Ki67 Assessment by qRT-PCR in OncotypeDx ® Breast Recurrent Score®: Low Correlation With IHC Assessment of ki67 and Prognostic Impact of ki67 RNA Level of Expression.

Background: Breast cancers expressing high levels of Ki67 are associated with poor outcomes. To provide individualized patient care, reliability of prognostic and predictive information deriving from Ki67 assessment is essential. 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, and aim to predict the benet of chemotherapy and the risk of distant recurrence. 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 . Methods: In a cohort of 98 patients with early ER+/HER2- breast cancers, we obtained: Recurrence score ® , RNA level of Ki67 expression measured when assessing RS ® and Ki67 tumor labelling by immunohistochemistry (Ki67 IHC ). The Ki67 RNA was compared to the Ki67 IHC by Kappa Cohen test. With a mean follow-up of 57 months, the association between the event free survival (EFS) and Ki67 status was measured using R package survival. Results: This population was essentially composed of no special type tumor (91%), N0 or Nmic (71%), grade 1 (62%), and tumor size pT1c (1-2 cm) (58%). The RS ® values were <18 in 38% (n=37), 18-30 in 51% (n=50) and >30 in 11% (n=11) of the patients. A low agreement of 0.24 was found by Cohen’s kappa test between Ki67 IHC and Ki67 RNA . 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). Conclusion:

Conclusion: 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.

Background
In terms of incidence, prevalence and mortality, breast cancer is in rst 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 speci c 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 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][5][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][9][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]. 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 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

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 [24]. The statistical comparison between IHC and ODX ® was realized by Cohen's kappa test.
Statistical analyzes were performed using R version 3.6.2 [25]. The survival follow-up was analyzed by the package survival version 3.  (Figure 2A) unlike Ki67IHC (p=0.25) ( Figure 2B). However, we did not observe any signi cant difference on EFS according to the three RS® groups risk (p=0.4) (Figure 3). Using the age categories from the TAILORx study [27], the association between EFS and Ki67RNA 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 Ki67IHC and EFS was observed (p>0.20).
3.4. Ki67RNA association with RS® As expected, Ki67RNA levels were signi cantly associated with RS® (p=5.10-4) ( Figure 4A). To a lesser extent, an association between ODX® test with Ki67IHC status has also been observed (p=0.013) ( Figure  4B). Supplementary data 4 shows the distribution of Ki67IHC score and Ki67RNA level according to patient age, tumor grade and size, PR status, nodal status, Recurrence score, treatment and events.  [20,21] and is less reproducible than the quanti cation 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 Ki67IHC informs us about the tumor speci c proliferation index and the Ki67RNA would re ect the proliferation of the tumor and its microenvironment. For all the reasons mentioned above, the association between RS® and Ki67IHC status is lower but remains signi cant (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 con rmed the e ciency 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 [27], WSG Plan B [29] and analyses from a prospective epidemiological database [30]). 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 [31], ESMO [32], and ASCO [33]). 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 Ki67RNAhigh was signi cantly associated with the occurrence of events (p=0.02) but we did not nd any association between Ki67IHC 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 Ki67RNA evaluation has a prognostic impact as RS®.

Correlation between Ki67RNA and RS®
As expected in our cohort, the RS® obtained is strongly associated with the Ki67RNA (p=5.  which is one of the components of this test ( Figure 4A). To optimize RS® interpretation, the TAILORx subgroup study shows a bene t 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 [34].
Recently in San Antonio Breast cancer symposium, the results of RxPONDER study were presented [35]. 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 signi cantly bene t from chemotherapy [35]. 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 bene t in routine care have been shown in 20 decision-impact studies [36][37][38]. However, in many places, the oncotype score remains inaccessible or not reimbursed [7,28,38]. This is why the evaluation of the level of Ki67RNA by a molecular biology techniques [39,40] could be a low-cost prognostic alternative in some countries. But this hypothesis will have to be validated during a prospective translational study.