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.

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 specic 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 RNAhigh tumors were signicantly associated with the occurrence of events (p=0.02). On the other hand, we did not nd 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.


Introduction
In terms of incidence, prevalence and mortality, breast cancer is in rst place worldwide in women 1  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 bene t. 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 bene t 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-speci c reference values 15  However, little is known about variability in IHC Ki67-labelling results between routine pathology labs 20,21 and its potential in uence 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 < 18 in 38% (n = 37), 18-30 in 51% (n = 50) and > 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.

Agreement between Ki67 IHC and Ki67 RNA
The Ki67 IHC positivity rate of > 20% 23 was used to de ne for the "high-risk" tumor group. We showed by ROC curve analysis an optimal threshold at 6.35 with a speci city 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. 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%) (  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 signi cantly associated with the occurrence of events (p = 0.02) but we did not nd 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 con rm 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. 4A). 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 in Genomic Health report could also be helpful for therapeutic decisions on borderline situation.

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 scienti c 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