Loss Of Heterozygosity In Region Research Articles

Update on the Cytogenetics and Molecular Genetics of Chordoma

Chordoma is a rare mesenchymal tumour of complex biology for which only histologic and immunohistochemical criteria have been defined, but no biomarkers predicting the clinical outcome and response to treatment have yet been recognised. We herein review the interdisciplinary information achieved by epidemiologists, neurosurgeons and basic scientists on chordoma, usually a sporadic tumour, which also includes a small fraction of familial cases. Main focus is on the current knowledge of the genetic alterations which might pinpoint candidate genes and molecular mechanisms shared by sporadic and familiar chordomas. Due to the scarcity of the investigated tumour specimens and the multiple chromosome abnormalities found in tumours with aberrant karyotypes, conventional cytogenetics and Fluorescence In Situ Hybridization failed to detect recurrent chordoma-specific chromosomal rearrangements. Genome-wide approaches such as Comparative Genomic Hybridization (CGH) are yet at an initial stage of application and should be implemented using BAC arrays either genome-wide or targeting selected genomic regions, disclosed by Loss of Heterozygosity (LOH) studies. An LOH region was shown by a systematic study on a consistent number of chordomas to encompass 1p36, a genomic interval where a candidate gene was suggested to reside. Despite the rarity of multiplex families with chordoma impaired linkage studies, a chordoma locus could be mapped to chromosome 7q33 by positive lod score in three independent families. The role in chordomagenesis of the Tuberous Sclerosis Complex (TSC) genes has been proved, but the extent of involvement of TSC1 and TSC2 oncosuppressors in chordoma remains to be assessed. In spite of the scarce knowledge on the genetics and molecular biology of chordoma, recent initiation of clinical trials using molecular-targeted therapy, should validate new molecular targets and predict the efficacy of a given therapy. Comparative genetic and biomolecular studies should enhance the molecular taxonomy of chordoma which might have a prognostic significance and better orient the therapeutic options.

Fine mapping and evaluation of candidate genes for cervical cancer on 11q23

We previously showed that loss of heterozygosity (LOH) at 11q23 is a common genetic alteration in cervical cancer (CC) and that it correlates with extensive invasion of lymph-vascular spaces. In the current study, we looked for allelic loss in paired normal/tumor genomic DNA from 121 cervical tumors by using 20 well-mapped microsatellite markers on 11q. LOH at one or more loci was observed in 81 (66.9%) tumors. The deletion patterns in tumors are complex. However, at least three LOH islands could be defined between D11S614 and D11S4167. We also genotyped 11 CC cell lines and analyzed the results using the homozygosity mapping-of-deletions method. Five of the 11 cell lines showed continuous homozygosity that extended through 11q23.3-11q24.1. We used a candidate-gene approach to screen candidate tumor-suppressor genes (TSGs) that were localized in that region. Intragenic changes in the entire coding sequence of four candidate genes (RNF26, USP2, POU2F3, and TRIM29) in the region and a proposed TSG (PPP2R1B) centromeric to the region were evaluated. The expression status of USP2, POU2F3, TRIM29, and another proposed TSG that is telomeric to the region (BCSC1) also was examined. We identified previously described single-nucleotide polymorphisms (SNPs), several novel variants, and three rare SNPs in the five candidate genes. Decreased expression of POU2F3 and TRIM29 was found in some cervical tumors and CC cell lines. Our results indicate that a major region of LOH in cervical cancer exists within a 3.6-Mb stretch of DNA on 11q23.3-q24.1 and that somatic mutations in RNF26, USP2, TRIM29, POU2F3, or PPP2R1B probably are not important for cervical carcinogenesis.

Loss of heterozygosity on chromosome 10q associated with malignancy and prognosis in astrocytic tumors, and discovery of novel loss regions

Certain tumor suppressor genes (TSG) residing on human chromosome 10q are implicated in astrocytic tumors. We thoroughly examined loss of heterozygosity (LOH) on chromosome 10q in astrocytic tumors to determine the extent of deletion and their relation to prognostic variables of patients. We analyzed 63 astrocytic tumors, including 9 diffuse astrocytomas, 36 anaplastic astrocytomas, and 18 glioblastomas. DNAs from tumors and leukocytes were analyzed for LOH at 18 microsatellite loci by polymerase chain reaction using fluorescence-labeled primers. Then correlation between LOH and clinicopathological variables was examined statistically. Twenty-four (66.7%) anaplastic astrocytomas and 15 (83.3%) glioblastomas had at least one LOH on chromosome 10q. However, diffuse astrocytomas exhibited no LOH. Nineteen tumors (10 anaplastic astrocytomas and 9 glioblastomas) were believed to have a total loss of one chromosome 10. Analyses on 20 tumors with interstitial LOH revealed that most of the high LOH regions matched the location of known TSGs, while some novel LOH regions were found preferentially in anaplastic astrocytoma. The median survivals of the total, partial, and no loss groups were 10.1, 14.8, and 46.8 months, respectively, indicating a significant difference in the survivals of these groups (P=0.0289). Thus, analyzing chromosome 10q loss is helpful for diagnosing malignancy in astrocytic tumors and for predicting patients' survival. Our data also suggested that there are novel TSGs for anaplastic astrocytoma at 10q24 and 10q26.

Loss of heterozygosity analysis in uterine cervical adenocarcinoma

Objective. Uterine cervical adenocarcinoma (CAC) is a rare form of cervical cancer, constituting only 5–8% of all cervical epithelial malignancies. Loss of heterozygosity (LOH) analysis of CAC was undertaken to identify alterations of chromosomal loci that may play important roles in the development of this tumor type. Methods. We analyzed loss of heterozygosity (LOH) using a total of 50 markers on 20 chromosomal arms in 37 cases of microdissected CAC DNA. Results. LOH of >40% was observed on 2p (50%), 3p (45%), 9p (45%), 11q (46%), 17p (57%), 17q (44%), 18q (57%), and 19p (44%). LOH of 30–40% was observed on 6p (38%), 6q (40%), and 10q (31%). Overall, mean LOH was 34% and fractional allelic loss (FAL) was 0.34. High-level and low-level microsatellite instability (MSI) was shown in four cases (11%) and six cases (16%), respectively. Frequency of LOH on10q was significantly higher in endometrioid-type than endocervical-type adenocarcinoma (71% versus 20%; P < 0.05). Conversely, 6q LOH was higher in endocervical type than endometrioid type (0% versus 60%; P < 0.05). 19p13.3 has been reported to be frequently deleted in adenoma malignum, a histological subtype of CAC. To define the critical regions of LOH in CAC in general, we further performed deletion mapping of 19p using 13 markers. Unlike adenoma malignum, multiple regions on 19p appeared to be important loci of LOH for CAC. Conclusion. CACs develop with frequent LOH of multiple chromosomal arms, which may be related to its aggressive clinical behavior and poor prognosis. LOH of 10q may be unique to endometrioid-type CAC.

Extensive analysis of the 7q31 region in human prostate tumors supports TES as the best candidate tumor suppressor gene.

Loss of heterozygosity (LOH) on chromosome arm 7q31 is found in many prostate tumors. Such alterations are generally associated with inactivation of tumor suppressor genes. It has been shown previously that the main region of LOH at 7q31 spans the interval between the D7S486 and D7S2460 microsatellite loci, which contains several candidate tumor suppressor genes (TSG) such as TES, CAV2, CAV1, MET, CAPZA2, ST7 and WNT2. We tested 41 human sporadic prostate tumors for 7q31 LOH by using 5 polymorphic markers overlapping the critical region and used a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay to study the expression of the 7 candidate TSGs located in this genomic region. We found that CAV1, CAV2, MET and TES mRNA expression was lower in prostate tumors than in normal prostate tissues. Our immunohistochemical results and previously published data on the compartmental expression of these messenger RNAs in stromal and epithelial cells suggest that TES is the best candidate tumor suppressor gene at 7q31.

Loss of heterozygosity analysis in uterine cervical adenocarcinoma

<h2>Abstract</h2> <i>Objective</i>. Uterine cervical adenocarcinoma (CAC) is a rare form of cervical cancer, constituting only 5–8% of all cervical epithelial malignancies. Loss of heterozygosity (LOH) analysis of CAC was undertaken to identify alterations of chromosomal loci that may play important roles in the development of this tumor type. <i>Methods</i>. We analyzed loss of heterozygosity (LOH) using a total of 50 markers on 20 chromosomal arms in 37 cases of microdissected CAC DNA. <i>Results</i>. LOH of >40% was observed on 2p (50%), 3p (45%), 9p (45%), 11q (46%), 17p (57%), 17q (44%), 18q (57%), and 19p (44%). LOH of 30–40% was observed on 6p (38%), 6q (40%), and 10q (31%). Overall, mean LOH was 34% and fractional allelic loss (FAL) was 0.34. High-level and low-level microsatellite instability (MSI) was shown in four cases (11%) and six cases (16%), respectively. Frequency of LOH on10q was significantly higher in endometrioid-type than endocervical-type adenocarcinoma (71% versus 20%; <i>P</i> < 0.05). Conversely, 6q LOH was higher in endocervical type than endometrioid type (0% versus 60%; <i>P</i> < 0.05). 19p13.3 has been reported to be frequently deleted in adenoma malignum, a histological subtype of CAC. To define the critical regions of LOH in CAC in general, we further performed deletion mapping of 19p using 13 markers. Unlike adenoma malignum, multiple regions on 19p appeared to be important loci of LOH for CAC. <i>Conclusion</i>. CACs develop with frequent LOH of multiple chromosomal arms, which may be related to its aggressive clinical behavior and poor prognosis. LOH of 10q may be unique to endometrioid-type CAC.

Clustering of minimal deleted regions reveals distinct genetic pathways of human hepatocellular carcinoma.

Systematic scan and statistical analysis of loss of heterozygosity (LOH) has been widely used to define chromosomal aberrations in various cancers for cloning of tumor suppressor genes and for development of prognostic markers. However, the establishment of novel strategies is needed, so that the nonrandom but heterogeneous chromosomal aberration data could provide significant insights into our understanding of molecular pathogenesis of cancers. After comprehensive allelotyping of recurrent allelic losses with 441 highly informative microsatellite markers and overlapping LOH regions on human hepatocellular carcinoma (HCC) chromosomes, 33 minimal deleted regions (MDRs) were revealed. Five and 15 of the 33 MDRs have physical intervals in less than 5 and 10 Mb, respectively, with the smallest MDR9p1 of 2.2 Mb located at 9p21.3-p21.2. Statistical and Kaplan-Meier survival analysis revealed a significant association between the loss of MDR15q1 (15q21.1-q22.2) and the HCC patient survival (adjusted P = 0.033). After cluster analysis of 33 MDRs that represented LOH profiles of each HCC tissue based on clinicopathological features and p53 mutations, two major genetic pathways, low-stage and advanced-stage HCC, were uncovered based on high concordance of MDR clusters. We propose that the definition of genome-wide MDRs on the cancer genome not only narrows down the location of existing tumor suppressor genes to facilitate positional candidate cloning and develop potential prognostic markers after statistical association of MDRs with clinicopathological features but also dissects genetic interactions and pathways of chromosomal aberrations in tumorigenesis.

High-resolution single-nucleotide polymorphism array and clustering analysis of loss of heterozygosity in human lung cancer cell lines.

Chromosomal loss of heterozygosity (LOH) is a common mechanism for the inactivation of tumor suppressor genes in human epithelial cancers. Hybridization to single-nucleotide polymorphism (SNP) arrays is an efficient method to detect genome-wide cancer LOH. Here, we survey LOH patterns in a panel of 33 human lung cancer cell lines using SNP array hybridization containing 1500 SNPs. We compared the LOH patterns generated by SNP array hybridization to those previously obtained by 399 microsatellite markers and find a high degree of concordance between the two methods. A novel informatics platform, dChipSNP, was used to perform hierarchical tumor clustering based on genome-wide LOH patterns. We demonstrate that this method can separate non-small-cell and small-cell lung cancer samples based on their shared LOH. Furthermore, we analysed seven human lung cancer cell lines using a novel 10 000 SNP array and demonstrate that this is an efficient and reliable method of high-density allelotyping. Using this array, we identified small regions of LOH that were not detected by lower density SNP arrays or by standard microsatellite marker panels.

Definition of a region of loss of heterozygosity at chromosome 11q23.3-25 in head and neck squamous cell carcinoma using laser capture microdissection technique.

To date, loss of heterozygosity (LOH) studies on HNSCC have had limited success in identifying a confined region of loss on chromosome 11q partially due to the heterogeneous nature of tumor tissue examined. Additionally, little is known about the role of the 11q allelic deletion in HNSCC tumorigenesis and current reports are conflicting. The aim of this study was to better define LOH at distal 11q by using combination of a pure cell population procured by laser capture microdissection (LCM) and subsequent sensitive PCR amplification of polymorphic microsatellites. This study analyzed HNSCC for LOH using a panel of 5 microsatellite markers spanning 11q23-25. Thirty-four paired DNA samples from tumor and autologous normal tissue were harvested by LCM technique to ensure a pure cell population for PCR amplification. Approximately 2000 to 3000 cells were procured from each sample. Twenty-one of 34 cases (62%, P < 0.001) showed LOH on at least one of the loci examined. The highest frequency of LOH was found at the 11q23.3-25 segment, with 44% at marker D11S968 and 35% at marker D11S1316. A distinct novel region of frequent LOH at 11q23.3-25, defined by D11S1316 and D11S968, was identified. No allelic loss was found in any normal squamous tissue samples. To study LOH in HNSCC, combination of pure cell population procurement by LCM and sensitive PCR provides a more accurate approach than the conventional method using a bulk of heterogeneous tissue. A novel region of LOH at 11q23.3-25 was defined. LOH in this region may harbor putative tumor suppressor gene(s) critical for HNSCC. Furthermore, these allelic losses were not found in any non-neoplastic squamous tissue samples, clarifying prior discrepant data.

Candidate regions of tumor suppressor locus on chromosome 9q31.1 in gastric cancer.

Loss of heterozygosity (LOH) is an important event of tumorigenesis. In gastric cancer, we found a novel region of LOH in chromosome 9q having about 800 kb deletions at 9q31.1. The microsatellite marker D9S938 in that region exhibiting the highest LOH frequency, 56.5%. In addition, the LOH at 9q31.1 did not show any relationship to either histologic types or stages of gastric cancers, and several genes were predicted in the remaining allele by in silico methods. These data suggest that the deletion at 9q31.1 would be common in both differentiated-type and undifferentiated-type gastric cancers. Furthermore, this deletion was found in the primary tumors of early-stage gastric cancer, indicating that loss of function of predicted genes appears to be associated with the tumorigenesis of gastric cancer.

Mapping of candidate region for chordoma development to 1p36.13 by LOH analysis.

Various cytogenetic and molecular findings indicate 1p36 loss as a consistent change in sporadic and inherited chordoma, a rare embryogenetic neoplasm arising from notochord remnants. We studied 27 sporadic chordomas by means of loss of heterozygosity (LOH) of 31 microsatellites localized to the 1p36.32-36.11 region, and restricted the minimal LOH interval shared by 85% of the tumours to 1p36.13. We also used RT-PCR analysis to investigate the role of the candidate genes CASP9, EPH2A, PAX7, DAN and DVL1, which were selected on the basis of the physical mapping of the LOH region and their plausible oncosuppressor function. RT-PCR analysis showed the presence of DAN and PAX7 transcript fragments of the expected size in all of 8 chordoma samples, whereas the CASP9-specific fragment was observed in only 3 and EPH2A was absent in one. Smaller than expected DVL1 transcripts were found in 4 tumours as well as in their normal counterpart (nucleus pulposus), which also showed a typically sized transcript. Sequencing revealed the skipping of 3 exons in the smallest DVL1 fragment, thus leading to a frameshift and predicting a truncated DVL1 gene product. Our study of the largest cohort of chordoma patients recruited so far indicates a common molecular lesion at 1p36.13, and suggests that the CASP9, EPH2A and DVL1 genes may play an onco-suppressing role and be involved in the development of chordoma.

Genetic mapping of allelic loss on chromosome 6q within heterogeneous prostate carcinoma.

A number of genetic events have been reported in prostate carcinogenesis, including frequent loss of heterozygosity (LOH) on chromosomes 8q, 10q, 16q and 18q. In samples of heterogeneous, multifocal prostate carcinomas, we focused on chromosome 6q using PCR-based techniques with 15 microsatellite markers to identify the specific 6q deletion within tumors. LOH of one or more polymorphic markers was detected in 10 of 21 tumors (48%). Two of these 10 tumors demonstrated LOH in all cancerous foci at specific loci and 4 tumors showed deletion in one focus. Different deletion patterns were found in 3 tumors when different polymorphic markers were used. In 90% of tumors showing LOH in one or more foci, however, two common regions of LOH were identified; one at 1.81 cM on 6q15-16.3 between markers D6S1631 and D6S1056, and the other at 5.11 cM on 6q16-21 between markers D6S424 and D6S283. By RT-PCR analysis, the TAK1 gene located at these loci did not correlate with LOH status, indicating that TAK1 is not a target gene in prostate carcinoma. The 6q deletion occurs heterogeneously and LOH was more frequent in tumors of higher pathological stages, implying that this alteration is a late event in prostate carcinogenesis. Because prostate carcinomas are genetically multicentric and of multifocal origin, it remains unclear whether the foci containing 6q deletions specifically expand within tumors or to what extent they contribute to the histological heterogeneity characteristic of the disease.

Detailed characterization of a homozygously deleted region corresponding to a candidate tumor suppressor locus at distal 17p13.3 in human lung cancer.

17p13.3 is one of the chromosomal regions most frequently affected by allelic loss in a variety of human neoplasms including lung cancer. A number of loss of heterozygosity (LOH) analyses have suggested the existence of a tumor suppressor gene at 17p13.3, distal to the p53 locus at 17p13.1. In the present study, we characterized a homozygous deletion at 17p13.3 in a small cell lung cancer cell line by constructing a bacterial artificial chromosome (BAC) contig and a restriction map surrounding the region, as well as by utilizing publicly available draft sequences. We defined the breakpoint, assigned and analysed two known genes, 14-3-3 epsilon and CRK, and a novel gene LOST1 within or at the end of the homozygous deletion of about 170 kb in size. Marked reduction of LOST1 expression was detected in 69% (11/16) of lung cancer specimens by quantitative real-time RT-PCR, while significant DNA hypermethylation was observed at the 5' end of the LOST1 gene in four of six lung cancer cell lines with negligible LOST1 expression. We also show here that a polymorphic marker D17S1174, which resides within the homozygous deletion, was apparently located in the middle of the minimum LOH region, providing further supportive evidence for the presence of a tumor suppressor gene(s) in this region.

Fine mapping of Wilms' tumors with 16q loss of heterozygosity localizes the putative tumor suppressor gene to a region of 6.7 megabases.

The aim of this study was to more precisely map the region of 16q loss of heterozygosity (LOH) in Wilms' tumors and to examine the expression of putative tumor suppressor. We performed polymerase chain reaction-based LOH analysis on the 185 sample pairs from 21 to 80 megabases (Mb) on chromosome 16q. Expression of two candidate tumor suppressor genes located within the identified consensus region of 16q LOH was examined by immunohistochemistry. We identified 16q LOH in 7 (4%) of 185 Wilms' tumors not previously thought to demonstrate such genetic loss. The smallest common region of genetic loss was located between 67.3 and 74.0 Mb on chromosome 16. Within this 6.7-Mb region, there reside only three recognized tumor suppressor genes: E-cadherin, P-cadherin, and E2F4. E-cadherin demonstrates statistically significantly reduced expression in Wilms' tumors with 16q LOH. We have localized the consensus region of 16q LOH in Wilms' tumor to a 6.7-Mb locus and have identified three candidate Wilms' tumor suppressor genes within this narrowed region. Our data support E-cadherin as a candidate tumor suppressor gene in Wilms' tumor; however, further studies are needed to definitively prove its role as the tumor suppressor gene associated with 16q LOH.

Characterization of the common fragile site FRA9E and its potential role in ovarian cancer.

Common fragile sites (CFSs) are regions of profound genomic instability that have been hypothesized to play a role in cancer. The major aim of this study was to locate a fragile region associated with ovarian cancer. Differential display (DD)-PCR analysis comparing normal ovarian epithelial cultures and ovarian cancer cell lines identified pregnancy-associated plasma protein-A (PAPPA) because of its frequent loss of expression (LOE) in ovarian cancer cell lines. PAPPA is localized to human chromosome 9q32-33.1, a region associated with significant loss of heterozygosity (LOH) in ovarian tumors (>50%) and in close proximity to the FRA9E CFS. FISH analysis determined that PAPPA was contained within the distal end of FRA9E. Characterization of FRA9E determined that aphidicolin-induced instability extended over 9 Mb, identifying FRA9E as the largest CFS characterized to date. Comprehensive LOH analysis revealed several distinct peaks of LOH within FRA9E. Semiquantitative RT-PCR analysis of 16 genes contained within FRA9E indicated that genes showing LOE in ovarian tumors coincided with regions of high LOH. PAPPA displayed the most significant loss (72%). This study provides evidence to suggest that instability within FRA9E may play an important role in the development of ovarian cancer and lends further support for the hypothesis that CFSs may be causally related to cancer.

Identification of RUNX1/AML1 as a classical tumor suppressor gene.

Based on our previous results indicating the presence of a tumor suppressor gene (TSG), chromosome 21 was analysed for loss of heterozygosity (LOH) in 18 patients with acute myeloid leukemia (17, AML-M0; one, AML-M1). Allelotyping at polymorphic loci was performed on purified material, allowing unequivocal detection of allelic loss and homozygous deletions. Six AML-M0 patients shared a common region of LOH harboring a single gene: RUNX1 (AML1), the most frequent site of translocations in acute leukemia and a well-known fusion oncogene. Fluorescence in situ hybridization allowed the identification of deletions with breakpoints within RUNX1 in two patients as the cause of LOH. In the four others the LOH pattern and the presence of two karyotypically normal chromosomes 21 were in line with mitotic recombination. Further molecular and cytogenetic analyses showed that this caused homozygosity of primary RUNX1 mutations: two point mutations, a partial deletion and, most significantly, a complete deletion of RUNX1. These findings identify RUNX1 as a classical TSG: both alleles are mutated or absent in cancer cells from four of the 17 AML-M0 patients examined. In contrast to AML-M0, the AML-M1 patient was trisomic for chromosome 21 and has two mutated and one normal RUNX1 allele, suggesting that the order of mutagenic events leading to leukemia may influence the predominant tumor type.

Analysis of the candidate 8p21 tumour suppressor, BNIP3L, in breast and ovarian cancer.

Loss of heterozygosity (LOH) on the short arm of chromosome 8, at 8p12–p23, is one of the most frequent genetic events in both breast and ovarian cancer, suggesting the location of a shared tumour suppressor gene. Microcell-mediated chromosome transfer of chromosome 8 suppresses tumorigenicity and growth of colorectal and prostate cancer cell lines, further supporting the presence of a tumour suppressor gene on 8p. We have taken a candidate gene approach to try to identify this tumour suppressor gene at 8p12–p23. BNIP3L, which has sequence homology to pro-apoptotic proteins and the ability to suppress colony formation in soft agar, is located at 8p21, within a region of ovarian cancer LOH, breast cancer LOH and prostate cancer metastasis suppression. BNIP3L expression was assessed by both RT–PCR and Northern blot analysis in breast and ovarian cancer cell lines and found to be expressed at similar levels relative to expression in their respective normal epithelial cell lines. Genetic analysis of BNIP3L in 40 primary ovarian and 25 primary breast tumours identified one somatic, intronic mutation in one ovarian tumour, as well as several polymorphisms, including one resulting in an amino-acid substitution. These data suggest that BNIP3L is unlikely to be the target of 8p LOH in ovarian or breast cancer.

Loss of heterozygosity analysis of mouse pulmonary adenomas induced by coal tar.

Manufactured gas plant (MGP) residues, commonly known as coal tars, were generated several decades ago as a byproduct of residential and industrial gas production from the distillation of coal. Previous short-term exposure studies have shown MGP residues to be tumorigenic in mouse liver and lung. In order to gain further insight into carcinogenesis by complex mixtures of environmental chemicals containing known carcinogenic polycyclic aromatic hydrocarbons, we investigated mouse pulmonary tumors for loss of heterozygosity (LOH) as a result of multiple exposure to MGP residues. Twenty mouse lung adenomas produced in (C57BL/6 x C3H)F1 hybrid mice and manually microdissected were selected to examine genome-wide allelic losses at 58 microsatellite loci. Regions of chromosomes 1, 4, 5, 8, and 11 were affected in 30-40% of tumors. The elevated rates of allelic imbalance in these chromosomes may indicate the location of unknown tumor suppressor genes significant to neoplastic transformation in mouse lung tissues. Laser capture microdissection-based LOH analysis of pulmonary adenomas showed that contamination of nonneoplastic tissues was not masking the allelic losses in the manually microdissected tumor analysis. The low frequency of chromosome instability in these tumors, measured by means of inter-simple sequence repeat PCR, suggests the presence of discrete regions of LOH instead of extensive structural rearrangements.

Human pituitary adenomas infrequently contain inactivation of retinoblastoma 1 gene and activation of cyclin dependent kinase 4 gene.

Components of cyclinD1/cyclin-dependent kinase 4 (CDK4)/p16INK4a/pRb pathway are the frequent target of many tumor types. We examined the role of retinoblastoma susceptibility gene (RB1) and the CDK4 gene in human pituitary tumorigenesis. For the RB1 gene, pRb expression and loss of heterozygosity (LOH) on 13q in pituitary adenomas were analysed. Immunostaining of pRb revealed lack of expression in 1 of 29 pituitary adenomas. In 4 of 31 pituitary adenomas, allelic imbalances including LOH of RB1 on 13q14 were detected. Three of 4 pituitary adenomas, in which one adenoma lacked pRb expression, had a common LOH region at least from D13S219 on 13q12.3-q13 to D13S265 on 13q31-32. Interphase fluorescence in situ hybridization with a probe of RB1 showed 2 copies of RB1 gene suggesting that mitotic recombination events, not deletion or chromosome loss, led to LOH in the 3 pituitary adenomas analyzed. All 27 exons, intron-exon boundaries, and essential promoter region of RB1 gene were then sequenced in genomic DNA from 4 pituitary adenomas with allelic imbalance on 13q14 including one adenoma without pRb expression and 3 adenomas with pRb expression. Any somatic mutations, insertions, or microdeletions in the RB1 gene were not detected in 4 pituitary adenomas. Methylation sensitive (MS)-polymerase chain reaction (PCR) and bisulfite sequencing analysis revealed hypomethylated status of CpG islands in the promoter region of the RB1 genes of 4 pituitary adenomas. In addition, activating mutations of CDK4 gene, which is a component of cyclinD1/CDK4/p16INK4a/pRb pathway, were not detected in 31 pituitary adenomas. Based on these results, it is concluded that somatic mutations of the RB1 gene or CDK4 gene do not appear to play a major role in pituitary tumorigenesis. This supports the presence of potential tumor suppressor gene(s) on 13q12.3-q13 to 13q31-32 in pituitary adenomas.

The minimum LOH region defined on chromosome 17p13.3 in human hepatocellular carcinoma with gene content analysis

Hepatocellular carcinoma (HCC) is one of the most common human cancers in Asia. Previous studies have shown that in addition to aberrations of the p53 gene on chromosome 17p13.1, other gene(s) on chromosome 17p13.3 may also play a role in HCC. To detect the status of loss of heterozygosity (LOH) in HCC and to determine the minimum region of LOH on 17p13.3, we analyzed 22 paired HCC and non-cancerous liver samples with 14 polymorphic markers plus TP53 (p53 gene) as a comparison. The data revealed a high level of LOH (>68%) in a minimum region between D17S1866 and D17S1574, spanning over a 1.5 Mb region. Genomic library screening using markers in the region has resulted in the isolation of a cluster of BAC/PAC clones. We created a physical map in this region. Using large-scale genome sequencing, gene annotation, cDNA screening, and exon trapping, we identified 17 known genes and 13 novel genes in the minimum region. The function of these genes was analyzed and the possibility of several putative tumor suppressor genes was discussed.