Rare among Rare: Phenotypes of Uncommon CMT Genotypes

(1) Background: Charcot–Marie–Tooth disease (CMT) is the most frequent form of inherited chronic motor and sensory polyneuropathy. Over 100 CMT causative genes have been identified. Previous reports found PMP22, GJB1, MPZ, and MFN2 as the most frequently involved genes. Other genes, such as BSCL2, MORC2, HINT1, LITAF, GARS, and autosomal dominant GDAP1 are responsible for only a minority of CMT cases. (2) Methods: we present here our records of CMT patients harboring a mutation in one of these rare genes (BSCL2, MORC2, HINT1, LITAF, GARS, autosomal dominant GDAP1). We studied 17 patients from 8 unrelated families. All subjects underwent neurologic evaluation and genetic testing by next-generation sequencing on an Ion Torrent PGM (Thermo Fischer) with a 44-gene custom panel. (3) Results: the following variants were found: BSCL2 c.263A > G p.Asn88Ser (eight subjects), MORC2 c.1503A > T p.Gln501His (one subject), HINT1 c.110G > C p.Arg37Pro (one subject), LITAF c.404C > G p.Pro135Arg (two subjects), GARS c.1660G > A p.Asp554Asn (three subjects), GDAP1 c.374G > A p.Arg125Gln (two subjects). (4) Expanding the spectrum of CMT phenotypes is of high relevance, especially for less common variants that have a higher risk of remaining undiagnosed. The necessity of reaching a genetic definition for most patients is great, potentially making them eligible for future experimentations.

CD8+ cell somatic mutations in multiple sclerosis patients and controls—Enrichment of mutations in STAT3 and other genes implicated in hematological malignancies

Somatic mutations have a central role in cancer but their role in other diseases such as common autoimmune disorders is not clear. Previously we and others have demonstrated that especially CD8+ T cells in blood can harbor persistent somatic mutations in some patients with multiple sclerosis (MS) and rheumatoid arthritis. Here we concentrated on CD8+ cells in more detail and tested (i) how commonly somatic mutations are detectable, (ii) does the overall mutation load differ between MS patients and controls, and (iii) do the somatic mutations accumulate non-randomly in certain genes? We separated peripheral blood CD8+ cells from newly diagnosed relapsing MS patients (n = 21) as well as matched controls (n = 21) and performed next-generation sequencing of the CD8+ cells’ DNA, limiting our search to a custom panel of 2524 immunity and cancer related genes, which enabled us to obtain a median sequencing depth of over 2000x. We discovered nonsynonymous somatic mutations in all MS patients’ and controls’ CD8+ cell DNA samples, with no significant difference in number between the groups (p = 0.60), at a median allelic fraction of 0.5% (range 0.2–8.6%). The mutations showed statistically significant clustering especially to the STAT3 gene, and also enrichment to the SMARCA2, DNMT3A, SOCS1 and PPP3CA genes. Known activating STAT3 mutations were found both in MS patients and controls and overall 1/5 of the mutations were previously described cancer mutations. The detected clustering suggests a selection advantage of the mutated CD8+ clones and calls for further research on possible phenotypic effects.

Differential Gene Expression in Bladder Tumors from Workers Occupationally Exposed to Arylamines

Occupational exposure to the arylamines benzidine and β-naphthylamine increase bladder cancer risk up to 100-fold, making them some of the most powerful human carcinogens. We hypothesize that tumors arising in people with occupational exposures have different patterns of gene expression than histologically similar tumors from people without such exposures. In a case-case study, we compare gene expression in 22 formalin-fixed paraffin-embedded (FFPE) bladder tumors from men with high-level occupational exposure to arylamines to that in 26 FFPE bladder tumors from men without such exposure. Gene expression analysis was performed on the NanoString nCounter system using a PanCancer Progression Panel comprised of 740 cancer progression-related genes and a custom panel of 69 arylamine- and bladder cancer-related genes which were chosen from in vitro studies. Although fold differences were small, there was evidence of differential expression by exposure status for 17 genes from the Progression Panel and 4 genes from the custom panel. In total, 10 genes showed dose-response association at a p < 0.01 , of which 4 genes (CD46, NR4A1, BAX, and YWHAZ) passed a false discovery rate (FDR) q value cutoff of 0.05 but were not significant after Bonferroni correction. Overall, we find limited evidence for differentially expressed genes in pathways related to DNA damage signaling and epithelial-to-mesenchymal transition (EMT).

Multiple TET2 Mutations As a New Biological Clue for Differentiating Oligomonocytic Chronic Myelomonocytic Leukemia from Myelodysplastic Syndromes

Introduction. Oligomonocytic chronic myelomonocytic leukemia (OM-CMML) is defined as those myelodysplastic syndromes (MDS) or MDS/MPN unclassifiable cases with relative monocytosis (≥ 10% monocytes) and a total monocyte count of 0.5-&lt;1x10 9/L. These patients show similar clinical, genomic and immunophenotypic features than overt CMML, although the majority of them are currently classified as MDS following WHO 2017 classification. TET2 is the most prevalent mutated gene in CMML and it is frequent to find multiple TET2 mutations in the same patient. Objective. We investigated TET2 mutations in 42 OM-CMML, their diagnostic value and their correlation with immunophenotypic pattern, and compared them with that observed in 54 CMML and 86 MDS. Patients and methods. Samples were collected from 182 patients: 42 OM-CMML, 54 overt CMML, and 86 MDS. Molecular characterization was performed by next-generation sequencing (NGS) using a custom panel (QIAseq Custom DNA Panels, Qiagen) including the whole codifying region of 25 myeloid-associated genes and sequenced using Illumina technology. Multiparametric flow cytometry (FC) analysis of monocyte subsets was performed on whole PB collected on EDTA. Cell surface staining of 2 × 10 6 cells was performed and at least 500 000 total events were acquired per tube (FACS Canto II; BD Biosciences). The FC strategy analysis, the 5-tube experimental panel, and the NGS custom panel are described in Calvo & Garcia-Gisbert, Blood Adv 2020. Results. Patients with multiple TET2 (multi-TET2) mutations (2 or more) were identified with similar percentages in OM-CMML (17/42, 40.5%) and CMML (29/54, 53.7%) while they were infrequent in MDS (5/86, 5.8%) (P&lt;0.001). In MDS patients, 4/5 with multi-TET2 showed a percentage of monocytes &gt;10% though they were not considered OM-CMML since the monocyte count was below 500/microL. CMML and OM-CMML patients with multi-TET2 showed predominantly 2 TET2 mutations (CMML: 20/29, 69%; OM-CMML: 14/17, 82%;). The distribution of patients with &gt;2 TET2 mutations was similar in CMML and OM-CMML. We did not observe differences among the frequency of nonsense, frameshift or missense TET2 mutations, nor between early or late-truncating (early: aa 1-1128, late: 1128-1936) TET2 mutations between OM-CMML and CMML.TET2 variant allele frequency (VAF) was significantly lower in MDS (median 11.83%) than in OM-CMML and CMML (P&lt;0.001 in both comparisons). OM-CMML and CMML showed similar TET2 VAF (medians 41.4% and 40.3%). In those patients with TET2 VAF &gt;55% or a sum of muti-TET2 mutations VAF above 55% (17 OM, 19 CMML, 2 MDS), we could infer a biallelic alteration in the same clone (Coltro, Leukemia 2020). We did not observe differences in the frequency of OM-CMML showing biallelic TET2 mutations when compared to CMML.The main clinical characteristics of CMML patients (overt and OM-CMML), grouped by TET2 mutational status (unmutated: TET2 WT; 1 TET mutation: TET2 single; ≥2 mutations: multi-TET2) are depicted in Table 1. As shown, we did not observed significant differences when comparing TET2 single to TET2 WT or multi-TET2 Of note, multi-TET2 patients showed a higher percentage of PB and BM monocytes, BM promonocytes and more dysgranulopoiesis than TET2 WT patients.Patients with TET2 WT presented a lower number of mutated genes than TET single and multi-TET2 patients (P=0.019 and P=0.029). In line with previous studies, IDH mutations and TET2 mutations where mutually exclusive (P=0.001). Co-mutation of SRSF2 and TET2, the well-accepted gene signature of CMML, was found in 25/96 patients (26.0%, 13 OM-CMML, 12 CMML) in 10 cases co-mutated with TET2 single and in 15 cases with multi-TET2.We assessed the proportion of OM-CMML patients with MO1&gt;94% since this has been shown as very sensitive and specific feature of CMML. In TET2 mutated OM-CMML, we observed a higher proportion of patients with MO1&gt;94% (90% mut. vs 40% WT; P=0.001) and CD56 expression on monocytes (70% mut. vs 30% WT; P=0.025). We observed a trend (non-significant) difference in the proportion of patients showing these features when comparing TET2 single vs multi-TET2 OM-CMML (single, CD56: 57.1%, MO1&gt;94%: 85.7%; multi-TET2, CD56: 81.3%, MO1&gt;94%: 93.8%). Conclusion. The high occurrence of multiple TET2 mutations in overt and OM-CMML and its infrequency in MDS is a new biological clue for supporting the consideration of OM-CMML in the continuum of CMML. Figure 1 Figure 1. Disclosures Salar: Celgene: Consultancy, Speakers Bureau; Gilead: Research Funding; Roche: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau. Bellosillo: Roche: Research Funding, Speakers Bureau; Qiagen: Consultancy, Speakers Bureau; Thermofisher Scientific: Consultancy, Speakers Bureau.

Single Cell Mutation Analysis Delineates Clonal Architecture in Leukemic Transformation of Myeloproliferative Neoplasms

Introduction. Myeloproliferative neoplasms (MPN) are clonal disorders of hematopoietic stem cells that include polycythemia vera, essential thrombocythemia, and primary myelofibrosis. 10-20% of MPN pts transform to secondary acute myeloid leukemia (sAML), unresponsive to conventional therapy and associated with dismal outcome (Dunbar A, 2020). In addition to somatic driver mutations affecting JAK2, CALR or MPL, several additional variants are harbored by MPN pts inb chronic phase, and a restricted set of them were associated with risk of leukemic evolution (Vannucchi AM, Leukemia 2013; Tefferi A, Blood ASdv 2016) However, the molecular mechanisms underlying leukemic transformation remain largely unknown. Although bulk next generation sequencing (NGS) highlights the overall mutation landscape, it cannot distinguish which mutations occur in the same clone(s), nor elucidate the order of mutations or resolve clonal complexity. Conversely, single-cell sequencing (SCS) might allow to resolve clonal heterogeneity and reconstruct clonal phylogenies at each disease phase (Parenti, NPJ Prec Onc 2021)). Aim: To delineate the clonal landscape of sAML, we performed single-cell mutational profiling in 10 pts with MPNs who progressed to sAML. Methods. There were 2 set of samples/approaches: (i) 15 paired samples (chronic (CP)/blast phase (BP)) from 7 pts were analyzed using the Mission-Bio Tapestri platform and the Myeloid panel in order to target SNVs and indels across 45 myeloid genes with 312 amplicons. In one pt we also analyzed an intermediate phase corresponding to progression from PV to PPV-MF before BP development. (ii) 7 further paired CD34+ samples from 3 pts were analyzed using a 239-amplicon custom panel including 29 genes frequently mutated in myeloid neoplasms. SCS libraries were sequenced on Illumina Novaseq. Data were processed by using Mission Bio's Tapestri Pipeline and analyzed with Mission Bio's Tapestri Insights software package and R software. CNV analysis was performed by using an integrated pipeline for multiomics analysis (Mosaic, Mission Bio) Results. (i) A total of 78,354 single cells were sequenced (average 5,223) using Tapestri Myeloid panel, with an average of 28,303 reads per cell and coverage of 97X. SCS was able to identify 17 low-frequency variants not detected in bulk analysis; however, it failed to discriminate homopolymeric regions including the ASXL1 G646Wfs*12. (ii) A total of 25,417 single cells were sequenced (average 3636) using a custom panel, with a coverage of 186X and an average Allele Dropout Rate of 8.6%. This panel was able to identify ASXL1 G646Wfs*12 variant. Overall, we found a significant correlation of variant allele frequency (VAF) measured by bulk and SCs approach (R =0.84, p&lt;.0001). Epigenetic variants (i.e. ASXL1, TET2, EZH2) account for around half of the mutations and affect a large fraction of CP cells (3 representative samples in Fig.1). In 8/10 pts, leukemic clone emerged from a driver mutation-positive cells (JAK2V617F n=4; CALR Type1 n=4). In all pts we are able to identify at least 3 mutated clones and in 7 pts the dynamics of the clones allowed to identify the one(s) responsible for evolution to sAML. In 7/10 pts, the leukemic clones were already detectable at low frequency (&lt;2%) at CP and became dominant in BP; these low-frequency clones were missed by bulk sequencing. Furthermore, SCS revealed acquisition of 3 mutually exclusive mutations in RAS pathway in one pt: two NRAS mutations and a KRAS mutation. Copy number variation (CNV) could be assessed in 4 pts. Of note, during progression to sAML, we found single cells with amplification of ETV6 (&gt;20 copies in 2 pts), NRAS (8 copies in 2pts) and BRAF (8 copies in 2pts). Other subclonal ploidy abnormalities were also observed in RUNX1, EZH2, U2AF1 and ZRSR2 (5-18 copies). Conclusions. Together, these data suggest that MPN present a complex clonal combination evolving over time. SCS allows to resolve this milieu, that is largely missed by conventional bulk NGS, in particularly SCS identifies rare leukemia-driving clones that were already present in chronic phase and describes their dynamics during leukemic progression. Leukemic transformation after MPN is a highly heterogeneous process with mutations and CNVs acquired in different genes and different clones. Overall, our findings provide further insights into the pathogenesis of AML transformation of MPN. Supported by AIRC, Mynerva project no.21267 Figure 1 Figure 1. Disclosures Vannucchi: BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Germline Predisposition to Pediatric Cancer, from Next Generation Sequencing to Medical Care

Knowledge about genetic predisposition to pediatric cancer is constantly expanding. The categorization and clinical management of the best-known syndromes has been refined over the years. Meanwhile, new genes for pediatric cancer susceptibility are discovered every year. Our current work shares the results of genetically studying the germline of 170 pediatric patients diagnosed with cancer. Patients were prospectively recruited and studied using a custom panel, OncoNano V2. The well-categorized predisposing syndromes incidence was 9.4%. Likely pathogenic variants for predisposition to the patient’s tumor were identified in an additional 5.9% of cases. Additionally, a high number of pathogenic variants associated with recessive diseases was detected, which required family genetic counseling as well. The clinical utility of the Jongmans MC tool was evaluated, showing a high sensitivity for detecting the best-known predisposing syndromes. Our study confirms that the Jongmans MC tool is appropriate for a rapid assessment of patients; however, the updated version of Ripperger T criteria would be more accurate. Meaningfully, based on our findings, up to 9.4% of patients would present genetic alterations predisposing to cancer. Notably, up to 20% of all patients carry germline pathogenic or likely pathogenic variants in genes related to cancer and, thereby, they also require expert genetic counseling. The most important consideration is that the detection rate of genetic causality outside Jongmans MC et al. criteria was very low.

Peripheral T-cell lymphoma: Molecular profiling recognizes subclasses and identifies prognostic markers

Peripheral T-cell lymphoma (PTCL) is a clinically aggressive disease, with a poor response to therapy and a low overall survival rate of around 30% after 5 years. We have analyzed a series of 105 cases with a diagnosis of PTCL using a customized NanoString platform that includes 208 genes associated with T-cell differentiation, oncogenes and tumor suppressor genes, deregulated pathways and stromal cell subpopulations. A comparative analysis of the various histological types of PTCL (angioimmunoblastic T-cell lymphoma, AITL; PTCL-with T follicular helper phenotype, PTCL-TFH; PTCL-not otherwise signified, PTCL-NOS) showed that specific sets of genes were associated with each of the diagnoses. These included TFH markers, cytotoxic markers and genes whose expression was a surrogate for specific cellular subpopulations, including follicular dendritic cells, mast cells and genes belonging to precise survival (NF-κB) and other pathways. Furthermore, the mutational profile was analyzed using a custom panel that targeted 62 genes in 76 cases distributed in AITL, PTCL-TFH and PTCL-NOS. The main differences between the three nodal PTCL classes involved the RHOAG17V mutations (p&lt;0.0001), which were approximately twice as frequent in AITL (34.09%) as in PTCL-TFH (16.66%) cases, but were not detected in PTCL-NOS. A multivariate analysis identified gene sets that allowed the series of cases to be stratified into different risk groups. This study supports and validates the current division of PTCL into these three categories, identifies sets of markers that can be used for a more precise diagnosis, and recognizes the expression of B-cell genes as an IPI-independent prognostic factor for AITL.

Diagnostics of hereditary cancer syndromes by NGS. Process of creating a database.

Background:more than 500 thousand new cases of malignant neoplasms are registered annually in the Russian Federation, of which more than 50 thousand new cases are due to hereditary forms.Improving the diagnosis of these diseases will make it possible to detect tumors in the early stages and take timely preventive and therapeutic measures. Aims:creation of a database and development of software for NGS data analysis for the prevention and early diagnosis of hereditary forms of oncological diseases. Methods:the present study used 636 DNA samples obtained from cancer patients with a high hereditary risk or a burdened family history. DNA was isolated from blood lymphocytes. DNA libraries were prepared with a KAPA Target Enrichment Custom Panel (Roche). The panel included probes for targeted enrichment of the coding region of 44 genes. NGS was performed on the MiSeq platform (Illumina). Results:we identified 65 pathogenic/ probably pathogenic nucleotide sequence variants in 96 patients in theATM, BLM, BRCA1, BRCA2, CHEK2, EPCAM, MEN1, MLH1, MSH2, MSH3, MSH6, MUTYH, PALB2, TP53genes. We also identified 2858 nucleotide sequence variants of unknown clinical significance. Conclusions:we have created a local database that contains both genetic variants and clinical and anamnestic data. The database contains 4763 nucleotide sequence variants at the moment, among which 2522 are unique variants identified in a single patient.

Targeted Long-Read Sequencing Reveals Comprehensive Architecture, Burden and Transcriptional Signatures from HBV-Associated Integrations and Translocations in HCC Cell Lines

Hepatitis B virus (HBV) can integrate into the chromosomes of infected hepatocytes, creating potentially oncogenic lesions that can lead to hepatocellular carcinoma (HCC). However, our current understanding of integrated HBV DNA architecture, burden and transcriptional activity is incomplete due to technical limitations. A combination of genomics approaches was used to describe HBV integrations and corresponding transcriptional signatures in three HCC cell lines: huH-1, PLC/PRF/5 and Hep3B. To generate high coverage long-read sequencing data, a custom panel of HBV-targeting biotinylated oligonucleotide probes was designed. Targeted long-read DNA sequencing captured entire HBV integration events within individual reads, revealing that integrations may include deletions and inversions of viral sequences. Surprisingly, all three HCC cell lines contain integrations that are associated with host chromosomal translocations. In addition, targeted long-read RNA sequencing allowed for the assignment of transcriptional activity to specific integrations and resolved the contribution of overlapping HBV transcripts. HBV transcripts chimeric with host sequences were resolved in their entirety and often included >1000bp of host sequence. This study provides the first comprehensive description of HBV integrations and associated transcriptional activity in three commonly utilized HCC-derived cell lines. The application of novel methods sheds new light on the complexity of these integrations, including HBV bidirectional transcription, nested transcripts, silent integrations and host genomic rearrangements. The observation of multiple HBV-associated chromosomal translocations gives rise to the hypothesis that HBV may be a driver of genetic instability and provides a potential new mechanism for HCC development. Importance HCC-derived cell lines have served as practical models to study HBV biology for decades. These cell lines harbor multiple HBV integrations and express only HBV surface antigen (HBsAg). To date, an accurate description of the integration burden, architecture and transcriptional profile of these cell lines has been limited due to technical constraints. We have developed a targeted long-read sequencing assay which reveals the entire architecture of integrations in these cell lines. In addition, we identified five chromosomal translocations with integrated HBV DNA at the inter-chromosomal junctions. Incorporation of long-read RNA-Seq data indicated that many integrations and translocations were transcriptionally silent. The observation of multiple HBV-associated translocations has strong implications regarding the potential mechanisms for the development of HBV-associated HCC.

Updating the Genetic Landscape of Inherited Retinal Dystrophies

Inherited retinal dystrophies (IRD) are a group of diseases characterized by the loss or dysfunction of photoreceptors and a high genetic and clinical heterogeneity. Currently, over 270 genes have been associated with IRD which makes genetic diagnosis very difficult. The recent advent of next generation sequencing has greatly facilitated the diagnostic process, enabling to provide the patients with accurate genetic counseling in some cases. We studied 92 patients who were clinically diagnosed with IRD with two different custom panels. In total, we resolved 53 patients (57.6%); in 12 patients (13%), we found only one mutation in a gene with a known autosomal recessive pattern of inheritance; and 27 patients (29.3%) remained unsolved. We identified 120 pathogenic or likely pathogenic variants; 30 of them were novel. Among the cone-rod dystrophy patients, ABCA4 was the most common mutated gene, meanwhile, USH2A was the most prevalent among the retinitis pigmentosa patients. Interestingly, 10 families carried pathogenic variants in more than one IRD gene, and we identified two deep-intronic variants previously described as pathogenic in ABCA4 and CEP290. In conclusion, the IRD study through custom panel sequencing demonstrates its efficacy for genetic diagnosis, as well as the importance of including deep-intronic regions in their design. This genetic diagnosis will allow patients to make accurate reproductive decisions, enroll in gene-based clinical trials, and benefit from future gene-based treatments.