Loss Of Heterozygosity Research Articles

Analysis of X chromosome inactivation and prenatal diagnosis for a Chinese pedigree with loss of heterozygosity at Xq22.1q22.3

To explore the correlation between skewed X chromosome inactivation (XCI) and clinical phenotype of a Chinese pedigree with loss of heterozygosity at Xq22.1q22.3. A pedigree diagnosed at Taizhou Hospital on November 10, 2021 was selected as the study subject. G-banded chromosomal karyotyping and copy number variation sequencing (CNV-seq) were carried out to analyze the amniotic fluid and peripheral blood samples from the couple. XCI was detected by PCR amplification of CAG repeats in exon 1 of androgen receptor gene before and after the digestion with methylation-sensitive restriction enzyme Hpa II. Correlation between the genotype and clinical phenotype was analyzed. The karyotypes of the pregnant woman and the fetus were both determined as 46,X,del(X)(q22), and the result of CNV-seq was seq[hg19]del(X)(q22.1q22.3) chrX: g.10046000_105740000del, suggesting that both had harbored a 5.28 Mb deletion on the X chromosome. No obvious abnormality was found in the husband. XCI analysis showed that the activity ratio of the two X chromosomes of the pregnant woman and her fetus was 0 : 100. The X chromosome harboring the q22.1q22.3 deletion was completely inactivated, and the inactivated X chromosome of the fetus was derived from its mother. The fetus has harbored a maternally derived inactivated X chromosome del(X)(q22) , and its phenotype is closely associated with the activity of the abnormal X chromosome. Pedigree XCI analysis combined with the clinical phenotype has facilitated recognition of the maternal phenotype and prognosis of female fetus with loss of heterozygosity at Xq22.1q22.3.

Correlation of 1p/16q loss of heterozygosity and 1p gain with clinicopathological characteristics and prognosis in Wilms tumor

Objective: To investigate the relationship between 1p/16q loss of heterozygosity (LOH) and 1p gain in Wilms tumor and their clinicopathologic characteristics and prognosis. Methods: A total of 175 Wilms tumor samples received from the Department of Pathology, Beijing Children's Hospital from September 2019 to August 2022 were retrospectively analyzed. The histopathologic type and presence of lymph node involvement were evaluated by two pathologists. The clinical data including patients'gender, age, tumor location, preoperative chemotherapy, and tumor stage were summarized. Fluorescence in situ hybridization (FISH) was done to detect 1p/16q LOH and 1p gain and their correlation with the clinicopathological features and prognosis were analyzed. Results: Among the 175 samples, 86 cases (49.1%) were male and 89 (50.9%) were female. The mean age was (3.5±2.9) years, and the median age was 2.6 years. There were 26 (14.9%) cases with 1p LOH, 28 (16.0%) cases with 16q LOH, 10 (5.7%) cases of LOH at both 1p and 16q, and 53 (30.3%) cases with 1q gain. 1q gain was significantly associated with 1p LOH (P<0.01) and 16q LOH (P<0.01). There were significant differences (P<0.01) between 1q gain, 1p LOH and 16q LOH among different age groups. The rate of 16q LOH in the high-risk histopathological subtype (50.0%) was significantly higher than that in the intermediate-risk subtype (13.6%, P<0.05). The frequency of 1q gain, 1p LOH, and 16q LOH in children with advanced clinical stages (Ⅲ and Ⅳ) was significantly higher than that in children with early clinical stages (Ⅰ and Ⅱ). 1q gain, 1p LOH, and 16q LOH showed no significant correlation with gender, unilateral or bilateral disease, chemotherapy, or lymph node metastasis. The progression-free survival (PFS) time for patients with 1q gain and 1p LOH was significantly shorter than those without these aberrations (P<0.05). Additionally, the PFS time of patients with 16q LOH was slightly shorter than those with normal 16q, although the difference was not statistically significant. Patients with stage Ⅲ to Ⅳ disease exhibiting 1q gain or 1p LOH had a significantly higher relative risk of recurrence, metastasis, and mortality. Conclusions: 1p/16q LOH and 1q gain are associated with age, high-risk histological type, and clinical stage in Wilms tumor. 1q gain and 1p LOH are significantly correlated with the prognosis of Wilms tumor.

High-resolution genomic profiling and locus-specific FISH in subcutaneous and visceral adipose tissue of obese patients.

Obesity is known as a heterogeneous and multifactorial disease. The distribution of body fat is crucial for the development of metabolic complications. Comprehensive genetic analyses on different fat tissues are rare but necessary to provide more detailed information. Therefore, we performed genetic analyses of three patients with obesity using high resolution genome wide SNP array (blood, visceral fat tissue) and fluorescence in situ hybridization (FISH) analyses (visceral and subcutaneous fat tissue). Altogether, we identified 31 small Copy Number Variations (losses: 1p31.1, 1p22.2, 1q21.3, 2q34, 2q37.1, 3q28, 6p25.3, 7q31.33, 7q33, 8p23.3, 10q22.3, 11p15.4, 11p15.1, 11p14.2, 11p12, 13q12.3, 15q11.2-q13.1, 15q13.3, 20q13.2, 22q11.21; gains: 2q22.1-q22.2, 3p14.3, 4p16.3, 4q32.2, 6q27, 7p14.3, 7q34, 11p12, 12p11.21, 16p11.2-p11.1, 17q21.31) and 289 small copy-neutral Loss of Heterozygosity (cn-LOH). For the chromosomal region 15q11.2-q13.1, we detected a microdeletion (Prader-Willi-Syndrome) in one patient. Interestingly, we identified chromosomal SNP differences between EDTA-blood and visceral fat tissue (deletion and gain). Small losses of 7q31.33, 7q33, 11p14.2, 11p12, 13q12.3 as well as small gain of 7q34 were detected only in fat tissue and not in blood. Furthermore, FISH analyses on 7q31.33, 7q33 and 11p12 revealed differences between subcutaneous and visceral fat tissue. Generally, the deletions were detected more frequent in visceral fat tissue. Predominantly detected cn-LOH vs. CNV suggests a meaning of these cn-LOH for the pathogenesis of obesity. We conclude that the SNP array and FISH analyses used is applicable to generate more information for basic research on difficult cell subpopulations (e.g., visceral adipose tissue) and could opens up new diagnostic aspects in the field of obesity. Altogether, the significance of these mostly not yet described genetic aberrations in different fat tissues needs to confirmed in a larger series.

Investigating the Impact of Tumor Biology and Social Determinants on Time to Diagnosis and Stage at Presentation of Wilms Tumor.

Delays in diagnosis and time to diagnosis generally are used interchangeably in cancer disparity research, but these terms may have important differences. Although these terms are related, we hypothesize that time to diagnosis is determined by the aggressiveness of the tumor based on intrinsic factors such as tumor biology, whereas delays in diagnosis are caused by extrinsic factors such as socioeconomic status, leading to presentation at higher stage of disease due to barriers of care. We conducted a retrospective study of 306 patients diagnosed with Wilms tumor at Children's Hospital Colorado between 1971 and 2016 identifying patient barriers as extrinsic markers and using unfavorable histology and loss of heterozygosity as markers of aggressive tumor biology. Multivariable logistic regression was performed. Patients with Medicaid were more likely to present greater than 4 days after initial symptoms compared to those with private insurance, and those with housing concerns were more likely to be diagnosed greater than 9 days from initial symptoms. Tumor biology was noted to be associated with higher stage at diagnosis, but patient barriers were not. These findings suggest the interplay between tumor biology, patient barriers, diagnostic timing, and stage at diagnosis is more complex, multifactorial, and in need of further study.

Abstract B106: A minimal driver pair for a spontaneous high-grade serous ovarian cancer mouse model: Myc and p53-R270H

Abstract Genetically engineered mouse models (GEMM) have fundamentally changed how ovarian cancer etiology, early detection, and treatment can be understood. One challenge of the last decade has been to provide evidence for which cells are responsible for the origin of epithelial ovarian cancer. Different promoters, including Pax8, Lgr5, Ogvp1, and others have allowed for expression of transgenes in specific epithelial compartments in mice. Ovgp1 has been shown to be specific for fallopian tube epithelium (FTE), which most HGSOC is now thought to originate from. Pairing Ovgp1 with multiple homozygous deletions of powerful tumor suppressors like Brca1, Trp53, Rb1, and Nf1 yields murine disease after a year of age. However, human tumors do not contain homozygous deletions of these many tumor suppressors. Others have utilized Pax8 to knock out Trp53 and Pten to yield tumors at 3-8 months of age, but human tumors rarely have PTEN knockouts and Pax8 drives kidney and thyroid expression as well. We postulated that it is possible to strictly mimic human HGSOC genetics in a mouse model and nonetheless drive oncogenesis within the lifespan of a mouse. Here, we describe the OvTrpMyc model, Trp53em1Jdel_Tg(Ovgp1-Trp53*R270H-Myc), in which p53-R270H and Myc expression are driven by the Ovgp1 promoter. The transgene was inserted at the endogenous Trp53 locus, predicting that this design would allow for loss of the opposing wild-type chromosome 11 (syntenic to chromosome 17 in humans) to better mimic human HGSOC genetics. This model generated lethal disease at a median of 15.6 months of age. Dissected metastatic tumors were peritoneal and expressed Pax8, consistent with human disease. Immunohistochemical staining of the fallopian tube, ovary, and uterus indicated clear nuclear staining of p53 and Myc on the FTE. Notably, pockets of intraepithelial metastases expressing p53 and Myc were also found on the uterine epithelium. Cells grown ex vivo from uterus or fallopian tube dissections were analyzed for transcriptomes. RNA-seq revealed a clustering of these extracted cancer cells with human HGSOC and p53 mutant serous endometrial cancers. Loss of heterozygosity at the p53 locus was universal in these ex vivo grown cancer cells, as was extensive aneuploidy. The OvTrpMyc model requires only a single transgene for its breeding strategy, better enabling cancer detection, prevention, and treatment experiments. Taken together, the OvTrpMyc model of p53-R270H mutation and Myc expression driven by Ovgp1 is sufficient to recapitulate cancer hallmarks of HGSOC and, surprisingly, p53 mutant serous uterine cancer as well. Citation Format: Joe R Delaney. A minimal driver pair for a spontaneous high-grade serous ovarian cancer mouse model: Myc and p53-R270H [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B106.

Abstract A020: NAD+ Boosting through NRH supplementation enhances treatment efficacy in EOC

Abstract Background: Dihydronicotinamide riboside (NRH), the reduced form of nicotinamide riboside (NR), is a recently identified, naturally occurring precursor of arguably the most crucial cofactor for cellular function, Nicotinamide Adenine Dinucleotide (NAD+). Recent investigation suggests that NRH is more adept at increasing NAD+ stores than traditional NAD precursors, and such extreme NAD+ boosting via NRH supplementation induces cytotoxicity in certain cellular contexts. It has also been shown that the lack of functional BRCA protein in epithelial ovarian cancer (EOC) directly impacts intracellular NAD+ levels. Given that altered cellular metabolism and DNA repair mechanisms are central alterations in EOC, and these processes are functionally dependent on NAD+, we sought to assess whether NRH supplementation in EOC cell lines enhanced cellular cytotoxicity alone and in combination with standard therapeutic agents. Methods: We supplemented 3 ovarian cancer cell lines (SKOV3, OVCAR8 and ES2) with NRH and traditional NAD precursor, NA (nicotinic acid) alone and in combination with carboplatin, paclitaxel, combination, and olaparib. Cytotoxicity was measured via CellTiter-Glo. RNA sequencing of EOC cell lines was performed to determine the differential expression of NAD+ anabolic and catabolic enzymes. Of note, all three cell lines are known to be BRCA1 &amp;2 wildtype while OVCAR8 cells are hypermethylated and harbor loss of heterozygosity (LOH high) at the BRCA1 loci. Results: Significant cytotoxicity was noted in NRH treated cells (~40 %) with minimal cell death in the NA treated lines. Strikingly, NRH supplementation enhanced carboplatin cytotoxicity by 25-50 % depending on cell line, paclitaxel cytotoxicity by 10-25 %, and the combination by 10-35 % whereas NA supplementation had no effect on the baseline treatment induced cytotoxicity. Interestingly, the effect of NRH supplementation with olaparib differed between the ES2 and OVCAR8 cell lines, with a significant 20 % increase in cell death in ES2 vs OVCAR8 cells with a negligible increase in cell death. PARP inhibition by olaparib requires NAD+. RNA sequencing revealed an increased expression in NAMPT, an enzyme involved in the NAD+ salvage pathway in the ES2 cell line relative to the OVCAR8, suggesting that the ES2 cells harbor a baseline NAD+ deficit rendering them more vulnerable to PARP inhibition following NAD+ boosting. This is also consistent with the OVCAR8 data suggesting that hypermethylated BRCA1 results in a relative increase in NAD+ to the extent that NRH supplementation does not enhance the efficacy of PARP inhibition. Conclusions: Here we show that in the context of EOC, exploitation of the NAD+ bioenergetic phenotype through NRH supplementation is a biologically feasible strategy to enhance the response of traditional therapy with potentially minimal toxicity. This data suggests several potential mechanisms by which cellular NAD+ availability belies treatment efficacy and resistance and highlights the potential utility of NAD+ metabolomics as a biomarker to guide treatment decisions. Citation Format: Kevin Lee, Tanvi Joshi, Catherine Lyons, Mary Howard Singleton, Elizabeth Catranis, Sagar Chokshi, Luciana Madiera da Silva, Marie Migaud, Jennifer Scalici. NAD+ Boosting through NRH supplementation enhances treatment efficacy in EOC [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A020.

Abstract B018: MYC is sufficient to generate mid-life high-grade serous ovarian and serous endometrial carcinomas in a BRCA wild type p53-R270H mouse model

Abstract Genetically engineered mouse models (GEMM) have fundamentally changed how cancer etiology, early detection, and treatment can be understood. Mouse models have been developed that support the fallopian tube as the origin of some ovarian cancers. However, serous endometrial cancer is a much rarer gynecologic cancer with fewer models available. The genetics of serous endometrial cancer are very similar that of high-grade serous ovarian cancer. The most common mutation found in both high-grade serous ovarian cancer and serous uterine cancer is p53 and in humans, MYC is amongst the most amplified genes in both tyeps of cancer. We developed a GEMM of high-grade serous ovarian cancer by using a fallopian tube epithelium specific promoter Ovgp1 to express MYC and dominant negative mutant p53-R270H. Female mice developed lethal cancer at an average of 15.1 months. Histopathological examination of mice revealed HGSOC characteristics including nuclear p53 and nuclear MYC in clusters of cells within the fallopian tube epithelium and ovarian surface epithelium. Unexpectedly, nuclear p53 and MYC clustered cell expression was also identified in the uterine luminal epithelium, possibly from intraepithelial metastasis from the fallopian tube epithelium. Extracted tumor cells exhibited strong loss of heterozygosity at the p53 locus, leaving the mutant allele. Copy number alterations in these cancer cells were prevalent, disrupting a large fraction of genes. Transcriptome profiles most closely matched human HGSOC and serous endometrial cancer. Histological and transcriptomic findings are consistent with the hypothesis that serous uterine cancer may originate from the fallopian tube epithelium. Citation Format: Alexandra Blackman, Amy Rees, Rob Bowers, Christian Jones, Silvia Vaena, Shelby Carter, Evan Villamor, Della Evans, George Fullbright, David Long, Laura Spruill, Martin Romeo, Kristi Helke, Anthony Emanuel, Joe Delaney. MYC is sufficient to generate mid-life high-grade serous ovarian and serous endometrial carcinomas in a BRCA wild type p53-R270H mouse model [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B018.

The NF1+/- Immune Microenvironment: Dueling Roles in Neurofibroma Development and Malignant Transformation.

Neurofibromatosis type 1 (NF1) is a common genetic disorder resulting in the development of both benign and malignant tumors of the peripheral nervous system. NF1 is caused by germline pathogenic variants or deletions of the NF1 tumor suppressor gene, which encodes the protein neurofibromin that functions as negative regulator of p21 RAS. Loss of NF1 heterozygosity in Schwann cells (SCs), the cells of origin for these nerve sheath-derived tumors, leads to the formation of plexiform neurofibromas (PNF)-benign yet complex neoplasms involving multiple nerve fascicles and comprised of a myriad of infiltrating stromal and immune cells. PNF development and progression are shaped by dynamic interactions between SCs and immune cells, including mast cells, macrophages, and T cells. In this review, we explore the current state of the field and critical knowledge gaps regarding the role of NF1(Nf1) haploinsufficiency on immune cell function, as well as the putative impact of Schwann cell lineage states on immune cell recruitment and function within the tumor field. Furthermore, we review emerging evidence suggesting a dueling role of Nf1+/- immune cells along the neurofibroma to MPNST continuum, on one hand propitiating PNF initiation, while on the other, potentially impeding the malignant transformation of plexiform and atypical neurofibroma precursor lesions. Finally, we underscore the potential implications of these discoveries and advocate for further research directed at illuminating the contributions of various immune cells subsets in discrete stages of tumor initiation, progression, and malignant transformation to facilitate the discovery and translation of innovative diagnostic and therapeutic approaches to transform risk-adapted care.

Analytical Validation of Loss of Heterozygosity and Mutation Detection in Pancreatic Fine-Needle Aspirates by Capillary Electrophoresis and Sanger Sequencing.

Pancreatic cystic disease, including duct dilation, represents precursor states towards the development of pancreatic cancer, a form of malignancy with relatively low incidence but high mortality. While most of these cysts (>85%) are benign, the remainder can progress over time, leading to malignant transformation, invasion, and metastasis. Cytologic diagnosis is challenging, limited by the paucity or complete absence of cells representative of cystic lesions and fibrosis. Molecular analysis of fluids collected from endoscopic-guided fine-needle aspiration of pancreatic cysts and dilated duct lesions can be used to evaluate the risk of progression to malignancy. The basis for the enhanced diagnostic utility of molecular approaches is the ability to interrogate cell-free nucleic acid of the cyst/duct and/or extracellular fluid. The allelic imbalances at tumor suppressor loci and the selective oncogenic drivers are used clinically to help differentiate benign stable pancreatic cysts from those progressing toward high-grade dysplasia. Methods are discussed and used to determine the efficacy for diagnostic implementation. Here, we report the analytical validation of methods to detect causally associated molecular changes integral to the pathogenesis of pancreatic cancer from pancreatic cyst fluids.

Ataxia-Telangiectasia Mutated Loss-of-Function Displays Variant and Tissue-Specific Differences across Tumor Types.

Mutations in the ATM gene are common in multiple cancers, but clinical studies of therapies targeting ATM-aberrant cancers have yielded mixed results. Refinement of ATM loss of function (LOF) as a predictive biomarker of response is urgently needed. We present the first disclosure and preclinical development of a novel, selective ATR inhibitor, ART0380, and test its antitumor activity in multiple preclinical cancer models. To refine ATM LOF as a predictive biomarker, we performed a comprehensive pan-cancer analysis of ATM variants in patient tumors and then assessed the ATM variant-to-protein relationship. Finally, we assessed a novel ATM LOF biomarker approach in retrospective clinical data sets of patients treated with platinum-based chemotherapy or ATR inhibition. ART0380 had potent, selective antitumor activity in a range of preclinical cancer models with differing degrees of ATM LOF. Pan-cancer analysis identified 10,609 ATM variants in 8,587 patient tumors. Cancer lineage-specific differences were seen in the prevalence of deleterious (Tier 1) versus unknown/benign (Tier 2) variants, selective pressure for loss of heterozygosity, and concordance between a deleterious variant and ATM loss of protein (LOP). A novel ATM LOF biomarker approach that accounts for variant classification, relationship to ATM LOP, and tissue-specific penetrance significantly enriched for patients who benefited from platinum-based chemotherapy or ATR inhibition. These data help to better define ATM LOF across tumor types in order to optimize patient selection and improve molecularly targeted therapeutic approaches for patients with ATM LOF cancers.

The genomic landscape of transposable elements in yeast hybrids is shaped by structural variation and genotype-specific modulation of transposition rate.

Transposable elements (TEs) are major contributors to structural genomic variation by creating interspersed duplications of themselves. In return, structural variants (SVs) can affect the genomic distribution of TE copies and shape their load. One long-standing hypothesis states that hybridization could trigger TE mobilization and thus increase TE load in hybrids. We previously tested this hypothesis (Hénault et al., 2020) by performing a large-scale evolution experiment by mutation accumulation (MA) on multiple hybrid genotypes within and between wild populations of the yeasts Saccharomyces paradoxus and Saccharomyces cerevisiae. Using aggregate measures of TE load with short-read sequencing, we found no evidence for TE load increase in hybrid MA lines. Here, we resolve the genomes of the hybrid MA lines with long-read phasing and assembly to precisely characterize the role of SVs in shaping the TE landscape. Highly contiguous phased assemblies of 127 MA lines revealed that SV types like polyploidy, aneuploidy, and loss of heterozygosity have large impacts on the TE load. We characterized 18 de novo TE insertions, indicating that transposition only has a minor role in shaping the TE landscape in MA lines. Because the scarcity of TE mobilization in MA lines provided insufficient resolution to confidently dissect transposition rate variation in hybrids, we adapted an in vivo assay to measure transposition rates in various S. paradoxus hybrid backgrounds. We found that transposition rates are not increased by hybridization, but are modulated by many genotype-specific factors including initial TE load, TE sequence variants, and mitochondrial DNA inheritance. Our results show the multiple scales at which TE load is shaped in hybrid genomes, being highly impacted by SV dynamics and finely modulated by genotype-specific variation in transposition rates.

Tumor Predisposing Post-Zygotic Chromosomal Alterations in Bladder Cancer-Insights from Histologically Normal Urothelium.

Bladder urothelial carcinoma (BLCA) is the 10th most common cancer with a low survival rate and strong male bias. We studied the field cancerization in BLCA using multi-sample- and multi-tissue-per-patient protocol for sensitive detection of autosomal post-zygotic chromosomal alterations and loss of chromosome Y (LOY). We analysed 277 samples of histologically normal urothelium, 145 tumors and 63 blood samples from 52 males and 15 females, using the in-house adapted Mosaic Chromosomal Alterations (MoChA) pipeline. This approach allows identification of the early aberrations in urothelium from BLCA patients. Overall, 45% of patients exhibited at least one alteration in at least one normal urothelium sample. Recurrence analysis resulted in 16 hotspots composed of either gains and copy number neutral loss of heterozygosity (CN-LOH) or deletions and CN-LOH, encompassing well-known and new BLCA cancer driver genes. Conservative assessment of LOY showed 29%, 27% and 18% of LOY-cells in tumors, blood and normal urothelium, respectively. We provide a proof of principle that our approach can characterize the earliest alterations preconditioning normal urothelium to BLCA development. Frequent LOY in blood and urothelium-derived tissues suggest its involvement in BLCA.

An immunogenetic basis for lung cancer risk.

Cancer risk is influenced by inherited mutations, DNA replication errors, and environmental factors. However, the influence of genetic variation in immunosurveillance on cancer risk is not well understood. Leveraging population-level data from the UK Biobank and FinnGen, we show that heterozygosity at the human leukocyte antigen (HLA)-II loci is associated with reduced lung cancer risk in smokers. Fine-mapping implicated amino acid heterozygosity in the HLA-II peptide binding groove in reduced lung cancer risk, and single-cell analyses showed that smoking drives enrichment of proinflammatory lung macrophages and HLA-II+ epithelial cells. In lung cancer, widespread loss of HLA-II heterozygosity (LOH) favored loss of alleles with larger neopeptide repertoires. Thus, our findings nominate genetic variation in immunosurveillance as a critical risk factor for lung cancer.

Precision needle-punch tumor enrichment from paraffin blocks improves the detection of clinically actionable genomic alterations and biomarkers.

While many molecular assays can detect mutations at low tumor purity and variant allele frequencies, complex biomarkers such as tumor mutational burden (TMB), microsatellite instability (MSI), and genomic loss of heterozygosity (gLOH) require higher tumor purity for accurate measurement. Scalable, quality-controlled, tissue-conserving methods to increase tumor nuclei percentage (TN%) from tumor specimens are needed for complex biomarkers and hence necessary to maximize patient matching to approved therapies or clinical trial enrollment. We evaluated the clinical utility and performance of precision needle-punch enrichment (NPE) compared with traditional razor blade macroenrichment of tumor specimens on molecular testing success. Pathologist-directed NPE was performed manually on formalin-fixed, paraffin embedded (FFPE) blocks. Quality control of target capture region and quantity of residual tumor in each tissue block was determined via a post-enrichment histologic slide recut. Resultant tumor purity and biomarker status were determined by the computational analysis pipeline component of the FDA-approved next-generation sequencing (NGS) assay, FoundationOne®CDx. Following NPE implementation for real-world clinical samples, assay performance and biomarker (MSI, TMB, gLOH) detection were analyzed. In real-world clinical samples, enrichment rate via NPE was increased to ~50% over a 2.5-year period, exceeding the prior use of razor blade macro-enrichment (<30% of cases) prior to NPE implementation due to proven efficacy in generating high quality molecular results from marginal samples and the ease of use for both pathologist and histotechnologists. NPE was associated with lower test failures, higher computational tumor purity, and higher rates of successful TMB, MSI and gLOH determination when stratified by pre-enriched (incipient) tumor nuclei percentage. In addition, challenging cases in which tumor content was initially insufficient for testing were salvaged for analysis of biomarker status, gene amplification/deletion, and confident mutant or wild-type gene status determination. Pathologist-directed precision enrichment from tissue blocks (aka NPE) increases tumor purity, and consequently, yields a greater number of successful tests and complex biomarker determinations. Moreover, this process is rapid, safe, inexpensive, scalable, and conserves patient surgical pathology material. NPE may constitute best practice with respect to enriching tumor cells from low-purity specimens for biomarker detection in molecular laboratories.

Genomic epidemiology of antifungal resistance in human and avian isolates of Candida albicans: a pilot study from the One Health perspective.

Stress-induced genomic changes in Candida albicans contribute to the adaptation of this species to various environmental conditions. Variations of the genome composition of animal-origin C. albicans strains are largely unexplored and drug resistance or other selective pressures driving the evolution of these yeasts remained an intriguing question. Comparative genome analysis was carried out to uncover chromosomal aneuploidies and regions with loss of heterozygosity (LOH), two mechanisms that manage genome plasticity. We detected aneuploidy only in human isolates. Bird-derived isolates showed LOH in genes commonly associated with antifungal drug resistance similar to human isolates. Our study suggests that environmental fungicide usage might exert selective pressure on C. albicans infecting animals, thus contributing to the spread of potentially resistant strains between different hosts.

Identification of Germline FOXE1 and Somatic MAPK Pathway Gene Alterations in Patients with Malignant Struma Ovarii, Cleft Palate and Thyroid Cancer.

Germline variants in the FOXE1 transcription factor have been associated with thyroid ectopy, cleft palate (CP) and thyroid cancer (TC). Here, we aimed to clarify the role of FOXE1 in Portuguese families (F1 and F2) with members diagnosed with malignant struma ovarii (MSO), an ovarian teratoma with ectopic malignant thyroid tissue, papillary TC (PTC) and CP. Two rare germline heterozygous variants in the FOXE1 promoter were identified: F1) c.-522G>C, in the proband (MSO) and her mother (asymptomatic); F2) c.9C>T, in the proband (PTC), her sister and her mother (CP). Functional studies using rat normal thyroid (PCCL3) and human PTC (TPC-1) cells revealed that c.9C>T decreased FOXE1 promoter transcriptional activity in both cell models, while c.-522G>C led to opposing activities in the two models, when compared to the wild type. Immunohistochemistry and RT-qPCR analyses of patients' thyroid tumours revealed lower FOXE1 expression compared to adjacent normal and hyperplastic thyroid tissues. The patient with MSO also harboured a novel germline AXIN1 variant, presenting a loss of heterozygosity in its benign and malignant teratoma tissues and observable β-catenin cytoplasmic accumulation. The sequencing of the F1 (MSO) and F2 (PTC) probands' tumours unveiled somatic BRAF and HRAS variants, respectively. Germline FOXE1 and AXIN1 variants might have a role in thyroid ectopy and cleft palate, which, together with MAPK pathway activation, may contribute to tumours' malignant transformation.

Pan-Cancer Interrogation of MUTYH Variants Reveals Biallelic Inactivation and Defective Base Excision Repair Across a Spectrum of Solid Tumors.

Biallelic germline pathogenic variants of the base excision repair (BER) pathway gene MUTYH predispose to colorectal cancer (CRC) and other cancers. The possible association of heterozygous variants with broader cancer susceptibility remains uncertain. This study investigated the prevalence and consequences of pathogenic MUTYH variants and MUTYH loss of heterozygosity (LOH) in a large pan-cancer analysis. Data from 354,366 solid tumor biopsies that were sequenced as part of routine clinical care were analyzed using a validated algorithm to distinguish germline from somatic MUTYH variants. Biallelic germline pathogenic MUTYH variants were identified in 119 tissue biopsies. Most were CRCs and showed increased tumor mutational burden (TMB) and a mutational signature consistent with defective BER (COSMIC Signature SBS18). Germline heterozygous pathogenic variants were identified in 5,991 biopsies and their prevalence was modestly elevated in some cancer types. About 12% of these cancers (738 samples: including adrenal gland cancers, pancreatic islet cell tumors, nonglioma CNS tumors, GI stromal tumors, and thyroid cancers) showed somatic LOH for MUTYH, higher rates of chromosome 1p loss (where MUTYH is located), elevated genomic LOH, and higher COSMIC SBS18 signature scores, consistent with BER deficiency. This analysis of MUTYH alterations in a large set of solid cancers suggests that in addition to the established role of biallelic pathogenic MUTYH variants in cancer predisposition, a broader range of cancers may possibly arise in MUTYH heterozygotes via a mechanism involving somatic LOH at the MUTYH locus and defective BER. However, the effect is modest and requires confirmation in additional studies before being clinically actionable.

Abstract B002: Profiling intra-tumor heterogeneity and chromosomal instability in malignant peripheral nerve sheath tumors

Abstract Malignant peripheral nerve sheath tumors (MPNSTs) are rare soft-tissue sarcomas that manifest on peripheral nerves, constituting 2% of all sarcomas. They arise both sporadically and in 8% to 13% of individuals with neurofibromatosis type 1 (NF1), where they represent the major cause of morbidity and mortality. While previous genomic analyses of MPNSTs have been conducted on small cohorts, often limited to single samples per patient and lacking comprehensive resolution, the extent of intra-tumor heterogeneity and the contribution of chromosomal instability to MPNST development remain unclear. To address these limitations, we developed a multi-omics integration pipeline and applied it to integrate bulk Next Generation Sequencing (NGS), single-cell RNA sequencing, single-cell DNA sequencing, spatial transcriptomics (ST), and Laser Capture Microsection (LCM) information. 35 tumor samples were collected from 10 patients with MPNST, and for each sample, data were generated using the above five techniques. Overall, we detected copy-number aberrations recurrently found in MPNSTs, including events such as loss of heterozygosity (LOH), whole genome doubling (WGD) and chromosome 7 and 8 amplifications. Subclonal copy number alterations were called by the Battenberg algorithm, followed by SNV clustering using Dirichlet Process-based methods. Applying the pigeonhole principle, we constructed tumor phylogenetic trees. Intriguingly, our findings indicate that for patient 2.3, primary tumor samples PT_1 and PT_2 displayed a close phylogenetic relationship, while local recurrences LR_2 and LR_3 represent a different branch of the phylogenetic tree. Following stringent quality control and cell type annotation, we leveraged normal cells identified within the snRNA data as controls to infer copy number changes in tumor cells through inferCNV. Notably, in patient 2.16, we identified significant copy number gains on chromosomes 5, 7, 8, and 15. Comparing the inferred copy number changes derived from snRNA with those obtained through Battenberg copy number profiles showed a remarkable congruence, thereby reinforcing the robustness of our results. To reveal communication networks among tumor microenvironment (TME) cells, we used iTalk to visualize ligand-receptor interactions between distinct cell types. For Patient 2.3, macrophages emerged as key players, releasing cytokines such as IL-16 and IL-18, which exerted their influence on receptors in tumor cells. Additionally, T cells engaged in checkpoint factors, including TNFSF4 and CD28, interacting with receptors on tumor cells, such as TRAF2 and LGALS9. Our research revealed that MPNSTs are primarily driven by chromosomal instability and intra-tumor heterogeneity is shaped by TME interactions. We enhanced our understanding of the tumors' evolutionary histories and intra-tumor heterogeneity down to single-cell resolution. By shedding light on the genomic landscape of MPNSTs, we aspire to make profound contributions to the field of sarcoma research, ultimately improving patient outcomes. Citation Format: Yixiao Cheng, Haixi Yan, Maxime Tarabichi, Zhihui Zhang, Chunxu Gao, Tom Lesluyes, Adrienne Flanagan, Peter Van Loo. Profiling intra-tumor heterogeneity and chromosomal instability in malignant peripheral nerve sheath tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B002.

Abstract A036: The genetic landscape of head and neck cancer using brush biopsy

Abstract Oral potentially malignant disorders (OPMDs) are characterized by the presence of dysplasia and DNA copy number aberrations (CNAs) and subsequently progress to oral squamous cell carcinoma (OSCC). OSCC has a 5-year survival rate of only about 50% and varies greatly depending on site and stage. The combination of next-generation sequencing and advanced computational data analysis approaches has revolutionized our understanding of the genomic underpinnings of cancer development and progression. This can reveal individual targetable alterations, mutational load, complex mutation signatures, and tumor-specific antigens, which might inform the utilization of targeted therapies and early diagnosis. Oral brush biopsy represents an attractive non-invasive advantage in the characterization of premalignant and malignant oral lesions. Still, a sufficient number of abnormal cells is mandatory to diagnose. The current study tested the possibility of recreating the genomic landscape of oral cancer using brush biopsy. Materials and methods: As a pilot study to address this question, we analyzed matched tissue and brush biopsy of a patient with synchronous OPMD and OSCC. Brush and tissue biopsy was taken from normal epithelium, OPMD, and OSCC. We scrutinized the evolution and heterogeneity of these three brush/tissue biopsy pairs. The techniques used include whole exome sequencing (WES), mutational signature analysis to evaluate smoking and other aberrant signatures, copy number analysis (using ASCAT), and subclonal architecture reconstruction (using DPclust). Results: To analyze intra-tumoral heterogeneity (ITH), ASCAT and DPclust were used to call copy number aberrations (CNA) and cluster somatic SNVs based on cancer cell fraction (CCF), respectively. We observed a large number of shared SNVs between the OPMD and OSCC lesions suggesting a common evolutionary origin. After adjusting the ploidy and purity based on DPClust results, ASCAT profiles revealed differences in aberrant tumor cell fraction, gains and loss of heterozygosity, and copy number neutral events between the samples. It showed almost similar profiles between the respective lesions' brush and tissue samples, suggesting that brush biopsy can recapitulate the genomic landscape. The OPMD and OSCC lesions showed loss of heterozygosity in chromosome 9 and chromosome 17, which play a potential role in tumorigenesis with loss of function of P53. We inferred the subclonal architecture of each sample, illuminating the clonal relationships between samples and distinct lineages of precursor clones developing independently. Conclusion: Collectively, these results emphasize the possibility of using brush biopsy to reconstruct the genomic characterization of OPMD and OSCC show prognostic potential for diagnosis and intervention strategies, and accelerate progress in precision cancer care. Citation Format: Evit John, Tom Lesluyes, Xiao Zhao, Peter Van Loo. The genetic landscape of head and neck cancer using brush biopsy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A036.

Comprehensive analysis of targetable alterations in urachal cancer by NGS.

663 Background: Urachal cancer (UrC) is a rare genitourinary cancer with molecular drivers commonly found in colorectal cancer and harbors fewer actionable alterations. Although prior studies have identified genomic alterations associated with UrC, these lack comprehensive genomic profiling, often using targeted panels, and lack RNA interrogation. This study presents detailed characterization of molecular profiles and the tumor microenvironment of real-world UrC patient samples. Methods: UrC samples (n = 42) were profiled using next-generation sequencing (NGS) of DNA and RNA. Prevalence was calculated for pathogenic SNVs/Indels and copy number amplifications, dMMR/MSI status assessed by IHC and NGS, PD-L1 expression measured by IHC, and TMB-H defined as ≥ 10 mut/Mb. Immune cell fractions of TME were estimated using RNA deconvolution. Mann-Whitney U, chi-square, and Fisher exact tests were applied where appropriate, with p-values adjusted for multiple comparisons. Histological features were reviewed by a genitourinary pathologist. Results: UrC samples were collected from 19 males and 23 females with a median age at sample collection was 58.5 years (range: 24-86 years). Pathology review revealed the majority were adenocarcinomas (n=41/42, 97.6%), including mucinous (n=17/41, 41.5%) and enteric adenocarcinomas (n=10/41, 24.4%). Pathogenic variants were most commonly observed in TP53 (n = 35, 83.3%), KRAS (n=15, 36%), GNAS (n=5, 12%), SMAD4 (n=4, 10%), PIK3CA (n=4, 10%), STK11 (n=3, 7%), BRCA2 (n=3, 7%), ARID1A (n=2, 5%), PTEN (n=2, 5%), ERBB2 (n=2, 5%), BRAF (n=2, 5%), and APC (n=2, 5%). MAPK pathway alterations were present in 52.3% (n=22) overall, with increased MAPK pathway activation in MAPK-altered UrC (median MPAS: 0.77 vs -0.61, p = 0.066). Loss of heterozygosity was prevalent (n = 9, 29%). While no UrC tumors had dMMR/MSI, 5% (n=2) were TMB-H and 9% (n=3) were PD-L1+. The mean fraction of T cells – CD8 (0.4%) and monocytes (0.0%) represented a smaller fraction UrC TME composition compared to macrophages M1 (5.19%) and neutrophil (6.4%). The median NK cell fraction was significantly higher in MAPK-altered UrC patients (3.5 vs 2.7%, p = 0.022), yet a transcriptional signature of response to immunotherapy (IFN-g) was similar between MAPK-altered and MAPK-WT UrC patients (median IFN: -0.366 vs -0.384, p = 0.052). Conclusions: This study provides a comprehensive molecular characterization of UrC and the associated immune landscape. While MAPK and DNA repair gene alterations were common, UrC tumors rarely harbored predictive markers of response to immunotherapy, suggesting limited efficacy in this patient population. However, the recurrence of MAPK alterations and associated pathway activation in UrC warrants further investigation of MAPK-targeted therapies in prospective clinical trials.