Ipilimumab and nivolumab combined with anthracycline-based chemotherapy in metastatic hormone receptor-positive breast cancer: a randomized phase 2b trial.

BACKGROUND: Immune checkpoint inhibitors have shown minimal clinical activity in hormone receptor-positive metastatic breast cancer (HR+mBC). Doxorubicin and low-dose cyclophosphamide are reported to induce immune responses and counter regulatory T cells (Tregs). Here, we report the efficacy and safety of combined programmed cell death protein-1/cytotoxic T-lymphocyte-associated protein 4 blockade concomitant with or after immunomodulatory chemotherapy for HR+mBC. METHODS: Patients with HR+mBC starting first-/second- line chemotherapy (chemo) were randomized 2:3 to chemotherapy (pegylated liposomal doxorubicin 20 mg/m2 every second week plus cyclophosphamide 50 mg by mouth/day in every other 2-week cycle) with or without concomitant ipilimumab (ipi; 1 mg/kg every sixth week) and nivolumab (nivo; 240 mg every second week). Patients in the chemo-only arm were offered cross-over to ipi/nivo without chemotherapy. Co-primary endpoints were safety in all patients starting therapy and progression-free survival (PFS) in the per-protocol (PP) population, defined as all patients evaluated for response and receiving at least two treatment cycles. Secondary endpoints included objective response rate, clinical benefit rate, Treg changes during therapy and assessment of programmed death-ligand 1 (PD-L1), mutational burden and immune gene signatures as biomarkers. RESULTS: Eighty-two patients were randomized and received immune-chemo (N=49) or chemo-only (N=33), 16 patients continued to the ipi/nivo-only cross-over arm. Median follow-up was 41.4 months. Serious adverse events occurred in 63% in the immune-chemo arm, 39% in the chemo-only arm and 31% in the cross-over-arm. In the PP population (N=78) median PFS in the immune-chemo arm was 5.1 months, compared with 3.6 months in the chemo-only arm, with HR 0.94 (95% CI 0.59 to 1.51). Clinical benefit rates were 55% (26/47) and 48% (15/31) in the immune-chemo and chemo-only arms, respectively. In the cross-over-arm (ipi/nivo-only), objective responses were observed in 19% of patients (3/16) and clinical benefit in 25% (4/16). Treg levels in blood decreased after study chemotherapy. High-grade immune-related adverse events were associated with prolonged PFS. PD-L1 status and mutational burden were not associated with ipi/nivo benefit, whereas a numerical PFS advantage was observed for patients with a high Treg gene signature in tumor. CONCLUSION: The addition of ipi/nivo to chemotherapy increased toxicity without improving efficacy. Ipi/nivo administered sequentially to chemotherapy was tolerable and induced clinical responses. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Identifier: NCT03409198.

Circulating Tumor Cells in Breast Cancer Patients Treated by Neoadjuvant Chemotherapy: A Meta-analysis.

Background: We conducted a meta-analysis in nonmetastatic breast cancer patients treated by neoadjuvant chemotherapy (NCT) to assess the clinical validity of circulating tumor cell (CTC) detection as a prognostic marker. Methods: We collected individual patient data from 21 studies in which CTC detection by CellSearch was performed in early breast cancer patients treated with NCT. The primary end point was overall survival, analyzed according to CTC detection, using Cox regression models stratified by study. Secondary end points included distant disease-free survival, locoregional relapse-free interval, and pathological complete response. All statistical tests were two-sided. Results: Data from patients were collected before NCT (n = 1574) and before surgery (n = 1200). CTC detection revealed one or more CTCs in 25.2% of patients before NCT; this was associated with tumor size (P < .001). The number of CTCs detected had a detrimental and decremental impact on overall survival (P < .001), distant disease-free survival (P < .001), and locoregional relapse-free interval (P < .001), but not on pathological complete response. Patients with one, two, three to four, and five or more CTCs before NCT displayed hazard ratios of death of 1.09 (95% confidence interval [CI] = 0.65 to 1.69), 2.63 (95% CI = 1.42 to 4.54), 3.83 (95% CI = 2.08 to 6.66), and 6.25 (95% CI = 4.34 to 9.09), respectively. In 861 patients with full data available, adding CTC detection before NCT increased the prognostic ability of multivariable prognostic models for overall survival (P < .001), distant disease-free survival (P < .001), and locoregional relapse-free interval (P = .008). Conclusions: CTC count is an independent and quantitative prognostic factor in early breast cancer patients treated by NCT. It complements current prognostic models based on tumor characteristics and response to therapy.

Tracing the origin of disseminated tumor cells in breast cancer using single-cell sequencing.

BACKGROUND: Single-cell micro-metastases of solid tumors often occur in the bone marrow. These disseminated tumor cells (DTCs) may resist therapy and lay dormant or progress to cause overt bone and visceral metastases. The molecular nature of DTCs remains elusive, as well as when and from where in the tumor they originate. Here, we apply single-cell sequencing to identify and trace the origin of DTCs in breast cancer. RESULTS: We sequence the genomes of 63 single cells isolated from six non-metastatic breast cancer patients. By comparing the cells' DNA copy number aberration (CNA) landscapes with those of the primary tumors and lymph node metastasis, we establish that 53% of the single cells morphologically classified as tumor cells are DTCs disseminating from the observed tumor. The remaining cells represent either non-aberrant "normal" cells or "aberrant cells of unknown origin" that have CNA landscapes discordant from the tumor. Further analyses suggest that the prevalence of aberrant cells of unknown origin is age-dependent and that at least a subset is hematopoietic in origin. Evolutionary reconstruction analysis of bulk tumor and DTC genomes enables ordering of CNA events in molecular pseudo-time and traced the origin of the DTCs to either the main tumor clone, primary tumor subclones, or subclones in an axillary lymph node metastasis. CONCLUSIONS: Single-cell sequencing of bone marrow epithelial-like cells, in parallel with intra-tumor genetic heterogeneity profiling from bulk DNA, is a powerful approach to identify and study DTCs, yielding insight into metastatic processes. A heterogeneous population of CNA-positive cells is present in the bone marrow of non-metastatic breast cancer patients, only part of which are derived from the observed tumor lineages.

Next-generation sequencing of disseminated tumor cells.

Disseminated tumor cells (DTCs) detected in the bone marrow have been shown as an independent prognostic factor for women with breast cancer. However, the mechanisms behind the tumor cell dissemination are still unclear and more detailed knowledge is needed to fully understand why some cells remain dormant and others metastasize. Sequencing of single cells has opened for the possibility to dissect the genetic content of subclones of a primary tumor, as well as DTCs. Previous studies of genetic changes in DTCs have employed single-cell array comparative genomic hybridization which provides information about larger aberrations. To date, next-generation sequencing provides the possibility to discover new, smaller, and copy neutral genetic changes. In this study, we performed whole-genome amplification and subsequently next-generation sequencing to analyze DTCs from two breast cancer patients. We compared copy-number profiles of the DTCs and the corresponding primary tumor generated from sequencing and SNP-comparative genomic hybridization (CGH) data, respectively. While one tumor revealed mostly whole-arm gains and losses, the other had more complex alterations, as well as subclonal amplification and deletions. Whole-arm gains or losses in the primary tumor were in general also observed in the corresponding DTC. Both primary tumors showed amplification of chromosome 1q and deletion of parts of chromosome 16q, which was recaptured in the corresponding DTCs. Interestingly, clear differences were also observed, indicating that the DTC underwent further evolution at the copy-number level. This study provides a proof-of-principle for sequencing of DTCs and correlation with primary copy-number profiles. The analyses allow insight into tumor cell dissemination and show ongoing copy-number evolution in DTCs compared to the primary tumors.

Persistence of disseminated tumor cells after neoadjuvant treatment for locally advanced breast cancer predicts poor survival.

INTRODUCTION: Presence of disseminated tumor cells (DTCs) in bone marrow (BM) and circulating tumor cells (CTC) in peripheral blood (PB) predicts reduced survival in early breast cancer. The aim of this study was to determine the presence of and alterations in DTC- and CTC-status in locally advanced breast cancer patients undergoing neoadjuvant chemotherapy (NACT) and to evaluate their prognostic impact. METHODS: Bone marrow and peripheral blood were collected before NACT (BM1: n = 231/PB1: n = 219), at surgery (BM2: n = 69/PB2: n = 71), and after 12 months from start of NACT (BM3: n = 162/PB3: n = 141). Patients were included from 1997 to 2003 and followed until 2009 (or ten years follow-up). DTC- and CTC-status were determined by morphological evaluation of immunocytochemically detected cytokeratin-positive cells. The prognostic significance of DTCs/CTCs was assessed by univariate and multivariate Cox-regression analyses. RESULTS: Before NACT, DTCs and CTCs were detected in 21.2% and 4.9% of the patients, respectively. At surgery, 15.9% and 1.4% had DTC- and CTC-presence, compared to 26.5% and 4.3% at 12 months from start of NACT. Of patients for whom DTC results both before NACT and at 12 months were available, concordant results were observed in 68%, and 14 out of 65 had positive DTC-status at both time points. Presence of ≥ 1 DTC 12 months from start of NACT, but not at other time points, predicted reduced disease-free survival (DFS; HR 2.3, p = 0.003), breast cancer-specific survival (BCSS; HR 3.0, p < 0.001) and overall survival (OS; HR 2.8, p < 0.001). Before NACT, presence of ≥ 3 DTCs was also associated with unfavorable outcome, and reduced BCSS was observed for CTC-positive patients (HR 2.2, p = 0.046). In multivariate analysis, DTC status (

High-resolution analyses of copy number changes in disseminated tumor cells of patients with breast cancer.

The presence of disseminated tumor cells (DTCs) in bone marrow (BM) identifies breast cancer patients with less favorable outcome. Furthermore, molecular characterization is required to investigate the malignant potential of these cells. This study presents a single-cell array comparative genomic hybridization (SCaCGH) method providing molecular analysis of immunomorphologically detected DTCs. The resolution limit of the method was estimated using the cancer cell line SK-BR-3 on 44 and 244k arrays. The technique was further tested on 28 circulating tumor cells and four hematopoietic cells (HCs) from peripheral blood (n = 8 patients). The SCaCGH method was finally applied to 24 DTCs, three immunopositive cells morphologically classified as probable HCs from breast cancer patients and five HC controls from BM (n = 7 patients plus n = 1 healthy donor). The frequency of copy number changes of the DTCs revealed similarities with primary breast tumor samples. Three of the patients had available profiles for DTCs and the corresponding tumor tissue from primary surgery. More than two-third of the analyzed DTCs disclosed equivalent changes, both to each other and to the corresponding primary disease, whereas the rest of the cells showed balanced profiles. The probable HCs revealed either balanced profiles (n = 2) or changes comparable to the tumor tissue and DTCs (n = 1), indicating morphological overlap between HCs and DTCs. Similar aberration patterns were visible in DTCs collected at diagnosis and at 3 years relapse-free follow-up. SCaCGH may be a powerful tool for the molecular characterization of DTCs.