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Mosaic mutations in normal tissues can occur early in embryogenesis and be associated with hereditary cancer syndromes when affecting cancer susceptibility genes (CSG). Their contribution to apparently sporadic cancers is currently unknown. Analysis of paired tumor/blood sequencing data of 35,310 patients with cancer revealed 36 pathogenic mosaic variants affecting CSGs, most of which were not detected by prior clinical genetic testing. These CSG mosaic variants were consistently detected at varying variant allelic fractions in microdissected normal tissues (n = 48) from distinct embryonic lineages in all individuals tested, indicating their early embryonic origin, likely prior to gastrulation, and likely asymmetrical propagation. Tumor-specific biallelic inactivation of the CSG affected by a mosaic variant was observed in 91.7% (33/36) of cases, and tumors displayed the hallmark pathologic and/or genomic features of inactivation of the respective CSGs, establishing a causal link between CSG mosaic variants arising in early embryogenesis and the development of apparently sporadic cancers.Significance:Here, we demonstrate that mosaic variants in CSGs arising in early embryogenesis contribute to the oncogenesis of seemingly sporadic cancers. These variants can be systematically detected through the analysis of tumor/normal sequencing data, and their detection may affect therapeutic decisions as well as prophylactic measures for patients and their offspring.See related commentary by Liggett and Sankaran, p. 889.This article is highlighted in the In This Issue feature, p. 873

Supplementary Data from Cancer-Causative Mutations Occurring in Early Embryogenesis

Purpose:Ductal carcinoma in situ (DCIS) is a nonobligate precursor of invasive breast cancer. Here, we sought to investigate the level of intralesion genetic heterogeneity in DCIS and the patterns of clonal architecture changes in the progression from DCIS to invasive disease.Experimental Design:Synchronous DCIS (n = 27) and invasive ductal carcinomas of no special type (IDC-NSTs; n = 26) from 25 patients, and pure DCIS (n = 7) from 7 patients were microdissected separately and subjected to high-depth whole-exome (n = 56) or massively parallel sequencing targeting ≥410 key cancer-related genes (n = 4). Somatic genetic alterations, mutational signatures, clonal composition, and phylogenetic analyses were defined using validated computational methods.Results:DCIS revealed genetic alterations similar to those of synchronously diagnosed IDC-NSTs and of non-related IDC-NSTs from The Cancer Genome Atlas (TCGA), whereas pure DCIS lacked PIK3CA mutations. Clonal decomposition and phylogenetic analyses based on somatic mutations and copy number alterations revealed that the mechanisms of progression of DCIS to invasive carcinoma are diverse, and that clonal selection might have constituted the mechanism of progression from DCIS to invasive disease in 28% (7/25) of patients. DCIS displaying a pattern of clonal selection in the progression to invasive cancer harbored higher levels of intralesion genetic heterogeneity than DCIS where no clonal selection was observed.Conclusions:Intralesion genetic heterogeneity is a common feature in DCIS synchronously diagnosed with IDC-NST. DCIS is a nonobligate precursor of IDC-NST, whose mechanisms of progression to invasive breast cancer are diverse and vary from case to case.

Supplementary Figure S3 shows the copy number profiles of ductal carcinomas in situ (DCIS) and invasive ductal carcinomas of no special type (IDC-NST), and frequency of most commonly mutated genes in DCIS, compared to luminal B IDC-NSTs, and according to ER/HER2 status.

Supplementary Figure S1. ER, PR, and HER2 mRNA and protein levels across endocrine-sensitive (parental) and endocrine-resistant BC cell lines. Supplementary Figure S2. Reduced sensitivity to CDK4/6 inhibition is associated with IFN-signaling in ER+ and endocrine-resistant derivative BC cell lines. Supplementary Figure S3. The cyclin D-CDK4/6-Rb axis is altered in ER+ cell lines with acquired resistance to palbociclib. Supplementary Figure S4. Effect of acute IFN-signaling activation on sensitivity to palbociclib in ER+ BC cell lines. Supplementary Figure S5. Proteomic profiles of MCF7 P, MCF7 EDR, and their PalboR derivatives. Supplementary Figure S6. Relationship between IFN-signaling and Rb status in ER+/HER2- BC. Supplementary Figure S7. DNMT1 mRNA levels in palbociclib-sensitive and PalboR MCF7 and T47D cell lines. Supplementary Figure S8. Mutations of DNA damage response genes are not associated with IFN-signaling in ER+ BC. Supplementary Figure S9. Levels of ER and ER signalling in palbociclib-sensitive and PalboR MCF7 and T47D cell lines. Supplementary Figure S10. HALLMARK gene sets significantly up-regulated in ER+/HER2- BC patients stratified by the status (+/-) of RBsig and IRPS. Supplementary Figure S11. Impact of selected IRPS genes on relapse-free survival of ER+ BC patients.

Supplementary Table S2 shows the non-synonymous somatic mutations identified in synchronous DCIS and IDC-NSTs subjected to whole-exome sequencing or MSK-IMPACT targeted massively parallel sequencing and/or targeted amplicon sequencing.

Supplementary Table S1. Details of antibodies used in this study. Supplementary Table S2. Q-PCR primers. Supplementary Table S3. HALLMARK gene-set enrichment analysis (GSEA) of baseline tumor samples from the NeoPalANA trial. Supplementary Table S4. Palbociclib IC50 values in MCF7 P, MCF7 EDR, T47D P-LM, and T47D EDR palbociclib-resistant (PalboR)-LM cell lines. Supplementary Table S5. Top-20 Gene Ontology (GO) terms significantly enriched in PalboR cell lines. Supplementary Table S6. HALLMARK gene sets significantly up-regulated in cell lines with acquired resistance to palbociclib. Supplementary Table S7. RPPA of MCF7 P, MCF7 EDR, and their PalboR derivatives.

Supplemental Materials and Methods and Materials present a complete description of methods, reagents, and statistics required to reproduce the experiments in the paper.

Table S5: Genes in the chr6q23-24 region, association with CN levels and pCR.

Purpose:The heterogeneity of response to anti-HER2 agents represents a major challenge in patients with HER2-positive breast cancer. To better understand the sensitivity and resistance to trastuzumab and lapatinib, we investigated the role of copy number aberrations (CNA) in predicting pathologic complete response (pCR) and survival outcomes in the NeoALTTO trial.Experimental Design:The neoadjuvant phase III NeoALTTO trial enrolled 455 patients with HER2-positive early-stage breast cancer. DNA samples from 269 patients were assessed for genome-wide copy number profiling. Recurrent CNAs were found with GISTIC2.0.Results:CNA estimates were obtained for 184 patients included in NeoALTTO. Among those, matched transcriptome and whole-exome data were available for 154 and 181 patients, respectively. A significant association between gene copy number and pCR was demonstrated for ERBB2 amplification. Nevertheless, ERBB2 amplification ceased to be predictive once ERBB2 expression level was considered. GISTIC2.0 analysis revealed 159 recurrent CNA regions. Lower copy number levels of the 6q23-24 locus predicted absence of pCR in the whole cohort and in the estrogen receptor–positive subgroup. 6q23-24 deletion was significantly more frequent in TP53 wild-type (WT) compared with TP53-mutated, resulting in copy number levels significantly associated with lack of pCR only in the TP53 WT subgroup. Interestingly, a gene-ontology analysis highlighted several immune processes correlated to 6q23-24 copy number.Conclusions:Our analysis identified ERBB2 copy number as well as 6q23-24 CNAs as predictors of response to anti–HER2-based treatment. ERBB2 expression outperformed ERBB2 amplification. The complexity of the 6q23-24 region warrants further investigation.

Purpose:Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (CDK4/6i) are highly effective against estrogen receptor–positive (ER+)/HER2− breast cancer; however, intrinsic and acquired resistance is common. Elucidating the molecular features of sensitivity and resistance to CDK4/6i may lead to identification of predictive biomarkers and novel therapeutic targets, paving the way toward improving patient outcomes.Experimental Design:Parental breast cancer cells and their endocrine-resistant derivatives (EndoR) were used. Derivatives with acquired resistance to palbociclib (PalboR) were generated from parental and estrogen deprivation–resistant MCF7 and T47D cells. Transcriptomic and proteomic analyses were performed in palbociclib-sensitive and PalboR lines. Gene expression data from CDK4/6i neoadjuvant trials and publicly available datasets were interrogated for correlations of gene signatures and patient outcomes.Results:Parental and EndoR breast cancer lines showed varying degrees of sensitivity to palbociclib. Transcriptomic analysis of these cell lines identified an association between high IFN signaling and reduced CDK4/6i sensitivity; thus an “IFN-related palbociclib-resistance Signature” (IRPS) was derived. In two neoadjuvant trials of CDK4/6i plus endocrine therapy, IRPS and other IFN-related signatures were highly enriched in patients with tumors exhibiting intrinsic resistance to CDK4/6i. PalboR derivatives displayed dramatic activation of IFN/STAT1 signaling compared with their short-term treated or untreated counterparts. In primary ER+/HER2− tumors, the IRPS score was significantly higher in lumB than lumA subtype and correlated with increased gene expression of immune checkpoints, endocrine resistance, and poor prognosis.Conclusions:Aberrant IFN signaling is associated with intrinsic resistance to CDK4/6i. Experimentally, acquired resistance to palbociclib is associated with activation of the IFN pathway, warranting additional studies to clarify its involvement in resistance to CDK4/6i.

Table S1: Clinicopathological characteristics of the patients. Table S2: List of 49 genes associated with CNAs in breast cancer from the COSMIC Cancer Gene Census database. Table S3: Gene ontology analysis of the genes correlated with the genome instability index. Table S4: Gene ontology analysis of the genes anti-correlated with the genome instability index.

Purpose: We sought to determine whether sequencing analysis of circulating cell-free DNA (cfDNA) in patients with prospectively accrued endometrial cancer captures the mutational repertoire of the primary lesion and allows for disease monitoring. Experimental Design: Peripheral blood was prospectively collected from 44 newly diagnosed patients with endometrial cancer over a 24-month period (i.e., baseline, postsurgery, every 6 months after). DNA from the primary endometrial cancers was subjected to targeted next-generation sequencing (NGS) of 468 cancer-related genes, and cfDNA to a high-depth NGS assay of 129 genes with molecular barcoding. Sequencing data were analyzed using validated bioinformatics methods. Results: cfDNA levels correlated with surgical stage in endometrial cancers, with higher levels of cfDNA being present in advanced-stage disease. Mutations in cfDNA at baseline were detected preoperatively in 8 of 36 (22%) patients with sequencing data, all of whom were diagnosed with advanced-stage disease, high tumor volume, and/or aggressive histologic type. Of the 38 somatic mutations identified in the primary tumors also present in the cfDNA assay, 35 (92%) and 38 (100%) were detected at baseline and follow-up, respectively. In 6 patients with recurrent disease, changes in circulating tumor DNA (ctDNA) fraction/variant allele fractions in cfDNA during follow-up closely mirrored disease progression and therapy response, with a lead time over clinically detected recurrence in two cases. The presence of ctDNA at baseline (P < 0.001) or postsurgery (P = 0.014) was significantly associated with reduced progression-free survival. Conclusions: cfDNA sequencing analysis in patients with endometrial cancer at diagnosis has prognostic value, and serial postsurgery cfDNA analysis enables disease and treatment response monitoring. See related commentary by Grant et al., p. 305

Purpose: The heterogeneity of response to anti-HER2 agents represents a major challenge in patients with HER2-positive breast cancer. To better understand the sensitivity and resistance to trastuzumab and lapatinib, we investigated the role of copy number aberrations (CNA) in predicting pathologic complete response (pCR) and survival outcomes in the NeoALTTO trial. Experimental Design: The neoadjuvant phase III NeoALTTO trial enrolled 455 patients with HER2-positive early-stage breast cancer. DNA samples from 269 patients were assessed for genome-wide copy number profiling. Recurrent CNAs were found with GISTIC2.0. Results: CNA estimates were obtained for 184 patients included in NeoALTTO. Among those, matched transcriptome and whole-exome data were available for 154 and 181 patients, respectively. A significant association between gene copy number and pCR was demonstrated for ERBB2 amplification. Nevertheless, ERBB2 amplification ceased to be predictive once ERBB2 expression level was considered. GISTIC2.0 analysis revealed 159 recurrent CNA regions. Lower copy number levels of the 6q23-24 locus predicted absence of pCR in the whole cohort and in the estrogen receptor–positive subgroup. 6q23-24 deletion was significantly more frequent in TP53 wild-type (WT) compared with TP53-mutated, resulting in copy number levels significantly associated with lack of pCR only in the TP53 WT subgroup. Interestingly, a gene-ontology analysis highlighted several immune processes correlated to 6q23-24 copy number. Conclusions: Our analysis identified ERBB2 copy number as well as 6q23-24 CNAs as predictors of response to anti–HER2-based treatment. ERBB2 expression outperformed ERBB2 amplification. The complexity of the 6q23-24 region warrants further investigation.

Inherited mutations in BRCA1, BRCA2, and PALB2 cause a high risk of breast cancer. Here, we conducted parallel conditional knockout (CKO) of Brca1, Palb2, and Brca2, individually and in combination, along with one copy of Trp53, in the mammary gland of nulliparous female mice. We observed a functional equivalence of the three genes in their basic tumor-suppressive activity, a linear epistasis of Palb2 and Brca2, but complementary roles of Brca1 and Palb2 in mammary tumor suppression, as combined ablation of either Palb2 or Brca2 with Brca1 led to delayed tumor formation. Whole-exome sequencing (WES) revealed both similarities and differences between Brca1 and Palb2 or Brca2 null tumors. Analyses of mouse mammary glands and cultured human cells showed that combined loss of BRCA1 and PALB2 led to high levels of reactive oxygen species (ROS) and increased apoptosis, implicating oxidative stress in the delayed tumor development in Brca1;Palb2 double CKO mice. The functional complementarity between BRCA1 and PALB2/BRCA2 and the role of ROS in tumorigenesis require further investigation.

Purpose: Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (CDK4/6i) are highly effective against estrogen receptor–positive (ER+)/HER2− breast cancer; however, intrinsic and acquired resistance is common. Elucidating the molecular features of sensitivity and resistance to CDK4/6i may lead to identification of predictive biomarkers and novel therapeutic targets, paving the way toward improving patient outcomes. Experimental Design: Parental breast cancer cells and their endocrine-resistant derivatives (EndoR) were used. Derivatives with acquired resistance to palbociclib (PalboR) were generated from parental and estrogen deprivation–resistant MCF7 and T47D cells. Transcriptomic and proteomic analyses were performed in palbociclib-sensitive and PalboR lines. Gene expression data from CDK4/6i neoadjuvant trials and publicly available datasets were interrogated for correlations of gene signatures and patient outcomes. Results: Parental and EndoR breast cancer lines showed varying degrees of sensitivity to palbociclib. Transcriptomic analysis of these cell lines identified an association between high IFN signaling and reduced CDK4/6i sensitivity; thus an “IFN-related palbociclib-resistance Signature” (IRPS) was derived. In two neoadjuvant trials of CDK4/6i plus endocrine therapy, IRPS and other IFN-related signatures were highly enriched in patients with tumors exhibiting intrinsic resistance to CDK4/6i. PalboR derivatives displayed dramatic activation of IFN/STAT1 signaling compared with their short-term treated or untreated counterparts. In primary ER+/HER2− tumors, the IRPS score was significantly higher in lumB than lumA subtype and correlated with increased gene expression of immune checkpoints, endocrine resistance, and poor prognosis. Conclusions: Aberrant IFN signaling is associated with intrinsic resistance to CDK4/6i. Experimentally, acquired resistance to palbociclib is associated with activation of the IFN pathway, warranting additional studies to clarify its involvement in resistance to CDK4/6i.

Graphical abstract, Nearly half of the human genome is occupied by repetitive sequences of ancient virus-like genetic elements. The largest class, comprising 17% of the genome, belong to the type 1 Long INterspersed Elements (LINE-1) and are the only class capable of autonomous propagation in the genome. When epigenetic silencing mechanisms of LINE-1 fail, the proteins encoded by LINE-1 engage in reverse transcription to make new copies of their own or other DNAs that are pasted back into the genome. To elucidate how LINE-1 is dysregulated as a result of carcinogen exposure, we developed a computational model of key elements in the LINE-1 lifecycle, namely, the role of cytosolic ribonuclease (RNase), RNA interference (RNAi) by the antisense ORF0 RNA, and sequestration of LINE-1 products into stress granules and multivesicular structures. The model showed that when carcinogen exposure is represented as either a sudden increase in LINE-1 mRNA count, or as an increase in mRNA transcription rate, the retrotransposon copy number exhibits a distinct threshold behavior above which LINE-1 enters a positive feedback loop that allows the cDNA copy number to grow exponentially. We also found that most of the LINE-1 RNA was degraded via the RNAase pathway and that neither ORF0 RNAi, nor the sequestration of LINE-1 products into granules and multivesicular structures, played a significant role in regulating the retrotransposon’s life cycle. Several aspects of the prediction agree with experimental results and indicate that the model has significant potential to inform future experiments related to LINE-1 activation.

Histologic special types of breast cancer (BC) account for ~20% of BCs. Large sequencing studies of metastatic BC have focused on invasive ductal carcinomas of no special type (IDC-NSTs). We sought to define the repertoire of somatic genetic alterations of metastatic histologic special types of BC. We reanalyzed targeted capture sequencing data of 309 special types of BC, including metastatic and primary invasive lobular carcinomas (ILCs; n = 132 and n = 127, respectively), mixed mucinous (n = 5 metastatic and n = 14 primary), micropapillary (n = 12 metastatic and n = 8 primary), and metaplastic BCs (n = 6 metastatic and n = 5 primary), and compared metastatic histologic special types of BC to metastatic IDC-NSTs matched according to clinicopathologic characteristics and to primary special type BCs. The genomic profiles of metastatic and primary special types of BC were similar. Important differences, however, were noted: metastatic ILCs harbored a higher frequency of genetic alterations in TP53, ESR1, FAT1, RFWD2, and NF1 than primary ILCs, and in CDH1, PIK3CA, ERBB2, TBX3, NCOR1, and RFWD2 than metastatic IDC-NSTs. Metastatic ILCs displayed a higher mutational burden, and more frequently dominant APOBEC mutational signatures than primary ILCs and matched metastatic IDC-NSTs. ESR1 and NCOR mutations were frequently detected in metastatic mixed mucinous BCs, whereas PIK3CA and TP53 were the most frequently altered genes in metastatic micropapillary and metaplastic BCs, respectively. Taken together, primary and metastatic BCs histologic special types have remarkably similar repertoires of somatic genetic alterations. Metastatic ILCs more frequently harbor APOBEC mutational signatures than primary ILCs and metastatic IDC-NSTs.

Alpelisib is a selective inhibitor of PI3Kα, shown to improve outcomes for PIK3CA mutant, hormone receptor positive (HR+) metastatic breast cancers (MBC) when combined with antiestrogen therapy. To uncover mechanisms of resistance, we conducted a detailed, longitudinal analysis of tumor and plasma circulating tumor DNA among such patients from a phase I/II trial combining alpelisib with an aromatase inhibitor (AI) (NCT01870505). The trial's primary objective was to establish safety with maculopapular rash emerging as the most common grade 3 adverse event (33%). Among 44 evaluable patients, the observed clinical benefit rate was 52%. Correlating genetic alterations with outcome, we identified loss-of-function PTEN mutations in 25% of patients with resistance. ESR1 activating mutations also expanded in number and allele fraction during treatment and were associated with resistance. These data indicate that genomic alterations that mediate resistance to alpelisib or antiestrogen may promote disease progression and highlight PTEN loss as a recurrent mechanism of resistance to PI3Kα inhibition.

Introduction: Germline pathogenic variants in genes in the homologous recombination (HR) pathway such as BRCA1, BRCA2, ATM, PALB2, RAD51C and RAD51D confer an increased risk of breast and ovarian cancers. Screening for germline variants in these cancer susceptibility genes is critical as prophylactic measures and monitoring can be performed in these individuals to minimize their risk of developing breast and/or ovarian cancers. More recently, it has been recognized that cancers arising in carriers of germline pathogenic (P)/ likely pathogenic (LP) variants in HR genes often show a homologous recombination (HR) deficient (HRD) phenotype and can be targeted with platinum agents or PARP inhibitors. RAD51B is a RAD51 paralog that binds to RAD51C and functions as a heterodimer in the HR pathway. Whilst RAD51B germline variants have been reported in isolated cases of breast and ovarian cancers, it is unclear as to whether RAD51B P/LP germline variants would confer predisposition to these cancer types. Materials and Methods: We screened 9,287 consecutive unselected cancer patients who consented for both tumor and germline testing using the MSK-IMPACT platform for the presence of RAD51B germline truncating variants. Selected RAD51B germline mutant breast cancers identified by MSK-IMPACT were subjected to whole-exome sequencing (WES) analysis to determine the dominant mutational signatures using DeconstructSigs. Functional assays were performed to ascertain the impact of RAD51B loss on HR DNA repair and PARP inhibitor sensitivity using genome editing methods in non-malignant breast epithelial cell lines. Results: Out of the 9,287 cancer patients, we detected likely pathogenic loss of function RAD51B germline variants in 11 patients (0.12%), which was similar to the frequency detected in the gnomAD database (0.09%). All female carriers of RAD51B loss of function variants (n=8) had breast or ovarian cancers (8/1,619 breast or ovarian cancers, 0.5%). The observed carrier frequencies of germline truncating variants in RAD51B was statistically enriched in breast and ovarian cancer patients compared to individuals in the gnomAD database (0.5% vs 0.09% P=0.0003; odds ratio = 5.06 (95% CI: 2.1-10.3)). Although segregation studies were not available, 9/11 of the RAD51B germline loss of function variant carriers had a personal or family history of breast or ovarian cancers. All five breast and ovarian cancers from RAD51B mutation carriers investigated by WES were found to harbor RAD51B bi-allelic inactivation through loss of heterozygosity of the RAD51B wild-type allele. In addition, these five cases were found to display genomic features of HRD including high large-scale state transition scores and a dominant mutational signature 3. CRISPR/Cas9 genome editing of RAD51B in a non-malignant breast epithelial cell model revealed that RAD51B deficient cells display PARP inhibitor sensitivity similar to that reported in BRCA1 and BRCA2 deficient cell lines. Conclusion: RAD51B loss-of-function germline variants confer susceptibility to breast and ovarian cancer development. Breast and ovarian cancers occurring in the context of RAD51B germline mutations harbor bi-allelic inactivation of RAD51B through loss-of-heterozygosity of the wild-type allele, genomic features of HRD and are likely sensitive to PARP inhibition. Albeit rarely germline mutated, RAD51B should be considered as an addition to clinical germline testing panels for hereditary breast and ovarian cancer syndrome patients. Citation Format: Diana SReis-FilhoReis-Filho Mandelker, Semanti Mukherjee, Jeremy Setton, Pier Selenica, Yelena Kemel, Ozge Ceyhan-Birsoy, Margaret Sheehan, Kaitlyn Tkachuk, David N Brown, Simon Powell, Britta Weigelt, Mark E Robson, Nadeem Riaz, Kenneth Offit, Jorge S Reis-Filho. RAD51B loss-of-function variants confer susceptibility to hereditary breast and ovarian cancers and result in tumors with genomic features of homologous recombination repair defects [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-09-01.

Background: Male breast cancer is rare, with approximately 2,600 cases diagnosed annually in the United States. Data are scant regarding the genomics and pathophysiology of male breast cancer, especially in the metastatic setting, requiring most treatment recommendations in male breast cancer to be made by inference from breast cancer in women. Methods: We performed prospective genomic profiling of primary and metastatic tumor samples from men with breast cancer treated at Memorial Sloan Kettering Cancer Center using the MSK-IMPACT targeted-DNA-sequencing panel for somatic mutations. Comprehensive demographic, clinical, and pathologic data were collected on all included patients. Statistics are descriptive. Results: Genomic sequencing was performed on 45 samples from 41 men (31 primary samples and 14 from metastatic sites). Median age at time of sample collection was 61 years, with a range of 27-92 years. Thirty-seven (90.2%) men had ER+/HER2- breast cancer, 3 (7.3%) had ER+/HER2+ breast cancer and 1 (2.4%) had triple negative disease. Thirty-nine (95.1%) had ductal carcinoma, and no cases of lobular carcinoma were identified. Forty patients underwent germline testing, and 12 (30%) were found to have pathogenic germline mutations (6 BRCA2 mutations, 2 BRCA1 mutations [one of whom had a concurrent CHEK2 mutation], and one mutation each in PALB2, MUTYH, and MSH6). Overall, the pattern of genomic alterations in male breast cancer was similar that in women. Twelve (29.3%) patients had PIK3CA mutations, 9 (22%) had GATA3 mutations, 3 (7.3%) had TP53 mutations, 3 (7.3%) had ARID1A mutations, 3 (7.3%) had KMT2C mutations, 2 (4.9%) had FOX1A mutations, 2 (4.9%) had RB1 mutations, and 2 (4.9%) had TERT promoter hotspot mutations. Eleven (26.8%) patients had CCND1 amplification, 8 (19.5%) had MYC amplification, 6 (14.6%) had FGFR1 amplification, and 5 (12.2%) had MDM2 amplification. All other findings were present in ≤ 1 patient. All included patients had normal mutational burden, and all samples were microsatellite stable. PIK3CA mutations occurred in 33% of primary samples vs. 15% of metastatic samples, CCND1 amplification occurred in 23% of primary samples vs. 38% of metastatic samples, and TERT hotspot promoter mutations were found only in metastatic samples. Of note, we observed a single ESR1 D538G mutation in the metastatic sample of a patient with significant prior exposure to aromatase inhibitors in the adjuvant and metastatic settings. We further found concurrent ERBB2 mutation and amplification in the post-treatment metastatic samples of an ER+/HER2- patient, who was treated with neratinib for 14 weeks with clinical response. Lastly, we report a heavily pretreated patient with metastatic secretory breast carcinoma who was found to have an ETV6-NTRK3 fusion gene. This patient was treated with a first-generation TRK inhibitor and continues to exhibit an ongoing clinical response at 8.6 months. Conclusions: Based on our data, the overall genomic landscape of male breast cancer appears comparable to that of breast cancer in women, as has been previously reported. However, despite the small number of metastatic cases examined, several previously unreported and treatment-informing signatures were discovered, especially in those patients with less common male breast cancer variants. Further study is warranted to confirm these findings in a larger cohort. Citation Format: Joshua Z Drago, Cristian Serna-Tamayo, Carlos H Dos Anjos, David N Brown, Shanu Modi, Komal Jhaveri, David B Solit, Tiffany A Traina, Sarat Chandarlapaty, Jorge S Reis-Filho, Mark E Robson, Ayca Gucalp, Pedram Razavi. Genomic profiling of primary and metastatic breast cancer in men [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-17-01.

Estrogen receptor alpha (ERα) drives mammary gland development and breast cancer (BC) growth through an evolutionarily conserved linkage of DNA binding and hormone activation functions. Therapeutic targeting of the hormone binding pocket is a widely utilized and successful strategy for breast cancer prevention and treatment. However, resistance to this endocrine therapy is frequently encountered and may occur through bypass or reactivation of ER-regulated transcriptional programs. We now identify the induction of a novel ERα isoform, ERα-LBD, that is encoded by an alternative ESR1 transcript and lacks the activation function and DNA binding domains. Despite lacking the transcriptional activity, ERα-LBD is found to promote breast cancer growth and resistance to the ERα antagonist fulvestrant. ERα-LBD is predominantly localized to the cytoplasm and mitochondria of BC cells and leads to enhanced glycolysis, respiration and stem-like features. Intriguingly, ERα-LBD expression and function does not appear to be restricted to cancers that express full length ERα but also promotes growth of triple negative breast cancers and ERα-LBD transcript (ESR1-LBD) is also present in BC samples from both ERα(+) and ERα(−) human tumors. These findings point to ERα-LBD as a potential mediator of breast cancer progression and therapy resistance.SIGNIFICANCE STATEMENTEndocrine resistant and metastatic breast cancer (BC) is a clinically significant problem. Our study of fulvestrant resistant cancer cells led to the discovery of a novel ERα isoform which we call ERα-LBD. Encoded by a truncated transcript variant (ESR1-LBD) and lacking the N-terminal domains (activation of transcription and DNA binding), ERα-LBD displays a unique role in BC tumorigenesis and progression by mechanisms that may involve metabolic and cell growth advantages, stemness and therapy resistance. Importantly, ESR1-LBD is preferentially expressed in human breast tumor tissues and may be used as prognostic marker in BC.

Pathogenic germline mutations in the RAD51 paralog genes RAD51C and RAD51D, are known to confer susceptibility to ovarian and triple-negative breast cancer. Here, we investigated whether germline loss-of-function variants affecting another RAD51 paralog gene, RAD51B, are also associated with breast and ovarian cancer. Among 3422 consecutively accrued breast and ovarian cancer patients consented to tumor/germline sequencing, the observed carrier frequency of loss-of-function germline RAD51B variants was significantly higher than control cases from the gnomAD population database (0.26% vs 0.09%), with an odds ratio of 2.69 (95% CI: 1.4–5.3). Furthermore, we demonstrate that tumors harboring biallelic RAD51B alteration are deficient in homologous recombination DNA repair deficiency (HRD), as evidenced by analysis of sequencing data and in vitro functional assays. Our findings suggest that RAD51B should be considered as an addition to clinical germline testing panels for breast and ovarian cancer susceptibility.

Mosaic mutations in normal tissues can occur early in embryogenesis and be associated with hereditary cancer syndromes when affecting cancer susceptibility genes (CSG). Their contribution to apparently sporadic cancers is currently unknown. Analysis of paired tumor/blood sequencing data of 35,310 patients with cancer revealed 36 pathogenic mosaic variants affecting CSGs, most of which were not detected by prior clinical genetic testing. These CSG mosaic variants were consistently detected at varying variant allelic fractions in microdissected normal tissues (n = 48) from distinct embryonic lineages in all individuals tested, indicating their early embryonic origin, likely prior to gastrulation, and likely asymmetrical propagation. Tumor-specific biallelic inactivation of the CSG affected by a mosaic variant was observed in 91.7% (33/36) of cases, and tumors displayed the hallmark pathologic and/or genomic features of inactivation of the respective CSGs, establishing a causal link between CSG mosaic variants arising in early embryogenesis and the development of apparently sporadic cancers. Significance: Here, we demonstrate that mosaic variants in CSGs arising in early embryogenesis contribute to the oncogenesis of seemingly sporadic cancers. These variants can be systematically detected through the analysis of tumor/normal sequencing data, and their detection may affect therapeutic decisions as well as prophylactic measures for patients and their offspring. See related commentary by Liggett and Sankaran, p. 889. This article is highlighted in the In This Issue feature, p. 873

Background: Despite therapeutic advancements, a substantial number of patients with treated early-stage invasive lobular carcinoma (ILC) of the breast will still relapse as late as 20 years after the initial tumor diagnosis, thus making follow-up a challenging task. Here, using patient-matched primary and metastatic tumors from patients with ILC, we aimed to decipher the evolution of lobular breast cancer through time and explore metastatic dissemination events and underlying heterogeneity. Materials and Methods: We retrospectively identified 38 patients with metastatic ILC, of which 6 with de novo metastatic disease. In total, 99 formalin-fixed paraffin embedded tissue samples of primary tumor and metastasis, together with well-annotated clinicopathological data, were available. Low coverage whole-genome sequencing was performed to infer somatic copy number aberrations (CNA). Phylogenetic reconstruction was performed with Integer Linear Program for the Copy-Number Tree Problem algorithm. Results: Reconstruction of the phylogenetic trees showed different patterns of dissemination. For patients with multiple metastatic samples sequenced (4/4 patients), the metastases were found to be seeded from a common metastatic precursor. For a subset of patients with multiple primaries (5/10 patients), distant metastases were seeded from specific clones inside the primary tumor. Detailed analysis of known driver genes showed that alterations in common driver genes such as TP53, MYC and CCND1 were shared between primary tumor and metastases in a large proportion of the patients (47%, 32% and 21% of patients respectively) whilst CNA in other driver genes including FGFR2, PTEN or AKT2 among others, were frequently private in the metastases. Interestingly, we observed that in approximately 30% of the patients, the metastases harbored less CNA than the matched primary tumor, resulting in shorter metastatic branches and possibly indicating a decelerated tumor progression. Further analysis of the genomic and clinicopathological characteristics showed that these patients were mostly de novo metastatic (p-value=0.002), metastasizing primarily to the liver or bone and had significantly more aberrations in NF1, AKT1 and FGFR (p-value=0.04, 0.01 and 0.03 respectively), when compared to the rest of the cohort. Finally, most of the patients (63%) had a lower number of CNA accumulated after the metastatic lineage separation compared to the common trunk, implying a late dissemination event. Conclusions: In this study, we described different metastatic dissemination events taking place in ILC. Our analyses allowed us to identify a subset of tumors characterized by slow tumor progression, indicating potential tumor dormancy, as well as evidence of late dissemination evidence for most of the patients, thus highlighting the importance of early detection through cancer screening. These findings further shed light on how ILCs evolves through time and could potentially influence the clinical management of invasive lobular breast cancer. Citation Format: Danai Fimereli, David Venet, Mattia Rediti, David N Brown, Bram Boeckx, Marion Maetens, Samira Majjaj, Ghizlane Rouas, Maria Capra, Giuseppina Bonizzi, Federica Contaldo, Christine Galant, Martine Piccart, Giancarlo Pruneri, Denis Larsimont, Diether Lambrechts, Christine Desmedt, Françoise Rothé, Christos Sotiriou. Portraying tumor evolution of lobular breast cancer through phylogenetic analysis [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD14-05.

Purpose:Invasive mucinous adenocarcinoma (IMA) is a unique subtype of lung adenocarcinoma, characterized genomically by frequent KRAS mutations or specific gene fusions, most commonly involving NRG1. Comprehensive analysis of a large series of IMAs using broad DNA- and RNA-sequencing methods is still lacking, and it remains unclear whether molecular subtypes of IMA differ clinicopathologically.Experimental Design:A total of 200 IMAs were analyzed by 410-gene DNA next-generation sequencing (MSK-IMPACT; n = 136) or hotspot 8-oncogene genotyping (n = 64). Driver-negative cases were further analyzed by 62-gene RNA sequencing (MSK-Fusion) and those lacking fusions were further tested by whole-exome sequencing and whole-transcriptome sequencing (WTS).Results:Combined MSK-IMPACT and MSK-Fusion testing identified mutually exclusive driver alterations in 96% of IMAs, including KRAS mutations (76%), NRG1 fusions (7%), ERBB2 alterations (6%), and other less common events. In addition, WTS identified a novel NRG2 fusion (F11R–NRG2). Overall, targetable gene fusions were identified in 51% of KRAS wild-type IMAs, leading to durable responses to targeted therapy in some patients. Compared with KRAS-mutant IMAs, NRG1-rearranged tumors exhibited several more aggressive characteristics, including worse recurrence-free survival (P < 0.0001).Conclusions:This is the largest molecular study of IMAs to date, where we demonstrate the presence of a major oncogenic driver in nearly all cases. This study is the first to document more aggressive characteristics of NRG1-rearranged IMAs, ERBB2 as the third most common alteration, and a novel NRG2 fusion in these tumors. Comprehensive molecular testing of KRAS wild-type IMAs that includes fusion testing is essential, given the high prevalence of alterations with established and investigational targeted therapies in this subset.

Aims Micropapillary variant of mucinous carcinoma of the breast (MPMC) is a rare histological form of oestrogen receptor (ER)-positive invasive carcinoma that is characterised by micropapillary clusters of tumour cells in lakes of extracellular mucin. The aims of this study were to determine the genetic alterations underpinning MPMCs, and to determine whether they overlap with those of mucinous carcinomas and/or invasive micropapillary carcinomas. Methods and results DNA from five MPMCs was subjected to whole-exome sequencing. Somatic mutations, copy number alterations and mutational signatures were determined with state-of-the-art bioinformatics methods. No mutations in genes significantly mutated in breast cancer, including TP53, PIK3CA, GATA3, and MAP3K1, were detected. We identified copy number alterations that have been reported in invasive micropapillary carcinomas, such as recurrent gains in 1q, 6p, 8q, and 10q, and recurrent losses in 16q, 11q, and 13q, as well as a recurrent 8p12-8p11.2 amplification encompassing FGFR1. Like mucinous carcinomas, three of the five MPMCs analysed lacked PIK3CA mutations, 1q gains, and 16q losses, which are the hallmark genetic alterations of ER-positive breast cancers, whereas two MPMCs harboured 16q losses and/or a complex pattern of copy number alterations similar to those found in breast-invasive micropapillary carcinomas. Conclusions MPMCs are heterogeneous at the genetic level; some tumours show a pattern of somatic genetic alterations similar to those of mucinous carcinomas, whereas others resemble invasive micropapillary carcinomas at the genetic level. These findings suggest that MPMCs may not constitute one histological subtype, but rather a convergent phenotype that can stem from mucinous carcinomas or invasive micropapillary carcinomas.

Background Noninvasive genotyping using plasma cell-free DNA (cfDNA) has the potential to obviate the need for some invasive biopsies in cancer patients while also elucidating disease heterogeneity. We sought to develop an ultra-deep plasma next-generation sequencing (NGS) assay for patients with non-small-cell lung cancers (NSCLC) that could detect targetable oncogenic drivers and resistance mutations in patients where tissue biopsy failed to identify an actionable alteration. Patients and methods Plasma was prospectively collected from patients with advanced, progressive NSCLC. We carried out ultra-deep NGS using cfDNA extracted from plasma and matched white blood cells using a hybrid capture panel covering 37 lung cancer-related genes sequenced to 50 000× raw target coverage filtering somatic mutations attributable to clonal hematopoiesis. Clinical sensitivity and specificity for plasma detection of known oncogenic drivers were calculated and compared with tissue genotyping results. Orthogonal ddPCR validation was carried out in a subset of cases. Results In 127 assessable patients, plasma NGS detected driver mutations with variant allele fractions ranging from 0.14% to 52%. Plasma ddPCR for EGFR or KRAS mutations revealed findings nearly identical to those of plasma NGS in 21 of 22 patients, with high concordance of variant allele fraction (r = 0.98). Blinded to tissue genotype, plasma NGS sensitivity for de novo plasma detection of known oncogenic drivers was 75% (68/91). Specificity of plasma NGS in those who were driver-negative by tissue NGS was 100% (19/19). In 17 patients with tumor tissue deemed insufficient for genotyping, plasma NGS identified four KRAS mutations. In 23 EGFR mutant cases with acquired resistance to targeted therapy, plasma NGS detected potential resistance mechanisms, including EGFR T790M and C797S mutations and ERBB2 amplification. Conclusions Ultra-deep plasma NGS with clonal hematopoiesis filtering resulted in de novo detection of targetable oncogenic drivers and resistance mechanisms in patients with NSCLC, including when tissue biopsy was inadequate for genotyping.

Aims Low-grade serous carcinomas (LGSCs) and their precursors serous borderline tumours (SBTs) characteristically harbour mutations in BRAF, KRAS or NRAS but rarely in TP53, whereas high-grade serous carcinomas (HGSCs) are characterised by frequent TP53 mutations but rare BRAF, KRAS or NRAS mutations. In a small subset of cases, LGSCs and/or SBTs develop into high-grade tumours, including HGSCs and poorly differentiated carcinomas (PDCs). Here, we sought to define the repertoire of somatic genetic alterations in low-grade serous tumours and synchronous or metachronous high-grade adnexal carcinomas. Methods and results DNA extracted from five SBTs/LGSCs and synchronous or metachronous HGSCs/PDCs and matched normal tissue was subjected to massively parallel sequencing targeting all exons and selected non-coding regions of 341 cancer-related genes. The low-grade and high-grade tumours from a given case were related, and shared mutations and copy number alterations. Progression from low-grade to high-grade lesions was observed, and involved the acquisition of additional mutations and/or copy number alterations, or shifts from subclonal to clonal mutations. Only two (an HGSC and a PDC) of the five high-grade tumours investigated harboured TP53 mutations, whereas NRAS and KRAS hotspot mutations were seen in two HGSCs and one HGSC, respectively. Conclusions Our results suggest that progression from SBT to HGSC may take place in a subset of cases, and that at least some of the rare HGSCs lacking TP53 mutations may be derived from a low-grade serous precursor.

AIMS To characterise the genetic alterations in adult primary uterine rhabdomyosarcomas (uRMSs) and to investigate whether these tumours are genetically distinct from uterine carcinosarcomas (UCSs). METHODS AND RESULTS Three tumours originally diagnosed as primary adult pleomorphic uRMS were subjected to massively parallel sequencing targeting 468 cancer-related genes and RNA-sequencing. Mutational profiles were compared with those of UCSs (n = 57) obtained from The Cancer Genome Atlas. Sequencing data analyses were performed using validated bioinformatic approaches. Pathogenic TP53 mutations and high levels of genomic instability were detected in the three cases. uRMS1 harboured a likely pathogenic YTHDF2-FOXR1 fusion. uRMS2 harboured a PPP2R1A hotspot mutation and amplification of multiple genes, including WHSC1L1, FGFR1, MDM2, and CCNE1, whereas uRMS3 harboured an FBXW7 hotspot mutation and an ANKRD11 homozygous deletion. Hierarchical clustering of somatic mutations and copy number alterations revealed that these tumours initially diagnosed as pleomorphic uRMSs and UCSs were similar. Subsequent comprehensive pathological re-review of the three uRMSs revealed previously unidentified minute pan-cytokeratin-positive atypical glands in one case (uRMS3), favouring its reclassification as UCS with extensive rhabdomyosarcomatous overgrowth. CONCLUSIONS Adult pleomorphic uRMSs harbour TP53 mutations and high levels of copy number alterations. Our findings underscore the challenge in discriminating between uRMS and UCS with rhabdomyosarcomatous differentiation.

OBJECTIVE: To characterize and compare the molecular subtypes and profiles of prospectively-accrued newly-diagnosed early- and advanced-stage endometrial cancers (ECs). METHODS: EC patients consented to an IRB-approved protocol of massively parallel sequencing of 410–468 cancer-related genes; 175 ECs of 7 histologic types (n=135 FIGO stages I/II, n=40 FIGO stages III/IV) were included. Previously reported sequencing data from 99 additional advanced-stage ECs were retrieved for comparisons. RESULTS: Irrespective of histologic type, all 175 ECs could be stratified into the molecular subtypes, with 75 (43%) being of p53 wild-type, 49 (28%) MMR-deficient, 39 (22%) p53 abnormal and 12 (7%) of POLE molecular subtypes. Subtype distribution, mutational and copy number profiles varied according to histologic type. In endometrioid ECs, genetic alterations varied according to histologic grade. Potential therapeutic targets, including high tumor mutational burden, ERBB2 amplification and PIK3CA hotspot mutations, were found across histologic types in 63% (n=110) of all ECs. Compared to their early-stage counterparts, advanced-stage endometrioid ECs had a significantly higher fraction of genome altered (median 0.1% vs 12%, p0.05) and PIK3CA mutations (46% vs 27%, p>0.05). Whole-genome doubling was found in advanced- but not early-stage carcinosarcomas and clear cell carcinomas. CONCLUSIONS: Our findings demonstrate the molecular heterogeneity within and across histologic types of EC and the increased genomic complexity of advanced-stage ECs. Molecular subtypes are present across EC histologic types and may help stratify EC patients for prognostic and therapeutic purposes.

Introduction: Invasive lobular carcinoma (ILC) is the most frequent special histologic subtype of breast cancer (BC). ILC is identifiable by pathologic assessment given its distinctive discohesive growth pattern, largely caused by CDH1 inactivation. Compared to common forms of BC, ILCs display lower responses to chemotherapy and selective estrogen receptor modulators. The low interobserver agreement for the diagnosis of ILC, however, renders the inclusion of histologic subtyping in therapeutic decision-making challenging. Artificial intelligence (AI)-based algorithms hold promise for improving pathologic diagnosis; their performance, however, depends on the ground truth labeling used. Here, we seek to develop an AI-based methodology for detection of ILC using ‘CDH1 biallelic mutations’ (i.e., mutation + loss-of-heterozygosity of the wild-type allele or two pathogenic somatic mutations) as ground truth, reasoning that in BC, >95% of CDH1 bi-allelic inactivation is found in ILCs.Materials and methods: We developed a convolutional neural network system to detect CDH1 biallelic genetic inactivation (AI-CDH1) using whole slide images (WSI) of 1,100 primary BCs with available targeted sequencing data. The model was trained using a 10-fold cross-validation method to detect biallelic mutations. The mean number of positive and negative samples in the training set was 85.2 (SD=2.57) and 562.8 (SD=10.51) per fold, respectively. The evaluation set consisted of a mean of 14.2 (SD=2.04) positive and 93.8 (SD=9.13) negative samples. We evaluated the performance of the AI-CDH1 classifier to predict the lobular phenotype and CDH1 status using original and revised labels, following a histopathologic re-review of the histologic type and CDH1 status curation. The latter was conducted by incorporating information on biallelic CDH1 inactivation beyond CDH1 mutations (homozygous deletions, deleterious structural rearrangements, and loss-of-heterozygosity and gene promoter methylation).Results: The AI-CDH1 classifier predicted biallelic CDH1 mutations with an area under the curve (AUC)=0.944 (95 CI: 0.925-0.963), sensitivity=91.6% and specificity=85.9%, PPV=49.8%, NPV=98.5% and accuracy=86.7%, and the original ‘lobular phenotype’ with an AUC=0.941 (95 CI: 0.922-0.960), sensitivity=89%, specificity=86.7%, PPV=55.6%, NPV=97.7% and accuracy=87.1%. Review of the CDH1 gene status revealed that 7/957 BCs lacking CDH1 biallelic mutations harbored biallelic CDH1 inactivation by promoter methylation, homozygous deletions or structural rearrangements. The AI-CDH1 classifier detected all seven reclassified BCs and predicted the revised CDH1 biallelic inactivation with an AUC=0.948 (95 CI: 0.930-0.966), sensitivity=92%, specificity=86.5%, PPV=52.3%, NPV=98.5% and accuracy=87.2%. Upon histologic re-review, which resulted in reclassification of 36/927 non-lobular BCs as ‘lobular’ and 5/173 ‘lobular’ BCs as ‘non-lobular’, the AI-CDH1 classifier detected the ‘lobular phenotype’ with an AUC=0.953 (95 CI: 0.935-0.971), sensitivity=90.7%, specificity=89.7%, PPV=66.8%, NPV=97.7% and accuracy=89.9%. Using the revised histologic re-classification and CDH1 biallelic inactivation status labels, the AI-CDH1 classifier predicted the lobular phenotype irrespective of CDH1 status (P>0.05).Conclusions: By training a machine learning system to detect ‘CDH1 biallelic mutations’, as ground truth rather than histologic diagnosis of lobular carcinoma, which might be confounded by human subjectivity, we developed an AI-based system that can detect ILCs accurately, providing a new paradigm for the development of AI-based cancer classification systems. Citation Format: Jorge S Reis-Filho, Fresia Pareja, Fatemeh Derakhshan, David N Brown, Jillian Sue, Pier Selenica, Yi Kan Wang, Arnaud Da Cruz Paula, Monami Banerjee, Zahra Ebrahimzadeh, Manuel Isava, Matthew Lee, Ran Godrich, Adam Casson, Ruben Padron, George Shaikovski, Alexander van Eck, Antonio Marra, Higinio Dopeso, Hannah Y Wen, Edi Brogi, Matthew G Hanna, Chris Kanan, Jeremy D Kunz, Felipe C Geyer, Carla Leibowitz, David Klimstra, Leo Grady, Thomas J Fuchs. An artificial intelligence-based predictor of CDH1 biallelic mutations and invasive lobular carcinoma [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD11-01.

Metaplastic breast carcinoma (MBC) and uterine carcinosarcoma (UCS) are rare aggressive cancers, characterized by an admixture of adenocarcinoma and areas displaying mesenchymal/sarcomatoid differentiation. In this study, we investigate whether MBCs and UCSs harbor similar patterns of genetic alterations and mutational signatures using whole‐exome sequencing data. In addition, we examine whether the different histologic components of MBCs and UCSs are clonally related., 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.

Alpelisib is a selective inhibitor of PI3Kα, shown to improve outcomes for PIK3CA mutant, hormone receptor positive (HR+) metastatic breast cancers (MBC) when combined with antiestrogen therapy. To uncover mechanisms of resistance, we conducted a detailed, longitudinal analysis of tumor and plasma circulating tumor DNA among such patients from a phase I/II trial combining alpelisib with an aromatase inhibitor (AI) (NCT01870505). The trial’s primary objective was to establish safety with maculopapular rash emerging as the most common grade 3 adverse event (33%). Among 44 evaluable patients, the observed clinical benefit rate was 52%. Correlating genetic alterations with outcome, we identified loss-of-function PTEN mutations in 25% of patients with resistance. ESR1 activating mutations also expanded in number and allele fraction during treatment and were associated with resistance. These data indicate that genomic alterations that mediate resistance to alpelisib or antiestrogen may promote disease progression and highlight PTEN loss as a recurrent mechanism of resistance to PI3Kα inhibition.

Circulating cell-free DNA (cfDNA) from blood plasma of cancer patients can be used to interrogate somatic tumor alterations non-invasively or when adequate tissue is unavailable. We have developed and clinically implemented MSK-ACCESS (Analysis of Circulating cfDNA to Evaluate Somatic Status), an NGS assay for detection of very low frequency somatic alterations in select exons and introns of 129 genes. Analytical validation demonstrated 92% sensitivity in de-novo mutation calling down to 0.5% allele frequency and 99% for a priori mutation profiling. To evaluate the performance and utility of MSK-ACCESS, we report results from the first 681 prospective blood samples (617 patients) that underwent clinical analysis to guide patient management. Somatic mutations, copy number, and/or structural variants were detected in 73% of the samples, and 56% of these circulating-tumor DNA (ctDNA) positive samples had clinically actionable alterations. The utilization of matched white blood cell sequencing allowed retention of somatic alterations while filtering out over 10,000 germline and clonal hematopoiesis variants, thereby greatly enhancing the specificity of the assay. Taken together, our experience illustrates the importance of analyzing a matched normal sample when interpreting cfDNA results and highlights the potential of cfDNA profiling to guide treatment selection, monitor treatment response, and identify mechanisms of treatment resistance.

Purpose: Ductal carcinoma in situ (DCIS) is a nonobligate precursor of invasive breast cancer. Here, we sought to investigate the level of intralesion genetic heterogeneity in DCIS and the patterns of clonal architecture changes in the progression from DCIS to invasive disease. Experimental Design: Synchronous DCIS (n = 27) and invasive ductal carcinomas of no special type (IDC-NSTs; n = 26) from 25 patients, and pure DCIS (n = 7) from 7 patients were microdissected separately and subjected to high-depth whole-exome (n = 56) or massively parallel sequencing targeting ≥410 key cancer-related genes (n = 4). Somatic genetic alterations, mutational signatures, clonal composition, and phylogenetic analyses were defined using validated computational methods. Results: DCIS revealed genetic alterations similar to those of synchronously diagnosed IDC-NSTs and of non-related IDC-NSTs from The Cancer Genome Atlas (TCGA), whereas pure DCIS lacked PIK3CA mutations. Clonal decomposition and phylogenetic analyses based on somatic mutations and copy number alterations revealed that the mechanisms of progression of DCIS to invasive carcinoma are diverse, and that clonal selection might have constituted the mechanism of progression from DCIS to invasive disease in 28% (7/25) of patients. DCIS displaying a pattern of clonal selection in the progression to invasive cancer harbored higher levels of intralesion genetic heterogeneity than DCIS where no clonal selection was observed. Conclusions: Intralesion genetic heterogeneity is a common feature in DCIS synchronously diagnosed with IDC-NST. DCIS is a nonobligate precursor of IDC-NST, whose mechanisms of progression to invasive breast cancer are diverse and vary from case to case.

Granular cell tumors (GCTs) are rare tumors that can arise in multiple anatomical locations, and are characterized by abundant intracytoplasmic granules. The genetic drivers of GCTs are currently unknown. Here, we apply whole-exome sequencing and targeted sequencing analysis to reveal mutually exclusive, clonal, inactivating somatic mutations in the endosomal pH regulators ATP6AP1 or ATP6AP2 in 72% of GCTs. Silencing of these genes in vitro results in impaired vesicle acidification, redistribution of endosomal compartments, and accumulation of intracytoplasmic granules, recapitulating the cardinal phenotypic characteristics of GCTs and providing a novel genotypic–phenotypic correlation. In addition, depletion of ATP6AP1 or ATP6AP2 results in the acquisition of oncogenic properties. Our results demonstrate that inactivating mutations of ATP6AP1 and ATP6AP2 are likely oncogenic drivers of GCTs and underpin the genesis of the intracytoplasmic granules that characterize them, providing a genetic link between endosomal pH regulation and tumorigenesis., Granular cell tumors (GCTs) are rare tumors that arise in multiple anatomical locations. Here, the authors investigate the genomics of GCTs, finding inactivating somatic mutations in ATP6AP1 or ATP6AP2 in 72% of the 82 GCTs analyzed. In vitro manipulation of these genes recapitulated GCT phenotypes in cellular models.

The advent of next generation sequencing technologies has boosted the interest in exploring the role of fusion genes in the development and progression of solid tumors. In breast cancer, most of the detected gene fusions seem to be "passenger" events while the presence of recurrent and driver fusions is still under study. We performed RNA sequencing in 55 well-characterized breast cancer samples and 10 adjacent normal breast tissues, complemented by an analysis of SNP array data. We explored the presence of fusion genes and defined their association with breast cancer subtypes, clinical-pathologic characteristics and copy number aberrations. Overall, 370 fusions were detected across the majority of the samples. HER2+ samples had significantly more fusions than triple negative and luminal subtypes. The number of fusions was correlated with histological grade, Ki67 and tumor size. Clusters of fusion genes were observed across the genome and a significant correlation of fusions with copy number aberrations and more specifically amplifications was also revealed. Despite the large number of fusion events, only a few were recurrent, while recurrent individual genes forming fusions with different partners were also detected including the estrogen receptor 1 gene in the previously detected ESR1-CCDC170 fusion. Overall we detected novel gene fusion events while we confirmed previously reported fusions. Genomic hotspots of fusion genes, differences between subtypes and small number of recurrent fusions are the most relevant characteristics of these events in breast cancer. Further investigation is necessary to comprehend the biological significance of these fusions.

Background In the NeoALTTO trial, the combination of trastuzumab and lapatinib for the neoadjuvant treatment of HER2 positive early breast cancer patients (pts) nearly doubled the rate of pathological complete response (pCR) compared with either anti-HER2 treatment alone. In the same trial, pCR was shown to be a surrogate for long-term outcome. Expression of ERBB2, ESR1 and immune signatures were the main drivers of pCR (Fumagalli et al. JAMA Oncol 2016). The main aim of the current study was to investigate the relevance of copy number aberrations (CNAs) for pCR and event-free survival (EFS). Methods For 271 out of the 455 pts enrolled in the study with sufficient tumour content, DNA was hybridized on CytoScan HD arrays. The log2 ratio intensities and B allele frequencies were segmented jointly using the multitrack segmentation algorithm. Integer level estimates of total copy number and major allele were obtained using GAP. Recurrent CNAs were found with GISTIC2. The genome instability index (GII) was defined as the median absolute deviation of the normalized copy number. Tumor infiltrating lymphocytes (TILs) and gene expression were obtained as described previously. The correlations between differents parameters were assessed using Spearman correlations (ρ). The Mann-Whitney test was used to relate binary and numerical features. EFS analysis was performed using the Cox proportional hazard model. Results CNAs estimates were obtained for 185 out of 271 pts. CNA distribution was similar to the ones observed in HER2+ pts from the METABRIC and the TCGA datasets. There were 64% of diploid pts, 26% of triploid and 9% of tetraploid or more. Aneuploidy level was not associated with pCR or EFS. Of interest, there were many significant differences in CNA profiles between ER+ and ER- pts. Those differences mirror those observed between ER+ and ER- among HER2- pts in TCGA (ρ=63%) and METABRIC (ρ=56%). ERBB2 amplification was predictive of high pCR (p=0.0007), albeit less so than ERBB2 expression, and ceased to be significant correcting for ERBB2 expression. The pCR rate increased with the GII (p=0.03), independently of ERBB2 amplification. The effect was stronger in ER+ patients (p=0.01). GISTIC analysis revealed 159 recurrent CNA regions. Amplification of 2 regions on 6q23-24 was significantly associated with higher pCR (p=0.00005 and p=0.00087, FDR=0.006 and 0.05). The most significant segment of 6q23-24 contained 39 genes, some whose expression level also predicted pCR (e.g. MAP3K5, p=10-4). Gene ontology analysis of the genes correlated with this segment highlighted the category 'response to interferon-alpha' (p=4.3x10-7). No amplified region or gene was found to be predictive of EFS, after multiple testing correction. Conclusions The amplification of ERBB2 was shown to be predictive of pCR, however its expression was more predictive. High genomic instability was associated with higher rate of pCR in ER+ subgroup. Of interest, a novel amplified region, involving chromosome 6 was shown to be predictive of pCR, which may warrant further investigation. Citation Format: Sotiriou C, Rothé F, Maetens M, Fumagalli D, Brown DN, Salgado R, Bradbury I, Pusztai L, Harbeck N, Ignatiadis M, Sarp S, de la Pena L, de Azambuja E, Huober J, Nuciforo P, Baselga J, Piccart M, Loi S, Venet D. Copy number aberration analysis to predict response to neoadjuvant anti-HER2 therapy: Results from the NeoALTTO phase III trial [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 GS1-04.