Proteomics-derived basal biomarker DNA-PKcs is associated with intrinsic subtype and long-term clinical outcomes in breast cancer

Precise biomarkers are needed to guide better diagnostics and therapeutics for basal-like breast cancer, for which DNA-dependent protein kinase catalytic subunit (DNA-PKcs) has been recently reported by the Clinical Proteomic Tumor Analysis Consortium as the most specific biomarker. We evaluated DNA-PKcs expression in clinically-annotated breast cancer tissue microarrays and correlated results with immune biomarkers (training set: n = 300; validation set: n = 2401). Following a pre-specified study design per REMARK criteria, we found that high expression of DNA-PKcs was significantly associated with stromal and CD8 + tumor infiltrating lymphocytes. Within the basal-like subtype, tumors with low DNA-PKcs and high tumor-infiltrating lymphocytes displayed the most favourable survival. DNA-PKcs expression by immunohistochemistry identified estrogen receptor-positive cases with a basal-like gene expression subtype. Non-silent mutations in PRKDC were significantly associated with poor outcomes. Integrating DNA-PKcs expression with validated immune biomarkers could guide patient selection for DNA-PKcs targeting strategies, DNA-damaging agents, and their combination with an immune-checkpoint blockade

The immune microenvironment and relation to outcome in patients with advanced breast cancer treated with docetaxel with or without gemcitabine

Preclinical studies suggest that some effects of conventional chemotherapy, and in particular, gemcitabine, are mediated through enhanced antitumor immune responses. The objective of this study was to use material from a randomized clinical trial to evaluate whether patients with preexisting immune infiltrates responded better to treatment with gemcitabine + docetaxel (GD) compared to docetaxel alone. Formalin fixed, paraffin-embedded breast cancer tissues from SBG0102 phase 3 trial patients randomly assigned to treatment with GD or docetaxel were used. Immunohistochemical staining for CD8, FOXP3, LAG3, PD-1, PD-L1 and CD163 was performed. Tumor infiltrating lymphocytes (TILs) and tumor associated macrophages were evaluated. Prespecified statistical analyses were performed in a formal prospective-retrospective design. Time to progression was primary endpoint and overall survival secondary endpoint. Correlations between biomarker status and endpoints were evaluated using the Kaplan-Meier method and Cox proportional hazards models. Biomarker data was obtained for 237 patients. There was no difference in treatment effect according to biomarker status for the whole cohort. In planned subgroup analysis by PAM50 subtype, in non-luminal (basal-like and HER2E) breast cancers FOXP3 was a significant predictor of treatment effect with GD compared to docetaxel, with a HR of 0.22 (0.09-0.52) for tumors with low FOXP3 compared to HR 0.92 (0.47-1.80) for high FOXP3 TILs (Pinteraction = 0.01). Immune biomarkers were not predictive of added benefit of gemcitabine in a cohort of mixed breast cancer subtypes. However, in non-luminal breast cancers, patients with low FOXP3+ TILs may have significant benefit from added gemcitabine

Phospho-Ser784-VCP is required for DNA damage response and is associated with poor prognosis of chemotherapy-treated breast cancer

Spatiotemporal protein reorganization at DNA damage sites induced by genotoxic chemotherapies is crucial for DNA damage response (DDR), which influences treatment response by directing cancer cell fate. This process is orchestrated by valosin-containing protein (VCP), an AAA+ ATPase that extracts polyubiquinated chromatin proteins and facilitates their turnover. However, because of the essential and pleiotropic effects of VCP in global proteostasis, it remains challenging practically to understand and target its DDR-specific functions. We describe a DNA-damage-induced phosphorylation event (Se

Clinical utility of biomarkers for basal-like breast cancer

The genomic subtyping of breast cancers into luminal A, luminal B, human epidermal growth factor receptor-2 (Her2)-Enriched and basal-like has remarkably advanced breast cancer diagnosis, treatment, and outcome. However, comparatively few advancements have been made in identifying biomarkers that can tailor treatments for the aggressive basal-like subtype. This group is clinically approximated as triple negative breast cancer (TNBC), characterized by immunohistochemical negativity for estrogen receptor, progesterone receptor, and Her2. However, this definition identifies a biologically heterogenous group, highlighting the complexity of guiding therapeutic choices for TNBC including those with basal-like molecular biology. My research goals have included identifying improved diagnostic biomarkers that can guide better therapeutic options for these aggressive cancers in a clinically-applicable manner using immunohistochemistry, RNA expression, and proteomic profiling. First, applying an optimized immunohistochemical panel, defined by nestin positivity or INPP4B negativity, on 239 specimens from the phase III SBG0102 clinical trial, I was able to identify those with a basal-like gene expression subtype and to predict which metastatic breast cancer patients benefit from gemcitabine chemotherapy. Then, using a 770-gene RNA panel targeting multiple biological mechanisms and additional 30-custom genes related to capecitabine metabolism on 111 TNBC specimens from the phase III FinXX adjuvant capecitabine trial, I found that genes and metagenes related to immune response (cytotoxic cells, PDL2), endothelial, mast cells and 38 individual genes are the most significantly associated with capecitabine benefit. Finally, to characterize the heterogeneity of basal-like and triple negative breast cancers beyond current genomic classifications, I used a method called SP3-Clinical Tissue Proteomics, compatible with routine clinical specimens, for comprehensive protein profiling which revealed distinct subgroups within basal-like and triple negative breast cancers. Specifically, within 88 TNBC samples, four proteomic clusters were evident, which had features of “basal-immune hot”, “basal-immune cold”, “mesenchymal”, and “luminal”, each with distinct clinical outcomes. This work sets a foundation for developing clinically-applicable tests that can tackle the diversity of basal-like and triple negative breast cancers using standard formalin-fixed pathology clinical trial materials. The findings presented may guide the selection of breast cancer patients for clinical trial evaluation of existing chemotherapy or emerging therapies.Medicine, Faculty ofGraduat

Tumor Copy Number Alteration Burden as a Predictor for Resistance to Immune Checkpoint Blockade across Different Cancer Types

Immune checkpoint blockade (ICB) benefits only a subset of advanced cancer patients, and predictive biomarkers for immunotherapy response are needed. Recently, copy number alteration (CNA) burden has been proposed to predict ICB resistance. We assessed this finding using the publicly accessible data for 1661 ICB-treated patients whose tumors were profiled by MSK-IMPACT, an approved targeted assay in clinical care. We tested the hypothesis that the continuous increase in CNA burden is associated with poor overall survival following ICB. In addition, we hypothesized that the combinatorial biomarkers of tumor mutational burden (TMB) and CNA burden would better stratify patients for immune status and ICB response. Of the 1661 cases, 79% (n = 1307) were treated with anti PD-1/PD-L1 and the remaining 21% (n = 354) with anti CTLA-4 or the combination of both. In a multivariate analysis, increase in CNA burden was associated with poor overall survival [HR = 1.52, 95% CI (1.01–2.30), p = 0.04]. The combination of biomarkers TMB and CNA burden stratified patients into four clinically distinct subsets among which “LowTMB/HighCNA” showed the worst survival (p < 0.0001). The four patient subsets had unique CNA profiles and enriched pathways, which could predict transcriptional and phenotypic effects related to immune signaling and CD8+ T-cell abundance in the tumor microenvironment. CNA burden was associated with poor overall survival in patients receiving ICB and could improve patient stratification when incorporated with TMB. These findings may guide patient selection for immunotherapy or alternative strategies

Heterogeneity of triple negative breast cancer : Current advances in subtyping and treatment implications

As the field of translational ‘omics has progressed, refined classifiers at both genomic and proteomic levels have emerged to decipher the heterogeneity of breast cancer in a clinically-applicable way. The integration of ‘omics knowledge at the DNA, RNA and protein levels is further expanding biologic understanding of breast cancer and opportunities for customized treatment, a particularly pressing need in clinically triple negative tumors. For this group of aggressive breast cancers, work from multiple groups has now validated at least four major biologically and clinically distinct omics-based subtypes. While to date most clinical trial designs have considered triple negative breast cancers as a single group, with an expanding arsenal of targeted therapies applicable to distinct biological pathways, survival benefits may be best realized by designing and analyzing clinical trials in the context of major molecular subtypes. While RNA-based classifiers are the most developed, proteomic classifiers proposed for triple negative breast cancer based on new technologies have the potential to more directly identify the most clinically-relevant biomarkers and therapeutic targets. Phospho-proteomic data further identify targetable signalling pathways in a unique subtype-specific manner. Single cell profiling of the tumor microenvironment represents a promising way to allow a better characterization of the heterogeneity of triple negative breast cancer which could be integrated in a spatially resolved context to build an ecosystem-based patient classification. Multi-omic data further allows in silico analysis of genetic and pharmacologic screens to map therapeutic vulnerabilities in a subtype-specific context. This review describes current knowledge about molecular subtyping of triple negative breast cancer, recent advances in omics-based genomics and proteomics diagnostics addressing the diversity of this disease, key advances made through single cell analysis approaches, and developments in treatments including targeted therapeutics being tested in major clinical trials.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofReviewedFacult

Immune infiltrates in the breast cancer microenvironment : detection, characterization and clinical implication

Although unlike melanoma, breast cancer is not generally viewed as a highly immunogenic cancer, recent studies have described a rich tumor immune microenvironment in a subset of breast cancers. These immune infiltrates, comprised cells from the innate and adaptive immune response, can be detected and characterized in biopsy specimens and have prognostic value. Tumor-infiltrating lymphocytes (TILs) represent the majority of mononuclear immune infiltrates in the breast tumor microenvironment and can be easily identified in formalin-fixed paraffinembedded tissues after standard hematoxylin & eosin staining. High levels of TILs are most common in HER2+ and basal-like subtypes where they are associated with good prognosis and with response to certain therapies such as the anti-HER2 antibody trastuzumab. International collaborative efforts are underway to standardize the assessment of TILs so as to facilitate their implementation as a breast cancer biomarker. Using immunohistochemistry to further characterize TILs, recent reports describe the presence of important lymphocyte populations including CD8+ cytotoxic, FOXP3+ regulatory, and CD4+ helper and follicular T cells which have overlapping associations with prognosis and response to therapies. Moreover, recently identified immune checkpoint markers (PD-1, PD-L1) are present in some breast cancers, implying some cases might be especially amenable to immune checkpoint inhibitor treatment strategies which are being evaluated in a number of active clinical trials.Medicine, Faculty ofPathology and Laboratory Medicine, Department ofReviewedFacultyPostdoctoralGraduat

Neither Tumor-Infiltrating Lymphocytes nor Cytotoxic T Cells Predict Enhanced Benefit from Chemotherapy in the DBCG77B Phase III Clinical Trial

Recent studies have shown that immune infiltrates in the tumor microenvironment play a role in response to therapy, with some suggesting that patients with immunogenic tumors may receive increased benefit from chemotherapies. We evaluated this hypothesis in early breast cancer by testing the interaction between immune biomarkers and chemotherapy using materials from DBCG77B, a phase III clinical trial where high-risk premenopausal women were randomized to receive chemotherapy or no chemotherapy. Tissue microarrays were evaluated for tumor-infiltrating lymphocytes (TILs) assessed morphologically on hematoxylin and eosin-stained slides, and by immunohistochemistry for CD8, FOXP3, LAG-3, PD-1 and PD-L1. Following REMARK reporting guidelines, data analyses were performed according to a prespecified statistical plan, using 10-year invasive disease-free survival as the endpoint. Differences in survival probabilities between biomarker groups were evaluated by Kaplan–Meier and Cox proportional hazard ratio analyses and prediction for treatment benefit by an interaction test. Our results showed that stromal TILs were associated with an improved prognosis (HR = 0.93; p-value = 0.03), consistent with previous studies. However, none of the immune biomarkers predicted benefit from chemotherapy in the full study set nor within major breast cancer subtypes. Our study indicates that primary tumors with higher immune infiltration do not derive extra benefit from cyclophosphamide-based cytotoxic chemotherapy.Medicine, Faculty ofNon UBCPathology and Laboratory Medicine, Department ofReviewedFacultyResearche

Nestin and INPP4B markers in breast cancer

In a formal prospective-retrospective analysis of the phase III SBG0102 clinical trial randomizing metastatic breast cancer patients to gemcitabine-docetaxel or to single agent docetaxel, patients with basal-like tumors by PAM50 gene expression had significantly better overall survival in the gemcitabine arm. By immunohistochemistry (IHC), triple negative status was not predictive, but more specific biomarkers have since become available defining basal-like by nestin positivity or loss of inositol-polyphosphate-4-phosphate (INPP4B). Here, we evaluate their capacity to identify which patients benefit from gemcitabine in the metastatic setting. Nestin and INPP4B staining and interpretation followed published methods. A prespecified statistical plan evaluated the primary hypothesis that patients with basal-like breast cancer, defined as “nestin+ or INPP4B-”, would have superior overall survival on gemcitabine-docetaxel when compared to docetaxel. Interaction tests, Kaplan-Meier curves and forest plots were used to assess prognostic and predictive capacities of biomarkers relative to treatment. Among 239 cases evaluable for this study, 36 (15%) had been classified as basal-like by PAM50. “Nestin+ or INPP4B-” was observed in 41 (17%) of the total cases and was significantly associated with PAM50 basal-like subtype. Within an estimated median follow-up of 13 years, patients assigned as IHC basal “nestin+ or INPP4B-” had significantly better overall survival on gemcitabine-docetaxel versus docetaxel monotherapy (HR=0.31, 95%CI: 0.16-0.60), whereas no differences were observed for other patients (HR=0.99), P-interaction<0.01. In the metastatic setting, women with IHC basal breast cancers defined as “nestin+ or INPP4B-” have superior overall survival when randomized to gemcitabine-containing chemotherapy compared to docetaxel alone. These findings need to be validated using larger prospective-retrospective phase III clinical trials series.Medicine, Faculty ofNon UBCPathology and Laboratory Medicine, Department ofReviewedFacultyResearcherGraduat