Concomitant Loss Of Heterozygosity Research Articles

Driver mutation zygosity is a critical factor in predicting clonal hematopoiesis transformation risk

Clonal hematopoiesis (CH) can be caused by either single gene mutations (eg point mutations in JAK2 causing CHIP) or mosaic chromosomal alterations (e.g., loss of heterozygosity at chromosome 9p). CH is associated with a significantly increased risk of hematologic malignancies. However, the absolute rate of transformation on an annualized basis is low. Improved prognostication of transformation risk is urgently needed for routine clinical practice. We hypothesized that the co-occurrence of CHIP and mCAs at the same locus (e.g., transforming a heterozygous JAK2 CHIP mutation into a homozygous mutation through concomitant loss of heterozygosity at chromosome 9) might have important prognostic implications for malignancy transformation risk. We tested this hypothesis using our discovery cohort, the UK Biobank (n = 451,180), and subsequently validated it in the BioVU cohort (n = 91,335). We find that individuals with a concurrent somatic mutation and mCA were at significantly increased risk of hematologic malignancy (for example, In BioVU cohort incidence of hematologic malignancies is higher in individuals with co-occurring JAK2 V617F and 9p CN-LOH; HR = 54.76, 95% CI = 33.92–88.41, P < 0.001 vs. JAK2 V617F alone; HR = 44.05, 95% CI = 35.06–55.35, P < 0.001). Currently, the ‘zygosity’ of the CHIP mutation is not routinely reported in clinical assays or considered in prognosticating CHIP transformation risk. Based on these observations, we propose that clinical reports should include ‘zygosity’ status of CHIP mutations and that future prognostication systems should take mutation ‘zygosity’ into account.

A Clinicopathological and Molecular Analysis of Fumarate Hydratase (FH)-deficient Renal Cell Carcinomas with Heterogeneous Loss of FH Expression

Aims. Fumarate hydratase (FH)-deficient renal cell carcinoma (RCC) is a rare aggressive renal malignancy associated with hereditary leiomyomatosis and RCC syndrome (HLRCC). Tumors exhibiting heterogeneous (ie, patchy) FH loss by immunohistochemistry have rarely been described, may be diagnostically challenging, and have never been the focus of a study. We aimed to investigate the FH mutational status of FH-deficient RCC with heterogeneous versus complete FH loss, to characterize additional genetic drivers, and to evaluate 2SC immunohistochemistry in this setting. Methods and Results. We studied FH-deficient RCC with heterogeneous (n = 3) and complete (n = 4) FH loss. Targeted next-generation sequencing (NGS) was performed on all tumors. No patients had a known history of HLRCC. All tumors had histological features within the morphologic spectrum described for FH-deficient RCC. All 7 tumors were immunoreactive for 2SC. Molecularly, all 7 tumors revealed multiple hits involving the FH locus resulting in complete loss of wild-type alleles. All tumors with heterogeneous FH loss harbored FH missense variants within domain 2 of the protein, and each had concomitant copy neutral loss of heterozygosity (CN-LOH). In complete FH loss tumors, FH alterations included splice variants with concomitant loss of heterozygosity (n = 2) and homozygous gene deletions (n = 2). Other non-recurrent alterations included biallelic alterations of TP53, NF2, SMAD4 and activation of PIK3CA. Conclusions. Our series highlights how heterogeneous FH loss (patchy positive staining) is an important staining pattern to recognize since it is compatible with a diagnosis of FH-deficient RCC and should prompt additional ancillary studies (confirmatory 2SC immunohistochemistry and/or molecular testing) and genetic evaluation.

Abstract LB-075: CDK12-mutated lethal prostate cancers: How hot are these tumors

Abstract Background: Genomic analyses describing the molecular landscape of primary prostate cancer (PCa) and metastatic castration-resistant prostate cancer (mCRPC) have identified recurrent CDK12 alterations in 2-4% of primary PCa and 4-11% of mCRPC. These aberrations, as well as high lymphocyte tumor infiltration, could be clinically relevant as putative biomarkers of response to immunotherapies. However, there are few studies looking into whether CDK12 biallelic mutant cases are immune ‘hot'. Here, we describe a cohort of patients with CDK12 aberration and studied the tumor infiltration of those cancers. Methods: Patients with mCRPC and available diagnostic archival and/or CRPC tumor samples were evaluated. Mutation analysis involved custom designed targeted sequencing on MiSeq sequencer. Loss of heterozygosity (LOH) was assessed for the CDK12 cases from exome sequencing using ASCAT, which include single copy lost or copy neutral LOH. Tumor infiltrating lymphocytes were assessed initially with CD3 IHC, using a deep learning-based AI analysis approach. We then subtyped the TILs using a multiplex IF approach, classifying lymphocytes using CD4, CD8 and FOXP3 positivity. Results: Overall 913 samples (between Feb/15 and Oct/19) were sequenced by targeted NGS, 42 patients presented with pathogenic alterations in CDK12 (4.6%), 27 had bi-allelic alterations, 14 mono-allelic, 1 likely biallelic. In these cases, we identified 39 frameshift alterations, 10 missense mutations mainly involving the kinase domain; with 5 having concomitant LOH. One case showed deep deletion of CDK12 and five presented with additional aberrations in other DNA repair related genes. CDK12 biallelic alterations were present in all 3 cases with both diagnostic and matched mCRPC samples available. We studied T cell infiltration in 100 (23 CDK12 biallelic alterations, 7 monoallelic, 70 controls) samples selected from within the initial targeted NGS cohort. Median intratumoral CD3+ cell density was significantly higher in CDK12 biallelic loss samples compared to matched controls in diagnostic biopsies (271.4 vs 104.683 cells/mm2, p=0.026). A similar trend was seen in mCRPC samples (142.130 vs 51.75 cells/mm2, p=0.36). Intratumoral CD4+ cell infiltration was again significantly higher in CDK12 biallelic loss samples compared to controls (98.864 vs 6.188 cells/mm2, p=0.014). Conclusions: In our analysis we show that a proportion of mCRPC patients harbor defects in CDK12, these are often alterations involving both alleles, likely present from the time of diagnosis. A majority of these CDK12 altered cancers has high CD3 infiltration compared to controls. We envision that these CDK12 aberrant ‘hot' tumors could represent a subset of mCRPC likely to respond to immune-checkpoint inhibition. Citation Format: Bora Gurel, Pasquale Rescigno, Wei Yuan, Rita Pereira, Mateus Crespo, Mattia Rediti, Ines Figueiredo, Maialen Barrero, Diletta Bianchini, Maria D. Fenor de la Maza, Khobe Chandran, Juliet Carmichael, Alec Paschalis, Adam Sharp, George Seed, Ruth Riisnaes, Claudia Bertan, Suzanne Carreira, Johann S. De Bono. CDK12-mutated lethal prostate cancers: How hot are these tumors [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 LB-075.

Abstract 2487: Functional characterization of the BRCA2 variant, K3326X

Abstract Individuals with germline mutations in the breast cancer susceptibility gene, BRCA2, have an approximate 70% risk of developing breast cancer, a 30% risk of developing ovarian cancer, a 20-fold increased risk of developing prostate cancer, and a 10-fold risk of developing pancreatic cancer during their lifetime. Loss of function germline mutations in BRCA2 affect its role in the homologous recombination (HR) DNA repair pathway leading to significant genomic instability. In addition to deleterious truncating mutations, several sequence variants, collectively called Variants of Unknown Significance (VUS), have been identified and are distributed along its length. One such variant is rs11571833, a nonsense mutation in the last exon (c.9976A&amp;gt;T, K3326X), resulting in the loss of the C-terminal 93 amino acid residues in BRCA2. This truncated variant has been previously described as a polymorphism that does not increase susceptibility to breast and ovarian cancers, and as a neutral unclassified variant non-deleterious to its function. However, recent studies identified K3326X to be enriched in breast cancer cases and to increase the risk for lung, pancreatic, ovarian, and upper aero-digestive tract cancers. Preliminary data, obtained from the Rutgers Cancer Institute of New Jersey breast cancer case-control study, identified K3326X enrichment in 1.25% of cases compared to 0.7% of controls. Several of the carriers had second primaries and displayed a trend toward increased number of family members diagnosed with colon cancer. Notably, K3326X was also identified in 1.38% (11 of 796) of our histologically-diverse cohort of genomically-profiled tumors that included cancers of the breast, ovarian/fallopian tube, lung, vulvar, cancer of unknown primary, and one breast cancer case having prolonged response to platinum-based therapy. Thus, K3326X may represent a functional loss of wild type BRCA2 function, as we observe concomitant loss of heterozygosity at this locus. In a preliminary study, we evaluated the K3326X variant, in vitro, in a functional DR-GFP-based reporter assay measuring HR. Our data reveal the BRCA2 K3326X variant to be impaired in the HR pathway indicating a loss of wild-type protein function. We will also evaluate cell viability of the K3326X variant in the presence of DNA damaging drugs like cisplatin, poly-ADP ribose polymerase inhibitors and mitomycin C. Future studies will also incorporate a retrospective evaluation of tumor specimens that have undergone comprehensive genomic profiling. These data would indicate that BRCA2 K3326X represents a functional hypomorphic variant that may have implications in therapeutic approaches and cancer risk evaluations across a spectrum of tumor types. Citation Format: Srilatha R. Simhadri, Sonia C. Dolfi, Atul Kulkarni, Bing Xia, Shridar Ganesan, Kim M. Hirshfield. Functional characterization of the BRCA2 variant, K3326X [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2487. doi:10.1158/1538-7445.AM2017-2487

The Transient Inactivation of the Master Cell Cycle Phosphatase Cdc14 Causes Genomic Instability in Diploid Cells of Saccharomyces cerevisiae.

Genomic instability is a common feature found in cancer cells . Accordingly, many tumor suppressor genes identified in familiar cancer syndromes are involved in the maintenance of the stability of the genome during every cell division and are commonly referred to as caretakers. Inactivating mutations and epigenetic silencing of caretakers are thought to be the most important mechanisms that explain cancer-related genome instability. However, little is known of whether transient inactivation of caretaker proteins could trigger genome instability and, if so, what types of instability would occur. In this work, we show that a brief and reversible inactivation, during just one cell cycle, of the key phosphatase Cdc14 in the model organism Saccharomyces cerevisiae is enough to result in diploid cells with multiple gross chromosomal rearrangements and changes in ploidy. Interestingly, we observed that such transient loss yields a characteristic fingerprint whereby trisomies are often found in small-sized chromosomes, and gross chromosome rearrangements, often associated with concomitant loss of heterozygosity, are detected mainly on the ribosomal DNA-bearing chromosome XII. Taking into account the key role of Cdc14 in preventing anaphase bridges, resetting replication origins, and controlling spindle dynamics in a well-defined window within anaphase, we speculate that the transient loss of Cdc14 activity causes cells to go through a single mitotic catastrophe with irreversible consequences for the genome stability of the progeny.

Abstract B117: Analysis of secondary mutations in BRCA1/2 genes as a mechanism of resistance to PM01183 in BRCA-mutation carriers.

Abstract Introduction: PM01183 is a modified analogue of trabectedine currently being evaluated in clinical trials, including germline BRCA mutation carriers. PM01183 binds to the DNA minor groove forming PM01183-DNA adducts that provoke DNA double strand (ds) breaks. The homologous recombination (HR) DNA repair pathway is the major mechanism involved in the repair of these DNA ds breaks, and BRCA mutated cells, which are HR deficient, show great sensitivity to these agents. In BRCA carriers, the somatic loss of the wild type copy of BRCA through loss-of-heterozygosity (LOH) is proposed to be an early event in tumorigenesis. Genomic characterization of somatic BRCA status can yield valuable information for therapeutic decisions and the development of new therapeutic strategies in BRCA carriers. Secondary mutations in BRCA1/2 genes restoring the gene and the HR function have been identified in up to 46% of cisplatin-resistant BRCA-associated ovarian tumors. Secondary mutations have also been identified in breast cancers with acquired resistance to olaparib. Based on the sensitivity of BRCA mutated cells to ds DNA-damaging, we hypothesized that BRCA genetic reversion could appear as one of the mechanism of resistance to PM01183. Methods: Patients with metastatic BRCA-associated breast cancer treated at our institution with PM01183 were enrolled in this study after signing the specifically designed, IRB-approved informed consent form. A blood sample was drawn prior to receiving treatment. Tumor biopsies were performed at baseline and at the time of progression to PM01183 from two patients exhibiting an initial clinical response. Whole exome sequencing (Illumina) was performed on DNA extracted from blood and from fresh-frozen paired tumor samples in order to analyze BRCA1/2 status, BRCA1/2 genetic reversion, and to detect novel genetic events acquired at disease progression. Findings: Sequencing of the BRCA genes in blood confirmed that one patient carried a germline mutation in BRCA1 (c5123C&amp;gt;A), with concomitant LOH found in both tumor samples. The second patient was confirmed a BRCA2-mutation carrier (9610 C&amp;gt;T), without LOH of BRCA2. Secondary mutations in BRCA1/2 genes were not identified in either tumor after progression to PM01183. Other de novo genetic events upon PM01183 progression are under review. Conclusions: Secondary functional-reverting mutations in BRCA1/2 genes have not been identified as a mechanism of resistance to PM01183 in any of the two patients analyzed. LOH of BRCA was not found in the metastatic disease of one patient, thus warranting further investigation of the mechanisms of tumorigenesis in BRCA tumors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B117. Citation Format: Cristina Cruz, Ana Vivancos, Ginevra Caratu, Yasir Ibrahim, Violeta Serra, Judith Balmana. Analysis of secondary mutations in BRCA1/2 genes as a mechanism of resistance to PM01183 in BRCA-mutation carriers. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B117.

Identification of SomaticVHLGene Mutations in Sporadic Head and Neck Paragangliomas in Association With Activation of the HIF-1α/miR-210 Signaling Pathway

Head and neck paragangliomas (HNPGLs) arise from parasympathetic paraganglias and 35% to 45% are hereditary caused by mutations in succinate dehydrogenase (SDH) genes. The connection between SDH and tumor development is unclear. The most accepted hypothesis proposes a central role for the pseudohypoxic (pHx) pathway activated by hypoxia-inducible factor (HIF). Paradoxically, we showed that activation of HIF in HNPGLs is restricted to a subset of HNPGLs lacking SDH mutations. These tumors overexpress HIF-1α protein and target genes and the HIF-inducible microRNA miR-210 (pHx-HNPGLs). The present study aimed at unraveling the SDH-independent mechanisms involved in the activation of HIF in HNPGLs. The VHL gene was analyzed in 53 tumors by gene sequencing, multiplex-ligation-dependent probe amplification, and quantitative PCR. The miR-210, HIF-1α, and CA9 levels were used as markers of the pHx gene signature. Meta-analysis of the transcriptome of pHx-HNPGLs was performed using the Oncomine platform. Assays in cells lacking functional pVHL and HIF-1α were performed to analyze the role of pVHL/HIF-1α on miR-210 expression. We identified, for the first time, somatic VHL mutations in HNPGLs. These were found in 2 of 4 pHx-HNPGLs with concomitant loss of heterozygosity in one of them; but not in non-pHx-HNPGLs. Meta-analysis of the transcriptome of pHx-HNPGLs revealed that these tumors are highly related to clear cell renal cell carcinoma. Cell-based assays showed that loss of pVHL lead to upregulation of miR-210 mainly via HIF-1α activation. VHL, involved in tumorigenesis of PGLs and clear cell renal cell carcinomas, may be an important player in the pathogenesis of sporadic HNPGLs via activation of an HIF-1α/miR-210 pHx pathway.

Genetic and epigenetic alterations of the LKB1 gene and their associations with mutations in TP53 and EGFR pathway genes in Korean non-small cell lung cancers

IntroductionLiver kinase 1 (LKB1) plays a critical barrier role in lung tumorigenesis by controlling initiation, differentiation and metastasis. We searched for genetic and epigenetic alterations of the LKB1 gene in Korean non-small cell lung cancers (NSCLCs) and correlated the results with clinicopathological features. We also investigated the relationship between genetic and epigenetic alterations of LKB1 and mutations in the TP53 gene and epidermal growth factor receptor (EGFR) pathway genes. MethodsA total of 159 NSCLCs were analyzed for loss of heterozygosity (LOH) at microsatellite loci D19S886, and D19S878. Mutations and methylation status of LKB1 were examined by direct sequencing and a methylation-specific polymerase chain reaction, respectively. ResultsA somatic mutation was found in one of the 159 tumors. LOH and promoter methylation was detected in 19.5% (31/159) and 13.2% (21/159) of the tumors, respectively. Four of the 159 tumors had concomitant LOH and methylation of LKB1. In total, 30.2% of the 159 NSCLCs harbored LKB1 LOH or promoter methylation, which were correlated with down-regulation of gene expression. LKB1 LOH was more frequent in males, smokers, and tumors with a TP53 mutation than in females, never-smokers, and tumors without a TP53 mutation, respectively. However, no significant correlation between LKB1 alterations and mutations in EGFR pathway genes was found. ConclusionThese results suggest that the prevalence of LKB1 genetic and epigenetic alterations in NSCLCs vary depending on patient ethnicity. Our results show that LKB1 alterations often occur simultaneously with mutations in EGFR pathway genes.

LOH 19q indicates shorter disease progression-free interval in low-grade oligodendrogliomas with EMP3 methylation

We previously described a cohort of gradeII oligodendroglioma (OII) patients, in whom the loss of heterozygosity (LOH) 19q was present in the subgroup at a higher risk of relapse. In this study, we evaluated the CpG methylation of the putative tumor suppressor epithelial membrane protein3 (EMP3, 19q13.3) gene promoter in the same OII cohort, to investigate whether a correlation could be found between EMP3 cytogenetic and epigenetic loss and higher risk of relapse. Twenty-three tumor samples from OII patients were collected over a period of 10years. Seventeen glioblastoma (GBM) samples (2 of which were relapses) were collected from 15patients. The EMP3, O6-methylguanine methyltransferase (MGMT) and cyclooxygenase2 (COX2) promoter methylation, evaluated by methylation-specific PCR, and the isocitrate dehydrogenase1 (IDH1) mutation, identified by sequencing, were compared between the OII and GBM histotypes. The EMP3 promoter methylation was correlated with the analysis of LOH19q, performed by microsatellite amplification, in OII patients. Disease progression-free interval was evaluated in the OII patients with the EMP3 methylation with either LOH19q or conserved chromosome 19 arms. The EMP3 and MGMT promoter methylation was more frequent in OII than in GBM patients, and the IDH1 mutation was absent in GBM. The COX2 promoter was unmethylated in both histotypes. Both LOH+/- 19q OII patients showed EMP3 hypermethylation. Concomitant LOH19q and EMP3 gene promoter methylation was observed in the OII patients at a higher risk of relapse. Our results suggest that a total (cytogenetic and epigenetic) functional loss of both EMP3 alleles accounts for the reduced disease progression-free interval in OII patients. Although the small sample size limits the strength of this study, our results support testing this hypothesis in larger cohorts of patients, considering the methylation of the EMP3 gene promoter together with LOH19q as an indication for treatment with adjuvant therapy abinitio in order to improve the overall survival of OII patients.

Concomitant loss of heterozygosity at the BRCA1 and FHIT genes as a prognostic factor in sporadic breast cancer

Previous studies have shown loss of heterozygosity (LOH) at the BRCA1 and FHIT genes in sporadic primary breast cancer. The aim of this study was to evaluate concomitant LOH at the BRCA1 and FHIT genes in sporadic breast cancer and investigate its influence on patient survival. Loss of heterozygosity was determined using microsatellite markers. The analysis on the informative cases ( n = 72) indicated LOH at both the BRCA1 and FHIT loci in 25 cases (35%), the absence of LOH at both loci in 23 cases (32%), and the presence of LOH at one of the loci in 24 cases (33%). The concomitant LOH was associated with poor prognostic factors, such as large tumors ( P = 0.01), axillary nodal involvement ( P < 0.01), histologic grade III ( P < 0.01), vascular invasion ( P = 0.01), and negative hormone receptor ( P = 0.02). After a median follow-up period of 48 months, the concomitant LOH group had the shortest survival ( P < 0.02 by log-rank test; P < 0.05 by Cox model; hazard ratio of 4.87), compared with patients without LOH. These data suggest that concomitant allelic losses of the BRCA1 and FHIT genes are associated with more aggressive breast tumors.

Homozygous loss of BHD causes early embryonic lethality and kidney tumor development with activation of mTORC1 and mTORC2.

Germline mutations in the BHD/FLCN tumor suppressor gene predispose patients to develop renal tumors in the hamartoma syndrome, Birt-Hogg-Dubé (BHD). BHD encodes folliculin, a protein with unknown function that may interact with the energy- and nutrient-sensing AMPK-mTOR signaling pathways. To clarify BHD function in the mouse, we generated a BHD knockout mouse model. BHD homozygous null (BHD(d/d)) mice displayed early embryonic lethality at E5.5-E6.5, showing defects in the visceral endoderm. BHD heterozygous knockout (BHDd(/+)) mice appeared normal at birth but developed kidney cysts and solid tumors as they aged (median kidney-lesion-free survival = 23 months, median tumor-free survival = 25 months). As observed in human BHD kidney tumors, three different histologic types of kidney tumors developed in BHD(d/+) mice including oncocytic hybrid, oncocytoma, and clear cell with concomitant loss of heterozygosity (LOH), supporting a tumor suppressor function for BHD in the mouse. The PI3K-AKT pathway was activated in both human BHD renal tumors and kidney tumors in BHD(d/+) mice. Interestingly, total AKT protein was elevated in kidney tumors compared to normal kidney tissue, but without increased levels of AKT mRNA, suggesting that AKT may be regulated by folliculin through post translational or post-transcriptional modification. Finally, BHD inactivation led to both mTORC1 and mTORC2 activation in kidney tumors from BHD(d/+) mice and human BHD patients. These data support a role for PI3K-AKT pathway activation in kidney tumor formation caused by loss of BHD and suggest that inhibitors of both mTORC1 and mTORC2 may be effective as potential therapeutic agents for BHD-associated kidney cancer.

Multiple schwannomas: report of two cases

In this paper authors present two cases of multiple schwannomas without the features of neurofibromatosis (NF). The authors retrospectively reviewed the hospital charts, radiology films, operative notes and pathology slides of these two patients. There was no family history of neurofibromatosis. The two patients had contrast enhanced MRI, which was negative for vestibular schwannomas. Both underwent surgical excision of symptomatic lesions. Histopathology confirmed these lesions as schwannomas. Molecular genetic analysis in case 1 demonstrated two distinct mutations of the NF2 gene in two different schwannomas, with concomitant loss of heterozygosity in both tumours. In contrast peripheral blood lymphocytes did not reveal mutations of NF2. The authors recommend surgery for symptomatic lesions. Asymptomatic tumours can be monitored. Regular follow up is essential as they may develop fresh lesions at any time. The relevant literature is discussed.

Intratumoral heterogeneity in microsatellite alterations in BRCA1 and PTEN regions in sporadic colorectal cancer.

Chromosome regions 17q21 (BRCA1) and 10q23 (PTEN) have been found deleted in colorectal cancer. We studied the frequency of loss of heterozygosity (LOH) in these 2 regions in 214 patients with only 1 sample per tumor and in 100 patients with several samples per tumor. Three microsatellite markers of each region were used for the LOH test. The polymerase chain reaction product was electrophoresed in 8% polyacrylamide gels, and band intensity was shown by silver staining. The proportions of LOH in the two regions were 38.4% for 17q21 and 30.8% for 10q23 in the group of 214 and were 47.7% for 17q21 and 34.7% for 10q23 in the group of 100. We found a high correlation between the LOH in both regions (P <.001), where 81% of LOH in 10q23 region was matched by concomitant LOH in 17q21. In the group of tumors with several samples (group of 100), 39% and 68% did not present LOH in the 17q21 and 10q23 regions, respectively, in all of their tumor samples. However, in the 20 patients with LOH in both regions in the group of 100 (several samples per tumor), all samples with LOH in 10q23 also had LOH in 17q21, whereas not all samples with LOH in 17q21 had LOH in 10q23. These results show that colorectal cancer is highly heterogeneous, at least for these tumors markers, and suggest a sequential acquisition pattern of these anomalies during tumor growth, in which changes in 17q21 could occur before those in 10q23.

Hypermethylation of the tumor suppressor gene RASSFIA and frequent concomitant loss of heterozygosity at 3p21 in cervical cancers

Loss of heterozygosity (LOH) at chromosome 3p21 is frequent in cervical cancers. The candidate tumor suppressor gene, RASSF1A located at 3p21.3, is found to be inactivated in several major human cancers, implicating its significance in carcinogenesis. We aimed to investigate the status of RASSF1A in cervical cancers. The mutation and methylation status of RASSF1A were analysed in 4 cervical cancer cell lines, 50 primary cervical cancers including 33 squamous cell carcinoma (SCC), 17 adenocarcinoma (AC) and 11 normal controls. The primary cancer samples were also detected for LOH at 3p21 and human papillomavirus (HPV). Hypermethylation of RASSF1A was detected in 30% of SCC, 12% of AC and in 1 of the 4 cancer cell lines but was absent in all normal cases. Methylation of the cancer cell line was associated with loss of gene expression, which was restored by demethylation. About 67% (8 of 12) of hypermethylated primary cancers showed concomitant LOH at 3p21. No somatic mutation was found in all primary cancer samples or cell lines but 2 cases showed germline polymorphism at codon 133. Oncogenic HPV DNAs were found in most cancer samples. No correlation was detected between RASSF1A-hypermethylation or LOH at 3p21 and age of patient, HPV genotype, tumor grade and stage. Hypermethylation of RASSF1A occurs in a subset of cervical cancers, among which concomitant LOH at 3p21 is common. The results supported that RASSF1A may be one of the cervical cancer-related tumor suppressor genes located at 3p21 regions.

Detection of loss of heterozygosity at RAD51, RAD52, RAD54 and BRCA1 and BRCA2 loci in breast cancer: pathological correlations.

Loss of heterozygosity (LOH) in loci of the 15q15.1, 12p13, 1p32, 17q21 and 13q12–13 regions may collaborate in the inactivation of RAD51, RAD52, RAD54, BRCA1, BRCA2 and possibly other genes implicated in the repair of double-stranded DNA and in DNA recombination. We investigate allelic losses in microsatellites of the RAD51, RAD52, RAD54, BRCA1 and BRCA2 regions, and their correlations with nine pathologic parameters in 127 breast carcinomas. The LOH analysis was performed by amplifying DNA by PCR, using 15 markers of the 15q15.1, 12p13.3, 1p32, 17q21 and 13q12–13 regions. LOH was found in the RAD51 region in 32% of tumours, in the RAD52 region in 16%, in RAD54 in 20% and in the BRCA1 and BRCA2 regions in 49% and 44% respectively. Significant correlations between one or more regions with concomitant LOH and pathologic parameters were observed with respect to age (P = 0.008), oestrogen receptor content (P = 0.03), progesterone receptors (P = 0.003), higher grade (P = 0.001), more advanced stage (P = 0.004) and peritumoural vessel involvement (P < 0.0001). The number of cases in which LOH was observed simultaneously in two or more regions was always higher than expected on the basis of their statistical probability, and curiously, the three patients with LOH at five regions concomitantly were under the age of 30 years. These results suggest that LOH at these regions could be related to breast cancer, and probably to a poor tumour prognosis. © 1999 Cancer Research Campaign

P16MTS1/CDK4I mutations and concomitant loss of heterozygosity at 9p21-23 are frequent events in squamous cell carcinoma of the larynx.

We have examined the presence of p16MTS1/CDK4I gene deletions, mutations and methylation status, and 9p21-23 deletions in a series of 46 squamous cell carcinomas of the larynx and paired normal mucosa previously characterized for cyclin D1 gene amplification and overexpression. pRb expression was also examined by immunohistochemistry. p16MTS1/CDK4I mutations were found in 10/46 (22%) carcinomas and hypermethylation in 2/31 (7%). Loss of heterozygosity at 9p21-23 was found in 24 out of 42 (57%) carcinomas examined. All p16MTS1/CDK4I mutated cases and the two hypermethylated carcinomas showed 9p21-23 loss of heterozygosity. The loss of heterozygosity correlated with advanced local invasion (P=0.0045), lymph node metastases (P=0.0326), stage IV of the tumors (P=0.0058), and existence of cyclin D1 amplification/overexpression (P < 0.03). Only one out of 37 carcinomas was negative for pRb expression. No alterations in p16 gene or 9p21-23 loss of heterozygosity were detected in this case. These findings indicate that p16MTS1/CDK4I is frequently inactivated by gene mutation, hypermethylation, and allelic deletions in a significant subset of squamous cell carcinomas of larynx. Since 9p21-23 loss of heterozygosity was more frequently detected than p16MTS1/CDK4I mutations, and mutated carcinomas invariably had loss of heterozygosity, allelic losses probably precede the p16MTS1/CDK4I mutations. Their association with cyclin D1 deregulation in advanced carcinomas could indicate a possible cooperative effect in the progression of these neoplasms.

Deletional, mutational, and methylation analyses of CDKN2 (p16/MTS1) in primary and metastatic prostate cancer

The tumor suppressor gene CDKN2 (p16/MTS1) resides on chromosome 9p21 and encodes a 16 kDa inhibitor of the cyclin-dependent kinases. Inactivation of CDKN2 by homozygous deletion, point mutation, and recently described aberrant methylation in the 5' promoter region may increase progression through the cell cycle in tumors. In this study, we examine the CDKN2 gene for the presence of inactivating alterations in human prostate cancer. Sequence analysis of cell lines revealed no mutation in LNCaP, PC3, and TSU-PR1 and a missense mutation, GAC-->TAC (asp to tyr), in exon 2 of the DU145 cell line at codon 76. No mutations were identified in three primary prostate cancers or in seven lymph node metastases. Loss of heterozygosity (LOH) was analyzed by analysis of microsatellite markers in the vicinity of the CDKN2 gene. LOH was detected in 12 (20%) of 60 primary tumors at one or more loci and in 13 (46%) of 28 metastases. Methylation analysis of the CpG-rich promoter region revealed a dense methylation of CDKN2 in cell lines PC3, PPC1, and TSU-PR1, and this was found to correlate with a lack of mRNA expression by reverse transcription-polymerase chain reaction. A demethylating agent, 5-aza-2'-deoxycytidine, induced reexpression when cells were exposed in vitro. DU145 and LNCaP expressed the CDKN2 transcript and were unmethylated in the promoter region. Three of twenty-four (13%) primary prostate cancers and 1 of 12 metastatic tumors demonstrated promoter methylation. No normal prostate tissues were methylated at the CDKN2 gene promoter. One tumor was found to contain concomitant LOH and promoter methylation indicative of biallelic inactivation. A comprehensive analysis of CDKN2 in prostate cancer reveals that point mutations are infrequent, but gene deletion and methylation combine to inactivate CDKN2 in a subset of tumors. Moreover, alterations in this gene may represent a late event in prostate cancer progression.

Analysis of location and integrity of the human PITSLRE (p58(cdc2L1)) genes in neuroblastoma cell genomes

The family of PITSLRE kinase genes, located in chromosome 1p36, has recently been associated with neuroblastoma tumorigenesis. In order to evaluate the role of these genes as putative tumor suppressor genes, we have analyzed the integrity of the coding region in primary tumors and its location relative to a neuroblastoma consensus deletion. A subset of aggressive neuroblastoma tumors with allelic loss of different parts of chromosome 1p were investigated. Single-strand conformation polymorphism (SSCP), heteroduplex (HD) and sequencing analysis of tumor DNA did not reveal any significant changes in the coding region. In particular, a primary tumor with an interstitial allelic deletion in 1p36 did not reveal concomitant loss of heterozygosity of the PITSLRE gene region when analyzed with a C/T DNA sequence polymorphism in exon 5 of PITSLRE1. FISH analysis on neuroblastoma cell lines with small interstitial deletions and with a balanced translocation in 1p36 revealed that the PITSLRE gene cluster was localized distal to the neuroblastoma consensus deletion. against an involvement of the PITSLRE genes in neuroblastoma tumorigenesis.

Loss of heterozygosity on chromosomes 17 and 18 in breast carcinoma: two additional regions identified.

The loss of heterozygosity (LOH) at specific regions of the human genome in tumor DNA is recognized as evidence for a tumor-suppressor gene located within the corresponding region of the homologous chromosome. Restriction fragment length polymorphism analysis of a panel of primary human breast tumor DNAs has led to the identification of two additional regions on chromosomes 17q and 18q that frequently are affected by LOH. Deletions of each of these regions have a significant correlation with clinical parameters that are associated with aggressive breast carcinomas. Previous restriction fragment length polymorphism analysis of this panel of tumors has uncovered several other frequently occurring mutations. LOH on chromosome 18q frequently occurs in tumors with concomitant LOH of loci on chromosomes 17p and 11p. Similarly, tumors having LOH on 17q also have LOH on chromosomes 1p and 3p. This suggests that certain combinations of mutations may collaborate in the development and malignant progression of breast carcinomas.

Genetic structure and heterozygosity in the kuru region, Eastern Highlands of New Guinea

Genetic structure of 20 of the 37 linguistic groups in the Eastern Highlands of New Guinea including the kuru region is analyzed using information on blood groups and serum protein polymorphisms. The average individual is heterozygous at 28.6% of loci and the average number of alleles per locus is 1.234. Coefficients of kinship for linguistic groups range from 0.005 for the sweet potato cultivating North Fore to 0.075 for the isolated Pawaians whose dietary staple is sago and who depend more on hunting and gathering. As one selects linguistic groups with smaller and smaller population size and increasing isolation, one finds that kinship coefficients rise as much as tenfold, but there is no concomitant loss of heterozygosity or trend toward fixation of alleles. Genetic relationships established by genetic distance trees and by principal components analysis are comparable and are consistent with other anthropological observations.