The quantity of tumor-infiltrating lymphocytes (TILs) in breast cancer (BC) is a robust prognostic factor for improved patient survival, particularly in triple-negative and HER2-overexpressing BC subtypes. Although T cells are the predominant TIL population, the relationship between quantitative and qualitative differences in T cell subpopulations and patient prognosis remains unknown. We performed single-cell RNA sequencing (scRNA-seq) of 6,311 T cells isolated from human BCs and show that significant heterogeneity exists in the infiltrating T cell population. We demonstrate that BCs with a high number of TILs contained CD8 T cells with features of tissue-resident memory T (T) cell differentiation and that these CD8 T cells expressed high levels of immune checkpoint molecules and effector proteins. A CD8 T gene signature developed from the scRNA-seq data was significantly associated with improved patient survival in early-stage triple-negative breast cancer (TNBC) and provided better prognostication than CD8 expression alone. Our data suggest that CD8 T cells contribute to BC immunosurveillance and are the key targets of modulation by immune checkpoint inhibition. Further understanding of the development, maintenance and regulation of T cells will be crucial for successful immunotherapeutic development in BC.

Cancer derived microarray data sets are routinely produced by various platforms that are either commercially available or manufactured by academic groups. The fundamental difference in their probe selection strategies holds the promise that identical observations produced by more than one platform prove to be more robust when validated by biology. However, cross-platform comparison requires matching corresponding probe sets. We are introducing here sequence-based matching of probes instead of gene identifier-based matching. We analyzed breast cancer cell line derived RNA aliquots using Agilent cDNA and Affymetrix oligonucleotide microarray platforms to assess the advantage of this method. We show, that at different levels of the analysis, including gene expression ratios and difference calls, cross-platform consistency is significantly improved by sequence- based matching. We also present evidence that sequence-based probe matching produces more consistent results when comparing similar biological data sets obtained by different microarray platforms. This strategy allowed a more efficient transfer of classification of breast cancer samples between data sets produced by cDNA microarray and Affymetrix gene-chip platforms.

Concordance between the four programmed death ligand 1 immunohistochemical assays when performed and scored as intended show that apart from 28-8 and 22C3, they cannot be used interchangeably in clinical practice. A protocol was successfully developed to use 22C3 on an alternate platform, which may help to overcome some barriers to implementation.

IntroductionBasal-like breast cancers behave more aggressively despite the presence of a dense lymphoid infiltrate. We hypothesised that immune suppression in this subtype may be due to T regulatory cells (Treg) recruitment driven by hypoxia-induced up-regulation of CXCR4 in Treg.MethodsImmunoperoxidase staining for FOXP3 and CXCL12 was performed on tissue microarrays from 491 breast cancers. The hypoxia-associated marker carbonic anhydrase IX (CA9) and double FOXP3/CXCR4 staining were performed on sections from a subset of these cancers including 10 basal-like and 11 luminal cancers matched for tumour grade.ResultsHigh Treg infiltration correlated with tumour CXCL12 positivity (OR 1.89, 95% CI 1.22 to 2.94, P = 0.004) and basal phenotype (OR 3.14, 95% CI 1.08 to 9.17, P = 0.004) in univariate and multivariate analyses. CXCL12 positivity correlated with improved survival (P = 0.005), whereas high Treg correlated with shorter survival for all breast cancers (P = 0.001), luminal cancers (P < 0.001) and basal-like cancers (P = 0.040) that were confirmed in a multivariate analysis (OR 1.61, 95% CI 1.02 to 2.53, P = 0.042). In patients treated with hormone therapy, high Treg were associated with a shorter survival in a multivariate analysis (OR 1.78, 95% CI 1.01 to 3.15, P = 0.040). There was a tendency for luminal cancers to show CXCL12 expression (102/138, 74%) compared to basal-like cancers (16/27, 59%), which verged on statistical significance (P = 0.050). Up-regulation of CXCR4 in Treg correlated with the basal-like phenotype (P = 0.029) and tumour hypoxia, as indicated by CA9 expression (P = 0.049).ConclusionsOur data show that in the setting of hypoxia and CXCR4 up-regulation in Treg, CXCL12 expression may have the negative consequence of enhancing Treg recruitment and suppressing the anti-tumour immune response.

The majority of deaths from breast cancer occur following the development of metastatic disease, a process inhibited by b-blockers in preclinical studies. This phase II randomized controlled trial evaluated the effect of preoperative b-blockade with propranolol on biomarkers of metastatic potential and the immune cell profile within the primary tumor of patients with breast cancer.Patients and Methods: In this triple-blind placebo-controlled clinical trial, 60 patients were randomly assigned to receive an escalating dose of oral propranolol (n ¼ 30; 80-160 mg daily) or placebo (n ¼ 30) for 7 days prior to surgery. The primary endpoint investigated the effect of propranolol on prometastatic and proinflammatory gene expression within the primary tumor.Results: Propranolol downregulated primary tumor expression of mesenchymal genes (P ¼ 0.002) without affecting epithelial gene expression (P ¼ 0.21). Bioinformatic analyses implicated downregulation of Snail/Slug (P ¼ 0.03), NF-kB/Rel (P < 0.01), and AP-1 (P < 0.01) transcription factors in structuring the observed transcriptome alterations, and identified changes in intratumoral neutrophil, natural killer cell, and dendritic cell recruitment (all P < 0.01). Patients with clinical evidence of drug response (lowered heart rate and blood pressure) demonstrated elevated tumor infiltration of CD68 þ macrophages and CD8 þ T cells.Conclusions: One week of b-blockade with propranolol reduced intratumoral mesenchymal polarization and promoted immune cell infiltration in early-stage surgically-resectable breast cancer. These results show that b-blockade reduces biomarkers associated with metastatic potential, and support the need for larger phase III clinical trials powered to detect the impact of b-blockade on cancer recurrence and survival.See related commentary by Blaes et al., p. 1781

Purpose: The immune microenvironment of breast ductal carcinoma in situ (DCIS) has yet to be fully explored, and the relationship of immune cells to genetic features of DCIS is unknown.Experimental Design: We quantified tumor associated lymphocytes (TIL) and evaluated PD-L1 protein levels by immunohistochemistry in a cohort of pure DCIS (138 and 79 cases, respectively), some of which had copy number (n ¼ 55) and mutation data (n ¼ 20).Results: TILs were identified in the stroma surrounding DCIS (119/138, 86%) and present at a median TIL score of 5% (range, 0%-90%). Most DCIS were negative for tumor cell PD-L1 staining (89%), but 25% of cases were positive for immune cell staining. We observed that, as in invasive breast cancer, TILs and PD-L1 positivity were significantly greater in high-grade (P ¼ 0.002/0.035), ER-negative (P ¼ 0.02/0.02), and ERBB2-amplified tumors (P < 0.001/0.048). Comedo necrosis was significantly positively associated with TILs (P < 0.0001) but not with PD-L1. The TILs score was significantly higher in cases with TP53 mutation (P ¼ 0.03) but not with PIK3CA or GATA3 mutation. In the cases with copy number data, both the fraction of the genome altered and the number of telomeric imbalances were significantly positively correlated with TILs (both P < 0.001). This result strongly contrasted with invasive breast cancer data, where aneuploidy was not correlated to TIL levels.Conclusions: Although a small cohort, our data suggest a preliminary model by which the progression of DCIS to invasive carcinoma may involve an altered relationship of tumor copy number with the immune microenvironment, possibly by the immunoediting of the tumor. Clin Cancer Res; 23(17); 5210-7.Ó2017 AACR.

Breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) is a rare form of T-cell lymphoma that occurs after implantation of breast prostheses. We performed comprehensive next generation sequencing based genomic characterization of 11 cases of BIA-ALCL including sequence variant detection on 180 genes frequently mutated in haematological malignancy, genome-wide copy number assessment, structural variant detection involving the T-cell receptor loci and TRB deep-sequencing. We observed sequence variants leading to JAK/STAT activation in 10 out of 11 patients. We also observed germline TP53 mutations in two cases. In addition we detected a recurrent copy number loss involving RPL5 as well as copy number amplifications involving TNFRSF11A [RANK] (in 2 cases), MYC, P2RX7, TMEM119 and PDGFRA. In summary, our comprehensive genomic characterisation of 11 cases of BIA-ALCL has provided insight into potential pathobiological mechanisms (JAK/STAT, MYC and TP53) as well as identifying targets for future therapeutic intervention (TNFRSF11A, PDGFRA) in this rare entity.

The spectrum of genomic alterations in ductal carcinoma in situ (DCIS) is relatively unexplored, but is likely to provide useful insights into its biology, its progression to invasive carcinoma and the risk of recurrence. DCIS (n = 20) with a range of phenotypes was assessed by massively parallel sequencing for mutations and copy number alterations and variants validated by Sanger sequencing. PIK3CA mutations were identified in 11/20 (55%), TP53 mutations in 6/20 (30%), and GATA3 mutations in 9/20 (45%). Screening an additional 91 cases for GATA3 mutations identified a final frequency of 27% (30/111), with a high proportion of missense variants (8/30). TP53 mutations were exclusive to high grade DCIS and more frequent in PR-negative tumors compared with PR-positive tumors (P = 0.037). TP53 mutant tumors also had a significantly higher fraction of the genome altered by copy number than wild-type tumors (P = 0.005), including a significant positive association with amplification or gain of ERBB2 (Po 0.05). The association between TP53 mutation and ERBB2 amplification was confirmed in a wider DCIS cohort using p53 immunohistochemistry as a surrogate marker for TP53 mutations (P = 0.03). RUNX1 mutations and MAP2K4 copy number loss were novel findings in DCIS. Frequent copy number alterations included gains on 1q, 8q, 17q, and 20q and losses on 8p, 11q, 16q, and 17p. Patterns of genomic alterations observed in DCIS were similar to those previously reported for invasive breast cancers, with all DCIS having at least one bona fide breast cancer driver event. However, an increase in GATA3 mutations and fewer copy number changes were noted in DCIS compared with invasive carcinomas. The role of such alterations as prognostic and predictive biomarkers in DCIS is an avenue for further investigation.