The long and winding road to biomarkers for immunotherapy: a retrospective analysis of samples from patients with triple-negative breast cancer treated with pembrolizumab.

BACKGROUND: Immune checkpoint blockade (ICB) in combination with chemotherapy improves outcome of patients with triple-negative breast cancer (TNBC) in metastatic and early settings. The identification of predictive biomarkers able to guide treatment decisions is challenging and currently limited to programmed death-ligand 1 (PD-L1) expression and high tumor mutational burden (TMB) in the advanced setting, with several limitations. MATERIALS AND METHODS: We carried out a retrospective analysis of clinical-pathological and molecular characteristics of tumor samples from 11 patients with advanced TNBC treated with single-agent pembrolizumab participating in two early-phase clinical trials: KEYNOTE-012 and KEYNOTE-086. Clinical, imaging, pathological [i.e. tumor-infiltrating lymphocytes (TILs), PD-L1 status], RNA sequencing, and whole-exome sequencing data were analyzed. We compared our results with publicly available transcriptomic data from TNBC cohorts from TCGA and METABRIC. RESULTS: Response to pembrolizumab was heterogeneous: two patients experienced exceptional long-lasting responses, six rapid progressions, and three relatively slower disease progression. Neither PD-L1 nor stromal TILs were significantly associated with response to treatment. Increased TMB values were observed in tumor samples from exceptional responders compared to the rest of the cohort (P = 3.4 × 10-4). Tumors from exceptional responders were enriched in adaptive and innate immune cell signatures. Expression of regulatory T-cell markers (FOXP3, CCR4, CCR8, TIGIT) was mainly observed in tumors from responders except for glycoprotein-A repetitions predominant (GARP), which was overexpressed in tumors from rapid progressors. GARP RNA expression in primary breast tumors from the public dataset was significantly associated with a worse prognosis. CONCLUSIONS: The wide spectrum of clinical responses to ICB supports that TNBC is a heterogeneous disease. Tumors with high TMB respond better to ICB. However, the optimal cut-off of 10 mutations (mut)/megabase (Mb) may not reflect the complexity of all tumor subtypes, despite its approval as a tumor-agnostic biomarker. Further studies are required to better elucidate the relevance of the tumor microenvironment and its components as potential predictive biomarkers in the context of ICB.

MicroRNA profiling and identification of let-7a as a target to prevent chemotherapy-induced primordial follicles apoptosis in mouse ovaries.

Cancer treatments as cyclophosphamide and its active metabolites are highly gonadotoxic leading to follicle apoptosis and depletion. Considering the risk of subsequent infertility, fertility preservation is recommended. Beside the germ cells and gametes cryopreservation options, ovarian pharmacological protection during treatment appears to be very attractive. Meanwhile, the advances in the field of oncology have brought microRNAs into spotlight as a potential feature of cancer treatment. Herein, we investigated miRNAs expressions in response to chemotherapy using postnatal-day-3 (PND3) mouse ovaries. Our results revealed that several miRNAs are differently expressed during chemotherapy exposure. Amongst them, let-7a was the most profoundly downregulated and targets genes involved in crucial cellular processes including apoptosis. Thus we developed a liposome-based system to deliver the let-7a mimic in whole PND3 ovaries in vitro. We showed that let-7a mimic prevented the upregulation of genes involved in cell death and reduced the chemotherapy-induced ovarian apoptosis, suggesting that it can be an interesting target to preserve ovarian function. However, its impact on subsequent follicular development has to be further elucidated in vivo using an appropriate delivery system. In this study, we demonstrated that miRNA replacement approaches can be a useful tool to reduce chemotherapy-induced ovarian damage in the future.

Trastuzumab versus observation for HER2 nonamplified early breast cancer with circulating tumor cells (EORTC 90091-10093, BIG 1-12, Treat CTC): a randomized phase II trial.

Background: Trastuzumab improves the outcome of women with HER2 positive breast cancer. We aimed to assess whether trastuzumab decreases the detection rate of circulating tumor cells (CTCs) in women with high risk, HER2 nonamplified, early breast cancer. Patients and methods: The EORTC 90091-10093 BIG 1-12 Treat CTC is a phase II trial, conducted in 70 hospitals and 6 CTC laboratories across 5 European countries. Patients with centrally confirmed HER2 nonamplified breast cancer and ≥1 centrally confirmed CTC per 15 ml of blood by CellSearch® following surgery and (neo)adjuvant chemotherapy were randomized (1 : 1) to 6 cycles of trastuzumab intravenously versus 18 weeks of observation. Randomization was stratified for center, locally confirmed estrogen receptor status and adjuvant versus neoadjuvant chemotherapy. The primary end point was rate of detection of ≥1 CTC per 15 ml of blood at week 18. Secondary end points were invasive disease-free survival (iDFS) and cardiac safety. Results: Between 30 April 2013 and 17 October 2016, 1317 patients were screened; 95 (7.2%) had detectable CTC(s), and 63 (4.8%) were randomized to trastuzumab (n = 31) or observation (n = 32). Fifty-eight patients were assessable for the primary end point, 29 in each arm. In 9 of the 58 patients, CTC(s) were still detected at week 18 : 5 in the trastuzumab and 4 in the observation arm (one-sided Fisher's exact test, P = 0.765). An Independent Data Monitoring Committee recommended stopping further accrual for futility for the primary end point. Median follow-up at database lock was 13 months (IQR 4-16.5). The 1-year iDFS was 93.8% (95% CI 77.3-98.4) in the observation versus 84.8% (95% CI 63.4-94.2) in the trastuzumab arm. No grade 2-4 cardiac events were observed in the trastuzumab arm. Conclusion: Trastuzumab does not decrease the detection rate of CTCs in HER2 nonamplified, nonmetastatic breast cancer.

Unravelling triple-negative breast cancer molecular heterogeneity using an integrative multiomic analysis.

Background: Recent efforts of genome-wide gene expression profiling analyses have improved our understanding of the biological complexity and diversity of triple-negative breast cancers (TNBCs) reporting, at least six different molecular subtypes of TNBC namely Basal-like 1 (BL1), basal-like 2 (BL2), immunomodulatory (IM), mesenchymal (M), mesenchymal stem-like (MSL) and luminal androgen receptor (LAR). However, little is known regarding the potential driving molecular events within each subtype, their difference in survival and response to therapy. Further insight into the underlying genomic alterations is therefore needed. Patients and methods: This study was carried out using copy-number aberrations, somatic mutations and gene expression data derived from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) and The Cancer Genome Atlas. TNBC samples (n = 550) were classified according to Lehmann's molecular subtypes using the TNBCtype online subtyping tool (http://cbc.mc.vanderbilt.edu/tnbc/). Results: Each subtype showed significant clinic-pathological characteristic differences. Using a multivariate model, IM subtype showed to be associated with a better prognosis (HR = 0.68; CI = 0.46-0.99; P = 0.043) whereas LAR subtype was associated with a worst prognosis (HR = 1.47; CI = 1.0-2.14; P = 0.046). BL1 subtype was found to be most genomically instable subtype with high TP53 mutation (92%) and copy-number deletion in genes involved in DNA repair mechanism (BRCA2, MDM2, PTEN, RB1 and TP53). LAR tumours were associated with higher mutational burden with significantly enriched mutations in PI3KCA (55%), AKT1 (13%) and CDH1 (13%) genes. M and MSL subtypes were associated with higher signature score for angiogenesis. Finally, IM showed high expression levels of immune signatures and check-point inhibitor genes such as PD1, PDL1 and CTLA4. Conclusion: Our findings highlight for the first time the substantial genomic heterogeneity that characterize TNBC molecular subtypes, allowing for a better understanding of the disease biology as well as the identification of several candidate targets paving novel approaches for the development of anticancer therapeutics for TNBC.

Somatic mutation, copy number and transcriptomic profiles of primary and matched metastatic estrogen receptor-positive breast cancers.

BACKGROUND: Estrogen receptor-positive (ER+) breast cancers (BCs) constitute the most frequent BC subtype. The molecular landscape of ER+ relapsed disease is not well characterized. In this study, we aimed to describe the genomic evolution between primary (P) and matched metastatic (M) ER+ BCs after failure of adjuvant therapy. MATERIALS AND METHODS: A total of 182 ER+ metastatic BC patients with long-term follow-up were identified from a single institution. P tumor tissue was available for all patients, with 88 having matched M material. According to the availability of tumor material, samples were characterized using a 120 mutational hotspot qPCR, a 29 gene copy number aberrations (CNA) and a 400 gene expression panels. ESR1 mutations were assayed by droplet digital PCR. Molecular alterations were correlated with overall survival (OS) using the Cox proportional hazards regression models. RESULTS: The median follow-up was 6.4 years (range 0.5-26.6 years). Genomic analysis of P tumors revealed somatic mutations in PIK3CA, KRAS, AKT1, FGFR3, HRAS and BRAF at frequencies of 41%, 6%, 5%, 2%, 1% and 2%, respectively, and CN amplification of CCND1, ZNF703, FGFR1, RSF1 and PAK1 at 23%, 19%, 17%, 12% and 11%, respectively. Mutations and CN amplifications were largely concordant between P and matched M (>84%). ESR1 mutations were found in 10.8% of the M but none of the P. Thirteen genes, among which ESR1, FOXA1, and HIF1A, showed significant differential expression between P and M. In P, the differential expression of 18 genes, among which IDO1, was significantly associated with OS (FDR < 0.1). CONCLUSIONS: Despite the large concordance between P and matched M for the evaluated molecular alterations, potential actionable targets such as ESR1 mutations were found only in M. This supports the importance of characterizing the M disease. Other targets we identified, such as HIF1A and IDO1, warrant further investigation in this patient population.

Plasma circulating tumor DNA as an alternative to metastatic biopsies for mutational analysis in breast cancer.

BACKGROUND: Molecular screening programs use next-generation sequencing (NGS) of cancer gene panels to analyze metastatic biopsies. We interrogated whether plasma could be used as an alternative to metastatic biopsies. PATIENTS AND METHODS: The Ion AmpliSeq™ Cancer Hotspot Panel v2 (Ion Torrent), covering 2800 COSMIC mutations from 50 cancer genes was used to analyze 69 tumor (primary/metastases) and 31 plasma samples from 17 metastatic breast cancer patients. The targeted coverage for tumor DNA was ×1000 and for plasma cell-free DNA ×25 000. Whole blood normal DNA was used to exclude germline variants. The Illumina technology was used to confirm observed mutations. RESULTS: Evaluable NGS results were obtained for 60 tumor and 31 plasma samples from 17 patients. When tumor samples were analyzed, 12 of 17 (71%, 95% confidence interval (CI) 44% to 90%) patients had ≥1 mutation (median 1 mutation per patient, range 0-2 mutations) in either p53, PIK3CA, PTEN, AKT1 or IDH2 gene. When plasma samples were analyzed, 12 of 17 (71%, 95% CI: 44-90%) patients had ≥1 mutation (median 1 mutation per patient, range 0-2 mutations) in either p53, PIK3CA, PTEN, AKT1, IDH2 and SMAD4. All mutations were confirmed. When we focused on tumor and plasma samples collected at the same time-point, we observed that, in four patients, no mutation was identified in either tumor or plasma; in nine patients, the same mutations was identified in tumor and plasma; in two patients, a mutation was identified in tumor but not in plasma; in two patients, a mutation was identified in plasma but not in tumor. Thus, in 13 of 17 (76%, 95% CI 50% to 93%) patients, tumor and plasma provided concordant results whereas in 4 of 17 (24%, 95% CI 7% to 50%) patients, the results were discordant, providing complementary information. CONCLUSION: Plasma can be prospectively tested as an alternative to metastatic biopsies in molecular screening programs.

Semiautomated isolation and molecular characterisation of single or highly purified tumour cells from CellSearch enriched blood samples using dielectrophoretic cell sorting.

BACKGROUND: Molecular characterisation of single circulating tumour cells (CTCs) holds considerable promise for predictive biomarker assessment and to explore CTC heterogeneity. We evaluate a new method, the DEPArray system, that allows the dielectrophoretic manipulation and isolation of single and 100% purified groups of CTCs from pre-enriched blood samples and explore the feasibility of their molecular characterisation. METHODS: Samples containing known numbers of two cell populations were used to assess cell loss during sample loading. Cultured breast cancer cells were isolated from spiked blood samples using CellSearch CTC and Profile kits. Single tumour cells and groups of up to 10 tumour cells were recovered with the DEPArray system and subjected to transcriptional and mutation analysis. RESULTS: On average, 40% cell loss was observed when loading samples to the DEPArray system. Expected mutations in clinically relevant markers could be obtained for 60% of single recovered tumour cells and all groups of tumour cells. Reliable gene expression profiles were obtained from single cells and groups of up to 10 cells for 2 out of 3 spiked breast cancer cell lines. CONCLUSION: We describe a semiautomated workflow for the isolation of small groups of 1 to 10 tumour cells from whole blood samples and provide proof of principle for the feasibility of their comprehensive molecular characterisation.