Contralateral breast cancers: Independent cancers or metastases?

A cancer in the contralateral breast in a woman with a previous or synchronous breast cancer is typically considered to be an independent primary tumor. Emerging evidence suggests that in a small subset of these cases the second tumor represents a metastasis. We sought to investigate the issue using massively parallel sequencing targeting 254 genes recurrently mutated in breast cancer. We examined the tumor archives at Memorial Sloan Kettering Cancer Center for the period 1995-2006 to identify cases of contralateral breast cancer where surgery for both tumors was performed at the Center. We report results from 49 patients successfully analyzed by a targeted massively parallel sequencing assay. Somatic mutations and copy number alterations were defined by state-of-the-art algorithms. Clonal relatedness was evaluated by statistical tests specifically designed for this purpose. We found evidence that the tumors in contralateral breasts were clonally related in three cases (6%) on the basis of matching mutations at codons where somatic mutations are rare. Clinical data and the presence of similar patterns of gene copy number alterations were consistent with metastasis for all three cases. In three additional cases, there was a solitary matching mutation at a common PIK3CA locus. The results suggest that a subset of contralateral breast cancers represent metastases rather than independent primary tumors. Massively parallel sequencing analysis can provide important evidence to clarify the diagnosis. However, given the inter-tumor mutational heterogeneity in breast cancer, sufficiently large gene panels need to be employed to define clonality convincingly in all cases.

Bi-allelic alterations in DNA repair genes underpin homologous recombination DNA repair defects in breast cancer.

Homologous recombination (HR) DNA repair-deficient (HRD) breast cancers have been shown to be sensitive to DNA repair targeted therapies. Burgeoning evidence suggests that sporadic breast cancers, lacking germline BRCA1/BRCA2 mutations, may also be HRD. We developed a functional ex vivo RAD51-based test to identify HRD primary breast cancers. An integrated approach examining methylation, gene expression, and whole-exome sequencing was employed to ascertain the aetiology of HRD. Functional HRD breast cancers displayed genomic features of lack of competent HR, including large-scale state transitions and specific mutational signatures. Somatic and/or germline genetic alterations resulting in bi-allelic loss-of-function of HR genes underpinned functional HRD in 89% of cases, and were observed in only one of the 15 HR-proficient samples tested. These findings indicate the importance of a comprehensive genetic assessment of bi-allelic alterations in the HR pathway to deliver a precision medicine-based approach to select patients for therapies targeting tumour-specific DNA repair defects. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Pleomorphic lobular carcinoma in situ of the breast: a single institution experience with clinical follow-up and centralized pathology review.

PURPOSE: The natural history of pleomorphic lobular carcinoma in situ (PLCIS) remains largely unknown. METHODS: A pathology database search (1995-2012) was performed to identify patients diagnosed with an LCIS variant. Patients with synchronous breast cancer and/or no evidence of pleomorphism were excluded. Original slides were re-evaluated by three pathologists to identify a consensus cohort of PLCIS. Borderline lesions with focal atypia were classified as LCIS with pleomorphic features (LCIS-PF). Clinical data were obtained from medical records. RESULTS: From 233 patients, we identified 32 with an LCIS variant diagnosis and no concurrent breast cancer. Following review, 16 cases were excluded due to lack of pleomorphism. The remaining 16 were classified as PLCIS (n = 11) and LCIS-PF (n = 5). 12/16 patients were treated with surgical excision ± chemoprevention. Patients with a prior breast cancer history and those having mastectomy were excluded from outcome analysis. Among the remaining 7 patients with PLCIS/LCIS-PF, 4/7 (57%) developed ipsilateral breast cancer at a median follow-up of 67 months. Median age at the time of breast cancer diagnosis was 56 years old and median time from PLCIS/LCIS-PF to cancer diagnosis was 59 months (range 45-66 months). The four cancers included 1 invasive lobular carcinoma (ILC), 1 microinvasive ILC, 1 invasive ductal carcinoma, and 1 ductal carcinoma in situ. CONCLUSIONS: We confirm that PLCIS in isolation is indeed a rare entity, further contributing to the difficulty in determining the actual risk conferred by this lesion. Long-term follow-up data on larger cohorts are needed to define standardized management and outcomes for patients with PLCIS.

Microglandular adenosis associated with triple-negative breast cancer is a neoplastic lesion of triple-negative phenotype harbouring TP53 somatic mutations.

Microglandular adenosis (MGA) is a rare proliferative lesion of the breast composed of small glands lacking myoepithelial cells and lined by S100-positive, oestrogen receptor (ER)-negative, progesterone receptor (PR)-negative, and HER2-negative epithelial cells. There is evidence to suggest that MGA may constitute a non-obligate precursor of triple-negative breast cancer (TNBC). We sought to define the genomic landscape of pure MGA and of MGA, atypical MGA (AMGA) and associated TNBCs, and to determine whether synchronous MGA, AMGA, and TNBCs would be clonally related. Two pure MGAs and eight cases of MGA and/or AMGA associated with in situ or invasive TNBC were collected, microdissected, and subjected to massively parallel sequencing targeting all coding regions of 236 genes recurrently mutated in breast cancer or related to DNA repair. Pure MGAs lacked clonal non-synonymous somatic mutations and displayed limited copy number alterations (CNAs); conversely, all MGAs (n = 7) and AMGAs (n = 3) associated with TNBC harboured at least one somatic non-synonymous mutation (range 3-14 and 1-10, respectively). In all cases where TNBCs were analyzed, identical TP53 mutations and similar patterns of gene CNAs were found in the MGA and/or AMGA and in the associated TNBC. In the MGA/AMGA associated with TNBC lacking TP53 mutations, somatic mutations affecting PI3K pathway-related genes (eg PTEN, PIK3CA, and INPP4B) and tyrosine kinase receptor signalling-related genes (eg ERBB3 and FGFR2) were identified. At diagnosis, MGAs associated with TNBC were found to display subclonal populations, and clonal shifts in the progression from MGA to AMGA and/or to TNBC were observed. Our results demonstrate the heterogeneity of MGAs, and that MGAs associated with TNBC, but not necessarily pure MGAs, are genetically advanced, clonal, and neoplastic lesions harbouring recurrent mutations in TP53 and/or other cancer genes, supporting the notion that a subset of MGAs and AMGAs may constitute non-obligate precursors of TNBCs.

Clonal relationships between lobular carcinoma in situ and other breast malignancies.

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.

Infiltrating epitheliosis of the breast: characterization of histological features, immunophenotype and genomic profile.

AIMS: Infiltrating epitheliosis is a rare complex sclerosing lesion (CSL) of the breast, characterized by infiltrating ducts immersed in a scleroelastotic stroma and filled with cells having architectural and cytological patterns reminiscent of those of usual ductal hyperplasia. In this study we sought to define the molecular characteristics of infiltrating epitheliosis. METHODS AND RESULTS: Eight infiltrating epitheliosis, adjacent breast lesions (one usual ductal hyperplasia, one papilloma, one micropapillary ductal carcinoma in situ and one low-grade adenosquamous carcinoma), and corresponding normal breast tissue from each case were microdissected and subjected to massively parallel sequencing analysis targeting all coding regions of 254 genes mutated recurrently in breast cancer and/or related to DNA repair. Mutations in components of the PI3K pathway were found in all infiltrating epitheliosis samples, seven of which harboured PIK3CA hotspot mutations, while the remaining case displayed a PIK3R1 somatic mutation. CONCLUSIONS: Somatic mutations affecting PI3K pathway genes were found to be highly prevalent in infiltrating epitheliosis, suggesting that these lesions may be neoplastic rather than hyperplastic. The landscape of somatic genetic alterations found in infiltrating epitheliosis is similar to that of radial scars/CSLs, suggesting that infiltrating epitheliosis may represent one end of this spectrum of lesions.

Evaluating Magnetic Seed Localization in Targeted Axillary Dissection for Node-Positive Early Breast Cancer Patients Receiving Neoadjuvant Systemic Therapy: A Comprehensive Review and Pooled Analysis.

Background/Objectives: De-escalation of axillary surgery is made possible by advancements in both neoadjuvant systemic therapy (NST) and in localisation technology for breast lesions. Magseed®, developed in 2013 by Dr. Michael Douk of Cambridge, United Kingdom, is a wire-free localisation technology that facilitates the localisation and retrieval of lymph nodes for staging. Targeted axillary dissection (TAD), which entails marked lymph node biopsy (MLNB) and sentinel lymph node biopsy (SLNB), has emerged as the preferred method to assess residual disease in post-NST node-positive patients. This systematic review and pooled analysis evaluate the performance of Magseed® in TAD. Methods: The search was carried out in PubMed and Google Scholar. An assessment of localisation, retrieval rates, concordance between MLNB and SLNB, and pathological complete response (pCR) in clinically node-positive patients post NST was undertaken. Results: Nine studies spanning 494 patients and 497 procedures were identified, with a 100% successful deployment rate, a 94.2% (468/497) [95% confidence interval (CI), 93.7-94.7] localisation rate, a 98.8% (491/497) retrieval rate, and a 68.8% (247/359) [95% CI 65.6-72.0] concordance rate. pCR was observed in 47.9% (220/459) ) [95% CI 43.3-52.6] of cases. Subgroup analysis of studies reporting the pathological status of MLNB and SLNB separately revealed an FNR of 4.2% for MLNB and 17.6% for SLNB (p = 0.0013). Mean duration of implantation was 37 days (range: 0-188). Conclusions: These findings highlight magnetic seed localisation's efficacy in TAD for NST-treated node-positive patients, aiding in accurate axillary pCR identification and safe de-escalation of axillary surgery in excellent responders.

Is there a role for routine screening MRI in women with LCIS?

Women with lobular carcinoma in situ (LCIS) have an elevated breast cancer risk, yet the benefit of MRI screening is unclear. We examined cancer detection rates with mammography alone versus mammography plus MRI in this high-risk population. From a prospectively maintained, single-institution database, we identified 776 patients diagnosed with LCIS after the adoption of screening MRI in April 1999. In addition to annual mammography and breast exam, MRI was used at the discretion of the physician and patient. Kaplan-Meier methods and landmark analyses at 1, 2, and 3 years following LCIS diagnosis were performed to compare rates of cancer detection with or without MRI. MRI screening was performed in 455 (59 %) patients (median, 3/patient). Median time from LCIS diagnosis to first MRI was 9 months (range 0.3-137 months). Patients undergoing MRI were younger (p < 0.0001), premenopausal (p < 0.0001), and more likely to have ≥1 first-degree relative with breast cancer (p = 0.009). At a median follow-up of 58 months, 98/776 (13 %) patients developed cancer. The crude cancer detection rate in both screening groups was 13 %. MRI was not associated with earlier stage, smaller size, or node negativity. Landmark analyses at 1, 2, and 3 years after LCIS diagnosis failed to demonstrate increased cancer detection rates among women having MRI (p = 0.23, 0.26, and 0.13, respectively). Although a diagnosis of LCIS remains a significant risk factor for breast cancer, the routine use of MRI does not result in increased cancer detection rates (short-term), nor does it result in earlier stage at diagnosis, illustrating the importance of defining optimal screening strategies for high-risk patients based on tumor biology rather than numerical risk.

Cadherin-catenin complex dissociation in lobular neoplasia of the breast.

E-cadherin (E-CD) inactivation with loss of E-CD-mediated cell adhesion is the hallmark of lesions of the lobular phenotype. E-CD is typically absent by immunohistochemistry in both lobular carcinoma in situ (LCIS) and invasive lobular lesions, suggesting it occurs early in the neoplastic process. In laboratory models, downstream post-transcriptional modifiers such as TWIST and SNAIL contribute to the dissociation of the intracellular component of the cadherin-catenin complex (CCC), resulting in tumor progression and invasion. We hypothesized that complete CCC dissociation may play a role in lobular neoplasia progression. Here we explore the relationship between loss of E-CD and dissociation of the CCC in pure LCIS and LCIS associated with invasive cancer. Fresh-frozen tissues were obtained from 36 patients undergoing mastectomy for pure LCIS (n = 11), LCIS with ILC (n = 18) or LCIS with IDC (n = 7). Individual lesions were subject to laser-capture microdissection and gene-expression analysis (Affymetrix HG-U133A 2.0). Immunohistochemistry for ER,PR,HER2, E-CD,N-CD,α-,ß-, and phosphoß-catenin, TWIST, and SNAIL were evaluated in normal, in situ, and invasive components from matched formalin-fixed paraffin-embedded samples (n = 36). CCC-dissociation was defined as negative membranous E-CD, α- and ß-catenin expression. E-CD was negative in all LCIS and ILC lesions, and positive in all normal and IDC lesions. Membranous α and ß-catenin expressions decreased with the transition from LCIS to ILC (pure LCIS 82%; LCIS w/ILC 28%; ILC 0%), while TWIST expression increased (pure LCIS low; LCIS w/ILC moderate; ILC high). Gene expression paralleled IHC-staining patterns with a stepwise downregulation of E-CD, α and ß-catenins from normal to LCIS to invasive lesions, and increasing expression of TWIST from normal to LCIS to ILC. Loss of E-CD expression is an early event in lobular neoplasia. Decreasing membranous catenin expression in tandem with increasing levels of TWIST across the spectrum of lobular lesions suggests that CCC dissociation is a progressive process.

Is there a low-grade precursor pathway in breast cancer?

BACKGROUND: Newly proposed models of breast tumorigenesis suggest that low- and high-grade lesions have distinct tumor progression pathways. Our objective was to examine the relationship between histologic grade and molecular subtype in women with lobular carcinoma in situ (LCIS) and ductal carcinoma in situ (DCIS) who developed subsequent ipsilateral invasive breast cancers. METHODS: Patients who underwent surveillance for classical LCIS (1994-2007) and those followed after lumpectomy±radiation for DCIS (1991-2004) who developed subsequent ipsilateral invasive cancers and had available tissue blocks were included. ER/PR/HER2 surrogates were used for molecular subtype. RESULTS: Material was available for 27 patients with classical LCIS who developed ipsilateral invasive cancer (12 invasive ductal cancer [IDC], 14 invasive lobular, 1 mixed), and 26 patients with DCIS (12 low-grade [LG], 14 high-grade [HG]) who developed ipsilateral IDC. No difference in age at diagnosis or median time to invasive cancer existed between groups with LCIS and DCIS. When stratified by grade, 0 of 12 LG-DCIS developed LG-IDC (3 grade II; 9 grade III), and only 1 of 12 LCIS patients who developed IDC had LG-IDC. Thirteen (93%) patients with HG-DCIS developed HG-IDC. In contrast, molecular subtype was maintained in 23 of 27 (85%) cases of LCIS and in 18 of 26 (69%) cases of DCIS. CONCLUSIONS: These data do not support a low-grade precursor pathway characterized by LCIS and LG-DCIS. ER/PR and HER2 status have a high rate of concordance between in situ and subsequent invasive lesions. Additional studies of metachronous in situ and invasive lesions are needed to better understand pathways of breast tumorigenesis.

Lobular Carcinomas In Situ Display Intralesion Genetic Heterogeneity and Clonal Evolution in the Progression to Invasive Lobular Carcinoma.

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.

Biomarkers That Predict Sensitivity to Heat Shock Protein 90 Inhibitors.

INTRODUCTION: Heat shock protein (HSP) 90, a viable target for cancer treatment, mediates the maturation and stabilization of client oncoproteins. HSP90 inhibitors (HSP90i) are potentially active in a variety of tumors, but therapeutic benefit is confirmed in only a small subset. We explored potential biomarkers across multiple studies of HSP90i in advanced solid tumors. PATIENTS AND METHODS: Archived tumor specimens from patients treated with HSP90i in 7 different phase I/II trials at Memorial Sloan Kettering Cancer Center were identified. Tumor tissue was tested using immunohistochemistry; estrogen, progesterone, and androgen receptors ≥ 1% positive and < 1% negative; HSP90 and HSP70: 0, 1 + negative, and 2+, 3 + positive; phosphatase and tensin homolog: 0 negative, 1 reduced, and 2 positive; HER2: 0, 1 + negative, 2 + equivocal, 3 + positive; and epidermal growth factor receptor: 0 negative, and 1+, 2+, 3 + positive. The expression of the biomarker panel was correlated with clinical benefit (CB) (defined by overall response [ORR] or CB by the "8-week" scan) using Fisher exact test. RESULTS: Adequate tissue was available for 51 of 158 patients (32%), including 10 different solid tumors. Of these, 71% (36 of 51) and 51% (26 of 51) patients met the criteria to assess CB by best ORR or by the "8-week scan" assessment, respectively. Breast was the most frequent tumor. The mean duration of HSP90i therapy was 55 days (range, 16-411 days). There were 16 responses (4 partial response; 12 stable disease); 13 of 16 responses strongly correlated with HER2-positive status (P = .001). CONCLUSION: Our findings suggest HER2 as a sensitive client and perhaps the only effective biomarker for sensitivity to these HSP90i.

Targeted capture massively parallel sequencing analysis of LCIS and invasive lobular cancer: Repertoire of somatic genetic alterations and clonal relationships.

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.

The Genomic Landscape of Male Breast Cancers.

PURPOSE: Male breast cancer is rare, and its genomic landscape has yet to be fully characterized. Lacking studies in men, treatment of males with breast cancer is extrapolated from results in females with breast cancer. We sought to define whether male breast cancers harbor somatic genetic alterations in genes frequently altered in female breast cancers. EXPERIMENTAL DESIGN: All male breast cancers were estrogen receptor-positive, and all but two were HER2-negative. Fifty-nine male breast cancers were subtyped by immunohistochemistry, and tumor-normal pairs were microdissected and subjected to massively parallel sequencing targeting all exons of 241 genes frequently mutated in female breast cancers or DNA-repair related. The repertoires of somatic mutations and copy number alterations of male breast cancers were compared with that of subtype-matched female breast cancers. RESULTS: Twenty-nine percent and 71% of male breast cancers were immunohistochemically classified as luminal A-like or luminal B-like, respectively. Male breast cancers displayed a heterogeneous repertoire of somatic genetic alterations that to some extent recapitulated that of estrogen receptor (ER)-positive/HER2-negative female breast cancers, including recurrent mutations affecting PIK3CA (20%) and GATA3 (15%). ER-positive/HER2-negative male breast cancers, however, less frequently harbored 16q losses, and PIK3CA and TP53 mutations than ER-positive/HER2-negative female breast cancers. In addition, male breast cancers were found to be significantly enriched for mutations affecting DNA repair-related genes. CONCLUSIONS: Male breast cancers less frequently harbor somatic genetic alterations typical of ER-positive/HER2-negative female breast cancers, such as PIK3CA and TP53 mutations and losses of 16q, suggesting that at least a subset of male breast cancers are driven by a distinct repertoire of somatic changes. Given the genomic differences, caution may be needed in the application of biologic and therapeutic findings from studies of female breast cancers to male breast cancers. Clin Cancer Res; 22(16); 4045-56. ©2016 AACR.

Lobular Carcinoma in Situ: A 29-Year Longitudinal Experience Evaluating Clinicopathologic Features and Breast Cancer Risk.

PURPOSE: The increased breast cancer risk conferred by a diagnosis of lobular carcinoma in situ (LCIS) is poorly understood. Here, we review our 29-year longitudinal experience with LCIS to evaluate factors associated with breast cancer risk. PATIENTS AND METHODS: Patients participating in surveillance after an LCIS diagnosis are observed in a prospectively maintained database. Comparisons were made among women choosing surveillance, with or without chemoprevention, and those undergoing bilateral prophylactic mastectomies between 1980 and 2009. RESULTS: One thousand sixty patients with LCIS without concurrent breast cancer were identified. Median age at LCIS diagnosis was 50 years (range, 27 to 83 years). Fifty-six patients (5%) underwent bilateral prophylactic mastectomy; 1,004 chose surveillance with (n = 173) or without (n = 831) chemoprevention. At a median follow-up of 81 months (range, 6 to 368 months), 150 patients developed 168 breast cancers (63% ipsilateral, 25% contralateral, 12% bilateral), with no dominant histology (ductal carcinoma in situ, 35%; infiltrating ductal carcinoma, 29%; infiltrating lobular carcinoma, 27%; other, 9%). Breast cancer incidence was significantly reduced in women taking chemoprevention (10-year cumulative risk: 7% with chemoprevention; 21% with no chemoprevention; P < .001). In multivariable analysis, chemoprevention was the only clinical factor associated with breast cancer risk (hazard ratio, 0.27; 95% CI, 0.15 to 0.50). In a subgroup nested case-control analysis, volume of disease, which was defined as the ratio of slides with LCIS to total number of slides reviewed, was also associated with breast cancer development (P = .008). CONCLUSION: We observed a 2% annual incidence of breast cancer among women with LCIS. Common clinical factors used for risk prediction, including age and family history, were not associated with breast cancer risk. The lower breast cancer incidence in women opting for chemoprevention highlights the potential for risk reduction in this population.

In situ single-cell analysis identifies heterogeneity for PIK3CA mutation and HER2 amplification in HER2-positive breast cancer.

Detection of minor, genetically distinct subpopulations within tumors is a key challenge in cancer genomics. Here we report STAR-FISH (specific-to-allele PCR-FISH), a novel method for the combined detection of single-nucleotide and copy number alterations in single cells in intact archived tissues. Using this method, we assessed the clinical impact of changes in the frequency and topology of PIK3CA mutation and HER2 (ERBB2) amplification within HER2-positive breast cancer during neoadjuvant therapy. We found that these two genetic events are not always present in the same cells. Chemotherapy selects for PIK3CA-mutant cells, a minor subpopulation in nearly all treatment-naive samples, and modulates genetic diversity within tumors. Treatment-associated changes in the spatial distribution of cellular genetic diversity correlated with poor long-term outcome following adjuvant therapy with trastuzumab. Our findings support the use of in situ single cell-based methods in cancer genomics and imply that chemotherapy before HER2-targeted therapy may promote treatment resistance.

Gene expression profiling of lobular carcinoma in situ reveals candidate precursor genes for invasion.

PURPOSE: 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. METHODS: 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. RESULTS: Unsupervised hierarchical clustering of 40 LCIS samples identified 2 clusters of LCIS distinguished by 6431 probe sets (p < 0.001). 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 (p < 0.001) 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. CONCLUSION: 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.

PI3K pathway activation in high-grade ductal carcinoma in situ--implications for progression to invasive breast carcinoma.

PURPOSE: To assess the prevalence of phosphoinositide 3-kinase (PI3K) pathway alterations in pure high-grade ductal carcinoma in situ (DCIS) and DCIS associated with invasive breast cancer (IBC), and to determine whether DCIS and adjacent IBCs harbor distinct PI3K pathway aberrations. EXPERIMENTAL DESIGN: Eighty-nine cases of pure high-grade DCIS and 119 cases of high-grade DCIS associated with IBC were characterized according to estrogen receptor (ER) and HER2 status, subjected to immunohistochemical analysis of PTEN, INPP4B, phosphorylated (p)AKT and pS6 expression, and to microdissection followed by Sequenom genotyping of PIK3CA and AKT1 hotspot mutations. RESULTS: Alterations affecting the PI3K pathway were found in a subset of pure DCIS and DCIS adjacent to IBC. A subtype-matched comparison of pure DCIS and DCIS adjacent to IBC revealed that PIK3CA hotspot mutations and pAKT expression were significantly more prevalent in ER-positive/HER2-negative DCIS adjacent to IBC (P values, 0.005 and 0.043, respectively), and that in ER-negative/HER2-positive cases INPP4B loss of expression was more frequently observed in pure DCIS (a P value of 0.013). No differences in the parameters analyzed were observed in a pairwise comparison of the in situ and invasive components of cases of DCIS and adjacent IBC. Analysis of the PIK3CA-mutant allelic frequencies in DCIS and synchronous IBC revealed cases in which PIK3CA mutations were either restricted to the DCIS or to the invasive components. CONCLUSION: Molecular aberrations affecting the PI3K pathway may play a role in the progression from high-grade DCIS to IBC in a subset of cases (e.g., a subgroup of ER-positive/HER2-negative lesions).

Hotspot activating PRKD1 somatic mutations in polymorphous low-grade adenocarcinomas of the salivary glands.

Polymorphous low-grade adenocarcinoma (PLGA) is the second most frequent type of malignant tumor of the minor salivary glands. We identified PRKD1 hotspot mutations encoding p.Glu710Asp in 72.9% of PLGAs but not in other salivary gland tumors. Functional studies demonstrated that this kinase-activating alteration likely constitutes a driver of PLGA.

Progression from ductal carcinoma in situ to invasive breast cancer: revisited.

Ductal carcinoma in situ (DCIS) is an intraductal neoplastic proliferation of epithelial cells that is separated from the breast stroma by an intact layer of basement membrane and myoepithelial cells. DCIS is a non-obligate precursor of invasive breast cancer, and up to 40% of these lesions progress to invasive disease if untreated. Currently, it is not possible to predict accurately which DCIS would be more likely to progress to invasive breast cancer as neither the significant drivers of the invasive transition have been identified, nor has the clinical utility of tests predicting the likelihood of progression been demonstrated. Although molecular studies have shown that qualitatively, synchronous DCIS and invasive breast cancers are remarkably similar, there is burgeoning evidence to demonstrate that intra-tumor genetic heterogeneity is observed in a subset of DCIS, and that the process of progression to invasive disease may constitute an 'evolutionary bottleneck', resulting in the selection of subsets of tumor cells with specific genetic and/or epigenetic aberrations. Here we review the clinical challenge posed by DCIS, the contribution of the microenvironment and genetic aberrations to the progression from in situ to invasive breast cancer, the emerging evidence of the impact of intra-tumor genetic heterogeneity on this process, and strategies to combat this heterogeneity.