Accurate identification of tumor-derived somatic variants in plasma circulating cell-free DNA (cfDNA) requires understanding the various biologic compartments contributing to the cfDNA pool. We sought to define the technical feasibility of a high-intensity sequencing assay of cfDNA and matched white-blood cell (WBC) DNA covering a large genomic region (508 genes, 2Mb, >60,000X raw-depth) in a prospective study of 124 metastatic cancer patients, with contemporaneous matched tumor tissue biopsies, and 47 non-cancer controls. The assay displayed a high sensitivity and specificity, allowing for de novo detection of tumor-derived mutations and inference of tumor mutational burden, microsatellite instability, mutational signatures and sources of somatic mutations identified in cfDNA. The vast majority of cfDNA mutations (81.6% in controls and 53.2% in cancer patients) had features consistent with clonal hematopoiesis (CH). This cfDNA sequencing approach revealed that CH constitutes a pervasive biological phenomenon emphasizing the importance of matched cfDNA-WBC sequencing for accurate variant interpretation.

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Major depressive disorder (MDD), is a prevalent mood disorder that associates with differential prefrontal brain expression patterns1. Treatment of MDD includes a variety of biopsychosocial approaches, but in medical practice, antidepressant drugs are the most common treatment for depressive episodes, and not surprisingly, they are among the most prescribed medications in North America2,3. While they are clearly effective, particularly for moderate to severe depressive episodes, there is important variability in how individuals respond to antidepressant treatment. Failure to respond has important individual, economic and social consequences for patients and their families4. Several lines of evidence demonstrate that genes are regulated through the activity of microRNAs (miRNAs), which act as fine–tuners and on–off switches in gene expression patterns5–7. Here we report on complementary studies using postmortem human brain samples, cellular assays and samples from clinical trials of depressed patients, and show that miR-1202, a miRNA specific to primates and enriched in the human brain, is differentially expressed in depressed individuals. Additionally, miR-1202 regulates the expression of the Metabotropic Glutamate Receptor 4 (GRM4) gene and predicts antidepressant response at baseline. These results suggest that miR-1202 is associated with the pathophysiology of depression and is a potential target for novel antidepressant treatments.

Purpose Metaplastic breast carcinoma (MBC) is a rare and aggressive histologic type of breast cancer predominantly of triple-negative phenotype, and characterized by the presence of malignant cells showing squamous and/or mesenchymal differentiation. We sought to define the repertoire of somatic genetic alterations and the mutational signatures of MBCs. Experimental Design Whole-exome sequencing was performed in 35 MBCs, with 16, ten and nine classified as harboring chondroid, spindle and squamous metaplasia as the predominant metaplastic component. The genomic landscape of MBCs was compared to that of triple-negative invasive ductal carcinomas of no special type (IDC-NSTs) from The Cancer Genome Atlas. Wnt and PI3K/AKT/mTOR pathway activity was assessed using a quantitative PCR assay. Results MBCs harbored complex genomes with frequent TP53 (69%) mutations. In contrast to triple-negative IDC-NSTs, MBCs more frequently harbored mutations in PIK3CA (29%), PIK3R1 (11%), ARID1A (11%), FAT1 (11%) and PTEN (11%). PIK3CA mutations were not found in MBCs with chondroid metaplasia. Compared to triple-negative IDC-NSTs, MBCs significantly more frequently harbored mutations in PI3K/AKT/mTOR pathway-related (57% vs 22%) and canonical Wnt pathway-related (51% vs 28%) genes. MBCs with somatic mutations in PI3K/AKT/mTOR or Wnt pathway-related genes displayed increased activity of the respective pathway. Conclusion MBCs are genetically complex and heterogeneous, and are driven by a repertoire of somatic mutations distinct from that of triple-negative IDC-NSTs. Our study highlights the genetic basis and the importance of PI3K/AKT/mTOR and Wnt pathway dysregulation in MBCs, and provides a rationale for the metaplastic phenotype and the reported responses to PI3K/AKT/mTOR inhibitors in these tumors.

Phyllodes tumours (PTs) are breast fibroepithelial lesions that are graded based on histological criteria as benign, borderline or malignant. PTs may recur locally. Borderline PTs and malignant PTs may metastasize to distant sites. Breast fibroepithelial lesions, including PTs and fibroadenomas, are characterized by recurrent MED12 exon 2 somatic mutations. We sought to define the repertoire of somatic genetic alterations in PTs and whether these may assist in the differential diagnosis of these lesions. We collected 100 fibroadenomas, 40 benign PTs, 14 borderline PTs and 22 malignant PTs. Six, 6 and 13 benign, borderline and malignant PTs respectively and their matched normal tissue were subjected to targeted massively parallel sequencing (MPS) using the MSK-IMPACT sequencing assay. Recurrent MED12 mutations were found in 56% of PTs; in addition, mutations affecting cancer genes (e.g. TP53, RB1, SETD2 and EGFR) were exclusively detected in borderline and malignant PTs. We found a novel recurrent clonal hotspot mutation in the TERT promoter (−124 C>T) in 52% and TERT gene amplification in 4% of PTs. Laser capture microdissection revealed that these mutations were restricted to the mesenchymal component of PTs. Sequencing analysis of the entire cohort revealed that the frequency of TERT alterations increased from benign (18%), to borderline (57%) and to malignant PTs (68%; P<0.01), and TERT alterations were associated with increased levels of TERT mRNA (P<0.001). No TERT alterations were observed in fibroadenomas. An analysis of TERT promoter sequencing and gene amplification distinguished PTs from fibroadenomas with a sensitivity and a positive predictive value of 100% (CI 95.38%–100%) and 100% (CI 85.86%–100%), respectively, and a sensitivity and a negative predictive value of 39% (CI 28.65%–51.36%) and 68% (CI 60.21%–75.78%), respectively. Our results suggest that TERT alterations may drive the progression of PTs, and may assist in the differential diagnosis between PTs and fibroadenomas.

Synchronous early-stage endometrioid endometrial carcinomas (EECs) and endometrioid ovarian carcinomas (EOCs) are associated with a favorable prognosis and have been suggested to represent independent primary tumors rather than metastatic disease. We subjected sporadic synchronous EECs/EOCs from five patients to whole-exome massively parallel sequencing, which revealed that the EEC and EOC of each case displayed strikingly similar repertoires of somatic mutations and gene copy number alterations. Despite the presence of mutations restricted to the EEC or EOC in each case, we observed that the mutational processes that shaped their respective genomes were consistent. High-depth targeted massively parallel sequencing of sporadic synchronous EECs/EOCs from 17 additional patients confirmed that these lesions are clonally related. In an additional Lynch Syndrome case, however, the EEC and EOC were found to constitute independent cancers lacking somatic mutations in common. Taken together, sporadic synchronous EECs/EOCs are clonally related and likely constitute dissemination from one site to the other.

BRCA1 and BRCA2 are involved in homologous recombination (HR) DNA repair and are germ-line cancer pre-disposition genes that result in a syndrome of hereditary breast and ovarian cancer (HBOC). Whether germ-line or somatic alterations in these genes or other members of the HR pathway and if mono- or bi-allelic alterations of HR-related genes have a phenotypic impact on other cancers remains to be fully elucidated. Here, we perform a pan-cancer analysis of The Cancer Genome Atlas (TCGA) data set and observe that bi-allelic pathogenic alterations in homologous recombination (HR) DNA repair-related genes are prevalent across many malignancies. These bi-allelic alterations often associate with genomic features of HR deficiency. Further, in ovarian, breast and prostate cancers, bi-allelic alterations are mutually exclusive of each other. The combination of these two properties facilitates reclassification of variants of unknown significance affecting DNA repair genes, and may help personalize HR directed therapies in the clinic.

Key Points• Programmed death ligands 1 and 2 are rearranged at a frequency of 20% in PMBCL.The pathogenesis of primary mediastinal large B-cell lymphoma (PMBCL) is incompletely understood. Recently, specific genotypic and phenotypic features have been linked to tumor cell immune escape mechanisms in PMBCL. We studied 571 B-cell lymphomas with a focus on PMBCL. Using fluorescence in situ hybridization here, we report that the programmed death ligand (PDL) locus (9p24.1) is frequently and specifically rearranged in PMBCL (20%) as compared with diffuse large B-cell lymphoma, follicular lymphoma, and Hodgkin lymphoma. Rearrangement was significantly correlated with overexpression of PDL transcripts. Utilizing high-throughput sequencing techniques, we characterized novel translocations and chimeric fusion transcripts involving PDLs at base-pair resolution. Our data suggest that recurrent genomic rearrangement events underlie an immune privilege phenotype in a subset of B-cell lymphomas. (Blood. 2014;123(13):2062-2065 IntroductionPrimary mediastinal large B-cell lymphoma (PMBCL) is an aggressive disease known to share certain genotypic and phenotypic features with classic Hodgkin lymphoma (CHL) and diffuse large B-cell lymphoma (DLBCL).1,2 However, the complete landscape of genetic alterations involved in PMBCL pathogenesis has yet to be fully elucidated.3 Among the most common chromosomal alterations in PMBCL are amplifications of chromosome 9p and translocations involving CIITA (16p13.13).4-7 These aberrations have been suggested to affect tumor-microenvironment interactions resulting in immune privilege. 7,8 Here, we demonstrate that rearrangements involving immune cell anergy-inducing programmed death ligand (PDL) 1 (CD274) and 2 (PDCD1LG2) are recurrent in and characteristic of PMBCL. Furthermore, we show such rearrangements are correlated with elevated transcript levels, and we characterize novel translocations identified using high-throughput sequencing. Study designWe studied 571 primary B-cell lymphoma samples in conjunction with 17 established B-cell-derived cell lines. Using in-house bacterial artificial chromosome probes, fluorescence in situ hybridization (FISH) or FISH combined with CD30 immunofluorescence (in the case of CHL specimens) was performed to characterize the PDL locus.7,9,10 These cases were also analyzed with Epstein-Barr virus (EBV)-encoded RNA in situ hybridization. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for CD274 and PDCD1LG2 transcript expression on a subset of cases from the FISH cohort (N 5 76). Surface PDCD1LG2 expression of the cell lines was determined via flow cytometry. To characterize the Hodgkin lymphoma cell lines found to be rearranged by FISH, 1 whole-transcriptome sequencing (RNA-seq) library (CHL-derived L-428) was reanalyzed, and 1 new wholegenome library (L-428) and 2 new RNA-seq libraries (CHL-derived L-1236 and nodular lymphocyte predominant Hodgkin lymphoma-derived DEV) were sequenced.7 This study was approved by the BC Cancer Agency ...