Ana Maria Gonzalez-Angulo

Purpose: Metastatic breast cancer (MBC) is not curable and there is a growing interest in personalized therapy options. Here we report molecular profiling of MBC focusing on molecular evolution in actionable alterations. Experimental Design: Sixty-two patients with MBC were included. An analysis of DNA, RNA, and functional proteomics was done, and matched primary and metastatic tumors were compared when feasible. Results: Targeted exome sequencing of 41 tumors identified common alterations in TP53 (21; 51%) and PIK3CA (20; 49%), as well as alterations in several emerging biomarkers such as NF1 mutations/deletions (6; 15%), PTEN mutations (4; 10%), and ARID1A mutations/deletions (6; 15%). Among 27 hormone receptor–positive patients, we identified MDM2 amplifications (3; 11%), FGFR1 amplifications (5; 19%), ATM mutations (2; 7%), and ESR1 mutations (4; 15%). In 10 patients with matched primary and metastatic tumors that underwent targeted exome sequencing, discordances in actionable alterations were common, including NF1 loss in 3 patients, loss of PIK3CA mutation in 1 patient, and acquired ESR1 mutations in 3 patients. RNA sequencing in matched samples confirmed loss of NF1 expression with genomic NF1 loss. Among 33 patients with matched primary and metastatic samples that underwent RNA profiling, 14 actionable genes were differentially expressed, including antibody–drug conjugate targets LIV-1 and B7-H3. Conclusions: Molecular profiling in MBC reveals multiple common as well as less frequent but potentially actionable alterations. Genomic and transcriptional profiling demonstrates intertumoral heterogeneity and potential evolution of actionable targets with tumor progression. Further work is needed to optimize testing and integrated analysis for treatment selection.

Altered cellular metabolism is a hallmark of cancer. It is increasingly recognized that selected tumors are dependent on oxidative phosphorylation (OXPHOS). However, the role of OXPHOS in TNBC is not well understood. We performed RNA sequencing in pre-treatment biopsies from 43 patients with operable triple negative breast cancer (TNBC) who received sequential taxane- and anthracycline-based neoadjuvant chemotherapy. At a median follow-up of 63 months, 14 patients recurred and 12 patients died. At a false discovery rate of 0.05, 33 genes were differentially expressed between the patients who did and did not have a subsequent recurrence. Ingenuity pathway analysis demonstrated that one of the top canonical pathways that differed was higher expression of oxidative phosphorylation signature (p=5.89E-0.7). The patients who recurred had significantly higher levels of mitochondrial genes: MT-ND1 (adjusted p or FDR-BH; q=0.007); MT-ND5 (q=0.03) and MT-ND4 (q=0.04). Further, 21 genes were differentially expressed between patients based on survival, including MT-ND5 (q=0.001); MT-ND4 (q=0.005), MT-ND4L (q=0.015), MT-ND6 (q=0.018), and MT-ATP6 (P=0.03). Top canonical pathway that was differentially expressed based on survival was oxidative phosphorylation (p=9.98E-10). We therefore sought to determine the efficacy of IACS-10759, a novel inhibitor of OXPHOS, in 10 different TNBC patient-derived xenografts representing different gene expression based Lehmann TNBC subtypes. Growth inhibition was observed in multiple subtypes, with regression in one basal-like 1(BL1) 1 model, and stabilization of growth in multiple BL1 and immunomodulatory expression subtypes. Taken together, our data suggests that high OXPHOS is associated with higher recurrence and lower survival. OXPHOS is a promising target in several TNBC subtypes. A Phase I trial of IACS-10759, a potent inhibitor of complex I of oxidative phosphorylation, in leukemia is ongoing and planned in TNBC and other solid tumors. Citation Format: Funda Meric-Bernstam, Kurt Evans, Xiaofeng Zheng, Xiaoping Su, Erkan Yuca, Stephen Scott, Argun Akcakanat, Naoto Ueno, Bora Lim, Jennifer Litton, Vicente Valero, Fraser Symmans, Gabriel Hortobagyi, Charles Perou, Debu Tripathy, Guilio Draetta, Joe Marszalek, Ana Maria Gonzalez-Angulo, Stacy Moulder. Oxidative phosphorylation as a target in triple negative breast cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4970. doi:10.1158/1538-7445.AM2017-4970

1017 Background: To understand the mechanisms underlying the evolution of tumors in the process of metastasis, we studied 61 paired primary-relapse BC from the GEICAM ConvertHER study. While some of the metastases maintained the clinical (ER/PR and HER2 status) and/or intrinsic subtype (defined by expression arrays) of the original tumor (concordant), others exhibited a subtype shift (discordant). We aimed to identify the genomic alterations driving the metastases and, particularly, their relationship with the subtype switch. Methods: We detected the somatic variants (mutations and copy number alterations (CNAs)) affecting 202 genes across the 61 sample pairs via targeted sequencing. We employed the Cancer Genome Interpreter (cancergenomeinterpreter.org), a bioinformatics approach to identify the alterations most likely driving tumorigenesis, and subsequently identified those whose cancer cell fraction markedly changed in the metastases. We explored the clonal remodeling in metastasis comparing the cell fractions of driver mutations in both concordant and discordant tumors. Results: We found that 156 genes had 747 somatic mutations and 171 genes suffered 1042 somatic CNAs in the 61 studied tumor pairs. We identified a median of 11 and 9 mutations in primaries and metastases, respectively. Several frequent BC mutational drivers, such as TP53, PIK3CA, MLL3, MAP3K1, and NOTCH2 were amongst the more frequently changed their cancer cell fraction in metastases with respect to primaries. We found that driver mutations of discordant tumors exhibited a significantly higher increase of clonal cell fraction. Moreover, whether the clonal status of a driver mutation was conserved in the metastasis was significantly associated to whether the tumor maintains its clinical subtype but not its intrinsic subtype. Conclusions: Our results suggest that a shift in the clinical subtype of BC undergoing metastasis is accompanied by more significant changes at the genomic level than those suffered by tumors that maintain their clinical subtype. This remodeling of the landscape of drivers could open new therapeutic opportunities to specifically target discordant BC.

The cytokine TWEAK and its receptor, Fn14, have emerged as potentially valuable targets for cancer therapy. Granzyme B (GrB)–containing Fn14-targeted constructs were generated containing either the Fn14 ligand TWEAK (GrB-TWEAK) or an anti-Fn14 humanized single-chain antibody (GrB-Fc-IT4) as the targeting moieties. Both constructs showed high affinity and selective cytotoxicity against a panel of Fn14-expressing human tumor cells including triple-negative breast cancer (TNBC) lines. Cellular expression of the GrB inhibitor PI-9 in target cells had no impact on the cytotoxic effect of either construct. Cellular expression of MDR1 showed no cross-resistance to the fusion constructs. GrB-TWEAK and GrB-Fc-IT4 activated intracellular caspase cascades and cytochrome c–related proapoptotic pathways consistent with the known intracellular functions of GrB in target cells. Treatment of mice bearing established HT-29 xenografts with GrB-TWEAK showed significant tumor growth inhibition compared with vehicle alone (P < 0.05). Both GrB-TWEAK and GrB-Fc-IT4 displayed significant tumor growth inhibition when administered to mice bearing orthotopic MDA-MB-231 (TNBC) tumor xenografts. The Cancer Genome Atlas analysis revealed that Fn14 mRNA expression was significantly higher in TNBC and in HER2-positive disease (P < 0.0001) compared with hormone receptor–positive breast cancer, and in basal-like 2 tumors (P = 0.01) compared with other TNBC molecular subtypes. IHC analysis of a 101 patient TNBC tumor microarray showed that 55 of 101 (54%) of tumors stained positive for Fn14, suggesting that this may be an excellent potential target for precision therapeutic approaches. Targeting Fn14 using fully human, GrB-containing fusion constructs may form the basis for a new class of novel, potent, and highly effective constructs for targeted therapeutic applications. Mol Cancer Ther; 13(11); 2688–705. ©2014 AACR.

This paper aimed to look at the clinical impact of the biological heterogeneity of HER2-positive breast cancer by evaluating the molecular features and survival outcomes of the intrinsic subtypes within HER2-positive disease by the American Society of Clinical Oncology (ASCO)/College of American Pathologists (CAP) guidelines within the TCGA and METABRIC cohorts. They found that compared with HER2-negative breast cancer, HER2-positive breast cancer had a higher frequency of the HER2-enriched subtype (47.0% vs 7.1%) and a lower frequency of luminal A (10.7% vs 39.0%) and basal-like (14.1% vs 23.4%) subtypes. In each subtype, only 0.3% to 3.9% of genes were found differentially expressed between clinical HER2-positive and HER2-negative tumors. In clinically HER2-positive tumors, HER2 gene and protein expression was statistically significantly higher in the HER2 enriched and basal-like subtypes than either luminal subtype. They concluded that the when the intrinsic subtypes are taken into account, clinical HER2-positivity does not translate into large changes downstream signaling pathways, or in patient survival in the absence of anti-HER2 therapy.

This neoadjuvant chemotherapy (NAC) 2×2 factorial, open-label, randomized phase II trial was designed to evaluate the role of additional carboplatin and/or bevacizumab in triple-negative breast cancer (TNBC). A total of 443 patients with stage II to III TNBC received weekly paclitaxel for 12 weeks, followed by dose-dense doxorubicin plus cyclophosphamide (ddAC) for four cycles, and were randomly assigned to concurrent carboplatin every three weeks for four cycles concomitant with paclitaxel and/or bevacizumab every two weeks throughout chemotherapy. The endpoints were the effects on pathologic complete response (pCR) in the breast (ypT0/is), pCR in the breast/axilla (ypT0/isN0), treatment delivery, and safety. The authors found that only carboplatin (54% vs. 41%; P=0.0029) significantly increased pCR breast/axilla (one-sided P value). No synergism was demonstrated between the two agents. Patients assigned to either carboplatin or bevacizumab were less likely to complete chemotherapy without skipped doses, dose modification, or early discontinuation from toxicities. Grade ≥3 toxicities were as expected with the additional agents. This is the second NAC study that demonstrates that the addition of carboplatin to NAC increased pCR rates, but whether this will improve relapse-free or overall survival is unknown and will require definitive studies targeting specific patient populations.

This large cohort study was designed to evaluate the role of contralateral prophylactic mastectomy (CPM) and survival benefit in young patients with estrogen receptor (ER)-negative breast cancer. They selected 14,627 women ≤45 years of age with stage I-II breast cancer who underwent unilateral mastectomy or CPM from 2004 to 2006. At a median follow-up of 6.1 years and after adjusting for other prognostic factors, there was no difference in survival (HR=0.93; p=0.39). This paper provides data that support not to use CPM in young ER-negative patients with early-stage breast cancer. However, consideration of BRCA status is important before decisions are made.

Recent data suggest that tumor-infiltrating lymphocytes (TILs) are associated with outcome in triple-negative breast cancer (TNBC). In this study, the investigators did a validation of the prognostic role of TILs in primary TNBCs in two adjuvant phase III trials. They found that most of the evaluable cancers had TILs. At a median follow-up of 10.6 years, higher stroma TIL scores were associated with a better prognosis. Multivariable analysis showed that stromal TILs were an independent prognostic marker of disease-free survival (DFS), distant recurrence-free interval (DRFI), and overall survival (OS). This paper validates stromal TILs as a robust prognostic factor in TNBC treated with current chemotherapy regimens.

This paper combines the results of two phase 3 trials that randomly assigned premenopausal women with hormone receptor-positive breast cancer to exemestane plus ovarian suppression or tamoxifen plus ovarian suppression for 5 years. Combined data from 4690 patients at median follow-up of 68 months showed that the disease-free survival at 5 years was 91.1% in the exemestane-ovarian suppression group and 87.3% in the tamoxifen-ovarian...

T-cell infiltration (tumor-infiltrating lymphocytes ([ILs]) in estrogen receptor (ER)-negative breast cancer correlates with better survival. The authors used data from four trials with over 12,000 patients and measured cytotoxic (CD8+) and regulatory (forkhead box protein 3 [FOXP3+]) T cells in available tissues. They found that in ER-negative breast cancer (triple-negative and HER2-positive), presence of CD8+ T cells within the tumor was associated with a 28% reduction in the hazard of breast cancer-specific mortality (BCSM), and CD8+ T cells within the stroma with a 21% reduction in BCSM. In ER-positive HER2-positive breast cancers, CD8+ T cells within the tumor were associated with a 27% reduction in BCSM and in ER-negative breast cancer, they were associated with greater benefit from anthracycline regimens. This study on a large cohort confirms that an intact immune system is important in breast cancer outcomes.