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While regulatory T (Treg) cells are traditionally viewed as professional suppressors of antigen presenting cells and effector T cells in both autoimmunity and cancer, recent findings of distinct Treg cell functions in tissue maintenance suggest that their regulatory purview extends to a wider range of cells and is broader than previously assumed. To elucidate tumoral Treg cell ‘connectivity’ to diverse tumor-supporting accessory cell types, we explored immediate early changes in their single-cell transcriptomes upon punctual Treg cell depletion in experimental lung cancer and injury-induced inflammation. Before any notable T cell activation and inflammation, fibroblasts, endothelial and myeloid cells exhibited pronounced changes in their gene expression in both cancer and injury settings. Factor analysis revealed shared Treg cell-dependent gene programs, foremost, prominent upregulation of VEGF and CCR2 signaling-related genes upon Treg cell deprivation in either setting, as well as in Treg cell-poor versus Treg cell-rich human lung adenocarcinomas. Accordingly, punctual Treg cell depletion combined with short-term VEGF blockade showed markedly improved control of PD-1 blockade-resistant lung adenocarcinoma progression in mice compared to the corresponding monotherapies, highlighting a promising factor-based querying approach to elucidating new rational combination treatments of solid organ cancers.

Supplementary Table S2: Comparison of clonal relatedness of the lesions analyzed between Begg et al. (2), Sakr et al. (45) and this study.

Purpose:Lobular carcinoma in situ (LCIS) is a preinvasive lesion of the breast. We sought to define its genomic landscape, whether intralesion genetic heterogeneity is present in LCIS, and the clonal relatedness between LCIS and invasive breast cancers.Experimental Design: We reanalyzed whole-exome sequencing (WES) data and performed a targeted amplicon sequencing validation of mutations identified in 43 LCIS and 27 synchronous more clinically advanced lesions from 24 patients [9 ductal carcinomas in situ (DCIS), 13 invasive lobular carcinomas (ILC), and 5 invasive ductal carcinomas (IDC)]. Somatic genetic alterations, mutational signatures, clonal composition, and phylogenetic trees were defined using validated computational methods.Results:WES of 43 LCIS lesions revealed a genomic profile similar to that previously reported for ILCs, with CDH1 mutations present in 81% of the lesions. Forty-two percent (18/43) of LCIS were found to be clonally related to synchronous DCIS and/or ILCs, with clonal evolutionary patterns indicative of clonal selection and/or parallel/branched progression. Intralesion genetic heterogeneity was higher among LCIS clonally related to DCIS/ILC than in those nonclonally related to DCIS/ILC. A shift from aging to APOBEC-related mutational processes was observed in the progression from LCIS to DCIS and/or ILC in a subset of cases.Conclusions:Our findings support the contention that LCIS has a repertoire of somatic genetic alterations similar to that of ILCs, and likely constitutes a nonobligate precursor of breast cancer. Intralesion genetic heterogeneity is observed in LCIS and should be considered in studies aiming to develop biomarkers of progression from LCIS to more advanced lesions.

Supplementary Fig. S1: Repertoire of somatic mutations, clonal composition and phylogenetic tree analysis of cases 1-3 and cases 5-9.

Supplementary Fig. S3: Repertoire of somatic mutations, clonal composition and phylogenetic tree analysis of cases 19-24.

Excel file containing Supplementary Data File 1: Non-synonymous somatic mutations identified by whole-exome sequencing and amplicon sequencing in the LCIS, DCIS, ILCs and IDCs studied.

Supplementary Table S1: Sequencing statistics.

Supplementary Fig. S2: Repertoire of somatic mutations, clonal composition and phylogenetic tree analysis of cases 11-18.

Supplementary Fig. S4: Clonal relatedness between lobular carcinoma in situ (LCIS), invasive lobular carcinoma (ILC), ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC), Shannon and Gini-Simpson Indices measure of intra-lesion genetic heterogeneity, and truncal and branch mutations affecting cancer genes. A) Pairwise comparison of the clonality index between samples based on the presence of synonymous and non-synonymous somatic mutations. The color gradient represents the CI (Clonal Index) of the probability that a given pair of samples is clonally-related based on the number of shared mutations and the frequency of each shared mutation in ILCs breast cancers from The Cancer Genome Atlas breast cancer study (31). B) Barplot showing the proportion of LCIS harboring intra-lesion genetic heterogeneity in the LCIS clonally-related to DCIS/ILC (red bar) and LCIS not clonally-related to DCIS/ILC (gray bar) (p-value = 0.0016, Fisher's exact test). Boxplots illustrating the distribution of the Shannon diversity index (C) and the Gini-Simpson diversity index (D) for each lesion type (LCIS, DICS, ILC and IDC). E-F) Truncal and branch mutations affecting cancer genes as defined by Kandoth et al. (24), the Cancer Gene Census (25) and/ or Lawrence et al. (26). Venn diagrams representing the overlap between genes affected by truncal or branch mutations and cancer genes. Hypergeometric test P-value and representation factors are shown below the respective Venn diagrams.

File containing Supplementary Methods, Legends for Supplementary Tables S1-S2, Legends for Supplementary Figures S1-S4 and Legends for the Supplementary Data File 1.

The immunosuppressive function of regulatory T (Treg) cells is dependent on continuous expression of the transcription factor Foxp3. Foxp3 loss of function or induced ablation of Treg cells results in a fatal autoimmune disease featuring all known types of inflammatory responses with every manifestation stemming from Treg cell paucity, highlighting a vital function of Treg cells in preventing fatal autoimmune inflammation. However, a major question remains whether Treg cells can persist and effectively exert their function in a disease state, where a broad spectrum of inflammatory mediators can either inactivate Treg cells or render innate and adaptive pro-inflammatory effector cells insensitive to suppression. By reinstating Foxp3 protein expression and suppressor function in cells expressing a reversible Foxp3 null allele in severely diseased mice, we found that the resulting single pool of rescued Treg cells normalized immune activation, quelled severe tissue inflammation, reversed fatal autoimmune disease and provided long-term protection against them. Thus, Treg cells are functional in settings of established broad-spectrum systemic inflammation and are capable of affording sustained reset of immune homeostasis.

Intestinal health relies on the immunosuppressive activity of CD4+ regulatory T (Treg) cells1. Expression of the transcription factor Foxp3 defines this lineage, and can be induced extrathymically by dietary or commensal-derived antigens in a process assisted by a Foxp3 enhancer known as conserved non-coding sequence 1 (CNS1)2–4. Products of microbial fermentation including butyrate facilitate the generation of peripherally induced Treg (pTreg) cells5–7, indicating that metabolites shape the composition of the colonic immune cell population. In addition to dietary components, bacteria modify host-derived molecules, generating a number of biologically active substances. This is epitomized by the bacterial transformation of bile acids, which creates a complex pool of steroids8 with a range of physiological functions9. Here we screened the major species of deconjugated bile acids for their ability to potentiate the differentiation of pTreg cells. We found that the secondary bile acid 3β-hydroxydeoxycholic acid (isoDCA) increased Foxp3 induction by acting on dendritic cells (DCs) to diminish their immunostimulatory properties. Ablating one receptor, the farnesoid X receptor, in DCs enhanced the generation of Treg cells and imposed a transcriptional profile similar to that induced by isoDCA, suggesting an interaction between this bile acid and nuclear receptor. To investigate isoDCA in vivo, we took a synthetic biology approach and designed minimal microbial consortia containing engineered Bacteroides strains. IsoDCA-producing consortia increased the number of colonic RORγt-expressing Treg cells in a CNS1-dependent manner, suggesting enhanced extrathymic differentiation. The secondary bile acid 3β-hydroxy-deoxycholic (isodeoxycholic) acid, produced by gut bacteria, promotes the generation of colonic extrathymic regulatory T cells, whose immunosuppressive activities are known to be essential for intestinal health.

Inflammation of non-barrier immunologically quiescent tissues is associated with a massive influx of blood-borne innate and adaptive immune cells. Cues from the latter are likely to alter and expand the spectrum of states observed in cells that are constitutively resident. However, local communications between immigrant and resident cell types in human inflammatory disease remain poorly understood. Here, we explored heterogeneity of synovial fibroblasts (FLS) in inflamed joints of rheumatoid arthritis (RA) patients using paired single cell RNA and ATAC sequencing (scRNA/ATAC-seq), multiplexed imaging, and spatial transcriptomics along with in vitro modeling of cell extrinsic factor signaling. These analyses suggest that local exposures to myeloid and T cell derived cytokines, TNFα, IFNγ, IL-1β, or lack thereof, drive six distinct FLS states some of which closely resemble fibroblast states in other disease-affected tissues including skin and colon. Our results highlight a role for concurrent, spatially distributed cytokine signaling within the inflamed synovium.

Regulatory T (Treg) cells expressing the transcription factor Foxp3 are an essential suppressive T cell lineage of dual origin: Foxp3 induction in thymocytes and mature CD4

Purpose: Lobular carcinoma in situ (LCIS) is a preinvasive lesion of the breast. We sought to define its genomic landscape, whether intralesion genetic heterogeneity is present in LCIS, and the clonal relatedness between LCIS and invasive breast cancers. Experimental Design: We reanalyzed whole-exome sequencing (WES) data and performed a targeted amplicon sequencing validation of mutations identified in 43 LCIS and 27 synchronous more clinically advanced lesions from 24 patients [9 ductal carcinomas in situ (DCIS), 13 invasive lobular carcinomas (ILC), and 5 invasive ductal carcinomas (IDC)]. Somatic genetic alterations, mutational signatures, clonal composition, and phylogenetic trees were defined using validated computational methods. Results: WES of 43 LCIS lesions revealed a genomic profile similar to that previously reported for ILCs, with CDH1 mutations present in 81% of the lesions. Forty-two percent (18/43) of LCIS were found to be clonally related to synchronous DCIS and/or ILCs, with clonal evolutionary patterns indicative of clonal selection and/or parallel/branched progression. Intralesion genetic heterogeneity was higher among LCIS clonally related to DCIS/ILC than in those nonclonally related to DCIS/ILC. A shift from aging to APOBEC-related mutational processes was observed in the progression from LCIS to DCIS and/or ILC in a subset of cases. Conclusions: Our findings support the contention that LCIS has a repertoire of somatic genetic alterations similar to that of ILCs, and likely constitutes a nonobligate precursor of breast cancer. Intralesion genetic heterogeneity is observed in LCIS and should be considered in studies aiming to develop biomarkers of progression from LCIS to more advanced lesions.

Effective antiviral immunity requires generation of T and B lymphocytes expressing the transcription factor T-bet, a regulator of type 1 inflammatory responses. Using T-bet expression as an endogenous marker for cells participating in a type 1 response, we report coordinated interactions of T-bet-expressing T and B lymphocytes based on their dynamic co-localization at the T cell zone and B follicle boundary (T-B boundary) and germinal centers (GC) during lung influenza infection. We demonstrate that the assembly of this circuit takes place in distinct anatomical niches within the draining lymph node, guided by CXCR3 that enables positioning of T(H)1 cells at the T-B boundary. The encounter of B and T(H)1 cells at the T-B boundary enables IFNγ produced by the latter to induce IgG2c class switching. Within GCs, T-bet(+) T(FH) cells represent a specialized stable sub-lineage required for GC growth, but dispensable for IgG2c class switching. Our studies show that during respiratory viral infection, T-bet-expressing T and B lymphocytes form a circuit assembled in a spatiotemporally controlled manner that acts as a functional unit enabling a robust and coherent humoral response tailored for optimal antiviral immunity.

The immunosuppressive function of regulatory T (Treg) cells is dependent on continuous expression of the transcription factor Foxp3. Foxp3 loss-of-function or induced ablation of Treg cells results in a fatal autoimmune disease featuring all known types of inflammatory responses with every manifestation stemming from Treg cell paucity, highlighting a vital function of Treg cells in preventing fatal autoimmune inflammation. However, a major question remains whether Treg cells can persist and effectively exert their function in a disease state, where broad spectrum of inflammatory mediators can either inactivate Treg cells or render innate and adaptive pro-inflammatory effector cells insensitive to suppression. By reinstating Foxp3 protein expression and suppressor function in cells expressing a reversible Foxp3 null allele in severely diseased mice, we found that the resulting single pool of “redeemed” Treg cells normalized immune activation, quelled severe tissue inflammation, reversed fatal autoimmune disease and provided long-term protection against them. Thus, Treg cells are functional in settings of established broad spectrum systemic inflammation and are capable of affording sustained reset of the immune homeostasis.

Significance Anorexia or decreased appetite is a common feature of infection. The effects of acute nutritional stress on effector T cells, which are required for pathogen-specific responses and protective immunological memory, are not fully understood. We show that reduced food intake during infection decreases the numbers of effector T cells in a manner dependent on the Farnesoid X Receptor (FXR). FXR was previously implicated in controlling hepatic responses to fasting. Our findings suggest that FXR functions in effector T cells to promote coherent physiological responses to decreased feeding, allowing organisms to scale their immune responses according to food availability., Reduced nutrient intake is a widely conserved manifestation of sickness behavior with poorly characterized effects on adaptive immune responses. During infectious challenges, naive T cells encountering their cognate antigen become activated and differentiate into highly proliferative effector T cells. Despite their evident metabolic shift upon activation, it remains unclear how effector T cells respond to changes in nutrient availability in vivo. Here, we show that spontaneous or imposed feeding reduction during infection decreases the numbers of splenic lymphocytes. Effector T cells showed cell-intrinsic responses dependent on the nuclear receptor Farnesoid X Receptor (FXR). Deletion of FXR in T cells prevented starvation-induced loss of lymphocytes and increased effector T cell fitness in nutrient-limiting conditions, but imparted greater weight loss to the host. FXR deficiency increased the contribution of glutamine and fatty acids toward respiration and enhanced cell survival under low-glucose conditions. Provision of glucose during anorexia of infection rescued effector T cells, suggesting that this sugar is a limiting nutrient for activated lymphocytes and that alternative fuel usage may affect cell survival in starved animals. Altogether, we identified a mechanism by which the host scales immune responses according to food intake, featuring FXR as a T cell-intrinsic sensor.

ST2 expression was largely dispensable for T reg cell accumulation and maintenance in tissues at steady state. However, ST2 deficiency limited to T reg cells was important in limiting the size of IL-17A–producing γδT cells in a mouse model of neuroinflammation., ST2, the receptor for the alarmin IL-33, is expressed by a subset of regulatory T (T reg) cells residing in nonlymphoid tissues, and these cells can potently expand upon provision of exogenous IL-33. Whether the accumulation and residence of T reg cells in tissues requires their cell-intrinsic expression of and signaling by ST2, or whether indirect IL-33 signaling acting on other cells suffices, has been a matter of contention. Here, we report that ST2 expression on T reg cells is largely dispensable for their accumulation and residence in nonlymphoid organs, including the visceral adipose tissue (VAT), even though cell-intrinsic sensing of IL-33 promotes type 2 cytokine production by VAT-residing T reg cells. In addition, we uncovered a novel ST2-dependent role for T reg cells in limiting the size of IL-17A–producing γδT cells in the CNS in a mouse model of neuroinflammation, experimental autoimmune encephalomyelitis (EAE). Finally, ST2 deficiency limited to T reg cells led to disease exacerbation in EAE.

Green et al. find that T cell–derived amphiregulin facilitates lung tumor progression seemingly independently of the immune response, suggesting that T cells can promote tumor growth through the production of factors normally associated with tissue repair., The involvement of effector T cells and regulatory T (T reg) cells in opposing and promoting solid organ carcinogenesis, respectively, is viewed as a shifting balance between a breach versus establishment of tolerance to tumor or self-antigens. We considered that tumor-associated T cells might promote malignancy via distinct mechanisms used by T cells in nonlymphoid organs to assist in their maintenance upon injury or stress. Recent studies suggest that T reg cells can participate in tissue repair in a manner separable from their immunosuppressive capacity. Using transplantable models of lung tumors in mice, we found that amphiregulin, a member of the epidermal growth factor family, was prominently up-regulated in intratumoral T reg cells. Furthermore, T cell–restricted amphiregulin deficiency resulted in markedly delayed lung tumor progression. This observed deterrence in tumor progression was not associated with detectable changes in T cell immune responsiveness or T reg and effector T cell numbers. These observations suggest a novel “nonimmune” modality for intratumoral T reg and effector T cells in promoting tumor growth through the production of factors normally involved in tissue repair and maintenance.

Adaptive immune responses are tailored to different types of pathogens through differentiation of naive CD4 T cells into functionally distinct subsets of effector T cells (T helper 1 (TH1), TH2, and TH17) defined by expression of the key transcription factors T-bet, GATA3, and RORγt, respectively. Regulatory T (Treg) cells comprise a distinct anti-inflammatory lineage specified by the X-linked transcription factor Foxp3 (refs 2, 3). Paradoxically, some activated Treg cells express the aforementioned effector CD4 T cell transcription factors, which have been suggested to provide Treg cells with enhanced suppressive capacity. Whether expression of these factors in Treg cells-as in effector T cells-is indicative of heterogeneity of functionally discrete and stable differentiation states, or conversely may be readily reversible, is unknown. Here we demonstrate that expression of the TH1-associated transcription factor T-bet in mouse Treg cells, induced at steady state and following infection, gradually becomes highly stable even under non-permissive conditions. Loss of function or elimination of T-bet-expressing Treg cells-but not of T-bet expression in Treg cells-resulted in severe TH1 autoimmunity. Conversely, following depletion of T-bet- Treg cells, the remaining T-bet+ cells specifically inhibited TH1 and CD8 T cell activation consistent with their co-localization with T-bet+ effector T cells. These results suggest that T-bet+ Treg cells have an essential immunosuppressive function and indicate that Treg cell functional heterogeneity is a critical feature of immunological tolerance.

A substantial proportion of tumors consist of genotypically distinct subpopulations of cancer cells. This intratumor genetic heterogeneity poses a substantial challenge for the implementation of precision medicine. Single-cell genomics constitutes a powerful approach to resolve complex mixtures of cancer cells by tracing cell lineages and discovering cryptic genetic variations that would otherwise be obscured in tumor bulk analyses. Because of the chemical alterations that result from formalin fixation, single-cell genomic approaches have largely remained limited to fresh or rapidly frozen specimens. Here we describe the development and validation of a robust and accurate methodology to perform whole-genome copy-number profiling of single nuclei obtained from formalin-fixed paraffin-embedded clinical tumor samples. We applied the single-cell sequencing approach described here to study the progression from in situ to invasive breast cancer, which revealed that ductal carcinomas in situ show intratumor genetic heterogeneity at diagnosis and that these lesions may progress to invasive breast cancer through a variety of evolutionary processes.

Levine et al. investigate the extent to which regulatory T cells with either a monoclonal T cell receptor (TCR) or random TCR repertoire in place of their developmentally selected specificities maintain TCR-dependent gene expression and immunosuppressive function., The regulatory T cell (T reg cell) T cell receptor (TCR) repertoire is highly diverse and skewed toward recognition of self-antigens. TCR expression by T reg cells is continuously required for maintenance of immune tolerance and for a major part of their characteristic gene expression signature; however, it remains unknown to what degree diverse TCR-mediated interactions with cognate self-antigens are required for these processes. In this study, by experimentally switching the T reg cell TCR repertoire to a single T reg cell TCR, we demonstrate that T reg cell function and gene expression can be partially uncoupled from TCR diversity. An induced switch of the T reg cell TCR repertoire to a random repertoire also preserved, albeit to a limited degree, the ability to suppress lymphadenopathy and T helper cell type 2 activation. At the same time, these perturbations of the T reg cell TCR repertoire led to marked immune cell activation, tissue inflammation, and an ultimately severe autoimmunity, indicating the importance of diversity and specificity for optimal T reg cell function.

Intestinal health relies on the immunosuppressive activity of CD4

PURPOSE: Lobular carcinoma in situ (LCIS) is a pre-invasive lesion of the breast. We sought to define its genomic landscape, whether intra-lesion genetic heterogeneity is present in LCIS, and the clonal relatedness between LCIS and invasive breast cancers. EXPERIMENTAL DESIGN: We reanalyzed whole-exome sequencing (WES) data and performed a targeted amplicon sequencing validation of mutations identified in 43 LCIS and 27 synchronous more clinically-advanced lesions from 24 patients (nine ductal carcinomas in situ (DCIS), 13 invasive lobular carcinomas (ILCs) and five invasive ductal carcinomas (IDCs)). Somatic genetic alterations, mutational signatures, clonal composition and phylogenetic trees were defined using validated computational methods. RESULTS: WES of 43 LCIS lesions revealed a genomic profile similar to that previously reported for ILCs, with CDH1 mutations present in 81% of the lesions. Forty-two percent (18/43) of LCIS were found to be clonally-related to synchronous DCIS and/or ILCs, with clonal evolutionary patterns indicative of clonal selection and/or parallel/branched progression. Intra-lesion genetic heterogeneity was higher among LCIS clonally-related to DCIS/ILC than in those non-clonally related to DCIS/ILC. A shift from aging to APOBEC-related mutational processes was observed in the progression from LCIS to DCIS and/or ILC in a subset of cases. CONCLUSIONS: Our findings support the contention that LCIS has a repertoire of somatic genetic alterations similar to that of ILCs, and likely constitutes a non-obligate precursor of breast cancer. Intra-lesion genetic heterogeneity is observed in LCIS and should be considered in studies aiming to develop biomarkers of progression from LCIS to more advanced lesions.

The RANK/RANKL axis emerges as a key regulator of breast cancer initiation, progression, and metastasis. RANK-c is a RANK receptor isoform produced through alternative splicing of the TNFRSF11A (RANK) gene and a dominant-negative regulator of RANK-induced nuclear factor-κB (NF-κB) activation. Here we report that RANK-c transcript is expressed in 3.2% of cases in The Cancer Genome Atlas breast cancer cohort evenly between ER-positive and ER-negative cases. Nevertheless, the ratio of RANK to RANK-c (RANK/RANK-c) is increased in ER-negative breast cancer cell lines compared to ER-positive breast cancer cell lines. In addition, forced expression of RANK-c in ER-negative breast cancer cell lines inhibited stimuli-induced NF-κB activation and attenuated migration, invasion, colony formation, and adhesion of cancer cells. Further, RANK-c expression in MDA-MB-231 cells inhibited lung metastasis and colonization in vivo. The RANK-c-mediated inhibition of cancer cell aggressiveness and nuclear factor-κB (NF-κB) activation in breast cancer cells seems to rely on a RANK-c/TNF receptor-associated factor-2 (TRAF2) protein interaction. This was further confirmed by a mutated RANK-c that is unable to interact with TRAF2 and abolishes the ability to attenuate NF-κB activation, migration, and invasion. Additional protein interaction characterization revealed epidermal growth factor receptor (EGFR) as a novel interacting partner for RANK-c in breast cancer cells with a negative effect on EGFR phosphorylation and EGF-dependent downstream signaling pathway activation. Our findings further elucidate the complex molecular biology of the RANKL/RANK system in breast cancer and provide preliminary data for RANK-c as a possible marker for disease progression and aggressiveness.

Background Recent evidence suggests that lobular carcinoma in situ (LCIS) can be a clonal precursor of invasive breast cancers of both the ductal and lobular phenotypes. We sought to confirm these findings with an extensive study of fresh frozen breast specimens from women undergoing mastectomy. Methods Patients with a history of LCIS presenting for therapeutic mastectomy were identified prospectively. Frozen tissue blocks were collected, screened for lesions of interest, and subjected to microdissection and DNA extraction. Copy number profiling, whole-exome sequencing, or both were performed. Clonal relatedness was assessed using specialized statistical techniques developed for this purpose. Results After exclusions for genotyping failure, a total of 84 lesions from 30 patients were evaluated successfully. Strong evidence of clonal relatedness was observed between an LCIS lesion and the invasive cancer for the preponderance of cases with lobular carcinoma. Anatomically distinct in situ lesions of both ductal and lobular histology were also shown to be frequently clonally related. Conclusions These data derived from women with LCIS with or without invasive cancer confirm that LCIS is commonly the clonal precursor of invasive lobular carcinoma and that distinct foci of LCIS frequently share a clonal origin, as do foci of LCIS and ductal carcinoma in situ. Electronic supplementary material The online version of this article (doi:10.1186/s13058-016-0727-z) contains supplementary material, which is available to authorized users.

Purpose Lobular carcinoma in situ (LCIS) has been proposed as a non-obligate precursor of invasive lobular carcinoma (ILC). Here we sought to define the repertoire of somatic genetic alterations in pure LCIS and in synchronous LCIS and ILC using targeted massively parallel sequencing. Methods DNA samples extracted from microdissected LCIS, ILC and matched normal breast tissue or peripheral blood from 30 patients were subjected to massively parallel sequencing targeting all exons of 273 genes, including the genes most frequently mutated in breast cancer and DNA repair-related genes. Single nucleotide variants and insertions and deletions were identified using state-of-the-art bioinformatics approaches. Results The constellation of somatic mutations found in LCIS (n = 34) and ILC (n = 21) were similar, with the most frequently mutated genes being CDH1 (56% and 66%, respectively), PIK3CA (41% and 52%, respectively) and CBFB (12% and 19%, respectively). Among 19 LCIS and ILC synchronous pairs, 14 (74%) had at least one identical mutation in common, including identical PIK3CA and CDH1 mutations. Paired analysis of independent foci of LCIS from 3 breasts revealed at least one common mutation in each of the 3 pairs ( CDH1 , PIK3CA , CBFB and PKHD1L1 ). Conclusion LCIS and ILC have a similar repertoire of somatic mutations, with PIK3CA and CDH1 being the most frequently mutated genes. The presence of identical mutations between LCIS–LCIS and LCIS–ILC pairs demonstrates that LCIS is a clonal neoplastic lesion, and provides additional evidence that at least some LCIS are non-obligate precursors of ILC.

Nature 546, 421–425 (2017); 10.1038/nature22360 In this Letter, author Alejandra Mendoza was incorrectly spelled Alejandra Medoza. The original Letter has been corrected online.

Lobular carcinoma in situ (LCIS) is both a risk indicator and non-obligate precursor of invasive lobular carcinoma (ILC). We sought to characterize the transcriptomic features of LCIS and ILC, with a focus on the identification of intrinsic molecular subtypes of LCIS and the changes involved in the progression from normal breast epithelium to LCIS and ILC.Fresh-frozen classic LCIS, classic ILC, and normal breast epithelium (N) from women undergoing prophylactic or therapeutic mastectomy were prospectively collected, laser-capture microdissected, and subjected to gene expression profiling using Affymetrix HG-U133A 2.0 microarrays.Unsupervised hierarchical clustering of 40 LCIS samples identified 2 clusters of LCIS distinguished by 6431 probe sets (p0.001). Genes identifying the clusters included proliferation genes and other genes related to cancer canonical pathways such as TGF beta signaling, p53 signaling, actin cytoskeleton, apoptosis and Wnt-Signaling pathway. A supervised analysis to identify differentially expressed genes (p0.001) between normal epithelium, LCIS, and ILC, using 23 patient-matched triplets of N, LCIS, and ILC, identified 169 candidate precursor genes, which likely play a role in LCIS progression, including PIK3R1, GOLM1, and GPR137B. These potential precursor genes map significantly more frequently to 1q and 16q, regions frequently targeted by gene copy number alterations in LCIS and ILC.Here we demonstrate that classic LCIS is a heterogeneous disease at the transcriptomic level and identify potential precursor genes in lobular carcinogenesis. Understanding the molecular heterogeneity of LCIS and the potential role of these potential precursor genes may help personalize the therapy of patients with LCIS.

Metaplastic breast carcinoma is a rare and aggressive histologic type of breast cancer, preferentially displaying a triple-negative phenotype. We sought to define the transcriptomic heterogeneity of metaplastic breast cancers on the basis of current gene expression microarray-based classifiers, and to determine whether these tumors display gene copy number profiles consistent with those of BRCA1-associated breast cancers. Twenty-eight consecutive triple-negative metaplastic breast carcinomas were reviewed, and the metaplastic component present in each frozen specimen was defined (ie, spindle cell, squamous, chondroid metaplasia). RNA and DNA extracted from frozen sections with tumor cell content >60% were subjected to gene expression (Illumina HumanHT-12 v4) and copy number profiling (Affymetrix SNP 6.0), respectively. Using the best practice PAM50/claudin-low microarray-based classifier, all metaplastic breast carcinomas with spindle cell metaplasia were of claudin-low subtype, whereas those with squamous or chondroid metaplasia were preferentially of basal-like subtype. Triple-negative breast cancer subtyping using a dedicated website (http://cbc.mc.vanderbilt.edu/tnbc/) revealed that all metaplastic breast carcinomas with chondroid metaplasia were of mesenchymal-like subtype, spindle cell carcinomas preferentially of unstable or mesenchymal stem-like subtype, and those with squamous metaplasia were of multiple subtypes. None of the cases was classified as immunomodulatory or luminal androgen receptor subtype. Integrative clustering, combining gene expression and gene copy number data, revealed that metaplastic breast carcinomas with spindle cell and chondroid metaplasia were preferentially classified as of integrative clusters 4 and 9, respectively, whereas those with squamous metaplasia were classified into six different clusters. Eight of the 26 metaplastic breast cancers subjected to SNP6 analysis were classified as BRCA1-like. The diversity of histologic features of metaplastic breast carcinomas is reflected at the transcriptomic level, and an association between molecular subtypes and histology was observed. BRCA1-like genomic profiles were found only in a subset (31%) of metaplastic breast cancers, and were not associated with a specific molecular or histologic subtype.

Introduction: Lobular carcinoma in situ (LCIS) is considered both a risk factor and non-obligate precursor of invasive breast cancer. We sought to determine the genomic landscape of LCIS and the mutational processes involved in the clonal evolution and progression from LCIS to ductal carcinoma in situ (DCIS) and invasive lobular carcinoma (ILC). Methods: Patients with a history of LCIS undergoing therapeutic or prophylactic mastectomy were prospectively enrolled in an IRB approved protocol. Frozen tissue blocks were collected, screened for lesions of interest (LCIS, DCIS, ILC, invasive ductal carcinomas (IDC)) and subjected to microdissection and DNA/RNA extraction. Matched germline DNA was available for all cases. Whole exome sequencing was performed on a HiSeq2000 and data were aligned to the reference human genome and processed using GATK. Single nucleotide variants (SNVs) and small insertions/deletions were identified using MuTect and Varscan, respectively. Purity and ploidy estimates were calculated using ABSOLUTE. Clonal frequencies were estimated using Pyclone and the clonal structure of each sample was reconstructed using SubcloneSeeker. Shannon index and Simpson index metrics were used to calculate heterogeneity levels. Mutational signatures were defined according to their mutational trinucleotide context, and the expression levels of APOBEC gene family members were assessed by quantitative reverse transcription (qRT)-PCR. Results: 30 LCIS, 10 ILCs, 7 DCIS and 5 IDCs from 15 patients qualified for data analysis. CDH1 was the most frequently mutated gene and found to be targeted by mutations in 26 LCIS samples (23 somatic, 3 germline). The repertoire of somatic mutations in LCIS was similar to that of luminal A breast cancers, with the exception of the significantly higher frequency of CDH1 mutations and the lower prevalence of TP53 mutations. ILCs were clonally related to at least one LCIS in 10 patients, and in 3/7 patients, DCIS was clonally related to at least one LCIS. Clonal composition analysis revealed that the presence of a minor clone(s) in LCIS, and the levels of intra-tumor genetic heterogeneity were significantly higher in LCIS clonally related with DCIS/ILC than in LCIS unrelated to DCIS/ILC. In two cases, a minor LCIS subclone constituted the major clone in the associated DCIS/ILC. A comparative analysis of the mutational signatures in the truncal and branch mutations of these cases revealed that whilst the truncal mutations displayed an aging signature, branch mutations were enriched for the APOBEC signature. qRT-PCR analysis demonstrated that cases displaying the APOBEC signature also harbored significantly higher levels of APOBEC3B expression than samples with the aging signature. Conclusions: LCIS displays intra-lesion genetic heterogeneity, and the progression from LCIS to DCIS or ILC may involve the selection of clones resulting from distinct mutational processes during clonal evolution. Our findings also suggest that cytodine deamination driven by the overexpression of APOBEC3B may drive the progression of LCIS to DCIS/ILC in a subset of cases. Citation Format: Reis-Filho JS, Schizas M, Piscuoglio S, Sakr RA, Ng CKY, Lim RS, Carniello JVS, Towers R, Martelotto L, Giri DD, de Andrade VP, Viale A, Solit DB, Weigelt B, King TA. Lobular carcinoma in situ displays intra-lesion genetic heterogeneity and its progression to invasive disease involves clonal selection and variations in mutational processes. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr S4-04.

BACKGROUND: Germline CDH1 mutations are responsible for the increased risk of both gastric cancer and invasive lobular breast cancer (ILC) in families with hereditary diffuse gastric cancer syndrome; yet germline CDH1 mutations in women with ILC without a family history (FH) of gastric cancer are rare. Lobular carcinoma in situ (LCIS) is both a risk factor and non-obligate precursor of ILC and recent data suggest that germline CDH1 mutations may be present in up to 8% of patients with bilateral LCIS +/- ILC; raising questions about the role of genetic testing in this context. The purpose of this study was to determine the frequency of germline CDH1 mutations in a large prospectively followed cohort of patients with pathologically confirmed bilateral LCIS. METHODS: Patients with a biopsy proven history of LCIS, entering surveillance or presenting for surgery (prophylactic or therapeutic mastectomy), between 2005 and 2013 were prospectively identified and enrolled in IRB approved protocols at Memorial Sloan-Kettering Cancer Center for the collection of tissue and/or germline DNA (IRB 01-135, 99-030). All biopsies were reviewed to confirm LCIS and mastectomy specimens were subject to extensive sampling of all quadrants. Cases with confirmed bilateral LCIS were chosen for the primary analysis. Cases where bilateral mastectomy tissue sampling confirmed only unilateral LCIS were included for comparison. Germline DNA was anonymized and analyzed for CDH1 mutations using targeted capture sequencing with baits for all exons of CDH1 on HiSeq2000. Germline single nucleotide variants were called using GATK HaplotypeCaller and insertions/deletions by Varscan and Scalpel. Mutations were manually inspected using the Integrative Genomics Viewer (IGV). Clinical data were abstracted prior to anonymization. RESULTS: Germline DNA was available for 114 patients; 78 underwent bilateral mastectomy for breast cancer (BC), 8 chose prophylactic mastectomy and 28 patients with biopsy proven bilateral LCIS were identified in surveillance. Following mastectomy, tissue sampling confirmed bilateral LCIS in 67/86 (78%) patients, and ruled out bilateral LCIS in 19 patients; yielding 95 patients with bilateral LCIS for the primary analysis. Median age at LCIS diagnosis for bilateral and unilateral cases respectively was 48yrs (range 36-70) and 44 yrs (range 38-63). One patient with bilateral LCIS also reported a FH of gastric cancer. Pathogenic germline CDH1 mutations (D72N (missense) and E35* (nonsense)) were identified in 2/95 (2%) patients with bilateral LCIS, one of whom also had invasive breast cancer (ILC). A germline CDH1 mutation was not identified in the patient with bilateral LCIS and a FH of gastric cancer, nor were CDH1 mutations identified among the 19 patients with unilateral LCIS. CONCLUSIONS: In this cohort of 95 patients with pathologically documented bilateral LCIS +/- BC, the overall frequency of CDH1 germline mutations was 2%; considerably lower than previously reported. To our knowledge this is the largest series to address this question and these findings do not support germline testing for CDH1 mutations in women with bilateral LCIS. Citation Format: Reyes SA, Sakr RA, Schizas M, Towers R, Park AY, Ng CKY, Weigelt B, Reis-Filho JS, King TA. Germline CDH1 mutations in lobular carcinoma in situ. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-06-02.

INTRODUCTION: Lobular carcinoma in situ (LCIS) is considered both a risk factor and non-obligate precursor of low-grade estrogen receptor-positive breast cancer. We sought to define the mutational repertoire, subclone complexity and heterogeneity of LCIS, and whether invasive lobular carcinomas (ILCs) would stem from specific subclones within a LCIS. METHODS: Patients with a history of LCIS undergoing therapeutic or prophylactic mastectomy were prospectively enrolled in an IRB approved protocol. Frozen tissue blocks were collected, screened for lesions of interest (LCIS, ductal carcinoma in situ (DCIS), ILC, invasive ductal carcinoma (IDC)) and subject to microdissection and DNA extraction. Matched germline DNA was available for all cases. Whole exome sequencing was performed on a HiSeq2000 (Illumina) and data were aligned to the reference human genome hg19 and processed using GATK. SNVs were called using MuTect, and indels were called using a combination of Varscan and Strelka. Purity and ploidy estimates were calculated by ABSOLUTE. Clonal frequencies were estimated using Pyclone. RESULTS: 30 LCIS, 10 ILCs, 6 IDCs and 7 DCIS from 15 patients qualified for data analysis, resulting in 18 LCIS-ILC pairs, 22 LCIS-LCIS pairs, 12 LCIS-DCIS pairs, and 14 LCIS-IDC pairs for comparison. 9/18 (50%) LCIS-ILC pairs and 8/22 (36%) LCIS-LCIS pairs were clonally related, supported by several shared mutations (median 18, range 7-81 for LCIS-ILC; median 14, range 5-22 for LCIS-LCIS). All related LCIS-ILC pairs and 6/8 related LCIS-LCIS pairs shared a pathogenic CDH1 mutation; 75% of related LCIS-ILC pairs also shared a PIK3CA hotspot mutation. 7/12 (58%) LCIS-DCIS pairs were found to be clonally related but the number of shared mutations was generally lower than that found in LCIS-ILC pairs (median 9, range 2-11). No evidence of a clonal relationship was found in any of the LCIS-IDC pairs tested. Clonal composition analysis revealed that samples of LCIS display intra-lesion genetic heterogeneity in the form of the presence of a minor clone in 70% of cases. In one case, the LCIS minor subclone (∼15%) constituted the major clone in the ILC and in another case the LCIS minor subclone constituted the major clone in the associated DCIS. The majority of the clonally related lesions were located in the same quadrant of the breast, however evidence of clonality was found in 5 LCIS-LCIS and 3 LCIS-DCIS pairs located in separate quadrants of the breast. CONCLUSIONS: Intra-lesion genetic heterogeneity is a common phenomenon in LCIS. The dominant clone of a LCIS may not always be the clone directly involved in the progression to malignancy. The spatial relationships of clonally related lesions in this study suggest that anatomy does not always infer clonality, as lesions located in separate quadrants of the breast may be clonally related. Citation Format: Michail Schizas, Rita A. Sakr, Britta Weigelt, Charlotte KY Ng, Jose Victor S. Carniello, Dilip Giri, Salvatore Piscuoglio, Luciano G. Martelotto, Russell Towers, Victor P. Andrade, Raymond Lim, David B. Solit, Jorge S. Reis-Filho, Tari A. King. Whole exome sequencing reveals heterogeneity within lobular carcinoma in situ (LCIS) and clonal selection in the progression to malignant lesions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2971. doi:10.1158/1538-7445.AM2015-2971

Correction to: Modern Pathology advance online publication, 21 November 2014; doi:10.1038/modpathol.2014.142 The title of this paper is incorrect due to a post-acceptance error. The correct title is ‘Metaplastic breast carcinomas display genomic and transcriptomic heterogeneity.’ The authors and editors apologize for the confusion this error has caused.