Private Mutations Research Articles

The Personalized Inherited Signature Predisposing to Non-Small-Cell Lung Cancer in Non-Smokers.

Lung cancer (LC) continues to be an important public health problem, being the most common form of cancer and a major cause of cancer deaths worldwide. Despite the great bulk of research to identify genetic susceptibility genes by genome-wide association studies, only few loci associated to nicotine dependence have been consistently replicated. Our previously published study in few phenotypically discordant sib-pairs identified a combination of germline truncating mutations in known cancer susceptibility genes in never-smoker early-onset LC patients, which does not present in their healthy sib. These results firstly demonstrated the presence of an oligogenic combination of disrupted cancer-predisposing genes in non-smokers patients, giving experimental support to a model of a "private genetic epidemiology". Here, we used a combination of whole-exome and RNA sequencing coupled with a discordant sib's model in a novel cohort of pairs of never-smokers early-onset LC patients and in their healthy sibs used as controls. We selected rare germline variants predicted as deleterious by CADD and SVM bioinformatics tools and absent in the healthy sib. Overall, we identified an average of 200 variants per patient, about 10 of which in cancer-predisposing genes. In most of them, RNA sequencing data reinforced the pathogenic role of the identified variants showing: (i) downregulation in LC tissue (indicating a "second hit" in tumor suppressor genes); (ii) upregulation in cancer tissue (likely oncogene); and (iii) downregulation in both normal and cancer tissue (indicating transcript instability). The combination of the two techniques demonstrates that each patient has an average of six (with a range from four to eight) private mutations with a functional effect in tumor-predisposing genes. The presence of a unique combination of disrupting events in the affected subjects may explain the absence of the familial clustering of non-small-cell lung cancer. In conclusion, these findings indicate that each patient has his/her own "predisposing signature" to cancer development and suggest the use of personalized therapeutic strategies in lung cancer.

Genomic alterations associated with resistance and circulating tumor DNA dynamics for early detection of progression on CDK4/6 inhibitor in advanced breast cancer.

Combined CDK4/6 inhibitor (CDK4/6i) and endocrine therapy significantly improves outcome for patients with estrogen receptor-positive (ER+) metastatic breast cancer, but drug resistance and thus disease progression inevitably occur. Herein, we aimed to identify genomic alterations associated with combined CDK4/6i and endocrine therapy resistance, and follow the levels of specific mutations in longitudinal circulating tumor DNA (ctDNA) for early detection of progression. From a cohort of 86 patients with ER+ metastatic breast cancer we performed whole exome sequencing or targeted sequencing of paired tumor (N = 8) or blood samples (N = 5) obtained before initiation of combined CDK4/6i and endocrine therapy and at disease progression. Mutations in oncogenic genes at progression were rare, while amplifications of growth-regulating genes were more frequent. The most frequently acquired alterations observed were PIK3CA and TP53 mutations and PDK1 amplification. Longitudinal ctDNA dynamics of mutant PIK3CA or private mutations revealed increased mutation levels at progression in 8 of 10 patients (80%). Impressively, rising levels of PIK3CA-mutated ctDNA were detected 4-17 months before imaging. Our data add to the growing evidence supporting longitudinal ctDNA analysis for real-time monitoring of CDK4/6i response and early detection of progression in advanced breast cancer. Further, our analysis suggests that amplification of growth-related genes may contribute to combined CDK4/6i and endocrine therapy resistance.

Spatial intra-tumour heterogeneity and treatment-induced genomic evolution in oesophageal adenocarcinoma: implications for prognosis and therapy

BackgroundOesophageal adenocarcinoma (OAC) is a highly heterogeneous cancer with poor survival. Standard curative treatment is chemotherapy with or without radiotherapy followed by oesophagectomy. Genomic heterogeneity is a feature of OAC and has been linked to treatment resistance.MethodsWhole-genome sequencing data from 59 treatment-naïve and 18 post-treatment samples from 29 OAC patients was analysed. Twenty-seven of these were enrolled in the DOCTOR trial, sponsored by the Australasian Gastro-Intestinal Trials Group. Two biopsies from each treatment-naïve tumour were assessed to define ‘shared’ (between both samples) and ‘private’ (present in one sample) mutations.ResultsMutational signatures SBS2/13 (APOBEC) and SBS3 (BRCA) were almost exclusively detected in private mutation populations of treatment-naïve tumours. Patients presenting these signatures had significantly worse disease specific survival. Furthermore, mutational signatures associated with platinum-based chemotherapy treatment as well as high platinum enrichment scores were only detected in post-treatment samples. Additionally, clones with high putative neoantigen binding scores were detected in some treatment-naïve samples suggesting immunoediting of clones.ConclusionsThis study demonstrates the high intra-tumour heterogeneity in OAC, as well as indicators for treatment-induced changes during tumour evolution. Intra-tumour heterogeneity remains a problem for successful treatment strategies in OAC.

Evolutionary and functional analyses of LRP5 in archaic and extant modern humans

BackgroundThe human lineage has undergone a postcranial skeleton gracilization (i.e. lower bone mass and strength relative to body size) compared to other primates and archaic populations such as the Neanderthals. This gracilization has been traditionally explained by differences in the mechanical load that our ancestors exercised. However, there is growing evidence that gracilization could also be genetically influenced.ResultsWe have analyzed the LRP5 gene, which is known to be associated with high bone mineral density conditions, from an evolutionary and functional point of view. Taking advantage of the published genomes of archaic Homo populations, our results suggest that this gene has a complex evolutionary history both between archaic and living humans and within living human populations. In particular, we identified the presence of different selective pressures in archaics and extant modern humans, as well as evidence of positive selection in the African and South East Asian populations from the 1000 Genomes Project. Furthermore, we observed a very limited evidence of archaic introgression in this gene (only at three haplotypes of East Asian ancestry out of the 1000 Genomes), compatible with a general erasing of the fingerprint of archaic introgression due to functional differences in archaics compared to extant modern humans. In agreement with this hypothesis, we observed private mutations in the archaic genomes that we experimentally validated as putatively increasing bone mineral density. In particular, four of five archaic missense mutations affecting the first β-propeller of LRP5 displayed enhanced Wnt pathway activation, of which two also displayed reduced negative regulation.ConclusionsIn summary, these data suggest a genetic component contributing to the understanding of skeletal differences between extant modern humans and archaic Homo populations.

Clonal evolution and relapse in early-stage follicular lymphoma - a tree with many branches†.

Follicular lymphoma (FL) is an indolent B-cell neoplasm characterised by multistep evolution from premalignant precursor cells carrying the hallmark t(14;18) translocation in the majority of cases. In a new article in The Journal of Pathology, samples of relapsed early-stage FL - primary manifestation and relapse with or without transformation - initially treated with radiotherapy only, were studied for clonal relationships and evolution. Using somatic mutations and the rearranged immunoglobulin sequences as markers, the majority of paired lymphoma samples showed so-called branched evolution from a common, possibly premalignant progenitor cell, with both shared and private mutations. In addition, clonally unrelated cases were identified. This and previous studies with similar findings clearly document that relapse or transformation of FL in many instances not necessarily represents a linear progression of disease due to acquisition of additional mutations and therapy resistance, but rather new outgrowths derived from a pool of clonally related, long-lived, and low proliferating precursor cells, or even unrelated second neoplasms. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Genetic intratumour heterogeneity and clonal evolution in extramammary Paget's disease.

All tumour cells in a patient have shared and non-shared genetic alterations, and the diversity of mutations is described as intratumoural heterogeneity (ITH). Multiregion sequencing is a genome sequencing analytical technique used for multiple, spatially-separated biopsy tissues that may further our understanding of ITH and tumour evolution. Although genetic mutations in extramammary Paget's disease (EMPD) have recently been detected by next-generation sequencing analysis, there have been no reports of ITH based on multiregion sequencing in EMPD. Thus, we clarified the landscape of ITH and tumour evolution in EMPD. We performed whole-exome sequencing on 35 tissues (30 tumour tissues and five normal skin samples as a paired control), collected from five patients with EMPD. The rate of private mutations was significantly higher than that of ubiquitous and shared mutations. Ubiquitous mutations were not present in driver genes, and most driver genes exhibited private and shared mutations. The most frequent base substitution was C>T in almost all lesions, and most mutational signatures corresponded to signature 1, 2, 3, and 8. The types of proposed aetiology in most lesions were based on age and AID/APOBEC family and BRCA1/BRCA2 mutations. Evolutionary trees were characterized by short trunks and long branches due to the extremely high ratio of private mutations. In contrast, pathogenic factors, such as base substitutions, mutational signatures, and proposed aetiology, were shared. Tumour evolution in EMPD appears to be characterized by a high level of genetic ITH with shared background factors.

Human genetics of face recognition: discovery of MCTP2 mutations in humans with face blindness (congenital prosopagnosia).

Face recognition is important for both visual and social cognition. While prosopagnosia or face blindness has been known for seven decades and face-specific neurons for half a century, the molecular genetic mechanism is not clear. Here we report results after 17 years of research with classic genetics and modern genomics. From a large family with 18 congenital prosopagnosia (CP) members with obvious difficulties in face recognition in daily life, we uncovered a fully cosegregating private mutation in the MCTP2 gene which encodes a calcium binding transmembrane protein expressed in the brain. After screening through cohorts of 6589, we found more CPs and their families, allowing detection of more CP associated mutations in MCTP2. Face recognition differences were detected between 14 carriers with the frameshift mutation S80fs in MCTP2 and 19 noncarrying volunteers. Six families including one with 10 members showed the S80fs-CP correlation. Functional magnetic resonance imaging found association of impaired recognition of individual faces by MCTP2 mutant CPs with reduced repetition suppression to repeated facial identities in the right fusiform face area. Our results have revealed genetic predisposition of MCTP2 mutations in CP, 76 years after the initial report of prosopagnosia and 47 years after the report of the first CP. This is the first time a gene required for a higher form of visual social cognition was found in humans.

Unveiling the population genetic structure of Iranian horses breeds by whole-genome resequencing analysis.

Preserving genetic diversity is pivotal for enhancing genetic improvement and facilitating adaptive responses to selection. This study focuses on identifying key genetic variants, including single nucleotide polymorphisms (SNPs), insertion/deletion polymorphisms (INDELs), and copy number variants (CNVs), while exploring the genomic evolutionary connectedness among seven Iranian horses representing five indigenous breeds: Caspian, Turkemen, DareShuri, Kurdish, and Asil. Using whole-genome resequencing, we generated 2.7Gb of sequence data, with raw reads ranging from 1.2Gb for Caspian horses to 0.38Gb for Turkoman horses. Post-filtering, approximately 1.9Gb of reads remained, with ~ 1.5Gb successfully mapped to the horse reference genome (EquCab3.0), achieving mapping rates between 76.4% (Caspian) and 98.35% (Turkoman). We identified 2,909,816 SNPs in Caspian horses, constituting around 0.1% of the genome. Notably, 71% of these SNPs were situated in intergenic regions, while 8.5 and 6.8% were located upstream and downstream, respectively. A comparative analysis of SNPs between Iranian and non-Iranian horse breeds showed that Caspian horses had the lowest number of shared SNPs with Turkoman horses. Instead, they showed a closer genetic relationship with DareShuri, Quarter, Arabian, Standardbred, and Asil breeds. Hierarchical clustering highlighted Caspian horses as a distinct cluster, underscoring their distinctive genomic signature. Caspian horses exhibit a unique genetic profile marked by an enrichment of private mutations in neurological genes, influencing sensory perception and awareness. This distinct genetic makeup shapes mating preferences and signifies a separate evolutionary trajectory. Additionally, significant non-synonymous single nucleotide polymorphisms (nsSNPs) in reproductive genes offer intervention opportunities for managing Caspian horses. These findings reveal the population genetic structure of Iranian horse breeds, contributing to the advancement of knowledge in areas such as conservation, performance traits, climate adaptation, reproduction, and resistance to diseases in equine science.

Abstract 5659: Origin of synchronous or metachronous multiple pancreatic cancers

Abstract [Introduction] The majority of patients with pancreatic cancer are inoperable at the time of diagnosis and even those cases with successful tumor resection for early-stage tumors frequently had second tumors within the remaining pancreas shortly after the surgery, contributing to a poor clinical outcome. Additionally, synchronous multiple pancreatic cancers have been frequently documented. One of the long-standing issues about such synchronous and/or metachronous multiple pancreatic cancers is their clonal origins. This study is designed to elucidate the origin of multiple pancreatic cancers through an unbiased detection of somatic mutations in primary and metachronous cancers, along with adjacent precursor lesions. [Methods] Serially obtained formalin-fixed paraffin-embedded surgical specimens from 20 patients who had undergone curative surgery for an early-stage pancreatic cancer were subjected to laser microdissection for the enrichment of tumor and precancerous lesions, from which DNA was extracted and analyzed for somatic mutations using whole-exome sequencing (WES) with matched normal DNA. The investigation into the shared and private mutations across different samples aimed to unveil the clonal evolution history of these lesions. [Results] We analyzed 20 patients with multiple pancreatic cancers of 2 metachronous and synchronous, 16 metachronous, and 2 synchronous tumors. In metachronous cases, all patients exhibited margin-negative pathology for the primary cancer, with a median interval of 38.9 months (ranging from 7 to 85 months) between the initial and second surgery. In addition, a total of 20 pancreatic intraepithelial neoplasia (PanIN) from 5 of these cases were analyzed. We identified a median of 66 (range: 31-232) and 20 (14-42) somatic mutations in cancer and PanIN lesions, respectively. None of the patients have known pathogenic germline variants. All samples had one or more driver mutations. In most cases (N=19), sharing many somatic mutations, multiple tumors had a common clonal origin. In another synchronous case, the synchronous tumors shared only one mutation, suggesting that they were clonally independent tumors. By contrast, none of the mutations other than hotspot KRAS mutations were shared between precursor and cancer lesions. While multiple independent clones were observed in precancerous lesions, most of multiple cancers originated from a common ancestor. Moreover, negative pathology of the margins at the initial surgery suggested that those multiple lesions arose from distant dissemination or metastasis, rather than contiguous, intraductal invasion. [Conclusions] In conclusion, our study suggests that even in early pancreatic cancer, dissemination within the pancreas may occur, contributing to the development of synchronous and metachronous cancers. Citation Format: Tomonori Hirano, Nobuyuki Kakiuchi, Yasuhide Takeuchi, Takayuki Anazawa, Kazuyuki Nagai, Toshihiko Masui, Sachiko Minamiguchi, Hironori Haga, Takeshi Tanaka, Atsuhiro Masuda, Yuzo Kodama, Norimitsu Uza, Hiroshi Seno, Satoru Miyano, Hiroko Tanaka, Seishi Ogawa. Origin of synchronous or metachronous multiple pancreatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5659.

Neutral diversity in experimental metapopulations

New automated and high-throughput methods allow the manipulation and selection of numerous bacterial populations. In this manuscript we are interested in the neutral diversity patterns that emerge from such a setup in which many bacterial populations are grown in parallel serial transfers, in some cases with population-wide extinction and splitting events. We model bacterial growth by a birth–death process and use the theory of coalescent point processes. We show that there is a dilution factor that optimises the expected amount of neutral diversity for a given number of cycles, and study the power law behaviour of the mutation frequency spectrum for different experimental regimes. We also explore how neutral variation diverges between two recently split populations by establishing a new formula for the expected number of shared and private mutations. Finally, we show the interest of such a setup to select a phenotype of interest that requires multiple mutations.

High-Grade, Nonsarcomatoid Chromophobe Renal Cell Carcinoma: A Series of 22 Cases With Novel Molecular Features on a Subset

Chromophobe renal cell carcinoma (ChRCC) is the third most common subtype of renal cell carcinoma, and typically exhibits indolent behavior, though a rare subset can exhibit high-grade morphological features and are associated with a poor prognosis. Although there is limited data on the molecular characteristics of metastatic and sarcomatoid ChRCC, the molecular features of high-grade non-sarcomatoid ChRCC remain unexplored. Herein, we characterize 22 cases of ChRCC with high-grade, non-sarcomatoid components. High-grade ChRCC frequently demonstrated advanced stage at diagnosis (64% ≥pT3a or N1), with regions of extrarenal extension, nodal metastases, and vascular invasion consisting solely of high-grade ChRCC morphologically. We performed spatially guided panel-based DNA sequencing on 11 cases, comparing high- and low-grade regions (n = 22 samples). We identified recurring somatic alterations emblematic of ChRCC, including deletions of chromosomes 1, 2, 6, 10, 13, 17, and 21 in 91% (10/11) of cases and recurring mutations in TP53 (81.8%, n = 9/11) and PTEN (36.4%, n = 4/11). Notably, though PTEN and TP53 alterations were found in both high- and low- grade regions, private mutations were identified in 3 cases, indicating convergent evolution. Finally, we identified recurring RB1 mutations in 27% (n = 3) of high-grade regions leading to selective protein loss by immunohistochemistry not observed in adjacent low-grade regions. This finding was confirmed in the TCGA cohort where 2/66 cases contained RB1 mutations and demonstrated unequivocal high-grade, non-sarcomatoid morphology. We also detected multiple chromosomal gains confined to the high-grade regions, consistent with imbalanced chromosome duplication. These findings broaden our understanding of the molecular pathogenesis of ChRCC and suggest that subclonal RB1 mutations can drive the evolution to high-grade, non-sarcomatoid ChRCC.

A New Tool for Probabilistic Assessment of MPS Data Associated with mtDNA Mixtures.

Mitochondrial (mt) DNA plays an important role in the fields of forensic and clinical genetics, molecular anthropology, and population genetics, with mixture interpretation being of particular interest in medical and forensic genetics. The high copy number, haploid state (only a single haplotype contributed per individual), high mutation rate, and well-known phylogeny of mtDNA, makes it an attractive marker for mixture deconvolution in damaged and low quantity samples of all types. Given the desire to deconvolute mtDNA mixtures, the goals of this study were to (1) create a new software, MixtureAceMT™, to deconvolute mtDNA mixtures by assessing and combining two existing software tools, MixtureAce™ and Mixemt, (2) create a dataset of in-silico MPS mixtures from whole mitogenome haplotypes representing a diverse set of population groups, and consisting of two and three contributors at different dilution ratios, and (3) since amplicon targeted sequencing is desirable, and is a commonly used approach in forensic laboratories, create biological mixture data associated with two amplification kits: PowerSeq™ Whole Genome Mito (Promega™, Madison, WI, USA) and Precision ID mtDNA Whole Genome Panel (Thermo Fisher Scientific by AB™, Waltham, MA, USA) to further validate the software for use in forensic laboratories. MixtureAceMT™ provides a user-friendly interface while reducing confounding features such as NUMTs and noise, reducing traditionally prohibitive processing times. The new software was able to detect the correct contributing haplogroups and closely estimate contributor proportions in sequencing data generated from small amplicons for mixtures with minor contributions of ≥5%. A challenge of mixture deconvolution using small amplicon sequencing is the potential generation of spurious haplogroups resulting from private mutations that differ from Phylotree. MixtureAceMT™ was able to resolve these additional haplogroups by including known haplotype/s in the evaluation. In addition, for some samples, the inclusion of known haplotypes was also able to resolve trace contributors (minor contribution 1-2%), which remain challenging to resolve even with deep sequencing.

TFH lymphoma and associated clonal hematopoiesis

T‑follicular helper (TFH) cell lymphoma (TFHL) is alymphoma of mature Tcells with phenotypic characteristics and gene expression signature of TFH cells. The lymphoma harbors recurrent mutations of RHOAG17V, IDH2R172, TET2 and DNMT3A. Whereas RHOAG17V and IDH2R172 are almost exclusively found in this entity, TET2 and DNMT3A mutations occur in abroad variety of hematological neoplasms and are the most frequently affected genes in clonal hematopoiesis (CH). CH in humans shows aprogression rate to overt hematologic neoplasia of about 0.5to 1% per year, depending on clone size, number of mutations and affected genes. In 2018, the first case was described in which alymphoid (TFHL) and myeloid (acute myeloid leukemia) neoplasm arose from acommon mutated progenitor cell with shared mutations and additional private mutations. In recent years, further studies showed in up to 70% of patients with TFHL the occurrence of identical mutations of TET2 and/or DNMT3A in the myeloid cells, irrespective of bone marrow involvement, indicating aprominent role of CH in the pathogenesis of TFHL. In up to 18%, these patients show also additional synchronous or metachronous overt myeloid neoplasms, often with private myelodysplastic-type mutations, most often myelodysplastic syndrome, chronic myelomonocytic leukemia and acute myeloid leukemia. Recently, there is also evidence for two distinct lymphoid neoplasms arising from CH. TFH lymphoma cases with antecedent or concomitant hematologic neoplasm often show high variant allelic frequencies of TET2 and often more than one mutation, suggesting arole for surveillance in these patients.

Integrated molecular and multiparametric MRI mapping of high-grade glioma identifies regional biologic signatures

Sampling restrictions have hindered the comprehensive study of invasive non-enhancing (NE) high-grade glioma (HGG) cell populations driving tumor progression. Here, we present an integrated multi-omic analysis of spatially matched molecular and multi-parametric magnetic resonance imaging (MRI) profiling across 313 multi-regional tumor biopsies, including 111 from the NE, across 68 HGG patients. Whole exome and RNA sequencing uncover unique genomic alterations to unresectable invasive NE tumor, including subclonal events, which inform genomic models predictive of geographic evolution. Infiltrative NE tumor is alternatively enriched with tumor cells exhibiting neuronal or glycolytic/plurimetabolic cellular states, two principal transcriptomic pathway-based glioma subtypes, which respectively demonstrate abundant private mutations or enrichment in immune cell signatures. These NE phenotypes are non-invasively identified through normalized K2 imaging signatures, which discern cell size heterogeneity on dynamic susceptibility contrast (DSC)-MRI. NE tumor populations predicted to display increased cellular proliferation by mean diffusivity (MD) MRI metrics are uniquely associated with EGFR amplification and CDKN2A homozygous deletion. The biophysical mapping of infiltrative HGG potentially enables the clinical recognition of tumor subpopulations with aggressive molecular signatures driving tumor progression, thereby informing precision medicine targeting.

A novel mutation in EROS (CYBC1) causes chronic granulomatous disease

Chronic Granulomatous Disease (CGD) is an inborn error of immunity characterised by opportunistic infection and sterile granulomatous inflammation. CGD is caused by a failure of reactive oxygen species (ROS) production by the phagocyte NADPH oxidase. Mutations in the genes encoding phagocyte NADPH oxidase subunits cause CGD. We and others have described a novel form of CGD (CGD5) secondary to lack of EROS (CYBC1), a highly selective chaperone for gp91phox. EROS-deficient cells express minimal levels of gp91phox and its binding partner p22phox, but EROS also controls the expression of other proteins such as P2X7. The full nature of CGD5 is currently unknown. We describe a homozygous frameshift mutation in CYBC1 leading to CGD. Individuals who are heterozygous for this mutation are found in South Asian populations (allele frequency = 0.00006545), thus it is not a private mutation. Therefore, it is likely to be the underlying cause of other cases of CGD.

Ophthalmic findings in patients with autosomal recessive lamellar ichthyosis due to TGM1 mutations in an isolated population

PurposeTo describe the ocular clinical characteristics of a group of Mexican patients with lamellar ichthyosis (LI) arising from TGM1 pathogenic variants.MethodsOphthalmological exploration, pedigree analysis and genetic screening were performed in patients with an established clinical diagnosis of lamellar ichthyosis from families located in a small community in the Southeast of Mexico.ResultsNine patients with LI in five families were identified. There were six affected females. All patients (9/9) demonstrated eye lid abnormalities with eight patients showing lid margin abnormalities. Madarosis was present in only three individuals and corneal scarring was documented in two. All nine individuals carried biallelic TGM1 variants, either homozygously or as compound heterozygous.ConclusionOcular anomalies are common in individuals with TGM1-related LI. The occurrence of a variety of private or rare mutations hampers the identification of a genotype–phenotype correlation for ocular anomalies in this disorder.

Mutation Analysis of SARS-CoV-2 Variants Isolated from Symptomatic Cases from Andhra Pradesh, India.

There has been a continuous evolution in the SARS-CoV-2 genome; therefore, it is necessary to monitor the shifts in the SARS-CoV-2 variants. This study aimed to detect various SARS-CoV-2 variants circulating in the state of Andhra Pradesh, India. The study attempted to sequence the complete S-gene of SARS-CoV-2 of 104 clinical samples using Sanger's method to analyze and compare the mutations with the global prevalence. The method standardized in this study was able to amplify the complete length of the S-gene (3822 bp). The resulting nucleotide and amino acid mutations were analyzed and compared with the local and global SARS-CoV-2 databases using Nextclade and GISAID tools. The Delta variant was the most common variant reported in the present study, followed by the Omicron variant. A variant name was not assigned to thirteen samples using the Nextclade tool. There were sixty-nine types of amino acid substitutions reported (excluding private mutations) throughout the spike gene. The T95I mutation was observed predominantly in Delta variants (15/38), followed by Kappa (3/8) and Omicron (1/31). Nearly all Alpha and Omicron lineages had the N501Y substitution; Q493R was observed only in the Omicron lineage; and other mutations (L445, F486, and S494) were not observed in the present study. Most of these mutations found in the Omicron variant are located near the furin cleavage site, which may play a role in the virulence, pathogenicity, and transmission of the virus. Phylogenetic analysis showed that the 104 complete CDS of SARS-CoV-2 belonged to different phylogenetic clades like 20A, 20B, 20I (Alpha), 21A (Delta), 21B (Kappa), 21I (Delta), 21J (Delta), and 21L (Omicron).

Metastatic pattern of ovarian cancer delineated by tracing the evolution of mitochondrial DNA mutations

Ovarian cancer (OC) is the most lethal gynecologic tumor and is characterized by a high rate of metastasis. Challenges in accurately delineating the metastatic pattern have greatly restricted the improvement of treatment in OC patients. An increasing number of studies have leveraged mitochondrial DNA (mtDNA) mutations as efficient lineage-tracing markers of tumor clonality. We applied multiregional sampling and high-depth mtDNA sequencing to determine the metastatic patterns in advanced-stage OC patients. Somatic mtDNA mutations were profiled from a total of 195 primary and 200 metastatic tumor tissue samples from 35 OC patients. Our results revealed remarkable sample-level and patient-level heterogeneity. In addition, distinct mtDNA mutational patterns were observed between primary and metastatic OC tissues. Further analysis identified the different mutational spectra between shared and private mutations among primary and metastatic OC tissues. Analysis of the clonality index calculated based on mtDNA mutations supported a monoclonal tumor origin in 14 of 16 patients with bilateral ovarian cancers. Notably, mtDNA-based spatial phylogenetic analysis revealed distinct patterns of OC metastasis, in which a linear metastatic pattern exhibited a low degree of mtDNA mutation heterogeneity and a short evolutionary distance, whereas a parallel metastatic pattern showed the opposite trend. Moreover, a mtDNA-based tumor evolutionary score (MTEs) related to different metastatic patterns was defined. Our data showed that patients with different MTESs responded differently to combined debulking surgery and chemotherapy. Finally, we observed that tumor-derived mtDNA mutations were more likely to be detected in ascitic fluid than in plasma samples. Our study presents an explicit view of the OC metastatic pattern, which sheds light on efficient treatment for OC patients.

Correspondence of Yolk Sac and Embryonic Genotypes in F0 Mouse CRISPants.

CRISPR-mediated genome editing in vivo can be accompanied by prolonged stability of the Cas9 protein in mouse embryos. Then, genome edited variant alleles will be induced as long as Cas9 protein is active, and unmodified wildtype target loci are available. The corollary is that CRISPR-modified alleles that arise after the first zygotic cell division potentially could be distributed asymmetrically to the cell lineages that are specified early during morula and blastocyst development. This has practical implications for the investigation of F0 generation individuals, as cells in embryonic and extraembryonic tissues, such as the visceral yolk sac, might end up inheriting different genotypes. We here investigated the hypothetically possible scenarios by genotyping individual F0 CRISPants and their associated visceral yolk sacs in parallel. In all cases, we found that embryonic genotype was accurately reflected by yolk sac genotyping, with the two tissues indicating genetic congruence, even when the conceptus was a mosaic of cells with distinct allele configurations. Nevertheless, low abundance of a variant allele may represent a private mutation occurring only in the yolk sac, and in those rare cases, additional genotyping to determine the mutational status of the embryo proper is warranted.

Abstract A040: Multi-site sequencing of lethal prostate cancer reveals metastatic dissemination by independent and early-branching clones

Abstract Primary prostate cancer (PCa) is frequently multifocal (genetically independent(GI) clones) and is further characterized by extensive intratumoral heterogeneity (genetically related clones). In metastatic castrate resistant PCa (mCRPC) monoclonal and polyclonal seeding of metastatic sites by related clones has been reported. However, the limited sequencing depth and/or sampling of sites is insufficient to rule out alternative evolutionary routes to drug resistance and metastatic dissemination. Here, we performed deep multi-site sequencing of 22 mCRPC patients to investigate the clonal architecture of mCRPC. Methods We obtained and profiled 81 metastatic biopsies from 22 patients using RNA and whole-exome sequencing (average depth of 600x). For each tumor we reconstructed its clonal phylogeny based on an integrated analysis of mutations (SNV) and copy-number variation (CNV) based on an explicit evolutionary model. Cancer Cell Fractions (CCF) were computed by adjusting variant allele frequencies (VAFs) for tumor purity and CNV multiplicity, followed by joint identification of clones through clustering of variants with similar CCFs and phylogenetic reconstruction using PhylogicNDT, which implements a nonparametric Drichlet process model. To assess overall genetic similarity between metastatic sites (and clones) we quantified the proportions of shared somatic mutations (present in all sites) and private mutations (mutations observed in only one site). Results We noted a high degree of genetic heterogeneity across metastatic sites, with a median of 35% (IQR:[21% - 51%]) shared somatic mutations, and 41% (IQR:[31% - 62%]) site-private mutations. No anatomical site was enriched for private mutations (anova p-value=0.747). Next, we characterized the clonal architecture across metastatic sites. We found a median of 10 clones per patient (IQR:[7.25-14.25]) and a median of 5.5 clones(IQR:[4-8]) per site with an average of 77 new synonymous and non-synonymous mutations per site. The number of subclones did not differ between metastatic sites as grouped by anatomical locations (anova P-value = 0.432). Strikingly, for 5 patients (23%) we found evidence for GI clones or extremely early branching clones metastasizing to different sites. In 2 of the 5 cases genetically unrelated clones metastasized to different sites, shared no common mutations and had distinct CNV profiles. For the remaining 3 cases metastatic sites only a median of 3% of mutations were shared across all sites, but displayed similar CNV profiles, indicating that chromosomal instability drove mutagenesis and preceded the acquisition of driver mutations. Conclusion In line with prior results our data confirms the high heterogeneity of mCRPC. We find metastatic sites to be multi-clonal with evidence of cross-site seeding. The limited number of truncal mutations, patterns of early-branching, suggest the need for multiple biopsies to fully characterize a patient's mCRPC. Our results also demonstrate, for the first time, that mCRPC can metastasize from more than one GI clone in the primary tissue. Citation Format: Noshad Hosseini, Zachery Reichert, Rohit Mehra, Arul Chinnaiyan, Marcin Cieslik. Multi-site sequencing of lethal prostate cancer reveals metastatic dissemination by independent and early-branching clones [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A040.