Loss Of Heterozygosity Analysis Research Articles

Correlation of allele-specific HLA loss of heterozygosity in patients with cancer with driver mutations.

e14658 Background: While immunotherapies have revolutionized cancer treatment, the limited predictability of response and resistance mechanisms present challenges to the development of novel therapeutics for solid tumors. Many of these use a sensitive and specific T cell receptor (TCR)-HLA:peptide interaction as a mechanism of action and can be impacted by HLA loss of heterozygosity (LOH). Allele-agnostic HLA LOH analyses assessing loss of any MHC class I allele are relevant for HLA-agnostic approaches such as immune checkpoint inhibitors; however, context-specific studies remain limited. For example, adoptive TCR T cell therapies and bi-specific TCR T cell engagers target neoantigen peptides derived from oncogenic driver mutations, such as those of KRAS and TP53, presented by a specific HLA allele. Thus, allele-agnostic analyses may overestimate the frequency and relevance of HLA LOH for allele-specific modalities. Methods: A real-world comprehensive genomic profiling dataset consisting of 78,418 cases (1), germline heterozygous for one or more HLA class I locus, was analyzed for HLA LOH in the context of frequent driver mutations (KRAS G12C, G12D, G12V and TP53 R175H), the corresponding HLA alleles (A*02:01, A*03:01, and A*11:01), and within tumor subtypes. Results: Allele-agnostic HLA LOH was observed in 16.6% of all samples and varied between indications. Notably, allele-specific loss of HLAs A*02:01, A*03:01, and A*11:01 across all samples was much lower than allele-agnostic loss (6.3%, 6.6%, and 6.3% cases, respectively). Although, a trend for increased allele-specific HLA LOH was observed when analyzed by driver mutation, the frequency remained low. Specifically, across all cases, A*02:01, A*03:01, and A*11:01 allele-specific LOH ranged between 5.9% - 9.7% in relation to KRAS G12C, G12D, and G12V compared to 5.9% - 6.2% for wild-type (WT). Moreover, it did not achieve statistical significance for any HLA-KRAS mutation pairs in colorectal (CRC), non-small cell lung (NSCLC), and pancreatic (PDAC) cancers, except for A*11:01 and KRAS G12C in NSCLC, which was lower in mutated cases (5.3% for G12C vs 10.1% for WT KRAS, p=0.045). Both allele-agnostic and A*02:01-specific HLA LOH in TP53 WT samples were lower than average. The TP53 R175H mutation was associated with limited increase in HLA-A*02:01 LOH (from 4.4% for WT to 8.1% for TP53 R175H) and varied between indications, e.g. no significant increase was observed in R175H-positive breast cancer and NSCLC cases contrary to CRC and PDAC. Conclusions: Based on this real-world dataset, over 90% of cases retain the TCR-targeted allele and, therefore, preserve the molecular complex necessary for HLA/driver mutation-based therapies. Notably, the database is enriched for advanced cancer patients. Further studies should test correlations with treatments and whether tumors in earlier lines of treatment have an even lower rate of HLA LOH. 1. Montesion et al., PMID: 33127846.

In-house molecular diagnosis of diffuse glioma updating the revised WHO classification by a platform of the advanced medical care system, Senshin-Iryo.

Since the World Health Organization (WHO) 2016 revision, the number of molecular markers required for diffuse gliomas has increased, placing a burden on clinical practice. We have established an in-house, molecular diagnostic platform using Senshin-Iryo, a feature of Japan's unique healthcare system, and partially modified the analysis method in accordance with the WHO 2021 revision. Herein, we review over a total 5 years of achievements using this platform. Analyses of IDH, BRAF, and H3 point mutations, loss of heterozygosity (LOH) on 1p/19q and chromosomes 10 and 17, and MGMT methylation were combined into a set that was submitted to Senshin-Iryo as "Drug resistance gene testing for anticancer chemotherapy" and was approved in August 2018. Subsequently, in October 2021, Sanger sequencing for the TERT promoter mutation was added to the set, and LOH analysis was replaced with multiplex ligation-dependent probe amplification (MLPA) to analyze 1p/19q codeletion and newly required genetic markers, such as EGFR, PTEN, and CDKN2A from WHO 2021. Among the over 200 cases included, 54 were analyzed after the WHO 2021 revision. The laboratory has maintained a diagnostic platform where molecular diagnoses are confirmed within 2 weeks. Initial expenditures exceeded the income from patient copayments; however, it has gradually been reduced to running costs alone and is approaching profitability. After the WHO 2021 revision, diagnoses were confirmed using molecular markers obtained from Senshin-Iryo in 38 of 54 cases (70.1%). Among the remaining 16 patients, only four (7.4%) were diagnosed with diffuse glioma, not elsewhere classified, which was excluded in 12 cases where glioblastoma was confirmed by histopathological diagnosis. Our Senshin-Iryo trial functioned as a salvage system to overcome the transition period between continued revisions of WHO classification that has caused a clinical dilemma in the Japanese healthcare system.

A custom next-generation sequencing panel for 1p/19q codeletion and mutational analysis in gliomas.

The World Health Organization has updated their classification system for the diagnosis of gliomas, combining histological features with molecular data including isocitrate dehydrogenase 1 and codeletion of chromosomal arms 1p and 19q. 1p/19q codeletion analysis is commonly performed by fluorescence in situ hybridization (FISH). In this study, we developed a 57-gene targeted next-generation sequencing (NGS) panel including 1p/19q codeletion detection mainly to assess diagnosis and potential treatment response in melanoma, gastrointestinal stromal tumor, and glioma patients. Loss of heterozygosity analysis was performed using the NGS method on 37 formalin-fixed paraffin-embedded glioma tissues that showed 1p and/or 19q loss determined by FISH. Conventional methods were applied for the validation of some glioma-related gene mutations. In 81.1% (30 of 37) and 94.6% (35 of 37) of cases, 1p and 19q were found to be in agreement whereas concordance for 1p/19q codeletion and no 1p/19q codeletion was found in 94.7% (18 of 19) and 94.4% (17 of 18) of cases, respectively. Overall, comparing NGS results with those of conventional methods showed high concordance. In conclusion, the NGS panel allows reliable analysis of 1p/19q codeletion and mutation at the same time.

Ubtf- Tandem Duplications Are Recurrent Somatic Genetic Events in Adult AML and Associate with Additional Structural Variants and a Distinct Gene Expression Profile (Alliance)

Background The gene upstream binding transcription factor ( UBTF ) encodesa nucleolar protein that is a key regulator of the epigenetic status of ribosomal DNA and RNA transcription. Tandem duplications (TDs) of the UBTF gene have been initially identified in pediatric patients (pts) with acute myeloid leukemia (AML) and were recently also reported as relatively rare, but recurrent genetic events in adult AML based on targeted re-sequencing efforts (1.2%-3%). Their presence was associated with dysplastic bone marrow features, and co-occurrence of select genetic features, including FLT3-internal tandem duplications (ITDs) and WT1 mutations, and inferior outcome. However, associated biologic features and affected pathways have not yet been thoroughly investigated, and may provide further insights into leukemogenesis and identify potentially targetable vulnerabilities. Thus, we set out to assess the frequency of UBTF-TDs in a large cohort of adult AML pts (n=807) treated on frontline protocols of the Alliance for Clinical Trials in Oncology with complete RNA sequencing data, comprehensive targeted mutation profiling, centrally reviewed cytogenetic data and clinical information available, and to gain further insights into UBTF-TDs via comprehensive integrated genomic profiling. Methods UBTF-TD detection was performed via the RNAseq-based RNAmut and EnFusion algorithm. To gain insights into associated molecular features, all UBTF-TD + pts underwent paired tumor-normal whole-exome sequencing. To understand associated pathways and downstream effects, differential gene expression (DGE) and gene set enrichment analyses (GSEA) were performed. Results UBTF-TDs were detected in 6/807 pts (0.7%). In line with previous studies, these 6 UBTF-TD + pts were young, with a median age of 35y (range: 19-59y). Three pts had extramedullary disease, 2 with skin and 1 with gingival involvement. All but 1 of the 6 pts were cytogenetically normal. Clinically, pts did poorly after treatment with intensive chemotherapy induction, with a CR rate of only 33%. No pt was alive 2.5 y after diagnosis. In paired tumor/normal exome sequencing, UBTF-TD + pts had a median mutation burden of 21 non-synonymous variants in coding genes (range: 17-27), with a notable presence of larger insertions/deletions (indels), loss of heterozygosity (LOH) or TDs as co-occurring somatic genetic lesions (median: 4, range: 2-5). There was a 100% co-occurrence of UBTF-TDs with FLT3-ITDs. In addition, pts harbored larger indels in WT1 (n=2), BPTF, CEBPA, Corf53, KRTAP5-7, NFE2, PTPRN2, RNF144B, SOWAHC and ZCCHC14. Also, analysis of copy-number variations and LOH revealed LOH in 5 pts, in chromosomes 13 (n=3), 15 (n=1) and 18 (n=1), suggestive of a propensity to the acquisition of structural variants. In unsupervised clustering analysis of the RNAseq data, UBTF-TD + pts clustered with FLT3-ITD + pts but formed a relatively distinct subcluster in principal component and t-distributed stochastic neighbor embedding analyses. In supervised clustering comparing 6 UBTF-TD +/ FLT3-ITD + and 196 UBTF-TD -/ FLT3-ITD + pts, 2 UBTF-TD - pts clustered together with the UBTF-TD +/ FLT3-ITD + group, suggesting either presence of an undetected TD or genetic phenocopy. UBTF expression was higher in UBTF-TD pts. DGE analysis of UBTF-TD +/ FLT3-ITD + and UBTF-TD -/ FLT3-ITD + pts (Fig 1) showed distinct upregulation of the locus of the stem cell and chemoresistance marker ALDH1A3, including the associated long non-coding RNAs AC015712.7/AC015712.2 in UBTF-TD + pts, which was confirmed by a comparison of those pts to AML pts who had neither TD (n=605). GSEA indicated a downregulation of inflammation pathways and several metabolic pathways of UBTF-TD +/ FLT3-ITD + compared to FLT3-ITD + pts (Fig 2). Conclusions UBTF-TDs are rare, but recurrent genetic events in adult AML, associated with high chemotherapy resistance. Our results highlight a susceptibility of those pts to the acquisition of structural variants and suggest a distinct associated biology, with upregulation of ALDH1A3 as possible contributing resistance mechanism. Support: U10CA180821, U10CA180882, U24CA196171; Clinicaltrials.gov Identifiers: NCT00048958 (CALGB 8461), NCT00899223 (CALGB 9665), NCT00900224 (CALGB 20202); https://acknowledgments.alliancefound.org

E2F1, DIAP1, and the presence of a homologous chromosome promote while JNK inhibits radiation-induced loss of heterozygosity in Drosophila melanogaster.

Loss of heterozygosity (LOH) can occur when a heterozygous mutant cell loses the remaining wild-type allele to become a homozygous mutant. LOH can have physiological consequences if, for example, the affected gene encodes a tumor suppressor. We used fluorescent reporters to study the mechanisms of LOH induction by X-rays, a type of ionizing radiation (IR), in Drosophila melanogaster larval wing discs. IR is used to treat more than half of patients with cancer, so understanding its effects is of biomedical relevance. Quantitative analysis of IR-induced LOH at different positions between the telomere and the centromere on the X chromosome showed a strong sex dependence and the need for a recombination-proficient homologous chromosome, whereas, paradoxically, position along the chromosome made little difference in LOH incidence. We propose that published data documenting high recombination frequency within centromeric heterochromatin on the X chromosome can explain these data. Using a focused screen, we identified E2F1 as a key promotor of LOH and further testing suggests a mechanism involving its role in cell-cycle regulation. We leveraged the loss of a transcriptional repressor through LOH to express transgenes specifically in cells that have already acquired LOH. This approach identified JNK signaling and apoptosis as key determinants of LOH maintenance. These studies reveal previously unknown mechanisms for the generation and elimination of cells with chromosome aberrations after exposure to IR.

Newly discovered genomic mutation patterns in radiation-induced small intestinal tumors of ApcMin/+ mice.

Among the small intestinal tumors that occur in irradiated mice of the established mouse model B6/B6-Chr18MSM-F1 ApcMin/+, loss of heterozygosity analysis can be utilized to estimate whether a deletion in the wild-type allele containing the Adenomatous polyposis coli (Apc) region (hereafter referred to as Deletion), a duplication in the mutant allele with a nonsense mutation at codon 850 of Apc (Duplication), or no aberration (Unidentified) has occurred. Previous research has revealed that the number of Unidentified tumors tends to increase with the radiation dose. In the present study, we investigated the molecular mechanisms underlying the development of an Unidentified tumor type in response to radiation exposure. The mRNA expression levels of Apc were significantly lower in Unidentified tumors than in normal tissues. We focused on epigenetic suppression as the mechanism underlying this decreased expression; however, hypermethylation of the Apc promoter region was not observed. To investigate whether deletions occur that cannot be captured by loss of heterozygosity analysis, we analyzed chromosome 18 using a customized array comparative genomic hybridization approach designed to detect copy-number changes in chromosome 18. However, the copy number of the Apc region was not altered in Unidentified tumors. Finally, gene mutation analysis of the Apc region using next-generation sequencing suggested the existence of a small deletion (approximately 3.5 kbp) in an Unidentified tumor from a mouse in the irradiated group. Furthermore, nonsense and frameshift mutations in Apc were found in approximately 30% of the Unidentified tumors analyzed. These results suggest that radiation-induced Unidentified tumors arise mainly due to decreased Apc expression of an unknown regulatory mechanism that does not depend on promoter hypermethylation, and that some tumors may result from nonsense mutations which are as-yet undefined point mutations.

The prognostic value of HLA loss of heterozygosity after allogeneic hematopoietic stem cell transplantation in children with relapsed acute leukemia

The loss of a patient-specific HLA haplotype on the surface of the blast cell population is one of the ways a tumor can evade the immune surveillance of donor cells. This phenomenon is observed in approximately 30 % of relapses in patients with hematologic malignancies who underwent partially mismatched allogeneic hematopoietic stem cell transplantation (HSCT). In this study, for the first time, a large cohort of pediatric patients (n = 80) with relapsed acute myeloid (AML) or acute lymphoblastic (ALL) leukemia after allogeneic HSCT was analyzed with the help of the STR method (highlypolymorphic microsatellite marker analysis) using 6 HLA haplotype markers. The study was approved by the Independent Ethics Committee and the Scientific Council of the I. P. Pavlov First Saint Petersburg State Medical University of Ministry of Healthcare of the Russian Federation. Loss of heterozygosity (LOH) was observed in 18 / 80 (22 %) relapsed patients with various types of acute leukemia: out of these, 8 / 44 (18 %) patients had B-cell ALL, 4 / 10 (40 %) patients – T-cell ALL and 6 / 25 (24 %) patients – AML. All relapses with LOH were observed in patients who had undergone haploidentical HSCT, and were found to occur later than relapses without loss of the HLA haplotype (median time to relapse: 8.8 months vs 6.2 months, p = 0.043). In the patients treated with haploidentical HSCT (n = 61), we assessed factors increasing the risk of LOH at relapse. The number of previous therapy lines in the patients with AML (n = 17) and acute or chronic graft-versus-host disease in the patients with ALL (n = 44) were associated with an increasedrisk of genetic loss of the HLA haplotype (p = 0.008 and p = 0.015 respectively). A relapse following the second allogeneic HSCT was associated with LOH in 4 / 5 (80 %) patients, p = 0.008. The prognosis of the patients with LOH was extremely poor. At a median follow-up of 6 months, the overall survival from relapse was 22 % in the LOH group and 37 % in the non-LOH group. The median overall survival was 4.5 months (95 % confidence interval 3–NA) and 10.3 months (95 % confidence interval 5.7–16.1) respectively, p = 0.063. Among the patients with LOH, the best survival rates were observed in those who had undergone a repeat allogeneic HSCT from a different donor. Thus, the analysis of LOH is an important tool for determining the prognosis and further treatment in pediatric patients with acute leukemias. We strongly recommend that this diagnostic test should be included into standard testing of patients after partially-mismatched allogeneic HSCT.

Interstitial deletion of the Apc locus in β-catenin-overexpressing cells is a signature of radiation-induced intestinal tumors in C3B6F1 ApcMin/+ mice†.

Recent studies have identified interstitial deletions in the cancer genome as a radiation-related mutational signature, although most of them do not fall on cancer driver genes. Pioneering studies in the field have indicated the presence of loss of heterozygosity (LOH) spanning Apc in a subset of sporadic and radiation-induced intestinal tumors of ApcMin/+ mice, albeit with a substantial subset in which LOH was not detected; whether copy number losses accompany such LOH has also been unclear. Herein, we analyzed intestinal tumors of C3B6F1 ApcMin/+ mice that were either left untreated or irradiated with 2Gy of γ-rays. We observed intratumor mosaicism with respect to the nuclear/cytoplasmic accumulation of immunohistochemically detectable β-catenin, which is a hallmark of Apc+ allele loss. An immunoguided laser microdissection approach enabled the detection of LOH involving the Apc+ allele in β-catenin-overexpressing cells; in contrast, the LOH was not observed in the non-overexpressing cells. With this improvement, LOH involving Apc+ was detected in all 22 tumors analyzed, in contrast to what has been reported previously. The use of a formalin-free fixative facilitated the LOH and microarray-based DNA copy number analyses, enabling the classification of the aberrations as nondisjunction/mitotic recombination type or interstitial deletion type. Of note, the latter was observed only in radiation-induced tumors (nonirradiated, 0 of 8; irradiated, 11 of 14). Thus, an analysis considering intratumor heterogeneity identifies interstitial deletion involving the Apc+ allele as a causative radiation-related event in intestinal tumors of ApcMin/+ mice, providing an accurate approach for attributing individual tumors to radiation exposure.

Ataxia-Telangiectasia Mutated Loss of Heterozygosity in Melanoma.

ATM germline pathogenic variants were recently found enriched in high-risk melanoma patients. However, ATM loss of heterozygosity (LOH) has never been investigated in melanoma and, therefore, a causal association with melanoma development has not been established yet. The purpose of this study was to functionally characterize 13 germline ATM variants found in high-risk melanoma patients-and classified by in silico tools as pathogenic, uncertain significance, or benign-using multiple assays evaluating ATM/pATM expression and/or LOH in melanoma tissues and cell lines. We assessed ATM status by Immunohistochemistry (IHC), Western Blot, Whole-Exome Sequencing/Copy Number Variation analysis, and RNA sequencing, supported by Sanger sequencing and microsatellite analyses. For most variants, IHC results matched those obtained with in silico classification and LOH analysis. Two pathogenic variants (p.Ser1135_Lys1192del and p.Ser1993ArgfsTer23) showed LOH and complete loss of ATM activation in melanoma. Two variants of unknown significance (p.Asn358Ile and p.Asn796His) showed reduced expression and LOH, suggestive of a deleterious effect. This study, showing a classic two-hit scenario in a well-known tumor suppressor gene, supports the inclusion of melanoma in the ATM-related cancer spectrum.

A case of early onset adenocarcinoma associated with colorectal polyposis with an unknown germline mutation

BackgroundTypically, in cases of adenomatous polyposis, colorectal cancer develops in the third or fourth decade of life. We report the case of a female patient with colorectal polyposis who developed adenocarcinoma at 8 years of age.Case presentationAn 8-year-old girl was admitted with a 4-year history of occasional bloody stools. Colonoscopy revealed colon polyposis and histopathological assessment confirmed a well-differentiated adenocarcinoma in the adenomatous polyps, so laparoscopy-assisted proctocolectomy was performed in the lithotomy position by a simultaneous abdominal and anal approach. To completely resect the rectal mucosa, excision was commenced just distal to the dentate line. After the mucosal resection up to the peritoneal reflection level, an inverted muscular cuff was cut circumferentially, and the terminal ileum was pulled through the muscular cuff and anastomosed to the anal canal. Histopathology revealed multiple adenomatous polyps and scattered well-differentiated tubular adenocarcinomas (tub1) in the adenomatous polyps and the non-polypoid mucosal lesions. Because complete resection was achieved, additional adjuvant chemotherapy was not administered. Polymerase chain reaction (PCR)-direct sequencing of the entire coding region and the exon–intron junctions, and real-time PCR of DNA extracted from blood cells, revealed no mutations of either APC or MUTYH. No deletions, duplications, translocations or inversions of APC, MUTYH and GREM1 genes were found using multiplex ligation-dependent probe amplification (MLPA) and G-banding analysis. Multi-gene panels sequencing for polyposis syndromes or hereditary colorectal cancers, and trio-whole exome sequencing was conducted. However, no candidate pathogenic variants of genes were detected in de novo dominant or autosomal recessive model. Somatic mutation of APC was not detected in 4 polyps by loss of heterozygosity analysis at a single nucleotide polymorphism in intron 14. The patient has remained disease-free for 5 years. Currently, the patient is on loperamide and passes stool 5 times/day without any soiling.ConclusionsThe genetic analysis suggests that she may have a germline mutation at unscreened region of these genes or in unidentified FAP gene. The patient will be carefully followed up for residual rectal carcinoma and for the development of other cancers.

Succinate dehydrogenase and MYC-associated factor X mutations in pituitary neuroendocrine tumours.

Pituitary neuroendocrine tumours (PitNETs) associated with paragangliomas or phaeochromocytomas are rare. SDHx variants are estimated to be associated with 0.3–1.8% of PitNETs. Only a few case reports have documented the association with MAX variants. Prolactinomas are the most common PitNETs occurring in patients with SDHx variants, followed by somatotrophinomas, clinically non-functioning tumours and corticotrophinomas. One pituitary carcinoma has been described. SDHC, SDHB and SDHA mutations are inherited in an autosomal dominant fashion and tumorigenesis seems to adhere to Knudson’s two-hit hypothesis. SDHD and SDHAF2 mutations most commonly have paternal inheritance. Immunohistochemistry for SDHB or MAX and loss of heterozygosity analysis can support the assessment of pathogenicity of the variants. Metabolomics is promising in the diagnosis of SDHx-related disease. Future research should aim to further clarify the role of SDHx and MAX variants or other genes in the molecular pathogenesis of PitNETs, including pseudohypoxic and kinase signalling pathways along with elucidating epigenetic mechanisms to predict tumour behaviour.

Fumarate hydratase gene germline variants and mosaicism associated with pheochromocytoma and paraganglioma.

Fumarate hydratase (FH) catalyzes the conversion of fumaric acid to L-malic acid. Heterozygous variants of the human fumarate hydratase gene (FH) predispose to hereditary leiomyomatosis and renal cell cancer and, rarely, pheochromocytoma/paraganglioma (PPGL). No mosaic variant in FH has been reported yet. Using next-generation sequencing, five individuals with FH variants were found in 319 PPGL patients. Immunohistochemistry staining and loss of heterozygosity analysis in tumor tissues were performed to determine the pathogenicity of the variants. Deep targeted sequencing was performed on the peripheral blood DNA of a pheochromocytoma (PCC) patient with uterine leiomyomas. Finally, two of the five variants were found to be pathogenic. A germline variant (c.817G>A, p.Ala273Thr) was found in a patient with a PPGL family history. A mosaic variant (c.206G>A, p.Gly69Asp) with an allelic ratio of 5% in blood DNA was confirmed in the PCC patient with uterine leiomyomas. No metastatic PPGL was observed in the two PPGL patients with FH pathogenic variants. In summary, we report mosaicism in FH and the first PPGL pedigree with an FH pathogenic germline variant. Both germline variantsand mosaicism should be taken into account during genetic testing.

Clinicopathological and mutational analysis of esophageal basaloid squamous cell carcinoma.

Esophageal basaloid squamous cell carcinoma (EBSCC) is a poorly differentiated variant of esophageal squamous cell carcinoma (ESCC). We aimed to investigate the clinicopathological and molecular biological characteristics of EBSCC and enrolled 58 patients with EBSCCs. Clinicopathological factors including age, sex, tumor size and location, gross tumor type (superficial, protrusive, ulcerative, and unclassifiable), lymphovascular invasion, infiltrative growth, intramural invasion, TNM stage, and dominant histological type were examined. EBSCCs were classified into four types (solid, cribri, microcystic, and tubular) according to the dominant histology. Next-generation sequencing (NGS) of a cancer hotspot panel was performed in 19 cases. NGS identified TP53 as the most frequently mutated gene, and copy number variation analysis revealed the most frequent loss of heterozygosity (LOH) at the ataxia telangiectasia mutated (ATM) and retinoblastoma 1 (RB1) loci. Target sequencing for TP53 was performed for the remaining 39 cases. We also performed LOH analysis for TP53, ATM, and RB1 and immunohistochemical staining for p53, ATM, and Rb in all cases. The rates of TP53 mutations and LOH and p53 aberrant expression were high (79.3%, 63.2%, and 72.4%, respectively); however, the frequencies were similar to those reported for ESCC. LOH rates of the RB1 and ATM loci were also high (55.3% and 67.2%, respectively). Overall survival rate was 66.5%, and recurrence-free survival rate was 55.0%. Only conventional clinicopathological factors had a prognostic impact in EBSCC; the microcystic type had the poorest prognosis. Our findings could be useful in developing novel treatment strategies for EBSCC.

Tumor and Constitutional Sequencing for Neurofibromatosis Type 1.

PURPOSENF1 variants in tumors are important to recognize, as multiple mechanisms may give rise to biallelic variants. Both deletions and copy-neutral loss of heterozygosity (LOH) are potential mechanisms of NF1 loss, distinct from point mutations, and additional genes altered may drive different tumor types. This study investigates whether tumors from individuals with neurofibromatosis type 1 (NF1) demonstrate additional gene variants and detects NF1 second hits using paired germline and somatic sequencing. In addition, rare tumor types in NF1 may also be characterized by tumor sequencing.MATERIALS AND METHODSSequences of 529 cancer driver genes were analyzed in 6,381 tumors, yielding 391 NF1-mutated tumors in which NF1 LOH analysis was performed. Driver genes were evaluated by tumor type including malignant peripheral nerve sheath tumors and gliomas.RESULTSNF1 LOH was seen in 133 of 391 tumor samples in the cohort. Individuals with NF1 had more prevalent copy-neutral LOH (P < .0001), suggesting somatic intrachromosomal recombination. Osteosarcoma in NF1 also had NF1 LOH and additional p53 alteration. NF1 second hit data from tumors were informative for inferring deleteriousness of missense variants that were conflicting in ClinVar, potentially helping to add to NF1 annotation. Although criteria for evaluating germline and somatic variants are different, deleterious effects on NF1 function may be shared.CONCLUSIONSequencing of NF1-associated tumors demonstrated a spectrum of second hits in NF1 and the prevalence of copy-neutral LOH. Future work may be aimed at further understanding of LOH mechanisms and strategies to mitigate tumor risk.

Case Review: Whole-Exome Sequencing Analyses Identify Carriers of a Known Likely Pathogenic Intronic BRCA1 Variant in Ovarian Cancer Cases Clinically Negative for Pathogenic BRCA1 and BRCA2 Variants.

Background: Detecting pathogenic intronic variants resulting in aberrant splicing remains a challenge in routine genetic testing. We describe germline whole-exome sequencing (WES) analyses and apply in silico predictive tools of familial ovarian cancer (OC) cases reported clinically negative for pathogenic BRCA1 and BRCA2 variants. Methods: WES data from 27 familial OC cases reported clinically negative for pathogenic BRCA1 and BRCA2 variants and 53 sporadic early-onset OC cases were analyzed for pathogenic variants in BRCA1 or BRCA2. WES data from carriers of pathogenic BRCA1 or BRCA2 variants were analyzed for pathogenic variants in 10 other OC predisposing genes. Loss of heterozygosity analysis was performed on tumor DNA from variant carriers. Results: BRCA1 c.5407-25T>A intronic variant, identified in two affected sisters and one sporadic OC case, is predicted to create a new splice effecting transcription of BRCA1. WES data from BRCA1 c.5407-25T>A carriers showed no evidence of pathogenic variants in other OC predisposing genes. Sequencing the tumor DNA from the variant carrier showed complete loss of the wild-type allele. Conclusions: The findings support BRCA1 c.5407-25T>A as a likely pathogenic variant and highlight the importance of investigating intronic sequences as causal variants in OC families where the involvement of BRCA1 is highly suggestive.

Value of the loss of heterozygosity to BRCA1 variant classification

At least 10% of the BRCA1/2 tests identify variants of uncertain significance (VUS) while the distinction between pathogenic variants (PV) and benign variants (BV) remains particularly challenging. As a typical tumor suppressor gene, the inactivation of the second wild-type (WT) BRCA1 allele is expected to trigger cancer initiation. Loss of heterozygosity (LOH) of the WT allele is the most frequent mechanism for the BRCA1 biallelic inactivation. To evaluate if LOH can be an effective predictor of BRCA1 variant pathogenicity, we carried out LOH analysis on DNA extracted from 90 breast and seven ovary tumors diagnosed in 27 benign and 55 pathogenic variant carriers. Further analyses were conducted in tumors with PVs yet without loss of the WT allele: BRCA1 promoter hypermethylation, next-generation sequencing (NGS) of BRCA1/2, and BRCAness score. Ninety-seven tumor samples were analyzed from 26 different BRCA1 variants. A relatively stable pattern of LOH (65.4%) of WT allele for PV tumors was observed, while the allelic balance (63%) or loss of variant allele (15%) was generally seen for carriers of BV. LOH data is a useful complementary argument for BRCA1 variant classification.

Loss of heterozygosity in CHEK2-associated breast cancer

Background. CHEK2-associated neoplasms account for a significant proportion of hereditary breast cancer (BC) in Russia. The phenomenon of somatic deletion of the normal allele of a gene affected by a hereditary mutation, or loss of heterozygosity (LOH), is a frequent mechanism of complete inactivation of the corresponding protein, which is realized during the development of hereditary breast carcinomas. The contribution of the LOH phenomenon to the pathogenesis of CHEK2-dependent tumors is poorly understood, and almost all available data concern only one type of mutations - CHEK2 1100delC.&#x0D; The aim of the study was to characterize the frequency of LOH in breast tumor tissues from carriers of the three types of CHEK2 alterations: CHEK2 1000delC, CHEK2 IVS2+1G&gt;A, and CHEK2 del5395.&#x0D; Materials and methods. LOH analysis was performed in a group of 50 breast cancer cases from women carrying CHEK2 1000delC (n = 19), CHEK2 IVS2+1G&gt;A (n = 12), and CHEK2 del5395 (n = 19) mutations. Detection of LOH was carried out using a combination of methods that directly analyze the mutation locus (allele-specific PCR, Sanger sequencing, digital droplet PCR), and assess the status of single nucleotide polymorphisms surrounding the CHEK2 gene (digital droplet PCR).Results. The frequency of the LOH phenomenon in the studied cohort reached 27/50 (54%). Loss of heterozygosity was observed in 10/19 (52.6%) CHEK2 1000delC-associated, 6/12 (50%) CHEK2 IVS2+1G&gt;A-associated, and 11/19 (57.9%) CHEK2 del5395-associated tumors. In one carcinoma from a carrier of the CHEK2 IVS2+1G&gt;A alteration, the loss of mutated allele was confirmed. The main clinical and pathological characteristics were compared between tumors with loss and retention of heterozygosity. This comparison did not reveal any significant differences.Conclusion. Loss of heterozygosity is observed in about half of breast carcinomas arising in CHEK2 mutation carriers; the frequency of this phenomenon does not differ between three types of CHEK2 genetic defects.

ABCL-261: STR Profiling Reveals Tumor Genome Instability in Primary Mediastinal B-Cell Lymphoma

<h3>Context</h3> Molecular factors to determine the prognosis for primary mediastinal B-cell lymphoma (PMBCL) are not described yet. In this regard, the loss of heterozygosity (LOH) in STR loci and elevated microsatellite alteration at selected tetranucleotides (EMAST) in the tumor genome are of interest. <h3>Objective</h3> To analyze the frequency of LOH and EMAST in tumor cells of PMBCL patients at diagnosis and to check possible association with disease features. <h3>Design</h3> STR profiles of the DNA of tumor cells were analyzed in a cohort of 72 PMBCL patients undergoing EPOCH regimen treatment at the National Research Center for Hematology (Moscow, Russia). <h3>Setting</h3> DNA was isolated from tumor biopsy samples taken at diagnosis. Control DNA samples were taken from the blood of patients in complete remission or from the buccal epithelium. STR profiles for LOH and EMAST analysis were assessed by PCR with the COrDIS Plus multiplex kit for amplification of 19 STR markers and amelogenin loci (Gordiz Ltd, Russia). The fragment analysis was performed on an ABI3130 Genetic Analyzer. <h3>Patients</h3> Inclusion criterion: <i>de novo</i>-diagnosed PMBCL patients. Exclusion criterion: pre-treatment. <h3>Main Outcomes Measures</h3> Progression-free survival (PFS) and overall survival (OS) at 24 months from diagnosis were used to assess the independent impact of genetic instability as a risk factor. <h3>Results</h3> LOH was detected in 37 of 72 patients (51.4%). EMAST was found in 40 patients (55.5%); 24 had a combination of EMAST with LOH. For all EMAST-positive patients, the number of involved loci was no more than 4 out of 19 (EMAST-low, less than one-third of the panel of microsatellite markers). It was shown that OS and PFS were the highest in patients with a stable STR tumor profile. <h3>Conclusions</h3> STR profiling of PMBCL tumor genome revealed LOHs associated with deletions or uniparental disomy and detected EMAST attributed to failure of DNA mismatch repair. The high incidence of LOH and EMAST in PMBCL can be associated with standard therapy failure in a significant number of patients. <h3>Funding</h3> This study was supported by Rakfond grant 2/2020.

Molecular Characteristics of Lymphocyte-predominant Triple-negative Breast Cancer.

Tumor-infiltrating lymphocytes (TILs) are considered a prognostic marker for triple-negative breast cancer (TNBC). Immune checkpoint inhibitor (ICI)-based treatments are more effective for tumors with PD-L1-positive TILs, suggesting crucial roles of TILs in the local tumor immunity. However, factors attracting TILs are still largely unknown. Focusing on tumor antigenicity, we examined TNBC samples to identify the characteristics of TIL-high tumors. Nine treatment-naïve TNBCs (TIL-high: five, TIL-low: four) were subjected to next-generation sequencing (NGS). Loss of heterozygosity (LOH) of PTEN was also analyzed. A variety of copy number variations were observed, and no genes differed significantly between TIL-high and -low groups. However, PTEN loss was more frequently observed in the TIL-high group: 60% compared to 25% in TIL-low tumors. NGS correlated well with LOH analysis in identifying PTEN loss. All three tumors with PTEN loss in the TIL-high group showed high PD-L1. All nine samples were microsatellite-stable. Frequent PTEN loss and high expression of PD-L1 in TIL-high TNBC suggest that PTEN mutation may be a biomarker for ICIs.

Abstract PD10-12: Genomic analysis from the talazoparib beyond BRCA clinical trial: Homologous recombination (HR) deficiency scores, loss-of-heterozygosity and mutations in non-BRCA1/2 mutant tumors with other HR mutations

Abstract Background: Talazoparib, a PARP1 inhibitor, is active in germline BRCA1/2 mutant advanced HER2-negative breast cancer, but its activity beyond BRCA1/2 less well understood. The Talazoparib Beyond BRCA clinical trial treated 20 patients with germline (g) or somatic (s) mutations in HR pathways genes other than BRCA1/2, which included PALB2, CHEK2, ATM, BRIP1, RAD50, ATR, PTEN, and FANCA. Talazoparib treatment was associated with a 31% overall response rate in patients with breast cancer, including tumor regression from baseline as best response in 6 out of 6 patients treated gPALB2 mutations. We now present the results of additional genomic studies to further characterize treatment responses and resistance mechanisms. Methods: Primary and metastatic tumor biopsies were assessed using the commercially-available next-generation sequencing HRD assay (Myriad). Panel gene sequencing of tumors was performed for 108 genes associated with genomic instability. Loss-of-heterozygosity (LOH) was assessed in tumors. Tumor/normal exome sequencing identified pre-treatment somatic variants. Circulating-tumor DNA at baseline and disease progression was assessed by targeted panel sequencing and plasma whole exome sequencing. Results: Of the 18 HRD assays performed, 3 failed and were thus excluded. Seven patients had HRD analysis performed on both primary and metastatic tumors. For these patients the HRD score was significantly higher in the metastasis versus the primary (means 46.2 versus 36.5, p = 0.018 by paired t-test). By panel sequencing of tumors (n=18) the most common alterations detected included mutations in PIK3CA (n=8), PALB2 (n=6), ATM (n=5), KRAS (n=4), PTEN (n=5) and TP53 (n=4). In all cases except one, the HR-associated mutation used as entry criteria were detected. LOH analysis showed that of tumors with gPALB2 mutations, 3 of the 6 had LOH for PALB2, and an additional two tumors had 2 independent PALB2 mutations suggesting bi-allelic inactivation. The one remaining tumor had an uncertain LOH result due to test failure. Thus, it is likely that most, if not all, of the tumors in our cohort with gPALB2 mutations had complete inactivation of PALB2 gene function. Other detected mutations that were associated with LOH included all sTP53 mutations (n=4), all gCHEK2 mutations (n=3), gFANCA (n=1), all sRB1 mutations (n=3) and NF1 (n=1). Of the three gATM mutations one had LOH, while the others had two independent mutations, suggestive of bi-allelic inactivation. sATM mutations were associated with LOH in a breast cancer and with 2 independent (possibly bi-allelic) mutations in a non-breast cancer. Additional results from ctDNA targeted sequencing and plasma whole exome sequencing of baseline and progression samples will be presented. Conclusions: In this proof-of-concept phase II study, talazoparib demonstrated activity in HER2-negative advanced breast cancer patients with a HR pathway mutation beyond BRCA1/2, especially in patients with gPALB2 mutations and high HRD scores. These additional genomic analyses provide evidence that HRD scores may be significantly higher in metastatic samples compared to primary tumors. Also, we detected either LOH or bi-allelic inactivation for most HR-associated mutations used to determine eligibility. These findings may inform selection of patients for PARP inhibitor monotherapy beyond BRCA1/2. Citation Format: Joshua J Gruber, Anosheh Afghahi, Alyssa Hatton, Wyatt Gross, Jessica Foran, Alex McMillan, James M Ford, Melinda L Telli. Genomic analysis from the talazoparib beyond BRCA clinical trial: Homologous recombination (HR) deficiency scores, loss-of-heterozygosity and mutations in non-BRCA1/2 mutant tumors with other HR mutations [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD10-12.