Biological subtyping of early breast cancer : a study comparing RT-qPCR with immunohistochemistry

The biological subtype of breast cancer influences the selection of systemic therapy. Distinction between luminal A and B cancers depends on consistent assessment of Ki-67, but substantial intra-observer and inter-observer variability exists when immunohistochemistry (IHC) is used. We compared RT-qPCR with IHC in the assessment of Ki-67 and other standard factors used in breast cancer subtyping. RNA was extracted from archival breast tumour tissue of 769 women randomly assigned to the FinHer trial. Cancer ESR1, PGR, ERBB2 and MKI67 mRNA content was quantitated with an RT-qPCR assay. Local pathologists assessed ER, PgR and Ki-67 expression using IHC. HER2 amplification was identified with chromogenic in situ hybridization (CISH) centrally. The results were correlated with distant disease-free survival (DDFS) and overall survival (OS). qPCR-based and IHC-based assessments of ER and PgR showed good concordance. Both low tumour MKI67 mRNA (RT-qPCR) and Ki-67 protein (IHC) levels were prognostic for favourable DDFS [hazard ratio (HR) 0.42, 95 % CI 0.25-0.71, P = 0.001; and HR 0.56, 0.37-0.84, P = 0.005, respectively] and OS. In multivariable analyses, cancer MKI67 mRNA content had independent influence on DDFS (adjusted HR 0.51, 95 % CI 0.29-0.89, P = 0.019) while Ki-67 protein expression had not any influence (P = 0.266) whereas both assessments influenced independently OS. Luminal B patients treated with docetaxel-FEC had more favourable DDFS and OS than those treated with vinorelbine-FEC when the subtype was defined by RT-qPCR (for DDFS, HR 0.52, 95 % CI 0.29-0.94, P = 0.031), but not when defined using IHC. Breast cancer subtypes approximated with RT-qPCR and IHC show good concordance, but cancer MKI67 mRNA content correlated slightly better with DDFS than Ki-67 expression. The findings based on MKI67 mRNA content suggest that patients with luminal B cancer benefit more from docetaxel-FEC than from vinorelbine-FEC.Peer reviewe

Comparison of immunohistochemistry with PCR for assessment of ER, PR, and Ki-67 and prediction of pathological complete response in breast cancer

Background: Proliferation may predict response to neoadjuvant therapy of breast cancer and is commonly assessed by manual scoring of slides stained by immunohistochemistry (IHC) for Ki-67 similar to ER and PgR. This method carries significant intra- and inter-observer variability. Automatic scoring of Ki-67 with digital image analysis (qIHC) or assessment of MKI67 gene expression with RT-qPCR may improve diagnostic accuracy. Methods: Ki-67 IHC visual assessment was compared to the IHC nuclear tool (AperioTM) on core biopsies from a randomized neoadjuvant clinical trial. Expression of ESR1, PGR and MKI67 by RT-qPCR was performed on RNA extracted from the same formalin-fixed paraffin-embedded tissue. Concordance between the three methods (vIHC, qIHC and RT-qPCR) was assessed for all 3 markers. The potential of Ki-67 IHC and RT-qPCR to predict pathological complete response (pCR) was evaluated using ROC analysis and non-parametric Mann-Whitney Test. Results: Correlation between methods (qIHC versus RT-qPCR) was high for ER and PgR (spearman´s r = 0.82, p < 0.0001 and r = 0.86, p < 0.0001, respectively) resulting in high levels of concordance using predefined cut-offs. When comparing qIHC of ER and PgR with RT-qPCR of ESR1 and PGR the overall agreement was 96.6 and 91.4%, respectively, while overall agreement of visual IHC with RT-qPCR was slightly lower for ER/ESR1 and PR/PGR (91.2 and 92.9%, respectively). In contrast, only a moderate correlation was observed between qIHC and RT-qPCR continuous data for Ki-67/MKI67 (Spearman’s r = 0.50, p = 0.0001). Up to now no predictive cut-off for Ki-67 assessment by IHC has been established to predict response to neoadjuvant chemotherapy. Setting the desired sensitivity at 100%, specificity for the prediction of pCR (ypT0ypN0) was significantly higher for mRNA than for protein (68.9% vs. 22.2%). Moreover, the proliferation levels in patients achieving a pCR versus not differed significantly using MKI67 RNA expression (Mann-Whitney p = 0.002), but not with qIHC of Ki-67 (Mann-Whitney p = 0.097) or vIHC of Ki-67 (p = 0.131). Conclusion: Digital image analysis can successfully be implemented for assessing ER, PR and Ki-67. IHC for ER and PR reveals high concordance with RT-qPCR. However, RT-qPCR displays a broader dynamic range and higher sensitivity than IHC. Moreover, correlation between Ki-67 qIHC and RT-qPCR is only moderate and RT-qPCR with MammaTyper® outperforms qIHC in predicting pCR. Both methods yield improvements to error-prone manual scoring of Ki-67. However, RT-qPCR was significantly more specific

Identifying the Molecular Mechanisms Contributing to Progression, Metastasis, and Death in Low-grade Non-muscle-invasive Bladder Cancer: A Case Report

Transitional cell carcinoma of the bladder is a common malignancy with an estimated 549 393 new cases occurring in 2018 alone. Both non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) show high recurrence and progression rates, and therefore impose a great burden on patients and health care systems. Current risk stratification and therapy strategies are predominantly based on clinical and histopathological findings for tumor stage and grade. The chemoresistance and metastasis of low-grade tumors suggest an incomplete understanding of disease mechanisms, despite numerous studies on differentiating molecular subtypes of bladder cancer to identify tumor drivers and potential therapeutic targets. We present a highly unusual course for a low-grade bladder tumor leading to metastasis and death, for which we used postmortem histopathological and molecular analyses to evaluate targetable alterations in key signaling pathways driving the underlying tumor biology. (C) 2021 The Authors. Published by Elsevier B.V. on behalf of European Association of Urology

Predictive value of ultra-high ESR1 mRNA expression in early breast cancer.

Abstract Background Quantitative determination of estrogen receptor mRNA expression in luminal breast tumors is predictive for benefit from adjuvant tamoxifen compared to placebo treatment as has been shown in the large randomized NSABP B-14 trial, while protein determination by IHC or LBA is not (Kim et al JCO 2011). Interestingly, the ultrahigh expression of ESR1 mRNA (above ER score 10 by Oncotype test) has been indicative for tamoxifen benefit. This predictive cut-off value of mRNA expression is significantly higher than the diagnostic cut-off (at ER score 6.5). Here we tested wether the ultrahigh expression of ESR1 mRNA determined by commercial MammaTyper® testing is predictive for survival after neoadjuvant chemotherapy treatment of advanced breast cancer. Materials and Methods Pretreatment core cut biopsies from n=54 patients with PBC treated within a randomized phase II trial (2) of anthracyline/taxane based NAC with available clinical follow-up information were examined. RNA was extracted from the FFPE sections and ESR1 mRNA from each section was measured by commercial assays. For technical comparison of ESR1 mRNA values by Oncotype DX versus MammaTyper® from n=113 surgical samples were analyzed by both commercial assays in a blinded fashion. Statistical analysis was performed using the SAS JMP® 9.0.0 software. Results Quantification of ESR1 mRNA expression after RNA extraction from separate slices of 113 primary breast tumors and determination by different commercial RT-qPCR assays resulted in high correlation of continuous expression results (Spearman r=0,85; p&amp;lt;0,0001). The rate of ESR1 mRNA negative cases by both methods by predefined diagnostic cut-offs was low in this cohort (1/113 and 6/113, respectively) resulting in high concordance for positive ER status by both methods.The median expression of ER score and ESR1 40-DDCq was high (10,2 and 39,8, respectively) and almost exactly at the predictive ER score cut-off. Hence, the Tamoxifen benefit cut-off of ER score 10 by Oncotype is comparable with a 40-DDCqvalue of 39,6 for ESR1 mRNA determination by MammaTyper®, which resembles an ESR1 mRNA expression 3fold above the diagnostic cut-off. In the independent chemotherapy cohort theoptimal discrimination for overall survival could be achieved by an elevated ESR1 mRNA expression exactly at 39,6 resulting in 100% overall survival for ultra-high expressors and 75 % overall survival for lower ESR1 mRNA expression after 5 years (p=0,006). Conclusion Previous data suggest that ultrahigh expression of ESR1 mRNA is predictive for improved overall survival and tamoxifen benefit (1). Here we show that ultrahigh expression of ESR1 mRNA is also prognostic in more advanced breast tumors after neoadjuvant chemotherapy. These findings validate the importance of quantitative determination of estrogen receptor expression and substantiate the understanding of receptor expression being a continuous determinant with indication specific cut-off values. Ultrahigh expression of ESR1 seems to identify a distinct subset of luminal breast tumors with superior prognosis and benefit from tamoxifen treatment. These findings warrant further investigation, which are currently being done in independent large breast cancer cohorts. Citation Format: Wirtz RM, Scheffen I, Marme F, Laible M, Sclombs K, Schumacher C, Schneeweiss A, Eidt S, Sinn H-P. Predictive value of ultra-high ESR1 mRNA expression in early breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-05-25.</jats:p

Prognostic Impact of mRNA Expression Levels of HER1–4 (ERBB1–4) in Patients with Locally Advanced Rectal Cancer

Background. No predictive or prognostic biomarker is available for patients with locally advanced rectal cancer (LARC) undergoing perioperative chemoradiotherapy (CRT). Members of the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases EGFR (HER1, ERBB1), HER2 (ERBB2), HER3 (ERBB3), and HER4 (ERBB4) are therapeutic targets in several cancers. The analysis was performed to assess expression levels and study the potential prognostic impact for disease-free and overall survival in patients with LARC. Patients and Methods. ERBB1–4 mRNA expression and tumor proliferation using Ki-67 (MKI67) mRNA were evaluated using RT-quantitative PCR in paraffin-embedded tumor samples from 86 patients (median age: 63) treated with capecitabine or 5-fluorouracil-based CRT within a phase 3 clinical trial. Results. A positive correlation of HER4 and HER2, HER3 and HER2, and HER4 and HER3 with each other was observed. Patients with high mRNA expression of ERBB1 (EGFR, HER1) had significantly increased risk for recurrence and death. Patients with high mRNA expression of MKI67 had reduced risk for relapse. Conclusion. This analysis suggests a prognostic impact of both ERBB1 and MKi67 mRNA expression in LARC patients treated with capecitabine or fluorouracil-based chemoradiotherapy