Somatic Hotspot Mutations Research Articles

Somatic DICER1-Mutant Benign Thyroid Nodules in Adults: A Group of Follicular Nodular Disease With Continuous Growth.

Germline DICER1 mutations cause familial multinodular goiter (MNG). However, the prevalence of somatic DICER1 mutations in non-MNG benign thyroid nodules and their characteristics remain unknown. Adult-onset thyroid nodules with a pathological diagnosis were genotyped via targeted sequencing. DICER1-mutant nodules were assessed clinically and pathologically. Organoids were established to investigate follicular formation and growth. Transcriptomic analysis was conducted to evaluate transcriptional features, which were validated by immunofluorescence. Among 931 adult-onset thyroid nodules, we identified 13 benign thyroid nodules with DICER1 hotspot mutations. The majority harbored a somatic DICER1 hotspot mutation with a somatic DICER1 truncating variant. Clinically, 38.5% of the DICER1-mutant nodules exhibited substantial growth. DICER1-mutant nodules with durations longer than 2 years were substantially enlarged (P=.0448). Pathologically, all DICER1-mutant nodules were defined as thyroid follicular nodular disease (TFND). The TFND nodules with DICER1 mutations grew faster than those with wild-type DICER1. Organoid culture of a DICER1-mutant nodule revealed increased active follicular formation. Compared with the normal thyroid tissues, the DICER1-mutant nodules had comparable thyroid differentiation scores, significantly higher ERK scores (P=.0141) and lower epithelial‒mesenchymal transition scores (P=.0001). Moreover, the expression of genes related to follicular polarity, such as CDH16, SLC5A5, TSHR and TPO, was downregulated in the DICER1-mutant nodules. Somatic DICER1 two-hit mutations represent a notable percentage in adult TFND patients, and DICER1-mutant benign thyroid nodules were characterized by continuous growth.

Melanoma-specific mutation hotspots in distal, non-coding, promoter-interacting regions implicate novel candidate driver genes.

To develop targeted treatments, it is crucial to identify the full spectrum of genetic drivers in melanoma, including those in non-coding regions. However, recent efforts to explore non-coding regions have primarily focused on gene-adjacent elements such as promoters and non-coding RNAs, leaving intergenic distal regulatory elements largely unexplored. We used Hi-C chromatin contact data from melanoma cells to map distal, non-coding, promoter-interacting regulatory elements genome-wide in melanoma. Using this "promoter-interaction network", alongside whole-genome sequence and gene expression data from the Pan Cancer Analysis of Whole Genomes, we developed multivariate linear regression models to identify distal somatic mutation hotspots that affect promoter activity. We identified eight recurrently mutated hotspots that are novel, melanoma-specific, located in promoter-interacting distal regulatory elements, alter transcription factor binding motifs, and affect the expression of genes (e.g., HSPB7, CLDN1, ADCY9 and FDXR) previously implicated as tumour suppressors/oncogenes in various cancers. Our study suggests additional non-coding drivers beyond the well-characterised TERT promoter in melanoma, offering new insights into the disruption of complex regulatory networks by non-coding mutations that may contribute to melanoma development. Furthermore, our study provides a framework for integrating multiple levels of biological data to uncover cancer-specific non-coding drivers.

Clinical Utility of CTNNB1 (c.121A>G) Mutation in Circulating Tumor DNA in Egyptian Patients with Hepatocellular Carcinoma: A Case-Control Study

Abstract Background Hepatocellular carcinoma (HCC) is the most common malignancy of the liver and with 782,000 new cases annually and 600,000 deaths/year globally. In Egypt, HCC causes a significant public health problem with a rising incidence as it is strongly linked to viral hepatitis mainly HCV. CTNNB1 mutations is one of the most common genetic alterations in HCC. Our aim is to investigate the clinical utility of CTNNB1 (rs121913412) single nucleotide variation (SNV) in ctDNA in patients with early and late-stage HCC in comparison to chronic HCV patients and healthy controls. Methods All participants were subjected to history taking, clinical evaluation, laboratory investigations including liver profile, kidney function tests, alfa-fetoprotein. Additionally, complete blood counts and coagulation profile were done. Child and Model for End-Stage Liver Disease (MELD) scores were calculated, BCLC staging was done for each patient and radiological protocol for HCC (spiral triphasic CT or MRI). Genotyping of rs121913412 CTNNB1 (c.121A>G) on extracted DNA from blood samples was done using Real Time PCR System. Results Thirty patients with HCC on top of HCV were recruited including 14 early HCC and 16 with advanced HCC in addition to non-HCC group including 10 HCV-positive patients and 10 healthy controls. All participants have undergone laboratory investigations and radiological protocol for HCC cases. Genotyping of rs121913412 CTNNB1 (c.121A>G) in peripheral blood samples using RT-PCR for all cases was done. The genotype AA was detected in 27out of 30 (30%) of the cases while 3 HCC cases out of 30 (10 %) were positive for the CTNNB1 p.T41A mutation; AG phenotype (heterozygous), no cases showed GG phenotype. In HCV- positive patients’ group and healthy controls none were positive for the CTNNB1 mutation all showed wild AA genotype. No significant differences in the genotype (AA, AG, GG) distribution between HCC and non-HCC groups (χ2=3.619, p = 0.0571). The frequency of AA vs AG + GG genotype was considerably higher in cases when compared to controls however no statistical significance was detected (χ2=3.619, p = 0.0571). Distribution of A vs G allele in cases compared to controls showed statistically significant difference (χ2=4.571, p = 0.0325). Conclusion In this study we were not able to prove the clinical utility of CTNNB1 rs121913412 in Egyptian HCC patients. Further large multi-centric studies are needed to explore the clinical utility of ctDNA in assessment of tumor-specific hotspot somatic mutations in genes including CTNNB1, TERT and TP53 genes that can be identified in the peripheral blood of HCC patients. Comparison studies utilizing both ctDNA and DNA extracted from HCC tissue samples are recommended.

Histone H3 Variants in the Multiverse of Cancer

Our understanding of cancer genomes has allowed for the integration of molecular data into histopathological classifications for routine stratification of patients. In the last 10–15 years, thanks to this systematic implementation of large-scale sequencing, the identification of hotspot somatic mutations in histone genes came into the limelight, underscoring the concept of oncohistones. As drivers in pediatric brain tumors, and in several other types of cancers, oncohistones brought a “new dimension of Strange” into the cancer multiverse, to paraphrase Marvel. An integrative approach to cancer complexity as a multidimensional projection is urgently needed to consider all relevant etiological, developmental, and evolutionary components. Here, we discuss recent progress on histone variants and chaperones, their regulation and alterations in cancers, the available in vivo models, and current treatment strategies. More specifically, we adopt a view through the lens of tissue-specific differences and means for genome expression and integrity maintenance.

Delineating the mechanistic relevance of the TP53 gene and its mutational impact on gene expression and patients’ survival in bladder cancer

Bladder carcinoma (BLCA) is a widespread urological malignancy causing significant global mortality, often hindered by delayed diagnosis and limited treatments. BLCA frequently exhibits TP53 mutations, playing a pivotal role in its pathogenesis and underscoring the potential of targeting TP53 as a therapeutic approach for this prevalent urological malignancy. Tumor tissues from 50 bladder cancer patients were used for mutational analysis in TP53's mutation-rich exons (5, 7, & 8). The gene expression of the TP53 gene, along with a TP53-target gene B‐cell translocation gene 2 (BTG2) was also assessed in the cDNA samples from the same BLCA tissues and 15 urine controls of healthy people. The analysis revealed 22 % of patients with somatic hotspot mutations, 18 % with pathogenic missense mutations, and 12 % with intronic variants. Patients with somatic mutations exhibited the worst prognosis, supported by survival analysis from The Cancer Genome Atlas (TCGA) BLCA data. Interestingly, H296Y missense mutation correlated with higher TP53 expression and improved survival, while intronic SNPs were linked to worse outcomes. Additionally, upregulated BTG2 expression in mutated patients was observed which was correlated with poor prognosis, emphasizing the role of TP53 mutations in bladder cancer progression. The multivariate analysis highlighted the predictive power of TP53 mutations, with a high frequency of high-grade tumors (78.57 %) in mutated patients, underscoring their role in cancer progression. In conclusion, this study emphasizes the crucial role of TP53 mutations in bladder cancer patients from Bangladesh.

Prevalence and effect of PIK3CA H1047R somatic mutation among Indian head and neck cancer patients

PIK3CA is one among the several mutated genes in cancer, including head and neck squamous cell carcinoma (HNSCC). H1047R is a hotspot somatic mutation in PIK3CA that occurs most frequently in several forms of cancers. Distribution of PIK3CA H1047R mutation in Indian HNSCC patients was screened and its effect on disease progression and response to treatment was analysed in this study. Genomic DNA was extracted from tumour biopsies of HNSCC patients (n = 48) and polymerase chain reaction coupled restriction fragment length polymorphism (PCR-RFLP) technique was used to screen for the mutation. Overall survival (OS) and Progression-free survival (PFS) of the patients were calculated in order to study effect of this mutation on survival and response to treatment respectively. Results showed that irrespective of patients’ criteria, twenty-five patients (52 %) carried a heterozygous form of mutation (His/Arg) and the rest (48 %) were wild type (His/His). The mean OS of the cohort with the mutation was 20.451 months (SE ± 1.710 months) while 26.31 months (SE ± 2.431) was in wild type population. PFS of the patients with the mutation was 18.612 months (SE ± 2.072), and for the wild type population, it was 26.31 months (SE ± 2.431). These observations suggest that Indian HNSCC patients with PIK3CA H1047R mutation have poor prognosis.

Clinicopathological analysis of 22 Müllerian adenosarcomas and the sequencing of DICER1 mutation

BackgroundMüllerian adenosarcoma, a rare malignancy, presents diagnostic and therapeutic challenges. In this study, we conducted an analysis of the clinicopathological characteristics of 22 adenosarcomas, with a particular focus on screening for DICER1 hot mutations.MethodsThe cohort consisted of patients with adenosarcoma who were registered at the West China Second Hospital between the years 2020 and June 2022. Sanger sequencing was employed to screen for somatic Hotspot mutations in the RNase IIIb domain of DICER1 in the 22 adenosarcomas.ResultsOnly one patient exhibited a DICER1 mutation that was not a DICER1 Hotspot mutation. Among the 22 patients, all underwent total hysterectomy with bilateral salpingo-oophorectomy, and 14 out of these 22 patients received adjuvant treatment.ConclusionIn summary, our study of 22 Müllerian adenosarcomas focused on the clinicopathological features and the presence of DICER1 Hotspot mutations. Although our findings did not reveal any DICER1 mutations in the studied samples, this negative result provides valuable information for the field by narrowing down the genetic landscape of adenosarcomas and highlighting the need for further research into alternative molecular pathways driving this malignancy.

Tissue-specific enhancer-gene maps from multimodal single-cell data identify causal disease alleles.

Translating genome-wide association study (GWAS) loci into causal variants and genes requires accurate cell-type-specific enhancer-gene maps from disease-relevant tissues. Building enhancer-gene maps is essential but challenging with current experimental methods in primary human tissues. Here we developed a nonparametric statistical method, SCENT (single-cell enhancer target gene mapping), that models association between enhancer chromatin accessibility and gene expression in single-cell or nucleus multimodal RNA sequencing and ATAC sequencing data. We applied SCENT to 9 multimodal datasets including >120,000 single cells or nuclei and created 23 cell-type-specific enhancer-gene maps. These maps were highly enriched for causal variants in expression quantitative loci and GWAS for 1,143 diseases and traits. We identified likely causal genes for both common and rare diseases and linked somatic mutation hotspots to target genes. We demonstrate that application of SCENT to multimodal data from disease-relevant human tissue enables the scalable construction of accurate cell-type-specific enhancer-gene maps, essential for defining noncoding variant function.

Impact of Somatic Second Hit and Comutations in Myeloid Neoplasms with Germline DDX41 Mutations

Background: Germline DDX41 mutations are the most common genetic predisposition to myeloid neoplasms (MN) and have been suggested to represent a distinct MN subtype. A second somatic DDX41 mutation (second hit) is known to occur in most patients, with a clear predominance for R525H. However, its impact on disease progression and survival remains elusive. Additionally, comutation patterns are not well defined across all MN subtypes, with limited evidence regarding their prognostic significance. Methods: We performed a retrospective cohort study to evaluate outcomes of DDX41-mutant MN, focusing on the impact of co-occurring mutations, including second hit DDX41 lesions. We included 100 patients with at least 1 DDX41 variant and a MN or precursor condition. DDX41 mutation was identified by an in-house NGS myeloid gene panel. Skin biopsy was the most frequent DNA source for germline testing. When this was not available, we applied a validated model to predict the nature of DDX41 variants based on VAF, public germline databases and known hotspot somatic mutations. Kaplan-Meier analysis and Cox models were used for survival analyses. Observations were censored for transplant. Results: Overall, the median age at diagnosis was 71 years (IQR 65-79) and 66% were male. The underlying diagnosis was MDS in 43% of cases, AML in 47% and other MN in 10% (including chronic myelomonocytic leukemia, myeloproliferative neoplasms and clonal cytopenia of undetermined significance). Most patients had a normal karyotype (77%, 72/93). In the MDS cohort 36% (14/39) had higher-risk (HR) disease (IPSS-R>3.5) and 30% of AML patients (14/46) had adverse risk disease (ELN 2022). Median overall survival (OS) for patients with HR-MDS or AML was 24.6 months (95% CI 7.7-37.4). Out of 169 identified DDX41 variants, 51% were predicted as germline, corresponding to 86% of all patients carrying a germline variant. Twenty-one patients had a DDX41 mutation confirmed in germline DNA, and all would have been predicted by the model. In our cohort, most MN patients carrying a germline DDX41 mutation had a somatic second hit (71%, 61/86) but the presence of a second hit did not impact OS (logrank p=0.72) and its prevalence was not significantly different in patients with lower-risk (LR)-MDS, HR-MDS or AML (p=0.45). This suggests that, albeit the occurrence of a second hit seems related to developing a MN, its presence is not necessarily associated with progression nor worse survival. There was a numerically worse median OS in those harboring the R525H second hit (33 vs 89 months), but the survival distribution was not significantly different (logrank p=0.28). However, an increasing R525H prevalence was noted from LR-MDS to HR-MDS to AML (33%, 67%, 77%; p=0.004). Among patients with germline DDX41 variants, the presence of at least one somatic mutation beyond DDX41 (comutation) was associated with significantly worse OS in univariate analysis (HR 4.26, 95% CI 1.16-15.67, p=0.03) but did not retain significance when diagnosis was added to the model (HR 2.43, 95% CI 0.57-10.36, p=0.23). Yet, the distribution of the proportion of patients with at least one comutation between LR-MDS, HR-MDS and AML was significantly different (p=0.03), with an increase from LR-MDS to the other 2 categories. The same results held true for the presence of 2 or more mutations. The top 5 comutations were TET2 (14%), ASXL1 (13%), SRSF2 (11%), DNMT3A (10%) and CUX1 (10%). Interestingly, there was a significantly lower prevalence of CUX1 in MDS than in AML (2% vs 17%, p=0.02) and a tendency for increased frequency from LR-MDS to HR-MDS to AML (0% vs 7% vs 17%, p=0.07), highlighting a potential role in disease progression. There was no significant difference when comparing the clone sizes of second hit DDX41 mutations and comutations (median VAF 14% vs 14%, p= 0.78). Conclusions: While most patients with germline DDX41 MN harbor a somatic second hit, its presence was not predictive of survival and its frequency was not differently distributed across the MDS-AML continuum. The presence of additional comutations equally lacked survival impact. However, the frequency of patients with any somatic comutation, the frequency of R525H second hit, and the occurrence of CUX1 mutation increased from MDS to AML, placing these as candidate drivers of leukemic progression in germline DDX41 MN. Moreover, similar clone sizes of somatic DDX41 mutations and comutations favors co-occurrence of these events.

TERT promoter mutations and gene amplification in endometrial cancer

ObjectiveTo assess the clinicopathologic, molecular profiles, and survival outcomes of patients with endometrial carcinomas (ECs) harboring telomerase reverse transcriptase (TERT) hotspot mutations or gene amplification. MethodsECs harboring somatic TERT promoter hotspot mutations or gene amplification (TERT-altered) were identified from 1944 ECs that underwent clinical tumor-normal sequencing from 08/2016–12/2021. Clinicopathologic variables, somatic mutation profiles, and survival outcomes of TERT-alt and TERT-wild-type EC were assessed. ResultsWe identified 66 TERT-altered ECs (43 TERT-mutated and 23 TERT-amplified), representing 3% of the unselected ECs across histologic subtypes. Most TERT-altered ECs were of copy number (CN)-high/TP53abn molecular subtype (n = 40, 60%), followed by microsatellite-unstable (MSI-H) or CN-low/no specific molecular profile (NSMP)(n = 13, 20% each). TERT-amplified and TERT-mutated ECs were molecularly distinct, with TERT-amplified ECs being more genomically instable and more frequently harboring TP53 and PPP2R1A alterations (q < 0.1). Compared to TERT-wild-type ECs, TERT-altered ECs were more commonly of CN-H/TP53abn molecular subtype (31% vs 57%, p = 0.001), serous histology (10% vs 26%, p = 0.004), and were significantly enriched for TP53, CDKN2A/B, and DROSHA somatic genetic alterations (q < 0.1). Median progression-free survival was 18.7 months (95% CI 11.8–not estimable [NE]) for patients with TERT-altered EC and 80.9 months (65.8-NE) for patients with TERT-wild-type EC (HR 0.33, 95% CI 0.21–0.51, p < 0.001). Similarly, median overall survival was 46.7 months (95% CI 30-NE) for TERT-altered EC patients and not reached for TERT-wild-type EC patients (HR 0.24, 95% CI 0.13–0.44, p < 0.001). ConclusionTERT-altered ECs, although rare, are enriched for CN-high/TP53abn tumors, TP53, CDKN2A/B and DROSHA somatic mutations, and independently predict worse survival outcomes.

Somatic ARAF mutations in pediatric Langerhans cell histiocytosis: clinicopathologic, genetic and functional profiling.

ARAF mutations have been identified in a limited subset of patients with Langerhans cell histiocytosis (LCH), a rare disorder characterized by abnormal proliferation of Langerhans cells. LCH is primarily instigated by mutations in the mitogen-activated protein kinase (MAPK) signaling pathway, with BRAFV600E and MAP2K1 mutations constituting most cases. ARAF mutations in LCH highlight the heterogeneity of the disease and provide insights into its underlying molecular mechanisms. However, the occurrence of ARAF-positive LCH cases is extremely rare, with only two reported globally. Although they may be linked to a more aggressive form of LCH and a more severe clinical progression, the clinical significance and functional consequences of these mutations remain uncertain. We performed next-generation sequencing (NGS) to explore driver mutations in 148 pediatric LCH patients and recognized a series of mutations, including an identical novel somatic ARAF mutation, c.1046_1051delAGGCTT (p.Q349_F351delinsL), in four pediatric LCH patients. It was considered an ARAF hotspot mutation. All reported ARAF-positive patients worldwide exhibited characteristic pathological features of LCH, albeit with involvement across multiple systems. In vitro functional studies showed that this mutation could trigger the MAPKinase pathway and phosphorylate its downstream effectors MEK1/2 and ERK1/2 (relatively weaker than BRAFV600E). Over-activation of mutant A-Raf kinase could be inhibited by the BRAF inhibitor vemurafenib. LCH is uncommon, and ARAF mutation is even rarer. In our study, we have identified a novel hotspot somatic ARAF mutation, which has been verified through functional analysis to be an activating mutation. LCH patients with ARAF mutation typically have an unfavorable prognosis due to limited treatment experiences, although they do not exhibit a high relapse rate. To aid in the development of personalized treatment approaches and prognostic markers for LCH patients, it is recommended to conduct typical pathological and immunohistochemical examinations, as well as genetic tests utilizing a targeted gene panel or whole exome sequencing (WES), for LCH diagnosis, thereby promoting the use of inhibitor treatment strategies.

Protein Tyrosine Phosphatase Non-Receptor 11 (PTPN11/Shp2) as a Driver Oncogene and a Novel Therapeutic Target in Non-Small Cell Lung Cancer (NSCLC)

PTPN11 encodes the SHP2 protein tyrosine phosphatase that activates the mitogen-activated protein kinase (MAPK) pathway upstream of KRAS and MEK. PTPN11/Shp2 somatic mutations occur frequently in Juvenile myelomonocytic leukaemia (JMML); however, the role of mutated PTPN11 in lung cancer tumourigenesis and its utility as a therapeutic target has not been fully addressed. We applied mass-spectrometry-based genotyping to DNA extracted from the tumour and matched the normal tissue of 356 NSCLC patients (98 adenocarcinomas (LUAD) and 258 squamous cell carcinomas (LUSC)). Further, PTPN11 mutation cases were identified in additional cohorts, including TCGA, Broad, and MD Anderson datasets and the COSMIC database. PTPN11 constructs harbouring PTPN11 E76A, A72D and C459S mutations were stably expressed in IL-3 dependent BaF3 cells and NSCLC cell lines (NCI-H1703, NCI-H157, NCI-H1299). The MAPK and PI3K pathway activation was evaluated using Western blotting. PTPN11/Shp2 phosphatase activity was measured in whole-cell protein lysates using an Shp2 assay kit. The Shp2 inhibitor (SHPi) was assessed both in vitro and in vivo in a PTPN11-mutated cell line for improved responses to MAPK and PI3K targeting therapies. Somatic PTPN11 hotspot mutations occurred in 4/98 (4.1%) adenocarcinomas and 7/258 (2.7%) squamous cells of 356 NSCLC patients. Additional 26 PTPN11 hotspot mutations occurred in 23 and 3 adenocarcinomas and squamous cell carcinoma, respectively, across the additional cohorts. Mutant PTPN11 significantly increased the IL-3 independent survival of Ba/F3 cells compared to wildtype PTPN11 (p &lt; 0.0001). Ba/F3, NCI-H1703, and NCI-H157 cells expressing mutant PTPN11 exhibited increased PTPN11/Shp2 phosphatase activity and phospho-ERK1/2 levels compared to cells expressing wildtype PTPN11. The transduction of the PTPN11 inactivating mutation C459S into NSCLC cell lines led to decreased phospho-ERK, as well as decreased phospho-AKT in the PTPN11-mutated NCI-H661 cell line. NCI-H661 cells (PTPN11-mutated, KRAS-wild type) were significantly more sensitive to growth inhibition by the PI3K inhibitor copanlisib (IC50: 13.9 ± 4.7 nM) compared to NCI-H1703 (PTPN11/KRAS-wild type) cells (IC50: &gt;10,000 nM). The SHP2 inhibitor, in combination with the PI3K targeting therapy copanlisib, showed no significant difference in tumour development in vivo; however, this significantly prevented MAPK pathway induction in vitro (p &lt; 0.0001). PTPN11/Shp2 demonstrated the in vitro features of a driver oncogene and could potentially sensitize NSCLC cells to PI3K inhibition and inhibit MAPK pathway activation following PI3K pathway targeting.

Reclassification of two germline DICER1 splicing variants leads to DICER1 syndrome diagnosis

DICER1 syndrome is an inherited condition associated with an increased risk of developing hamartomatous and neoplastic lesions in diverse organs, mainly at early ages. Germline pathogenic variants in DICER1 cause this condition. Detecting a variant of uncertain significance in DICER1 or finding uncommon phenotypes complicate the diagnosis and can negatively impact patient care. We present two unrelated patients suspected to have DICER1 syndrome. Both females (aged 13 and 15 years) presented with multinodular goiter (thyroid follicular nodular disease) and ovarian tumours. One was diagnosed with an ovarian Sertoli-Leydig cell tumour (SLCT) and the other, with an ovarian juvenile granulosa cell tumour, later reclassified as a retiform variant of SLCT. Genetic screening showed no germline pathogenic variants in DICER1. However, two potentially splicing variants were found, DICER1 c.5365-4A>G and c.5527+3A>G. Also, typical somatic DICER1 RNase IIIb hotspot mutations were detected in the thyroid and ovarian tissues. In silico splicing algorithms predicted altered splicing for both germline variants and skipping of exon 25 was confirmed by RNA assays for both variants. The reclassification of the ovarian tumour, leading to recognition of the association with DICER1 syndrome and the characterization of the germline intronic variants were all applied to recently described DICER1 variant classification rules. This ultimately resulted in confirmation of DICER1 syndrome in the two teenage girls.

Base-resolution UV footprinting by sequencing reveals distinctive damage signatures for DNA-binding proteins

Decades ago, it was shown that proteins binding to DNA can quantitatively alter the formation of DNA damage by UV light. This established the principle of UV footprinting for non-intrusive study of protein-DNA contacts in living cells, albeit at limited scale and precision. Here, we perform deep base-resolution quantification of the principal UV damage lesion, the cyclobutane pyrimidine dimer (CPD), at select human promoter regions using targeted CPD sequencing. Several transcription factors exhibited distinctive and repeatable damage signatures indicative of site occupancy, involving strong (up to 17-fold) position-specific elevations and reductions in CPD formation frequency relative to naked DNA. Positive damage modulation at some ETS transcription factor binding sites coincided at base level with melanoma somatic mutation hotspots. Our work provides proof of concept for the study of protein-DNA interactions at individual loci using light and sequencing, and reveals widespread and potent modulation of UV damage in regulatory regions.

Familial Clonal Hematopoiesis in a Long Telomere Syndrome

BackgroundTelomere shortening is a well-characterized cellular aging mechanism, and short telomere syndromes cause age-related disease. However, whether long telomere length is advantageous is poorly understood.MethodsWe examined the clinical and molecular features of aging and cancer in persons carrying heterozygous loss-of-function mutations in the telomere-related gene POT1 and noncarrier relatives.ResultsA total of 17 POT1 mutation carriers and 21 noncarrier relatives were initially included in the study, and a validation cohort of 6 additional mutation carriers was subsequently recruited. A majority of the POT1 mutation carriers with telomere length evaluated (9 of 13) had long telomeres (>99th percentile). POT1 mutation carriers had a range of benign and malignant neoplasms involving epithelial, mesenchymal, and neuronal tissues in addition to B- and T-cell lymphoma and myeloid cancers. Five of 18 POT1 mutation carriers (28%) had T-cell clonality, and 8 of 12 (67%) had clonal hematopoiesis of indeterminate potential. A predisposition to clonal hematopoiesis had an autosomal dominant pattern of inheritance, as well as penetrance that increased with age; somatic DNMT3A and JAK2 hotspot mutations were common. These and other somatic driver mutations probably arose in the first decades of life, and their lineages secondarily accumulated a higher mutation burden characterized by a clocklike signature. Successive generations showed genetic anticipation (i.e., an increasingly early onset of disease). In contrast to noncarrier relatives, who had the typical telomere shortening with age, POT1 mutation carriers maintained telomere length over the course of 2 years.ConclusionsPOT1 mutations associated with long telomere length conferred a predisposition to a familial clonal hematopoiesis syndrome that was associated with a range of benign and malignant solid neoplasms. The risk of these phenotypes was mediated by extended cellular longevity and by the capacity to maintain telomeres over time. (Funded by the National Institutes of Health and others.)

Abstract 3488: Truncated FGFR2 - a clinically actionable oncogene in multiple cancers

Abstract Human cancers frequently bear driver alterations in genes encoding receptor tyrosine kinases (RTKs), which has led to effective therapeutics targeting oncogenic signaling of RTK variants. Somatic hotspot mutations and structural amplifications and fusions affecting fibroblast growth factor receptor 2 (FGFR2) likewise occur in multiple tumor types including breast cancer. However, clinical responses to FGFR inhibitors have remained variable, emphasizing a need to better understand which FGFR2 alterations are oncogenic and therapeutically targetable. We applied transposon-based screening and tumor modelling in the mouse mammary gland to uncover truncation of the last exon (E18) of Fgfr2 as a potent driver mutation. Mouse and human FGFR2-E18 encodes the C-terminus of this RTK. Human oncogenomic datasets revealed a plethora of somatic FGFR2 alterations potentially causing transcription of E18-truncated FGFR2. These alterations were comprised of canonical in-frame fusions as well as diverse FGFR2 variants of unknown significance (VUS), which included non-canonical rearrangements, E1-E17 partial amplifications, and E18 nonsense and frameshift mutations. Functional in vitro and in vivo interrogation of a compendium of E18-truncated and full-length FGFR2 variants pinpointed FGFR2-E18 truncation as single-driver alteration in cancer. In contrast, the oncogenic competence of FGFR2 full-length amplifications depended on a distinct cooperating driver gene landscape. Notably, gradual truncation and site-directed mutagenesis of Fgfr2-E18 identified a novel 2-amino-acid motif within the C-terminus critical for kinase domain binding and suppression of oncogenic FGFR2 signaling. Aberration of this motif conspired with the loss of the receptor internalization motif to fully phenocopy oncogenicity of E18-truncated Fgfr2. These data suggest that genomic alterations that generate stable E18-truncated FGFR2 variants are actionable therapeutic targets, which we confirmed in preclinical mouse and human tumor models, and in a clinical trial. Thus, we uncovered a novel paradigm in oncogenic FGFR2 signaling and propose that breast and other cancers harboring any FGFR2 variant that truncates E18 should be considered for FGFR-targeted therapies. Citation Format: Daniel Kaspar Zingg, Jinhyuk Bhin, Julia Yemelyanenko, Sjors M. Kas, Catrin Lutz, Chi-Chuan Lin, Sjoerd Klarenbeek, Jessica K. Lee, Ian M. Silverman, Stefano Annunziato, Marieke van de Ven, Siraj M. Ali, Timothy C. Burn, Shridar Ganesan, Lodewyk F. Wessels, Jos Jonkers. Truncated FGFR2 - a clinically actionable oncogene in multiple cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3488.

Prospective Identification of Prognostic Hot-Spot Mutant Gene Signatures for Leukemia: A Computational Study Based on Integrative Analysis of TCGA and cBioPortal Data.

The advantage of an increasing amount of bioinformatics data on leukemias intrigued us to explore the hot-spot mutation profiles and investigate the implications of those hot-spot mutations in patient survival. We retrieved somatic mutations and their distribution in protein domains through data analysis of The Cancer Genome Atlas and cBioPortal databases. After determining differentially expressed mutant genes related to leukemia, we further conducted principal component analysis and single-factor Cox regression analyses. Moreover, survival analysis was performed for the obtained candidate genes, followed by a multi-factor Cox proportional hazard model method for the impacts of the candidate genes on the survival and prognosis of patients with leukemia. At last, the signaling pathways involved in leukemia were investigated by gene set enrichment analysis. There were 223 somatic missense mutation hot-spots identified with pertinence to leukemia, which were distributed in 41 genes. Differential expression in leukemia was witnessed in 39 genes. We found a close correlation between seven genes and the prognosis of leukemia patients, among which, three genes could significantly influence the survival rate. In addition, among these three genes, CD74 and P2RY8 were highlighted due to close pertinence with survival conditions of leukemia patients. Finally, data suggested that B cell receptor, Hedgehog, and TGF-beta signaling pathways were enriched in low-hazard patients. In conclusion, these data underline the involvement of hot-spot mutations of CD74 and P2RY8 genes in survival status of leukemia patients, highlighting their as novel therapeutic targets or prognostic indicators for leukemia patients. Summary of Graphical Abstract: We identified 223 leukemia-associated somatic missense mutation hotspots concentrated in 41 different genes from 2297 leukemia patients in the TCGA database. Differential analysis of leukemic and normal samples from the TCGA and GTEx databases revealed that 39 of these 41 genes showed significant differential expression in leukemia. These 39 genes were subjected to PCA analysis, univariate Cox analysis, survival analysis, multivariate Cox regression analysis, GSEA pathway enrichment analysis, and then the association with leukemia survival prognosis and related pathways were investigated.

DICER1-associated central nervous system sarcoma with neural lineage differentiation: a case report

BackgroundDICER1-associated central nervous system sarcoma (DCS) without evidence of other cancer-related syndromes is rare. Though the morphology of DCS was highly variable, the immunophenotype was predominant myogenic phenotype. Other lineage markers were consistently negative.Case presentationWe report a case of DCS with neurogenic differentiation proved by immunohistochemical staining and whole-exome sequencing (WES). An 8-year-old female patient presented with 8-day history of headache, nausea and vomiting. Magnetic resonance imaging (MRI) revealed a heterogeneous mass in the left parietal lobe. The patient underwent the craniotomy via left parietal approach to resect the tumor completely. Histologically, the tumor predominately showed fibrosarcoma-like spindle cells with obvious cytoplasmic eosinophilic globules. Immunohistochemically, the tumor stained positively for DICER1, Desmin, and several neurogenic markers. DICER1 somatic hotspot mutation was confirmed by WES, as well as TP53 and RAF1 mutations which were commonly found in DCS, and other sarcoma-associated genes including AR, AXL and ETV5 mutations. Subsequently, the result of Gene Ontology (GO) analysis showed that the mutated genes in this case were involved in neuron development. All of these findings indicated the diagnosis of DCS with neurogenic differentiation. Postoperatively, the patient received high-dose radiotherapy (60 Gy) and chemotherapy. There was no MRI evidence of tumor recurrence at the 21-month postoperative follow-up.ConclusionsThis unusual DCS case with neuronal differentiation is an important addition to the immuno-phenotypic spectrum of DCS. Although the prognosis for DCS is poor, gross tumor resection with high dose radiotherapy and chemotherapy may assist in prolonging survival.

Prevalence and breakdown of non-small cell lung cancer BRAF driver mutations in a large UK cohort

BRAF inhibitors have been shown in clinical trials to improve patient outcomes in non-small cell lung cancer (NSCLC) patients harbouring selected BRAF driver mutations with a limited side effect profile, and therefore show potential as therapeutics in clinical practice. To utilise BRAF inhibitors effectively, understanding the prevalence of BRAF mutations within the local patient population is crucial, especially since NSCLC driver mutation rates have been observed to vary in different populations around the world. We interrogated a clinical archive of next generation sequencing (NGS) data representative of 7 years of routine UK practice in the National Health Service (NHS) to investigate the frequency of BRAF mutations, the breakdown of mutation classes and co-occurrence of other oncogenic driver mutations.Tissue biopsies from NSCLC cases referred to the Sarah Cannon Molecular Diagnostics Laboratory between January 2015 and February 2022 from multiple centres across UK were included in this study. Somatic mutation hotspots in relevant cancer-associated genes were analysed using amplicon/ion-torrent based NGS assays, and all NSCLC samples which harboured recognised BRAF driver mutations were identified through a combination of automated and manual data retrieval. Data regarding any other detected mutations and basic demographic information were also collected.Over the 7-year period, 5384 NSCLC samples were sequenced, with BRAF mutation identified in 185 (3.44%) of cases. These 185 cases represented a total of 73 Class I BRAF mutations (39.5%), 61 Class II mutations (33.0%) and 51 Class III mutations (27.6%). Of the 73 identified Class I mutations, 69 (69/185, 37.3%) were V600E and four (4/185, 2.16%) were non-V600E mutations. Five V600E cases had co-mutations (5/185, 2.7%). Various other known driver mutations were also identified in these 185 tumour samples, with KRAS (18/185, 9.73%) and PIK3CA (7/185, 3.78%) occurring at the highest frequency.This is the first large cohort-level study in the UK to profile the breakdown of BRAF-positive NSCLC biopsy samples using NGS in routine clinical practice. This study defines the proportion of NSCLC patients that may be expected to benefit from BRAF inhibitors and highlights the utility of using NGS as a diagnostic tool to improve targeted therapy stratification for NSCLC patients.

Clinical and pathological characteristics of familial melanoma with germline TERT promoter variants

Around 10% of melanoma occurs in patients with a suspected familial predisposition. TERT promoter mutations are the most common somatic hotspot mutations in human cancers. However, only two families with germline mutations have been identified to date. We present detailed histological, clinical, and molecular pathologic analyses of affected patients and details of newly identified individuals in one of these previously reported families. TERT (NM_198253.3) Chr.5:1,295,161T&gt;C (c.-57 T&gt;C) promoter variants were detected in all melanoma-affected (n= 18) and one non-diseased family member. The median age at diagnosis was 30 years (n= 18, range 16-46 years, 2 unknown). While most primary melanomas arose on the upper extremities (n= 7, 21%) and were superficial spreading melanoma (SSM, n= 8, 24%), many primary melanomas also originated from non-UV-exposed mucosal (n= 2, 6%) and acral (n= 4, 12%) locations. One SSM sample harbored a Chr.5:1,295,228C&gt;T TERT promoter mutation in addition to the germline Chr.5:1,295,161T&gt;C variant, arguing additional pathway activation can support tumor pathogenesis. Patients treated with BRAF inhibitor and/or immune checkpoint inhibition (ICI) showed responses, although of limited duration. One mucosal melanoma harbored both a KIT copy number gain and an activating c.1727 p.Leu576Pro mutation. Following the modest response to ICI, subsequent KIT inhibitor (imatinib) therapy demonstrated an ongoing complete pathological response (currently 7months).