Purpose Recent studies suggest that tumor-infiltrating lymphocytes (TILs) are associated with disease-free (DFS) and overall survival (OS) in operable triple-negative breast cancer (TNBC). We seek to validate the prognostic impact of TILs in primary TNBCs in two adjuvant phase III trials conducted by the Eastern Cooperative Oncology Group (ECOG). Patients and methods Full-face hematoxylin and eosin–stained sections of 506 tumors from ECOG trials E2197 and E1199 were evaluated for density of TILs in intraepithelial (iTILs) and stromal compartments (sTILs). Patient cases of TNBC from E2197 and E1199 were randomly selected based on availability of sections. For the primary end point of DFS, association with TIL scores was determined by fitting proportional hazards models stratified on study. Secondary end points were OS and distant recurrence–free interval (DRFI). Reporting recommendations for tumor marker prognostic studies criteria were followed, and all analyses were prespecified. Results The majority of 481 evaluable cancers had TILs (sTILs, 80%; iTILs, 15%). With a median follow-up of 10.6 years, higher sTIL scores were associated with better prognosis; for every 10% increase in sTILs, a 14% reduction of risk of recurrence or death (P = .02), 18% reduction of risk of distant recurrence (P = .04), and 19% reduction of risk of death (P = .01) were observed. Multivariable analysis confirmed sTILs to be an independent prognostic marker of DFS, DRFI, and OS. Conclusion In two national randomized clinical trials using contemporary adjuvant chemotherapy, we confirm that stromal lymphocytic infiltration constitutes a robust prognostic factor in TNBCs. Studies assessing outcomes and therapeutic efficacies should consider stratification for this parameter.

Prognostic Value of Tumor-Infiltrating Lymphocytes in Triple-Negative Breast Cancers From Two Phase III Randomized Adjuvant Breast Cancer Trials: ECOG 2197 and ECOG 1199

Emerging targets in cancer immunotherapy

A Comprehensive Immunologic Portrait of Triple-Negative Breast Cancer

Treatment strategies for patients with advanced solid tumors have traditionally been based on three different paradigms: surgery, cytotoxics (chemotherapy or radiation therapy) and targeted therapies. Immunotherapy has emerged as a novel treatment paradigm in our armamentarium. Unfortunately, most patients still do not benefit from immunotherapy. These patients often have “cold tumors” characterized by a paucity of effector T cells in the tumor microenvironment, low mutational load, low neoantigen burden and often an immunosuppressive tumor microenvironment. TIGIT is an immunoreceptor inhibitory checkpoint that has been implicated in tumor immunosurveillance. Expression of TIGIT has been demonstrated in both NK cells and T cells and plays a role in their activation and maturation. TIGIT competes with immunoactivator receptor CD226 (DNAM-1) for the same set of ligands: CD155 (PVR or poliovirus receptor) and CD112 (Nectin-2 or PVRL2). TIGIT’s role in tumor immunosurveillance is analogous to the PD-1/PD-L1 axis in tumor immunosuppression. Both TIGIT and PD-1 are upregulated in a variety of different cancers. Anti-TIGIT antibodies have demonstrated synergy with anti-PD-1/PD-L1 antibodies in pre-clinical models. Currently, there are multiple first-in-man phase I trials hoping to exploit this new pathway and improve response rates with existing immunotherapies.

TIGIT: a novel immunotherapy target moving from bench to bedside

Recent research has shown that TOP2A plays an important role in the tumorigenesis of many malignancies, such as breast cancer, ovarian cancer, and prostate cancer. However, few studies have been conducted on TOP2A expression and functions in colon cancer. In the present study, we found that TOP2A expression was obviously elevated in colon cancer tissues compared to adjacent non-cancerous tissues. Depletion of TOP2A in HCT116 and SW480 colon cancer cells by transfection of specific small interfering RNA significantly suppressed proliferation and inhibited invasion of cells, even induced apoptosis as indicated by both MTT assay, Annexin V/propidium iodide staining, and Transwell assay. Furthermore, we explored the underlying mechanisms. Knockdown of TOP2A not only affects the expression of cell apoptosis-related (Bcl-2 and Bax) and invasion-related proteins (MMP-2 and MMP-9), but also reduced the phosphorylation levels of ERK and AKT. In conclusion, we showed that TOP2A was upregulated in colon cancer tissue samples and that TOP2A may serve as an oncogene in colon cancer.

Proliferation and invasion of colon cancer cells are suppressed by knockdown of TOP2A.

Although obesity is a risk factor for breast cancer, little effort has been made in the identification of druggable molecular alterations in obese–breast cancer patients. Tumors are controlled by their surrounding microenvironment, in which the adipose tissue is a main component. In this work, we intended to describe molecular alterations at a transcriptomic and protein–protein interaction (PPI) level between obese and non-obese patients. Gene expression data of 269 primary breast tumors were compared between normal-weight (BMI   30, n = 139) patients. No significant differences were found for the global breast cancer population. However, within the luminal A subtype, upregulation of 81 genes was observed in the obese group (FC ≥ 1.4). Next, we explored the association of these genes with patient outcome, observing that 39 were linked with detrimental outcome. Their PPI map formed highly compact cluster and functional annotation analyses showed that cell cycle, cell proliferation, cell differentiation, and cellular response to extracellular stimuli were the more altered functions. Combined analyses of genes within the described functions are correlated with poor outcome. PPI network analyses for each function were to search for druggable opportunities. We identified 16 potentially druggable candidates. Among them, NEK2, BIRC5, and TOP2A were also found to be amplified in breast cancer, suggesting that they could act as strategic players in the obese-deregulated transcriptome. In summary, our in silico analysis describes molecular alterations of luminal A tumors and proposes a druggable PPI network in obese patients with potential for translation to the clinical practice.

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Functional transcriptomic annotation and protein–protein interaction network analysis identify NEK2, BIRC5, and TOP2A as potential targets in obese patients with luminal A breast cancer

Background: Most patients with early stage triple negative breast cancer (TNBC) receive adjuvant chemotherapy. Activation of the immune system is associated with tumor response and may help identify TNBC with favorable outcome. Methods: Gene expression data were obtained from the GEO Dataset GDS2250/GSE3744. Affymetrix CEL files were downloaded and analyzed with Affymetrix Transcriptome Analysis Console 3.0. Functional genomics was implemented with David Bioinformatics Resources 6.8. Data contained at Oncomine were used to identify genes upregulated in basal-like cancer compared to normal breast tissue. Data contained at cBioportal were used to assess for molecular alterations. The KMPlotter online tool, METABRIC and GSE25066 datasets were used to associate gene signatures with clinical outcome. Results: 1564 upregulated genes were identified as differentially expressed between normal and basal-like tumors. Of these, 16 genes associated with immune function were linked with clinical outcome. HLA-C, HLA-F, HLA-G and TIGIT were associated with both improved relapse-free survival (RFS) and overall survival (OS). The combination of HLA-F/TIGIT and HLA-C/HLA-F/TIGIT showed the most favorable outcome (HR for RFS 0.44, p<0.001; HR for OS 0.22, p<0.001; and HR for RFS 0.46, p<0.001; HR for OS 0.15, p<0.001; respectively). The association of HLA-C/HLA-F with outcome was confirmed using the METABRIC and GSE25066 datasets. No copy number alterations of these genes were identified. Conclusion: We describe a gene signature associated with immune function and favorable outcome in basal-like breast cancer. Incorporation of this signature in prospective studies may help to stratify risk of early stage TNBC. © 2017 Martinez-Canales et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Transcriptomic immunologic signature associated with favorable clinical outcome in basal-like breast tumors.

Although early stage ovarian cancer is in most cases a curable disease, some patients relapse even with appropriate adjuvant treatment. Therefore, the identification of patient and tumor characteristics to better stratify risk and guide rational drug development is desirable. Using transcriptomic functional annotation followed by protein-protein interacting (PPI) network analyses, we identified functions that were upregulated and associated with detrimental outcome in patients with early stage ovarian cancer. Some of the identified functions included cell cycle, cell division, signal transduction/protein modification, cellular response to extracellular stimuli or transcription regulation, among others. Genes within these functions included AURKA, AURKB, CDK1, BIRC5, or CHEK1 among others. Of note, the histone-lysine N-methyltransferase (EZH2) and the ubiquitin-conjugating enzyme E2C (UBE2C) genes were found to be upregulated and amplified in 10% and 6% of tumors, respectively. Of note, EZH2 and UBE2C were identified as principal interacting proteins of druggable networks. In conclusion, we describe a set of genes overexpressed in ovarian cancer with potential for therapeutic intervention including EZH2 and UBE2C.

/pdf/functional-transcriptomic-annotation-and-protein-protein-56aq21f1e0.pdf

Functional transcriptomic annotation and protein–protein interaction analysis identify EZH2 and UBE2C as key upregulated proteins in ovarian cancer

Basal-like breast cancer is an incurable disease with limited therapeutic options, mainly due to the frequent development of anti-cancer drug resistance. Therefore, identification of druggable targets to improve current therapies and overcome these resistances is a major goal. Targeting DNA repair mechanisms has reached the clinical setting and several strategies, like the inhibition of the CHK1 kinase, are currently in clinical development. Here, using a panel of basal-like cancer cell lines, we explored the synergistic interactions of CHK1 inhibitors (rabusertib and SAR020106) with approved therapies in breast cancer and evaluated their potential to overcome resistance. We identified a synergistic action of these inhibitors with agents that produce DNA damage, like platinum compounds, gemcitabine, and the PARP inhibitor olaparib. Our results demonstrated that the combination of rabusertib with these chemotherapies also has a synergistic impact on tumor initiation, invasion capabilities, and apoptosis in vitro. We also revealed a biochemical effect on DNA damage and caspase-dependent apoptosis pathways through the phosphorylation of H2AX, the degradation of full-length PARP, and the increase of caspases 3 and 8 activity. This agent also demonstrated synergistic activity in a platinum-resistant cell line, inducing an increase in cell death in response to cisplatin only when combined with rabusertib, while no toxic effect was found on non-tumorigenic breast tissue-derived cell lines. Lastly, the combination of CHK1 inhibitor with cisplatin and gemcitabine resulted in more activity than single or double combinations, leading to a higher apoptotic effect. In conclusion, in our study we identify therapeutic options for the clinical development of CHK1 inhibitors, and confirm that the inhibition of this kinase can overcome acquired resistance to cisplatin.

https://www.mdpi.com/1422-0067/21/23/9034/pdf

Sandra Martinez-Canales

Papers

Functional transcriptomic annotation and protein–protein interaction network analysis identify NEK2, BIRC5, and TOP2A as potential targets in obese patients with luminal A breast cancer

Transcriptomic immunologic signature associated with favorable clinical outcome in basal-like breast tumors.

Functional transcriptomic annotation and protein–protein interaction analysis identify EZH2 and UBE2C as key upregulated proteins in ovarian cancer

Checkpoint Kinase 1 Pharmacological Inhibition Synergizes with DNA-Damaging Agents and Overcomes Platinum Resistance in Basal-Like Breast Cancer