Loss Of Heterozygosity Profiles Research Articles

Abstract 2328: Next-generation sequencing-based regression algorithm to determine homologous recombination deficiency scores in a pan-cancer cohort

Abstract Background: Homologous recombination deficiency (HRD) inhibits double strand breaks from being repaired in DNA, leading to cancer cells failing to recover themselves and cell death. Previous studies found that HRD-positive ovarian cancer patients had more significant clinical benefits from poly ADP-ribose polymerase inhibitors (PARPi) treatment. For accurate detection of HRD, next-generation sequencing (whole exome, whole genome) was used to identify large-scale genomic aberrations including telomeric allelic imbalance (HRD-TAI score), loss of heterozygosity profiles (HRD-LOH score), and large-scale state transitions (HRD-LST score). So far, many studies (HRDetect, SigMA, CHORD and shallowHRD) have been conducted to accurately determine HRD in a pan-cancer cohort. As the study progressed, it has been revealed that PARPi is effective when applied to patients with HRD in not only ovarian cancer but also breast cancer, prostate cancer, and pancreatic cancer. Purpose: To develop an algorithm that accurately predict HRD score in patients with ovarian cancer, breast cancer, prostate cancer, and pancreatic cancer utilizing a machine learning algorithm. Methods: We used whole-genome sequencing (WGS) data of 710 samples from 309 patients, whole-exome sequencing (WES) data of 4,650 samples from 2,193 patients from the pan-cancer cohort of the TCGA (TCGA-OV, TCGA-BRCA, TCGA-PRAD, and TCGA-PAAD). The HRD-TAI/HRD-LST/HRD-LOH scores were calculated through structural variation analyses. After data cleaning processes, machine-learning models were trained and tested on 228 out of 2,502 samples, and validation was performed on 1,248 out of 2,502 samples from the TCGA dataset. Results: To assess the performance of the machine-learning regressor, the concordance between predictions and annotations was quantified by calculating the R squared (𝑅;;;^2). As a result of training using machine learning algorithm, we achieved a high 𝑅;;;^2 (0.904) with a RMSE (root mean squared error) score (7.649) for a pan-cancer cohort. Conclusions: Our regressor was robust and accurate when applied to 4 cancer types. Using our systematic pan-cancer analysis, we found novel insights into the mechanisms of HRD across cancer types with potential contribution to clinical practice. Citation Format: Boram Choi, Youn Jin Choi. Next-generation sequencing-based regression algorithm to determine homologous recombination deficiency scores in a pan-cancer cohort [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 2328.

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.

Immune profiling of oral pre-malignant lesions (OPLs): An Erlotinib Prevention of Oral Cancer (EPOC) study biobank analysis.

1545 Background: We previously demonstrated that high-risk loss of heterozygosity (LOH) profiles (i.e., 3p14/9p21 LOH) and EGFR gene copy number gain (CNG) in OPLs were associated with inferior oral cancer-free survival (OCFS) in patients enrolled in the randomized EPOC trial. Herein, we performed comprehensive immune profiling of OPLs and correlated the findings with molecular features and outcomes, using the prospectively collected and clinically annotated EPOC biobank. Methods: We evaluated OPL specimens by multiplex immunofluorescence using the Opal 7-color fIHC Kit and the Vectra multispectral microscope / inForm Cell Analysis software. Markers included AE1/AE3 pancytokeratins, PD-L1 (clone E1L3N), CD3, CD8, and CD68. Wilcoxon rank-sum and Fisher’s exact tests were used to assess the associations between binary markers and continuous and categorical variables, respectively. Cox model was used to investigate associations of markers with OCFS. Results: The cohort included 188 OPL patients with hyperkeratosis/hyperplasia (18%), mild/moderate (44%), or severe dysplasia (5%); 65% had high-risk LOH profiles. The 5-year OCFS was 72.3% (median follow-up of 50 months). PD-L1 expression in > 1% of epithelial cells occurred in 28% of OPLs. Intraepithelial CD3+, CD3+/CD8+, CD68+, and CD68+/PD-L1+ cells were detected in 100%, 88%, 88%, and 54% of the samples, respectively. OPLs with high-risk LOH profiles had increased epithelial PD-L1 expression (P = 0.007), intraepithelial CD68+/PD-L1+ cells (P = 0.002), and a trend towards more CD3+/CD8+ cells in the stroma (P = 0.06) but not in the epithelium (P = 0.97), compared with low-risk LOH OPLs. Increased epithelial PD-L1 expression was associated with inferior OCFS on univariate (P = 0.023), and multivariate analysis including LOH status and EGFR CNG as co-variates (P = 0.018). Conclusions: High-risk OPLs defined by LOH profiles had increased PD-L1 expression in epithelial cells and intraepithelial macrophages, as well as stromal CD3+/CD8+ immune infiltration. Higher PD-L1 expression was associated with increased oral cancer risk. The findings may support evaluation of (PD-1-targeted) immunoprevention strategies in high-risk OPLs.

Erlotinib and the Risk of Oral Cancer: The Erlotinib Prevention of Oral Cancer (EPOC) Randomized Clinical Trial.

Standard molecularly based strategies to predict and/or prevent oral cancer development in patients with oral premalignant lesions (OPLs) are lacking. To test if the epidermal growth factor receptor inhibitor erlotinib would reduce oral cancer development in patients with high-risk OPLs defined by specific loss of heterozygosity (LOH) profiles. Secondary objectives included prospective determination of LOH as a prognostic marker in OPLs. The Erlotinib Prevention of Oral Cancer (EPOC) study was a randomized, placebo-controlled, double-bind trial. Accrual occurred from November 2006 through July 2012, with a median follow-up time of 35 months in an ambulatory care setting in 5 US academic referral institutions. Patients with OPLs were enrolled in the protocol, and each underwent LOH profiling (N = 379); they were classified as high-risk (LOH-positive) or low-risk (LOH-negative) patients based on their LOH profiles and oral cancer history. The randomized sample consisted of 150 LOH-positive patients. Oral erlotinib treatment (150 mg/d) or placebo for 12 months. Oral cancer-free survival (CFS). A total of 395 participants were classified with LOH profiles, and 254 were classified LOH positive. Of these, 150 (59%) were randomized, 75 each to the placebo and erlotinib groups. The 3-year CFS rates in placebo- and erlotinib-treated patients were 74% and 70%, respectively (hazard ratio [HR], 1.27; 95% CI, 0.68-2.38; P = .45). The 3-year CFS was significantly lower for LOH-positive compared with LOH-negative groups (74% vs 87%, HR, 2.19; 95% CI, 1.25-3.83; P = .01). Increased EGFR gene copy number correlated with LOH-positive status (P < .001) and lower CFS (P = .01). The EGFR gene copy number was not predictive of erlotinib efficacy. Erlotinib-induced skin rash was associated with improved CFS (P = .01). In this trial, LOH was validated as a marker of oral cancer risk and found to be associated with increased EGFR copy number (the target of the intervention). Erlotinib did not, however, improve CFS in high-risk patients with LOH-positive or high-EGFR-gene-copy-number OPLs. These results support incorporation of LOH testing as a prognostic tool in routine clinical practice but do not support erlotinib use in this setting. clinicaltrials.gov Identifier: NCT00402779.

Abstract A11: NGS-based tumor genomic profiling to identify ovarian cancer patients who benefit from the PARP inhibitor rucaparib.

Abstract Background: PARP inhibitors (PARPi) are synthetically lethal to tumor cells with homologous recombination deficiency (HRD). HRD can result from deleterious BRCA1/2 mutations (BRCAmut) or other mechanisms that have not been fully elucidated. Regardless of mechanism, HRD leads to a common phenotype of genome-wide loss of heterozygosity (LOH). It has been hypothesized that this genomic phenotype can be used to identify BRCA wild-type (BRCAwt) HRD tumors likely sensitive to PARPi. Using comprehensive next generation sequencing (NGS)-based tumor genomic profiling, we developed an HRD assay for potential use as a companion diagnostic for rucaparib in high-grade ovarian cancer (HGOC) by combining tumor BRCA1/2 status and quantification of genomic LOH. Methods: In the phase 2 study ARIEL2 Part 1 (NCT01891344), pre-treatment screening biopsies and archival formalin-fixed paraffin embedded tumor specimens were profiled using Foundation Medicine's NGS-based HRD assay, which detects all classes of genomic alterations, including base substitutions, insertions/deletions, and homozygous deletions in BRCA1/2. Genomic LOH was assessed by sequencing &amp;gt;3,500 evenly-distributed single nucleotide polymorphisms across the genome and quantifying the extent of genomic LOH. A pre-specified genomic LOH cutoff was determined using publicly available SNP array data of ovarian tumors to predict platinum sensitivity as a surrogate marker for PARPi sensitivity. Response was assessed by RECIST v1.1 and GCIG CA-125 response criteria. Results: As of July 1 2015, 195 archival tumor and 152 screening biopsy samples (142 matched pairs) from 206 HGOC patients enrolled (204 patients treated) in ARIEL2 Part 1 were successfully profiled using the NGS-based HRD assay. Some screening biopsies were not suitable for successful NGS-based HRD assessment primarily because of insufficient tumor nuclei or inadequate tumor volume. Most matched pairs of archival and pre-trial screening samples exhibited similar genomic LOH profiles (r=0.86); however, 14% of screening samples had higher genomic LOH compared with archival samples collected more than one year earlier. All BRCA1/2 germline and somatic mutated tumors had high genomic LOH in the screening samples. Receiver operating characteristic analysis of genomic LOH showed utility in identifying RECIST/CA-125 responders to rucaparib (AUC=0.72, p&amp;lt;1e-4), with slightly better predictive utility using screening samples compared to archival samples (AUC=0.72 vs 0.69). Using the pre-specified genomic LOH cutoff, high genomic LOH tumors were detected in 54% of evaluable BRCAwt patients; significantly different overall response rates were found in patients with high vs low genomic LOH tumors (48% vs 26%; chi-square p=0.0074). Conclusions: We developed an NGS-based HRD assay that assesses tumor BRCA1/2 and genomic LOH to prospectively identify HGOC patients who may benefit from rucaparib treatment. The optimized NGS-based HRD assay will be prospectively tested in the ongoing portion of the phase 2 study (ARIEL2 Part 2, NCT01891344) and a phase 3 maintenance study (ARIEL3, NCT01968213) that will investigate rucaparib in HGOC. Citation Format: Iain A. McNeish, Kevin K. Lin, James X. Sun, Sandra Goble, Amit Oza, Robert L. Coleman, Clare L. Scott, Gottfried Konecny, Anna V. Tinker, David M. O'Malley, Rebecca Kristeleit, Ling Ma, James D. Brenton, Katherine Bell-McGuinn, Ana Oaknin, Alexandra Leary, Elaina Mann, Heidi Giordano, Roman Yelensky, Mitch Raponi, Elizabeth Swisher. NGS-based tumor genomic profiling to identify ovarian cancer patients who benefit from the PARP inhibitor rucaparib. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A11.

Genomic profiling of CHEK2*1100delC-mutated breast carcinomas.

BackgroundCHEK2*1100delC is a moderate-risk breast cancer susceptibility allele with a high prevalence in the Netherlands. We performed copy number and gene expression profiling to investigate whether CHEK2*1100delC breast cancers harbor characteristic genomic aberrations, as seen for BRCA1 mutated breast cancers.MethodsWe performed high-resolution SNP array and gene expression profiling of 120 familial breast carcinomas selected from a larger cohort of 155 familial breast tumors, including BRCA1, BRCA2, and CHEK2 mutant tumors. Gene expression analyses based on a mRNA immune signature was used to identify samples with relative low amounts of tumor infiltrating lymphocytes (TILs), which were previously found to disturb tumor copy number and LOH (loss of heterozygosity) profiling. We specifically compared the genomic and gene expression profiles of CHEK2*1100delC breast cancers (n = 14) with BRCAX (familial non-BRCA1/BRCA2/CHEK2*1100delC mutated) breast cancers (n = 34) of the luminal intrinsic subtypes for which both SNP-array and gene expression data is available.ResultsHigh amounts of TILs were found in a relatively small number of luminal breast cancers as compared to breast cancers of the basal-like subtype. As expected, these samples mostly have very few copy number aberrations and no detectable regions of LOH. By unsupervised hierarchical clustering of copy number data we observed a great degree of heterogeneity amongst the CHEK2*1100delC breast cancers, comparable to the BRCAX breast cancers. Furthermore, copy number aberrations were mostly seen at low frequencies in both the CHEK2*1100delC and BRCAX group of breast cancers. However, supervised class comparison identified copy number loss of chromosomal arm 1p to be associated with CHEK2*1100delC status.ConclusionsIn conclusion, in contrast to basal-like BRCA1 mutated breast cancers, no apparent specific somatic copy number aberration (CNA) profile for CHEK2*1100delC breast cancers was found. With the possible exception of copy number loss of chromosomal arm 1p in a subset of tumors, which might be involved in CHEK2 tumorigenesis. This difference in CNAs profiles might be explained by the need for BRCA1-deficient tumor cells to acquire survival factors, by for example specific copy number aberrations, to expand. Such factors may not be needed for breast tumors with a defect in a non-essential gene such as CHEK2.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1880-y) contains supplementary material, which is available to authorized users.

Randomized placebo-controlled trial (RCT) of erlotinib for prevention of oral cancer (EPOC).

6007 Background: Loss of heterozygosity (LOH) profiles predict oral cancer (OC) risk in patients (pts) with oral premalignant lesions (OPL). We conducted a randomized, multi-center, double-blind, placebo-controlled, personalized medicine trial of the EGFR inhibitor erlotinib in pts with OPLs defined as high risk by molecular criteria. Our primary hypothesis was that erlotinib would improve oral cancer-free survival (OCFS) in the high-risk, LOH+ population. Methods: Pts with histological evidence of an OPL (with or without a prior history of invasive OC) underwent LOH profiling at 3p14, 9p21, 4q, 8p, 11p, 13q, 17p. LOH+ pts were defined as those with prior OC and LOH at 3p14 and/or 9p21; or those without prior OC and LOH at 3p14 and/or 9p21 plus an additional chromosomal site. LOH- pts received no treatment. LOH+ pts were stratified by history of OC, then randomized (1:1) to erlotinib 150 mg po daily for 12 months or placebo and were assessed every 3-6 months for development of invasive OC. Primary endpoin...

Profiles of genomic instability in high-grade serous ovarian cancer predict treatment outcome.

High-grade serous cancer (HGSC) is the most common cancer of the ovary and is characterized by chromosomal instability. Defects in homologous recombination repair (HRR) are associated with genomic instability in HGSC, and are exploited by therapy targeting DNA repair. Defective HRR causes uniparental deletions and loss of heterozygosity (LOH). Our purpose is to profile LOH in HGSC and correlate our findings to clinical outcome, and compare HGSC and high-grade breast cancers. We examined LOH and copy number changes using single nucleotide polymorphism array data from three HGSC cohorts and compared results to a cohort of high-grade breast cancers. The LOH profiles in HGSC were matched to chemotherapy resistance and progression-free survival (PFS). LOH-based clustering divided HGSC into two clusters. The major group displayed extensive LOH and was further divided into two subgroups. The second group contained remarkably less LOH. BRCA1 promoter methylation was associated with the major group. LOH clusters were reproducible when validated in two independent HGSC datasets. LOH burden in the major cluster of HGSC was similar to triple-negative, and distinct from other high-grade breast cancers. Our analysis revealed an LOH cluster with lower treatment resistance and a significant correlation between LOH burden and PFS. Separating HGSC by LOH-based clustering produces remarkably stable subgroups in three different cohorts. Patients in the various LOH clusters differed with respect to chemotherapy resistance, and the extent of LOH correlated with PFS. LOH burden may indicate vulnerability to treatment targeting DNA repair, such as PARP1 inhibitors.

Patterns of genomic loss of heterozygosity predict homologous recombination repair defects in epithelial ovarian cancer

Background:Defects in BRCA1, BRCA2, and other members of the homologous recombination pathway have potential therapeutic relevance when used to support agents that introduce or exploit double-stranded DNA breaks. This study examines the association between homologous recombination defects and genomic patterns of loss of heterozygosity (LOH).Methods:Ovarian tumours from two independent data sets were characterised for defects in BRCA1, BRCA2, and RAD51C, and LOH profiles were generated. Publically available data were downloaded for a third independent data set. The same analyses were performed on 57 cancer cell lines.Results:Loss of heterozygosity regions of intermediate size were observed more frequently in tumours with defective BRCA1 or BRCA2 (P=10−11). The homologous recombination deficiency (HRD) score was defined as the number of these regions observed in a tumour sample. The association between HRD score and BRCA deficiency was validated in two independent ovarian cancer data sets (P=10−5 and 10−29), and identified breast and pancreatic cell lines with BRCA defects.Conclusion:The HRD score appears capable of detecting homologous recombination defects regardless of aetiology or mechanism. This score could facilitate the use of PARP inhibitors and platinum in breast, ovarian, and other cancers.

Loss of Heterozygosity (LOH) Profiles—Validated Risk Predictors for Progression to Oral Cancer

A major barrier to oral cancer prevention has been the lack of validated risk predictors for oral premalignant lesions (OPL). In 2000, we proposed a loss of heterozygosity (LOH) risk model in a retrospective study. This paper validated the previously reported LOH profiles as risk predictors and developed refined models via the largest longitudinal study to date of low-grade OPLs from a population-based patient group. Analysis involved a prospective cohort of 296 patients with primary mild/moderate oral dysplasia enrolled in the Oral Cancer Prediction Longitudinal Study. LOH status was determined in these OPLs. Patients were classified into high-risk or low-risk profiles to validate the 2000 model. Risk models were refined using recursive partitioning and Cox regression analyses. The prospective cohort validated that the high-risk lesions (3p and/or 9p LOH) had a 22.6-fold increase in risk (P = 0.002) compared with low-risk lesions (3p and 9p retention). Addition of another 2 markers (loci on 4q/17p) further improved the risk prediction, with five-year progression rates of 3.1%, 16.3%, and 63.1% for the low-, intermediate-, and high-risk lesions, respectively. Compared with the low-risk group, intermediate- and high-risk groups had 11.6-fold and 52.1-fold increase in risk (P < 0.001). LOH profiles as risk predictors in the refined model were validated in the retrospective cohort. Multicovariate analysis with clinical features showed LOH models to be the most significant predictors of progression. LOH profiles can reliably differentiate progression risk for OPLs. Potential uses include increasing surveillance for patients with elevated risk, improving target intervention for high-risk patients while sparing a large number of low-risk patients from needless screening and treatment.

Clinicopathological Characteristics and Molecular Analyses of Multifocal Intraductal Papillary Mucinous Neoplasms of the Pancreas

To examine the clinicopathologic features and clonal relationship of multifocal intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. Intraductal papillary mucinous neoplasms are increasingly diagnosed cystic precursor lesions of pancreatic cancer. Intraductal papillary mucinous neoplasms can be multifocal and a potential cause of recurrence after partial pancreatectomy. Thirty four patients with histologically documented multifocal IPMNs were collected and their clinicopathologic features catalogued. In addition, thirty multifocal IPMNs arising in 13 patients from 3 hospitals were subjected to laser microdissection followed by KRAS pyrosequencing and loss of heterozygosity (LOH) analysis on chromosomes 6q and 17p. Finally, we sought to assess the clonal relationships among multifocal IPMNs. We identified 34 patients with histologically documented multifocal IPMNs. Synchronous IPMNs were present in 29 patients (85%), whereas 5 (15%) developed clinically significant metachronous IPMNs. Six patients (18%) had a history of familial pancreatic cancer. A majority of multifocal IPMNs (86% synchronous, 100% metachronous) were composed of branch duct lesions, and typically demonstrated a gastric-foveolar subtype epithelium with low or intermediate grades of dysplasia. Three synchronous IPMNs (10%) had an associated invasive cancer. Molecular analysis of multiple IPMNs from 13 patients demonstrated nonoverlapping KRAS gene mutations in 8 patients (62%) and discordant LOH profiles in 7 patients (54%); independent genetic alterations were established in 9 of the 13 patients (69%). The majority of multifocal IPMNs arise independently and exhibit a gastric-foveolar subtype, with low to intermediate dysplasia. These findings underscore the importance of life-long follow-up after resection for an IPMN.

Testing clonality of three and more tumors using their loss of heterozygosity profiles

Cancer patients often develop multiple malignancies that may be either metastatic spread of a previous cancer (clonal tumors) or new primary cancers (independent tumors). If diagnosis cannot be easily made on the basis of the pathology review, the patterns of somatic mutations in the tumors can be compared. Previously we have developed statistical methods for testing clonality of two tumors using their loss of heterozygosity (LOH) profiles at several candidate markers. These methods can be applied to all possible pairs of tumors when multiple tumors are analyzed, but this strategy can lead to inconsistent results and loss of statistical power. In this work we will extend clonality tests to three and more malignancies from the same patient. A non-parametric test can be performed using any possible subset of tumors, with the subsequent adjustment for multiple testing. A parametric likelihood model is developed for 3 or 4 tumors, and it can be used to estimate the phylogenetic tree of tumors. The proposed tests are more powerful than combination of all possible pairwise tests.

Mutational profiling of sporadic versus toxin-associated brain cancer formation: Initial findings using loss of heterozygosity profiling

The role of environmental and occupational toxin exposure as a cause of or contributing factor for cancer development and progression is incompletely understood. A unique signature of specific mutational change to discriminate toxin-exposed from sporadic cancer is generally sought but not often encountered. We report an approach to better understand cancer causality based on the measurement of the cumulative DNA damage (via loss of heterozygosity) over a defined genomic region (chromosome 3) that is applicable to archival, fixative-treated tissue and cytology specimens of cancer. Our method was applied to (1) a cohort of 10 brain tumor subjects (9 gliomas, 1 hemangioblastoma) with potential exposure to chlorinated solvents and (2) a control cohort of sporadic brain cancer controls (7 gliomas, 1 hemangioblastoma). We show that brain tumors arising in potentially toxin-exposed subjects bear a significantly higher level of passenger LOH mutations compared to sporadic cancer controls. The methodology utilized tissue microdissection, PCR amplification and capillary electrophoresis (fragment analysis for LOH determination, DNA sequencing for specific point mutations), and examined a panel of 15 microsatellite markers distributed along both arms of chromosome 3 that aimed at capturing passenger mutational change accrued during stages of clonal expansion of neoplastic cells. This proof-of-principle study using mutational profiling for passenger LOH mutational damage provides support for the utility of this approach and further studies in order to differentiate between genotoxin-associated versus sporadic (unexposed) cancer development.

Integrative genomic analysis reveals somatic mutations in pheochromocytoma and paraganglioma

Pheochromocytomas and paragangliomas are neuroendocrine tumors that occur in the context of inherited cancer syndromes in ∼30% of cases and are linked to germline mutations in the VHL, RET, NF1, SDHA, SDHB, SDHC, SDHD, SDHAF2 and TMEM127 genes. Although genome-wide expression studies have revealed some of the mechanisms likely to be involved in pheochromocytoma/paraganglioma tumorigenesis, the complete molecular distinction of all subtypes of hereditary tumors has not been solved and the genetic events involved in the generation of sporadic tumors are unknown. With these purposes in mind, we investigated 202 pheochromocytomas/paragangliomas, including 75 hereditary tumors, using expression profiling, BAC array comparative genomic hybridization and somatic mutation screening. Gene expression signatures defined the hereditary tumors according to their genotype and notably, led to a complete subseparation between SDHx- and VHL-related tumors. In tumor tissues, the systematic characterization of somatic genetic events associated with germline mutations in tumor suppressor genes revealed loss of heterozygosity (LOH) in a majority of cases, but also detected point mutations and copy-neutral LOH. Finally, guided by transcriptome classifications and LOH profiles, somatic mutations in VHL or RET genes were identified in 14% of sporadic pheochromocytomas/paragangliomas. Overall, we found a germline or somatic genetic alteration in 45.5% (92/202) of the tumors in this large series of pheochromocytomas/paragangliomas. Regarding mutated genes, specific molecular pathways involved in tumorigenesis mechanisms are identified. Altogether, these new findings suggest that somatic mutation analysis is likely to yield important clues for personalizing molecular targeted therapies.

Clonality: an R package for testing clonal relatedness of two tumors from the same patient based on their genomic profiles

If a cancer patient develops multiple tumors, it is sometimes impossible to determine whether these tumors are independent or clonal based solely on pathological characteristics. Investigators have studied how to improve this diagnostic challenge by comparing the presence of loss of heterozygosity (LOH) at selected genetic locations of tumor samples, or by comparing genomewide copy number array profiles. We have previously developed statistical methodology to compare such genomic profiles for an evidence of clonality. We assembled the software for these tests in a new R package called 'Clonality'. For LOH profiles, the package contains significance tests. The analysis of copy number profiles includes a likelihood ratio statistic and reference distribution, as well as an option to produce various plots that summarize the results. Bioconductor (http://bioconductor.org/packages/release/bioc/html/Clonality.html) and http://www.mskcc.org/mskcc/html/13287.cfm.

Carcinoma Ex Spiradenoma/Cylindroma Confirmed by Immunohistochemical and Molecular Loss-of-Heterozygosity Profiling

We present a case of spiradenoma/cylindroma with admixed carcinoma of unknown origin, resolved using immunohistochemical and molecular loss-of-heterozygosity (LOH) profiling. The patient, a woman in her mid-70s, initially presented with separate mammary (ductal) carcinomas of the right and left breasts that were treated with radical mastectomies. For 9 years, the patient remained disease free until complaining of a slow-growing skin nodule on the lower back that was excised under clinical suspicion of metastatic mammary carcinoma. Histopathological exam revealed a benign eccrine spiradenoma/cylindroma and an intermixed carcinoma, with a differential diagnosis of either primary eccrine carcinoma or mammary carcinoma metastatic to the spiradenoma/cylindroma. Histological features and immunohistochemical staining favored eccrine carcinoma but not unequivocally; therefore, LOH profiles were performed on archival paraffin block tissue from the 3 neoplastic lesions (4 components). The mammary carcinomas showed disparate LOH at 5 of 7 (right breast) and 4 of 7 (left breast) informative genetic loci, establishing these carcinomas as separate primary neoplasms. Both the spiradenoma/cylindroma and eccrine carcinoma revealed no LOH at the tested loci, establishing the unknown carcinoma as an independent carcinoma arising within a spiradenoma/cylindroma. This neoplasm is referred to in the literature as carcinoma ex spiradenoma/cylindroma and spiradenocylindrocarcinoma.

Genome-wide detection of loss of heterozygosity and copy number variation in a human lung large cell carcinoma cell line by affymetrix single-nucleotide polymorphism array 500K.

Loss of heterozygosity (LOH) and Copy number copy number variation (CNV) of DNA sequences is a common feature of cancer genomes, which is thought to be linked to tumorigenesis and progression. High-density singlenucleotide polymorphism (SNP) genotyping array are able to provided a genotype and copy number information with genome-wide coverage, which is suitable for the analysis of complex genetic alterations present in cancer. Thus a human lung large cell carcinoma cell line NL9980 was assayed for the global profile of LOH and CNV. Genomic DNA from the cell line was screened for LOH and CNV using Affymetrix GeneChip(R) Human Mapping array 500K Set. The hybridization intensity data of 500 000 SNP loci was analyzed using Affymetrix proprietary software for genotyping and copy number of each locus, and a genome-wide map of LOH and CNV of the cell line was constructed. The SNP call rate of array Nsp I (-262K) was 95.14%, and the rate of Sty I (-238K) was 97.15%. The both call rates of the components of 500K array set were in excess of 93%, the cardinal quality control standard. LOH profiles of the sample were across all chromosomes, and most of CN gains and losses regions were found on chromosomes such as 2, 3, 4, 5, 7, 10, 11, and 18. The results have shown that there were complex genetic alterations present in NL9980. And it is possible to achieve high performance outcomes using Affymetrix SNP array 500K to interrogate LOH and CNV in lung cancer genome. This advance of highresolution with allelic information should substantially improve the ability to further understanding of the genetic basis of lung cancers.

Narrowing of the regions of allelic losses of chromosome 1p36 in meningioma tissues by an improved SSCP analysis

Mapping loss of heterozygosity (LOH) regions in the genomes of tumor tissues is a practical approach for identifying genes whose loss is related to tumorigenesis. Conventional LOH analyses using microsatellite or single nucleotide polymorphism (SNP) markers require the simultaneous examination of tumor- and matched normal-DNA. Here, we improved the previously developed SNP-based LOH assay using single strand conformation polymorphism (SSCP) analysis, so that LOH in tumor samples heavily contaminated with normal DNA can now be precisely estimated, even when matched normal DNA is not available. We demonstrate the reliability of the improved SSCP-based LOH detection method, called the LOH estimation by quantitative SSCP analysis using averaged control (LOQUS-AC), by comparing the results with those of the previous "LOH estimated by quantitative SSCP assay" (LOQUS) method. Using the LOQUS-AC assay, LOH was detected at a high consistency (98.1%) with the previous LOQUS method. We then applied this new method to characterize LOH profiles in 130 meningiomas, using 68 SNPs (i.e., a mean inter-SNP interval of 441 kbp) that are evenly distributed throughout chromosome 1p36. Benign, atypical and anaplastic meningiomas exhibited 1p36 LOH at frequencies of 48.39, 84.62 and 100.00%, respectively, using LOQUS-AC. Subsequently, we detected a candidate common LOH region on 1p36.11 that might harbor tumor suppressor genes related to malignant progression of meningioma.

Clonal relationships between epidermotropic metastatic melanomas and their primary lesions: a loss of heterozygosity and X-chromosome inactivation-based analysis

Loss of heterozygosity (LOH) has previously been demonstrated at multiple chromosome microsatellites in primary and metastatic melanomas. Epidermotropic metastases of melanoma are unique in their varied histopathologic appearance, which can mimic a primary lesion. Our objective was to compare LOH profiles in primary and epidermotropic metastatic melanoma to delineate their clonal relationship. We examined the pattern of allelic loss in the primary melanomas of nine patients in addition to the 21 corresponding epidermotropic metastatic melanomas (average 2.3 metastases per patient). DNA samples were prepared from formalin-fixed, paraffin-embedded tissue sections using laser capture microdissection. Eight DNA microsatellite markers on six different chromosomes were analyzed: D1S214 (1p), D6S305 (6q), D9S171 (9p), D9S157 (9p), IFNA (9p), D10S212 (10q), D11S258 (11q), D18S70 (18q). In addition, X-chromosome inactivation analysis was performed in tumors from four women. LOH was seen in 67% (6/9) of primary melanomas and 81% (17/21) of epidermotropic metastatic melanomas. The most frequent allelic losses in informative cases occurred at 10q (33%), 9p (22%), and 11q (22%) in primary melanomas, and at 10q (50%), 1p (44%), and 6q (39%) in epidermotropic metastatic melanomas. Primary lesions demonstrating LOH had concordant allelic loss in at least one locus in a corresponding epidermotropic metastatic melanoma in 83% (5/6) of cases. X-chromosome analysis showed nonrandom inactivation in 75% (3/4) and 71% (5/7) of primary melanoma and epidermotropic metastatic melanoma cases, respectively. Our LOH and X-chromosome inactivation analysis data suggest that epidermotropically metastatic melanomas are clonally related to their primary lesion in many cases. Our data also indicated that some cases diagnosed as epidermotropic metastatic melanoma might be divergent clones or new primaries rather than metastatic disease.

Genomic assessments of the frequent loss of heterozygosity region on 8p21.3∼p22 in head and neck squamous cell carcinoma

Most human cancers are characterized by genetic instabilities. Chromosomal aberrations include segments of allelic imbalance identifiable by loss of heterozygosity (LOH) at polymorphic loci, which may be used to implicate regions harboring tumor suppressor genes. Here we performed whole-genome LOH profiling on 41 human head and neck squamous cell carcinoma (HNSCC) cell lines. Several frequent LOH regions were identified on chromosomal arms 3p, 4p, 4q, 5q, 8p, 9p, 10p, 11q, and 17p. A genomic region of ∼7 Mb located at 8p21.3∼p22 exhibits the most frequent LOH (87.9%), which suggests that this region harbors one or more important tumor suppressor genes. Mitochondrial tumor suppressor gene 1 ( MTUS1) is a recently identified candidate tumor suppressor gene that resides in this region. Consistent downregulation in expression was observed in HNSCC for MTUS1 as measured by real-time quantitative reverse transcriptase-polymerase chain reaction. Sequence analysis of MTUS1 gene in HNSCC revealed several important sequence variants in the exon regions of this gene. Thus, our results suggest that MTUS1 is one of the candidate tumor suppressor genes for HNSCC residing at 8p21.3∼p22. The identification of these candidate genes will facilitate the understanding of tumorigenesis of HNSCC.