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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.

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.

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.

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.

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.

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.

PURPOSE: CDK4/6 inhibitors are highly effective against ER+/HER2- breast cancer (BC); however, intrinsic and acquired resistance is common. Elucidating the molecular features of sensitivity and resistance to CDK4/6i’s may lead to identification of predictive biomarkers and novel therapeutic targets, paving the way toward improving patient outcomes. EXPERIMENTAL DESIGN: Parental BC 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 BC lines showed varying degrees of sensitivity to palbociclib. Transcriptomic analysis of these cell lines identified an association between high interferon (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 to 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. CONCLUSION: 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.

Background Epidemiological evidence indicates a clear relationship between pregnancy and breast cancer (BC) risk. However, little is known regarding the impact of pregnancy on BC biology. DNA copy number aberrations (CNAs) play an important role in breast carcinogenesis. BC during pregnancy is a rare disease but yet could serve as a good model to study the impact of pregnancy on BC biology. Methods We retrospectively included 54 pregnant and 113 non-pregnant BC patients matched for age and stage with complete clinico-pathological, gene expression and 5-year follow-up data. CNAs were assessed using Affymetrix OncoScan FFPE arrays. We identified the CNAs associated with pregnancy using a multivariate logistic regression adjusted for classical clinico-pathological features. We further evaluated their impact on gene expression. Results After quality control, CNA profiles were obtained for 38 pregnant and 87 non-pregnant BC patients. We identified 13 regions with copy number gains, 11 of which were more frequently gained in pregnant compared to non-pregnant controls and 5 regions with copy number loss, 3 of which were more frequently lost in pregnant patients (p≤0.05). Of interest, we identified 4 genes previously identified as driver event associated with CNAs in breast cancer (S. Nik-Zainal et al, Nature 2016). AKT1 and CDKN2A/B were more frequently gained in the pregnant compared to the non-pregnant (23.7% vs. 8.0%, p=0.068 and 18.4% vs. 4.6% p=0.036) and ARID1B was less frequently gained in the pregnant cohort (2.6% vs. 13.8%, p=0.02). Interestingly, PAPPA which had been previously identified as a pregnancy-dependent oncogene (Takabatake Y. et al, EMBO Mol Med. 2016) was also more frequently gained in the pregnant compared to the non-pregnant patients (21.1% vs 5.8%, p=0.03). We next evaluated the effect of these CNAs on their own gene expression levels and found that AKT1 and CDKN2A/B CNAs were affected by gene-dosage effect. Conclusions In this study, we were able to identify several genomic alterations associated with pregnancy that could further elucidate the impact of pregnancy on BC risk. Moreover, by combining CNAs with gene expression, we were able to identify genes whose expression were associated with CNAs and therefore could be considered potential drivers of this rare disease. Citation Format: Nguyen B, Brown DN, Rothé F, Desmedt C, Majjaj S, Pruneri G, Peccatori F, Azim Jr HA, Sotiriou C. Interrogating the impact of pregnancy on breast cancer biology using DNA copy number profiling [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 P1-05-17.

This abstract was withdrawn by the authors.

Introduction: There is evidence to suggest that the biology of breast cancer (BC) might vary according to age, as BCs in elderly patients might be associated with accumulation of DNA damage and BCs in younger individuals might be enriched for genetic alterations affecting cancer predisposition genes. Whether the genetic landscape of BC in elderly individuals differs is yet to be determined. Here, we sought to describe the spectrum of somatic genetic alterations and mutational signatures in BC according to age. Materials and Methods: We conducted a re-analysis of two cohorts of BC: i) MSK-IMPACT targeted sequencing cohort (primary, n=918; metastatic, n=1,000) and ii) whole-exome sequencing data of primary BCs from The Cancer Genome Atlas (TCGA) BC study (n=1087). BCs of younger ( Results: Primary BCs in elderly and younger patients, matched by ER/HER2 status, from the MSK-IMPACT (n=220 and n=440) and TCGA (n=290, each) cohorts, and metastatic BCs from elderly (n=98) and younger (n=196) patients from the MSK-IMPACT cohort, matched by ER/HER2 status, were compared. In both cohorts of primary BCs, PIK3CA, TP53 and CDH1 were the most frequently mutated genes. Compared to younger patients, primary BCs in elderly patients from the MSK-IMPACT cohort displayed a lower frequency of mutations in TP53 (23% vs 34%, P Conclusion: Our findings show that BCs in elderly patients harbor a lower frequency of somatic mutations in TP53, AKT1 and MAP2K4 than younger individuals. In contrast, BCs in elderly patients more frequently harbor somatic mutations in the transcription factors RUNX1 and NCOR1. Bi-allelic alterations affecting HRD-related genes was numerically more frequent in younger than in older patients. As expected, BCs in elderly patients show a numerically higher incidence of the aging signature, whereas primary BCs affecting younger patients displayed a higher frequency of the APOBEC signature than primary BCs from elderly individuals. Citation Format: Pier Selenica, Fresia Pareja, Audree Tadros, Lorenzo Ferrando, David N Brown, Hong Zhang, Pedram Razavi, Diana Mandelker, Mark E Robson, Sarat Chandarlapaty, Britta Weigelt, Jorge S Reis-Filho. Genomic landscape of breast cancer occurring in elderly individuals [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 P4-05-08.

Introduction: Mucinous carcinoma of the breast (MCB) is a rare histologic form of estrogen receptor (ER)-positive/HER2-negative breast cancer characterized by tumor cells floating in lakes of mucin. As compared to invasive ductal carcinoma of no special type (IDC-NST), we have recently shown that MCBs harbor a lower frequency of PIK3CA mutations and lack concurrent 1q gains and 16q losses, hallmark genetic alterations of ER-positive breast cancer. Despite their distinctive phenotype and previous efforts to characterize their genomic landscape by whole-genome, whole-exome and RNA-sequencing, no pathognomonic somatic mutation or fusion gene underpinning MCBs or the mucinous phenotype have been identified. In this study we sought to determine whether MCBs would be defined by specific epigenetic changes. Materials and methods: Thirty-six MCBs were subjected to DNA methylation profiling using Infinium MethylationEPIC arrays. Following quantile normalization, low quality probe filtering, and data background and dye bias correction using the ‘noob’ algorithm, methylation profiling data of MCBs were compared to those of ER-positive/HER2-negative IDC-NSTs from The Cancer Genome Atlas (TCGA) lacking concurrent 1q gains/16q losses or PIK3CA hotspot mutations and matched according to age and menopausal status at a 1:2 ratio (n=72). An enrichment analysis of the differentially methylated targets in gene promoters and enhancers was conducted using Minfi and MethylGSEA R packages. A subset of 12 MCBs of this cohort was subjected to RNA-sequencing and compared to age, menopausal status and molecular features-matched ER-positive/HER2-negative IDC-NSTs from TCGA (1:2 ratio; n=24) using gene set enrichment analysis (GSEA). Results: Enrichment analysis of differentially methylated probes revealed a significant enrichment of targets in promoters and enhancers of mucin-encoding genes and in genes of the mTOR signaling pathway between MCBs and matched IDC-NSTs from TCGA (P Conclusions: Taken together, our data suggest that MCBs display a high expression of mucin-encoding genes, due to hypomethylation of promoters and enhancers and could provide the basis for their distinctive phenotype. Moreover, our findings suggest that despite the lack of genetic alterations affecting genes of the PI3K/mTOR signaling pathway in MCBs, mTOR signaling might be constitutively active in these tumors via epigenetic mechanisms, supporting the notion that, in the absence of pathognomonic genetic alterations, a disruption of the epigenetic landscape is a critical driver in the development of this rare breast cancer type. Citation Format: Fresia Pareja, Lorenzo Ferrando, Edaise M da Silva, Arnaud Da Cruz Paula, Anthe Stylianou, David N Brown, Pier Selenica, Jonathan Serrano, Hannah Y Wen, Hong Zhang, Edi Brogi, Larry Norton, Matjia Snuderl, Jorge S Reis-Filho, Britta Weigelt. Methylation profiling of mucinous breast cancer [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 P4-05-11.

Introduction: Breast cancers (BCs) with microsatellite instability (MSI)/DNA mismatch repair (MMR) deficiency are rare. Their identification is key, given that these tumors might be sensitive to immune checkpoint blockade. Here we sought to define the characteristics of primary and metastatic MSI-high (MSI-H) BCs using massively parallel sequencing. Materials and methods: To define MSI, MSISensor was applied to MSK-IMPACT targeted sequencing data of 918 primary BCs (pBCs) and 1,000 metastatic BCs (mBCs), and MSISensor and MANTIS to whole-exome sequencing (WES) data of 1,084 pBCs from The Cancer Genome Atlas (TCGA) BC project. BCs were classified as MSI-H if they displayed an MSISensor score ≥3.5 for WES or ≥10 for targeted sequencing data, or a MANTIS score ≥0.4 for WES data. The dominant mutational signature was inferred using SigMA in cases harboring at least 5 single nucleotide variants (SNVs). Mann-Whitney U and Fisher’s exact t-tests were employed for statistical analyses. Results: MSISensor classified 0.3% (3/903) of pBCs and 0.3% (3/965) of the mBCs subjected to MSK-IMPACT sequencing as MSI-H. The MSI-H pBCs were invasive ductal carcinomas (IDC), preferentially ER-positive/HER2-negative (66.6%). Compared to MSS pBCs, MSI-H pBCs displayed a higher tumor mutational burden (TMB; 11.2 vs 2.6 somatic mutations) and were enriched for dominant MMR mutational signatures 20 or 26 (33% vs 1.1%). MSI-H mBCs were all ER-positive/HER2-negative IDCs, displayed similar TMBs, percentage of small insertions and deletions (indels) and fraction of the genome altered (FGA) as compared to MSS mBCs. Only one of the three MSI-H mBCs had enough SNVs for inference of a dominant mutational signature, which was aging. MSISensor and MANTIS classified 1.5% (11/735) and 1.8% (15/836) of pBCs from TCGA as MSI-H. We observed a moderate agreement between MSISensor and MANTIS (Cohen’s Kappa = 0.552) with 1% (7/735) of pBCs being classified as MSI-H by both algorithms. pBCs from TCGA classified as MSI by either MSISensor or MANTIS were IDCs (90.9% and 85.7%, respectively) and invasive lobular carcinoma (ILC; 9.1% and 14.3%, respectively), and were either ER-positive/HER2-negative (45.5% and 42.9%, respectively) or ER-negative/HER2-negative (45.5% and 42.9%, respectively). These MSI-H pBCs (TCGA) displayed a higher TMB (MSISensor: 12.7 vs 0.8; MANTIS: 3.6 vs 0.8; both P Conclusions: We observed a moderate agreement between different MSI detection algorithms in the classification of pBCs as MSI-H based on WES analysis. pBCs classified as MSI-H were of common histologic types, either ER-positive/HER2-negative or ER-negative/HER2-negative, and displayed features characteristic of MSI, including a high TMB, high percentage of indels and an enrichment for MMR deficiency mutational signatures. mBCs classified as MSI-H by targeted capture sequencing were found to be of ER-positive/HER2-negative phenotype. Citation Format: Pier Selenica, Fresia Pareja, Lorenzo Ferrando, David N Brown, Nadeem Riaz, Britta Weigelt, Joshua Z Drago, Mark E Robson, Pedram Razavi, Sarat Chandarlapaty, Jorge S Reis-Filho. Massively parallel sequencing analysis of microsatellite instability in breast cancer [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 P4-05-01.

PURPOSE Endometrial cancer (EC) is not considered a component of the hereditary breast and ovarian cancer syndrome but can arise in patients with germline BRCA1/2 (g BRCA1/2) mutations. Biallelic BRCA1/2 alterations are associated with genomic features of homologous recombination DNA repair deficiency (HRD) in cancer. We sought to determine if ECs in g BRCA1/2 mutation carriers harbor biallelic alterations and/or features of HRD. METHODS Of 769 patients with EC who underwent germline panel testing, 10 pathogenic g BRCA1/2 mutation carriers were identified, and their tumor- and normal-derived DNA was subjected to massively parallel sequencing targeting at least 410 cancer-related genes. Three g BRCA1/2-associated ECs were identified in 232 ECs subjected to whole-exome sequencing by The Cancer Genome Atlas. Somatic mutations, copy number alterations, loss of heterozygosity, microsatellite instability (MSI), and genomic HRD features were assessed. RESULTS Of the 13 patients included who had EC, eight harbored pathogenic g BRCA1 mutations and five harbored g BRCA2 mutations. Eight (100%) and two (40%) ECs harbored biallelic BRCA1 and BRCA2 alterations through loss of heterozygosity of the wild-type allele. All ECs harbored somatic TP53 mutations. One monoallelic/sporadic g BRCA2-associated EC had MLH1 promoter methylation and was MSI high. High large-scale state transition scores, a genomic feature of HRD, were found only in ECs with bi- but not monoallelic BRCA1/2 alterations. The Signature Multivariate Analysis HRD signature Sig3 was enriched in biallelic g BRCA1/2 ECs, and the three ECs from The Cancer Genome Atlas with BRCA1 biallelic alterations subjected to whole-exome sequencing displayed a dominant HRD-related mutational signature 3. CONCLUSION A subset of g BRCA1/2-associated ECs harbor biallelic BRCA1/2 alterations and genomic features of HRD, which may benefit from homologous recombination–directed treatment regimens. ECs in BRCA2 mutation carriers might be sporadic and even MSI high, and may potentially benefit from immune-checkpoint inhibition.

Aims Melanocytic naevi are benign lesions of the skin or mucosa that may constitute non-obligate precursors of malignant melanoma, particularly when they show lentiginous and dysplastic features. The aim of this study was to investigate the repertoire of somatic genetic alterations in melanocytic naevi. Methods and results DNA extracted from 12 melanocytic naevi and DNA from matching normal tissue were separately microdissected and subjected to targeted massively parallel sequencing of ≥300 cancer genes. A median of 5.5 (range 1-12) non-synonymous somatic mutations were detected, with 10 cases harbouring mutually exclusive BRAF V600E (6/12) or NRAS (4/12) clonal hotspot mutations. One of the two cases lacking BRAF and NRAS mutations was a dysplastic naevus harbouring an HRAS Q61L hotspot mutation. Analysis of the laser-capture microdissected components of a naevus synchronously diagnosed with in-situ and invasive malignant melanoma revealed a truncal, clonal BRAF V600E mutation, and the acquisition of a CDKN2A homozygous deletion in the invasive component, in conjunction with additional clonal mutations affecting NF2, FAT4 and KDR in both in-situ and invasive malignant components. Conclusion Melanocytic naevi harbour recurrent BRAF V600E or NRAS hotspot mutations with low mutational burdens. Our findings also show that progression from naevi to malignant melanoma may be driven by the acquisition of additional genetic alterations, including CDKN2A homozygous deletions.

Pathogenic germline variants in checkpoint kinase 2 (CHEK2), which plays pivotal roles in DNA damage response and cell cycle regulation, confer an increased breast cancer (BC) risk. Here, we investigated the phenotypic and genomic characteristics of 33 BCs from CHEK2 germline mutation carriers (16 high-risk variants and 17 low-risk p.Ile157Thr variants). CHEK2-associated BCs from patients with high-risk germline variants were largely hormone receptor-positive (87%, 13/15), and 81% (13/16) exhibited loss of heterozygosity (LOH) of the CHEK2 wild-type allele. Conversely, CHEK2-associated BCs from patients with the low-risk p.Ile157Thr variant displayed less-frequent loss of heterozygosity (5/17, 29%) and higher levels of CHEK2 protein expression than those with high-risk germline variants. CHEK2-associated BCs lacked a dominant mutational signature 3, a genomics feature of homologous recombination DNA repair deficiency (HRD). Our findings indicate that CHEK2-associated BCs are generally hormone receptor-positive and lack HRD-related mutational signatures, recapitulating the features of ATM-associated BCs. Specific CHEK2 germline variants may have a distinct impact on tumor biology.

Aims Polymorphous adenocarcinoma (PAC) usually follows an indolent course, but some cases may show recurrences and high-grade features. The genetic events associated with recurrences and high-grade versions are yet to be defined. Our aim was to determine the genetic underpinning of recurrent PACs of the salivary gland and the repertoire of somatic genetic alterations in cases with high-grade histology. Methods and results Four PACs from three patients, including one case with matching primary and recurrent tumours, one de-novo high-grade PAC, and a PAC that transformed to a high-grade tumour following multiple recurrences, were subjected to targeted sequencing (Memorial Sloan Kettering Mutation Profiling of Actionable Cancer Targets assay) or whole-exome sequencing. Both matching primary and recurrent tumours, and the de-novo high-grade PAC, harboured clonal PRKD1 E710D hotspot mutations, whereas the PAC that underwent high-grade transformation upon recurrence, which was wild-type for PRKD1, harboured a PRKD2 rearrangement. The PACs analysed here also harboured mutations targeting cancer genes such as PIK3CA, SETD2, ARID1A, and NOTCH2. A clonal decomposition analysis of the matching primary and recurrent PACs revealed that a minor subclone from the primary tumour became dominant in the recurrent tumour following a clonal selection evolutionary pattern. Conclusions Our findings demonstrate that recurrent and high-grade PACs are underpinned by PRKD1 E710D hotspot mutations or PRKD2 rearrangements, and that recurrences of PACs may stem from the selection of pre-existing subclones in the primary tumour.

Mucinous carcinoma of the breast (MCB) is a rare histologic form of estrogen receptor (ER)-positive/HER2-negative breast cancer (BC) characterized by tumor cells floating in lakes of mucin. We assessed the genomic landscape of 32 MCBs by whole-exome sequencing and/or RNA-sequencing. GATA3 (23.8%), KMT2C (19.0%), and MAP3K1 (14.3%) were the most frequently mutated genes in pure MCBs. In addition, two recurrent but not pathognomonic fusion genes, OAZ1-CSNK1G2 and RFC4-LPP, were detected in 3/31 (9.7%) and 2/31 (6.5%) samples, respectively. Compared with ER-positive/HER2-negative common forms of BC, MCBs displayed lower PIK3CA and TP53 mutation rates and fewer concurrent 1q gains and 16q losses. Clonal decomposition analysis of the mucinous and ductal components independently microdissected from five mixed MCBs revealed that they are clonally related and evolve following clonal selection or parallel evolution. Our findings indicate that MCB represents a genetically distinct ER-positive/HER2-negative form of BC.

Cyclin dependent kinase 4/6 (CDK4/6) inhibitors (CDK4/6i) are effective in breast cancer; however, drug resistance is frequently encountered and poorly understood. We conducted a genomic analysis of 348 estrogen receptor-positive (ER+) breast cancers treated with CDK4/6i and identified loss-of-function mutations affecting FAT1 and RB1 linked to drug resistance. FAT1 loss led to marked elevations in CDK6, the suppression of which restored sensitivity to CDK4/6i. The induction of CDK6 was mediated by the Hippo pathway with accumulation of YAP and TAZ transcription factors on the CDK6 promoter. Genomic alterations in other Hippo pathway components were also found to promote CDK4/6i resistance. These findings uncover a tumor suppressor function of Hippo signaling in ER+ breast cancer and establish FAT1 loss as a mechanism of resistance to CDK4/6i.

AimsMost benign breast fibroepithelial lesions (FEL) in adults harbour recurrent somatic MED12 exon 2 mutations and rare TERT promoter hotspot mutations. We sought to determine the frequency of MED12 exon 2 and TERT promoter hotspot mutations in fibroadenomas (FA) and benign phyllodes tumours (BePT) in adolescents and young adults.MethodsDNA from 21 consecutive FAs and eight consecutive BePTs in adolescents and young adults was subjected to Sanger sequencing of the exon 2 of MED12 and the TERT promoter hotspot locus.ResultsWe identified MED12 exon 2 mutations in 62% and 88% of FAs and BePTs, respectively, and no TERT promoter hotspot mutations. The majority of the MED12 exon 2 mutations identified were in-frame deletions (60%).ConclusionsAs in adults, benign FELs in juvenile patients harbour recurrent MED12 exon 2 mutations.

Solid pseudopapillary neoplasms (SPNs) are rare and relatively indolent tumors of the pancreas. While primary SPNs can be surgically resected, there are currently no therapies available for patients with advanced stage disease. Given that these tumors frequently carry CTNNB1 hotspot (recurrently mutated loci in a gene) mutations resulting in β-catenin nuclear accumulation, it has been speculated that the Wnt pathway may be a driver in this disease. Here, we present a comprehensive "multi-omics" study where the genome, transcriptome, and methylome of SPNs were analyzed. We found that SPNs are characterized by a low-complexity genome where somatic mutations in CTNNB1, present in 100% of the cases, are the only actionable genomic lesions. Compared to more common subtypes of pancreatic tumors (adenocarcinomas and pancreatic neuroendocrine tumors), SPNs show high expression levels of genes belonging to the Wnt pathway. Their methylome was consistent with an epithelial cell origin and a general upregulation of Wnt pathway genes. Clinical studies to evaluate the exquisite sensitivity of SPNs to inhibitors of the Wnt pathway are warranted.

Background: Local and regional lymph node metastases are associated with worse prognosis in a variety of cancers, including breast cancer. However, to date it still remains debated whether axillary lymph node metastases can seed distant metastases or whether they merely represent a surrogate marker for tumor aggressiveness. Here, using multiple matched samples from primary tumor, axillary lymph nodes and distant metastases from breast cancer patients, we explored to which extent lymph nodes are transit points for further dissemination events to distant organs and assessed their role in disease progression using copy number phylogenetic reconstruction. Samples and Methods: We retrospectively identified 30 estrogen receptor-positive lymph node-positive metastatic breast cancer patients with 238 formalin-fixed paraffin-embedded tissue samples available together with well-annotated clinico-pathological and outcome data. Eligible patients had at least one primary tumor, one axillary lymph node metastasis, one distant metastatic sample as well as histologically normal tissue sample as germline reference. Low coverage whole genome sequencing was performed to infer somatic copy number aberrations. After rigorous filtering of the raw data, copy number phylogenetic reconstruction was performed with Integer Linear Program for the Copy-Number Tree Problem algorithm. Bootstrapping of the samples and of the segmented copy number data was performed to assess the robustness of the phylogenetic tree classification. Results: Phylogenetic tree analyses revealed that two different routes of metastatic dissemination occur in breast cancer. Unexpectedly, in only 24% of the patients, lymph nodes and distant metastases shared a common origin implying that dissemination occurred via the axillary lymphatic system whereas in the majority of the cases (76%) lymph node and distant metastases showed a distinct origin suggesting a direct seeding from the primary tumor to a distant organ. Of note, histological grade was the only clinic-pathological feature significantly associated with the route of dissemination, low grade tumors being associated with the common origin dissemination route (p=0.04). Interestingly, the patients sharing a common origin dissemination route were found to have significantly worse prognosis than the remaining patients (p=5.3x10−4), despite being low grade. Bootstrapping analyses showed identical topology as the original phylogeny in about 80% of the cases demonstrating the robustness of our method. Conclusions: In this study, we showed for the first time that the predominant route of metastatic dissemination operating in estrogen receptor-positive breast cancer is the direct seeding of tumor cells from the primary tumor to distant organ, independently of lymph node metastasis. These findings shed light on the routes on which tumor cells metastasize and could potentially improve the clinical management of lymph node-positive breast cancer patients. Citation Format: David Venet, David Brown, Danai Fimereli, Bram Boeckx, Marion Maetens, Samira Majjaj, Ghizlane Rouas, Maria Capra, Giuseppina Bonizzi, Federica Contaldo, Christine Galant, Martine Piccart, Giancarlo Pruneri, Denis Larsimont, Diether Lambrechts, Françoise Rothé, Christine Desmedt, Christos Sotiriou. Phylogenetic reconstruction of advanced breast cancer reveals two different routes of metastatic dissemination associated with distinct clinical outcome [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 PD8-02.

Introduction: Breast cancer (BC) is heterogeneous and comprises diverse entities with distinctive phenotypes, biology and clinical presentations. There are 21 special histologic types, accounting for approximately 20% of invasive BCs. Large-scale sequencing studies have focused preferentially on common histologic forms, and data on the genomic landscape of special histologic types, particularly in the metastatic setting, are scarce. Here, we sought to define the repertoire of somatic genetic alterations of metastatic special histologic types of BC. Materials and methods: We conducted a reanalysis of MSK-IMPACT targeted sequencing data of 346 special histologic types of BC from Razavi et al (Cancer Cell 2018), including invasive lobular (ILC; primary, n=129; metastasis, n=150), mucinous (primary n=11, metastasis n=5), micropapillary (primary n=8, metastasis n=13) and metaplastic carcinomas (primary, n=6; metastasis, n=11). Somatic single nucleotide variants (SNVs) and copy number alterations (CNAs) were inferred using a validated bioinformatics pipeline, and mutational signatures were assessed with SigMA. Genetic alterations of metastatic special histologic type BCs were compared to those of primary tumors and metastatic invasive ductal carcinomas of no special type (IDC-NSTs) matched by age, menopausal status and estrogen receptor (ER)/HER2 status at a 1:2 ratio for ILCs and at a 1:3 ratio for the remaining BCs. Results: The most frequently mutated genes in metastatic ILCs were CDH1 (71%), PIK3CA (49%), ESR1 (20%) and KMT2C (19%). ESR1 mutations were more frequent in metastatic than in primary ILCs (20% vs 3%; P0.05). Metastatic mucinous BCs had a numerically higher frequency of mutations in ESR1 (40% vs 6%; P>0.05) and GATA3 (40% vs 0%; P>0.05), and lower frequency of TP53 mutations (20% vs 53%; P>0.05) than matched metastatic IDC-NSTs. PIK3CA and TP53 were the most recurrently mutated genes in metastatic micropapillary BCs and were numerically more frequent in metastatic than in primary tumors (PIK3CA, 61% vs 25%; TP53, 46% vs 25%; P>0.05). PIK3CA was more frequently mutated in metastatic micropapillary BCs than IDC-NSTs (61% vs 23%; P Conclusions: The repertoire of somatic genetic alterations of primary and metastatic special histologic type BCs markedly overlaps. Nonetheless, there is an enrichment of mutations in ESR1 in metastatic BCs of typically ER-positive special histologic type. Metastatic BCs of special histologic type show an enrichment of similar genetic alterations as primary tumors, such as a higher frequency of mutations in CDH1, FOXA1 and TBX3 in ILCs, a lower frequency of PIK3CA and TP53 mutations in mucinous BCs and a higher frequency of PIK3CA and TP53 mutations in micropapillary and metaplastic BCs. Citation Format: Fresia Pareja, Simon SK Lee, Francisco Beca, Lorenzo Ferrando, Pier Selenica, David N Brown, Hannah Y Wen, Hong Zhang, Edi Brogi, Pedram Razavi, Sarat Chandarlapaty, Britta Weigelt, Jorge S Reis-Filho. The genomic landscape of metastatic special histologic types of breast cancer [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 PD8-06.

Background: The CDK4/6 inhibitors (i) palbociclib (Palbo), ribociclib, and abemaciclib remarkably improved the outcome of patients with metastatic ER+/HER2- breast cancer (BC) and are now under clinical investigation in early BC. Despite high efficacy, de novo and acquired resistance to CDK4/6i is common. Elucidating the molecular basis for sensitivity and resistance to CDK4/6i is crucial to identify predictive biomarkers and novel therapeutic targets to improve patient outcome. Materials and Methods: MCF7, T47D and ZR75-1 parental (P) BC cells and their derivatives made resistant to tamoxifen, estrogen deprivation (EDR), or fulvestrant were used. The P and EDR models of MCF7 and T47D cells were chronically exposed to increasing concentrations of Palbo to generate derivatives with acquired resistance to Palbo (PalboR). The transcriptomic profiles of P, endocrine-resistant (EndoR) and PalboR models were determined by RNA-seq. IC50s were determined by exposing MCF7, T47D, and ZR75-1 P and EndoR lines (n=12) to increasing concentrations of Palbo. Cell growth was assessed by methylene blue staining, and changes in the mRNA and protein levels of key cell cycle molecules were assessed by RT-PCR and Western blot, respectively. Gene expression data from the Cancer Dependency Map (DepMap), baseline tumors from the NeoPalAna (NCT01723774) and neoMONARCH (NCT02441946) neoadjuvant trials, as well as the TCGA and METABRIC datasets were interrogated for correlations of gene signatures and patient outcome (by KMPlot). Results: Palbo treatment resulted in a dose-dependent inhibition of the growth of P and EndoR BC cell lines, with varying degree of sensitivity among the models. GSEA analysis comparing the least sensitive (IC50>350nM) vs. the most sensitive (IC50 Citation Format: Carmine De Angelis, Xiaoyong Fu, Maria Letizia Cataldo, Agostina Nardone, Valerie M. Jansen, Jamunarani Veeraraghavan, Sarmistha Nanda, Lanfang Qin, Vidyalakshmi Sethunath, Resel Pereira, Matteo Benelli, Ilenia Migliaccio, Luca Malorni, Joshua Donaldson, Pier Selenica, David N. Brown, Britta Weigelt, Jorge S. Reis-Filho, Ben H. Park, Sara A. Hurvitz, Dennis J. Slamon, Mothaffar F. Rimawi, Rinath Jeselsohn, Kent Osborne, Rachel Schiff. High levels of interferon-response gene signatures are associated with de novo and acquired resistance to CDK4/6 inhibitors in ER+ breast cancer [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 GS2-01.

Breast cancer diagnosed during pregnancy (BCP) is a rare and highly challenging disease. To investigate the impact of pregnancy on the biology of breast cancer, we conducted a comparative analysis of a cohort of BCP patients and non-pregnant control patients by integrating gene expression, copy number alterations and whole genome sequencing data. We showed that BCP exhibit unique molecular characteristics including an enrichment of non-silent mutations, a higher frequency of mutations in mucin gene family and an enrichment of mismatch repair deficiency mutational signature. This provides important insights into the biology of BCP and suggests that these features may be implicated in promoting tumor progression during pregnancy. In addition, it provides an unprecedented resource for further understanding the biology of breast cancer in young women and how pregnancy could modulate tumor biology., SCOPUS: ar.j, info:eu-repo/semantics/published

Although invasive ductal breast cancer (IDC) represents the most common histological type of breast cancer, minor subtypes exist such as mucinous breast cancer (MuBC). MuBC are distinguished by tumor cells floating in extracellular mucin. MuBC patients are generally older and associated with a favorable prognosis. To unravel the molecular architecture of MuBC, we applied low-pass whole-genome sequencing and microscopic evaluation of stromal tumor infiltrating lymphocytes to 30 MuBC from a retrospective institutional cohort. We further analyzed two independent datasets from the International Cancer Genomics Consortium and The Cancer Genome Atlas. Genomic data (n = 26 MuBC, n = 535 estrogen receptor [ER] positive/HER2-negative IDC), methylation data (n = 28 MuBC, n = 529 ER-positive/HER2-negative IDC), and transcriptomic data (n = 27 MuBC, n = 467 ER-positive/HER2-negative IDC) were analyzed. MuBC was characterized by low tumor infiltrating lymphocyte levels (median = 0.0%, average = 3.4%, 95% confidence interval = 1.9% to 4.9%). Compared with IDC, MuBC had a lower genomic instability (P =. 01, two-sided Mann-Whitney U test) and a decreased prevalence of PIK3CA mutations (39.7% in IDC vs 6.7% in MuBC, P =. 01 in the International Cancer Genomics Consortium; and 34.8% vs 0.0%, P =. 02 in The Cancer Genome Atlas, two-sided Fisher's exact test). Finally, our report identifies aberrant DNA methylation of MUC2 as a possible cause of extracellular production of mucin in MuBC., SCOPUS: ar.j, info:eu-repo/semantics/published

Invasive lobular breast cancer (ILC) is the second most common histological subtype of breast cancer after invasive ductal cancer (IDC). Here, we aimed at evaluating the prevalence, levels, and composition of tumor-infiltrating lymphocytes (TILs) and their association with clinico-pathological and outcome variables in ILC, and to compare them with IDC., SCOPUS: ar.j, info:eu-repo/semantics/published

Several studies using genome-wide molecular techniques have reported various degrees of genetic heterogeneity between primary tumours and their distant metastases. However, it has been difficult to discern patterns of dissemination owing to the limited number of patients and available metastases. Here, we use phylogenetic techniques on data generated using whole-exome sequencing and copy number profiling of primary and multiple-matched metastatic tumours from ten autopsied patients to infer the evolutionary history of breast cancer progression. We observed two modes of disease progression. In some patients, all distant metastases cluster on a branch separate from their primary lesion. Clonal frequency analyses of somatic mutations show that the metastases have a monoclonal origin and descend from a common 'metastatic precursor'. Alternatively, multiple metastatic lesions are seeded from different clones present within the primary tumour. We further show that a metastasis can be horizontally cross-seeded. These findings provide insights into breast cancer dissemination., SCOPUS: ar.j, info:eu-repo/semantics/published

CDK4/6 inhibitors (CDK4/6i) have proven benefits in estrogen receptor-positive (ER+) metastatic breast cancer (MBC), however the activity is variable among patients and biomarkers of sensitivity are lacking. We sought to determine whether any genomic alterations might characterize extreme responders and non-responders from over 500 patients with ER+ MBC who received CDK4/6i plus aromatase inhibitors or fulvestrant. We first analyzed a cohort of 348 patients who has MSK-IMPACT sequencing performed on a pre-treatment tumor sample and asked whether any somatic mutation or copy number alteration was associated with altered progression-free survival (PFS) on subsequent CDK4/6i-based therapy. As expected, common alterations in CCND1, PIK3CA or ESR1 were not associated with altered PFS on CDK4/6i. Also as anticipated, RB1 loss was associated with resistance to CDK4/6i (median PFS: 3.6 months, 95% confidence interval [CI]: 2.2, not reached, p=0.00041). Unexpectedly, alterations in components of the Hippo pathway (FAT1, YAP, LATS1/2, and NF2) were mutually exclusive with each other and with RB1 and were collectively associated with resistance to CDK4/6i in ER+ MBC (Hazard Radio [HR]: 3.6; 95% CI: 1.7, 7.8, p=0.0044). FAT1 was the most frequently mutated gene in the Hippo pathway with alterations resulting in FAT1 loss associated with a markedly shortened PFS (median: 2.4 months, HR: 11.1; 95% CI: 4.65, 26.62; p=2.2x10-11). No alterations were clearly linked with extremely prolonged PFS on CDK4/6i. Additional analyses from a larger cohort of patients with genomic sequencing prior to starting CDK4/6i are now underway and will be presented. To further understand the mechanisms of Hippo pathway regulation of cell cycle, cell line and patient derived xenograft models were utilized. These studies revealed that different mechanisms of suppression of Hippo signaling such as FAT1 knockdown (CRISPR or shRNA) and NF2 knockdown (shRNA) lead to YAP activation and YAP-dependent CDK4/6i resistance. Moreover, in all cases, resistance was mediated by upregulation of CDK6 as knockdown of CDK6 prevented drug resistance. Finally, we have sought to understand the implications of these distinct mechanisms of resistance to CDK4/6i. Clinical follow up on subsequent lines of therapy and duration of treatment after CDK4/6i (e.g. chemotherapy or other hormone therapy) are now being analyzed based on class of resistance alteration and will be presented. In conclusion, RB1 loss and Hippo pathway alterations mediate resistance to CDK4/6i in patients with ER+ MBC, nominating these as potential biomarkers for this class of drugs. Citation Format: Pedram Razavi, Qing Li, Zhiqiang Li, David N. Brown, Christina Ping, Ronglai Shen, Francisco Sanchez-Vega, Nikolaus D. Schultz, Jose Baselga, Maurizio Scaltriti, Jorge S. Reis-Filho, Sarat Chandarlapaty. Genomic profiling of metastatic breast cancer identifies the Hippo pathway to be a key determinant of CDK4/6 inhibitor sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 927.

1009 Background: Genomic profiling of ER+ metastatic breast cancer (MBC) has revealed highly prevalent genomic alterations (e.g. ESR1, NF1, ERBB2) associated with exposure to antiestrogen therapy and endocrine resistance. It is not known whether any such acquired genomic alterations are observed after exposure to now standard CDK4/6 inhibitors (CDK4/6i). Methods: To identify genomic alterations associated with acquired resistance to CDK4/6i + antiestrogen combinations, we prospectively performed tumor and matched normal sequencing on 1059 ER+ breast cancers from 845 MBC patients collected prior to (n = 838) or post-treatment with CDK4/6i (n = 221), including 110 pre- and post-treatment pairs. We performed gene enrichment analyses to identify the oncogenic mutations and copy number alterations that were more frequent in post-CDK4/6i samples compared to CDK4/6i-naïve samples and further compared these results to those of post-hormone alone therapy datasets. Results: The post-CDK4/6i samples were collected following exposure to CDK4/6i plus aromatase inhibitors (51%), plus fulvestrant (28%), or multiple/other (21%). Along with alterations previously associated with resistance to hormonal therapy alone, our analysis identified multiple genes to be specifically enriched in the post-CDK4/6i tumors. Among these, loss-of-function alterations in RB1 were significantly enriched in the post-CDK4/6i compared to CDK4/6i-naïve samples (7.9% vs. 2.7%, p-value = 1.5e-5). The majority of the RB1 mutations in the post-CDK4/6i tumors had loss of heterozygosity (LOH), while LOH was uncommon in the CDK4/6i-naive tumors. Additionally, multiple alterations in effectors of PI3K/AKT signaling (excluding PIK3CA), cell cycle (such as CDKN2A loss) and in Hippo signaling were more frequent in the post-CDK4/6i tumors. Conclusions: This large clinico-genomic analysis of the post- and pre-CDK4/6i MBCs reveals multiple genomic lesions to be enriched after exposure to CDK4/6i, highlighting therapy-related genomic evolution as a recurrent phenomenon and identifying unique genomic subsets that have strong potential to alter the benefit of subsequent lines of therapies.

BACKGROUND: We have previously shown that TILs are predictive of benefit to trastuzumab and chemotherapy in HER2+ BC in the FinHER study, an adjuvant, phase III study in early-stage BC, where HER2+ patients were randomized to 9 weeks of trastuzumab or no trastuzumab in addition to chemotherapy (Loi et al, ASCO 2012). We sought to further confirm this positive association as well as to understand the composition of TILs. METHODS: The association between TILs and response to trastuzumab with chemotherapy (epirubicin/cyclophosphamide with docetaxel with or without capecitabine) was evaluated in 156 HER2+ patients from the neoadjuvant GeparQuattro trial. The primary correlative endpoint was the association between TILs (quantified using the same method as previously published) with pathological complete response rates (pCR), adjusted for clinicopathological characteristics. To understand the composition of TILs, correlations between TILs and gene expression levels of 13 pre-defined immune markers were evaluated from 202 HER2+ samples from the FinHER study. These represented T and B cell infiltration (CD3D, IGKC), Th1 (IFNG, CD8A), chemoattractants (CXCL9, CXCL13), immunosuppression (VEGFA, FOXP3, IDO1) and T-cell checkpoint receptors and ligands (PD-1, PD-L1, CTLA-4, CD80). Prognostic associations and interactions with trastuzumab were studied in Cox regression models for distant-disease free survival (DDFS). Preclinical mice models of HER2 mammary cancer were used to investigate combination therapies. RESULTS: In the GeparQuattro trial data, each 10% increment in TILs was associated with higher rates of pCR (adjusted OR:1.14 95%CI:1.01-1.29;P = 0.037) after neoadjuvant trastuzumab and chemotherapy supporting the findings from the FinHER study. Gene expression analyses using the FinHER samples revealed IDO1 and CXCL13 were most highly correlated with TILs (R = 0.58 and 0.51;p We next postulated that enhancing T-cell responses would be synergistic with trastuzumab. Trastuzumab in combination with several inhibitors of T-cell negative regulation (anti-CTLA4, anti-PD-1, anti-PD-L1) resulted in higher tumor regressions compared with monotherapy in a mouse transplant model of HER2+ mammary cancer. In particular, anti-PD1 (P = 0.02) and anti-PD-L1 (P = 0.008) were most effective. In a BALB/c-MMTV-neu transgenic mice mouse model (i.e. immune tolerant to HER2 antigen), the combination of trastuzumab and anti-PD1 antibody could significantly delay mammary gland tumor formation (p CONCLUSIONS: We confirm that TILs are associated with higher responses to trastuzumab and chemotherapy. Furthermore, tumor-mediated immunosuppression is evident in the lymphocytic infiltrate with PD-1 and IDO1 significantly predictive of trastuzumab benefit. The addition of a T-cell checkpoint inhibitor in a preclinical model significantly enhanced trastuzumab responses. These combinations warrant evaluation in the clinical setting. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S1-05.

Purpose Invasive lobular breast cancer (ILBC) is the second most common histologic subtype after invasive ductal breast cancer (IDBC). Despite clinical and pathologic differences, ILBC is still treated as IDBC. We aimed to identify genomic alterations in ILBC with potential clinical implications. Methods From an initial 630 ILBC primary tumors, we interrogated oncogenic substitutions and insertions and deletions of 360 cancer genes and genome-wide copy number aberrations in 413 and 170 ILBC samples, respectively, and correlated those findings with clinicopathologic and outcome features. Results Besides the high mutation frequency of CDH1 in 65% of tumors, alterations in one of the three key genes of the phosphatidylinositol 3-kinase pathway, PIK3CA, PTEN, and AKT1, were present in more than one-half of the cases. HER2 and HER3 were mutated in 5.1% and 3.6% of the tumors, with most of these mutations having a proven role in activating the human epidermal growth factor receptor/ERBB pathway. Mutations in FOXA1 and ESR1 copy number gains were detected in 9% and 25% of the samples. All these alterations were more frequent in ILBC than in IDBC. The histologic diversity of ILBC was associated with specific alterations, such as enrichment for HER2 mutations in the mixed, nonclassic, and ESR1 gains in the solid subtype. Survival analyses revealed that chromosome 1q and 11p gains showed independent prognostic value in ILBC and that HER2 and AKT1 mutations were associated with increased risk of early relapse. Conclusion This study demonstrates that we can now begin to individualize the treatment of ILBC, with HER2, HER3, and AKT1 mutations representing high-prevalence therapeutic targets and FOXA1 mutations and ESR1 gains deserving urgent dedicated clinical investigation, especially in the context of endocrine treatment.

SQLE encodes squalene epoxidase, a key enzyme in cholesterol synthesis. SQLE has sporadically been reported among copy-number driven transcripts in multi-omics cancer projects. Yet, its functional relevance has never been subjected to systematic analyses. Here, we assessed the correlation of SQLE copy number (CN) and gene expression (GE) across multiple cancer types, focusing on the clinico-pathological associations in breast cancer (BC). We then investigated whether any biological effect of SQLE inhibition could be observed in BC cell line models. Breast, ovarian, and colorectal cancers showed the highest CN driven GE among 8,783 cases from 22 cancer types, with BC presenting the strongest one. SQLE overexpression was more prevalent in aggressive BC, and was an independent prognostic factor of unfavorable outcome. Through SQLE pharmacological inhibition and silencing in a panel of BC cell lines portraying the diversity of SQLE CN and GE, we demonstrated that SQLE inhibition resulted in a copy-dosage correlated decrease in cell viability, and in a noticeable increase in replication time, only in lines with detectable SQLE transcript. Altogether, our results pinpoint SQLE as a bona fide metabolic oncogene by amplification, and as a therapeutic target in BC. These findings could have implications in other cancer types., SCOPUS: ar.j, info:eu-repo/semantics/published

Standard treatment for clinical stage II/III rectal adenocarcinoma (READ) includes a combination of chemotherapy with pyrimidine analogues, such as capecitabine, and radiation therapy, followed by surgery after 6 to 12 weeks. Currently no predictors of benefit from neoadjuvant chemoradiotherapy (NACT) exist, and transcriptomic studies, prevalently focused on coding transcripts or miRNAs have so far failed to find clinically useful gene signatures. In the present analysis we assessed the expression levels of 8,127 long non-coding RNAs (lncRNAs), poorly studied in the context of READ, to infer whether they could stratify pre-treatment READ samples according to patients' pathological complete response (pCR). Methods: We collected and analysed with Agilent SurePrint G3 Human v2 8x60K microarrays, a consecutive series of 49 specimens from READ patients undergoing NACT with a combination of capecitabine (825 mg/m2) and radiation (50.4 Gy) cycles for 6 weeks. We performed a regularized variable selection regression (“glmnet” R package) to identify potential lncRNAs predicting pCR. We then analysed, with a bivariate correlation, the top lncRNAs predictors with the expression of 20,560 coding transcripts represented in the microarrays we used, and performed pathway enrichment analysis using Ingenuity® Pathway Analysis (Qiagen, Inc.). Results: Out of 49 patients for whom we could obtain snap-frozen biopsies with complete clinical and pathological data, we analysed 30 patients, 12 with minor or absent pathological response and 18 with major or complete response to NACT. We identified a signature of 11 lncRNAs able to successfully stratify responder vs. non-responder patients. By investigating the correlation of these 11 lncRNAs with coding transcripts, we identified one of them, lnc-ZNF-366-6, presenting an unusually high correlation with more then 1136 coding genes (FDR < 0.01). By gene enrichment analysis we found an enrichment in cancer-related pathways, especially alterations in solid and colon adenocarcinoma pathways (p < 0.01), as well as mitochondrial dysfunction, oxidative phosphorylation and sirtuin signalling pathways (p < 0.00001), defined by the positively and negatively correlated variables with lnc-ZNF-366-6. Moreover, we identified potential drivers of disease or therapeutic targets, either correlated or anti-correlated with lnc-ZNF-366-6. Conclusions: LncRNAs have the potential to classify responder versus non-responder READ patients undergoing NACT. Lnc-ZNF-366-6 deserves further experimental validation and functional analyses to clarify its exact contribution to resistance to DNA damaging agents, such as radiation, and to inhibitors of DNA/RNA synthesis, such as capecitabine. Citation Format: Lorenzo Ferrando, Gabriella Cirmena, A Garuti, Stefano Scabini, Federica Grillo, Luca Mastracci, Edoardo Isnaldi, Ciro Marrone, Roberto Murialdo, David Norman Brown, Roberto Fiocca, Emanuele Romairone, Alberto Ballestrero, Gabriele Zoppoli. Long non-coding RNA ZNF-366-6 predicts lack of response to neoadjuvant chemoradiotherapy in locally advanced rectal adenocarcinoma and presents meaningful biological associations with potential coding cancer driver transcripts related to gastrointestinal cancer pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2465.

Background: Invasive lobular breast cancer (ILC) is the second most common histological subtype of breast cancer, is mostly estrogen receptor-positive (ER+) and has distinct clinico-pathological features when compared to ER+ invasive ductal carcinoma (IDC). In this study, we aimed to characterize the genomic alterations defining ILC in a large cohort of ILC patients with long-term follow up (FU). Material & methods: In 499 centrally histologically confirmed ILC patients (median FU= 9.8 years) we analyzed mutational data gathered from targeted sequencing of 360 cancer genes at an average coverage of 106X (alignment done with BWA, substitutions and indels –further referred to as mutations- called with Caveman and Pindel). Matched normal DNA was available for 242 patients. Genome-wide copy number (CN) data were available for 178 patients. E-cadherin (CDH1) and beta-catenin (CTNNB1) stains were carried out using the DakoTM antibodies. Invasive disease free survival (IDFS) was considered as primary survival endpoint. Results: A median of 6 [range:0-38] non-silent mutations was identified across the primary tumors of all patients. The most frequently mutated genes (>3%) are listed in Table 1. Of those, CDH1, PIK3CA, TBX3, FOXA1 and the chromatin-related genes MLL2, MLL3, ARID1A and ARID1B were more frequently mutated in our ER+/HER2- ILC (n= 451) compared to the ER+/HER2- IDC (n=266) from The Cancer Genome Atlas, whereas GATA3, TP53 and MAP3K1 were less frequently mutated. Samples with a CDH1 mutation were associated with changes at the protein level: 97.5% displayed a complete loss of the protein, associated with cytoplasmic staining for CTNNB1, compared to only 63% of the CDH1 non-mutated tumors. CDH1 mutated tumors were further characterized by increased mutational frequencies of the ERBB-genes: 15.4% for the CDH1 mutated tumors versus 3% in the CDH1 non-mutated tumors, most of those mutations being described in the literature as activating the pathway. Almost all tumors (97%) with CN data had a heterozygous loss of CDH1. The special alveolar, solid and trabecular lobular histotypes were associated with specific CN alterations and mutations. Tumors with mutated ARID1A or ATM were associated with worse IDFS at the univariate level and ARID1A remained significant in a multivariate analysis including standard parameters (HR =2.07, p=0.003). At the CN level, ATM and ARID1A losses, as well as HER2 and VEGFA gains/amplifications were associated with decreased IDFS, all but ARID1A holding significance at the multivariate level (HR_HER2=2.41, HR_VEGFA= 1.99, HR_ATM= 1.79, all p Conclusion: This is the first and largest study to our knowledge to report genomic alterations present in ILC and their association with survival. This work therefore opens new avenues for a better understanding of the disease and its clinical management. Table 1: List of the most frequently mutated genesGene %Gene %Gene %Gene %CDH1 62.9MAP3K1 7USP9X 4.4EPHB6 3.8PIK3CA 44.9BRCA2 6.6ATR 4.2MED12L 3.6MLL3 15.8ERBB2 6.4COL22A1 4.2PTEN 3.6TBX3 13.2ARID1B 5.6MED13 4.2ERBB3 3.2FOXA1 9.6ATM 4.8NOTCH1 4.2ROS1 3.2MLL2 9.4MLL 4.6AKT1 4.2BPTF 3.2ARID1A 8.4MYO5B 4.4MYO3A 4BRCA1 3.2GATA3 8EP300 4.4NF1 4IRS2 3.2TP53 7.4RUNX1 4.4EP400 3.8NOTCH4 3 Citation Format: Christine Desmedt, Gunes Gundem, Gabriele Zoppoli, Giancarlo Pruneri, Elia Biganzoli, Marco Fornili, Debora Fumagalli, Françoise Rothé, David Brown, Peter Van Loo, Sylvain Brohée, Delphine Vincent, Naima Keddoumi, Samira Majjaj, Ghizlane Rouas, Thomas Van Brussel, Diether Lambrechts, Otto Metzger, Christine Galant, François Bertucci, Martine Piccart, Denis Larsimont, Giuseppe Viale, Peter J Campbell, Christos Sotiriou. Characterization and clinical relevance of the genomic alterations defining lobular breast cancer [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 S2-05.

Multifocal breast cancer (MFBC), defined as multiple synchronous unilateral lesions of invasive breast cancer, is relatively frequent and has been associated with more aggressive features than unifocal cancer. Here, we aimed to investigate the genomic heterogeneity between MFBC lesions sharing similar histopathological parameters. Characterization of different lesions from 36 patients with ductal MFBC involved the identification of non-silent coding mutations in 360 protein-coding genes (171 tumour and 36 matched normal samples). We selected only patients with lesions presenting the same grade, ER, and HER2 status. Mutations were classified as 'oncogenic' in the case of recurrent substitutions reported in COSMIC or truncating mutations affecting tumour suppressor genes. All mutations identified in a given patient were further interrogated in all samples from that patient through deep resequencing using an orthogonal platform. Whole-genome rearrangement screen was further conducted in 8/36 patients. Twenty-four patients (67%) had substitutions/indels shared by all their lesions, of which 11 carried the same mutations in all lesions, and 13 had lesions with both common and private mutations. Three-quarters of those 24 patients shared oncogenic variants. The remaining 12 patients (33%) did not share any substitution/indels, with inter-lesion heterogeneity observed for oncogenic mutation(s) in genes such as PIK3CA, TP53, GATA3, and PTEN. Genomically heterogeneous lesions tended to be further apart in the mammary gland than homogeneous lesions. Genome-wide analyses of a limited number of patients identified a common somatic background in all studied MFBCs, including those with no mutation in common between the lesions. To conclude, as the number of molecular targeted therapies increases and trials driven by genomic screening are ongoing, our findings highlight the presence of genomic inter-lesion heterogeneity in one-third, despite similar pathological features. This implies that deeper molecular characterization of all MFBC lesions is warranted for the adequate management of those cancers., SCOPUS: ar.j, FLWOA, info:eu-repo/semantics/published

Background Over the past years, tumor derived alterations in plasma has emerged as a promising cancer biomarker. The clinical applications envisioned with liquid biopsies range from monitoring of treatment response to disease progression and acquired resistance. However, the analysis of circulating tumor DNA (ctDNA) is challenging owing to the small fraction of tumor specific DNA relative to background levels of non-tumor derived DNA and therefore requires highly specific and sensitive techniques. Experimental procedures The Ion AmpliSeqTM Cancer Hotspot Panel v2, covering 2,800 COSMIC mutations from 50 cancer genes was used to analyze 31 primary tumor and metastatic lesions along with 19 matched plasma samples from 13 breast cancer (BC) patients receiving neoadjuvant treatment and 6 advanced stage metastatic BC patients. Digital PCR (dPCR) experiments were set up using mutation-specific assays (Biorad) for the detection of 13 hotspot mutations in PIK3CA, TP53, PTEN, AKT1, CDKN2A and STK11 genes previously identified in the solid tumor samples using next generation sequencing (NGS). Results The specificity and sensitivity of the mutation-specific dPCR assays were tested in dilution curves using synthetic oligonucleotides and tumor samples bearing the mutation of interest. Mutations could be robustly detected in plasma samples using dPCR with a sensitivity as low as 0.1% and a sensitivity of 0.5% using NGS. All mutations previously identified using NGS were validated using dPCR in tumor (n = 27) as well as in plasma samples (n = 5). Moreover, dPCR identified 4 additional mutations in 4 plasma samples in the neoadjuvant setting and 4 in the metastatic setting, 2 in tumor and 2 in plasma samples. The mutant allelic frequencies (MAF) of these newly identified mutations were very low (range 0.2%-1.8%) close to the detection limit of the technology. Through the use of dPCR, the detection rate of plasma ctDNA increased from 8% using NGS to 38% in the neoadjuvant setting and from 67% using NGS to 100% in the metastatic setting thereby highlighting the greater sensitivity of the dPCR technology. The MAF assessed by NGS and dPCR were highly correlated both in the neoadjuvant and metastatic (Spearman ρ = 0.84 and ρ = 0.97) settings, a higher concordance between NGS and dPCR being observed when the MAF are high. Conclusions The use of dPCR allows the detection of low-frequency tumor-specific mutations in plasma with a greater sensitivity compared to NGS providing the high accuracy and sensitivity essential for liquid biopsies. Citation Format: Françoise Rothé, Ghizlane Rouas, Michelle Coureau, Marion Maetens, David Brown, Christos Sotiriou, Michail Ignatiadis. Comparison of NGS and digital PCR for the detection of point mutations in breast tumor and plasma samples. [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 3968.