Modification of Ki67 on Prognostic Model of Extranodal NK/T Cell Lymphoma

Modification of Ki67 on Prognostic Model of Extranodal NK/T Cell Lymphoma | 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 Modification of Ki67 on Prognostic Model of Extranodal NK/T Cell Lymphoma ShiShou Wu, Wenfeng Zhang, Liling Song, Lei Jiang, Ping Yang, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3862754/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: To explore the value of Ki67 in the prognostic evaluation of extranodal NK / T cell lymphoma and the modification effect of NK cell lymphoma prognostic risk index (PINK) and Nomogram modified risk index (NRI). Methods : A retrospective analysis of the clinical data of 106 patients with extranodal nasal NK/T cell lymphoma diagnosed in Yantai Yuhuangding Hospital and the affiliated Hospital of Nantong University from 2008 to 2020. Ki67 immunohistochemical staining and Quantitative Dot Blot (QDB) which is an absolute protein quantitative detection were performed on pathological paraffin specimens, and the R4.1.0 and SPSS were used to analyze the data and determine the threshold. Results: A total of 106 patients,75/106 (70.7%) patients were male and 31/106(29.3%) were female. The onset age of patients ranged from 14 to 86 years old, and the median onset age was 60 years old. As of September 10, 2020, 105 patients were followed up, 54/105(51.4%) patients were died, 51/105 (48.6%) patients were survived, and the median survival time was 330 days. Immunohistochemical results showed that patients with Ki67 proliferation index > 60% or MIB1 (Ki67) > 11.9 nmol/g had worse prognosis. The data were modified by Ki67 (IHC) and Ki67 (QDB), and the survival analysis of PINK and NRI models showed that there were statistically significant differences among the risk groups (P < 0.001). Compared with Ki67 (IHC), Ki67 (QDB) modified PINK and NRI models could significantly improve their predictive ability (AUC PINK , 0.67 vs 0.74; C-index PINK , 0.68 vs 0.75; AUC NRI , 0.74 vs 0.80; C-index NRI , 0.69 vs 0.71). Conclusions: The results of Ki67 by QDB could significantly enhance the predictive ability of PINK and NRI models. PINK model modified by Ki67 QDB can more accurately describe the prognosis of ENKTCL patients. The absolute quantitative detection technology of QDB protein is expected to be applied to clinical laboratory detection to achieve individualized and accurate diagnosis and treatment of tumors. NK/T-cell lymphoma Prognosis Ki-67 QDB Figures Figure 1 Figure 2 Figure 3 INTRODUCTION Extranodal nasal NK/T cell lymphoma (ENKTCL) is a non-Hodgkin's lymphoma with high invasiveness and heterogeneity. Despite the combination of chemotherapy and radiotherapy, the recurrence rate and mortality of ENKTCL are still on the rise. It is particularly important to how to accurately identify patients with high prognosis in the early stage of ENKTCL. NK cell lymphoma prognosis index (PINK) and Nomogram improved risk index (NRI) prognosis models are increasingly recognized by clinicians. However, only clinical factors are included in PINK and NRI models, and no prognostic model based on clinical and pathological parameters has been reported at present. In this study, 106 patients with ENKTCL were used to verify PINK and NRI, and to explore the modifying effect of Ki67 on prognosis model. PATIENTS AND METHODS Patient Selection From January 1, 2008 to June 30, 2020, 106 patients with extranodal nasal NK/T cell lymphoma treated in Yantai YuHuangding Hospital and the affiliated Hospital of Nantong University were studied. All cases were confirmed by pathological biopsy HE staining, immunohistochemical staining and in situ hybridization staining. Methods The Immunohistochemical Staining of Ki67 (clone number: MIB-1) was Carried out by EnVision Method. The antibodies used were purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.. Immunohistochemical interpretation method: Each immunohistochemical staining section was set as an external control, and the staining results were interpreted by three pathologists and averaged. The positive signal of Ki67 immunohistochemical staining was marked by brown-yellow granules or lumpy granules in the nucleus, which can be identified as positive tumor cells. At high magnification, positive cells in 10 fields were counted randomly and averaged. Experimental Steps of Quantitative Dot Blot (QDB) Detection QDB test was performed according to the method reported by Yu et al [ 1 ] . Statistical Analysis In this study, SPSS 19.0, R 4.1. 0 software were used for statistical analysis. P < 0.05 was statistically significant. Kaplan-Meier method was used for survival analysis, and Log-rank test was used to compare the differences among different groups. Hmisc, survival ROC [ 2 ] and Time-dependent ROC analysis packets [ 3 ] were used to calculate the area under C-index and ROC curves. RESULTS Patients Characteristics A total of 106 patients, 75/106 (70.7%) patients were male and 31/106(29.3%) were female. The onset age of patients ranged from 14 to 86 years old, and the median onset age was 60 years old. Among 106 patients, 69/106(65.1%)of them occurred in nasal cavity, 8/106༈7.5%༉occurred in skin, 7/106༈6.6%༉occurred in gastrointestinal tract, 8/106༈7.5%༉occurred in pharynx, 3/106 (2.8%) occurred in lung, 3/106 (2.8%) occurred in oral cavity, 1/106 occurred in paranasal sinuses, abdominal wall, liver, neck, breast, parotid gland, vocal cords and penis (each accounted for 0.9%). Effect of Ki67 on prognosis of ENKTCL Patients The results of Kaplan-Meier survival analysis showed that patients with positive rate of Ki67 > 60% by immunohistochemistry had poor prognosis (Fig. 1 A). The absolute quantitative results of QDB protein showed that patients with MIB-1 (Ki67) > 11.9 nmol/g in tumor tissue had poor prognosis (Fig. 1 B). Validation of Predictive Ability of NRI/PINK Prognosis Model 106 patients with NK/T-cell lymphoma were divided into low-risk group, middle-risk group and high-risk group by PINK scoring rule (P < 0.05). The prognosis difference of each risk group was statistically significant (P < 0.05). AUC (area under ROC curve) = 0.68 C-index was 0.70 (Fig. 2 A 3A/C). 106 patients with NK/T cell lymphoma were divided into middle and low risk groups by NRI scoring rules (P < 0.05). The prognosis difference of each risk group was statistically significant (P < 0.05). AUC = 0. 72C-index was 0.70 (Fig. 2B3B/D). Modification of Predictive Ability of NRIPINK Prognostic Model by the Absolute Quantitative Results of Ki67 Protein The results of Ki67 immunohistochemistry and protein quantification were added to the existing score factors of PINK, and 106 patients were regrouped as shown in Fig. 2 C/E and Fig. 3 A/C. All patients were divided into low-risk group, middle-risk group and high-risk group, and the prognosis difference of each risk group was statistically significant (P < 0.05). The area under ROC curve of PINK model modified by Ki67 was AUC PINK+Ki67(IHC) = 0.67, AUC PINK+Ki67(QDB) = 0.74, C-index PINK+Ki67(IHC) = 0.68 and C-index PINK+Ki67(QDB) = 0.75. The results of Ki67 immunohistochemistry and protein quantification were added to the existing NRI scoring factors. 106 patients were regrouped as shown in Fig. 2 D/F and Fig. 3 B/D. All patients were divided into middle and low risk groups, middle and high risk groups, high risk groups and extremely high risk groups. The prognosis of each risk group was statistically significant (P < 0.05). The area under ROC curve of NRI model modified by Ki67 was AUC NRI+Ki67(IHC) = 0.74, AUC NRI+Ki67(QDB) = 0.80, C-index NRI+Ki67(IHC) = 0.69 and C-index NRI+Ki67(QDB) = 0.71. DISCUSSION Ann Arbor staging was the most controversial prognostic model of ENKTCL [ 4 – 6 ] . It was a primary nodal lymphoma-based staging method for Hodgkin's lymphoma and was also used for non-Hodgkin's lymphoma staging. However, ENKTCL was different from other types. Its lesions were usually confined to nasal cavity, invaded adjacent structures or soft tissues of tumor, which belonged to Ann Arbor stage I and II patients. Therefore, Ann Arbor staging system could not correctly evaluate the effect of local tumor invasion on prognosis. Therefore, in recent years, some scholars had tried to establish a new prognosis evaluation model by increasing or decreasing individual significant factors based on the age, Ann Arbor stage, LDH, ECOG score and distant lymph node metastasis included in IPI score [ 7 – 8 ] such as NK lymphoma prognosis index (PINK) and Nomogram improved risk index (NRI). The results of this study could validate the above prognostic model, but the patients' prognostic risk grouping was not clear, and it was difficult to identify patients with poor prognosis early and accurately, which was easy to delay treatment. Both PINK and NRI prognostic models were clinical parameters. ENKTCL prognostic model based on clinical and pathological parameters was the development direction. Ki67 was a pathological marker that reflects the proliferation of tumor cells. It hadd been confirmed that Ki67 was a prognostic factor of ENKTCL, and the determination of its critical value was still controversial. Results [ 9 – 10 ] showed that Ki67 ≥ 50% or Ki67 ≥ 65% was a poor prognostic factor. This study showed that Ki67 proliferative index > 60% was a poor prognostic factor for ENKTCL. All the above thresholds were limited by semi-quantitative detection determined by immunohistochemical staining results. QDB was a method for detecting the protein expression level of high-throughput samples by simplifying the experimental process of Western Blotting(Western Blot, WB) [ 1 , 11 – 12 ] . In the experimental process, only one antibody suitable for immunohistochemistry was used, but QDB technique can not only supplement the shortcomings that immunohistochemistry can not be absolutely quantified, but also avoided the influence of staining quality and interpretation error of immunohistochemistry [ 13 ] . Based on previous studies, QDB technology had a clear role in providing absolute quantification of protein in high-throughput research. It could reduce the bias of protein in quantitative level and was expected to become an important experimental technology for clinical pathology laboratory detection. The group of tumor samples were detected by QDB protein and statistical analysis showed that Ki67 > 11.9 nmol/g were all ENKTCL patients with adverse prognostic factors. It was consistent with the immunohistochemical results in previous studies and this study. Based on the data, the results showed that PINK and NRI could predict the prognosis of the patients. However, NRI model had insufficient ability to accurately identify patients with poor prognosis in early stage. Although PINK model can predict patients, the area under ROC curve was only 0.68, the generalization was poor, and the reliability of prediction results was insufficient. Based on the above experimental statistical results, the modified PINK and NRI models with Ki67 immunohistochemistry and QDB results still had outstanding predictive effect. The prognosis of each risk group was significantly different, especially with the modification of Ki67 QDB, the prediction of the new model was further improved, and the AUC of PINK + Ki67(QDB) model or NRI + Ki67(QDB) model was significantly higher than that model of PINK + Ki67(IHC) or NRI + Ki67(IHC). Both the PINK model、NRI model and the modified new model can predict the prognosis of patients in this study, but neither NRI model nor modified NRI model can accurately predict the prognosis of all patients, especially it was impossible to accurately predict the prognosis trend of patients in middle and high-risk groups, high-risk groups and extremely high-risk groups at the early stage of illness. The advantage of modified PINK model over NRI model was not only that it can accurately predict the prognosis of patients in the early stage of illness, PINK model modified by Ki67 (QDB) has higher AUC and C index than PINK model modified by Ki67 (QDB). Therefore, the new PINK prognosis model based on clinical and pathological parameters can predict and described the prognosis of ENKTCL patients more accurately by incorporating Ki67(QDB) into PINK model with a threshold of 11.6 nmol/g. CONCULUSION Ki67 QDB protein absolute quantitative detection results can significantly enhance the predictive ability of PINKNRI model, PINK model modified by Ki67 QDB can more accurately describe the prognosis of ENKTCL patients. QDB protein absolute quantitative detection technology was expected to be applied to clinical laboratory detection to achieve individualized and accurate diagnosis and treatment of tumors. Declarations Disclosure of conflict of interest None. Data Availability Statements The data that support the findings of this study are available on request from the first author. The data are not publicly available due to privacy or ethical restrictions. Author Contribution Guohua Yu, Jiandi Zhang and Shishou Wu designed the study and drafted the manuscript. Wenfeng Zhang, Lei Jiang and Liling Song participated in immunohistochemical staining. Ping Yang searched the literatures and performed the histological evaluation. Licai An, Yuanfeng Zhang , Yunjun Wang, Yu Pan ​and Yuan Gao participated in providing the clinical information. Yifei Liu and Guohua Yu participated in revising the manuscript. All authors read and approved the final manuscript. All authors have agreed to authorship and order of authorship for this manuscript and that all authors have the appropriate permissions and rights to the reported data. Acknowledgements In this study, all methods were carried out in accordance with relevant guidelines and regulations. The clinical data of all patients were based on objective facts. All statistical methods were carried out in accordance with international uniform rules. References Yu Guohua Z, Wenfeng Z. Developing a routine lab test for absolute quantification of HER2 in FFPE breast cancer tissues using Quantitative Dot Blot (QDB) method.[J]. Sci Rep. 2020;10:12502. Wan Jiaming G, Cheng F, Hongpeng, et al. Autophagy-Related Long Non-coding RNA Is a Prognostic Indicator. for Bladder Cancer [J] Front Oncol. 2021;11:647236. Zhang X-P, Yuan-Xing G, Shuai X, et al. A novel online calculator to predict early recurrence and long-term survival of patients with resectable pancreatic ductal adenocarcinoma after pancreaticoduodenectomy: A multicenter study[. J] Int J Surg. 2022;106:106891. Wang Ke-feng et al. Chang Bo-yang,Chen Xiao-qin,. A prognostic model based on pretreatment platelet lymphocyte ratio for stage IE/IIE upper aerodigestive tract extranodal NK/T cell lymphoma, nasal type.[J].Med Oncol, 2014, 31: 318. Guan Pujun D, Tian Z, Li et al. Staging challenges in extranasal and juvenile extranodal NK/T-cell lymphoma.[J].Leukemia, 2020, 34: 3428–3431. Huang J-J, Ying-Jie Z, Yi X, et al. A novel prognostic model for extranodal natural killer/T-cell lymphoma. [J] Med Oncol. 2012;29:2183–90. Yang Y, Zhang Y-J, Zhu Y et al. Prognostic nomogram for overall survival in previously untreated patients with extranodal NK/T-cell lymphoma, nasal-type: a multicenter study.[J].Leukemia, 2015, 29: 1571–7. Kim SJ, Yoon DH, Jaccard A, et al. A prognostic index for natural killer cell lymphoma after non-anthracycline-based treatment: a multicentre, retrospective analysis. Lancet Oncol. 2016;17:389–400. Kim SJ, Kim BS, Choi CW, et al. Ki-67 expression is predictive of prognosis in patients with stage I/II extranodal NK/T-cell lymphoma. nasal type [J] Ann Oncol. 2007;18:1382–7. Hung SY-JWP-NC, et al. Extranodal NK/T-cell lymphoma, nasal type: Clinical features, outcome, and prognostic factors in 101 cases[. J] Eur J Haematol. 2018;101:379–88. Tian Geng T, Fangrong YC et al. Quantitative dot blot analysis (QDB), a versatile high throughput immunoblot method.[J].Oncotarget, 2017, 8: 58553–58562. Zhang Wenfeng Y, Guohua, Zhang Y, et al. Quantitative Dot Blot (QDB) as a universal platform for absolute quantification of tissue biomarkers[. J] Anal Biochem. 2019;576:42–7. Qi Xiaoying Z, Yunyun Z, Yuan et al. High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB).[J].J Vis Exp, 2018, 10.3791/56885 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3862754","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":267154910,"identity":"02c73bb1-c1aa-4472-b763-0e5a7d352ec9","order_by":0,"name":"ShiShou 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08:59:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3862754/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3862754/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49766969,"identity":"4acdffdd-e908-45f6-b8c3-8195188db495","added_by":"auto","created_at":"2024-01-17 17:07:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":152816,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical staining results showed that ENKTCL patients with Ki67 \u0026gt; 60% had worse prognosis(A) and QDB results showed that patients with MIB-1 (Ki67) \u0026gt; 11.9 nmol/g had worse prognosis(B).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3862754/v1/eb4365e36cfb49cf033aa78f.png"},{"id":49766171,"identity":"92ca2c54-f6c0-42d3-86bf-f501a1fd2204","added_by":"auto","created_at":"2024-01-17 16:59:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":499363,"visible":true,"origin":"","legend":"\u003cp\u003ePINK model(A) showed there was significant difference of prognosis between low-risk group, middle-risk group and high-risk group(P \u0026lt; 0.05); NRI model(B) showed there was significant difference of prognosis between low-risk group, middle-risk group and high-risk group(P \u0026lt; 0.05); Ki67 (IHC) modified PINK model(C) showed the prognosis of each risk group was significantly different (P \u0026lt; 0.05); Ki67 (IHC) results modified NRI model(D) showed the prognosis of each risk group was significantly different (P \u0026lt; 0.05); Ki67 (QDB) modified PINK model(E) showed the prognosis of each risk group was significantly different (P \u0026lt; 0.05); Ki67 (QDB) modified NRI model(F) showed the prognosis of each risk group was significantly different (P \u0026lt; 0.05); Log-rank test.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3862754/v1/d1f9c3f0f50855d50c4ab007.png"},{"id":49766970,"identity":"48cfce1b-f337-4788-a1ea-2a59aaca54fe","added_by":"auto","created_at":"2024-01-17 17:07:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":116799,"visible":true,"origin":"","legend":"\u003cp\u003eROC curves and AUCs (A) of PINK model, PINK+Ki67(IHC) model and PINK+Ki67(QDB) model are respectively 0.68, 0.67 and 0.74. The C-index (C) of the three models is respectively 0.70, 0. 68 and 0.75. ROC curves and AUCs(B) of NRI model, NRI+Ki67(IHC) model and NRI+Ki67(QDB) model are respectively 0.72, 0.74 and 0.80; The C-index (D) of the three models is respectively 0.70, 0.69 and 0.71.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3862754/v1/e0090c06ef0a22d190d844c3.png"},{"id":49777838,"identity":"82a37bf6-f376-4bd3-818c-b43b77fa910b","added_by":"auto","created_at":"2024-01-17 21:52:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":915311,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3862754/v1/3d2e6405-4ac8-40a6-b1e6-310f41b1a406.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Modification of Ki67 on Prognostic Model of Extranodal NK/T Cell Lymphoma","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eExtranodal nasal NK/T cell lymphoma (ENKTCL) is a non-Hodgkin's lymphoma with high invasiveness and heterogeneity. Despite the combination of chemotherapy and radiotherapy, the recurrence rate and mortality of ENKTCL are still on the rise. It is particularly important to how to accurately identify patients with high prognosis in the early stage of ENKTCL. NK cell lymphoma prognosis index (PINK) and Nomogram improved risk index (NRI) prognosis models are increasingly recognized by clinicians. However, only clinical factors are included in PINK and NRI models, and no prognostic model based on clinical and pathological parameters has been reported at present. In this study, 106 patients with ENKTCL were used to verify PINK and NRI, and to explore the modifying effect of Ki67 on prognosis model.\u003c/p\u003e"},{"header":"PATIENTS AND METHODS","content":"\u003cp\u003ePatient Selection\u003c/p\u003e \u003cp\u003eFrom January 1, 2008 to June 30, 2020, 106 patients with extranodal nasal NK/T cell lymphoma treated in Yantai YuHuangding Hospital and the affiliated Hospital of Nantong University were studied. All cases were confirmed by pathological biopsy HE staining, immunohistochemical staining and in situ hybridization staining.\u003c/p\u003e \u003cp\u003eMethods\u003c/p\u003e \u003cp\u003eThe Immunohistochemical Staining of Ki67 (clone number: MIB-1) was Carried out by EnVision Method.\u003c/p\u003e \u003cp\u003eThe antibodies used were purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd..\u003c/p\u003e \u003cp\u003eImmunohistochemical interpretation method: Each immunohistochemical staining section was set as an external control, and the staining results were interpreted by three pathologists and averaged. The positive signal of Ki67 immunohistochemical staining was marked by brown-yellow granules or lumpy granules in the nucleus, which can be identified as positive tumor cells. At high magnification, positive cells in 10 fields were counted randomly and averaged.\u003c/p\u003e \u003cp\u003eExperimental Steps of Quantitative Dot Blot (QDB) Detection\u003c/p\u003e \u003cp\u003eQDB test was performed according to the method reported by Yu et al\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eIn this study, SPSS 19.0, R 4.1. 0 software were used for statistical analysis. P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was statistically significant. Kaplan-Meier method was used for survival analysis, and Log-rank test was used to compare the differences among different groups. Hmisc, survival ROC\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e and Time-dependent ROC analysis packets\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e were used to calculate the area under C-index and ROC curves.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003ePatients Characteristics\u003c/p\u003e \u003cp\u003eA total of 106 patients, 75/106 (70.7%) patients were male and 31/106(29.3%) were female. The onset age of patients ranged from 14 to 86 years old, and the median onset age was 60 years old. Among 106 patients, 69/106(65.1%)of them occurred in nasal cavity, 8/106༈7.5%༉occurred in skin, 7/106༈6.6%༉occurred in gastrointestinal tract, 8/106༈7.5%༉occurred in pharynx, 3/106 (2.8%) occurred in lung, 3/106 (2.8%) occurred in oral cavity, 1/106 occurred in paranasal sinuses, abdominal wall, liver, neck, breast, parotid gland, vocal cords and penis (each accounted for 0.9%).\u003c/p\u003e \u003cp\u003eEffect of Ki67 on prognosis of ENKTCL Patients\u003c/p\u003e \u003cp\u003eThe results of Kaplan-Meier survival analysis showed that patients with positive rate of Ki67\u0026thinsp;\u0026gt;\u0026thinsp;60% by immunohistochemistry had poor prognosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The absolute quantitative results of QDB protein showed that patients with MIB-1 (Ki67)\u0026thinsp;\u0026gt;\u0026thinsp;11.9 nmol/g in tumor tissue had poor prognosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eValidation of Predictive Ability of NRI/PINK Prognosis Model\u003c/p\u003e \u003cp\u003e106 patients with NK/T-cell lymphoma were divided into low-risk group, middle-risk group and high-risk group by PINK scoring rule (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The prognosis difference of each risk group was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). AUC (area under ROC curve)\u0026thinsp;=\u0026thinsp;0.68 C-index was 0.70 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA 3A/C).\u003c/p\u003e \u003cp\u003e106 patients with NK/T cell lymphoma were divided into middle and low risk groups by NRI scoring rules (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The prognosis difference of each risk group was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). AUC\u0026thinsp;=\u0026thinsp;0. 72C-index was 0.70 (Fig.\u0026nbsp;2B3B/D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eModification of Predictive Ability of NRIPINK Prognostic Model by the Absolute Quantitative Results of Ki67 Protein\u003c/p\u003e \u003cp\u003eThe results of Ki67 immunohistochemistry and protein quantification were added to the existing score factors of PINK, and 106 patients were regrouped as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC/E and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA/C. All patients were divided into low-risk group, middle-risk group and high-risk group, and the prognosis difference of each risk group was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The area under ROC curve of PINK model modified by Ki67 was AUC\u003csub\u003ePINK+Ki67(IHC)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.67, AUC\u003csub\u003ePINK+Ki67(QDB)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.74, C-index\u003csub\u003ePINK+Ki67(IHC)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.68 and C-index\u003csub\u003ePINK+Ki67(QDB)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.75.\u003c/p\u003e \u003cp\u003eThe results of Ki67 immunohistochemistry and protein quantification were added to the existing NRI scoring factors. 106 patients were regrouped as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD/F and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB/D. All patients were divided into middle and low risk groups, middle and high risk groups, high risk groups and extremely high risk groups. The prognosis of each risk group was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The area under ROC curve of NRI model modified by Ki67 was AUC\u003csub\u003eNRI+Ki67(IHC)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.74, AUC\u003csub\u003eNRI+Ki67(QDB)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.80, C-index\u003csub\u003eNRI+Ki67(IHC)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.69 and C-index\u003csub\u003eNRI+Ki67(QDB)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.71.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eAnn Arbor staging was the most controversial prognostic model of ENKTCL\u003csup\u003e[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. It was a primary nodal lymphoma-based staging method for Hodgkin's lymphoma and was also used for non-Hodgkin's lymphoma staging. However, ENKTCL was different from other types. Its lesions were usually confined to nasal cavity, invaded adjacent structures or soft tissues of tumor, which belonged to Ann Arbor stage I and II patients. Therefore, Ann Arbor staging system could not correctly evaluate the effect of local tumor invasion on prognosis. Therefore, in recent years, some scholars had tried to establish a new prognosis evaluation model by increasing or decreasing individual significant factors based on the age, Ann Arbor stage, LDH, ECOG score and distant lymph node metastasis included in IPI score \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e such as NK lymphoma prognosis index (PINK) and Nomogram improved risk index (NRI). The results of this study could validate the above prognostic model, but the patients' prognostic risk grouping was not clear, and it was difficult to identify patients with poor prognosis early and accurately, which was easy to delay treatment. Both PINK and NRI prognostic models were clinical parameters. ENKTCL prognostic model based on clinical and pathological parameters was the development direction. Ki67 was a pathological marker that reflects the proliferation of tumor cells. It hadd been confirmed that Ki67 was a prognostic factor of ENKTCL, and the determination of its critical value was still controversial. Results\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e showed that Ki67\u0026thinsp;\u0026ge;\u0026thinsp;50% or Ki67\u0026thinsp;\u0026ge;\u0026thinsp;65% was a poor prognostic factor. This study showed that Ki67 proliferative index\u0026thinsp;\u0026gt;\u0026thinsp;60% was a poor prognostic factor for ENKTCL. All the above thresholds were limited by semi-quantitative detection determined by immunohistochemical staining results. QDB was a method for detecting the protein expression level of high-throughput samples by simplifying the experimental process of Western Blotting(Western Blot, WB)\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. In the experimental process, only one antibody suitable for immunohistochemistry was used, but QDB technique can not only supplement the shortcomings that immunohistochemistry can not be absolutely quantified, but also avoided the influence of staining quality and interpretation error of immunohistochemistry\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Based on previous studies, QDB technology had a clear role in providing absolute quantification of protein in high-throughput research. It could reduce the bias of protein in quantitative level and was expected to become an important experimental technology for clinical pathology laboratory detection. The group of tumor samples were detected by QDB protein and statistical analysis showed that Ki67\u0026thinsp;\u0026gt;\u0026thinsp;11.9 nmol/g were all ENKTCL patients with adverse prognostic factors. It was consistent with the immunohistochemical results in previous studies and this study. Based on the data, the results showed that PINK and NRI could predict the prognosis of the patients. However, NRI model had insufficient ability to accurately identify patients with poor prognosis in early stage. Although PINK model can predict patients, the area under ROC curve was only 0.68, the generalization was poor, and the reliability of prediction results was insufficient. Based on the above experimental statistical results, the modified PINK and NRI models with Ki67 immunohistochemistry and QDB results still had outstanding predictive effect. The prognosis of each risk group was significantly different, especially with the modification of Ki67 QDB, the prediction of the new model was further improved, and the AUC of PINK\u0026thinsp;+\u0026thinsp;Ki67(QDB) model or NRI\u0026thinsp;+\u0026thinsp;Ki67(QDB) model was significantly higher than that model of PINK\u0026thinsp;+\u0026thinsp;Ki67(IHC) or NRI\u0026thinsp;+\u0026thinsp;Ki67(IHC).\u003c/p\u003e \u003cp\u003eBoth the PINK model、NRI model and the modified new model can predict the prognosis of patients in this study, but neither NRI model nor modified NRI model can accurately predict the prognosis of all patients, especially it was impossible to accurately predict the prognosis trend of patients in middle and high-risk groups, high-risk groups and extremely high-risk groups at the early stage of illness. The advantage of modified PINK model over NRI model was not only that it can accurately predict the prognosis of patients in the early stage of illness, PINK model modified by Ki67 (QDB) has higher AUC and C index than PINK model modified by Ki67 (QDB). Therefore, the new PINK prognosis model based on clinical and pathological parameters can predict and described the prognosis of ENKTCL patients more accurately by incorporating Ki67(QDB) into PINK model with a threshold of 11.6 nmol/g.\u003c/p\u003e"},{"header":"CONCULUSION","content":"\u003cp\u003eKi67 QDB protein absolute quantitative detection results can significantly enhance the predictive ability of PINKNRI model, PINK model modified by Ki67 QDB can more accurately describe the prognosis of ENKTCL patients. QDB protein absolute quantitative detection technology was expected to be applied to clinical laboratory detection to achieve individualized and accurate diagnosis and treatment of tumors.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eDisclosure of conflict of interest\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003ch2\u003eData Availability Statements\u003c/h2\u003e\n\u003cp\u003eThe data that support the findings of this study are available on request from the first author. The data are not publicly available due to privacy or ethical restrictions.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eGuohua Yu, Jiandi Zhang and Shishou Wu designed the study and drafted the manuscript. Wenfeng Zhang, Lei Jiang and Liling Song participated in immunohistochemical staining. Ping Yang searched the literatures and performed the histological evaluation. Licai An, Yuanfeng Zhang , Yunjun Wang, Yu Pan ​and Yuan Gao participated in providing the clinical information. Yifei Liu and Guohua Yu participated in revising the manuscript. All authors read and approved the final manuscript. All authors have agreed to authorship and order of authorship for this manuscript and that all authors have the appropriate permissions and rights to the reported data.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003e In this study, all methods were carried out in accordance with relevant guidelines and regulations. The clinical data of all patients were based on objective facts. All statistical methods were carried out in accordance with international uniform rules.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYu Guohua Z, Wenfeng Z. Developing a routine lab test for absolute quantification of HER2 in FFPE breast cancer tissues using Quantitative Dot Blot (QDB) method.[J]. Sci Rep. 2020;10:12502.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWan Jiaming G, Cheng F, Hongpeng, et al. Autophagy-Related Long Non-coding RNA Is a Prognostic Indicator. for Bladder Cancer [J] Front Oncol. 2021;11:647236.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X-P, Yuan-Xing G, Shuai X, et al. A novel online calculator to predict early recurrence and long-term survival of patients with resectable pancreatic ductal adenocarcinoma after pancreaticoduodenectomy: A multicenter study[. J] Int J Surg. 2022;106:106891.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Ke-feng et al. Chang Bo-yang,Chen Xiao-qin,. A prognostic model based on pretreatment platelet lymphocyte ratio for stage IE/IIE upper aerodigestive tract extranodal NK/T cell lymphoma, nasal type.[J].Med Oncol, 2014, 31: 318.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuan Pujun D, Tian Z, Li et al. Staging challenges in extranasal and juvenile extranodal NK/T-cell lymphoma.[J].Leukemia, 2020, 34: 3428\u0026ndash;3431.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang J-J, Ying-Jie Z, Yi X, et al. A novel prognostic model for extranodal natural killer/T-cell lymphoma. [J] Med Oncol. 2012;29:2183\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Zhang Y-J, Zhu Y et al. Prognostic nomogram for overall survival in previously untreated patients with extranodal NK/T-cell lymphoma, nasal-type: a multicenter study.[J].Leukemia, 2015, 29: 1571\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim SJ, Yoon DH, Jaccard A, et al. A prognostic index for natural killer cell lymphoma after non-anthracycline-based treatment: a multicentre, retrospective analysis. Lancet Oncol. 2016;17:389\u0026ndash;400.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim SJ, Kim BS, Choi CW, et al. Ki-67 expression is predictive of prognosis in patients with stage I/II extranodal NK/T-cell lymphoma. nasal type [J] Ann Oncol. 2007;18:1382\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHung SY-JWP-NC, et al. Extranodal NK/T-cell lymphoma, nasal type: Clinical features, outcome, and prognostic factors in 101 cases[. J] Eur J Haematol. 2018;101:379\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian Geng T, Fangrong YC et al. Quantitative dot blot analysis (QDB), a versatile high throughput immunoblot method.[J].Oncotarget, 2017, 8: 58553\u0026ndash;58562.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Wenfeng Y, Guohua, Zhang Y, et al. Quantitative Dot Blot (QDB) as a universal platform for absolute quantification of tissue biomarkers[. J] Anal Biochem. 2019;576:42\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQi Xiaoying Z, Yunyun Z, Yuan et al. High Throughput, Absolute Determination of the Content of a Selected Protein at Tissue Levels Using Quantitative Dot Blot Analysis (QDB).[J].J Vis Exp, 2018, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3791/56885\u003c/span\u003e\u003cspan address=\"10.3791/56885\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"NK/T-cell lymphoma, Prognosis, Ki-67, QDB","lastPublishedDoi":"10.21203/rs.3.rs-3862754/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3862754/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e To explore the value of Ki67 in the prognostic evaluation of extranodal NK / T cell lymphoma and the modification effect of NK cell lymphoma prognostic risk index (PINK) and Nomogram modified risk index (NRI).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: A retrospective analysis of the clinical data of 106 patients with extranodal nasal NK/T cell lymphoma diagnosed in Yantai Yuhuangding Hospital and the affiliated Hospital of Nantong University from 2008 to 2020. Ki67 immunohistochemical staining and Quantitative Dot Blot (QDB) which is an absolute protein quantitative detection were performed on pathological paraffin specimens, and the R4.1.0 and SPSS were used to analyze the data and determine the threshold.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e A total of 106 patients,75/106 (70.7%) patients were male and 31/106(29.3%) were female. The onset age of patients ranged from 14 to 86 years old, and the median onset age was 60 years old. As of September 10, 2020, 105 patients were followed up, 54/105(51.4%) patients were died, 51/105 (48.6%) patients were survived, and the median survival time was 330 days. Immunohistochemical results showed that patients with Ki67 proliferation index \u0026gt; 60% or MIB1 (Ki67) \u0026gt; 11.9 nmol/g had worse prognosis. The data were modified by Ki67 (IHC) and Ki67 (QDB), and the survival analysis of PINK and NRI models showed that there were statistically significant differences among the risk groups (P \u0026lt; 0.001). Compared with Ki67 (IHC), Ki67 (QDB) modified PINK and NRI models could significantly improve their predictive ability (AUC\u003csub\u003ePINK\u003c/sub\u003e, 0.67 vs 0.74; C-index\u003csub\u003ePINK\u003c/sub\u003e, 0.68 vs 0.75; AUC\u003csub\u003eNRI\u003c/sub\u003e, 0.74 vs 0.80; C-index\u003csub\u003eNRI\u003c/sub\u003e, 0.69 vs 0.71).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThe results of Ki67 by QDB could significantly enhance the predictive ability of PINK and NRI models. PINK model modified by Ki67 QDB can more accurately describe the prognosis of ENKTCL patients. The absolute quantitative detection technology of QDB protein is expected to be applied to clinical laboratory detection to achieve individualized and accurate diagnosis and treatment of tumors.\u003c/p\u003e","manuscriptTitle":"Modification of Ki67 on Prognostic Model of Extranodal NK/T Cell Lymphoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-17 16:59:23","doi":"10.21203/rs.3.rs-3862754/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"fd62bdeb-5834-4f42-90c0-bd0d6a903df7","owner":[],"postedDate":"January 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-20T02:59:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-17 16:59:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3862754","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3862754","identity":"rs-3862754","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}