The complete sequence and comparative analysis of ape sex chromosomes.

Apes possess two sex chromosomes-the male-specific Y chromosome and the X chromosome, which is present in both males and females. The Y chromosome is crucial for male reproduction, with deletions being linked to infertility1. The X chromosome is vital for reproduction and cognition2. Variation in mating patterns and brain function among apes suggests corresponding differences in their sex chromosomes. However, owing to their repetitive nature and incomplete reference assemblies, ape sex chromosomes have been challenging to study. Here, using the methodology developed for the telomere-to-telomere (T2T) human genome, we produced gapless assemblies of the X and Y chromosomes for five great apes (bonobo (Pan paniscus), chimpanzee (Pan troglodytes), western lowland gorilla (Gorilla gorilla gorilla), Bornean orangutan (Pongo pygmaeus) and Sumatran orangutan (Pongo abelii)) and a lesser ape (the siamang gibbon (Symphalangus syndactylus)), and untangled the intricacies of their evolution. Compared with the X chromosomes, the ape Y chromosomes vary greatly in size and have low alignability and high levels of structural rearrangements-owing to the accumulation of lineage-specific ampliconic regions, palindromes, transposable elements and satellites. Many Y chromosome genes expand in multi-copy families and some evolve under purifying selection. Thus, the Y chromosome exhibits dynamic evolution, whereas the X chromosome is more stable. Mapping short-read sequencing data to these assemblies revealed diversity and selection patterns on sex chromosomes of more than 100 individual great apes. These reference assemblies are expected to inform human evolution and conservation genetics of non-human apes, all of which are endangered species.

Real-world performance of integrative clinical genomics in pediatric precision oncology.

Despite significant improvement in the survival of pediatric cancer patients, treatment outcomes for high-risk, relapsed, and refractory cancers remain unsatisfactory. Moreover, prolonged survival is frequently associated with long-term adverse effects due to intensive multimodal treatments. Accelerating the progress of pediatric oncology requires both therapeutic advances and strategies to mitigate the long-term cytotoxic side effects, potentially through targeting specific molecular drivers of pediatric malignancies. In this report, we present the results of integrative genomic and transcriptomic profiling of 230 patients with malignant solid tumors (the "primary cohort") and 18 patients with recurrent or otherwise difficult-to-treat nonmalignant conditions (the "secondary cohort"). The integrative workflow for the primary cohort enabled the identification of clinically significant single-nucleotide variants, small insertions/deletions, and fusion genes, which were found in 55% and 28% of patients, respectively. For 38% of patients, molecularly informed treatment recommendations were made. In the secondary cohort, known or potentially driving alteration was detected in 89% of cases, including a suspected novel causal gene for patients with inclusion body infantile digital fibromatosis. Furthermore, 47% of findings also brought therapeutic implications for subsequent management. Across both cohorts, changes or refinements to the original histopathological diagnoses were achieved in 4% of cases. Our study demonstrates the efficacy of integrating advanced genomic and transcriptomic analyses to identify therapeutic targets, refine diagnoses, and optimize treatment strategies for challenging pediatric and young adult malignancies and underscores the need for broad implementation of precision oncology in clinical settings.

Integrative NGS testing reveals clonal dynamics of adverse genomic defects contributing to a natural progression in treatment-naïve CLL patients.

Large-scale next-generation sequencing (NGS) studies revealed extensive genetic heterogeneity, driving a highly variable clinical course of chronic lymphocytic leukaemia (CLL). The evolution of subclonal populations contributes to diverse therapy responses and disease refractoriness. Besides, the dynamics and impact of subpopulations before therapy initiation are not well understood. We examined changes in genomic defects in serial samples of 100 untreated CLL patients, spanning from indolent to aggressive disease. A comprehensive NGS panel LYNX, which provides targeted mutational analysis and genome-wide chromosomal defect assessment, was employed. We observed dynamic changes in the composition and/or proportion of genomic aberrations in most patients (62%). Clonal evolution of gene variants prevailed over the chromosomal alterations. Unsupervised clustering based on aberration dynamics revealed four groups of patients with different clinical behaviour. An adverse cluster was associated with fast progression and early therapy need, characterized by the expansion of TP53 defects, ATM mutations, and 18p- alongside dynamic SF3B1 mutations. Our results show that clonal evolution is active even without therapy pressure and that repeated genetic testing can be clinically relevant during long-term patient monitoring. Moreover, integrative NGS testing contributes to the consolidated evaluation of results and accurate assessment of individual patient prognosis.

Low-burden TP53 mutations in CLL: clinical impact and clonal evolution within the context of different treatment options.

Patients with chronic lymphocytic leukemia (CLL) bearing TP53 mutations experience chemorefractory disease and are therefore candidates for targeted therapy. However, the significance of low-burden TP53 mutations with <10% variant allele frequency (VAF) remains a matter for debate. Herein, we describe clonal evolution scenarios of low-burden TP53 mutations, the clinical impact of which we analyzed in a "real-world" CLL cohort. TP53 status was assessed by targeted next-generation sequencing (NGS) in 511 patients entering first-line treatment with chemo- and/or immunotherapy and 159 patients in relapse before treatment with targeted agents. Within the pretherapy cohort, 16% of patients carried low-burden TP53 mutations (0.1% to 10% VAF). Although their presence did not significantly shorten event-free survival after first-line therapy, it affected overall survival (OS). In a subgroup with TP53 mutations of 1% to 10% VAF, the impact on OS was observed only in patients with unmutated IGHV who had not received targeted therapy, as patients benefited from switching to targeted agents, regardless of initial TP53 mutational status. Analysis of the clonal evolution of low-burden TP53 mutations showed that the highest expansion rates were associated with fludarabine, cyclophosphamide, and rituximab regimen in both first- and second-line treatments (median VAF increase, 14.8× and 11.8×, respectively) in contrast to treatment with less intense treatment regimens (1.6×) and no treatment (0.8×). In the relapse cohort, 33% of patients carried low-burden TP53 mutations, which did not expand significantly upon targeted treatment (median VAF change, 1×). Sporadic cases of TP53 mutations' clonal shifts were connected with the development of resistance-associated mutations. Altogether, our data support the incorporation of low-burden TP53 variants in clinical decision making.

Tumor and microenvironment response but no cytotoxic T-cell activation in classic Hodgkin lymphoma treated with anti-PD1.

Classic Hodgkin lymphoma (cHL) is the cancer type most susceptible to antibodies targeting programmed cell death protein 1 (PD1) and is characterized by scarce Hodgkin and Reed-Sternberg cells (HRSCs), perpetuating a unique tumor microenvironment (TME). Although anti-PD1 effects appear to be largely mediated by cytotoxic CD8+ T cells in solid tumors, HRSCs frequently lack major histocompatibility complex expression, and the mechanism of anti-PD1 efficacy in cHL is unclear. Rapid clinical responses and high interim complete response rates to anti-PD1 based first-line treatment were recently reported for patients with early-stage unfavorable cHL treated in the German Hodgkin Study Group phase 2 NIVAHL trial. To investigate the mechanisms underlying this very early response to anti-PD1 treatment, we analyzed paired biopsies and blood samples obtained from NIVAHL patients before and during the first days of nivolumab first-line cHL therapy. Mirroring the rapid clinical response, HRSCs had disappeared from the tissue within days after the first nivolumab application. The TME already shows a reduction in type 1 regulatory T cells and PD-L1+ tumor-associated macrophages at this early time point of treatment. Interestingly, a cytotoxic immune response and a clonal T-cell expansion were not observed in the tumors or peripheral blood. These early changes in the TME were distinct from alterations found in a separate set of cHL biopsies at relapse during anti-PD1 therapy. We identify a unique very early histologic response pattern to anti-PD1 therapy in cHL that is suggestive of withdrawal of prosurvival factors, rather than induction of an adaptive antitumor immune response, as the main mechanism of action.

Evolution of TP53 abnormalities during CLL disease course is associated with telomere length changes.

BACKGROUND: Telomeres are protective structures at chromosome ends which shorten gradually with increasing age. In chronic lymphocytic leukemia (CLL), short telomeres have been associated with unfavorable disease outcome, but the link between clonal evolution and telomere shortening remains unresolved. METHODS: We investigated relative telomere length (RTL) in a well-characterized cohort of 198 CLL patients by qPCR and focused in detail on a subgroup 26 patients who underwent clonal evolution of TP53 mutations (evolTP53). In the evolTP53 subgroup we explored factors influencing clonal evolution and corresponding changes in telomere length through measurements of telomerase expression, lymphocyte doubling time, and BCR signaling activity. RESULTS: At baseline, RTL of the evolTP53 patients was scattered across the entire RTL spectrum observed in our CLL cohort. RTL changed in the follow-up samples of 16/26 (62%) evolTP53 cases, inclining to reach intermediate RTL values, i.e., longer telomeres shortened compared to baseline while shorter ones prolonged. For the first time we show that TP53 clonal shifts are linked to RTL change, including unexpected RTL prolongation. We further investigated parameters associated with RTL changes. Unstable telomeres were significantly more frequent among younger patients (P = 0.032). Shorter telomeres were associated with decreased activity of the B-cell receptor signaling components p-ERK1/2, p-ZAP-70/SYK, and p-NFκB (P = 0.04, P = 0.01, and P = 0.02, respectively). CONCLUSIONS: Our study revealed that changes of telomere length reflect evolution in leukemic subclone proportion, and are associated with specific clinico-biological features of the explored cohort.

GLASS: assisted and standardized assessment of gene variations from Sanger sequence trace data.

MOTIVATION: Sanger sequencing is still being employed for sequence variant detection by many laboratories, especially in a clinical setting. However, chromatogram interpretation often requires manual inspection and in some cases, considerable expertise. RESULTS: We present GLASS, a web-based Sanger sequence trace viewer, editor, aligner and variant caller, built to assist with the assessment of variations in 'curated' or user-provided genes. Critically, it produces a standardized variant output as recommended by the Human Genome Variation Society. AVAILABILITY AND IMPLEMENTATION: GLASS is freely available at http://bat.infspire.org/genomepd/glass/ with source code at https://github.com/infspiredBAT/GLASS. CONTACT: nikos.darzentas@gmail.com or malcikova.jitka@fnbrno.cz. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A novel germline mutation in GP1BA gene N-terminal domain in monoallelic Bernard-Soulier syndrome.

Mutations in the GP1BA gene have been associated with platelet-type von Willebrand disease and Bernard-Soulier syndrome. Here, we report a novel GP1BA mutation in a family with autosomal dominant macrothrombocytopenia and mild bleeding. We performed analyses of seven family members. Using whole-exome sequencing of germline DNA samples, we identified a heterozygous single-nucleotide change in GP1BA (exone2:c.176T>G), encoding a p.Leu59Arg substitution in the N-terminal domain, segregating with macrothrombocytopenia. This variant has not been previously reported. We also analysed the structure of the detected sequence variant in silico. In particular, we used the crystal structure of the human platelet receptor GP Ibα N-terminal domain. Replacement of aliphatic amino-acid Leu 59 with charged, polar and larger arginine probably disrupts the protein structure. An autosomal dominant mode of inheritance, a family history of mild bleeding episodes, aggregation pattern in affected individuals together with evidence of mutation occurring in part of the GP1BA gene encoding the leucine-rich repeat region suggest a novel variant causing monoallelic Bernard-Soulier syndrome.

Transcript Isoform Diversity of Y Chromosome Ampliconic Genes of Great Apes Uncovered Using Long Reads and Telomere-to-Telomere Reference Genome Assemblies.

Y chromosomes of great apes harbor Ampliconic Genes (YAGs)-multi-copy gene families (BPY2, CDY, DAZ, HSFY, PRY, RBMY, TSPY, VCY, and XKRY) that encode proteins important for spermatogenesis. Previous work assembled YAG transcripts based on their targeted sequencing but not using reference genome assemblies, potentially resulting in an incomplete transcript repertoire. Here we used the recently produced gapless telomere-to-telomere (T2T) Y chromosome assemblies of great ape species (bonobo, chimpanzee, human, gorilla, Bornean orangutan, and Sumatran orangutan) and analyzed RNA data from whole-testis samples for the same species. We generated hybrid transcriptome assemblies by combining targeted long reads (Pacific Biosciences), untargeted long reads (Pacific Biosciences) and untargeted short reads (Illumina)and mapping them to the T2T reference genomes. Compared to the results from the reference-free approach, average transcript length was more than two times higher, and the total number of transcripts decreased three times, improving the quality of the assembled transcriptome. The reference-based transcriptome assemblies allowed us to differentiate transcripts originating from different Y chromosome gene copies and from their non-Y chromosome homologs. We identified two sources of transcriptome diversity-alternative splicing and gene duplication with subsequent diversification of gene copies. For each gene family, we detected transcribed pseudogenes along with protein-coding gene copies. We revealed previously unannotated gene copies of YAGs as compared to currently available NCBI annotations, as well as novel isoforms for annotated gene copies. This analysis paves the way for better understanding Y chromosome gene functions, which is important given their role in spermatogenesis.

Comprehensive genetic analysis by targeted sequencing identifies risk factors and predicts patient outcome in Mantle Cell Lymphoma: results from the EU-MCL network trials.

Recent studies highlighted genetic aberrations associated with prognosis in Mantle Cell lymphoma (MCL), yet comprehensive testing is not implemented in clinical routine. We conducted a comprehensive genomic characterization of 180 patients from the European MCL network trials by targeted sequencing of peripheral blood DNA using the EuroClonality(EC)-NDC assay. The IGH::CCND1 fusion was identified in 94% of patients, clonal IGH-V-(D)-J rearrangements in all, and 79% had ≥1 somatic gene mutation. The top mutated genes were ATM, TP53, KMT2D, SAMHD1, BIRC3 and NFKBIE. Copy number variations (CNVs) were detected in 83% of patients with RB1, ATM, CDKN2A/B and TP53 being the most frequently deleted and KLF2, CXCR4, CCND1, MAP2K1 and MYC the top amplified genes. CNVs and mutations were more frequently observed in older patients with adverse impact on prognosis. TP53mut, NOTCH1mut, FAT1mut TRAF2del, CDKN2A/Bdel and MAP2K1amp were linked to inferior failure-free (FFS) and overall survival (OS), while TRAF2mut, EGR2del and BCL2amp related to inferior OS only. Genetic complexity (≥3 CNVs) observed in 51% of analysed patients was significantly associated with impaired FFS and OS. We demonstrate that targeted sequencing from peripheral blood and bone marrow reliably detects diagnostically and prognostically important genetic factors in MCL patients, facilitating genetic characterization in clinical routine.

Precision immuno-oncology approach for four malignant tumors in siblings with constitutional mismatch repair deficiency syndrome.

Constitutional mismatch repair deficiency (CMMRD) is a rare syndrome characterized by an increased incidence of cancer. It is caused by biallelic germline mutations in one of the four mismatch repair genes (MMR) genes: MLH1, MSH2, MSH6, or PMS2. Accurate diagnosis accompanied by a proper molecular genetic examination plays a crucial role in cancer management and also has implications for other family members. In this report, we share the impact of the diagnosis and challenges during the clinical management of two brothers with CMMRD from a non-consanguineous family harbouring compound heterozygous variants in the PMS2 gene. Both brothers presented with different phenotypic manifestations and cancer spectrum. Treatment involving immune checkpoint inhibitors significantly contributed to prolonged survival in both patients affected by lethal gliomas. The uniform hypermutation also allowed immune-directed treatment using nivolumab for the B-cell lymphoma, thereby limiting the intensive chemotherapy exposure in this young patient who remains at risk for subsequent malignancies.

Insights into IGH clonal evolution in BCP-ALL: frequency, mechanisms, associations, and diagnostic implications.

Introduction: The malignant transformation leading to a maturation arrest in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) occurs early in B-cell development, in a pro-B or pre-B cell, when somatic recombination of variable (V), diversity (D), and joining (J) segment immunoglobulin (IG) genes and the B-cell rescue mechanism of VH replacement might be ongoing or fully active, driving clonal evolution. In this study of newly diagnosed BCP-ALL, we sought to understand the mechanistic details of oligoclonal composition of the leukemia at diagnosis, clonal evolution during follow-up, and clonal distribution in different hematopoietic compartments. Methods: Utilizing high-throughput sequencing assays and bespoke bioinformatics we identified BCP-ALL-derived clonally-related IGH sequences by their shared 'DNJ-stem'. Results: We introduce the concept of 'marker DNJ-stem' to cover the entirety of, even lowly abundant, clonally-related family members. In a cohort of 280 adult patients with BCP-ALL, IGH clonal evolution at diagnosis was identified in one-third of patients. The phenomenon was linked to contemporaneous recombinant and editing activity driven by aberrant ongoing DH/VH-DJH recombination and VH replacement, and we share insights and examples for both. Furthermore, in a subset of 167 patients with molecular subtype allocation, high prevalence and high degree of clonal evolution driven by ongoing DH/VH-DJH recombination were associated with the presence of KMT2A gene rearrangements, while VH replacements occurred more frequently in Ph-like and DUX4 BCP-ALL. Analysis of 46 matched diagnostic bone marrow and peripheral blood samples showed a comparable clonal and clonotypic distribution in both hematopoietic compartments, but the clonotypic composition markedly changed in longitudinal follow-up analysis in select cases. Thus, finally, we present cases where the specific dynamics of clonal evolution have implications for both the initial marker identification and the MRD monitoring in follow-up samples. Discussion: Consequently, we suggest to follow the marker DNJ-stem (capturing all family members) rather than specific clonotypes as the MRD target, as well as to follow both VDJH and DJH family members since their respective kinetics are not always parallel. Our study further highlights the intricacy, importance, and present and future challenges of IGH clonal evolution in BCP-ALL.

The Complete Sequence and Comparative Analysis of Ape Sex Chromosomes.

Apes possess two sex chromosomes-the male-specific Y and the X shared by males and females. The Y chromosome is crucial for male reproduction, with deletions linked to infertility. The X chromosome carries genes vital for reproduction and cognition. Variation in mating patterns and brain function among great apes suggests corresponding differences in their sex chromosome structure and evolution. However, due to their highly repetitive nature and incomplete reference assemblies, ape sex chromosomes have been challenging to study. Here, using the state-of-the-art experimental and computational methods developed for the telomere-to-telomere (T2T) human genome, we produced gapless, complete assemblies of the X and Y chromosomes for five great apes (chimpanzee, bonobo, gorilla, Bornean and Sumatran orangutans) and a lesser ape, the siamang gibbon. These assemblies completely resolved ampliconic, palindromic, and satellite sequences, including the entire centromeres, allowing us to untangle the intricacies of ape sex chromosome evolution. We found that, compared to the X, ape Y chromosomes vary greatly in size and have low alignability and high levels of structural rearrangements. This divergence on the Y arises from the accumulation of lineage-specific ampliconic regions and palindromes (which are shared more broadly among species on the X) and from the abundance of transposable elements and satellites (which have a lower representation on the X). Our analysis of Y chromosome genes revealed lineage-specific expansions of multi-copy gene families and signatures of purifying selection. In summary, the Y exhibits dynamic evolution, while the X is more stable. Finally, mapping short-read sequencing data from >100 great ape individuals revealed the patterns of diversity and selection on their sex chromosomes, demonstrating the utility of these reference assemblies for studies of great ape evolution. These complete sex chromosome assemblies are expected to further inform conservation genetics of nonhuman apes, all of which are endangered species.

Bioinformatic strategies for the analysis of genomic aberrations detected by targeted NGS panels with clinical application.

Molecular profiling of tumor samples has acquired importance in cancer research, but currently also plays an important role in the clinical management of cancer patients. Rapid identification of genomic aberrations improves diagnosis, prognosis and effective therapy selection. This can be attributed mainly to the development of next-generation sequencing (NGS) methods, especially targeted DNA panels. Such panels enable a relatively inexpensive and rapid analysis of various aberrations with clinical impact specific to particular diagnoses. In this review, we discuss the experimental approaches and bioinformatic strategies available for the development of an NGS panel for a reliable analysis of selected biomarkers. Compliance with defined analytical steps is crucial to ensure accurate and reproducible results. In addition, a careful validation procedure has to be performed before the application of NGS targeted assays in routine clinical practice. With more focus on bioinformatics, we emphasize the need for thorough pipeline validation and management in relation to the particular experimental setting as an integral part of the NGS method establishment. A robust and reproducible bioinformatic analysis running on powerful machines is essential for proper detection of genomic variants in clinical settings since distinguishing between experimental noise and real biological variants is fundamental. This review summarizes state-of-the-art bioinformatic solutions for careful detection of the SNV/Indels and CNVs for targeted sequencing resulting in translation of sequencing data into clinically relevant information. Finally, we share our experience with the development of a custom targeted NGS panel for an integrated analysis of biomarkers in lymphoproliferative disorders.

LYmphoid NeXt-Generation Sequencing (LYNX) Panel: A Comprehensive Capture-Based Sequencing Tool for the Analysis of Prognostic and Predictive Markers in Lymphoid Malignancies.

B-cell neoplasms represent a clinically heterogeneous group of hematologic malignancies with considerably diverse genomic architecture recently endorsed by next-generation sequencing (NGS) studies. Because multiple genetic defects have a potential or confirmed clinical impact, a tendency toward more comprehensive testing of diagnostic, prognostic, and predictive markers is desired. This study introduces the design, validation, and implementation of an integrative, custom-designed, capture-based NGS panel titled LYmphoid NeXt-generation sequencing (LYNX) for the analysis of standard and novel molecular markers in the most common lymphoid neoplasms (chronic lymphocytic leukemia, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, and mantle cell lymphoma). A single LYNX test provides the following: i) accurate detection of mutations in all coding exons and splice sites of 70 lymphoma-related genes with a sensitivity of 5% variant allele frequency, ii) reliable identification of large genome-wide (≥6 Mb) and recurrent chromosomal aberrations (≥300 kb) in at least 20% of the clonal cell fraction, iii) the assessment of immunoglobulin and T-cell receptor gene rearrangements, and iv) lymphoma-specific translocation detection. Dedicated bioinformatic pipelines were designed to detect all markers mentioned above. The LYNX panel represents a comprehensive, up-to-date tool suitable for routine testing of lymphoid neoplasms with research and clinical applicability. It allows a wide adoption of capture-based targeted NGS in clinical practice and personalized management of patients with lymphoproliferative diseases.

Consistent B Cell Receptor Immunoglobulin Features Between Siblings in Familial Chronic Lymphocytic Leukemia.

Key processes in the onset and evolution of chronic lymphocytic leukemia (CLL) are thought to include chronic (antigenic) activation of mature B cells through the B cell receptor (BcR), signals from the microenvironment, and acquisition of genetic alterations. Here we describe three families in which two or more siblings were affected by CLL. We investigated whether there are immunogenetic similarities in the leukemia-specific immunoglobulin heavy (IGH) and light (IGL/IGK) chain gene rearrangements of the siblings in each family. Furthermore, we performed array analysis to study if similarities in CLL-associated chromosomal aberrations are present within each family and screened for somatic mutations using paired tumor/normal whole-genome sequencing (WGS). In two families a consistent IGHV gene mutational status (one IGHV-unmutated, one IGHV-mutated) was observed. Intriguingly, the third family with four affected siblings was characterized by usage of the lambda IGLV3-21 gene, with the hallmark R110 mutation of the recently described clinically aggressive IGLV3-21R110 subset. In this family, the CLL-specific rearrangements in two siblings could be assigned to either stereotyped subset #2 or the immunogenetically related subset #169, both of which belong to the broader IGLV3-21R110 subgroup. Consistent patterns of cytogenetic aberrations were encountered in all three families. Furthermore, the CLL clones carried somatic mutations previously associated with IGHV mutational status, cytogenetic aberrations and stereotyped subsets, respectively. From these findings, we conclude that similarities in immunogenetic characteristics in familial CLL, in combination with genetic aberrations acquired, point towards shared underlying mechanisms behind CLL development within each family.

Validation of the EuroClonality-NGS DNA capture panel as an integrated genomic tool for lymphoproliferative disorders.

Current diagnostic standards for lymphoproliferative disorders include multiple tests for detection of clonal immunoglobulin (IG) and/or T-cell receptor (TCR) rearrangements, translocations, copy-number alterations (CNAs), and somatic mutations. The EuroClonality-NGS DNA Capture (EuroClonality-NDC) assay was designed as an integrated tool to characterize these alterations by capturing IGH switch regions along with variable, diversity, and joining genes of all IG and TCR loci in addition to clinically relevant genes for CNA and mutation analysis. Diagnostic performance against standard-of-care clinical testing was assessed in a cohort of 280 B- and T-cell malignancies from 10 European laboratories, including 88 formalin-fixed paraffin-embedded samples and 21 reactive lesions. DNA samples were subjected to the EuroClonality-NDC protocol in 7 EuroClonality-NGS laboratories and analyzed using a bespoke bioinformatic pipeline. The EuroClonality-NDC assay detected B-cell clonality in 191 (97%) of 197 B-cell malignancies and T-cell clonality in 71 (97%) of 73 T-cell malignancies. Limit of detection (LOD) for IG/TCR rearrangements was established at 5% using cell line blends. Chromosomal translocations were detected in 145 (95%) of 152 cases known to be positive. CNAs were validated for immunogenetic and oncogenetic regions, highlighting their novel role in confirming clonality in somatically hypermutated cases. Single-nucleotide variant LOD was determined as 4% allele frequency, and an orthogonal validation using 32 samples resulted in 98% concordance. The EuroClonality-NDC assay is a robust tool providing a single end-to-end workflow for simultaneous detection of B- and T-cell clonality, translocations, CNAs, and sequence variants.

Recurrent gene mutations detected in canine mast cell tumours by next generation sequencing.

Genetic causes of canine mast cell tumours (MCTs), except for mutations in the KIT gene detected in some MCTs, are generally unknown. We used whole exome sequencing to reveal mutation spectra in canine MCTs. We detected somatic mutations in 87 genes including 10 genes recognized as human cancer drivers. Besides KIT, 14 other genes were recurrently mutated. Subsequently, we performed next generation sequencing of a panel of 50 selected genes in additional MCT samples. In this group, the most frequently altered gene was GNB1 showing a recurrent dinucleotide substitution at position of Gly116 in 30% of the MCT samples (n = 6/20) and Ile80 substitution accompanied by a splice region mutation in one case. We extended the study by analysis of the above mentioned GNB1 regions in additional MCT samples by Sanger sequencing, and assessed the overall prevalence of GNB1 mutations to 17.3% (n = 14/81), which is similar to the prevalence of KIT alterations. Our results indicate that GNB1 mutations are probably involved in canine MCT pathogenesis in both cutaneous and subcutaneous MCT cases. As opposed to KIT alterations, the presence of GNB1 mutations did not negatively affect survival times, and our data even showed a trend towards positive prognosis. If our results are confirmed in a larger number of MCTs, an extension of molecular testing of canine MCTs by GNB1 analysis would help to refine the molecular stratification of MCTs, and become useful for targeted treatment strategies.

Assessment of Tumor Mutational Burden in Pediatric Tumors by Real-Life Whole-Exome Sequencing and In Silico Simulation of Targeted Gene Panels: How the Choice of Method Could Affect the Clinical Decision?

BACKGROUND: Tumor mutational burden (TMB) is an emerging genomic biomarker in cancer that has been associated with improved response to immune checkpoint inhibitors (ICIs) in adult cancers. It was described that variability in TMB assessment is introduced by different laboratory techniques and various settings of bioinformatic pipelines. In pediatric oncology, no study has been published describing this variability so far. METHODS: In our study, we performed whole exome sequencing (WES, both germline and somatic) and calculated TMB in 106 patients with high-risk/recurrent pediatric solid tumors of 28 distinct cancer types. Subsequently, we used WES data for TMB calculation using an in silico approach simulating two The Food and Drug Administration (FDA)-approved/authorized comprehensive genomic panels for cancer. RESULTS: We describe a strong correlation between WES-based and panel-based TMBs; however, we show that this high correlation is significantly affected by inclusion of only a few hypermutated cases. In the series of nine cases, we determined TMB in two sequentially collected tumor tissue specimens and observed an increase in TMB along with tumor progression. Furthermore, we evaluated the extent to which potential ICI indication could be affected by variability in techniques and bioinformatic pipelines used for TMB assessment. We confirmed that this technological variability could significantly affect ICI indication in pediatric cancer patients; however, this significance decreases with the increasing cut-off values. CONCLUSIONS: For the first time in pediatric oncology, we assessed the reliability of TMB estimation across multiple pediatric cancer types using real-life WES and in silico analysis of two major targeted gene panels and confirmed a significant technological variability to be introduced by different laboratory techniques and various settings of bioinformatic pipelines.