Loss Of Heterozygosity For Chromosome Research Articles

Genome-wide loss of heterozygosity predicts aggressive, treatment-refractory behavior in pituitary neuroendocrine tumors

Pituitary neuroendocrine tumors (PitNETs) exhibiting aggressive, treatment-refractory behavior are the rare subset that progress after surgery, conventional medical therapies, and an initial course of radiation and are characterized by unrelenting growth and/or metastatic dissemination. Two groups of patients with PitNETs were sequenced: a prospective group of patients (n = 66) who consented to sequencing prior to surgery and a retrospective group (n = 26) comprised of aggressive/higher risk PitNETs. A higher mutational burden and fraction of loss of heterozygosity (LOH) was found in the aggressive, treatment-refractory PitNETs compared to the benign tumors (p = 1.3 × 10−10 and p = 8.5 × 10−9, respectively). Within the corticotroph lineage, a characteristic pattern of recurrent chromosomal LOH in 12 specific chromosomes was associated with treatment-refractoriness (occurring in 11 of 14 treatment-refractory versus 1 of 14 benign corticotroph PitNETs, p = 1.7 × 10−4). Across the cohort, a higher fraction of LOH was identified in tumors with TP53 mutations (p = 3.3 × 10−8). A machine learning approach identified loss of heterozygosity as the most predictive variable for aggressive, treatment-refractory behavior, outperforming the most common gene-level alteration, TP53, with an accuracy of 0.88 (95% CI: 0.70–0.96). Aggressive, treatment-refractory PitNETs are characterized by significant aneuploidy due to widespread chromosomal LOH, most prominently in the corticotroph tumors. This LOH predicts treatment-refractoriness with high accuracy and represents a novel biomarker for this poorly defined PitNET category.

Loss of heterozygosity for chromosomes 16q in Wilms tumors predicts outcomes: A meta-analysis.

The research findings suggest that the prognosis of children with Wilms tumor (WT) is affected by various factors. Some scholars have indicated that loss of heterozygosity (LOH) on chromosome 16q is associated with a poor prognosis in patients with WT. To further elucidate this relationship, we conducted a meta-analysis. This meta-analysis was registered in INPLASY (INPLASY2023100060). We systematically searched databases including Embase, PubMed, Web of Science, Cochrane, and Google Scholar up to May 31, 2020, for randomized trials reporting any intrapartum fetal surveillance approach. The meta-analysis was performed within a frequentist framework, and the quality and network inconsistency of trials were assessed. Odds ratios and 95%CIs were calculated to report the relationship between event-free survival and 16q LOH in patients with WT. Eleven cohort studies were included in this meta-analysis to estimate the relationship between event-free survival and 16q LOH in patients with WT (I2 = 25%, P < 0.001). As expected, 16q LOH can serve as an effective predictor of event-free survival in patients with WT (risk ratio = 1.95, 95%CI: 1.52-2.49, P < 0.001). In pediatric patients with WT, there exists a partial correlation between 16q LOH and an unfavorable treatment prognosis. Clinical detection of 16q chromosome LOH warrants increased attention to the patient's prognosis.

Abstract 5211: Somatic copy number variation as prognostic marker for recurrence in never-smokers with early-stage lung adenocarcinoma

Abstract Objective: Lung cancer in never-smokers has a distinct genomic profile with low tumor mutation burden, more tumor promotor mutations and fewer somatic copy number variations (CNV) than smokers. CNVs have shown to be associated with poor outcome in advanced stage disease. However, the relationship between CNV and clinicopathologic features on prognosis in early-stage lung cancer is undefined. To address this knowledge gap, we assessed the impact of CNV on recurrence in never-smokers who underwent curative resection for pathological stage I-II lung adenocarcinoma using an integrated genomic and clinicopathological analysis. Methods: We analyzed a cohort of 210 never-smokers with stage I-II lung adenocarcinoma treated from 2014 to 2022 who met the inclusion criteria (no neoadjuvant therapy, pathological stage I-II, complete resection, next-generation sequencing available). The patient cohort was stratified using unsupervised hierarchical clustering of arm-level CNVs. We assessed cumulative incidence of recurrence (CIR) between CNV groups using competing risk regression analysis, adjusting for SUVmax, pathologic stage, lymphovascular invasion, IASLC grade, and tumor mutation burden. Results: Of the 210 patients, 159 patients were female (76%), and patients were primarily from Caucasian (n=140, 67%) or Asian decent (n=50, 24%). Based on arm-level CNV clustering, the cohort was stratified into three groups: CNV low (n=86, 41%), CNV moderate (n=75, 36%) and CNV high (n=49, 23%). The CNV low tumors were characterized by a low CNV burden. Conversely, the CNV moderate tumors primarily exhibited gains in chromosomes 1q, 5p and 7p, along with loss of heterozygosity in chromosome 9p, 9q and 13. CNV high tumors were characterized by whole-genome doubling. Whole-genome doubling was significantly more common in CNV high tumors (n=33, 66%) compared to CNV low (n=2, 2.3%) and CNV moderate tumors (n=2, 2.7%) (p&amp;lt;.001). EGFR (n=145, 69%) and TP53 (n=61, 29%) were the most frequently altered genes, with EGFR alterations being significantly more prevalent in CNV high tumors (n=41, 84%) compared to CNV low tumors (n=46, 53%) (p&amp;lt;.001). In total, 42 patients (20%) developed recurrences. The CIR was lower in the CNV low group (5y-CIR, 14%, 95%CI: 6.5%-25%) compared to the CNV moderate (5y-CIR, 31%, 95%CI: 18%-44%) and CNV high group (5y-CIR, 48%, 95%CI: 27%-66%) (p=.004), with adjusted hazard ratios of 2.3 (95%CI: 0.93-5.71; p=0.074) and 2.65 (95%CI: 1.07-6.60; p=.037), respectively. The CIR for locoregional (n=11, 5%) and distant recurrence (n=31, 15%) remained lower in the CNV low group compared to the combined CNV moderate &amp; high groups (Gray test p=.020 and p=.049, respectively). Conclusion: In never-smokers who underwent curative treatment of pathological stage I-II lung adenocarcinoma, copy number status seems a predictor of recurrence, with CNV low tumors associated with the best prognosis. Citation Format: Stijn Vanstraelen, Allison Reiner, Brooke H. Mastrogiacomo, Kay See Tan, Joseph Dycoco, Bernard J. Park, Prasad S. Adusumilli, Matthew J. Bott, David R. Jones, Gaetano Rocco. Somatic copy number variation as prognostic marker for recurrence in never-smokers with early-stage lung adenocarcinoma [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 5211.

CRISPR/Cas9-mediated editing of ACTB in induced pluripotent stem cells: A model for investigating human ACTB loss-of-function and genetic adaptive responses

Heterozygous beta-actin (ACTB) indel and nonsense mutations are linked to developmental disorders. We generated two CRISPR/Cas9 human induced pluripotent stem cell (iPSC) lines, WTSIi018-B-19 and WTSIi018-B-20, carrying heterozygous and homozygous indel mutations in ACTB exon 4. Both iPSCs exhibited normal cell morphology, expression of pluripotency markers, and the ability to differentiate into the three primary germ layers. While iPSCs with a heterozygous ACTB mutation maintain genome integrity, homozygous mutants showed a loss of heterozygosity in chromosome three. These mutants provide a powerful model to study the onset, progression, and complex interplay of genetic compensation and phenotypic variation of ACTB-related diseases.

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.

Abstract PO-015: 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 and show prognostic potential for diagnosis and intervention strategies and accelerate progress in precision cancer care. Citation Format: Evit John, Tom Lesluyes, Toby Baker, Xiao Zhao, Peter Van Loo. The genetic landscape of head and neck cancer using brush biopsy [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-015.

Embryonal rhabdomyosarcoma of oropharynx in a 65-year-old male with indolent clinical presentation and unusual morphology

Abstract Introduction/Objective Embryonal rhabdomyosarcoma (ERMS) is a malignant primitive mesenchymal tumor which commonly affects children and adolescence. Although head and neck area is a common location for ERMS, oropharynx is rarely involved. ERMS in adults often present with stage IV disease and show a relatively poor prognosis. Loss of heterozygosity in 11p15.5 is one of the common genetic findings in sporadic ERMS. Methods/Case Report A 65-year-old male with no significant medical history, presented with pharyngodynia and was found to have a polypoid mass originating from the pharyngeal surface of the soft palate and extends into the vallecula. He reported having this mass for a decade and having intermittent episodes of similar oropharyngeal symptoms which were usually resolved with antibiotics. Results (if a Case Study enter NA) Excisional biopsy shows a 2.4 cm polypoid mass with atypical cellular proliferation in submucosa. Tumor cells demonstrate ovoid to round nuclei and eosinophilic cytoplasm. No mitotic figure was found and Ki67 proliferation index was extremely low (&amp;lt;1%). Immunostains showed that the tumor cells are positive for Desmin and MyoD1. Scattered cells are positive for Myogenin. Due to the unusual clinical presentation and low proliferation activity, late onset of fetal rhabdomyoma was also considered in the differential diagnosis. The specimen was sent for chromosomal microarray which showed chromosomal gains and losses including loss of heterozygosity in chromosome 11. This finding together with cytologic atypia are mostly compatible with the diagnosis of ERMS. Post-biopsy MRI and PET-CT did not reveal any residual disease or distant metastasis. Patient has been followed up for 6 months after biopsy with no sign of recurrence. Conclusion Our case indicates that ERMS in adult can present with an indolent clinical process and low proliferating activity. Diagnosis can be challenging in this scenario. Chromosomal microarray is a useful tool to help confirm the diagnosis.

O-090 Correcting a PLCζ mutation in the human germ line to overcome hereditary infertility

Abstract Study question Can clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing result in the correction of a single base pair substitution that causes male infertility? Summary answer CRISPR/Cas9 administration during intracytoplasmic sperm injection (ICSI) leads to correction attempts of mutant phospholipase C zeta (PLCζ), howeverc loss-of-heterozygosity (LOH). What is known already Failed fertilization after ICSI can be caused by mutations in the sperm-related oocyte factor PLCζ which can be overcome by assisted oocyte activation (AOA). In this way, children may inherit the infertility-causing mutation. Mutation transmission can be overcome through CRISPR/Cas9 delivery during ICSI. In previous studies using CRISPR/Cas9 in the human germline for mutation correction, loss-of-heterozygosity (LOH, loss of the allele of one of the parents) was observed. Two different explanations were given, namely partial or complete paternal chromosomal loss or the correction of the mutation by using the maternal wild-type allele instead of the exogeneous supplied repair template. Study design, size, duration We injected a gRNA-Cas9 protein complex to target the PLCζ mutant allele, a repair template harboring the desired nucleotide substitution and an additional synonymous variant to track template usage, together with patient’s sperm. To overcome fertilization failure, AOA was applied during ICSI. After a culture period of maximal 6 days the embryos were collected. At day 3, some embryos were dissociated in individual blastomeres. The extracted DNA was analyzed through different genetic sequencing techniques. Participants/materials, setting, methods Donated sperm of a patient experiencing complete fertilization failure after routine ICSI, harboring a heterozygous base pair substitution in PLCZ1 (c.136-1G&amp;gt;C), was utilized. Sperm was injected in donated in vitro matured oocytes or in vivo matured oocytes containing clusters of smooth endoplasmic reticulum. Next-generation sequencing was used to assess correction potential. Short tandem repeat (STR) and single nucleotide polymorphism (SNP) assays were used to determine whether the sperm contained the mutation and to evaluate LOH. Main results and the role of chance CRISPR/Cas9 injections had no significant impact (p &amp;gt; 0.05) on embryonic development. Due to the heterozygous nature of the mutation, 47% (27/58) of the embryos originated from mutated sperm injection. The CRISPR components showed a high specificity with absence of insertions/deletions in 97% of the embryos originating from wild-type sperm (n = 31). Embryos originating from mutant sperm (n = 27) fall into three categories:(1) 22% showed the untargeted mutant allele, (2) 52% showed additional mutagenesis and (3) 26% showed the wild-type allele, which could be explained by correction. Mosaicism, defined as various editing events, was present in 17% (1), 21% (2) and 71% (3) of the embryos. The low occurrence of the synonymous variant, incorporated in the repair template, suggests that the template is not used during correction attempts. In only 29% (2/7) and 14% (1/7) of the ‘corrected embryos’, respectively long (&amp;gt;18Mb) or medium width LOH (4Mb) was observed through STR analysis. SNP analysis in closer proximity showed in 71% (5/7) of the embryos LOH, even in the absence of LOH through STR, suggesting also the occurrence of short width LOH. These results will be studied in more detail before definitive conclusions can be made. Chromosomal LOH will be studied by ddRADseq. Limitations, reasons for caution The occurrence of mosaicism and LOH might complicate the use of traditional CRISPR/Cas9 in human embryos and should be studied in detail to draw definite conclusions on its potential future use. To this end, genomic data have been produced from both individual blastomeres and whole-embryos which will be further analyzed. Wider implications of the findings Our findings demonstrate caution to use CRISPR/Cas9 to correct mutations in the germ line. They seem to contradict other reports that show predominant lack of mosaicism and presence of long width LOH. A deeper evaluation will be undertaken to define the length and type of LOH in this study. Trial registration number Not Applicable

2. Giant cell glioblastoma with massive loss of heterozygosity: A new molecularly defined subtype

Giant cell glioblastoma (GCG) is a rare histologic variant of glioblastoma that is characterized by numerous bizarre, multinucleate giant cells and abundant reticulin deposition. GCG occurs in younger patients (mean age, 44 years) and generally has a better prognosis than other variants of IDH-wildtype glioblastoma. Although prior studies have identified frequent TP53 alterations in these tumors, the genomics of GCG remain poorly characterized. Herein, we describe an inter-institutional analysis of seven patients ranging from age 9 to 68 of variable race and gender diagnosed with GCG. Across 100% (7/7) of specimens in our cohort, chromosomal microarray analysis and targeted next generation sequencing consistently identified near haploidization / massive loss of heterozygosity (LOH). Remarkably, the genomic profile of these tumors showed LOH of chromosomes 3, 4, 6, 8, 13, 14, 15, 17, 22 in all cases (7/7), and LOH of chromosomes 1, 2, 5, 9, 10, 11, 18, 19 in 6/7 cases. Notably, chromosome 7 always retained heterozygosity. We propose that this genomic profile represents a distinct subtype of GCG. These findings provide evidence of an unusual mechanism of tumorigenesis and could have the potential to reveal new tumor vulnerability.

Integrated genomic profiling and modelling for risk stratification in patients with advanced oesophagogastric adenocarcinoma

ObjectivePrognosis of patients with advanced oesophagogastric adenocarcinoma (mEGAC) is poor and molecular determinants of shorter or longer overall survivors are lacking. Our objective was to identify molecular features and develop...

Genetic and clinical landscape of breast cancers with germline BRCA1/2 variants

The genetic and clinical characteristics of breast tumors with germline variants, including their association with biallelic inactivation through loss-of-heterozygosity (LOH) and second somatic mutations, remain elusive. We analyzed germline variants of 11 breast cancer susceptibility genes for 1,995 Japanese breast cancer patients, and identified 101 (5.1%) pathogenic variants, including 62 BRCA2 and 15 BRCA1 mutations. Genetic analysis of 64 BRCA1/2-mutated tumors including TCGA dataset tumors, revealed an association of biallelic inactivation with more extensive deletions, copy neutral LOH, gain with LOH and younger onset. Strikingly, TP53 and RB1 mutations were frequently observed in BRCA1- (94%) and BRCA2- (9.7%) mutated tumors with biallelic inactivation. Inactivation of TP53 and RB1 together with BRCA1 and BRCA2, respectively, involved LOH of chromosomes 17 and 13. Notably, BRCA1/2 tumors without biallelic inactivation were indistinguishable from those without germline variants. Our study highlights the heterogeneity and unique clonal selection pattern in breast cancers with germline variants.

Abstract 2702: Analysis of low-pass sequencing data reveals extensive loss-of-heterozygosity in circulating multiple myeloma cells

Abstract Background: Clonal evolution and heterogeneity are among the factors that make treatment of Multiple Myeloma (MM) challenging, and disease monitoring requires invasive bone marrow biopsies. Tumor heterogeneity has previously been demonstrated at copy-number level, showing patterns of recurring Copy-Number Alterations (CNA). Conversely, Loss-of-Heterozygosity (LoH) profiling has been investigated less extensively. Presence of genomic patterns of LoH has been associated with Homologous Recombination Deficiency (HRD) in MM, suggesting a role for therapy with PARP inhibitors. Here we report the genome-wide LoH profiling in single-CMMCs isolated from enriched peripheral blood of four different MM patients using a non-invasive approach that combines CellSearch® and DEPArray™ technology. Methods: CMMCs were obtained from peripheral blood of four MM patients using CellSearch for enrichment and DEPArray NxT for isolation. CMMCs enrichment was obtained using a custom kit with anti-CD138 or anti-CD138/CD38 antibody-conjugated ferrofluids for positive enrichment and CD38-PE, CD19/CD45-APC immunofluorescent staining for detection. After single cell isolation, Ampli1™ Whole Genome Amplification (WGA) kit was used to amplify single-cell genomic DNA. Whole genome sequencing libraries were prepared from WGA products using Ampli1™ LowPass kit and low-pass sequencing was performed on HiSeq 2500 Illumina® platform. Genome-wide single-cell Low-Pass Copy Number Alteration (LPCNA) analysis was performed using the cloud-based bioinformatic suite MSBiosuite. Single-cell genome-wide LoH profiles were obtained by a custom algorithm based on the ratio between mono- and bi-allelic loci in non-overlapping segments of uniform copy-number. Results: Out of 315 cells analyzed from the four MM patients, 244 (77%) showed LoH events. Extensive LoH regions were detected in all patients, with 231 cells showing copy-neutral LoH events and 121 showing LoH events in copy-gain regions. Globally, 460 copy-neutral LoHs, and 292 LoHs in copy-gain regions were detected. Two highly conserved copy-neutral events (9p; 16q) were detected in one of the patients analyzed, suggesting they are early events. Interestingly, the first of the 2 events includes CDKN2A, a tumor suppressor which has been implicated in predisposition to MM. Notably, in a second patient, chromosome 11 is associated both to a copy-neutral LoH and to an LoH event in a copy-gain segment including the CCND1 gene, whose amplification in MM has been associated with multidrug resistance expression. Conclusions: Here we present a non-invasive workflow for the molecular characterization of single CMMCs isolated from peripheral blood. Specifically, we reported the presence of chromosomal or sub-chromosomal LoH events in all MM patients, suggesting a pervasive phenomenon in MM which deserves to be further explored as potential therapy target. Citation Format: Claudio Forcato, Alberto Ferrarini, Genny Buson, Paola Tononi, Marianna Garonzi, Valentina del Monaco, Andrea Raspadori, Steven Gross, Francesca Fontana, Gianni Medoro, Mark Connelly, Nicolò Manaresi. Analysis of low-pass sequencing data reveals extensive loss-of-heterozygosity in circulating multiple myeloma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2702.

Investigation of Cervical Tumor Biopsies for Chromosomal Loss of Heterozygosity (LOH) and Microsatellite Instability (MSI) at the HLA II Locus in HIV-1/HPV Co-infected Women.

Background: A subgroup of women who are co-infected with human immunodeficiency virus type 1 (HIV-1) and human papillomavirus (HPV) progress rapidly to cervical disease regardless of high CD4 counts. Chromosomal loss of heterozygosity (LOH) and microsatellite instability (MSI) are early frequent genetic alterations occurring in solid tumors. Loss of an allele or part of a chromosome can have multiple functional effects on immune response genes, oncogenes, DNA damage-repair genes, and tumor-suppressor genes. To characterize the genetic alterations that may influence rapid tumor progression in some HIV-1-positive women, the extent of LOH and MSI at the HLA II locus on chromosome 6p in cervical tumor biopsy DNA samples with regard to HIV-1/HPV co-infection in South African women was investigated.Methods: A total of 164 women with cervical disease were recruited for this study, of which 74 were HIV-1-positive and 90 were HIV-1-seronegative. DNA from cervical tumors and matched buccal swabs were used for analyses. Six fluorescently-labeled oligonucleotide primer pairs in a multiplex PCR amplification were used to study LOH and MSI. Pearson chi-squared test for homogeneity of proportions using an exact p value, a two-proportion Z-score test, ROC curves and a logistic regression model were used for statistical analyses. All p-values were corrected for false discovery rate (FDR) using the Benjamini-Hochberg test and the adjusted p-values (q-values) were reported. All tests were significant when both p and q < 0.05.Results: Tumor DNA from HIV-1/HPV co-infected women demonstrated a higher frequency of LOH/MSI at the HLA II locus on chromosome 6p21.21 than tumor DNA from HIV-1-seronegative women (D6S2447, 74.2 vs. 42.6%; p = 0.001, q = 0.003), D6S2881 at 6p21.31 (78.3 vs. 42.9%; p = 0.002, q = 0.004), D6S2666 at 6p21.32 (79 vs. 57.1%; p = 0.035, q = 0.052), and D6S2746, at 6p21.33 (64.3 vs. 29.4%; p < 0.001, q < 0.001), respectively.Conclusions: HPV infection alone can induce LOH/MSI at the HLA II locus in cervical tumor DNA, whereas HIV-1 co-infection exacerbates it, suggesting that this may accelerate cervical disease progression in a subgroup of HIV-1-positive women.

An immune response-associated genomic model to predict early resistance to PD1/PD-L1 blockade among real-world TMB-high Chinese cancer patients.

e14167 Background: TMB-high was used as a favorable biomarker to predict the response to anti-PD-1/PD-L1 therapy, but a substantial portion of the TMB-high patients suffered from early resistance with limited clinical benefits but potential severe adverse effects. Methods: Whole-exome sequencing were performed in more than 200 advanced cancer patients, who received anti-PD-1 therapy alone or in combination with chemotherapy and targeted therapy across 20 provinces of China, with quarterly follow-ups. The genomic profiles of the patients with progressive disease (PD) and complete remission (CR) outcomes were individually analyzed and the potential resistance and hypersensitive biomarkers were identified. These biomarkers were formulated into an immune response-associated genomic (IRAG) model based on XGBoost algorithm to predict the sensitivity to PD-1 blockage and early resistance among TMB-high patients. Results: Non-synonymous somatic mutations (CTNNB1, B2M, PD-L1 and HLA class I genes), somatic gene amplifications (MDM2/4, CDK4, CCND1/FGF3/FGF4/FGF19, FLT3 and MYC), chromosome 9p21 deletion, and loss of heterozygosity in chromosome 6 HLA-I region were identified as potential resistance biomarkers in the PD group. TMB-high, microsatellite instability (MSI), TMB-low with indel-proportion high, POLE loss-of-function mutations, Epstein-Barr virus infection and DNA mismatch repair-deficient germline and somatic mutations were identified as potential hypersensitivity biomarkers in the CR group. By combining all genomic resistance and hypersensitivity biomarkers, we developed a quantitative IRAG model and separated the patients into three groups: TMB-low, TMB-high/IRAG-low and TMB-high/IRAG-high. The PFS in TMB-high/IRAG-low group was characterized with early clinical resistance, similar to the TMB-low group (median: 3.8 vs. 4.2 months, p = 0.22). The reanalysis of the patient set with known clinical outcomes showed that the TMB-high/IRAG-high group had a significantly longer PFS than the other two groups. (median: 10 vs. 3.8, 4.2 months, p &lt; 0.001). The TMB-high/IRAG-high group clearly showed higher chromosomal stability with fewer somatic copy number alterations. Conclusions: We developed a model based on one single genomic assay (whole-exome) to successfully identify those TMB-high patients who suffered from early resistance to PD-1 inhibitors. Larger cohort and independent analyses are necessary to verify the validity of these results.

Loss of heterozygosity by SCRaMbLEing.

Genetic variation drives phenotypic evolution within populations. Genetic variation can be divided into different forms according to the size of genomic changes. However, study of large-scale genomic variation such as structural variation and aneuploidy is still limited and mainly based on the static, predetermined feature of individual genomes. Here, using SCRaMbLE, different levels of loss of heterozygosity (LOH) events including short-range LOH, long-range LOH and whole chromosome LOH were detected in evolved strains. By contrast, using rapid adaptive evolution, aneuploidy was detected in the adaptive strains. It was further found that deletion of gene GLN3, long-range LOH in the left arm of synthetic chromosome X, whole chromosome LOH of synthetic chromosome X, and duplication of chromosome VIII (trisomy) lead to increased rapamycin resistance in synthetic yeast. Comparative analysis of genome stability of evolved strains indicates that the aneuploid strain has a higher frequency of degeneration than the SCRaMbLEd strain. These findings enrich our understanding of genetic mechanism of rapamycin resistance in yeast, and provide valuable insights into yeast genome architecture and function.

Next Generation Sequencing of Sporadic Vestibular Schwannoma: Necessity of Biallelic NF2 Inactivation and Implications of Accessory Non-NF2 Variants.

1) Describe the genetic alterations discovered in a series of sporadic vestibular schwannomas (VS). 2) Identify if more clinically aggressive variants possess different genetic alterations compared to more indolent-behaving VS. Fresh frozen tumor and matched peripheral blood leukocytes from 23 individuals with sporadic VS were analyzed using whole-exome sequencing, tumor whole transcriptome expression profiling (mRNA-Seq), and tumor mate-pair analysis. Source cases included tumors with fast preoperative growth, giant tumors in young patients, tumors with macrocystic change, recurrent tumors following radiation or microsurgery, and indolent small tumors with minimal or no growth before surgery. Somatic and germ-line alterations of the NF2 gene and beyond the NF2 locus were identified and analyzed using complementing analyses. Biallelic somatic events involving the NF2 gene were discovered in every analyzed tumor specimen with no concurrent NF2 variants identified in matching peripheral blood specimens. Thirteen tumors showed loss of one chromosome 22 (ch22), 4 tumors showed copy-neutral 22q loss of heterozygosity, and 31 unique small variants in the NF2 gene were discovered. Of the latter, 10 were essential splice site, 11 frame shift, 7 stop gain, 2 missense, and 1 in-frame mutation. No other common or recurring NF2 mutations were identified. However, several other notable large chromosomal aberrations were discovered including 2 tumors with loss of a chromosome 21, 3 with loss of an X or Y chromosome, 1 with copy-neutral loss of heterozygosity in chromosome 15, and 1 with loss of 18p and 16q. All of these other major chromosomal abnormalities only occurred in tumors demonstrating a more aggressive phenotype. To date, few studies have used whole-exome sequencing, mate-pair analysis, and RNA-seq to profile genome-wide alterations in sporadic VS. Using high-throughput deep sequencing, "two-hit" alterations in the NF2 gene were identified in every tumor and were not present in peripheral blood supporting that all events were somatic. Type of NF2 gene alteration and accessory mutations outside the NF2 locus may predict phenotypic expression and clinical course.

Towards a Universal Law Controlling all Human Cancer Chromosome LOH Deletions, Perspectives in Prostate and Breast Cancers screening

Background: Global analysis of 3 human genomes of increasing levels of evolution (Neanderthal/Sapiens Build34/Sapiens hg38) reveals 2 levels of numerical constraints controlling, structuring and optimizing these genome's DNA sequences. A global constraint - called for Human Genome Optimum - optimizes the genome at its global scale. The same operator applied to each of the 24 individual chromosomes reveals a hierarchical structure of these 24 chromosomes. Methods: We analyze how this HGO genomic optimum is perturbed by hundred single or multiple LOH (Loss of Heterozygosity) deletions relating to different chromosomes and cancers. Results: The generic law highlighted is stated as follows an LOH deletion affects a chromosome upstream of the HGO point (chromosomes 4 13). In the chromosomal spectrum, this deletion degrades the genomic optimum of the cancer genome. When an LOH deletion affects a chromosome downstream of the HGO point (chromosomes 19 22) in the chromosomal spectrum, this deletion improves the genomic optimum of the cancer genome. The exhaustive analysis of the 240 LOHs for the following 6 cases: Chromosome 13 (breast cancer), chromosome 5 (breast cancer), chromosome 10 (glioblastoma cancer), chromosome 1 (colorectal cancer), chromosome 1 (neuroblastoma cancer) and chromosome 16 (prostate cancer) obey this law in 227 cases and do not obey this law for 13 cases (success rate of our law=94.58%). In this article we will detail this type of analysis on 153 LOH relating to breast and prostate tumors affecting respectively chromosome 13, chromosome 5 (breast) and chromosome 16 (prostate). In this detailed study, the HGO law described here is verified in 143 cases out of 153 or 93.46% of favorable cases. Conclusion: The main application of this fundamental discovery will be the genomic characterization and classification of tumors, making it possible to predict the dangerousness and even the pathogenicity.

Fenton reaction-induced renal carcinogenesis in Mutyh-deficient mice exhibits less chromosomal aberrations than the rat model.

Oxidative stress including iron excess has been associated with carcinogenesis. The level of 8-oxoguanine, a major oxidatively modified base in DNA, is maintained very low by three distinct enzymes, encoded by OGG1, MUTYH and MTH1. Germline biallelic inactivation of MUTYH represents a familial cancer syndrome called MUTYH-associated polyposis. Here, we used Mutyh-deficient mice to evaluate renal carcinogenesis induced by ferric nitrilotriacetate (Fe-NTA). Although the C57BL/6 background is cancer-resistant, a repeated intraperitoneal administration of Fe-NTA induced a high incidence of renal cell carcinoma (RCC; 26.7%) in Mutyh-deficient mice in comparison to wild-type mice (7.1%). Fe-NTA treatment also induced renal malignant lymphoma, which did not occur without the Fe-NTA treatment in both the genotypes. Renal tumor-free survival after Fe-NTA treatment was marginally different (P = 0.157) between the two genotypes. Array-based comparative genome hybridization analyses revealed, in RCC, the loss of heterozygosity in chromosomes 4 and 12 without p16INKA inactivation; these results were confirmed by a methylation analysis and showed no significant difference between the genotypes. Lymphomas showed a preference for genomic amplifications. Dlk1 inactivation by promoter methylation may be involved in carcinogenesis in both tumors. Fe-NTA-induced murine RCCs revealed significantly less genomic aberrations than those in rats, demonstrating a marked species difference.

Increased LOH due to Defective Sister Chromatid Cohesion Is due Primarily to Chromosomal Aneuploidy and not Recombination.

Loss of heterozygosity (LOH) is an important factor in cancer, pathogenic fungi, and adaptation to changing environments. The sister chromatid cohesion process (SCC) suppresses aneuploidy and therefore whole chromosome LOH. SCC is also important to channel recombinational repair to sister chromatids, thereby preventing LOH mediated by allelic recombination. There is, however, insufficient information about the relative roles that the SCC pathway plays in the different modes of LOH. Here, we found that the cohesin mutation mcd1-1, and other mutations in SCC, differentially affect the various types of LOH. The greatest effect, by three orders of magnitude, was on whole chromosome loss (CL). In contrast, there was little increase in recombination-mediated LOH, even for telomeric markers. Some of the LOH events that were increased by SCC mutations were complex, i.e., they were the result of several chromosome transactions. Although these events were independent of POL32, the most parsimonious way to explain the formation of at least some of them was break-induced replication through the centromere. Interestingly, the mcd1-1 pol32Δ double mutant showed a significant reduction in the rate of CL in comparison with the mcd1-1 single mutant. Our results show that defects in SCC allow the formation of complex LOH events that, in turn, can promote drug or pesticide resistance in diploid microbes that are pathogenic to humans or plants.

P207 Genetic alterations in nasopharyngeal carcinoma tissues

Aim Nasopharyngeal Carcinoma is the most-studied Virus associated cancer. EBV has been shown to induce loss of heterozygosity in chromosome 11. Here we aim to examine if EBV-positive nasopharyngeal carcinoma (NPC) show genetic alterations that impact the tissue identity using STR assay. Methods Fourteen untreated NPC patients were analyzed in this study by comparing 15 STR loci polymorphisms from both nasopharyngeal cancerous tissues and blood samples taken from the same individual. After DNA extraction, MSI and tissue identity testing targeting 15 STR loci were performed using AmpFlSTR® Identifiler® Plus PCR Amplification Kit and analyzed using Genetic Analyzer 3130. Results The data show that genetic alterations (MSI and/or LOH) were observed in 11/14 (78.5%) of NPC patient tissues. MSI was observed in 6 samples (42.8%) and LOH was observed in 7 samples (50.00%), while both LOH and MSI were detectable in 3 samples (21.4%). MSI alone was observed in five markers (32.5%): D51S11, CSF1P0, D19S433, TP0X and D18S51 whereas LOH was observed in 32.5% of markers: D7S820 (7q21.11), D3S1358 (3p21.31), TH0l (11p15.5), D16S539 (16q24.1), D2S1338 (2q35) respectively. Both MSI and LOH were observed in vWA (6.25%). Neither MSI nor LOH was detected in D8S1179, D13S317, 5S818, FGA and Amelogenin markers. Conclusions In this study, we show for the first time that nasopharyngeal carcinoma (NPC) show multiple genetic alterations in the tissue identity and open the door for further investigations in understanding the relation between these alterations and NPC diagnostics and prognostics.