Detection Of Loss Of Heterozygosity Research Articles

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.

Detection of Biallelic Loss of DNA Repair Genes in Formalin-Fixed, Paraffin-Embedded Tumor Samples Using a Novel Tumor-Only Sequencing Panel

Patient selection for synthetic lethal-based cancer therapy may be improved by assessment of gene-specific loss of heterozygosity (LOH) and biallelic loss of function (LOF). This report describes SyNthetic lethal Interactions for Precision Diagnostics (SNiPDx), a targeted next-generation sequencing (NGS) panel for detection of LOH and biallelic LOF alterations in 26 target genes focused on DNA damage response pathways, in tumor-only formalin-fixed, paraffin-embedded (FFPE) samples. NGS was performedacross all exons of these 26 genes and encompassed a total of 7632 genome-wide single-nucleotide polymorphisms on genomic DNA from 80 FFPE solid tumor samples. The Fraction and Allele-Specific Copy Number Estimates from Tumor Sequencing algorithm was optimized to assess tumor purity and copy number based on heterozygous single-nucleotide polymorphisms. SNiPDx demonstrated high sensitivity (95%) and specificity (91%) for LOH detection compared with whole genome sequencing. Positive agreement with local NGS-based testing in the detection of genetic alterations was 95%. SNiPDx detected 93% of biallelic ATM LOF mutations, 100% of ATM single-nucleotide variants and small insertions/deletions, and 100% of all ATM LOH status events identified by orthogonal NGS-based testing. SNiPDx is a novel, clinically feasible test for analysis of allelic status in FFPE tumor samples, which demonstrated high accuracy when compared with other NGS-based approaches in clinical use.

Prospective BASECAMP-1 experience in patients with gastrointestinal (GI) cancer: Identifying patients with human leukocyte antigen (HLA) loss of heterozygosity (LOH) for a future therapeutic trial exploiting LOH as a tumor vulnerability.

209 Background: Metastatic colorectal (CRC), pancreatic (PANC), and gastroesophageal cancers are the leading causes of GI cancer–related mortality (5-y survival: 15%, 3%, and 5%-6%, respectively) (ACS 2022). HLA LOH is a recurrent mechanism of immune escape observed in 15%-20% of GI cancers (Hecht R., ASCO GI 2022). The Tmod platform is a logic-gated chimeric antigen receptor (CAR) T-cell modular system, comprising a carcinoembryonic antigen (CEA)- or mesothelin (MSLN)-targeting CAR activator and a separate HLA-A*02-targeting blocker receptor. Both in vitro/in vivo, Tmod CAR T therapy kills cells with HLA-A*02 LOH (tumor) without harming cells with retained HLA-A*02 expression (normal). However, HLA-A*02 LOH can only be therapeutically exploited if patients are identifiable through a feasible and timely clinical workflow. Methods: We established a biobanking protocol (BASECAMP-1, NCT04981119) to determine whether HLA-A*02 LOH patients can be prospectively identified. Patients with CRC, PANC, or non-small cell lung cancer (NSCLC), and a high risk for incurable relapse, were screened first using a standard HLA assay. Heterozygous HLA-A*02 positive tumor samples were then assessed for LOH using a bioinformatic algorithm applied via the Tempus xT platform. Results: As of Sep 1, 2022, 83 patients were consented at 4 institutions. HLA status was obtained from 70 patients and 28 were identified as HLA-A*02:01 heterozygous (40%; expected frequency based on USA NMDP data, 27.6%). LOH results were available for 16 patients; 4 LOH-positive patients were identified (25%, 2 PANC, 2 NSCLC). The LOH assay sensitivity declines below a tumor purity of 40% (Hecht R., ASCO GI 2022). Six patients had a tumor purity of 20% (all with PANC, a tumor known for high stromal content), limiting possible LOH detection. The impact of tumor purity on LOH sensitivity was highlighted in a patient with a low initial sample tumor purity (30%) that resulted in a 41% probability of HLA-A*02:01 LOH (below positive threshold). A second sample with a higher tumor purity (70%), obtained from formalin-fixed, paraffin-embedded sections, resulted in a 92% probability of HLA-A*02:01 LOH (positive). Conclusions: BASECAMP-1 prospective identification of HLA-A*02 LOH is feasible in the real-world setting. The frequencies of the HLA-A*02 allele and of HLA-A*02 LOH in this cohort mirrored expected population frequencies. LOH results can be obtained within a clinically feasible workflow and timeframe, although samples with a < 40% tumor purity have a reduced sensitivity for LOH detection, an issue recurrently observed in patients with PANC. The BASECAMP-1 strategy enables prospective identification of appropriate patients for future therapeutic clinical trials using Tmod CEA and MSLN logic-gated CAR T cells. Clinical trial information: NCT04981119 .

Abstract 2801: Detection of biallelic loss of DNA repair genes in formalin-fixed, paraffin embedded (FFPE) tumor samples using a novel tumor-only sequencing panel with error correction

Abstract Background: Loss-of-function (LOF) mutations in DNA damage response (DDR) tumor suppressor genes are compensated for by functional redundancies, exposing synthetic lethal (SL) interactions and opportunities for targeted therapy. Patient selection for SL-based therapy may be improved by assessment of gene-specific loss of heterozygosity (LOH) and biallelic LOF, neither of which is routinely reported by existing targeted sequencing panels. The Synthetic Lethal Interactions for Precision Diagnostics (SNiPDx) targeted sequencing panel features a novel bioinformatic analysis pipeline that enables accurate genome-wide determination of allele-specific copy number, estimation of tumor ploidy and purity, and detection of single nucleotide and indel variants in target genes focused on DDR pathways, all from tumor-only samples. Here we describe the development and accuracy of SNiPDx for detection of LOH and bi-allelic LOF genetic alterations in FFPE samples. Methods: Genomic DNA (>50 ng) was extracted from FFPE samples of multiple solid tumor types (n = 43). Next-generation sequencing was performed on anchored multiplex PCR libraries, constructed using probes that incorporate unique molecular identifiers and span 26 genes and 5,000 genome-wide common germline single-nucleotide polymorphisms (SNPs). Unmatched non-tumor samples (n = 24) were used to generate a reference baseline dataset. The FACETS algorithm, optimized to account for differential DNA fragmentation across samples, was used to assess copy number imbalance in heterozygous SNPs and to quantify tumor purity. Allele fractions at each heterozygous SNP were used to estimate allelic imbalances across chromosomal regions. A reference dataset was derived from matched FFPE tumor samples by whole genome sequencing (WGS) and analysis of sequence data using 3 complementary algorithms. Allele-specific copy number analysis and tumor purity estimation from SNiPDx and WGS data were compared. Results: Copy number was evaluable in 605 genes from 24 matched tumor samples that passed quality control filters. Median sequencing depth across samples by SNiPDx and WGS were 1346x and 18.6x, respectively. LOH detection by SNiPDx was reproducible (100%) across 170 genes from 7 samples sequenced and analyzed in duplicate. A strong correlation was observed between sample purity estimates by WGS and SNiPDx (Pearson’s r = 0.81, p < 0.001). Compared with WGS-derived calls, the sensitivity and specificity of LOH detection by SNiPDx were 95% and 90%, respectively, rising to 97% and 91% in regions with LOH agreement by all 3 WGS algorithms, and to 99% and 97% in diploid regions with no subclonal alterations. b The SNiPDx panel is a novel clinical test for biallelic loss in FFPE tumor-only samples with high accuracy as validated through concordance with a WGS-derived dataset. Citation Format: Dominik Glodzik, Pier Selencia, Ryan Rogge, Ian M. Silverman, Michael Zinda, Maria Koehler, Robert D. Daber, Verity Johnson, Jorge S. Reis-Filho, Victoria Rimkunas. Detection of biallelic loss of DNA repair genes in formalin-fixed, paraffin embedded (FFPE) tumor samples using a novel tumor-only sequencing panel with error correction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2801.

Detection of homologous recombination deficiency (HRD) in cell-free DNA (cfDNA) using an amplicon-based next-generation sequencing (NGS) assay.

3042 Background: Homologous recombination (HR) deficiency is characterized by tumor genomic instability, often due to alterations in BRCA1/2 and other HR-related genes. HRD predicts sensitivity to PARP inhibitors (PARPi) in prostate, ovarian and breast cancers. For the subset of cancers that have genomic instability without detectable alterations in HR genes, profiling biomarkers of HRD such as loss of heterozygosity (LOH) could identify additional HRD positive (HRD+) patients that may benefit from PARPi. Here, we present a novel cfDNA NGS assay that can detect tumor LOH non-invasively to improve assessment of HRD+ status. Methods: An amplicon-based HRD NGS assay covering > 1000 SNPs was developed to detect LOH both globally and on the gene level in cfDNA. Analytical validation was done using LOH+ BRCA-mutant cell lines in limiting dilution admixtures. Clinical performance was assessed by benchmarking findings from 46 tumor tissue DNA samples against results from an orthogonal NGS-based genomic scarring assay. Clinical utility of the HRD assay was evaluated in 75 cfDNA samples, including 72% (54/75) from BRCA-associated cancers (36 breast, 12 prostate, 4 ovarian, 2 pancreatic) and 28% from other cancer types. All cfDNA samples were previously clinically tested by an NGS assay that included BRCA1/2 (LiquidHALLMARK). A subset of cfDNA samples identified as HRD+ were further assessed for alterations in 26 key HR genes ( ARID1A, ATM, ATR, ATRX, BAP1, BARD1, BRCA1, BRCA2, BRIP1, CDK12, CHEK1, CHEK2, FANCA, FANCC, FANCG, FANCL, MRE11, NBN, PALB2, PTEN, RAD51, RAD51B, RAD51C, RAD51D, RAD54L, and XRCC2). Results: In analytical validation, the assay could detect HRD in admixtures with as low as 18% tumor fraction. In tissue samples, overall concordance of HRD status with the orthogonal test was 91.3% (42/46), with a positive percent agreement of 94.4% (34/36) and negative percent agreement of 80.0% (8/10). In cfDNA, 26.7% (20/75) of samples were HRD+, including 29.6% of BRCA-associated cancers (16/54; 12 breast, 2 prostate, 1 ovarian, and 1 pancreatic) and 19.1% (4/21) of other cancer types. In the three HRD+ breast cancers with pathogenic BRCA mutations, the assay also identified BRCA-specific LOH. Nine HRD+ breast cancers (two BRCA+ and seven BRCA-) were further analyzed for alterations in 26 HR genes. Of the two BRCA+ HRD+ breast cancers, one harbored an additional PTEN loss-of-function (LOF) mutation. Of the seven BRCA- HRD+ breast cancers, one harbored biallelic PALB2 and CDK12 LOF mutations, while another harbored a CDK12 LOF mutation. Conclusions: LOH detection in cfDNA provides additional diagnostic yield of HRD+ status in multiple cancer types, even in the absence of pathogenic HR gene alterations. Further clinical studies to evaluate the utility of HRD detection in cfDNA using LOH and to determine concordance with tumor tissue are ongoing.

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.
 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>A, and CHEK2 del5395.
 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>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>A-associated, and 11/19 (57.9%) CHEK2 del5395-associated tumors. In one carcinoma from a carrier of the CHEK2 IVS2+1G>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.

Abstract 399: Pan-cancer survey of HLA loss of heterozygosity using a robustly validated NGS-based machine learning algorithm

Abstract HLA loss of heterozygosity (LOH) is increasingly being recognized as an important immune escape mechanism in response to checkpoint inhibitor therapy. HLA LOH reduces the repertoire of neoantigens displayed on the cell surface of cancer cells, limiting the efficacy of the immune system to detect and eliminate them. Though highly accurate HLA LOH detection algorithms are needed to allow clinical utility, the field lacks robust, allele-specific validation approaches. Moreover, algorithms of unknown sensitivity and specificity have led to significant discrepancies in the estimated occurrence of HLA LOH as an immune escape mechanism across tumor types. To address these challenges, we have developed a machine learning algorithm to detect HLA LOH (DASH - Deletion of Allele-Specific HLAs), established the accuracy of the algorithm with an allele-specific PCR validation strategy, investigated the frequencies of HLA LOH across 14 tumor types in a cohort of over 800 patients and observed allele-specific neoantigen expansion in response to immunotherapy. To build DASH, we profiled 279 patients on the ImmunoID NeXT Platform to create a training dataset. Our novel features, which account for allele-specific differences in exome probe capture and capitalize on our whole exome platform by including information about copy number alterations in the regions flanking the HLA genes, were used to train an XGBoost model. Orthogonal, allele-specific validation was required to accurately assess sensitivity and specificity for clinical utility. Thus, we profiled over 30 paired tumor-normal cell lines on the ImmunoID NeXT Platform® and identified cell lines with HLA LOH. Using in silico mixtures, we found 100% sensitivity and specificity for tumors with at least 36% tumor purity. Next, we designed a digital PCR (dPCR) assay using patient-specific, allele-specific primers that target a single HLA allele while avoiding all other HLA alleles and tested the limit of detection of the assay in the same cell lines. Then, we performed dPCR with patient-specific primers on 20 tumor and normal sample pairs and found 94% sensitivity. After establishing the high sensitivity and specificity of DASH, we profiled over 800 patients spanning 14 tumor types on the ImmunoID NeXT Platform. We found that over 25% of patients in the majority of tumor types had at least one HLA LOH event. Further, we observed that novel neoantigens that arose during checkpoint treatment were significantly more likely to bind to deleted HLA alleles as compared to the remaining HLA alleles in a head and neck carcinoma cohort treated with anti-PD-1 therapy, shedding light on the mechanism of immune escape in response to checkpoint inhibitors. In summary, we introduced an HLA LOH detection method, performed allele-specific validation, exposed widespread HLA across tumor types and observed the mechanism of immune escape in response to immunotherapy. Citation Format: Rachel Marty Pyke, Datta Mellacheruvu, Charles Abbott, Steven Dea, Eric Levy, Simo V. Zhang, Nikita Bedi, A. Dimitrios Colevas, Devayani Bhave, Manju Chinnappa, Gabor Bartha, John Lyle, John West, Michael Snyder, John Sunwoo, Richard Chen, Sean Michael Boyle. Pan-cancer survey of HLA loss of heterozygosity using a robustly validated NGS-based machine learning algorithm [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 399.

Abstract 6678: Sensitive HLA loss of heterozygosity detection reveals allele-specific neoantigen expansion as resistance mechanism to anti-PD-1 therapy

Abstract Loss of heterozygosity (LOH) in the HLA locus is increasingly being recognized as an important mechanism of immune escape and a proposed biomarker for immunotherapy response. Neoantigens that bind to a deleted HLA allele will no longer be presented to the immune system, potentially allowing subclones with these deletions to escape immune surveillance. Despite interest in the field, few methods exist to detect HLA LOH, and their sensitivity is not well understood. Moreover, the mechanistic impact of HLA LOH in response to immune checkpoint inhibitors (ICI) remains unexplored. Here, using a novel tool to detect HLA LOH, DASH (Deletion of Allele-Specific HLAs), we reveal allele-specific neoantigen expansion in response to ICIs in a head and neck carcinoma cohort and the widespread occurrence of HLA LOH across several tumor types. We performed exome sequencing with ImmunoID NeXT, which enhances coverage of the HLA locus, on tumor and normal samples from 260 patients to create a training dataset for our model. For each patient, we mapped reads to each of their allele-specific HLAs and manually annotated LOH. Then, using purity, ploidy and two novel features (normalized b-allele frequency and allele-specific coverage ratios), we trained an XGBoost model. To evaluate our tool, we compared DASH predictions on held out tumor samples to deletion calls from a standard copy number tool in the regions flanking each HLA gene (91% concordance, 0.73 F1-score), ascertained our sensitivity by diluting cell line data with known HLA LOH to imitate variable purity (100% accuracy in samples above 17% purity) and confirmed the functional impact of HLA LOH using immunopeptidomics data of tumor samples (average of 47% fewer unique peptides binding to lost alleles than kept). To explore the mechanistic impact of HLA LOH in response to ICIs, we studied a cohort of nine head and neck carcinoma patients who received a single dose of nivolumab, and sequenced pre- and posttreatment tumor biopsies for each patient. With DASH, we detected HLA LOH in four of the patients, pretreatment. For these patients, we found a significant posttreatment expansion of neoantigens predicted to bind to the deleted HLA alleles in comparison to the pretreatment biopsy (p=0.046, Wilcoxon signed-rank), revealing the evolutionary force of HLA LOH as a resistance mechanism during ICI therapy. To assess the pervasiveness of HLA LOH across tumor types, we applied DASH to over 500 pretreatment tumors across 13 tumor types and found highly variable frequencies of HLA LOH across the tumor types. In summary, we developed a sensitive method to detect HLA LOH and exposed neoantigen expansion to deleted HLA alleles in response to ICI therapy, emphasizing the limitations of deleted alleles to ignite an immune response. Moreover, we found widespread occurrences of HLA LOH across tumor types, highlighting the importance of accurate HLA LOH detection as a pan-cancer biomarker. Citation Format: Rachel Marty Pyke, Charles Abbott, Dattatreya Mellacheruvu, Simo V. Zhang, Nikita Bedi, A. Dimitrios Colevas, John Sunwoo, John West, Richard Chen, Sean Michael Boyle. Sensitive HLA loss of heterozygosity detection reveals allele-specific neoantigen expansion as resistance mechanism to anti-PD-1 therapy [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 6678.

Abstract 729: Landscape of homologous recombination repair (HRR) mutations in prostate cancer profiled by ctDNA next-generation sequencing

Abstract Background: PARP inhibition can cause synthetic lethality and increased therapeutic sensitivity in patients with HRR deficiency (HRD), which can be detected through the molecular profiling of HRR genes. Prostate cancer has a high prevalence of HRD (20-25%, Athie 2019). High failure rates for tissue biopsy in metastatic prostate cancer patients (25-75%) (Ross 2005, Spritzer 2013) pose challenges for HRD profiling, underscoring the need for a non-invasive, ctDNA alternative. Copy number loss, a frequent cause of HRD, is further difficult to call due to signal dilution by cell-free leukocytic DNA. We developed a pipeline that detects loss-of-function SNV/Indels, structural rearrangements, and gene deletions to identify HRD on GuardantOMNITM, a 500-gene liquid biopsy panel. We present its performance across >620 prostate cancer GuardantOMNITM samples. Methods: Samples from 627 prostate cancer patients were processed on GuardantOMNITM RUO, with median unique coverage of 4982 molecules sequenced to 20,000x read depth. Somatic and germline SNVs and small indels were called using the Guardant bioinformatics pipeline (Helman 2018). A novel HRD module was developed to annotate pathogenic SNV/Indels and identify structural rearrangements, gene-level homozygous deletions, loss-of-heterozygosity (LOH) and genome-wide LOH, comprising of a novel CNV (Barbacioru 2019) and de-novo fusion caller (Gnerre, submitted). Loss-of-function variants were analyzed in 24 HRD genes. Results: Pathogenic alterations in HRD genes were called in 260/620 (42%) prostate cancer samples with ctDNA detected: 28% of all samples had a pathogenic somatic or germline SNV/Indel, 20% had a homozygous deletion, 3.4% had a rearrangement involving an HRD gene. The majority of SNV/Indels occurred in BRCA2 (31% of all 158 SNV/Indels) and ATM (22%), similar to tissue (Dahwan 2016), but mutations also occurred across an additional 21 genes, including CDK12 (8%), CHEK2 (5%) and NBN (3.8%). Of prostate patients with a germline BRCA1/2 SNV/Indel and sufficient tumor shedding for LOH detection (max MAF>10%), 10/19 (52%) also had LOH, compared to 86% in tissue (Jonsson 2019). Homozygous deletions were enriched in BRCA2 (6.9% of all samples), ATM (4.4%) and CHEK1 (2.3%). Rearrangements, including fusions and multi-exonic deletions, accounted for 6.5% of inactivating HRD mutations detected. In total, 24% of prostate samples had a biallelic inactivation involving an SNV, Indel or deletion. Conclusion: We demonstrate in a prostate cancer cohort that GuardantOMNITM ctDNA profiling calls all classes of mutations contributing to HRD, with relative prevalence of alterations consistent with those in tissue. CfDNA presents a potential alternative for identifying patients who may benefit from PARP or cisplatin/platinum therapies, expanding the prevalence from 28% using small variants to 42% with the complete HRD biomarker set. Citation Format: Jennifer Yen, Sante Gnerre, Catalin Barbacioru, Elena Helman, Ohad Manor, Arielle Yablonovitch, Ravi Vijaya Satya, Leo Liu, Jennifer Saam, Stephen Fairclough, Becky Nagy, Richard Lanman, Darya Chudova, AmirAli Talasaz. Landscape of homologous recombination repair (HRR) mutations in prostate cancer profiled by ctDNA next-generation sequencing [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 729.

HLA genotyping in synovial sarcoma: Identifying HLA-A*02 and its association with clinical outcome.

e23560 Background: Patients (pts) with synovial sarcoma (SS) and an HLA-A*02 genotype whose tumors express NY-ESO-1 may be eligible for clinical trials of adoptive T cell therapy. We reasoned that a next generation tumor sequencing platform utilizing matched normal DNA (MSK-IMPACT) could accurately identify HLA genotype. Although HLA-A*02 is necessary for some adoptive T cell therapies, the prognosis of this genotype on clinical outcome has not been described in SS. Methods: Pts with metastatic SS who consented to screen for a clinical trial of engineered T cells had high-resolution HLA genotyping performed with a Clinical Laboratory Improvement Amendments (CLIA)-certified test. Where feasible, HLA genotype and loss-of-heterozygosity (LOH) of HLA alleles were determined from IMPACT samples. Overall survival (OS) was estimated in three overlapping cohorts and stratified by HLA-A*02 status: pts treated with anthracyclines or alkylators in the first line, pazopanib in the second line or beyond, and all pts from time of metastasis. Results: 66 pts with SS were screened, but not treated with T cells; 30% (n = 20) were HLA-A*02-positive on a CLIA-certified outside test. 23 pts had HLA genotyping both by IMPACT and an outside laboratory, 22 (96%) of whom had concordant results. 3 pts had LOH of at least 1 HLA allele, including one with LOH of HLA*02:01 in the primary tumor. Among pts treated chemotherapy (n = 36) or pazopanib (n = 37), OS did not significantly differ between HLA-A*02-positive or negative pts. Univariable analyses of OS from the time of metastasis in the whole cohort identified primary tumor size and time to metastasis as variables significantly associated with outcome (hazard ratio (HR) 1.2, 95% confidence interval (CI) 1.123 – 1.345 [P < 0.001] and HR 0.99, 95% CI 0.976 – 0.999 [P = 0.032], respectively). HLA-A*02-positive status and age did not reach the significance threshold (HR 1.95, 95% CI 0.995 – 3.813 [P = 0.052] and HR 1.021, 95% CI 0.999 – 1.044 [P = 0.061], respectively). Multivariable analysis found older age and larger tumor size were independently associated with significantly shorter OS (HR 1.03, 95% CI 1.002 – 1.049 [P = 0.037] and HR 1.2, 95% CI 1.127 – 1.37 [P < 0.001], respectively). Conclusions: Targeted exome panels like IMPACT that utilize matched tumor-normal DNA may accurately identify HLA genotype. Detection of LOH at HLA loci may identify a subgroup of pts who would be refractory to treatment with HLA-A*02-restricted engineered T cells. HLA-A*02 status was not associated with a statistically significant survival difference in pts with metastatic SS.

Abstract A19: HLA allele-specific loss of heterozygosity detection using augmented exome capture approach

Abstract Loss of human leukocyte antigen (HLA) is of increasing interest as a mechanism of cancer immune evasion and biomarker for cancer immunotherapy response. Each cancer patient has six class I HLA alleles that are capable of presenting a set of tumor-specific neoantigens. However, HLA alleles are often deleted in tumors, resulting in a loss of heterozygosity (LOH). When LOH occurs, neoantigen presentation is significantly impaired, potentially facilitating tumor immune evasion. Given the biologic impact of HLA LOH, there is a need for robust algorithms that can detect allele-specific HLA LOH in tumor samples. Here, we describe a novel computational approach to detect HLA LOH from exome sequencing, demonstrate the robustness of the method, and apply the method to calculate the frequencies of HLA LOH in key cancer types. We performed exome sequencing with augmented HLA region capture on the ImmunoID NeXT platform for tumor and normal samples of 184 patients across several cancer types and identified 430 nonhomozygous HLA genes. Next, we extracted the reads mapping to a custom HLA database and mapped them on to the patient-specific HLA alleles. For each allele, we calculated two key features: 1) the tumor b-allele frequency normalized by the native b-allele frequency and 2) the allele-specific tumor to normal coverage ratio. Using these two features, along with tumor purity and ploidy values, we trained a random forest model on a subset of the HLA genes (n=300). While standard copy-number variant (CNV) tools are unable to detect LOH in the polymorphic HLA genes, they can accurately measure deletions in their flanking regions, which we used to validate the accuracy of our allele-specific HLA LOH algorithm. In our test set (n=130), we found a high concordance between our allele-specific deletion calls and the generic deletion calls (94% accuracy, 0.85 F1 Score). When we constrained our test set to samples with high tumor content (>50%, n=40), we saw even stronger concordance (98% accuracy, 0.95 F1 Score). The only discordant call was a focal deletion within an HLA gene that was detected by our algorithm but missed by the CNV tool. Next, we ran our algorithm on patient samples of different cancer indications. For non-small cell lung cancer, we found a high frequency of patients affected by LOH (35%, 9 of 26), which is similar to frequencies previously reported in the literature. Furthermore, we found a lower frequency of melanoma tumors with LOH (15%, 7 of 48). In conclusion, we developed a novel algorithm to call allele-specific HLA LOH on the ImmunoID NeXT exome sequencing platform that augments coverage in the polymorphic HLA locus and demonstrated overall robust performance. The relatively high frequency of LOH events we detected in the melanoma and lung cancer samples suggests the importance of LOH analysis to inform cancer immunotherapy biomarker studies and personalized cancer therapies that depend on neoantigen presentation. Citation Format: Rachel Marty Pyke, Charles Abbott, Simo V. Zhang, Datta Mellacheruvu, John West, Richard Chen, Sean Michael Boyle. HLA allele-specific loss of heterozygosity detection using augmented exome capture approach [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A19.

Detection of Loss of Heterozygosity in Tk-Deficient Mutants from L5178Y Tk+/--3.7.2C Mouse Lymphoma Cells.

The mouse lymphoma assay (MLA), a forward mutation assay using the Tk+/--3.7.2C clone of the L5178Y mouse lymphoma cell line and the Thymidine kinase (Tk) gene, has been widely used as an in vitro genetic toxicity assay for more than four decades. The MLA can evaluate the ability of mutagens to induce a wide range of genetic events including both gene mutations and chromosomal mutations and has been recommended as one component of several genotoxicity test batteries. Tk-deficient mutants often exhibit chromosomal abnormalities involving the distal end of chromosome 11 where the Tk gene is located, in mice, and the type of chromosome alteration can be analyzed using a loss of heterozygosity (LOH) approach. LOH has been considered an important event in human tumorigenesis and can result from any of the following several mechanisms: large deletions, mitotic recombination, and chromosome loss. In this chapter, the authors describe the procedures for the detection of LOH in the Tk mutants from the MLA, and apply LOH analysis for understanding the types of genetic damage that is induced by individual chemicals.

The Efficiency of SNP-Based Microarrays in the Detection of Copy-Neutral Events at 15q11.2 and 11p15.5 Loci.

Prader-Willi, Angelman, Beckwith-Wiedemann, and Russell-Silver are imprinting syndromes. In this study, we aimed to compare the efficiency of single nucleotide polymorphism (SNP) microarray analysis with methylation-specific Multiplex ligation-dependent probe amplification (MS-MLPA) in the detection of uniparental disomy in these syndromes. The patient samples with regions of loss of heterozygosity (LOH), covering 15q11.2 and 11p15.5 critical loci, were analyzed with MS-MLPA to demonstrate the efficiency of SNP microarray in the detection of uniparental disomy (UPD). In a total of seven patients, LOH covering 15q11.2 and 11p15.5 critical loci was detected. Two (28.6%) of these seven patients showed aberrant methylation (suggesting UPD) in MS-MLPA. SNP microarray is a useful tool in the detection of LOH; however, it should be used with caution, since false-positive or false-negative LOH results can be obtained. Although methylation analysis is recommended as the first tier test in the diagnosis of most of the imprinting disorders, combining methylation analysis with SNP microarray can enhance our evaluation process.

Abstract 5226: Genomic analysis of pancreatic ductal adenocarcinoma in a patient with MUTYH-associated polyposis

Abstract Biallelic pathogenic germline variants in the DNA repair glycosylase, MUTYH, cause MUTYH-associated polyposis, characterised by an increased susceptibility to colorectal adenomas and carcinomas secondary to defective base excision repair. We report a patient diagnosed with Stage IIB distal pancreatic ductal adenocarcinoma (PDAC) at the age of 45 years. Prior colonoscopy and gastroscopy noted three colonic tubular adenomas and a gastric fundic gland polyp. The patient was consented to whole genome and transcriptome sequencing of the PDAC and matched normal blood DNA through the British Columbia Personalized Onco-Genomics (POG) program. Analysis of germline and somatic variants including single nucleotide variants, copy number determination, loss of heterozygosity detection and mutational signatures was undertaken. Expression fold-changes were calculated against Illumina BodyMap pancreatic tissue averages and compared against The Cancer Genome Atlas PDAC cases. Germline analysis revealed biallelic mutations in the MUTYH gene. In light of this patient's personal and family history of adenomatous colon polyps, clinic-initiated panel testing of 14 cancer susceptibility genes, including MUTYH, via Illumina sequencing with reflex Sanger confirmation revealed the same biallelic MUTYH changes. Analysis of the patient's PDAC revealed a base excision repair pathway signature, demonstrated by an increased frequency of C:G>A:T transversions, consistent with deficient MUTYH activity. This is the first association of germline MUTYH biallelic pathogenic variants with PDAC and provides evidence of the contribution of aberrant MUTYH function to the genomic landscape of a PDAC. Detection of the base excision repair mutational signature may be a sensitive way to screen tumors for aberrant MUTYH function that can reveal potential germline MUTYH-related cancer susceptibility, and allow inference of pathogenicity of detected MUTYH variants, which may have cancer prevention and therapeutic implications. Citation Format: Kasmintan A. Schrader, Carolyn Chu’ng, Eric Zhao, Hui-li Wong, Yaoqing Shen, Martin Jones, Tom Thomson, Howard Lim, Sean Young, Carol Cremin, Robert Holt, Peter Eirew, Joanna Karasinska, Jacquie Schein, Yongjun Zhao, Andy Mungall, Richard Moore, Yussanne Ma, Alexandra Fok, Robyn Roscoe, Stephen Yip, Gillian Mitchell, Aly Karsan, Steven Jones, David Schaeffer, Janessa Laskin, Marco Marra, Daniel Renouf. Genomic analysis of pancreatic ductal adenocarcinoma in a patient with MUTYH-associated polyposis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5226.

Monoallelic and biallelic deletions of 13q14 in a group of CLL/SLL patients investigated by CGH Haematological Cancer and SNP array (8x60K).

BackgroundDeletion of 13q14 is the most common cytogenetic change in chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and is detected in about 50 % of patients by fluorescence in situ hybridization (FISH), which can reveal presence of del(13)(q14) and mono- or biallelic deletion status without information about the size of the lost region. Array-comparative genomic hybridization (aCGH) and single nucleotide polymorphism (SNP) can detect submicroscopic copy number changes, loss of heterozygosity (LOH) and uniparental disomy (UPD) regions. The purpose of this study was detection of the size of del(13)(q14) deletion in our group of patients, comparing the size of the monoallelic and biallelic deletions, detection of LOH and UPD regions.ResultsWe have investigated 40 CLL/SLL patients by karyotype, FISH and CGH and SNP array. Mutational status was of immunoglobulin heavy-chain variable-region (IGVH) was also examined. The size of deletion ranged from 348,12 Kb to 38.97 Mb. Detected minimal deleted region comprised genes: TRIM13, miR-3613, KCNRG, DLEU2, miR-16-1, miR-15a, DLEU1. The RB1 deletions were detected in 41 % of cases. The average size in monoallelic 13q14 deletion group was 7,2 Mb while in biallelic group was 4,8 Mb. In two cases 13q14 deletions were located in the bigger UPD regions.ConclusionsOur results indicate that bigger deletion including RB1 or presence of biallelic 13q14 deletion is not sufficient to be considered as adverse prognostic factor in CLL/SLL. CytoSure Haematological Cancer and SNP array (8x60k) can precisely detect recurrent copy number changes with known prognostic significance in CLL/SLL as well as other chromosomal imbalances. The big advantage of this array is simultaneous detection of LOH and UPD regions during the same test.

Cross-laboratory validation of the OncoScan® FFPE Assay, a multiplex tool for whole genome tumour profiling.

BackgroundAdoption of new technology in both basic research and clinical settings requires rigorous validation of analytical performance. The OncoScan® FFPE Assay is a multiplexing tool that offers genome-wide copy number and loss of heterozygosity detection, as well as identification of frequently tested somatic mutations.MethodsIn this study, 162 formalin fixed paraffin embedded samples, representing six different tumour types, were profiled in triplicate across three independent laboratories. OncoScan® formalin fixed paraffin embedded assay data was then analysed for reproducibility of genome-wide copy number, loss of heterozygosity and somatic mutations. Where available, somatic mutation data was compared to data from orthogonal technologies (pyro/sanger sequencing).ResultsCross site comparisons of genome-wide copy number and loss of heterozygosity profiles showed greater than 95% average agreement between sites. Somatic mutations pre-validated by orthogonal technologies showed greater than 90% agreement with OncoScan® somatic mutation calls and somatic mutation concordance between sites averaged 97%.ConclusionsReproducibility of whole-genome copy number, loss of heterozygosity and somatic mutation data using the OncoScan® assay has been demonstrated with comparatively low DNA inputs from a range of highly degraded formalin fixed paraffin embedded samples. In addition, our data shows examples of clinically-relevant aberrations that demonstrate the potential utility of the OncoScan® assay as a robust clinical tool for guiding tumour therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-015-0079-z) contains supplementary material, which is available to authorized users.

The anaplastic lymphoma kinase as an oncogene in solid tumors

Loss of heterozygosity (LOH) of tumor suppressor genes is a frequent event in tumorigenesis. LOH is most often analyzed by microsatellite typing, but here we offer a fast and efficient method for simultaneous SNP genotyping and mutation scanning, which can also be used for LOH detection. High resolution melting (HRM) provides simple variant detection, and can be adopted for a wide range of applications. When a melting profile for a specific SNP is determined, the screening can be done without the need for sequencing, and only the melting profiles differing from the established melting profiles should be sequenced. LOH of PTCH1 gene is often found in a series of different tumors, for example basal cell carcinoma (BCC) and ovarian carcinoma (OC). In this study LOH was detected in 50 % of BCC and 27.27 % of OC, and the detection rates of microsatellite typing and HRM were comparable. Both methods depended only on the heterozygosity of the loci analyzed, but HRM offers an additional advantage of detection of all sequence variants in the gene of interest.

SNP arrays: comparing diagnostic yields for four platforms in children with developmental delay.

BackgroundMolecular karyotyping is now the first-tier genetic test for patients affected with unexplained intellectual disability (ID) and/or multiple congenital anomalies (MCA), since it identifies a pathogenic copy number variation (CNV) in 10-14% of them. High-resolution microarrays combining molecular karyotyping and single nucleotide polymorphism (SNP) genotyping were recently introduced to the market. In addition to identifying CNVs, these platforms detect loss of heterozygosity (LOH), which can indicate the presence of a homozygous mutation or uniparental disomy. Since these abnormalities can be associated with ID and/or MCA, their detection is of particular interest for patients whose phenotype remains unexplained. However, the diagnostic yield obtained with these platforms is not confirmed, and the real clinical value of LOH detection has not been established.MethodsWe selected 21 children affected with ID, with or without congenital malformations, for whom standard genetic analyses failed to provide a diagnosis. We performed high-resolution SNP array analysis with four platforms (Affymetrix Genome-Wide Human SNP Array 6.0, Affymetrix Cytogenetics Whole-Genome 2.7 M array, Illumina HumanOmni1-Quad BeadChip, and Illumina HumanCytoSNP-12 DNA Analysis BeadChip) on whole-blood samples obtained from children and their parents to detect pathogenic CNVs and LOHs, and compared the results with those obtained on a moderate resolution array-based comparative genomic hybridization platform (NimbleGen CGX-12 Cytogenetics Array), already used in the clinical setting.ResultsWe identified a total of four pathogenic CNVs in three patients, and all arrays successfully detected them. With the SNP arrays, we also identified a LOH containing a gene associated with a recessive disorder consistent with the patient’s phenotype (i.e., an informative LOH) in four children (including two siblings). A homozygous mutation within the informative LOH was found in three of these patients. Therefore, we were able to increase the diagnostic yield from 14.3% to 28.6% as a result of the information provided by LOHs.ConclusionsThis study shows the clinical usefulness of SNP arrays in children with ID, since they successfully detect pathogenic CNVs, identify informative LOHs that can lead to the diagnosis of a recessive disorder. It also highlights some challenges associated with the use of SNP arrays in a clinical laboratory.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-014-0070-0) contains supplementary material, which is available to authorized users.

Analysis for loss of heterozygosity on chromosome arm 13q by STR analysis or SNP sequencing can replace analysis of FLT3-ITD to detect patients with prognostically adverse AML.

We aimed at developing novel assays for Loss of Heterozygosity (LOH) detection on 13q as a substitute for FLT3-ITD analysis to identify acute myeloid leukemia (AML) patients with high risk of shorter survival. To this aim, we first analyzed a selected cohort of 185 patients with (n = 138) or without (n = 47) FLT3-ITD by short tandem repeat (STR) analysis for 13q LOH. In 46 of 138 FLT3-ITD positive cases, a FLT3-ITD/FLT3wt ratio of ≥ 1 was measured indicating LOH. Applying analysis with a combination of five different STR markers, a threshold of an STR allelic ratio of >65% allows the identification of LOH in 40/46 (87%) samples. Survival analysis revealed significantly inferior outcome in patients with LOH as detected by STR analysis (event free survival (EFS): no LOH: 13.3 months versus LOH: 4.5 months, p < 0.001; overall survival (OS): no LOH: 35.8 months versus LOH: 9.7 months, p = 0.001). In multivariate Cox regression analysis, 13q LOH was found to be an independent adverse parameter for EFS and OS (p = 0.003 and p < 0.001, respectively) besides age and white blood cell count. Thus, the prognostic impact of 13q LOH is nearly identical to the one of FLT3-ITD/FLT3wt load of ≥ 1 and thus is a feasible alternative to identify respective patients with high risk AML. In a second approach, a cohort of 91 patients was subjected to a proof-of-principle study of applying single nucleotide polymorphism analysis by next generation amplicon sequencing for detection of LOH. 53/91 cases had LOH and 50 were identified with this new method resulting in a positive detection rate of 94.3%.

Single cell analysis exposes intratumor heterogeneity and suggests that FLT3-ITD is a late event in leukemogenesis

FMS-like tyrosine kinase 3 receptor-internal tandem duplication (FLT3-ITD) commonly occurs in acute myeloid leukemia and is considered rare in acute lymphocytic leukemia. Acute leukemia has poor prognosis, mainly due to relapse. Standard FLT3-ITD diagnostic techniques are based on genomic polymerase chain reaction and have recently incorporated GeneScan (Applied Biosystems, Foster City, CA) to identify variations of the FLT3 gene. As this is an average-based assay utilized in a heterogeneous leukemic cell population, we hypothesized that cells of acute leukemia, considered FLT3-ITD-negative by standard methods, could possess a fraction of FLT3-ITD-positive cells. The present study employed single cell mutation analysis to evaluate the FLT3-ITD status in newly diagnosed acute myeloid leukemia (n = 5) and acute lymphocytic leukemia (n = 3) patients. A total of 541 single leukemic cells and 36 mononuclear cells from healthy volunteers were analyzed. Seven patients, considered FLT3-ITD-negative according to bulk DNA analysis, appeared to possess a small fraction of FLT3-ITD-positive cells based on single cell analysis. Moreover, this approach revealed the heterogeneity of the tumor as evident by different FLT3-ITD mutations present in the same patient. The presence of a minor clone carrying FLT3-ITD in almost all patients tested provides evidence that this lesion is a common late event in leukemogenesis. Additionally, 3 relapsed patients demonstrated loss of heterozygosity of the normal allele, affecting 25%-100% of the cells found to be FLT3-ITD-positive. Though further clinical testing is warranted, these findings may have implications on the prognostic significance of FLT3-ITD and the use of targeted therapy.