Stromal MED12 exon 2 mutations in complex fibroadenomas of the breast

© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ. http://creativecommons.org/licenses/by-nc/4.0/This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.Aims: Here we explore the presence of mediator complex subunit 12 (MED12) exon 2 and telomerase reverse transcriptase (TERT) promoter hotspot mutations in complex fibroadenomas (CFAs) of the breast. Methods: The stromal components from 18 CFAs were subjected to Sanger sequencing of MED12 exon 2 and the TERT promoter hotspot loci. The epithelial and stromal components of two MED12 mutated CFAs were subjected to laser capture microdissection, and Sanger sequencing of MED12 exon 2, TERT promoter and PIK3CA exons 9 and 20, separately. Results: MED12 exon 2 mutations were identified in the stroma of 17% of CFAs. The analyses of epithelial and stromal components, microdissected separately, revealed that MED12 mutations were restricted to the stroma. No TERT promoter or PIK3CA mutations in exons 9 and 20 were detected in analysed CFAs. Conclusions: Like conventional fibroadenomas, MED12 exon 2 mutations appear to be restricted to the stromal component of CFAs, supporting the notion that CFAs are stromal neoplasms.This study was funded by the Breast Cancer Research Foundation. BW is funded by a Cycle for Survival grant, CS by a Fundação para a Ciência e Tecnologia grant (SFRH/BDE/110544/2015). FP is partially funded by a K12 CA184746 grant. The research reported in this paper was supported in part by a Cancer Centre Support Grant of the National Institutes of Health/National Cancer Institute (grant No P30CA008748).info:eu-repo/semantics/publishedVersio

Whole‐exome sequencing and RNA sequencing analyses of acinic cell carcinomas of the breast

AimsAcinic cell carcinoma of the breast (ACC) is a rare histologic form of triple‐negative breast cancer (TNBC). Despite its unique histology, targeted sequencing analysis has failed to identify recurrent genetic alterations other than those found in common forms of TNBC. Here, we subjected three breast ACCs to whole‐exome and RNA‐sequencing, seeking to define whether they would harbor a pathognomonic genetic alteration.Methods and ResultsTumor and normal DNA and RNA samples from three breast ACCs were subjected to whole‐exome sequencing. Somatic mutations, copy number alterations, mutational signatures and fusion genes were determined using state‐of‐the‐art bioinformatics methods. Our analyses revealed TP53 hotspot mutations associated with loss of heterozygosity of the wild‐type allele in two cases. Mutations affecting homologous recombination (HR) DNA repair‐related genes were found in two cases, and an MLH1 pathogenic germline variant was detected in one case. In addition, copy number analysis revealed the presence of a somatic BRCA1 homozygous deletion and focal amplification of 12q14.3‐12q21.1, encompassing MDM2, HMGA2, FRS2 and PTPRB. No oncogenic in‐frame fusion transcript was identified in the three breast ACCs analyzed.ConclusionsNo pathognomonic genetic alterations were detected in the ACCs analyzed. These tumors have somatic genetic alterations similar to those of common forms of TNBC and may display HR deficiency or microsatellite instability. These findings provide further insights as to why ACCs which are usually clinically indolent may evolve into or in parallel with high‐grade TNBC

Comprehensive genomic profiling of cell-free circulating tumor DNA detects response to Ribociclib plus Letrozole in a patient with metastatic breast cancer

© 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).Analysis of cell-free circulating tumor DNA obtained by liquid biopsy is a non-invasive approach that may provide clinically actionable information when conventional tissue biopsy is inaccessible or infeasible. Here, we followed a patient with hormone receptor-positive and human epidermal growth factor receptor (HER) 2-negative breast cancer who developed bone metastases seven years after mastectomy. We analyzed circulating cell-free DNA (cfDNA) extracted from plasma using high-depth massively parallel sequencing targeting 468 cancer-associated genes, and we identified a clonal hotspot missense mutation in the PIK3CA gene (3:178952085, A > G, H1047R) and amplification of the CCND1 gene. Whole-exome sequencing revealed that both alterations were present in the primary tumor. After treatment with ribociclib plus letrozole, the genetic abnormalities were no longer detected in cfDNA. These results underscore the clinical utility of combining liquid biopsy and comprehensive genomic profiling to monitor treatment response in patients with metastasized breast cancer.This work was supported by Fundação para a Ciência e Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Ensino Superior, Portugal, through Fundos do Orçamento de Estado to Instituto de Medicina Molecular João Lobo Antunes (LA/P/0082/2020), and FCT/FEDER/POR Lisboa 2020, Programa Operacional Regional de Lisboa, PORTUGAL 2020 (LISBOA-01-0145-FEDER-016394), and FEDER/POR Lisboa 2020-Programa Operacional Regional de Lisboa, PORTUGAL 2020 (Infogene, 045300). C.S. was a recipient of a FCT fellowship (SFRH/BDE/110544/2015). This work was funded in part by the National Institutes of Health (NIH)/National Cancer Institute (NCI) Cancer Center Support Grant (P30 CA008748; MSK). J.S.R-F. and B.W. are funded in part by the NIH/NCI P50 CA247749 01 grant and a Breast Cancer Research Foundation grant. J.S.R.-F. is also funded in part by a Susan G Komen leadership grant, and B.W. by a Cycle for Survival grant.info:eu-repo/semantics/publishedVersio

Genomic profiling of primary and recurrent Adult Granulosa Cell Tumors of the Ovary

Adult-type granulosa cell tumor (aGCT) is a rare malignant ovarian sex cord-stromal tumor, harboring recurrent FOXL2 c.C402G/p.C134W hotspot mutations in 97% of cases. These tumors are considered to have a favorable prognosis, however aGCTs have a tendency for local spread and late recurrences, which are associated with poor survival rates. We sought to determine the genetic alterations associated with aGCT disease progression. We subjected primary non-recurrent aGCTs (n = 7), primary aGCTs that subsequently recurred (n = 9) and their matched recurrences (n = 9), and aGCT recurrences without matched primary tumors (n = 10) to targeted massively parallel sequencing of ≥410 cancer-related genes. In addition, three primary non-recurrent aGCTs and nine aGCT recurrences were subjected to FOXL2 and TERT promoter Sanger sequencing analysis. All aGCTs harbored the FOXL2 C134W hotspot mutation. TERT promoter mutations were found to be significantly more frequent in recurrent (18/28, 64%) than primary aGCTs (5/19, 26%, p = 0.017). In addition, mutations affecting TP53, MED12, and TET2 were restricted to aGCT recurrences. Pathway annotation of altered genes demonstrated that aGCT recurrences displayed an enrichment for genetic alterations affecting cell cycle pathway-related genes. Analysis of paired primary and recurrent aGCTs revealed that TERT promoter mutations were either present in both primary tumors and matched recurrences or were restricted to the recurrence and absent in the respective primary aGCT. Clonal composition analysis of these paired samples further revealed that aGCTs display intra-tumor genetic heterogeneity and harbor multiple clones at diagnosis and relapse. We observed that in a subset of cases, recurrences acquired additional genetic alterations not present in primary aGCTs, including TERT, MED12, and TP53 mutations and CDKN2A/B homozygous deletions. Albeit harboring relatively simple genomes, our data provide evidence to suggest that aGCTs are genetically heterogeneous tumors and that TERT promoter mutations and/or genetic alterations affecting other cell cycle-related genes may be associated with disease progression and recurrences

TERT promoter hotspot mutations and gene amplification in metaplastic breast cancer.

Metaplastic breast cancers (MBCs) are characterized by complex genomes, which seem to vary according to their histologic subtype. TERT promoter hotspot mutations and gene amplification are rare in common forms of breast cancer, but present in a subset of phyllodes tumors. Here, we sought to determine the frequency of genetic alterations affecting TERT in a cohort of 60 MBCs with distinct predominant metaplastic components (squamous, 23%; spindle, 27%; osseous, 8%; chondroid, 42%), and to compare the repertoire of genetic alterations of MBCs according to the presence of TERT promoter hotspot mutations or gene amplification. Forty-four MBCs were subjected to: whole-exome sequencing (WES; n = 27) or targeted sequencing of 341-468 cancer-related genes (n = 17); 16 MBCs were subjected to Sanger sequencing of the TERT promoter, TP53 and selected exons of PIK3CA, HRAS, and BRAF. TERT promoter hotspot mutations (n = 9) and TERT gene amplification (n = 1) were found in 10 of the 60 MBCs analyzed, respectively. These TERT alterations were less frequently found in MBCs with predominant chondroid differentiation than in other MBC subtypes (p = 0.01, Fisher's exact test) and were mutually exclusive with TP53 mutations (p < 0.001, CoMEt). In addition, a comparative analysis of the MBCs subjected to WES or targeted cancer gene sequencing (n = 44) revealed that MBCs harboring TERT promoter hotspot mutations or gene amplification (n = 6) more frequently harbored PIK3CA than TERT wild-type MBCs (n = 38; p = 0.001; Fisher's exact test). In conclusion, TERT somatic genetic alterations are found in a subset of TP53 wild-type MBCs with squamous/spindle differentiation, highlighting the genetic diversity of these cancers

The genetic landscape of metaplastic breast cancers and uterine carcinosarcomas.

Metaplastic breast carcinoma (MBC) and uterine carcinosarcoma (UCS) are rare aggressive cancers, characterized by an admixture of adenocarcinoma and areas displaying mesenchymal/sarcomatoid differentiation. We sought to define whether MBCs and UCSs harbor similar patterns of genetic alterations, and whether the different histologic components of MBCs and UCSs are clonally related. Whole-exome sequencing (WES) data from MBCs (n = 35) and UCSs (n = 57, The Cancer Genome Atlas) were reanalyzed to define somatic genetic alterations, altered signaling pathways, mutational signatures, and genomic features of homologous recombination DNA repair deficiency (HRD). In addition, the carcinomatous and sarcomatous components of an additional cohort of MBCs (n = 11) and UCSs (n = 6) were microdissected separately and subjected to WES, and their clonal relatedness was assessed. MBCs and UCSs harbored recurrent genetic alterations affecting TP53, PIK3CA, and PTEN, similar patterns of gene copy number alterations, and an enrichment in alterations affecting the epithelial-to-mesenchymal transition (EMT)-related Wnt and Notch signaling pathways. Differences were observed, however, including a significantly higher prevalence of FAT3 and FAT1 somatic mutations in MBCs compared to UCSs, and conversely, UCSs significantly more frequently harbored somatic mutations affecting FBXW7 and PPP2R1A as well as HER2 amplification than MBCs. Genomic features of HRD and biallelic alterations affecting bona fide HRD-related genes were found to be more prevalent in MBCs than in UCSs. The distinct histologic components of MBCs and UCSs were clonally related in all cases, with the sarcoma component likely stemming from a minor subclone of the carcinoma component in the samples with interpretable chronology of clonal evolution. Despite the similar histologic features and pathways affected by genetic alterations, UCSs differ from MBCs on the basis of FBXW7 and PPP2R1A mutations, HER2 amplification, and lack of HRD, supporting the notion that these entities are more than mere phenocopies of the same tumor type in different anatomical sites

Local iron homeostasis in the breast ductal carcinoma microenvironment

Abstract BACKGROUND: While the deregulation of iron homeostasis in breast epithelial cells is acknowledged, iron-related alterations in stromal inflammatory cells from the tumor microenvironment have not been explored. METHODS: Immunohistochemistry for hepcidin, ferroportin 1 (FPN1), transferrin receptor 1 (TFR1) and ferritin (FT) was performed in primary breast tissues and axillary lymph nodes in order to dissect the iron-profiles of epithelial cells, lymphocytes and macrophages. Furthermore, breast carcinoma core biopsies frozen in optimum cutting temperature (OCT) compound were subjected to imaging flow cytometry to confirm FPN1 expression in the cell types previously evaluated and determine its cellular localization. RESULTS: We confirm previous results by showing that breast cancer epithelial cells present an 'iron-utilization phenotype' with an increased expression of hepcidin and TFR1, and decreased expression of FT. On the other hand, lymphocytes and macrophages infiltrating primary tumors and from metastized lymph nodes display an 'iron-donor' phenotype, with increased expression of FPN1 and FT, concomitant with an activation profile reflected by a higher expression of TFR1 and hepcidin. A higher percentage of breast carcinomas, compared to control mastectomy samples, present iron accumulation in stromal inflammatory cells, suggesting that these cells may constitute an effective tissue iron reservoir. Additionally, not only the deregulated expression of iron-related proteins in epithelial cells, but also on lymphocytes and macrophages, are associated with clinicopathological markers of breast cancer poor prognosis, such as negative hormone receptor status and tumor size. CONCLUSIONS: The present results reinforce the importance of analyzing the tumor microenvironment in breast cancer, extending the contribution of immune cells to local iron homeostasis in the tumor microenvironment context.info:eu-repo/semantics/publishedVersio