Non-invasive lobular neoplasia of the breast: Morphologic features, clinical presentation, and management dilemmas.

The designation of noninvasive lobular neoplasia applies to atypical epithelial proliferations composed of noncohesive cells secondary to loss or functional alteration of E-cadherin-mediated cell adhesion. The morphologic spectrum of noninvasive lobular neoplasia encompasses atypical lobular hyperplasia (ALH) and classic lobular carcinoma in situ (classic LCIS) and two LCIS variants, namely florid LCIS (F-LCIS) and pleomorphic LCIS (P-LCIS), as defined in the World Health Organization (WHO) Classification of Tumors of the Breast 5th ed. Herein, we review the morphologic, immunohistochemical, and molecular features of noninvasive lobular neoplasia, with special emphasis on F-LCIS and P-LCIS. We also review imaging features, management at core needle biopsy, upgrade rates at surgical excision, and clinical management dilemmas.

Apocrine Variant of Pleomorphic Lobular Carcinoma In Situ: Further Clinical, Histopathologic, Immunohistochemical, and Molecular Characterization of an Emerging Entity.

To date, the apocrine variant of lobular carcinoma in situ (AP-LCIS) has been cursorily described as a subtype of lobular carcinoma in situ (LCIS). We retrospectively reviewed 34 cases of AP-LCIS (including 23 associated with invasive lobular carcinoma) to fully characterize it. AP-LCIS typically presented with screen-detected calcifications in older women (mean age: 65 y) and was characterized by distended terminal duct lobular units with relatively large "pleomorphic" cells, central necrosis, and calcifications. AP-LCIS cells exhibited abundant eosinophilic occasionally granular cytoplasm, hyperchromatic nuclei, and prominent nucleoli. Synchronous classic and/or florid LCIS was identified in 24/34 (70%) AP-LCIS, and in 9/11 (82%) pure AP-LCIS. Most (68%) cases of AP-LCIS were estrogen receptor-positive (50% strongly), 35% were progesterone receptor-positive, 26% were human epidermal growth factor 2-positive, 18% demonstrated high-proliferation rate (Ki67: >15%), and 90% were androgen receptor-positive. Aurora kinase A, immunoreactive in 38% of AP-LCIS cases, was not significantly associated with recurrence, development of invasion, or nodal positivity (P>0.05). Compared with conventional (nonapocrine) pleomorphic lobular carcinoma in situ (P-LCIS), aurora kinase A was expressed in a significantly greater proportion of P-LCIS (100%). AP-LCIS and P-LCIS did not otherwise differ in clinicopathologic features. Next-generation sequencing utilizing the Oncomine Comprehensive Panel v2, performed on 27 AP-LCIS cases, showed no specific molecular findings. In a mean follow-up of 57 months, 2 (of 11, 18%) pure AP-LCIS cases recurred (2 both in situ and invasive) and none metastasized or proved fatal. AP-LCIS should be regarded as another high-grade LCIS similar to P-LCIS in many respects, and pending additional studies should be managed similarly.

Genomic profiling of pleomorphic and florid lobular carcinoma in situ reveals highly recurrent ERBB2 and ERRB3 alterations.

Pleomorphic LCIS (P-LCIS) and florid LCIS (F-LCIS) are morphologic variants distinguished from classic LCIS by marked nuclear pleomorphism and/or an expansile growth pattern with or without necrosis. Given the rarity of these LCIS variants, little data exist regarding their molecular pathogenesis, natural history, and optimal management. The purpose of this study was to genomically profile LCIS variants to gain further insight into their biology. Nineteen cases of pure LCIS variants (17 P-LCIS, 2 F-LCIS) diagnosed on core needle biopsy at our institution from 2006 to 2017 were included, five of which were upgraded to invasive cancer at excision. Macrodissected lesions were analyzed by a hybrid-capture next generation sequencing assay that surveyed exonic sequences of 447 genes for mutations and copy number variations (CNVs) and 191 regions across 60 genes for structural rearrangements. LCIS variants were all confirmed as E-cadherin negative by immunohistochemistry. Receptor profiles among the 17 P-LCIS cases included HR+/HER2- (nine cases), HR+/HER2+ (three cases), HR-/HER2+ (two cases), and HR-/HER2- (three cases). The two F-LCIS cases were HR+/HER2- and HR+/HER2+. All LCIS variants had genetic alterations consistent with a lobular phenotype including 1q gain (16 cases), 16q loss (18 cases), and CDH1 mutations (18 cases). Highly recurrent ERBB2 alterations were noted including mutations (13 cases) and amplifications (six cases). Other significant alterations included mutations in PIK3CA (six cases), RUNX1 (four cases), ERBB3 (four cases), and CBFB (three cases), as well as amplification of CCND1 (five cases). A TP53 mutation was identified in one case of HR-/HER2+ P-LCIS with signet ring cell features that lacked 1q gain and 16q loss. P-LCIS and F-LCIS contain genetic alterations characteristic of lobular neoplasia; however, these LCIS variants are distinguished from classical LCIS reported in the literature by their highly recurrent ERBB2 alterations.

Synergistic association of FOXP3+ tumor infiltrating lymphocytes with CCL20 expressions with poor prognosis of primary breast cancer: A retrospective cohort study.

Studies have shown that forkhead/winged helix transcription factor P3 (FOXP3) tumor infiltrating lymphocytes (TILs) are intimately associated with invasion and survival of many invasive tumors. The inflammatory chemokine ligand 20 (CCL20) and its receptor CCR6 were found to be associated with tumor prognosis in some studies. Although increases in FOXP3 TILs infiltration and CCL20 expression have been revealed in several malignancies, their correlation in human breast tumors is as yet unclear.Surgically resected samples from 156 patients with invasive breast cancer (BC) were assessed for the expression of FOXP3 and CCL20 by immunohistochemistry. Correlation between their expressions and the association with clinicopathological characteristics and patient's prognosis were studied. Forty pairs of fresh BC and their nontumor adjacent tissues (NATs) in BC were carried out by real-time quantitative PCR (qRT-PCR) to evaluate the correlation between FOXP3 and CCL20 mRNA expression.CCL20 and FOXP3 TILs mRNA expression in tumor tissue demonstrated a high correlation (rs = 0.359, P < .001) in this cohort of breast cancer patients. Both elevated CCL20 expression and FOXP3 TILs infiltration were significantly correlated with high histological grade, positive human epidermal growth factor receptor-2 (HER2), high Ki67 index, and axillary lymph node metastases. Tumors with concomitant high expressions of both markers had the worst prognosis. Multivariate analysis showed that these 2 markers were independent predictors of overall survival. The patients with axillary lymph node metastases with the concomitant CCL20 high expression and increased FOXP3 TILs infiltration had the worst overall survival (OS) (P < .001), In lymph node-negative breast cancer patients, the status of CCL20 and FOXP3 was not related to OS (P = .22).The results suggest that CCL20 and FOXP3 TILs may have synergistic effects, and their upregulated expressions may lead to immune evasion in breast cancer. Combinatorial immunotherapeutic approaches aiming at blocking CCL20 and depleting FOXP3 might improve therapeutic efficacy in breast cancer patients.

Ki67 index in intrinsic breast cancer subtypes and its association with prognostic parameters.

OBJECTIVES: Ki67 is the most commonly used marker to evaluate proliferative index in breast cancer, however no cutoff values have been clearly defined for high ki67 index. Cancer management should be according to loco-regional profile; therefore, we aimed to determine ki67 index in 1951 cases of intrinsic breast cancer subtypes and its association with other prognostic parameters in our set up. RESULTS: Triple negative breast cancers showed highest ki67 index (mean 50.9 ± 23.7%) followed by Her2neu (mean 42.6 ± 21.6%) and luminal B cancers (mean 34.9 ± 20.05%). Metaplastic and medullary breast cancers significantly showed higher ki67 index as compared to ductal carcinoma, NOS. No significant association of ki67 index was noted with any of the histologic parameters in different subtypes of breast cancer expect for tumor grade. Although, ki67 index is a valuable biomarker in breast cancer, however no independent prognostic significance of ki67 could be established in our study.

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.

Clinicopathologic characterization of breast carcinomas in patients with non-BRCA germline mutations: results from a single institution's high-risk population.

As multigene panel testing for hereditary cancer syndromes becomes commonplace, germline mutations in genes other than BRCA1/2 are increasingly identified in breast cancer patients. While histopathologic features of BRCA-mutated breast cancers have been well-characterized, less is known about non-BRCA-related hereditary cancers. We herein investigate the clinicopathologic characteristics of breast cancers in women with non-BRCA germline mutations. Out of 612 women who underwent germline testing, 16 (2.6%) women with 18 cancers had mutations in non-BRCA genes: ATM, CHEK2, PALB2, TP53, BMPR1A, BRIP1, MUTYH, and RAD50. An additional 2 cancers were identified in a woman with a diagnosis of Bloom syndrome (BLM mutation) who was not germline tested. Average age at diagnosis was 50 (range: 27-77), and 65% had no personal cancer history. The majority (79%) of tumors were grade 1 to 2; 35% were either lobular or ductal with lobular features. Stromal responses varied from absent to desmoplastic to sclerotic; 69% of cases had an in situ component. With the exception of a brisk lymphocytic response in BLM- and TP53-mutated cancers, lymphocytic infiltration was mild or absent. In summary, the majority of non-BRCA-related hereditary breast cancers represent the patient's sentinel malignancy. Lobular features were seen in a subset, and high-grade, immunogenic carcinomas were uncommon except in the setting of BLM and TP53 mutations. Overall, these findings demonstrate a range of involved genes in non-BRCA mutation carriers with breast cancer and histopathologic heterogeneity in the associated cancers, arguing against use of histomorphology to inform panel testing algorithms.

Breast carcinoma in sclerosing adenosis: a clinicopathological and immunophenotypical analysis on 206 lesions.

AIMS: To fully elucidate the clinicopathological features of breast carcinoma in sclerosing adenosis (SA-BC). METHODS: Clinical and histological characteristics of 206 SA-BCs from 180 patients were retrospectively evaluated. Immunohistochemical phenotype was examined. The clinicopathological relevance of the topographical pattern of SA-BCs was analysed. RESULTS: Overall, up to 46 patients (25.6%) had contralateral cancer, either SA associated or not. Of 99 cases who underwent core needle biopsy (CNB), 36 were underestimated as adenosis or atypical ductal hyperplasia at CNB, 5 invasive cases were misinterpreted as in situ carcinomas, whereas 4 ductal carcinoma in situ (DCIS) cases were overdiagnosed as invasive carcinoma. Microscopically, 163 tumours were in situ, including 136 DCIS, 19 lobular carcinomas in situ (LCIS) and 8 mixed DCIS/LCIS; of these carcinomas in situ (CIS), 37 had microinvasion. The DCIS group exhibited low, intermediate and high grades in 53.7%, 34.6% and 11.8% of cases, respectively, mostly with solid (43.4%) or cribriform (41.9%) pattern. Forty out of 43 invasive cases were invasive ductal carcinoma (IDC), mostly DCIS predominant. Immunophenotypically, luminal A phenotype was identified in 55.1%, 63.2% and 45.0% of DCIS, LCIS and IDC cases, respectively. Topographical type A group (carcinoma being entirely confined to SA, n=176) was characterised by smaller size, less invasiveness, lower grade and more frequency of luminal A immunophenotype compared with type B group (≥ 50% but not all of the carcinomatous lesion being located in SA, n=30) (all P<0.05). CONCLUSIONS: CIS, especially non-high-grade DCIS, represents the most common variant of SA-BC, and luminal A is the most predominant immunophenotype. CNB assessment might be challenging in some SA-BCs. The topographical pattern has great clinicopathological relevance. Careful evaluation of the contralateral breast and long-term follow-up for patients with SA-BC is necessary given its high prevalence of bilaterality.

Mixed ductal-lobular carcinomas: evidence for progression from ductal to lobular morphology.

Mixed ductal-lobular carcinomas (MDLs) show both ductal and lobular morphology, and constitute an archetypal example of intratumoural morphological heterogeneity. The mechanisms underlying the coexistence of these different morphological entities are poorly understood, although theories include that these components either represent 'collision' of independent tumours or evolve from a common ancestor. We performed comprehensive clinicopathological analysis of a cohort of 82 MDLs, and found that: (1) MDLs more frequently coexist with ductal carcinoma in situ (DCIS) than with lobular carcinoma in situ (LCIS); (2) the E-cadherin-catenin complex was normal in the ductal component in 77.6% of tumours; and (3) in the lobular component, E-cadherin was almost always aberrantly located in the cytoplasm, in contrast to invasive lobular carcinoma (ILC), where E-cadherin is typically absent. Comparative genomic hybridization and multiregion whole exome sequencing of four representative cases revealed that all morphologically distinct components within an individual case were clonally related. The mutations identified varied between cases; those associated with a common clonal ancestry included BRCA2, TBX3, and TP53, whereas those associated with clonal divergence included CDH1 and ESR1. Together, these data support a model in which separate morphological components of MDLs arise from a common ancestor, and lobular morphology can arise via a ductal pathway of tumour progression. In MDLs that present with LCIS and DCIS, the clonal divergence probably occurs early, and is frequently associated with complete loss of E-cadherin expression, as in ILC, whereas, in the majority of MDLs, which present with DCIS but not LCIS, direct clonal divergence from the ductal to the lobular phenotype occurs late in tumour evolution, and is associated with aberrant expression of E-cadherin. The mechanisms driving the phenotypic change may involve E-cadherin-catenin complex deregulation, but are yet to be fully elucidated, as there is significant intertumoural heterogeneity, and each case may have a unique molecular mechanism. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

A comprehensive analysis of polymorphic variants in steroid hormone and insulin-like growth factor-1 metabolism and risk of in situ breast cancer: Results from the Breast and Prostate Cancer Cohort Consortium.

We assessed the association between 1,414 single nucleotide polymorphisms (SNPs) in genes involved in synthesis and metabolism of steroid hormones and insulin-like growth factor 1, and risk of breast cancer in situ (BCIS), with the aim of determining whether any of these were disease specific. This was carried out using 1,062 BCIS cases and 10,126 controls as well as 6,113 invasive breast cancer cases from the Breast and Prostate Cancer Cohort Consortium (BPC3). Three SNPs showed at least one nominally significant association in homozygous minor versus homozygous major models. ACVR2A-rs2382112 (ORhom = 3.05, 95%CI = 1.72-5.44, Phom = 1.47 × 10-4 ), MAST2-rs12124649 (ORhom = 1.73, 95% CI =1.18-2.54, Phom = 5.24 × 10-3 ), and INSR-rs10500204 (ORhom = 1.96, 95% CI = 1.44-2.67, Phom =1.68 × 10-5 ) were associated with increased risk of BCIS; however, only the latter association was significant after correcting for multiple testing. Furthermore, INSR-rs10500204 was more strongly associated with the risk of BCIS than invasive disease in case-only analyses using the homozygous minor versus homozygous major model (ORhom = 1.78, 95% CI = 1.30-2.44, Phom = 3.23 × 10-4 ). The SNP INSR-rs10500204 is located in an intron of the INSR gene and is likely to affect binding of the promyelocytic leukemia (PML) protein. The PML gene is known as a tumor suppressor and growth regulator in cancer. However, it is not clear on what pathway the A-allele of rs10500204 could operate to influence the binding of the protein. Hence, functional studies are warranted to investigate this further.

TIMP-3 mRNA expression levels positively correlates with levels of miR-21 in in situ BC and negatively in PR positive invasive BC.

BACKGROUND: Breast carcinomas (BC) belong to a heterogeneous group of malignant diseases. Correct categorization of BC based on molecular biomarkers has a very important role in deciding the proper course of therapy for each patient. It has been already shown that the decrease of TIMP metalloproteinase inhibitor 3 (TIMP-3) together with overexpression of microRNA-21 (miR-21) might be involved in the process of BC invasion. This is the first study that examined relationship among miR-21, TIMP-3 mRNA and TIPM-3 protein levels in BC groups formed according to invasiveness. METHODS: In this study, we used 46 breast cancer samples. Estrogen and progesterone receptor (ER, PR) protein levels were evaluated by immunohistochemistry (IHC) method. TIMP-3 mRNA expression was examined by two-step real-time quantitative PCR (qRT-PCR). Western blot analysis was performed for 16 samples. RESULTS: Statistically significant differences in TIMP-3 expression levels between invasive groups were discovered in ER positive (ER+) (p=0.015), Her-2 negative (p=0.026) subgroups, and patients without lymph-node metastasis (p=0.039). Interestingly, significant positive correlation was detected between miR-21 and TIMP-3 mRNA levels (P<0.001, ρ=0.949) in the group of in situ tumors. TIMP-3 mRNA expression levels highly negatively correlated with levels of miR-21 in PR+ invasive BCs (p=0.007, ρ=-0.641). TIMP-3 protein levels negatively correlated with miR-21 levels in pure invasive BCs. CONCLUSION: These data suggest that signaling pathways involved in formation and progression of BCs in groups formed according to invasiveness might be different. Our findings propose that TIMP-3 mRNA expression levels could be significant prognostic parameter, but within specific BC subtypes.

Characterization of HER2 gene amplification heterogeneity in invasive and in situ breast cancer using bright-field in situ hybridization.

The aims of this study were to evaluate and compare the HER2 gene amplification status in invasive and adjacent in situ breast carcinoma, using bright-field in situ hybridization, and to document the possible presence of HER2 genetic heterogeneity (HER2-GH) in both components. A cohort of 100 primary invasive carcinomas (IC) associated with carcinoma in situ (CIS) were evaluated for HER2 gene amplification by SISH according to the 2013 ASCO/CAP HER2 guideline. A second cohort of all the cases with HER2-GH since the introduction of the updated ASCO/CAP HER2 guideline was also characterized, and an evaluation of the HER2 gene amplification in the CIS component, if present, was also done. In the first cohort, the HER2 amplification in the IC was negative in 87% of the cases and positive in 13% of the cases, without the presence of HER2-GH. All the cases had an associated CIS with the same HER2 status as IC, with four cases of CIS presenting HER2-GH. In the CIS, we observed a significant relationship of HER2 gene amplification with high nuclear grade. In the four cases with HER2-GH in CIS, two cases presented HER2 gene amplification in the IC. The second cohort included 12 cases with HER2-GH in a total of 1243 IC cases (0.97%). Additionally, we identified two cases associated with non-amplified CIS. HER2-GH is a rare event in IC and can already be present in CIS, not being an important step in the acquisition of invasive features.

Current Concepts in Diagnosis, Molecular Features, and Management of Lobular Carcinoma In Situ of the Breast With a Discussion of Morphologic Variants.

CONTEXT: - Lobular carcinoma in situ (LCIS) refers to a neoplastic proliferation of cells that characteristically shows loss of E-cadherin expression and has long been regarded as a risk factor for invasive breast cancer. Long-term outcome studies and molecular data have also implicated LCIS as a nonobligate precursor to invasive carcinoma. In the past few decades, pleomorphic and florid LCIS have been recognized as morphologic variants of LCIS with more-aggressive histopathologic features, less-favorable biomarker profiles, and more-complex molecular features compared with classic LCIS. There is still a lack of consensus regarding certain aspects of managing patients with LCIS. OBJECTIVES: - To review recently published literature on LCIS and to provide an overview of the current morphologic classification of LCIS, recent molecular advances, and trends in patient management. DATA SOURCES: - Sources included peer-reviewed, published journal articles in PubMed (US National Library of Medicine, Bethesda, Maryland) and published guidelines from the National Comprehensive Cancer Network (Fort Washington, Pennsylvania). CONCLUSIONS: - Lobular carcinoma in situ represents a marker for increased risk of breast cancer, as well as a nonobligate precursor to invasive carcinoma. Morphologic variants of LCIS-florid and pleomorphic LCIS-are genetically more-complex lesions and are more likely to be associated with invasive carcinoma. Further investigation into which molecular alterations in LCIS are associated with progression to invasive carcinoma is needed to help guide medical and surgical management.

Breast and renal cancer-Derived endothelial colony forming cells share a common gene signature.

BACKGROUND: Neovascularisation supports the metastatic switch in many aggressive solid cancers. Tumour neovessels are mostly lined by endothelial cells sprouting from nearby capillaries, but they could also be contributed by circulating endothelial progenitor cells (EPCs). However, scant information is available about tumour-derived EPCs. METHODS: We carried out the first thorough, unbiased comparison of phenotype, function and genotype of normal versus tumour-derived endothelial colony forming cells (ECFCs), a truly endothelial EPC subtype. We used healthy donors-derived ECFCs (N-ECFCs) as control for breast cancer (BC)- and renal cell carcinoma (RCC)-derived ECFCs. RESULTS: We found that both BC- and RCC-ECFCs belong to the endothelial lineage. Normal and tumour-derived ECFCs did not differ in terms of proliferative and tubulogenic rates. However, RCC-ECFCs were more resistant to rapamycin-induced apoptosis, whereas BC-ECFCs were more sensitive as compared with N-ECFCs. Gene expression profiling revealed 382 differentially expressed genes (DEGs; 192 upregulated and 150 downregulated) and 71 DEGs (33 upregulated, 38 downregulated) when comparing, respectively, BC- and RCC-ECFCs with N-ECFCs. Nonetheless, BC- and RCC-derived ECFCs shared 35 DEGs, 10 of which were validated by qRT-PCR; such 35 DEGs are organised in a gene network centred on FOS. CONCLUSION: These results provide the first clear-cut evidence that BC- and RCC-derived ECFCs exhibit an altered gene expression profile as compared with N-ECFCs; yet, they share a common gene signature that could highlight novel and more specific targets to suppress tumour vascularisation.

PIK3CA mutations are common in lobular carcinoma in situ, but are not a biomarker of progression.

BACKGROUND: Lobular carcinoma in situ (LCIS) is a non-invasive breast lesion that is typically found incidentally on biopsy and is often associated with invasive lobular carcinoma (ILC). LCIS is considered by some to be a risk factor for future breast cancer rather than a true precursor lesion. The aim of this study was to identify genetic changes that could be used as biomarkers of progression of LCIS to invasive disease using cases of pure LCIS and comparing their genetic profiles to LCIS which presented contemporaneously with associated ILC, on the hypothesis that the latter represents LCIS that has already progressed. METHODS: Somatic copy number aberrations (SCNAs) were assessed by SNP array in three subgroups: pure LCIS, LCIS associated with ILC and the paired ILC. In addition exome sequencing was performed on seven fresh frozen samples of LCIS associated with ILC, to identify recurrent somatic mutations. RESULTS: The copy number profiles of pure LCIS and LCIS associated with ILC were almost identical. However, four SCNAs were more frequent in ILC than LCIS associated with ILC, including gain/amplification of CCND1. CCND1 protein over-expression assessed by immunohistochemical analysis in a second set of samples from 32 patients with pure LCIS and long-term follow up, was associated with invasive recurrence (P = 0.02, Fisher's exact test). Exome sequencing revealed that PIK3CA mutations were as frequent as CDH1 mutations in LCIS, but were not a useful biomarker of LCIS progression as they were as frequent in pure LCIS as in LCIS associated with ILC. We also observed heterogeneity of PIK3CA mutations and evidence of sub-clonal populations in LCIS irrespective of whether they were associated with ILC. CONCLUSIONS: Our data shows that pure LCIS and LCIS co-existing with ILC have very similar SCNA profiles, supporting the hypothesis that LCIS is a true precursor lesion. We have provided evidence that over-expression of CCND1 may identify a subgroup of patients with pure LCIS who are more likely to develop invasive disease, in contrast to PIK3CA mutations, which occur too early in lobular tumorigenesis to be informative.

Changes in miR-221/222 Levels in Invasive and In Situ Carcinomas of the Breast: Differences in Association with Estrogen Receptor and TIMP3 Expression Levels.

BACKGROUND: Breast cancer (BC) is a heterogeneous group of diseases that still represents a major cause of death in the female population. MicroRNAs (miRNAs, miRs), such as miR-221 and miR-222, have been shown to be involved in BC pathology by acting via its target genes such as tissue inhibitor of metalloproteinase 3 (TIMP3). OBJECTIVES: The main goals of this study were to find differences in miR-221/222 levels of expression in BC groups based on invasiveness, and to investigate the association with estrogen receptor (ER), TIMP3 messenger RNA (mRNA) levels, and clinicopathological characteristics of patients and tumors. METHODS: In this study, we measured levels of miR-221/222 in 63 breast tissue samples by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) using TaqMan® technology and immunohistochemistry. RESULTS: miR-221/222 levels varied significantly across groups based on invasiveness (P < 0.001). In in situ tumors, miR-221 and miR-222 were negatively associated with ER (P = 0.001, r = -0.714, and P = 0.013, r = -0.585, respectively). In invasive breast carcinomas associated with non-invasive tumors, miR-222 was inversely associated with ER (P = 0.039, r = -0.620). Pure invasive BCs showed a positive correlation of miR-221 and miR-222 with TIMP3 mRNA levels (P = 0.008, r = 0.508, and P = 0.010, r = 0.497, respectively). CONCLUSION: An increase in miR-221/222 might be an important event for in situ carcinoma formation, and miR-221/222 may be important molecules that highlight potential differences between invasive breast carcinomas associated with non-invasive and pure invasive BCs.

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.

GT198 Expression Defines Mutant Tumor Stroma in Human Breast Cancer.

Human breast cancer precursor cells remain to be elucidated. Using breast cancer gene product GT198 (PSMC3IP; alias TBPIP or Hop2) as a unique marker, we revealed the cellular identities of GT198 mutant cells in human breast tumor stroma. GT198 is a steroid hormone receptor coactivator and a crucial factor in DNA repair. Germline mutations in GT198 are present in breast and ovarian cancer families. Somatic mutations in GT198 are present in ovarian tumor stromal cells. Herein, we show that human breast tumor stromal cells carry GT198 somatic mutations and express cytoplasmic GT198 protein. GT198(+) stromal cells share vascular smooth muscle cell origin, including myoepithelial cells, adipocytes, capillary pericytes, and stromal fibroblasts. Frequent GT198 mutations are associated with GT198(+) tumor stroma but not with GT198(-) tumor cells. GT198(+) progenitor cells are mostly capillary pericytes. When tested in cultured cells, mutant GT198 induces vascular endothelial growth factor promoter, and potentially promotes angiogenesis and adipogenesis. Our results suggest that multiple lineages of breast tumor stromal cells are mutated in GT198. These findings imply the presence of mutant progenitors, whereas their descendants, carrying the same GT198 mutations, are collectively responsible for forming breast tumor microenvironment. GT198 expression is, therefore, a specific marker of mutant breast tumor stroma and has the potential to facilitate diagnosis and targeted treatment of human breast cancer.

Prediction of heterogeneity in breast cancer immunophenotype at ductal carcinoma in situ stage?

INTRODUCTION: Ductal carcinoma in situ (DCIS) is considered a heterogeneous lesion at the molecular level. However, there is a paucity of literature about the existence of molecular subtypes in DCIS which can predict their biological behavior at the preinvasive stage. MATERIALS AND METHODS: Precise prevalence of molecular subtypes of pure DCIS and DCIS component of infiltrating duct carcinoma (IDC) was evaluated using immunohistochemistry and correlated with known prognostic factors. RESULTS: DCIS cases were classified as luminal A (46.6% in each group), luminal B (pure DCIS 20% and DCIS component of IDC 13.3%), HER2 overexpressing, basal and nonbasal (pure DCIS 3.3% and 26.6% and DCIS component of IDC 3.3% and 33.3%, respectively), and triple negative, nonbasal (pure DCIS and DCIS component of IDC 3.3% each). The molecular phenotype of DCIS correlated well with that of the coexisting IDC. CONCLUSIONS: This study demonstrated molecular heterogeneity in DCIS; however, similar molecular phenotypes were seen in the coexisting IDC suggesting that DCIS is a precursor lesion and can predict phenotype of the invasive component. This also suggests that the invasiveness of DCIS is not dependent solely on the molecular character of the tumor epithelial cells, but factors such as tumor microenvironment may play a role.