Intratumor genetic heterogeneity and clonal evolution to decode endometrial cancer progression

Endometrial cancer; Clonal evolution; MutationCáncer endometrial; Evolución clonal; MutaciónCàncer d'endometri; Evolució clonal; MutacióAnalyzing different tumor regions by next generation sequencing allows the assessment of intratumor genetic heterogeneity (ITGH), a phenomenon that has been studied widely in some tumor types but has been less well explored in endometrial carcinoma (EC). In this study, we sought to characterize the spatial and temporal heterogeneity of 9 different ECs using whole-exome sequencing, and by performing targeted sequencing validation of the 42 primary tumor regions and 30 metastatic samples analyzed. In addition, copy number alterations of serous carcinomas were assessed by comparative genomic hybridization arrays. From the somatic mutations, identified by whole-exome sequencing, 532 were validated by targeted sequencing. Based on these data, the phylogenetic tree reconstructed for each case allowed us to establish the tumors’ evolution and correlate this to tumor progression, prognosis, and the presence of recurrent disease. Moreover, we studied the genetic landscape of an ambiguous EC and the molecular profile obtained was used to guide the selection of a potential personalized therapy for this patient, which was subsequently validated by preclinical testing in patient-derived xenograft models. Overall, our study reveals the impact of analyzing different tumor regions to decipher the ITGH in ECs, which could help make the best treatment decision.We thank all those at the Translational Research Laboratory of the MD Anderson Cancer Center Madrid for their invaluable help with this study. Tissue samples were obtained with the support of the MD Anderson Foundation Biobank (record number B.0000745, ISCIII National Biobank Record), the “Xarxa Catalana de Bancs de Tumors” and “Plataforma de Biobancos” ISCIII (PT13/0010/0014, B.000609). This study has been supported by the Spanish Ministry of Economy and Innovation (PID2019-104644RB-I00 (GMB), the Instituto de Salud Carlos III (ISCIII, CIBERONC, CB16/12/00295 - GMB-, CB16/12/00328 -EC, AGM- and CB16/12/00231 -XMG- [all partly supported by FEDER funds]) and by the AECC Scientific Foundation (FC_AECC PROYE19036MOR -GMB- and Coordinated groups 2018 -XMG, AGM, GMB-). SO is funded by an AECC-postdoctoral grant (2020). JSR-F and BW are funded in part by the Breast Cancer Research Foundation and in part by the NIH/NCI P50 CA247749 01 grant. Research reported in this publication was supported in part by a Cancer Center Support Grant of the NIH/NCI (Grant No. P30CA008748; MSK). We thank the Eurofins Megalab laboratory for helping us to perform the analysis of DNA HPV detection

Homologous recombination DNA repair defects in PALB2-associated breast cancers

Mono-allelic germline pathogenic variants in the Partner And Localizer of BRCA2 (PALB2) gene predispose to a high-risk of breast cancer development, consistent with the role of PALB2 in homologous recombination (HR) DNA repair. Here, we sought to define the repertoire of somatic genetic alterations in PALB2-associated breast cancers (BCs), and whether PALB2-associated BCs display biallelic inactivation of PALB2 and/or genomic features of HR-deficiency (HRD). Twenty-four breast cancer patients with pathogenic PALB2 germline mutations were analyzed by whole-exome sequencing (WES, n = 16) or targeted capture massively parallel sequencing (410 cancer genes, n = 8). Somatic genetic alterations, loss of heterozygosity (LOH) of the PALB2 wild-type allele, largescale state transitions (LSTs) and mutational signatures were defined. PALB2-associated BCs were found to be heterogeneous at the genetic level, with PIK3CA (29%), PALB2 (21%), TP53 (21%), and NOTCH3 (17%) being the genes most frequently affected by somatic mutations. Bi-allelic PALB2 inactivation was found in 16 of the 24 cases (67%), either through LOH (n = 11) or second somatic mutations (n = 5) of the wild-type allele. High LST scores were found in all 12 PALB2-associated BCs with bi-allelic PALB2 inactivation sequenced by WES, of which eight displayed the HRD-related mutational signature 3. In addition, bi-allelic inactivation of PALB2 was significantly associated with high LST scores. Our findings suggest that the identification of bi-allelic PALB2 inactivation in PALB2- associated BCs is required for the personalization of HR-directed therapies, such as platinum salts and/or PARP inhibitors, as the vast majority of PALB2-associated BCs without PALB2 bi-allelic inactivation lack genomic features of HRD.. The authors thank Heather Thorne, Eveline Niedermayr, all the kConFab research nurses and staff, the heads and staff of the Family Cancer Clinics, and the Clinical Follow Up Study (which has received funding from the NHMRC, the National Breast Cancer Foundation, Cancer Australia, and the National Institute of Health (USA)) for their contributions to this resource, and the many families who contribute to kConFab. Research reported in this paper was supported in part by the Breast Cancer Research Foundation and the Sarah Jenkins Fund, a Cancer Center Support Grant of the National Institutes of Health/National Cancer Institute (grant No. P30CA008748; MSK), a grant of the Ministry of Health of the Czech Republic (NV15-29959A), Charles University projects PROGRES Q28/LF1 and SVV2019/260367, an HIR Grant UM.C/HlR/ MOHE/06 from the Ministry of Higher Education, Malaysia, and the National Health and Medical Research Council, Australia (NHMRC, Project Grant APP1029974). kConFab is supported by a grant from the National Breast Cancer Foundation, and previously by the NHMRC, the Queensland Cancer Fund, the Cancer Councils of New South Wales, Victoria, Tasmania, and South Australia, and the Cancer Foundation of Western Australia. W.D.F. was funded in part by Susan G Komen. A.L. was supported by the China Scholarship Council. T.N.-D. is an Early Career Fellow of the National Breast Cancer Foundation and M.S. is a NHMRC Senior Research Fellow of the National Health and Medical Research Council. M.T. was funded by the National Institute for Health Research (NIHR) Cambridge Biomedical Research Centre, Addenbrooke’s Hospital and European Union Seventh Framework Program (2007–2013)/European Research Council (310018). S.P. was supported by the Swiss National Science Foundation (Ambizione grant number: PZ00P3_168165). J.S.R-F. is partly funded by the Breast Cancer Research Foundation and Britta Weigelt by Cycle for Survival

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

MYBL1 rearrangements and MYB amplification in breast adenoid cystic carcinomas lacking the MYB–NFIB fusion gene

Breast adenoid cystic carcinoma (AdCC), a rare type of triple-negative breast cancer, has been shown to be driven by MYB pathway activation, most often underpinned by the MYB–NFIB fusion gene. Alternative genetic mechanisms, such as MYBL1 rearrangements, have been reported in MYB–NFIB-negative salivary gland AdCCs. Here we report on the molecular characterization by massively parallel sequencing of four breast AdCCs lacking the MYB–NFIB fusion gene. In two cases, we identified MYBL1 rearrangements (MYBL1–ACTN1 and MYBL1–NFIB), which were associated with MYBL1 overexpression. A third AdCC harboured a high-level MYB amplification, which resulted in MYB overexpression at the mRNA and protein levels. RNA-sequencing and whole-genome sequencing revealed no definite alternative driver in the fourth AdCC studied, despite high levels of MYB expression and the activation of pathways similar to those activated in MYB–NFIB-positive AdCCs. In this case, a deletion encompassing the last intron and part of exon 15 of MYB, including the binding site of ERG-1, a transcription factor that may downregulate MYB, and the exon 15 splice site, was detected. In conclusion, we demonstrate that MYBL1 rearrangements and MYB amplification probably constitute alternative genetic drivers of breast AdCCs, functioning through MYBL1 or MYB overexpression. These observations emphasize that breast AdCCs probably constitute a convergent phenotype, whereby activation of MYB and MYBL1 and their downstream targets can be driven by the MYB–NFIB fusion gene, MYBL1 rearrangements, MYB amplification, or other yet to be identified mechanisms. Copyright © 2017 Pathological Society of Great Britain and Ireland

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

Phyllodes tumors with and without fibroadenoma-like areas display distinct genomic features and may evolve through distinct pathways

Breast fibroepithelial lesions (fibroadenomas and phyllodes tumors) are underpinned by recurrent; MED12; exon 2 mutations, which are more common in fibroadenomas and benign phyllodes tumors.; TERT; promoter hotspot mutations have been documented in phyllodes tumors, and found to be more frequent in borderline and malignant lesions. Several lines of evidence suggest that a subset of phyllodes tumors might arise from fibroadenomas. Here we sought to investigate the genetic differences between phyllodes tumors with fibroadenoma-like areas vs. those without. We retrieved data for 16 borderline/ malignant phyllodes tumors, including seven phyllodes tumors with fibroadenoma-like areas and nine phyllodes tumors without fibroadenoma-like areas, which had been previously subjected to targeted capture massively parallel sequencing. Whilst; MED12; exon 2 mutations were significantly more frequent in tumors with fibroadenoma-like areas (71 vs. 11%), an enrichment in genetic alterations targeting; bona fide; cancer genes was found in those without fibroadenoma-like areas, in particular in; EGFR; mutations and amplifications (78 vs. 14%). No significant difference in the frequency of; TERT; genetic alterations was observed (71% in cases with fibroadenoma-like areas vs 56% in those without fibroadenoma-like areas). Our data suggest that the development of phyllodes tumors might follow two different evolutionary pathways: a; MED12; -mutant pathway that involves the progression from a fibroadenoma to a malignant phyllodes tumor; and a; MED12; -wild-type pathway, where malignant phyllodes tumors arise de novo through the acquisition of genetic alterations targeting cancer genes. Additional studies are warranted to confirm our observations and define whether the outcome differs between both pathways

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

Homologous recombination DNA repair defects in PALB2-associated breast cancers.

Mono-allelic germline pathogenic variants in the Partner And Localizer of BRCA2 (PALB2) gene predispose to a high-risk of breast cancer development, consistent with the role of PALB2 in homologous recombination (HR) DNA repair. Here, we sought to define the repertoire of somatic genetic alterations in PALB2-associated breast cancers (BCs), and whether PALB2-associated BCs display bi-allelic inactivation of PALB2 and/or genomic features of HR-deficiency (HRD). Twenty-four breast cancer patients with pathogenic PALB2 germline mutations were analyzed by whole-exome sequencing (WES, n = 16) or targeted capture massively parallel sequencing (410 cancer genes, n = 8). Somatic genetic alterations, loss of heterozygosity (LOH) of the PALB2 wild-type allele, large-scale state transitions (LSTs) and mutational signatures were defined. PALB2-associated BCs were found to be heterogeneous at the genetic level, with PIK3CA (29%), PALB2 (21%), TP53 (21%), and NOTCH3 (17%) being the genes most frequently affected by somatic mutations. Bi-allelic PALB2 inactivation was found in 16 of the 24 cases (67%), either through LOH (n = 11) or second somatic mutations (n = 5) of the wild-type allele. High LST scores were found in all 12 PALB2-associated BCs with bi-allelic PALB2 inactivation sequenced by WES, of which eight displayed the HRD-related mutational signature 3. In addition, bi-allelic inactivation of PALB2 was significantly associated with high LST scores. Our findings suggest that the identification of bi-allelic PALB2 inactivation in PALB2-associated BCs is required for the personalization of HR-directed therapies, such as platinum salts and/or PARP inhibitors, as the vast majority of PALB2-associated BCs without PALB2 bi-allelic inactivation lack genomic features of HRD.ER