Genome-wide detection of LOH in prostate cancer using human SNP microarray technology

Loss of heterozygosity (LOH) of chromosomal regions is crucial in tumor progression. In this study we assessed the potential of the Affymetrix GeneChip HuSNP mapping assay for detecting genome-wide LOH in prostate tumors. We analyzed two human prostate cell lines, P69SV40Tag (P69) and its tumorigenic subline, M12, and 11 prostate cancer cases. The M12 cells showed LOH in chromosomes 3p12.1-p22.1, 11q22.1-q24.2, 19p13.12, and 19q13.42. All of the prostate cases with informative single-nucleotide polymorphism (SNP) markers showed LOH in 1p31.2, 10q11.21, 12p13.1, 16q23.1-q23.2, 17p13.3, 17q21.31, and 21q21.2. Additionally, a high percentage of cases showed LOH at 6p25.1-p25.3 (75%), 8p22-p23.2, and 10q22.1 (70%). Several tumor suppressor genes (TSGs) have been mapped in these loci. These results demonstrate that the HuSNP mapping assay can serve as an alternative to comparative genomic hybridization for assessing genome-wide LOH and can identify chromosomal regions harboring candidate TSGs implicated in prostate cancer.     

Asbestos,chromosomal deletions,and tumor suppressor gene alterations in human malignant mesothelioma

Exposure to the carcinogen asbestos is considered to be a major factor contributing to the development of most malignant mesotheliomas (MMs). We highlight the role of asbestos in MM and summarize cytogenetic and molecular genetic findings in this malignancy. The accumulation of numerous clonal chromosomal deletions in most MMs suggests a multistep process of tumorigenesis, characterized by the loss and/or inactivation of multiple tumor suppressor genes (TSGs). Cytogenetic and loss of heterozygosity (LOH) analyses of MMs have demonstrated frequent deletions of specific sites within chromosome arms 1p, 3p, 6q, 9p, 13q, 15q, and 22q. Furthermore, TSGs within two of these regions, i.e., p16/CDKN2A-p14^ARF at 9p21 and NF2 at 22q12, are frequently altered in MMs. Homozygous deletion appears to be the major mechanism affecting p16/CDKN2A-p14^ARF, whereas inactivating mutations coupled with allelic loss occur at the NF2 locus. Finally, recent studies have demonstrated the presence and expression of simian virus 40 (SV40) in many MMs. SV40 large T antigen has been shown to inactivate the TSG products Rb and p53, suggesting the possibility that asbestos and SV40 could act as cocarcinogens in MM. The frequent occurrence of homozygous deletions of p16/CDKN2A-p14^ARF and the ability of SV40 Tag to bind TSG products suggest that perturbations of both Rb- and p53-dependent growth-regulatory pathways are critically involved in the pathogenesis of MM. J. Cell. Physiol. 180:150–157, 1999. © 1999 Wiley-Liss, Inc.     

Genetic aberration in primary hepatocellular carcinoma：correlation between p53 gene mutation and loss—of—heterozygosity on chromosome 16q21—q23 and 9p21—p23

To elucidate the molecular pathology underlying the development of hepatocellular carcinoma (HCC),we used 41 highly polymorphic microsatellite markers to examine 55 HCC and corresponding non-tumor liver tissues on chromosome 9,16 and 17.Loss-of-heterozygosity(LOH) is observed with high frequency on chromosomal region 17p13(36k/55,65%),9q21-p23(28/55,51%),16q21-23(27/55,49%) in tumors.Meanwhile,microsatellite instability is rarely found in these microsatellite loci.Direct sequencing was performed to detect the tentative mutation of tumor wuppressor genes in these regions:p53,MTS1/p16,and CDH1/E-cadherin.Wihin exon 5-9 of p53 gene,14 out of 55 HCC specimens(24%) have somatic mutations,and nucleotide deletion of this gene is reported in HCC for the first time.Mutation in MTS1/p16 is found only in one tumor case.We do not find mutations in CDH1/E-cadherin.Furthermore,a statistically significant correlation is present between p53 gene mutation and loss of chromosome region 16q21-q23 and 9p21-p23,which indicates that synergism between p53 inactivation and deletion of 16q21-q23 and 9p21-p23 may play a role in the pathogenesis of HCC.     

Identification of novel subregions of LOH in gastric cancer and analysis of the HIC1 and TOB1 tumor suppressor genes in these subregions

Previously, we identified 3 overlapping regions showing loss of heterozygosity (LOH, R(1)-R(3) from 11 to 30 cM) on chromosome 17 in 45 primary gastric cancers (GCs). The data indicated the presence of tumor suppressor genes (TSGs) on chromosome 17 involved in GC. Among the putative TSGs in these regions, HIC1 (in SR(1)) and TOB1 (in SR(3)) remain to be examined in GC. By immunohistochemistry (IHC), methylation-specific PCR (MSP) and western blot, we evaluated the expression and regulation status for HIC1 and TOB1 protein in GC. We narrowed down the deletion intervals on chromosome 17 and defined five smaller LOH subregions, SR(1)-SR(5) (0.54 to 3.42 cM), in GC. We found that HIC1 had downregulated expression in 86% (91/106) and was methylated in 87% (26/30) of primary GCs. Of the primary GCs showing downregulation of HIC1 protein, 75% (18/24) had methylated HIC1 gene. TOB1 was either absent or expressed at reduced levels in 75% (73/97) of the GC samples. In addition, a general reduction was found in total and the ratio of unphosphorylated to phosphorylated TOB1 protein levels in the differentiated GC cell lines. Further analysis revealed significant simultaneous downregulation of both HIC1 and TOB1 protein in GC tissue microarray samples (67%, 52/78) and in primary GCs (65%, 11/17). These results indicate that silencing of HIC1 and TOB1 expression is a common occurrence in GC and may contribute to the development and progression of the disease.     

Genetic and Epigenetic Alterations in Primary–Progressive Paired Oligodendroglial Tumors

The aim of the present study was to identify genetic and epigenetic alterations involved in the progression of oligodendroglial tumors. We characterized 21 paired, World Health Organization (WHO) grade II and III oligodendroglial tumors from patients who received craniotomies for the partial or complete resection of primary and secondary oligodendroglial tumors. Tumor DNA was analyzed for alterations in selected genetic loci (1p36, 9p22, 10q23–24, 17p13, 19q13, 22q12), isocitrate dehydrogenase 1 (IDH1), isocitrate dehydrogenase 2 (IDH2) and the CpG island methylation status of critical tumor-related genes (MGMT, P16, DAPK, PTEN, RASSF1A, Rb1). Alterations of these markers were common early in the tumorigenesis. In the primary tumors we identified 12 patients (57.1%) with 1p36 deletions, 17 (81.0%) with 19q13 deletions, 9 (42.9%) with 1p36/19q13 codeletions, 11 (52.3%) with 9p22 deletions, and 12 (57.1%) with IDH1 mutation. Epigenetic analysis detected promoter methylation of the MGMT, P16, DAPK, PTEN, RASSF1A, and Rb1 genes in 38.1%, 19.0%, 38.1%, 33.3%, 66.7%, and 14.3% of primary tumors, respectively. After progression, additional losses of 1p, 9p, 10q, 17p, 19q and 22q were observed in 3 (14.3%), 1 (4.8%), 3 (14.3%), 2 (9.5%), 1 (4.8%) and 3 (14.3%) cases, respectively. Additional methylations of the MGMT, P16, DAPK, PTEN, RASSF1A, and RB1 promoters was observed in 4 (19.0%), 2 (9.5%), 0 (0%), 6 (28.6%), 2(9.5%) and 3 (14.3%) cases, respectively. The status of IDH1 mutation remained unchanged in all tumors after progression. The primary tumors of three patients with subsequent progression to high-grade astrocytomas, all had 9p deletion, intact 1p, intact 10q and unmethylated MGMT. Whether this may represent a molecular signature of patients at-risk for the development of aggressive astrocytomas needs further investigation.     

Spinophilin acts as a tumor suppressor by regulating Rb phosphorylation

The scaffold protein Spinophilin (SPN) is a regulatory subunit of phosphatase1a located at 17q21.33. This region is frequently associated with microsatellite instability and LOH containing a relatively high density of known tumor suppressor genes, including BRCA1. Several linkage studies have suggested the existence of an unknown tumor suppressor gene distal to BRCA1. Spn may be this gene, but the mechanism through which this gene makes its contribution to cancer has not been described. In this study, we aimed to determine how loss of Spn may contribute to tumorigenesis. We explored the contribution of SPN to PP1a-mediated Rb regulation. We found that the loss of Spn downregulated PPP1CA and PP1a activity, resulting in a high level of phosphorylated Rb and increased ARF and p53 activity. However, in the absence of p53, reduced levels of SPN enhanced the tumorigenic potential of the cells. Furthermore, the ectopic expression of SPN in human tumor cells greatly reduced cell growth. Taken together, our results demonstrate that the loss of Spn induces a proliferative response by increasing Rb phosphorylation, which, in turn, activates p53, thereby neutralizing the proliferative response. We suggest that Spn may be the tumor suppressor gene located at 17q21.33 acting through Rb regulation.     

Molecular genetic analysis of the intratumoral clonal heterogeneity of colorectal adenocarcinomas

Molecular genetic analysis of allelic deletions from the loci containing the tumor suppressor genes p16, p15, p19 (9p21), RB1 (13p14), PTEN (10q23), and TP53 (17p13); microsatellite instability; and activating mutations of K-RAS (codons 12 and 13) was performed in four different segments of sporadic colorectal cancer (CRC) in 11 patients. Intratumoral genetic heterogenity was detected in 9 out of 11 (81%) colorectal adenocarcinomas and was morphologically validated. Analysis of different segments of one tumor reported that not only intratumoral heterogeneity, but also the order of the appearance and distribution of molecular anomalies during tumorigenesis in sporadic CRC. K-RAS point mutations and anomalies of the p16-RB1-cyclin D pathway were assumed to occur prior to microsatellite instability and PTEN deletions during tumor progression.     

Candidate Luminal B Breast Cancer Genes Identified by Genome,Gene Expression and DNA Methylation Profiling

Breast cancers (BCs) of the luminal B subtype are estrogen receptor-positive (ER+), highly proliferative, resistant to standard therapies and have a poor prognosis. To better understand this subtype we compared DNA copy number aberrations (CNAs), DNA promoter methylation, gene expression profiles, and somatic mutations in nine selected genes, in 32 luminal B tumors with those observed in 156 BCs of the other molecular subtypes. Frequent CNAs included 8p11-p12 and 11q13.1-q13.2 amplifications, 7q11.22-q34, 8q21.12-q24.23, 12p12.3-p13.1, 12q13.11-q24.11, 14q21.1-q23.1, 17q11.1-q25.1, 20q11.23-q13.33 gains and 6q14.1-q24.2, 9p21.3-p24,3, 9q21.2, 18p11.31-p11.32 losses. A total of 237 and 101 luminal B-specific candidate oncogenes and tumor suppressor genes (TSGs) presented a deregulated expression in relation with their CNAs, including 11 genes previously reported associated with endocrine resistance. Interestingly, 88% of the potential TSGs are located within chromosome arm 6q, and seven candidate oncogenes are potential therapeutic targets. A total of 100 candidate oncogenes were validated in a public series of 5,765 BCs and the overexpression of 67 of these was associated with poor survival in luminal tumors. Twenty-four genes presented a deregulated expression in relation with a high DNA methylation level. FOXO3, PIK3CA and TP53 were the most frequent mutated genes among the nine tested. In a meta-analysis of next-generation sequencing data in 875 BCs, KCNB2 mutations were associated with luminal B cases while candidate TSGs MDN1 (6q15) and UTRN (6q24), were mutated in this subtype. In conclusion, we have reported luminal B candidate genes that may play a role in the development and/or hormone resistance of this aggressive subtype.     

The onset of p53 loss of heterozygosity is differentially induced in various stem cell types and may involve the loss of either allele

p53 loss of heterozygosity (p53LOH) is frequently observed in Li-Fraumeni syndrome (LFS) patients who carry a mutant (Mut) p53 germ-line mutation. Here, we focused on elucidating the link between p53LOH and tumor development in stem cells (SCs). Although adult mesenchymal stem cells (MSCs) robustly underwent p53LOH, p53LOH in induced embryonic pluripotent stem cells (iPSCs) was significantly attenuated. Only SCs that underwent p53LOH induced malignant tumors in mice. These results may explain why LFS patients develop normally, yet acquire tumors in adulthood. Surprisingly, an analysis of single-cell sub-clones of iPSCs, MSCs and ex vivo bone marrow (BM) progenitors revealed that p53LOH is a bi-directional process, which may result in either the loss of wild-type (WT) or Mut p53 allele. Interestingly, most BM progenitors underwent Mutp53LOH. Our results suggest that the bi-directional p53LOH process may function as a cell-fate checkpoint. The loss of Mutp53 may be regarded as a DNA repair event leading to genome stability. Indeed, gene expression analysis of the p53LOH process revealed upregulation of a specific chromatin remodeler and a burst of DNA repair genes. However, in the case of loss of WTp53, cells are endowed with uncontrolled growth that promotes cancer.Heterozygosity, caused by a mutation in a single allele of a tumor suppressor gene (TSG), is one of the first steps in malignant transformation.¹ Often, TSGs undergo loss of the wild-type (WT) allele, designated as loss of heterozygosity (LOH).^{2, 3, 4} Patients with the rare cancer predisposition Li-Fraumeni syndrome (LFS), carrying germ-line heterozygous p53 mutations,⁵ apparently exhibit normal development yet later in adult life develop a wide spectrum of tumors; predominantly sarcomas,^{6, 7, 8} where 40–60% of tumors exhibit WT p53 loss of heterozygosity (p53LOH).⁸Giving that cancer development could be associated with stemness deregulation challenges, the notion that the occurrence of p53LOH in stem cells (SCs) may contribute to the emergence of cancer SCs. Genomic fidelity is a hallmark of SCs.⁹ The genome of embryonic stem cells (ESCs) is extremely stable, whereas adult stem cells (ASCs) exhibit a less stable genome.¹⁰ Genetic deregulation in ASCs was shown to be associated with tumor development.^{11, 12, 13} Mesenchymal stem cells (MSCs) that acquire mutations in oncogenes/TSGs such as p53 may function as tumor-initiating cells leading to de-novo sarcomagensis.^{14, 15, 16, 17} Furthermore, MSCs isolated from young mice, aged in culture acquired clinically relevant p53 mutations.¹⁸ In all, these findings suggest a link between p53 inactivation in SCs and tumorigenesis.Although induced pluripotent stem cells (iPSCs) seemed to represent ESCs,^{19, 20} several studies questioned the assumption that iPSCs are as genomically stable as ESCs.^{21, 22, 23, 24} p53 was found to have a major role in the generation of iPSCs both in attenuating reprogramming and controlling the quality of the reprogrammed cells.^{25, 26} An additional role of p53 during reprogramming may be an indirect effect on cell proliferation²⁷ and on the restriction of mesenchymal–epithelial transition during the early phases of reprogramming.²⁸ Importantly, Mutp53 cells exhibiting a fully reprogrammed iPSC phenotype in vitro, form malignant tumors in vivo, instead of the benign teratomas induced by the WTp53-iPSCs.²⁵ As p53 is the guardian of the genome, it is important to investigate how p53LOH would affect genome stability and tumorigenicity of iPSCs.The availability of in vitro SC p53LOH models (iPSCs, MSCs) can help decipher the role of p53LOH in cancer initiation. Indeed, the incidence of p53LOH was found to be extremely different between these SCs. Surprisingly, we found that reprograming of heterozygous p53 (HZp53) fibroblasts, which frequently undergo p53LOH, gave rise to iPSC clones, most of which retained their HZp53 status and exhibited features of normal WTp53-iPSCs. However, p53LOH process is robust in MSCs. Interestingly, single-cell sub-cloning of iPSCs, MSCs and ex vivo bone marrow (BM) progenitors revealed that, in addition to the loss of the WTp53, loss of the Mutp53 allele also takes place. Of note, this bi-directional p53LOH occurred in an age-dependent manner linking LOH to aging and tumorigenesis. Surprisingly, most of the p53LOH events in BM progenitors preferred the loss of the Mutp53 allele. Taken together, our results of a bi-directional p53LOH process, accompanied by a burst of DNA repair pathways, may suggest that p53LOH can be regarded as a DNA repair event. In the case of a DNA repair-orientated productive LOH process, where the Mutp53 allele is lost, cells are rescued of tumorigenesis. However, when the WTp53 allele is lost, cells become prone to tumor initiation.     

Loss of heterozygosity and microsatellite instability in tumor-associated stromal cells and tumor epithelium of prostate cancer

In the past decade, intense studies of the tumor microenvironment yielded ample data testifying to the critical role of stroma in carcinogenesis. Genetic lesions accumulate not only in the tumor epithelium, but also in tumor-associated fibroblasts. The epithelial and stromal components of prostate cancer (PC) and prostatic intraepithelial neoplasia (PIN) were isolated by laser capture microdissection and subjected to microsatellite analysis of chromosome regions 8p22, 13q14, and 16q23. The frequency of allele alterations in the epithelium was 48% for 8p22, 72% for 13q14, and 37% for 16q23. Slightly higher frequencies of the loss of heterozygosity (LOH) and microsatellite instability in these loci were observed in tumor-associated stroma. Molecular alterations were also found in both epithelial (16–27%) and stromal (8–22%) components in PIN. LOH on chromosomes 16 and 13 in the epithelium was significantly associated with the Gleason score, PC stage, and metastasis into regional lymph nodes. Thus, multiple genetic aberrations occur in the stromal component of PC as frequently as in the tumor epithelium.     

Genome-Wide Analysis of Loss of Heterozygosity in Breast Infiltrating Ductal Carcinoma Distant Normal Tissue Highlights Arm Specific Enrichment and Expansion across Tumor Stages

Studies have shown concurrent loss of heterozygosity (LOH) in breast infiltrating ductal carcinoma (IDC) and adjacent or distant normal tissue. However, the overall extent of LOH in normal tissue and their significance to tumorigenesis remain unknown, as existing studies are largely based on selected microsatellite markers. Here we present the first autosome-wide study of LOH in IDC and distant normal tissue using informative loci deduced from SNP array-based and sequencing-based techniques. We show a consistently high LOH concurrence rate in IDC (mean = 24%) and distant normal tissue (m = 54%), suggesting for most patients (31/33) histologically normal tissue contains genomic instability that can be a potential marker of increased IDC risk. Concurrent LOH is more frequent in fragile site related genes like WWOX (9/31), NTRK2 (10/31), and FHIT (7/31) than traditional genetic markers like BRCA1 (0/23), BRCA2 (2/29) and TP53 (1/13). Analysis at arm level shows distant normal tissue has low level but non-random enrichment of LOH (topped by 8p and 16q) significantly correlated with matched IDC (Pearson r = 0.66, p = 3.5E-6) (topped by 8p, 11q, 13q, 16q, 17p, and 17q). The arm-specific LOH enrichment was independently observed in tumor samples from 548 IDC patients when stratified by tumor size based T stages. Fine LOH structure from sequencing data indicates LOH in low order tissues non-randomly overlap (∼67%) with LOH that usually has longer tract length (the length of genomic region affected by LOH) in high order tissues. The consistent observations from multiple datasets suggest progressive LOH in the development of IDC potentially through arm-specific pile up effect with discernible signature in normal tissue. Our finding also suggests that LOH detected in IDC by comparing to paired adjacent or distant normal tissue are more likely underestimated.     

Immunogenomics Analysis Reveals that TP53 Mutations Inhibit Tumor Immunity in Gastric Cancer

Although immunotherapy continues to demonstrate efficacy in a variety of refractory cancers, currently, no any immunotherapeutic strategy is clinically used for gastric cancer (GC) except its microsatellite instable subtype. Thus, it is important to identify molecular biomarkers for predicting the responders to GC immunotherapy. TP53 mutations frequently occur in GC and are associated with unfavorable clinical outcomes in GC. We performed a comprehensive characterization of the associations between TP53 mutations and immune activities in GC based on two large-scale GC cancer genomics data. We compared expression and enrichment levels of 787 immune-related genes and 23 immune gene-sets among TP53-mutated GCs, TP53‐wildtype GCs, and normal tissue, and explored the correlations between p53-mediated pathways and immune activities in GC. Strikingly, almost all analyzed immune gene-sets were significantly downregulated in enrichment levels in TP53-mutated GCs compared to TP53‐wildtype GCs. These less active immune pathways and cell types in TP53-mutated GCs included 15 immune cell types and function, tumor-infiltrating lymphocytes, regulatory T cells, immune checkpoint, cytokine and cytokine receptor, human leukocyte antigen, pro‐inflammatory, and parainflammation. Moreover, we identified a number of p53-mediated pathways and proteins that were significantly associated with immune activities in GC. Furthermore, we demonstrated that the TP53 mutation itself could result in the depressed immune activities in GC and other cancer types. We revealed that chromosomal instability was an important mechanism for the depressed tumor immunity in TP53-mutated cancers. Finally, we showed that immune cell infiltration and immune activities were likely positively associated with survival prognosis in GC. Our findings suggest that p53 may play an important role in activating tumor immunity in GC and other cancer types and that the TP53 mutation status could be useful in stratifying cancer patients responsive to a certain immunotherapy.     

Potential use of loss of heterozygosity in pleural effusions of breast cancer metastases using the microsatellite marker of the 16q22.1 region of the CDH1 gene

OBJECTIVE: To evaluate the presence of allelic loss in 16q22.1, including the locus of E-cadherin, in pleural effusions in breast cancer patients. STUDY DESIGN: Molecular analysis of DNA was performed using a DNA extraction kit (NucleoSpin, Macherey-Nagel, Germany). Loss of heterozygosity (LOH) in primary tumors and pleural effusions was analyzed using a microsatellite marker of the CDH1 gene, D16S265, described in previous studies. LOH was evaluated by radioactive polymerase chain reaction assay in 17 samples of pleural effusions and breast tissues (primary tumors and nonneoplastic adjacent tissue) from breast cancer patients: 7 positive for neoplastic cells, 6 suspected and 4 cases without evidence of neoplastic cells in the effusions. RESULTS: Thirteen cases (76%) were informative. LOH was detected in 5 cases (38.5%). In 3 of them LOH was detected only in the cytologic sample, and in 2 of them LOH was detected in the primary tumor and cytologic sample. CONCLUSION: Results show that LOH in the CDH1 gene can identify tumor cells in pleural effusions when morphologic analysis is difficult.     

Clinical Implications of Rabphillin-3A-Like Gene Alterations in Breast Cancer

For the rabphillin-3A-like (RPH3AL) gene, a putative tumor suppressor, the clinical significance of genetic alterations in breast cancers was evaluated. DNA and RNA were extracted from formalin-fixed, paraffin-embedded (FFPE) cancers and matching normal tissues. DNA samples were assessed for loss of heterozygosity (LOH) at the 17p13.3 locus of RPH3AL and the 17p13.1 locus of the tumor suppressor, TP53. RPH3AL was sequenced, and single nucleotide polymorphisms (SNPs) were genotyped. RNA samples were evaluated for expression of RPH3AL, and FFPE tissues were profiled for its phenotypic expression. Alterations in RPH3AL were correlated with clinicopathological features, LOH of TP53, and patient survival. Of 121 cancers, 80 had LOH at one of the RPH3AL locus. LOH of RHP3AL was associated with nodal metastasis, advanced stage, large tumor size, and poor survival. Although ~50% were positive for LOH at the RPH3AL and TP53 loci, 19 of 105 exhibited LOH only at the RPH3AL locus. Of these, 12 were non-Hispanic Caucasians (Whites), 15 had large tumors, and 12 were older (>50 years). Patients exhibiting LOH at both loci had shorter survival than those without LOH at these loci (log-rank, P = 0.014). LOH at the TP53 locus alone was not associated with survival. Analyses of RPH3AL identified missense point mutations in 19 of 125 cases, a SNP (C>A) in the 5’untranslated region at -25 (5’UTR-25) in 26 of 104, and a SNP (G>T) in the intronic region at 43 bp downstream to exon-6 (intron-6-43) in 79 of 118. Genotype C/A or A/A of the SNP at 5’UTR-25 and genotype T/T of a SNP at intron-6-43 were predominantly in Whites. Low levels of RNA and protein expression of RPH3AL were present in cancers relative to normal tissues. Thus, genetic alterations in RPH3AL are associated with aggressive behavior of breast cancers and with short survival of patients.     

17p13 (p53 locus), 5q21 (APC locus) and 9p21 (p16 locus) allelic deletions are frequently found in oral exfoliative cytology cells from smoker patients with non-small-cell lung cancer

Molecular cytogenetic and LOH analyses of non-small cell lung cancer (NSCLC) have shown frequent allelic deletions in a variety of chromosomes where tumour suppressor genes are located. Allelic loss at 9p21 (p16 locus), 17p13 (p53) and 5q21(APC) has been frequently described in NSCLC and has also been described in premalignant epithelial lesions of the bronchus and normal bronchial cells. These findings suggest that a tissue field of somatic genetic alterations precedes the histopathological phenotypic changes of carcinoma. Similar changes have been described in oral and laryngeal epithelial tumours associated with smoke exposure. We previously reported frequent LOH at 5q21, 9p21 and TP53 in tumor cells and peritumoral normal bronchial cells from surgically resected NSCLC. We now analyze 96 cases of normal oral exfoliative cytology in which normal epithelial cells were obtained: 43 cases from smoker patients with NSCLC diagnosis, 33 smoker patients with no evidence of malignancy and 20 non-smoker patients with no evidence of tumour. All groups had a similar age and sex distribution. PCR amplification was performed utilising the specific markers D5S346, D9S157 and TP53. In normal oral mucosae cells from patients with NSCLC, we found that 21% of the informative cases showed LOH at any of the three analyzed loci distributed as follows: 14.3% of the informative cases showed LOH at 5q21, 7.7% at 9p21 and 22.2% at TP53. Within the smoker risk group only one case (4% of the informative cases) showed LOH at TP53, while no LOH was found at 5q21 or 9p21. No LOH was found in non-smokers. In conclusion, our results show that a significant number of patients with NSCLC have LOH at TP53, 5q21 and 9p21 in normal oral mucosae, while LOH at these loci is unusual in similar cells obtained from patients with no evidence of malignancy. Our study demonstrates that LOH studies can detect smoker patients with a mutated genotype in normal epithelial cells. Further prospective studies may confirm whether LOH studies can detect patients with a higher risk of NSCLC.     

The Genomic Landscape of TP53 and p53 Annotated High Grade Ovarian Serous Carcinomas from a Defined Founder Population Associated with Patient Outcome

High-grade ovarian serous carcinomas (HGSC) are characterized by TP53 mutations and non-random patterns of chromosomal anomalies, where the nature of the TP53 mutation may correlate with clinical outcome. However, the frequency of common somatic genomic events occurring in HGSCs from demographically defined populations has not been explored. Whole genome SNP array, and TP53 mutation, gene and protein expression analyses were assessed in 87 confirmed HGSC samples with clinical correlates from French Canadians, a population exhibiting strong founder effects, and results were compared with independent reports describing similar analyses from unselected populations. TP53 mutations were identified in 91% of HGSCs. Anomalies observed in more than 50% of TP53 mutation-positive HGSCs involved gains of 3q, 8q and 20q, and losses of 4q, 5q, 6q, 8p, 13q, 16q, 17p, 17q, 22q and Xp. Nearly 400 regions of non-overlapping amplification or deletion were identified, where 178 amplifications and 98 deletions involved known genes. The subgroup expressing mutant p53 protein exhibited significantly prolonged overall and disease-free survival as compared with the p53 protein null subgroup. Interestingly, a comparative analysis of genomic landscapes revealed a significant enrichment of gains involving 1q, 8q, and 12p intervals in the subgroup expressing mutant p53 protein as compared with the p53 protein null subgroup. Although the findings show that the frequency of TP53 mutations and the genomic landscapes observed in French Canadian samples were similar to those reported for samples from unselected populations, there were differences in the magnitude of global gains/losses of specific chromosomal arms and in the spectrum of amplifications and deletions involving focal regions in individual samples. The findings from our comparative genomic analyses also support the notion that there may be biological differences between HGSCs that could be related to the nature of the TP53 mutation.     

3p21, 5q21, 9p21 and 17p13 allelic deletions accumulate in the dysplastic spectrum of laryngeal carcinogenesis and precede malignant transformation

A tissue field of somatic genetic alterations precede the histopathological phenotypic changes of carcinoma. Loss of Heterozygosity (LOH) at the sites of known or putative tumor suppressor genes is a common genetic abnormality detected in precancerous conditions. These genomic changes could be of potential use in the diagnosis and prognosis of pre-malignant laryngeal lesions. Recently the concept of laryngeal intraepithelial neoplasia (LIN) was introduced. To evaluate patients with an increased risk of developing invasive laryngeal carcinoma via a dysplasia-carcinoma progression we investigated 102 microdissected cell populations. Cell populations were procured from 15 laryngectomy specimens with different peritumoral histological changes adjacent to the squamous cell carcinoma cells and 15 laryngeal endoscopic biopsies with no evidence of malignant transformation in a 6-10-year follow-up period. Histological diagnoses were subdivided into keratosis without dysplasia (KWD), with mild dysplasia (LIN 1), with moderate dysplasia (LIN 2), and with severe dysplasia or carcinoma in situ (LIN 3). Microsatellite analysis was performed with the aim of studying LOH of 5q21 (APC), 9p21 (p16), 3p21 and 17p13 (p53) chromosomal regions. Frequent allelic losses were found in carcinoma cells at p53 (54%), p16 (66%), 3p21(87%) and 5q21(58%). Identical LOH patterns were determined in 100% of the LIN3 peritumoral cells, 60% of LIN2, 50% of LIN 1 and 25% of KWD. In contrast, histologically normal mucosae, KWD and LIN1 lesions without malignant progression showed no allelic loss. These results show that dysplasia correlates with LOH at 3p21, 5q21, 9p21 and 17p13 in early laryngeal carcinogenesis. These genomic changes in pre-malignant laryngeal lesions could be of potential use as markers for cancer risk assessment.     

A Common Region of Deletion on Chromosome 17q in Both Sporadic and Familial Epithelial Ovarian Tumors Distal to BRCA1

Linkage analysis in familial breast and ovarian cancer and studies of allelic deletion in sporadic ovarian tumors have identified a region on chromosome 17q containing a candidate tumor-suppressor gene (referred to as BRCA1) of likely importance in ovarian carcinogenesis. We have examined normal and tumor DNA samples from 32 patients with sporadic and 8 patients with familial forms of the disease, for loss of heterozygosity (LOH) at 21 loci on chromosome 17 (7 on 17p and 14 on 17q). LOH on 17p was 55% (22/40) for informative 17pl3.1 and 17pl3.3 markers. When six polymorphic markers flanking the familial breast/ovarian cancer susceptibility locus on 17ql2-q21 were used, LOH was 58% (23/40), with one tumor showing telomeric retention. Evaluation of a set of markers positioned telomeric to BRCA1 resulted in the highest degree of LOH, 73% (29/40), indicating that a candidate locus involved in ovarian cancer may reside distal to BRCA1. Five of the tumors demonstrating allelic loss for 17q markers were from individuals with a strong family history of breast and ovarian cancer. More important, two of these tumors (unique patient number [UPN] 57 and UPN 79) retained heterozygosity for all informative markers spanning the BRCA1 locus but showed LOH at loci distal to but not including the anonymous markers CMM86 (D17S74) and 42D6 (D17S588), respectively. Deletion mapping of seven cases (two familial and five sporadic) showing limited LOH on 17q revealed a common region of deletion, distal to GH and proximal to D17S4, that spans −25 cM. These results suggest that a potential tumor-suppressor gene involved in both sporadic and familial ovarian cancer may reside on the distal portion of chromosome 17q and is distinct from the BRCA1 gene.     

Spinophilin: a new tumor suppressor at 17q21

The scaffold protein spinophilin (SPN) is a regulatory subunit of phosphatase 1a (PP1a) located at 17q21.33. This region is frequently associated with microsatellite instability and LOH and contains a relatively high density of known tumor suppressor genes, and several unidentified candidate tumor suppressor genes located distal to BRCA1. Spn is located in this locus and proposed to be a new tumor suppressor. Loss of Spn induces a proliferative response by increasing pRb phosphorylation, which in turn activates p53, thereby, neutralizing the proliferative response. The absence of p53 bypasses this barrier and enhances the malignant phenotype. Furthermore, the ectopic expression of SPN in human tumor cells from different types of malignancies greatly reduced cell growth. Spn knock-out mice had decreased lifespan with increased cellular proliferation in tissues such as the mammary ducts and early appearance of tumors. Furthermore, the combined loss of Spn and mutant p53 activity led to increased mammary carcinomas, confirming the functional relationship between p53 and Spn. In human tumors, Spn is absent in 20% and reduced in another 37% of human lung tumors. Spn reduction correlates with malignant grade and p53 mutations. Furthermore, Spn mRNA is lost in a percentage of renal carcinomas and lung adenocarcinomas. Finally, lower levels of Spn mRNA correlate with higher grade of ovarian carcinoma and chronic myelogenous leukemia. Therefore, Spn may be the tumor suppressor gene that is located at 17q21.33 and that its tumor suppressive function is dependent on the absence of p53.