Establishment and Characterization of Preclinical Model of Primary Ovarian Squamous Cell Carcinoma

Establishment and Characterization of Preclinical Model of Primary Ovarian Squamous Cell Carcinoma | 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 Article Establishment and Characterization of Preclinical Model of Primary Ovarian Squamous Cell Carcinoma Chenyang Xu, Weiming Xu, Conghui Wu, Chen Xu, Zhengxiao Ma, Jiamin Wang, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5682512/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Jul, 2025 Read the published version in Human Cell → Version 1 posted 4 You are reading this latest preprint version Abstract Primary ovarian squamous cell carcinoma (POSCC) represents an exceedingly rare subtype of epithelial ovarian cancer (EOC) characterized by a cryptic etiology and insidious onset. The rarity and high mortality associated with pure primary ovarian squamous cell carcinoma (SCC) make it challenging to conduct large randomized controlled studies. Furthermore, there are currently no commercially available ovarian SCC cell lines for research purposes, necessitating the urgent establishment of novel lines. To our knowledge, this study reports the first preclinical model of primary ovarian squamous cell carcinoma (POSCC), denoted as ZOC254. We have detailed the establishment and characterization of ZOC254, derived from a 64-year-old female patient, which preserves the primary tumor's original traits across various levels during prolonged in vitro expansion. Whole-exome sequencing (WES) of both the primary tumor and derived cell line revealed homologous recombination deficiency (HRD) and high tumour mutational burden (TMB). ZOC254 also exhibited the PIK3CA: p.E542K mutation associated with targeted therapy. The effectiveness of olaparib, everolimus, and conventional chemotherapeutic agents for ovarian cancer was preliminarily assessed on the growth of patient-derived cells (PDC). The POSCC cell line and derived xenograft transplantation model reported in this study serve the purpose of broadening the resources accessible for preclinical investigations into ovarian squamous carcinoma. ovarian squamous cell carcinoma cell line establishment TMB HRD targeted therapy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Ovarian cancer is the eighth leading cause of cancer-related mortality in women worldwide [1, 2] primarily due to its dismal prognosis, attributed to 65% of cases being diagnosed at an advanced stage of invasive disease [3]. Ovarian carcinomas are classified into five primary types - high-grade serous, low-grade serous, clear cell, endometrioid, and mucinous ovarian cancer - comprising more than 95% of cases, based on histopathological and molecular genetic profiles [4, 5]. In contrast, Primary ovarian squamous cell carcinoma (POSCC) is very rare and the incidence is less than 1% [3, 6, 7]. In the 2014 edition of the World Health Organization (WHO) classification of female reproductive system tumors, primary ovarian squamous cell carcinoma (POSCC) ceased to have a distinct classification and was grouped under ovarian germ cell tumors. Subsequently, in the 2020 WHO edition, it was reclassified as somatic tumors originating from teratomas, primarily due to their association with malignancies arising from mature cystic teratomas [8, 9]. Due to its unclear pathogenesis, etiology, and non-specific clinical manifestations, Primary ovarian squamous cell carcinoma (POSCC) is frequently misdiagnosed as a benign tumor, resulting in delayed treatment [10]. The 5-year overall survival rate was 31.2%, with a disease-free survival rate of 31.6% [7]. Understanding the biological characteristics of the primary cell model is crucial for elucidating the disease's pathogenesis and developing novel treatment strategies. The quality and characteristics of cell line models are essential for obtaining reliable results in preclinical cancer research, where established cancer cell lines play a pivotal role. Human malignant tumor cell lines offer a rich source of experimental material [11, 12]. The rarity of malignant transformation in mature ovarian teratomas, with rates ranging from approximately 0.46% to 4.00%, poses challenges in establishing a dependable cell line for ovarian squamous cell carcinoma, resulting in a scarcity of disease-specific models that impede fundamental research [13]. Existing ovarian cancer cell lines are predominantly composed of high-grade serous carcinoma from the ovary and squamous cell carcinoma cell lines linked to gynecologic malignancies, primarily originating in the cervix [14]. Ovarian-associated squamous cell carcinoma primarily originates from the malignant transformation of mature cystic teratomas, characterized by a mixture of various dermal differentiation components [15]. This underscores the significance of creating novel and meticulously characterized cancer cell lines for preclinical investigations. Homologous recombination deficiency (HRD), which occurs in approximately 50% of epithelial ovarian cancers (EOCs), represents a crucial therapeutic target [16]. While the use of PARP inhibition as a monotherapy has been extensively explored in various cancers, including prostate, pancreatic, breast, and ovarian cancers, its application in treating squamous cell carcinoma has been sparsely documented [17, 18]. BRCA1/2 play crucial roles in the homologous recombination repair pathway, and patients harboring BRCA1/2 mutations may benefit from first-line maintenance therapy with Poly ADP-ribose polymerase inhibitors (PARPi), such as olaparib [19, 20]. Pharmacological research investigating the efficacy of olaparib against POSCC has the potential to significantly advance clinical practice in terms of both diagnosis and intervention. In the current study, we generated a novel strain of POSCC cells , which is derived from a patient with histologically confirmed ovarian SCC. The results of our detailed its clinical and molecular characteristics and the applicability of patient-derived xenografts (PDX). Furthermore, the utility of the PARP inhibitor, olaparib, was investigated in drug sensitivity studies in vitro . Our collective findings may be helpful in providing insights into modeling therapies for the management of this disease. To our knowledge, this is the first study to report a cell line developed from a POSCC patient and investigate the genetic background of this rare ovarian cancer type with the aid of whole-exome sequencing. MATERIALS AND METHODS Patient and histological sample assessment This study was approved by the Ethics Committee of ZheJiang Cancer Hospital, (Hangzhou, Zhejiang, China; IRB-2015-208). Following admission, the patient signed an informed consent form for use of bio-specimens for scientific research. The patient was diagnosed with malignant tumor (squamous cell carcinoma and necrosis were visible in the section) by ice pathology during the operation, and underwent standard comprehensive staging surgery, including the whole uterus, bilateral adnexa, greater omentum, and abdominal paraaortic lymph nodes. Diagnosis of POSCC was based on internationally approved criteria[21]. For histological analysis, surgically resected samples were fixed in 10% buffered formalin for 48 h, sliced into 1 cm x 1 cm x 0.3 cm squares, and placed in the embedding box. Tissue blocks were routinely embedded in paraffin and cut into 4 µm sections using a Leica RM 2155 microtome. After performing necessary processing, slides were incubated with primary antibody ER and P16 of Roche (Shanghai, China) ready to use (RTU), P53 and PAX-8 (1:200 dilution) of LBP Medicine Science and Technology (Guangzhou, China), PR, Ki-67 and WT-1 (RTU) of DAKO (Denmark), p40（1:200 dilution）of ZSJO-Bio (Beijing, China), p63（1:200 dilution）of MAIXIN BIOTECH（Fuzhou, China）. Including MLH1, PMS2, MSH2, MSH6，immunohistochemistry has been previously described[22]. Hematoxylin and eosin (H&E) and immunohistochemical staining were performed in the pathology department of Zhejiang Cancer Hospital. The histopathology of tissues was evaluated by three independent pathologists. Establishing the ZOC254 cell line and primary cell cultures Fresh ovary specimens were washed two to three times with saline. Bleeding and necrotic areas of tissue were removed using a sterile scalpel. Biopsies were incubated for 10 min at 4°C with PBS containing 500 units/mL penicillin and 500 µg/mL streptomycin. Tissues were sliced into 1 mm x 1 mm sections and incubated with 5 mL of 0.1% (w/v) collagenase (Sigma -Aldrich, St. Louis, MO, USA) and 0.125% (w/v) trypsin-EDTA (Sigma) for 4 h at 37°C with shaking. Fresh DMEM/F12 culture medium (Gibco Laboratories, Grand Island, USA) supplemented with 10% (v/v) FBS (Gibco) and 1% (v/v) penicillin-streptomycin (Gibco) was used to terminate digestion. Dissociated cells were gently passed through a cell strainer (70 µm), centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The culture medium used was DMEM/F12 (Gibco) containing 10% FBS, penicillin (100 units/mL) and streptomycin (100 mg/mL) (Gibco) with incubation at 5% CO 2 and 37°C. Fibroblasts were removed via differential phase digestion and adherence[23]. After repeated passage, a novel POSCC cell line, ZOC254, was successfully established over more than 30 generations. Short tandem repeat analysis DNA from ZOC254 cells and corresponding leukocytes from donor patient was extracted using the AxyPrep Multisource Genomic Miniprep DNA (Axygen, New York, USA). Short tandem repeat (STR) analysis at 21STR loci using ABI 3730XL genetic analyzer (Shanghai Biowing Applied Biotechnology Co. Ltd., Shanghai) was performed to verify cell authenticity and unicity according to our previous studies[24].The STR profiles were then compared with the profiles in public cell banks, including the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and Japanese Collection of Research Bioresources (JCRB) for reference. Twenty-one STR loci (D5S818, D13S317, D7S820, D163539, VWA, TH01, AMEL, TPOX, CSF1PO, D12S391, FGA, D2S1338, D21S11, D18S51, D8S1179, D3S1358, D6S1043, PENTAE, D19S433, PENTAD and D1S1656) were matched for DNA fingerprinting analysis. Karyotypic analysis Karyotype analysis of tumor cells was conducted following optimized cytogenetic protocols for neoplastic specimens. Fresh tumor tissues obtained via surgical resection or biopsy were mechanically dissociated and enzymatically treated with collagenase to obtain single-cell suspensions, while liquid tumors (e.g., leukemia/lymphoma) were directly processed. Cells were cultured in RPMI 1640 medium supplemented with 15% fetal bovine serum, L-glutamine, and antibiotics at 37°C in 5% CO₂ for 24–48 hours. Metaphase arrest was induced using 0.1 μg/mL colcemid for 60–90 minutes, adjusted for tumor proliferation rates. Hypotonic treatment with 0.075 M KCl (15 minutes, 37°C) and fixation in methanol: acetic acid (3:1) were performed, with repeated washes to remove cellular debris. Chromosome spreads were prepared on chilled slides and aged overnight. G-banding was optimized using 0.025% trypsin (30–60 seconds) followed by Giemsa staining. A minimum of 30 metaphases were analyzed using MetaSystems’ automated platform, with clonal abnormalities defined per ISCN 2023 (≥2 cells for constitutional changes; ≥3 cells for acquired aberrations). Normal stromal cells were excluded via morphology and banding pattern comparison. Ethical approval and informed consent were obtained for human specimens, with biosafety protocols followed for handling neoplastic materials. Flow Cytometry Viable cells were harvested (1 × 10⁶ cells/sample), centrifuged to pellet cells (800rpm, 5 min), followed by supernatant removal. The pellet was washed once with phosphate-buffered saline (PBS) under identical centrifugation conditions. Triplicate biological replicates were processed to ensure statistical validity. Use the Cell Cycle Staining Kit (MULTI SCIENCES, China) according to manufacturer guidelines. Add 1 mL DNA Staining Solution and 10 μL Permeabilization Solution to the cell pellet. Vortex mix thoroughly for 5–10 sec to ensure homogeneous staining. Incubate the mixture at room temperature (20–25°C) for 30 min in light-protected conditions. Flow cytometric analysis was performed using the lowest acquisition rate setting to ensure signal resolution. Whole-exome sequencing In order to comprehensively analyze targeted drug selection related gene mutations immunotherapy related genomic indicators (TNB/TMB/HLA/MSI, etc.), genetic risk related germline mutations. Cell pellet sample of ZOC254 at passage 13 and matched peripheral white blood cell were screened with Whole-exome sequencing (WES) technology on an Gene+Seq-2000 sequencing platform (Geneplus Technology, Beijing) following the manufacturer’s instructions and the procedure. The types of gene mutation contained point or frameshift mutation, sites of mutation (codon, exon, functional domain, or conserved area), copy number variants (CNVs), fusion variant, insertion and deletion mutations. To evaluate the status of loss of heterozygosity (LOH), telomere allele imbalance (TAI) and large segment recombination abnormality (LST) in tumor tissue samples, HRD score ≥ 34 was positive and HRD score < 34 was negative. The names of genes are officially named in NCBI-Gene (Official Symbol). Detection results of gene polymorphism of chemotherapeutic drugs and hints of drug useings Classification is according to the PharmGKB website http://www.pharmgkb.org/page/clinAnnLevels. Determination of cell proliferation and growth kinetics The xCELLigence RTCA-DP device (Agilent, Santa Clara, CA, USA) was used to examine the cell growth rate in real time according to the manufacturer’s instructions. Electronic microtiter plates were seeded with 2.0 × 10 3 cells (E-Plate; Agilent) in each well, and proliferation was measured for a week. Cell density measurements were performed in quadruplicate with programmed signal detection every 30 min. The data was exported in excel format, analyses were performed using Prism 8.02 (GraphPad Software, San Diego, CA, USA). Data obtained during the logarithmic growth phase were subjected to natural logarithmic transformation of cellular index (ln [CI]) at each temporal measurement point. Subsequent linear regression analysis yielded the kinetic equation ln [CI] = kt + C, where k represents the proliferation rate constant. Population doubling time (DT) was ultimately calculated through the fundamental relationship DT = ln (2)/k. Colony formation assay ZOC254 cells in the logarithmic growth stage were inoculated (500, 1500 and 4500 cells per plate) in six-well plates. After two weeks of incubation, cells were washed twice with PBS, fixed with 4% paraformaldehyde universal tissue fixator for 20 min, and stained with 0.1% crystal violet for 15 min. Dishes were washed at least three times with PBS. Images were obtained and the number of cell clones with > 50 cells counted. Wound healing assay ZOC254 cells were seeded into ibidi Culture-Insert 2 Well (ibidi, Germany) at the density of 500,000 cells/well and incubated overnight. A defined 500 µm cell free gap was left behind after the insert’s removal, and the cells were washed with PBS. At the following day, the cells were incubated in serum-free DMEM/F12 medium, and the images were captured at 0, 6, 12 and 24 h. In vitro sensitivity test The reagents used in this study were purchased from Chinese suppliers: Olaparib (HY-10162, MedChemExpress) and Everolimus (HY-10218, MedChemExpress) was purchased from Haoxinbio (Hangzhou, China). Stock solutions (10 mM) were reconstituted in DMSO for all the drugs. Cisplatin Injection (Hansoh Pharma, Lianyungang, China), Carboplatin for Injection (Qilu Pharma, Jinan, China) and Paclitaxel Injection (Huiyu Pharma, Neijiang, China). Cell chemosensitivity was measured with the Cell Counting Kit-8 (CCK-8) assay. ZOC254 cells at the logarithmic growth stage were collected, and 8×10 5 cells per well seeded in 96-well plates (Costar, Corning Incorporated, New York, USA) 24 h prior to drug treatment. Next, cells were treated with different concentrations of olaparib for 72 h. Every 24 h, cells in each well were mixed with CCK-8 (APE X BIO, Houston, USA) reagent and incubated at 37°C for 1-4 h and absorbance at 450 nm was measured. IC 50 values were calculated and cell killing inhibition curves were plotted using Prism 8.02. Tumorigenicity assay All animal experiments were approved by Zhejiang Cancer Hospital Animal Use and Care Committee (Hangzhou, Zhejiang, China; [2015]-1-7号（科）) and where relevant conform to national guidelines for animal usage in research. Experimental animals were fed in a SPF sterile laminar flow chamber. Cells (1 × 10 6 /200 µL) were suspended in matrix gel and subcutaneously injected into the backs of female BALB/c nude mice (4 weeks old, 17–20 g in weight) purchased from Harm Slyke Laboratory Animals Co., Ltd. (license no. Scxk2012-0002). Tumor sizes were monitored every three days and the tumor growth curves and animal weights recorded accordingly. Tumor volume (V) was calculated using the formula: V (mm 3 )= 1/2(length × width 2 ). At a maximal tumor volume of 2000 mm 3 , mice were euthanized. Statistical analysis Statistical analyses were performed using SPSS software (version 26.0, Chicago, IL). All experiments were repeated at least three times and data were expressed as means ±SD. The statistical significance of differences between groups was calculated using the Student’s t-test. Differences were considered statistically significant at P<0.05. RESULTS The cell lines are stable experimental models for POSCC 1 Clinical and diagnostic features The patient (64 years old; female) presented at ZheJiang Cancer Hospital without obvious inducement and symptoms of frequent urination (most obvious at night), endless urine, difficult stool, fatigue, and abdominal distension, but no abdominal pain. Preoperative assessments were conducted, revealing elevated levels of serological tumor markers such as CA125, CA199, or CEA, along with increased levels of serum squamous cell carcinoma antigen (SCC-Ag). Abdominal, pelvic CT and urinary tract US were used to detect a solid abdominal pelvic cystic mass (about 16.5×9.2cm), which was considered a teratoma and secondary renal water (the right renal pelvis, the right ureter was slightly extended). Following total hysterectomy and bilateral adnexectomy, the patient was diagnosed with moderately differentiated ovarian squamous cell carcinoma (SCC), which extended from the adventitial membrane of the right lateral uterine wall to the deep muscularis. Diagnosis of POSCC was supported by HE and immunohistochemical staining ( Figure 1 A ; p40-positive, p63-positive, P53-positive, Ki-67-positive (5%), WT-1-negative, PR-negative, ER-negative, PAX-8-negative（ Figure 1B-I ). Additional imaging modalities including CT scans and ultrasound of the chest, upper abdomen, and kidneys did not detect any distant metastases beyond the pelvic mass. Following surgical resection, there was no evidence of metastatic squamous cell carcinoma in the cervix. No concurrent precursor lesions were identified in the bilateral ovariectomized specimens, leading to the definitive diagnosis of primary pure squamous carcinoma of the ovary. In this case, the patient was subjected to radical resection and only received symptomatic support (anti-inflammatory, hemostatic and hypoglycemic) therapy after surgery. Chemotherapy and targeted therapy were rejected [25, 26]. 2 Establishment and histological characteristics of a novel POSCC cell line ZOC254 Fresh surgically resected ovarian cancer tissue was processed and cultured in time, and a certain purity of ovarian cell line was obtained after multiple gradient digestion and passage, which was named ZOC254. The ZOC254 cell line retained the histological features of the primary tumor, in particular, strong positivity for p40 ( Figure 2B ) and p63 ( Figure 2C ), as well as nuclear accumulation of P53 ( Figure 2D ). The newly generated cell line was highly proliferative (80%), determined based on the Ki-67-positivity rate ( Figure 2E ) [27]. Cells were positive for Wilms tumor protein (WT-1) ( Figure 2F ), which is potentially useful in identifying the primary sites of serous carcinoma [28, 29]. Progesterone receptor (PR) ( Figure 2G ) and estrogen receptor (ER) ( Figure 2H ), predictive biomarkers of endocrine therapy, were not expressed, indicating that the patient would not benefit from endocrine therapy [30]. Pax-8 ( Figure 2I ), a reliable marker to identify cancer cells of gyneduct origin [31], was not expressed in the ZOC254 cell line. The growth and morphology of the ZOC254 were photographed under an inverted microscope, during long-term cultivation in vitro . The cells were passaged > 30 generations, and there was no obvious change in morphology during cell passage ( Figure 2J) . The cells were flat, polygonal and oval and had formed an epithelial-like monolayer. The nucleoli of cells were obvious, a few were multinucleated, and the nucleo-plasma ratio was large. To systematically evaluate their antigen-secreting properties, we performed the quantitative assessment of tumor markers （serum SCC, CA125, and CA19-9）with the cell culture supernatant using clinical laboratory assays. The results showed that SCC (23 ng/mL) and CA125 (93.99 U/mL) were both higher than normal serum reference values by Abbott ALINIQ direct chemiluminescence immunoassay, and CA19-9 (10.2 U/mL) was measured by Roche Cobas e801. 3 Authenticity of the ZOC254 cell line Short tandem repeats (STR), also known as microsatellite DNA, refer to a type of DNA sequence formed by tandem repetition on chromosomes with several base pairs as the core unit (2-6 base pairs, 10-60 repeats, and a gene fragment of less than 400 base pairs). The authenticity of the ZOC254 cell line was verified through short tandem repeat (STR) profiling, which provided information on the true origin and unique profile of these cells with respect to those present in the reference databases ( Table 1 ). We found that the characteristics of ZOC254 cell line are consistent with tissues, and the results demonstrate clear and consistent genetic profiles that confirm the cell line's authenticity. STR sequence retrieval was performed in the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) database. No matching sites of these cells were detected in the database, suggestive of a new cell line and excluded the possibility of cross-contamination with other cell lines. Moreover, no multiple alleles were found, indicating no single cells and no other cell contamination. The matching degree of STR genotype between the ZOC254 cell line and paired parent tissue was 90%. According to the ATCC Standards Committee identification guidelines (ANSI/ATCC ASN-0002 2011), a matching degree ≥80% is considered the same source. The resulting tumors recapitulated the histologic features of squamous cell carcinoma (SCC), including keratinization, as confirmed by H&E staining ( Figure 3A) . Importantly, the immunohistochemical (IHC) profile of the xenograft tumors closely matched that of the original patient tissue and patient-derived cells ( Figure 3B-I) . We also have performed microsatellite marker analysis on the primary tumor tissue, ZOC254 cell line, and patient-derived xenografts ( Figure S2 ), and the results show consistent profiles across all three sample types. To further analyze the ZOC254 cell line, we conducted a karyotype analysis on it. Karyotypic analysis of ZOC254 cells revealed chromosomal abnormalities across 51 analyzed metaphase spreads ( Figure 3 J ) . The majority of cells (84.3%, 43/51) exhibited triploid-to-tetraploid chromosomal counts (3n-4n), while we also observed a minor population of hypo-triploid cells (13.7%, 7/51) and a single hyper-tetraploid cell (>4n). Notably, these numerical abnormalities were accompanied by Multiple structural aberrations, such as chromosomal rearrangements, deletions, and derivative chromosomes, suggesting genomic instability characteristic of this aggressive tumor type. We employed flow cytometry to analyze the cell cycle distribution of ZOC254 cells. The results revealed a distinctive cell cycle distribution profile, with 34% of cells in G0/G1 phase, 50.1% in S phase, and 13.3% in G2/M phase ( Figure 3K-L) . This pattern reveals a predominant S-phase population, suggesting active DNA replication and proliferative capacity characteristic of this tumor cell line. These findings confirm that our model maintains the essential histopathological and immunohistochemical features of this rare ovarian squamous cell carcinoma, thereby providing a reliable preclinical platform for further investigation. 4 Function experiment of ZOC254 cell line in vitro and in vivo To assess the biological properties of ZOC254, we first analyzed its growth rate. The doubling time of ZOC254 was calculated to be approximately 30 hours (Figure 4A) . Subsequently, we investigated the migratory behavior of ZOC254 using a wound scratch assay, tracking cell movement towards the wound site. Our findings demonstrated efficient wound healing over time, indicating robust cell migration capabilities (Figure 4B) . Additionally, the cell line exhibited the capacity to form clones in relation to the density of initially seeded cells ( Figure 4C ). The tumor formation ability of the ZOC254 cell line was evaluated via nude mouse inoculation ( Figure 4D ). After 24 days of inoculation, growth of ZOC254 xenografts was observed in nude mice. The collective results support the utility of our cell line in the construction of animal models, drug screening and efficacy evaluation. Basic molecular characteristics of ZOC254 WES was used to clarify the gene genetic characteristics of ZOC254. Exon sequencing revealed the presence of 509 somatic mutations, 0 germline pathogenic or suspected pathogenic mutations and 0 gene fusions. The copy number variation distribution and panoramic display of somatic cells are presented in Figure 5 . In terms of immune prediction, ZOC254 displayed TMB-H(421Muts) and MSS, and HLA LOH was negative. Noteworthy, cells were positive for HRD and 54.8% displayed the targeted therapy-related mutation, PIK3CAc.1624G>A (p.E542K) supporting the potential utility of the targeted drugs, olaparib and everolimus[32]. The status of heterozygosity loss (LOH), telomere allele imbalance (TAI) and large segment recombination abnormality (LST) in the genome of tumor tissue samples were evaluated to determine the index of HRD status. HRD score ≥34 was positive. HRD score A (p.E542K) is a missense mutation, which causes the 542nd amino acid of the protein encoded by the PIK3CA gene to mutate from glutamic acid to lysine. Olaparib and everolimus are potential therapeutic targets for ZOC254 Olaparib has been FDA-approved for treating ovarian cancer patients carrying BRCA1/2 germline mutations (SOLO1), breast cancer patients with BRCA1/2 germline mutations (OlympiAD), and for first-line maintenance therapy in pancreatic cancer patients with BRCA1/2 germline mutations (POLO) [33, 34]. Preclinical studies have demonstrated that inhibitors targeting the PI3K pathway, including mTOR inhibitors, can suppress the activity of the primary PI3K pathway in PIK3CA mutant tumors [35]. Human tumor cells harboring activating mutations in the PI3K pathway have exhibited responsiveness to everolimus in both in vivo and in vitro experiments [36]. Based on the results of WES, we focused on the drug sensitivity of ZOC254 cell line to olaparib and everolimus. We also examined the antitumor effects of first-line chemotherapy agents, including cisplatin, carboplatin and paclitaxel. The median inhibition concentration (IC 50 ) values of ZOC254 cells were summarized ( Table 2 ) and growth curves for two anticancer drugs, olaparib and everolimus ( Figure 6 ) are shown. The IC50 values of olaparib at 24h, 48h, and 78 h were 17.99 μM, 10.97 μM, and 8.83 μM, respectively. The IC50 values of everolimus at 24h, 48h, and 78 h were 216.31 μM, 47.71 μM, and 44.48 μM, respectively. Our results indicate that inhibition of ZOC254 cell proliferation is enhanced with the drug concentration or time elapsed. Growth curves for three first-line chemotherapy agents are shown in Figure S1 . DISCUSSION This study presents the initial preclinical model of POSCC. The etiology and tissue origin of ovarian squamous cell carcinomas remain unclear. Both primary and metastatic squamous cell carcinomas of the ovary have been identified, with the latter most commonly associated with metastasis from cervical or vaginal squamous cell carcinomas [37, 38]. Primary ovarian squamous cell carcinoma is rare and often accompanied by precursor lesions. Malignant transformation of mature cystic teratoma (MCT) is frequently reported [39]. Consequently, most POSCCs exhibit well-differentiated ectoderm, endoderm, and mesoderm tissues [40]. In contrast, pure primary squamous carcinoma lacks an MCT component and signifies the carcinogenesis of epithelial cells on the ovarian surface [41]. The patient in this study received a diagnosis of squamous cell carcinoma through histopathology and immunohistochemistry. No apparent lesions were detected in the kidneys, bladder, or bilateral vaginal appendages before and during the operation. Absence of squamous cell carcinoma was noted in the cervix of the surgically resected specimen, thereby excluding metastatic squamous cell carcinoma. Bilateral ovariectomized specimens did not exhibit precursor lesions such as mature cystic teratoma, Brenner tumor, or endometriosis [41]. POSCC typically lacks PAX8 expression aligns with its non-Müllerian origin (e.g., teratoma-derived or metaplastic squamous differentiation)[42-44]. This pattern contrasts with PAX8 expression in serous or endometrioid carcinomas, which typically originate from the fallopian tube or ovarian surface epithelium. The observed discrepancy in WT-1 immunoreactivity between the original tumor (negative) and cultured ZOC254 cells (positive) likely reflects the partial WT-1 positivity in vitro likely represents culture-induced artifact or minor subpopulation expansion, while the consistent negativity in tumor/xenografts underscores the true biological signature of this ovarian SCC[45]. In contrast, primary cell culture conditions might selectively promote the survival or proliferation of a minor subpopulation with WT-1 expression, possibly representing a less differentiated or transitional state. Immunohistochemically, tumor cells were positive to p40，p63 and negative for WT-1，PAX-8，which further ruled out high-grade serous carcinoma, urothelial carcinoma, and teratoma malignancies，leading to a final diagnosis of POSCC. Large randomized controlled studies are challenging to conduct due to the rarity and high mortality rates associated with POSCC [46]. Most existing reports consist of systematic reviews and analyses of published data underscoring the need to expand the current repository of POSCC literature [39]. Hence, in this instance, ZOC254 exhibits a stable profile and remains unaffected by other dermal differentiated cells, making it an excellent model for studying ovarian squamous cell carcinoma. The PDC and PDX recapitulated the histologic features of POSCC, including keratinization, as confirmed by H&E staining. Importantly, the IHC profile closely matched that of the original patient tissue, further validating the genetic stability and model reliability of this rare ovarian SCC model. The patient-derived cells maintain the fundamental genetic characteristics of the original tumor through both in vitro culture and in vivo xenograft passages. These cells were directly isolated from patient-derived tumor tissues without genetic modification or immortalization treatment, which allows them to maintain critical biological features of the original tumor microenvironment[47-49]. This approach offers distinct advantages, including preservation of natural tumor heterogeneity (subpopulations with varying degrees of malignancy) and avoidance of accumulated genetic mutations observed in immortalized cell lines during long-term in vitro culture[47, 50, 51]. Furthermore, this approach provides a more reliable model for experimental applications such as drug sensitivity testing and therapeutic efficacy evaluation[52, 53]. The consistency between primary tumor and xenografts validates our model's ability to faithfully recapitulate the patient's tumor biology. Clinically, HRD represents a crucial therapeutic target in EOC. PARP inhibitors have shown significant clinical advantages in HRD ovarian cancers, particularly in cases of BRCA1/2-mutated HGSOCs [54]. Nevertheless, there is a lack of research examining the impact of PARP inhibitors on POSCC. Ovarian cancer often exhibits a strong familial predisposition associated with hereditary mutations in BRCA1 or BRCA2 [55, 56]. Nevertheless, our patient did not exhibit any deleterious or suspected deleterious BRCA1/2 mutations. Although mutated BRCA genes are primarily linked to HRD, other crucial homologous recombination repair (HRR) proteins could also be mutated or functionally impaired, thereby potentially broadening the therapeutic avenues for PARP inhibitors [57]. Through WES, we can assess the presence of loss of heterozygosity (LOH), telomere allele imbalance (TAI), and large segment recombination abnormality (LST) in tumor tissue samples to ascertain the status of HRD. The initial PARP inhibitor, olaparib, received FDA approval for treating ovarian cancers bearing BRCA mutations [58]. Olaparib has demonstrated notable clinical advantages as a maintenance regimen for women with HGSOC exhibiting HRD, including cases attributable to BRCA1/2 mutations [59]. In comparison to the commercial ovarian cell lines A2780 and SKOV3, olaparib demonstrated a more potent inhibition of proliferation in pure primary ovarian SCCs at equivalent concentrations [60] , as assessed through in vitro drug sensitivity testing. It is believed that the mammalian target of rapamycin (mTOR) plays a crucial role in controlling cell growth, cell cycle advancement, and tumorigenesis [61]. With the in-depth research on the effective antitumor effect of everolimus combined with 5-AZa-2-deoxycytidine on clear cell ovarian carcinoma by inhibiting COL6A3-Akt-mTOR pathway[62], the research of everolimus on ovarian cancer has entered the phase II[63]. Based on the results of WES, cells displayed the targeted therapy-related mutation, PIK3CA:p.E542K. This mutation has been recorded 1412 times in the COSMIC database and has been detected in colorectal cancer, breast cancer, and urinary tract tumors. E542K is located in the conserved helical domain of the protein and is a hotspot mutation of the PIK3CA gene. In vitro studies have shown that E542K exhibits very strong oncogenic activity in cell survival rate tests and colony formation experiments. In functional studies on PIK3CA pathway activation, E542K Significantly increases the phosphorylation level of downstream AKT, etc., and exerts a strong activation effect on the PI3K pathway[64, 65]. Drug sensitivity analyses revealed the median inhibitory concentration (IC50) values of ZOC254 cells, providing valuable guidance for targeted therapy in ovarian squamous cell carcinoma. Overall, we have successfully established and characterized a POSCC cell line, leading to the enrichment of the ovarian cancer cell population. Homologous recombination deficiency (HRD) was identified through whole-exome sequencing (WES), offering insights into the potential efficacy of targeted PARP inhibitor therapy. Furthermore, this study represents the initial reported use of olaparib in treating POSCC. References H. Xu, R. Zou, F. Li, J. Liu, N. Luan, S. Wang, L. Zhu, MRPL15 is a novel prognostic biomarker and therapeutic target for epithelial ovarian cancer, Cancer Med 10(11) (2021) 3655-3673. J.Q. Wang, Z.X. Wu, Y. Yang, J.S. Li, D.H. Yang, Y.F. Fan, Z.S. Chen, Establishment and Characterization of a Novel Multidrug Resistant Human Ovarian Cancer Cell Line With Heterogenous MRP7 Overexpression, Front Oncol 11 (2021) 731260. C. Zhang, T. 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Genotyping results of STR and Amelogenin loci in the ZOC254 cell line and derived tissues Loci ZOC254 cell line Patient’s tissue Allele1 Allele2 Allele3 Allele1 Allele2 Allele3 D5S818 12 12 12 12 D13S317 10 10 9 10 D7S820 10 11 10 11 D16S539 9 9 9 9 VWA 14 16 14 16 TH01 9 9 9 9 AMEL X X X X TPOX 8 9 8 9 CSF1PO 11 12 11 12 D12S391 20 20 20 20 FGA 19 19 19 23 D2S1338 18 18 18 22 D21S11 29 30 29 30 D18S51 20 20 14 20 D8S1179 10 11 10 11 D3S1358 18 18 15 18 D6S1043 18 18 18 18 PENTAE 11 11 5 11 D19S433 15.2 15.2 15.2 15.2 PENTAD 11 12.1 11 12 D1S1656 15 16 Supplementary Files FigureSupplement25.4.3.docx Cite Share Download PDF Status: Published Journal Publication published 07 Jul, 2025 Read the published version in Human Cell → Version 1 posted Reviewers agreed at journal 08 Apr, 2025 Reviewers invited by journal 07 Apr, 2025 Editor assigned by journal 05 Apr, 2025 First submitted to journal 03 Apr, 2025 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. 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University","correspondingAuthor":false,"prefix":"","firstName":"Zhengxiao","middleName":"","lastName":"Ma","suffix":""},{"id":439834164,"identity":"543b2ff6-6dc8-4ee8-b10a-0e4437f14d28","order_by":5,"name":"Jiamin Wang","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jiamin","middleName":"","lastName":"Wang","suffix":""},{"id":439834165,"identity":"e3b1c3b9-34b1-4457-a39f-007045ac0fbf","order_by":6,"name":"Yue Zhuo","email":"","orcid":"","institution":"Wenzhou-Kean University","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Zhuo","suffix":""},{"id":439834166,"identity":"36d8970f-a338-4bd8-a023-24d5f7adf7fb","order_by":7,"name":"Xinan Cai","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xinan","middleName":"","lastName":"Cai","suffix":""},{"id":439834167,"identity":"b49f4c53-f4df-4a78-9126-47a19e020ab2","order_by":8,"name":"Yuting Zhang","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuting","middleName":"","lastName":"Zhang","suffix":""},{"id":439834168,"identity":"89f93d5c-d680-4a46-b55f-c27ddcd18373","order_by":9,"name":"Yingqi Lyu","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yingqi","middleName":"","lastName":"Lyu","suffix":""},{"id":439834169,"identity":"d91950fc-4583-4fef-87ca-1a1ca3148175","order_by":10,"name":"Jinchao Wang","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jinchao","middleName":"","lastName":"Wang","suffix":""},{"id":439834170,"identity":"0ad5d0c7-b7c0-446e-989b-7fda05f7c1d5","order_by":11,"name":"Minran Huang","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Minran","middleName":"","lastName":"Huang","suffix":""},{"id":439834171,"identity":"951021b6-3799-4cba-a228-4aae952cd807","order_by":12,"name":"Shu Sun","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Shu","middleName":"","lastName":"Sun","suffix":""},{"id":439834172,"identity":"2235ea95-1e4c-448b-b988-adbd7730c19b","order_by":13,"name":"Tingting Feng","email":"","orcid":"","institution":"Zhejiang Cancer Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tingting","middleName":"","lastName":"Feng","suffix":""},{"id":439834173,"identity":"bdcd1c33-e517-4469-aa94-9ac2f12e8b51","order_by":14,"name":"Lisha Ying","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lisha","middleName":"","lastName":"Ying","suffix":""},{"id":439834174,"identity":"8a520ef5-5a07-4786-8154-9b4c33fa6e8b","order_by":15,"name":"Dan Su","email":"","orcid":"","institution":"Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Su","suffix":""}],"badges":[],"createdAt":"2024-12-20 09:16:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5682512/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5682512/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s13577-025-01253-4","type":"published","date":"2025-07-07T15:57:39+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80281794,"identity":"26e4cbff-8e33-4dbb-a3f1-f92fff2ac803","added_by":"auto","created_at":"2025-04-10 06:05:34","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":124970,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHE and immunohistochemical staining of patient tissues. (A) \u003c/strong\u003eEvaluation of histological features of tumor tissues via H\u0026amp;E staining. (\u003cstrong\u003eB-I)\u003c/strong\u003e Immunohistochemical staining of p40, p63, P53, Ki-67, WT-1, PR, ER, and PAX-8. The scale bar corresponds to 100 µm.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5682512/v1/cda0bc2ab75a831924762b9f.jpg"},{"id":80279230,"identity":"bdda97d6-9045-43de-b22c-027664a02688","added_by":"auto","created_at":"2025-04-10 05:33:34","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":463297,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic characterization of ZOC254 cell line. (A) \u003c/strong\u003eEvaluation of histological features of ZOC254 via H\u0026amp;E staining. (\u003cstrong\u003eB-I)\u003c/strong\u003eImmunohistochemical staining of p40, p63, P53, Ki-67, WT-1, PR, ER, and PAX-8. The scale bar corresponds to 100 µm. \u003cstrong\u003e(J) \u003c/strong\u003edepict the optical morphology of ZOC254 cell lines P10, P20 and P30, respectively. ZOC254 cells were selected for subculture and the growth of living cells observed under an optical microscope (100×).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5682512/v1/81225007a85aba0523b6dd11.jpg"},{"id":80280442,"identity":"20cc19e0-3a15-4f88-b53c-3181f2b1acd3","added_by":"auto","created_at":"2025-04-10 05:41:34","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":216675,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHE and immunohistochemical staining of patient-derived xenograft, as well as karyotype analysis and cell cycle analysis of zoc254 cell line.\u003c/strong\u003e \u003cstrong\u003e(A) \u003c/strong\u003eEvaluation of histological features of patient-derived xenografts via H\u0026amp;E staining. \u003cstrong\u003e(B-I)\u003c/strong\u003eImmunohistochemical staining of p40, p63, P53, Ki-67, WT-1, PR, ER, and PAX-8. The scale bar corresponds to 100 µm. \u003cstrong\u003e(J)\u003c/strong\u003eThe Chromosome Karyotype Analysis Report of ZOC254 cells.\u003cstrong\u003e (K) \u003c/strong\u003eRepresentative image of cell cycle peaks of ZOC254 cell line. \u003cstrong\u003e(L)\u003c/strong\u003e Percentage plot of individual cell cycle of ZOC254 cells.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5682512/v1/084a7f48c82e74e127834b52.jpg"},{"id":80280443,"identity":"335bc973-f8f0-4fbc-adea-5b6844da326a","added_by":"auto","created_at":"2025-04-10 05:41:34","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":59746,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProliferation, cloning and migration ability of ZOC254. (A)\u003c/strong\u003e Cell index of ZOC254 detected automatically and regularly with the RTCA Station. (\u003cstrong\u003eB) \u003c/strong\u003eThe initial cell numbers were 500, 1,500 and 4,500, respectively. (\u003cstrong\u003eC)\u003c/strong\u003e Wound scratch assay of ZOC254. Yellow solid line indicates the edges. \u003cstrong\u003e(D) \u003c/strong\u003eTumor formation ability of the ZOC254 cell line determined based on changes in tumor volume, measured every 3 days.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5682512/v1/9fc46dc46690f616215e7cc6.jpg"},{"id":80280446,"identity":"bcd93322-4e70-4a38-b762-e9d50f9c48d4","added_by":"auto","created_at":"2025-04-10 05:41:34","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":176752,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCopy number variation distribution and panoramic display of somatic cells. \u003c/strong\u003eAccording to the whole-exome sequencing data of tumor and normal paired tissues, variations in chromosome copy number in the whole exome range were obtained. Humans are diploid organisms. In a normal genome, each autosomal chromosome has two copies. In the above image, the innermost layer indicates loss of heterozygosity, the secondary inner layer represents the copy number variations at the chromosome level, inward and outward, and further deviation of the blue line value from the baseline correlates with more significant loss of chromosome copy number variation. Further deviation of the red line value from baseline corresponds to more significant chromosome copy number amplification.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5682512/v1/7c7d4de1c533557743e5ea2f.jpg"},{"id":80279237,"identity":"a7895f86-c09d-44dd-a38a-eb06a96c2ea7","added_by":"auto","created_at":"2025-04-10 05:33:34","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":48131,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOlaparib and Everolimus inhibits growth of ZOC54. \u003c/strong\u003eGraph showing the inhibitory effects of olaparib on the proliferation of ZOC254. Average results from three independent experiments are presented. SD signifies standard deviation (n=3).\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5682512/v1/56eb073a226b47c724bd67d7.jpg"},{"id":86699498,"identity":"07f5bed4-acb7-4232-bd2b-c690f6832a23","added_by":"auto","created_at":"2025-07-14 16:10:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1945532,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5682512/v1/b24127bd-b4ca-4dde-8089-c9019b7f5ee0.pdf"},{"id":80279244,"identity":"9af8de3d-7f0a-4dfc-8b11-c5d314b03e8d","added_by":"auto","created_at":"2025-04-10 05:33:35","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":1792917,"visible":true,"origin":"","legend":"","description":"","filename":"FigureSupplement25.4.3.docx","url":"https://assets-eu.researchsquare.com/files/rs-5682512/v1/4832af250484366108e82383.docx"}],"financialInterests":"","formattedTitle":"Establishment and Characterization of Preclinical Model of Primary Ovarian Squamous Cell Carcinoma","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eOvarian cancer is the eighth leading cause of cancer-related mortality in women worldwide [1, 2] primarily due to its dismal prognosis, attributed to 65% of cases being diagnosed at an advanced stage of invasive disease [3].\u0026nbsp;Ovarian carcinomas are classified into five primary types - high-grade serous, low-grade serous, clear cell, endometrioid, and mucinous ovarian cancer - comprising more than 95% of cases, based on histopathological and molecular genetic profiles\u0026nbsp;[4, 5]. In contrast, Primary ovarian squamous cell carcinoma (POSCC) is very rare and the incidence\u0026nbsp;is less than 1%\u0026nbsp;[3, 6, 7].\u0026nbsp;In the 2014 edition of the World Health Organization (WHO) classification of female reproductive system tumors, primary ovarian squamous cell carcinoma (POSCC) ceased to have a distinct classification and was grouped under ovarian germ cell tumors. Subsequently, in the 2020 WHO edition, it was reclassified as somatic tumors originating from teratomas, primarily due to their association with malignancies arising from mature cystic teratomas\u0026nbsp;[8, 9]. Due to its unclear pathogenesis, etiology, and non-specific clinical manifestations, Primary ovarian squamous cell carcinoma (POSCC) is frequently misdiagnosed as a benign tumor, resulting in delayed treatment\u0026nbsp;[10]. The 5-year overall survival rate was 31.2%, with a disease-free survival rate of 31.6%\u0026nbsp;[7]. Understanding the biological characteristics of the primary cell model is crucial for elucidating the disease\u0026apos;s pathogenesis and developing novel treatment strategies.\u003c/p\u003e\n\u003cp\u003eThe quality and characteristics of cell line models are essential for obtaining reliable results in preclinical cancer research, where established cancer cell lines play a pivotal role. Human malignant tumor cell lines offer a rich source of experimental material [11, 12]. The rarity of malignant transformation in mature ovarian teratomas, with rates ranging from approximately 0.46% to 4.00%, poses challenges in establishing a dependable cell line for ovarian squamous cell carcinoma, resulting in a scarcity of disease-specific models that impede fundamental research [13]. Existing ovarian cancer cell lines are predominantly composed of high-grade serous carcinoma from the ovary and squamous cell carcinoma cell lines linked to gynecologic malignancies, primarily originating in the cervix [14]. Ovarian-associated squamous cell carcinoma primarily originates from the malignant transformation of mature cystic teratomas, characterized by a mixture of various dermal differentiation components [15]. This underscores the significance of creating novel and meticulously characterized cancer cell lines for preclinical investigations.\u003c/p\u003e\n\u003cp\u003eHomologous recombination deficiency (HRD), which occurs in approximately 50% of epithelial ovarian cancers (EOCs), represents a crucial therapeutic target [16]. While the use of PARP inhibition as a monotherapy has been extensively explored in various cancers, including prostate, pancreatic, breast, and ovarian cancers, its application in treating squamous cell carcinoma has been sparsely documented [17, 18]. BRCA1/2 play crucial roles in the homologous recombination repair pathway, and patients harboring BRCA1/2 mutations may benefit from first-line maintenance therapy with Poly ADP-ribose polymerase inhibitors (PARPi), such as olaparib [19, 20].\u0026nbsp;Pharmacological research investigating the efficacy of olaparib against POSCC has the potential to significantly advance clinical practice in terms of both diagnosis and intervention.\u003c/p\u003e\n\u003cp\u003eIn the current study, we generated a novel strain of POSCC cells , which is derived from a patient with histologically confirmed ovarian SCC. The results of our detailed its clinical and molecular characteristics and the applicability of patient-derived xenografts (PDX). Furthermore, the utility of the PARP inhibitor, olaparib, was investigated in drug sensitivity studies \u003cem\u003ein vitro\u003c/em\u003e. Our collective findings may be helpful in providing insights into modeling therapies for the management of this disease. To our knowledge, this is the first study to report a cell line developed from a POSCC patient and investigate the genetic background of this rare ovarian cancer type with the aid of whole-exome sequencing.\u0026nbsp;\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003ePatient and histological sample assessment\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of ZheJiang Cancer Hospital, (Hangzhou, Zhejiang, China; IRB-2015-208). Following admission, the patient signed an informed consent form for use of bio-specimens for scientific research.\u0026nbsp;The patient was diagnosed with malignant tumor (squamous cell carcinoma and necrosis were visible in the section) by ice pathology during the operation, and underwent standard comprehensive staging surgery, including the whole uterus, bilateral adnexa, greater omentum, and abdominal paraaortic lymph nodes.\u0026nbsp;Diagnosis of POSCC was based on internationally approved criteria[21].\u0026nbsp;For histological analysis, surgically resected samples were fixed in 10% buffered formalin for 48 h, sliced into 1 cm x 1 cm x 0.3 cm squares, and placed in the embedding box. Tissue blocks were routinely embedded in paraffin and cut into 4 µm sections using a Leica RM 2155 microtome. After performing necessary processing, slides were incubated with primary antibody ER and P16 of Roche (Shanghai, China) ready to use (RTU), P53 and PAX-8 (1:200 dilution) of LBP Medicine Science and Technology (Guangzhou, China), PR, Ki-67 and WT-1 (RTU) of DAKO (Denmark), p40（1:200 dilution）of ZSJO-Bio (Beijing, China), p63（1:200 dilution）of MAIXIN BIOTECH（Fuzhou, China）. Including MLH1, PMS2, MSH2, MSH6，immunohistochemistry has been previously described[22]. Hematoxylin and eosin (H\u0026amp;E) and immunohistochemical staining were performed in the pathology department of Zhejiang Cancer Hospital.\u0026nbsp;The histopathology of tissues was evaluated by three independent pathologists.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEstablishing the ZOC254 cell line and primary cell cultures\u003c/p\u003e\n\u003cp\u003eFresh ovary specimens were washed two to three times with saline. Bleeding and necrotic areas of tissue were removed using a sterile scalpel. Biopsies were incubated for 10 min at 4°C with PBS containing 500 units/mL penicillin and 500 µg/mL streptomycin.\u0026nbsp;Tissues were sliced into 1 mm x 1 mm sections and incubated with 5 mL of 0.1% (w/v) collagenase\u0026nbsp;(Sigma -Aldrich, St. Louis, MO, USA) and 0.125% (w/v) trypsin-EDTA\u0026nbsp;(Sigma) for 4 h at 37°C with shaking.\u0026nbsp;Fresh DMEM/F12 culture medium (Gibco Laboratories, Grand Island, USA) supplemented with 10% (v/v) FBS (Gibco) and 1% (v/v) penicillin-streptomycin (Gibco) was used to terminate digestion.\u0026nbsp;Dissociated cells were\u0026nbsp;gently passed through a cell strainer (70 µm), centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The culture medium used was DMEM/F12 (Gibco) containing 10% FBS, penicillin (100 units/mL) and streptomycin (100 mg/mL) (Gibco) with incubation at 5% CO\u003csub\u003e2\u003c/sub\u003e and 37°C.\u0026nbsp;Fibroblasts were removed via differential phase digestion and adherence[23].\u0026nbsp;After repeated passage, a novel POSCC cell line, ZOC254, was successfully established over more than 30 generations.\u003c/p\u003e\n\u003cp\u003eShort tandem repeat analysis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDNA from ZOC254 cells and corresponding leukocytes from donor patient was extracted using the AxyPrep Multisource Genomic Miniprep DNA (Axygen,\u0026nbsp;New York, USA). Short tandem repeat (STR) analysis at 21STR loci using ABI 3730XL genetic analyzer (Shanghai Biowing Applied Biotechnology Co. Ltd., Shanghai) was performed to verify cell authenticity and unicity according to our previous studies[24].The STR profiles were then compared with the profiles in public cell banks, including the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and Japanese Collection of Research Bioresources (JCRB) for reference. Twenty-one STR loci (D5S818, D13S317, D7S820, D163539, VWA, TH01, AMEL, TPOX, CSF1PO, D12S391, FGA, D2S1338, D21S11, D18S51, D8S1179, D3S1358, D6S1043, PENTAE, D19S433, PENTAD and D1S1656) were matched for DNA fingerprinting analysis.\u003c/p\u003e\n\u003cp\u003eKaryotypic analysis\u003c/p\u003e\n\u003cp\u003eKaryotype analysis of tumor cells was conducted following optimized cytogenetic protocols for neoplastic specimens. Fresh tumor tissues obtained via surgical resection or biopsy were mechanically dissociated and enzymatically treated with collagenase to obtain single-cell suspensions, while liquid tumors (e.g., leukemia/lymphoma) were directly processed. Cells were cultured in RPMI 1640 medium supplemented with 15% fetal bovine serum, L-glutamine, and antibiotics at 37°C in 5% CO₂\u0026nbsp;for 24–48 hours. Metaphase arrest was induced using 0.1 μg/mL colcemid for 60–90 minutes, adjusted for tumor proliferation rates. Hypotonic treatment with 0.075 M KCl (15 minutes, 37°C) and fixation in methanol: acetic acid (3:1) were performed, with repeated washes to remove cellular debris. Chromosome spreads were prepared on chilled slides and aged overnight. G-banding was optimized using 0.025% trypsin (30–60 seconds) followed by Giemsa staining. A minimum of 30 metaphases were analyzed using MetaSystems’ automated platform, with clonal abnormalities defined per ISCN 2023 (≥2 cells for constitutional changes; ≥3 cells for acquired aberrations). Normal stromal cells were excluded via morphology and banding pattern comparison. Ethical approval and informed consent were obtained for human specimens, with biosafety protocols followed for handling neoplastic materials.\u003c/p\u003e\n\u003cp\u003eFlow Cytometry\u003c/p\u003e\n\u003cp\u003eViable cells were harvested (1 × 10⁶\u0026nbsp;cells/sample), centrifuged to pellet cells (800rpm, 5 min), followed by supernatant removal. The pellet was washed once with phosphate-buffered saline (PBS) under identical centrifugation conditions. Triplicate biological replicates were processed to ensure statistical validity. Use the Cell Cycle Staining Kit (MULTI SCIENCES, China) according to manufacturer guidelines. Add 1 mL DNA Staining Solution and 10 μL Permeabilization Solution to the cell pellet. Vortex mix thoroughly for 5–10 sec to ensure homogeneous staining. Incubate the mixture at room temperature (20–25°C) for 30 min in light-protected conditions. Flow cytometric analysis was performed using the lowest acquisition rate setting to ensure signal resolution.\u003c/p\u003e\n\u003cp\u003eWhole-exome sequencing\u003c/p\u003e\n\u003cp\u003eIn order to comprehensively analyze targeted drug selection related gene mutations immunotherapy related genomic indicators (TNB/TMB/HLA/MSI, etc.), genetic risk related germline mutations. Cell pellet sample of ZOC254 at passage 13 and matched peripheral white blood cell were screened with Whole-exome sequencing (WES) technology on an Gene+Seq-2000 sequencing platform (Geneplus Technology, Beijing)\u0026nbsp;following the manufacturer’s instructions and the procedure. The types of gene mutation contained point or frameshift mutation, sites of mutation (codon, exon, functional domain, or conserved area), copy number variants (CNVs), fusion variant, insertion and deletion mutations. To evaluate the status of loss of heterozygosity (LOH), telomere allele imbalance (TAI) and large segment recombination abnormality (LST) in tumor tissue samples, HRD score ≥ 34 was positive and HRD score \u0026lt; 34 was negative.\u0026nbsp;The names of genes are officially named in NCBI-Gene (Official Symbol).\u0026nbsp;Detection results of gene polymorphism of chemotherapeutic drugs and hints of drug useings Classification is according to the PharmGKB website http://www.pharmgkb.org/page/clinAnnLevels.\u003c/p\u003e\n\u003cp\u003eDetermination of cell proliferation and growth kinetics\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe xCELLigence RTCA-DP device (Agilent, Santa Clara, CA, USA) was used to examine the cell growth rate in real time according to the manufacturer’s instructions. Electronic microtiter plates were seeded with 2.0 × 10\u003csup\u003e3\u003c/sup\u003e cells (E-Plate; Agilent) in each well, and proliferation was measured for a week. Cell density measurements were performed in quadruplicate with programmed signal detection every 30 min. The data was exported in excel format, analyses were performed using Prism 8.02 (GraphPad Software, San Diego, CA, USA).\u0026nbsp;Data obtained during the logarithmic growth phase were subjected to natural logarithmic transformation of cellular index (ln [CI]) at each temporal measurement point. Subsequent linear regression analysis yielded the kinetic equation ln [CI] = kt + C, where k represents the proliferation rate constant. Population doubling time (DT) was ultimately calculated through the fundamental relationship DT = ln (2)/k.\u003c/p\u003e\n\u003cp\u003eColony formation assay\u003c/p\u003e\n\u003cp\u003eZOC254 cells in the logarithmic growth stage were inoculated (500, 1500 and 4500 cells per plate) in six-well plates. After two weeks of incubation, cells were washed twice with PBS, fixed with 4% paraformaldehyde universal tissue fixator for 20 min, and stained with 0.1% crystal violet for 15 min. Dishes were washed at least three times with PBS. Images were obtained and the number of cell clones with \u0026gt; 50 cells counted.\u003c/p\u003e\n\u003cp\u003eWound healing assay\u003c/p\u003e\n\u003cp\u003eZOC254 cells were seeded into ibidi Culture-Insert 2 Well (ibidi, Germany) at the density of 500,000 cells/well and incubated overnight. A defined 500 µm cell free gap was left behind after the insert’s removal, and the cells were washed with PBS. At the following day, the cells were incubated in serum-free DMEM/F12 medium, and the images were captured at 0, 6, 12 and 24 h.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIn vitro\u0026nbsp;\u003c/em\u003esensitivity test\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe reagents used in this study were purchased from Chinese suppliers: Olaparib (HY-10162, MedChemExpress) and Everolimus (HY-10218, MedChemExpress) was purchased from Haoxinbio (Hangzhou, China). Stock solutions (10 mM) were reconstituted in DMSO for all the drugs. Cisplatin Injection (Hansoh Pharma, Lianyungang, China), Carboplatin for Injection (Qilu Pharma, Jinan, China) and Paclitaxel Injection (Huiyu Pharma, Neijiang, China). Cell chemosensitivity was measured with the Cell Counting Kit-8 (CCK-8) assay.\u0026nbsp;ZOC254 cells at the logarithmic growth stage were collected, and 8×10\u003csup\u003e5\u0026nbsp;\u003c/sup\u003ecells per well seeded in 96-well plates (Costar, Corning Incorporated, New York, USA) 24 h prior to drug treatment. Next, cells were treated with different concentrations of olaparib for 72 h. Every 24 h, cells in each well were mixed with CCK-8 (APE\u003csub\u003eX\u003c/sub\u003eBIO, Houston, USA) reagent and incubated at 37°C for 1-4 h and absorbance at 450 nm was measured. IC\u003csub\u003e50\u003c/sub\u003e values were calculated and cell killing inhibition curves were plotted using Prism 8.02.\u003c/p\u003e\n\u003cp\u003eTumorigenicity assay\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;All animal experiments were approved by Zhejiang Cancer Hospital Animal Use and Care Committee (Hangzhou, Zhejiang, China; [2015]-1-7号（科）) and where relevant conform to national guidelines for animal usage in research.\u0026nbsp;Experimental animals were fed in a SPF sterile laminar flow chamber. Cells (1 × 10\u003csup\u003e6\u003c/sup\u003e /200 µL) were suspended in matrix gel and subcutaneously injected into the backs of female BALB/c nude mice (4 weeks old, 17–20 g in weight) purchased from Harm Slyke Laboratory Animals Co., Ltd. (license no. Scxk2012-0002). Tumor sizes were monitored every three days and the tumor growth curves and animal weights recorded accordingly.\u0026nbsp;Tumor volume (V) was calculated using the formula: V (mm\u003csup\u003e3\u003c/sup\u003e)= 1/2(length × width\u003csup\u003e2\u003c/sup\u003e). At a maximal tumor volume of 2000 mm\u003csup\u003e3\u003c/sup\u003e, mice were euthanized.\u003c/p\u003e\n\u003cp\u003eStatistical analysis\u003c/p\u003e\n\u003cp\u003eStatistical analyses were performed using SPSS software (version 26.0, Chicago, IL). All experiments were repeated at least three times and data were expressed as means ±SD. The statistical significance of differences between groups was calculated using the Student’s t-test. Differences were considered statistically significant at P\u0026lt;0.05.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eThe cell lines are stable experimental models for POSCC\u003c/p\u003e\n\u003cp\u003e1 Clinical and diagnostic features\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe patient (64 years old; female) presented at ZheJiang Cancer Hospital without obvious inducement and symptoms of frequent urination (most obvious at night), endless urine, difficult stool, fatigue, and abdominal distension, but no abdominal pain. Preoperative assessments were conducted, revealing elevated levels of serological tumor markers such as CA125, CA199, or CEA, along with increased levels of serum squamous cell carcinoma antigen (SCC-Ag). Abdominal, pelvic CT and urinary tract US were used to detect a solid abdominal pelvic cystic mass (about 16.5×9.2cm), which was considered a teratoma and secondary renal water (the right renal pelvis, the right ureter was slightly extended). Following total hysterectomy and bilateral adnexectomy, the patient was diagnosed with moderately differentiated ovarian squamous cell carcinoma (SCC), which extended from the adventitial membrane of the right lateral uterine wall to the deep muscularis. Diagnosis of POSCC was supported by HE and immunohistochemical staining (\u003cstrong\u003eFigure 1\u003c/strong\u003e\u003cstrong\u003eA\u003c/strong\u003e;\u0026nbsp;p40-positive, p63-positive, P53-positive, Ki-67-positive (5%), WT-1-negative, PR-negative, ER-negative, PAX-8-negative（\u003cstrong\u003eFigure 1B-I\u003c/strong\u003e).\u0026nbsp;Additional imaging modalities including CT scans and ultrasound of the chest, upper abdomen, and kidneys did not detect any distant metastases beyond the pelvic mass. Following surgical resection, there was no evidence of metastatic squamous cell carcinoma in the cervix. No concurrent precursor lesions were identified in the bilateral ovariectomized specimens, leading to the definitive diagnosis of primary pure squamous carcinoma of the ovary.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this case, the patient was subjected to radical resection and only received symptomatic support (anti-inflammatory, hemostatic and hypoglycemic) therapy after surgery. Chemotherapy and targeted therapy were rejected [25, 26].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2 Establishment and histological characteristics of a novel POSCC cell line ZOC254\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFresh surgically resected ovarian cancer tissue was processed and cultured in time, and a certain purity of ovarian cell line was obtained after multiple gradient digestion and passage, which was named ZOC254. The ZOC254 cell line retained the histological features of the primary tumor, in particular, strong positivity for p40 (\u003cstrong\u003eFigure 2B\u003c/strong\u003e) and p63 (\u003cstrong\u003eFigure 2C\u003c/strong\u003e), as well as nuclear accumulation of P53 (\u003cstrong\u003eFigure 2D\u003c/strong\u003e). The newly generated cell line was highly proliferative (80%), determined based on the Ki-67-positivity rate (\u003cstrong\u003eFigure 2E\u003c/strong\u003e) [27]. Cells were positive for Wilms tumor protein (WT-1) (\u003cstrong\u003eFigure 2F\u003c/strong\u003e), which is potentially useful in identifying the primary sites of serous carcinoma [28, 29].\u0026nbsp;Progesterone receptor (PR) (\u003cstrong\u003eFigure 2G\u003c/strong\u003e) and estrogen receptor (ER) (\u003cstrong\u003eFigure 2H\u003c/strong\u003e), predictive biomarkers of endocrine therapy, were not expressed, indicating that the patient would not benefit from endocrine therapy\u0026nbsp;[30]. Pax-8 (\u003cstrong\u003eFigure 2I\u003c/strong\u003e), a reliable\u0026nbsp;marker to\u0026nbsp;identify cancer cells of gyneduct origin\u0026nbsp;[31], was not expressed in the ZOC254 cell line. The growth and morphology of the ZOC254 were photographed under an inverted microscope,\u0026nbsp;during long-term cultivation in \u003cem\u003evitro\u003c/em\u003e.\u0026nbsp;The cells were passaged \u0026gt; 30 generations, and there was no obvious change in morphology during cell passage (\u003cstrong\u003eFigure 2J)\u003c/strong\u003e.\u0026nbsp;The cells were flat, polygonal and oval and had formed an epithelial-like monolayer. The nucleoli of cells were obvious, a few were multinucleated, and the nucleo-plasma ratio was large.\u003c/p\u003e\n\u003cp\u003eTo systematically evaluate their antigen-secreting properties, we performed the quantitative assessment of tumor markers （serum SCC, CA125, and CA19-9）with the cell culture supernatant using clinical laboratory assays. The results showed that SCC (23 ng/mL) and CA125 (93.99 U/mL) were both higher than normal serum reference values by Abbott ALINIQ direct chemiluminescence immunoassay, and CA19-9 (10.2 U/mL) was measured by Roche Cobas e801.\u003c/p\u003e\n\u003cp\u003e3 Authenticity of the ZOC254 cell line\u003c/p\u003e\n\u003cp\u003eShort tandem repeats (STR), also known as microsatellite DNA, refer to a type of DNA sequence formed by tandem repetition on chromosomes with several base pairs as the core unit (2-6 base pairs, 10-60 repeats, and a gene fragment of less than 400 base pairs). The authenticity of the ZOC254 cell line was verified through short tandem repeat (STR) profiling, which provided information on the true origin and unique profile of these cells with respect to those present in the reference databases (\u003cstrong\u003eTable 1\u003c/strong\u003e).\u0026nbsp;We found that the characteristics of ZOC254 cell line are consistent with tissues, and the results demonstrate clear and consistent genetic profiles that confirm the cell line's authenticity. STR sequence retrieval was performed in the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) database. No matching sites of these cells were detected in the database, suggestive of a new cell line and\u0026nbsp;excluded the possibility of cross-contamination with other cell lines. Moreover, no multiple alleles were found, indicating no single cells and no other cell contamination. The matching degree of STR genotype between the ZOC254 cell line and\u0026nbsp;paired parent tissue was 90%. According to the ATCC Standards Committee identification guidelines (ANSI/ATCC ASN-0002 2011), a matching degree ≥80% is considered the same source.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe resulting tumors recapitulated the histologic features of squamous cell carcinoma (SCC), including keratinization, as confirmed by H\u0026amp;E staining (\u003cstrong\u003eFigure 3A)\u003c/strong\u003e. Importantly, the immunohistochemical (IHC) profile of the xenograft tumors closely matched that of the original patient tissue and patient-derived cells (\u003cstrong\u003eFigure 3B-I)\u003c/strong\u003e. We also have performed microsatellite marker analysis on the primary tumor tissue, ZOC254 cell line, and patient-derived xenografts (\u003cstrong\u003eFigure S2\u003c/strong\u003e), and the results show consistent profiles across all three sample types.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further analyze the ZOC254 cell line, we conducted a karyotype analysis on it. Karyotypic analysis of ZOC254 cells revealed chromosomal abnormalities across 51 analyzed metaphase spreads (\u003cstrong\u003eFigure 3\u003c/strong\u003e\u003cstrong\u003eJ\u003c/strong\u003e\u003cstrong\u003e)\u003c/strong\u003e.\u0026nbsp;The majority of cells (84.3%, 43/51) exhibited triploid-to-tetraploid chromosomal counts (3n-4n), while we also observed a minor population of hypo-triploid cells (13.7%, 7/51) and a single hyper-tetraploid cell (\u0026gt;4n). Notably, these numerical abnormalities were accompanied by Multiple structural aberrations, such as chromosomal rearrangements, deletions, and derivative chromosomes, suggesting genomic instability characteristic of this aggressive tumor type.\u003c/p\u003e\n\u003cp\u003eWe employed flow cytometry to analyze the cell cycle distribution of ZOC254 cells. The results revealed a distinctive cell cycle distribution profile, with 34% of cells in G0/G1 phase, 50.1% in S phase, and 13.3% in G2/M phase (\u003cstrong\u003eFigure 3K-L)\u003c/strong\u003e. This pattern reveals a predominant S-phase population, suggesting active DNA replication and proliferative capacity characteristic of this tumor cell line.\u003c/p\u003e\n\u003cp\u003eThese findings confirm that our model maintains the essential histopathological and immunohistochemical features of this rare ovarian squamous cell carcinoma, thereby providing a reliable preclinical platform for further investigation.\u003c/p\u003e\n\u003cp\u003e4 Function experiment of ZOC254 cell line\u003cem\u003e\u0026nbsp;in vitro\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;in vivo\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the biological properties of ZOC254, we first analyzed its growth rate. The doubling time of ZOC254 was calculated to be approximately 30 hours \u003cstrong\u003e(Figure 4A)\u003c/strong\u003e.\u0026nbsp;Subsequently, we investigated the migratory behavior of ZOC254 using a wound scratch assay, tracking cell movement towards the wound site. Our findings demonstrated efficient wound healing over time, indicating robust cell migration capabilities\u003cstrong\u003e\u0026nbsp;(Figure 4B)\u003c/strong\u003e. Additionally, the cell line exhibited the capacity to form clones in relation to the density of initially seeded cells (\u003cstrong\u003eFigure 4C\u003c/strong\u003e). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe tumor formation ability of the ZOC254 cell line was evaluated via nude mouse inoculation (\u003cstrong\u003eFigure 4D\u003c/strong\u003e).\u0026nbsp;After 24 days of inoculation, growth of ZOC254 xenografts was observed in nude mice.\u0026nbsp;The collective results support the utility of our cell line in the construction of animal models, drug screening and efficacy evaluation.\u003c/p\u003e\n\u003cp\u003eBasic molecular characteristics of ZOC254\u003c/p\u003e\n\u003cp\u003eWES was used to clarify the gene genetic characteristics of ZOC254. Exon sequencing revealed the presence of 509 somatic mutations, 0 germline pathogenic or suspected pathogenic mutations and 0 gene fusions.\u0026nbsp;The copy number variation distribution and panoramic display of somatic cells are presented in \u003cstrong\u003eFigure 5\u003c/strong\u003e. In terms of immune prediction, ZOC254 displayed TMB-H(421Muts) and MSS, and HLA LOH was negative. Noteworthy, cells were positive for HRD and 54.8% displayed the targeted therapy-related mutation, PIK3CAc.1624G\u0026gt;A (p.E542K) supporting the potential utility of the targeted drugs, olaparib and everolimus[32]. The status of heterozygosity loss (LOH), telomere allele imbalance (TAI) and large segment recombination abnormality (LST) in the genome of tumor tissue samples were evaluated to determine the index of HRD status. HRD score ≥34 was positive. HRD score \u0026lt;34 was negative.\u0026nbsp;In this case, LOH score was 21, TAI score was 6, LST12, so HRD score was 39 (positive).\u0026nbsp;PIK3CAc.1624G\u0026gt;A (p.E542K)\u0026nbsp;is a missense mutation, which causes the 542nd amino acid of the protein encoded by the PIK3CA gene to mutate from glutamic acid to lysine.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eOlaparib and everolimus are potential therapeutic targets for ZOC254\u003c/p\u003e\n\u003cp\u003eOlaparib has been FDA-approved for treating ovarian cancer patients carrying BRCA1/2 germline mutations (SOLO1), breast cancer patients with BRCA1/2 germline mutations (OlympiAD), and for first-line maintenance therapy in pancreatic cancer patients with BRCA1/2 germline mutations (POLO) [33, 34]. Preclinical studies have demonstrated that inhibitors targeting the PI3K pathway, including mTOR inhibitors, can suppress the activity of the primary PI3K pathway in PIK3CA mutant tumors [35]. Human tumor cells harboring activating mutations in the PI3K pathway have exhibited responsiveness to everolimus in both in \u003cem\u003evivo\u003c/em\u003e and in \u003cem\u003evitro\u003c/em\u003e experiments [36]. Based on the results of WES, we focused on the drug sensitivity of ZOC254 cell line to olaparib and everolimus. We also examined the antitumor effects of first-line chemotherapy agents, including cisplatin, carboplatin and paclitaxel. The median inhibition concentration (IC\u003csub\u003e50\u003c/sub\u003e) values of ZOC254 cells were summarized (\u003cstrong\u003eTable 2\u003c/strong\u003e) and growth curves for two anticancer drugs, olaparib and everolimus (\u003cstrong\u003eFigure 6\u003c/strong\u003e) are shown. The IC50 values of olaparib at 24h, 48h, and 78 h were 17.99 μM, 10.97 μM, and 8.83 μM, respectively. The IC50 values of everolimus at 24h, 48h, and 78 h were 216.31 μM, 47.71 μM, and 44.48 μM, respectively. Our results indicate that inhibition of ZOC254 cell proliferation is enhanced with the drug concentration or time elapsed. Growth curves for three first-line chemotherapy agents are shown in \u003cstrong\u003eFigure S1\u003c/strong\u003e.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis study presents the initial preclinical model of POSCC. The etiology and tissue origin of ovarian squamous cell carcinomas remain unclear. Both primary and metastatic squamous cell carcinomas of the ovary have been identified, with the latter most commonly associated with metastasis from cervical or vaginal squamous cell carcinomas [37, 38]. Primary ovarian squamous cell carcinoma is rare and often accompanied by precursor lesions. Malignant transformation of mature cystic teratoma (MCT) is frequently reported [39]. Consequently, most POSCCs exhibit well-differentiated ectoderm, endoderm, and mesoderm tissues [40]. In contrast, pure primary squamous carcinoma lacks an MCT component and signifies the carcinogenesis of epithelial cells on the ovarian surface [41].\u0026nbsp;The patient in this study received a diagnosis of squamous cell carcinoma through histopathology and immunohistochemistry. No apparent lesions were detected in the kidneys, bladder, or bilateral vaginal appendages before and during the operation. Absence of squamous cell carcinoma was noted in the cervix of the surgically resected specimen, thereby excluding metastatic squamous cell carcinoma. Bilateral ovariectomized specimens did not exhibit precursor lesions such as mature cystic teratoma, Brenner tumor, or endometriosis\u0026nbsp;[41].\u003c/p\u003e\n\u003cp\u003ePOSCC typically lacks PAX8 expression aligns with its non-M\u0026uuml;llerian origin (e.g., teratoma-derived or metaplastic squamous differentiation)[42-44]. This pattern contrasts with PAX8 expression in serous or endometrioid carcinomas, which typically originate from the fallopian tube or ovarian surface epithelium. The observed discrepancy in WT-1 immunoreactivity between the original tumor (negative) and cultured ZOC254 cells (positive) likely reflects the partial WT-1 positivity in \u003cem\u003evitro\u003c/em\u003e likely represents culture-induced artifact or minor subpopulation expansion, while the consistent negativity in tumor/xenografts underscores the true biological signature of this ovarian SCC[45]. In contrast, primary cell culture conditions might selectively promote the survival or proliferation of a minor subpopulation with WT-1 expression, possibly representing a less differentiated or transitional state. Immunohistochemically, tumor cells were positive to p40，p63 and negative for WT-1，PAX-8，which further ruled out high-grade serous carcinoma, urothelial carcinoma, and teratoma malignancies，leading to a final diagnosis of POSCC. Large randomized controlled studies are challenging to conduct due to the rarity and high mortality rates associated with POSCC [46]. Most existing reports consist of systematic reviews and analyses of published data\u0026nbsp;\u0026nbsp;underscoring the need to expand the current repository of POSCC literature\u0026nbsp;[39]. Hence, in this instance, ZOC254 exhibits a stable profile and remains unaffected by other dermal differentiated cells, making it an excellent model for studying ovarian squamous cell carcinoma.\u003c/p\u003e\n\u003cp\u003eThe PDC and PDX recapitulated the histologic features of POSCC, including keratinization, as confirmed by H\u0026amp;E staining. Importantly, the IHC profile closely matched that of the original patient tissue, further validating the genetic stability and model reliability of this rare ovarian SCC model. The patient-derived cells maintain the fundamental genetic characteristics of the original tumor through both \u003cem\u003ein vitro\u003c/em\u003e culture and \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003exenograft passages.\u0026nbsp;These cells were directly isolated from patient-derived tumor tissues without genetic modification or immortalization treatment, which allows them to maintain critical biological features of the original tumor microenvironment[47-49]. This approach offers distinct advantages, including preservation of natural tumor heterogeneity (subpopulations with varying degrees of malignancy) and avoidance of accumulated genetic mutations observed in immortalized cell lines during long-term\u003cem\u003e\u0026nbsp;in vitro\u003c/em\u003e culture[47, 50, 51]. Furthermore, this approach provides a more reliable model for experimental applications such as drug sensitivity testing and therapeutic efficacy evaluation[52, 53]. \u0026nbsp;The consistency between primary tumor and xenografts validates our model\u0026apos;s ability to faithfully recapitulate the patient\u0026apos;s tumor biology.\u003c/p\u003e\n\u003cp\u003eClinically, HRD represents a crucial therapeutic target in EOC. PARP inhibitors have shown significant clinical advantages in HRD ovarian cancers, particularly in cases of BRCA1/2-mutated HGSOCs [54]. Nevertheless, there is a lack of research examining the impact of PARP inhibitors on POSCC. Ovarian cancer often exhibits a strong familial predisposition associated with hereditary mutations in BRCA1 or BRCA2 [55, 56].\u0026nbsp;Nevertheless, our patient did not exhibit any deleterious or suspected deleterious BRCA1/2 mutations. Although mutated BRCA genes are primarily linked to HRD, other crucial homologous recombination repair (HRR) proteins could also be mutated or functionally impaired, thereby potentially broadening the therapeutic avenues for PARP inhibitors\u0026nbsp;[57]. Through WES, we can assess the presence of loss of heterozygosity (LOH), telomere allele imbalance (TAI), and large segment recombination abnormality (LST) in tumor tissue samples to ascertain the status of HRD. The initial PARP inhibitor, olaparib, received FDA approval for treating ovarian cancers bearing BRCA mutations\u0026nbsp;[58]. Olaparib has demonstrated notable clinical advantages as a maintenance regimen for women with HGSOC exhibiting HRD, including cases attributable to BRCA1/2 mutations\u0026nbsp;[59]. In comparison to the commercial ovarian cell lines A2780 and SKOV3, olaparib demonstrated a more potent inhibition of proliferation in pure primary ovarian SCCs at equivalent concentrations\u0026nbsp;[60]\u0026nbsp;, as assessed through in \u003cem\u003evitro\u003c/em\u003e drug sensitivity testing. It is believed that the mammalian target of rapamycin (mTOR) plays a crucial role in controlling cell growth, cell cycle advancement, and tumorigenesis\u0026nbsp;[61].\u0026nbsp;With the in-depth research on the effective antitumor effect of everolimus combined with 5-AZa-2-deoxycytidine on clear cell ovarian carcinoma by inhibiting COL6A3-Akt-mTOR pathway[62], the research of everolimus on ovarian cancer has entered the phase II[63].\u0026nbsp;Based on the results of\u0026nbsp;WES,\u0026nbsp;cells displayed the targeted therapy-related mutation, PIK3CA:p.E542K. This mutation has been recorded 1412 times in the COSMIC database and has been detected in colorectal cancer, breast cancer, and urinary tract tumors.\u0026nbsp;E542K is located in the conserved helical domain of the protein and is a hotspot mutation of the PIK3CA gene. In \u003cem\u003evitro\u003c/em\u003e studies have shown that E542K exhibits very strong oncogenic activity in cell survival rate tests and colony formation experiments. In functional studies on PIK3CA pathway activation, E542K Significantly increases the phosphorylation level of downstream AKT, etc., and exerts a strong activation effect on the PI3K pathway[64, 65]. Drug sensitivity analyses revealed the median inhibitory concentration (IC50) values of ZOC254 cells, providing valuable guidance for targeted therapy in ovarian squamous cell carcinoma.\u003c/p\u003e\n\u003cp\u003eOverall, we have successfully established and characterized a POSCC cell line, leading to the enrichment of the ovarian cancer cell population. Homologous recombination deficiency (HRD) was identified through whole-exome sequencing (WES), offering insights into the potential efficacy of targeted PARP inhibitor therapy. Furthermore, this study represents the initial reported use of olaparib in treating POSCC.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eH. Xu, R. Zou, F. Li, J. Liu, N. Luan, S. Wang, L. Zhu, MRPL15 is a novel prognostic biomarker and therapeutic target for epithelial ovarian cancer, Cancer Med 10(11) (2021) 3655-3673.\u003c/li\u003e\n\u003cli\u003eJ.Q. Wang, Z.X. Wu, Y. Yang, J.S. Li, D.H. Yang, Y.F. Fan, Z.S. Chen, Establishment and Characterization of a Novel Multidrug Resistant Human Ovarian Cancer Cell Line With Heterogenous MRP7 Overexpression, Front Oncol 11 (2021) 731260.\u003c/li\u003e\n\u003cli\u003eC. Zhang, T. 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Scott, Identification of Variant-Specific Functions of PIK3CA by Rapid Phenotyping of Rare Mutations, Cancer research 75(24) (2015) 5341-54.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1.\u0026nbsp;\u003c/strong\u003eGenotyping results of STR and Amelogenin loci in the ZOC254 cell line and derived tissues\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"543\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" style=\"width: 73px;\"\u003e\n \u003cp\u003eLoci\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 249px;\"\u003e\n \u003cp\u003eZOC254 cell line\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" style=\"width: 220px;\"\u003e\n \u003cp\u003ePatient\u0026rsquo;s tissue\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003eAllele1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eAllele2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eAllele3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eAllele1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eAllele2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eAllele3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eD5S818\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eD13S317\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eD7S820\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eD16S539\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eVWA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eTH01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eAMEL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eTPOX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eCSF1PO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eD12S391\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eFGA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eD2S1338\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eD21S11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eD18S51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eD8S1179\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eD3S1358\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 103px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e \u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n 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dbi2X4PvfL3wY3jG2bDKrpdNH598O5c9h0+puBHm92Obb+I0pZiGP/HGQtQJUOwo/towPwohag9G39TPIp0qRcNFYkPUKXjFC6c+IUTdgqkHpjeZlhCipkhsiKkKc4m8ceFe8fitlOOvIYQoHgQGc/srr7yy+Qj4fL8QNaVev/oqGgY4KeHAK6EhRN2C9R3cob5fv372LcTkIMuGEEIIIQpFlg0hhBBCFIrEhhBCCCEKRWJDCCGEEIUisSGEEEKIQpHYEEIIIUShSGwIIYQQolAkNoQQQghRKBIbQgghhCgUiQ0hhBBCFIrEhhBCCCEKRWJDCCGEEIUisSGEEEKIQpHYEEIIIUShSGwIIYQQolAkNoQQQghRKBIbQgghhCgUiQ0hhBBCFIrEhhBCCCEKRWJDCCGEEIUisSGEEEKIQpHYEEIIIUShNFqx8cADD9inIfLxxx+Hs88+O7Rr1y4888wzaWjxXH755YXd74033gg9evQI00wzTZhrrrnCCSeckO6pHPKBOE3pfKgrkGbya0qknbp0wAEH2P2mFpR5ysZ3332XhjQOqBvUB9Je18t5p06d7FPb8MxvvvlmuzbloK7BM6J+8IycUmENmUYpNnjATZs2DZtttpkVThrI7CdfWCnIhK2yyioVx/Cbwu376iIfffRR+mvKsO+++4YHH3zQ8qU2QTSsv/764fjjjw9jx44N+++/fzjjjDOssubJ33vcuHFTPB8aI0sssUR4+eWX063ioUw0RgFZiiZNmtj3Dz/8YN+NhXwZoE1/+OGH0626x/fffz/JMyoV1lBpdGIDYbDwwguHDh062PZDDz0Unn76afvdrFkz65iOOeYY2wY6tEUXXTTcdtttoXfv3iFJEvvccsstYdSoUWHuuedOj6w7tGnTJqy55prp1pTl9NNPD9dee22tWo1uv/12q5DLL798aN68ud2DZ8B2nhNPPDH9NXXzoS5AGSefvKwXCXnNs5lSnHvuuemvf6He0nhPyXjUBcj7OeaYI92q29De8qkNsmWAZ87gsa5CW7XSSiulWxMoFdaQaVRiAyV87LHHhj333DMNmYA3xquuuqpVXAehwYi6bdu2VkGyhZnjaNwGDx4cPv300zRUAB3+TjvtNMXN2VisZMVo+GC96t+/f7olGiMqA/WPRiU2UMKbbLJJ2SMfhAkjaiwalZ2DHwGWktoAq0up6Ryfe8+G8Y0vAmHl+DD4+Xyc7BRSFkSW7yt1bbb9PI7DLyKLizcsEtXhPhVci29PH3i6eQ7g9ywFcfLGp7LjsLb4vbL3AYSR+xzw4blWJ5aqOsfD/APZ/HbzL3ntvih8mJ7z6SGuRf4QRnyz8fdnwnU8jOv7/Uv5qxDGeT5HzDX5zSc//VRV2pzssyOOH374YbqnckhDdjoye91y4sf522+/vf1ee+217RqkjzzLnpuF63g8+c5a3crJf8/DUvnveH5xf/bzLPw6jqenuvtwbY4hrDI4zvOR87C8OpVdz+POh7gA+/NhTja9+Q/Pivtk2wl+c73K4HrkEcdBTfI3C8eVKgNZyFuPm98vS2XPoiqqKkeQjT/f+TpVLpyXjRt51iBIGgljx45NSO5ZZ52VhkwM+6IQSbeSJI6QLWxKZ9H+++9v9+zZs2caMoH7778/2W677ez34MGD7Zinn37atkkT2xzjsC97DHB+Pj35sNdffz1p27ZtxXkDBgyw/Z5vbJNP5Ccf4lsqTzmG61QFaWzfvr3lNfi9uGYWT191lDrO84F7eZr4TZjfl3QQD+4PHNesWbOJykOe6s5hv+etH0PeZtML5BHHcTzhbHMMsO15wnXzz4R8yt4/fy/PDz+PMI8TeUA5Yh/3JO5OOfnBPsLyceJTGaSH/V5ePM5e1suNn5/n9wZ+58sykEee56SLNHCMP4Pq8t/z0Mt59j7EFzy/OMavQ3y5lsO+7H6O5xjfzt6Hekw8PZ/ycF+OzT8fwvhd1fX83Oy1S4VxDmE8A/A2h7Q7/PZn52nKPpMspJN95ImXo3w8+V0qf0vBcRyTv5/H0eOdTwdU9Swqg+M5juM5Ll+OuH52P8fn4+dpzZIPI83Exa/LM/b8qu9U3jI0MLxwegXNw77sQ/Xjp/SDpqBS2CisWdjOFkCOcTiHuGYbi1KVsZzCnq2ojldG4Ph83PLHg1e2yiowaWF/ViBBqYamVLxLUeo4z4fsffJ5Q37m0+SCpLIGr5xzSLvnHWGUpXx+sC9bJv0ajseVtGWpLKxUGfZ0gudRNh75fKsubf7sSpWT7HXycK5fw+GcbJzLiV+pdEH+OL+f1xsgbYR5HMrN/+rKT1ZYAOXY6wz3yu/3TtDv7dcsVZfycK3Kno/HqarrEU5eZcmH8UzycSaM4xy2s9cnn/3+lcE52eft8awqf0tR2TGEkRdZCPO0lfMs8pRbjrL7Kb/spxw4+fIJ+TDSk49fOWWiPtBoplGee+45+1566aXtu67CdM1xxx1nUwKYKgFzI7g/CW994AjnZsjVVlvNwmsDHF8xUWLC88/w4cNtOon44HCJmQ/TnscPM3Qen1p677337DuPT7HwFkOWXXbZxb5r26scT/U8P/30k33fddddlt/ZNPO2C1QW/3LO4VleeeWV9nuFFVYIsWGZZDqO58jzxAxMnvo1iqbUtKCboqtL29ChQ+03Pk5ZKptqdHCIi22OfVOGMG9TtkpRVfzKxctQ1g+LvPY4QLn5X6r8eJtCfmE2z0L6uDYQj9gRTZSfnTt3tn15833r1q3TX6XheK61zjrrpCETqMxBtLrrVcacc85Z0v8pdoTprxC6du1qecb0Am0R+Ty5zshV5W9NKZUXvJUGNXkWTnXliLJD+0je+DX9xYHKyndlLLnkkvadnZIq1b7WRxrlq6/l4K+TvfTSS/Y9Jenevbt9u7f1VVddFXbYYQf77SAyeEuGN2IeeeSRNLR2iOraKlL+442JVyAqF42Ni6HJYfz48emvCZRqdIri7bffTn8FEwKl0lxVRS/nHBqjOAq136UEFIKNhuXwww+3DoRr1gWqSps33NnGt1xoQOmY7777bnMkjqPcdM/UYUrlP+kslZ+XXXZZekR5eH2ZXBFRLrxmHkfrFX4cdMS0hfvss49tAx3uvffeG4YNG2ZtUd7noy7xyiuvpL9q71nkKXXNcvyYsiCyX3zxxdCxY8ew5ZZbWtksx5+kPiCxUQl0EnSmKNaajqj+KzTidFCMLunIqczZEQOVmsbg8ccft9dAJ6fRrwrWysiTdYbiflTM119/3dZWoEJMLmPGjEl/TcyUfpUv62DnkPdVPftyzuE3ZYjniaNrdh/Htm/f3vKTfKxLI5hy0lbTRpBzceg77bTTbOQ/uaPgmlIqnty/NvO/1KAEIeP5hdDkfnnyTobl8s4776S/ioH2r2/fvuHVV1+1kTpv5WFxzS4LADxD3tS74YYbwplnnlmlY2ddYXKfRWXlyCl1/uQ8XwQH+Txy5EizMJH3bkWuzzQaseHrLdTENHfxxRfbN6OeUoUTCC+igjFqADry3XbbzX47iA9M2G4KrinZtPgo1dluu+1M5OQr1pAhQ+wbi4qfz/0vuOACs3RkOyHw684///z2ncdHtJ7Hjk9tbLTRRvYN+TjWNpiDSUO24YDzzz+/wqyZp5xzyMObbrrJhBkfOjZGK95wMCWBEMlbraY21aVtwQUXtO1Slpqq8Lo3pUSV13l/m8mhDJOO2sp/RqFcJz+yxzJJZ+xl/aSTTrJvZ3I6Iq9PTN38F7J1qlTbRn3mHpQBRuhMCeWFRja9LAuAGJlS04CTy+Q8i+rKEc8YK1CvXr0mykvqedZ6Wg7ku7eliA7yn7JVzpt9dZ1GIzZ8FFWq4/KOldFJVkFSgQYPHmwNL34RPHgvTHyzfeCBB4YjjjjCwoAwn7fLVkamGwjjuxzoyKkYzC9mO15A7RJX4k08KPTAeh9+Tx/5ZEdA3klQ0SjQiCSflyXepB2LCYUbHwOfj8Xs7Q0y+UeaPZ/oQKho+U4ZsyWWocqsLqSP0T4dlsefa1Jhe/bsWXEe6cOPBEqNLLK4NYS0cSzX8/RnRaYLGi8LTFsRV3xVEHekmW+uV5kfQnXncH9GJEcddVR6RrBXqMlbBB1x86k6BAkQb4Qk0PjxGT16tG1ny63nA6NOJ1su/XepMuDneIMGfm3Pl+rShlhAONH4+rMjTm4y5toehyz+fChr/s05HMt1yJNy4uf5xjPlXD/Wz/X88Y6eMubpoEyzn/JXTv6XU35Yt4c6QH54neF+7lfBvVzEU5c8HghtX7vH73PppZfad2VQL6gfpIlr+PP2eHOdbLkvdT2eLXEhfTwDpmmBa3hekl7PN/8Qb56TP1uOJ8zjQP57Z14KjuF5+zOHcvK3FKXKgD93zwvw+/h3Oc8iT3XlCLAC0U8wnUS7ymfjjTeeaE2nfPmEUmEMbv05+HeDWJwwqtZGAx7csdCkWxNgm2zIfs7KeWrjIcy5eBz7MXjRc1z+7YL8uYA3MecD3+V6F+MlXep67lFNPPC8Jg7EhzD2cY7Hk082zXhHE5Y9lv3ZOJFerufHZT3F8cL2fXz47R7ZWcirUnHPwzGer9wr6xFOPPw+/sk/vyyeD1yP9JTKh8ryJoquirwpN+5VnZO/PniYf8DfIiDtpNc94wkvJ/58SuVT/ji2Ob+cMKguP8hrynJ2P9finGx5ycI5fj++uQfp9OdVk/j5vTkf8seRJ5CPpx/v/Nf8JwyoAx4HrpWv48TD08oxxIkwqOyaVcE52Wtxbco+9/U0VXY90unncix43vBMwNsYPtlrZa/HfbP7efaeplJkr8GnJvlbimwZKFUHSoVBVc+iMtjv9/O8ykPb5flBvLPtItseBz6ks1QYcc6GlypL9ZVp+BMT1ShA8ceHZ/9fo7IR63+BUQKjwVhYJlqSFzWMMxwKmTigimtryd66CCqdUT1zjkXksxCiWGjLcEJ1i7BD3e7WrVuNHR+FaFRiAzAD8gYHjpW1DUICMydigjdEvEIyfZLN5vx2QwMzO6+wVmaWFELUXTDd48hbWRtF/fapMCHKpdG9jYLjJfPmRVQW9zPAGZD5usZYIUlzly5dJDSEqKe4zwSiAr8OBlF8ECEMpPDrEqKmNDrLhkMlYk2HvJmwtqBi4ryEB3djsWwUnadCiCkDdfn666+vcM5m+pkBFA7PlTl9C1EVjVZsFA1igzcJGB1kfTYIZ52BhuyzIYQQQmTRol4FgaBAaADrZPjrdXzn180QQgghGjISG7UI720zRcInu/QtooM5T8J9WwghhGgsaBpFCCGEEIUiy4YQQgghCkViQwghhBCFIrEhhBBCiEKR2BBCCCFEoUhsCCGEEKJQJDaEEEIIUSgSG0IIIYQoFIkNIYQQQhSKxIYQQgghCkViQwghhBCFIrEhhBBCiEKR2BBCCCFEoUhsCCGEEKJQJDaEEEIIUSgSG0IIIYQoFIkNIYQQQhSKxIYQQgghCkViQwghhBCFIrEhhBBCiEKR2BBCCCFEoUhsCCGEEKJQJDaEEEIIUSjTJJH0d4PklVdeCW+++WaYbrrp0hAhhBCi4UF3vsEGG4TWrVunIXWHBi82dthhh/D444+H6aefPg0RQgghGh7/+9//woknnhgOPPDANKTu0ODFxg8//BB++eWXMM0006QhQgghRMOkWbNmYdZZZ0236g4NXmwIIYQQYuoiB1EhhBBCFIrEhhBCCCEKRWJDCCGEEIUinw0hhJgK/P777/ZaPs580047rb22SFibNm3MyS/L119/Hb744ovw119/2b6FFlrInN7//PPPMNtss9kxOMOPGTPGrgVcb9555w1zzz23bXPuiBEjwt9//x0WW2yxMMsss1i4EFMCiQ0hhJgKIA769u0brrrqqvD999+b6Nhtt93C4YcfHtq1a2fHjBo1Klx00UX2+v4yyywTllhiCRMYCA+EyTbbbBO22GILO3b//fcPAwYMsN/OkCFDQufOncOvv/4aTj755PDtt9+aOPnpp5/CxRdfHOaaa670SCGKRWJDCCGmIt26dQt33nlnaN++fXjsscdC06ZNLfztt98OPXr0CCNHjgw33HCDCQv4559/whNPPGHC5Nxzzw3bbrutiQiuw3pCWDxYb2HRRRcNF154YZh99tnDKaecYr/ffffdMMMMM4RVV101bLTRRmHgwIF2TSGKRj4bQggxFfEFB1nl2Fc6HjduXNhrr73CO++8Ew499NAKoQFMk3Ts2DGcfvrpNiUCd999ty3mNGzYMBMsTz75ZLjyyitNaPz2229mPZlxxhnDfPPNZxYUVpjE6vHxxx/b+UIUjcSGEEJMRUoZlxEPL774ogkDLBCl6NSpU1hxxRXNF2Pw4MFhq622MuvIkUceGV599dX0qGCCBZ8PRApWD+7H7y+//NJ8RoSYEkhsCCFEHeOee+6x7yZNmoSWLVva7zwtWrQwHw7ExOjRo236hamX888/P2yyySbm6wHuWJrFnUi/++47+xaiaCQ2hBCijoHDKGCFqM6tbvnll7e3TPDtePDBB8PWW29t559wwgnhvffeq5ia0b9sEFMTiQ0hhKhjLLLIIvaNTwZvn1QFIoIPlo31118/3HzzzfZGC/8T6vnnn7dXXH36xPHfOIsKMSWQ2BBCiDpG165d7Ztpjqz/RZas1YM3VBwExEEHHWTra+B8uvTSS4eZZ57Z9mWP5xVY1tsQYkogsSGEEFMR959wCwXg/MmrrcBrr6yLkQWxcNNNN4WPPvoofPjhhyYo+vTpU+GbgbjAeXS11Vazhb1wMv3jjz/sg+BgjY6lllrKjhFiSjDdKbyALYQQYorCFMn7778f+vXrZ+tkwOqrrx5atWplr6mut956Yfz48eH+++83n4x55pnHLBX85lVWfDFwBP30009tga6HH37Y3jph8a+hQ4eGVVZZxa4Biy++uIXxKuyPP/5oDqjnnHOOhQsxJdCiXkIIMRUYO3asLbbFAlzuUzHTTDOFvffe2wQDEMYbJvfdd5+9VYLYQCBssMEGE02BvPTSS+HWW281IbHsssuGDTfc0KwdWXAWvf76680qwrodvCYrxJRCYkMIIYQQhSKfDSGEEEIUisSGEEIIIQpFYkMIIYQQhSKxIYQQQohCkdgQQgghRKFIbAghhBCiUPTqqxBCTGVohllHg4W7qvt/JT/88EN466237N/Pgy877v9wzfnmm2/CqFGjbA0P//8qCyywQGjWrJmdI8SURJYNIYSYivz222+hV69e4cYbb6xYrrwqfv755zBkyBBb2MtXCUV85Ondu7ftZ/Gu/fbbL7zxxhvhlVdesf+b8sknn6RHCTFlkNgQQoipBKuH0vmPGTMmHHLIIbZCaHVgnTj77LNtJVGWNceCwZLmWbBoIChYkRQOO+wwExxbbbVVWHvttUP37t3DZ599ZvuEmBJIbAghxFRiwIAB4aGHHjIx4P+ZFcsFS5n/+uuvts2/imeb8Cxt27a1/40CgwYNsuOcN99805Yr53+hQHbapEePHmGOOeYIRx11lP1/FiGmBBIbQggxFfjyyy/NQtGmTRv7fybA/y3B0tGiRYtw5plnWhj/P4XtI4880radOeecM3Tp0sV+88/Z3LrBNfi39Ouuu679l1fIuubh67HxxhuH22+/3f55mxBTAokNIYSYCgwfPjx8/vnnFf90zXFnTv938f7t4Q5Colu3bhX/kO2WW26xb6670EILmRipzP/f7/nggw/atxBFI7EhhBBTgRdffNG+F110UfuuKVgwmA7ZYYcdbHvYsGH2H2Jfe+21sNJKK03ydkoW3khh2oZjf//99zRUiOKQ2BBCiKnAt99+a9//dfUBnD4RD/x7+X79+oXx48eHpZZaypxPK4N78ubLTz/9ZKJFiKKR2BBCiKmAO4ROO215zXBlxy2//PKhY8eO9pvXZ1mrA6tGVkTkX6n17XJetRWiNpDYEEKIqQDWBxg3bpx9Oy4AXCzgSApMd+D4+eGHH9q2w/G77767/W7atGno3Lmz/c5Oo+StJ/iBYPngjRZ/PVaIIpHYEEKIqUCHDh1MKOBnUYprrrkmbL311qFVq1ahSZMm4dZbbw3nnXeenfP444+Hd999NzzxxBPmOIplY4kllrAplebNm9sUzbPPPlvxNsq9994bPvroI/sNrLGB4FhjjTWq9O0QorbQcuVCCDEVwHKx/fbb29sjrO7JmhisrbHrrruacMDywCqhG264YbjhhhvsmJ133tmEx6WXXlrhd7H//vuHdu3ahTvvvNNeo11hhRXszZTnn3++YpEwroUIOfHEE+0cFvi6++677ZhFFlnEjhGiSCQ2hBBiKvHee++FLbbYIhx33HFhr732stVAEQy8kspKn0WAhQMRwxLpPv0iRNFIbDQQMLE++eST9k+cWMKYRmTJJZe0fZhpWamQEQ2jp3333TcsvPDCtq+u89VXX9kojXlq4kyDzOt+pWCk9thjj9lvnOY4Voi6DmX29NNPD6eddpqtEsqqoPw+4YQT0iNqD5YxZ+XQ5ZZbLhx//PGaQhFTjOlOYXk6Ue/BRMo/YzrnnHPCc889ZyMmVhfE4x2nMZYlxhS75557WkdcHxoZ/gsmoz7EEsLp5JNPDgsuuKD9c6lStGzZ0oQJc93MY++0007pHiHqLlgyKNP4YPD/Tr7//ntbrIuFuWobfDyYZtl7773LfgtGiNpApa2BQEeLcxgLBOFdzgI/Rx99tM0LIzgwybJ//fXXr/ZfWNcVHnnkEfsvlQipAw880CwXXbt2TfdOCism+sqI9SWNQgBvpiCO+R48eHBYZ5110j21C20AAl6IKY0sGw0I/svj/PPPbx9ekWN1QP4B01prrWWOZziD4QHvHfHXX39tc8OPPvqovV6H1YApGF7Fu/nmm22Fw/fff99GXvwPBbZbt25t/1vhhRdeCA888IBdh/tlefrpp00YsLgQUx9VjaCII45t/G8HBJP/46iXXnopXHTRReHjjz82wYQT2zbbbFOxP8s777wT7rjjDpsm4niEFks4s7LiyJEjzdpBfpA2hBf341j+0+Zdd91VsY/40tC//vrrttiRO87xm3ziH2YRXxz1yEtEHXEmL7gm2z/88EOYe+657fpCCCEmIMtGA4JOeZZZZrF/7rTaaquZtzpakk4yP9LnVTisHfynSDpIzLaYVpl+oEP/9NNPzWP9gAMOsOvxz6HwkscPYuDAgWZpwPeDURKdMzBV06dPH9uHIMEBjVf1KuPyyy+3qR7Ow4LRqVMnExmAgMEjH4gPr/+Vgn8+tdFGG5lgQFgwhZQFcXHttddaWm677bZw/vnnhx133NHmqxEE1113ne1DdGBBIU6ki5UYgXzYfPPNw7HHHmuLJSFqyCdeHSROTz31lOUNAmPo0KE2TdWYhAZvOfAKpT766FM3PnXVDVMOog0IOj78NujsGYHTSeL3wMI9dKqM+HEOQ5DQ2fJfJemAcUijo0Ug8P7+iiuuaE5rLA5E4cVKwfwxvh6ce9ZZZ9kreAiNZ555JlxxxRUmQnBQXW+99Wzf9ddfbz4W+JAQFxcODj4l/FdK4uaWlfbt24eVV17ZrAhYGuj4EQKICcRA9t9kA8szcw3SiDWFe2y66aYmrkj7fffdZxWP1wuxbrAWASKCf+fNlAs+LDjS8k1ce/fuXXE+59x0002WR+QVVhWccDkfcYEI4zfxI2+xiJBf5C+CpjE43iEycWz87bffNP8vRB2AgRvt/2abbZaG1B3UQjRQEAwXX3yxWQV41Y138XE8806B9+3xTGdumMV/sIowSsUbHtimo+Z8xAidM9Dhcw5vhPjKg0wzgP+7aqwlCA+mdVj1kOvnwQLBwkNYCxA5XBchgwNbdvEhoAKV0sQc9+abb9r/hcCyAHnnUc7jfGB9AhzwEBG8ncMaBKS5Kjw/8rj1AsdV0oi15KSTTgqHHnpoo/HwJ+94bpQFnJD10Uefqf+prk2basTGWDQQYgefXHrppenWBPr27UsvbZ842k/++uuvdE+SxI492W233ZK77rormXnmmZMoLJKnn37a9j3yyCPJjDPOmMTCm/zxxx/JZ599Ztdo0aJF8sYbbySxA086duxoYYcddpids8MOO9j2uuuum1x++eVJ//79k6uvvjoZMWKE7c8SR8R2bJcuXWx7zJgxScuWLS0sChELi4LAtrnP+PHjLSzL/fffb/sXW2yx5LvvvrOw3r17WxhpBeLZvXt3CzvmmGMszGFfjx49bN/JJ59sYZ06dbJtwuH5559PoiBK2rdvn3z66afJHnvskTRv3jx54YUXbP+ff/6Z7LLLLnYOnyjMkh9//NH2CSGEmIAsGw2IUr4C+BrEzjDd+pdBgwbZwj44QWK5wJIBk+Nv4NYSnCUd/BqYYmDEX2pdDPfB8PvFsmgfrA3zzjuvhTkcUypeTP0Algt8TbKUOr5UPLgnVGaNWH311W21RvKJKQPSiKUGnxhgCofpFD6MKph+YipKCCHEv0hsNCDocPGxyEKne+6559r0QnYffgV00ryBwSummN7Y9n97XaqzBhcFCAzvoF1s4MOBWMCPAx8IpjlwJsUBNQ9+GLymi6MlUxWjR4+2t2Bqsr4AUxh0+p9//rmtUQCswAgsBkZaSIcv2ez7HOLt00NM+TANhM9LFt40Ofzww8PBBx9s+XjEEUeEFi1aVIgbfFJ4KwcfDvw1fGpBCCHEv+jV1wbCkCFDzA+Djh7nSpwtHTo/FvKhQ+f/LND50jnz5gf+FquuuqqN0OlwGcFz7gUXXBBefvllEyi8+srbIogS/BMYweOvgZBwfw2sI6xKyL3osHlLhE4Yx0pet82D/wRrCuC0iqMh8Uc8IIIQAKQDnxPECD4gXHellVaayAJBHDgH/xDSggj44IMPwnfffWev6OIDwvG8QksYr/qSvqyzKvfCT4QPr80ifBA9pHvLLbc0nxDeZmGhMJxFeTMHfw/SiDMpjqnEn7dW8EPBnwUHrcbityGEEOWgt1EaCLz2SYdKJ8eInbc6sjBNgjBAKLCfNwjomOebbz5z0qQj5oO1gf2sXYFVgPPo6Bmxu2UEsUInzfH8xiLCWyVuJcDxFKsC186vwZFn7NixFneEAyIAZ1FAZGCd4NVV7kucEDNupchCPDie+3E+ggiHUa7J9RER/OY/YBJH1uDIQnw5DiHCGy5YYqgWpAnh0aNHD3OudYsF+xEXCBAsMUBekvdLL720FhQTQogcEhtCVAH/owILDZYPLEaABQh/F/axcJgQQoiqkdgQogpYP4N1RRAVWD6wsDCFwnQLC5gxVSOEEKJqJDaEqAKqB9MwLFHO9AnigmkYFjjTdIkQQpSHxIYQQgghCkWvvgohhBCiUCQ2hBBCCFEoEhtCCCGEKBSJDSGEEEIUisSGEEIIIQpFYkMIIYQQhaJXX4UQdQKW0+d/6rBM/RprrGH/ZK+yfwjI2if8LxqWkmcp/csuu8zCWY5+2223DbPPPrtt13f4v0B33nlnWHfddcOCCy5oYSzfz/8Deu2112ytl7XWWsv+0SLceuuttpQ+y/zzv3v49wMNCf59wi233BK+/PJLW2DP4R8jduzY0f6vE8eQP/xfI/5dwuabb16x+N7dd98dRowYYf++YOONN7b/zySmEIgNIYSYmowdOzbp1KkTAx/7zDbbbMmFF16Y7p2YH3/8MYkdRdK2bdskChQL69Wrl50333zzJZ9//rmFNQQuuOACS9djjz1m23/88Uey3377WdoHDRqU9OvXL2nZsmVy8cUX2/5HH300mWOOOeycwYMHW1hDgufdrFmzinLinygekiFDhtgx5557bjLddNNZeBSrSdeuXZPx48fbvmeffTZp0aKF7Rs4cKCFiSmDplGEEFOdq6++Osw111y2BDwrtDKijx2pjWCzYMU46aST7D8Q80/33PLB6J5RPv+oLzvirc/ce++94YQTTrDf/l+ESTf/dZj/1LzTTjuFAw44ICy00EKWb/wDQiwc/ENCKPVPC+s7/HfnZZZZJvTu3dv+QzT/GXqzzTYL7dq1s/88/cILL4QozOz/Fm2xxRa2AvBdd91lFjNYccUVwwILLGC/tQLwlEViQwgxVcEETieJ4DjxxBNDHJlaOP9/hv/Gm4WOg//Am59eoVMBwhEkTDE89dRT9t+N6yNMAcRReMV/UnZYMh/YN3z4cPuNEGGagLSTD54XiK4vvvgiPPzww/afkRsCpPW2224LJ598cjj88MPDwQcfbOKKf4yIuCIf+vfvH4477rhw+eWX23+Shmw5yuYP+UL+jBkzxsJEcUhsCCGmKvw33fXWW8/m0YFRKtDR+igdGNUjTDp37lzRYZRi4MCBoVOnTnbNs88+Ow2tPyAQGJ3vuuuuFXnirLDCCuaHMGrUqLDbbrtZ+uhIL7zwQjvW84WOF0GC/wp5wbHffvut7avP8EznnXfedCuY5Wvo0KGha9euts3/LMLSA5Qf93Np3bq1fWdBrPEPFskfrETkuygOiQ0hRJ3Cp0522WWXMPfcc9vvkSNH2ofOwa0aeesGI1VGqDhFYmKn42Uqpj5ZNxBTOEDi1NiyZUtzdgSfGlp22WXDddddF9q2bRveeeedcOyxx4aVV145bLjhhrafNLt1Z9y4cTbN0KZNm/DQQw+ZtaehwbRK8+bNTYTl+fXXX81Zds011zRnUMfLDeKLcoKT6BNPPGEOx6I4JDaEEHWKQYMG2QgWfwT48ccfzVrBqBWzt49AERHZ0SgdMx00c/itWrWyOfk//vjDOt76AqZ/3qRhOuCDDz6wN08AEfX333/bb6ZSFllkkQqBMXjw4HDKKafYb59KASxAWImaNm1q2+RFQ4O0r7POOpNMN8HTTz9t5eWss86qeBsFPH94W2fxxRcPc8wxh203xPypS0hsCCHqDMzHY95m3t07SUbwvNq69tpr27TKkUceaeFYOphqoBN2B0osAHS4bhGgsx49erT9rusgnkj/PvvsY06MvP7rYmqPPfawUTxTSUyNMJJn+mD33Xe3/YgUXgf2fADygo7VO1emXhoSCImPPvrIrEB5EGennnqq+f906NAhDZ2YfP58/vnn9i2KQWJDCFEnwOTNSPWaa64xxz4cQTFvzzbbbPa2yeqrr24dh68dgV/C0ksvbR2sdxh5CK8vlg06P6ZJWCuC9PLtPhu8gdGsWTMTHMCIHA477DATZYzKeYMnO7WUz5P6ZOEpB/xaEJN5MYH4ZHpkv/32C3vttZeF8TYK67g0pvypa0hsCCGmOrx5wtsFjM6vv/56m0Lp3r17eOutt2wUz4j++eefN9O4Txkw145jJHjH4Z0JAoTffOrLK44IC3xMXn755fDMM8+EBx98sMJB9oILLjAB5tYet1KweBcsvPDC5hiJRcfzgG8EDB9oaK96IjZ43dXzwLn00ktNtL7yyitWjnD+ZLEz0u9lAhp6/tQ1JDaEEFMVOkjWk2B1xzfeeCNccsklNo3CmglLLrlketS/uB8DzpQOr8QSjmn99ttvN2HCftae4BXY+gjp8ekgTysOskyj4ETK6J2pAhxAr7jiChMiDzzwQMV0AOt0YBnyqRgcIHGabAgwTTJkyJBJplBIP6/F4mSMnw/l6MYbb7Q3ePDbQLQiaAFrx7BhwyqEG+UN64coBi1XLoSYqtCRYrGgI/RRJs0SnQOLVzVp0sTCHDoa3qygc2W6AbM5nQzTKnTOjFCZeuFtDK7DYmG8keDXri8wNcLrq0yPEH/eugDyC+sH+UDerLTSShWvgyKsmH4iDzgOh1neunBfDpwp8/lZH8FpmDLA1FrW+fPtt98OH3744URWCsoH01M41SJCWQ6f163JXyxHnj+UHfyCSjmbiv+OxIYQQgghCkXTKEIIIYQoFIkNIYQQQhSKxIYQQgghCkViQwghhBCFIrEhhBBCiEKR2BBCCCFEoUhsCCGEEKJQJDaEEEIIUSgSG0IIIYQoFIkNIYQQQhSKxIYQQgghCkViQwghhBCFIrEhhBBCiEJp8P/19Z577gmffPJJvfv30kIIIURN8H+Tv/LKK6chdYcGLzZWW2218NJLL6VbQgghRMPluOOOC3379k236g4NXmzcfPPN4YUXXgjTTz99GiKEEEI0PLBs7LDDDqF9+/ZpSN2hwYsNIYQQQkxd5MgghBBCiEKR2BBCCCFEoUhsCCGEEKJQJDaEEEIIUSgSG0IIIYQoFIkNIYQQQhSKxIYQQgghCkViQwghhBCFIrEhhBBCiEKR2BBCCCFEoUhsCCGEEKJQJDZEWfzxxx/27/pvv/328Ntvv6WhAj7++ONw9dVXhzfeeCMNEUIIkUViQ5TFiy++GLp06RIuv/zyMPPMM6ehAsaOHRsOPvjgcPjhh4dff/01DRVCCOFIbFQC/6q3sn+IWx/+US7xr02uueYa+z7ooIPCNNNMY78nF/KP+FUWx3w4x0+JPPd41ZRVV101dO7cOTz++OMmyoQQQkyM/sV8hm+//dbM4c8++2xo1qyZTR3MMMMMoVu3bqFTp04VI/oHH3wwnHTSSeHHH38MhxxyiH2mNq+//no44IADQteuXcNRRx0VrrrqqnDRRReFv//+O5x99tlh8803T4+sOeRLhw4dbNT+wgsvhFatWqV7Jubdd98NvXv3DgMHDgxzzDFHGjoBitkxxxwT7r333rDiiiuG2WabzcJHjBgRfvjhh4p4brnllmYlePXVVy18pplmCksvvbTth/XXX9/S2KJFC9uGP//80ywuPDue0T777BN23HHHMOOMM9r+//3vf+HSSy81wcR9yacddtghTDvtBK3Nc+zXr1947bXX7By/xuqrr277gfiPHDnS8nTTTTedJD9vueWW0KNHj3DEEUeE8847Lw0VtQnPcdSoUVb+KBfAs3///fdtai8rgnleiy22mNXjUnz44Yfh888/D/PPP39o165dmG666dI9wcoaU2PsX3DBBW1/feGLL76wMp6vf7/88ou1EbRnK6ywQkXdqIzRo0eHjz76KDRt2jQstdRSFfkNb775Zsn8Jq/mm2++NKR+8c0331j95tkvssgiYYEFFkj3lObLL7+0/CHdbdu2tXKU5aeffrIyRh6xn3xs9MTMEpF33nkniZ1aEgtFEjsOC/v555+Tww47DDGWxA4q+f333y0c4kjWwo8//vg0ZOpy/vnnW3zat2+fjB071sLatGljYddff71tTy5RfCWxcUq6d++exAY/DZ2UU045xe43dOjQNORfYmOXbLjhhslpp52WxIpoYVFoJLGS2jlrrrlmRbxh//33t3D2jxkzJhk3blxy6qmnWthqq62WxIqcHpkkUeAksVLbPv9k0+zn+Sd2LMmNN96Y7k2SXXbZxcJffvlle8Y829jYJLHDsf3E86CDDkrmnnvuSa7tfPbZZ7Zv+eWXT0NEbfLWW28le+21l9VR8tqJArHiuWQ/UUjaOXmiOLHyQHk78cQT7Xq9evWqKNd8x4FEsu6661rdXnbZZZM+ffrYeXWZKIySk08+OYnCIHnooYfS0Al89dVXSRTCyaGHHmp5uO+++yZRYKd7J4W6EYV2csIJJ1g+ReGefPfdd7bv+++/TxZeeOFJ8juKkeTuu++2Y+ob99xzT7LSSisle+65Z3L00UfbM48Dl3TvpNx2221JFGzWRpGnyy233ERtwhNPPJHEQZG1M5TDODhJPv3003Rv40ViI0IFonJRaY499tg0dAJUSgoT+6h8TpcuXSYJm5rEkUhy4IEHJnfccUcakiRxRGZxvOGGG9KQyYPGh+v07NkzDZmUODKoEDfbb799GvovcSSQDBw4cCLBRqPH8bPOOmvyyCOPpKETIF/Zh9ggbRBHHckmm2xi4Xz/+uuvtu+4444z8fHMM88kSyyxhO3fcsst7RwEA/v5fvrppyvyxOMYR8pJ8+bNTUzFUaGFuXC79dZbbZtGlEZ6lllmsfBBgwZZeBYEFPeOIzvLC1E7/PPPP1YWWrZsaXnfqlWrivIAN910k+U5ApFnTr1EKCJss2XN4ZlynYMPPti2ERxs9+/f37avvfZa2/ZBhA82CK+rELfFF1/c4kkZffTRR9M9E/KPsr7kkktaOxdH48m8885rdaIUb775ptUFBD3cfPPNdl06VnjsscfsWhtvvHGy1VZbJV27drVyjwBhcFbfQARQtmjjXYBRjpo0aVIx2MhC/jEgRaQC+UtbRLlE8CGEzzzzTGuP7rvvPiuL5N+FF15oxzdm5LMRGTx4sE0PAHPvWTBHrrzyyvY7VjwzrZYCE2+siOH00083c3rs+MN7771n+9yBcIsttjDz/p133hn22GOPEJW0TRc4mDqjag5RXYett97arhVHFLYPczGmfUz4sSE0Mx5TOUxNfPDBByGO1sLXX39t1xs/fryd4zANwrQK8bruuuvM9FcT3OmxKtMi/gpuQn3yySfD22+/bb+dOeec06aj3BzLVFQcDdjvXXbZJWy00Ub22ykVR6Y9mEKBhx9+2O4ThUKIHYaZKtdaay1zYgU/PzasNq2EaZSpoG222cbCfX9snMP0009v+Rs7ljBu3LjwySef2DTN8ssvb8fERtXu0bp1a9suBVMvscENf/31l02/idoBHxqeeRR7tp0tF+yjTFDvhgwZYm9L3XXXXVYWYmc4kenfuf/+++3b962yyir2TRtAvaEcg+/v2LGjfV922WWT5c8zJcBnKIoHmwrK1xvahoceeijMPffcYfbZZw9zzTWXfdOW0S7keeqpp6wu+LQSUy7U6yjSQuxIbd8VV1xh9S+KcGvLyKPY4VZMjdYnyIOvvvrK2mjqPTAdRFmgPc4TxYlNkTD1ylQb0yTkLVO+hFNu4iDKyiB5sswyy9h59XV6qTZp9GKDBiTr1FfKH8ELDIVr+PDh9jvPBRdcEOJoKkSFHHbbbTdrnOKIIsTRhBXGOFKwDpZKTkFmbn/YsGGhe/fuVomZK9x5553tjQaECH4KdHA0IsSROdaVVlrJGg46WRpVXkO95JJLTJBwfbbZn+/sxowZY0IjKniL24ABA9I95eECq1TjDaSRueI+ffpYg8NvGv4sdMY0dEAlPuOMM2zel7nOQw891MLLYaGFFkp/BXsWXDfbyPlctIuXWWed1QSJ4/vXXntt++bZ4MNB40qDuu6664ann37afDCY83d4BlV1NnR63IvGPjuXLf4bPBfEfhw9pyH/Qp5Tf+JIOw2Z4DdEeXSRkMc7EEQhMJigXDNfT8fj4d5pu+8DQr5U51wXIP3t27efyO/EIV10hOQVaeLDcXSapQZO/lq7p58yjb8BbQx+HPiuIeodOl2Enov8+gb1n0EUbRbt7qOPPhqeeeYZGwhm67+DEKEdo01l4Eh7yyvvnLv44ovb4IaBFeXovvvus76FQaIPchozjV5sYJGgcSoHKqBbGvLgiEZhY6RMx48jFuIEB0igo6eS0zEywkfAYKVATd900022j46Xgk9hdWsKBZ8KDe7shiMjTlsIGhwVESbekdOo5Ds7RAr3xWrDKJ7zKktHKVyAVdbZvvXWW1Zpcd70Tv6GG24wK0EpsLKQLsAyU6ojqQzygJEZ5C048NJLL1nebrvttmnIvyDonn/+ectbHESdY4891t6yQYjQcOD45aOccqFsUJb8t6hdeHalyHewQ4cOtTLi9SePWzIos44/L+oIdQlwgoTK7lvX8LKXBwtoZTCiz7PsssvaNw6TfGhPvN6XKtfPPfecdbC0MfURrJEXXnihtbu0q5tttpm1ZViVfWCSBWdhjqetxhqO1Q2LJwOtbFnE4RyRQd/yxBNPmCBr7DR6sYEooHBVhXfeHItoKAVmXrziMZfdcccdVjFLdUBciwoMPi3BlAONGm9M0BnS0VFAOY7z85WczhYBQOfOtAsjj8oaG/AGk0oxzzzz2P3KFVjQpEkT+66s4aKBxyIAjHBII3nxwAMPWFgWwnnrBJjW2Hvvve13uSAwfv75Z/udtWgApl2mrqjoNIB5MKEz8u3fv78JOuf888+3EcqgQYNMKHIPprJefvnl9IjqwbzMaIcyUpkFSBQLo0mmVPJToVkYgVJX33nnnbDffvvZ21E8O+otnQvTn+xnOoWpT0anQFlzQV+fqMoaV8oCR5ty5pln2tTprrvuatOuWHSwcOTfcAHaH6y5tCv1Fd46w1rDVCtlCOsGgqKyvMOCjWhlquT333+3toPjs9CuXXnllTYoZCqL9qTU4Kgx0ejFBhXOO0oopfZ9FIQKrmzEhHn2lFNOCSeccIKZNLEglBoJgIe76GDag3hgpmTqBRXMPGypxgCIx+Q0fAgSPjScdIrlgkhBtbuVJguv0zEiIO000jT23tHTebtZ2uHVUTplpj969uxpjZiDybe6dSqygodXaB0sNX379rWpp3XWWcc6kKz1hvOY6jrrrLOsoaAxJd8ZwZ522mmWp1hDrr32WhOUiDHSUi4IIIQOwjX7Wq6YcvBa7CuvvGK+UZXB88GyxxQcHYBb32gDsIjQoSJGKRdHHnmkXRPw26lJnakrZOtXnlL7EFxYG6nT5APbWFYZGCy66KLpURNgaokyz1RWfYXni38PbS+DC6aygdfXS7VFTD8hTBAZlCF86wCxkfW/o41l4MXUNu0BbR5TNY2ZRi82gLUd3AyISs2CiHA/DebxKnP0wezGGhOoXhQv1gTEAqIjj4sIzJTA9AsiADXs6zVgreAapaZFuKYLlTwcmz/ej6VxoANGyddkJMI0R5s2bUx05S0imFFJN1MjKHmcPt0HA+sMlhrnkUcesZES4DvCNFIW/FBcnGTTkP2NvwogKFwkknfEgU6GxgIRgfMaQggQd5g56TAQFDQUxBdxQ2fD8e7XgZnU19fINsbZfC2V91hMEDCV+QqI4sGSxjQAwtHh2VOHs9MhiAYENPUIPyrqNFYOh3CsjpQrOhysiDW1wNUVaFscL7+M2CnbtFNAuSWtDscxYODj7SEWHwYIWZiypD1AiNRXmArBykX7TxvQq1cvKz+0QwgQwI/F2yX87LA8r7HGGmYdxfeM8kM74u057YuDPw1Ty1g767P1pzaQ2IhgJWAhKjoaFodyj3QKDSN2CiONDWZVxxsv/3aHSBo8OjIaOc73ztY7Kyo2BZwCzCgMBYw6pgAjajiPxae8kuMl7WLHzXo4duH0lcUrA/HJm/8YpQDCgP28dVHViCcPyhxrAP4M/oYN0FDz8UbLwYxNnpJGRgjEjfjyxgxhNPS8IeJQSZlaQYgwFwpucqQRdKc+fFs4Bg95Rl0IMsBagqMsz4oGg3BGpf42ybnnnmtOsVg9aCAYvV588cUWR/w7GLFhxSBfaTwxe9JIZBfuIt7ueIvXeR7EDeTfqhG1g8+HU4fyfhoOdXCDDTaYqFNkoTfEAg7ZWZjOY76dToKyk3cGpJzj1IfFiulNTOx1mWyeZH/jg4J4psxi7aNtoF2ibFMPaZ8QJAy4vJ4B7Q7TKIh7OlT256Gt4zruQ1UfcWsV7TIgCHyKlWdO24+ApV5zjLeb3gZwLG0Ovnq0kzj006b49BttG2WNgar73DVaYucmUsaMGWPvSPNeeqxoyXbbbZfEEXhy++23T/TOfuxg7T1r1oCII+wkigZbeyEq4mSnnXZKXn31VVvzgu0rrrjCzmGhqxlnnNHWlOC9eK676qqrViwgxvVjB2lrVcRO0a7Bu+y8/x0rvB3DO+3ck+vus88+FYtjsT5EHNXbvthwJLHztXDiHzvxJAqlJI74bVEuFqSZHKL4Ye7H0gdvv/22LX4TO2pbFCtWKAtnAaw99tjD4hFHh/b+OYt9RQGVxMbNwqJwsXUrWERn9913TzbbbDNbK4G1EWJnb2sbrLzyynYs+UG6eR7EP4qEidaxYOGmOCqx+3A8H96b5115IB+JJ/decMEFbX9sUCz/o3iwY6IYs3SxVkO3bt1sH+mDKHbsHXn2RVFl12GhowsuuKDifN6p57orrrhixeJHonagXviiapS/KCStzviaCA4LslEnKQ9ZKGOc54utRaFv5ZEyEYWJLVjnxI7YrsO6Huyn/r344ovp3roL60H069fPFpDiw6JlX375Zbp3wiJTrIXBejlRdFu7wyKGwNo35E/sCG39EtaRoP3o0KGDlWfymjqQ59tvv7W6EAVHGlI/IR0swEXbef/991t+0D74OiSDBw+2BQOjgE1Gjhxp9XuttdaytiQOSi3fybu+ffvaOkC+dhDrndCGsV4J7aG31Y0ZLVdeAkb/jAIwl5dy9mO07SMsNz+Wmi7JgtMRI35GXczfcV3O4TrlwGPiXihxfmMt4DfnM1ohzmxnfzuEkR7CKhsVVgeqnhEg12aaBIXPb/LI48Jv7sU2c73EzePKff3enr8O5/nxwMjL48r5HMs3x3BsFq7Feflwh/hAZfuz+H3yz9wtGh5H0sN1/TgsPlijmBbi3XpRezD1h8MvliUvP9QD5sOz5nvmy5m2y1swmCrDd4iRKa87uyMf8+74/GTrLeUOqyTgKIx/lpfJugwOnUyPev5QPrFEMh3rMAWKMydlljex3FKDxRErIVZdphhxbmckzjQBH0bspcASy+gd60cpx9H6BFZU0o0Vg/xZb731bDqUNgMLBtZu/LjigMTKA9YwjsfRnnaQqXPOASyj5DP+bVh8mOrF6bac9qehI7ExheBNCXwKqLw4CuXfpBBCCCEaKpJbUwC8ufE18FE6b2EwYhNCCCEaA7JsTAEwa+IolJ0W8Pf6hRBCiIaOxIYQQgghCkXTKEIIIYQoFIkNIYQQQhSKxIYQQgghCkViQwghhBCFIrEhhBBCiEKR2BBCCCFEoUhsCCGEEKJQJDaEEEIIUSgSG0IIIYQoFIkNIYQQQhSKxIYQQgghCkViQwghhBCFIrEhhBBCiEKR2BBCCCFEoUhsCCGEEKJQJDaEEEIIUSgSG0IIIYQoFIkNIYQQQhSKxIYQQgghCkViQwghhBCFIrEhhBBCiEKR2BBCCCFEoUhsCCGEEKJQJDaEEEIIUSgSG0IIIYQoFIkNMdX4888/wyGHHBI23HDD8NFHH6WhokjGjBkT1l9//XDEEUekIUIIUTwSG2Kq8frrr4dLLrkk/O9//wtt2rRJQ0WRzDfffGGWWWaxfH/uuefSUCGEKJbpTomkv0Xk77//Dp988kl45ZVXwgcffBB++umn0KRJk5AkSfj999/DjDPOmB5Zcz799FO77ujRo8M///wTmjVrlu6pXV588cUwcuTI8MUXX1jc6VwmBywPCALS3rRp0zS09ujbt294+eWXw6mnnhpWWGGFNPRf/vrrr/D8889bWsizsWPHhnnnnTdMO+0EjfzOO++EN954I3z22Wc2Yp9//vlt3y+//GL5/Oabb4Zff/01zDnnnGH8+PFh+umnD9NNN52d63z33XfhtddeC2+//bbl1wwzzGBp/fbbb8Nss82WHjUx5Av3xRoz00wzhdlnnz3dMynEmfgT72mmmSYNncC4cePCV199FeaYY46J9v38888Wd8ohz66yeAD5wmfuueeuuAbPi/jx7IhbNn4cQxpvvfXWMOuss4bNNtss3SOEEMUxTWyYkvR3o+f9998PZ5xxhjXUa621Vlh66aXDH3/8YY32559/Hnr27Bk22mij9OiaQ+e47bbbhnfffdfM2Oedd166p3a5+OKL7fp0vHfffXfZHQqdHGldffXVrWPm3K5du1qab7/99loVHL/99ltYb731LE8QBosvvni651/o1K+99tpw4YUXhvfee8+E3o033hi6d+9u+59++mkTLOw76aSTwm677WaCgefUunVrG8Xz3BASCLt+/fqZ8ADSOnDgwHDdddeZVWXttde2jp0yQCdPGPfNg3g56KCD7DwE43LLLRcuuugiS0seys72228fRo0aZQLQhQ5iaMiQIeGCCy4Iyy67bLjiiissHBAge++9d7jttttse+WVVw6XX365fedBCG+11VYmKO+66y67PnE688wzw6uvvmoC7sknnwwnn3yypc/5+OOPwzrrrBPmmWeeMHTo0NC8efN0TzGQZ4MGDQrPPvtsWH755e05Ze+JEOS5vvXWWyaa9txzz7Dgggume4NZvqgrlBWEEgMCnucxxxxjz/iHH34IN9xwgz27li1bhr322svCK4N8ozw/9dRToVWrVmHnnXcOSyyxRLp3wjNg/+OPPx4WWWSRsMcee9j31IZ8uv76620abKmllkpDJ9STe+65J9x///2Wb/vss4+VfwfRTnlCuM8888xh1113Dcsss0y6d1I4nudB/lAPeB7Z/KRcs5+yRb6RP4jp+gBl6ZxzzrE2jnzMQv5Srzt27DhR/uYhf+644w6zDDLY2GWXXaweO+y/5ZZbwmOPPVZRfrLPo9GC2BBJEgtOEhueJDZiyUMPPZSGTuCll15KFlpooSRW6DRk8tluu+0Qd8nRRx+dhtQ+scNNYqOTRMEwSVqqInZ+yQ477JDEDsu2Ywdmcd1ggw2SH3/80cJqi9gxJLGzS2InmMRKnoaWpnfv3kkUThYXnlEUJ+meJIkNXjJgwIB0K7H4R5GYfP3112lIksTGJVl11VWT2InYduxsKp7D4YcfnsTG08KBfVEgJN26dUtDJiZ25ElsqJIo4JI55pjDrkEaooBIj5gAeXjCCSdU7I+NnIVHoZlsvfXWFemJDbmFO5QLjt9kk02SKCLsmC222CLdOzFRlNh+4urXj52KXTuKGdvu0aNHsthiiyWxQ7Zt4NgoNpIo3pIoxNLQYogdYRI7N4unf6IwS77//nvbP3bsWItLFFNJFI3J5ptvniywwAJJFPy2H6JQq8hr/3AOfPnll0n79u1tm7zl2osuumgyYsQI25/n22+/TaIQtzzjPlyrXbt2SRSltp/4HHnkkRPtjx1PMnr0aNs/NSCNl156aRI7NIvPo48+mu6ZwHHHHVeRL3yiAEiioLR9v//+exLFXbLwwgtbPh544IGWl4888ojtz8Pz4pjs9aJAtHyBKPSSAw44YKL9q622WvLVV1/Z/rpO//79Lc5RwKchk+ZvFAnpnkkhf6K4SOaff34ro4ceemgy66yzJlHopUdMqMPZ/CH/4oAj3dt4kc9G5JtvvrHRKqb4WJFCbOjTPRNYZZVVQuzwbFTlMDrCPI4Jn1G6w1QLI1lM25joAVM6x2dhFEoYo2gUdR5GK4xAuT6/Hcz7hHF9RufEmWswenTic7VPHo7B9J+/J9c566yzwlFHHWWjOtLFN5adwYMHh9NPP91M/eQTaeOepJN08zt773JhNBo7HBtZVjUNAZj+sVy0aNHC7rfvvvvaN2C98ecSGw2bfmAEzOgOiwn5cPDBB4d11123Ih+xhjDyYFTGFE52agwrAXmRHek65FMUnSE29jaKxIICWFOiuLHfzgMPPBBeeOGFdOtfGLkff/zxoUOHDmnIv1AeVlxxxfDwww+HKBJthA4+lZWFEe6HH35ov8kfn0K5+eabzbrhI1GmlpgO5HoOZY98Jz/y5bK2wYKDRezBBx8Mu+++u8XziSeeCM8884ztxyLjI2jyHKsH1qg+ffrYKBSwTGFlwpJF3pD311xzje2jfA4fPjxEQRWWXHJJG7VTfnnGpepAHDiEHXfc0SwXXIeyQ1niHGC0iqWD/YzeKXNYIvPPd0qBFefee++1dGL5yUPamYq77777zNLj1jm2gbzFOhjFRoiC2/KX8kHZpY7nocxSznluOG/zvLD08pwgihTLV57nfvvtZ/UPq92wYcNsf12GskM7AsQbqsvfPFjnqHtY5rBqUt4op1gPsViyn3yl/h922GFmISb/yK/GjsRGhPlrzM6AWboUnTt3rjCVcXxUr1aJMZ9vvPHG1sHBjz/+GHbaaacQR0Vh//33N1MajSjHgBdyGgQ6LUzmdDzZjglBwHTLscceax/MmHTyQGFec8017fpxFG9mvzjytakSphPyeCdEZxNH8dZIkxY6X2/wuR9mQSoeAod0kR4qENMANDoIDRp7zK+YBIkXUyz83mabbUw41AQ6OSrpXHPNlYZUDmZJnguCj/TQwJIWwslP71QwrdOZA2Zl8gkTJ3lNx02nR/rokIFnUkrokLfkfx6O3W677Sp8KLg+cF1EisMzQoBsueWWaci/0HmttNJKNoWRh2uQ3+5j41MfWX8MoEzRiPEWj8N+BCANP9ChZPHy7dD5QKkOuTZhegyx2qlTJ/t2Eecih2eTpW3btpZeOjvEIw04HR3PGpFEuaWsu0PxiBEj7NvzB9FBPjIF6GI/C6Zz8h8oL6Sfa3oYU4Zez3ne5CNxJ15TA9KMcMah16cAs1D2zj///LD55pubwPLpNq+PlHeHPMSsz5QHdcjbrCxMQyHsaCMQ3Qw4gMEBUG6YaiZPEITua1W0aP2vUM6oG/nn6PnL1HOp/M3j5c0hLxdddFEb2OD7Rf5QzjfddNOAOyTTNVBqQNnYaPRigw6WuUynsvlrGkA6VjpdOl+EAoWJ+Tg6bSomUFl9/h6HUAoZCpjCjiXAG0Wut/XWW4ezzz7bFDUqmApLg8Bv5ou5JnPVjFIYzdPw0Qi0b9++4hqMUJijZ/6UETNkOyY6Yzoh9iEosNwglKgYnMf9mEd3vw4qB42Jd9TAHC337tatW0UnhTgiToz0UfF0DjUha62pDjoEPog3PsAcNHOvdLoO89GIIPIFuAdz+TwPLB5YMEg3lh2oal6fZ1aKbN76yLBLly4V98TqRHliBOnCMg9podzlyV4bsIiBWziAUT7lxf1WHM7F6sWzyl8H8h1vZXGrbZj7diFGx4hTKvicuG8Tb8DSwDOlzJIOyhUWCDoERAGd6dVXX11h9XBh5tZFnjEdCM/Gj8nixyNgGBRQrnl+WO6AMsTz4b5YOxk89OjRo2L/1IK8KPVcEVeIJuCZen66CCA9QH6Sx+StP3vSloeO0ztd/BEo19zXRQxi0ffzXF2suyipi1AvKENYIfz556ksf/P4+V62yE+3rNLWYzH0toBBCWUe6nL+TCkkNmKjXxPVycgUFUyHDHTW4GZY8MqMeXadddYJAwYMMJMsBdU7GR/RUzhpeFHdmMvpFHHaa9eunVVq9lNgMQGyL9tY0DAADQsFG4HC9AqNrUNjSoPDKIQREGLK00unSyME3vmTHhrb6kB4YNlAXEF2KqkciAfpKFd0eJxIh1uJGMnddNNNE02DMOKio8g68tKoYvJF8JEftQUdFtMedEqA+RmrF/lCA+TPmriXk6d5sM5QfugUgUbzzjvvNKsLafYGz69fVdryDamPRMtpYGsLzP18sMi5mON5UXaZ3kCk8ay8TpEmBCHPGYdnOlZG41j6XNwjkukUsbohJDmffKeeVJU23oJilEqeMXhAlDqUSSxB1GmuhcBFqNd1sAJhDWLAsMEGG1gY1jGcRkkP7Qr543WOfKsKyjNTcDwjhF4eyiPtDRY8pmjqIjw/BiYM0rBYOpSPyYGBCwMs2k7KBPnpbWi23QWsSww4eRal8q+x0ejFBo22j9bBC05l0IDR2DOdgOAoZar1jgWFi6igkaSwZ/FjEAI+MqEDYL6aEQjCxAsv9+R4Gj//nYWOm86NDp/GwcUI+LGYWddYYw0TSlQUjuEzuZ0Nogv8+u5DUS6MihBRnOedSzmQn5h5yVMaQ94EcQsAjS0j2tVWW806aj7uG8FzQozRefloj+MnF6almN+/8sorK4QjnSUmVBp4fCWYhgIsX1g6So20K4O3N2ioeFPFR0d0qJQ5Gnauf+6551o4oge/FEbf+QbP8RGv44uo5cOLBD8ZnjtCweOJBQ0rIeKQt4OYwuNZIeIYNSO2sFZh4SO/EZuUW6YDmariPIQ417nsssss3xDT5JFbVEpBPiJwmIqi82Uw4IKZusez8/2EX3rppbavLoOFEf8pplw87Uw3UVaZrkUIU3cQCexnQFMVPC/KB+WsVLmi3CHWGMRkBX9dgvpDe4cIYxDibQV+FFipawrCjfKG4CV/KLP4TlEfs29QAc8DMUKeV2ZRaUw0erEBjJS9A6JDKgUdhXesVC7mfvmUWh/CKWeel4aODpLKipWgqkrrnU4eBBIKHsGBCMiLEbj++utNbKDuacArGwUjPsoRIH6+H1tTJ1GmpIgLFdUbgHLBEZCOxUWaWxAQLnQabFO5GXHh0Ob+F8SZTshf+8P3oTKqEkA4C/IaLeWAuX46N5wIyXfKEaIRIZS12vCMSj2XUiAqacjpeBFVlA+mTygbdLT565MuOkQsYYy6uI8LTr8neeYw/86IlM6mlO9IETCnzbPhNV6ePXH2MsRInPKJgPB6iB+QP1+H8n/iiSdaQ08aXOgzasTCRcPv5ZGpj+qEFOXPrX1cL/u8gEEIYpaOlg66LsMgBQsQ+cM0K3nr4pZnj1jCCZI0I7LJX8pKZVAGEXhMVWJ55Vpez4DyTn7379/f2rns86xLYDFGLPE6K35f1CNAAEyutQo/JPIT8UYZIW8Qve6PBPjKUHYQfrQ3dTV/piQSGxFG/bvvvrv9RgnnrRV0PFQ6TMAo4hNOOMHC8HXIOwyBN3g+p18Kn6OmkaPDpYHAMQ3xQgEmDtyDhoFjUc7sy1Z4bxzdGkLHQcXnGBcNmErZTyPEtTBf41cAHk/wTolr0mnTOXqH5dfKkt03OdAxkn80aozgawqmSRpX8LjTUdFhMVJ2EB3kLdYHxBYd9mmnnWYdF2Z3rB95OL+yhoj8Oe644yz9PCM6ySOPPNIc7hCtjKLomBAE3AcYdTPi9rld8szzz78dRARmfeJH2SI9vXr1sukDyin3ROxgomXED/gc+MibKQrwaRKeI502DmsO1i86C4RSVX4rtQX5QePOB6FL40y68lZERp5MOSI+8NEAN1dnpzqxEtJR+hSew0gTh2nKhvsbkVc8I3eeZlSKhcjLDKNRLJCURSxDiFMcfB0cAKlX2fyra1AmaZ+wbmFBAxxkvZ47CD4EBILDyw6DBPLNncUB4cpxlF/KFnlFPUGgAqN17ueOv+xHGFN26xoIUd4uwu+JvPEyg/UR65lTWTtGu+prruShL8BChnimTXBoZ2mbCMMvDyiD7mTbaIkFRURiRUl69uyZxMKYxApka0zEwmTvXMeOOomjMjsuKuUkdpS0VLZewkEHHWS/Y0NV8f57LNwWxjoSr7/+esW6FcD6DrHTsXUfnnrqqeTkk09OWrRokQwdOjQ9Iklig5nEziaJo2db+yJ2CBYGsWFJokK366+//vrJyy+/bHGInWgShZId89BDDyWzzz67HROVdRI7l4o1A2KHaO+Bx445iUIkueGGG+yc2OAkcQRn4XvssYetgxAbFDsnNuzJ888/n8TGy9bvIIy1CqJQSBZZZBHb3nbbbe0d/JoQG0Q797DDDktDJobrscZJhw4dkuOPPz755ptv0j0TiJ1Wsu+++yaxwtt2FC1J7ECTVVZZJbnyyittPY4HHnjA8imOwuwYh/AovJI2bdoksWG1fIyCwfKcZxIb1vTIfyE+J510ksU5+yEfYweWHvUvXJf9sfOsWAeDNMSGK4nCsWIfZSo2alZOYocx0bX5xJG3PcM8rD/C/uw6G7EjSLp27ZpE8Wz3YQ2EfNo9DV6mioZ1GSjPrIHBGiKxkbc64kRRbmulREGYRGFm6204lDPiGsVHxVomPCPy1omNeBKFjNXBOIKtWGMConCz8ylDHBeFvK2LEAWcrcvBs6YcUJZZK4LyPNtssyV9+vRJ3nzzTcsr9n/00UfpFacOr776asVaI97OONRT0kQayV/KP98O+UZdox7HUflE60jETtCuGQcztt4E0A42adIkiR21XYeyus0221SsR0P7Qfviz5M1Urp372776jLEn7agVNmnrfD8zeZPFLrWLlI2vQxEMWb9A/m52GKLTVL3qZc8jyis7cM6MFEgV9TRxopWEM3BXDbrALhfA06YmOBw1HQwcTMiR/VjccAawqgIBc0oDpWPIsbCwKtmOPi5MxajN66FykUtM+rEvJefcsGBjTk/FDcjOfcGZxTDtAAjDd4ZZ9SOrwBzyzgvYfLFvMmrsvzmw/v3jFwwKXItPKPx6CeOeGgTd0abxA21TjqwBDBVwTGY/zARMtrmLRScq7AYMCrGKkERwgqDo2RNRsqMVhnFMlonbnmzLiMs8haLEnEgfYyksjDCZ+4eUyW/scow0sA6geWCuWmeH34ceRiBsD4Ax5FORrj4eJCnpUY6WKqID9/kKxAvzPCMpPNmf9LEm0Q8W0ZVXJO3hniuWGHYpoxg7mdOnXxlZE+8stfHCoS1I+/UxhoKeNlTNjCLe5yxauAUhymX/CJNvo+RPukjzvhAVDfV8F9h2oRyQfnKgomZNJFe8oP8px6wBgbl0aEuYjnCwZH1b3j1mLJH3SMPuQ6Oj+Qjb+iw39+QAN72YhTPtArTCIceeqhZVSh7PG9GvliOsCoyMsdqiR8OI372U3Z46yw7DTUloQ4Qb8oRdZ66hskeh2dGzTx/nj3HZWE/Uxyc5+klrZSz7Gqf+HPg0MxUCRYfjqe9yj8vXjVnyglHWqwEPK8sWDl83Zm6CnlEGSKd1GPSUV3+cixLC9CuYfGiHcV6RhtAe8TbiPQBDg7ctAXU4Sy8DYgVpDEjsVHPoFNHbGAmpfD66671FTo/Ki4CoeiOT0yYVkEgIianhL8G4o+OLjtdRJNDA40QRNT7NuKhFJjtEfEIbYQIx/r1GBzwm7BSb1dwDuZ/0ooIQZSwTSfDNh2vT28Bx+NMSD75/sl9c6E2IL5MsxIfdzIk7gh/BjI4OSKy8/mL7xbPmPxHOJE/vj5MFo5luoV9nIOwJ7+z1wP2IabJG+pr/n5MT2RFYl2EePLsEQKIB+JbXf4C+UPZpCyQn5xPfmZFrUN7xkAynz+Uv/xgpLEhsVHPwArACBCVzWiDkdvUbAyFEEKI6phYvoo6D1MvqGpMxowycF4SQggh6jKybAghhBCiUGTZEEIIIUShSGwIIYQQolAkNoQQQghRKBIbQgghhCgUiQ0hhBBCFIrEhhBCCCEKRWJDCCGEEIUisSGEEEKIQpHYEEIIIUShSGwIIYQQolAkNoQQQghRKBIbQgghhCgUiQ0hhBBCFIrEhhBCCCEKRWJDCCGEEIUisSGEEEKIQpHYEEIIIUShSGwIIYQQolAkNoQQQghRKBIbQgghhCgUiQ0hhBBCFIrEhhBCCCEKRWJDCCGEEIUisSGEEEKIQpHYEEIIIUShSGyIsvjrr7/C22+/HUaMGBGSJElD6xf//POPxf+9995LQ4QQQkwJJDZEWTzyyCNh2WWXDb179w7TTDNNGlr/OPbYY8OKK64YXnrppTRECCFE0UhsiLK48sor7Xvfffe17/rItNNOG/bee+/wxx9/hP79+6ehQgghimaapL7axAvis88+C3feeWd46623wiyzzBIWWWSR0L59+9CkSZPQtGnTsOiii4ZHH3003HbbbeHQQw8NSy21VHpmzRk/fnw4/fTTw4cffhhat24djj/++DDvvPOme6uGKQEe3XTTTWfbr776qnWgu+++e1hzzTUtrLb44IMPwrrrrhtatmwZHnvssTDXXHOleyZAPMgv8m3UqFEV+bTaaqvZ9Mtiiy0WrrrqqvDmm29WTMHMPvvs9s211ltvvbDOOutYfsMbb7wRLrjggvC///3P0jnTTDPZh7SS35tttpk9F+fUU08NI0eOtGOxusw666x27jzzzGPHrr322hXWmO+//962f/jhh/Diiy+GBRZYwMJF/QChOMMMM5hwzPL777+HH3/80fY1a9Zskv0OZeTXX3+17yxszzbbbHY+cL0///wzzDzzzGHGGWe0sPoM9ZB0ez0gvdSTUmnjOOpPFuotdZD8APKGusQ1qO/1HdJLuqeffnprh2pivSVvKXvkA3nkYb/99puVJ2/XGj2xEIlIrDzJeeedl8SGKtlwww2TJ554Ivnqq6+S4cOHJ3vuuSc9ZHLffffZsZ06dbLtffbZx7b/C/fee69da8EFF0xGjBiRhlbP448/nlxxxRXpVpIcfPDBdp0uXbokseKkobVDFBF27QMOOCAN+ZfYadu9YyOURKGTvP7668mXX36ZRFGSbLTRRpafUawkv/zyS3LsscfadaJoSB566KEkCoTkmGOOse0NNtgg+fTTT+2asaJavs8333x2/HbbbZeMHj06eeCBB5Ill1wyiUIlufrqq+1YiBU9OeSQQ+xY4sG9uX67du0s7MQTT0xi45oenSQ9evSw8KFDh6Yhoq7z7bffJpdeeqmVqc8//zwNncAdd9xh5WennXZKNt98cyuHH3/8cbp3Yt59910rk7GTTWLHYJ/YGSTNmzdPohi2Y4YMGZJEUWvhUQgnzz//vIXXVyj72267bRI7UEsvaY+Dp+Tll19Oj/iXKCCSOFixOpnNn9gJJ7fffrsd88wzzyTdunVLdtxxx2SttdZKLrzwwiR2rLavPhIHmNZuRuGUtGnTJunXr1+6p2q+++675KKLLkrigCbZcsstkzhIsnDa8Y033tjyjvbq+uuvn6j9aaxIbKT07dvXOqA11ljDKlyWOJpKtt566+Suu+6ybTq6OGpPHn30Udv+L7z66qt234UXXtg633KgMC+++OJJ796905Akefjhh5NVVlklufnmm9OQ2uOss86yOPbv3z8NmQCiYJdddrF9u+22m21n+eKLL6wTeOedd2z72muvtWNpyLyhoxJ27drVwulIfvrpJwsfP358ssIKK1g4nYfz4IMPWmMZRwwmgpzLLrvMjqVhfOuttyzstNNOqwjLCjkaR8IvvvjiNETUZQYMGJAsu+yy9sxatWplwtN59tlnrSx0797dtqlDc801lwnUUqL7tttus/K30EIL2WeRRRaxjpQBBgMOhAUdRMuWLa2z4J6Uw2+++Sa9Qv2DPKETJb20M4h4hFkcyadH/AtijPpCPvvxc845Z7LAAgvYYGDUqFEWvvbaayd///13RV0655xz0ivUL2jrERrzzjtvMsccc1haGLAwsKmK1157LVlxxRUtL2hzvd2inCA0WrRokTRt2tSuh7BDoDV25LMRwVkwVhb7jU8CZtgsmBoPO+ywECtpiJ1kiJ1o6Ny5c4UpFpPiLbfcEvr06WNTBbzxEEfyITaS5lAZR2QhFrZw9tln2/TARx99ZOdVBqZipmrOOOOMEDvM8Nxzz6V7Qogjdrs393jiiSfsHrHBtamHLbbYwsycmIAHDRpk0wtRKNjUBlMcUVCF2PGnVyqfcePG2Xd26gJuuummEFW7TYnsv//+ZoLMQn4dfPDBFVM9sbzZN/hvzJVRqNjvoUOHhijo7Df4MdnzmM5ZddVVzUx55plnVph7S13b44M509MAsQG172yYqJvwfGMnYE69kH3OQN2gLHh4u3btQvPmzUPsDKwO5vnyyy+tnH3yySchdp7h448/tmk8ph4xeVN/Bw4caMdR/yifr7/+evj888/TK9Q/7rvvvnDPPfdYekk37RftQxQV6RH/8v7774fLL7/cpnb9+DigsDoXO1ZrR5hqZoqSqapNN93UvqNwt6nJ+gbpjEIzfPXVVzZFPP/881v7+fjjj6dHTArt94477hii6A133HFHiMLWptmB6WymsqPosH3kE9PlTNk2emIlbfRgyicr+GRHwFkYtTPyYWqlY8eOdmwUJrYPqwiWjhdeeCGJoiSJHa+FM4riOEZSRx99dDJs2LBkscUWS5ZeeukkFlg7JmvZwPQbO0BTxpjlnnzyySRW8mS22WarGMVzHqMKzuE+jFq+/vprG/0TttVWW1lcmUZwZY1ydyvBMsssYybpmrDrrrva6I/4ONxjk002sWu2bt16EquGg1WID1xzzTV2PPkRBZ6FAaMpwvnst99+Fvbzzz8nyy+/vIVhNcmyzTbbWDimb7diYPokjFFZbDDtWTG6JYxRK9dzeA6EY/kQ9YMbb7zRnlnsDCaybGCiJpwpAp4n03iMUnv27JkeMTFM+2V5++23zWryyiuv2DajfTd50xYwvcn1uW59ZOzYsdY20eZgzaAO0l5UBu1PFupNhw4dkihAbDsOYCw/sPRi2SA/sTQytVkfR+9M/3jbRXp8ipWp18pgOpljsPjmofzQ9gDtnk+5Y3lt7DR6y0YsaOYA6eRH5w7hjHxw4FxiiSUszJ3QUMSMfLB0HH744SFWbAtfeeWV7RvHoVg5w/rrrx+iiDDrwtVXX237snA9RgfEh1E3joycwwjtwQcftGNi4xfmnntu+41zIyM51POSSy5pYYzEsCTEjrrCCZNR4f3332/hjEqwitQERpaxrKRbE4iCJYwZM8Z++z1LgVWIT1UwKsAKArFxtHtxzcpwCwsWIJzUsmBlYkTKaCOKmHDEEUeYcyrOf04+LaLu4xasPJtvvnnYdttt7ZliWcQhuHv37uGUU05Jj5iYOeecM/01AayCODm65YTRvpc9rkkZox62bdvWwuobcQAUhg8fbm3ODTfcYKPu2AGapbUUeWdPLKZYQmi3oFWrVvbN6B4LAHnFh7avsmdUl8ESnG3zScNMM81kVuJSYCXGossxtKUHHXRQuPDCCyusOpQf+gmg/NC/kKdRsFlYY6bRiw0qiheOcokK2L6900J8UCHjSD9cd911YYcddrBw309n653dcsstZ99MgeShYGKqZPGsIUOG2DdmXsiahH36JovHyeEYPw5TJw0qaeU4OuSawBsjnJeddkAYVSYwagoVHE9wIK+Ip+ddKeJoy76JQ17I0HDE0Uno16+fmdjPPfdcE2hZEDRQ0+cu6h5Mefbq1Susssoqtk09ZKoRc3Z1UD8Q4d26dbMyl4fpE94yOO644yqEe32DwQqDAqaH4ug6LL744jbFdOCBB1bUg6pgOhchxptogJCjfiHkTz75ZMtr6i/1MD/9XN9AMNAu77XXXjZtVArKFWWCQSdvI5JmBpj+Sn0WpqF4S++QQw6xNYoaO41ebNA5LbPMMulWqBitl4N35ryyihJmbo4KSCEEb8DoOL3zREkD832V8dNPP4VjjjnGCr6/CutWlKo64eogPvgvMB9dE3w0g/hxiJf7PlDJvv76a/s9OWCdcCHDK7PV4fPndABt2rSx3w75hMUHSwn7S3UivoIowk7UbxAEiAWsiPj70Cky6qb+UNargnJEJ7zBBhukIf9Cx3PeeedZ3cZaUl9hkEFdpbPDrwrxgI8K6eZTFQww7r777tC1a9c0ZILl45prrjFrLv5TWD44DsvPf1kGoC6AfxtWaXzdKgPfDsDayyv922+/vVma8c/ILxSIfx7LJpx44olpSOOm0YsNoMB4h0qhKQUWBh9RewfmAgCLBA6irJnBaBknKsxtvp/j/Rwch8DNtlkQPow2EC5Mm+y6664VZs1sp1lKcJTqVEvBuaUsI1WBGMNsmBUbsN9++1kaSdMDDzyQhk4MHXve6pLHKymNojdslYkqKjsjBmCqhCmkPNUJMszKoNFG/YepAZwaEQxY8JhKAaYJspa4UuA4ibj1ac8sOIyzTgxiAxAwDQHW8/EppuraAeolx6yxxhppyAQYMDG1RDvl+cLoPjsdUd/AaRiLD4sXYsll0MngMY87gjo4I3vf4f0DeB+AAz9tJxa3Ug7LjQmJjQgNDt7UdOw0Xg8//HC6Z4K6pxCef/75FYXPOzM6ePafcMIJNjfas2fP0KVLFzMpUij9OAqhd5A+mmDeNAvXohI/+eST4ZVXXrHrMnrwDh4fCSwAVGi3jjAPiziiQXBhwzfX8o+HZamuM85Dp4z59emnn57I8kMaEFhcn/zJvunCVA0m1qxvSlZ0+G+83ZnzJF1cC18Tx4/JNopXXHGFmSbxIMe87fixpC17nzzcj+e51lpraUGveoSXYcp0tjzjNwA+pUf5YdTZokULm7ocNmyYPWum1fJQz1lMjs4gC20A9RSxz7w8S/XffPPN6d76BWb/jh07TjQY4I0LLEHUa8TCxhtvHI488shJfC6watA25q2HwLEnnXRSuPXWW80PBOtSfYWBJG8K7rHHHla+sNYgEpguob1DVGEVIs1M12EtwvrMfsoig03KHEIOmGKiP+HNRtpBngFWk6rapUZBbJxFCgtJ8ebFSiutlOy8887m0R47QHuHPHZSdkzscJNYAemtbeEfFv866qijkiOOOMI8mFkECI9viBXZjsNTm329evWyt04OPfRQ84KODaUtDMYxLCjDWy2xw7Y3VtiOnWkycOBAe++bd/+j8LDr3n///Ukc0dsxeOPzFgvrg3Adrs9CYbHy2PoDhPGGDOngN5/tt9++5Dv2VREbFjuX+GSJFcjeSWcxpdVXX93e0IniKznjjDNsESa81YHf7du3r4gDb9vwhg7e2iwQxCJlUVTYsbydwnMgfRzLu+ykYa+99rJFhM4880xbUMeJQqfibZvYWCSxcbB1R0rBwm0cV1/XBWhsUCZiY15RfnkDKbsYFQtwRaFha7Xw5sS5555r62NQJoCFpziPOpUliglbY8PrlMMbZayRwDnZT319m+Cmm26y+LOQGesDsX4NddHbqEsuucT2x86yoo0D6i2Le9G+ZGEBPeo7dZZn0adPH1uwr75Cevytuuync+fOtv+6666zbcoU7RKwvhHtDOsO0f7zBhwLFgLtUhSwk1yPN+gaO1quPAfqMxYY+zCiRrEy/+8jJ8KZz2W6BNWKyQ0rBscyasbbnXljFDKKmblMzsdky9we4WxzPkqZkRPX5nxUMn4QTKXgB+FvaGCCw8sZk52DjwTnM0rBcsI21gHC8FVAbfPhfny4vv/mkaPCa+IgSdpwnIqdus1l5yEvsL642idf3N8EGEGRt+7QSfqIK/nFVEg2LowaMEEyMiW+nMcIlmPI27zHPO/Kkz6/NnP1OG65U5vD/aIwtLTwhoq/1SPqLpR91prhG8uily0c8rBuUZaxuOFHQBmkDjAVx9sTHPvUU0/Z+jK8scII3MFsfvvtt5vFw83/lA+OxZKRtXZQjxipZq1u9QXqwo033mj/XoF0kmf4bPg0LnUhin6bSsLfzNs56iuWQ0b42XYHyyLTkDi6M23Fm2HU0foKz5q310i3p4O2hila1gjCskH+YAVlio42hvLANAmWH8oJlqEoJqx9okyxrlL2epRLrByszdGYkdgoEBcbdLo0bvIREEII0RiRz0ZBMBpH5QKj/WuvvdZG/kIIIURjQ5aNgsDUhtMo0wWY0zDNrb766hOZJIUQQojGgMSGEEIIIQpF0yhCCCGEKBSJDSGEEEIUisSGEEIIIQpFYkMIIYQQhSKxIYQQQohCkdgQQgghRKFIbAghhBCiUCQ2hBBCCFEoEhtCCCGEKBSJDSGEEEIUisSGEEIIIQpFYkMIIYQQhSKxIYQQQohCkdgQQgghRKFIbAghhBCiUCQ2hBBCCFEoEhtCCCGEKBSJDSGEEEIUisSGEEIIIQpFYkMIIYQQhSKxIYQQQohCkdgQQgghRKFIbAghhBCiUCQ2hBBCCFEoEhtCCCGEKBSJDTFFGD16dDjooIPCJZdckoY0Hv78889wxhlnhJNOOin8/vvvaagQQjQeJDbEFGHgwIGhX79+4X//+18a0niYccYZwxdffBFOO+20cMcdd6ShQgjReJgmiaS/GyX//PNP+Prrr8M000wTpp9++kB2MPokfNppp7WOgk+zZs3SM6Y848ePDz///HOYYYYZwiyzzBJmm222ivDpppsuzDrrrLZdV/nxxx/DGmusYR3u22+/HRZYYIHw66+/hm+//dbiTt4jQv744w9Lz+yzz24fnkdl8JzIkyZNmqQh/8L9yBvyimtz3b/++svybc4556z0usRp7NixYY455rBPHo/zXHPNVfK+QLmh/Pi9neeffz5ssMEGoWPHjiY4ZppppnSPEEI0fBq92KBjOP/888M111wTPv30U+tkOnfuHOaee+7wzTffhCFDhoRWrVpZJzm1eO6558LOO+8cPv7443DkkUeGc889N7z//vthk002CUsttVS46aabpqoYqo7HHnvM4tq9e/dw4403mqB48803bWrh4YcfNmGw7LLLhnXWWcemW1599dWw4oorhkMPPTSsu+666VUmhvP32Wcfu17btm3T0Ak89NBDZkUZOnSoiRiusfTSS1ueIRx32mkni0tWdLz33ntmeUDkfP/992G33XazcuC89dZbFl8Ey7hx48K+++4bNtxww3TvBD766KMwYMCAMGbMmHD55ZdPJEgQIQiuV155Jbz77rthscUWS/cImqAnnngifPDBB2GRRRax51VKjP3yyy/hhRdeCKuuumqlYo9n99JLL1ldps64MM/C/Tjmtddes3rOc0SE5vnpp5/Cs88+GzbeeGMrs/UByvv9999v7cKiiy4a/v77b6sPo0aNmigNhK+22mphpZVWSkMmgCjneMowgy2HcOon9dRBxJM/yy+/fGjevHkaWrehHj744INWzsijPJSNBx54ICy88MLWZlQH+UIbRn32Ok3e0oZRVhlwkM8rr7zyRPnZKEFsiCTp1asXoiuJo+5kxIgRaWiS3HPPPcnaa6+dbk09dtllF4vfIYccYtuxI7XthRZaKBk5cqSF1VUuvPBCi2sUSWnIBH788cdkiSWWsH2x87awWFGTnj17WtiCCy440bPIEoWBHXPRRRelIRNDnrCfzwUXXGBhn332WbLmmmta2FFHHZX8+eefFv71118nsdFNtt12W9s+55xzktgJJbEzsu2xY8daPKO4se0zzzwzmWuuuZIoGmw7dnBWfuabbz67dseOHZMooGxflv3228/2U6bEBGLnmJx44olJbJQtb2aYYYaKfHZip5/cdtttSRRrVt55jnlix5dEgZkss8wyyZZbbplcccUVSRxIpHv/5ddff0369OmTxA4gmXnmme2eG2ywgZVF59tvv02uvfbaZIUVVkhip2NxrC/cfvvtlqYbbrjBtr/88stkueWWs7D8Jw6k7JgsUWQkrVu3Lnn8I488YsdE0ZfEDjuJIszqweuvv27h9QHiHUVXEgcFacjEUM6iQE0uvfTSNKRqyOdZZ501GTRoUBoyoW2iH7n44outHJKfffv2Tfc2XuSzkeIjpZgn9mGEgLqPDVdYffXVTa3ycfjNcShlPr7tsE14Fq5ZHZxTznGx0wxXX321jeDbtWtX8n7VQXyzacpvZyFOle2rjtg52Pfiiy9u3w6qn6kh8DSj/jfffHOzMDAaYwSa54cffghXXXWV/b755ptt2iRP1mrhzyWKlxA7NtuHdcj9J5566ikbifiojREK0ymxIbFtRopYRXykwzcjaN/PCC826BONlLJTKA7WGsACIiaA9YlnjPPsWmutZSNFLHWPP/647afMHX/88WZJYiqKcpK3MuCAG4WcWcJ22GEHe6577bVXSevIsGHDbCTOqPOuu+6y8oblcPjw4bafaTLKyOGHHx5iJ2rxKfUs6yIvvvhiOPDAA+235xHWUH4fc8wxZrmLnV7YaqutrM1gxJ3nww8/NOsuec7xUViHjTbaKLRv377CCsI1dt999xDFh1mG88+jruLWUMpUqTjTBhx22GFmQYtCNA2tHMojxzO9isXUw7CA0tbtvffeVg7pP8jLzz//3I5prEhspGQ7ajojzLCYvIGGkMJ5wAEHmPkNE9s222xjnZ43Xj169DDfDxrDONo2M323bt3CLbfcYtcAKuj8889vlR1T+vbbb29z+HH0EX777Tebztl6663DLrvsEo499libxslD40jnFkfmVqjjqN06c6YqiAf+EHSkhDO9wD2IVxzxmCmPYzATAg1rHM3bNFGXLl2sMtCok0Y6XPbDrbfeauk94YQTQv/+/e1eNQFzNJV3nnnmSUP+xYVAtkGn8vIhraQnD6ZbTOlA5aaRLRc6fMzL4ILlmWeese88dEA0TIgR8DLi394htmzZ0p5bqcY7C88eJle0NUTo2K677rrQu3dvex5MZ+CLg9AHygX7TjnlFNvOCnrnrLPOsmnQI444wjrJynxygHKzxRZbVPxG7FJPXAgTH663//7723Z9gbp7zz33TOJrRL1DFJMm6u9xxx1nPkcICKaQSsE0A20Lx9MOzTvvvNZh+lQJHSztC/lc6nnURWgDb7vttknyx2H69r777jNfq3L45JNPTKTmYZBC+0zb4e0S+UQ5qy+irCgkNnJQeXBk5O0JH4G61aNXr15WQenc6biptFgYGNUyEqLTQfUziuCDqt11110rOiUECcKC6zJH/d1334Unn3zS/EF4JRR/DEZo5513njWee+655yRWDjo6RmyoZkYhjLgRKswrt27d2ioNfgykg4rFyJ/rvPPOOzbyRt336dPH/A7o/BkJkl7ShN8H89cILeLINaikjAa4PufR0RNWLsSfdFLRqpqzJA1YKEgPoykaNvKkQ4cO6REToCMivxgpeAeBhadcuK43ODQY5APfeYgreUlaS4k+4FwEp8MouCrciiP+BT8W6g3MN998JnxxGl5ooYUsjOfAM6vMJ4lyikihTmARo6GvSsy1aNHCvqkPdKQ8M+oqdQcYoVI+KvMJqYsg5rHm0L7k8wlrRNbiRpnFYoOzcinwy8gKfEQMecUAyeF58KmqPtclsFTQDjL4KzXgoU1hP2n08lEVtFPkN1bvpk2bpqETYDCHBZVj8PvC8sygFcHsg43GisRGDiojqp0GLD8tQWHxEQ+mXhoqOkcsHYgOOiasE6hjRk1Ucho/H0F75aTyYjnAmfDkk0+2yn3hhRfaKITGgQK/yiqr2AgDy0kp3GyXtQj4iG699dazUd6mm25q25g6EUo4RnIM4gLLAeSvwzbxRGgQRmdKZT311FNt5MT3kksuaceWA/ejE6EDyOdnFpz1NttsM5uieOONN8wxF0tSfjTw8ssv20gUM7A3gAg3zikHOnx/e4f4YIKvrHMi/eRDZfHO5n05cC/gmmJSEJxYNOjw8iKzsmfAs0co08Dfe++9Vja7du0aRowYkR5RmnPOOScMHjzYrssInutkqU/PCOsplgfaFC/LlQkB0kmaGTyUAyN0yi3PJAvXqC95hFUXEbDMMstMkj+k4c4777T2m/3VDRgYPJHfa6+9tllIfTDobQHl8Nprr7VyyKCNtYUQzgzqGjsSGzmwViAYmIooNRLFXI44YBoEMYBlgjctABFBg0lHznQEc78o6bxPAdYROuA2bdqYCGDkzDF0rHjPUwH4TcXAElFTfOTuFSDrj0Ilo0FHQJQi24DQoFChGDF+9dVXJlYwrzLNUhMYbSF43DReio4dO5rII/+ZvmFEkIf4IL4QC3Qw7gNB/jH/Xg48H95+AYQVeeMiLQ/PgH15weOQl5XtK4XP2VZmym3sML3Hs8U6WK6Q4y0hoLNFQDBViTn86KOPrhB3pbjiiitM0C6xxBL2XC6++OJqO5q6CCNy6jJtCZ0bgxvAnF9KROMjw0CmXMvN3XffbYMAH5TUNxggMQj0zp92CLC2AmWO/QxgaFO8zLCf9iYP+cExCAgsn57HtFl+PN+0+506dbL24dFHHzXrWX0RZ0UhsZGDgkKjg1XAHQIZ3furr8wp0+kC5n4KMI5tgMrlfEy6jJZQuHTSNGpZMBVn50vzDWt2u7KOsCq8UJcq3FybCpJviPyefPtv0kJlwTLDtA2VjN90BjUBKwQgxiqDRgCfByolDBo0yKaDspDXI0eODE8//bSJEYQYjmvAvLQ3IFVBI+ziDxM+1iRGPZDNd9JOR8R8NZYryO4HnmPejFoVmK+B64qJ4bniV8AU4gorrJCGVo93Hu7QxzNFsODUixNvZTCQwAKIdZHj6Tgwp9cnKKOIbKZwKad0qC6+DjnkkAoHaIdyz2ua2223XRpSNbR7+CW4hbQ+grWL6TKm6hg4uR8F7QxtBmKN3+ynnfLBHedg8cjD9Dp5y2CRQacPoHDMxWrE+eQX4g/Ry7GANQSrbGNGYiPFR6h0KFRiCp+LCJwQ8VR2MNMyOiWcBsvPxYeAcBotnJFo0JhPzYsNREm2s8fng/u5CODjTpX4hmTJd3il8GPyx7KdD3NzIgKLDyqf+3Mc6WLtAyoolQzHV8BEXZNRIGkgX5gqcbMjZOPj31ROTLwIJRxz6YQcKj8Oboi8s88+26wsbCPImLsv1Tjk4S0EronYw8QJ66+/vn27tcfjyLMFLDvgo0aHOeAsnpf+nQWhxtwtnUJ+XZDGDnlD2aKBx+8AcEKuSiw4mK3BnxmDAUbh1D2eA+WUKTa/Fm9nICwcrGN0Gljr6vJaNZVBp8Z0LdOviGafIiQ9eQdQhANvedHeONR1BgHZPHEY9TNC97dQ6iMMFMgfnjMilvQDlgmmqxEYTJ/4fs8/xARWVmANHqwegGDx6/Fxiw/tN/lN+4IA5rq0S5RprEiU8XLKc4MmNuqNnt9++y3ZbbfdMAMksVNM4qjI1mD46quvkuuuuy6JnYO9n52lc+fOyWKLLWbHZImFy64zyyyzJFGUJHvttVcSGzvbF1W07YuFL4kq2MKc2JnbGgPXXHNN8tJLLyWxEE+03gDX4tzdd9/dtjmf7dh52fEQO00LO+CAA2z74IMPtu2OHTva9q233prEBtjCooCwMNaKmH/++W2dA+7BugLs57jYgdtxsYIlzz77rK15wf36TsY746yRQJ5E8WLbUdAlsZGze3O/2KHbehUwfPhwuw/h6667bhI7CMu7WMktLznX+fTTT209Do6NFbwirx9//HEL4xNHLra+wrBhw5LYOFsa44jHjgOeNXndoUMHi8Nhhx1meenxiQ1ysuOOOybrrLOOhZG//I4jRdsfO7pk3LhxyU477WT3Y10D1vnIQnzIU64jJua0006z9TOOOOII++y8886WxzwXZ9CgQZa3rVq1srUjHMpK06ZNkyhokyjykyeffDKJjXzFmimsqcF5Xbp0sbVPKMuse0BdiOIxufHGG6388HyyUMb9fjz/+gDpX2WVVSzerBeRhzaOtV6yUA9od1if5LvvvktDJ0B5pg0pBWtucB512tebqetEEZDEAYTlD+1sHvoBz7+rrrrKwh599FFbd4N2Y8yYMRaWxdsez28vp74eEs+E8hmFiLVVjZnpTvF3yhopzOEzLcAbCYw4GSkx8mVukw+mL0Y+vEKaHflgPkcJ4yiUBWsIow3ULZYJ3i7B1B/z2l4b5RqobZw0mWd2Jc1oAwsA765jMeE8VDEjNPxCeD2T62IB4Bz3XcCLnjRgIsWE6qNmlDRWCUYmTOug6LEsECeuwyidkbureywpqHlehcWiwRQBcSaOqHnWvGBag5EnTrLEqyZwLSwPqHw84YkL+c7ogftzT0ZY5AOOboxQiSOjA54B5kni7g6ixJV5UqwfPAvSxLV4duT99ddfb6MXro3FgnlT9uFzgz9OdrTGvXkV0M8jz7CeuGc6ee77mUbiuv7GDDByJh7cx0fIhBEnrgV4o5MG/FJq6vPSkKFM8Ro3U2CUez7kE6+nYnGiXOIbhbkbZ2xG4pR76iT5jNM2I0pWvWTkyBQbVjT8rng+mNG5JuWFN8MoB5S9KDZsmhMr3emnn27z60CZog5iYmc0S7mlzFLPajJlNjWgfPIWG2+8YX3NrvaJuR8ndHzJqF8O1jassDi14+/iI3/aQ/KFcu3WI+Ae5A/TAlh7sQJTL8mfct7kmJrQJlK/acvwQ8lbjbGCYcHFustUCO020+eUUdqhPfbYY5KVZrHI8VKB5zd1GysR01WAjwfb1HucUBszjX65cjFlwCmTDoEOAGfQxgQmfoQrDTONWKlplsYKZmd8cWjMs+DEiKhDRON/geDDZE1zhQM2+zFnOwhrFgdDaLDcuQsDBAhC3TsCOgbuiT8C4pQOx0Uj4GPFFE72fohIrlnXRSLljIEJDtO8zYMgc8gHhDrpQBw4iAcGLkwDZAW45xsizKcKgIENz4Pz/DrkD1MR+c67roFQRYwixhhE5Z8n9ZNlCkg75YtBIQKE/EHY+to+WZhqIv34Cvnr04gapp4RbIg4RIsGGBIbQgghhCgYDbGEEEIIUSgSG0IIIYQoFIkNIYQQQhSKxIYQQgghCkViQwghhBCFIrEhhBBCiEKR2BBCCCFEoUhsCCGEEKJQJDaEEEIIUSgSG0IIIYQoFIkNIYQQQhSKxIYQQgghCkViQwghhBCFIrEhhBBCiEKR2BBCCCFEoUhsCCGEEKJQJDaEEEIIUSgSG0IIIYQoFIkNIYQQQhSKxIYQQgghCkViQwghhBCFIrEhhBBCiEKR2BBCCCFEoUhsCCGEEKJQJDaEEEIIUSgSG0IIIYQoFIkNIYQQQhSKxIYQQgghCkViQwghhBCFIrEhhBBCiEKZJomkvxslP//8c+jfv3+Ybrrp7EN2/Pnnn2H66acPiy22WFhjjTVC8+bN06Mnjx9//DHcfPPN4ddffw2zzjpr2Guvvez6L730UhgxYkTo0qVLaNKkSXr0lOGff/4Jd999d/j444/DTDPNFDbffPOwyCKLpHuFEEKI2qPRWzboaFdeeeUwaNCgcNhhh4VLLrnERMaoUaPC9ttvH9Zff/3w3HPPpUdPHgiJ7777Lhx55JHh2GOPDf/73/9M5BxyyCFhl112CTfeeGN65AQGDhxogqdIpp122jDvvPOGU045JRx66KHhjTfeSPcIUb/47bffwrnnnht23nnnsO+++4aXX3453VM51K8HH3wwHH300eHrr79OQyfw/PPPh7333jvssMMOoUePHtYmMFCoz9DGkD+77bZbuP/++9PQ0jDguu666+z4PfbYIzzwwAPpnkmhndxggw3KyvO6zO23325tMZ8777wzDa0cBo/kz+677x7uuuuuNHQCP/zwQzjuuOPCiiuuGLp37x5eeOGFdE8jB8uGSJJYqbDwJMstt1wyfvx4C9twww0trEOHDsk///xjYZPLo48+msw444xJFB5JbLiSv/76K4kVP5lnnnmSRx55JD0qSa666qpkiSWWSKIgSUOKIza4SYsWLZJpppkmuffee9NQIeoP1MuDDz44WW211ZIo4JMLLrggWWihhZK33347PWJiqHf33Xdf0qlTJyv3Sy+9dDJmzJh0b5K8+uqrydxzz51069Yt+eOPP5KHH37Y2oCTTz45PaL+0a9fv2SBBRZI3nnnnWTYsGFJy5YtkzvuuCPdOykXXXRR0rZt2+Szzz5LotBImjdvnkRhlu79l19++SWJYszy5/HHH09D6x9RWCWtWrVKhg8fnrz44ovJfPPNl1x//fXp3kmhjaaMjRgxInnqqaeSueaaK4lixfZF4VuRJ/7hepWVx8aEfDZSYl5UfGN5gJlnntm+P//88/D999/bb0ZBQ4cODbFzDm+++WbFsQ6jrGeffdamKGIBq7gu0xZObORs+mTTTTcNZ599dojiIvz+++/h9NNPDwceeGD45ptvTC1zDMQG0iwPXPOxxx4LY8eOtXAgLozQYqMY3nrrrRAbExu5cOxXX32VHhVsuiQ2MCE2pmmIEPWfV155xaZBl1pqqTDbbLPZtCfl/vLLL0+PmJhx48aFDz74wOozdRMLH/XRYcRK/WJKMQ4OwqqrrhratGkTbrvttvSI+gVtyaWXXhpat24d2rVrF1ZYYQVr1y6++OKS1hry7qyzzrKp4wUXXNDylanf8847z9qhLFGUhHvuucd+Z/OwPsEUN3kRBUNYcsklLb3NmjWzMMpKHo4/44wzLE+wgC+66KIhilPLH9p42v44YA2jR48OAwYMCLPPPnv48ssvrd1u7Ehs5KDxoZHBzOoCI46arPLdeuutZlb96KOPbKqDho2K7FB5MeNSCRELHTp0sIYQspWRe9CgYcLFrInZjnt+++23JjpoBLm3x2GnnXYK11xzTfjwww/t/ltttZWJB3jttdfCNttsE+JIzeIURxvhiCOOCI8++mj4+++/7ZjBgwebX0gc2YSDDjrI4gf1tYEQwkFsIPjxtwLKNHXpiSeeCOPHj7ewLNRj6gfTA6VAsMDVV19tIv6nn36yjqNjx44WXt8YOXJkeO+998IMM8xgeeP58+STT1onmOf9998PX3zxxST5+frrr4dPP/3UwoC2ENHStm3bNKR+QpooQ6Qxmz/Dhw+fKL0O+Un7j88d+PEMLGlf27dvHw4++ODQqlUrm5JZffXV7bjsYLOxIrGRgw6fUdGOO+5o85F8n3POObbv2muvtUaMAnTiiSeauLjgggusQYIzzzzTfD/23HPP0KdPHyuE9913n4kHCqWDCECoLLvssrZNQUSArLfeeraNambOmP3MhTKqQmUfddRR5l+CD4nPox5zzDFmEQHUMwIF5Y0IosBTCfbff38bnZ188skmSphPpFHxCiNEfYWONA8dJeUe4V0Z+VG6g58G9c5FvvttULfrIwxQgPaHdsgtreD7stBh5iE/GRy5bws+CLRH3bp1q/edKMIBys2fd999N/31L+QPwhZROsccc5g1A2hf+XDtVVZZxcIaMxIbORAI888/v01nICwY4WBOBMxiL774YlhooYXMFAt07jRqNE5MrcwyyyxmhuOcRx55xMxxFLb8NArkK6pbIrKsu+66Vrl79eplIw437eFw6mBNQbzQ8CJ2Nt5443RPCEOGDLFzcIQlzlQKrCfPPPNMeoQQ9RfqXymynUZNYKoBCyHmceo0ZnEGIFUJl7rMH3/8kf6alFJCobL8BDpV8oJRPxZZt5bUZ6pKb6kyVNXxeZiSYiocR1La58aOxEYOlOlmm21mVgZMhIgPBwFBAeQNDhoin++lEuKBPGbMGPPZcEHA/CiN1n8BUyXziHiTI2YQDZCt5BzDmzSAb0fWp+OTTz6xb6ZdECKc37Nnz7DMMstMdoMsRF2htsswpnOsltT/U0891eoW0ylYAxsDVeUn01VYbJm6ZSr2/PPPN58QwNel1LRMfaZUXtSkvPFGD1YOLOP0FY0d5UAO1H4pCwPwChyvwq600kr2upePDCiAzPXiKARYRJy8A2lNwTS30UYbmXPnPvvsUzGnnB9RzDPPPPYqK41l1jmuadOm9s0opHfv3uG0006zaRecmP5r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Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"ovarian squamous cell carcinoma, cell line establishment, TMB, HRD, targeted therapy","lastPublishedDoi":"10.21203/rs.3.rs-5682512/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5682512/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Primary ovarian squamous cell carcinoma (POSCC) represents an exceedingly rare subtype of epithelial ovarian cancer (EOC) characterized by a cryptic etiology and insidious onset. The rarity and high mortality associated with pure primary ovarian squamous cell carcinoma (SCC) make it challenging to conduct large randomized controlled studies. Furthermore, there are currently no commercially available ovarian SCC cell lines for research purposes, necessitating the urgent establishment of novel lines. To our knowledge, this study reports the first preclinical model of primary ovarian squamous cell carcinoma (POSCC), denoted as ZOC254. We have detailed the establishment and characterization of ZOC254, derived from a 64-year-old female patient, which preserves the primary tumor's original traits across various levels during prolonged in vitro expansion. Whole-exome sequencing (WES) of both the primary tumor and derived cell line revealed homologous recombination deficiency (HRD) and high tumour mutational burden (TMB). ZOC254 also exhibited the PIK3CA: p.E542K mutation associated with targeted therapy. The effectiveness of olaparib, everolimus, and conventional chemotherapeutic agents for ovarian cancer was preliminarily assessed on the growth of patient-derived cells (PDC). The POSCC cell line and derived xenograft transplantation model reported in this study serve the purpose of broadening the resources accessible for preclinical investigations into ovarian squamous carcinoma.","manuscriptTitle":"Establishment and Characterization of Preclinical Model of Primary Ovarian Squamous Cell Carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-10 05:33:29","doi":"10.21203/rs.3.rs-5682512/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-04-08T08:10:02+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-08T03:50:42+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-05T04:29:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Human Cell","date":"2025-04-04T01:24:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"human-cell","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"huce","sideBox":"Learn more about [Human Cell](http://link.springer.com/journal/13577)","snPcode":"13577","submissionUrl":"https://www.editorialmanager.com/huce/default2.aspx","title":"Human Cell","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"7f4522bd-5ec5-4ae6-ba9f-c6c9988fc1e7","owner":[],"postedDate":"April 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-14T16:06:18+00:00","versionOfRecord":{"articleIdentity":"rs-5682512","link":"https://doi.org/10.1007/s13577-025-01253-4","journal":{"identity":"human-cell","isVorOnly":false,"title":"Human Cell"},"publishedOn":"2025-07-07 15:57:39","publishedOnDateReadable":"July 7th, 2025"},"versionCreatedAt":"2025-04-10 05:33:29","video":"","vorDoi":"10.1007/s13577-025-01253-4","vorDoiUrl":"https://doi.org/10.1007/s13577-025-01253-4","workflowStages":[]},"version":"v1","identity":"rs-5682512","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5682512","identity":"rs-5682512","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}