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Research objective Previously, we reported the negative correlation between pathological complete response (pCR) and HER2 positive breast cancer exhibiting amplified HER2 gene tumor cells without HER2 protein overexpression (HER2 epigenetic intratumoral heterogeneity) among trastuzumab-based neoadjuvant chemotherapy treated patients. Our objective in this study was to elucidate if tumor cells with HER2 epigenetic intratumoral heterogeneity express HER2 RNA using a HER2 RNA in situ hybridization (ISH) method. Materials and methods Formalin-fixed, paraffin-embedded (FFPE) sections of breast cancer biopsy samples were investigated for HER2 RNA expression at the individual cell level using a HER2 RNA ISH assay. RNA preservation in tissue sections was examined using a peptidylprolyl isomerase B (PPIB) RNA ISH assay. Three groups of cases were examined: 1) HER2 negative breast cancer cases (HER2 RNA ISH negative control group). 2) HER2 positive breast cancer cases with homogeneous HER2 positive tumor cells (HER2 RNA ISH positive control; pCR group) 3) HER2 positive breast cancer cases with HER2 epigenetic intratumoral heterogeneity (a mixture of HER2 gene and protein positive tumor cells and HER2 gene positive tumor cells without HER2 protein expression; incomplete response group) Consecutive sections of HER2 RNA ISH slides were stained for HER2 gene and protein concurrently on the same tissue section using HER2 gene-protein assay (GPA) which is a combination of FDA-approved HER2 immunohistochemical (HER2 protein) and HER2 dual ISH (HER2 gene and chromosome 17 centromere) assays. Analyses of HER2 RNA expression in individual cells was microscopically evaluated and matched to HER2 GPA slides. Results RNA preservation was confirmed in tissue sections of all three groups by a PPIB RNA ISH assay. Tumor cells of HER2 negative breast cancer cases (negative control group) lacked HER2 RNA ISH signal while HER2 gene and protein positive tumor cells of homogeneous breast cancer cases (positive control group) demonstrated high HER2 RNA expression levels. HER2 gene and protein positive tumor cells of HER2 positive intratumoral heterogeneity cases showed high HER2 RNA expression. However, amplified HER2 gene breast cancer cells without HER2 protein overexpression of HER2 positive intratumoral heterogeneity cases also showed high levels of HER2 RNA expression. Thus, revealing in cases with intratumoral heterogeneity, transcription of HER2 RNA occurs, but translation of HER2 protein is altered by some mechanism(s) in tumor cells. Conclusions Transcription of HER2 RNA was observed in breast tumor cells with amplified HER2 gene but absence of HER2 protein overexpression (HER2 epigenetic intratumoral heterogeneity) of patients who showed incomplete response to neoadjuvant trastuzumab therapy. Our study suggests that inconsistent HER2 protein translation in breast cancer with HER2 epigenetic heterogeneity might be the primary resistance mechanism to trastuzumab-based neoadjuvant chemotherapy. Citation Format: Nitta H, Horii R, Murillo A, Portier B, Akiyama F. Breast cancer HER2 epigenetic intratumoral heterogeneity results from lack of HER2 protein translation [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-06-02.

While combinations of immune checkpoint (ICP) inhibitors and neo-adjuvant chemotherapy (NAC) have begun to be tested in patients with breast cancer (BC), chemotherapeutic effects on ICP expression in circulating T cells are still unclear. This information could help design future clinical trials including the selection of the best ICP inhibitors to be incorporated into NAC and finding predictive/prognostic biomarkers. Peripheral blood samples and/or tumor specimens before and after NAC were obtained from twenty-four women with operable BC. Using flow cytometry, the expression of CTLA4, PD-1, Lag3, OX40, and Tim3 on circulating T lymphocytes before and at the end of NAC were measured. Differences in the percentage of CD4+ and CD8+ T cells expressing various checkpoint receptors pre- and post-NAC were determined by a paired t-test. This data showed decreased ICP expression after NAC by circulating CD4+ T cells, including significant decreases in CTLA4 (p Citation Format: Dionisia M. Quiroga, Andrew Stiff, Christopher McQuinn, Zaibo Li, Hiroaki Nitta, Himanshu Savardekar, Brooke Benner, Bhuvaneswari Ramaswamy, Maryam Lustberg, Rachel Layman, Erin Macrae, Mahmoud Kassem, Nicole Williams, Sagar Sardesai, Jeffrey VanDeusen, Daniel Stover, Mathew Cherian, Thomas Mace, Lianbo Yu, Megan Duggan, William E. Carson, Robert Wesolowski. Analysis of immune checkpoint receptor expression by circulating T cells and tumor specimens in patients pre- and post-neoadjuvant chemotherapy for operable breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5514.

Background: The eligibility of patients for HER2-targeted therapies is determined by the evaluation of HER2 gene amplification and HER2 protein overexpression. The gene-protein assay (GPA, Ventana Medical Systems, Inc., USA) is a new method for simultaneous evaluation of HER2 immunohistochemistry (IHC) and dual in situ hybridization (DISH) using a single tissue section. In this study, we investigated the relationship between HER2 IHC and DISH results evaluated by GPA. In addition, we analyzed the correlation between HER2 status and prognosis of invasive breast cancer patients. Patients and Methods: In this study, invasive carcinoma tissues of 280 consecutive patients treated in Saitama Cancer Center in 2000-2001 (median follow-up: 130 months) were examined. Among HER2 positive initial samplings, no patients originally received adjuvant trastuzumab therapy. However, 76% of HER2 positive cases of recurrence received trastuzumab therapy. GPA was performed on a section of routinely processed primary tumor and the status of HER gene and protein were separately evaluated in whole areas of tumor sections using the following FDA criteria: DISH (negative: HER2/CEN17 < 2, positive: HER2/CEN17 ≥ 2.0) and IHC (score 0 to 3+). In IHC score 2+ patients group, final HER2 positivity was decided according to DISH results using criteria of FDA criteria. Recurrence-free survival (RFS) and cancer-specific survival (CSS) stratified by IHC and DISH results were analyzed. In addition, patterns of heterogeneity were grouped according to the following 4 phenotypic and genotypic types: A) IHC 2+/DISH+; B) IHC 2+/DISH-; C) IHC 1+ & 0/DISH+; and D) IHC 1+ & 0/DISH-. The presence of heterogeneity in relation to prognosis was analyzed in the IHC 0 & 1+/DISH- group. Results: The HER2 IHC 3+ group (27.5%), both with or without trastuzumab therapy, had significantly worse survival than HER2 IHC 1+ & 0 group (RFS: P=0.0039; CSS: P=0.0362) and HER2 DISH+ group (27.5%) had significantly worse survival than HER2 DISH- group (RFS: P=0.0056; CSS: P=0.0497). HER2 positive group defined by FDA criteria had significantly worse RFS than HER2 negative group (P=0.0211). HER2 IHC 1+ & 0/DISH+ group had significantly worse RFS than IHC 1+ & 0/DISH- group (P=0.0208). In the HER2 IHC 1+ & 0/ DISH- group, patients with heterogeneity (33 cases) had significantly worse survival than those without heterogeneity (RFS: P=0.0176; CSS: P=0.0199). Conclusions: HER2 GPA technology might be useful for evaluating the discrepancy and heterogeneity of HER2 IHC and DISH results at single cell levels simultaneously and the presence of HER2 tumor cell heterogeneity might be a potent prognostic factor in HER2 negative breast cancer patients. Further clinical research must be conducted for clarification of the relationship between the presence of HER2 intratumoral heterogeneity and the effectiveness of HER2-targeted therapies. Citation Format: Sasagu Kurozumi, Mary Padilla, Masafumi Kurosumi, Hiroshi Matsumoto, Yuji Hayashi, Katsunori Tozuka, Shigenori E Nagai, Kenichi Inoue, Hanako Oba, Jun Horiguchi, Izumi Takeyoshi, Jim Ranger-Moore, Eslie Dennis, Hiroaki Nitta. Utility of simultaneous HER2 protein and gene assessment for the evaluation of discrepancy and intratumoral heterogeneity of HER2 status and the prediction of prognosis in invasive breast cancer using the gene-protein assay (GPA) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-04-10.