The Great Majority of Homologous Recombination Repair-Deficient Tumors Are Accounted for by Established Causes.

Background: Gene-agnostic genomic biomarkers were recently developed to identify homologous recombination deficiency (HRD) tumors that are likely to respond to treatment with PARP inhibitors. Two machine-learning algorithms that predict HRD status, CHORD, and HRDetect, utilize various HRD-associated features extracted from whole-genome sequencing (WGS) data and show high sensitivity in detecting patients with BRCA1/2 bi-allelic inactivation in all cancer types. When using only DNA mutation data for the detection of potential causes of HRD, both HRDetect and CHORD find that 30-40% of cases that have been classified as HRD are due to unknown causes. Here, we examined the impact of tumor-specific thresholds and measurement of promoter methylation of BRCA1 and RAD51C on unexplained proportions of HRD cases across various tumor types. Methods: We gathered published CHORD and HRDetect probability scores for 828 samples from breast, ovarian, and pancreatic cancer from previous studies, as well as evidence of their biallelic inactivation (by either DNA alterations or promoter methylation) in HR-related genes. ROC curve analysis evaluated the performance of each classifier in specific cancer. Tenfold nested cross-validation was used to find the optimal threshold values of HRDetect and CHORD for classifying HR-deficient samples within each cancer type. Results: With the universal threshold, HRDetect has higher sensitivity in the detection of biallelic inactivation in BRCA1/2 than CHORD and resulted in a higher proportion of unexplained cases. When promoter methylation was excluded, in ovarian carcinoma, the proportion of unexplained cases increased from 26.8 to 48.8% for HRDetect and from 14.7 to 41.2% for CHORD. A similar increase was observed in breast cancer. Applying cancer-type-specific thresholds led to similar sensitivity and specificity for both methods. The cancer-type-specific thresholds for HRDetect reduced the number of unexplained cases from 21 to 12.3% without reducing the 96% sensitivity to known events. For CHORD, unexplained cases were reduced from 10 to 9% while sensitivity increased from 85.3 to 93.9%. Conclusion: These results suggest that WGS-based HRD classifiers should be adjusted for tumor types. When applied, only ∼10% of breast, ovarian, and pancreas cancer cases are not explained by known events in our dataset.

Data sharing to improve concordance in variant interpretation across laboratories: results from the Canadian Open Genetics Repository.

BACKGROUND: This study aimed to identify and resolve discordant variant interpretations across clinical molecular genetic laboratories through the Canadian Open Genetics Repository (COGR), an online collaborative effort for variant sharing and interpretation. METHODS: Laboratories uploaded variant data to the Franklin Genoox platform. Reports were issued to each laboratory, summarising variants where conflicting classifications with another laboratory were noted. Laboratories could then reassess variants to resolve discordances. Discordance was calculated using a five-tier model (pathogenic (P), likely pathogenic (LP), variant of uncertain significance (VUS), likely benign (LB), benign (B)), a three-tier model (LP/P are positive, VUS are inconclusive, LB/B are negative) and a two-tier model (LP/P are clinically actionable, VUS/LB/B are not). We compared the COGR classifications to automated classifications generated by Franklin. RESULTS: Twelve laboratories submitted classifications for 44 510 unique variants. 2419 variants (5.4%) were classified by two or more laboratories. From baseline to after reassessment, the number of discordant variants decreased from 833 (34.4% of variants reported by two or more laboratories) to 723 (29.9%) based on the five-tier model, 403 (16.7%) to 279 (11.5%) based on the three-tier model and 77 (3.2%) to 37 (1.5%) based on the two-tier model. Compared with the COGR classification, the automated Franklin classifications had 94.5% sensitivity and 96.6% specificity for identifying actionable (P or LP) variants. CONCLUSIONS: The COGR provides a standardised mechanism for laboratories to identify discordant variant interpretations and reduce discordance in genetic test result delivery. Such quality assurance programmes are important as genetic testing is implemented more widely in clinical care.

A novel mouse model of PMS2 founder mutation that causes mismatch repair defect due to aberrant splicing.

Hereditary non-polyposis colorectal cancer, now known as Lynch syndrome (LS) is one of the most common cancer predisposition syndromes and is caused by germline pathogenic variants (GPVs) in DNA mismatch repair (MMR) genes. A common founder GPV in PMS2 in the Canadian Inuit population, NM_000535.5: c.2002A>G, leads to a benign missense (p.I668V) but also acts as a de novo splice site that creates a 5 bp deletion resulting in a truncated protein (p.I668*). Individuals homozygous for this GPV are predisposed to atypical constitutional MMR deficiency with a delayed onset of first primary malignancy. We have generated mice with an equivalent germline mutation (Pms2c.1993A>G) and demonstrate that it results in a splicing defect similar to those observed in humans. Homozygous mutant mice are viable like the Pms2 null mice. However, unlike the Pms2 null mice, these mutant mice are fertile, like humans homozygous for this variant. Furthermore, these mice exhibit a significant increase in microsatellite instability and intestinal adenomas on an Apc mutant background. Rectification of the splicing defect in human and murine fibroblasts using antisense morpholinos suggests that this novel mouse model can be valuable in evaluating the efficacy aimed at targeting the splicing defect in PMS2 that is highly prevalent among the Canadian Inuits.

Circulating tumor DNA is readily detectable among Ghanaian breast cancer patients supporting non-invasive cancer genomic studies in Africa.

Circulating tumor DNA (ctDNA) sequencing studies could provide novel insights into the molecular pathology of cancer in sub-Saharan Africa. In 15 patient plasma samples collected at the time of diagnosis as part of the Ghana Breast Health Study and unselected for tumor grade and subtype, ctDNA was detected in a majority of patients based on whole- genome sequencing at high (30×) and low (0.1×) depths. Breast cancer driver copy number alterations were observed in the majority of patients.

Current gene panels account for nearly all homologous recombination repair-associated multiple-case breast cancer families.

It was hypothesized that variants in underexplored homologous recombination repair (HR) genes could explain unsolved multiple-case breast cancer (BC) families. We investigated HR deficiency (HRD)-associated mutational signatures and second hits in tumor DNA from familial BC cases. No candidates genes were associated with HRD in 38 probands previously tested negative with gene panels. We conclude it is unlikely that unknown HRD-associated genes explain a large fraction of unsolved familial BC.

Molecular characterization of DICER1-mutated pituitary blastoma.

Pituitary blastoma (PitB) has recently been identified as a rare and potentially lethal pediatric intracranial tumor. All cases that have been studied molecularly possess at least one DICER1 pathogenic variant. Here, we characterized nine pituitary samples, including three fresh frozen PitBs, three normal fetal pituitary glands and three normal postnatal pituitary glands using small-RNA-Seq, RNA-Seq, methylation profiling, whole genome sequencing and Nanostring® miRNA analyses; an extended series of 21 pituitary samples was used for validation purposes. These analyses demonstrated that DICER1 RNase IIIb hotspot mutations in PitBs induced improper processing of miRNA precursors, resulting in aberrant 5p-derived miRNA products and a skewed distribution of miRNAs favoring mature 3p over 5p miRNAs. This led to dysregulation of hundreds of 5p and 3p miRNAs and concomitant dysregulation of numerous mRNA targets. Gene expression analysis revealed PRAME as the most significantly upregulated gene (500-fold increase). PRAME is a member of the Retinoic Acid Receptor (RAR) signaling pathway and in PitBs, the RAR, WNT and NOTCH pathways are dysregulated. Cancer Hallmarks analysis showed that PI3K pathway is activated in the tumors. Whole genome sequencing demonstrated a quiet genome with very few somatic alterations. The comparison of methylation profiles to publicly available data from ~ 3000 other central nervous system tumors revealed that PitBs have a distinct methylation profile compared to all other tumors, including pituitary adenomas. In conclusion, this comprehensive characterization of DICER1-related PitB revealed key molecular underpinnings of PitB and identified pathways that could potentially be exploited in the treatment of this tumor.

Investigating the causal role of MRE11A p.E506* in breast and ovarian cancer.

The nuclease MRE11A is often included in genetic test panels for hereditary breast and ovarian cancer (HBOC) due to its BRCA1-related molecular function in the DNA repair pathway. However, whether MRE11A is a true predisposition gene for HBOC is still questionable. We determined to investigate this notion by dissecting the molecular genetics of the c.1516G > T;p.E506* truncating MRE11A variant, that we pinpointed in two unrelated French-Canadian (FC) HBOC patients. We performed a case-control study for the variant in ~ 2500 breast, ovarian, and endometrial cancer patients from the founder FC population of Quebec. Furthermore, we looked for the presence of second somatic alterations in the MRE11A gene in the tumors of the carriers. In summary, these investigations suggested that the identified variant is not associated with an increased risk of developing breast or ovarian cancer. We finally performed a systematic review for all the previously reported MRE11A variants in breast and ovarian cancer. We found that MRE11A germline variants annotated as pathogenic on ClinVar often lacked evidence for such classification, hence misleading the clinical management for affected patients. In summary, our report suggests the lack of clinical utility of MRE11A testing in HBOC, at least in the White/Caucasian populations.

Founder BRCA1/BRCA2/PALB2 pathogenic variants in French-Canadian breast cancer cases and controls.

Inherited germline pathogenic variants are responsible for ~5% of breast cancer globally. Through rapid expansion and isolation since immigration in the early 17th century, French Canadians are a relatively genetically homogenous founder population and therefore represent a unique demographic for genetic contributions to disease. To date, twenty variants in BRCA1, BRCA2, and PALB2 that predispose families to breast and ovarian cancer have been identified as recurring in the French-Canadian founder population. Our objective was to evaluate the clinical efficacy and validity of targeted genetic testing for these variants in Montreal French Canadians. A total of 555 breast cancer cases unselected for family history or age of diagnosis were genotyped, along with 1940 controls without a personal or family history of cancer. A Sequenom genotyping assay identified a pathogenic variant in 0.2% (5 of 1940) of cancer-free controls, and 3.8% (21/555) of breast cancer cases. Almost 10% (12/113) of early onset cases were heterozygous for founder BRCA1 or BRCA2 pathogenic variant. Of twenty variants tested, only seven were identified in this study. The option of providing this test as population-based screening is discussed.

DGCR8 microprocessor defect characterizes familial multinodular goiter with schwannomatosis.

BACKGROUNDDICER1 is the only miRNA biogenesis component associated with an inherited tumor syndrome, featuring multinodular goiter (MNG) and rare pediatric-onset lesions. Other susceptibility genes for familial forms of MNG likely exist.METHODSWhole-exome sequencing of a kindred with early-onset MNG and schwannomatosis was followed by investigation of germline pathogenic variants that fully segregated with the disease. Genome-wide analyses were performed on 13 tissue samples from familial and nonfamilial DGCR8-E518K-positive tumors, including MNG, schwannomas, papillary thyroid cancers (PTCs), and Wilms tumors. miRNA profiles of 4 tissue types were compared, and sequencing of miRNA, pre-miRNA, and mRNA was performed in a subset of 9 schwannomas, 4 of which harbor DGCR8-E518K.RESULTSWe identified c.1552G>A;p.E518K in DGCR8, a microprocessor component located in 22q, in the kindred. The variant identified is a somatic hotspot in Wilms tumors and has been identified in 2 PTCs. Copy number loss of chromosome 22q, leading to loss of heterozygosity at the DGCR8 locus, was found in all 13 samples harboring c.1552G>A;p.E518K. miRNA profiling of PTCs, MNG, schwannomas, and Wilms tumors revealed a common profile among E518K hemizygous tumors. In vitro cleavage demonstrated improper processing of pre-miRNA by DGCR8-E518K. MicroRNA and RNA profiling show that this variant disrupts precursor microRNA production, impacting populations of canonical microRNAs and mirtrons.CONCLUSIONWe identified DGCR8 as the cause of an unreported autosomal dominant mendelian tumor susceptibility syndrome: familial multinodular goiter with schwannomatosis.FUNDINGCanadian Institutes of Health Research, Compute Canada, Alex's Lemonade Stand Foundation, the Mia Neri Foundation for Childhood Cancer, Cassa di Sovvenzioni e Risparmio fra il Personale della Banca d'Italia, and the KinderKrebsInitiative Buchholz/Holm-Seppensen.

SWI/SNF-Compromised Cancers Are Susceptible to Bromodomain Inhibitors.

The antitumor activity of bromodomain and extraterminal motif protein inhibitors (BETi) has been demonstrated across numerous types of cancer. As such, these inhibitors are currently undergoing widespread clinical evaluation. However, predictive biomarkers allowing the stratification of tumors into responders and nonresponders to BETi are lacking. Here, we showed significant antiproliferative effects of low dosage BETi in vitro and in vivo against aggressive ovarian and lung cancer models lacking SMARCA4 and SMARCA2, key components of SWI/SNF chromatin remodeling complexes. Restoration of SMARCA4 or SMARCA2 promoted resistance to BETi in these models and, conversely, knockdown of SMARCA4 sensitized resistant cells to BETi. Transcriptomic analysis revealed that exposure to BETi potently downregulated a network of genes involved in receptor tyrosine kinase (RTK) signaling in SMARCA4/A2-deficient cells, including the oncogenic RTK HER3. Repression of signaling downstream of HER3 was found to be an important determinant of response to BETi in SMARCA4/A2-deficient cells. Overall, we propose that BETi represent a rational therapeutic strategy in poor-prognosis, SMARCA4/A2-deficient cancers. SIGNIFICANCE: These findings address an unmet clinical need by identifying loss of SMARCA4/A2 as biomarkers of hypersensitivity to BETi.

Author Correction: Germline HAVCR2 mutations altering TIM-3 characterize subcutaneous panniculitis-like T cell lymphomas with hemophagocytic lymphohistiocytic syndrome.

In the version of this article originally published, the main-text sentence "In three patients of European ancestry, we identified the germline variant encoding p.Ile97Met in TIM-3, which was homozygous in two (P12 and P13) and heterozygous in one (P15) in the germline but with no TIM-3 plasma membrane expression in the tumor" misstated the identifiers of the two homozygous individuals, which should have been P13 and P14. The error has been corrected in the HTML, PDF and print versions of the paper.

Germline HAVCR2 mutations altering TIM-3 characterize subcutaneous panniculitis-like T cell lymphomas with hemophagocytic lymphohistiocytic syndrome.

Subcutaneous panniculitis-like T cell lymphoma (SPTCL), a non-Hodgkin lymphoma, can be associated with hemophagocytic lymphohistiocytosis (HLH), a life-threatening immune activation that adversely affects survival1,2. T cell immunoglobulin mucin 3 (TIM-3) is a modulator of immune responses expressed on subgroups of T and innate immune cells. We identify in ~60% of SPTCL cases germline, loss-of-function, missense variants altering highly conserved residues of TIM-3, c.245A>G (p.Tyr82Cys) and c.291A>G (p.Ile97Met), each with specific geographic distribution. The variant encoding p.Tyr82Cys TIM-3 occurs on a potential founder chromosome in patients with East Asian and Polynesian ancestry, while p.Ile97Met TIM-3 occurs in patients with European ancestry. Both variants induce protein misfolding and abrogate TIM-3's plasma membrane expression, leading to persistent immune activation and increased production of inflammatory cytokines, including tumor necrosis factor-α and interleukin-1ß, promoting HLH and SPTCL. Our findings highlight HLH-SPTCL as a new genetic entity and identify mutations causing TIM-3 alterations as a causative genetic defect in SPTCL. While HLH-SPTCL patients with mutant TIM-3 benefit from immunomodulation, therapeutic repression of the TIM-3 checkpoint may have adverse consequences.

Implementing Motor Unit Number Index (MUNIX) in a large clinical trial: Real world experience from 27 centres.

OBJECTIVE: Motor Unit Number Index (MUNIX) is a quantitative neurophysiological method that reflects loss of motor neurons in Amyotrophic Lateral Sclerosis (ALS) in longitudinal studies. It has been utilized in one natural history ALS study and one drug trial (Biogen USA) after training and qualification of raters. METHODS: Prior to testing patients, evaluators had to submit test-retest data of 4 healthy volunteers. Twenty-seven centres with 36 raters measured MUNIX in 4 sets of 6 different muscles twice. Coefficient of variation of all measurements had to be <20% to pass the qualification process. MUNIX COV of the first attempt, number of repeated measurements and muscle specific COV were evaluated. RESULTS: COV varied considerably between raters. Mean COV of all raters at the first measurements was 12.9% ±â€¯13.5 (median 8.7%). Need of repetitions ranged from 0 to 43 (mean 10.7 ±â€¯9.1, median 8). Biceps and first dorsal interosseus muscles showed highest repetition rates. MUNIX variability correlated considerably with variability of compound muscle action potential. CONCLUSION: MUNIX revealed generally good reliability, but was rater dependent and ongoing support for raters was needed. SIGNIFICANCE: MUNIX can be implemented in large clinical trials as an outcome measure after training and a qualification process.

Atypical tuberous sclerosis complex presenting as familial renal cell carcinoma with leiomyomatous stroma.

We report an atypical tuberous sclerosis complex (TSC) phenotype presenting as familial multiple renal cell carcinomas (RCCs) with (angio)leiomyomatous stroma (RCCLS) (5/7 familial RCCs) on a background of multiple angiomyolipomas, hypopigmented skin macules, and absence of neurological anomalies. In the index case and three relatives, germline genetic testing identified a heterozygous TSC2 missense pathogenic variant [c.2714 G > A, (p.Arg905Gln)], a rare TSC-associated alteration which has previously been associated with a milder TSC phenotype. Whole-exome sequencing of five RCCs from the index case and one RCC from his mother demonstrated either unique tumour-specific deleterious second hits in TSC2 or significant allelic imbalance at the TSC2 gene locus (5/6 RCCs). This study confirms the key tumourigenic role of tumour-specific TSC2 second hits in TSC-associated RCCs and supports the notion that RCCLS may be strongly related to abnormalities of the mTOR pathway.

Functionally Null RAD51D Missense Mutation Associates Strongly with Ovarian Carcinoma.

RAD51D is a key player in DNA repair by homologous recombination (HR), and RAD51D truncating variant carriers have an increased risk for ovarian cancer. However, the contribution of nontruncating RAD51D variants to cancer predisposition remains uncertain. Using deep sequencing and case-control genotyping studies, we show that in French Canadians, the missense RAD51D variant c.620C>T;p.S207L is highly prevalent and is associated with a significantly increased risk for ovarian high-grade serous carcinoma (HGSC; 3.8% cases vs. 0.2% controls). The frequency of the p.S207L variant did not significantly differ from that of controls in breast, endometrial, pancreas, or colorectal adenocarcinomas. Functionally, we show that this mutation impairs HR by disrupting the RAD51D-XRCC2 interaction and confers PARP inhibitor sensitivity. These results highlight the importance of a functional RAD51D-XRCC2 interaction to promote HR and prevent the development of HGSC. This study identifies c.620C>T;p.S207L as the first bona fide pathogenic RAD51D missense cancer susceptibility allele and supports the use of targeted PARP-inhibitor therapies in ovarian cancer patients carrying deleterious missense RAD51D variants. Cancer Res; 77(16); 4517-29. ©2017 AACR.

Impaired RASGRF1/ERK-mediated GM-CSF response characterizes CARD9 deficiency in French-Canadians.

BACKGROUND: Caspase recruitment domain-containing protein 9 (CARD9) deficiency is an autosomal recessive primary immunodeficiency conferring human susceptibility to invasive fungal disease, including spontaneous central nervous system candidiasis (sCNSc). However, clinical characterization of sCNSc is variable, hindering its recognition. Furthermore, an in-depth understanding of the bases for this susceptibility has remained elusive. OBJECTIVES: We sought to comprehensively characterize sCNSc and to dissect the mechanisms by which a hypomorphic CARD9 mutation causes susceptibility to Candida species. METHODS: We describe the clinical and radiologic findings of sCNSc caused by CARD9 deficiency in a French-Canadian cohort. We performed genetic, cellular, and molecular analyses to further decipher its pathophysiology. RESULTS: In our French-Canadian series (n = 4) sCNSc had onset in adulthood (median, 38 years) and was often misinterpreted radiologically as brain malignancies; 1 patient had additional novel features (eg, endophthalmitis and osteomyelitis). CARD9 deficiency resulted from a hypomorphic p.Y91H mutation and allelic imbalance established in this population through founder effects. We demonstrate a consistent cellular phenotype of impaired GM-CSF responses. The ability of CARD9 to complex with B-cell CLL/lymphoma 10 (BCL10) and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is intact in our series, arguing against its involvement in susceptibility to fungi. Instead, we show that the p.Y91H mutation impairs the ability of CARD9 to complex with Ras protein-specific guanine nucleotide-releasing factor 1 (RASGRF1), leading to impaired activation of nuclear factor κB and extracellular signal-regulated kinase (ERK) in monocytes and subsequent GM-CSF responses. Successful treatment of a second patient with adjunctive GM-CSF bolsters the clinical relevance of these findings. CONCLUSIONS: Hypomorphic CARD9 deficiency caused by p.Y91H results in adult-onset disease with variable penetrance and expressivity. Our findings establish the CARD9/RASGRF1/ERK/GM-CSF axis as critical to the pathophysiology of sCNSc.

Ovarian embryonal rhabdomyosarcoma is a rare manifestation of the DICER1 syndrome.

Embryonal rhabdomyosarcoma (ERMS), a soft tissue sarcoma, is one of the most common pediatric cancers. Certain ERMSs are associated with the DICER1 syndrome, a tumor predisposition syndrome caused by germ-line DICER1 mutations. Characteristic somatic mutations have also been identified in DICER1-associated tumor types. These "hotspot" mutations affect the catalytic activity of the DICER1 ribonuclease IIIb domain. Primary ovarian ERMS (oERMS) is extremely rare. We present a case of a 6-year-old girl with an oERMS harboring 2 DICER1 mutations. The girl also exhibited other DICER1 phenotypes: cystic nephroma (CN) and multinodular goiter. Somatic investigations of the CN identified a hotspot DICER1 mutation different from that in the oERMS. Significantly, the CN presented at 12 years of age, which is much older than the previously reported age range of susceptibility. This report documents the occurrence of DICER1 mutations in a case of oERMS, expanding the spectrum of DICER1-associated tumors.

Germline RECQL mutations are associated with breast cancer susceptibility.

Several moderate- and high-risk breast cancer susceptibility genes have been discovered, but more are likely to exist. To discover new breast cancer susceptibility genes, we used 2 populations (from Poland and Quebec, Canada) and applied whole-exome sequencing in a discovery phase (n = 195), followed by validation. We identified rare recurrent RECQL mutations in each population. In Quebec, 7 of 1,013 higher-risk breast cancer cases and 1 of 7,136 newborns carried the c.634C>T (p.Arg215*) variant (P = 0.00004). In Poland, 30 of 13,136 unselected breast cancer cases and 2 of 4,702 controls carried the c.1667_1667+3delAGTA (p.K555delinsMYKLIHYSFR) variant (P = 0.008). RECQL is implicated in resolving stalled DNA replication forks to prevent double-stranded DNA (dsDNA) breaks. This function is related to that of other known breast cancer susceptibility genes, many of which are involved in repairing dsDNA breaks. We conclude that RECQL is a breast cancer susceptibility gene.