Articles de revues : « Variante somatica »

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Bennett, Mark F., Michael S. Hildebrand, Sayaka Kayumi, Mark A. Corbett, Sachin Gupta, Zimeng Ye, Michael Krivanek et al. « Evidence for a Dual-Pathway, 2-Hit Genetic Model for Focal Cortical Dysplasia and Epilepsy ». Neurology Genetics 8, n^o 1 (25 janvier 2022) : e0652. http://dx.doi.org/10.1212/nxg.0000000000000652.

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Background and ObjectivesThe 2-hit model of genetic disease is well established in cancer, yet has only recently been reported to cause brain malformations associated with epilepsy. Pathogenic germline and somatic variants in genes in the mechanistic target of rapamycin (mTOR) pathway have been implicated in several malformations of cortical development. We investigated the 2-hit model by performing genetic analysis and searching for germline and somatic variants in genes in the mTOR and related pathways.MethodsWe searched for germline and somatic pathogenic variants in 2 brothers with drug-resistant focal epilepsy and surgically resected focal cortical dysplasia (FCD) type IIA. Exome sequencing was performed on blood- and brain-derived DNA to identify pathogenic variants, which were validated by droplet digital PCR. In vitro functional assays of a somatic variant were performed.ResultsExome analysis revealed a novel, maternally inherited, germline pathogenic truncation variant (c.48delG; p.Ser17Alafs*70) in NPRL3 in both brothers. NPRL3 is a known FCD gene that encodes a negative regulator of the mTOR pathway. Somatic variant calling in brain-derived DNA from both brothers revealed a low allele fraction somatic variant (c.338C>T; p.Ala113Val) in the WNT2 gene in 1 brother, confirmed by droplet digital PCR. In vitro functional studies suggested a loss of WNT2 function as a consequence of this variant. A second somatic variant has not yet been found in the other brother.DiscussionWe identify a pathogenic germline mTOR pathway variant (NPRL3) and a somatic variant (WNT2) in the intersecting WNT signaling pathway, potentially implicating the WNT2 gene in FCD and supporting a dual-pathway 2-hit model. If confirmed in other cases, this would extend the 2-hit model to pathogenic variants in different genes in critical, intersecting pathways in a malformation of cortical development. Detection of low allele fraction somatic second hits is challenging but promises to unravel the molecular architecture of FCDs.

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Ura, Hiroki, Sumihito Togi et Yo Niida. « Dual Deep Sequencing Improves the Accuracy of Low-Frequency Somatic Mutation Detection in Cancer Gene Panel Testing ». International Journal of Molecular Sciences 21, n^o 10 (16 mai 2020) : 3530. http://dx.doi.org/10.3390/ijms21103530.

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Cancer gene panel testing requires accurate detection of somatic mosaic mutations, as the test sample consists of a mixture of cancer cells and normal cells; each minor clone in the tumor also has different somatic mutations. Several studies have shown that the different types of software used for variant calling for next generation sequencing (NGS) can detect low-frequency somatic mutations. However, the accuracy of these somatic variant callers is unknown. We performed cancer gene panel testing in duplicate experiments using three different high-fidelity DNA polymerases in pre-capture amplification steps and analyzed by three different variant callers, Strelka2, Mutect2, and LoFreq. We selected six somatic variants that were detected in both experiments with more than two polymerases and by at least one variant caller. Among them, five single nucleotide variants were verified by CEL nuclease-mediated heteroduplex incision with polyacrylamide gel electrophoresis and silver staining (CHIPS) and Sanger sequencing. In silico analysis indicated that the FBXW7 and MAP3K1 missense mutations cause damage at the protein level. Comparing three somatic variant callers, we found that Strelka2 detected more variants than Mutect2 and LoFreq. We conclude that dual sequencing with Strelka2 analysis is useful for detection of accurate somatic mutations in cancer gene panel testing.

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Das, Kingshuk, Amber Carter, Brandie Heald, Scott T. Michalski, Sarah M. Nielsen, Nhu Ngo, Sara Elrefai et al. « Integrated germline and somatic cancer testing provides opportunity to identify cancer risk and resolve variant origins. » Journal of Clinical Oncology 40, n^o 16_suppl (1 juin 2022) : 10589. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.10589.

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10589 Background: Germline and somatic genetic testing are established tools for the management of cancer patients. Somatic testing is primarily used to inform therapy and germline testing is used to diagnose hereditary cancer predisposition syndromes. While somatic testing can detect germline variants, the interpretation and reporting algorithms are optimized to predict therapeutic efficacy. As a result, germline variants may be missed or only interpreted in context of their potential to act as a therapeutic target. We retrospectively reviewed a series of patients who received both germline and somatic testing to examine the opportunities for concurrent germline testing to improve somatic reporting. Methods: Our study reviewed data from 43 patients with solid cancer diagnoses who were otherwise unselected and underwent testing with a 435-gene somatic genetic test and an 83-gene germline test. The most frequent cancers were pancreatic (18), ovarian (8), and prostate (7). Results: Out of the 43 patients, 7 (16%) harbored a pathogenic or likely pathogenic germline variant (PGV) in a cancer susceptibility gene. PGVs were identified in MLH1, MSH6, CHEK2, PALB2, CDKN2A, NBN, and MUTYH. Notably, 3 of these genes ( CHEK2, PALB2, MUTYH) were not considered therapeutic targets, and therefore were only included as ancillary findings near the end of the preliminary somatic test reports (generated prior to integration of germline test results). In addition, 40 of 43 (93%) patients had at least one variant detected by somatic testing in at least one of the germline panel genes (mean number variant genes = 4.1, maximum = 10); all of these variants were within the reportable range of the germline assay, and therefore germline test results were able to resolve their germline versus somatic origins. The genes that most frequently had somatic variants identified were TP53 (79% of patients), CDKN2A (37%), SMAD4 (30%), and FLCN (21%). Conclusions: Due to the size of commonly ordered somatic gene panels, there is a high probability of detecting variants in hereditary cancer predisposition genes (> 90% of patients in this study) that can provide either therapy or cancer risk information or both. Given that a significant proportion (16% in this study) of cancer patients harbor PGVs (which can further inform treatment, disease surveillance, preventive measures, and risk assessment for family members), it is crucial to resolve the somatic versus germline origin of these variants. Since interpretation and reporting algorithms for somatic testing are optimized for therapy prediction, and variables such as specimen tumor purity, tumor ploidy, and variant allele fraction render estimates of variant origin unreliable for diagnostic purposes, it is important to take advantage of germline testing concurrently in patients receiving somatic testing to glean this critical information.

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Tsuchida, Naomi, Yosuke Kunishita, Yuri Uchiyama, Yohei Kirino, Makiko Enaka, Yukie Yamaguchi, Masataka Taguri et al. « Pathogenic UBA1 variants associated with VEXAS syndrome in Japanese patients with relapsing polychondritis ». Annals of the Rheumatic Diseases 80, n^o 8 (31 mars 2021) : 1057–61. http://dx.doi.org/10.1136/annrheumdis-2021-220089.

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ObjectivesTo determine clinical and genetic features of individuals with relapsing polychondritis (RP) likely caused by pathogenic somatic variants in ubiquitin-like modifier activating enzyme 1 (UBA1).MethodsFourteen patients with RP who met the Damiani and Levine criteria were recruited (12 men, 2 women; median onset age (IQR) 72.1 years (67.1–78.0)). Sanger sequencing of UBA1 was performed using genomic DNA from peripheral blood leukocytes or bone marrow tissue. Droplet digital PCR (ddPCR) and peptide nucleic acid (PNA)-clamping PCR were used to detect low-prevalence somatic variants. Clinical features of the patients were investigated retrospectively.ResultsUBA1 was examined in 13 of the 14 patients; 73% (8/11) of the male patients had somatic UBA1 variants (c.121A>C, c.121A>G or c.122T>C resulting in p.Met41Leu, p.Met41Val or p.Met41Thr, respectively). All the variant-positive patients had systemic symptoms, including a significantly high prevalence of skin lesions. ddPCR detected low prevalence (0.14%) of somatic variant (c.121A>C) in one female patient, which was subsequently confirmed by PNA-clamping PCR.ConclusionsGenetic screening for pathogenic UBA1 variants should be considered in patients with RP, especially male patients with skin lesions. The somatic variant in UBA1 in the female patient is the first to be reported.

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Fujita, Atsushi, Takefumi Higashijima, Hiroshi Shirozu, Hiroshi Masuda, Masaki Sonoda, Jun Tohyama, Mitsuhiro Kato et al. « Pathogenic variants of DYNC2H1, KIAA0556, and PTPN11 associated with hypothalamic hamartoma ». Neurology 93, n^o 3 (13 juin 2019) : e237-e251. http://dx.doi.org/10.1212/wnl.0000000000007774.

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ObjectiveIntensive genetic analysis was performed to reveal comprehensive molecular insights into hypothalamic hamartoma (HH).MethodsThirty-eight individuals with HH were investigated by whole exome sequencing, target capture-based deep sequencing, or single nucleotide polymorphism (SNP) array using DNA extracted from blood leukocytes or HH samples.ResultsWe identified a germline variant of KIAA0556, which encodes a ciliary protein, and 2 somatic variants of PTPN11, which forms part of the RAS/mitogen-activated protein kinase (MAPK) pathway, as well as variants in known genes associated with HH. An SNP array identified (among 3 patients) one germline copy-neutral loss of heterozygosity (cnLOH) at 6p22.3–p21.31 and 2 somatic cnLOH; one at 11q12.2–q25 that included DYNC2H1, which encodes a ciliary motor protein, and the other at 17p13.3–p11.2. A germline heterozygous variant and an identical somatic variant of DYNC2H1 arising from cnLOH at 11q12.2–q25 were confirmed in one patient (whose HH tissue, therefore, contains biallelic variants of DYNC2H1). Furthermore, a combination of a germline and a somatic DYNC2H1 variant was detected in another patient.ConclusionsOverall, our cohort identified germline/somatic alterations in 34% (13/38) of patients with HH. Disruption of the Shh signaling pathway associated with cilia or the RAS/MAPK pathway may lead to the development of HH.

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Wu, Yanqing, Wenzhe Fan, Miao Xue, Yiyang Tang, Suo Peisu, Bo Yang, Tanxiao Huang, Jing Zhang et Jiaping Li. « TP53 pathogenic variants with low allele fraction in germline genetic testing. » Journal of Clinical Oncology 40, n^o 16_suppl (1 juin 2022) : 10600. http://dx.doi.org/10.1200/jco.2022.40.16_suppl.10600.

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10600 Background: Blood or saliva DNA generally considered to be representative of germline genome in genetic cancer risk assessment. However white blood cells from these samples may also include somatic origin DNA due to postzygotic variation or, most commonly, clonal hematopoiesis (CH). Low variant allele fraction (VAF) found in germline genetic testing suggest the possibility of somatic variant and may lead to misinterpretation of genetic risk. TP53, of which germline pathogenic variants are associated with Li-Fraumeni syndrome (LFS), is frequently mutated in CH. This analysis investigated characteristics of TP53 pathogenic variants with low VAF. Methods: We reviewed the prevalence and distribution of TP53 pathogenic variants(PVs) detected in 11,277 advanced cancer patients who underwent clinical testing with a clinical NGS pan-cancer panel. Potential somatic PVs were defined as variants with low VAF from 10% to 35%. The VAF were evaluated in matched tumor tissue samples if available. Results: TP53 pathogenic variants were detected in 36 (0.32%) patients from blood or saliva samples, VAF between 10% and 35% were identified in 8(22.22%) patients and 7 of them were performed NGS sequencing in matched tumor tissue samples. The average VAF of tissue samples were 9.31times lower than blood or saliva samples(21.69% vs 3.88%). Conclusions: TP53 pathogenic variants with low allele fraction in blood or saliva samples indicate the possibility of somatic variant, a reduced VAF in matched tumor tissue samples may contribute to confirmation for suspicion of somatic origin.

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Brown, Natasha J., Zimeng Ye, Chloe Stutterd, Sureshni I. Jayasinghe, Amy Schneider, Saul Mullen, Simone A. Mandelstam et Michael S. Hildebrand. « Somatic IDH1 variant (p.R132C) in an adult male with Maffucci syndrome ». Molecular Case Studies 7, n^o 6 (29 septembre 2021) : a006127. http://dx.doi.org/10.1101/mcs.a006127.

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Maffucci syndrome is a rare, highly variable somatic mosaic condition, and well-known cancer-related gain-of-function variants in either the IDH1 or IDH2 genes have been found in the affected tissues of most reported individuals. Features include benign enchondroma and spindle-cell hemangioma, with a recognized increased risk of various malignancies. Fewer than 200 affected individuals have been reported; therefore, accurate estimates of malignancy risk are difficult to quantify and recommended surveillance guidelines are not available. The same gain-of-function IDH1 and IDH2 variants are also implicated in a variety of other benign and malignant tumors. An adult male presented with several soft palpable lesions on the right upper limb. Imaging and histopathology raised the possibility of Maffucci syndrome. DNA was extracted from peripheral blood lymphocytes and tissue surgically resected from a spindle-cell hemangioma. Sanger sequencing and droplet digital polymerase chain reaction (PCR) analysis of the IDH1 gene were performed. We identified a somatic mosaic c.394C > T (p.R132C) variant in exon 5 of IDH1, in DNA derived from hemangioma tissue at ∼17% variant allele fraction. This variant was absent in DNA derived from blood. This variant has been identified in the affected tissue of most reported individuals with Maffucci syndrome. Although this individual has a potentially targetable variant, and there is a recognized risk of malignant transformation in this condition, a decision was made not to intervene with an IDH1 inhibitor. The reasons and prospects for therapy in this condition are discussed.

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Khanna, Shivani, Steven Brad Maron, Leah Chase, Samantha Lomnicki, Sonia Kupfer et Daniel V. T. Catenacci. « Suspected and confirmed germline variants from tumor-only somatic sequencing of 864 gastrointestinal malignancies. » Journal of Clinical Oncology 37, n^o 15_suppl (20 mai 2019) : e13131-e13131. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e13131.

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e13131 Background: Targeted tumor-only somatic sequencing informs therapies and is becoming a routine part of cancer care. It also identifies patients with possible germline variants who require confirmatory genetic testing. The aim was to identify patients with suspected and confirmed germline variants whose GI tumors underwent somatic sequencing. Methods: 864 patients with GI tumors who had Foundation One (FO) somatic sequencing from 4/2003-3/2018 were evaluated. Inclusion criteria for suspected germline variants were: a) allele frequency ≥ 35% in hereditary cancer genes and b) pathogenic variants by FO and/or ClinVar. Variants in commonly mutated somatic genes ( TP53, KRAS, CDKN2A) were excluded in patients over age 40. Recommendation of genetic evaluation and germline test results were recorded. Patient, family, and tumor characteristics were compared using univariate analysis. Results: 199 of 864 patients had suspected germline pathogenic variants. 50 patients were recommended genetic evaluation, and 26 patients underwent genetic testing. A germline pathogenic variant was confirmed in 15 patients. Among all patients suspected to have germline variants, 8% were confirmed by genetic testing. Patients under age 40 and those with family cancer history were more often referred for testing (Table). Patients with variants in BRCA1, MLH1, MSH2, PMS2, POLE and TP53 were more often referred for testing. Conclusions: A quarter of patients carried a somatic pathogenic variant with allele frequency ≥35% in a hereditary cancer gene. 25% of these patients were recommended for genetic evaluation. Younger patients and those with family history were more often referred. 8% of patients with suspected germline variants were confirmed by genetic testing. These results provide “real world” experience in using somatic only tumor testing to identify patients with germline pathogenic variants who then might benefit from future cancer screening and genetic testing in family members. Comparison of characteristics by recommendation to undergo genetic testing based on somatic tumor sequencing results. [Table: see text]

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Michalski, Scott T., Daniel Esteban Pineda Alvarez, Meaghan Russell, Shan Yang, Guru Sonpavde et Edward D. Esplin. « Tumor sequencing with germline genetic testing : Identification of patients with hereditary cancer and precision treatment eligibility. » Journal of Clinical Oncology 37, n^o 15_suppl (20 mai 2019) : 1580. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.1580.

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1580 Background: Cancer is a fundamentally genetic disease, as such, somatic and germline mutation analysis is used in the comprehensive assessment of patients with cancer. Studies report that approximately 10% of patient’s tumors have clinically significant variants known to predispose to hereditary cancer, with medical implications for both patients and family members. We retrospectively reviewed a series of patients where providers suspected a somatic variant also existed in the germline and followed up with clinical germline genetic testing. We report the rate of concordance between germline and somatic results and their clinical impact. Methods: Our study used de-identified data from 1043 consecutive patients who underwent somatic genetic testing followed by germline testing with NGS-based hereditary cancer gene panels. Results: Somatic results most frequently prompting germline testing included variants in BRCA2 (290), BRCA1 (174), TP53 (158), ATM (70), MLH1 (65), APC (65), PMS2 (61), MSH6 (58), PTEN (54) and CDH1 (42). In 364/1043 cases (35%) the variant was detected as likely pathogenic or pathogenic (LP/P) in the germline. Genes confirmed as germline variants in 60-100% of cases included: FANCA, AXIN2, RAD50, MUTYH, BLM, PALB2, CHEK2, FANCD2, MITF, SDHB. Variants in: FH, BRCA2, RET, ATM, SDHA, BRIP1, MSH2, BRCA1, BAP1, EGFR and RAD51D confirmed in the germline in 25-60%. Variants were rarely germline for TP53 (3%), APC (3%), PTEN (2%) and none in CDKN2A, NF1 and STK11. In 24 (2%) cases a LP/P germline variant was detected but not reported in the tumor. Conclusions: Approximately ⅓ of patients suspected to have hereditary risk after tumor testing had LP/P germline variants. Notably, some genes had a high probability of variants occurring in the germline, while others were primarily seen in tumors. Interestingly, 6% of the germline variants were not included on the somatic report due to technical and gene content differences in either assays or due to differences of clinical classification between somatic and germline testing. Adding germline results to somatic testing may inform options for precision treatment, prevention, or early detection of, secondary malignancies and guide genetic counseling of family members.

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Sancho-Galán, Pau, Antonio Amores-Arrocha, Víctor Palacios et Ana Jiménez-Cantizano. « Preliminary Study of Somatic Variants of Palomino Fino (Vitis vinifera L.) Grown in a Warm Climate Region (Andalusia, Spain) ». Agronomy 10, n^o 5 (4 mai 2020) : 654. http://dx.doi.org/10.3390/agronomy10050654.

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Vegetative propagation of Vitis vinifera cultivars over hundreds of years has led to the accumulation of a large number of somatic variants of the same grapevine variety. These variants are now considered a working tool to cope with changing environmental conditions as a result of, among others, global warming. In this work, three somatic variants of the major grapevine variety of the South West (SW) of Andalusia (Spain), Palomino Fino, have been genetically and morphologically characterized, as well as their grape musts from two different vintages. The genetic analysis at 22 microsatellite loci confirmed the identity of the three somatic variants that presented the same genotype as Palomino Fino, while the morphological study showed differences between the three somatic variants and Palomino Fino, highlighting the somatic variant Palomino Pelusón. Regarding the physicochemical analysis of its musts, differences were also observed between the somatic variants and Palomino Fino. As a result of all of the above, the use of grapes from somatic variants can be a viable and natural alternative for the production of quality wines in warm climate areas. On the other hand, promoting the cultivation of the somatic variants could contribute to preventing the loss of Palomino Fino intraspecific variability.

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Chin, Diana, Matthew A. Kutny, Jonathan Grim, Robert B. Gerbing, Kristen Miller, Jason E. Farrar, Jaime M. Guidry Auvil et al. « Comprehensive Genomic and Transcript Profiling of CBL Gene in Childhood AML : A Report from Children's Oncology Group Studies AAML03P1, AAML0531 and COG/NCI Target AML Initiative ». Blood 126, n^o 23 (3 décembre 2015) : 170. http://dx.doi.org/10.1182/blood.v126.23.170.170.

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Abstract The Casitas B-Lineage Lymphoma (CBL) gene encodes for an E3 ubiquitin ligase that targets activated receptor tyrosine kinases for degradation. Mutations of the CBL gene have been described in juvenile myelomonocytic leukemia (JMML) but less is known about mutations and variants of CBL in de novo AML. We previously reported that somatic mutations of CBL are rare in pediatric AML. In this report we present a comprehensive evaluation of genomic and transcript variants of CBL including novel deletion events as well as transcript variants which, in combination with somatic mutations, account for over 6% of pediatric AML with extreme association with inv(16) and favorable outcome. Initial assessment of CBL transcript in a cohort of 100 patients identified previously reported deletion of exon 8 (CBL ΔE8, N=2) associated with CBL splice mutations as well as a novel whole exon 8 and 9 deletion variant (CBL ΔE8+9, N=3) without identifiable underlying somatic alterations. Long distance PCR, as well as custom Nanostring CNV array evaluation revealed a genomic deletion underlying this transcript variant. Subsequent whole genome sequencing as part of COG/NCI TARGET AML initiative, identified discrete genomic deletions of 1998, 3588 and 6189 bp across exon 8 and 9, leading to the generation of this novel variant. We evaluated the functional consequence of the novel CBL ΔE8+9 deletion variant by expressing it in IL3-dependent Ba/F3 cell line. Compared to control cells, Ba/F3 cells expressing CBL ΔE8+9 demonstrated cytokine independent growth. A comprehensive profiling of CBL variants was conducted in 796 pediatric de novo AML patients by transcript profiling (transcript variants) or by exome capture sequencing (somatic mutations including point mutations and smaller indels). All patients were treated on Children's Oncology Group studies AAML03P1 (N=167) and AAML0531 (N=629) and presence of CBL variants was correlated with disease characteristics and clinical outcome. Of the 796 patient specimens tested, 50 patients (6.3%) had one of 3 distinct CBL variants; transcript variant (N=28), somatic mutation (N=14), or dual transcript variant and somatic mutation (N=8). All cases of CBL ΔE8+9 were associated with a corresponding genomic deletion. Out of 14 cases of CBL ΔE8 and 1 case of CBL ΔE9, only 4 cases (27%) had a splice site mutation identified as the underlying mechanism of splice variant. Presence of CBL variants was correlated with clinical characteristics and outcome. Those with CBL variants had a significantly higher prevalence of inv(16) compared with CBL wild type (WT) (37% vs. 13%, p<0.001). This association differed by CBL variant type; 44% transcript variants and 50% dual variants had inv(16) compared to 14% somatic mutations and 13% CBL WT (p<0.001). NPMc+ was more prevalent in those with CBL somatic mutations (29%) than transcript variant (4%), dual variant (0%) or CBL WT (8%) (p=0.035). Similarly, genetic risk groups differed between CBL variants vs. WT (Low risk 70% vs 39%, p=<0.001; Standard risk 22% vs. 46%, p=0.001; High risk 8% vs. 15%, p=0.196). Clinical characteristics including gender, age, race and ethnicity were not significantly different. FAB morphologic assessment revealed an enrichment for the M4 subtype in CBL variant vs. WT (53% vs. 23%, P<.001) which is likely accounted for by the association of inv(16) with this morphologic group. Patients with CBL variants had a 100% clinical remission rate by end of induction II compared to 89% for CBL WT patients (p=0.014). Survival from study entry was similar between CBL mutant vs. WT patients (5 year OS 72% vs. 66%, p=0.24; 5 year EFS 61% vs. 50%, p=0.11). Due to the strong association of CBL mutation with core binding factor leukemia, we assessed whether CBL variant was prognostic of outcome within this favorable risk group, but there was no significant difference in outcomes. Variants of the CBL gene in pediatric AML include genetic mutations with and without whole exon deletions. These CBL variants are highly associated with low risk AML but do not provide independent risk prognosis. The cooperating events of CBL variants in core binding factor leukemia deserve greater study. Our initial analysis of the transcript variants in a cell line model suggest that these large exon 8+9 deletions represent important oncogenic events. The authors would like to gratefully acknowledge the important contributions of the late Dr. Robert Arceci to the AML TARGET initiative. Disclosures No relevant conflicts of interest to declare.

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Peculis, Raitis, Vita Rovite, Kaspars Megnis, Inga Balcere, Austra Breiksa, Jurijs Nazarovs, Janis Stukens et al. « Whole exome sequencing reveals novel risk genes of pituitary neuroendocrine tumors ». PLOS ONE 17, n^o 8 (26 août 2022) : e0265306. http://dx.doi.org/10.1371/journal.pone.0265306.

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Somatic genetic alterations in pituitary neuroendocrine tumors (PitNET) tissues have been identified in several studies, but detection of overlapping somatic PitNET candidate genes is rare. We sequenced and by employing multiple data analysis methods studied the exomes of 15 PitNET patients to improve discovery of novel factors involved in PitNET development. PitNET patients were recruited to the study before PitNET removal surgery. For each patient, two samples for DNA extraction were acquired: venous blood and PitNET tissue. Exome sequencing was performed using Illumina NexSeq 500 sequencer and data analyzed using two separate workflows and variant calling algorithms: GATK and Strelka2. A combination of two data analysis pipelines discovered 144 PitNET specific somatic variants (mean = 9.6, range 0–19 per PitNET) of which all were SNVs. Also, we detected previously known GNAS PitNET mutation and identified somatic variants in 11 genes, which have contained somatic variants in previous WES and WGS studies of PitNETs. Noteworthy, this is the third study detecting somatic variants in gene RYR1 in the exomes of PitNETs. In conclusion, we have identified two novel PitNET candidate genes (AC002519.6 and AHNAK) with recurrent somatic variants in our PitNET cohort and found 13 genes overlapping from previous PitNET studies that contain somatic variants. Our study demonstrated that the use of multiple sequencing data analysis pipelines can provide more accurate identification of somatic variants in PitNETs.

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Moody, Emily W., Jennie Vagher, Whitney Espinel, David Goldgar, Kelsi J. Hagerty et Amanda Gammon. « Comparison of Somatic and Germline Variant Interpretation in Hereditary Cancer Genes ». JCO Precision Oncology, n^o 3 (décembre 2019) : 1–8. http://dx.doi.org/10.1200/po.19.00144.

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PURPOSE To compare the classification of genetic variants reported on tumor genomic profiling (TGP) reports with germline classifications on clinical test results and ClinVar. Results will help to inform germline testing discussions and decisions in patients with tumor variants in genes that are relevant to hereditary cancer risk. PATIENTS AND METHODS This study compared somatic and germline classifications of small nucleotide variants in the following genes: BRCA1, BRCA2, CHEK2, PALB2, ATM, MLH1, MSH2, MSH6, and PMS2. Somatic classifications were taken from reports from a single commercial TGP laboratory of tests ordered by providers at Huntsman Cancer Institute between March 2014 and June 2018. Somatic variant interpretations were compared with classifications from germline test results as well as with ClinVar interpretations. RESULTS Of the 623 variants identified on TGP, 353 had a definitive classification in ClinVar, and 103 were assayed with a germline test, with 66 of the variants tested observed in germline. Analysis of somatic variants of uncertain significance listed on TGP reports determined that 22% had a different interpretation compared with ClinVar and that 32% differed from the interpretation on a germline test result. Pathogenic variants on TGP test results were found to differ 13% and 5% of the time compared with ClinVar interpretations and germline test results, respectively. CONCLUSION These results suggest that TGP variants are often classified differently in a germline context. Differences may be due to different processes in variant interpretation between somatic and germline laboratories. These results are important for health care providers to consider when making decisions about additional testing for hereditary cancer risks.

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Smith, Kyle S., Vinod K. Yadav, Shanshan Pei, Daniel A. Pollyea, Craig T. Jordan et Subhajyoti De. « SomVarIUS : somatic variant identification from unpaired tissue samples ». Bioinformatics 32, n^o 6 (20 novembre 2015) : 808–13. http://dx.doi.org/10.1093/bioinformatics/btv685.

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Abstract Motivation: Somatic variant calling typically requires paired tumor-normal tissue samples. Yet, paired normal tissues are not always available in clinical settings or for archival samples. Results: We present SomVarIUS, a computational method for detecting somatic variants using high throughput sequencing data from unpaired tissue samples. We evaluate the performance of the method using genomic data from synthetic and real tumor samples. SomVarIUS identifies somatic variants in exome-seq data of ∼150 × coverage with at least 67.7% precision and 64.6% recall rates, when compared with paired-tissue somatic variant calls in real tumor samples. We demonstrate the utility of SomVarIUS by identifying somatic mutations in formalin-fixed samples, and tracking clonal dynamics of oncogenic mutations in targeted deep sequencing data from pre- and post-treatment leukemia samples. Availability and implementation: SomVarIUS is written in Python 2.7 and available at http://www.sjdlab.org/resources/ Contact: subhajyoti.de@ucdenver.edu Supplementary information: Supplementary data are available at Bioinformatics online.

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Chowdhury, Murad, Brent S. Pedersen, Fritz J. Sedlazeck, Aaron R. Quinlan et Ryan M. Layer. « Searching thousands of genomes to classify somatic and novel structural variants using STIX ». Nature Methods 19, n^o 4 (avril 2022) : 445–48. http://dx.doi.org/10.1038/s41592-022-01423-4.

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AbstractStructural variants are associated with cancers and developmental disorders, but challenges with estimating population frequency remain a barrier to prioritizing mutations over inherited variants. In particular, variability in variant calling heuristics and filtering limits the use of current structural variant catalogs. We present STIX, a method that, instead of relying on variant calls, indexes and searches the raw alignments from thousands of samples to enable more comprehensive allele frequency estimation.

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Gilmore, G. L., J. Q. Yang, K. B. Marcu et B. K. Birshtein. « Absence of somatic mutation in the variable region of MPC 11 variants expressing a different heavy chain isotype. » Journal of Immunology 139, n^o 2 (15 juillet 1987) : 619–24. http://dx.doi.org/10.4049/jimmunol.139.2.619.

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Abstract Somatic mutation of immunoglobulin variable regions contributes to the diversity of the immune response. Some investigators have postulated that somatic mutation is coupled to isotype switching; others have presented evidence that the two processes are not linked. By using in vitro variants of the MPC 11 mouse myeloma cell line that produce heavy chains of different isotypes, we have examined several VH regions at the nucleotide level using a variety of different sequencing procedures. We have found no evidence of somatic mutation and conclude that the processes of somatic mutation and isotype switching may be independent.

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Koehler, Viktoria Florentine, Pia Adam, Carmina Teresa Fuss, Linmiao Jiang, Elke Berg, Karin Frank-Raue, Friedhelm Raue et al. « Treatment of RET-Positive Advanced Medullary Thyroid Cancer with Multi-Tyrosine Kinase Inhibitors—A Retrospective Multi-Center Registry Analysis ». Cancers 14, n^o 14 (13 juillet 2022) : 3405. http://dx.doi.org/10.3390/cancers14143405.

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Background: RET (rearranged during transfection) variants are the most prevalent oncogenic events in medullary thyroid cancer (MTC). In advanced disease, multi-tyrosine kinase inhibitors (MKIs) cabozantinib and vandetanib are the approved standard treatment irrespective of RET status. The actual outcome of patients with RET-positive MTC treated with MKIs is ill described. Methods: We here retrospectively determined the RET oncogene variant status with a targeted DNA Custom Panel in a prospectively collected cohort of 48 patients with advanced MTC treated with vandetanib and/or cabozantinib at four German referral centers. Progression-free survival (PFS) and overall survival (OS) probabilities were estimated using the Kaplan-Meier method. Results: In total, 44/48 (92%) patients had germline or somatic RET variants. The M918T variant was found in 29/44 (66%) cases. In total, 2/32 (6%) patients with a somatic RET variant had further somatic variants, while in 1/32 (3%) patient with a germline RET variant, additional variants were found. Only 1/48 (2%) patient had a pathogenic HRAS variant, and no variants were found in 3 cases. In first-line treatment, the median OS was 53 (95% CI (95% confidence interval), 32–NR (not reached); n = 36), and the median PFS was 21 months (12–39; n = 33) in RET-positive MTC patients. In second-line treatment, the median OS was 18 (13–79; n = 22), and the median PFS was 3.5 months (2–14; n = 22) in RET-positive cases. Conclusions: RET variants were highly prevalent in patients with advanced MTC. The treatment results in RET-positive cases were similar to those reported in unselected cohorts.

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Lim, Tristan L., David B. Lieberman, Adam R. Davis, Ryan Hausler, Ashkan Bigdeli, Yimei Li, Jacquelyn Powers et al. « Germline POT1 Variants Can Predispose to a Variety of Hematologic Neoplasms ». Blood 136, Supplement 1 (5 novembre 2020) : 2–4. http://dx.doi.org/10.1182/blood-2020-134160.

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Germline mutations in the shelterin component protection of telomeres 1 (POT1) were recently found to be associated with familial chronic lymphocytic leukemia (CLL), melanoma, glioma, and several other familial cancer syndromes. The role of POT1 mutations in myeloid neoplasms and other hematologic malignancies, however, remains unknown. To explore the role of POT1 variants in hematologic neoplasms, we analyzed POT1 variants in 3323 consecutive patients who underwent next-generation sequencing (NGS) of a panel of hematologic malignancy-associated genes at our institution and characterized the clinical and pathological characteristics of patients with germline and somatic POT1 mutations. Of 3323 consecutive patients who underwent NGS, 2770 patients had a hematologic malignancy (lymphoid n = 1299, myeloid n = 934, and both lymphoid and myeloid n = 537), while 553 patients were evaluated for non-malignant cytopenias. All 57 patients (2.06%) carrying either a POT1 disease-associated variant or variant of uncertain significance had a hematologic malignancy compared to no identified POT1 variants in 553 patients with benign cytopenias (OR = 23.5, p < 0.001), suggesting that the presence of POT1 variants was predictive of a hematologic malignancy. Of 57 patients, 33 had lymphoid malignancies, 23 had myeloid neoplasms, and 2 had a lymphoid and myeloid neoplasm (Fig 1). Patient variants were classified as germline or somatic using constitutional DNA sequencing, POT1 emergence/disappearance over time, or POT1 maintenance in remission. In the absence of these data, likely germline or likely somatic designations were made by assessing variant allele frequencies against clinical/pathologic characteristics. 18 patients (33%) were found to have germline or likely germline POT1 variants (29% and 42% in the lymphoid and myeloid malignancy groups, respectively). Another 6 patients (11%) had variants whose germline status could not be determined. Of the 17 unique germline POT1 variants, 10 were missense and located within mapped functional protein domains, while 7 were classified as predicted loss-of-function (pLOF) due to a disruption of start, premature stop, frameshift, or spice site alteration. Patients with hematological malignancies had a ~5-8x increased odds of having a germline pLOF POT1 variant compared to cancer-free individuals in the Genome Aggregation Database (gnomAD, n = 113,108 exomes, OR = 7.5, p < 0.001) or in the Penn Medicine BioBank (PMBB, n = 7877, OR = 5.0, p = 0.010), with a prevalence of 0.25% compared to 0.03% and 0.05%, respectively. Germline pLOF POT1 variants were significantly more enriched in patients with myeloid (gnomAD: OR = 6.1, p = 0.02) and lymphoid (gnomAD: OR = 9.8, p < 0.001; PMBB: OR = 6.5, p = 0.004) malignancies. In 33 patients with lymphoid malignancies and POT1 variants, the most common diagnoses were CLL/SLL (n = 21, germline n = 6, somatic n = 12), CD5- CD10- indolent B cell neoplasms (n = 4, germline n = 1, somatic n = 3), and multiple myeloma (n = 3, all somatic) (Table 1). Lymphoid malignancies with a germline POT1 variant had a relative paucity of additional mutations; in contrast, somatic POT1 variants frequently co-occurred with other mutations, most commonly with TP53 (Fig 2, n = 5, 23%). Among 23 patients with myeloid malignancies, patients with germline POT1 variants developed malignancies at a significantly younger age compared to those whose POT1 variants were somatic (median age 59.5 vs 70.5 years, p = 0.04). The most common diagnosis in patients with myeloid neoplasms carrying germline POT1 variants was MPN (germline n = 5, somatic n = 1). AML, MDS/MPN, and MDS occurred in 4, 3, and 1 patients respectively. All patients with myeloid neoplasms had additional disease-associated mutations, with the most common co-occurring variants in TET2 (n = 7), JAK2 (n = 6, co-occurring with 50% of germline POT1 myeloid variants), and NRAS (n = 6). In conclusion, this is the first comprehensive analysis of POT1 variants in an unselected hospital-based population undergoing molecular testing for variants associated with hematologic malignancies. Our results show that the presence of POT1 variants is predictive of having a hematologic neoplasm and that over 30% of POT1 variants in hematologic malignancy patients are germline. Our study expands the spectrum of POT1-associated familial neoplasms and highlights the needs for better recognition of familial hematologic cancer syndromes. Disclosures No relevant conflicts of interest to declare.

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Hutter, Stephan, Niroshan Nadarajah, Manja Meggendorfer, Wolfgang Kern, Torsten Haferlach et Claudia Haferlach. « Whole Genome Sequencing in Routine Hematologic Samples : How to Proceed Analyses Best When Germline Controls Are Missing ? » Blood 132, Supplement 1 (29 novembre 2018) : 5275. http://dx.doi.org/10.1182/blood-2018-99-113294.

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Abstract Background: The human genome is very heterogeneous on the individual level which challenges interpretation of whole genome sequencing (WGS) data. In order to reduce complexity in tumor genetics WGS of a tumor is performed together with WGS of "normal" tissue from the respective patient (i.e. fingernails, skin biopsy, hair, buccal swaps) which is used as the germline sequence (tumor/matched normal approach, TMNA). This approach allows the extraction of somatic mutations acquired in the tumor through sophisticated algorithms. In routine diagnostics, especially in hematological neoplasms, "normal" tissue representing the germline sequence is usually not available, which prohibits the standard use of somatic tumor/normal variant calling tools. Aims: On the road to implement WGS into routine diagnostics we tested a TMNA in comparison to a tumor/unmatched normal approach (TUNA), where pooled genomic DNA (Promega, Fitchburg, WI) was used instead of a matched normal. Cohorts and Methods: 9 samples from patients with hematological neoplasms (7 AML, 2 ALL) were sequenced at diagnosis on Illumina HiSeqX machines (Illumina, San Diego, CA), along with complete remission samples to serve as matched normals for the TMNA. For comparison, a mixture of genomic DNA from multiple anonymous donors was used as "normal" for the TUNA. Read mapping and somatic variant calling was performed using the tools Isaac3 and Strelka2, respectively. Statistical differences between groups were assessed by two-sided Mann-Whitney tests. Results: The TMNA produced a median of 17,700 somatic variant calls, while the TUNA produced 419,000. This 24-fold disparity is mainly due to residual germline variants missed by the TUNA. A large fraction of TMNA variants (57%) was located in regions of known low confidence variant calling (as defined by the Genome in a Bottle Consortium) and likely contain mostly artifacts. After removing these regions from analysis a median of 7,700 and 331,000 variants remained in the TMNA and TUNA datasets, respectively. In order to eliminate germline variants, the gnomAD population database was queried and any present variants were discarded. As expected, this removed over 95% of all variants from the TUNA dataset, but also 41% from the TMNA dataset. The latter might be attributed to common germline variants falsely being called as somatic by the TMNA and/or somatic mutations occurring at polymorphic sites. After this filtering step a median of 3,770 and 15,500 variants remained in the TMNA and TUNA datasets, respectively. This 4-fold disparity in variant number is most likely caused by rare germline variation remaining in the TUNA dataset. Of the remaining TMNA variants only 65% could be found within the larger TUNA dataset. A major factor governing this observation was variant allele frequency (VAF). Variants that overlapped between both datasets had on average higher VAFs than those unique to the TMNA (p < 2.2x10-16). Further inspection of the VAF distribution among samples revealed a bimodal or nearly bimodal distribution for all samples. All distributions shared a sharp peak centered on a VAF of 10%, which was unexpected given the estimated tumor fractions of the samples predict VAFs of 25% and higher. Variants in this lower part of the distribution (arbitrarily defined as VAFs < 20%) constitute on average 50% of all variants in a TMNA sample, with extremes reaching 95% in 2 samples. These low frequency variants show distinctly lower mapping qualities than variants with VAFs ≥ 20% (p < 2.2x10-16), i.e. they reside in regions of elevated mapping ambiguity which potentially leads to the creation of artefacts. Analyzing the overlap of only the higher VAF variants we find that 97.4% of all TMNA variants can also be found in the TUNA dataset. Conclusions: Comparing tumor samples to matched normal material from the respective patient is the preferred approach for somatic variant calling in WGS data, however even with modern algorithms false positives due to technical artifacts seem to be highly abundant. A deeper understanding of the nature of these artifacts is crucial for developing appropriate filtering schemes and improving variant calling algorithms. In the absence of a matched normal using a TUNA can uncover the vast majority (97.4%) of high-quality variants found in a TMNA, however distinguishing true somatic variants from residual rare germline variation in a TUNA remains a major challenge. Disclosures Hutter: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

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Subbotina, T. N., I. E. Maslyukova, A. A. Faleeva, P. A. Nikolaeva, A. S. Khazieva, E. A. Dunaeva, K. O. Mironov et al. « Using the Minor Variant Finder software to identify and quantify the allelic burden level of somatic mutations in oncohematologic diseases ». Oncohematology 15, n^o 2 (16 juillet 2020) : 85–91. http://dx.doi.org/10.17650/1818-8346-2020-15-2-85-91.

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Background. There are problems related to both quantitative assessment of an allele burden level of a mutant gene and interpretation of results in DNA samples with the burden level of the mutant allele less than 15–20 %, when using Sanger sequencing for analyzing somatic mutations. Applied Biosystems (USA) has developed new software Minor Variant Finder, which allows determining mutations with the allele burden level from 5 %.The objective: to determine the allele burden level and identification of minor variants of somatic mutations in the ASXL1, JAK2 genes and BCR-ABL oncogene using Minor Variant Finder software in patients with myeloproliferative neoplasms.Materials and methods. The level of mutant allele burden for 15 patients with myeloproliferative neoplasms was determined by the identified mutations using the Minor Variant Finder software, after analysis of point somatic mutations in the ASXL1, JAK2 genes and BCR-ABL oncogene by Sanger sequencing.Results. The allele burden level in all 5 ASXL1-positive samples and BCR-ABL-positive sample was determined as higher than 20 % using the Minor Variant Finder software. The allele burden level in 2 cases was higher than 20 % and in 7 cases lower than 20 %, when we analyzed 9 JAK2-positive samples.Conclusion. Minor Variant Finder software can be used to estimate the allele burden level and to identify minor variants of somatic mutations in the ASXL, JAK2 and BCR-ABL genes.

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Shao, Xiangqiang, Shruti Rao, Coumarane Mani, Jason Saliba, Rong He, Chimene Kesserwan, Arpad Danos et al. « Expert Curation of Somatic FLT3 Variants By the Clingen Somatic Hematologic Cancer Taskforce (ClinGen HCT) ». Blood 138, Supplement 1 (5 novembre 2021) : 4387. http://dx.doi.org/10.1182/blood-2021-153546.

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Abstract Clinical significance of somatic gene variants needs to be comprehensively characterized for their diagnostic, prognostic and/or therapeutic actionability in patient management. However, challenges remain due to discrepancies in interpretation and reporting of these somatic variants among different testing labs. Therefore, standardized curation, clinical interpretation and reporting of somatic variants in hematologic cancers is critical. To address this issue, the Hematologic Cancer Taskforce (HCT), composed of 52 multi-disciplinary experts including oncologists, molecular pathologists, lab directors, genomic scientists and biocurators, was formed in January 2020 within the ClinGen Somatic Cancer Clinical Domain Working Group (CDWG) with a goal to undertake systematic curation and evidence-based clinical interpretation of genes/somatic variants associated with hematologic malignancies. In collaboration with the Clinical Interpretation of Variants in Cancer (CIViC) (civicdb.org) knowledgebase, HCT members curate, edit, and verify Evidence Items for each variant extracted from peer-reviewed publications. Monthly discussions based on these Evidence Items lead to the preparation of variant Assertions, which summarize the state of the field consensus variant interpretation and include tiering based on the AMP/ASCO/CAP guidelines (PMID: 27993330). FMS-like tyrosine kinase 3 (FLT3) encodes a class III receptor tyrosine kinase expressed in hematopoietic cells. FLT3 mutations, including both internal tandem duplication (ITD) and mutations in the tyrosine kinase domain (TKD), are the most common mutations in acute myeloid leukemia (AML), occurring in approximately 30% of all AML cases. Implementing FLT3 tyrosine kinase inhibitors (TKIs) in different treatment regimens for FLT3 mutated AML patients has led to significantly improved overall survival. Type I FLT3 inhibitors, including midostaurin, gilteritinib, sunitinib, lestaurtinib, and crenolanib, bind to the ATP-binding site when the receptor is in active conformation. Type II FLT3 inhibitors, including sorafenib, ponatinib, and quizartinib, interact with a hydrophobic region directly adjacent to the ATP-binding domain that is only accessible when the receptor is inactive, which prevents receptor activation. Generally in AML cells, type I FLT3 inhibitors prevent activity for both ITD and TKD mutations, while Type II inhibitors target ITD but lack efficiency against TKD mutations. The development of TKD mutations in AML cells with ITD have proved to be a mechanism of acquired, or secondary resistance to Type II FLT3 inhibitors. The HCT is piloting curation assessments of FLT3 alterations, including ITD, TKD and non-TKD variants, in AML. So far, the HCT has curated 75 evidence items for FLT3 somatic variants. FLT3-ITD, as well as D835 and I836 were asserted as tier 1 level A variants based on the prediction of response to gilteritinib in relapsed/refractory AML (PMIDs: 27993330, 31665578, 28645776, 28516360, 27908881). Recent curation activities are focused on FLT3 D839G and N676K, as clinical trials using large AML patient cohorts are lacking in their ability to validate drug response/resistance associations of these two TKD variants due to their low frequency. Functional studies showed both variants result in increased proliferation and protection from apoptosis, supporting the oncogenic potential of these two variants (PMIDs: 26891877, 2468088). FLT3 D839G combined with ITD confers resistance to pexidartinib and ponatinib, both Type II FLT3 inhibitors (PMIDs: 25847190, 23430109). FLT3 N676K predicts response to the Type I FLT3 inhibitor, gilteritinib, when N676K is present alone or in combination with ITD. Interestingly, FLT3 N676K in the absence of ITD predicts response to sorafenib, a Type II FLT3 inhibitor (PMIDs: 32040554, 32984009). However, these results are mostly derived from in vitro studies. Two separate Tier II, Level D Assertions have been submitted for FLT3-ITD&D839G for its response to pexidartinib and ponatinib, and more evidence is being collected to form an Assertion for FLT3 N676K. The complexity of the prediction of response/resistance associated with FLT3 D839G and N676K supports the importance of evidence-based curation and collection for these variants in the context of the overall mutation profile, disease context and specific FLT3 TKIs to clearly define their clinical impact. Disclosures Pullarkat: Stemline Therapeutics: Honoraria.

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Kaur, Pushpinder, Daniel Campo, Tania B. Porras, Alexander Ring, Janice Lu, Yvonne Chairez, Yunyun Su, Irene Kang et Julie E. Lang. « A Pilot Study for the Feasibility of Exome-Sequencing in Circulating Tumor Cells Versus Single Metastatic Biopsies in Breast Cancer ». International Journal of Molecular Sciences 21, n^o 14 (8 juillet 2020) : 4826. http://dx.doi.org/10.3390/ijms21144826.

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The comparison of the landscape of somatic alterations in circulating tumor cells (CTCs) versus metastases is challenging. Here, we comprehensively characterized the somatic landscape in bulk (amplified and non-amplified), spike-in breast cancer cells, CTCs, and metastases from breast cancer patients using whole-exome sequencing (WES). We determined the level of genomic concordance for somatic nucleotide variants (SNVs), copy number alterations (CNAs), and structural variants (SVs). The variant allele fractions (VAFs) of somatic variants were remarkably similar between amplified and non-amplified cell line samples as technical replicates. In clinical samples, a significant fraction of somatic variants had low VAFs in CTCs compared to metastases. The most frequently recurrent gene mutations in clinical samples were associated with an elevated C > T mutational signature. We found complex rearrangement patterns including intra- and inter-chromosomal rearrangements, singleton, and recurrent gene fusions, and tandem duplications. We observed high molecular discordance for somatic alterations between paired samples consistent with marked heterogeneity of the somatic landscape. The most prevalent copy number calls were focal deletion events in CTCs and metastases. Our results demonstrate the feasibility of an integrated workflow for the identification of a complete repertoire of somatic alterations and highlight the intrapatient genomic differences that occur between CTCs and metastases.

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Kueffner, Robert, Hui Li, Kakit Cheung, Marc Fink, Zachry Soens, Jinlian Wang, Osman Siddiqui et al. « VONC : A solution for the clinical assessment of somatic genomic alterations. » Journal of Clinical Oncology 37, n^o 15_suppl (20 mai 2019) : e13155-e13155. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e13155.

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e13155 Background: Next generation sequencing (NGS) technology is transforming the diagnosis and treatment of cancer. However, the massive scale of data has overwhelmed pathologists who need streamlined tools to process this data, automate report generation and minimize human errors. Methods: We developed the Variant interpretation station for ONCology, VONC, as an end-to-end solution for moving from NGS whole exome and transcriptome data to actionable clinical reports that support cancer diagnosis, prognosis, and personalized treatment strategies for solid and hematologic malignancies. Results: VONC integrates all steps for moving from raw NGS data, variant calling and LIMS, to comprehensive annotation of variants. The main functional feature of VONC is a transparent process that effectively combines automated and expert curation to identify clinically relevant and actionable driver variants. VONC also enables efficient management of multi-group, -role, -system and -site curation processes. In contrast to current tools, VONC handles all somatic and constitutional genomic alterations including SNV, indel, CNV, fusion, splicing, and gene expression. Key data sources include 1) 350,000 variants for 50 tumor types across 57,000 sequenced cancer patients; 2) variant frequencies estimated from 1.5M cancer patients; 3) expert curated literature evidence from 16,818 papers covering 26,496 alterations spanning 2,448 cancer driver genes; and 4) curated database of FDA-approved drugs and recruiting clinical trials. VONC presents a prioritized list of variants in oncogenes and tumor suppressors through functional (literature-based) and structure-based (hotspots) algorithms. This is coupled to all supporting information necessary for clinical decision making. Curators can quickly screen variant type, QC metrics, and frequency in sequencing cohorts of cancer patients as well as healthy subjects. Within minutes, variants can be triaged and annotated with FDA approved, NCCN guidelines reported, or literature supported therapeutics, including resistance and contraindicated. Conclusions: VONC is a clinically-ready tool with an intuitive end-user interface tailored for the rapid assessment of variants in cancer patients, to facilitate personalized cancer medicine in a high-throughput laboratory.

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Chuang, Huai-Chia, Wei-Ting Hung, Yi-Ming Chen, Pu-Ming Hsu, Jeng-Hsien Yen, Joung-Liang Lan et Tse-Hua Tan. « Genomic sequencing and functional analyses identify MAP4K3/GLK germline and somatic variants associated with systemic lupus erythematosus ». Annals of the Rheumatic Diseases 81, n^o 2 (5 octobre 2021) : 243–54. http://dx.doi.org/10.1136/annrheumdis-2021-221010.

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ObjectivesMAP4K3 (GLK) overexpression in T cells induces interleukin (IL)-17A production and autoimmune responses. GLK overexpressing T-cell population is correlated with severity of human systemic lupus erythematosus (SLE); however, it is unclear how GLK is upregulated in patients with SLE.MethodsWe enrolled 181 patients with SLE and 250 individuals without SLE (93 healthy controls and 157 family members of patients with SLE) in two independent cohorts from different hospitals/cities. Genomic DNAs of peripheral blood mononuclear cells were subjected to next-generation sequencing to identify GLK gene variants. The functional consequences of the identified GLK germline or somatic variants were investigated using site-directed mutagenesis and cell transfection, followed by reporter assays, mass spectrometry, immunoblotting, coimmunoprecipitation, and in situ proximity ligation assays.ResultsWe identified 58 patients with SLE from Cohort #1 and #2 with higher frequencies of a somatic variant (chr2:39 477 124 A>G) in GLK 3′-untranslated region (UTR); these patients with SLE showed increased serum anti-double-stranded DNA levels and decreased serum C3/C4 levels. This somatic variant in 3′-UTR enhanced GLK mRNA levels in T cells. In addition, we identified five patients with SLE with GLK (A410T) germline variant in Cohort #1 and #2, as well as two other patients with SLE with GLK (K650R) germline variant in Cohort #1. Another GLK germline variant, A579T, was also detected in one patient with SLE from Cohort #2. Both GLK (A410T) and GLK (K650R) mutants inhibited GLK ubiquitination induced by the novel E3 ligase makorin ring-finger protein 4 (MKRN4), leading to GLK protein stabilisation.ConclusionsMultiple GLK germline and somatic variants cause GLK induction by increasing mRNA or protein stability in patients with SLE.

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Chuang, Huai-Chia, Wei-Ting Hung, Yi-Ming Chen, Pu-Ming Hsu, Jeng-Hsien Yen, Joung-Liang Lan et Tse-Hua Tan. « Genomic sequencing and functional analyses identify MAP4K3/GLK germline and somatic variants associated with systemic lupus erythematosus ». Annals of the Rheumatic Diseases 81, n^o 2 (5 octobre 2021) : 243–54. http://dx.doi.org/10.1136/annrheumdis-2021-221010.

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ObjectivesMAP4K3 (GLK) overexpression in T cells induces interleukin (IL)-17A production and autoimmune responses. GLK overexpressing T-cell population is correlated with severity of human systemic lupus erythematosus (SLE); however, it is unclear how GLK is upregulated in patients with SLE.MethodsWe enrolled 181 patients with SLE and 250 individuals without SLE (93 healthy controls and 157 family members of patients with SLE) in two independent cohorts from different hospitals/cities. Genomic DNAs of peripheral blood mononuclear cells were subjected to next-generation sequencing to identify GLK gene variants. The functional consequences of the identified GLK germline or somatic variants were investigated using site-directed mutagenesis and cell transfection, followed by reporter assays, mass spectrometry, immunoblotting, coimmunoprecipitation, and in situ proximity ligation assays.ResultsWe identified 58 patients with SLE from Cohort #1 and #2 with higher frequencies of a somatic variant (chr2:39 477 124 A>G) in GLK 3′-untranslated region (UTR); these patients with SLE showed increased serum anti-double-stranded DNA levels and decreased serum C3/C4 levels. This somatic variant in 3′-UTR enhanced GLK mRNA levels in T cells. In addition, we identified five patients with SLE with GLK (A410T) germline variant in Cohort #1 and #2, as well as two other patients with SLE with GLK (K650R) germline variant in Cohort #1. Another GLK germline variant, A579T, was also detected in one patient with SLE from Cohort #2. Both GLK (A410T) and GLK (K650R) mutants inhibited GLK ubiquitination induced by the novel E3 ligase makorin ring-finger protein 4 (MKRN4), leading to GLK protein stabilisation.ConclusionsMultiple GLK germline and somatic variants cause GLK induction by increasing mRNA or protein stability in patients with SLE.

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O’Sullivan, Brian, et Cathal Seoighe. « vcfView : An Extensible Data Visualization and Quality Assurance Platform for Integrated Somatic Variant Analysis ». Cancer Informatics 19 (janvier 2020) : 117693512097237. http://dx.doi.org/10.1177/1176935120972377.

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Motivation: Somatic mutations can have critical prognostic and therapeutic implications for cancer patients. Although targeted methods are often used to assay specific cancer driver mutations, high throughput sequencing is frequently applied to discover novel driver mutations and to determine the status of less-frequent driver mutations. The task of recovering somatic mutations from these data is nontrivial as somatic mutations must be distinguished from germline variants, sequencing errors, and other artefacts. Consequently, bioinformatics pipelines for recovery of somatic mutations from high throughput sequencing typically involve a large number of analytical choices in the form of quality filters. Results: We present vcfView, an interactive tool designed to support the evaluation of somatic mutation calls from cancer sequencing data. The tool takes as input a single variant call format (VCF) file and enables researchers to explore the impacts of analytical choices on the mutant allele frequency spectrum, on mutational signatures and on annotated somatic variants in genes of interest. It allows variants that have failed variant caller filters to be re-examined to improve sensitivity or guide the design of future experiments. It is extensible, allowing other algorithms to be incorporated easily. Availability: The shiny application can be downloaded from GitHub ( https://github.com/BrianOSullivanGit/vcfView ). All data processing is performed within R to ensure platform independence. The app has been tested on RStudio, version 1.1.456, with base R 3.6.2 and Shiny 1.4.0. A vignette based on a publicly available data set is also available on GitHub.

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Jürgens, Lara, Felix Manske, Elvira Hubert, Tabea Kischka, Lea Flötotto, Oliver Klaas, Victoria Shabardina, Christoph Schliemann, Wojciech Makalowski et Klaus Wethmar. « Somatic Functional Deletions of Upstream Open Reading Frame-Associated Initiation and Termination Codons in Human Cancer ». Biomedicines 9, n^o 6 (29 mai 2021) : 618. http://dx.doi.org/10.3390/biomedicines9060618.

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Upstream open reading frame (uORF)-mediated translational control has emerged as an important regulatory mechanism in human health and disease. However, a systematic search for cancer-associated somatic uORF mutations has not been performed. Here, we analyzed the genetic variability at canonical (uAUG) and alternative translational initiation sites (aTISs), as well as the associated upstream termination codons (uStops) in 3394 whole-exome-sequencing datasets from patient samples of breast, colon, lung, prostate, and skin cancer and of acute myeloid leukemia, provided by The Cancer Genome Atlas research network. We found that 66.5% of patient samples were affected by at least one of 5277 recurrent uORF-associated somatic single nucleotide variants altering 446 uAUG, 347 uStop, and 4733 aTIS codons. While twelve uORF variants were detected in all entities, 17 variants occurred in all five types of solid cancer analyzed here. Highest frequencies of individual somatic variants in the TLSs of NBPF20 and CHCHD2 reached 10.1% among LAML and 8.1% among skin cancer patients, respectively. Functional evaluation by dual luciferase reporter assays identified 19 uORF variants causing significant translational deregulation of the associated main coding sequence, ranging from 1.73-fold induction for an AUG.1 > UUG variant in SETD4 to 0.006-fold repression for a CUG.6 > GUG variant in HLA-DRB1. These data suggest that somatic uORF mutations are highly prevalent in human malignancies and that defective translational regulation of protein expression may contribute to the onset or progression of cancer.

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Baer, Constance Regina, Niroshan Nadarajah, Claudia Haferlach, Wolfgang Kern et Torsten Haferlach. « A Study on Paired Tissue Sequencing in Hematologic Diseases to Distinguish Somatic from Germline Sequence Variants in Routine Diagnostics ». Blood 128, n^o 22 (2 décembre 2016) : 5511. http://dx.doi.org/10.1182/blood.v128.22.5511.5511.

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Abstract Background: Genetic testing is an integral part of modern diagnostics. Sequencing genomes or exomes in different consortia revealed novel aberrations with importance to hematologic classification, prognosis or therapy. However, a high number of low frequency variants were also found in healthy populations. This challenged the distinction between population variation (polymorphism) and disease associated changes based on early databases with limited extent. For diagnostic purposes, the distinction of somatic (acquired) variants from rare germline variants allows moving towards personalized genetic characterization including molecular markers for individual follow-up. Aim: 1) present an approach to distinguish between somatic or germline variants by comparison with matched tissue (buccal swap, nails), 2) define diagnostically relevant patterns for variant classification or database use. Patients and Methods: Variants were initially classified in a three-tier system: (A) Protein truncating variants (PTV) or changes with strong evidence in literature (e.g. JAK2V617F) were defined as actionable/disease associated. (B) Criteria for polymorphism were met, if population frequencies were available from two sources (1000 Genomes, ExAC). (C) Remaining, critical variants were sequenced in germline DNA (ACMG guidelines, Richards, 2015). We selected 88 patients with critical variants in peripheral blood (PB, n=29) or bone marrow (BM, n=59) and available DNA from buccal swaps (n=40), nails (n=31) or both (n=17). Samples were received for routine diagnostic assessment (suspected diagnosis: myelodysplastic syndrome or chronic myelomonocytic leukemia [n=56], myeloproliferative neoplasm [n=8], acute myeloid leukemia [n=6] or B cell malignancy [n=18]). From PB or BM, 829 analysis by Sanger-, 454- (Roche, Branford, CT) or Illumina sequencing (San Diego, CA) were performed (1-49 [median 6] genes/patient). Results: In 88 patients, we identified 74 actionable/disease associated changes, 67 polymorphisms and one or two variants (n=96) per patient that could not be classified in the previous categories, requiring matched germline DNA sequencing. We found that 35% (34/96) of these variants were also present in germline, although not listed in common polymorphism databases. Consequently, theses variants do not qualify as markers for clonality and follow-up. Of note, 19% of nails and 14% of all buccal swabs received in our laboratory were not analyzable due to low DNA amounts (not included in cohort). Importantly, DNA from both sources can contain low levels of somatic mutations. Next, we compared somatic and germline variants in terms of predicted effects on function, variant burden and population frequency, to identify patterns with relevance to future categorization. Firstly, predicted as damaging by PolyPhen algorithm were significantly more somatic (92%, 49/53) than germline variants (61%, 19/31, p<0.001). Most variants (excluding PTVs) were found in TET2 (n=25). Of 11 confirmed somatic variants, 10 were located in conserved domains, while none of the germline variants was located in these domains. Secondly, germline variants had a median burden of 50% (40-59%) or 98% in one case, which is the expected result for variants derived from either one or both alleles. For somatic variants, burdens were observed between 2% and 100% (median 40%), representing the varying degree of malignant cells in PB or BM. For comparison, disease associated variants showed a similar distribution: 3-90% (median: 40%). Thirdly, we compared variants to ExAC data, the largest available set of exonic variants in healthy individuals (over 60,000). Only 14/34 (41%) germline variants were found in the ExAC data (population frequencies <0.1%). However, 3/62 (5%) of our somatic variants also occurred in the ExAC set. Conclusions: A growing number of sequencing data outdated the traditional distinction between polymorphism and mutation. By comparison with DNA from buccal swabs or nails, we showed that somatic and germline variants have different global patterns (e.g. variant burden, predicted function), but the decision in individual cases based on in silico data can be misleading. Only sequencing germline DNA distinguishes somatic from germline variants on a personalized level and allows strategies to define germline variants potentially contributing to tumorigenesis in future studies. Disclosures Baer: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Other: Part Owner MLL Munich Leukemia Laboratory.

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Eng, Whitney, Christopher L. Sudduth, Dennis J. Konczyk, Patrick J. Smits, Amir H. Taghinia, Steven J. Fishman, Ahmad Alomari, Denise M. Adams et Arin K. Greene. « Parkes Weber syndrome with lymphedema caused by a somatic KRAS variant ». Molecular Case Studies 7, n^o 6 (4 octobre 2021) : a006118. http://dx.doi.org/10.1101/mcs.a006118.

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Parkes Weber syndrome is a vascular malformation overgrowth condition typically involving the legs. Its main features are diffuse arteriovenous fistulas and enlargement of the limb. The condition has been associated with pathogenic germline variants in RASA1 and EPHB4. We report two individuals with Parkes Weber syndrome of the leg and primary lymphedema containing a somatic KRAS variant (NM_004985.5:c.35G > A; p.Gly12Asp). KRAS variants, which cause somatic intracranial and extracranial arteriovenous malformations, also result in Parkes Weber syndrome with lymphatic malformations.

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Eng, Whitney, Christopher L. Sudduth, Dennis J. Konczyk, Patrick J. Smits, Amir H. Taghinia, Steven J. Fishman, Ahmad Alomari, Denise M. Adams et Arin K. Greene. « Parkes Weber syndrome with lymphedema caused by a somatic KRAS variant ». Molecular Case Studies 7, n^o 6 (4 octobre 2021) : a006118. http://dx.doi.org/10.1101/mcs.a006118.

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Parkes Weber syndrome is a vascular malformation overgrowth condition typically involving the legs. Its main features are diffuse arteriovenous fistulas and enlargement of the limb. The condition has been associated with pathogenic germline variants in RASA1 and EPHB4. We report two individuals with Parkes Weber syndrome of the leg and primary lymphedema containing a somatic KRAS variant (NM_004985.5:c.35G > A; p.Gly12Asp). KRAS variants, which cause somatic intracranial and extracranial arteriovenous malformations, also result in Parkes Weber syndrome with lymphatic malformations.

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Faienza, Maria F., Mariangela Chiarito, Fulvia Baldinotti, Domenico Canale, Carmela Savino, Guglielmo Paradies, Domenico Corica et al. « NR5A1 Gene Variants : Variable Phenotypes, New Variants, Different Outcomes ». Sexual Development 13, n^o 5-6 (2019) : 258–63. http://dx.doi.org/10.1159/000507411.

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NR5A1 (nuclear receptor subfamily 5 group A member 1) is a transcriptional regulator of adrenal and gonadal development and function. Heterozygous and homozygous NR5A1 mutations have been described in people with 46,XY disorders of sex development (DSD). The clinical, endocrine, and genetic features of four 46,XY subjects with NR5A1 genetic variants (2 sisters, 2 boys) from 3 unrelated families are reported. All subjects presented with hypergonadotropic hypogonadism and abnormal pubertal progression. Markers of Sertoli cell function were more affected than those of Leydig cell function. Genetic investigation demonstrated the presence of different heterozygous NR5A1 genetic variants. In the boys, pathogenetic NR5A1 gene variants were found that had been previously reported. The 2 sisters carried a new genetic variant in exon 4, and in silico analysis and ACMG classification indicated its pathogenicity. The data confirmed that NR5A1 gene mutations may present with variable genital phenotypes. Anyway, reproductive function was always impaired. Any clinical or endocrine data seem to be unable to differentiate these patients from other 46,XY DSD cases, suggesting that molecular analysis must be warranted. In subjects with NR5A1 mutations, different decisions in sex assignment may permit satisfying somatic and psychological outcome, but any option requires hormonal substitutive therapy from adolescence onward.

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Saliba, Jason, Gordana Raca, Angshumoy Roy, Ian King, Shamini Selvarajah, Xinjie Xu, Rashmi Kanagal-Shamanna et al. « Abstract 1192 : The Clinical Genome Resource (ClinGen) somatic cancer clinical domain working group ». Cancer Research 82, n^o 12_Supplement (15 juin 2022) : 1192. http://dx.doi.org/10.1158/1538-7445.am2022-1192.

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Abstract Interpretation of the clinical significance of somatic gene variants in cancer remains a major challenge in cancer diagnosis, prognosis and treatment response prediction. We will report on progress and plans of the Clinical Genome Resource (ClinGen) Somatic Cancer Clinical Domain Working Group (CDWG). The CDWG membership consists of over 150 multi-disciplinary experts in cancer biology, oncology, pathology, genetics, genomics and informatics. The mission of the ClinGen Somatic Cancer CDWG is to facilitate the development of data curation guidelines and standards to determine the clinical significance of somatic alterations in cancer, thereby enhancing the usability, dissemination and implementation of cancer somatic changes in the ClinGen resource and other knowledgebases including CIViC, ClinVar, and the Variant Interpretation for Cancer Consortium (VICC) MetaKB. Our goal is to create high-quality assertions of the clinical significance of specific somatic variants in cancer by leveraging the CIViC curation interface, adapting the germline procedures of ClinGen to somatic variant interpretation, and implementing the interoperability standards of the Global Alliance for Genomics and Health (GA4GH). The ClinGen Somatic effort is overseen by the Somatic CDWG and reports progress to the overall ClinGen consortium. There are Somatic Cancer subdomains focused on particular clinically important domains of cancer variant interpretation including three Task Forces (covering Pediatric Cancer, Hematologic Cancer, and Solid Tumors) and a growing number of Somatic Cancer Variant Curation Expert Panels (SC-VCEPs). To improve quality and consistency of clinical interpretations, each candidate somatic cancer VCEP must complete a four step approval process adapted from ClinGen’s work in Germline disease domains. The Somatic CDWG works to ensure that each group is aware of available training materials and detailed standard operating procedures. Each SC-VCEP also coordinates with the ClinGen Cancer Variant Interpretation Committee (CVI) whose goal is to support development of granular specifications for the AMP/ASCO/CAP guidelines for somatic variant interpretation. New SC-VCEPs are anticipated to focus on specific clinically relevant genes, pathways, disease entities, variant classes or treatment modalities. Currently, three SC-VCEPs have begun to work through the four step process (focused on FGFR mutations, NTRK fusions, and FLT3 mutations respectively), and two more SC-VCEPs are in the planning stage (Histone H3 and Ph-like ALL). To date, ClinGen Somatic groups have contributed 619 evidence lines into CIViC from 353 published papers and 21 assertions of clinical significance. Input from the AACR community is critical for the establishment of new SC-VCEPs that address areas of variant interpretation with the greatest need. Citation Format: Jason Saliba, Gordana Raca, Angshumoy Roy, Ian King, Shamini Selvarajah, Xinjie Xu, Rashmi Kanagal-Shamanna, Laveniya Satgunaseelan, David Meredith, Mark Evans, Alanna Church, Panieh Terraf, Yassmine Akkari, Heather E. Williams, Wan-Hsin Lin, Chimene Kesserwan, Deborah I. Ritter, Kilannin Krysiak, Arpad Danos, Alex Wagner, Marilyn M. Li, Dmitriy Sonkin, Jonathan S. Berg, Sharon E. Plon, Heidi L. Rehm, Shashikant Kulkarni, Ramaswamy Govindan, Obi L. Griffith, Malachi Griffith, on behalf of the ClinGen Somatic CDWG. The Clinical Genome Resource (ClinGen) somatic cancer clinical domain working group [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1192.

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Kanagal-Shamanna, Rashmi, Shruti Rao, Panieh Terraf, Gordana Raca, Jason Saliba, Arpad Danos, Coumarane Mani et al. « Expert Curation of Somatic Variants in Hematological Malignancies By the Clingen Somatic Hematological Cancer Taskforce (ClinGen HCT) ». Blood 136, Supplement 1 (5 novembre 2020) : 23. http://dx.doi.org/10.1182/blood-2020-143028.

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Hematological malignancies comprise a genetically heterogeneous spectrum of diseases caused by abnormal proliferation or maturation of a variety of hematological cell lineages. Genomic abnormalities including chromosomal translocations, copy number variations and sequence level gene mutations underlie the pathogenesis of these disorders and frequently serve as important diagnostic, prognostic and/or therapeutic markers. However, the substantial discrepancy in interpretation and reporting of these genomic abnormalities among testing labs creates challenges for patient management. Therefore, standardizing the curation, clinical interpretation and reporting of somatic alterations within the context of their diagnostic, prognostic and therapeutic significance in hematological cancers is critical. In January 2020, the ClinGen Somatic Cancer Clinical Domain working group formed the Hematological Cancer Taskforce (HCT) with a goal to undertake systematic curation and evidence-based clinical interpretation of genes/somatic variants associated with hematological malignancies. The HCT has recruited 32 multi-disciplinary experts including oncologists, molecular pathologists, clinical lab directors, genomic scientists and biocurators with expertise in hematological malignancies. In collaboration with the Clinical Interpretation of Variants in Cancer (CIViC) (civicdb.org) knowledgebase, variants from peer-reviewed publications are curated with editor review for clinical utility as evidence items. Monthly discussions based on these evidence items lead to the creation of summary variant assertions using the AMP/ASCO/CAP guidelines (Li M, et al., Journal of Molecular Diagnostics, 2017). The HCT is currently focused on expert curation and clinical interpretation of somatic variants in FLT3 (internal tandem duplication, tyrosine kinase domain and non tyrosine kinase domain variants) in acute myeloid leukemia (AML). Expert curation of gene fusions in Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) in collaboration with the ClinGen somatic pediatric cancer taskforce is currently underway. To date, the HCT has curated 45 evidence items from clinical and pre-clinical studies on the aforementioned genes/variants. In addition, three AMP Tier I, level A variant assertions of FLT3-ITD, D835 and I836, which predict response to Gilteritinib, an FDA-approved drug for relapsed or refractory AML, have been curated. In the future, the HCT plans to extend its focus on curation of BCR-ABL1 kinase domain mutations in chronic myeloid leukemia (CML). Based on the initial pilot curation phase, the HCT will develop gene-specific recommendations to standardize the reporting and interpretation of somatic variants to better assist clinical decisions and apply to become official ClinGen Somatic Expert Panels in each of these gene-disease domains. Disclosures Blombery: Novartis: Consultancy; Invivoscribe: Honoraria; Amgen: Consultancy; Janssen: Honoraria.

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Aydemirli, M. D., K. van der Tuin, F. J. Hes, A. M. W. van den Ouweland, T. van Wezel, E. Kapiteijn et H. Morreau. « A unique case of two somatic APC mutations in an early onset cribriform-morular variant of papillary thyroid carcinoma and overview of the literature ». Familial Cancer 19, n^o 1 (9 octobre 2019) : 15–21. http://dx.doi.org/10.1007/s10689-019-00146-4.

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Abstract We report a case of a 22-year-old female patient who was diagnosed with a cribriform-morular variant of papillary thyroid carcinoma (CMV-PTC). While at early ages this thyroid cancer variant is highly suggestive for familial adenomatous polyposis (FAP), there was no family history of FAP. In the tumor biallelic, inactivating APC variants were identified. The patient tested negative for germline variants based on analysis of genomic DNA from peripheral blood leukocytes. Somatic mosaicism was excluded by subsequent deep sequencing of leukocyte and normal thyroid DNA using next generation sequencing (NGS). This report presents a rare sporadic case of CMV-PTC, and to the best of our knowledge the first featuring two somatic APC mutations underlying the disease, with an overview of CMV-PTC cases with detected APC and CTNNB1 pathogenic variants from the literature.

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te Paske, Iris B. A. W., José Garcia-Pelaez, Anna K. Sommer, Leslie Matalonga, Teresa Starzynska, Anna Jakubowska, Laura Valle et al. « A mosaic PIK3CA variant in a young adult with diffuse gastric cancer : case report ». European Journal of Human Genetics 29, n^o 9 (1 juin 2021) : 1354–58. http://dx.doi.org/10.1038/s41431-021-00853-6.

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AbstractHereditary diffuse gastric cancer (HDGC) is associated with germline deleterious variants in CDH1 and CTNNA1. The majority of HDGC-suspected patients are still genetically unresolved, raising the need for identification of novel HDGC predisposing genes. Under the collaborative environment of the SOLVE-RD consortium, re-analysis of whole-exome sequencing data from unresolved gastric cancer cases (n = 83) identified a mosaic missense variant in PIK3CA in a 25-year-old female with diffuse gastric cancer (DGC) without familial history for cancer. The variant, c.3140A>G p.(His1047Arg), a known cancer-related somatic hotspot, was present at a low variant allele frequency (18%) in leukocyte-derived DNA. Somatic variants in PIK3CA are usually associated with overgrowth, a phenotype that was not observed in this patient. This report highlights mosaicism as a potential, and understudied, mechanism in the etiology of DGC.

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Merker, Jason D., Kelly Devereaux, A. John Iafrate, Suzanne Kamel-Reid, Annette S. Kim, Joel T. Moncur, Stephen B. Montgomery et al. « Proficiency Testing of Standardized Samples Shows Very High Interlaboratory Agreement for Clinical Next-Generation Sequencing–Based Oncology Assays ». Archives of Pathology & ; Laboratory Medicine 143, n^o 4 (30 octobre 2018) : 463–71. http://dx.doi.org/10.5858/arpa.2018-0336-cp.

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Context.— Next-generation sequencing–based assays are being increasingly used in the clinical setting for the detection of somatic variants in solid tumors, but limited data are available regarding the interlaboratory performance of these assays. Objective.— To examine proficiency testing data from the initial College of American Pathologists (CAP) Next-Generation Sequencing Solid Tumor survey to report on laboratory performance. Design.— CAP proficiency testing results from 111 laboratories were analyzed for accuracy and associated assay performance characteristics. Results.— The overall accuracy observed for all variants was 98.3%. Rare false-negative results could not be attributed to sequencing platform, selection method, or other assay characteristics. The median and average of the variant allele fractions reported by the laboratories were within 10% of those orthogonally determined by digital polymerase chain reaction for each variant. The median coverage reported at the variant sites ranged from 1922 to 3297. Conclusions.— Laboratories demonstrated an overall accuracy of greater than 98% with high specificity when examining 10 clinically relevant somatic single-nucleotide variants with a variant allele fraction of 15% or greater. These initial data suggest excellent performance, but further ongoing studies are needed to evaluate the performance of lower variant allele fractions and additional variant types.

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Arnaud, Pauline, Hélène Morel, Olivier Milleron, Laurent Gouya, Christine Francannet, Antoine Da Costa, Carine Le Goff, Guillaume Jondeau, Catherine Boileau et Nadine Hanna. « Unsuspected somatic mosaicism for FBN1 gene contributes to Marfan syndrome ». Genetics in Medicine 23, n^o 5 (25 janvier 2021) : 865–71. http://dx.doi.org/10.1038/s41436-020-01078-6.

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Abstract Purpose Individuals with mosaic pathogenic variants in the FBN1 gene are mainly described in the course of familial screening. In the literature, almost all these mosaic individuals are asymptomatic. In this study, we report the experience of our team on more than 5,000 Marfan syndrome (MFS) probands. Methods Next-generation sequencing (NGS) capture technology allowed us to identify five cases of MFS probands who harbored a mosaic pathogenic variant in the FBN1 gene. Results These five sporadic mosaic probands displayed classical features usually seen in Marfan syndrome. Combined with the results of the literature, these rare findings concerned both single-nucleotide variants and copy-number variations. Conclusion This underestimated finding should not be overlooked in the molecular diagnosis of MFS patients and warrants an adaptation of the parameters used in bioinformatics analyses. The five present cases of symptomatic MFS probands harboring a mosaic FBN1 pathogenic variant reinforce the fact that apparently asymptomatic mosaic parents should have a complete clinical examination and a regular cardiovascular follow-up. We advise that individuals with a typical MFS for whom no single-nucleotide pathogenic variant or exon deletion/duplication was identified should be tested by NGS capture panel with an adapted variant calling analysis.

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Froyen, Guy, Marie Le Mercier, Els Lierman, Karl Vandepoele, Friedel Nollet, Elke Boone, Joni Van der Meulen et al. « Standardization of Somatic Variant Classifications in Solid and Haematological Tumours by a Two-Level Approach of Biological and Clinical Classes : An Initiative of the Belgian ComPerMed Expert Panel ». Cancers 11, n^o 12 (16 décembre 2019) : 2030. http://dx.doi.org/10.3390/cancers11122030.

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In most diagnostic laboratories, targeted next-generation sequencing (NGS) is currently the default assay for the detection of somatic variants in solid as well as haematological tumours. Independent of the method, the final outcome is a list of variants that differ from the human genome reference sequence of which some may relate to the establishment of the tumour in the patient. A critical point towards a uniform patient management is the assignment of the biological contribution of each variant to the malignancy and its subsequent clinical impact in a specific malignancy. These so-called biological and clinical classifications of somatic variants are currently not standardized and are vastly dependent on the subjective analysis of each laboratory. This subjectivity can thus result in a different classification and subsequent clinical interpretation of the same variant. Therefore, the ComPerMed panel of Belgian experts in cancer diagnostics set up a working group with the goal to harmonize the biological classification and clinical interpretation of somatic variants detected by NGS. This effort resulted in the establishment of a uniform, two-level classification workflow system that should enable high consistency in diagnosis, prognosis, treatment and follow-up of cancer patients. Variants are first classified into a tumour-independent biological five class system and subsequently in a four tier ACMG clinical classification. Here, we describe the ComPerMed workflow in detail including examples for each step of the pipeline. Moreover, this workflow can be implemented in variant classification software tools enabling automatic reporting of NGS data, independent of panel, method or analysis software.

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Sürün, Bilge, Charlotta P. I. Schärfe, Mathew R. Divine, Julian Heinrich, Nora C. Toussaint, Lukas Zimmermann, Janina Beha et Oliver Kohlbacher. « ClinVAP : a reporting strategy from variants to therapeutic options ». Bioinformatics 36, n^o 7 (12 décembre 2019) : 2316–17. http://dx.doi.org/10.1093/bioinformatics/btz924.

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Abstract Motivation Next-generation sequencing has become routine in oncology and opens up new avenues of therapies, particularly in personalized oncology setting. An increasing number of cases also implies a need for a more robust, automated and reproducible processing of long lists of variants for cancer diagnosis and therapy. While solutions for the large-scale analysis of somatic variants have been implemented, existing solutions often have issues with reproducibility, scalability and interoperability. Results Clinical Variant Annotation Pipeline (ClinVAP) is an automated pipeline which annotates, filters and prioritizes somatic single nucleotide variants provided in variant call format. It augments the variant information with documented or predicted clinical effect. These annotated variants are prioritized based on driver gene status and druggability. ClinVAP is available as a fully containerized, self-contained pipeline maximizing reproducibility and scalability allowing the analysis of larger scale data. The resulting JSON-based report is suited for automated downstream processing, but ClinVAP can also automatically render the information into a user-defined template to yield a human-readable report. Availability and implementation ClinVAP is available at https://github.com/PersonalizedOncology/ClinVAP. Supplementary information Supplementary data are available at Bioinformatics online.

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Kraft, Ira Lignugaris, Amy M. Trottier, George F. Steinhardt, Nifang Niu, Pankhuri Wanjari, Wenjun Chen, Jeremy Segal et Lucy A. Godley. « Using sequential next-generation sequencing assays to identify germline cancer predisposition variants. » Journal of Clinical Oncology 38, n^o 15_suppl (20 mai 2020) : 1581. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.1581.

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1581 Background: Next-generation sequencing (NGS) increasingly guides clinical care in hematological malignancies by identifying DNA mutations that change dynamically over time. Clinical samples contain variable numbers of malignant and non-malignant cells. So, careful interpretation is required to determine if a particular variant is somatic, germline, or clonal hematopoietic in origin. Methods: The University of Chicago uses a targeted NGS assay of ~1200 genes, reporting 150 as a clinical test. We aimed to identify individuals with hereditary predisposition by detecting persistent variants on sequential assays regardless of disease state. Results: 943 NGS assays from July 2017 – Feb. 2020 on 711 patients [ages 1 mo – 95 yrs, median 65 yrs] were included. 2,320 variants in 33 genes were identified with 144 patients having the same variant identified on more than one assay. Single nucleotide variants (SNVs) with variant allele frequency (VAF) ≥ 0.3 were prioritized. The first candidate gene identified with potential germline SNVs was CSF3R. 28 unique SNVs in CSF3R were found, 14 were confirmed as germline, 6 somatic, and 8 were unconfirmed due to lack of available tissue. At least 2 confirmed germline CSF3R variants were likely deleterious based on functional testing. Sequential SNVs were quantified using the coefficient of variation, characterizing each by change in VAF over time. Using a worst-case-scenario analysis, in which unconfirmed variants were not counted as germline, a computer algorithm was designed to identify potential germline variants (specificity 0.89, PPV 0.75). Via an iterative method, the algorithm compares new assays to a pool of previously reported tests, flagging patients with potential germline mutations so that biopsies may be studied in the lab, records reviewed, and referrals placed to genetic counselors. To date, 61 patients with 89 likely germline variants have been identified. Known hereditary hematological malignancy genes, such as ATM, ASXL1, CHEK2, DDX41, TSC1, and RUNX1, had the most variants identified. Limitations include the challenge in distinguishing variants that do not change over time, reliance on a targeted NGS panel, and normalizing VAF data prior to analysis. Conclusions: These data highlight the utility of NGS of bone marrow and peripheral blood samples to identify patients suspected of having germline DNA variants. In addition to identifying known predisposition syndromes, one may discover new inherited cancer syndromes and help guide clinical practice in real time.

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Leonardi, Emanuela, Mariagrazia Bellini, Maria C. Aspromonte, Roberta Polli, Anna Mercante, Claudia Ciaccio, Elisa Granocchio et al. « A Novel WAC Loss of Function Mutation in an Individual Presenting with Encephalopathy Related to Status Epilepticus during Sleep (ESES) ». Genes 11, n^o 3 (24 mars 2020) : 344. http://dx.doi.org/10.3390/genes11030344.

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WAC (WW Domain Containing Adaptor With Coiled-Coil) mutations have been reported in only 20 individuals presenting a neurodevelopmental disorder characterized by intellectual disability, neonatal hypotonia, behavioral problems, and mildly dysmorphic features. Using targeted deep sequencing, we screened a cohort of 630 individuals with variable degrees of intellectual disability and identified five WAC rare variants: two variants were inherited from healthy parents; two previously reported de novo mutations, c.1661_1664del (p.Ser554*) and c.374C>A (p.Ser125*); and a novel c.381+2T>C variant causing the skipping of exon 4 of the gene, inherited from a reportedly asymptomatic father with somatic mosaicism. A phenotypic evaluation of this individual evidenced areas of cognitive and behavioral deficits. The patient carrying the novel splicing mutation had a clinical history of encephalopathy related to status epilepticus during slow sleep (ESES), recently reported in another WAC individual. This first report of a WAC somatic mosaic remarks the contribution of mosaicism in the etiology of neurodevelopmental and neuropsychiatric disorders. We summarized the clinical data of reported individuals with WAC pathogenic mutations, which together with our findings, allowed for the expansion of the phenotypic spectrum of WAC-related disorders.

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Correa, Ricardo, Mihail Zilbermint, Annabel Berthon, Stephanie Espiard, Maria Batsis, Georgios Z. Papadakis, Paraskevi Xekouki et al. « The ARMC5 gene shows extensive genetic variance in primary macronodular adrenocortical hyperplasia ». European Journal of Endocrinology 173, n^o 4 (octobre 2015) : 435–40. http://dx.doi.org/10.1530/eje-15-0205.

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ObjectivePrimary macronodular adrenal hyperplasia (PMAH) is a rare type of Cushing's syndrome (CS) that results in increased cortisol production and bilateral enlargement of the adrenal glands. Recent work showed that the disease may be caused by germline and somatic mutations in the ARMC5 gene, a likely tumor suppressor gene (TSG). We investigated 20 different adrenal nodules from one patient with PMAH for ARMC5 somatic sequence changes.DesignAll of the nodules were obtained from a single patient who underwent bilateral adrenalectomy. DNA was extracted by standard protocol and the ARMC5 sequence was determined by the Sanger method.ResultsSixteen of 20 adrenocortical nodules harbored, in addition to what appeared to be the germline mutation, a second somatic variant. The p.Trp476* sequence change was present in all 20 nodules, as well as in normal tissue from the adrenal capsule, identifying it as the germline defect; each of the 16 other variants were found in different nodules: six were frame shift, four were missense, three were nonsense, and one was a splice site variation. Allelic losses were confirmed in two of the nodules.ConclusionThis is the most genetic variance of the ARMC5 gene ever described in a single patient with PMAH: each of 16 adrenocortical nodules had a second new, ‘private,’ and – in most cases – completely inactivating ARMC5 defect, in addition to the germline mutation. The data support the notion that ARMC5 is a TSG that needs a second, somatic hit, to mediate tumorigenesis leading to polyclonal nodularity; however, the driver of this extensive genetic variance of the second ARMC5 allele in adrenocortical tissue in the context of a germline defect and PMAH remains a mystery.

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Carlston, Colleen M., Anne H. O'Donnell-Luria, Hunter R. Underhill, Beryl B. Cummings, Ben Weisburd, Eric V. Minikel, Daniel P. Birnbaum, Tatiana Tvrdik, Daniel G. MacArthur et Rong Mao. « Pathogenic ASXL1 somatic variants in reference databases complicate germline variant interpretation for Bohring-Opitz Syndrome ». Human Mutation 38, n^o 5 (21 mars 2017) : 517–23. http://dx.doi.org/10.1002/humu.23203.

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Simms, L. J., J. J. Prisciandaro, R. F. Krueger et D. P. Goldberg. « The structure of depression, anxiety and somatic symptoms in primary care ». Psychological Medicine 42, n^o 1 (20 juin 2011) : 15–28. http://dx.doi.org/10.1017/s0033291711000985.

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BackgroundObserved co-morbidity among the mood and anxiety disorders has led to the development of increasingly sophisticated dimensional models to represent the common and unique features of these disorders. Patients often present to primary care settings with a complex mixture of anxiety, depression and somatic symptoms. However, relatively little is known about how somatic symptoms fit into existing dimensional models.MethodWe examined the structure of 91 anxiety, depression and somatic symptoms in a sample of 5433 primary care patients drawn from 14 countries. One-, two- and three-factor lower-order models were considered; higher-order and hierarchical variants were studied for the best-fitting lower-order model.ResultsA hierarchical, bifactor model with all symptoms loading simultaneously on a general factor, along with one of three specific anxiety, depression and somatic factors, was the best-fitting model. The general factor accounted for the bulk of symptom variance and was associated with psychosocial dysfunction. Specific depression and somatic symptom factors accounted for meaningful incremental variance in diagnosis and dysfunction, whereas anxiety variance was associated primarily with the general factor.ConclusionsThe results (a) are consistent with previous studies showing the presence and importance of a broad internalizing or distress factor linking diverse emotional disorders, and (b) extend the bounds of internalizing to include somatic complaints with non-physical etiologies.

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MacFarland, Suzanne P., Kristin Zelley, Lea F. Surrey, Daniel Gallo, Minjie Luo, Pichai Raman, Gerald Wertheim, Stephen P. Hunger, Marilyn M. Li et Garrett M. Brodeur. « Pediatric Somatic Tumor Sequencing Identifies Underlying Cancer Predisposition ». JCO Precision Oncology, n^o 3 (décembre 2019) : 1–26. http://dx.doi.org/10.1200/po.19.00062.

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PURPOSE The diagnosis of cancer predisposition in pediatric patients with cancer is vital for treatment decisions, surveillance, and management of at-risk family members. Somatic tumor testing can identify potential underlying constitutional variants that confer increased cancer risk. Here, we report the characteristics of constitutional variants identified through tumor testing. MATERIALS AND METHODS Data were abstracted from medical record review of 1,023 patients who received in-house somatic tumor testing over a 28-month period. Patients were identified for testing using referral criteria developed as a collaboration between genomic diagnostics, pathology, and oncology. Characteristics of patients who underwent constitutional testing, including family history and variant loss of heterozygosity, were tracked. RESULTS From 1,023 patients who underwent somatic tumor sequencing in a 28-month period, 210 variants were identified in 141 patients (13.8%) that were concerning for cancer predisposition syndromes requiring intervention. A total of 73 variants in 41 patients have undergone clinical confirmatory testing thus far. Of these, 26 variants were confirmed to be constitutionally present (35.6%). Among patients tested, 23 (56.1%) of 41 total patients were diagnosed with a cancer predisposition syndrome. CONCLUSION Our data demonstrate that more than one third of variants in tumor somatic sequencing that were concerning for underlying cancer predisposition were constitutionally confirmed. Overall, somatic tumor testing identified potential cancer predisposition syndromes in pediatric patients, and some would not have been identified on the basis of clinical history alone.

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McCoy, Matthew, Shruti Rao, Shannon Cosgrove, Subha Madhavan, Shashikant Kulkarni, Xinjie Xu et Rashmi Kanagal-Shamanna. « Expert Variant Curation Combined with in-Silico analysis for Clinical Interpretation of BCL2 variants in Resistance to BCL2 Inhibitors in Chronic Lymphocytic Leukemia/ Small Lymphocytic Lymphoma ». Blood 136, Supplement 1 (5 novembre 2020) : 42–43. http://dx.doi.org/10.1182/blood-2020-143073.

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Venetoclax is an oral, highly selective, BCL2 inhibitor approved by the FDA for use in chronic lymphocytic leukemia/small lymphocytic lymphoma and acute myeloid leukemia. Despite favorable responses, multiple biological mechanisms lead to treatment resistance. One such mechanism includes somatic mutations in the BCL2 gene. Multiple lines of evidence suggest that hot-spot mutations in BCL2 such as Gly101Val induce treatment resistance by disrupting the binding of BCL2 to the BCL2 inhibitors such as venetoclax. Further, widespread use of high-throughput NGS technologies has identified multiple BCL2 mutations and additional concurrent molecular alterations at various variant allele frequencies in patients with progression while undergoing venetoclax therapy. In order to understand and determine the clinical significance of each of these mutations, careful expert curation and integration into somatic variant annotation AMP/ASCO/CAP guidelines is needed. Further, curation of those somatic variants that may not have sufficient functional evidence in literature may benefit from additional tools such as in silico analysis. To address these issues, we have undertaken an effort to integrate the contributions of a multidisciplinary expert panel (clinical laboratory diagnosticians, oncologists, biomedical informaticians and lab-based researchers) for curation of BCL2 variants in hematological malignancies under the umbrella of ClinGen, an NIH/NHGRI funded consortium to establish standards and centralized resources for assessing the clinical significance of gene variants. Within the ClinGen Somatic Cancer Clinical Domain Working Group (CDWG) ((https://www.clinicalgenome.org/working-groups/somatic/), the somatic hematological malignancy taskforce has identified 56 peer-reviewed publications on BCL2 inhibitors (Jan 2014 to June 2020). The functional evidence contained within these publications was curated using CIViC (Clinical Interpretation of Variants in Cancer, civicdb.org), an open access, crowdsourced aggregation of expert curated evidence. Only a fraction of the somatic variants identified in BCL2 has established functional evidence on variant induced disruption to Venetoclax inhibition. Curation of these remaining variants of unknown significance (VUS) only have in silico functional assays to provide evidence on their potential resistance to Venetoclax. The current curation guidelines do not consider in silico prediction as a strong line of evidence for the interpretation of somatic sequence variants, however this recommendation is meant to interpret generalized in silico predictors and not robust computational models of specific protein function. The latter are more comparable to an experimental functional assay, and provide curators with more trustworthy computational assessments of disruption to protein specific functions. In order to assess their potential to integrate and supplement experimental evidence, the interaction of Ventoclax with several drug resistant BCL2 variants was simulated using AutoDock Vina (J Comput Chem. 2010;31(2):455-61). Facilitated by the SNP2SIM workflow (BMC Bioinformatics. 2019;20(1):171), the relative impact on binding energy was compared to the wildtype system. The in silico binding assay accurately predicted resistance (Fig 1), and demonstrates the utility of applying these methods to the large number of VUS in BCL2. In conclusion, the evidence-based expert curation of BCL2 variants provides a standardized approach for reporting and interpretation across all labs. For those variants (Tier 3) with limited published evidence, computational models that can predict specific changes to functional protein interactions can provide additional tools to the expert curators. Development and incorporation of these tools into curation guidelines requires the refinement of the predictive models through focused validation studies. Disclosures No relevant conflicts of interest to declare.

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Weerts, Marjolein, Marcel Smid, John Foekens, Stefan Sleijfer et John Martens. « Mitochondrial RNA Expression and Single Nucleotide Variants in Association with Clinical Parameters in Primary Breast Cancers ». Cancers 10, n^o 12 (9 décembre 2018) : 500. http://dx.doi.org/10.3390/cancers10120500.

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The human mitochondrial DNA (mtDNA) encodes 37 genes, including thirteen proteins essential for the respiratory chain, and RNAs functioning in the mitochondrial translation apparatus. The total number of mtDNA molecules per cell (mtDNA content) is variable between tissue types and also between tumors and their normal counterparts. For breast cancer, tumors tend to be depleted in their mtDNA content compared to adjacent normal mammary tissue. Various studies have shown that primary breast tumors harbor somatic mtDNA variants. A decrease in mtDNA content or the presence of somatic variants could indicate a reduced mitochondrial function within breast cancer. In this explorative study we aimed to further understand genomic changes and expression of the mitochondrial genome within breast cancer, by analyzing RNA sequencing data of primary breast tumor specimens of 344 cases. We demonstrate that somatic variants detected at the mtRNA level are representative for somatic variants in the mtDNA. Also, the number of somatic variants within the mitochondrial transcriptome is not associated with mutational processes impacting the nuclear genome, but is positively associated with age at diagnosis. Finally, we observe that mitochondrial expression is related to ER status. We conclude that there is a large heterogeneity in somatic mutations of the mitochondrial genome within primary breast tumors, and differences in mitochondrial expression among breast cancer subtypes. The exact impact on metabolic differences and clinical relevance deserves further study.

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Singhal, Deepak, Christopher N. Hahn, Luke D. Moma, Li Yan A. Wee, Rakchha Chhetri, Milena Babic, Monika M. Kutyna et al. « Deleterious Germline Variants, Especially in the DNA Repair Pathway, Are Common in Patients with Non-Related Multiple Cancers, One of Them Being Hematological Malignancy ». Blood 134, Supplement_1 (13 novembre 2019) : 1704. http://dx.doi.org/10.1182/blood-2019-126462.

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Approximately 8% of un-selected pediatric (Zhang et al, NEJM 2015) and adult (Huang et al, Cell 2018) cancer patients have a deleterious germline (GL) variant. However, the frequency in patients with >2 non-related cancers, a combination of different hematological malignancies (HM) or a HM and a non-hematological malignancy, is not known. We hypothesize that genetic predisposition is higher in patients with multiple cancers, compared to historical literature for patients with a single cancer. Method: Clinical and laboratory information on 213 cancer patients, enrolled in the South Australian MDS (SA-MDS) registry (n=90) and University of Chicago clinic (n=123) were analyzed. Germline variants were identified by sequencing paired bone marrow and germline samples for 217-300 cancer related genes and annotated using American College of Medical Genetics (ACMG) 2015 guidelines as pathogenic or likely pathogenic (i.e. deleterious). Additionally, somatic mutations in 240 genes were identified in the SA-MDS cohort (n=90). Results: The median age at diagnosis was 64 years (interquartile range, 61-76 years). One-hundred and five patients had therapy-related myeloid neoplasm (T-MN), following treatment with chemo- and/or radiotherapy for an unrelated prior malignancy. While 100 patients had non-related multiple cancers (MC) one of them being HM without prior exposure to chemotherapy or radiotherapy, and, 8 patients had only myelodysplastic syndrome or acute myeloid leukemia. Overall, 20.5% (42/205) patients harbored 46 deleterious GL variants, annotated according to ACMG guidelines (Fig 1A). Most GL variants were in DNA repair pathway (26/46, 57%) followed by TP53, telomere maintenance and drug transport pathways (4/46, 9% each; Fig 1B). Three DDX41 variants were also seen. The frequency of patients with deleterious GL variants was similar in T-MN (21/105; 20%) and MC (21/100, 21%; Fig 1C). Similarly, the frequency of patients with deleterious GL variants in DNA repair pathways was also similar in T-MN (13/105, 12%) and MC (11/100, 11%). Of the patients with available cytogenetic data, complex cytogenetics were more frequent in patients with a deleterious GL variant compared to those without (11/28, 39% vs 28/125, 22%; p=0.06). At the time of analysis, somatic and germline mutation data on 240 genes was available in the SA-MDS cohort only (n=90). Somatic TP53 mutations were frequent in patients with deleterious GL variant (8/15, 53%) compared to patients without deleterious GL variant (11/75, 15%; p=0.003) (Fig 1D). Interestingly, 80% (4/5) patients with deleterious GL variant in a DNA repair pathway had a somatic TP53 mutation compared to 40% (4/10) with deleterious GL variant in non-DNA repair pathways. Conclusions: Our results from a large cohort show that patients with >2 non-related cancers have a higher frequency of deleterious germline variants compared to previous reports from patients with single cancer (20.5% vs 8%). Variants in the DNA repair pathway are the most common. The presence of deleterious germline variants is associated with genetic instability as evidenced by high frequency of complex karyotypes and somatic TP53 mutations. Disclosures Scott: Celgene: Honoraria. Godley:UpToDate, Inc.: Patents & Royalties: receives royalties from a coauthored article on inherited hematopoietic malignancies ; Invitae, Inc.: Membership on an entity's Board of Directors or advisory committees.

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Murley, Alexander G., Yu Nie, Zoe Golder, Michael John Keogh, Colin Smith, James W. Ironside et Patrick F. Chinnery. « High-Depth PRNP Sequencing in Brains With Sporadic Creutzfeldt-Jakob Disease ». Neurology Genetics 9, n^o 1 (19 janvier 2023) : e200054. http://dx.doi.org/10.1212/nxg.0000000000200054.

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Background and ObjectivesSporadic Creutzfeldt-Jakob disease (sCJD) has established genetic risk factors, but, in contrast to genetic and acquired CJD, the initial trigger for misfolded prion aggregation and spread is not known. In this study, we tested the hypotheses that pathologic somatic variants in the prion genePRNPare increased in sCJD, potentially leading to the seeding of misfolded prion protein.MethodsHigh-depth amplicon-based short read sequencing of thePRNPcoding region was performed on postmortem brain tissue from patients with a clinical and neuropathologic diagnosis of sCJD (n = 142), Alzheimer disease (AD) (n = 51) and controls with no clinical or neuropathologic diagnosis of a neurodegenerative disease (n = 71). Each DNA sample was sequenced twice, including independent PCR amplification, library preparation, and sequencing. We used RePlow to call somatic variants with high sensitivity and specificity and optimal sequence kernel association test to compare variant burden between groups.ResultsTwo sCJD cases had somatic (variant allele frequency 0.5–1%)PRNPvariants not previously identified, but with high in silico predicated pathogenicity. However, the pathogenicity of these variants is uncertain, as both located in the octapeptide repeat region where no point variations have previously been associated with sCJD. There was no overall difference in burden somaticPRNPin sCJD compared with controls and a lower burden compared with Alzheimer disease.DiscussionSomatic variants inPRNPare unlikely to play a major role in sCJD but may contribute to the disease mechanism in a minority of cases.

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Ricker, Charité, Erika Amundson, Sandra Algaze, Marcia Ciccone, Stephen Dong, Anishka D'souza, Kimberly Felicetti et al. « Assessing somatic and germline variants in cancer patients. » Journal of Clinical Oncology 39, n^o 15_suppl (20 mai 2021) : 10601. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.10601.

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10601 Background: The increasing integration of somatic and germline testing into oncology practice allows physicians to target oncologic therapy and identify those with cancer predisposition. We explored the impact of a somatic assay (liquid biopsy, LB) on the identification of patients appropriate for germline genetic testing. Methods: We identified a cohort of diverse cancer patients with LB to assess for targetable somatic gene variants at LAC+USC Medical Center between 2016 and 2020 (n= 467). To enrich the cohort for variants that may reflect germline findings, we focused on the 46 patients (9.9%) who had at least one variant identified with a cell-free DNA (cfDNA) fraction of 25.00% or greater. Retrospective chart review extracted demographics and medical history with variables related to cancer history and treatment. LB variants were classified based on whether germline confirmation was indicated and the results of germline tests, when done, were reviewed. Results: Table summarizes the characteristics of the 46 patients identified to have at least one variant on LB in ≤ 25% of the cfDNA. The most frequently mutated genes on LB were TP53 (n=18, 39%), KRAS (n=11, 24%), APC (n=8, 17%), BRCA2 (n=7, 15%), PIK3CA (n=6, 13.0%), and BRCA1 (n=4, 9%). Seventeen patients (40%) were referred for genetic counseling and 13 (30%) underwent germline testing of whom 10 (77%) carried pathogenic variants (PV). All germline PV were concordant with LB variants identified. Four patients with PV BRCA2 on LB and confirmed to be germline, had lung or biliary tract diagnoses, which are not part of the typical BRCA-tumor spectrum. Thirty-three patients were not referred for genetic counseling, though 24 (72%) had LB-identified variants in cancer predisposition genes and 18 (54%) merited a genetics referral. Among patients with germline mutations, three (23%) had targeted therapy and two (15%) had preventive surgery to address second primary cancer risk. Among the 467 patients with LB results, there were an additional 13 patients (not included in the enriched group) known to have a cancer predisposition gene PV. Only two (15%) had findings on LB reports that suggested germline testing would be indicated. Conclusions: While the purpose of somatic testing is to identify targeted therapy, it can provide germline insight, especially for patients not typically referred for genetic assessment. Further education and guidance is needed to assure that this aspect of somatic testing is appreciated by oncology providers and acted upon.[Table: see text]

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