Ki67 Assessment By qRT-PCR In OncotypeDx® Breast Recurrent Score®: Low Correlation With Immunohistochemistry Assessment of Ki67 And Prognostic Impact of Ki67 RNA Level of Expression.

Ki67 Assessment By qRT-PCR In OncotypeDx® Breast Recurrent Score®: Low Correlation With Immunohistochemistry Assessment of Ki67 And Prognostic Impact of Ki67 RNA Level of Expression. | 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 Ki67 Assessment By qRT-PCR In OncotypeDx ® Breast Recurrent Score ® : Low Correlation With Immunohistochemistry Assessment of Ki67 And Prognostic Impact of Ki67 RNA Level of Expression. Zohair Selmani, Chloé Molimard, Alexis Overs, Fernando Bazan, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-677106/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Breast cancers expressing high levels of Ki67 are associated with poor outcomes. Oncotype DX ® test was designed for ER+/HER2- early-stage breast cancers to help adjuvant chemotherapy decision by providing a Recurrent Score ® (RS ® ). RS ® measures the expression of 21 specific genes from tumor tissue, including Ki67 . The primary aim of this study was to assess the agreement between Ki67 RNA obtained with Oncotype DX ® RS ® and Ki67 IHC . Other objectives were to analyze the association between the event free survival (EFS) and the expression level of Ki67 RNA ; and association between RS ® and Ki67 RNA . Herein, we report a low agreement of 0.24 by Cohen’s kappa test between Ki67 IHC and Ki67 RNA in a cohort of 98 patients with early ER+/HER2- breast cancers. Moreover, Ki67 RNA high tumors were significantly associated with the occurrence of events ( p =0.02). On the other hand, we did not find any association between Ki67 IHC and EFS ( p =0.25). We observed of low agreement between expression level of Ki67 RNA and Ki67 protein labelling by IHC. Unlike Ki67 IHC and independently of the RS ® , Ki67 RNA could have a prognostic value. It would be interesting to better assess the prognosis and predictive value of Ki67 RNA measured by qRT-PCR. The Ki67 RNA in medical routine could be a good support in countries where Oncotype DX ® is not accessible. Molecular Biology Cancer Biology Breast cancer chemotherapy prognosis Oncotype Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction In terms of incidence, prevalence and mortality, breast cancer is in first place worldwide in women 1 . The vast majority of patients with breast cancer does not show detectable distant metastasis on diagnosis. Treatment of early breast cancer is based on surgical tumor resection with conditional lymph node dissection. Adjuvant systemic treatments including endocrine therapy (HT) and chemotherapy (CT) aim at the reduction of the distant recurrence rate and improvement of breast cancer specific survival. The decision of adjuvant therapeutic modalities is taken according to several prognostic and/or predictive factors including patient age, tumor size, histological type and grade, lymph node involvement and expression on the tumor of hormone receptors for estrogen (ER) progesterone (PR), and the human epidermal growth factor receptor 2 (HER2) as well as the percentage of tumor cells expressing the nuclear proliferation marker Ki67 2 . Genomic signatures were designed to give prognostic and predictive information to streamline adjuvant chemotherapy decision in ER-positive, HER2-negative breast cancer patients. Oncotype DX ® Breast (ODX ® ) is the most widely used molecular signature in this setting and is included in treatment guidelines for estimating both the risk of distant recurrence and predicting adjuvant chemotherapy benefit. ODX ® measures the RNA of 21 genes (16 cancer-associated genes and 5 housekeeping genes) and uses the expression pattern to calculate a recurrence score (RS ® ) that ranges between 0 and 100 3 . The RS ® result provides two types of information on tumor biology: (i) prognosis information: an estimate of the individual risk of distant cancer recurrence within 10 years, (ii) predictive information: an estimate of the likelihood of a benefit from chemotherapy 4–6 . Interestingly in breast cancer, Ki67 RNA (Ki67 RNA ) is a parameter analyzed by several molecular signatures such as PAM50 and ODX 7 . Furthermore, Ki67 is an interesting biomarker in early breast cancer and breast cancers expressing high levels of Ki67 are associated with poor outcomes 8–10 . To provide individualized patient care in the concept of precision medicine, reliability of prognostic and predictive information deriving from Ki67 value is essential 11,12 . Some studies 13,14 indicate that lowering in Ki67 expression after neoadjuvant endocrine treatment may predict long-term outcome. Nevertheless, substantial variability in Ki67 staining of breast cancer tissue by immunohistochemistry (IHC) and interpretation was found between 30 routine pathology labs. Clinical use of Ki67 staining for therapeutic decisions should be considered with caution and only fully aware of lab-specific reference values 15 . Ki67 staining lacks scoring standardization ; various studies have focused on assessment methodology standardization 16 , interobserver reproducibility 17 and digital image analysis of Ki67 staining 18,19 . However, little is known about variability in IHC Ki67-labelling results between routine pathology labs 20,21 and its potential influence on interpretation of Ki67 levels in breast cancer. When using Ki67 assessment by IHC in order to consider an indication of adjuvant chemotherapy 22 , clinicians should be aware of the low reproducibility of Ki67 scoring and its questionable analytical validity. In the present study, we analyzed 98 patients tested by the Oncotype DX ® Breast from June 2012 to April 2014. For this cohort, Ki67 RNA level obtained in patients’ Oncotype DX ® signatures were available. The primary aim of this study was to assess the agreement between Ki67 RNA and Ki67 staining by IHC (Ki67 IHC ). The other objectives were to analyze the association between the event free survival (EFS) and the expression level of Ki67 RNA in ODX ® signature; and association between RS ® and Ki67 RNA . Results Characteristics of the patient population (Table 1) Complete data sets from 98 breast cancer patients who underwent RS® testing were provided from 4 public treatment centers (public hospitals and university hospitals). The patients included were exclusively female and showed a wide age distribution (31 to 81 years) with a mean age of 57 years. The predominant tumor characteristics were no special type (NST) (91%), N0 or Nmic (71%), grade 1 (62%), and tumor size pT1c (1–2 cm) (58%). All patients had ER positive/ HER2 negative tumors. Table 1 shows the patient and disease characteristics of the full population. The RS® values were 30 in 11% (n = 11) of the patients. After surgery and collegial decision, all patients have received a treatment according the result of ODX® test (HT alone or CT-HT) in adjuvant situation. During the follow-up (57 months), we observed 19 events (19%): 3 local relapses, 10 metastatic relapses (bone, lung, liver and pancreas), 3 other cancers (contralateral breast, colorectal carcinoma and pancreatic carcinoma) and 3 deaths. Agreement between Ki67 IHC and Ki67 RNA The Ki67 IHC positivity rate of > 20% 23 was used to define for the “high-risk” tumor group. We showed by ROC curve analysis an optimal threshold at 6.35 with a specificity of 48% and a sensitivity of 84%. With this cut off, the Ki67 RNA high were > 6.35 and Ki67 RNA low were < 6.35 (Fig. 1 ). A correlation between Ki67 RNA expression and Ki67 IHC score was found (R = 0,36; p = 3.10 − 4 IC 95% [0.2–0.5]) (Supplementary data 2 ) . In the cohort, 29 tumors were Ki67 high and 29 were Ki67 low by the two methods, which represent a match between the results in 58 patients (61%) (Table 2 ). Discordances were observed in 37 patients (39%): 10 samples were Ki67 IHC low /Ki67 RNA high and 27 samples were Ki67 IHC high /Ki67 RNA low . An agreement of 0.24 was found by Cohen’s kappa test. Events-Free Survival among all patients according RS® groups and Ki67 status A significant relationship was observed between EFS and the level of Ki67 RNA expression ( p = 4.10 − 2 ) (Fig. 2 A) unlike Ki67 IHC ( p = 0.25) (Fig. 2 B). However, we did not observe any significant difference on EFS according to the three RS® groups risk ( p = 0.4) (Fig. 3 ). Using the age categories from the TAILORx study 24 , the association between EFS and Ki67 RNA status, was found only for women under 50 years ( p = 0.01) and not for women over 50 years ( p = 0.36) (Supplementary data 3). No association between Ki67 IHC and EFS was observed ( p > 0.20). Ki67 RNA association with RS® As expected, Ki67 RNA levels were significantly associated with RS® ( p = 5.10 − 4 ) (Fig. 4 A). To a lesser extent, an association between ODX® test with Ki67 IHC status has also been observed ( p = 0.013) (Fig. 4 B). Supplementary data 4 shows the distribution of Ki67 IHC score and Ki67 RNA level according to patient age, tumor grade and size, PR status, nodal status, Recurrence score, treatment and events. Discussion Study population In this study, we focused on the Ki67 RNA expression level of 98 patients. The majority of our data are consistent with the PONDx real life study 25 which found a female population over 50 (70%) with NST tumors (78%) ranging between 2–5 cm (89%), grade II (68%) and without lymph node involvement in 79% of cases. The PONDx cohort included more grade II than in our population (68% vs 29%) with a higher contingent of lobular carcinoma (13% vs 7%). In PONDx, RS ® results by prognostic category used between 2012–2014 were: 30: 10%. Compared to this real-life study, the local cohort shows an inversion of the proportions between populations at low risk (38% vs 54%) and those at intermediate risk (51% vs 36%). The few differences observed in the distribution of the risk groups established by the ODX ® test can be explained by an over-representation of grade II tumors in the PONDx study. Our cohort is comparable to this largest population for most epidemiologic parameters. Differences between Ki67 RNA and Ki67 IHC Comparisons of Ki67 status showed a discrepancy between the two different types of evaluation. Indeed, half of Ki67 RNA high (> 6.35) were found Ki67 IHC low (Table 2 ). The observed correlation between the two techniques, is therefore weak even if significant (R = 0.36, p = 3.10 -4 ; IC 95% [0.2–0.5]) (Supplementary data2). Technically, IHC shows greater variability 20,21 and is less reproducible than the quantification of mRNAs. Currently, there is no standardization for the interpretation of the Ki67 status by IHC 16 . As a consequence, it generates inter-site and inter-observer variability within the same site 17 . In contrast, the Oncotype test which is a molecular based and standardized test provides objective results that are independent of the observer. Furthermore, the ODX test analyzes the level of Ki67 messenger RNAs in the tumor sample. This analysis therefore considers the transcriptional status of the Ki67 gene level not only in tumor cells but also in associated cells such as lymphocytes, stromal or endothelial cells. Therefore, it is an overall estimate. By IHC, the protein expression is scored only on tumor cells. The two methods do not provide the same types of information on Ki67 status. These two types of evaluation of Ki67 are probably complementary because the Ki67 IHC informs us about the tumor specific proliferation index and the Ki67 RNA would reflect the proliferation of the tumor and its microenvironment. For all the reasons mentioned above, the association between RS ® and Ki67 IHC status is lower but remains significant ( p = 0.013). Events free survival analyses On the one hand in this study, adaptation of treatment based on RS ® permit to obtain equivalent EFS in populations with low and high risk of recurrence. We have modestly confirmed the efficiency of ODX ® test. Indeed, the lack of association between RS ® and EFS can be explained by the treatment (HT alone or CT-HT) performed according to the score. The clinical validity and utility of the RS ® has been demonstrated prospectively across multiple studies in breast cancer patients worldwide including multiple validation studies as well as long-term prospective studies (TAILORx 24 , WSG Plan B 26 and analyses from a prospective epidemiological database 27 ). Based on level Ia evidence, the Oncotype DX test has been incorporated in leading internationally-accepted clinical guidelines on the treatment of early breast cancer (St. Gallen 28 , ESMO 29 , and ASCO 30 ). This therapeutic adaptation will therefore smooth the differences between the categories of the ODX ® test and at the same time demonstrates the value of the information provided by its score. On the other hand, we were able to show that Ki67 RNA high was significantly associated with the occurrence of events ( p = 0.02) but we did not find any association between Ki67 IHC and EFS ( p = 0.25). Our data relate to a small population but leaves the possibility of a larger study in order to confirm these that Ki67 RNA evaluation has a prognostic impact as RS ® . Correlation between Ki67 RNA and RS ® As expected in our cohort, the RS® obtained is strongly associated with the Ki67 RNA ( p = 5.10 − 4 ) which is one of the components of this test (Fig. 4 A). To optimize RS® interpretation, the TAILORx subgroup study shows a benefit of CT in women ≤ 50 years old with an RS® of 16 to 25 ( p = 0.004). CT could be avoided in women over 50 years old with a RS® <26, in women 50 years old or less with an RS® <16 31 . Recently in San Antonio Breast cancer symposium, the results of RxPONDER study were presented 32 . And now, postmenopausal women with 1–3 positive nodes and RS® 0–25 can safetly forego adjuvant CT without compromising invasive disease-free survival. The premenopausal women with positive nodes and RS 0–25 likely significantly benefit from chemotherapy 32 . Oncotype DX® Breast enables relevant net reductions in chemotherapy use, sparing patients from serious toxicities 7 . On this other side, its clinical impact and pharmacoeconomic benefit in routine care have been shown in 20 decision-impact studies 33− 35 . However, in many places, the oncotype score remains inaccessible or not reimbursed 7,25,35 . This is why the evaluation of the level of Ki67 RNA by a molecular biology techniques 36,37 could be a low-cost prognostic alternative in some countries. But this hypothesis will have to be validated during a prospective translational study. In conclusion, we observed of low agreement between Ki67 RNA tumor level measured by qRT-PCR and Ki67 protein labelling by IHC. Substantial variability in Ki67 IHC of breast cancer tissue and interpretation have been shown and widely published. The clinical use of Ki67 labelling should be cautious and limited by the low reproducibility of Ki67 scoring and its questionable analytical validity. Unlike Ki67 IHC and independently of the RS ® , Ki67 RNA could have a prognostic value. The assessment of Ki67 RNA by qRT-PCR on breast cancer tumor would be feasible and cost effective. It would be of great clinical utility to better assess the prognosis and predictive value of Ki67 RNA . Presentation of Ki67 status in Genomic Health report could also be helpful for therapeutic decisions on borderline situation. Methods Ethics statement All procedures performed in studies involving human participants were in accordance with the ethical standards of the national research committee and with the 1964 Helsinki Declaration and its later amendments. In France, this search is considered like a non-interventional study according to European legislation. All patients were individually informed that their data should be used to scientific research. Informed and written consents were obtained from all subjects, or if subject are deceased, from a parent and/or legal guardian. All experimental protocols were approved by « comité de protection des personnes » (CCP) of Besançon, France. Patients and tumors characteristics Our study was an observational multicenter retrospective study collecting data on the real-life use of Oncotype DX Breast Recurrence Score ® test by physicians in clinical practice settings in France. Patients eligibility criteria for this analysis correspond to the population for which the Oncotype DX ® Breast Recurrence Score ® test is validated, i.e. adult patients with a recent first diagnosis of a single early invasive breast tumor with ER+/HER2- status, plus available documentation of lymph node involvement as either N0 (node negative), Nmic (micrometastatic) or N1 (1–3 positive nodes). The following data were documented: patient age and sex, conventional clinical and pathological disease characteristics including histologic type, tumor size and grade, nodal status, receptor status including ER, PR, and HER2, and the Ki67 proliferation marker by IHC. Between 2012 and 2014, the RS ® results were interpreted in three categories (low risk; intermediate risk; high risk with two cut-offs: 18 and 30). The event free survival (EFS) includes the local or metastatic relapse, other cancers and death with a mean follow-up of 57 months. Following the guidelines, the Oncotype DX ® Breast Recurrence Score ® test was realized after surgery and adjuvant treatments were adapted according to the RS ® . The RS ® values and the associated treatment (hormonal therapy plus/minus chemotherapy, or other modalities) were also collected. Immunohistochemistry analyses. The hormonal receptor status (ER and PR) was considered positive according to standardized European guidelines using a cut-off of ≥ 10% stained tumor cell nuclei. The Ki67 IHC evaluation was realized in 2 publics and 2 private pathological departments. In these structures, the Ki67 IHC was assessed with monoclonal antibody MIB1 16 . Based on recommendations established by St Gallen 2015, the Ki67 IHC high corresponding to Ki67 > 20%, defined “high-risk” subpopulations of tumors 23 . Ki67 RNA threshold determination and events statistical analyses Descriptive statistics were used to summarize clinicopathological characteristics. Variables were described by the size and rate. After normalization, levels of Ki67 RNA were distributed according to a Gaussian curve (Supplementary data 1). We have determined the optimal threshold using R package pROC version 1.16.1 38 . The statistical comparison between IHC and ODX® was realized by Cohen’s kappa test. Statistical analyzes were performed using R version 3.6.2 39 . The survival follow-up was analyzed by the package survival version 3.1-8 40 . Data availability The datasets generated and analyzed during this study (birthdate, admission date, discharge date, date of death…), are available from the corresponding author on reasonable request. Declarations Acknowledgements We thank all patients who agreed to participate in this observational study. Their also thank Genomic Health, Inc. (now Exact sciences) and Mahi Y. for the Ki67 RNA data. The authors are grateful to all participating physicians for their cooperation and for private and public anatomopathology departements (CH de Trevenans, Besancon Pathologie and Dr Chargeboeuf M.) for their participation in IHC analysis. Author information Affiliations 1 Department of Oncobiology, University Hospital of Besançon, Besançon, France. 2 Department of Medical Oncology, University Hospital of Besançon, Besançon, France. 3 Department of Anatomopathology, University Hospital of Besançon, Besançon, France. 4 Bourgogne Franche-Comté university, INSERM, UMR1098, Besançon, France. 5 ICANS, Strasbourg, France. 6 EA3181, UBFC, UFC, Besançon, France. Contributions Conception and design: EC, ZS. Development of methodology: ZS, EC. Acquisition of data : EC, ZS, FB, ED, LC, LM, NM, MJP, GM, JV, XP. Analysis and interpretation of data: ZS, AO, CM, JLP, JPF. Writing, review, and/or revision of the manuscript : ZS, CM, JLP, EC. Administrative, technical, or material support: EC, ZS, JLP, MPA, JPF, CB. Study supervision: EC, ZS. Corresponding author Correspondence to Zohair Selmani Additional information Competing interests ZS: Honoraria from Pfizer, Genomic Health EC: Honoraria from Novartis, Pfizer, Daiichi, Genomic Health FB: Honoraria from Roche, Novartis, Pfizer, Astra-Zeneca, Clovis, Daiichi. CB: advisory board from Bayer, BMS, msd, servier and research grant from Roche NM, JV, CM, AO, JLP, JPF, ED, LM, MPA, LC, MJP, GM, XP: have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Erratum Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA. Cancer J. Clin , 70 , 313–313 (2020). Curigliano, G. et al. De-escalating and escalating treatments for early-stage breast cancer: the St. Gallen International Expert Consensus Conference on the Primary Therapy of Early Breast Cancer 2017. Ann. Oncol. 28, 1700–1712(2017). Gluz, O. et al. 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In this population, 98 women were included from June 2012 to April 2014. All patients were tested by Oncotype DX ® Breast. The Nodal status was stratified in 3 groups: N0 (node negative), Nmic (micrometastatic) or N1 (1-3 positive nodes). The progesterone receptor status (PR) was considered positive according to standardized European guidelines using a cut-off of ≥10% stained tumor cell nuclei. All patients were ER positive and HER2 negative. The Ki67 IHC positivity was defined by the rate of >20% established by St Gallen 2015 recommendations. Almost, The Recurrence score results (RS ® ) are interpreted in three categories (low risk: RS ® <18; intermediate risk: RS ® = 18-30; high risk: RS ® ≥31). The events include the local or metastatic relapse, other cancers and death. HT: Hormonal therapy, CT: Chemotherapy, NA: not analysed , Table 2 . Agreement between Ki67RNA and Ki67IHC (n=95). Ki67 RNA High Ki67 RNA Low Ki67 IHC High 29 (30.5%) 27 (28%) Ki67 IHC Low 10 (11%) 29 (30.5%) Agreement of 0.24 by Cohen’s kappa test Additional Declarations Competing interest reported. ZS: Honoraria from Pfizer, Genomic Health EC: Honoraria from Novartis, Pfizer, Daiichi, Genomic Health FB: Honoraria from Roche, Novartis, Pfizer, Astra-Zeneca, Clovis, Daiichi. CB: advisory board from Bayer, BMS, msd, servier and research grant from Roche NM, JV, CM, AO, JLP, JPF, ED, LM, MPA, LC, MJP, GM, XP: have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 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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-677106","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":38852610,"identity":"4fb31e5b-a7dc-42da-ae9c-9afcb6ae2e8a","order_by":0,"name":"Zohair Selmani","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+ElEQVRIie3QvUoDQRDA8VmEqwau3RBJXmFk4Ygg3qskBLbaaEoLiwPh0ml74Etol/Jw4GxW0m6R5jhIncrKryOCxGKT2FnsvxiWgR8DCxAK/c9E2Q6MAdtn9rPt7TQb0sl+E7WXAJWHkv4dl0/uenmsnl8qKeZnaXx/UzdToEsfEYUesqlWmNgLLYXVo2JZKVUAnWYeciSR2ESMicOk+57zEJyJughv5LsSxYs1mw9GVWAiRf6Z9r8JeQmCAZ7kjCQ3pBQP+4iUmnhyyyitUQNhx6NHp5VC8pP2x+rGvHIaz+yJE/PztOfGdYNXfuLrzyAUCoVC230B941QNDJrcloAAAAASUVORK5CYII=","orcid":"","institution":"University of Franche-Comté","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Zohair","middleName":"","lastName":"Selmani","suffix":""},{"id":38852611,"identity":"7885a2df-c4ec-4352-af8c-e80692a90080","order_by":1,"name":"Chloé Molimard","email":"","orcid":"","institution":"Centre Hospitalier Universitaire de Besançon","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chloé","middleName":"","lastName":"Molimard","suffix":""},{"id":38852612,"identity":"d6e7b941-20dc-4ed5-b72f-0ef028a2f226","order_by":2,"name":"Alexis Overs","email":"","orcid":"","institution":"Centre Hospitalier Universitaire de Besançon","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Alexis","middleName":"","lastName":"Overs","suffix":""},{"id":38852613,"identity":"78aff661-e02b-4563-8f51-4ab14de65468","order_by":3,"name":"Fernando Bazan","email":"","orcid":"","institution":"Centre Hospitalier Universitaire de Besançon","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Fernando","middleName":"","lastName":"Bazan","suffix":""},{"id":38852614,"identity":"450d0a09-f687-426e-a8e5-ae0b0441c457","order_by":4,"name":"Loic Chaigneau","email":"","orcid":"","institution":"Centre Hospitalier Universitaire de Besançon","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Loic","middleName":"","lastName":"Chaigneau","suffix":""},{"id":38852615,"identity":"eb17b128-07fb-4140-91f2-d6eb2cd80a03","order_by":5,"name":"Erion Dobi","email":"","orcid":"","institution":"Centre Hospitalier Universitaire de Besançon","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Erion","middleName":"","lastName":"Dobi","suffix":""},{"id":38852616,"identity":"9a0594ec-c0ce-49e5-a78e-f62bafeb254b","order_by":6,"name":"Nathalie Meneveau","email":"","orcid":"","institution":"Centre Hospitalier Universitaire de Besançon","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nathalie","middleName":"","lastName":"Meneveau","suffix":""},{"id":38852617,"identity":"4198d4a3-544b-46f4-aeb4-7bf1f96a8326","order_by":7,"name":"Laura Mansi","email":"","orcid":"","institution":"Centre Hospitalier Universitaire de Besançon","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Mansi","suffix":""},{"id":38852618,"identity":"f9a83316-e9e7-4976-80fe-9661806ed9d3","order_by":8,"name":"Marie-Justine Paillard","email":"","orcid":"","institution":"Centre Hospitalier Universitaire de Besançon","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marie-Justine","middleName":"","lastName":"Paillard","suffix":""},{"id":38852619,"identity":"3f5f56a2-098f-4ce3-b537-13cedc9d6980","order_by":9,"name":"Guillaume Meynard","email":"","orcid":"","institution":"Centre Hospitalier Universitaire de Besançon","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Guillaume","middleName":"","lastName":"Meynard","suffix":""},{"id":38852620,"identity":"5cd81534-287a-44c5-98f6-14317a72200f","order_by":10,"name":"Julien Viot","email":"","orcid":"","institution":"University of Franche-Comté","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Julien","middleName":"","lastName":"Viot","suffix":""},{"id":38852621,"identity":"a2fe1b5a-7485-426e-a1e6-ae21dda51bba","order_by":11,"name":"Marie-Paule Algros","email":"","orcid":"","institution":"Centre Hospitalier Universitaire de Besançon","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Marie-Paule","middleName":"","lastName":"Algros","suffix":""},{"id":38852622,"identity":"0eb4e69e-8729-4165-96fc-476e8665cfa1","order_by":12,"name":"Christophe Borg","email":"","orcid":"","institution":"University of Franche-Comté","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Christophe","middleName":"","lastName":"Borg","suffix":""},{"id":38852623,"identity":"15917c4c-dcca-4e81-9778-f299c1ee644b","order_by":13,"name":"Jean-Paul Feugeas","email":"","orcid":"","institution":"University of Franche-Comté","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jean-Paul","middleName":"","lastName":"Feugeas","suffix":""},{"id":38852624,"identity":"01009242-85b1-4d7a-92c5-89083a795f41","order_by":14,"name":"Xavier Pivot","email":"","orcid":"","institution":"University of Strasbourg","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xavier","middleName":"","lastName":"Pivot","suffix":""},{"id":38852625,"identity":"fd81655a-e58b-494a-ac64-99b3de0ea240","order_by":15,"name":"Jean-Luc Prétet","email":"","orcid":"","institution":"University of Franche-Comté","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jean-Luc","middleName":"","lastName":"Prétet","suffix":""},{"id":38852626,"identity":"9caf94d6-3976-4b9a-abcc-cd5a75d730b7","order_by":16,"name":"Elsa Curtit","email":"","orcid":"","institution":"University of Franche-Comté","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Elsa","middleName":"","lastName":"Curtit","suffix":""}],"badges":[],"createdAt":"2021-07-01 10:44:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-677106/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-677106/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":11489706,"identity":"68e7853c-e238-4ddd-b392-8d5bce5aa98c","added_by":"auto","created_at":"2021-07-15 13:27:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":41921,"visible":true,"origin":"","legend":"Threshold determination, specificity and sensitivity of Ki67RNA.\nThreshold of Ki67RNA was determined using package pROC version 1.16.1 (pROC: an open-source package for R and S+ to analyze and compare ROC curves. BMC Bioinformatics, 12, p.77. DOI: 10.1186/1471-2105-12-77). An optimal threshold of Ki67RNA at 6.35 units was found with a specificity of 48% and sensitivity of 84%. ","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-677106/v1/9ea60f20f4fc547a3751de73.png"},{"id":11489707,"identity":"c297c2b9-2a6a-4e14-8e65-e674631560ae","added_by":"auto","created_at":"2021-07-15 13:27:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":52742,"visible":true,"origin":"","legend":"Events-Free Survival according Ki67 status by ODX® test and by immunochemistry.\nThe survival follow-up was analyzing by the package survival version 3.1-8 (26) . The events include the local or metastatic relapse, other cancers occurrence and death with a mean follow-up of 57 months. (A) Ki67RNA were analyzed according to two groups using the 6.35 units’ threshold. (B) The Ki67IHC were analyzed according to two groups using the rate of \u003e20%.\n","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-677106/v1/5795281819ecd89eda0e0422.png"},{"id":11489704,"identity":"3689231f-94aa-49f8-93e6-3eae7975c284","added_by":"auto","created_at":"2021-07-15 13:27:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":35467,"visible":true,"origin":"","legend":"Events-Free Survival among all patients according RS® groups.\nThe survival follow-up was analyzed by the package survival version 3.1-8 [26]. The events include the local or metastatic relapse, other cancers occurred and death with a mean follow-up of 57 months. The RS® are interpreted in three categories (low risk: RS® \u003c18; intermediate risk: RS®= 18-30; high risk: RS® \u003e30).\n","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-677106/v1/7daac444929f138852eb6d82.png"},{"id":11489703,"identity":"fe7974d4-0df4-449d-8a48-ddaa481ef69a","added_by":"auto","created_at":"2021-07-15 13:27:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":36394,"visible":true,"origin":"","legend":"Association between RS® and Ki67 status\nA. The association of RS® with Ki67RNA were analyzed according to the 6.35 units’ threshold. B. The association of RS® with Ki67IHC were analyzed using the rate of \u003e20%.\n","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-677106/v1/4544290e2eec442d3b89ed38.png"},{"id":15673961,"identity":"b913f050-76cb-4b17-9135-2397aed61ce5","added_by":"auto","created_at":"2021-11-18 14:20:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":541754,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-677106/v1/19780c5c-820a-4bc7-a901-e5eee8669460.pdf"},{"id":11489705,"identity":"cfedd08f-f57e-413e-be89-2bd0397da3b7","added_by":"auto","created_at":"2021-07-15 13:27:11","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":308246,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-677106/v1/84bea996090f1e6ea71b59bc.docx"}],"financialInterests":"Competing interest reported. ZS: Honoraria from Pfizer, Genomic Health \nEC: Honoraria from Novartis, Pfizer, Daiichi, Genomic Health\nFB: Honoraria from Roche, Novartis, Pfizer, Astra-Zeneca, Clovis, Daiichi.\nCB: advisory board from Bayer, BMS, msd, servier and research grant from Roche\nNM, JV, CM, AO, JLP, JPF, ED, LM, MPA, LC, MJP, GM, XP: have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.","formattedTitle":"\u003cp\u003eKi67 Assessment By qRT-PCR In OncotypeDx\u003csup\u003e®\u003c/sup\u003e Breast Recurrent Score\u003csup\u003e®\u003c/sup\u003e: Low Correlation With Immunohistochemistry Assessment of Ki67 And Prognostic Impact of Ki67 RNA Level of Expression.\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn terms of incidence, prevalence and mortality, breast cancer is in first place worldwide in women \u003csup\u003e1\u003c/sup\u003e. The vast majority of patients with breast cancer does not show detectable distant metastasis on diagnosis. Treatment of early breast cancer is based on surgical tumor resection with conditional lymph node dissection. Adjuvant systemic treatments including endocrine therapy (HT) and chemotherapy (CT) aim at the reduction of the distant recurrence rate and improvement of breast cancer specific survival. The decision of adjuvant therapeutic modalities is taken according to several prognostic and/or predictive factors including patient age, tumor size, histological type and grade, lymph node involvement and expression on the tumor of hormone receptors for estrogen (ER) progesterone (PR), and the human epidermal growth factor receptor 2 (HER2) as well as the percentage of tumor cells expressing the nuclear proliferation marker Ki67 \u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGenomic signatures were designed to give prognostic and predictive information to streamline adjuvant chemotherapy decision in ER-positive, HER2-negative breast cancer patients. Oncotype DX\u003csup\u003e\u0026reg;\u003c/sup\u003e Breast (ODX\u003csup\u003e\u0026reg;\u003c/sup\u003e) is the most widely used molecular signature in this setting and is included in treatment guidelines for estimating both the risk of distant recurrence and predicting adjuvant chemotherapy benefit. ODX\u003csup\u003e\u0026reg;\u003c/sup\u003e measures the RNA of 21 genes (16 cancer-associated genes and 5 housekeeping genes) and uses the expression pattern to calculate a recurrence score (RS\u003csup\u003e\u0026reg;\u003c/sup\u003e) that ranges between 0 and 100 \u003csup\u003e3\u003c/sup\u003e. The RS\u003csup\u003e\u0026reg;\u003c/sup\u003e result provides two types of information on tumor biology: (i) prognosis information: an estimate of the individual risk of distant cancer recurrence within 10 years, (ii) predictive information: an estimate of the likelihood of a benefit from chemotherapy \u003csup\u003e4\u0026ndash;6\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eInterestingly in breast cancer, Ki67 RNA (Ki67\u003csub\u003eRNA\u003c/sub\u003e) is a parameter analyzed by several molecular signatures such as PAM50 and ODX \u003csup\u003e7\u003c/sup\u003e. Furthermore, Ki67 is an interesting biomarker in early breast cancer and breast cancers expressing high levels of Ki67 are associated with poor outcomes \u003csup\u003e8\u0026ndash;10\u003c/sup\u003e. To provide individualized patient care in the concept of precision medicine, reliability of prognostic and predictive information deriving from Ki67 value is essential \u003csup\u003e11,12\u003c/sup\u003e. Some studies \u003csup\u003e13,14\u003c/sup\u003e indicate that lowering in Ki67 expression after neoadjuvant endocrine treatment may predict long-term outcome. Nevertheless, substantial variability in Ki67 staining of breast cancer tissue by immunohistochemistry (IHC) and interpretation was found between 30 routine pathology labs. Clinical use of Ki67 staining for therapeutic decisions should be considered with caution and only fully aware of lab-specific reference values \u003csup\u003e15\u003c/sup\u003e. Ki67 staining lacks scoring standardization ; various studies have focused on assessment methodology standardization \u003csup\u003e16\u003c/sup\u003e, interobserver reproducibility \u003csup\u003e17\u003c/sup\u003e and digital image analysis of Ki67 staining \u003csup\u003e18,19\u003c/sup\u003e. However, little is known about variability in IHC Ki67-labelling results between routine pathology labs \u003csup\u003e20,21\u003c/sup\u003e and its potential influence on interpretation of Ki67 levels in breast cancer. When using Ki67 assessment by IHC in order to consider an indication of adjuvant chemotherapy \u003csup\u003e22\u003c/sup\u003e, clinicians should be aware of the low reproducibility of Ki67 scoring and its questionable analytical validity.\u003c/p\u003e \u003cp\u003eIn the present study, we analyzed 98 patients tested by the Oncotype DX\u003csup\u003e\u0026reg;\u003c/sup\u003e Breast from June 2012 to April 2014. For this cohort, Ki67\u003csub\u003eRNA\u003c/sub\u003e level obtained in patients\u0026rsquo; Oncotype DX\u003csup\u003e\u0026reg;\u003c/sup\u003e signatures were available. The primary aim of this study was to assess the agreement between Ki67\u003csub\u003eRNA\u003c/sub\u003e and Ki67 staining by IHC (Ki67\u003csub\u003eIHC\u003c/sub\u003e). The other objectives were to analyze the association between the event free survival (EFS) and the expression level of Ki67\u003csub\u003eRNA\u003c/sub\u003e in ODX\u003csup\u003e\u0026reg;\u003c/sup\u003e signature; and association between RS\u003csup\u003e\u0026reg;\u003c/sup\u003e and Ki67\u003csub\u003eRNA\u003c/sub\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eCharacteristics of the patient population (Table\u0026nbsp;1)\u003c/p\u003e\n\u003cp\u003eComplete data sets from 98 breast cancer patients who underwent RS\u0026reg; testing were provided from 4 public treatment centers (public hospitals and university hospitals). The patients included were exclusively female and showed a wide age distribution (31 to 81 years) with a mean age of 57 years. The predominant tumor characteristics were no special type (NST) (91%), N0 or Nmic (71%), grade 1 (62%), and tumor size pT1c (1\u0026ndash;2 cm) (58%). All patients had ER positive/ HER2 negative tumors. Table\u0026nbsp;1 shows the patient and disease characteristics of the full population. The RS\u0026reg; values were \u0026lt;\u0026thinsp;18 in 38% (n\u0026thinsp;=\u0026thinsp;37), 18\u0026ndash;30 in 51% (n\u0026thinsp;=\u0026thinsp;50) and \u0026gt;\u0026thinsp;30 in 11% (n\u0026thinsp;=\u0026thinsp;11) of the patients. After surgery and collegial decision, all patients have received a treatment according the result of ODX\u0026reg; test (HT alone or CT-HT) in adjuvant situation.\u003c/p\u003e\n\u003cp\u003eDuring the follow-up (57 months), we observed 19 events (19%): 3 local relapses, 10 metastatic relapses (bone, lung, liver and pancreas), 3 other cancers (contralateral breast, colorectal carcinoma and pancreatic carcinoma) and 3 deaths.\u003c/p\u003e\n\u003cp\u003eAgreement between Ki67\u003csub\u003eIHC\u003c/sub\u003e and Ki67\u003csub\u003eRNA\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003eThe Ki67\u003csub\u003eIHC\u003c/sub\u003e positivity rate of \u0026gt;\u0026thinsp;20% \u003csup\u003e23\u003c/sup\u003e was used to define for the \u0026ldquo;high-risk\u0026rdquo; tumor group. We showed by ROC curve analysis an optimal threshold at 6.35 with a specificity of 48% and a sensitivity of 84%. With this cut off, the Ki67\u003csub\u003eRNA\u003c/sub\u003e\u003csup\u003ehigh\u003c/sup\u003e were \u0026gt;\u0026thinsp;6.35 and Ki67\u003csub\u003eRNA\u003c/sub\u003e\u003csup\u003elow\u003c/sup\u003e were \u0026lt;\u0026thinsp;6.35 (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). A correlation between Ki67\u003csub\u003eRNA\u003c/sub\u003e expression and Ki67\u003csub\u003eIHC\u003c/sub\u003e score was found (R\u0026thinsp;=\u0026thinsp;0,36; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e IC 95% [0.2\u0026ndash;0.5]) (Supplementary data 2\u003cspan class=\"Underline\" name=\"Emphasis\" type=\"Underline\"\u003e)\u003c/span\u003e. In the cohort, 29 tumors were Ki67\u003csup\u003ehigh\u003c/sup\u003e and 29 were Ki67\u003csup\u003elow\u003c/sup\u003e by the two methods, which represent a match between the results in 58 patients (61%) (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Discordances were observed in 37 patients (39%): 10 samples were Ki67\u003csub\u003eIHC\u003c/sub\u003e\u003csup\u003elow\u003c/sup\u003e/Ki67\u003csub\u003eRNA\u003c/sub\u003e\u003csup\u003ehigh\u003c/sup\u003e and 27 samples were Ki67\u003csub\u003eIHC\u003c/sub\u003e\u003csup\u003ehigh\u003c/sup\u003e/Ki67\u003csub\u003eRNA\u003c/sub\u003e\u003csup\u003elow\u003c/sup\u003e. An agreement of 0.24 was found by Cohen\u0026rsquo;s kappa test.\u003c/p\u003e\n\u003cp\u003eEvents-Free Survival among all patients according RS\u0026reg; groups and Ki67 status\u003c/p\u003e\n\u003cp\u003eA significant relationship was observed between EFS and the level of Ki67\u003csub\u003eRNA\u003c/sub\u003e expression (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.10\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA) unlike Ki67\u003csub\u003eIHC\u003c/sub\u003e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.25) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). However, we did not observe any significant difference on EFS according to the three RS\u0026reg; groups risk (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.4) (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eUsing the age categories from the TAILORx study \u003csup\u003e24\u003c/sup\u003e, the association between EFS and Ki67\u003csub\u003eRNA\u003c/sub\u003e status, was found only for women under 50 years (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01) and not for women over 50 years (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.36) (Supplementary data 3). No association between Ki67\u003csub\u003eIHC\u003c/sub\u003e and EFS was observed (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.20).\u003c/p\u003e\n\u003cp\u003eKi67\u003csub\u003eRNA\u003c/sub\u003e association with RS\u0026reg;\u003c/p\u003e\n\u003cp\u003eAs expected, Ki67\u003csub\u003eRNA\u003c/sub\u003e levels were significantly associated with RS\u0026reg; (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). To a lesser extent, an association between ODX\u0026reg; test with Ki67\u003csub\u003eIHC\u003c/sub\u003e status has also been observed (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.013) (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). Supplementary data 4 shows the distribution of Ki67\u003csub\u003eIHC\u003c/sub\u003e score and Ki67\u003csub\u003eRNA\u003c/sub\u003e level according to patient age, tumor grade and size, PR status, nodal status, Recurrence score, treatment and events.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eStudy population\u003c/p\u003e\n\u003cp\u003eIn this study, we focused on the Ki67\u003csub\u003eRNA\u003c/sub\u003e expression level of 98 patients. The majority of our data are consistent with the PONDx real life study \u003csup\u003e25\u003c/sup\u003e which found a female population over 50 (70%) with NST tumors (78%) ranging between 2\u0026ndash;5 cm (89%), grade II (68%) and without lymph node involvement in 79% of cases. The PONDx cohort included more grade II than in our population (68% vs 29%) with a higher contingent of lobular carcinoma (13% vs 7%). In PONDx, RS\u003csup\u003e\u0026reg;\u003c/sup\u003e results by prognostic category used between 2012\u0026ndash;2014 were: \u0026lt;18: 54%, 18\u0026ndash;30: 36%; \u0026gt;30: 10%. Compared to this real-life study, the local cohort shows an inversion of the proportions between populations at low risk (38% vs 54%) and those at intermediate risk (51% vs 36%).\u003c/p\u003e\n\u003cp\u003eThe few differences observed in the distribution of the risk groups established by the ODX\u003csup\u003e\u0026reg;\u003c/sup\u003e test can be explained by an over-representation of grade II tumors in the PONDx study. Our cohort is comparable to this largest population for most epidemiologic parameters.\u003c/p\u003e\n\u003cp\u003eDifferences between Ki67\u003csub\u003eRNA\u003c/sub\u003e and Ki67\u003csub\u003eIHC\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003eComparisons of Ki67 status showed a discrepancy between the two different types of evaluation. Indeed, half of Ki67\u003csub\u003eRNA\u003c/sub\u003e\u003csup\u003ehigh\u003c/sup\u003e (\u0026gt;\u0026thinsp;6.35) were found Ki67\u003csub\u003eIHC\u003c/sub\u003e\u003csup\u003elow\u003c/sup\u003e (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The observed correlation between the two techniques, is therefore weak even if significant (R\u0026thinsp;=\u0026thinsp;0.36, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.10\u003csup\u003e-4\u003c/sup\u003e; IC 95% [0.2\u0026ndash;0.5]) (Supplementary data2). Technically, IHC shows greater variability \u003csup\u003e20,21\u003c/sup\u003e and is less reproducible than the quantification of mRNAs. Currently, there is no standardization for the interpretation of the Ki67 status by IHC \u003csup\u003e16\u003c/sup\u003e. As a consequence, it generates inter-site and inter-observer variability within the same site \u003csup\u003e17\u003c/sup\u003e. In contrast, the Oncotype test which is a molecular based and standardized test provides objective results that are independent of the observer.\u003c/p\u003e\n\u003cp\u003eFurthermore, the ODX test analyzes the level of Ki67 messenger RNAs in the tumor sample. This analysis therefore considers the transcriptional status of the \u003cem\u003eKi67\u003c/em\u003e gene level not only in tumor cells but also in associated cells such as lymphocytes, stromal or endothelial cells. Therefore, it is an overall estimate. By IHC, the protein expression is scored only on tumor cells. The two methods do not provide the same types of information on Ki67 status. These two types of evaluation of Ki67 are probably complementary because the Ki67\u003csub\u003eIHC\u003c/sub\u003e informs us about the tumor specific proliferation index and the Ki67\u003csub\u003eRNA\u003c/sub\u003e would reflect the proliferation of the tumor and its microenvironment. For all the reasons mentioned above, the association between RS\u003csup\u003e\u0026reg;\u003c/sup\u003e and Ki67\u003csub\u003eIHC\u003c/sub\u003e status is lower but remains significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.013).\u003c/p\u003e\n\u003cp\u003eEvents free survival analyses\u003c/p\u003e\n\u003cp\u003eOn the one hand in this study, adaptation of treatment based on RS\u003csup\u003e\u0026reg;\u003c/sup\u003e permit to obtain equivalent EFS in populations with low and high risk of recurrence. We have modestly confirmed the efficiency of ODX\u003csup\u003e\u0026reg;\u003c/sup\u003e test. Indeed, the lack of association between RS\u003csup\u003e\u0026reg;\u003c/sup\u003e and EFS can be explained by the treatment (HT alone or CT-HT) performed according to the score. The clinical validity and utility of the RS\u003csup\u003e\u0026reg;\u003c/sup\u003e has been demonstrated prospectively across multiple studies in breast cancer patients worldwide including multiple validation studies as well as long-term prospective studies (TAILORx \u003csup\u003e24\u003c/sup\u003e, WSG Plan B \u003csup\u003e26\u003c/sup\u003e and analyses from a prospective epidemiological database \u003csup\u003e27\u003c/sup\u003e). Based on level Ia evidence, the Oncotype DX test has been incorporated in leading internationally-accepted clinical guidelines on the treatment of early breast cancer (St. Gallen \u003csup\u003e28\u003c/sup\u003e, ESMO \u003csup\u003e29\u003c/sup\u003e, and ASCO \u003csup\u003e30\u003c/sup\u003e). This therapeutic adaptation will therefore smooth the differences between the categories of the ODX\u003csup\u003e\u0026reg;\u003c/sup\u003e test and at the same time demonstrates the value of the information provided by its score.\u003c/p\u003e\n\u003cp\u003eOn the other hand, we were able to show that Ki67\u003csub\u003eRNA\u003c/sub\u003e\u003csup\u003ehigh\u003c/sup\u003e was significantly associated with the occurrence of events (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02) but we did not find any association between Ki67\u003csub\u003eIHC\u003c/sub\u003e and EFS (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.25).\u003c/p\u003e\n\u003cp\u003eOur data relate to a small population but leaves the possibility of a larger study in order to confirm these that Ki67\u003csub\u003eRNA\u003c/sub\u003e evaluation has a prognostic impact as RS\u003csup\u003e\u0026reg;\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eCorrelation between Ki67\u003csub\u003eRNA\u003c/sub\u003e and RS\u003csup\u003e\u0026reg;\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eAs expected in our cohort, the RS\u0026reg; obtained is strongly associated with the Ki67\u003csub\u003eRNA\u003c/sub\u003e (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e) which is one of the components of this test (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). To optimize RS\u0026reg; interpretation, the TAILORx subgroup study shows a benefit of CT in women\u0026thinsp;\u0026le;\u0026thinsp;50 years old with an RS\u0026reg; of 16 to 25 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004). CT could be avoided in women over 50 years old with a RS\u0026reg; \u0026lt;26, in women 50 years old or less with an RS\u0026reg; \u0026lt;16 \u003csup\u003e31\u003c/sup\u003e. Recently in San Antonio Breast cancer symposium, the results of RxPONDER study were presented \u003csup\u003e32\u003c/sup\u003e. And now, postmenopausal women with 1\u0026ndash;3 positive nodes and RS\u0026reg; 0\u0026ndash;25 can safetly forego adjuvant CT without compromising invasive disease-free survival. The premenopausal women with positive nodes and RS 0\u0026ndash;25 likely significantly benefit from chemotherapy \u003csup\u003e32\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOncotype DX\u0026reg; Breast enables relevant net reductions in chemotherapy use, sparing patients from serious toxicities \u003csup\u003e7\u003c/sup\u003e. On this other side, its clinical impact and pharmacoeconomic benefit in routine care have been shown in 20 decision-impact studies \u003csup\u003e33\u0026minus;\u0026thinsp;35\u003c/sup\u003e. However, in many places, the oncotype score remains inaccessible or not reimbursed \u003csup\u003e7,25,35\u003c/sup\u003e. This is why the evaluation of the level of Ki67\u003csub\u003eRNA\u003c/sub\u003e by a molecular biology techniques \u003csup\u003e36,37\u003c/sup\u003e could be a low-cost prognostic alternative in some countries. But this hypothesis will have to be validated during a prospective translational study.\u003c/p\u003e\n\u003cp\u003eIn conclusion, we observed of low agreement between Ki67\u003csub\u003eRNA\u003c/sub\u003e tumor level measured by qRT-PCR and Ki67 protein labelling by IHC. Substantial variability in Ki67\u003csub\u003eIHC\u003c/sub\u003e of breast cancer tissue and interpretation have been shown and widely published. The clinical use of Ki67 labelling should be cautious and limited by the low reproducibility of Ki67 scoring and its questionable analytical validity.\u003c/p\u003e\n\u003cp\u003eUnlike Ki67\u003csub\u003eIHC\u003c/sub\u003e and independently of the RS\u003csup\u003e\u0026reg;\u003c/sup\u003e, Ki67\u003csub\u003eRNA\u003c/sub\u003e could have a prognostic value. The assessment of Ki67\u003csub\u003eRNA\u003c/sub\u003e by qRT-PCR on breast cancer tumor would be feasible and cost effective. It would be of great clinical utility to better assess the prognosis and predictive value of Ki67\u003csub\u003eRNA\u003c/sub\u003e. Presentation of Ki67 status in Genomic Health report could also be helpful for therapeutic decisions on borderline situation.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eEthics statement\u003c/p\u003e\n\u003cp\u003eAll procedures performed in studies involving human participants were in accordance with the ethical standards of the national research committee and with the 1964 Helsinki Declaration and its later amendments. In France, this search is considered like a non-interventional study according to European legislation. All patients were individually informed that their data should be used to scientific research. Informed and written consents were obtained from all subjects, or if subject are deceased, from a parent and/or legal guardian. All experimental protocols were approved by \u0026laquo; \u003cem\u003ecomit\u0026eacute; de protection des personnes \u0026raquo;\u003c/em\u003e (CCP) of Besan\u0026ccedil;on, France.\u003c/p\u003e\n\u003cp\u003ePatients and tumors characteristics\u003c/p\u003e\n\u003cp\u003eOur study was an observational multicenter retrospective study collecting data on the real-life use of Oncotype DX Breast Recurrence Score\u003csup\u003e\u0026reg;\u003c/sup\u003e test by physicians in clinical practice settings in France. Patients eligibility criteria for this analysis correspond to the population for which the Oncotype DX\u003csup\u003e\u0026reg;\u003c/sup\u003e Breast Recurrence Score\u003csup\u003e\u0026reg;\u003c/sup\u003e test is validated, i.e. adult patients with a recent first diagnosis of a single early invasive breast tumor with ER+/HER2- status, plus available documentation of lymph node involvement as either N0 (node negative), Nmic (micrometastatic) or N1 (1\u0026ndash;3 positive nodes). The following data were documented: patient age and sex, conventional clinical and pathological disease characteristics including histologic type, tumor size and grade, nodal status, receptor status including ER, PR, and HER2, and the Ki67 proliferation marker by IHC. Between 2012 and 2014, the RS\u003csup\u003e\u0026reg;\u003c/sup\u003e results were interpreted in three categories (low risk; intermediate risk; high risk with two cut-offs: 18 and 30). The event free survival (EFS) includes the local or metastatic relapse, other cancers and death with a mean follow-up of 57 months. Following the guidelines, the Oncotype DX\u003csup\u003e\u0026reg;\u003c/sup\u003e Breast Recurrence Score\u003csup\u003e\u0026reg;\u003c/sup\u003e test was realized after surgery and adjuvant treatments were adapted according to the RS\u003csup\u003e\u0026reg;\u003c/sup\u003e. The RS\u003csup\u003e\u0026reg;\u003c/sup\u003e values and the associated treatment (hormonal therapy plus/minus chemotherapy, or other modalities) were also collected.\u003c/p\u003e\n\u003cp\u003eImmunohistochemistry analyses.\u003c/p\u003e\n\u003cp\u003eThe hormonal receptor status (ER and PR) was considered positive according to standardized European guidelines using a cut-off of \u0026ge;\u0026thinsp;10% stained tumor cell nuclei. The Ki67\u003csub\u003eIHC\u003c/sub\u003e evaluation was realized in 2 publics and 2 private pathological departments. In these structures, the Ki67\u003csub\u003eIHC\u003c/sub\u003e was assessed with monoclonal antibody MIB1 \u003csup\u003e16\u003c/sup\u003e. Based on recommendations established by St Gallen 2015, the Ki67\u003csub\u003eIHC\u003c/sub\u003e\u003csup\u003ehigh\u003c/sup\u003e corresponding to Ki67\u0026thinsp;\u0026gt;\u0026thinsp;20%, defined \u0026ldquo;high-risk\u0026rdquo; subpopulations of tumors \u003csup\u003e23\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eKi67\u003csub\u003eRNA\u003c/sub\u003e threshold determination and events statistical analyses\u003c/p\u003e\n\u003cp\u003eDescriptive statistics were used to summarize clinicopathological characteristics. Variables were described by the size and rate. After normalization, levels of Ki67\u003csub\u003eRNA\u003c/sub\u003e were distributed according to a Gaussian curve (Supplementary data 1). We have determined the optimal threshold using R package pROC version 1.16.1 \u003csup\u003e38\u003c/sup\u003e. The statistical comparison between IHC and ODX\u0026reg; was realized by Cohen\u0026rsquo;s kappa test.\u003c/p\u003e\n\u003cp\u003eStatistical analyzes were performed using R version 3.6.2 \u003csup\u003e39\u003c/sup\u003e. The survival follow-up was analyzed by the package survival version 3.1-8 \u003csup\u003e40\u003c/sup\u003e.\u003c/p\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003eData availability\u003c/h2\u003e\n \u003cp\u003eThe datasets generated and analyzed during this study (birthdate, admission date, discharge date, date of death\u0026hellip;), are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all patients who agreed to participate in this observational study.\u0026nbsp;Their also thank\u0026nbsp;Genomic Health, Inc.\u0026nbsp;(now Exact sciences) and Mahi Y. for the Ki67\u003csub\u003eRNA\u003c/sub\u003e data. The authors are grateful to all participating physicians for their cooperation and for private and public anatomopathology departements (CH de Trevenans, Besancon Pathologie and Dr Chargeboeuf M.) for their participation in IHC analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAffiliations\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eDepartment of Oncobiology, University Hospital of Besan\u0026ccedil;on, Besan\u0026ccedil;on, France. \u003csup\u003e2\u003c/sup\u003eDepartment of Medical Oncology, University Hospital of Besan\u0026ccedil;on, Besan\u0026ccedil;on, France. \u003csup\u003e3\u003c/sup\u003eDepartment of Anatomopathology, University Hospital of Besan\u0026ccedil;on, Besan\u0026ccedil;on, France. \u003csup\u003e4\u003c/sup\u003eBourgogne Franche-Comt\u0026eacute; university, INSERM, UMR1098, Besan\u0026ccedil;on, France. \u0026nbsp;\u003csup\u003e5\u003c/sup\u003eICANS, Strasbourg, France. \u003csup\u003e6\u003c/sup\u003eEA3181, UBFC, UFC, Besan\u0026ccedil;on, France.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConception and design:\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eEC, ZS.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDevelopment of methodology:\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eZS, EC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcquisition of data\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;:\u003c/em\u003e\u003c/strong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003eEC, ZS, FB, ED, LC, LM, NM, MJP, GM, JV, XP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAnalysis and interpretation of data:\u003c/em\u003e\u003c/strong\u003e ZS, AO, CM, JLP, JPF.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eWriting, review, and/or revision of the manuscript\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e ZS, CM, JLP, EC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAdministrative, technical, or material support:\u003c/em\u003e\u003c/strong\u003e EC, ZS, JLP, MPA, JPF, CB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStudy supervision:\u003c/em\u003e\u003c/strong\u003e EC, ZS.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Zohair Selmani\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eZS: Honoraria from Pfizer, Genomic Health\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEC: Honoraria from Novartis, Pfizer, Daiichi, Genomic Health\u003c/p\u003e\n\u003cp\u003eFB: Honoraria from Roche, Novartis, Pfizer, Astra-Zeneca, Clovis, Daiichi.\u003c/p\u003e\n\u003cp\u003eCB:\u0026nbsp;advisory board from Bayer, BMS, msd, servier and research grant from Roche\u003c/p\u003e\n\u003cp\u003eNM, JV, CM, AO, JLP, JPF, ED, LM, MPA, LC, MJP, GM, XP: have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eErratum Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. \u003cem\u003eCA. 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A Package for Survival Analysis in S_. version 2.38.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e. Patient and disease characteristics in the population of ER positive/HER2 negative patients.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn this population, 98 women were included from June 2012 to April 2014. All patients were tested by Oncotype DX\u003csup\u003e\u0026reg;\u003c/sup\u003e Breast. The Nodal status was stratified in 3 groups: N0 (node negative), Nmic (micrometastatic) or N1 (1-3 positive nodes). The progesterone receptor status (PR) was considered positive according to standardized European guidelines using a cut-off of \u0026ge;10% stained tumor cell nuclei. All patients were ER positive and HER2 negative. The Ki67\u003csub\u003eIHC\u003c/sub\u003e positivity was defined by the rate of \u0026gt;20% established by St Gallen 2015 recommendations. Almost, The Recurrence score results (RS\u003csup\u003e\u0026reg;\u003c/sup\u003e) are interpreted in three categories (low risk: RS\u003csup\u003e\u0026reg;\u003c/sup\u003e \u0026lt;18; intermediate risk: RS\u003csup\u003e\u0026reg;\u003c/sup\u003e = 18-30; high risk: RS\u003csup\u003e\u0026reg;\u003c/sup\u003e \u0026ge;31). The events include the local or metastatic relapse, other cancers and death.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eHT: Hormonal therapy, CT: Chemotherapy, NA: not analysed ,\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/83064_0857a92044b57365/83064_custom_files/img1626266965.png\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2\u003c/strong\u003e. Agreement between Ki67RNA and Ki67IHC (n=95).\u0026nbsp;\u003c/p\u003e\n\u003ctable cellpadding=\"0\" cellspacing=\"0\" width=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003eKi67\u003csub\u003eRNA\u003c/sub\u003e\u003csup\u003eHigh\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003eKi67\u003csub\u003eRNA\u003c/sub\u003e\u003csup\u003eLow\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003eKi67\u003csub\u003eIHC\u003c/sub\u003e\u003csup\u003eHigh\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e29 (30.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e27 (28%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003eKi67\u003csub\u003eIHC\u003c/sub\u003e\u003csup\u003eLow\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e10 (11%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.333333333333336%\"\u003e\n \u003cp\u003e29 (30.5%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cem\u003eAgreement of 0.24 by Cohen\u0026rsquo;s kappa test\u003c/em\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Breast cancer, chemotherapy, prognosis, Oncotype ","lastPublishedDoi":"10.21203/rs.3.rs-677106/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-677106/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBreast cancers expressing high levels of Ki67 are associated with poor outcomes. Oncotype DX\u003csup\u003e® \u003c/sup\u003etest was designed for ER+/HER2- early-stage breast cancers to help adjuvant chemotherapy decision by providing a Recurrent Score\u003csup\u003e®\u003c/sup\u003e (RS\u003csup\u003e®\u003c/sup\u003e). RS\u003csup\u003e® \u003c/sup\u003emeasures the expression of 21 specific genes from tumor tissue, including \u003cem\u003eKi67\u003c/em\u003e. The primary aim of this study was to assess the agreement between Ki67\u003csub\u003eRNA\u003c/sub\u003e obtained with Oncotype DX\u003csup\u003e® \u003c/sup\u003eRS\u003csup\u003e®\u003c/sup\u003e and Ki67\u003csub\u003eIHC\u003c/sub\u003e. Other objectives were to analyze the association between the event free survival (EFS) and the expression level of Ki67\u003csub\u003eRNA\u003c/sub\u003e; and association between RS\u003csup\u003e®\u003c/sup\u003e and Ki67\u003csub\u003eRNA\u003c/sub\u003e.\u003c/p\u003e\u003cp\u003eHerein, we report a low agreement of 0.24 by Cohen’s kappa test between Ki67\u003csub\u003eIHC\u003c/sub\u003e and Ki67\u003csub\u003eRNA\u003c/sub\u003e in a cohort of 98 patients with early ER+/HER2- breast cancers. Moreover, Ki67\u003csub\u003eRNA\u003c/sub\u003e\u003csup\u003ehigh \u003c/sup\u003etumors were significantly associated with the occurrence of events (\u003cem\u003ep\u003c/em\u003e=0.02). On the other hand, we did not find any association between Ki67\u003csub\u003eIHC\u003c/sub\u003e and EFS (\u003cem\u003ep\u003c/em\u003e=0.25).\u003c/p\u003e\u003cp\u003eWe observed of low agreement between expression level of Ki67\u003csub\u003eRNA\u003c/sub\u003e and Ki67 protein labelling by IHC. Unlike Ki67\u003csub\u003eIHC\u003c/sub\u003e and independently of the RS\u003csup\u003e®\u003c/sup\u003e, Ki67\u003csub\u003eRNA\u003c/sub\u003e could have a prognostic value. It would be interesting to better assess the prognosis and predictive value of Ki67\u003csub\u003eRNA\u003c/sub\u003e measured by qRT-PCR. The Ki67\u003csub\u003eRNA\u003c/sub\u003e in medical routine could be a good support in countries where Oncotype DX\u003csup\u003e®\u003c/sup\u003e is not accessible.\u003c/p\u003e","manuscriptTitle":"Ki67 Assessment By qRT-PCR In OncotypeDx® Breast Recurrent Score®: Low Correlation With Immunohistochemistry Assessment of Ki67 And Prognostic Impact of Ki67 RNA Level of Expression.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-07-15 13:27:09","doi":"10.21203/rs.3.rs-677106/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-10-12T08:42:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-08-25T07:17:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"0e686171-6f04-4967-b3fd-9fc8e3b3221b","date":"2021-08-16T15:33:09+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-08-12T14:20:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-08-09T09:22:46+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-07-12T08:23:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-07-12T08:11:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2021-07-01T10:41:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b774a544-d5e4-4a14-93d4-4acd2d29054a","owner":[],"postedDate":"July 15th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":5715225,"name":"Molecular Biology"},{"id":5715226,"name":"Cancer Biology"}],"tags":[],"updatedAt":"2022-02-16T08:44:14+00:00","versionOfRecord":[],"versionCreatedAt":"2021-07-15 13:27:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-677106","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-677106","identity":"rs-677106","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}