndax, Sun Pharma, Sanofi, Idera, Ultimovacs, Sandoz, Im-munocore, 4SC, Alk-ermes, Italfarmaco, Nektar, Boehringer-Ingelheim, Eisai, Regeneron, Daiichi Sankyo, Pfizer, Oncosec, Nouscom, Takis, Lunaphore. He also received research funding from Bristol Myers Squibb, Roche-Genentech, Array, Sanofi, and travel support from MSD. All outside the submitted work. All the other authors declare no competing interests. Funding. This research was supported by grants of the Italian Ministry of Health (IT-MOH) through“Ricerca Corrente” 2018-2020, RC4.1, and “Ricerca Corrente” M2/2. MLC is the recipient of a research fellowship by Fondazione Umberto Veronesi. Authors' contributions. Conception and design of the study: MLC, CMF and GM. Experiments carried out and acquisition of data: MLC, MB, MC and LL. Analysis and interpretation of data: MLC, SD’A, FDG and CMF. Statistical analysis: MLC, SM and CF. Drafting of the manuscript: MLC. Critical revision of the manuscript: CMF, GM, SM, PAA, SD’A, FDG and CF. All authors have read and agreed to the published version of the manuscript. Acknowledgements. Authors would like to thank Laura Bonmassar for skillful technical assistance, the nurses of the Department of Oncology and Dermatological Oncology IDI-IRCCS and of the Melanoma, Cancer Immunotherapy and Development Therapeutics Unit of Istituto Nazionale Tumori IRCCS Fondazione “G. Pascale” for their continuous help, and Fondazione Umberto Veronesi for the support of MLC research work. References Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat. Rev. Cancer. 2012. p. 252–64. Herzberg B, Fisher DE. Metastatic Melanoma and immunotherapy HHS Public Access. Clin Immunol. 2016;172:105–10. Larkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Cowey CL, Lao CD, et al. Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. N Engl J Med. 2015;373:23–34. Márquez-Rodas I, Cerezuela P, Soria A, Berrocal A, Riso A, González-Cao M, et al. Immune checkpoint inhibitors: Therapeutic advances in melanoma. Ann. Transl. Med. 2015. p. 267. Wan MT, Ming ME. Nivolumab versus ipilimumab in the treatment of advanced melanoma: a critical appraisal. Br J Dermatol. 2018;179:296–300. Buder-Bakhaya K, Hassel JC. Biomarkers for clinical benefit of immune checkpoint inhibitor treatment-A review from the melanoma perspective and beyond. Front Immunol. 2018;9. Yi M, Jiao D, Xu H, Liu Q, Zhao W, Han X, et al. Biomarkers for predicting efficacy of PD-1/PD-L1 inhibitors. Mol Cancer. 2018;17:1–14. Jia XH, Geng LY, Jiang PP, Xu H, Nan KJ, Yao Y, et al. The biomarkers related to immune related adverse events caused by immune checkpoint inhibitors. J Exp Clin Cancer Res. 2020;39:1–17. Madore J, Vilain RE, Menzies AM, Kakavand H, Wilmott JS, Hyman J, et al. PD-L1 expression in melanoma shows marked heterogeneity within and between patients: Implications for anti-PD-1/PD-L1 clinical trials. Pigment Cell Melanoma Res. 2015;28:245–53. Postow MA, Friedman CF, Postow MA. Emerging Tissue and Blood-Based Biomarkers that may Predict Response to Immune Checkpoint Inhibition. Curr Oncol Rep. 2016;18:21. Maibach F, Sadozai H, Seyed Jafari SM, Hunger RE, Schenk M. Tumor-Infiltrating Lymphocytes and Their Prognostic Value in Cutaneous Melanoma. Front Immunol. 2020;11:1–20. Goodman AM, Kato S, Bazhenova L, Patel SP, Frampton GM, Miller V, et al. Tumor mutational burden as an independent predictor of response to immunotherapy in diverse cancers. Mol Cancer Ther. 2017;16:2598–608. Capone M, Giannarelli D, Mallardo D, Madonna G, Festino L, Grimaldi AM, et al. Baseline neutrophil-to-lymphocyte ratio (NLR) and derived NLR could predict overall survival in patients with advanced melanoma treated with nivolumab. J Immunother Cancer. 2018;6:1–7. Bartlett EK, Flynn JR, Panageas KS, Ferraro RA, Jessica JM, Postow MA, et al. High neutrophil-to-lymphocyte ratio (NLR) is associated with treatment failure and death in patients who have melanoma treated with PD-1 inhibitor monotherapy. Cancer [Internet]. John Wiley and Sons Inc.; 2020 [cited 2021 Feb 17];126:76–85. Available from: https://pubmed.ncbi.nlm.nih.gov/31584709/ . Petrelli F, Ardito R, Merelli B, Lonati V, Cabiddu M, Seghezzi S, et al. Prognostic and predictive role of elevated lactate dehydrogenase in patients with melanoma treated with immunotherapy and BRAF inhibitors: A systematic review and meta-analysis. Melanoma Res. 2019;29:1–12. Diem S, Kasenda B, Spain L, Martin-Liberal J, Marconcini R, Gore M, et al. Serum lactate dehydrogenase as an early marker for outcome in patients treated with anti-PD-1 therapy in metastatic melanoma. Br J Cancer. 2016;114:256–61. Laino AS, Woods D, Vassallo M, Qian X, Tang H, Wind-Rotolo M, et al. Serum interleukin-6 and C-reactive protein are associated with survival in melanoma patients receiving immune checkpoint inhibition. J Immunother cancer. 2020;8:e000842. Turiello R, Capone M, Giannarelli D, Morretta E, Monti MC, Madonna G, et al. Serum CD73 is a prognostic factor in patients with metastatic melanoma and is associated with response to anti-PD-1 therapy. J Immunother cancer. 2020;8. Capone M, Fratangelo F, Giannarelli D, Sorrentino C, Turiello R, Zanotta S, et al. Frequency of circulating CD8 + CD73 + T cells is associated with survival in nivolumab-treated melanoma patients. J Transl Med. 2020;18. Chamoto K, Hatae R, Honjo T. Current issues and perspectives in PD-1 blockade cancer immunotherapy. Int J Clin Oncol. 2020;25:790–800. Haanen J, Ernstoff MS, Wang Y, Menzies AM, Puzanov I, Grivas P, et al. Autoimmune diseases and immune-checkpoint inhibitors for cancer therapy: review of the literature and personalized risk-based prevention strategy. 31. Ann Oncol: Elsevier Ltd.; 2020. pp. 724–44. Simonsen AB, Kaae J, Ellebaek E, Svane IM, Zachariae C. Cutaneous adverse reactions to anti–PD-1 treatment—A systematic review. J Am Acad Dermatol. 2020;83:1415–24. Rodrigues M, Ezzedine K, Hamzavi I, Pandya AG, Harris JE. New discoveries in the pathogenesis and classification of vitiligo. J Am Acad Dermatol. 2017;77:1–13. Parsad D, Sahni K. Stability in vitiligo: Is there a perfect way to predict it? J Cutan Aesthet Surg. 2013;6:75. Byrne KT, Turk MJ. New perspectives on the role of vitiligo in immune responses to melanoma. Oncotarget. 2011;2:684–94. Teulings HE, Limpens J, Jansen SN, Zwinderman AH, Reitsma JB, Spuls PI, et al. Vitiligo-like depigmentation in patients with stage III-IV melanoma receiving immunotherapy and its association with survival: A systematic review and meta-analysis. J Clin Oncol. 2015;33:773–81. Hua C, Boussemart L, Mateus C, Routier E, Boutros C, Cazenave H, et al. Association of vitiligo with tumor response in patients with metastatic melanoma treated with pembrolizumab. JAMA Dermatology. 2016;152:45–51. Nakamura Y, Tanaka R, Asami Y, Teramoto Y, Imamura T, Sato S, et al. Correlation between vitiligo occurrence and clinical benefit in advanced melanoma patients treated with nivolumab: A multi-institutional retrospective study. J Dermatol. 2017;44:117–22. Babai S, Voisin AL, Bertin C, Gouverneur A, Le-Louet H. Occurrences and Outcomes of Immune Checkpoint Inhibitors-Induced Vitiligo in Cancer Patients: A Retrospective Cohort Study. Drug Saf. 2020;43:111–7. Failla CM, Carbone ML, Fortes C, Pagnanelli G, D’atri S. Melanoma and vitiligo: In good company. Int J Mol Sci. 2019;20. Speeckaert R, Speeckaert M, De Schepper S, van Geel N. Biomarkers of disease activity in vitiligo: A systematic review. Autoimmun Rev. 2017;16:937–45. Wang XX, Wang QQ, Wu JQ, Jiang M, Chen L, Zhang CF, et al. Increased expression of CXCR3 and its ligands in patients with vitiligo and CXCL10 as a potential clinical marker for vitiligo. Br J Dermatol. 2016;174:1318–26. Maouia A, Sormani L, Youssef M, Helal AN, Kassab A, Passeron T. Differential expression of CXCL9, CXCL10, and IFN-γ in vitiligo and alopecia areata patients. Pigment Cell Melanoma Res. 2017;30:259–61. Elia G. MIG Th1 chemokine in Vitiligo. Clin Ter. 2018;169:E303–7. Abdallah M, El-Mofty M, Anbar T, Rasheed H, Esmat S, Al-Tawdy A, et al. CXCL-10 and Interleukin-6 are reliable serum markers for vitiligo activity: A multicenter cross-sectional study. Pigment Cell Melanoma Res. 2018;31:330–6. Rashighi M, Agarwal P, Richmond JM, Harris TH, Dresser K, Su MW, et al. CXCL10 is critical for the progression and maintenance of depigmentation in a mouse model of vitiligo. Sci Transl Med. 2014;6. Speeckaert R, Voet S, Hoste E, van Geel N. S100B Is a Potential Disease Activity Marker in Nonsegmental Vitiligo. J Invest Dermatol. 2017;137:1445–53. Birlea SA. S100B: Correlation with Active Vitiligo Depigmentation. J Invest Dermatol. 2017;137:1408–10. Acharya P, Mathur M. Interleukin-17 level in patients with vitiligo: A systematic review and meta-analysis. Australas J Dermatol. 2020;208–12. Singh RK, Lee KM, Vujkovic-Cvijin I, Ucmak D, Farahnik B, Abrouk M, et al. The role of IL-17 in vitiligo: A review. Autoimmun. Rev. 2016. p. 397–404. Honda Y, Okubo Y, Koga M. Relationship between levels of soluble interleukin-2 receptors and the types and activity of vitiligo. J Dermatol. 1997;24:561–3. Yeo UC, Yang YS, Park KB, Sung HT, Jung SY, Lee ES, et al. Serum concentration of the soluble interleukin-2 receptor in vitiligo patients. J Dermatol Sci. 1999;19:182–8. Speeckaert R, Lambert J, Van Geel N. Clinical significance of serum soluble CD molecules to assess disease activity in vitiligo. JAMA Dermatology. 2016;152:1194–200. ElGhareeb MI, El Mokadem S, El Sayed B, Khalifa N. Soluble CD27 and MIF as possible serum biomarkers of vitiligo activity in Egyptian patients in Sharkia Governorate. Dermatology Reports. 2019;11:3–5. Yan S, Shi J, Sun D, Lyu L. Current insight into the roles of microRNA in vitiligo. Mol Biol Rep. 2020;47:3211–9. Shi YL, Weiland M, Li J, Hamzavi I, Henderson M, Huggins RH, et al. MicroRNA expression profiling identifies potential serum biomarkers for non-segmental vitiligo. Pigment Cell Melanoma Res. 2013;26:418–21. Shi Q, Zhang W, Guo S, Jian Z, Li S, Li K, et al. Oxidative stress-induced overexpression of miR-25: The mechanism underlying the degeneration of melanocytes in vitiligo. Cell Death Differ. 2016;23:496–508. Gershenwald JE, Scolyer RA. Melanoma Staging: American Joint Committee on Cancer (AJCC) 8th Edition and Beyond. Ann. Surg. Oncol. 2018. p. 2105–10. Seymour L, Bogaerts J, Perrone A, Ford R, Schwartz LH, Mandrekar S, et al. iRECIST: guidelines for response criteria for use in trials testing immunotherapeutics. Lancet Oncol. 2017. p. e143–52. Persigehl T, Lennartz S, Schwartz LH. IRECIST: How to do it. Cancer Imaging. 2020. Malek TR, Castro I. Interleukin-2 Receptor Signaling: At the Interface between Tolerance and Immunity. Immunity. 2010. p. 153–65. Jiang T, Zhou C, Ren S. Role of IL-2 in cancer immunotherapy. Oncoimmunology. 2016;5. Damoiseaux J. The IL-2 – IL-2 receptor pathway in health and disease: The role of the soluble IL-2 receptor. Clin Immunol. 2020;218:108515. Atkins MB, Lotze MT, Dutcher JP, Fisher RI, Weiss G, Margolin K, et al. High-dose recombinant interleukin 2 therapy for patients with metastatic melanoma: Analysis of 270 patients treated between 1985 and 1993. J Clin Oncol. 1999;17:2105–16. Li Y, Strick-Marchand H, Lim AI, Ren J, Masse-Ranson G, Li D, et al. Regulatory T cells control toxicity in a humanized model of IL-2 therapy. Nat Commun. 2017;8:1–12. Boyman O, Sprent J. The role of interleukin-2 during homeostasis and activation of the immune system. Nat. Rev. Immunol. 2012. p. 180–90. Brusko TM, Wasserfall CH, Hulme MA, Cabrera R, Schatz D, Atkinson MA. Influence of Membrane CD25 Stability on T Lymphocyte Activity: Implications for Immunoregulation. PLoS One. 2009;4:e7980. Kohli K, Pillarisetty VG, Kim TS. Key chemokines direct migration of immune cells in solid tumors. Cancer Gene Ther. 2021. Groom JR, Luster AD. CXCR3 in T cell function. Exp. Cell Res. 2011. p. 620–31. Kastenmüller W, Brandes M, Wang Z, Herz J, Egen JG, Germain RN. Peripheral Prepositioning and Local CXCL9 Chemokine-Mediated Guidance Orchestrate Rapid Memory CD8 + T Cell Responses in the Lymph Node. Immunity. 2013;38:502–13. Kuo PT, Zeng Z, Salim N, Mattarollo S, Wells JW, Leggatt GR. The role of CXCR3 and its chemokine ligands in skin disease and cancer. Front. Med. 2018. Bridge JA, Lee JC, Daud A, Wells JW, Bluestone JA. Cytokines, chemokines, and other biomarkers of response for checkpoint inhibitor therapy in skin cancer. Front. Med. 2018. p. 351. Yamazaki N, Kiyohara Y, Uhara H, Iizuka H, Uehara J, Otsuka F, et al. Cytokine biomarkers to predict antitumor responses to nivolumab suggested in a phase 2 study for advanced melanoma. Cancer Sci. 2017;108:1022–31. Chow MT, Ozga AJ, Servis RL, Frederick DT, Lo JA, Fisher DE, et al. Intratumoral Activity of the CXCR3 Chemokine System Is Required for the Efficacy of Anti-PD-1 Therapy. Immunity. 2019;50:1498–512.e5. Khan S, Khan SA, Luo X, Fattah FJ, Saltarski J, Gloria-McCutchen Y, et al. Immune dysregulation in cancer patients developing immune-related adverse events. Br J Cancer. 2019;120:63–8. Koguchi Y, Hoen HM, Bambina SA, Rynning MD, Fuerstenberg RK, Curti BD, et al. Serum immunoregulatory proteins as predictors of overall survival of metastatic melanoma patients treated with ipilimumab. Cancer Res. 2015;75:5084–92. Tokunaga R, Zhang W, Naseem M, Puccini A, Berger MD, Soni S, et al. CXCL9, CXCL10, CXCL11/CXCR3 axis for immune activation – A target for novel cancer therapy. Cancer Treat. Rev. 2018. p. 40–7. Burns JM, Summers BC, Wang Y, Melikian A, Berahovich R, Miao Z, et al. A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development. J Exp Med. 2006;203:2201–13. Croce CM. Causes and consequences of microRNA dysregulation in cancer. Nat. Rev. Genet. 2009. p. 704–14. Chan SH, Wang LH. Regulation of cancer metastasis by microRNAs. J. Biomed. Sci. 2015. p. 1–12. Michniewicz AG, Czyz M. Role of mirnas in melanoma metastasis. Cancers (Basel). 2019;11. Motti ML, Minopoli M, Di Carluccio G, Ascierto PA, Carriero MV. Micrornas as key players in melanoma cell resistance to mapk and immune checkpoint inhibitors. Int J Mol Sci. 2020;21:1–16. Varrone F, Caputo E. The miRNAs role in melanoma and in its resistance to therapy. Int. J. Mol. Sci. 2020. Neagu M, Constantin C, Cretoiu SM, Zurac S. miRNAs in the Diagnosis and Prognosis of Skin Cancer. Front Cell Dev Biol. 2020;8:1–17. Mirzaei H, Gholamin S, Shahidsales S, Sahebkar A, Jafaari MR, Mirzaei HR, et al. MicroRNAs as potential diagnostic and prognostic biomarkers in melanoma. Eur J Cancer. 2016;53:25–32. Lorusso C, De Summa S, Pinto R, Danza K, Tommasi S. miRNAs as Key Players in the Management of Cutaneous Melanoma. Cells. 2020;9:415. Guo S, Guo W, Li S, Dai W, Zhang N, Zhao T, et al. Serum miR-16: A Potential Biomarker for Predicting Melanoma Prognosis. J Invest Dermatol. 2016;136:985–93. Ohira T, Naohiro S, Nakayama Y, Osaki M, Okada F, Oshimura M, et al. MiR-19b regulates hTERT mRNA expression through targeting PITX1 mRNA in melanoma cells. Sci Rep. 2015;5:1–9. Huo J, Zhang Y, Li R, Wang Y, Wu J, Zhang D. Upregulated microRNA-25 mediates the migration of melanoma cells by targeting DKK3 through the WNT/β-catenin pathway. Int J Mol Sci. 2016;17. Jiang Q-Q, Liu W-B. miR-25 Promotes Melanoma Progression by regulating RNA binding motif protein 47. médecine/sciences. 2018;34:59–65. Supplementary Files Additionaltable1.pdf Cite Share Download PDF Status: Published Journal Publication published 31 Mar, 2022 Read the published version in Scientific Reports → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-753314","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research","associatedPublications":[],"authors":[{"id":42269969,"identity":"80ba7f51-193e-4156-be6b-0ba827f78ff7","order_by":0,"name":"Maria Luigia Carbone","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDUlEQVRIiWNgGAWjYJCCAwwFDDwMzBBOAhAzPkhgk2Bgw6vFAFULswFYCz49QC1wANICtQKHFt32sw8P/DA4LCPvzvyA4UfNvTx+6eZjFQ/KLBj45BuwajE7k25wsMfgMI/hYTYDxp5jxcWSc46l3Ug4h9thZgfSGA7wgLQ0MxgwM7AlJG64kWN2I7ENj5bzzxgO/gFrYf/AzPAPpCX/WwFeLTfSGA6DbJFn5jFgZmwD28LGgF/LM4bDMgbpQPU8BQd7+xKKJWekGUsA/cLDxpaAw2FpzB/fVFjby/cf3/jgx7eEPH6J5Icff5TVyck3H8BuDQwYHADFKRLgwa8eCHBEwigYBaNgFIwCBgYAa2dX6vqk5U8AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-3350-1210","institution":"IDI-IRCCS","correspondingAuthor":true,"prefix":"","firstName":"Maria","middleName":"Luigia","lastName":"Carbone","suffix":""},{"id":42269970,"identity":"34d2413f-8d0e-4fc5-82a9-17e6548e3f67","order_by":1,"name":"Gabriele Madonna","email":"","orcid":"","institution":"Istituto Nazionale Tumori IRCCS Fondazione Pascale","correspondingAuthor":false,"prefix":"","firstName":"Gabriele","middleName":"","lastName":"Madonna","suffix":""},{"id":42269971,"identity":"4cd1cd7e-2e7c-481f-ade9-557ba7270902","order_by":2,"name":"Marianna Bove","email":"","orcid":"","institution":"Istituto Dermopatico dell'Immacolata IRCCS: Istituto Dermopatico dell'Immacolata Istituto di Ricovero e Cura a Carattere Scientifico","correspondingAuthor":false,"prefix":"","firstName":"Marianna","middleName":"","lastName":"Bove","suffix":""},{"id":42269972,"identity":"0101871a-fa75-44b4-869b-fe8e3b606423","order_by":3,"name":"Simona Mastroeni","email":"","orcid":"","institution":"Istituto Dermopatico dell'Immacolata IRCCS: Istituto Dermopatico dell'Immacolata Istituto di Ricovero e Cura a Carattere Scientifico","correspondingAuthor":false,"prefix":"","firstName":"Simona","middleName":"","lastName":"Mastroeni","suffix":""},{"id":42269973,"identity":"3712af40-da53-4317-a4f7-00b1a509af7a","order_by":4,"name":"Lauretta Levati","email":"","orcid":"","institution":"Istituto dermopatico dell'Immacolata IRCCS: Istituto dermopatico dell'Immacolata Istituto di Ricovero e Cura a Carattere Scientifico","correspondingAuthor":false,"prefix":"","firstName":"Lauretta","middleName":"","lastName":"Levati","suffix":""},{"id":42269974,"identity":"ea371f71-09c9-406b-9373-dc4540ce45d5","order_by":5,"name":"Mariaelena Capone","email":"","orcid":"","institution":"istituto nazionale tumori IRCCS fondazione Pascale","correspondingAuthor":false,"prefix":"","firstName":"Mariaelena","middleName":"","lastName":"Capone","suffix":""},{"id":42269975,"identity":"431cbcfc-be3b-4eb7-b8aa-8f6d7d85396e","order_by":6,"name":"Paolo Ascierto","email":"","orcid":"","institution":"Istituto Nazionale Tumori Istituto di Ricovero e Cura a Carattere Scientifico Fondazione Pascale: Istituto Nazionale Tumori IRCCS Fondazione Pascale","correspondingAuthor":false,"prefix":"","firstName":"Paolo","middleName":"","lastName":"Ascierto","suffix":""},{"id":42269976,"identity":"babef3ff-3481-499e-a785-4256f54f0b33","order_by":7,"name":"Federica De Galitiis","email":"","orcid":"","institution":"Istituto Dermopatico dell'Immacolata IRCCS: Istituto Dermopatico dell'Immacolata Istituto di Ricovero e Cura a Carattere Scientifico","correspondingAuthor":false,"prefix":"","firstName":"Federica","middleName":"","lastName":"De Galitiis","suffix":""},{"id":42269977,"identity":"db29b3b2-8e91-46e6-9fd1-a8c733c5a5cc","order_by":8,"name":"Stefania D'Atri","email":"","orcid":"","institution":"Istituto dermopatico dell'Immacolata IRCCS: Istituto dermopatico dell'Immacolata Istituto di Ricovero e Cura a Carattere Scientifico","correspondingAuthor":false,"prefix":"","firstName":"Stefania","middleName":"","lastName":"D'Atri","suffix":""},{"id":42269978,"identity":"8a2c4bfc-cc41-46ce-b6cc-b229cd3f6d55","order_by":9,"name":"Cristina Fortes","email":"","orcid":"","institution":"Istituto Dermopatico dell'Immacolata IRCCS: Istituto Dermopatico dell'Immacolata Istituto di Ricovero e Cura a Carattere Scientifico","correspondingAuthor":false,"prefix":"","firstName":"Cristina","middleName":"","lastName":"Fortes","suffix":""},{"id":42269979,"identity":"aa70e8cd-ee65-4ec8-a58b-1a2977da40ef","order_by":10,"name":"Cristina Maria Failla","email":"","orcid":"","institution":"Istituto dermopatico dell'Immacolata IRCCS: Istituto dermopatico dell'Immacolata Istituto di Ricovero e Cura a Carattere Scientifico","correspondingAuthor":false,"prefix":"","firstName":"Cristina","middleName":"Maria","lastName":"Failla","suffix":""}],"badges":[],"createdAt":"2021-07-26 11:05:42","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-753314/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-753314/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-022-09373-9","type":"published","date":"2022-03-31T11:53:46+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":11957629,"identity":"407e1370-a5ba-4fb9-a70d-ffec75bf4a99","added_by":"auto","created_at":"2021-07-30 15:25:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":119645,"visible":true,"origin":"","legend":"Serum levels of CD25 and CD27 in melanoma patients treated with CPIs. Protein amounts were analyzed by ELISA in serum samples of melanoma patients before (T0) and after 1 month (T1) or 3 months (T2) of treatment. Best overall response according to iRECIST criteria: iPD, progressive disease (n. 13 patients); iSD, stable disease (n. 16 patients); iPR, partial response (n. 17 patients); complete response, iCR (n. 11 patients). (a, b) CD25, (c, d) CD27 amount (pg/ml). Data are expressed as mean value ± standard error of the mean (SEM). *p \u003c 0.05, as assessed by Mann-Whitney U test to compare between-group differences; or by Wilcoxon signed-rank test to evaluate before-after treatment differences.","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-753314/v1/9cdb56bf42af82ae4ad73896.png"},{"id":11957630,"identity":"4e04e9b2-4852-45e2-bfba-63efa56e8f4c","added_by":"auto","created_at":"2021-07-30 15:25:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":163177,"visible":true,"origin":"","legend":"Serum levels of CXCL9, CXCL10, and CXCL11 in melanoma patients treated with CPIs. Chemokine levels were analyzed by ELISA in serum samples of melanoma patients before (T0) and after 1 month (T1) and 3 months (T2) of treatment. Best overall response according to iRECIST criteria: iPD, progressive disease (n. 13 patients); iSD, stable disease (n. 16 patients); iPR, partial response (n. 17 patients); complete response, iCR (n. 11 patients). (a, b) CXCL9, (c, d) CXCL10, (e, f) CXCL11 amount (pg/ml). Data are indicated as mean value ± standard error of the mean (SEM). *p \u003c 0.05, as assessed by Mann-Whitney U test to compare between-group differences; or by Wilcoxon signed-rank test to evaluate before-after treatment differences.","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-753314/v1/a0cdfd065d7b87ca322ac970.png"},{"id":11957632,"identity":"675718aa-2fe5-4b1f-b9bc-a40f0a5ab3ab","added_by":"auto","created_at":"2021-07-30 15:25:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":298379,"visible":true,"origin":"","legend":"Plasma miRNA expression in melanoma patients treated with CPIs. Quantitative RT-PCR analysis of (a) miR-19b, (b) miR-25, (c) miR-16, and (d) miR-574 expression in plasma samples obtained from melanoma patients, before (T0) and after 1 month (T1) and 3 months (T2) of treatment. Best overall response according to iRECIST criteria: iPD, progressive disease (n. 5 patients); iSD, stable disease (n. 8 patients); iPR/iCR, partial response/ immune complete response (n. 16 patients). The data were normalized to the level of cel-miR-39 in each sample and expressed as 2-ΔCtx104 values. Dots represent each patient values; largest bars, arithmetic mean; small bars, SEM. Stars indicate patients who had developed leukoderma at the data cut off.","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-753314/v1/aad7d82131eb008b9cfde23d.png"},{"id":19815713,"identity":"4f85b383-8479-4011-9509-43e1f5ca67fd","added_by":"auto","created_at":"2022-03-31 11:53:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1130882,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-753314/v1/ea6e02d8-422f-4f3f-8522-0f1365c91d83.pdf"},{"id":11957631,"identity":"c6ce6c86-4812-4cbf-a2bd-df6063ea8dbb","added_by":"auto","created_at":"2021-07-30 15:25:16","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":86233,"visible":true,"origin":"","legend":"","description":"","filename":"Additionaltable1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-753314/v1/75ada2c9923f8a1a83521a3c.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eVitiligo-Specific Soluble Biomarkers as Early Indicators of Response to Immune Checkpoint Inhibitors in Metastatic Melanoma Patients\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eIdentification of biomarkers for predicting response to therapies and monitoring disease progression is a rapidly growing field in cancer research. In the last decade, development of checkpoint inhibitors (CPIs) has represented the most significant breakthrough in cancer therapy, generating major clinical benefit in patients diagnosed with advanced metastatic cancer [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Despite the positive results obtained in around 40% of patients [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], therapy with anti-programmed death (PD)-1 antibodies is hampered by a substantial number of unresponsive individuals. Therefore, it is necessary to study prognostic biomarkers able to identify patients who would respond to therapy, so to avoid exposure of non-responders to potentially harmful side-effects, delaying introduction for them of alternative therapies [\u003cspan additionalcitationids=\"CR7\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Biomarkers reflecting tumor immune microenvironment and tumor cell-intrinsic features, directly obtained from tumor samples, have been already studied as potential biomarkers of response to CPIs in metastatic melanoma, i.e. intratumor programmed death-ligand (PD-L)1 expression, tumor-infiltrating lymphocytes, tumor mutational burden [\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, due to intratumor heterogeneity and dynamic changes in protein expression associated with the tumor microenvironment, these tissue biomarkers are not appropriate for monitoring tumor response during treatment. Additional biomarkers from peripheral blood samples have been studied in melanoma, e.g., neutrophil-to-lymphocyte ratio (NLR) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], serum lactate dehydrogenase (LDH) levels [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], cytokines and lymphocyte populations [interleukin (IL)-6, cluster of differentiation (CD)73, CD8\u003csup\u003e+\u003c/sup\u003eCD73\u003csup\u003e+\u003c/sup\u003e T cells] [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Data about these biomarkers could be easier obtainable and some of them have been already proposed for prognostic and diagnostic purposes.\u003c/p\u003e \u003cp\u003eDuring immunotherapy with CPIs, several immune-related adverse event (irAE) can be developed, in particular dermatologic toxicity associated with inflammatory and autoimmune responses [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These toxicities include vitiligo-like depigmentation or leukoderma and, to a lesser extent, psoriasis, bullous pemphigoid, and lichenoid reactions [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Leukoderma development is the only irAE associated with a favorable outcome in melanoma patients receiving immunotherapy with CPIs [\u003cspan additionalcitationids=\"CR26 CR27 CR28 CR29\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eVitiligo biomarkers, characterizing active vitiligo status early on and in the absence of clinical signs of the pathology, have been previously identified [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. They include an augmented presence in patient circulation of inflammatory molecules such as the chemokine (C-X-C motif) ligand (CXCL)9/MIG [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], CXCL10/IP-10 [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], and CXCL11/I-TAC [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], S100B [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], IL-17A [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], soluble forms of CD25/IL-2 receptor alpha (IL-2 Rα) [\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] and CD27/TNFRSF7 [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Moreover, some microRNAs (miRNAs), such as miR-16, miR-19b, and miR-25, were highly expressed in vitiligo patients, while miR-574 was downregulated, compared with healthy controls [\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eConsidering that leukoderma development has been described in melanoma patients receiving CPI immunotherapy and it has been associated with a positive response to therapy [\u003cspan additionalcitationids=\"CR26 CR27 CR28\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], there is the possibility that immunological mechanisms like those activated in vitiligo pathogenesis could be triggered also in melanoma patients responding to CPIs. If this would be the case, the same biomarkers that identify vitiligo active phase could also represent biomarkers of melanoma patient response to CPI immunotherapy. In this prospective, we analyzed several biomarkers of active vitiligo in serum and plasma samples derived from metastatic melanoma patients treated with anti-PD-1 inhibitors and correlated their expression levels with patient response to therapy.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003ePatients, treatment, and clinical assessment.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis study was conducted according to the Good Clinical Practice Guidelines and the Declaration of Helsinki. The study was approved by the Institutional Review Boards of Istituto Dermopatico dell\u0026rsquo;Immacolata (IDI)-IRCCS (510/3, April 2018), and Istituto Nazionale Tumori-IRCCS, Fondazione \u0026ldquo;G. Pascale\u0026rdquo; (20/14 oss, July 2014). All patients enrolled in the study provided written informed consent. This study included fifty-seven patients with unresectable metastatic melanoma, stage IIIc or IV based on American Joint Committee on Cancer (AJCC, 8th edition) staging [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], enrolled for treatment with CPIs (anti-PD-1) at IDI-IRCCS or Istituto Nazionale Tumori - Fondazione \u0026ldquo;G. Pascale\u0026rdquo; since June 2014. Patient characteristics, blood analysis, clinicopathologic and demographic information were collected. Peripheral blood samples have been sequentially collected before therapy onset and up to one-year therapy, disease progression or leukoderma appearance. Baseline evaluation included medical history, physical examination, and radiologic tumor assessment with computed tomography scans. Nivolumab (Opdivo\u0026reg;) was given at the dose of 240 mg every 2 weeks or 480 mg every 4 weeks, pembrolizumab (Keytruda\u0026reg;) at the dose of 200 mg every 3 weeks. Patients underwent physical examination and assessment of biochemical parameters monthly, whereas investigator-determined objective response was assessed radiologically with computed tomography scans approximately every 12 weeks after treatment initiation. Tumor response was classified as immune complete response (iCR), partial response (iPR), stable disease (iSD), or progressive disease (iPD), according to the immune response evaluation criteria in solid tumors (iRECIST) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. iPD includes both the unconfirmed progressive disease, a pseudo progression response to be confirmed in a re-evaluation follow-up visit after 4\u0026ndash;8 weeks, and confirmed progressive disease [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Therapy efficacy evaluation was based on best overall response (iBOR) and progression-free survival (PFS). iBOR was determined as best time point response according to iRECIST. PFS was defined as time from the anti-PD-1 treatment start until diseases progression or death; if no such an event occurred, the closing date was November 11th, 2020.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSerum preparation and molecules quantification by ELISA.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTotal blood samples were collected into vacutainer tubes (cat. no. 366881, BD Biosciences, Plymouth, UK), allowed to clot for 1 hour at 37\u0026deg;C, and centrifuged for 15 min at 1700 rcf at 4\u0026deg;C. Then, serum was aliquoted and stored at -80\u0026deg;C until use. Concentrations of human CD25/IL-2 Rα (cat. no. DY223, R\u0026amp;D Systems, Minneapolis, USA), CD27/TNFRSF7 (cat. no. DY382-05, R\u0026amp;D Systems, Minneapolis, USA), IL-17A (cat. no. DY317, R\u0026amp;D Systems, Minneapolis, USA), S100B (cat. no. DY1820-05, R\u0026amp;D Systems, Minneapolis, USA), CXCL9/MIG (cat. no. DY392, R\u0026amp;D Systems, Minneapolis, USA) and CXCL11/I-TAC (cat. no. DY672, R\u0026amp;D Systems, Minneapolis, USA) were measured using DuoSet ELISA kits (R\u0026amp;D Systems, Minneapolis, USA). CXCL10/IP-10 levels were evaluated with a BD Pharmingen kit (cat. no. 550926, San Diego, USA). The plates were analyzed in an ELISA iMark Microplate Reader (Bio-Rad, California, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCirculating total RNA extraction and quantitative miRNA real-time RT‑PCR.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTotal blood samples were collected into vacutainer sodium citrate tubes (cat. no. 367704, BD Biosciences, Plymouth, UK), centrifuged twice for 10 min at 1260 rcf at 4\u0026deg;C, and the plasma was aliquoted and stored at -80\u0026deg;C. Total RNA was extracted and purified from 200 \u0026micro;l of plasma using a miRNeasy Serum/Plasma kit (QIAGEN, Hilden, Germany), according to manufacturer\u0026rsquo;s instructions. A fixed volume of 2 \u0026micro;l of total RNA was used as input for consecutive reactions including poly(A) tailing, ligation, reverse transcription, and miR-Amp reaction with a TaqMan Advanced miRNA cDNA Synthesis kit (Applied Biosystems, CA, USA). The miRNA levels were then assessed by TaqMan Advanced miRNA Assay with TaqMan Fast Advanced miRNA master mix (Applied Biosystems, CA, USA). Each PCR plate was run in the QuantStudio 5 Real-Time PCR System (Applied Biosystems, CA, USA) according to manufacturer\u0026rsquo;s recommendations. The TaqMan Advanced miRNA probes (5'-phosphorylated) were as follows: hsa-miR-19b-3p (ID 478264_mir), hsa-miR-25-3p (ID 477994_mir), hsa-miR-16-5p (ID 477860_mir), hsa-miR-574-3p (ID 478163_mir). For quality control and analyses of miRNA expression, cel-miR-39-3p (ID 478293_mir, cat. no. A25576, Applied Biosystems, CA, USA) was used as an exogenous spike-in. Expression of miRNAs relative to cel-miR-39-3p was determined using the formula 2\u003csup\u003e\u0026minus;ΔCt\u003c/sup\u003e, where ΔC\u003csub\u003eT\u003c/sub\u003e = C\u003csub\u003eTmiRNA\u003c/sub\u003e - C\u003csub\u003eTmiR\u0026minus;39\u003c/sub\u003e, and C\u003csub\u003eT\u003c/sub\u003e (i.e., threshold cycle) indicates the fractional cycle number at which the amount of amplified target reaches a fixed threshold. To simplify data presentation, the relative expression values were multiplied by 10\u003csup\u003e4\u003c/sup\u003e. All determinations were performed at least three times, each in duplicate.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis.\u003c/h2\u003e \u003cp\u003eGroups based on treatment-response were compared using the Fisher's exact test for categorical variables, the non-parametric Mann-Whitney U test or the Kruskal-Wallis test for continuous variables. The Spearman\u0026rsquo;s coefficient was used to test correlation between levels of analyzed molecules at baseline. To evaluate before-after treatment differences, the Wilcoxon matched-pairs signed-rank test was used. For data analysis, the value of 0.01 pg/ml was assigned to samples with values under the lower limit of ELISA detection.\u003c/p\u003e \u003cp\u003eStatistical significance was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. All statistical analyses were conducted using GraphPad prism Software (La Jolla, CA, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePatients\u0026rsquo; characteristics and responses.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor this study, fifty-seven melanoma patients were analyzed. Considering the iBOR, 28 patients responded positively to CPI therapy with an iCR (n\u0026thinsp;=\u0026thinsp;11) or an iPR (n\u0026thinsp;=\u0026thinsp;17), 16 patients had no objective response (iSD), and 13 patients developed an iPD. The median follow-up time for PFS was 12.2 months (mean 17.1 month, ranging from 1.1 to 61.7 months). At 1 year, PFS was 57.7%, with a median PFS of 33.4 months for iCR, 20.7 for iPR, 9.7 for iSD, and 2.8 for iPD patients. There were no patients lost to follow-up. Fifteen patients developed leukoderma at a mean time of 6.8 months after CPI therapy starting (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003eCharacteristics and treatment outcomes of enrolled patients.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePatient\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eStage\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCheckpoint inhibitor\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eiBOR\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003ePFS\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eProgression\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eLeukoderma\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e1\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eIIIc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiCR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e10.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8.7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e2\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiCR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e40.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e3\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiCR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e13.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e4\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiCR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e12.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e5\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiCR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e21.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.6\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e6\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiCR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e33.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e7\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiCR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e37.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11.1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e8\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiCR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e37.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e9\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiCR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e48.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e10\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiCR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e13.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e11\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiCR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e62.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e12\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e40.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13.1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e13\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e26.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e14\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e20.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e15\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e19.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e16\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e7.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e17\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e11.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e18\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e10.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e19\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e33.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.1\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e20\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e9.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e21\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e8.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e22\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e42.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e23\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e36.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9.7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e24\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e39.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e25\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e38.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7.7\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e26\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e36.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6.9\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e27\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e13.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e28\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPR\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e12.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e29\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiSD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e7.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e30\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiSD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e12.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e31\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiSD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e18.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e32\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiSD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e11.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e33\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiSD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e5.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e34\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiSD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e5.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e35\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiSD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e36.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4.3\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e36\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiSD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e37\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiSD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e5.2\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e38\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiSD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e39\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiSD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e32.5\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e40\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiSD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e6.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e41\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiSD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e26.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e42\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiSD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e15.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e43\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiSD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2.8\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e44\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiSD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e17.3\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eno\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e45\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eIIIc\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e46\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e47\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1a\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e48\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.6\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e49\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.7\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e50\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e51\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.8\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e52\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e2.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e53\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e54\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e4.0\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e55\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e56\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003epembro\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e1.4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cspan class=\"BoldItalic\"\u003e57\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003enivo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eiPD\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"char\" char=\".\"\u003e\n\u003cp\u003e3.9\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eyes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"7\"\u003e\u003csup\u003ea\u003c/sup\u003eStaging before starting therapy: nivo, nivolumab; pembro, pembrolizumab.\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"7\"\u003e\u003csup\u003eb\u003c/sup\u003eiBOR, best overall response according to the iRECIST criteria: iCR, complete response; iPR, partial response; iSD, stable disease; iPD, progressive disease.\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"7\"\u003e\u003csup\u003ec\u003c/sup\u003eData cut off on 11th November 2020. PFS, progression-free survival (months).\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"7\"\u003e\u003csup\u003ed\u003c/sup\u003ePatients who had developed (months) or not (-) leukoderma at the cut off data.\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eWe analyzed patient characteristics and tumor-associated factors at the beginning of the treatment with anti-PD-1 antibodies. As shown in Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e, 37 patients were males (64.9%) and 20 females (35.1%). The mean age at treatment start was 67.3 years. Levels of circulating LDH were found high in 13 patients (26.5%) and were not assessed for 8 patients. The M staging of the extent of metastatization was stage IIIc-M1b for 21 patients (36.8%), and M1c-M1d for 36 patients (63.2%). In particular, stage IIIC for 2 patients, stage M1a for 12 patients (M1a(0) for 8, M1a(1) for 3, LDH not assessed for 1), stage M1b for 7 patients (M1b(0) for 5, M1b(1) for 2), stage M1c for 33 patients (M1c(0) for 20, M1c(1) for 8, LDH not assessed for 5), stage M1d for 3 patients (M1d(0) for 2, LDH not assessed for 1). BRAF gene mutation was detected in 15 patients (26.8%). Thirty-one patients (54.4%) were treated with pembrolizumab and 26 patients (45.6%) with nivolumab. First-line treatment was performed by 32 patients (56.1%), and second-to-fourth line by 25 patients (43.9%). Leukoderma was developed by 15 patients (26.3%). Of those, n\u0026thinsp;=\u0026thinsp;6 iCR patients (40.0%), n\u0026thinsp;=\u0026thinsp;7 iPR patients (46.7%), n\u0026thinsp;=\u0026thinsp;2 iSD patients (13.3%), none iPD patient. Clinical characteristics of the response groups were similar for sex, age, metastasis stage, serum LDH amount, BRAF mutation, drug administrated, and line of treatment. Patients who developed leukoderma were associated with a good response to CPI therapy (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003). Out of 15 patients with leukoderma, 12 (80%) developed it during a first-line treatment.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\u003ccaption\u003e\n\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n\u003cdiv class=\"CaptionContent\"\u003e\n\u003cp\u003ePatient characteristics, treatments, and leukoderma development.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/caption\u003e\n\u003cthead\u003e\n\u003ctr\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eCharacteristics\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eAll\u003c/p\u003e\n\u003cp\u003e(n\u0026thinsp;=\u0026thinsp;57)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eiPD\u003c/p\u003e\n\u003cp\u003e(n\u0026thinsp;=\u0026thinsp;13)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eiSD\u003c/p\u003e\n\u003cp\u003e(n\u0026thinsp;=\u0026thinsp;16)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eiPR\u003c/p\u003e\n\u003cp\u003e(n\u0026thinsp;=\u0026thinsp;17)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003eiCR\u003c/p\u003e\n\u003cp\u003e(n\u0026thinsp;=\u0026thinsp;11)\u003c/p\u003e\n\u003c/th\u003e\n\u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n\u003cth align=\"left\"\u003e\n\u003cp\u003e\u003cem\u003ep\u003c/em\u003e value\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n\u003c/th\u003e\n\u003c/tr\u003e\n\u003c/thead\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSex\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMale\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e37 (64.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9 (69.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9 (56.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12 (70.6)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7 (63.6)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eFemale\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e20 (35.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4 (30.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7 (43.7)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5 (29.4)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4 (36.4)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.866\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eAge, years\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emean (SD)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e67.3 (12.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e62.0 (12.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e67.1 (13.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e73.2 (12.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e64.7 (11.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003emedian (IQR)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e67 (59\u0026ndash;78)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e65 (54\u0026ndash;68)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e67 (60\u0026ndash;79)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e76 (67\u0026ndash;81)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e65 (56\u0026ndash;72)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.084\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026le;\u0026thinsp;65\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e22 (38.6)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6 (46.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7 (43.7)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4 (23.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5 (45.4)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u0026gt;\u0026thinsp;65\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e35 (61.4)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7 (53.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9 (56.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13 (76.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6 (54.6)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.509\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eSerum LDH\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNormal\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e36 (73.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8 (80.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10 (71.4)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11 (68.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7 (77.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eElevated\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13 (26.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2 (20.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4 (28.6)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5 (31.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2 (22.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.970\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eStage\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eIIIc-M1b\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e21 (36.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3 (23.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4 (25.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9 (52.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5 (45.4)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eM1c-M1d\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e36 (63.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10 (76.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12 (75.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8 (47.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6 (54.6)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.263\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eBRAF status\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eMutation\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15 (26.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5 (38.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e4 (25.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3 (18.7)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e3 (27.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo mutation\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e41 (73.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8 (61.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e12 (75.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13 (81.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8 (72.7)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.702\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eDrug\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003ePembrolizumab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e31 (54.4)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5 (38.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7 (43.7)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8 (47.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6 (54.6)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNivolumab\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e26 (45.6)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8 (61.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9 (56.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9 (52.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5 (45.4)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.907\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLine of treatment\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e1\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e32 (56.1)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5 (38.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7 (43.7)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e11 (64.7)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9 (81.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2\u0026ndash;4\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e25 (43.9)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e8 (61.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e9 (56.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6 (35.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2 (18.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e0.117\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eLeukoderma\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eNo\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e42 (73.7)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e13 (100.0)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e14 (87.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e10 (58.8)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e5 (45.4)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003eYes\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e15 (26.3)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e-\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e2 (12.5)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e7 (41.2)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e6 (54.6)\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd align=\"left\"\u003e\n\u003cp\u003e\u003cstrong\u003e0.003\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003ctfoot\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"12\"\u003eTotals may vary because missing values. Data are n (%) unless otherwise stated.\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"12\"\u003eAbbreviation: iPD, progressive disease; iSD, stable disease; iPR, partial response; iCR, complete response. SD, Standard Deviation; IQR, Interquartile Range; LDH, lactate dehydrogenase.\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd colspan=\"12\"\u003e\u003cem\u003ep\u003c/em\u003e value was calculated using the Fisher's exact test (\u003csup\u003ea\u003c/sup\u003e) or Kruskal-Wallis test (\u003csup\u003eb\u003c/sup\u003e).\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tfoot\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eIn a sub-group of 44 patients for whom blood analysis data were available (patients no. 1\u0026ndash;5, 7, 10, 14\u0026ndash;16, 18\u0026ndash;28, 30\u0026ndash;39, 42\u0026ndash;44, 46, 47, 49\u0026ndash;55, 57 of Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), we observed higher neutrophil count at the beginning of the therapeutic plan in iPD patients in respect with iCR patients (p\u0026thinsp;=\u0026thinsp;0.043; Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Moreover, our data indicated higher baseline values of NLR and derived NLR (dNLR) in iPD patients rather than in iCR patients, suggesting that these higher basal levels could associate with a tendency towards progression disease after treatment with CPIs (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3. Analysis of the relationship between neutrophils and lymphocytes before the start of immunotherapy with CPIs.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAqYAAACmCAYAAAAI5E22AAAgAElEQVR4AexdBbhVRRc1EWxUBBFbQOFHVBAUCyxKQmnpkhYR6Q4F6e6Q7u6Qlu5SEZAQ6e566//Wnjv3zL3v3hfw3uPGzPe9N+fOzJkzZ++ZOWv27L3nLthgKWApYClgKWApYClgKWApYCkQABS4KwDaYJtgKWApYClgKWApYClgKWApYCkAC0xtJ7AUsBSwFLAUsBSwFLAUsBQICApYYBoQbLCNsBSwFLAUsBSwFLAUsBSwFLDA1PYBSwFLAUsBSwFLAUsBSwFLgYCggAWmAcEG2whLAUsBSwFLAUsBSwFLAUsBC0xtH7AUsBSwFLAUsBSwFLAUsBQICApYYBoQbLCNsBSwFLAUsBSwFLAUsBSwFLDA1PYBSwFLAUsBSwFLAUsBSwFLgYCggAWmAcEG2whLAUsBSwFLAUsBSwFLAUsBC0xtH7AUsBSwFLAUsBSwFLAUsBQICApYYBoQbLCNsBSwFLAUsBSwFLAUsBSwFLDA1PYBSwFLAUsBSwFLAUsBSwFLgYCggAWmAcEG2whLAUsBSwFLAUsBSwFLAUsBC0xtH7AUsBSwFLAUsBSwFLAUsBQICApYYBoQbLCNsBSwFLAUsBSwFLAUsBSwFLDA1PYBSwFLAUsBSwFLAUsBSwFLgYCggAWmAcEG2whLAUsBSwFLAUsBSwFLAUsBC0xtH7AUsBSwFLAUsBSwFLAUsBQICAokADCNAGD/Qp8GwPnz5/DRRx/iiaRJkerZZ+1fGNDgmRQp8GzKlJbXYcBrjumUzzwjf3Z8h+78xvHMcW15HLo8NnlLXgdasMDUguY4WjgAp06fRO7cubBkyWIcOXIER44ctn8hS4MjOHz4P3xVsCBGjhxh+R2yfDbH8BG0bNkStWrVdPHbjvHQm+OOYM2a1fj000+xectmO65Delxz/B5Bzpw5Aw2XwgJTC0zjDJiePHUCefLkxq5dfwVcR7cNih8KlCheHPPmzY2fym2tAUeBbt26oVHjRgHXLtuguKPAwYMHkCdPHpw4eSLuKrU1BSwF8ufPH3Bts8DUAtM4B6bbt29zdXSrwhG6KhxARMRNFCtaFDNnzrD8Dot5BOjUqRPqN6hvfMjsGA+tMQ7s2bMbuXLlwqH/DtlxHdLjWrH3yy+/NMZzYFxaYBrSHS8hPxqAlphaYJqQdL9Tz7LANLQASUz6kQWmoc9zC0xDn8d6rFtgGkdSOU1QGwfe4LHANPB4Ep/jxALT8OI3+5IFpqHPcwtMQ5/H+rtggakFpiEvmbXANHwmNAVS7Fa+nuDDJbbANPTHuAWmoc9jPV9ZYGqBqQWmtg+EVB+wEtPw+YA5HzKrY6ppEaqxBabhM64tMLWgJKRAia9JOdQkpmrQhs8k5YunUaWFCjCNis86j7GmhU7Tv8MpDmaJaTjzLTZ9NBSAaUx4rcvoWNPI+7dOD8VYvas1fnJP7qHI5HB/p1ACpnpyupWY/SA2IVj7TagBU/LM5IXi4dWrV3HjxnUvhupyXsl+f+rywR6HAjAlk8iH2IRbKR+svA52YOrNV28+mPkRuHnzhplwG9fezwmG3+p1LTD1mPiDgXG2jZ4f66joESrAFPj7712oUKECevbogZs3b8ohARMmTkDnTp3QuXMndOnSGd27d0O3bl3Rvn07zJkzR5zN61nt2PFjmDJlslO+c2d069oVPbp3R6fOnTBx4gRxyaLLx5zGUdE/ofNCAJhGRGD8+HH45psSWLVqpYsdpCOwa9cuNGncGM2aN0O7du3Qs2dP/PvvQaxfvx579+6RMitX/o7efXqjU8eO0ifYL3r06I4unTujf/9+WLVyJa5eveKql1FC8yiunxeswBSYMX06SpQogcWLFwk/6Gt5xIjh6NTJ4F337q4x2xmLlyzG2bNn3Lz755+9GDbsV3Ts0MHF6y4ynnv17IlffmkvfP/tt4U4ceK46564pn1C1RfMwBTYuHEDSpYsieHDhyEiwqSZYgsXmbNmzUKtmjVljq9RowZ+atsGkyZNwrlz57B69Sr8sXMHLl68iOkzpqux3bmzjGn68O3Vq6d8A35p3w5Tp07B7r//dveR4BvfqukWmAb9xGx2dHvtORBDB5g2b94cd911F5ImfVwOC7h06RL27t2LokWLSDrz6tSpg4ULF0j81FNP4eWXXkL37t0FfFy+fBn/7PsHpUuVcpf/Mt+XWLp0KYYOHYq8efPi6WTJUOXbb3HgwIEg/ZAFOzAFzpw+jTSpUwuPChhOpvlxS5cuHRo0aIBDhw7J37hx45A/Xz5kzJgRO3fuEJ4dOXoU48aNxf333Sd1JH7gAYwZMxozZ85Ew0YN5ejOrFmzym/n6xXM80YwAlPgypUryJYtm/CI/jkpJTtz9gx27NiBNzNmlPT77rsPXbt2xYwZM1Cy5Dd47LHH8NabGTFhwgRhHUHLtm3b8E7mzC5eJ0avXr2wYsVyjBw5Uvx+PpgkCd7IkEGAj+J3MPI6WIGpojh5x/n5uVSpcPToEWfYAdi8aaOccvTQgw+CB4MsWLAAu3fvxvp162Tx+fHHH+PVV17B0qVLcP36dezfvx+1a38n9bHO8uXLY+Xvv0sfqVmzJpK6jt5u3boVrl+/FoQLT0UeC0wtMA3CzhvTyTV0gOmSJUvw9ttvoWLFCjh/7qx7cvulfXskuv9+PPLww/j111/d6dWrVcPdd92FJEmSYKLrQ8bM/v36gWCFH6x69eu5y586dQo5smeXCe/9bNmwb98/rryY0joQygU/MKX0pHnzZkiXPh1GjRolPLh27So4UfP8aHodMMPaNWsEeGzfsd2dfPLkCbz04ot4/LHH5Gxx82NI6ek9d9+Nxx59DEOGDHHdEwi8u9U2BCcw5a4HJaPp06dD3759XJI0xY7ixYrJwiJFiuRyFKdKjcDnn38uYzr500+LFE4zvFq1KsJTnjW+efNmnYzz588J+OU88Pxzz2OHu4/cKq3v1H3BCkxJL2Dy5MnI8L//oV69H3HtGsGiCiuWL5dxSoDZtGkTnewRT5o4EU88nhTLli11p0+fPk3me877Xbt2cafzon69etJHmDdkyGBX3p3i2608VzXZAlMLTC0wDfg+4DH3ePxo26YNHkiUSCaqgQMHuvMmTpwowISSs7JlyrjTuc2XJHFicIVe54c67nRezJs3D0888YR85Hh6ElfoKtzKBHMn7gl+YOrBENeP3bv/RrKnnhKJNrdmvQO389auW+tO/u+/Q3jxhRfcwHT//n3uPILcT3PkwH333osUyZNj9erVrrw7wa+4eGYwAlO+t/9QpHBhWWwSmJqApFXLVjLWOX6prqND5cqVcO8998giZM2aNTpZ4hYtWshYv//e+9Cliwli4oL2CVVHcANTD4a4fhw7ehSZ3n5bBAHZ3ntXFhEqy6SpSmlQv75xkh1E7YqCCM77HTr84lH94sWL8eSTT0penty5DX1Vs95AvlavY4FpwIOSQO5Egd620JCY/vnnH6KPtmTJYixbtgznDImpP2A6fvx4ASYEIBXKl3dPXlEB0zNnTuN/6dPj0UcewZNPPCm6TerGQOezbl9wA1PqflLfcP68eVi8aBE2btgg5Kf0mtuA/BilfvVVDBgwAGfOOLqGBJ48S1wH/8CUdALatGmNhx96SKRydb7/Xt8WpAvU4AOm3LZfvnwZ5syZLfymmoYpCfcHTFu0aI7EiRLJjkfHjh3dfIsKmLZp0wYPJXkQiRM9gNZtWrvv8VR50uMnUOPgBaYbN27ErJkzZSueEtIrVy4LDzp06CDjjztXjRo2dPHFF/0hku4dLlUdFqQ9gD9gyoUMF7FcvGT/+GNDQuur7kBMU6SwwNQC0yD9IMVkUIUCMKXh09/g9jq3fJIlS4atW7e4JjJAA1MCjeHDh7vTq1WrJuWTPv44Zs2a6U6PCphye/Grr74S0EKJaseOHVz3xYTWgVAm2IHpVdENJM/I61w5cwr9yRcuLu67514wj7x57733MHjwYFB32AnkARAdMB09erQsPh579FGR2ly8cMFVRSDwMLZtCEZgehPTpk7FU089KXz+4osvcN3wsmAC03UuSTiBa65cOaX8Cy+8gG1bt7rZbgLTTZs2udN5T86cOUV6xvlh/vz5QcrnYAWmEMPE9OnSCd+ef+45GZtkwieffCLzLMfgmLFjouGLZqka3xqYcruexo1m4KKTklSqb3iqB8R2XN2p8uptLDC1wNQC04DuA2qg/vRTW1lhU3dw+/Zt7rlIA1PqEzZu3FiMmX744Qc8kTQpMr7xBsaO0ZOeuiUqYMoSlStXdktY+MFT4U5NUrF9bnADU9L6woXzePutt+TD8vVXX7norxYnH374gWzxkrePPvywfNjy5smDTRs3GnyKHphSUsf+QpBLAzlad6sQW3oHQvngA6aa1l98oXRGCxYsGAmYElxwK79vv75Y9NtvKF68mEjJaAyzaJGy4ldST47ZSqKa8WzKlOBOCY0cKan7/vvv8fDDD+PJJ57AT23bBuG2ru5fwQtMyevy5crJeH79tdfEQ8KRI4dFt5Tjj38LFsRmweBITCltrV+/Hvbs2YM///wTffr0xvPPPy8LVxpOUl1A97XgkZCrFltgGtCgRA9MG9/awAoNiSmHasuWLQSUEEj4Aqb8+JQpXVpcCPXu1QsL5s8HDZq8Q2yAaYUKWgUgWPpf8APT48eP4a033xQ9XxOYko/Hjx9HvXr1kDJlStnOJc9pyPbqKy8bhi3RA9O5c+d4ANM9e3a7ukmw8NlsZ3AC00uXLuKzzz4VPvsCpuQrdYBr166Nnj17YED//rL9f/nyJS9eKWBKXXIC0yZNm+Lrr78W40ZK1DJnzoSNm/TChbeatAuW6+AGpvSEQoPDdK+/DhomUjXnheefF1BKYDpv3lwvnkbFFweYUuWqTJkyqFu3rtgGcPv+6aeTYfiwYbjuNrKKqq5AzFOksMA0KAdqIHaoQGxT+ABT761810xnfIhUSnTAtFTJkrLi5nbxTz//FIsJMxD4H5rAlJa8pu/RDRs2oGrVKqB1NrcCKV1T0m3yIHpgOnnyJLmP99I1kaOzHAg8jG0bQhOYElRSYrp2racxkxqQJo0UMKXxE/WQd+7cCfqyfTrZ0yJVf+6557DQbTBn3hdM16EDTI8dOyqGTgSp3LWQrfwxo2MxzzrAlOOeXjZOnzot+qRJHnhA6mzWrKmrPkbBxGe2VQULTIOOccHW0e5ke8MHmFIZ3rHK90VzNeCjAqaU4mTNkkX0D7mSp4GGCr7qC8S00ASmBKJjRuuPl4slAGiBSx+mBDHvZs0K8o8hOh1TWmdzIcP7alSv7lQYlHNhaANTxyrf33hzgCndRa1bt074ycMUHkzyoGz/0w8mD2dQwV89gZweOsCUY5OhUqWKsqCkAKBF8+bR8MaVLePTE5hqq/wtmzeDOqz8DnBsj3a5mrPAVNPu9uO7br+K6GoI5EFo2xZ3g8kCU4eWakxEBUzXrF4txlXcdir0dSHQtZAKwdInQxWYrgcNYpxAfqgwc+YM0BE7dQ81v/wDU94TAeqfUcJGVQDP06WChc9mOy0wNY2ftLsoGj7R+TpVAihJ+6pgQbdFuDMnmHQM5OvQAaY8qY2Bi36OP0o96T+axwyrYPJBpdBbCnXPddDGT7xXA1Pm0fiV2/v8oy0CD19Qwawz0K9Vi63ENCilBIHeuQKlfaEDTNu2bSMGDtQx3bXrT9eEA9DBPvXLuEoeNmyYOz3yx0dlDejXXyZD6iOZDvaZW/IbdUIJ/ev99ad+RqDwMibtCH5gyo8QjZ9olV+4UCFhGt2FPfLQQ+jfv7/BX3W5fv060V/jUbQ6UI+Nfkz5gaIUjXqrOhDI6v7Co0udEBP6BmKZ4ASmPJWHxk/kcyEXnzUv6ENY/MymSB6NyzZ1B9U6aIVNXm/a7Fjl8xjirO9mFdUcgphWLVvoRwTZFm9wA1P6kSaf07/+usfJTzRcTXR/IhmP/fv1NXjjXG7ZskVOezPHMB3sc77nOPZ2sP/dd7VkfueCJGfOL+QYU1VbII5dX21SrbXA1ALTIJukfHVmf2mhAUzPnjsrHy9ObrSy5VnqdBN04sQJUX5nOqWctMKlf0vlGN+TJkyj8UylihVlkqTEjC5LVq1eLVvCPIo0VapUqFq1qvvc9cjg1rPOwMsPbmBKt1ArV67EMymeER5lzpQJJ44fl0UCj4vNnj076L/yjz/+wNmzZ0HXQJzAeZQht/F5DjfP0549a5bbzyE/YDwBZv2GDejduzfSpk2L999/H5OnTHG+fkE9BwYfMCWfKM166aWXhM8ZMmTA7j275ZhSHgec7b33JP2BBxLJqVAXLlxwWdR7jj9K2WiRrV3JEYz06NEDLK/9oi5ZvFgOZuB4pxW342LMs67AG8tm+4IXmB47dgwfffih8JOO73lABsc5A/tBnz59ZAuei8imTZqIKga9ZKxfv14MWeklhSo7DDdu3MDpM6dBryuc7znv01vD4cOH3Yeh8Jvw4Ycfyo4I8ytVriT5UkFQjHPVUgtMg4JZ5iC11zGfREMDmHKSor+6bt27oXPnThgxYgSodzht2lT06NkD3bt3AyVmPXv1xOQpk3HkCM9j9uwnh/47JEfjSdnu3dC9R3epc8DAAejXrx9GDB8OnjDkBM/7vesLzN/BDUwvXryAcePHCY/Jp85dOsuWH63mx44dK14WeE46P2D1fvxRLPR5bCmPMWWgFG7psqUYOHCASFLI467dugpYISDp27cvli1damwb8q5g5LPZ5uADpjRkmzJ1Cjp26ijGK4znzpsrx4mOHTdWxmb37hyfXdCvX1/MmTPHZaBmvncE/vrrT4wcNRJdunaRejhH0GXQnLlzjG17yDnqPP60U+dOMuZpIKWCZ32B2xeCF5jyKGnyRY/nUaNHGac8KS7wcAwCVO5YlS9fDrVrf4fmzZphzJgxsgB1MQsnT57E9BnT5TvA+V7P+xMmjPc4YGPH9u3StyhNbdfuZ8yfPy+I+K2aaoFp0E/MwTK53Il2hgIwJd1uJXjTOzZ1eN8bLL+DG5j651DkPuC4DdJ3aR7p39HFunywx8EHTBUAjI4/vvK9eeWrjE7TZfVv71jnB0McrMCUtPUXNN0987m45J9n8F3Wswx/RVVO5wV6rN7KAlM3MwOdYbZ9zsCLKS1CBZjG9H3DvVywA1N//NOfIJ2vf5sfI50XbnGwAtNw49PtvG8wA9PYvLcd15oCFphaYGqstGIziIKhrAWmsQfzwcBXf20MVWDq731tOj9knTp1Qv0G9fU3LYTns3Dld7gA03Dlr/neahhbYGqBaQhP5BaYWmBqTnr2OvT6gwWmocdT73FqgWno81jz3ALTEAZkmsnhHltgGj4TGvu6lZiGF78Vz63ENNTneQtMw2dcW2BqgWnIS4stMA2fCc0C0/DitQZjVmIa+ny3wDT0eeyMZ0LTMN3KV6jc/g99Cly7fhX58n2Jffv2hv7L2jcUCtDtyqJFv1lqhAkFevbsiaYe54OHyYuH0WsePXoEefPmxbnz58LorcP3VQsUKBhwLx/vR5LOnDUTY8aMxqhRI0G/YvYvNGkwevRoDBjQH5kzZ0KHjh0wmjy3/A5ZGpDf9Ov44QcfoH79euIH0PI7NMe25it9PZYuXQoFChSw4ztE5zbO2926dUOmTJnA08rsPB66Y1rP4VmyZAk/YJrsqadQrFhRcWbL48LsX2jSoHy5cihevDjSpE6Dr7/+GhXKl7e8DuH+Xq5sWZQtWwbp06XDF198Iadc2bEdmmNb85UnmX3w/vt45513ULFCBeG/zrNxaPCeTueLFCmMNKlTo2hR+90O5X6t5/DXXksbfsD0mRQpPM6PDjgK2AbFGQXOnjuDvHnzgOeN2xAeFOBiZPbs2eHxsvYt0bVrVzRq3MhSIoQpsH//PuTOnRtHjh4N4be0r6YpkD9/fn0ZMHG8b+WnevZZHDtmO3jAcDweG3L6zCnkyZMbO3fuiMen2KoDiQLFixXD7NmzAqlJti3xSIHOnTujQcMG8fgEW/WdpgDPj8+VKxcOHzl8p5tin58AFAhL4ycC0yPuDq6twWwcepZ/1io/9Hga1Ti17qLCi9/sC9YqP/R5bq3yQ5/Hel5XqNcC05B3maQZHo6xBabhM6EpkBIRcRPFihbFzJkzXOv6cOz34fTOFpiG/hi3wDT0eaznLAtM7Ycr5EG5BabhM6FZYBpevHY+ZNbBvqZFqMYWmIbP2LbA1AJTC0ztIQsh1QfsVn74fMA0CLMS09DnuQWmoc9jZzwTmNmt/JD6MGvm2lgNZCsxDZ8JjX3eAtPw4rfiuZWYhvp8b4Fp+IxrJS+0wNQC0xCWGlpgGj4TmgWm4cVrDcasxDT0+W6Baejz2BnPVmKqZCwhDMw0s8M1tsA0fCY09nErMQ0vfiueW4lpqM/vFpiGz7iWadxu5YcPw0N98vL1fhaYhlf/tsA0vPhtgWl48NsC0/DgsxrPhKZ2K99u5YewxNgC0/CZ0NSkZt1F+VqghXKa3coP/TFugWno81jPUVZiqijgAUxdSe5IE4uxGcz0YLnW7Y+qvWYZfc04qnsCOc8C0+Dl3a30KysxDS9+s49YYBr6PLfANPR5rOd7hTusxDQS6FKEcf4rglESo4O61oS8ndhdY7yCP7Pt6om+2gycPXMG165fc7VFt03Hvu4JhLSo2meBafhMaOyLFpiGF78Vz62OaSDMw/HZBgtMA3tcy8zrhWF8fZd9pXn3G1XGAlMBpsDly5cwcOAAFCxYALly5USHjh00FXHlyhXMmDEDXxUsiM6dO+Ho0aOuPG+ixvY3cPXqFS+GxraO6MoDhw//h+rVqiFnzpxYtOg3H20Htm3birRp0qBxk8aSP3/+PDljvmrVqjh79oyPe6J7bkLkA9euXcWNG9f90NAC08Ce0OK6j1hgGl78Zv+xEtPQ57kFpoHJYwUL+A2+efOG8Q1W32VvAd6VK5ddOIKRv7lfFbHA1E0g4PTp03gnc2bcddddePDBBzF06BA3IQlcK1asiC2bNyMiwh9RY5MOzJ41C/PmzXU9Izb3xqYsP9YRKF6smLzXiBHDfTwP2LRpI55NmRI//FBH8v/66y888vDDSJ36VRw/fszHPbFpQ3yUBS5cOI8BAwfg4sULftpngan/CSA+eHKn67TANLz4zf5mgWno89wC08Djsfrkrly5EnPnzsWNGwSmKhCAFi1SBPPmamyj0v/44w+MHj0aFy9edJX09b1QWRaYGsCUzK/z/ffIkiULEt1/P55LlQokvA69+/TGoUOHDKLqHDNWk6WZ4nmt8vft24c3MmTArFkzPbN9/oqqzqjynMoqV6okwHT8+HFOolw591+9QumtCnv37kaqZ5/FW2+9iRMnjrtSWdb806XNWOebafo6qjyzjL72FbMOFerXq4evv/5a//Rqmyp38tQJkfxu377NVU63Ia5joxnuS+9nuDOMi6jKeOfxt6/gq9ztpvl6jpmm6zfT9LXOYxxdMMve7nVCAlNf7+Xd/tst4+t+pnk/J65++3qed91xVca73lv9nRDA1Hxnf+2MqkxUebo+s4y+1nnxFevnMPZ+hpkXk3xfZbzrvNXfcQVMvd/JX5tjUi4mZfT76rL6d1zHun4z9n6Gmaevb6WMvgcYP348unXrinPnzkmFxAzEM+fOnUWG//0Pc+fOwcZNG/H77ysknwKyeXPnoUGDBjh//ryrEbo+HatkC0zdA1Jt57dt0wbr1q1F+nTpcM/ddwt43PfPP0Ktrt274sCBAy6CAgcPHkT37t3RsmVLLFm8WNJvXL+OOXNmo2u3rpg5ayYuX76MsWPHolv3rm4Gbd26FR+8/76A32rVq2PKlCk4c+Y0xo4bK4zesmUzduzYjh9/rItly5ZKvRSXjxw5Eu3b/Ywe3bvj711/STrF5YsWL0L3Ht0xbfo0LFywQLbtO3XuZEgSgUoVK+L+++7DyJEjMGzYr/iuVi1sWL9etfnGdcybPw99+/XF7NmzJW3v3j3RAFMFAiZPnow2rVujffv2OGjQhtv/Awb0R7t2P6Nvnz747z8F6Km6MH36dKHPggXzpY0jRo5A9+7dhO58+N5/9qJv3z4YOHAgtm3dik4dO6Ju3R/w119/StuuXr2K1q1bIfEDD+Cdd95Bz549QQmvCrqDM04oiSkEvC9dukR4TJ5RNcIJqi1HDh/GkiWL3WX++GOnq4huM7dArmH58uXY9fcurzxVx4ED+zFhwniMGjUSy5cvcx7h7se6Ll+xLu4rz0xT5dgP+S6cWPi38vcVWLFiOfbu2a0rknjDhvUYOWqk9Htn5cz61Jhas2a13OeuZ+Xv0va4l8QnFDAFTp066aYNabR58yY4utzq3Q8ePODmN/vG7t1/u+imaQ1wnOk+wTL79qm5xlUQW7ZsQY8ePcBFsbO4Yq6uI6pY1xJVGZ2n6lz420KMGTsG48aNk3dUNegyjIGNGzdg9JjRmDlzpksnXd2r2gRR/eGcNmrUKMyaNcuLLmZdcXEd38AUsk3JMbdn7x5Fjki05zuflTF5+vQprzLq559//iFjyJXpxT/g3NkzMiZ+X7Ecy5ctA8dUhGyP8o6o6KRrjKqMrzx13+Qpk7Fz5w5XJboc5Hs0ceJEjBs31me/5fdo6tSp6NipIyjsIBhRQdcRl3FcAFO1w8bv3y+//CKgydVgL/oCx44dxbJly4RfHJtq3PF9GFTM75meG1mG/HWC+e7A8hXLDRU6XYdZxvta1+Sd7uu32jafNHkSOnXqiAkTJxjffV1e1bd48SLBGGPHjsGxo0dcD3HK8J369e0r9axetUo3wos+LA/pqyVKFDf4DsyZMwdPPfUUWrdqid69e2PQoEF48cUX3eqBukKC2R9//FH/9KpfJVtg6h706iPasGEDoQzF0E8+8QTuuetu5PvyS5y/cNjpywwAACAASURBVAH9+/fD/v37JZ8fmddfew1du3QRIJEieXJMmjRJts1HDB8u0smcX3yBmzdvonnzZvK7Qf36cu/mzZuRNWsWPPzQQ2jSpAmWLFkiovCGjRpKuZLffIPSpUvJNSWdDCWKF0eOHNnlQ/br0CF4/rnn3BPd3HlzkSRJEqRKlQo0BPj555+QJHFilCldWnQweX/lypWQKFEiVKpUET//pPLfevNNHD9xXNrMCYgqDPnz55fn8QPpX2IK0eusXr0aChYogG3btiFlypTInCmTdFROUp98kgOlSpUCP87NmjXFmxkzYvfu3fKs3r16yrOKFC4s9Kn1XS35zXYzcIv+3Xez4u677pJ3ICi999578Pnnn+HSpUs4f+6c0I3vmD37x+AESsCmgh5oagDFv8RUPZUqEI8+8ghSPvOMxD179nC1x4moCvLoo4/imWeeweOPPYbhw4c5mRERIFCtX78+HnnkEfCDoYJ+H4AAr2DBgiCgX79hPXLlygUupJygy/qOqYvrXx/XvAfgtkvKZ1PiqSefFBUPqnmwPzyYJAm6du3ifiQnH/bRTZs2yVj4/vvv3X2OhX79dSgeSJQIz6RIIX3k2WefRYoUKcDxsnbtGlc95rNv5zohgKlqcuNGjYTPfK/HHn0U7du3c9OEF1xgFC5cSPL4vsmeegozZkw33heypZUz5xdI+tjjSJ48udCIHzkduO2VMWNGfPzxRzLe+ZwBAwbobK8JPTLdyGtP3a/IZfSHluXq/fgjmjZtip1/7JSF4qeffIK9e/caz4OAr9KlSmHDhg3o368/atWqJWNSF+LitHDhwhg2bBi2b9+OcmXLolKlSrgiuvQs5a8Nt5oe/8CUggXO1QsWLnC9ptlWldSoUSM8nSyZsbBgGQVyBg8ZLHNz1apVXPcz0nWopPbt2uHRRx51zx+NGytdf5Wry0aOybfr17TBauR85zneeRA1MqqsjZ8w3tUuloEIETh3T5gwEatXr0apUiWxZu1aVxml8sa0TG+/jWzZssm35oMPPsCePf6Au/ezY/v7doEp3+k/UcUrXrw48uTJI/MYd9zU+OCrqXfnVY0aNfDYo48hRfIUSPr44xjQv7/73XnBb3qp0qVkDuc8xjly3NixRhn9fsB/h//DCy+8gNrf1/aRr8t5xtevX4vxPM0FcqmSJZElyzt4L9t78l4ff/QR9rqEaXwoJZXsnwSDO3bsQJ8+ffD+++/LHK8bRUzy1VcFUbJkScljf6dgyAm6jcRJl/HZZ5+J4EnlM0+FP/78Ay1atkDJUiVRrVo1EZbpPE1j4oNMmd4G7VhU0HU79VhgakwQ1CMlMD1/Xq3+evXqiYcefFA+rARHPXr2cEv+aExEMLhu/VpZXSZO9ACyZ88udJ47b44ALQJaht69e8nvJsZkU6ZMadx3770iLVHMAbp07SJS2o8++gj//ntQROWc3AmSCRq50mMgmCQA4gBjWL9+PR5//HHpUDdd3gNy5cwp9VPCxaC38ge6Pmzvvfuu5FM6zECww/YUKVJYfkcHTCmmZ5s6d+wo5UkPAmMOFIIV5mm1AYrz773nHnzvGpwcxMwnAGf46eef5HeHDur9KHnKl+9LkfBSB/f6jet47bW0eOXll3HgoJJYL168WCTOpUuXljrUP7ODq06eEMCU0rJu3bvh998pCVyOZcuXuVaSuj3A6tWr0Kt3L1ENYf6KFStw6RJ1bVQ7+c7c3uD7ctU5ZeoU13upfOrlvPvuu9A0YiYl3gQ88+f5GuD62ToGpk+fZqzcdbp3rCbeXj17guBr2fLlWCES098xbdpU/C99emzatEHaRiCdOnVqN8DkJM9+1aNHd8mn7m+9+vXQt29f/L5ypbzzqlWr8PPPP8uOARcZKni34VZ/Jwww5W5Gly5dhJfk99KlS3H+vNrO0vxcuHCBLGT5vuT3qlUrXYBdvxswc+YM0WOXMsuWYc2aNa4PEgTU8QO5fds2XL9+HXv27MZHH36IJ5ImlQVM9HQDKBnhYibqsiqXQPLNN980pC0QgMmF502X/hiluzSQpKRIh+wff4yOhqFoxQoVwLlNB+4EvfTSSyKFUWn6/eMqjk9gCnC3rGzZsjLfcs6J/A7AunXrxCF46ldf9VogK+NW7rJRCPDtt5U1WTyAKXlLgcIKzh8rloug4lQkyasveinANnrMGB/1+iqv04AjR44IcEj+dHLZsTMqQJUq34oAQ6cNGjQQWbNmdUvH+vXri06dO4sAgQuwCRMmyLewfPnyAoLUffpZcRHfLjCNkG/RUteijy1q/0t7PJg4MTZtVHOZHrf8nvfq1Ut4wXHLOf2Sh04ksHTZEvTr10/yKFnl+FULL/NdIQsGSpSfe+452b7W9FTPMsua18D8+fNlzo2ajgpwdunSWeYifkvIi9GjR8l3k4IoZQsDTJwwAWlSpxapvm4DhVafffqpfFu5AzlkyBD8sVPt4LGucuXKiiojx69nO5RtzEsvvoh/3OCX7acwYycKFymM2rVryy5mmzZt8MXnn2PSpImuOpyoXLlyYmiu6e7QRJWxwNQHMHWs0AFKgBLdd78A1M8//1zAAz8UXHE8+eSTMuFyG7NqlSpo3qyZUJVb+QRe+fPlk9+UnvF30yZN3JyhGJxb6yyrA7ffWa5Ro4Y6SeKGDZUkVa9gjh47KjoclGCx4xD4UQKXO3du94etfoP6UlefPr2lDgJTSiC59c6Q1aVHu94FTAmI2J6iRYtIfnTAlACebeU2O8OZ06fd0uSvv/5K8ubNnSN53JaiNDFHjhzym4CV93Klx9CmbRv5rT9wBDh58+aV1R+Nss6cPYPX0qaVv4P/HpR7qHJASVyJEiXkt/pnDnA1WOIXmPKpEejatSt2uVQrjMa4QSclV6TToUP/embLL7PNagKmNNxTYgrMmT1bpK0O0FDPzpw5kyiaOxWb9ZnXkBXuxIkTXEXNPPMaoPL62jVamunUTCX3/PnzicSAqZTuZs6cGVevXXUX4g4ApXyc3E6dPOG1/ayKNWrcSNRf3De5x6DZjlu5ThhgygXqdg9VDf0mbLPaefnll/Y4657UdT5jVYbjllLWa25JolkGWLVyJf50qa7onHVr18o4oiqOClHRCOJBxLdUwryPux83ZGwWdS1K9fPGjBkjksL169dJEnd+CEwdY0Pgp5/aymKFnkv+O3RIpP1ciJuhcuXKAnovXPBnpGi2J7bX8QVM1Tjo37+/gBouihctWuR6Ld1G4PixYyKB4txMYLp//75IZZhAEFCxYgWDLE4dBDmUTEYOuoy/mAuYbejew9yd8VdWpyvVhMGDB8m3IE2aNLLTp5/NnUB+S8aMHq2T8M8/e0UqSMkxw+zZs9x5+qJGjeqyCxI/W/q3D0zNbzrbTOnuK6+8jK1bt7heIUKAHL9BWnVPv5uKST8FNqmeRr5HDprGqiw9+XAep5Dohx9+MIqb5byvgXHjx2OWm8be+fo3ZI6d5xZKONV/++23eO2113DOtVjOnSs3ChQo4BQAMHXqFFGFo3oVJcCX3AbEqhif/9Zbb7kXIyZwrPLtt6LqeO2qnvfZJuC33xbi66++wg7ZKSkjO7oUxmmBmtkAejdKmjSp10JO1cNyFpi6P4rOVr7ZiTnQcufKJcDpvffeAydXrkzez5ZNLPcXLlxo0luuKZki8NLAlOCQv30BU0oeddDAtE2b1jpJYgeYDpLfBKb/+9//ZHuPkpoNGzfIZEJ3UNT9YaDbJz6TExCDBqZc3TIQmBLY6Y9ObIHpD3XqSP3UXfUO3G7ms7VVngamn376qRTVElMNTH9u97OU9wVMKdE9feZ0wALTXX/9JZJcriA5YLml7QQ10DZuWC/b4PxwUbd3p2tlqsrpiUaVpa4Sje68gSn1aCm91xJw/QyqB7zxRgYfE4gzyHXZIUMGy4Skfzux2QZ9rXOd39zmoeoKA7ebuB1DdQwzUD2BW1tcUHgG1Z6TJ0/Iom6te2tQ1x8XcXwDU8hHOm3atDL2uEWtx496V/WOv/32m0iy073+Gqi+42xxspQqM3nSJNkmpAFk8+bNRV/dqYMfSe03WdNF6bWyb0ybNs1FWp2nY1eyK6Lk2nEPp/N0WR0rnVBKzvQOhi7JscdxPMQ1h3zyySfI51ps6zLUvXzggQfkA79+3Top38cLmFL146GHHjIWKfrZcRHHHzClehMXhByTVGXxBUz1onTKlMngHOAPmH6SI4eHFFL3g6PHjghoT/lMSpE0r1z5uyatq6/4opFThFKq3n36OAnuK1/3MQ1i+0DhAHUKuQimCpoOnCPojUXbHzD98qWLIICtXMl7rtfPgNgEEMhwm1cFnRcX8e0DU6dN6op9u1vXrm6pIlOpU09hDxchVFGhfrcTFO04/yZLlky+R9wap8qTE/S7An//vUt2t6hmwV2FqIGpUwOvJk2eLDYfnqn8petXsaOGoPPUHdQ1pVodcQoFBJTWFyigVPR0nbRzoeoJbWScoN6Rv4kTuOOign6u0mel2kDu3Lmc29ztUkkUJFFK7NzPdF2HegaB8b333msI5nS+qsMCUzfB1EqS+pCOvy1FxL93/y0d8a0333L79KSuJidt6uJQgsoPNbe4uPrQElP6PWVo1bKllG3RvLmiOoBChQvJ9jZXGRSlM3A7mHV6A1NaujGdqwwGbvMTpGiLdG4BcpVLia4O33zzjUhAtR7ft5UrSx00SmB4N2tW2QrnNjTDwt8WyFY+JbkM1A3lIH377bdEKiuJBq2mTZ0q9adM8YwbLFH/ltvRg11b+ZMnqwlvxfLl8mxa4zGMHTNGflP/jKGOC+RyW0IHfgCpQ0qAc/7CedHn5Srw8JHDUoQGVDROK1OmjNBcAXLduXUc38ZPwO49u2VwUxpCfSPq5gxy6+ao/kMg2rlLF1DtgHrL3I51dJJ0W1VZf8CUupoCEoYM1iSSuHr16nj11VdwNJIyu6rPLExJ2/QZGtSYOWYbfF1DpL002OOEy3Do34MCrGpUr2ZWJNvTVNvwLZkF2CfoJ5jjRAVfz7vVtIQBplwkcFv22WdSitShT2+1K6HBxsZNm9ChQwcUK1ZM9Aaph6qNCnUZqna0a9dOJAwPPphEQI1jzMb310HTAlj5+++izhFTLxmUXNKgKnLQdarnXLx0AenSvS7btXphy3u2btkiuywEuJwTOR9wgWsGAl/2y6lTJoOLNO7K/FjXNGwA9MKc6gwqmM+/3ev4AKYEFn+DC2b2Uxp7RQamkC13bmsz0DD1VoApd1Fo6FmjenW8+MILuO/e+9DBpbLl+THXdHKR0BVxp6aflw6kytLlzZjj9hBat2kjW89UzfAGptRF5OKH85oOBECffvqJSP58j1vI9n/Tps6OoO+2m22JzXVcAFP1PH5rCUAbNmqEY26pJ/MgC5DOXTqjfLlyMk9Tz5S7BiqoMpSwcmxTqEJ7AYI7SkbNMtyBaP/LL26jsQ8//CBWwHTqtGmgEWLk4ItmLKXT1R3coWjdqpX84LilehV3Oi67VacA6oImf/pp2a1UdymJMXc+uMvSokVzL/1b9f4nTp6QHZIKFcqr2+S/5/P5/rSbOeL6Vuv6zXZSZYXqh4MH6++ZZx0WmLqYygFHhX7qcREsKutih1gL5s/HRx9+5AYAXE2lT59OJmXqhFarXs29HUOA9vrrr4vOJVckBJCcvAlmuYphoJSEaaVLl3FLXaiDybTvvqvl4ReMbaOkLdt770kbaST0+muvg6seBuq4UNcwQ4YM0iGov8jJpVbNmjKxctVE6S3r5gr/+PFjePWVV+S3dldF0ML8Dz54X6TCWsr57LMpfei0AfQ+QFB0z113CSCjsjNXauz8/OPzihQpgk2bN4suE0GN9mhA6/oXnn9eVqbU56EKAp/9Y9268j4nT53E22+/LWmURHC7ioCOA4lGPwwEeylTPoOXX35Z9H18byHFNzBVg1Ua5Noe4oAiOKX0SAXPMmz3xx9/LGogjuTU6Wf+gCm32J5O9pToZmqpBC2BKYnhCtbZXnWexy03bjv3698PQ4YOla3EmrVqYdDgwZLGPAcMsbW6Hd4xRAdJL4RYkh9EGuPQvZr5njNnzBC+RfaXq0qVL1/O0En0fs7t/o5vYMr2OYHqLjSmoFGhQ0fPMpxTKEmiAYSngZ5TD7fT0qROIxM+LYJV8KYFxDMF9d4j50MsuLnoJGjW/OaimVKdQYMHSRrz1rpUdxxeq9pobMMx6CyYINu5TKPkg/3rkYceRq1aNV3PVxF101mG8wfnTKo4EYhrzw0EAryHetOOuov3u93O77gHphQ0UDeQ0kgGStcITB0dU0qvT6FVq1bQVvgjRowQYOrJY6cv+JaYOvl8Dg10KIXngnu2242gSRs171GAQR4PHDQILVq0wDclvxF/zgSovXr3Fq8K6uAW1urcT51DLjK0lJ/zsDcw5bdHALbbmFQZutKYlcZwpI0Kul4lxChUqJCX4ZfOj4s4boAp3588pIEv+yztGGiv4byP65Lz219/IW+ePLLdbG73OyUg398PP/xQvkM07NWBuvhaFYI7H/6BqbqDKlJ63HJurlqtKmgQPHjIEOEz+e30vajoCZHYF8if30NtjCqGMkYNY1saDFO4Rc85KkTI4pOSUgqtWJ6LJY1XVD8Cjh0/JkIi38BUt01TgrFOM2Nl1U9hmmPMqfPVvRaYCuGUPhE/Lvyg0nDhgpcxA8lFi1luRerAbRvqIFF/UEseNQPpiL9z585iVER3HGPGjBYfX7pe+v6iwvGvv/4qSuScwGkMQeVlKj87Oh9kmAqcrGikQJcXpr4igSmtB6nLQjdFXL1zpaclsWyzHiyLflsohhSUaLFNnKQIMrmFxA8Qt3j4cfzzj53iKoTtIVhSQXce1SZ22unTpokOCetzdMiU/gu3iIYNHyZ1njljnh4FcCuXCv+kKdvM54ih0/Vr4iuWH0i2h7qOnBh4zT/TDdPKVSuFxs62sdk+1cb41TE1n6coxGNduWCpXfs7F80Y6XIqiSdxUTpCN2NOUO31DUxVHoHH86meE7+s/QcMQO9evWTbvGyZMk417mdFyLbzps2bhH40omvbto1YW9OLAmnKPNWPdPt8xarqwoUKgdt8Ouz7Z69PYMr+cM899wjPVVldJ08gOyzAxdGd1HlxFScEMNVtVW9HiQS3zajM7wTPMpS+0TKfizAVdD5jFSiV4+KLY1cFzzJcbNJrh7LAZgkzX239c/HCeYi83bZ9Gxo3biSGiJrfzFNSDM97WRuNFosVLYpnnkmBZs2ai+Elec42cf4iMKWKRs0aNVztU5EGptrDBBdkb7zxhnjh6Natm7xPutdfB9WgPBdP3m241d9xDUypQznTkPhDXPcRmGo9UAoK+EE3t7v5QSegc74P+n0UnfwDU89yBDI0ov3qq68MOusyEVI/bQqEx9u2iWpO4yZNsHXbVgFKm7dsErd6apvXuY+VUXruLBiBg/8eEGDqSPMhLgq9gSmNT7mYppcZJbBhbaxbhbY//YQZM02pofncuLiOG2Cq28yTG+kFgeCLrg5VMNupUjgmuK1Pt43+yvz7779iGMfdD4YD+/fLN8l1g0T0qqF3C1W6+awIAfTucbttm+yu0XUjxzD5TH5zB9N7zHv+Vrri3PGdNVP7RudzIIaZ9KRBIRO9bhAb8Dc9rETaxYiIEAESjadIH7oEc9qscFKWd97xUunxfB/PdvnKo4HXPHm+3lV17lFPs8DUY4J38UAib4L6yjPTeG3e451n/mY57+CdZtblnafvVemcMLXxk85xYn/3OiVUu83fvq692+OvXl0uujp85TPNX71meV/P0GlmnBASU/N5vAba/tQWWiVCtdoso1IoyapSxdt9jNpOiqxjqurlnZTITJ0yRXRZqc5A6ax/RXn1LP2fHyUtIddpKjbb532trC2pU33oX8d46+KF8yL1phW2GYYMHSKLJMdIS9cH0VUk2NHWop7jRZe7nTghgalupzIyoh6ZE3QeYxUqVqpoGDWa+U4Z7jA4bqd0GUpB9ssixre6hi7n1KOfyS1iR5KrUxmb9+hriPU9F4r0PUp1gRzZs4tVLcESJXDUKdZ64bo2Soboym2x+6hjBXK5yKbHAu7qPPXEkz7eSz/3duO4BKbqw0u3dB9+8AG+zJtXpGbkLaXAmTJlkp0uLgDefDOjHPFMqRr/qLNPTymUNmmvFJpGjKMHppoO3J0YDH78naDzGHsGWvQPcKsOmXme93DMFfr6a2mnfi9KQPle1EGsWVNJwrmYoEqSs3gELly8gOeff15c95lP4DUFCtooV+WZz42r67gEpg4NP//sM+Gd807e7YVswRcqFPUhLtwFoqocA3czqYOeP19+qZt9g0IIGu9StW/XLqUK5TkGnRbwavqMGeKb3DOVv7zbp3+rklSRo6BLBZ3HmCFC5gLuaBGYE/hxseEpEdVllR9bfocc3Vgnj6CWDvSV60FVt/+2me3Q1xCPJYkTJ5ZFn26fqkP9ssDUL7M1EQM9Bug4l7pdtJCOH6lEoNPAX/sSApiqgeQMTIi6BFeukQPbqULLVi3RsUMH1y/d/qiAqVNG10FXHLSS95Ri6HLOs3R5Stp9WdQ6bTfv1deQAx3oPsQ70LcuJ3dzQmndurW4JvGUHKk7vylRQvSP1S9df1zGCQFMNRV0uwEa+3hKyM0y6vqHunUj6R47dFBXtKalY3sznXpwdFa938vxvlNGt4OxZ6D+I9UEIgfzHvPaKUlg+dRTT4qLL51aoXx5kXg7eoYEhR3lw+vok3m2o06d70XFiLppKpjPi4vruAWm1LEdOWK4AAwuEvh+9MxC0Mltdkp4yBNuQbb7+WcB3NT9p/64/pDP9zhmWr21f2CqqatpQX3VEfIsneM5Pp1UXtFinlv6kYOujzGH6E1MGD9evCjwvWho2qBBfdEbJtAYPlwdVU3jHvo2NY16Ka2jtJw7NGagytvQIUMQ4dYX17nms+PiOi6Aqdk2dU3hAI/qjhx0m4FmTZuCxscquGgpP5wyVFXRbvy4EKSfcNKYnjkY87AeLgLoWP7wf/8ZdTl1uBIlmjFzJpZEqxvueS+l4ZTaRw6e5Zi/dMkSEWRxh1IFlmHwLEsjZnrd8M7jwRlUxdMecpz79P1Rxao2Gk9Tvc+XaghLWGDqZkZUxAzcPG7ZEGx8X6eODCAaVdy4cSsOlwP3HWPX8c33iH9gqvU9XaNXnKbXqlnLwyqd/nHNcPLkSVStVs19kpXzfhCFeep9Tfdwxq7fyamlZYsW4tz6SBRGTzR8o44uHbvTrQh1hjjJ0hME05hHn5wq6GeYsZKi0XUXLZQ9ywFcfVOS4hzTCzkEoa5LV9gpr4zE6Iw7stWy+bzbvY5/YMqtezPQZQrBF/UQdbh0WZ8FrVK43Ud+HzqkXJ2R39T7NgONyuiQ2gH0lFqcEXUfqn7oQFBI63DHcwhppoAHT+Yy+U0gya1KGmNqfmvjNafPmTRXVVGqQ5dfPI1NBfUMehugtS2ldDrQz2lkx/Eqd+KE8UiXLh3o5koF81lxdR2XwFS3Sb+dinfs3CE6pqt9uFDTJQkKXn7pJR+nZakSdBdF/6BOUM+iJNrxwKBy6YbNOWhBt4mx0iGkRwfyk1vw1DmuVLmy7IRwMUFrZwIP39Is5+m8ohEt5xpzK5/qX++/n00ORdGl+Sy6VnL0ZyE+PKkKZgaq99BATwWz3bd7fbvA1Gylvo4Qw+Wp0/RWtfLKo3MZU4efbrDo/kgH77mci5RqVaviH6+DKHR5xtzKd8AtU0x6ABs3bHCPW47V+vUboFnzZu55mvz2VBXU96uncMd02nR9eIdKo/77ag8fxip9z+7dcjAC/TCrwLoiB+6Y5Mmd28f3QUlT33kns+HNQbcnJrHSWaZuLv3jqmDep1IsMPXoJCaBgufanNi44le/g6f9ngM1Ltsdn8CUICgC3333nQxgAsn5C+aDEkPTt9y169dQoUIFsb7mpMNj26hb6myxOu9L0EEl90T33y8fHLWa1PlqwNIxNqUdZcuWMQChLqNjVZYAlNuKPLGMOnE8wUT/MY3+damfrIK+14yVkjq3Kalv5VlOvT+38uvX+xEnTp6UE7i4Xeht7cr7eEIVPUV41mE+Ky6u4xuYQtw/0fsFda8WLFggEhGqVOhAwMkteW55U/+Sunc0dKQRlA40mMmbN49YuNOqnWCCHyJtbMNy/+zbJ7rEPF2H3iuKFikip79xq5iGkJ7AQwFT6pGTpz753a8feGiC487Mm97UC78EetCgdNzRdWVrdFng+9q18V3t73DixAkBQzRcNPXdWZqGQdxWJL95oowTdD1xGccHMGX7nMDFPg1FqPuvgmc+02hdTG8blGA6ZVQ5Ahzq2XJ3wdnRUqU4LqgqQGMU9gWObUo2PevQ9II4QeeCQXjcv5+b1/zNP0o1qe9PLzH+6nBlSH+jf2ntn1TxWemifvbZp6JbzJOLuDOiPY1wd4Y6ijQ6pc9KqizR93XhQl+LSkBkl1q67bcT3w4wpWu763JSII2L6cGGQJNb7jxGO0L6tipDiTgNhzhH869t27YSa3pRCEG/2TR4ZF+gykurVi3lgA1PWus7lKcfqn1wYeoEkxb0//mbM25dc7Tib3/hKfmtjzw3xyLfi+OMrry+/qqg8Im8KPR1IVG7MT1y0Nqec/0335QwAKFqEY2N6Yedkl4e6MFFNoULPMFSBbO9vIYcfcqT7RwvHt5l/P2GzBt0TadtYJx3UnWzfgtM3ZOuP0IGQ7rqPs7/YGhzQrQxPoEp26+O9StatChogMQtPcfSnrnKBQdd6dDfJ09G6dG9u9uNiPeApA4h3UIReHAyUpIp9Rwalq1cuUqMUqZNn2ZIWaKio9Mjor7yVwfkBCq6UFHBu5wychvQvx/69O0rW86OdE+XVQCWhm7RO3vX99xqw3W9zAAAIABJREFUHP/AlJM9P048OYUGFHQ944QI8YIxauQI8cTBDx31PLU3Cs1vftx5AhsNXKp8W0U++lxwmIG6wJREcpuQHw1e008u3VRtdvvK9aaTWUNU15HvY1+bOWumfIxorOUEsyxw9coVDP11qFh/002WMspQpfmRIj1o9Eiw40h1mW/WE5fX8QVMdRshahQ89MS3ESjLAWtWr0azpk0MibdK50KFwIMHktCdEo1NHIt5dcwwvSfQRzCNE3mKlBN0G8zYyY3+yrzP+xriYYYO0Gl4p4IuoySi9NpBgz1n3Cqft5TCc/eFR1KzX/KvUsUKaNmyRRSAWNd9K/HtAVMKaihZLligIOhKiYsBRyLNN1fz9OxZsyBzedmycuwyLfNNulBwMGniRFBPnlbprJOSSbOMZz9X81GXzp3k9KWoyrkqiSYyaad4QZoLL6o5vOBOCVUI1M4psPefvQKw6TmCR5I6gfXROOq89E16XeGuTaeOHQwJLUuYz1X3MJVus3iaoaPa413O+zfkSPWaNWv4+QY6dVtgGono3sS0vyN3zGChSXwDU2cgyQj3+GfSyCPD9cPMV9eOFEwVUVIPlcdr53g4XV/kOuKaV44TZ3/PUm3x3OKOXNaR6kfOi7s2xzcwZdv9BfO9fJWJLp/3xKSMrtsse/vXPKjDW73Asz36Ger5vvjNvkLn2p79WN8XX3F8A1O22wz+3sN3GdLEpAevPXe09H0xfY6/599Kun42Y/N+lc4dIX0crSvFVc68z9e1WVdcXN8OMNXP1++k/Sfrdut8xjqY10zzVUaX9c43y+prXVb/jstY1+0vjhCvP96Sen/vpCXITm3+2qpK8LhTSpc9d/d83aOMOOmJKGq3capeC0w9Op0vgto0z04cTPRICGBKepjBH31iW0aX1/Xp34x1WkLE+rlRPUuXiaptukxU9dxuXkIAU7bRDP7arMtEl89yvsro+33Fvsrfbpp+Tkzq0WUZe5fXed7p8fU7IYAp284Q1Tu4ivgso/N07F2PTmfsnRffv/WzfT3HX55O9xX7qud20+IOmDot9tcmp4R/Xugy/urwTo9tee/7o/qt6/YV6/t0nv7tK9ZldOyrjHeaKnvyxHHX4ss73/wN8fnrLGjNPPNa1WmBaYJPBCYT7LX/wR8XtEkoYBoXbbV13H5fSChganl1+7yKKxomFDCNq/baemLfd+ICmFq6x57uMaFZ7IFs1O2wwNRF0ZgQ35aJujMFKn0sMA1Ovt1qf7LANLz4zX5igWno89wC09DnsZ7zLTC1wDTkpcUWmIbPhKZACvX3eIIRXeeooCc8G4dmX7DANDT5ao5XC0xDn8ea3xaY2g+XBaZ3QKdLD0Abx/1kayWmcU/TQO+nFpiGPs8tMA19Hut5JsyB6YUL51zA1EahTIGr164gX74vxU1FKL+nfTeHAjxhiqfS2BAeFOjRoweaNG0SHi8bpm/Jk8V40Ae9PtgQ+hQoUKBAwL3kXfHdIh4v1717dzlXlu4L7F9o0oDnBvfs2ROZM2VCu3Y/W36HeF8fOnQo+PfBBx/IaUw8ZtGO7dAc25qvw4YNA4/U5PGJvCb/dZ6NQ4P3nMfpB5mHTfDEIv62vA0N3nrzUc/hWbNmjW8YGOv64x2YPpMiBcqWK4fq1aujStWq9i9EaUD+litXDmnTpsE3JUtafocon/UYrlq1qpywkiFDBnyZNy9q1qxpx3aI87xGjRrIkSOHHM/La/YB3R9sHBq04DxeukwZpE2TBqVLl7bzeAj3cTr4r1qtKtKnSxdr4BjfN8Q7ME317LPGCR3x/Tq2/jtJgXPnz8rxj3/99eedbIZ9dgJSgEcG8vhXG8KDAl27dkXDxo3C42XD9C0PHNgPHn979PixMKVAeL12vnz5Au6FEwSYHjt2NOBe3DYo7ilw+swpOXN8547tcV+5rTEgKVC8WDE5jzkgG2cbFecU6Ny5Mxo0bBDn9doKA4cC//yzF7ly5cLhI4cDp1G2JfFGAetgP+Qt07W1WzjG1l1U+Fhzsn9bq/zw4rfiOfUP6zeob3wkw3GuC+V3tlb54TOu1TC2wNQC0xB2mWSBafhMaBaYhhevNRCz7qJCn+8WmIY+j53xTGhqgakFphaY2j4QIn3ASkzD5wPmfMisxFTTIlRjC0zDZ1xbialr6ydUB7N9LzL45KkTomO6ffs2y++QB+AWmIbPB0zPb1ZiGvo8t8A09HnsjGd+qK3ENOQ/1prh4RhbYBo+Exr7twWm4cVvxXMrMQ31ud0C0/AZ10p+ZIGpBaYhso3ra3K2wDR8JjQLTMOL13q8W4lp6PPdAtPQ57Eznq3EVMlYggCYqVWE539PRnrm+fqly+vYVxlfabp8MMYWmIbPhMb+aSWm4cVvxXMrMQ3GuTk2bbbANHzGtcIgVmIa8BJTYPfu3XIEW5++fTBgQH8cO+Y4Gf73338xZsxoMG/lqpU4cfIEFixYgIEDB6Bf/34YPmI4Nm7ciGvXrnoA8PPnz2PJ0iUYOGgg+vfvh1GjR2HylMkYNHgQRo0aiT17dhsoNTaTSCCVtcA0fCY09jsLTMOL34rnFpgG0pwbH22xwDR8xrUFpi7gFR8DKS7rBK5cuYJevXrh7rvukr9vvvkGV65clvZfvXoVw379FUWLFMGZM6dx/fp17N+/Dxn+9z/cddddcrzXhQsXEBHh2aYbN27gxInj4Jm0LNembRv8888/mDtvLjJnzoxkTz2FESOGu2jEyPP+4PhtgWlw8Cmu+pYFpuHFb/Ybu5Uf+jy3wDT0eay/AQpyWIlpUAAu4M8//8B7772HRx95BPfddx9aNG+uOAhg44YN6Nypk/v39WtXkePj7AI4W7du7U737NyuDpA3r5QbOnSIu9yYsWNw7z334JVXXsG/Bw+60nXHCab4TgNTTdLY0iwm98WkTGyf66/8rTxL38M4unqjKuPvXl/pdwqYxuRdfbWXaQz+8nR+dGWiuj+2eapFUbfJX53+2qnr9Jfvr76YpCc0MNXvEpO2+Sqj748PWvh6nr803Q5/+beaHh/13glgqt8jtnzS990q/eLjvlttU3Tvfqv1RvWOqk4LTKP8KERFwITMA9atW4shQ4eiSpUquP+++5A0aVKMHTtWuLhu7Vp0+OUX3Utw+fIlZP/4YwGcLVu2dKd7fmxUct48eaTcoEED3eV+//13PPXkk3jowQexZs1qV3pCvm9cPetOAVM3Kb0uonsvVfzGjetR3KezWJcO0dV7O/n6GWYcXX2q7PXr18ybvACYyrp584ZRJrp6o8u/E8DUaL77Mrp2Mt87eN/jna9/e5eLq99m/eZ1TOvX9zA27zHT9bWZf7vXCQVMddu945i237mP40L1e94bXYhp/bEpp59pPj8m9+v7zNi8T6VHRNw0Cpj5t3qd0MDUaL7HZVTt9yho/PC+x8jyuPQuFxe/9QNYlw4xqVeXVfOp53g269L5unxM6o6ujKrLAlOPSTQ6ot2pfGDlyt8xYeIEnDt3Fp999hnuvftuPJsypQBWSlNvF5j++utQ3bvQtm1bAavZ3nsPp0+fdqXfqXe/nefeCWCqyDVp0kRUqVoF1apWxfz582JAQwitqRs8aPBgDBw4EL/9ttDrPl33JAwYOACDBw/GkSNHvMpERS9X0Rj3eeCPP/5Ar949pT1LlizWFXiBD/OZwF9//YWePXtg2IjhohO9bZu3D1mAab379MLAQYPQp3dvnD17NhbvYT7PvE5oYAocO3oU7du1Q9myZdGiRXPs378/Bu+h1HPoW7dv3z444j7/W78LcO3aNfTt0wflypXD97VrY8WKFTGgvb6fsQ5mmr9rVXbatKno178/+vfvj//+O+SqwN89Zjpw9OgRtGrVCgcOmO8P2XFp07q10Kdt2zb477//YlGv+Qx/1wkBTFWTZ82aierVq+Pbb7/F9GnTYvEeqigX/D179sTo0aOFXkyl+tXIkSMxcMAAUDjAvyFDOP4HYPfuv6MYZ5oermbEYkxTZYt9a9CgQcbcxHp0nb5igP21d69eGDxokNy/YsVy4x7g1OlTYq8wYOBAjB8/XjfMKOOr3pikJSQwhfR99uVSpUqhZYsW2LVrl+td/LVVZY8dMwaVKlZEtWrVMHPmTD/vD8yePQv9+vbBoIHKvkONGX91m+m6SjPN37UqSz5wTJPXx48fd1Xg7x6mA5cuXRKB1NChQ0XIpW7S93COjcC4sWPQf0B/DP11KCZMGA9HCKHL3WqsnmaBaZSD8VaJG9f3EZiuxK/DfhWubd+2Da+8/LLom3788cfgB6VHj+6Ko0AMJKbuoqDE9N577sV3330nhlBVqnyLF154ASVLlsSWLZtdBeP6fRKqvoQHpjdv3kTfvn1RokRxNGnSBB9/9BEefPBBTJk8OQpaAmfPnEGxokXRq1dPHD58GP/8s1cmOX4IVCDNgHbt2qFq1ao4cOAACH6LFC6MkydPGGX80/bq1Suusv7LOB8nCP8///xzLF++DPv27QN1mydNnhTFsxTgzF+gAObMmSMT4bp16/DVVwWxfDk/YiqwL+fKlQvLli0VoFKjRg18+WVenDt3zlUiJu3zVSYhgakCYzVr1kTNmjVQt25dJE+eHO++m1XAqnoR322kIeLSpUvxSY4ceOKJJ4TXTnng4sWL+K72d/jwgw9QonhxvJ42LR5MkkR0yZ1yvup20k6fPoXLly7FEBQAXbt2RcWKFQVYcz4pWLCgD8Ds1O/0E6ZB3v+RRx7B9h3bVRMBAalVvv0W331XC3W+/x5PJE2Kzz79FGfPnnGV8VdfbNITBpiOGDESxYoVRePGjZE7Zy4kuj+RLAzVi0TVXi5CLqNevXooVqwYFi5ciBMnTrgkphAgRx1/ChleeP55mXufSZECie6/X8ZHdPXT/kCN/6jaoPOAvXv3IG/ePJg3by72HzggCwYavEb9HOD6tWsoXrw4nnzySTyXKhWeTpYMY8eOcd0H0JiWcxENaQ8ePIiGjRpCqZHpZ99OnFDAlAupA8iTOzdy5cyJEsWLia1FqlSpoEA4Xzfye3DO56Lr/WzZhMeZMmXCPXffg59++smrPLB921akTZsWKZ95BsmTP4133nnHazEXuX79TI7pS5cuetXprzwEVFevVlXGNHdXS5cuHY2gCTJnTJk6BenTp0OWLO/gqhhNO+9N25RmTZui7o91sW//Phw6dAgtW7bAjz/WBfNU8NemmKSrGiww9dHRdEcInFgB02HDh7kYD8yaPQtPP/00HnroIaROnRq//NLenRfdVr7aSlLbLQSm99xzD7p264qff/5ZJKXJkiXDmjVrXPXFpDMFapmEBqaQSXnixAluXnCwEoDkzJnTnRa5XwGUWFNCbYZ58+Yh23vZ3IBt/fp1ePHFF7Fjp/Px5wCuW/cH121R8QH4++9d6NBBq3xEXZaTbaGvv8YPP+i6galTpyJNmtQ4fNiX1Es1oXz58qhRs6b5GmjcqBG+KVFc0miclyN7djRp3Nhd5sKF86LP/NNPbV1pUbUtqryEA6aUGvy2cCFWr9aqLsDqVavwQKIHMHyYHqe+26q3cgf074/nn3sO+/b9Y7w36TwFHTp2EENGZlAa+f777wsgOHToX6Os7/pZoHef3li7NiZjWEnCCIo2bdroqhv4qmBBcMHgBP/P+v33FShRogRefeUV7Pxjp9zC7dy5c+Zg8+ZN7ioWLlwgakjTp8dG2ujvuTo9voEpcPz4MZFqUoqtQ5HCRZA5UyY4aje6PWYM0Di1cOHCyJM7j7EroGphv6eqFcf5of8O4b/D/8midPjwYfj0k0/AhaQKZp3mNXcn/hRpmG5X5LlFl1clSpcuhbJly7iLL168WMbe339TOsugy5sxsHjxIkydNlU8wrCtag5gGRU6dvgFFJLoQIn7yy+9jPnzYrJbZD7L13XCAdNuXbti4gRn/l67dq2A8Hz58rnHoyeNIN5wmjVr6p6nubAsXrwYEj/wADZu3KBJIsCNkssd27fJTte//x4EwaYKvt5bp6kS9MjDsRaT8hz7qV99Fbv++tNVHvji889Rv35992/P91DP4qKZ36xaNWogS5YsXsAU4DcobZq0YNt1OHb0CNKkflXeS6Xpdt9KrGqwwNTnQLwVgsbnPZGBKdnHQZQkcWIBk1zF6BA1MIWI6Q8ePCDFtY4pXUQxlPympNT3wQcfGG6p4vPd4rPuhAamEcbHhNTkuwEN6tcT6af8kH/e7wz06NFDgIez1QmRiL733rugVwUGAoUMGTK4pS1M+6V9Ozz/3PMxmOC4Lb8TTZs0kbp8TUpOGgSkPP7445gxfbqrPEBQlPzpZOjXv6+POlRSgfz58cknn7jv4QWlZWXLqI/hhg3r8cgjD4uLM7NQxQoVkD5dOpdEgDneNIrJ74QEpje9eK22wD788EMs+u0316v5a7PKHjduHF568UVDYqraz8mfY1gF1gFMnz4djz/2GJYuW2qk+6+fC9WopDy6b7Ky+vXqIUOG/+GS+5kQVYwXX3jBveXsmx8QaUzv3r3AxRjL79zpAFPloo5PUO9w8uRJWWitX7cuBu/g79280+MbmEaAxqT6HVwNR6dOHVGkSBGXtxP9jmbbVEmOt5defEk8oOh7dV2nTp00pGVObt0ffsDPIm3TaWa95jWwY8d29OjZUxeMYtzQ7eDfSPr444bkHSK9fuG559C+fTtXHWb9vFY7cA0aNMD6DeuN5ziXnJ/Sp08v/chJBbJn/xjflChhJHnXHdPfCQFMuYi4YgA/p9lcRL/88ssgv1Qw2w1ZfJ3xUHmDgLTHHntMBA66JtqCcJF+0l2PzjHr83WtynXp0hmLFsVsbuHOZ5Z33sF1w16hTetWeOmll0R9JPJ76OeqnIYNGog011NiCixdugRJkiTGb+52QOYvCsf4ffFfr64/uljVYIHpLX0AoyNuXOcDXLnRWt5hPASgcKuMW0HNmzVz5akBRqkU09V2gjsLlIQxTeuQfemyyqdeEwOt8N968025l3UrN1PMiet3Soj6Eh6YOpR2rri9tXfPHleCr/fmRLZDPhqFCxcS92AiaSlUCL/+qtQ3CFSyZskq0henZoDbLg8kSoQli7X+p1m/WRLYtesvtPIwhtP5ke+hCgHdk20yVvxsQ5asWVDGBTLV3Z73csuIEvjWrVpJNierL774wi2NmzRxovQtvRDSH2l6maCEgfrSkes1nxHVdcIBU91uJwb+2vWX6I05Bl1Rt5W6hp7AVJd3kcA95tTClGV3+5Vs6XtU3LlzJ6xevcozUX7pZzCGSIFoKJnvyy89ys6ePVO8f8ydO8eVbt6n7uVcQm8glMQvmD9PFlYamDp0UWVZyeYtm5U02ct1nSrrXX9Mf8c/MPUgjOsHd6+i7qvAls2b8fijj6JXz56yuCSdtJs/hz6sUL8rBMBycUNPKyroPB27kl0R29Cnr14omnm6vI4hi0GqWyxbvswsKNJ4Cih8z/Xse7/j4YcfFoPbzz/7TCSnZgVcbNKLi6N2pHKp5sKF9O3rjycEMHXopFrv/O7YoYNLeuhPgq2p4dxDwc9rr71m2AlAtsD5TebuBL+t7A+ez9L361jXq2Kq61H9KXLQ5RlzgXwR6dOljzSmJ06cKOo0VM1SwbzPvFaLVaoZeANT6kS/mzUrXn/9dexxfdPq1KkjO2CO0ZtZV2yvVcssMHVPCrElYEKWB8aNHYt69eu5Jg/9bIDbQe9kzix6XK7eJiuijG+8IQCAK9a/d+8WHUEanFSoUB7Zs2cXgMqtqffefVfKdezYQd+OBQvmC0jiIOLWLz9AKujnBkt8J4Cpog1pSyMD6pa1bq1AWtQ0BEaOGolHHn4EuXLlRPPmzTB//nw3T7h9xlU7dYHNwBX0Y48+ipEjR7iSHd4QIHHbmH/cnqF0hfXeMNKZB7Gi1fepan6sW1f0oKhHpAPLcosxZ84v/H7E+Jx69X4UkFmhfHlwi8tZSQOrVq1E4sQPoHbt2rpaiWlkQGC6desWV7puT2zihAamqm388NIwjDq4K9y6tNG1G2IE4xuYet8LdO/eDWXKlEaEzFckkWcZbilfu35VeH0z4qaM2+UrlgvfpQ9cu2pYgut7IfqJ1Hcjr8zADyDHv+mtw/OZEMMZvaCl+gF1Dz2BqXrOmTNnZLu6ePESxtambsPtxgkBTFUb2bepa80xRMmXE7zfQeWwT993772iItWlSxdQCJAtWzYsX+YPGEC2kfPmzet3zuWY1nxmezheevbqJSoFVJMRXos3jMhtatfuZyR/+mls266NEVU7v/76K7z77rt+dyu4bUtjHgK0NzJkwD13340OHZzvBXXd6cGFfcAMjRo1wnOiqqLnEO82xfR3QgJTs03qbWjY2LlzZ9ermfn+riF2Hzm/yClCBnVjhOjsU02g9nffyfc1ZcqUMh/qfM/xFYEbN9TcTb7yG9y1WxcBurwmn7kj4Xh30G3hLsY+URGqXdvzWzF7zmz5VoyfoA3T9D3esT9gynKQBS/nrTczZkSjhg0xbNivfvur9ztF/1tRwwJTrwk+esJ5MzG+f6uOxq1eDnTqbN0U8bx+LqWpa8S6lyylZSQNT6ik/0OdOqJTQv3R3n36iDI6dQAphZOTn5YsQZMmTQXsdOvWTYCL6hbAqJEj8W2Vb9G4SZNoJAO6HYEY3ylgCtn2IZ0zZlQLhEaNGmrSRgIVqs+pbPLt/vvuF1C4xq0jCFE0f+65VGJkYlQkExV922rJqtN/gW1bt4JbcHx206ZNxKKYEg+CxcaNG8nWGxceFy+ed1VJHqpAacfLL72Ef906jbQUvyrA9NNPc8D/6hjykSxQoIAAG0p/TPUEWnx++uknMkHSMIqBKgLUZ+VH88jhw64W3Ep/uhPAlDqaOwRoc4FAN2vLlmqpRlTvEFNgCtFd4wEa69frrVSzXkVvGs1xMUG+UvpMA46KFSuIlIYgqt6PdUXypYir74cYOLHdVatUcdFdRdyyIzDt26+Pke55Hw3xrl3lNjcwY8Z0AaaeFszqVs5ZdHNH8JLq2WfjGJwmFDClgc850TV9++23hTbVqlUV4K/eUtOGMfthhBj4pU2TRrbAuQNC62uqq3Bc7f1nr7rN/f1RP0uXKoVu3bp65em6AW4H00sD+Uz1LRpC5s+fXwwtueVc78cfxRA2sn4qxGtEiuTJ3XrAroeAwJTSMb6fCvp5OtYl6UXgjPSVJEmSuLe9eWrgI48+ijlu6boqz+8VFz173acI6vpiG985YLp161YxZKWk0DdtvN9FleK3c9bMGV73uH5CGZa+ljYt3s1KVS1z/mV9DBFy2iPVOshr7ohyV6Nc2bJyzTSqSC1coIUXuh20JfhbxpnnN4feAGbjkYcfxugxo93PcL4X+n71fKr3RJaY6rZBXFVS1Yu7dTzgxwlmPbdyrWqywNQ9MdwKERPmHjXJ8FnKwtNTwqXSFTu5yrpubBepVJ1n/uJKy9lWUjlKr82sT6UrXbGEeVffA+VWn32ngKlur9qaozUvJ4StW/xJAxWde3TvLmBx/br1sr1GwEnpNcPhI4fFuOT7OnVUYdd/Wtdym82xkNXPVvquPN3r5IkTYglMn7TUHaJVMC15mUflewUy9X2qYkp6ueV00FByZz+kgQMlOopPLKvvY6zcHFGqO3jwIMyaNQspUqTA/9KnN1QZINe0wqcOFC13KXV7/vnn5QN5e9L5OwFM1Xvz3XkkMC3z8+X70q9k06FXTICpkFQ8NYwaNUr98KC3Q3t+OIXXwtcTaNOmNbgNb/LaGd/6PrWAIlisXKmSq34VURJPYPrrUO1GzrmHkjrqu9Fo5/Lly7hw8YJ8qFgP9WNpKe68p0Mf6t7yY1aqVEnjWbreW40TCphGsHNJu8+cPSNAO1GiRG5w5v2+V69eFjUNLtDMwJ0N0pX2AU5Q9VKNigaQNGhSITJNOGfTGEuN3xMicevYqZOMcerwsg+wL3iOaVV/+1/agxb/O3bucB4NyHh+P9v7xvcg8nPV+6nbuBvEXTptGDllyhRQRYAGuWbgHEK9Y23P4Ekjf8/wlX4ngCl1ay/Lop4+xFXw1TYzTZWiig6PyfV/j8rhljr1xhe79TXNuiLENaQa0+TrCVDizQWgHtPsB5Gt9JU3DM7d3Lkyw4wZM0QgwKPHVfB8nsOfqCWmNPqkFJlHnTNmf+7iliizZn/1xiRdtcwC09siYkwIHRdlXP3IHXnXqTOYHtPgr6yu27senR5M8Z0HpqQiVSjo6WCqX9+HEKX2J594wm04Q/+kPOlL62ipj8E7KFnyGw/GEAByYvBtge1RVHSatO6nZ443T4FePXqISxz6LtSBH7zXXkuLqlVN6Zq+V5Xi5En3KpxQGVavWikuZqiX6khZmROBnTt3iESWupD0LjF+nDow4tYntjsLTPlWlHhTInb9upIk+n+X6ICpkA9z581Dv3791A/3f01zM3ZnygWBo3M4hpnneQ8Xsh999BG+/uorsxBGjx6FxIkT+zC2Un4eabFLIze6E6PHCUpXKI178803ZevaqYzPc0L16tWQMcMbTsJtz78JCEylrarpBw4ekPcd4tPVkpJiv/3WW5H0/Cg9pj4mdzC8w4ABA1C4UCEj2eSVvjayoazy+0bqHyyjy+sY4OKG0vHV7kNTqGN8DWlSp0Ghwvq5ury/WD2/RYsWAs75a+OG9eJtwVPtA+JykNJlx6jOX53RpSc0MFXv2KdPH9HhV7/0/6jaCmxYvw6du3QRUKvviMwL1qEC7Tn8uxLUpVRM39COfqiZZ7YJuHL5EtKlSyfuu8xS/FaQ/+vWRwe0/QFTBXqpsqON5dh/aLhK4Yjj1cNsT2yvVYstMI00gGNLSFve98ALBLrcCWBqTgWkAXDu7FlwS88BCipdlVTXNWvUkEmD+kE6UCc02VNPuScj+st8K2NGD4BHPbZ/Urb1AAAgAElEQVQ0qVPjnM9tOF2Tirll7mnta+ab/HJZ5T/2mEg9dSn6ViV4HjlyuCvJfA9ljJflncziRkffw5guUt544w0vzwFOCRpeFCxQwL0tfOv9KaGBqX4HTTuIw+ncuXJ58EiX8nyv6IHpqtWrMHz48Eh1RZaGefKBz+NHbMtW7YfYaYF3G5jD7V8Cyitu90RAx44dpc+eOqXd2Th1XLp8UaxzqXdIKRnBMx2Sp0r1rPjw3bzZ+7kOfbhNXSha8KXLxyROCGDqvLuin9q5yvT22+IsXeVG5kGlShXx1ltvGnqGymUbfZQOdRmb6vpYB0Ep9fWc+ny9vyvbFVEaOXiIc5y0Z655v7LK5/gdMUKPX+UKi0Cju9sPtvke+n5dq5PXtFlT9HL5WL548YIAoQYeroiArFmy4NvKlfXNPsCyrj+6OCGBqWru+PHjRDppNN5ov5mq264OFqHDedLDDEoP1ExRdKRKXs4vvohCVc68B+jXr68cqOOZyl+6DapeptC4ivSnvrkODRvUx+uvvWaoDugcxp51+N7K55w1SgQh8+bOdd9MVQT6buWco4JZV2yvVQ0WmHowJLZEtOU9O3Sg0eNOANOISECChmvly5UTn4Z64PKDskW29kkzoGuXLiJ1MP1iUupIJ+s8fYmBOob0Fblt21b5zX+UWPkzriKQ1CfK8JSon3/+SSRjvObqmX402bbLV7wdsbPmCBQsWAC1atVyP4sSl7feessAmMpdze7du6UM9eqoC8kPHXWYdeDWD7c0L1+5rJPcMYE11QPoqFmF2+lDCQtMPSXAfHYEylcoL5Ip/YKUKJBvVKfwHCvA+AkTZLs38lYnsHbdOjlh58plRTOqONDdD09y8bSUjRADCBqe8MNFvpK/6oCHxnLNk3r69+vn0iH3pi/EgIZbf47UHciVM5fotOv3oMoHt+kd3UWdo+I5c2aLXhuNg1SIPA7YbhqIOS7IvNtyK78TAphGfhe+Lx2Wm4dC0Lfnho0b3OOfBnFJkz7ucbgE3Wqle/11rxPbIOCEDtoj9wVPmtAKn+65NJ9/attWtlPVmB4sfJ4yZbJIQr37G/nCecj0Y0p1G1pYH3Kr7ESIKoGpTkDdWOdQBKri7Mb3338v6kUuZosfbRp26bBnz25ZMNOiXwXP9/BsW3R5CQVMVUvpOWSy6yARjmmOPRpt8nAEBu4y0D8p/Qur91Dt42l92qUUwSh3vUaOGCHb7VRvIU3MQLWOX4yjxL1pQlWcfn05pjlfDxYVmIYNG7h+D0KfPr1Bjwje9/EZ3IWigRJ3pXTIkT2HqAPo39Qpph2DL3WARg0aiCcCB9iSR8CypUvEWwtP6zMDXadpmkVuT3T8NfNVrRaYWmDqo2ObHSWYrxMamAK/r1gObuE1adJYLFl5WheNFfbu1cYOauDxNKQUyVOIlS9TaFRQIH8+fPb55wJYadHPbfE+ffsYFvAAHVlXrlxZFNx5dCSPRzwfSXmePAMO/fsvqP81bepUsRKdOXOG6KzyZB/+cSLhcakO2DB5zY/ln+AE8duiRdi0aZM4jXb86AGnTp4UPdRPPskhR2jymVRboE9D+lzlKTP8XafO9263KQRzNAQhPdq3by8fOOpMqWA+/1auEwqYqtbSOpvGXQR+NCzgAoEuXfgx04FW+vfee49hxa3ei5a2LVu1xEMPPoRFixa5jGjUfTRepL/B19KkAaVyb2Z8U6yhaUjibJdq+ihJNUEQraM1b8nX2bNmuX9PnjTR+DDqe9Xz2FbqPJYpXVpcXlGiVrZMWZjSUi5qqDLi22G68rlLw6/t253DH3jwA7f7eeAAARClbNyudoLZjlu9jm9gqvxUZn33XTEmpT9ZujqjcaDpbYLvROkg1R/Y53Vo174dcuTIIWN6w8aNIi2m2x4nKB4QYJYpVdpJ9vldghzGwCMgNZ85pqlnrn9zTC+W/nTdVZdJV+DA/v2iU8r72E4eu8l30oE+STNnzix64dq2gSc+0QKbElLyr0XLlgafVfvpVJ7qOjz1jm7Tan1Xy+M0wtsFKwR1PC2O3klUMN8rbq6pLtW1axfZFeIczkU4xx515HlYgHbhRVDKMU1jQwZu35NmBII8dIG0ypgxo5zmxQMUGLj79crLr6B4saKyg8ST1qiH6khXzXfgHRFirEiQrHkrY3q2M6bZD5w+6H0/xDMHj9ClMRRBLI9KJZ90oNCCY5rCCRW4AIvApYuXxP8s9ZH37N3jsrhn/Zxrbsp7U1Vg4W8L5VAIHg5D1RQatup6bp3fqgYLTH1OACaT7fWtd7I7TbuEB6Ynjh+TLQ0e2UYXI/wAeE4+auANGzZMPCY4Fplqe5AWrgSjlIx5+qFUEwPv5sqdW0aUjKjVLlN90Vo9K2b/fd/PU4mGjxguf55+G5WVfqtWLWXSc4yWIKfCUC9yyNDBmDRpEv5wOV1nO2gUsmrVKlAR31Ft8Nd+X22KKi2hgCnbANlWo7UsrV8HDhogEkXJkH+qDA1a+EHgR0UFGtHcFNA/ZswYOQZ4+owZxgeXrmamyclNBAF9+/WVP57kRCOxM35PinGeHPWVL/qpO5Yu+X971wEmVa399emzvL8+9WGjKBaKos9KkaI0lSaIDRQBFcHylN6kSu8svReli4AUQWmiVAFFmoBIUylK7yCg+/t/v5PJ3MzdmdmZ3Z3ZYSb5vt3cSU5yk3OS3JOTc06+wajRo2Rc6Usd1LiC+E+kZT03GyqY9airLql24FyPCzEM4gaNeres1/eI3yyfnufIM6bcNHIT2KRJE3Tv0V3cIjHNFw8QvHETpqVmHgAxQhs6bCjo+zTlNc8Kivesrw7JyEbXmlocCKdqfnLzwXnt2x6IQ/ak3r2EqaGRGwP1YqmqwWtGeWri30JdXUvKI3D209dXaqC2hJoeDYmp8rpAYz/Smn967tEd19RpUz2umYADB/bjvfff8x71c1M4YMAAUGKqy5BBHzRokHgcIQ65GZ81cwaaNG0Crpm+uqKB8JAajXW+v/Iqj8azFI7wRMUxdia8UtfinN7ilaomi+cVGjWRzlTB4rfG98pbVS83z/0H9AfpzY2RMpBmnr+2hJOm6reMaboRGQ7SLWz6B244OIw2Y8q2BQrudptwOs9MM591fqD6zfyMfjbbwWezfjMvULqGCZZv5qXnOZqMKdsZKJh9MGECpWsYna9/B4o1XEbG/t5l1m/mm+n62V++mWY+6zIZEUeaMWUbAwV3+004nWemmc86X8c6T/+OZKzfpWPzXTpNx8zzF8wy+jlUOA0fahwNxpRtCSW44dy//dURDIZ5kfoL1Bb9PjNfpzF2BzPPX76Gd8Ol5beqyzKmERsUaSGKLZOxkzQzGFNLw4ylYTj4jDZjGk7bLGxkxkU0GFNLu8jQLlS8RosxDbU9Fi5y4yHBGVP3cYvm+W0cXxg4eeoE6DPTUeaPr/7Z3qTEwCsvvyzHpylzbEo8YoD6ei1amjcxxWMvE7tPNMAqV64cDnrc0CU2NuK/97w0ItbCJZFuEB1Bb96yWRzV0vLZ/sUrDg5h2/af8dRTT2LZsqWW3nE/1g/hwMEDeK5yZXFrcshziYCd3/E6v9mvQ6L/SGMbPqu/eO5vIvbtkOhvly5dWq5TtTSO5zFwCAcPHhTfyJHmA8OtP+KMKR1BFytWTCxGeW+8/YtPHJQsVRLFihWVe4PprJ4WwpbW8UlroWtJ1TdatNMvZ+knS1t6x/n6RmaF13zST7Cd3/E5t0lXuqK6/bbbJLZ0jk86cw0vWbIEipcoLjeGhcs4Rho+4oxp9mzZMHPWTCxbtkysB2lBaP/iDwek7+w5s4U5HT9+vKV3nI/zpUuXis9IOq3u0aM7VqxYYed1nNN8+fIVqF+/vvjyXL58udDfruXxtZZzHZ8yZQro5/Wz6dPtOh7Hc3rp0mVYumwpShQvHmk+M+z6I86Y8ij/xInjYTfMFrj4MEDn8by73O3c+OLriW1xqBio9sorWLhwQajgFu4ix0C/fv3QslXLi7wXtvnBMLBv316UL18eR48dDQZm8+IEAwmrY/rHH797SGgt7CJnYZfZuLVW+fFLW39jy1rlJxa9OQasVX7809xa5cc/jfV6rtgy6y7KuouKoB81PdgyK7aMaeIsaIpJ4c1SVatUkYsN1BKXWWPPvjc6Y88yptHBc2aOZ8uYxj+N9fiyjKnCgGVMLWNqx0CcjAErMU2cD5jzIePVjs2aN/Os54x0no3jAxeWMY0POoYyHy1jahnTuF/ArcQ0cRY0LnqWMU0seiuaW8Y0lA/+xQxjGdPEmdeWMbWMqWVMrXQlrsaAZUwT5wOmGS17lB//NLeMafzT2JnPZMysjmlcfZg1cW2sJrKVmCbOgsYxbxnTxKK3ormVmMb7em8Z08SZ10peaBnTmGZMFZGc/2ohdn7rJ70w6d+pxRpex6nBM1/DXkyxZUwvTrqldYxZxjSx6M1xYiWm8U9zy5jGP431mq94EcuYxjDDdfLECezcuQO8J1j5XU0G3Vxt374Nu3f/hgMH9isqevpw5Mhhydu27WccPnIYJ0+eEP+dhGUdv/yyCydPnkzBZJ46ddILR9iNGzdg3bq1UtfZP8963sFID56LJbaM6cVHs/SMLcuYJha9OVYsYxr/NLeMafzTWK/7it2wjGnMMlvAnj17UL16dVx99dVISuotFPvhhx/w5JNP4vrrrkPuXLmw8ttvFSUB/Pzzz6hatQpeeeUVbN26FXv37sWHbdviphtvRNZbb0VSnyTs27cvBYPJtKSkJNyYJYvU2btXLwwbNgxVq1ZFkSKFkdS7N/7++y/Pe/QAuhhiy5gmzoKmmBTrLupimJcZ2UbLmMb/HLeMafzTWK8JljGNYUbLy2uia9cuuOSSS9CtW1dv4qjRo3HFP/+Jf15+OQoWyI89e/d48yZOGI8JEyZ4fy9dugT/uvpq3HzTTVi7dq0nXQ8CHZOp3YqrrrwSd911J44ePSJwhw8fxkMPPijv79+/X4Cyuo5YjDOTMfWSII140+WD4TUUmGDlQ8nT79BxKGVMGH/ldFqg2CwfznNmSUzd/Qi1zWY5f2XMfD77g8nINPN9odZrlgnUxlBgQn2fGy6ajGla+hFuGQ3v7mdG/tbv0HEodWtYHfsro/N07A8mLWmZxZimpR+6jBmbfTbT+WzmReLZfF+o9ZtlArUxFJhQ32fCqXqtxDTiA8NEeijPijDHjh3D6dOn5c5vMqa8+1uHmbNmommzprjzjjvwj0svReVnn/Uc0QOTP/kEkyZN1KD46quF+Pe11yJb1qxYtWqVJ93dDsjx/bXXXIPcuXPJsb+uoHnz5sKYPlm6NCiNUsFdPlZ/ZxZjqrBEtYu9xqYh9EVIMVhU1fj7L0qq/eEXkkeY5L9JF38w6U1T/fj9932i1uEhfhjvAs6ePYNDhw66yjg1uZ/UGEtru6PNmKrWX7hwHr/++gtOHD9mdCe1PijQP/88i/Pnz7nww7IMyVKv3ijqtMjROlk2qAcPHlCv8rTB//sUiO778YB9B/766y/89NMWqTvj15BoMaaqv8TNnj27Q8CPpiFAValdu3bi3Lk/PeUCjQ3i6gL27//Dz3gIVCbcdNUEvoMqX04IVo+COnL0sIxH/zRUMLw6dMuWzZ4xzbRg9YaalxmMqerP6VOnwlhfVRnzP+np4EDl/Pbrr541UUOGiodw4dR6+PPWrTh8+JB+mdEef/UpMKr07dq5I0AZT70/b5Xx4AD5qy/cNFWbZUwzZOKEi/xA8MDx48fR4oMPUPvNN/Hpp5/ijddfx2X/+IcPYzrpk0n44Yc1GDp0iEg5L7/sMjRu3EgoOmXKp5g40ZGYpoUxda5rBV599VVhTF9/7TUkJ7PdDIHaH2vpmcGYAr/99htq1qiBu++6C7flyCEqEdu3bw8Dd8CQIUNQoUIFnDp9KkA5oGvXrnjxxRdB5kCF1PDvAQuZfsCQwUPQvt2HGDhgANq3b4/TAdvjfjfflSzjuF7duvrFEs+bNxdPPlka5cuX8/5VrPgMqld/1dgUuesL5Xf0GdPPP/8chQs/hpy334578uZFr5698OefwRkQftg5Rvr06YOKzzwjTItCkO4j8P1336Fy5WdlDHEcde/WHRfkI6fwmvoc1CjXdQaLgd9//x3NmjXFoEGD0KpVK8yaNdNTQaBywJw5s1HE0/e8efOiR4+eIKOtAstB1inSn+N5+PDhUjdVjhyYQPWHmh4NxhQgQ1q7dm3ZuOfInh2VK1fGpk0/ptIPYMyYj/FYoUIyPh555BEs/GphgDIqmWv/c5Ure2AYpYYHDZoanM4Hxo4di9atWmLIkMFo27YteDKmgoYxY8VYs1333JMXOXJkR6mSJbFk6RKjDHDhwgXQO0LHTh2F1tWqVcPy5csNGLPOcJ+jy5hyI7VmzRq8++47aNSoUYjfPQgDW7deXZQqVQply5aRte3777/z4ADY8tMWWa85n7letPvww1TXCl/666pCwR+wa9cuNGrYUOZ0y5YtMPfLL1Ohh8ru378/OnXuJOqD3bt1w/lz3Dgz8L0QNcGGDRtg+IjhaPthWzRs0ABHjx6VPN/2htJON4yqxjKmqU58N+Ii+Rto26YNLr/sclnQz507J8yJmzGdMHECVqxQk75u3bpyrE9p59SpU/Hll1+kWWJKyerdd9+Fb775Bps3b8bAgQNx6y234NFHHsG6desyaOBFEn/uuqPNmAInTpxAnTp10Lx5MyxcuBBUgbju3/+Wj5Oza3W30/wN7Ni5A/fluw+FChbE6TOn/eAd+PHHH3HHHXeIvrHalRPMrMf9DNk1L1+2zE99blj+Bj7++GMUL17cy4zWq1cP77/3nqc8I3/ldBrwxRdzcN11/0ad2rW9ZSgdfPnll1H8iSdk0/Pqq9UkLlSwEPI/mh+nxDgvtbr1O9xxNBlTYPHixahUqRJGjBgBMtvU7Xar3fjD0Zkzp8W4kDrcN96YRYwSFYLYH+CXXbvQuHFjzJwxExs3bkTjRo3AzefkyZ8osKB4V3V8t3qVIa1348n8DZw/fx7PPfccevbsKfVTN71IkSJYuHBBgPcBS5cswbPPVhJmc968eXi1WjXpe9eunT1lFLNbrFgxzJk925tGRq1cubI4c+ZMgLrNtoXyHGnGFNLW+vXqoV69ujKnBw8ejBtvvBH35cuH3bu19NTdVmD+/Hlo0aIFVq1ciWVLl6JEiRK46847DSmTWQYgE0Oml6dTTjBh3M8QyRuZKBXc+e7fwPTPpqFw4cI4ekQxo2RMX6tZUyS1/uogk9apUye8/vrr+OKLLzBu3FhwE3LLzbdg/Yb13mZ26tQRL7zwgowlJn780UfImyevSMr91etvXgROiyZjqnDKtfvOO++UPnk7GXTeAV8vWoRnKz2Ll6tWxUsvvogGDRpAnyTs27sXzZo1E2HThg0b0L5dO5nT3LCFhh9gzZrvxeg5dXh1UlXm6adFoED4nTt34rFCBbF8ebD1H+jXrx8qVawopxwsRwEZx7AOhw4dQrFiRfHRRx9JEteOChXKy/znWFHBPe7C+a1qsIxp0MEWDkLTC6smBKUuNGriEShDly6dZcE3j/LJmH7zzdeSTwlr2TJlcOkll4jEptYbb2DGjOmKukAIR/kKlJb4ZKBy350LQ4cNFaaIH1mqCZw4ecJTX3r7GO3y0WdMFy1a5KPjS8SNGzcOV1xxBSjNViEQHiCeFChZevutt1GkcGE/jKliMIcNHYYa1auHxZhu3rwJHzRvnkob2DZK7o/hgf8+AO6edeAHMEuWLFi9OpBKiO4XQAnxwEEDUbZsWbxZq5auQo75uIFyh9atW8mmTKXresKNo8WYUkJ0XiROPKLV4e+//xamK1euu3HkiNLTDvax5YkI1XGcOhTu6XWDR6JmKFCgABo1bGAkBcMN0LlzJyxZstgDHxx22rSpouqjDCNVEX5UKRlLuelR0jFK2+gxRAf2vUL58iLhPeKRwM2YOUPWJOVRREHSO8hDDz8kG1+VEqxtoeRFnjFdtWolRo8apbsq8cyZM3HZPy7D8GHDPOlmW9X4oKGqc+zNzeRG3HrrrS5pI8tBPKwMHDhANmlPPfWkp05GZr3uZ6WCldSnTwjwAD2s5M//qEi5dIHt27bhthy3YeECf5sQzuNtwqxoeMY8rbv++uvRqlVLSeaxL/s1duwYAywZDzzwX9Su9aYnzd32cH5HkzFlu1TgCQ43bE4I1GbIhnro0KEOqOtp547tsuE0k8uUKSOnRCotUN1Oe8gDBN4smuWBMR9/jDx58hjrEOQUj6dU8HvyCVEhobCDAgkdvv7ma2TPnh2bPacDAwb0R66778axY1pCCixYsEB4hyVLTCm62Z5wntWbLWMadOKHg9D0wgKbN23C//3rX3jgv/d7j8T4gXHrmJIxXbz4Gz12sG3bNuTLd68c69PQiZIqHQIf5RMiWY4X+PTjxo2g1DVvnjxi/DRq5AjZ0fEoetHXizzVpbeP0S4fbcY0WXDHXaQK7C8kLXu2bCJdctL94QKYOHGi7Gp7dO+GAvnz+2VMR40eJbvn5s2aoWSJEn6YB/91U8+Px0fB26DazGNaGtfpDRDLUMeMx1BtWrf21MHI/S6lJ9e7d29hTilFeeON173wShVAvUOVhSxyJUoUx3ffrfbAuesM9Xe0GNNkYUzpnk0F3T4lZabUixIS3zwNo2Ng0qRJfhhT5uvgPHPD+VVACaauU8dk1np6T1VS0siB45so7aRk8y+v5w1g3Nix+M8NN/g9riaz6q/vY8aOAcf5Xo8OJk91uJ4NG+Z8sKdNnYonHn/cK0kK3DbdxtTiSDOmyTI+lR4wscX2KCkqJaadOzsS4pR9EVBvGRqYvvxyVVDqpIKqi89kSrn+169fXzYEumTKOk18ANxsDho82AFPMR81PLBi+XJcddVV4IZBB+q95smdG//73/88SRpexXQ56Ku+o8CKFS2Kt956S37MmD4d11x7LRZ4x6eCeeedd0SqTHsJFXzrDt43EzZzGFOe5oTKmM6ZM0ck4iNHjvTZsDl99KDAM374i6ed06drIZLZX/ezKtuvX19jPXbD6N8KtlzZsnIqpd/KmNLZW26+WU7kVLouw1jZp2T5z3+wdp02kqZnoN3CmPbpkyQwlKbSIJqnPjpwvme54QZ88EEoQg/znf6eVa2WMQ04kf0hLZJpkCO9rFmzykJx3LMjoTU+GVN+aHSgjqlbx4cMxE033YSrr7pKjvM1rGZM+cH47jut78J+UPJ2GKNHK0kAd/Pa+ElLa7iw8N3333+/6MSpOiOJg4yuO/qMqca7uSDt3v0r7r3nHvwQ9MgNWL9uHXr27CFVkIFMyZhCmFYtxaz7/vupMKZOa/hEA53OnTr5Jnp/adyrhC6dO8tY4odPB36ceQT/4osv6CS/jCmPnKdOnSIwFcpX8GFMnYLO+6ZP/0ykbek/CooeY5qyH+wPxO0aF29ltMYU3U93rDYhKSWmJpx6y3erV2P+vHnqR5D6PAAS8SNGyVbK4Fs/PzAPP/wwqrz0kg/ookVfydyf8ulkT7pvOZXom9a3b1+5SlD3napIVG+48oorQansjh07ZCysWLHCT51mXeE8R54xdRCj2wUcOnwIDz/0kMGM6Tx/saqBJwXcHKqg4YCvFi6ElrZRDYiSaidoOB07OXzauWsHRowc6ZsovzS8jiHv4GZjlffEQxUjE1PSr3ScZXVw6qEUuGiRIt7j3MmfTsall17q/a1LtG7dGtmyZTUYNV1HuHHmMKbVqr0SAmOqesuNOE87r7nmGmTJ8h906dLFq9bguwYoeH5vZ86a5UFVIHxoTKp48OBB+HalnjtmnlkesmmkVLPaK6+YQKA+POn/pV9dU+CDDz5Ajhw5sNM4BeLJLdVPqr/6qtRF9QDW7fhQp/racVH50zDqpWabwnlWpS1jGnChDweZGQGrJE00ZuGx/Ny5c8XSvmbNGvKBaNPGkVJ99NFo8MPhOwC4IxosUk7HcAH4+utFuPKKK3DDddfjW8PnKQ00qMfWrVs3qWbL5s2iq5oz5+1e3TRKRXicrI/0aaHo+86M6Hck68gMxtTdH2DsmDFiOBHYeExJYLp37+Y1/qGusS9jSsniMTF4OuCxmqa+ZzCJKTceK1d+Cx5F0iMDVQneeftteWYafeDyIgVlPKXbrSjM3TyZJtMVGRnT0qVKoXTpUsYRpS7HmJurX8S1mTaCKV++vIsx9YVnmVq1aolbNPVmMz/c52gypu62qda/9NKL+OyzaepH0LUlNcZUSZKpVnP7bbeJdOqwj6TNeT8ZBX7ovl2xwktrGifw+Jl0J62//XaFXM7h/kjSOvuG66/H2x7pl6fhciLDeU8DSyc473TXQ5gqVV7ybkhUvjoteOmll2Rd4qYmdYOhQO8IlB4NxtT9bmDGjBliyHI+FcND0obGXu+9954YvIwaNRLJMi4UVknTDh3ae41H6tSpHZQxJUNAPUE9pz/9dLIwFIrOq4TOVMtSfqfNdgPt2n0oDMTmLZsdkgJ4/vnnULBgQVEjUhlmOfez0nWsWLGi160g3RDSfaEvYwLUb9BA+kxDv9Trdb/H/B3LjKnTTl5es3r1alGx4txpa3yz9Xw4cfw4xo4bJ8wdfZQ7hsZOPXpu/fTTT3LqoWnbtGkTOX3Qc5oScOUhwiyr1Kh4auNIMBX2qSNMO5KJXq89upzKf/GFF3BHzpxi6OchmHhXoGohjTQZ2rVrJ/zAF3McvXF6XaGklac6TtB1hxurGixjGvTjES5S0wuvjtTJDD7y8MPo3qM7KLWkBR8Vp7l7oaUdd3K0wv9dnOWTkHyvClwAtS4jLW1pKckdDo9raOFPRXb+vVWnjnzwqAzPG6ZGDB8uO6P77suH8ePHe4/a+EH77/33I1vWW9G9e3ev7quePLEdZzZjqpgLHs0xeIIAABI2SURBVHkF/yDTKOEzw2oS6NihAwoWKKDJKjG9LdAvrQ4N6tcXRlH/9qUF8NOWLejWtSt6dO+O3r17yQfsmQoV5MKEnj16iP4yGQ/nWMYZR3Xr1cOdd/oypmRgS5cqLcYZju6cHvMchn+jX9++2OH1QAA8U7Ei+LF1goZX76IrKl7ikFKSZMKF+py5jOnnn89Cs6ZN8dcFuohhCNbu1BjTZPyx/w/ZoHLe8kP3v3ffZQddddNt2AWx/qY0nHTt26cPnn/uOTkWpiSHumldOncS4xrfdim9Mn+MKfVTw2FMqfrRtGlTsc523qGe+vbtI7rqPM2ht4qMs+BVuOAa16x5Mw9eGAXDe3rzIGpW3OCRCVEhcJ1kELf+vBXjxo9Dwfz5RbI4bZreuABkVB3jJYgleGDjJ2D9+nVo376d0Jm0pdtAGtHxRI2nLTzpoODCV/VA4YnleNGKmzF94YXnhTGlW6vg/VG5lKzNnv25BxagfnGrli1FoMIxuH79esyePRv57r1XBBu+60tgXAWm28XAmHrRIQ/8XtNm40eX54aDBw9i/oIFQmcKoKjHSo8GKmjc8FcyuOmgURnndJ+kJHDTS2EEn5nGDc0y7/fAKUv9fn+MKSWlZEx54pryfRCDLTdjyjWfvMMzz1SQInR1Rbrmzp1b9JJ5ykfvMDwVcexgdFvSEquWWcY0ootY2gjDBUJLOKhkzB0Jjzm5yHGHtWPHdlFKT2nlrYxntB88lqOBwt49e0QKunXrT+KvlDtqMi00ROACRkMXWoLT5yYV2Vm/abBAVymEpSK8loQFXkTS0udIlclMxpQ8RLJID2ko4bsQmP2FMGWUlPO4k8c7XNRp2HT/fffJIkKJw/p1a2XhmDVrFlgfYeiyJv+jj4o3Bm5YUr7Dk+SJwjnKb9u2DW655Wbs+kXXC9F3fazQY6AEzAm6L5QiTcdrr70mzJS0c9YsOfKjARSPkbgoq+CUoZV2aHpcukywOLMYU6pJ/Aq6ZDlwQPsADdZO5qXOmHqQJRGtY3mD257dgaRPJjSta0M7yqcO4b333gO6gzMDGU0ypnqTG3i+A7/+pvtuXpGsaMGNMjfXDPRewCNfWoGn/Binhq9A+dGUmCoMUR+UTIMKgdplpitIrt08haCeKQPXeDIbs+fMljnNOUKPBY8++iimTfssgMW/qkv/5/oe6lE+VYBuvflmbNi4QReXNYr6xTwJCU4TVYRCiwEDBnjLq3GhfCoPHz4MNWrUQNcuXUR3kuM1NGNLE1f+ni8GxtRsN3Dy1EkxYv5o9GgPrnS+gzpuYrlZ43dZBQ2jYweWTzwR5QlYyqDhGQP7D+wXIdP777/vAzpl6hS5MXLuXH9uo4D69euJxJTCAh3If1AwZkrDKXigpxCqnUybOkXcW5LhXbv2B08xsz3hPqsqLGMac4ypGlx6YPjG/vLchNcl/MHqvFDjQHW43xmrvzOLMVX4/XTyZDFmSoltX7zyWO6pp54SC3ZaatJyklJq7nqLFi0i/iJ5TKv845UFYcqVKyd6qzzmfaL4E6DyvQomLXzfTIfaHdq3902UX2YZ1bZJEyfgsssuMwySlDcA7qgpHfEXPh4zRgwA6MOvTNkyePrpp+VDnCtXLjxdpoyoDahyTv+rVKkSwLLZ3aZQfmcGY0pvGofkZGK7j8Ny9lS32Xx20mjoFlzH1IH96quvxLCIm0MHhzqfsW/gFcYcVymDu0wynq1UScaUCUup2w033CAbWZWu3+FbnuoivXr3wraft5rF5fmLL+eIGtEX3rEJkHnhFcspVZHMesN5jhZjqro3fcZ00MAlZdD4YU6g9gNUseAxOMPceXNRunRpmStqTpcF58odd+RE0aJFxaOKeo9Zn0rR/8nUDPFrDW6WUW2bN/dL0YGk5xAdzv15Vsbg22/V8SQpWPVD16F+0UUYmVuHgdW1mGVU2qBBytVgeP6b9fvccSwypmbfdf91u1VeieLFg7h4gxgZ0R1jYIZOv0PFtIg3T8ycXP1e1Q4KsIoWKYxyZcs4IGJgN1CuHfddQwiiynHjyPbQ36oOv/yySzx2aFdyGlbnMy5UqJAYUDppZnvCfVa1WMY04CISLkItfOAFObNwkxmMqZpY/PCSmTADJdPqWIv4cAIXEkqiz5w9g7Nnz8pfyxYtxH8sjSz4IXBgTnth3n3nHTFGotRLfSx88fzXX+fFopZWtfSHSmOYpk2aSBsk7fQpeVbH8mZZgK6DcubMCWfHr44Rb8ySBcuWLXUa731SlupUN9H9oK9KMqjUk2a/eOTnjBGlC0VpDY+HVDDbkJbnaDOmkNvZeAXwvr2OlIF94amFo+6gkWT2KRSrfF0O4ieVboTo8kcFs65kUbFxaHpGjnvpWobjTaf76hLr8rxEYbBcRXzEuB2G0hyqWDi3FTlt0R8n3kw3ccIEr066hlAnNgCP8Cl1dUt5ChTID3qW8NcPZ3zo9qUWR4MxVS0lYzbTsGhn6ulTJz2nS2ynGdy/VV6DBvVF15O/eArGeXHmrDOn6cGCHio4h5QxoG//SUOeqJGmpC19n3bv0UPGIenBdOVq0Lcc33fs+DHkyZMXvXv18jaUJ2XZsmXD5z6GODpb1wFs3LDB0B9W+TxtU7eSOXDM4QaY3lxGjhyhKzLmvYYNJ848xvT555/30wcjycPUmSl85ppMf82+t4T5QnHjyA1JINUWfhPU3D0tvnTpOpIed7iu6jmtVDZMXKp39OjRA/feey9Oee1CIG77uIaoddi3LfzFjTXHAo1RdVi6bKm4AnMMKfW7FARVR+hzmyeqKuj8tMaqFsuYWsY0nYtGWgdgNMplDmNKJ9TvvvuuOOOev2C+xKNGjgStVOmAn4HWzh06dPD+VtPR+d+6VSs89MADOCfXVTrp5hMZU7ptcYKJU2DTpk3o0bMHevXujaQ+SfI+uoXhjUO9k3rLB40+U1MatakauRCK3zvPC6hDSIV9bcRFpoU3g4wYMdyb5rRFPVFqSsbUCbqNypmz/zwNE24cTcZUGfbQ3yelk7yUYv78+eKEnP4d1e1ryeKPlJctLPG6d9N9Ukf51Pmj+owKKo/Hs5SMaD1Bup7iUbsjedR1MFY+M8eOG4tu3bsJXfv07QveQNOmTRsvrZnn3/8scOzoUdEFpL4jA32w8rYi7VmBaatWfgsaX1CCwsCPacOGDUV32ew7/dFOGD9eYMjMUOratElT+c1/u/fsRqVKFTNIp1j1P/I6psDUKVNETYU0pt9G/nGeN2nc2Hvd4+ezZoozcu1Ka+WqlWKUpjvPCxl4k55Db53jxLVqvYHHixVzEny+T8CGDevlVh7OX87jdu3bg0e2SUlJ6J2UJHYJoz8abWwofMcKJZnFSxT3upjr0rWLqNLo28rIDA/o3x/0sKCuOgaWLF4sfaf6EB3Ps+9Tp0wV+uvxoBu8afNmuXSCuuZOMNuQlufMYUzpqYK37zlBjbfDhw6K2g4NlBlowEadT9KGYf/+/WLHYeoSk3FbvGSxbDgIQyO2d955G5+k0PdU76Aggt5NunbjnE5Cn759UK9+PfmG8Jn079yliyEkMPHK+g+I3vDkyUrlhOp8VBHRbWYb1q1dC9opmJ5XKLjg5QA68Huh3YLpNB3TLoVSYV4YoILZhrQ+q5osY+oz8dOKTFsufElHNHAWbcaU1zTOwUMPPSgueOjMmq54eBXhbbflAI9idODxKfVynIVd40NB8OikfLlyfvyYEk4FMq/UTU3pBF3VRcaRH0kyGvyjxIQ7bfl9lGmHxdLfV5LptIM7dr6DhhX0RUmjBy66OlBakvXWrKJH6uu7VbWRDGz1GtXRqFFDXcR7bMS2lS9XHpMmTvTk6femJ44WY8qbVc6K1WuePLlRsGABPPII6fwwaDxIemu60t8wpYYtWnzg00/qBJPRy5cvn1zA4Pi2hDB22bNnEyMzfnx4PeDs2VpVg9W4cJT8N2jtK7QWuh4RScnxE0w7giMeWvuXpCla0QCHN3vxxp6OHTsYztJVvr7oY978eSKd520w/vrO8e5cGKCM+kqVKomOHTtiypQp4qydzI0Krn64+xXS70hLTHnD1zfyUX/woQeRP39+75y+/fbb0bVbV09fgDffrCW01gYvtevUlmPQmjVromevXujQsYNY6Pvvu0pt3KQxqlap4q3TTWsykLQtUDQ9IvYBlKDKbw+deTKT8hRE0ZEMD41faRRJiSY3m7u9VvPqBIBqRPRnzY0RDaVKlCwhLgMp6Zb17NFH5PifjBUDT2vWb9ggbog6dGwvGzSnAxlDY9o9UFd9r/fiiYyo138dXMvW/LBGXJ/RjRYlm5zvihaQo3fO6bp1lQ4ndTKpepEta1bUfO01fPhhW0O1SmGC+sM8gXr88cfRsVMnMTz1vWgkZVto5+E7p0/CnNNUo3FO4NzlIZtbMpbU42/dpjXGT3CuKmerhg0bJuOVpx46UMLKuc3NCU9SaIVPbwMqJIvaEr370Ak/x5HzPXC/P62/1ZssYxrS4pdWJNty7oU1ur+jzZgmi3Hab7t/FUkZXS3RoIzHOfQN6xylKp+ivA9dSR9TjhMuStxV80MSCGdUTKdhmv+PEOsMNaR8v16EWQNdSpl3PjvtURKcrV6dJN962Dca4Pke9ymYCxfOgQ7H1VGxbzmn/nDTo8WYJstHm8wn6So0Jp09tD54SBt58brPc+LGyUlTfeJxNxk4ftQcY0ONm/P4aesWrFi+TKywT5/RbtpIjUA4ST+tyeDw9hYayTmB71PSYbqnIb0oVQvYd48rM3P8sF66OOKfvgpT1R+oL+GkR5oxTZZ5SJzs+32f0FjTm7Ezfqn+slfUFvQRPI9zeQRKvHGsOyFw/8iIcM5Eks5sB6+6JcOlTz+c9wGbftzolaJx7v7ip+9c0xRTpNwc0kiSfVRrnO5p4H467wsFJroSUzLaHN+79/wmazefz5//00sT0pe4cy7RUAw9LwihvqjGi8KC6h/Xcao4cA7wRjDOCScEwoEDEfwpcHmehtDDhq9KAeGpbnBSJK5OW1Q6877//nus/UEbMzFFvYPfJXpQ8ZX6B3p/WtKlabIpUE+x8/+SSDeFRiXB/IdpItg4LQMrlspEnzFNfexq/JiQOs2Mdb6Z5n4OBcZdJtzf+h06dpcPlK7hAuXrdMYaNr1x9BhTs/X+n3VfzFydxthf0PnB8jRMJGLzve763Xnmb/dzsLKEdeen53fkGVN371L+1u03c5jmL2jYQLEuEyg/I9L1O3TsrtNM18+BYpZ1B3d96f0dXcbU3Rvnt+6Hk+KMZTONzxpWx+58fzAaNiNj873uegPlmenudgbLc9eflt+qfisxTTGA0oJMWyblRIwFnGQGYxoL/U7UNkSTMU1UHMdav6PBmMZanxOtPdFmTBMNv7HUX8uYelj/WCKKbUvGMriWMc1YfMb6+LSMaWLRm+PRMqbxT3PLmMY/jfW3JcEZUyqI2xD/GKArFt5a4fhui/8+J3oPeUf0ggXzEx0NCdP/fv36oWUr/751EwYJcd7Rvfv2gNcb08jLhvjHQKVKlWKukxHXMb0pSxZxvUJlZfrYs3/xiQO6x6EVceHHHsOkSRPFWbyldXzSmnSlQQJjOi3v1bMn1qz53s7tOF/fSGN6E6hRs4YY6K1aqcaAnefxM8959etnn00TR+60budvS9/4oa9JS67h/PN1gxgbPGrEGVO6naBjW7mhpkwZeeZv+xdnOOANRELnsuLo3dI7zugbYM4681vNczuv45fupLXzF7/9TOQxrG+Sc+a1pXO8jwfSOtZCxBnTWOuwbY/FgMWAxYDFgMWAxYDFgMVAbGLAMqaxSRfbKosBiwGLAYsBiwGLAYuBhMOAZUwTjuS2wxYDFgMWAxYDFgMWAxYDsYkBy5jGJl1sqywGLAYsBiwGLAYsBiwGEg4DljFNOJLbDlsMWAxYDFgMWAxYDFgMxCYGLGMam3SxrbIYsBiwGLAYsBiwGLAYSDgMWMY04UhuO2wxYDFgMWAxYDFgMWAxEJsYsIxpbNLFtspiwGLAYsBiwGLAYsBiIOEwYBnThCO57bDFgMWAxYDFgMWAxYDFQGxiwDKmsUkX2yqLAYsBiwGLAYsBiwGLgYTDgGVME47ktsMWAxYDFgMWAxYDFgMWA7GJAcuYxiZdbKssBiwGLAYsBiwGLAYsBhIOA5YxTTiS2w5bDFgMWAxYDFgMWAxYDMQmBv4fb0jna9tpdqIAAAAASUVORK5CYII=\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eNeutrophil count (\u0026micro;L); NLR, neutrophil-to-lymphocyte ratio (neutrophils / lymphocytes); dNLR, derived neutrophil-to-lymphocyte ratio [neutrophils / (leukocytes-neutrophils)]. Best overall response according to iRECIST criteria: iPD, progressive disease (n. 10 patients); iSD, stable disease (n. 13 patients); iPR, partial response (n. 14 patients); complete response, iCR (n. 7 patients). Data are expressed as mean value \u0026plusmn; standard error of the mean (SEM). (*) \u003cem\u003ep\u003c/em\u003e = 0.043 iCR versus iPD, assessed by Mann-Whitney U test.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCD25 and CXCL9 as potential early biomarkers of response to CPI therapy.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo search for potential biomarkers that could predict patient clinical response or that could represent early indicators of response to CPIs, selected biomarkers of the vitiligo active phase were analyzed in serum samples from patients before (T0) and after 1 month (T1) and 3 months (T2) of treatment. We analyzed circulating levels of CD25, CD27, CXCL9, CXCL10, CXCL11, S100B, and IL-17A. For the patients examined, the circulating amount of either S100B or IL-17A was below the ELISA detection level (data not shown).\u003c/p\u003e\n\u003cp\u003eRegarding CD25, iSD and iPR patients had significantly higher levels of CD25 at baseline than iPD patients (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). A tendency to have higher serum amounts of CD25 was observed also for iCR patients, but it did not reach statistical significance (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). Basal CD25 levels (T0) significantly increased at T1 and T2 for iPD patients, but values remained lower than the other patient groups at the same time point.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs far as CD27, iCR patients tended to have less circulating protein amount than other groups at all time-point analyzed. Statistical significance was observed only at T1 between iCR and iPD patients (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec, d).\u003c/p\u003e\n\u003cp\u003eCXCL9 was undetectable at T0 in the sera of iPD compared to other patients who had baseline higher chemokine circulating levels (significance was achieved at T0 for iSD and iCR versus iPD patients; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea, b). Moreover, the trend of lower circulating amount of CXCL9 in iPD patients compared to the other groups was maintained after 1 and 3 months of therapy (significance was achieved at T1 and T2 for iPR versus iPD patients). All three groups of patients showed CXCL9 serum basal levels (T0) that significantly increased after 1 month of therapy (T1). Interestingly, while for iPR and iCR patients CXCL9 levels continued to significantly growth at T2, for iPD and iSD patients the levels of CXCL9 decreased at T2.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCXCL10 amounts significantly increased at T1 compared to T0 for iSD, iPR and iCR patients, and a significative increment was also present for iCR patients at T2 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec, d). No significant differences in serum concentration among the three response groups of patients was observed. Similarly, CXCL11 concentrations among the patient groups was significantly different only at T1 for iCR versus iPD patients (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee, f). CXCL11 levels increased between T0 and T1 for iPD and iPR patients, and between T0 and T2 for iSD patients (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee, f). In iPD patients, the increment observed at T1 was followed by a reduction in CXCL11 levels at T2 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee, f).\u003c/p\u003e\n\u003cp\u003eSince both CD25 and CXCL9 levels could represent early biomarkers of response to CPI therapy, we also observed that no correlation was found between the two proteins levels (Spearman\u0026rsquo;s \u0026rho;\u0026thinsp;=\u0026thinsp;0.03, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.821).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emiR-19b, miR-25 and miR-16 as promising indicators of response to CPIs therapy.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor a subset of 29 patients, for whom plasma samples were available, circulating miRNAs, previously identified as biomarkers of vitiligo, were analyzed. Patients no. 1, 3\u0026ndash;5, 9\u0026ndash;11, 13\u0026ndash;18, 20, 27\u0026ndash;30, 32, 40\u0026ndash;47, 56, 57 of Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e were examined (iPD, 5 patients; iSD, 8 patients; and iPR/iCR, 16 patients; iPR and iCR were assembled due to the small number of patients analyzed). As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e and in Additional Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, although statistical significance was not achieved in the comparisons between patient groups or times of drugs administration, some trends towards significance were observed. Higher levels of miR-19b and miR-25 were present at all time points of treatment in the plasma of iSD and iPR/iCR patients in respect to iPD individuals. For miR-16, a higher amount was present in iSD patients at T1 ant T2 and in iPR/iCR patients at T2 compared to iPD patients at the corresponding time points. Furthermore, miR-19b, miR-25, and miR-16 showed an increment at T1 versus T0 in iSD and iPR/iCR patients, but not in iPD patients, whose values remained low. No major differences in the amount of miR-574 among the three patient groups and at any time of treatment were observed. Interestingly, regarding miR-19b, miR-25 and miR-16, most of the iPR/iCR outliers that fall outside the distribution of miRNA levels, were the same patients who developed leukoderma during CPI therapy (stars in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTo date, it is urgent to develop non-invasive methods predicting melanoma patient response or resistance to immunotherapy to better address them towards the most efficacious therapies. In this regard, the intent of our work was the identification of predictive and/or early biomarkers of response to anti-PD-1 antibodies, taking advantages of previously identified biomarkers of the autoimmune vitiligo pathology, since leukoderma represents an irAE that often occurs during CPI therapy in melanoma patients and correlates with a positive outcome.\u003c/p\u003e \u003cp\u003eWe confirmed in our patient subset the association between development of leukoderma and a favorable clinical response in melanoma patients [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Then, we analyzed specific vitiligo biomarkers as possible indicators of response to anti-PD-1 treatment in metastatic melanoma patients.\u003c/p\u003e \u003cp\u003eOur data suggest that, among the molecules that characterize the active phase of vitiligo, CD25 and CXCL9 represent independent predictive biomarkers of response to CPI treatment. In fact, higher levels of these proteins were observed at baseline (T0) in the serum of patients who respond positively or stably to CPI therapy compared to patients who advanced towards progression disease. In addition, a constant increment of CXCL9 circulating amounts during the therapeutic period could represent an early biomarker of response to CPIs.\u003c/p\u003e \u003cp\u003eCD25, the membrane IL-2 Rα that can be shed into a soluble molecule upon immune cell activation, plays a role in the regulation of T cell function and in the maintenance of immune tolerance [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Blockage of the PD-1/PD-L1 interaction leads to an early activation of T cells, inducing several distinct signal transduction pathways also involved in IL-2 expression [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. When high levels of IL-2 are available, cells that express CD25, i.e. natural killer cells and T lymphocytes, will be activated, leading to an important immune response towards the tumor. Indeed, high dose IL-2 treatment was the first immunotherapy approach used in advanced melanoma. However, IL-2 benefits were associated with substantial toxicity [\u003cspan additionalcitationids=\"CR55\" citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. In the present investigation, we observed that stable or responding patients had higher levels of soluble CD25 at baseline and in the first months of anti-PD-1 therapy compared with patients who did not respond to treatment. Thus, we hypothesize that the increment of serum CD25 in melanoma patients who respond to anti-PD-1 therapy could be an indicator of an IL-2-mediated T cell activation, predictive of an effective anti-tumor immune response [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In future studies, it would be interesting to deeply analyze the mechanism of action of the CD25/IL-2 pathway and its time schedule in the response to anti-PD-1 treatment, and to evaluate the possible beneficial effects of a combined therapy of low doses of IL-2 together with the anti-PD-1 treatment.\u003c/p\u003e \u003cp\u003eChemokines play key roles in the recruitment of effector immune cells into the tumor [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. In particular, CXCR3, the receptor for the interferon-inducible chemokines CXCL9, CXCL10, and CXCL11, is highly expressed on activated T cells and plays an essential role in T cell migration into lymphoid and peripheral tissues [\u003cspan additionalcitationids=\"CR60\" citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. A few studies have been already conducted on the circulating expression of CXCR3 ligands after CPI therapies [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. An increase of circulating CXCL9 and CXCL10 in melanoma patients was observed during anti-PD-1 treatment [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e], and correlated with treatment outcome [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. In our study, we observed that, during anti-PD-1 immunotherapy, CXCL9 increased in the serum of responder and stable patients compared to non-responder, suggesting that this chemokine could be an early indicator of a positive therapeutic response. Moreover, circulating CXCL9 was absent in non-responder patients before the beginning of the treatment and increased after anti-PD-1 treatment in the sera of responder and stable patients, suggesting that a high amount of CXCL9 is important to mount an effective T cells-mediated antitumor immune response. It would be interesting to study if patient pretreatment with a compound able to specifically induce CXCL9 expression could be helpful for obtaining a better response to anti-PD-1 antibodies as shown in the mouse model [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur data also indicate that CXCL10 significantly increases in stable and responder patients after the first month of therapy, suggesting an initial activation of the immune system fundamental for effective treatment response and tumor clearance. Conversely, elevated CXCL11 levels in blood have been previously linked to poorer outcome for CPI-treated melanoma [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. CXCL11 has distinct immunoregulatory functions from those of CXCL9 and CXCL10 [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. CXCL11 limits T cell effector functions through induction and/or recruitment of regulatory T cells, and it is capable of binding to CXCR7, commonly associated with tumor cells growth and invasiveness [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Indeed, we also observed that CXCL11 significantly increased in non-responder compared to complete responder patients after one month of CPI therapy.\u003c/p\u003e \u003cp\u003eOther potential biomarkers of response to CPIs could be represented by miRNAs. Expression profiles of miRNAs are frequently dysregulated during cancer progression, and they are also involved in the modulation of the immune microenvironment [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Altered expression of miRNAs frequently correlates with poor prognosis and/or inadequate response to treatments in all stages of malignant melanoma [\u003cspan additionalcitationids=\"CR72\" citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. Several circulating miRNAs have already been described as potential biomarkers of progression and prognosis in melanoma [\u003cspan additionalcitationids=\"CR75\" citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. In this work, we decided to analyze the miRNAs that characterize the vitiligo disease. We showed that miR-16, miR-19b, miR-25 levels have the tendency to increase in stable and responder melanoma patients, but not in non-responder ones. Moreover, in agreement to published data [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e], we observed that circulating miR-16 levels tended to decrease in non-responder melanoma patients after CPI therapy. The lack of statistical association in our study may be due to the low number of samples analyzed that will be increased in future investigations.\u003c/p\u003e \u003cp\u003eDespite the presence of literature data on the role of miR-19b and miR-25 in melanoma cells [\u003cspan additionalcitationids=\"CR79\" citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e], their expression levels have not been previously analyzed in patient plasma. Therefore, for the first time, our data indicate that in metastatic melanoma patients the levels of miR-19b and miR-25 increase in stable and responder patients after one month of CPI therapy. Most responder patients with high plasma levels of miR-19b, miR-25 and miR-16 were the same patients who developed leukoderma, further implying existence of a connection between the molecular mechanisms that drive the autoimmune response in vitiligo and those that induce response to CPIs.\u003c/p\u003e \u003cp\u003eFinally, among the patient characteristics already described as potential biomarkers of response to immunotherapy with CPIs, we confirmed in our patient subset that basal higher neutrophil count, NLR, and dNLR could be indicators of progression disease during CPI treatment [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur data confirm that leukoderma is associated with a favorable outcome in metastatic melanoma patients treated with CPIs and indicate that biomarkers of the active phase of vitiligo could represent promising indicators of response to CPIs. When validated in a larger cohort, dosage of CD25 and CXCL9 circulating levels before and in the first months of therapy could represent a tool for predicting patient response to anti-PD-1 antibodies. Finally, both CD25 and CXCL9 should be further investigated as therapeutic targets to overcome resistance to CPI treatment in metastatic melanoma.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCPIs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003echeckpoint inhibitors; PD-1:programmed death-1; PD-L1:programmed death-ligand 1; NLR:neutrophil-to-lymphocyte ratio; LDH:serum lactate dehydrogenase; irAE:immune-related adverse event; CXCL:(C-X-C motif) ligand; IL:interleukin; CD:cluster of differentiation; IL-2 Rα:IL-2 receptor alpha; miR:microRNA; AJCC:American Joint Committee on Cancer; iCR:complete response; iPR:partial response; iSD:stable disease; iPD:progressive disease; iRECIST:immune response evaluation criteria in solid tumors; iBOR:best overall response; PFS:progression-free survival; dNLR:derived NLR.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate.\u0026nbsp;\u003c/strong\u003eInformed consent was obtained from all patients under approved human research ethics committee protocols from the\u0026nbsp;Istituto Dermopatico dell\u0026rsquo;Immacolata IDI-IRCCS (510/3, April 2018) and the Istituto Nazionale Tumori-IRCCS - Fondazione \u0026ldquo;G. Pascale\u0026rdquo; (20/14 oss, July 2014 ).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication.\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials.\u0026nbsp;\u003c/strong\u003eAll data generated or analyzed during this study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests.\u0026nbsp;\u003c/strong\u003ePaolo A. Ascierto\u0026nbsp;has/had a consultant/advisory role for Bristol Myers Squibb, Roche-Genentech, Merck Sharp \u0026amp; Dohme, Novartis, Array, Merck Serono, Pierre-Fabre, Incyte, Medimmune, AstraZeneca, Syndax, Sun Pharma, Sanofi, Idera, Ultimovacs, Sandoz, Im-munocore, 4SC, Alk-ermes, Italfarmaco, Nektar, Boehringer-Ingelheim, Eisai, Regeneron, Daiichi Sankyo, Pfizer, Oncosec, Nouscom, Takis, Lunaphore. He also received research funding from Bristol Myers Squibb, Roche-Genentech, Array, Sanofi, and travel support from MSD. All outside the submitted work. All the other authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding.\u0026nbsp;\u003c/strong\u003eThis research was supported by grants of the Italian Ministry of Health (IT-MOH) through\u0026ldquo;Ricerca Corrente\u0026rdquo; 2018-2020,\u0026nbsp;RC4.1, and \u0026ldquo;Ricerca Corrente\u0026rdquo; M2/2.\u0026nbsp;MLC is the recipient of a research fellowship by Fondazione Umberto Veronesi.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions.\u0026nbsp;\u003c/strong\u003eConception and design of the study: MLC, CMF and GM. Experiments carried out and acquisition of data: MLC, MB, MC and LL. Analysis and interpretation of data: MLC, SD\u0026rsquo;A, FDG and CMF. Statistical analysis: MLC, SM and CF. Drafting of the manuscript: MLC. Critical revision of the manuscript: CMF, GM, SM, PAA, SD\u0026rsquo;A, FDG and CF. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements.\u0026nbsp;\u003c/strong\u003eAuthors would like to thank Laura Bonmassar for skillful technical assistance, the nurses of the\u0026nbsp;Department of Oncology and Dermatological Oncology IDI-IRCCS and\u0026nbsp;of the Melanoma, Cancer Immunotherapy and Development Therapeutics Unit of Istituto Nazionale Tumori IRCCS Fondazione \u0026ldquo;G. Pascale\u0026rdquo;\u0026nbsp;for their continuous help,\u0026nbsp;and Fondazione Umberto Veronesi for the support of MLC research work.\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat. Rev. Cancer. 2012. p.\u0026nbsp;252\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHerzberg B, Fisher DE. Metastatic Melanoma and immunotherapy HHS Public Access. Clin Immunol. 2016;172:105\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLarkin J, Chiarion-Sileni V, Gonzalez R, Grob JJ, Cowey CL, Lao CD, et al. Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma. N Engl J Med. 2015;373:23\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eM\u0026aacute;rquez-Rodas I, Cerezuela P, Soria A, Berrocal A, Riso A, Gonz\u0026aacute;lez-Cao M, et al. Immune checkpoint inhibitors: Therapeutic advances in melanoma. Ann. Transl. Med. 2015. p.\u0026nbsp;267.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWan MT, Ming ME. Nivolumab versus ipilimumab in the treatment of advanced melanoma: a critical appraisal. Br J Dermatol. 2018;179:296\u0026ndash;300.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuder-Bakhaya K, Hassel JC. Biomarkers for clinical benefit of immune checkpoint inhibitor treatment-A review from the melanoma perspective and beyond. Front Immunol. 2018;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYi M, Jiao D, Xu H, Liu Q, Zhao W, Han X, et al. Biomarkers for predicting efficacy of PD-1/PD-L1 inhibitors. Mol Cancer. 2018;17:1\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJia XH, Geng LY, Jiang PP, Xu H, Nan KJ, Yao Y, et al. The biomarkers related to immune related adverse events caused by immune checkpoint inhibitors. J Exp Clin Cancer Res. 2020;39:1\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMadore J, Vilain RE, Menzies AM, Kakavand H, Wilmott JS, Hyman J, et al. PD-L1 expression in melanoma shows marked heterogeneity within and between patients: Implications for anti-PD-1/PD-L1 clinical trials. Pigment Cell Melanoma Res. 2015;28:245\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePostow MA, Friedman CF, Postow MA. Emerging Tissue and Blood-Based Biomarkers that may Predict Response to Immune Checkpoint Inhibition. Curr Oncol Rep. 2016;18:21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaibach F, Sadozai H, Seyed Jafari SM, Hunger RE, Schenk M. Tumor-Infiltrating Lymphocytes and Their Prognostic Value in Cutaneous Melanoma. Front Immunol. 2020;11:1\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoodman AM, Kato S, Bazhenova L, Patel SP, Frampton GM, Miller V, et al. Tumor mutational burden as an independent predictor of response to immunotherapy in diverse cancers. Mol Cancer Ther. 2017;16:2598\u0026ndash;608.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCapone M, Giannarelli D, Mallardo D, Madonna G, Festino L, Grimaldi AM, et al. Baseline neutrophil-to-lymphocyte ratio (NLR) and derived NLR could predict overall survival in patients with advanced melanoma treated with nivolumab. J Immunother Cancer. 2018;6:1\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBartlett EK, Flynn JR, Panageas KS, Ferraro RA, Jessica JM, Postow MA, et al. High neutrophil-to-lymphocyte ratio (NLR) is associated with treatment failure and death in patients who have melanoma treated with PD-1 inhibitor monotherapy. Cancer [Internet]. John Wiley and Sons Inc.; 2020 [cited 2021 Feb 17];126:76\u0026ndash;85. Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmed.ncbi.nlm.nih.gov/31584709/\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePetrelli F, Ardito R, Merelli B, Lonati V, Cabiddu M, Seghezzi S, et al. Prognostic and predictive role of elevated lactate dehydrogenase in patients with melanoma treated with immunotherapy and BRAF inhibitors: A systematic review and meta-analysis. Melanoma Res. 2019;29:1\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDiem S, Kasenda B, Spain L, Martin-Liberal J, Marconcini R, Gore M, et al. Serum lactate dehydrogenase as an early marker for outcome in patients treated with anti-PD-1 therapy in metastatic melanoma. Br J Cancer. 2016;114:256\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaino AS, Woods D, Vassallo M, Qian X, Tang H, Wind-Rotolo M, et al. Serum interleukin-6 and C-reactive protein are associated with survival in melanoma patients receiving immune checkpoint inhibition. J Immunother cancer. 2020;8:e000842.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTuriello R, Capone M, Giannarelli D, Morretta E, Monti MC, Madonna G, et al. Serum CD73 is a prognostic factor in patients with metastatic melanoma and is associated with response to anti-PD-1 therapy. J Immunother cancer. 2020;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCapone M, Fratangelo F, Giannarelli D, Sorrentino C, Turiello R, Zanotta S, et al. Frequency of circulating CD8 + CD73 + T cells is associated with survival in nivolumab-treated melanoma patients. J Transl Med. 2020;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChamoto K, Hatae R, Honjo T. Current issues and perspectives in PD-1 blockade cancer immunotherapy. Int J Clin Oncol. 2020;25:790\u0026ndash;800.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaanen J, Ernstoff MS, Wang Y, Menzies AM, Puzanov I, Grivas P, et al. Autoimmune diseases and immune-checkpoint inhibitors for cancer therapy: review of the literature and personalized risk-based prevention strategy. 31. Ann Oncol: Elsevier Ltd.; 2020. pp.\u0026nbsp;724\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSimonsen AB, Kaae J, Ellebaek E, Svane IM, Zachariae C. Cutaneous adverse reactions to anti\u0026ndash;PD-1 treatment\u0026mdash;A systematic review. J Am Acad Dermatol. 2020;83:1415\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodrigues M, Ezzedine K, Hamzavi I, Pandya AG, Harris JE. New discoveries in the pathogenesis and classification of vitiligo. J Am Acad Dermatol. 2017;77:1\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eParsad D, Sahni K. Stability in vitiligo: Is there a perfect way to predict it? J Cutan Aesthet Surg. 2013;6:75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eByrne KT, Turk MJ. New perspectives on the role of vitiligo in immune responses to melanoma. Oncotarget. 2011;2:684\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeulings HE, Limpens J, Jansen SN, Zwinderman AH, Reitsma JB, Spuls PI, et al. Vitiligo-like depigmentation in patients with stage III-IV melanoma receiving immunotherapy and its association with survival: A systematic review and meta-analysis. J Clin Oncol. 2015;33:773\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHua C, Boussemart L, Mateus C, Routier E, Boutros C, Cazenave H, et al. Association of vitiligo with tumor response in patients with metastatic melanoma treated with pembrolizumab. JAMA Dermatology. 2016;152:45\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNakamura Y, Tanaka R, Asami Y, Teramoto Y, Imamura T, Sato S, et al. Correlation between vitiligo occurrence and clinical benefit in advanced melanoma patients treated with nivolumab: A multi-institutional retrospective study. J Dermatol. 2017;44:117\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBabai S, Voisin AL, Bertin C, Gouverneur A, Le-Louet H. Occurrences and Outcomes of Immune Checkpoint Inhibitors-Induced Vitiligo in Cancer Patients: A Retrospective Cohort Study. Drug Saf. 2020;43:111\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFailla CM, Carbone ML, Fortes C, Pagnanelli G, D\u0026rsquo;atri S. Melanoma and vitiligo: In good company. Int J Mol Sci. 2019;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpeeckaert R, Speeckaert M, De Schepper S, van Geel N. Biomarkers of disease activity in vitiligo: A systematic review. Autoimmun Rev. 2017;16:937\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang XX, Wang QQ, Wu JQ, Jiang M, Chen L, Zhang CF, et al. Increased expression of CXCR3 and its ligands in patients with vitiligo and CXCL10 as a potential clinical marker for vitiligo. Br J Dermatol. 2016;174:1318\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaouia A, Sormani L, Youssef M, Helal AN, Kassab A, Passeron T. Differential expression of CXCL9, CXCL10, and IFN-γ in vitiligo and alopecia areata patients. Pigment Cell Melanoma Res. 2017;30:259\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElia G. MIG Th1 chemokine in Vitiligo. Clin Ter. 2018;169:E303\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdallah M, El-Mofty M, Anbar T, Rasheed H, Esmat S, Al-Tawdy A, et al. CXCL-10 and Interleukin-6 are reliable serum markers for vitiligo activity: A multicenter cross-sectional study. Pigment Cell Melanoma Res. 2018;31:330\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRashighi M, Agarwal P, Richmond JM, Harris TH, Dresser K, Su MW, et al. CXCL10 is critical for the progression and maintenance of depigmentation in a mouse model of vitiligo. Sci Transl Med. 2014;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpeeckaert R, Voet S, Hoste E, van Geel N. S100B Is a Potential Disease Activity Marker in Nonsegmental Vitiligo. J Invest Dermatol. 2017;137:1445\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBirlea SA. S100B: Correlation with Active Vitiligo Depigmentation. J Invest Dermatol. 2017;137:1408\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAcharya P, Mathur M. Interleukin-17 level in patients with vitiligo: A systematic review and meta-analysis. Australas J Dermatol. 2020;208\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh RK, Lee KM, Vujkovic-Cvijin I, Ucmak D, Farahnik B, Abrouk M, et al. The role of IL-17 in vitiligo: A review. Autoimmun. Rev. 2016. p.\u0026nbsp;397\u0026ndash;404.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHonda Y, Okubo Y, Koga M. Relationship between levels of soluble interleukin-2 receptors and the types and activity of vitiligo. J Dermatol. 1997;24:561\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYeo UC, Yang YS, Park KB, Sung HT, Jung SY, Lee ES, et al. Serum concentration of the soluble interleukin-2 receptor in vitiligo patients. J Dermatol Sci. 1999;19:182\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpeeckaert R, Lambert J, Van Geel N. Clinical significance of serum soluble CD molecules to assess disease activity in vitiligo. JAMA Dermatology. 2016;152:1194\u0026ndash;200.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElGhareeb MI, El Mokadem S, El Sayed B, Khalifa N. Soluble CD27 and MIF as possible serum biomarkers of vitiligo activity in Egyptian patients in Sharkia Governorate. Dermatology Reports. 2019;11:3\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan S, Shi J, Sun D, Lyu L. Current insight into the roles of microRNA in vitiligo. Mol Biol Rep. 2020;47:3211\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi YL, Weiland M, Li J, Hamzavi I, Henderson M, Huggins RH, et al. MicroRNA expression profiling identifies potential serum biomarkers for non-segmental vitiligo. Pigment Cell Melanoma Res. 2013;26:418\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShi Q, Zhang W, Guo S, Jian Z, Li S, Li K, et al. Oxidative stress-induced overexpression of miR-25: The mechanism underlying the degeneration of melanocytes in vitiligo. Cell Death Differ. 2016;23:496\u0026ndash;508.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGershenwald JE, Scolyer RA. Melanoma Staging: American Joint Committee on Cancer (AJCC) 8th Edition and Beyond. Ann. Surg. Oncol. 2018. p.\u0026nbsp;2105\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeymour L, Bogaerts J, Perrone A, Ford R, Schwartz LH, Mandrekar S, et al. iRECIST: guidelines for response criteria for use in trials testing immunotherapeutics. Lancet Oncol. 2017. p.\u0026nbsp;e143\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePersigehl T, Lennartz S, Schwartz LH. IRECIST: How to do it. Cancer Imaging. 2020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalek TR, Castro I. Interleukin-2 Receptor Signaling: At the Interface between Tolerance and Immunity. Immunity. 2010. p.\u0026nbsp;153\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang T, Zhou C, Ren S. Role of IL-2 in cancer immunotherapy. Oncoimmunology. 2016;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDamoiseaux J. The IL-2 \u0026ndash; IL-2 receptor pathway in health and disease: The role of the soluble IL-2 receptor. Clin Immunol. 2020;218:108515.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAtkins MB, Lotze MT, Dutcher JP, Fisher RI, Weiss G, Margolin K, et al. High-dose recombinant interleukin 2 therapy for patients with metastatic melanoma: Analysis of 270 patients treated between 1985 and 1993. J Clin Oncol. 1999;17:2105\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Y, Strick-Marchand H, Lim AI, Ren J, Masse-Ranson G, Li D, et al. Regulatory T cells control toxicity in a humanized model of IL-2 therapy. Nat Commun. 2017;8:1\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoyman O, Sprent J. The role of interleukin-2 during homeostasis and activation of the immune system. Nat. Rev. Immunol. 2012. p.\u0026nbsp;180\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrusko TM, Wasserfall CH, Hulme MA, Cabrera R, Schatz D, Atkinson MA. Influence of Membrane CD25 Stability on T Lymphocyte Activity: Implications for Immunoregulation. PLoS One. 2009;4:e7980.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKohli K, Pillarisetty VG, Kim TS. Key chemokines direct migration of immune cells in solid tumors. Cancer Gene Ther. 2021.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGroom JR, Luster AD. CXCR3 in T cell function. Exp. Cell Res. 2011. p.\u0026nbsp;620\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKastenm\u0026uuml;ller W, Brandes M, Wang Z, Herz J, Egen JG, Germain RN. Peripheral Prepositioning and Local CXCL9 Chemokine-Mediated Guidance Orchestrate Rapid Memory CD8 + T Cell Responses in the Lymph Node. Immunity. 2013;38:502\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKuo PT, Zeng Z, Salim N, Mattarollo S, Wells JW, Leggatt GR. The role of CXCR3 and its chemokine ligands in skin disease and cancer. Front. Med. 2018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBridge JA, Lee JC, Daud A, Wells JW, Bluestone JA. Cytokines, chemokines, and other biomarkers of response for checkpoint inhibitor therapy in skin cancer. Front. Med. 2018. p.\u0026nbsp;351.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYamazaki N, Kiyohara Y, Uhara H, Iizuka H, Uehara J, Otsuka F, et al. Cytokine biomarkers to predict antitumor responses to nivolumab suggested in a phase 2 study for advanced melanoma. Cancer Sci. 2017;108:1022\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChow MT, Ozga AJ, Servis RL, Frederick DT, Lo JA, Fisher DE, et al. Intratumoral Activity of the CXCR3 Chemokine System Is Required for the Efficacy of Anti-PD-1 Therapy. Immunity. 2019;50:1498\u0026ndash;512.e5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan S, Khan SA, Luo X, Fattah FJ, Saltarski J, Gloria-McCutchen Y, et al. Immune dysregulation in cancer patients developing immune-related adverse events. Br J Cancer. 2019;120:63\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoguchi Y, Hoen HM, Bambina SA, Rynning MD, Fuerstenberg RK, Curti BD, et al. Serum immunoregulatory proteins as predictors of overall survival of metastatic melanoma patients treated with ipilimumab. Cancer Res. 2015;75:5084\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTokunaga R, Zhang W, Naseem M, Puccini A, Berger MD, Soni S, et al. CXCL9, CXCL10, CXCL11/CXCR3 axis for immune activation \u0026ndash; A target for novel cancer therapy. Cancer Treat. Rev. 2018. p.\u0026nbsp;40\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurns JM, Summers BC, Wang Y, Melikian A, Berahovich R, Miao Z, et al. A novel chemokine receptor for SDF-1 and I-TAC involved in cell survival, cell adhesion, and tumor development. J Exp Med. 2006;203:2201\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCroce CM. Causes and consequences of microRNA dysregulation in cancer. Nat. Rev. Genet. 2009. p.\u0026nbsp;704\u0026ndash;14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChan SH, Wang LH. Regulation of cancer metastasis by microRNAs. J. Biomed. Sci. 2015. p.\u0026nbsp;1\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMichniewicz AG, Czyz M. Role of mirnas in melanoma metastasis. Cancers (Basel). 2019;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMotti ML, Minopoli M, Di Carluccio G, Ascierto PA, Carriero MV. Micrornas as key players in melanoma cell resistance to mapk and immune checkpoint inhibitors. Int J Mol Sci. 2020;21:1\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVarrone F, Caputo E. The miRNAs role in melanoma and in its resistance to therapy. Int. J. Mol. Sci. 2020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNeagu M, Constantin C, Cretoiu SM, Zurac S. miRNAs in the Diagnosis and Prognosis of Skin Cancer. Front Cell Dev Biol. 2020;8:1\u0026ndash;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMirzaei H, Gholamin S, Shahidsales S, Sahebkar A, Jafaari MR, Mirzaei HR, et al. MicroRNAs as potential diagnostic and prognostic biomarkers in melanoma. Eur J Cancer. 2016;53:25\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLorusso C, De Summa S, Pinto R, Danza K, Tommasi S. miRNAs as Key Players in the Management of Cutaneous Melanoma. Cells. 2020;9:415.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuo S, Guo W, Li S, Dai W, Zhang N, Zhao T, et al. Serum miR-16: A Potential Biomarker for Predicting Melanoma Prognosis. J Invest Dermatol. 2016;136:985\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOhira T, Naohiro S, Nakayama Y, Osaki M, Okada F, Oshimura M, et al. MiR-19b regulates hTERT mRNA expression through targeting PITX1 mRNA in melanoma cells. Sci Rep. 2015;5:1\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuo J, Zhang Y, Li R, Wang Y, Wu J, Zhang D. Upregulated microRNA-25 mediates the migration of melanoma cells by targeting DKK3 through the WNT/β-catenin pathway. Int J Mol Sci. 2016;17.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang Q-Q, Liu W-B. miR-25 Promotes Melanoma Progression by regulating RNA binding motif protein 47. m\u0026eacute;decine/sciences. 2018;34:59\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"melanoma, vitiligo, immunotherapy, checkpoint inhibitor, anti-PD-1, pembrolizumab, nivolumab, chemokines, microRNA.","lastPublishedDoi":"10.21203/rs.3.rs-753314/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-753314/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eImmunotherapy with checkpoint inhibitors strongly improved the outcome of metastatic melanoma patients. However, not all the patients respond to treatment and identification of prognostic biomarkers able to select patients who would respond to this therapy is of outmost importance. Considering that development of vitiligo-like depigmentation in melanoma patients represents both an adverse event of immunotherapy and a favorable prognostic factor for overall survival, we analyzed known soluble biomarkers of vitiligo to validate them as early indicators of response to checkpoint inhibitors in metastatic melanoma.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods:\u003c/strong\u003e Fifty-seven patients with metastatic melanoma receiving anti-PD-1 checkpoint inhibitor immunotherapy were enrolled. Patient sera and plasma were evaluated for vitiligo biomarkers at pre-treatment and after 1 and 3 months of therapy. Patients were divided and analyzed according to the best overall response to treatment. Characteristic vitiligo proteins were analyzed by ELISA, while expression of circulating microRNAs, distinctive of vitiligo, was determined using real-time RT-PCR.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eBasal serum CD25 levels were higher in stable and responder melanoma patients and remained higher during the first 3 months of anti-PD-1 therapy compared to non-responder patients. The chemokine CXCL9 was absent in non-responder patients before therapy beginning. Moreover, an increase of CXCL9 levels was observed at 1 and 3 months of therapy for all patients, although higher CXCL9 amounts were present in stable and responder compared to non-responder patients. Finally, higher levels of miR-19b, miR-25 and miR-16 were observed after 1 month of therapy in plasma of stable and responder compared to non-responder patients.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eSerum levels of CD25 and CXCL9 before and during the first months of treatment could represent biomarkers of response to anti-PD-1 immunotherapy in metastatic melanoma patients. Plasmatic miR-19b, miR-25 and miR-16 could also represent possible early biomarkers of response to anti-PD-1 treatment, but they must be validated in a higher number of patients.\u003c/p\u003e","manuscriptTitle":"Vitiligo-Specific Soluble Biomarkers as Early Indicators of Response to Immune Checkpoint Inhibitors in Metastatic Melanoma Patients","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-07-30 15:25:14","doi":"10.21203/rs.3.rs-753314/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4b3bc5e8-3c76-4733-8bd6-bd449c26ade3","owner":[],"postedDate":"July 30th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":6102018,"name":"Biomedical Engineering"},{"id":6102019,"name":"Biomaterials"}],"tags":[],"updatedAt":"2022-03-31T11:53:46+00:00","versionOfRecord":{"articleIdentity":"rs-753314","link":"https://doi.org/10.1038/s41598-022-09373-9","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2022-03-31 11:53:46","publishedOnDateReadable":"March 31st, 2022"},"versionCreatedAt":"2021-07-30 15:25:14","video":"","vorDoi":"10.1038/s41598-022-09373-9","vorDoiUrl":"https://doi.org/10.1038/s41598-022-09373-9","workflowStages":[]},"version":"v1","identity":"rs-753314","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-753314","identity":"rs-753314","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}