A recurrent NTRK1 tyrosine kinase domain mutation pair is characteristic in a subset of dedifferentiated liposarcomas.

INTRODUCTION: Dedifferentiated liposarcoma (DDLPS) is a common form of liposarcoma with challenging treatment modalities. Pan-TRK immunopositivity can be often observed without NTRK gene fusion in soft tissue sarcomas with myogenic differentiation. Expression and the role of NTRK in DDLPS are under-studied. We sought to identify activating mutations of the NTRK genes. MATERIALS AND METHODS: 131 DDLPS patients were selected for pan-TRK immunohistochemistry and positive cases were analyzed by Sanger sequencing for NTRK1, NTRK2 and NTRK3 genes. Functional assays were performed using a lentiviral transduction system to study the effect of NTRK variants in fibroblast, immortalized fibroblast, and dedifferentiated liposarcoma cell lines. RESULTS: Out of the 131 DDLPS cases, 75 immunohistochemical staining positive cases, 46 were successfully Sanger sequenced. A recurrent somatic mutation pair in cis position (NGS) of the NTRK1 c.1810C>T (p.H604Y) and c.1838G>T (p.G613V) was identified in six cases (13%) that have never been reported in DDLPS. NTRK fusions were excluded in all six cases by FISH and NGS. The phospho-AKT immunopositivity among the six mutated cases suggested downstream activation of the NTRK signaling pathway. Functional assays showed no transforming effects, but resistance to first- and second-line TRK inhibitors of the p.G613V and p.H604Y variant. CONCLUSIONS: We detected (de novo/somatic) missense mutation variants in cis position of the NTRK1 gene in a subset of DDLPS indicating modifying mutations that may contribute to tumorigenesis in a subset of DDLPS. These variants beget resistance to TRK inhibitors indicating an interesting biomarker for other studies with TRK inhibitors.

Truncated titin is structurally integrated into the human dilated cardiomyopathic sarcomere.

Heterozygous (HET) truncating variant mutations in the TTN gene (TTNtvs), encoding the giant titin protein, are the most common genetic cause of dilated cardiomyopathy (DCM). However, the molecular mechanisms by which TTNtv mutations induce DCM are controversial. Here, we studied 127 clinically identified DCM human cardiac samples with next-generation sequencing (NGS), high-resolution gel electrophoresis, Western blot analysis, and super-resolution microscopy in order to dissect the structural and functional consequences of TTNtv mutations. The occurrence of TTNtv was found to be 15% in the DCM cohort. Truncated titin proteins matching, by molecular weight, the gene sequence predictions were detected in the majority of the TTNtv+ samples. Full-length titin was reduced in TTNtv+ compared with TTNtv- samples. Proteomics analysis of washed myofibrils and stimulated emission depletion (STED) super-resolution microscopy of myocardial sarcomeres labeled with sequence-specific anti-titin antibodies revealed that truncated titin was structurally integrated into the sarcomere. Sarcomere length-dependent anti-titin epitope position, shape, and intensity analyses pointed at possible structural defects in the I/A junction and the M-band of TTNtv+ sarcomeres, which probably contribute, possibly via faulty mechanosensor function, to the development of manifest DCM.

[Childhood brain tumors: diagnosis and therapy - comprehensive genomic profiling]. / Gyermekkori agydaganatok: diagnózis és terápia ­ komprehenzív genomikai profilozás.

With the advancement of molecular oncology, numerous new opportunities are available for the effective and efficient treatment of patients diagnosed with childhood brain tumors. This includes gene panel analysis aiding personalized treatment used in clinical trials, and the application of targeted therapy independent of tissue type (tumor agnostic therapy). Most personalized therapies inhibit certain kinases. In our review, we present the modern pathological diagnosis of childhood brain tumors, as well as the complex intracellular regulation of signal transduction pathways important from the point of view of clinical practice, and we describe their further targets defined on the basis of pharmacological characteristics of the pathway, based on international and our own results. Despite common mutations affecting kinases, personalized therapy is not available in many types of tumors. Through the example of childhood brain tumors, we demonstrate the expected future therapeutic significance of tyrosine kinases.

Novel RICTOR amplification harbouring entities: FISH validation of RICTOR amplification in tumour tissue after next-generation sequencing.

Alterations in mTOR signalling molecules, including RICTOR amplification, have been previously described in many cancers, particularly associated with poor prognosis. In this study, RICTOR copy number variation (CNV) results of diagnostic next-generation sequencing (NGS) were analysed in 420 various human malignant tissues. RICTOR amplification was tested by Droplet Digital PCR (ddPCR) and validated using the "gold standard" fluorescence in situ hybridisation (FISH). Additionally, the consequences of Rictor protein expression were also studied by immunohistochemistry. RICTOR amplification was presumed in 37 cases with CNV ≥ 3 by NGS, among these, 16 cases (16/420; 3.8%) could be validated by FISH, however, ddPCR confirmed only 11 RICTOR-amplified cases with lower sensitivity. Based on these, neither NGS nor ddPCR could replace traditional FISH in proof of RICTOR amplification. However, NGS could be beneficial to highlight potential RICTOR-amplified cases. The obtained results of the 14 different tumour types with FISH-validated RICTOR amplification demonstrate the importance of RICTOR amplification in a broad spectrum of tumours. The newly described RICTOR-amplified entities could initiate further collaborative studies with larger cohorts to analyse the prevalence of RICTOR amplification in rare diseases. Finally, our and further work could help to improve and expand future therapeutic opportunities for mTOR-targeted therapies.

Novel, clinically relevant genomic patterns identified by comprehensive genomic profiling in ATRX-deficient IDH-wildtype adult high-grade gliomas.

Glioblastomas are the most common IDH-wildtype adult high-grade gliomas, frequently harboring mutations in the TERT gene promoter (pTERT) and utilizing the subsequent telomerase overexpression for telomere length maintenance. However, some rare cases show loss of ATRX and use alternative mechanisms of telomere lengthening. In this study, we performed the first complex genomic analysis specifically concentrating on the latter subgroup. Comprehensive genomic profiling of 12 ATRX-deficient and 13 ATRX-intact IDH-wildtype adult high-grade gliomas revealed that ATRX and pTERT mutations are mutually exclusive. DNMT3A alterations were confined to ATRX-deficient, while PTEN mutations to ATRX-intact cases. RAS-MAPK pathway alterations, including NF1 mutations, were more characteristic in the ATRX-deficient group. Variants of genes related to homologous recombination repair showed different patterns of affected genes. Two ATRX-deficient tumors with high tumor mutational burden and mismatch repair deficiency were found. One of these contained a novel fusion involving the NTRK2 and LRRFIP2 genes, while the other showed loss of MSH2 and MSH6 without genetic alterations in the encoding genes suggesting an epigenetic background. Genetic characteristics of ATRX-deficient IDH-wildtype adult high-grade gliomas suggest that these tumors are particularly intriguing targets of potential future therapeutic interventions including immunotherapies combined with MAPK pathway inhibition and DNA repair inhibitors.

PersonALL: a genetic scoring guide for personalized risk assessment in pediatric B-cell precursor acute lymphoblastic leukemia.

BACKGROUND: Recurrent genetic lesions provide basis for risk assessment in pediatric acute lymphoblastic leukemia (ALL). However, current prognostic classifiers rely on a limited number of predefined sets of alterations. METHODS: Disease-relevant copy number aberrations (CNAs) were screened genome-wide in 260 children with B-cell precursor ALL. Results were integrated with cytogenetic data to improve risk assessment. RESULTS: CNAs were detected in 93.8% (n = 244) of the patients. First, cytogenetic profiles were combined with IKZF1 status (IKZF1normal, IKZF1del and IKZF1plus) and three prognostic subgroups were distinguished with significantly different 5-year event-free survival (EFS) rates, IKAROS-low (n = 215): 86.3%, IKAROS-medium (n = 27): 57.4% and IKAROS-high (n = 18): 37.5%. Second, contribution of genetic aberrations to the clinical outcome was assessed and an aberration-specific score was assigned to each prognostically relevant alteration. By aggregating the scores of aberrations emerging in individual patients, personalized cumulative values were calculated and used for defining four prognostic subgroups with distinct clinical outcomes. Two favorable subgroups included 60% of patients (n = 157) with a 5-year EFS of 96.3% (excellent risk, n = 105) and 87.2% (good risk, n = 52), respectively; while 40% of patients (n = 103) showed high (n = 74) or ultra-poor (n = 29) risk profile (5-year EFS: 67.4% and 39.0%, respectively). CONCLUSIONS: PersonALL, our conceptually novel prognostic classifier considers all combinations of co-segregating genetic alterations, providing a highly personalized patient stratification.

Next-Generation Sequencing-Based Genomic Profiling of Children with Acute Myeloid Leukemia.

Pediatric acute myeloid leukemia (AML) represents a major cause of childhood leukemic mortality, with only a limited number of studies investigating the molecular landscape of the disease. Here, we present an integrative analysis of cytogenetic and molecular profiles of 75 patients with pediatric AML from a multicentric, real-world patient cohort treated according to AML Berlin-Frankfurt-Münster protocols. Targeted next-generation sequencing of 54 genes revealed 17 genes that were recurrently mutated in >5% of patients. Considerable differences were observed in the mutational profiles compared with previous studies, as BCORL1, CUX1, KDM6A, PHF6, and STAG2 mutations were detected at a higher frequency than previously reported, whereas KIT, NRAS, and KRAS were less frequently mutated. Our study identified novel recurrent mutations at diagnosis in the BCORL1 gene in 9% of the patients. Tumor suppressor gene (PHF6, TP53, and WT1) mutations were found to be associated with induction failure and shorter event-free survival, suggesting important roles of these alterations in resistance to therapy and disease progression. Comparison of the mutational landscape at diagnosis and relapse revealed an enrichment of mutations in tumor suppressor genes (16.2% versus 44.4%) and transcription factors (35.1% versus 55.6%) at relapse. Our findings shed further light on the heterogeneity of pediatric AML and identify previously unappreciated alterations that may lead to improved molecular characterization and risk stratification of pediatric AML.

Case report: Complete and durable response to larotrectinib (TRK inhibitor) in an infant diagnosed with angiosarcoma harbouring a KHDRBS1-NTRK3 fusion gene.

Significant improvements in the survival rates of paediatric cancer have been achieved over the past decade owing to recent advances in therapeutic and diagnostic strategies. However, disease progression and relapse remain a major challenge for the clinical management of paediatric angiosarcoma. Comprehensive genomic profiling of these rare tumours using high-throughput sequencing technologies may improve patient stratification and identify actionable biomarkers for therapeutic intervention. Here, we describe the clinical, histopathological, immunohistochemical and molecular profile of a novel and precision medicine-informed case where a KHDRBS1-NTRK3 fusion determined by next-generation sequencing-based comprehensive genomic profiling led to complete and sustained remission (clinical and radiological response) in an otherwise incurable disease. Our patient represents the first paediatric angiosarcoma harbouring a targetable NTRK3 fusion in the literature and demonstrates the first example of targeting this alteration in angiosarcoma using larotrectinib, an NTRK inhibitor. Clinical and radiological remission was achieved in under two months of therapy, and the patient is currently in complete remission, 4 month after stopping larotrectinib therapy, which was given over 17 months with only mild side effects reported. Therefore, this remarkable case exemplifies the true essence of precision-based care by incorporating conventional pathology with the why, when, and how to test for rare oncogenic drivers and agnostic biomarkers in paediatric angiosarcoma.

Clinical sequencing identifies potential actionable alterations in a high rate of urachal and primary bladder adenocarcinomas.

OBJECTIVE: Administration of targeted therapies provides a promising treatment strategy for urachal adenocarcinoma (UrC) or primary bladder adenocarcinoma (PBAC); however, the selection of appropriate drugs remains difficult. Here, we aimed to establish a routine compatible methodological pipeline for the identification of the most important therapeutic targets and potentially effective drugs for UrC and PBAC. METHODS: Next-generation sequencing, using a 161 cancer driver gene panel, was performed on 41 UrC and 13 PBAC samples. Clinically relevant alterations were filtered, and therapeutic interpretation was performed by in silico evaluation of drug-gene interactions. RESULTS: After data processing, 45/54 samples passed the quality control. Sequencing analysis revealed 191 pathogenic mutations in 68 genes. The most frequent gain-of-function mutations in UrC were found in KRAS (33%), and MYC (15%), while in PBAC KRAS (25%), MYC (25%), FLT3 (17%) and TERT (17%) were recurrently affected. The most frequently affected pathways were the cell cycle regulation, and the DNA damage control pathway. Actionable mutations with at least one available approved drug were identified in 31/33 (94%) UrC and 8/12 (67%) PBAC patients. CONCLUSIONS: In this study, we developed a data-processing pipeline for the detection and therapeutic interpretation of genetic alterations in two rare cancers. Our analyses revealed actionable mutations in a high rate of cases, suggesting that this approach is a potentially feasible strategy for both UrC and PBAC treatments.

Potential role of MAP2K1 mutation in the trans-differentiation of interdigitating dendritic cell sarcoma: Case report and literature review.

A 5-year-old male child was diagnosed with interdigitating dendritic cell sarcoma (IDCS) during his maintenance therapy for B-cell precursor acute lymphoblastic leukemia (B-ALL). Multiplex lymph node involvements of the neck were found by positron emission tomography CT (PET-CT). Treatments, including surgical and chemotherapy, resulted in complete remission. Four years later, systemic bone infiltration was discovered. Surgical resection of the IV rib and intensive chemotherapy led to a complete morphological remission, and allogeneic bone marrow transplantation was performed. Comprehensive genomic profiling of the formalin fixed the tumor tissue, and the cryopreserved leukemic cells revealed several common alterations and divergent clonal evolution with a novel MAP2K1 mutation of the IDCS, which is responsible for the trans-differentiation of the common lymphoid-committed tumor progenitor.

Germline MUTYH mutations and high-grade gliomas: Novel evidence for a potential association.

There is growing body of evidence supporting the role of germline mutations in the pathogenesis of pediatric central nervous system (CNS) tumors, and the widespread use of next-generation sequencing (NGS) panels facilitates their detection. Variants of the MUTYH gene are increasingly recognized as suspected germline background of various extraintestinal malignancies, besides their well-characterized role in the polyposis syndrome associated with biallelic mutations. Using a multigene NGS panel (Illumina TruSight Oncology 500), we detected one H3 G34V- and one H3 K27M-mutant pediatric high-grade diffuse glioma, in association with c.1178G>A (p.G393D) and c.916C>T (p.R306C) MUTYH variants, respectively. Both MUTYH mutations were germline, heterozygous and inherited, according to the subsequent genetic testing of the patients and their first-degree relatives. In the H3 K27M-mutant glioma, amplifications affecting the 4q12 region were also detected, in association with KDR-PDGFRA, KIT-PDGFRA, and KDR-CHIC2 fusions, previously unreported in this entity. Among 47 other CNS tumors of various histological types tested with the same NGS panel in our institution, only one adult glioblastoma harbored MUTYH mutation. Together with a single previous report, our data raises the possibility of an association between germline MUTYH mutations and CNS malignancies, particularly in pediatric histone H3-mutant gliomas.

Foliate Lymphoid Aggregates as Novel Forms of Serous Lymphocyte Entry Sites of Peritoneal B Cells and High-Grade B Cell Lymphomas.

The cellular homeostasis of lymphoid tissues is determined by the continuous interactions of mobile hematopoietic cells within specialized microenvironments created by sessile stromal cells. In contrast to the lymph nodes and mucosal lymphoid tissues with well-defined entry and exit routes, the movement of leukocytes in the peritoneal cavity is largely unknown. In this study, we report that, in addition to the omental milky spots and fat-associated lymphoid clusters, in mice, the serous surface of the mesenteric adipose streaks contains lymphocyte-rich organoids comprised of a highly compacted leaf-like part connected to the adipose tissue that can also efficiently bind B cells and high-grade B cell lymphoma (diffuse large B cell lymphoma) cells. Denoted as foliate lymphoid aggregates (FLAgs), these structures show incomplete T/B segregation and a partially differentiated stromal architecture. LYVE-1-positive macrophages covering FLAgs efficiently bind i.p. injected normal B cells as well as different types of diffuse large B cell lymphoma cells. Within FLAgs, the lymphocytes compartmentalize according to their chemokine receptor pattern and subsequently migrate toward the mesenteric lymph nodes via the mesenteric lymphatic capillaries. The blood supply of FLAgs includes short vascular segments displaying peripheral lymph node addressin, and the extravasation of lymphocytes to the omental and mesenteric adipose tissues is partly mediated by L-selectin. The appearance of i.p. injected cells in mesenteric lymph nodes suggests that the mesentery-associated lymphatics may also collect leukocytes from the fat-associated lymphoid clusters and FLAgs, thus combining the mucosal and serous exit of mobile leukocytes and increasing the range of drainage sites for the peritoneal expansion of lymphoid malignancies.