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Abstract CDH1 (E-cadherin) bi-allelic inactivation is the hallmark alteration of breast invasive lobular carcinoma (ILC), resulting in its discohesive phenotype. A subset of ILCs, however, lack CDH1 genetic/epigenetic inactivation, and their genetic underpinning is unknown. Through clinical targeted sequencing data reanalysis of 364 primary ILCs, we identified 25 ILCs lacking CDH1 bi-allelic genetic alterations. CDH1 promoter methylation was frequent (63%) in these cases. Targeted sequencing reanalysis revealed 3 ILCs harboring AXIN2 deleterious fusions (n = 2) or loss-of-function mutation (n = 1). Whole-genome sequencing of 3 cases lacking bi-allelic CDH1 genetic/epigenetic inactivation confirmed the AXIN2 mutation and no other cell-cell adhesion genetic alterations but revealed a new CTNND1 (p120) deleterious fusion. AXIN2 knock-out in MCF7 cells resulted in lobular-like features, including increased cellular migration and resistance to anoikis. Taken together, ILCs lacking CDH1 genetic/epigenetic alterations are driven by inactivating alterations in other cell adhesion genes (CTNND1 or AXIN2), endorsing a convergent phenotype in ILC.

Abstract 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.

Summary: Background: Late distant recurrence is a challenge for the treatment of invasive lobular carcinoma (ILC) of the breast. Despite in-depth characterisation of primary ILC, the molecular landscape of metastatic ILC is still only partially understood. Methods: We retrospectively identified 38 ILC patients from the tissue banks of six European institutions. DNA extracted from patient matched primary and metastatic FFPE tissue blocks was whole genome sequenced to compute somatic copy number aberrations. This, in turn, was used to infer the evolutionary history of these patients. Findings: The data show different metastatic seeding patterns, with both an early and late divergence of the metastatic lineage observed in ILC. Additionally, cascading dissemination from a metastatic precursor was a dominant rule. Alterations in key cancer driver genes, such as TP53 or CCND1, were acquired early while additional aberrations were present only in the metastatic branch. In about 30% of the patients, the metastatic lineage harboured less aberrations than the primary tumour suggesting a period of tumour dormancy or prolonged adaptation at the distant site. This phenomenon was mostly observed in tumours from de novo metastatic patients. Interpretation: Our results provide insights into ILC evolution and offer potential paths for optimised ILC care. Funding: This work has received financial support from Les Amis de l'Institut Bordet, MEDIC, the Breast Cancer Research Foundation (BCRF) and the Belgian Fonds National de la Recherche Scientifique (F.R.S-FNRS).

Liquid biopsies allow the non-invasive detection of somatic mutations from tumours. Here, the authors develop and test MSK-ACCESS, an NGS-based clinical assay for identifying low frequency mutations in 129 genes and describe how it benefits patients in the clinic.

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.

Abstract 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.

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

Analysis of cell-free circulating tumor DNA obtained by liquid biopsy is a non-invasive approach that may provide clinically actionable information when conventional tissue biopsy is inaccessible or infeasible. Here, we followed a patient with hormone receptor-positive and human epidermal growth factor receptor (HER) 2-negative breast cancer who developed bone metastases seven years after mastectomy. We analyzed circulating cell-free DNA (cfDNA) extracted from plasma using high-depth massively parallel sequencing targeting 468 cancer-associated genes, and we identified a clonal hotspot missense mutation in the PIK3CA gene (3:178952085, A > G, H1047R) and amplification of the CCND1 gene. Whole-exome sequencing revealed that both alterations were present in the primary tumor. After treatment with ribociclib plus letrozole, the genetic abnormalities were no longer detected in cfDNA. These results underscore the clinical utility of combining liquid biopsy and comprehensive genomic profiling to monitor treatment response in patients with metastasized breast cancer.

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.

Granular cell tumors (GCTs) are rare tumors that arise in multiple anatomical locations. Here, the authors investigate the genomics of GCTs, finding inactivating somatic mutations in ATP6AP1 or ATP6AP2 in 72% of the 82 GCTs analyzed. In vitro manipulation of these genes recapitulated GCT phenotypes in cellular models.