Somatic Genetic Variants Research Articles

A somatic genetic clock for clonal species

Age and longevity are key parameters for demography and life-history evolution of organisms. In clonal species, a widespread life history among animals, plants, macroalgae and fungi, the sexually produced offspring (genet) grows indeterminately by producing iterative modules, or ramets, and so obscure their age. Here we present a novel molecular clock based on the accumulation of fixed somatic genetic variation that segregates among ramets. Using a stochastic model, we demonstrate that the accumulation of fixed somatic genetic variation will approach linearity after a lag phase, and is determined by the mitotic mutation rate, without direct dependence on asexual generation time. The lag phase decreased with lower stem cell population size, number of founder cells for the formation of new modules, and the ratio of symmetric versus asymmetric cell divisions. We calibrated the somatic genetic clock on cultivated eelgrass Zostera marina genets (4 and 17 years respectively). In a global data set of 20 eelgrass populations, genet ages were up to 1,403 years. The somatic genetic clock is applicable to any multicellular clonal species where the number of founder cells is small, opening novel research avenues to study longevity and, hence, demography and population dynamics of clonal species.

Pathogenic MTOR somatic variant causing focal cortical dysplasia drives hyperexcitability via overactivation of neuronal GluN2C N-methyl-D-aspartate receptors.

Genetic variations in proteins of the mechanistic target of rapamycin (mTOR) pathway cause a spectrum of neurodevelopmental disorders often associated with brain malformations and with intractable epilepsy. The mTORopathies are characterized by hyperactive mTOR pathway and comprise tuberous sclerosis complex (TSC) and focal cortical dysplasia (FCD) type II. How hyperactive mTOR translates into abnormal neuronal activity and hypersynchronous network remains to be better understood. Previously, the role of upregulated GluN2C-containing glutamate-gated N-methyl-D-aspartate receptors (NMDARs) has been demonstrated for germline defects in the TSC genes. Here, we questioned whether this mechanism would expand to other mTORopathies in the different context of a somatic genetic variation of the MTOR protein recurrently found in FCD type II. We used a rat model of FCD created by in utero electroporation of neural progenitors of dorsal telencephalon with expression vectors encoding either the wild-type or the pathogenic MTOR variant (p.S2215F). In this mosaic configuration, patch-clamp whole-cell recordings of the electroporated, spiny stellate neurons and extracellular recordings of the electroporated areas were performed in neocortical slices. Selective inhibitors were used to target mTOR activity and GluN2C-mediated currents. Neurons expressing the mutant protein displayed an excessive activation of GluN2C NMDAR-mediated spontaneous excitatory postsynaptic currents. GluN2C-dependent increase in spontaneous spiking activity was detected in the area of electroporated neurons in the mutant condition and was restricted to a critical time window between postnatal days P9 and P20. Somatic MTOR pathogenic variant recurrently found in FCD type II resulted in overactivation of GluN2C-mediated neuronal NMDARs in neocortices of rat pups. The related and time-restricted local hyperexcitability was sensitive to subunit GluN2C-specific blockade. Our study suggests that GluN2C-related pathomechanisms might be shared in common by mTOR-related brain disorders.

Overcoming Barriers to Discovery and Implementation of Equitable Pharmacogenomic Testing in Oncology.

Pharmacogenomics (PGx), the study of inherited genomic variation and drug response or safety, is a vital tool in precision medicine. In oncology, testing to identify PGx variants offers patients the opportunity for customized treatments that can minimize adverse effects and maximize the therapeutic benefits of drugs used for cancer treatment and supportive care. Because individuals of shared ancestry share specific genetic variants, PGx factors may contribute to outcome disparities across racial and ethnic categories when genetic ancestry is not taken into account or mischaracterized in PGx research, discovery, and application. Here, we examine how the current scientific understanding of the role of PGx in differential oncology safety and outcomes may be biased toward a greater understanding and more complete clinical implementation of PGx for individuals of European descent compared with other genetic ancestry groups. We discuss the implications of this bias for PGx discovery, access to care, drug labeling, and patient and provider understanding and use of PGx approaches. Testing for somatic genetic variants is now the standard of care in treatment of many solid tumors, but the integration of PGx into oncology care is still lacking despite demonstrated actionable findings from PGx testing, reduction in avoidable toxicity and death, and return on investment from testing. As the field of oncology is poised to expand and integrate germline genetic variant testing, it is vital that PGx discovery and application are equitable for all populations. Recommendations are introduced to address barriers to facilitate effective and equitable PGx application in cancer care.

The highs and lows of cyclic thrombocytopenia.

Cyclic thrombocytopenia (CTP) is characterized by periodic platelet oscillation with substantial amplitude. Most CTP cases have a thrombocytopenic background and are often misdiagnosed as immune thrombocytopenia with erratically effective treatment choices. CTP also occurs during hydroxyurea treatment in patients with myeloproliferative diseases. While the aetiology of CTP remains uncertain, here we evaluate historical, theoretical and clinical findings to provide a framework for understanding CTP pathophysiology. CTP retains the intrinsic oscillatory factors defined by the homeostatic regulation of platelet count, presenting as reciprocal platelet/thrombopoietin oscillations and stable oscillation periodicity. Moreover, CTP patients possess pathogenic factors destabilizing the platelet homeostatic system thereby creating opportunities for external perturbations to initiate and sustain the exaggerated platelet oscillations. Beyond humoral and cell-mediated autoimmunity, we propose recently uncovered germline and somatic genetic variants, such as those of MPL, STAT3 or DNMT3A, as pathogenic factors in thrombocytopenia-related CTP. Likewise, the JAK2 V617F or BCR::ABL1 translocation that drives underlying myeloproliferative diseases may also play a pathogenic role in hydroxyurea-induced CTP, where hydroxyurea treatment can serve as both a trigger and a pathogenic factor of platelet oscillation. Elucidating the pathogenic landscape of CTP provides an opportunity for targeted therapeutic approaches in the future.

Hotspots of Somatic Genetic Variation in Pituitary Neuroendocrine Tumors

The most common genetic drivers of pituitary neuroendocrine tumors (PitNETs) lie within mutational hotspots, which are genomic regions where variants tend to cluster. Some of these hotspot defects are unique to PitNETs, while others are associated with additional neoplasms. Hotspot variants in GNAS and USP8 are the most common genetic causes of acromegaly and Cushing’s disease, respectively. Although it has been proposed that these genetic defects could define specific clinical phenotypes, results are highly variable among studies. In contrast, DICER1 hotspot variants are associated with a familial syndrome of cancer predisposition, and only exceptionally occur as somatic changes. A small number of non-USP8-driven corticotropinomas are due to somatic hotspot variants in USP48 or BRAF; the latter is a well-known mutational hotspot in cancer. Finally, somatic variants affecting a hotspot in SF3B1 have been associated with multiple cancers and, more recently, with prolactinomas. Since the associations of BRAF, USP48, and SF3B1 hotspot variants with PitNETs are very recent, their effects on clinical phenotypes are still unknown. Further research is required to fully define the role of these genetic defects as disease biomarkers and therapeutic targets.

Dnmt3a-Mutant Haematopoietic Stem and Progenitor Cells Induce Mesenchymal Stromal Cell Senescence through the Production of Alarmins S100A8/A9

Clonal haematopoiesis (CH) is an age-related condition occurring due to somatic genetic variation in haematopoietic stem and progenitor cells (HSPCs), most frequently observed in the gene DNA methyltransferase DNMT3A. CH can be a precursor condition for haematologic malignancy, but the mechanisms driving progression of CH to malignancy are incompletely understood. Given that malignant cells are understood to reprogram their bone marrow (BM) microenvironment to create a self-reinforcing niche, we hypothesized that HSPCs carrying a CH-associated mutation in DNMT3A have microenvironment-remodelling properties that promote their clonal advantage. Using single-cell RNA-seq, we comprehensively captured bone and BM cells of haematopoietic and non-haematopoietic lineages in Dnmt3a R878H/+ mice in which the Dnmt3a mutation is present in the haematopoietic but not the non-haematopoietic compartments. In the non-haematopoietic fractions, we identified enrichment of a cellular senescence program, including transcriptional signatures of cell cycle arrest and expression of the canonical senescence-associated gene Cdkn1a (p21), in BM mesenchymal stromal cells (MSCs). The MSC cellular senescence phenotype caused by Dnmt3a-mutant haematopoiesis was confirmed at the protein level by increased expression of senescence-associated beta galactosidase (SA-β-gal) and the anti-apoptotic proteins BCL-2 and BCL-xL. In ex vivo co-culture assays, primary wild-type MSCs cultured with Dnmt3a-mutant HSPCs versus control HSPCs had increased SA-β-gal, Cdkn1a (p21), and Cdkn2a (p16) expression. Moreover, transwell assays demonstrated that MSC senescence induced by Dnmt3a-mutant HSPCs is cell contact-independent. To undercover the mechanisms driving MSC senescence, we analyzed soluble factors produced by Dnmt3a-mutant HSPCs. Conditioned mediafrom Dnmt3a-mutant HSPCs had significantly elevated levels of S100A8/A9, IL-6 and TNFα, which we confirmed were also elevated in BM fluid of Dnmt3a-mutant mice. In our single cell RNA-seq data, S100a8 and S100a9 were the top significantly increased genes in Dnmt3a-mutant versus control HSCs, making S100A8/A9 our top candidates for functional studies. We tested the direct effect of recombinant S100A8/A9 on primary wild-type MSCs and found that this was sufficient to induce SA-β-gal. Ongoing studies using the S100A8/A9 inhibitor Tasquinimod are examining the dependency of Dnmt3a-mutant hematopoiesis on alarmin production ex vivo and in vivo. Given that S100A8/A9 are heavily involved in the pathogenesis of acute inflammation and myeloproliferative neoplasms, induction of senescence through the alarmin axis is a mechanistic link between aging, clonal hematopoiesis and haematologic malignancy.

Work-Up and Treatment Strategies for Individuals with PIK3CA-Related Disorders: A Consensus of Experts from the Scientific Committee of the Italian Macrodactyly and PROS Association.

PIK3CA-related disorders encompass many rare and ultra-rare conditions caused by somatic genetic variants that hyperactivate the PI3K-AKT-mTOR signaling pathway, which is essential for cell cycle control. PIK3CA-related disorders include PIK3CA-related overgrowth spectrum (PROS), PIK3CA-related vascular malformations and PIK3CA-related non-vascular lesions. Phenotypes are extremely heterogeneous and overlapping. Therefore, diagnosis and management frequently involve various health specialists. Given the rarity of these disorders and the limited number of centers offering optimal care, the Scientific Committee of the Italian Macrodactyly and PROS Association has proposed a revision of the most recent recommendations for the diagnosis, molecular testing, clinical management, follow-up, and treatment strategies. These recommendations give insight on molecular diagnosis, eligible samples, preferable sequencing, and validation methods and management of negative results. The purpose of this paper is to promote collaboration between health care centers and clinicians with a joint shared approach. Finally, we suggest the direction of present and future research studies, including new systemic target therapies, which are currently under evaluation in several clinical trials, such as specific inhibitors that can be employed to downregulate the signaling pathway.

Abstract 16844: Identifying Splenic Radiomics Features Associated With Risk of Coronary Artery Disease

Introduction: Inflammation is a validated mechanism for residual coronary artery disease (CAD) risk via clinical trials and analysis of germline and somatic genetic variation in blood cells. While the spleen plays an important role in regulating inflammation, the role it plays in CAD is unclear. Hypothesis: We aim to test the hypothesis that radiographic splenic biomarkers may offer novel insights for CAD. Methods: We used deep learning to segment spleens from the abdominal MRIs of 38,474 patients in the UK Biobank. CAD was defined as history of coronary artery bypass grafting, myocardial infarction (MI), coronary artery angioplasty, or billing codes (OPCS-4: K40, K41, K45, K49, K50.2, K75). We extracted image features using the pyradiomics package. To identify image features associated with CAD, we used L1-regularized logistic regression models with the splenic features and clinical features used in the Pooled Cohort Equations (PCE) for outcomes of CAD and cardiovascular disease (CVD) events (defined as a composite of CAD, MI, and stroke outcomes). We computed the area under the receiver operating characteristic (AUROC) on a held-out 30% of the patient population and determined 95% confidence intervals using 1,000 Monte-Carlo resampled bootstraps. Results: Study participants had mean (SD) age 55.1 (7.5) years, 19,921 (51.8%) were women, 1,412 (3.7%) had CAD, and 2,473 (6.4%) had CVD events. The model to predict CAD using the splenic and PCE features had an AUROC of 0.84 (95% CI, 0.84-0.85), and 3 out of 10 significant covariates were splenic features. 1-SD increases in sphericity, minor axis length, and entropy in neighboring voxel gray levels were associated with 5.4% (95% CI, 5.1-5.7) higher, 13.3% (95% CI, 13.0-13.6) lower, and 10.5% (95% CI, 10.2-10.8) higher odds of CAD after adjustment. Results were consistent in the model to predict CVD events, and decreased minor axis length and increased texture variation were associated with increased risk. Model performance was lower for older age groups, who had higher incidences of CAD. Conclusions: This study leverages deep-learning methods across a population based cohort to highlight splenic features correlated with CAD and offers new opportunities to understand the role of the hematopoietic system in CAD.

Abstract 17773: Novel Partition Scores Identify Pathways Implicated in Clonal Hematopoiesis of Indeterminate Potential (CHIP) Mediated Heart Failure

Background: Peripheral blood somatic genetic variants such as those in clonal hematopoiesis of indeterminate potential (CHIP) increase the risk of both ischemic and non-ischemic cardiomyopathy but mechanisms beyond inflammation are unclear. Research question/Objectives: We sought to understand the prevalence and impact of CHIP and extended somatic variants (ExSV) using a new deep learning algorithm in heart failure patients on the outcome of all-cause mortality and leverage novel partition scores to identify pathways involved. Methods: CHIP and ExSV were evaluated using a novel proprietary deep learning algorithm in a heart failure cohort at Yale using whole exome sequencing and somatic variants identified were validated using UKB and TOPMED cohorts. Cox regression including somatic variants on the outcome of death was performed. Partition score analysis integrating patient demographics, co-morbidities, genomic and transcriptomic data was performed to identify traits and biological pathways implicated in heart failure Results: CHIP was highly prevalent in the 189 patient Yale heart failure cohort at 42% with mean age of 66. Fifty percent had diabetes and 46% had CAD. Using UKB and TOPMED cohorts, novel “CHIP-like” somatic variants were identified that conferred increased or decreased risk of heart failure. Some ExSV were associated with increased risk of death. Partition score assessment identified top traits associated with increased risk of heart failure, including LDL, triglycerides, diabetes, BMI and CAD and pathway analysis identified that metabolic pathways including cholesterol metabolism, glucose signaling and AMPK were implicated in the heart failure phenotype mediated by somatic variants (Table). Conclusions: CHIP and ExSV are prevalent in heart failure patients, impact the outcome of death and metabolic pathways are implicated in mediating the effects of somatic variants/CHIP in heart failure

CACSV: a computational web-sever that provides classification for cancer somatic genetic variants from different tissues

BackgroundUnderstanding the role and function of genetic variants is extremely important when analyzing and interpreting a myriad of human disease processes. For cancer in general, cell-specific genetic variants are ubiquitous and distinct tissues have significantly heterogenic genetic profiles. In clinical practice, only a few genetic variants have identifiable clinical utility. Finding clinically relevant genetic variants constitute a challenging process. In addition, there had been no reference protocol to provide guidance for cancer somatic genetic variants classification and interpretation. In 2017, the first version of a reference protocol was published by the Association for Molecular Pathology, the American Society of Clinical Oncology, and the College of American Pathologists. Previously, we incorporated the reference protocol into a computational method to expedite the process of identification of clinically relevant genetic variants. In this work, we developed a computational web-server to increase the accessibility and availability of clinically relevant genetic variants.ResultsOur work provides the clinical classification for ~ 3 million cancer genetic variants that are now publicly available in a shareable database on GitHub. We have developed a graphical user interface for the database to enhance the accessibility and ease-of-use.ConclusionCACSV provides an open-source for about 3 million cancer tissue-specific genetic variants with their assigned clinical annotations.

Impact of genome and epigenome on intratumor heterogeneity in colorectal cancer

Impact of genome and epigenome on intratumor heterogeneity in colorectal cancer

A familial case of <i>DICER1</i> syndrome in a patient with pleuropulmonary blastoma

Pleuropulmonary blastoma (PPB) is a very rare tumor of childhood that arises from the mesenchyme of the lung and is associated with mutations in the DICER1 gene. The present article describes a familial case of DICER1 syndrome in a patient aged 3 years and 11 months who underwent investigations and treatment for type II/III PPB of the lower lobe of the left lung at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology of Ministry of Healthcare of the Russian Federation. The patient's parents gave their consent to the use of their child's data, including photographs, for research purposes and in publications. The diagnosis was made based on the results of the pathology investigation of the tissue obtained by open biopsy. Furthermore, the diagnosis was confirmed by the review of histological specimens in the International PPB Registry (Minneapolis, USA). According to the data from the chest CT with intravenous contrast enhancement, the tumor was described as a mass consisting of cystic cavities of various calibers accumulating a contrast agent along the periphery, and a solid component. Specific combination treatment was based on the recommendations of the International PPB Registry and included chemotherapy according to the IVADo/IVA regimen and surgical treatment as a local control. Currently, specific treatment has been completed, the patient is alive with no signs of the disease, the follow-up period was 20 months. As regards the patient's family history, he has a sibling (older brother) who was diagnosed with a cystic nephroma at the age of 1 year and 4 months. Molecular genetic testing of the tumor tissues obtained from the patient (PPB) and his sibling (cystic nephroma) revealed pathogenic somatic variants in the DICER1 gene. Along with the somatic genetic variants, another pathogenic variant was found in both children, the germline status of which was confirmed. The germline variant in the DICER1 gene was inherited from the mother. Thus, the investigations confirmed a familial case of DICER1 syndrome. The case described in this article indicates that families in which DICER1 syndrome was found require special management aimed at early detection of tumors and non-tumor conditions associated with this syndrome in the carriers of pathogenic alleles.

Dissecting the Knowns for the Unknowns: Refining the Classification of Genomic Variations in Hematological Diseases By the Clingen Hematological Cancer Taskforce (HCT)

Genomic variants are important diagnostic, prognostic and/or therapeutic markers in hematological malignancies. However, substantial interlaboratorydiscrepancy in interpretation and reporting of these variants creates challenges. To provide a reliable clinical reference and assist the consistent interpretation of genomic variations, the Hematological Cancer Taskforce (HCT) was formed under the ClinGen Somatic Cancer Clinical Domain Working Group in 2020 with a goal to standardize the systematic curation, evidence-based clinical interpretation and reporting of somatic genetic variations found in hematological cancers. A team of multi-disciplinary experts, including genomic scientists, oncologists, hematopathologists, molecular pathologists, clinical lab directors, and biocurators with expertise in hematological malignancies, were recruited to evaluate the genomic alterations seen in hematological malignancies according to the AMP/ASCO/CAP and/or the ClinGen/CGC/VICC guidelines (PMIDs: 27993330, 35101336). The HCT identified FLT3 variants as the highest priority for curation and clinical interpretation efforts based on the immediate and strong clinically actionable implications of FDA-approved FLT3 inhibitors. A ClinGen FLT3 Somatic Cancer Variant Curation Expert Panel (SC-VCEP) was formed to focus on curation of FLT3 tyrosine kinase domain (TKD) variants and is now developing FLT3-specific classification guidelines. FLT3-ITD (internal tandem duplication) and TKD (D835/I836) are the most frequent FLT3 variants with well-established prognostic and therapeutic significance and predict response to Midostaurin and Gilteritinib, FDA-approved drugs for newly diagnosed and relapsed/ refractory AML, respectively. The FLT3SC-VCEP classified their assertions of the FLT3-ITD and D835 variants as Tier 1 - level A in CIViC following the AMP/ASCO/CAP guideline. The interpretation of the rest of FTL3 TKD variants including D835 were less consistent. A pilot assessment to apply the ClinGen/CGC/VICC oncogenicity standards has classified the following D835 variants as oncogenic (OS2, OS3, OP1, OP4): c.2505T>A (Asp835Glu), c.2503G>T (Asp835Tyr), c.2503G>C (Asp835His), and c.2504A>T (Asp835Val); one variant as likely oncogenic: c.2028C>A (Asn676Lys) (OS2, OM4, OP4); and one variant of uncertain significance (VUS): c.2503_2504delinsAT (Asp835Ile) (OM1, OP1, OP3, OP4). Assessment of additional FLT3 variants is underway with the goal to refine FLT3-specific rules to classify FLT3 variants using the AMP/ASCO/CAP guidelines and the ClinGen/CGC/VICC oncogenicity classification standards as a framework. Published evidence using FDA-approved tests simultaneously evaluating the TKD variants in amino acids D835 or I836 often do not specify the affected codon. Due to the high prevalence of D835 variants in AML, the clinical significance of I836 variants is much less studied. In addition to curation from the literature, the FLT3 SC-VCEP is leveraging their international expertise by performing a multi-institutional deidentified data collection for I836 variants from individual laboratories to assess therapeutic response and oncogenicity. Due to the critical need for consistent and standardized interpretation of genetic test results, the HCT aims at developing gene-specific recommendations to standardize the reporting and interpretation of somatic variants and plans to expand ClinGen SC-VCEPs in additional gene-disease domains.

The genomic landscape across 474 surgically accessible epileptogenic human brain lesions.

Understanding the exact molecular mechanisms involved in the etiology of epileptogenic pathologies with or without tumor activity is essential for improving treatment of drug-resistant focal epilepsy. Here, we characterize the landscape of somatic genetic variants in resected brain specimens from 474 individuals with drug-resistant focal epilepsy using deep whole-exome sequencing (>350×) and whole-genome genotyping. Across the exome, we observe a greater number of somatic single-nucleotide variants (SNV) in low-grade epilepsy-associated tumors (LEAT; 7.92 ± 5.65 SNV) than in brain tissue from malformations of cortical development (MCD; 6.11 ± 4 SNV) or hippocampal sclerosis (HS; 5.1 ± 3.04 SNV). Tumor tissues also had the largest number of likely pathogenic variant carrying cells. LEAT had the highest proportion of samples with one or more somatic copy number variants (CNV; 24.7%), followed by MCD (5.4%) and HS (4.1%). Recurring somatic whole chromosome duplications affecting Chromosome 7 (16.8%), chromosome 5 (10.9%), and chromosome 20 (9.9%) were observed among LEAT. For germline variant-associated MCD genes such as TSC2, DEPDC5, and PTEN, germline SNV were frequently identified within large loss of heterozygosity regions, supporting the recently proposed 'second hit' disease mechanism in these genes. We detect somatic variants in twelve established lesional epilepsy genes and demonstrate exome-wide statistical support for three of these in the etiology of LEAT (e.g., BRAF) and MCD (e.g., SLC35A2 and MTOR). We also identify novel significant associations for PTPN11 with LEAT and NRAS Q61 mutated protein with a complex MCD characterized by polymicrogyria and nodular heterotopia. The variants identified in NRAS are known from cancer studies to lead to hyperactivation of NRAS, which can be targeted pharmacologically. We identify large recurrent 1q21-q44 duplication including AKT3 in association with focal cortical dysplasia type 2a with hyaline astrocytic inclusions, another rare and possibly under-recognized brain lesion. The clinical genetic analyses showed that the numbers of somatic SNV across the exome and the fraction of affected cells were positively correlated with the age at seizure onset and surgery in individuals with LEAT. In summary, our comprehensive genetic screen sheds light on the genome-scale landscape of genetic variants in epileptic brain lesions, informs the design of gene panels for clinical diagnostic screening, and guides future directions for clinical implementation of epilepsy surgery genetics.

Gain-of-function defects in toll-like receptor 8 shed light on the interface between immune system and bone marrow failure disorders.

In this article, we will share lessons that patients with gain-of-function defects in Toll-like receptor 8 (TLR8-GOF) can teach us about the interface between bone marrow failure (BMF) disorders and inborn errors of immunity (IEI), subsequently referred to as “Interface Disorders”. TLR8-GOF is a relatively young entity (from a discovery standpoint) that—through both similar and dissimilar disease characteristics—can increase our understanding of interface disorders, for example, as it pertains to pathophysiology, the genetic mechanism of disease, and related diagnostics and therapeutics. From a genetics point of view, TLR8-GOF joins a growing list of (interface) disorders that can cause disease both with germline and somatic (mosaic) genetic variants. This not only has repercussions for the diagnostic workup of these disorders, inasmuch that routine genetic testing may miss somatic variants, but has therapeutic implications as well, for example, with the approach to curative treatment, such as hematopoietic stem cell transplantation. Following an introduction and schematic rendering of the interface, we will review the salient features of TLR8-GOF, with the understanding that the phenotype of this new disorder is likely not written in stone yet. In keeping with the principle of “Form Follows Function”, we will discuss specific immunological biomarkers that can be measured in clinical laboratories and highlight key disease features that pertain to TLR8-GOF, and can be found in several interface disorders. As can be seen from a schematic representation, the interface provides not only opportunities for learning and collaboration with respect to shared diagnostics but also the potential for drug repurposing and precision therapeutics. Ideally, collaboration also focuses on education and teaching, such that cross-fertilization and collaboration across these disciplines can create a framework for complementary research.

Germinal and somatic genetic variants of NF1 in neuroblastoma: own experience and literature review

Introduction. Neuroblastoma (NB) is the most common embryonic extracranial malignant neoplasm in children. The contribution of the NF1 gene to the development of NB is discussed in the literature, but there is no evidence of the pathogenetic role of NF1 gene aberrations in NB. According to various literature sources, the occurrence of pathogenic variants in the NF1 gene in the general cohort of patients with NB does not exceed 1–6 %.Materials and methods. The molecular genetic examination by next generation sequencing (NGS) was performed in 77 patients with NB during the period from April 2019 to July 2021 in the Laboratory of Molecular Oncology Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology, Immunology. The staging of patients was carried out within the framework of the international staging system, stratification into risk groups and therapy according to the protocol of the German Group for the study of NB GPOH NB-2004. International criteria of the response assessment system for patients with NB were used to assess the response to treatment. Calculations of event-free and overall survival by the Kaplan–Mayer method for the follow-up period up to 12.01.2022 were performed.Results and discussion. The cohort of patients included in the study was represented by patients with an initially unfavorable prognosis. Patients were divided into 3 groups: without pathogenic variants in genes belonging to the RAS-RAF-MEK pathway and TP53 – “RAS-/ TP53-” (n = 43), with clinically significant variants in the NF1 gene – “NF1+” (n = 12), clinically significant variants in the genes of RAS-RAF-MEK and TP53 pathway – “RAS+/TP53+” except NF1 (n = 22). The median age for the entire group of patients at the time of diagnosis was 41 months (0.1–173 months). Boys prevailed over girls with a ratio of 1.5:1. Patients with stage 4 of the disease according to the INSS classification prevailed – 81.8 % (63/77), high-risk groups according to the NB-2004 protocol – 77.9 % (60/77).In our study 13 clinically significant variants in NF1 were identified in 12 patients (15.6 %), of which 4 were germinal, 9 were somatic. The frequency of detection of pathogenic aberrations in the NF1 gene was much higher than the literature data, which can be associated with a selective cohort of studied patients with an unfavorable prognosis and patients with suspected hereditary genetic syndrome. The presentation of adverse events was observed in 83.3 % of patients, more often against the background of specific therapy (in 60 % of cases), which may be due to the rapid acquisition of NB chemoresistance, among other things. When comparing the three groups, it was shown that the frequency of objective responses to induction therapy was statistically significantly lower in the group of patients “NF1+”, when compared with other groups (p = 0.015; p = 0.024), which may also indicate the chemoresistance of NF1-aberrated NB. When analyzing survival there was no statistical difference between the compared groups.Conclusions. The data obtained by us do not allow us to consider the presence of genetic variants in NF1 separately as a prognostic factor, however, it can be assumed that a group of patients with an unfavorable prognosis may be enriched with cases with mutations in the NF1 gene. Refractory course of the disease/development of adverse events in the presence of genetic variants of NF1, causing the activation of the RASRAF- MEK signaling pathway, leads to the induction of tumor chemoresistance. The presence of clinical significance of aberrations in the NF1 gene does not lead to a statistically significant difference in prognosis when compared with patients with aberrations in other components of the RAS-RAF-MEK pathway, however, longer catamnestic follow-up of patients is necessary.Currently, there are no effective drugs for the treatment of NF1-associated NB in clinical practice that requires further study of the mechanisms of chemoresistance development in such patients. Understanding the molecular and genetic features of the course of NF1-associated NB can become the basis for the development of personalized therapy in the future.

Molecular Biomarkers in Cancer

Molecular cancer biomarkers are any measurable molecular indicator of risk of cancer, occurrence of cancer, or patient outcome. They may include germline or somatic genetic variants, epigenetic signatures, transcriptional changes, and proteomic signatures. These indicators are based on biomolecules, such as nucleic acids and proteins, that can be detected in samples obtained from tissues through tumor biopsy or, more easily and non-invasively, from blood (or serum or plasma), saliva, buccal swabs, stool, urine, etc. Detection technologies have advanced tremendously over the last decades, including techniques such as next-generation sequencing, nanotechnology, or methods to study circulating tumor DNA/RNA or exosomes. Clinical applications of biomarkers are extensive. They can be used as tools for cancer risk assessment, screening and early detection of cancer, accurate diagnosis, patient prognosis, prediction of response to therapy, and cancer surveillance and monitoring response. Therefore, they can help to optimize making decisions in clinical practice. Moreover, precision oncology is needed for newly developed targeted therapies, as they are functional only in patients with specific cancer genetic mutations, and biomarkers are the tools used for the identification of these subsets of patients. Improvement in the field of cancer biomarkers is, however, needed to overcome the scientific challenge of developing new biomarkers with greater sensitivity, specificity, and positive predictive value.

Abstract 1696: Cell-type specific mutational signatures as a proxy for cell-of-origin identification in lung cancer histological subtypes

Abstract Tumor evolution is dictated by the genetic, epigenetic and transcriptional landscape of the ‘cell-of-origin’ or the tumor initiating cell. While the genomic landscape of cancers has been extensively profiled, efforts to identify the cell-of-origin in individual tumors have been largely restricted to cell type specific activation of oncogenes in genetically engineered mouse models and correlative studies on gene expression patterns between normal tissue and tumor. Prior work from our lab has shown that highly expressed genes within a cell exhibit an overall increase in mutation rates as a consequence of transcription-coupled damage (TCD) which overwhelms transcription-coupled repair (TCR) relative to genes with low or medium level expressions. We therefore hypothesized that tumor cells retain a TCD-mutational signature representative of their cell-of-origin, enabling us to identify the cell-of-origin from the mutational landscape of human tumors.To test this hypothesis, we explored tumors originating from the lung which comprise comprise of distinct histological subtypes: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) that is further categorized into lung adenocarcinoma (LUAD), squamous cell lung cancer (LUSC) and large cell carcinoma (LCC). To identify lung epithelial cell type signatures, we first analyzed a publicly available single cell transcriptomic atlas of the normal human lung of 9,407 cells. For each of the 14 epithelial cell types defined in the lung atlas, we curated a gene signature of its respective unique and highly expressed genes. We next studied the somatic genetic variants in a dataset of 488 samples, which consisted of 230 LUAD tumors and 178 LUSC tumors from TCGA as well as 80 SCLC tumors from a previously published dataset. For each of the 488 tumors, we generated TCD-scores as a function of mutation frequency for each cell-type specific gene signature.Unsupervised hierarchical clustering on the 14 TCD-scores corresponding to the 14 epithelial cell types for each tumor, revealed heterogeneous patterns of cell-type specific mutational signatures. More specifically, LUAD samples were heterogeneously enriched for high AT2-specific, club-specific, ciliated-specific TCD-scores, while LUSC tumors were enriched for basal-specific and ciliated-specific TCD-scores. Together these data suggest that a tumor’s mutational profile can serve as a metric for the identification of the cell-of-origin. Further work will test the extension in different cancer types in other organs or a pan-cancer analysis. The identification of cell-of-origin for cancer subtypes would enable a better understanding of the tumor biology, and ultimately may lead to preventive therapies for individuals at high risk of developing cancer. Citation Format: Ayushi S. Patel, Itai Yanai. Cell-type specific mutational signatures as a proxy for cell-of-origin identification in lung cancer histological subtypes [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 1696.

Somatic mutational landscape of extracranial arteriovenous malformations and phenotypic correlations.

Somatic genetic variants may be the cause of extracranial arteriovenous malformations, but few studies have explored these genetic anomalies, and no genotype-phenotype correlations have been identified. The aim of the study was to characterize the somatic genetic landscape of extracranial arteriovenous malformations and correlate these findings with the phenotypic characteristics of these lesions. This study included twenty-three patients with extracranial arteriovenous malformations that were confirmed clinically and treated by surgical resection, and for whom frozen tissue samples were available. Targeted next-generation sequencing analysis of tissues was performed using a gene panel that included vascular disease-related genes and tumour-related genes. We identified a pathogenic variant in 18 out of 23 samples (78.3%). Pathogenic variants were mainly located in MAP2K1 (n = 7) and KRAS (n = 6), and more rarely in BRAF (n = 2) and RASA1 (n = 3). KRAS variants were significantly (P < 0.005) associated with severe extended facial arteriovenous malformations, for which relapse after surgical resection is frequently observed, while MAP2K1 variants were significantly (P < 0.005) associated with less severe, limited arteriovenous malformations located on the lips. Our study highlights a high prevalence of pathogenic somatic variants, predominantly in MAP2K1 and KRAS, in extracranial arteriovenous malformations. In addition, our study identifies for the first time a correlation between the genotype, clinical severity and angiographic characteristics of extracranial arteriovenous malformations. The RAS/MAPK variants identified in this study are known to be associated with malignant tumours for which targeted therapies have already been developed. Thus, identification of these somatic variants could lead to new therapeutic options to improve the management of patients with extracranial arteriovenous malformations.

Interval breast cancer is associated with interferon immune response

BackgroundThe aggressive nature of breast cancers detected between planned mammographic screens, so-called interval cancers, remains elusive. Here, we aim to characterise underlying molecular features of interval cancer. MethodsFrom 672 patients with invasive breast cancer, we analysed gene expression differences between 90 ‘true’ interval cancer cases (i.e. women with low-dense breasts defined as per cent mammographic density <25%) and 310 screen-detected tumours while accounting for PAM50 subtypes and thus overall tumour aggressiveness. We computed an interval cancer gene expression profile (IC-Gx) in a total of 2270 breast tumours (regardless of interval cancer status) and tested for association with expression-based immune subtypes in breast cancer. In addition, we investigated the contribution of inherited and somatic genetic variants in distinct features of interval cancer. ResultsWe identified 8331 genes nominally associated with interval cancer (P-value < 0.05, fold-change > 1.5). Gene set enrichment analysis showed immune-related pathways as key processes altered in interval cancer. Our IC-Gx, based on 47 genes with the strongest associations (false discovery rate < 0.05), was found to be associated mainly with immune subtypes involving interferon response. We isolated an interaction network of interval cancer and interferon genes for which a significant load of somatic and germline variants in class I interferon genes was observed. ConclusionWe identified novel molecular features of interval breast cancer highlighting interferon pathways as a potential target for prevention or treatment.