Repertoire Of Somatic Mutations Research Articles

Abstract 1692: Single-cell DNA sequencing from frozen endometrial tumors to address clonal evolution of somatic mutations

Abstract Background: Single-cell DNA sequencing with droplet microfluidics has enabled the characterization of genetic heterogeneity in hematologic malignancies and fresh solid tumors through the direct DNA sequencing of thousands of single cells from individual samples without the need of whole-genome amplification. The application of this technology in banked solid tumor samples has been rather limited to date, however. Here, we describe and validate a method to characterize clonal evolution of somatic mutations from frozen tumor specimens, utilizing endometrial carcinomas (ECs) as an example. Methods: Tissue cores were obtained from frozen ECs and the extracellular matrix was enzymatically digested. A subset of extracted nuclei were used for bulk DNA extraction and targeted sequencing. The remaining nuclei were encapsulated with cell lysis buffer, and the DNA was primed and barcoded with hydrogel beads containing 317 cancer gene amplicons using a Tapestri instrument (Mission Bio). Following droplet PCR, Illumina libraries were generated and sequenced on a NovaSeq6000. Sequencing reads were trimmed and mapped to the reference genome. Barcode sequences were error-corrected and cells were detected from barcodes based on number of reads assigned to each barcode and amplicon read completeness. Genotyping was performed using GATK and the allele drop-out (ADO) rate was calculated. Non-neoplastic cells were defined by the absence of somatic mutations. Allele-specific copy number (ASCN) profiles for each tumor cell were estimated by normalizing mean reads per cell per amplicon to reads of non-neoplastic cells. Results: We performed single-cell genotyping and ASCN profiling on two ECs with matched bulk sequencing data. For EC1, 5,004 tumor and 132 non-neoplastic cells were sequenced, for a 97% tumor purity. For EC2, 2,981 tumor and 165 non-neoplastic cells were obtained, for a 95% tumor purity. The average number of reads per amplicon per cell for EC1 and EC2 was 116 and 146 with an ADO rate of 6% and 8%, respectively. The data obtained from single-cell analysis were concordant with the matched bulk sequencing data in terms of clonal and subclonal mutations, and their respective cancer cell fraction. Single-cell sequencing further revealed the presence of loss of heterozygosity and emergence of minor subclones as late events in tumor evolution, which could not be inferred by bulk sequencing. In EC2, branch mutations displayed a spectrum consistent with microsatellite instability (MSI), reflecting its acquired MSI phenotype. Conclusions: Here we describe a robust method to perform single-cell targeted DNA sequencing with droplet microfluidics of individual nuclei isolated from frozen tissue samples, providing the opportunity to determine the repertoire of somatic mutations at single cell resolution and to infer cancer clonal architecture and dynamics utilizing banked solid cancer tissue samples. Citation Format: Arnaud Da Cruz Paula, Yingjie Zhu, Shirin Issa Bhaloo, Fresia Pareja, Timothy Hoang, Pier Selenica, Etta Hanlon, Thais Basili, Higinio Dopeso, Nadeem Abu-Rustum, Jorge Sergio Reis-Filho, Britta Weigelt. Single-cell DNA sequencing from frozen endometrial tumors to address clonal evolution of somatic mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1692.

Abstract 2949: Enabling single cell analysis of copy number variation in breast cancer

Abstract Breast cancer is arguably one of the solid tumors most comprehensively analyzed by massively parallel sequencing. Although the repertoire of clonal somatic genetic alterations in primary and metastatic breast cancers has been characterized, a complete characterization of the subclonal genetic alterations present in these cancers, as well as their clonal composition and clonal dynamics remain to be fully defined. Addressing these knowledge gaps is germane to the successful personalization of treatments for breast cancer patients. Single cell sequencing is uncovering many insights into the clonal evolution and heterogeneity of breast cancers and how this can be leveraged for clinical and therapeutic benefit. Bulk next generation sequencing has primarily focused on mutation profiling; however, there are several lines of evidence to demonstrate the biological and clinical importance of accurately detecting copy number alterations. Previous single cell sequencing studies focusing on copy number alterations were based on whole-genome amplification methods or could not concurrently define the repertoire of somatic mutations and copy number alterations in single cells. Here we report on a novel single cell DNA sequencing approach leveraging the Tapestri device and Insights bioinformatic pipeline to detect both copy number alterations and somatic mutations in individual cells derived from breast cancer specimens. The identification of targeted somatic genetic alterations at single cell resolution provides novel insights about the development, progression and therapeutic resistance of breast cancers, and enables the development of novel diagnostic tools and predictive biomarkers. Citation Format: Saurabh Parikh, Arnaud Da Cruz Paula, Shirin Issa Bhaloo, Alex Li, Britta Weigelt, Khushali Patel, Ania Wronski, Anup Parikh, Khushali Patel, Jorge Reis-Filho. Enabling single cell analysis of copy number variation in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2949.

Molecular profiling and molecular classification of endometrioid ovarian carcinomas

ObjectiveEndometrioid ovarian carcinomas (EOCs) comprise 5–10% of all ovarian cancers and commonly co-occur with synchronous endometrioid endometrial cancer (EEC). We sought to examine the molecular characteristics of pure EOCs in patients without concomitant EEC. MethodsEOCs and matched normal samples were subjected to massively parallel sequencing targeting 341–468 cancer-related genes (n = 8) or whole-genome sequencing (n = 28). Mutational frequencies of EOCs were compared to those of high-grade serous ovarian cancers (HGSOCs; n = 224) and EECs (n = 186) from The Cancer Genome Atlas, and synchronous EOCs (n = 23). ResultsEOCs were heterogeneous, frequently harboring KRAS, PIK3CA, PTEN, CTNNB1, ARID1A and TP53 mutations. EOCs were distinct from HGSOCs at the mutational level, less frequently harboring TP53 but more frequently displaying KRAS, PIK3CA, PIK3R1, PTEN and CTNNB1 mutations. Compared to synchronous EOCs and pure EECs, pure EOCs less frequently harbored PTEN, PIK3R1 and ARID1A mutations. Akin to EECs, EOCs could be stratified into the four molecular subtypes: 3% POLE (ultramutated), 19% MSI (hypermutated), 17% copy-number high (serous-like) and 61% copy-number low (endometrioid). In addition to microsatellite instability, a subset of EOCs harbored potentially targetable mutations, including AKT1 and ERBB2 hotspot mutations. EOCs of MSI (hypermutated) subtype uniformly displayed a good outcome. ConclusionsEOCs are heterogeneous at the genomic level and harbor targetable genetic alterations. Despite the similarities in the repertoire of somatic mutations between pure EOCs, synchronous EOCs and EECs, the frequencies of mutations affecting known driver genes differ. Further studies are required to define the impact of the molecular subtypes on the outcome and treatment of EOC patients.

Abstract P5-10-01: Using whole genome sequencing and somatic mutation signatures to unravel insight into familial breast cancer aetiology

Abstract Approximately 10-15% of breast cancers are associated with a strong family history of disease. Pathogenic variants in BRCA1, BRCA2 or other moderate to highly penetrant susceptibility genes (e.g. TP53, ATM, CHEK2, PALB2 and PTEN) account for a number of breast cancer families. However, for over 50% of families the underlying genetic contribution to their risk remains unknown (termed here as non-BRCA1/2). This has a profound impact for how individuals and their families are managed in the clinic. We applied whole genome sequencing (WGS) to determine whether somatic mutation analysis can reveal insight into the aetiology of familial breast cancer. The full repertoire of somatic mutations was evaluated in 26 BRCA1, 22 BRCA2 and 32 non-BRCA1/2 tumours; including SNPs, indels, copy number changes and structural rearrangements, and mutational signatures. Genomes were also analysed using the HRD Index and HRDetect, as predictors of homologous recombination deficiency. BRCA1, BRCA2 and non-BRCA1/2 tumours exhibited a different burden of mutations, a different spectrum of mutational signatures and different telomere length. Based on collective patterns of mutation signatures, tumours were classified as 'BRCA1-like', 'BRCA2-like' or 'non-BRCA1/2-like' with a 15% rate of tumour re-classification from their original clinical BRCA status. The results demonstrate the power of WGS to differentiate between BRCA1 and BRCA2 driven tumours; in the identification of double-pathogenic germline mutation carriers based on the resulting somatic mutation signature; and in the interpretation of BRCA unclassified variants. WGS of tumour genomes reveals fascinating insights into tumour aetiology and could compliment current genetic testing of breast cancer families. Citation Format: Simpson P, Nones K, Johnson J, Newell F, Patch A-M, Thorne H, Kazakoff S, De Luca X, Parsons M, Ferguson K, Reid L, McCart Reed A, Srihari S, Lakis V, Davidson A, Mukhopadhyay P, Holmes O, Xu Q, Wood S, Leonard C, Beasley J, Degasperi A, Nik-Zainal S, Ragan M, Spurdle A, Khanna KK, Lakhani S, Pearson J, Chenevix-Trench G, Waddell N. Using whole genome sequencing and somatic mutation signatures to unravel insight into familial breast cancer aetiology [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-10-01.

A Comparative Study of Genetic Profiles of Key Oncogenesis-Related Genes between Primary Lesions and Matched Lymph Nodes Metastasis in Lung Cancer.

Background: The genetic heterogeneity between primary and metastatic lesions has rarely studied.Purpose: This study aims to compare the mutation profiling of key cancer-related genes in primary lung cancers and their matched lymph nodes (LN) metastasis.Methods: The gene mutation profiling of both primary cancers and their matched LN metastasis was constructed using a hybridization capture-based massively parallel sequencing assay and the sequencing of 1,408 key cancer genes in 37 NSCLC patients.Results: There are differences in the repertoire of somatic mutations and CNVs between primary lesions and metastatic lymphatic lesions in NSCLC. More mutations were discovered in the primary lesion than LN metastasis lesion, indicating that tumor mutation burden is higher in primary lesion than that in LN metastasis lesion. The copy number profiles were largely preserved through the progression of the metastasis in lung cancer individually. Regarding to the mutation context, more prevailing of signature 1 in LN metastasis lesion was observed than that of primary lesion, indicating that the spontaneous deamination of cytosine may contribute to the accumulation of mutations during tumor development and immigration. Furthermore, the LN metastasis exclusive somatic mutation (LME-SMs) genes were enriched in peptidyl-tyrosine phosphorylation, peptidyl-tyrosine modification, protein tyrosine kinase activity, and transmembrane receptor protein kinase. The KEGG pathway analysis revealed that these genes might participate in the PI3K-Akt signaling pathway, MAPK signaling pathway and Ras signaling pathway.Conclusion: Since the heterogeneity of genetic profiling exists between primary lesion and LN metastasis lesion, multi-region lesion mutation profiling is required to fully understand the progression of NSCLC, and develop an appropriate therapeutic guide. Mutations in genes involved in cell proliferation, invasion and migration may be the genetic mechanism of metastasis.

Abstract P2-05-08: Mucinous breast carcinomas: A genomically distinct subtype of estrogen receptor-positive invasive breast cancers

Abstract Introduction: Mucinous carcinoma of the breast (MCB) is a rare histologic form of estrogen receptor (ER)-positive invasive carcinoma, accounting for up to 2% of breast cancers. MCBs are characterized by clusters of tumor cells floating in lakes of extracellular mucin, and are classified into mucinous A (paucicellular) and mucinous B (hypercellular) subtypes. Some MCBs are found admixed with invasive ductal carcinoma components, and then classified as mixed MCBs. The aims of this study were to determine the repertoire of somatic mutations of MCBs and to ascertain whether these genetic alterations are distinct from those identified in common forms of ER+/HER2- invasive breast cancers (IBCs). We also sought to determine whether the mucinous and ductal components of mixed MCBs would be clonally related. Materials and methods: Thirty MCBs including 25 pure MCBs (n=13 mucinous A, n=12 mucinous B) and five mixed MCBs were microdissected and subjected to whole exome sequencing. Each tumor component of mixed cases was microdissected and analyzed separately. Somatic mutations, copy number alterations and mutational signatures were defined using state-of-the-art bioinformatics methods. The mutational repertoire of MCBs was compared with that of ER+/HER2- IBCs (n = 240) from The Cancer Genome Atlas (TCGA) breast cancer study. Results: The genes most frequently mutated in MCBs were GATA3 (27%, 8/30, all frameshift mutations), KMT2C (13%, 4/30) and MAP3K1 (10%, 3/30). No significant differences were identified in single gene comparisons between mucinous A and mucinous B MCBs or between pure MCBs and the mucinous component of mixed MCBs (Fisher's exact tests, p>0.05). As compared to common forms of ER+/HER2- IBC, MCBs had a lower frequency of PIK3CA mutations (7% vs 42%, p<0.001) and a higher frequency of GATA3 mutations (27% vs 12%, p=0.04). Mucinous B MCBs had a higher frequency of KMT2C mutations than ER+/HER2- IBCs (25% vs 6%, p=0.04). Most MCBs displayed the mutational signature 1 (aging-related; 20/30, 67%), and no differences in the frequency of specific mutational signatures according to the type of MCBs were observed. Concurrent 1q gains and 16q losses, which are the hallmark genetic alterations of low-grade ER+/HER2- breast cancers, were not observed in pure MCBs, but were found in three of the five mixed MCBs analyzed. The mucinous and ductal components of all five mixed MCBs shared a median of 58% of somatic mutations (range 42%-64%), including clonal GATA3 frameshift mutations in two of them, as well as a similar pattern of copy number alterations, supporting their clonal relatedness. Additional somatic mutations found to be restricted to the ductal or mucinous components of all mixed MCBs analyzed were identified, including clonal missense mutations in PIK3C2B and PIK3R2 in the ductal component of one case, and a PIK3R5 missense mutation in the mucinous component of another case. Conclusions: The repertoire of somatic mutations in MCBs is distinct from that of common forms of ER+/HER2- IBCs. These differences include the lack of concurrent 1q gains/16q losses, a lower frequency of PIK3CA mutations and a higher frequency of GATA3 mutations in pure MCBs. Citation Format: Pareja F, Geyer FC, Piscuoglio S, Selenica P, Kumar R, Lim RS, Guerini-Rocco E, Marchio C, Mariani O, Ng CKY, Brogi E, Norton L, Vincent-Salomon A, Weigelt B, Reis-Filho JS. Mucinous breast carcinomas: A genomically distinct subtype of estrogen receptor-positive invasive breast cancers [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-05-08.

Abstract P2-03-01: Mutational landscape of breast cancers from PALB2 germline mutation carriers

Abstract Background: The PALB2 gene encodes the partner and localizer of the BRCA2 protein, which participates in homologous recombination during DNA repair via an interaction with BRCA1 and BRCA2. Germline mutations in PALB2 are associated with an increased risk of breast cancer, with a cumulative risk of 35% by age 70 in female PALB2 mutant carriers. The aims of this project were to characterize the genomic landscape of PALB2 breast cancers and define the differences in the repertoire of somatic genetic alterations and mutational signatures between PALB2, BRCA1 and BRCA2 breast cancers. Methods: Representative samples from fourteen breast cancers from patients with known PALB2 germline mutations (seven frame-shift (2 H1170fs, 3 K346fs, 1 T841fs and 1 L531fs), five truncating (3 W1038* and 2 Q775*) and two missense (W1140G and L35P)) were microdissected to ensure a tumor cell content of >70%. DNA samples from microdissected tumors and their matched normal counterparts were subjected to whole exome sequencing on an Illumina HiSeq2000 to a median depth of 118x (range 33-193x). Somatic single nucleotide variations were detected using MuTect, and small insertions and deletions were identified using Strelka and Varscan2. Using ABSOLUTE and FACETS, we investigated the presence of loss of heterozygosity (LOH) of the PALB2 wild-type allele in these tumors. In addition, the mutational signatures and large scale state transitions (LSTs) were defined. The repertoire of somatic mutations identified in PALB2 breast cancers was compared to that of breast cancers from BRCA1 (n=11) and BRCA2 (n=10) germline mutation carriers from The Cancer Genome Atlas study. Results: PALB2 breast cancers were found to harbor a median of 80 somatic mutations (range 22-286) and one somatic mutation (range 0-13) affecting known cancer genes. Somatic loss of the PALB2 wild-type allele was found in five cases, and in three additional cases, a second PALB2 somatic mutation likely constituted the second 'hit' (two with truncating mutations, Q479* and Q61*, and one with a Q921fs frameshift mutation). Six PALB2 breast cancers displayed the BRCA mutations signature; of these, five had PALB2 bi-allelic inactivation (three LOH of the wild-type allele and two a second PALB2 somatic mutation). 71% of the samples were found to have LSTs, including all cases with a BRCA mutational signature. A significant association between PALB2 bi-allelic inactivation and concurrent BRCA signature and high LST was observed (p=0.015). Breast cancers from PALB2 mutation carriers had fewer somatic TP53 mutations than BRCA1 breast cancers (3/14, 21% vs 9/11, 82%, p=0.004), but no difference in the repertoire of somatic mutations compared to that of BRCA2 breast cancers. Conclusions: PALB2 breast cancers were found to harbor pathogenic mutations in driver genes, including TP53, PIK3CA, NF1 and NCOR1, however lacked highly recurrent somatic mutations. Unlike BRCA1/2 breast cancers, the majority of breast cancers from PALB2 germline mutation carriers lacked LOH of the PALB2 wild-type allele. Importantly, however, an association between PALB2 bi-allelic inactivation and the BRCA mutational signature and LSTs was observed, providing additional evidence for a homologous recombination-deficient phenotype at least in a subset of PALB2 cancers. Citation Format: Burke KA, Berman S, Geyer FC, Piscuoglio S, Ng CK, Wen YH, Mannermaa A, Peterlongo P, Tondini C, Janatova M, Soo Hwang T, Ng P-S, Looi LM, Chenevix-Trench G, Southey MC, Weigelt B, Foulkes W, Tischkowitz M, Reis-Filho JS, PALB2 Interest Group. Mutational landscape of breast cancers from PALB2 germline mutation carriers [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-03-01.

Abstract S2-02: The landscape of somatic genetic alterations in BRCA1 and BRCA2 breast cancers

Abstract Introduction: Women carrying BRCA1 or BRCA2 germline mutations have a 45-80% lifetime risk of developing breast cancer (BC). BRCA1 and BRCA2 are perceived as bona fide tumor suppressor genes, whereby bi-allelic inactivation in tumor cells is required for tumorigenesis. Recent studies have indicated that loss of heterozygosity (LOH) of the wild-type allele of BRCA1 may be heterogeneous and constitute a late event. Therefore, additional somatic events prior to full BRCA1/2 inactivation may be required for tumorigenesis. Given that the somatic events that result in the development of BRCA1/2-BCs and their chronology are not understood, here we sought to define the genomic landscape of BRCA1/2-BCs and whether LOH of BRCA1/2 wild-type allele and/or mutations affecting additional tumor suppressor genes would be clonal or subclonal in these cancers. Methods: We retrieved 29 BRCA1-BCs and 10 BRCA2-BCs from the Pathology Departments of the authors' institutions. DNA extracted from microdissected tumor and normal breast samples was subjected to targeted capture massively parallel sequencing using either the MSK-IMPACT assay or an assay targeting all exons of 254 genes recurrently mutated in BC or related to DNA repair. Somatic single nucleotide variants, small insertions and deletions and copy number alterations affecting genes present in both sequencing assays (111 genes) were defined using state-of-the-art bioinformatics algorithms. ABSOLUTE and FACETS were employed to define clonal (i.e. present virtually in 100% of the cancer cells of a given case) and subclonal mutations and the presence of LOH of the BRCA1 and BRCA2 wild-type alleles. Results: Our analysis revealed bi-allelic inactivation of BRCA1 in 28 of 29 BRCA1-BCs (93% harbored LOH of the BRCA1 wild-type allele and 3% harbored a second somatic BRCA1 pathogenic mutation). The only BRCA1-BC lacking bi-allelic inactivation of BRCA1 was an estrogen receptor-positive lobular carcinoma, lacking genomic features consistent with homologous recombination DNA repair defects, diagnosed at 62 years of age. Bi-allelic inactivation of BRCA2 was found in all cases (100% of harbored LOH of the BRCA2 wild-type allele). A clonal somatic 'second hit' resulting in bi-allelic inactivation of BRCA1 or BRCA2 was detected in 76% and 100% of BRCA1-BCs and BRCA2-BCs, respectively. In BRCA1-BCs, TP53 mutations were detected in 76% of cases, and these mutations were found to be clonal in 58% of cases. The repertoire of somatic mutations affecting BRCA1-BCs included clonal somatic mutations or homozygous deletions of known tumor suppressor genes, such as PTEN, RB1, CDKN2A and NF1. In contrast, only 10% of the BRCA2-BCs harbored TP53 somatic mutations. Though clonal somatic mutations in several cancer genes were detected, 40% of BRCA2-BCs had no mutations affecting the cancer genes analyzed. Conclusions: Bi-allelic inactivation of BRCA1 and BRCA2 are frequent events in BRCA1-BCs and BRCA2-BCs, respectively. In a subset of BRCA1-BCs, however, the second 'hit' appeared to be subclonal, whereas mutations affecting TP53 and other tumor suppressor genes were clonal, supporting the notion that at least in a subset of these tumors, loss of the wild-type allele of BRCA1 may be preceded by inactivation of another tumor suppressor gene. Citation Format: Geyer FC, Burke KA, Macedo GS, Piscuoglio S, Ng CK, Martelotto LG, Papanastatiou AD, De Filippo MR, Schultheis AM, Brogi E, Robson M, Wen YH, Weigelt B, Schnitt SJ, Tung N, Reis-Filho JS. The landscape of somatic genetic alterations in BRCA1 and BRCA2 breast cancers [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S2-02.

Genetic analysis of microglandular adenosis and acinic cell carcinomas of the breast provides evidence for the existence of a low-grade triple-negative breast neoplasia family

Acinic cell carcinoma is an indolent form of invasive breast cancer, whereas microglandular adenosis has been shown to be a neoplastic proliferation. Both entities display a triple-negative phenotype, and may give rise to and display somatic genomic alterations typical of high-grade triple-negative breast cancers. Here we report on a comparison of previously published data on eight carcinoma-associated microglandular adenosis and eight acinic cell carcinomas subjected to targeted massively parallel sequencing targeting all exons of 236 genes recurrently mutated in breast cancer and/or DNA repair-related. Somatic mutations, insertions/ deletions, and copy number alterations were detected using state-of-the-art bioinformatic algorithms. All cases were of triple-negative phenotype. A median of 4.5 (1–13) and 4.0 (1–7) non-synonymous somatic mutations per carcinoma-associated microglandular adenosis and acinic cell carcinoma were identified, respectively. TP53 was the sole highly recurrently mutated gene (75% in microglandular adenosis versus 88% in acinic cell carcinomas), and TP53 mutations were consistently coupled with loss of heterozygosity of the wild-type allele. Additional somatic mutations shared by both groups included those in BRCA1, PIK3CA, and INPP4B. Recurrent (n=2) somatic mutations restricted to microglandular adenosis or acinic cell carcinomas included those affecting PTEN and MED12 or ERBB4, respectively. No significant differences in the repertoire of somatic mutations were detected between microglandular adenosis and acinic cell carcinomas, and between this group of lesions and 77 triple-negative carcinomas from The Cancer Genome Atlas. Microglandular adenosis and acinic cell carcinomas, however, were genetically distinct from estrogen receptor-positive and/or HER2-positive breast cancers from The Cancer Genome Atlas. Our findings support the contention that microglandular adenosis and acinic cell carcinoma are part of the same spectrum of lesions harboring frequent TP53 somatic mutations, and likely represent low-grade forms of triple-negative disease with no/minimal metastatic potential, of which a subset has the potential to progress to high-grade triple-negative breast cancer.

Abstract 134: Mutational landscape of breast cancers from PALB2 germline mutation carriers

Abstract Introduction: The PALB2 gene encodes the partner and localizer of BRCA2 protein, which interacts with BRCA1/2 and is involved in homologous recombination DNA repair. Germline mutations in PALB2 are associated with an increased risk of breast cancer, with a cumulative risk of 35% by age 70 in female PALB2 mutation carriers. Whether the PALB2 wild-type allele is lost in the development of PALB2 breast cancers has yet to be defined. Further, the repertoire of somatic genetic alterations in these tumors is currently unknown. In this study we sought to characterize the genomic landscape of PALB2 breast cancers and to define the differences in the repertoire of somatic genetic alterations and mutational signatures between PALB2 and BRCA1 and BRCA2 breast cancers. Material and Methods: Representative samples from nine breast cancers from patients with known PALB2 germline mutations were microdissected. DNA samples from microdissected tumors and matched normal counterparts were subjected to whole exome sequencing on an Illumina HiSeq2000. Somatic mutations were defined using MuTect and insertions and deletions using Strelka and Varscan2. Driver mutations were defined by state-of-the-art bioinformatics methods. Mutational signatures were defined using non-negative matrix factorization. Copy number alterations (CNAs) and regions with loss of heterozygosity were determined using FACETS. The mutational frequency of breast cancers from PALB2 germline mutation carriers was compared to that of breast cancers from BRCA1 (n = 11) and BRCA2 (n = 10) germline mutation carriers from The Cancer Genome Atlas study. Results: Three patients harbored germline frame-shift PALB2 mutations (2 S1169fs, 1 T841fs), five displayed truncating mutations (3 W1038* and 2 Q775*) and 1 harbored a missense mutation (W1140G, of uncertain significance). Somatic loss of the PALB2 wild-type allele was found in 3 cases, in 2 of which the loss was caused by CNAs and in 1 case it was caused by a somatic PALB2 Q479* mutation. A median of 65 somatic mutations (range 45-223) and a median of 1 driver mutation (range 0-3) were identified per tumor. Cancer genes mutated in PALB2 breast cancers included TP53 (n = 2), PIK3CA (n = 2), NF1 (n = 1) and NCOR1 (n = 1). Six cases displayed mutational signatures consistent with the aging process; the BRCA signature was not found in any of the cases analyzed. Breast cancers from PALB2 mutation carriers had fewer somatic TP53 mutations than BRCA1 breast cancers (2/9, 22% vs 9/11, 82%, p = 0.02). No difference in the repertoire of somatic mutations between PALB2 and BRCA2 breast cancers was observed. Conclusion: Unlike breast cancers from BRCA1 and BRCA2 mutation carriers, the majority of breast cancers from PALB2 mutation carriers lacked somatic loss of the wild-type allele and none displayed a BRCA mutational signature. No highly recurrently mutated gene was identified, but pathogenic mutations in driver genes (TP53, PIK3CA, NF1 and NCOR1) were found. Citation Format: Salvatore Piscuoglio, Charlotte KY Ng, Y Hannah Wen, Arto Mannermaa, Paolo Peterlongo, Carlo Tondini, Marketa Janatova, Teo Soo Hwang, Pei-Sze Ng, Lai-Meng Looi, William Foulkes, Georgia Chenevix-Trench, Britta Weigelt, Melissa C. Southey, Marc Tischkowitz, Jorge S. Reis-Filho, PALB2 Interest Group. Mutational landscape of breast cancers from PALB2 germline mutation carriers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 134.

Abstract 100: The landscape of somatic genetic alterations in BRCA1 and BRCA2 breast cancers

Abstract Background: BRCA1 or BRCA2 germline mutations account for a substantial proportion of hereditary breast cancers. The majority of BRCA1-associated breast cancers are of triple-negative phenotype (estrogen receptor (ER)-, progesterone receptor (PR)- and HER2-negative) and harbor TP53 somatic mutations. BRCA2-associated cancers are less homogeneous and often ER-positive. Systematic analyses of the profiles of somatic genetic alterations in BRCA1- and BRCA2-breast cancers have yet to be reported. Here we sought to determine the repertoire of somatic mutations and copy number alterations (CNAs) in breast cancers occurring in patients with BRCA1 or BRCA2 germline mutations.. Methods: Eleven BRCA1- and five BRCA2-associated breast cancers were microdissected. DNA was extracted from microdissected frozen tumor-normal pairs and subjected to targeted capture massively parallel sequencing using the MSK-IMPACT platform, which includes all exons and selected introns and regulatory regions of 410 key cancer genes. Somatic single nucleotide variants, small insertions and deletions, CNAs and the cancer cell fraction (CCF) of each alteration were defined using state-of-the-art bioinformatics algorithms. Results: 82% (9/11) and 18% (2/11) of BRCA1-breast cancers were triple-negative and ER-positive/HER2-negative, respectively. All BRCA2-cancers were ER-positive, of which one was HER2-positive. Sequencing analysis revealed a median of four (2-11) and two (0-6) non-synonymous somatic mutations in BRCA1- and BRCA2-breast cancers, respectively. Within BRCA1-breast cancers all but one (10/11, 91%) harbored TP53 clonal somatic mutations, and clonal somatic loss of the BRCA1 wild-type allele was found in 8 of these cases. The BRCA1-breast cancer lacking these alterations was ER-positive and the only case harboring a PIK3CA mutation. Additional clonal mutations identified in BRCA1-breast cancers included somatic mutations affecting EGFR and RB1. Subclonal mutations in known cancer genes (e.g. GATA3 and PTEN) were also identified, suggesting intra-tumor genetic heterogeneity. All BRCA2-breast cancers analyzed displayed clonal loss of the wild-type of BRCA2, but no gene was found to be recurrently mutated. Conclusions: BRCA1- and BRCA2-breast cancers are both characterized by clonal inactivation of the BRCA1 and BRCA2 wild-type alleles, respectively, which likely constitute early genetic events. Within BRCA1-breast cancers, TP53 mutations are highly recurrent and clonal, and may precede somatic loss of the BRCA1 wild-type allele, as the latter was subclonal in one case harboring a clonal TP53 somatic mutation. Citation Format: Gabriel S. Macedo, Kathleen A. Burke, Salvatore Piscuoglio, Charlotte K. Y. Ng, Felipe C. Geyer, Luciano G. Martelotto, Anastasios D. Papanastatiou, Maria R. De Filippo, Anne M. Schultheis, Edi Brogi, Mark E. Robson, Hannah Y. Wen, Britta Weigelt, Jorge S. Reis-Filho. The landscape of somatic genetic alterations in BRCA1 and BRCA2 breast cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 100.

Abstract P6-07-04: Distinct repertoires of somatic mutations affecting driver genes in mucinous and neuroendocrine carcinomas of the breast

Abstract Introduction: Mucinous carcinoma of the breast (MCB) is a rare histologic type, which accounts for approximately 2% of all invasive breast cancers (IBCs) and is characterized by clusters of tumor cells floating in large amounts of extracellular mucin. MCB comprises two main subtypes based on architectural and cytologic features: mucinous A (paucicellular) and mucinous B (hypercellular). Although MCBs are low-grade ER-positive/HER2-negative tumors of luminal molecular subtype, these cancers lack concurrent losses of 16q and gains of 1q, the hallmark genetic features of low-grade ER+/HER2- breast cancers, and have low levels of genetic instability. Neuroendocrine carcinoma of the breast (NCB) accounts for 2% - 5% of IBCs and displays morphologic features similar to those of neuroendocrine tumors of other organs. Previous transcriptomic analyses have revealed that NCBs and mucinous B, but not mucinous A, breast cancers display similar gene expression profiles. The aims of this study were to determine whether MCBs and NCBs share a similar repertoire of somatic genetic alterations and if these aberrations are distinct from those reported in common forms of ER+/HER2- IBCs. Material and methods: DNA extracted from microdissected MCBs (n=7 mucinous A, n=6 mucinous B), NCBs (n=14) and adjacent normal tissues were subjected to massively parallel sequencing targeting all exons of 254 genes most frequently mutated in IBC or related to DNA repair. Somatic point mutations were identified using MuTect and somatic insertions and deletions (indels) were defined using Strelka and Varscan2. We retrieved mutations in the same 254 genes in common forms of ER+/HER2- IBC (n=252) from The Cancer Genome Atlas (TCGA). Results: The most frequently mutated genes in MCBs were GATA3 (31% of cases, 4/13, all frame-shift indels), followed by KMT2C (MLL3) and MAP3K1 (both 23%). GATA3 and KMT2C (29%) were the most frequently mutated genes in mucinous A cancers, whereas MAP3K1 (33%) was the most frequently mutated gene in mucinous B cancers. ARID1A mutations were found in three of 14 (21%) NCBs, of which 2 were truncating mutations. A comparative analysis of the repertoire of somatic mutations found in mucinous A, mucinous B and NCBs did not reveal any statistically significant differences. As compared to common forms of ER+/HER2- IBCs, MCBs were found to have a significantly lower frequency of PIK3CA (8% vs 42%, p=0.02) mutations, which was particularly evident in mucinous A cancers (0% vs 42%, p=0.04). NCBs displayed significantly higher frequencies of somatic mutations affecting ARID1A (21% vs 2%, respectively, p=0.006), FOXA1 (14% vs 2%, respectively, p<0.05) and a lower frequency of PIK3CA somatic mutations (14% vs 42%, respectively, p<0.05) than common forms of ER+/HER2- IBCs. Conclusion: The frequency of mutations affecting bona fide breast cancer genes differed among mucinous A, mucinous B and NCBs. The repertoire of somatic mutations found in MCBs and NCBs differed from that of common forms of ER+/HER2- IBCs, in particular by the low frequency of somatic mutations affecting PIK3CA. Citation Format: Piscuoglio S, Ng CKY, Marchio C, Eberle CA, Guerini-Rocco E, Mariani O, Vincent-Salomon A, Reis-Filho JS, Weigelt B. Distinct repertoires of somatic mutations affecting driver genes in mucinous and neuroendocrine carcinomas of the breast. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-07-04.

Abstract S4-04: Lobular carcinoma in situ displays intra-lesion genetic heterogeneity and its progression to invasive disease involves clonal selection and variations in mutational processes

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

Abstract 3885: Mutational landscape and copy number alterations of mucinous breast carcinoma

Abstract Background: Mucinous breast carcinoma (MBC) is a special histologic type of breast cancer, comprising 2% of invasive breast carcinomas. These tumors have a distinctive histologic appearance, characterized by clusters of tumor cells floating in large amounts of extracellular mucin. MBCs are preferentially of low histologic grade and almost invariably express estrogen receptor (ER) and lack HER2 overexpression. MBCs, however, are likely to be unrelated to other forms of low-grade ER-positive/ HER-negative breast cancers, given that the hallmark features of low-grade ER-positive invasive carcinoma of no special type (IC-NST), namely 1q gains and 16q losses, are not found in MBCs. Here we sought to characterize the mutational landscape and copy number alterations (CNA) of MBCs by high-depth whole exome sequencing analysis. Material and Methods: Eighteen MBCs, of which 6 were mucinous A (paucicellular), 7 were mucinous B (hypercellular), and 5 were of mixed phenotype (mucinous admixed with adjacent IC-NST), were retrieved from the authors’ institutions. Sections from frozen blocks were microdissected to ensure a tumor cell content >60%. DNA samples extracted from tumor and matched normal tissues were subjected to high-depth (250x) whole exome sequencing. Single nucleotide variants (SNVs) were called using MuTect, and somatic insertions and deletions (indels) were called using Strelka and Varscan2. Potentially pathogenic mutations were defined using computational algorithms including PolyPhen-2, MutationTaster, Mutation Assessor, CHASM and FATHMM. Somatic CNAs were determined using VarScan2. Selected SNVs and indels were validated with Sanger sequencing. Results: A median of 30 (range 13-65) SNVs and indels were identified per MBC. The only significantly mutated gene as defined by MutSigCV (q<0.1) was GATA3, which was mutated in 39% of cases. The repertoire of somatic mutations found in MBCs was distinct from that of IC-NSTs of luminal molecular subtype analyzed in The Cancer Genome Atlas project. In fact, MBCs were found to harbor mutations in TP53 (0%) and PIK3CA (5.5%) at significantly lower frequencies than IC-NSTs of luminal subtype (19% and 40.6%, respectively; p<0.001). A comparison between mucinous subtypes (A and B) did not show any significant differences in terms of mutations and/ or CNAs, whereas mixed MBC/IC-NSTs were found to have more complex genomes. In mixed cases, the mucinous and adjacent IC-NST components shared mutations and CNAs; however, also CNAs and mutations restricted to one of the components were found. CNA analysis confirmed our previous observations that MBCs, contrary of low-grade ER-positive IC-NSTs, lack concurrent copy number gains of 1q and 16p and losses of 16q and 22q. Conclusions: The repertoire of somatic mutations and CNAs found in MBCs is distinct from that of luminal IC-NSTs, suggesting that MBCs likely evolve through genetic pathways distinct from those of low-grade ER-positive IC-NSTs. Citation Format: Salvatore Piscuoglio, Charlotte Ng, Carey A. Eberle, Elena Guerini-Rocco, Caterina Marchio, Odette Mariani, Anne Vincent-Salomon, Britta Weigelt, Jorge S. Reis-Filho. Mutational landscape and copy number alterations of mucinous breast carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3885. doi:10.1158/1538-7445.AM2015-3885

Abstract P2-03-08: Mutational landscape of metaplastic breast carcinomas

Abstract Introduction: Metaplastic breast carcinoma (MBC) is an aggressive histologic type of breast cancer, which preferentially displays a triple-negative phenotype. These tumors are characterized by the presence of malignant cells exhibiting differentiation towards squamous epithelium or mesenchymal elements, including spindle, chondroid, osseous and rhabdoid differentiation. Unlike other rare histologic types of breast cancer such as adenoid cystic and secretory carcinomas, which are underpinned by the MYB-NFIB and ETV6-NTRK3 fusion genes respectively, pathognomonic genetic alterations have not been identified in MBC. It has been suggested, however, that the frequency of PIK3CA somatic mutations would be significantly higher in MBCs than in other forms of triple-negative disease. Here we sought to characterize the mutational landscape of MBCs by means of high-depth whole exome sequencing analysis. Material and Methods: Twenty-one triple-negative MBCs were retrieved from the authors’ institutions. Representative sections from frozen blocks were microdissected to ensure tumor cell content greater than 50%. DNA samples extracted from microdissected tumor and matched peripheral blood leukocytes were subjected to high-depth (250x) whole exome sequencing on an Illumina GAIIx or HiSeq2000. Somatic point mutations were called using MuTect and somatic insertions and deletions (indels) were called using Strelka, Varscan2 and Haplotype Caller. Potentially pathogenic mutations were predicted using computational algorithms including PolyPhen-2, Mutation Taster, Mutation Assessor, CHASM and FATHMM. Significantly mutated genes were identified using MutSigCV. Pathway and network enrichment analysis of mutations was performed with Ingenuity Pathway Analysis and HOTNET. The genomic landscape of MBCs was compared with that of triple-negative breast cancers (TNBCs) analyzed as part of The Cancer Genome Atlas project. Results: A mean of 135 somatic non-synonymous point mutations and indels were identified per MBC. The most frequently mutated gene was TP53, found in 12/21 cases (57%), and the only significantly mutated gene as defined by MutSigCV (q<0.01). The repertoire of somatic mutations found in MBCs was qualitatively similar to that of TNBCs of no special type, and recurrently mutated genes were altered at similar frequencies in MBCs and TNBCs of no special type. When somatic mutations were annotated in pathways and networks, MBCs were found to have potentially pathogenic mutations affecting genes directly related to the PI3K pathway, including pathogenic non-synonymous mutations affecting PIK3CA, PIK3R1, PIK3R2, PIK3C2B, PIK3C2G and PTEN, significantly more frequently than TNBCs of no special type (10 out of 21 MBCs vs. 11 out of 62 TNBCs; Fisher's exact test p-value=0.0099). Conclusion: The majority (57%) of MBCs harbored non-synonymous mutations affecting TP53. While the frequencies of mutations affecting recurrently mutated genes in MBCs are similar to those found in other forms of TNBCs, MBCs significantly more frequently harbor mutations affecting PI3K pathway-related genes than TNBCs of no special type. Citation Format: Charlotte KY Ng, Britta Weigelt, Salvatore Piscuoglio, Y Hannah Wen, Maria R De Filippo, Luciano G Martelotto, Rachael Natrajan, Raymond Lim, Edi Brogi, Larry Norton, Anne Vincent-Salomon, Jorge S Reis-Filho. Mutational landscape of metaplastic breast carcinomas [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-03-08.

Abstract S6-06: The genomic landscape of male breast cancers

Abstract Background: Male breast cancer (MaBC) accounts for <1% of all breast cancers and its genomic landscape has yet to be characterized. The majority of MaBCs are ER-positive invasive ductal carcinomas of no special type and, unlike female breast cancers (FBC), rarely display HER2 gene amplification or a triple-negative phenotype. Given the relative rarity of MaBCs, treatment decisions for MaBC patients are often extrapolated from trials carried out with FBC patients. Here we employed targeted capture massively parallel sequencing to define the repertoire of somatic mutations and gene copy number alterations (CNAs) in MaBCs. Methods: Subtyping of the 64 MaBCs included in this study was performed by means of immunohistochemistry using the definitions described in the latest St. Gallen’s consensus report. DNA extracted from microdissected tumor and adjacent normal tissue were subjected to massively parallel targeting sequencing of all exons of 273 genes most frequently mutated in FBCs or directly related to DNA repair. Somatic mutations were defined using a combination of MuTect, SomaticSniper, MutationSeq and Haplotype Caller. Selected mutations were validated with Sequenom MassARRAY. CNAs were identified using Varscan2 and GISTIC2.0. Pathway and network analysis of mutations/CNAs was performed using Ingenuity Pathway Analysis and HOTNET. The genomic landscape of MaBCs was compared with that of FBCs of the same subtype analyzed as part of The Cancer Genome Atlas project. Results: All MaBCs were ER-positive and HER2-negative. Using the St. Gallen’s criteria, 37.5% and 62.5% were classified as luminal A-like or luminal B-like, respectively. The genes most frequently mutated in MaBCs were PIK3CA, GATA3, FLG and PLEC, with PIK3CA being the only significantly mutated gene as defined by MutSigCV (q=0.003). CNA analysis revealed recurrent gains of 1q and 8q and loss of 16q. GISTIC2.0 identified significantly recurrent high-level amplifications in 1q25.3, 8p11 (FGFR1, ZNF703), 8q24.3 (DEPTOR), 17q23 (PPM1D) and 15q26 (IGF1R) and deletions in 11q22 (ATM) and 21q22.12 (RUNX1). HOTNET analysis of the genes mutated and/or targeted by gene amplifications in MaBCs revealed significantly altered subnetworks involving genes related to DNA repair, PI3K and FGF signaling pathways. The most frequently mutated genes in luminal A-like MaBCs were PIK3CA and MLL3, while those of luminal B-like MaBCs were PIK3CA and GATA3. MLL3 and GATA3 mutations were only found in luminal A-like MaBCs (p=0.039) and luminal B-like MaBCs (p=0.048), respectively. Although the mutational landscapes of MaBCs and luminal FBCs were qualitatively similar, PIK3CA and TP53 were less frequently mutated in MaBCs (p<0.001), whereas genes found to be recurrently mutated in FBCs, such as MAP2K4 and NCOR1, were not mutated in MaBCs. Conclusions: MaBCs are preferentially of luminal subtype and are characterized by recurrent mutations in PIK3CA, GATA3, FLG and PLEC. Genetic alterations in MaBCs often target DNA repair and FGF signaling pathways. The known drivers of luminal FBCs appear to be less frequently altered in MaBCs. Given these important differences between MaBCs and FBCs, caution should be exercised in the extrapolation of biologic and clinical implications from studies in FBCs to the management of MaBCs. Citation Format: Salvatore Piscuoglio, Melissa Murray, Charlotte KY Ng, Elena Guerini Rocco, Luciano G Martelotto, Francois-Clement Bidard, Carey A Eberle, Nicola Fusco, Rita A Sakr, Leticia De Mattos-Arruda, Raymond Lim, Timour Baslan, James Hicks, Tari A King, Edi Brogi, Larry Norton, Britta Weigelt, Clifford A Hudis, Jorge S Reis-Filho. The genomic landscape of male breast cancers [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S6-06.

Abstract 927: Targeted capture next generation sequencing of fresh frozen lobular carcinoma in situ and invasive lobular cancer identifies a common repertoire of mutations

Abstract INTRODUCTION: Epidemiologic data suggest that lobular carcinoma in situ (LCIS) is a risk factor for invasive breast cancer, whereas analyses of copy number aberrations between synchronous LCIS and invasive lobular carcinoma (ILC) have provided evidence to support its role as a non-obligate precursor of ILC. Using fresh frozen (FF) tissue samples, we sought to define the repertoire of somatic mutations in LCIS and ILC, and to compare the mutational repertoire of synchronous LCIS and ILC, and synchronous foci of LCIS. METHODS: Patients with a biopsy proven history of LCIS, with or without ILC, undergoing surgery were prospectively enrolled on an IRB approved study and FF tissue blocks were collected. Representative lesions of LCIS and ILC were microdissected and DNA was extracted using QIAamp® DNA Micro kit (Qiagen®). Matched normal germline DNA or FF normal ducts were available for all cases. At least 50 ng of DNA was used for hybridization capture using a bait library (NimbleGen®) comprising 273 genes, either recurrently mutated in breast cancer or associated with DNA repair. Targeted massively parallel sequencing was performed on a HiSeq2000 (Illumina®). Sequencing data were aligned to the reference human genome hg19 and processed by GATK. Single nucleotide variants (SNVs) were defined on the basis of three mutation callers (i.e. Mutect, Mutationseq and GATK Haplotypecaller); insertions and deletions were identified using the GATK Haplotypecaller. RESULTS: 47 samples of LCIS and 21 samples of ILC from 30 patients (with matched normal samples) were qualified for data analysis. The constellation of somatic mutations found in LCIS and ILC was similar, with the most frequently mutated genes being PIK3CA, CDH1, HRNR and MAP3K1 found in 25% and 52%, 23% and 38%, 15% and 19% and 10% and 9% of samples, respectively. Paired analysis of synchronous LCIS and ILCs (n=21) revealed that PIK3CA and CDH1 mutations were shared in 24% and 28% of cases respectively. Overall, in 8 (38%) pairs, the LCIS and ILC had one identical mutation in common and in 7 (33%) pairs, there were two or more common identical mutations. In 5 pairs, there were no common mutations. Paired analysis of two independent foci of LCIS from 9 breasts revealed one common mutation in each of 4 (44%) pairs (CDH1, RUNX1, SPEN, ERBB2). Interestingly, the point mutation in ERBB2 (L755S) was found in two LCIS foci from a patient who underwent prophylactic mastectomy. CONCLUSION: The mutational spectrum of LCIS is highly similar to that of ILC. PIK3CA mutations were the most common somatic mutations in both LCIS and ILC. Further, in this study, 71% of LCIS-ILC pairs shared at least one identical mutation, providing strong circumstantial evidence that LCIS is a non-obligate precursor of ILC. The finding of shared mutations among independent foci of LCIS also raises questions about the clonal origin of these lesions. Citation Format: Rita A. Sakr, Jose V. Scarpa, Michael Schizas, Dilip Giri, Marina De Brot, Russell Towers, Charlotte KY Ng, Raymond Lim, Victor P. Andrade, Britta Weigelt, Jorge S. Reis-Filho, Tari A. King. Targeted capture next generation sequencing of fresh frozen lobular carcinoma in situ and invasive lobular cancer identifies a common repertoire of mutations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 927. doi:10.1158/1538-7445.AM2014-927