A BRCA1 deficient-like signature is enriched in breast cancer brain metastases and predicts DNA damage-induced poly (ADP-ribose) polymerase inhibitor sensitivity.

INTRODUCTION: There is an unmet clinical need for biomarkers to identify breast cancer patients at an increased risk of developing brain metastases. The objective is to identify gene signatures and biological pathways associated with human epidermal growth factor receptor 2-positive (HER2+) brain metastasis. METHODS: We combined laser capture microdissection and gene expression microarrays to analyze malignant epithelium from HER2+ breast cancer brain metastases with that from HER2+ nonmetastatic primary tumors. Differential gene expression was performed including gene set enrichment analysis (GSEA) using publicly available breast cancer gene expression data sets. RESULTS: In a cohort of HER2+ breast cancer brain metastases, we identified a gene expression signature that anti-correlates with overexpression of BRCA1. Sequence analysis of the HER2+ brain metastases revealed no pathogenic mutations of BRCA1, and therefore the aforementioned signature was designated BRCA1 Deficient-Like (BD-L). Evaluation of an independent cohort of breast cancer metastases demonstrated that BD-L values are significantly higher in brain metastases as compared to other metastatic sites. Although the BD-L signature is present in all subtypes of breast cancer, it is significantly higher in BRCA1 mutant primary tumors as compared with sporadic breast tumors. Additionally, BD-L signature values are significantly higher in HER2-/ER- primary tumors as compared with HER2+/ER + and HER2-/ER + tumors. The BD-L signature correlates with breast cancer cell line pharmacologic response to a combination of poly (ADP-ribose) polymerase (PARP) inhibitor and temozolomide, and the signature outperformed four published gene signatures of BRCA1/2 deficiency. CONCLUSIONS: A BD-L signature is enriched in HER2+ breast cancer brain metastases without pathogenic BRCA1 mutations. Unexpectedly, elevated BD-L values are found in a subset of primary tumors across all breast cancer subtypes. Evaluation of pharmacological sensitivity in breast cancer cell lines representing all breast cancer subtypes suggests the BD-L signature may serve as a biomarker to identify sporadic breast cancer patients who might benefit from a therapeutic combination of PARP inhibitor and temozolomide and may be indicative of a dysfunctional BRCA1-associated pathway.

Single-cell quantitative HER2 measurement identifies heterogeneity and distinct subgroups within traditionally defined HER2-positive patients.

Human epidermal growth factor receptor 2 (HER2) is an important biomarker for breast and gastric cancer prognosis and patient treatment decisions. HER2 positivity, as defined by IHC or fluorescent in situ hybridization testing, remains an imprecise predictor of patient response to HER2-targeted therapies. Challenges to correct HER2 assessment and patient stratification include intratumoral heterogeneity, lack of quantitative and/or objective assays, and differences between measuring HER2 amplification at the protein versus gene level. We developed a novel immunofluorescence method for quantitation of HER2 protein expression at the single-cell level on FFPE patient samples. Our assay uses automated image analysis to identify and classify tumor versus non-tumor cells, as well as quantitate the HER2 staining for each tumor cell. The HER2 staining level is converted to HER2 protein expression using a standard cell pellet array stained in parallel with the tissue sample. This approach allows assessment of HER2 expression and heterogeneity within a tissue section at the single-cell level. By using this assay, we identified distinct subgroups of HER2 heterogeneity within traditional definitions of HER2 positivity in both breast and gastric cancers. Quantitative assessment of intratumoral HER2 heterogeneity may offer an opportunity to improve the identification of patients likely to respond to HER2-targeted therapies. The broad applicability of the assay was demonstrated by measuring HER2 expression profiles on multiple tumor types, and on normal and diseased heart tissues.

Quantitative, fluorescence-based in-situ assessment of protein expression.

As companion diagnostics grow in prevalence and importance, the need for accurate assessment of in situ protein concentrations has increased. Traditional immunohistochemistry (IHC), while valuable for assessment of context of expression, is less valuable for quantification. The lack of rigorous quantitative potential of traditional IHC led to our development of an immunofluorescence-based method now commercialized as the AQUA technology. Immunostaining of tissue samples, image acquisition, and use of AQUA software allow investigators to quickly, efficiently, and accurately measure levels of expression within user-defined subcellular or architectural compartments. IHC analyzed by AQUA shows high reproducibility and demonstrates protein measurement accuracy similar to ELISA assays. The process is largely automated, eliminating potential error, and the resultant scores are exported on a continuous scale. There are now numerous published examples where observations made with this technology are not seen by traditional methods.

Re-evaluating early breast neoplasia.

Historically, histomorphological and epidemiological data suggested that atypical ductal hyperplasia and ductal carcinoma in situ are the earliest recognizable neoplastic stages of breast cancer progression. Over the past several years, detailed high-throughput molecular genetic, gene expression and epigenetic analyses have enhanced our understanding of these early neoplastic lesions and have re-shaped our view of human breast cancer progression to include multiple distinct pathways of evolution.

Antibody validation by quantitative analysis of protein expression using expression of Met in breast cancer as a model.

Expression of Met, the Hepatocyte Growth Factor receptor, has been shown to have prognostic value in numerous types of cancer including breast, gastric, cervical and head and neck carcinomas. However, traditional analyses of expression have shown variable results and a lack of reproducibility. The AQUA system is a method of quantitative in situ analysis of protein expression that allows the assessment of reproducibility of both antibodies and assay conditions. Here, we illustrate the necessity for antibody validation when assaying the prognostic value of a potential biomarker. Using five antibodies to the intracellular domain of the Met receptor and 10 cell line controls, we quantitatively assess reproducibility of protein expression. We show that many antibodies are not reproducible at a quantitative level from lot to lot or assay to assay, suggesting new criteria for antibody validation. We also build upon past literature addressing the prognostic value of Met in a cohort of 640 cases of invasive breast cancer on a tissue microarray. We show that high levels of expression of nuclear Met, as determined by antibodies to the intracellular domain and defined as nuclear by subcellular compartmental analysis, is associated with shorter disease-specific survival in breast cancer.

Met, the hepatocyte growth factor receptor, localizes to the nucleus in cells at low density.

Some breast cancer cases in our previous immunohistochemical studies show Met expression in the nucleus. Given nuclear localization of other receptor tyrosine kinases, we proceeded to investigate Met. Nuclear Met is seen in numerous cell lines and in germinal regions of many tissues using four unique antibodies. Cell fractionation reveals a 60-kDa band recognized by COOH-terminal Met antibodies that is present independent of hepatocyte growth factor treatment. Green fluorescent protein (GFP) fusion proteins of the cytoplasmic domain of Met transfected into HEK293 cells are found in the nucleus whereas the full-length Met-GFP fusion is membranous. Further deletions of the Met-GFP fusions identify a region of the juxtamembrane domain required for nuclear translocation. In a CaCo2 cell line model for epithelial maturation, we find that Met is initially nuclear, and then becomes membranous, after confluence. This work suggests processing of the Met receptor, analogous to ErbB4, resulting in the release of the cytoplasmic domain and its translocation to the nucleus in cells at low density.