Methylation of high-density CpG regions known as CpG Islands (CGIs) has been widely described as a mechanism associated with gene expression regulation. Aberrant promoter methylation is considered a hallmark of cancer involved in silencing of tumor suppressor genes and activation of oncogenes. However, recent studies have also challenged the simple model of gene expression control by promoter methylation in cancer, and the precise mechanism of and role played by changes in DNA methylation in carcinogenesis remains elusive. Using a large dataset of 672 matched cancerous and healthy methylomes, gene expression, and copy number profiles accross 3 types of tissues from The Cancer Genome Atlas (TCGA), we perform a detailed meta-analysis to clarify the interplay between promoter methylation and gene expression in normal and cancer samples. On the one hand, we recover the existence of a CpG island methylator phenotype (CIMP) with prognostic value in a subset of breast, colon and lung cancer samples, where a common subset of promoter CGIs hypomethylated in normal samples become hypermethylated. However, this hypermethylation is not accompanied by a decrease in expression of the corresponding genes, which are already lowly expressed in the normal genes. On the other hand, we identify tissue-specific sets of genes, different between normal and cancer samples, whose inter-individual variation in expression is significantly correlated with the variation in methylation of the 3’ flanking regions of the promoter CGIs. These subsets of genes are not the same in the different tissues, nor between normal and cancerous samples, but transcription factors are over-represented in all subsets. Our results suggest that epigenetic reprogramming in cancer does not contribute to cancer development via direct inhibition of gene expression through promoter hypermethylation. It may instead modify how the expression of a few specific genes, particularly transcription factors, are associated with DNA methylation variations in a tissue-dependent manner.

The Residual Cancer Burden (RCB) quantifies residual disease after neoadjuvant chemotherapy (NAC). Its predictive value has not been validated on large cohorts with long-term follow up. The objective of this work is to independently evaluate the prognostic value of the RCB index depending on BC subtypes (Luminal, HER2-positive and triple negative (TNBCs)). We retrospectively evaluated the RCB index on surgical specimens from a cohort of T1-T3NxM0 BC patients treated with NAC between 2002 and 2012. We analyzed the association between RCB index and relapse-free survival (RFS), overall survival (OS) among the global population, after stratification by BC subtypes. 717 patients were included (luminal BC (n = 222, 31%), TNBC (n = 319, 44.5%), HER2-positive (n = 176, 24.5%)). After a median follow-up of 99.9 months, RCB index was significantly associated with RFS. The RCB-0 patients displayed similar prognosis when compared to the RCB-I group, while patients from the RCB-II and RCB-III classes were at increased risk of relapse (RCB-II versus RCB-0: HR = 3.25 CI [2.1–5.1] p<0.001; RCB-III versus RCB-0: HR = 5.6 CI [3.5–8.9] p<0.001). The prognostic impact of RCB index was significant for TNBC and HER2-positive cancers; but not for luminal cancers (P_interaction = 0.07). The prognosis of RCB-III patients was poor (8-years RFS: 52.7%, 95% CI [44.8–62.0]) particularly in the TNBC subgroup, where the median RFS was 12.7 months. RCB index is a reliable prognostic score. RCB accurately identifies patients at a high risk of recurrence (RCB-III) with TNBC or HER2-positive BC who must be offered second-line adjuvant therapies.

Background: Breast cancer is a heterogeneous disease with different molecular subtypes that have varying responses to therapy. An ongoing challenge in breast cancer research is to distinguish high‐risk patients from good prognosis patients. This is particularly difficult in the low‐grade, ER‐positive luminal A tumors, where robust diagnostic tools to aid clinical treatment decisions are lacking. Recent data implicating chromatin regulators in cancer initiation and progression offers a promising avenue to develop new tools to help guide clinical decisions.Methods: Here we exploit a published transcriptome dataset and an independent validation cohort to correlate the mRNA expression of selected chromatin regulators with respect to the four intrinsic breast cancer molecular subtypes. We then perform univariate and multivariate analyses to compare the prognostic value of a panel of chromatin regulators to Ki67, a currently utilized proliferation marker.Results: Unsupervised hierarchical clustering revealed a gene cluster containing several histone chaperones and histone variants highly‐expressed in the proliferative subtypes (basal‐like, HER2‐positive, luminal B) but not in the luminal A subtype. Several chromatin regulators, including the histone chaperones CAF‐1 (subunits p150 and p60), ASF1b, and HJURP, and the centromeric histone variant CENP‐A, associated with local and metastatic relapse and poor patient outcome. Importantly, we find that HJURP can discriminate favorable and unfavorable outcome within the luminal A subtype, outperforming the currently utilized proliferation marker Ki67, as an independent prognostic marker for luminal A patients.Conclusions: The integration of chromatin regulators as clinical biomarkers, in particular the histone chaperone HJURP, will help guide patient substratification and treatment options for low‐risk luminal A breast carcinoma patients.

Purpose: High levels of tumor-infiltrating lymphocytes (TILs) before neoadjuvant chemotherapy (NAC) are associated with higher pathological complete response (pCR) rates, and better survival in TNBC and HER2-positive breast cancers (BCs). We investigated the value of TIL levels by evaluating lymphocyte infiltration before and after NAC. Patients and methods: We assessed stromal TIL levels in 716 pre- and post-treatment matched paired specimens, according to the guidelines of the international TIL working group. Results: Pre-NAC TIL levels were higher in tumors for which pCR was achieved than in cases with residual disease (33.9% versus 20.3%, p=0.001). This was observed in luminal tumors and TNBCs, but not in HER2-positive BCs, (p_Interaction =0.001). The association between pre-NAC TIL levels and pCR was non-linear in TNBCs (p=0.005). Mean TIL levels decreased after chemotherapy completion (pre-NAC TILs: 24.1% versus post-NAC TILs: 13.0%, p<0.001). This decrease was strongly associated with high pCR rates, and the variation of TIL levels was strongly inversely correlated with pre-NAC TIL levels (r=-0.80, p<0.001). Pre-NAC TILs and disease-free survival (DFS) were associated in a non-linear manner (p<0.001). High post-NAC TIL levels were associated with aggressive tumor characteristics and with impaired DFS in HER2-positive BCs (HR=1.04, CI [1.02-1.06], p=0.001), but not in luminal tumors or TNBCs (p_Interaction =0.04). Conclusion: The associations of pre and post-NAC TIL levels with response to treatment and DFS differ between BC subtypes. The characterization of immune subpopulations may improve our understanding of the complex interactions between pre- or post-NAC setting, BC subtype, response to treatment and prognosis.

Reciprocal interactions between tumor cells and their microenvironment vitally impact tumor progression. In this study, we show that GM-CSF produced by primary breast tumor cells induced the activation of plasmacytoid predendritic cells (pDC), a cell type critical to anti-viral immunity. pDC that expressed the GM-CSF receptor were increased in breast tumors compared with noninvolved adjacent breast tissue. Tumor-activated pDC acquired naïve CD4+ T-cell stimulatory capacity and promoted a regulatory Th2 response. Finally, the concomitant increase of GM-CSF and pDC was significantly associated with relatively more aggressive breast cancer subtypes. Our results characterize the first tumor-derived factor that can activate pDC to promote a regulatory Th2 response, with implications for therapeutic targeting of a tumor-immune axis of growing recognition in its significance to cancer. Cancer Res; 75(14); 2775–87. ©2015 AACR.