Reproducibility of mRNA-Based Testing of ESR1, PGR, ERBB2, and MKI67 Expression in Invasive Breast Cancer—A Europe-Wide External Quality Assessment

Estrogen receptor (ER), progesterone receptor (PgR), Ki-67, and HER2 immunohistochemistry (IHC) together with HER2 in situ hybridization (ISH) are utilized to classify invasive breast cancer (IBC) into predictive molecular subtypes. As IHC evaluation may be hampered by analytical errors, gene expression assays could offer a reliable alternative. In this first Europe-wide external quality assessment (EQA) study, we investigated performance of mRNA-based Xpert® Breast Cancer STRAT4 (CE-IVD) in five European laboratories. The cohort comprised ten pre-therapy IBC core biopsies diagnosed in the coordinating center (CC). STRAT4 binary (positive or negative) mRNA results of each marker (ESR1, PGR, ERBB2, MKI67) were compared with the gold standard IHC/ISH performed by the CC. Sensitivity, specificity, and accuracy of ESR1 and ERBB2 mRNA were 100% for all samples. In contrast, PGR expression was falsely negative for one case by two sites and MKI67 falsely negative for two cases (respectively by four and one sites). These cases had STRAT4 expression values close to assay cut-offs and immunohistochemically presented heterogeneous low positive PgR and heterogeneous Ki-67. Our EQA shows that STRAT4 mRNA assay may be a reproducible method to evaluate ER, PgR, HER2, and Ki-67 status. However, cases with expression values close to assay cut-offs should be carefully reviewed.

KRT20, KRT5, ESR1 and ERBB2 Expression Can Predict Pathologic Outcome in Patients Undergoing Neoadjuvant Chemotherapy and Radical Cystectomy for Muscle-Invasive Bladder Cancer

Patients with muscle-invasive bladder cancer (MIBC) that underwent neoadjuvant chemotherapy (NAC) prior to radical cystectomy (RC) show improved overall survival, especially those with pathological complete response (pCR). The response to NAC according to molecular subtypes has been discussed. Molecular targets such as estrogen receptor (ESR1) and human epidermal growth factor receptor 2 (ERBB2) play an important role in breast cancer management and have also been associated with urothelial bladder cancer. Hence, the association of Keratin 20 (KRT20) Keratin 5 (KRT5), ESR1, and ERBB2 mRNA expression in MIBC at transurethral resection (TUR-BT) with pCR after NAC was analyzed retrospectively. Formalin-fixed paraffin-embedded tumour tissue samples from TUR-BT of 54 patients (42 males, 12 females, median age of 64) with MIBC were analyzed for KRT20, KRT5, ESR1, and ERBB2 mRNA expression. After NAC, RC was performed, and the specimens were evaluated for pCR. Statistical analyses comprised nonparametric and chi2 testing, partition models, and Spearman correlation analyses. After NAC, 22 out of 54 patients (40.7%) had pCR. Tumours with an elevated expression of markers associated with luminal differentiation (KRT20, ERBB2, ESR1) were associated with a higher chance of pCR (55% vs. 15.8%, p = 0.009). Elevated ERBB2 expression was positively correlated with luminal expression features such as KRT20, and negatively with basal characteristics such as KRT5. Patients with MIBC showing a high expression of ERBB2, ESR1, or KRT20 have a significantly higher chance of pCR following NAC. These findings might improve patient selection for NAC in MIBC.

The mammalian ecdysoneless protein interacts with RNA helicase DDX39A to regulate nuclear mRNA export

The mammalian orthologue of Ecdysoneless (ECD) protein is required for embryogenesis, cell cycle progression, and mitigation of ER stress. Here, we identified key components of the mRNA export complexes as binding partners of ECD and characterized the functional interaction of ECD with key mRNA export-related DEAD BOX protein helicase DDX39A. We find that ECD is involved in RNA export through its interaction with DDX39A. ECD knockdown (KD) blocks mRNA export from the nucleus to the cytoplasm, which is rescued by expression of full length ECD but not ECD mutant that is defective in interaction with DDX39A. We have previously shown that ECD protein is overexpressed in ErbB2+ breast cancers (BC). In this study, we extended the analyses to two publically available BC mRNA TCGA and METABRIC data sets. In both dataset, ECD mRNA overexpression correlated with short patient survival, specifically ErbB2+ BC. In METABRIC dataset ECD overexpression also correlated with poor patient survival in TNBC . Further, ECD KD in ErbB2+BC cells led to a decrease in ErbB2 mRNA level due to a block in its nuclear export, and was associated with impairment of oncogenic traits. These findings provide a novel mechanistic insight into physiological and pathological function of ECD.

SATB1 and Her2 as predictive molecular and immunohistochemical markers for urothelial cell carcinoma of the bladder

Studying bladder cancer molecular biology revealed the presence of genetic alterations. So, detection of molecular biomarkers that help in monitoring the disease, evaluating the prognosis of the patients, and their response to therapy is needed. In this study, we investigated the expression and the prognostic significance of SATB-1 and ERBB2 mRNA and protein by quantitative RT-PCR and immunohistochemical analysis in urothelial bladder cancer cases and the surrounding normal bladder tissue. The correlations between the expression of both markers and the clinicopathological parameters were performed with further analysis of the correlation between the expression of SATB-1 and ERBB2. Compared to control, the expression of SATB-1 and ERBB2 mRNA and protein in cancer tissues were significantly up-regulated (p< 0.05). Also, a positive correlation between both markers was found (r= 0.53, p< 0.001). Moreover, elevated levels of both markers were significantly associated with the stage, lymph node involvement at both mRNA and protein levels (p< 0.001). In conclusion, there is a clinical significance of SATB-1 and ERBB2 as potential biomarkers for predicting bladder cancer patients of aggressive behavior and poor prognosis.

ERBB2 mRNA Expression and Response to Ado-Trastuzumab Emtansine (T-DM1) in HER2-Positive Breast Cancer

Trastuzumab emtansine (T-DM1) is approved for the treatment of human epidermal growth factor receptor 2 (HER2)-positive (HER2+) metastatic breast cancer (BC) and for residual disease after neoadjuvant therapy; however, not all patients benefit. Here, we hypothesized that the heterogeneity in the response seen in patients is partly explained by the levels of human epidermal growth factor receptor 2 gene (ERBB2) mRNA. We analyzed ERBB2 expression using a clinically applicable assay in formalin-fixed paraffin-embedded (FFPE) tumors (primary or metastatic) from a retrospective series of 77 patients with advanced HER2+ BC treated with T-DM1. The association of ERBB2 levels and response was further validated in 161 baseline tumors from the West German Study (WGS) Group ADAPT phase II trial exploring neoadjuvant T-DM1 and 9 in vitro BC cell lines. Finally, ERBB2 expression was explored in 392 BCs from an in-house dataset, 368 primary BCs from The Cancer Genome Atlas (TCGA) dataset and 10,071 tumors representing 33 cancer types from the PanCancer TCGA dataset. High ERBB2 mRNA was found associated with better response and progression-free survival in the metastatic setting and higher rates of pathological complete response in the neoadjuvant setting. ERBB2 expression also correlated with in vitro response to T-DM1. Finally, our assay identified 0.20–8.41% of tumors across 15 cancer types as ERBB2-high, including gastric and esophagus adenocarcinomas, urothelial carcinoma, cervical squamous carcinoma and pancreatic cancer. In particular, we identified high ERBB2 mRNA in a patient with HER2+ advanced gastric cancer who achieved a long-lasting partial response to T-DM1. Our study demonstrates that the heterogeneity in response to T-DM1 is partly explained by ERBB2 levels and provides a clinically applicable assay to be tested in future clinical trials of breast cancer and other cancer types.

Genomic-based predictive biomarkers to anti-HER2 therapies: A combined analysis of CALGB 40601 (Alliance) and PAMELA clinical trials.

571 Background: In HER2-positive breast cancer, new biomarkers of response are needed in order to direct multi-agent anti-HER2 combinations towards patients in whom they are truly needed. CALGB 40601 and PAMELA trials tested neoadjuvant dual HER2 blockade and included gene expression analysis aimed to evaluate different genomic biomarkers of trastuzumab and/or lapatinib benefit. Methods: Gene expression by mRNA sequencing (RNAseq) was performed on 265 and 142 pre-treatment tumors of the CALGB 40601 and the PAMELA clinical trials respectively. Intrinsic subtypes were determined by nCounter PAM50-predictor on the PAMELA samples. A new HER2-positive specific gene-centering method was trained on the PAMELA RNAseq data, and showed a higher concordance with PAM50 predictions obtained from nCounter platform. This method was then applied to CALGB 40601 samples. Results: In the combined cohort, the subtype distribution was 10% Luminal A, 8% Luminal B, 62% HER2-enriched (HER2-E), 10% Basal and 10% Normal-like. The pCR rate was significantly higher in HER2-E vs. not HER2-E subtypes (48.6% vs. 20.7%; P < 0.001). HER2-E subtype correlation, ERBB2 amplicon and B-cell genomic signatures were associated with pCR, while luminal signatures were associated with non-responders. In multivariate analysis HER2-E subtype, ERBB2 mRNA and IgG signature expression were independent predictors of response to paclitaxel + trastuzumab +/-lapatinib (OR = 1.98, OR = 1.51, OR = 1.48, respectively, P <0.05). The event free survival analysis at 5 years in the CALGB 40601 cohort showed a benefit of dual vs single anti-HER2-blockade (HR 0.35, P <0.05). Within the HER2-E, ERBB2-high and IgG–high subpopulations, there were also a benefit of dual vs. single anti-HER2 treatment (HR = 0.32, HR = 0.15, HR =0.15, respectively, P <0.05). Conclusions: Intrinsic subtype, ERBB2 mRNA levels, and IgG genomic signature are independent predictive biomarkers of response in the combined cohort. The clinical implementation of these biomarkers could help to design future escalation/de-escalation clinical trials in the HER2-positive neoadjuvant setting. Support: U10CA180821, U10CA180882, U24CA196171, P50-CA58223, GSK, SPORE, BCRF and SEOM. https://acknowledgments.alliancefound.org .