Pathogenesis of adenocarcinoma in Peutz-Jeghers syndrome

Peutz-Jeghers syndrome (PJS) is an autosomal dominant condition characterized by intestinal hamartomatous polyps, mucocutaneous melanin deposition, and increased risk of cancer. Families with PJS from the Johns Hopkins Polyposis Registry were studied to identify the molecular basis of this syndrome and to characterize the pathogenesis of gastrointestinal hamartomas and adenocarcinomas in PJS patients. Linkage analysis in the family originally described by Jeghers in 1949 and five other families confirmed linkage to 19p13.3 near a recently identified gene responsible for PJS. Germ-line mutations in this gene, STK11, were identified in all six families by sequencing genomic DNA. Analysis of hamartomas and adenocarcinomas from patients with PJS identified loss of heterozygosity (LOH) of 19p markers near STK11 in 70% of tumors. Haplotype analysis indicated that the retained allele carried a germ-line mutation, confirming that STK11 is a tumor suppressor gene. LOH of 17p and 18q was identified in an adenocarcinoma but not in hamartomas, implying that allelic loss of these two regions corresponds to late molecular events in the pathogenesis of cancer in PJS. The adenocarcinomas showing 17p LOH also demonstrated altered p53 by immunohistochemistry. None of the 18 PJS tumors showed microsatellite instability, LOH on 5q near APC, or mutations in codons 12 or 13 of the K-ras proto-oncogene. These data provide evidence that STK11 is a tumor suppressor gene that acts as an early gatekeeper regulating the development of hamartomas in PJS and suggest that hamartomas may be pathogenetic precursors of adenocarcinoma. Additional somatic mutational events underlie the progression of hamartomas to adenocarcinomas, and some of these somatic mutations are common to the later stages of tumor progression seen in the majority of colorectal carcinomas.     

Loss of LKB1 kinase activity in Peutz-Jeghers syndrome, and evidence for allelic and locus heterogeneity

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disease characterized by mucocutaneous pigmentation and hamartomatous polyps. There is an increased risk of benign and malignant tumors in the gastrointestinal tract and in extraintestinal tissues. One PJS locus has been mapped to chromosome 19p13.3; a second locus is suspected on chromosome 19q13.4 in a minority of families. The PJS gene on 19p13.3 has recently been cloned, and it encodes the serine/threonine kinase LKB1. The gene, which is ubiquitously expressed, is composed of 10 exons spanning 23 kb. Several LKB1 mutations have been reported in heterozygosity in PJS patients. In this study, we screened for LKB1 mutations in nine PJS families of American, Spanish, Portuguese, French, Turkish, and Indian origin and detected seven novel mutations. These included two frameshift mutations, one four-amino-acid deletion, two amino-acid substitutions, and two splicing errors. Expression of mutant LKB1 proteins (K78I, D176N, W308C, and L67P) and assessment of their autophosphorylation activity revealed a loss of the kinase activity in all of these mutants. These results provide direct evidence that the elimination of the kinase activity of LKB1 is probably responsible for the development of the PJS phenotypes. In two Indian families, we failed to detect any LKB1 mutation; in one of these families, we previously had detected linkage to markers on 19q13.3-4, which suggests locus heterogeneity of PJS. The elucidation of the molecular etiology of PJS and the positional cloning of the second potential PJS gene will further elucidate the involvement of kinases/phosphatases in the development of cancer-predisposing syndromes.     

Peutz-Jeghers syndrome: confirmation of linkage to chromosome 19p13.3 and identification of a potential second locus, on 19q13.4

Peutz-Jeghers syndrome (PJS) is an autosomal dominant disease with variable expression and incomplete penetrance, characterized by mucocutaneous pigmentation and hamartomatous polyposis. Patients with PJS have increased frequency of gastrointestinal and extraintestinal malignancies (ovaries, testes, and breast). In order to map the locus (or loci) associated with PJS, we performed a genomewide linkage analysis, using DNA polymorphisms in six families (two from Spain, two from India, one from the United States, and one from Portugal) comprising a total of 93 individuals, including 39 affected and 48 unaffected individuals and 6 individuals with unknown status. During this study, localization of a PJS gene to 19p13.3 (around marker D19S886) had been reported elsewhere. For our families, marker D19S886 yielded a maximum LOD score of 4.74 at a recombination fraction (theta) of .045; multipoint linkage analysis resulted in a LOD score of 7.51 for the interval between D19S886 and 19 pter. However, markers on 19q13.4 also showed significant evidence for linkage. For example, D19S880 resulted in a maximum LOD score of 3.8 at theta = .13. Most of this positive linkage was contributed by a single family, PJS07. These results confirm the mapping of a common PJS locus on 19p13.3 but also suggest the existence, in a minority of families, of a potential second PJS locus, on 19q13.4. Positional cloning and characterization of the PJS mutations will clarify the genetics of the syndrome and the implication of the gene(s) in the predisposition to neoplasias.     

STK11 mutations in Peutz-Jeghers syndrome and sporadic colon cancer

A potential tumor suppressor gene, STK11 , encoding a serine threonine kinase, has recently been identified on chromosome 19p13. Germ-line mutations of this gene have been found in patients with Peutz-Jeghers syndrome (PJS). To further investigate the relevance of STK11 mutations in PJS, we analyzed its coding sequence in nine patients and identified two deletions and three missense mutations. Because intestinal carcinomas have been observed to develop in association with PJS, we analyzed tumors from 71 patients for allelic deletions (loss of heterozygosity) and STK11 gene mutations, to elucidate the etiological role of STK11 gene in sporadic colorectal cancer. Loss of heterozygosity, evaluated using the microsatellite D19S886, was observed in 10 of 52 informative cases. No somatic mutations were detected except for a missense alteration in one tumor. Our data indicate the heterogeneity of PJS and the infrequent involvement of the STK11 gene in colorectal cancer.     

Frequent somatic mutations in serine/threonine kinase 11/Peutz-Jeghers syndrome gene in left-sided colon cancer

We analyzed somatic mutation and loss of heterozygosity (LOH) in the serine/threonine kinase 11 (STK11)/Peutz-Jeghers syndrome gene in 49 colorectal tumors in three different stages of a dysplasia-carcinoma sequence. We detected LOH in 10 of 19 (52.6%) informative colorectal cancers at loci D19S886 and/or D19S883, but no LOH was observed in 25 informative adenomas. We detected a total of 9 somatic mutations [7 of 13 (53.8%) left-sided colon cancers and 2 of 7 (28.6%) left-sided adenomas with high-grade dysplasia], but no mutations were detected in right-sided colon tumors. Of the nine mutations, one was a frameshift mutation (the same mutation detected in Peutz-Jeghers syndrome family previously), and the other eight were missense mutations. This results indicate that STK11 is a tumor suppressor gene and that genetic changes of STK11 play an important role in left-sided colon cancer carcinogenesis.     

Loss of heterozygosity at 7q31 is a frequent and early event in prostate cancer

It is widely accepted that an accumulation of genetic alterations plays an important role in the genesis of human cancers, but little is known about prostate cancer in this respect. Recent studies have identified regions on chromosome arms 8p, 10q, 16q, and 18q that are frequently deleted in human prostate cancer. We have previously described a loss of heterozygosity (LOH) at the Met locus on chromosome band 7q31 in a study of 20 localized prostate tumors. To determine whether a region on the 7q arm is important in the initiation and/or progression of prostate cancer, prostate tissue from 13 patients with confined prostate tumors, 17 with local extracapsular extension, and 13 with metastatic forms were analyzed for LOH, using a DNA probe for RFLP (pMetH) and 8 CA microsatellite repeats (7 on 7q21-q33 and 1 on 7p). Twenty (47%) of the 43 cases studied showed LOH at one or more 7q loci. The most frequently deleted region was chromosome 7q31.1-7q31.2, whereas the centromeric locus on 7q21 was generally conserved. The percentage of LOH was normally distributed around the D7S480 locus. Moreover, the rate of LOH in the 7q31 region was lower in metastatic tumors than in localized tumors. These results strongly suggest the presence of a tumor suppressor gene on the chromosome band 7q31 with an important role in the early stages of prostate cancer.     

Allelic losses on chromosomes 14, 10, and 1 in atypical and malignant meningiomas: a genetic model of meningioma progression

To investigate chromosomal events that underlie formation and progression of meningiomas, we have examined a set of 18 benign (WHO grade I), 15 atypical (grade II), and 13 anaplastic/malignant (grade III) meningiomas for loss of heterozygosity (LOH) on chromosomes 1p, 6p, 9q, 10q, and 14q. Frequent loss of loci on these chromosomes was seen in grade II and grade III tumors, specifically, 14q (II and III, 47 and 55%), 1p (40 and 70%), and 10q (27 and 40%). In contrast, LOH for these loci was infrequent in benign meningiomas, specifically, 14q (0%), 1p (11%), and 10q (12%). The smallest common regions of deletion that could be defined were 14q24-q32, 1p32-pter, and 10q24-qter. These observations indicate the likely presence of tumor suppressor genes in these regions that are involved in the development of WHO grade II and grade III meningiomas. Because LOH for loci on chromosomes 1p and 10q was found in tumors of all grades and because the frequency of LOH in all three regions increased with tumor grade, these results would support a model for the formation of aggressive meningiomas through tumor progression.     

Simplified guide for precise implant placement: a technical note

Loss of heterozygosity (LOH) at chromosome band 10q23 occurs frequently in a wide variety of human tumors. A recently identified candidate tumor suppressor gene, PTEN located on 10q23, is mutated in multiple advanced cancers. To explore whether PTEN is associated with human squamous cell carcinoma of the head and neck (SCCHN), DNAs from both normal muscle and tumor tissue in 19 SCCHN were used for detecting LOH at chromosome 10q23 and mutational analysis of PTEN by direct polymerase chain reaction (PCR)-DNA sequencing. LOH at 10q23 was identified in 6/15 SCCHN. Mutation of PTEN was identified in 3/19 SCCHN. Of these 3 patients, 2 had stage IV disease; the third patient, with recurrent, metastatic and stage III disease, showed a 36 bp germline heterozygous deletion within intron 7. Furthermore, a missense mutation at codon 501 (TCT --> TTT: Ser --> Phe) in exon 8 was also found in tumor from the same patient. Our results suggest that PTEN may play a role in the genesis of some SCCHNs.     

Comprehensive allelotyping of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common cancers in many parts of the world, however the molecular mechanisms underlying liver cell transformation remain obscure. A genome-wide scan of loss of heterozygosity (LOH) in tumors provides a powerful tool to search for genes involved in neoplastic processes. To identify recurrent genetic alterations in liver tumors, we examined DNAs isolated from 120 HCCs and their adjacent non tumorous parts for LOH using a collection of 195 microsatellite markers located roughly every 20 cM throughout 39 autosomal arms. The mean heterozygosity was 73%. Our findings provide additional support that LOH for loci on chromosomal arms 1p, 4q, 6q, 8p, 13q and 16p is significantly elevated in HCC. The highest percentage of LOH is found for a locus in 8p23 (42% of informative csaes). This corresponds to one of the most common genetic abnormalities reported to date in these tumors. In addition, high ratio of LOH (> or = 35%) is observed on chromosome arms which had not been implicated in previous studies, notably on 1q, 2q and 9q. No correlation was found between LOH of specific chromosomal regions and etiologic factors such as chronic infections with hepatitis B or C viruses. This first report of an extensive allelotypic analysis of HCC should help in identifying new genes whose loss of function contributes to the development of liver cancer.     

Multifocal renal cell carcinoma: evidence for a common clonal origin

The reported incidence of satellite tumor lesions in kidneys resected by radical nephrectomy for renal cell carcinoma (RCC) is 7-25%; however, genetic analyses of satellite tumors in comparison with those of main tumor lesions have not been performed well. In the present study, we investigated the incidence of loss of heterozygosity (LOH) at chromosome arms 3p, 6q, 8p, 9p, 9q, and 14q using 18 microsatellite markers in 10 nonpapillary RCCs of 50 mm or less in diameter and the accompanying satellite tumor lesions to evaluate the genetic alterations in main and satellite tumors. LOH was detected in 10, 3, 5, 3, 2, and 3 cases at chromosome arms 3p, 6q, 8p, 9p, 9q, and 14q, respectively. In addition, primary and satellite tumor lesions in 8 of 10 cases exhibited identical patterns of LOH on the 18 loci examined. In the remaining two cases, both main and satellite tumors demonstrated LOH on the common seven and three loci, respectively, whereas for another locus, LOH was observed only in the satellite tumor lesions. The similarity of LOH patterns detected in main and satellite tumor lesions indicates that the presence of satellite tumors might be the result of intrarenal metastasis from the main tumor lesion. These findings strongly suggest that even in case of small nonpapillary RCC, nephron-sparing surgery might carry the risk of failing to prevent postoperative local recurrence due to the incomplete resection of unrecognized satellite tumors with genetic alterations similar to those of the main tumor.     

Leukocyte migration in acute colonic inflammatory bowel disease: comparison of histological assessment and Tc-99m-HMPAO labeled leukocyte scan

Loss of heterozygosity (LOH) on chromosome 11 is frequently altered in various epithelial cancers. The present study was designed to investigate LOH on chromosome 11 in microdissected samples of normal prostatic epithelium and invasive carcinoma from the same patients. For this purpose, DNA was extracted from the microdissected normal and tumor cells of 38 prostate cancers, amplified by polymerase chain reaction PCR and analyzed for LOH on chromosome 11 using 9 different polymorphic DNA markers (D11S1307, D11S989, D11S1313, D11S898, D11S940, D11S1818, D11S924, D11S1336 and D11S912). LOH on chromosome 11 was identified in 30 of 38 cases (78%) with at least one marker. Four distinct regions of loss detected were: 1) at 11p15, at loci between D11S1307 and D11S989; 2) at 11p12, on locus D11S131 (11p12); 3) at 11q22, on loci D11S898, D11S940 and D11S1818; and 4) at 11q23-24, on loci between D11S1336 and D11S912. We found 25% of the tumors with LOH at 11p15; 39% had LOH at 11p12; 66% had LOH at 11q22; and 47% had LOH at 11q23-24. These deletions at 11p15, 11p12, 11q22 and 11q23-24 loci were not related to the stage or grade of the tumor.     

Analysis of PTEN and the 10q23 region in primary prostate carcinomas

Deletions involving chromosome 10q23 occur frequently in prostatic carcinomas. Recently, a novel tumour suppressor gene, PTEN, mapping to this interval, has been identified. Mutation or deletion of PTEN has been observed in a proportion of prostate cancer cell lines; however, primary prostate carcinomas have not been studied. We have investigated the involvement of PTEN in primary prostatic adenocarcinomas using a panel of 51 matched normal and prostate tumour DNAs. We first determined the proportion of tumours with allele loss at loci in 10q23 which span the region containing the PTEN gene. Our results show that LOH involving 10q23 is common in primary prostate carcinomas. Twenty-five of 51 (49%) tumours showed loss of heterozygosity (LOH) over the region spanning the PTEN locus. We next directly analysed the PTEN gene for mutations of the coding region using single strand conformation polymorphism (SSCP) and sequence analyses. Of those tumours with LOH, only a single tumour was found to carry a missense mutation in PTEN. No mutations in PTEN were identified in tumours without LOH. Our results suggest either that mutation of PTEN is a late event in prostate tumorigenesis, or that another tumour suppressor gene important in prostate cancer may lie close to PTEN in 10q23.     

Point mutation and homozygous deletion of PTEN/MMAC1 in primary bladder cancers

A new tumor suppressor gene PTEN/MMAC1 was recently isolated at chromosome 10q23 and found to be inactivated by point mutation or homozygous deletion in glioma, prostate and breast cancer. PTEN/MMAC1 was also identified as the gene predisposing to Cowden disease, an autosomal dominant cancer predisposition syndrome associated with an increased risk of breast, skin and thyroid tumors and occasional cases of other cancers including bladder and renal cell carcinoma. We screened 345 urinary tract cancers by microsatellite analysis and found chromosome 10q to be deleted in 65 of 285 (23%) bladder and 15 of 60 (25%) renal cell cancers. We then screened the entire PTEN/MMAC1 coding region for mutation in 25 bladder and 15 renal cell primary tumors with deletion of chromosome 10q. Two somatic point mutations, a frameshift and a splicing variant, were found in the panel of bladder tumors while no mutation was observed in the renal cell carcinomas. To screen for homozygous deletion, we isolated two polymorphic microsatellite repeats from genomic BAC clones containing the PTEN/MMAC1 gene. Using these new informative markers, we identified apparent retention at the gene locus indicative of homozygous deletion of PTEN/MMAC1 in four of 65 bladder and 0 of 15 renal cell tumors with LOH through chromosome 10q. Identification of the second inactivation event in six bladder tumors with LOH of 10q implies that the PTEN/MMAC1 gene is occasionally involved in bladder tumorigenesis. However, the low frequency of biallelic inactivation suggests that either PTEN/MMAC1 is inactivated by other mechanisms or it is not the only target of chromosome 10q deletion in primary bladder and renal cell cancer.     

Allelic deletion analyses of MMAC/PTEN and DMBT1 loci in gliomas: relationship to prognostic significance

The frequency of loss of heterozygosity (LOH) around MMAC/PTEN and DMBT1 loci and survival analyses based on the LOH status were assessed in 110 patients with different histological groups of gliomas. Twenty-six of the patients had anaplastic oligodendrogliomas, 31 had anaplastic astrocytomas, and 53 had glioblastomas multiforme (GM). At the DMBT1 locus, LOH was observed very frequently in all three histological groups, with no significant difference in the frequency of LOH among the three histological groups. At the MMAC/PTEN locus, patients with GM exhibited a significantly increased frequency of LOH (72%) compared with patients with anaplastic astrocytomas (29%) or anaplastic oligodendrogliomas (31%) (P < 0.0001). Kaplan-Meier survival plots showed that patients with LOH at the MMAC/PTEN locus had a significantly worse prognosis than did patients without LOH at the MMAC/PTEN locus [hazard ratio (LOH versus non-LOH), 2.65; 95% confidence interval (CI), 1.69-4.46; P < 0.0001]. Cox proportional hazards regression analysis, adjusted for age at surgery and histological grades (GM and non-GM), showed that LOH at the MMAC/PTEN locus was a significant predictor of shorter survival [hazard ratio (LOH versus non-LOH), 2.01; 95% CI, 1.1-3.5; P = 0.018). Our analysis failed to indicate a similar association between the frequency of LOH at the DMBT1 locus and patient survival [hazard ratio (LOH versus non-LOH), 2; 95% CI, 0.37-3.13; P = 0.2]. These results suggest that the DMBT1 gene may be involved early in the oncogenesis of gliomas, whereas alterations in the MMAC/PTEN gene may be a late event in the oncogenesis related to progression of gliomas and provide a significant prognostic marker for patient survival.     

Linkage of familial Wilms' tumor predisposition to chromosome 19 and a two-locus model for the etiology of familial tumors

Familial predisposition to Wilms' tumor (WT), a childhood kidney tumor, is inherited as an autosomal dominant trait. For most WT families studied, the 11p13 gene WT1 and genomic regions implicated in tumorigenesis in a subset of tumors can be ruled out as the site of the familial predisposition gene. Following a genome-wide genetic linkage scan, we have obtained strong evidence (log of the odds ratio = 4.0) in five families for an inherited WT predisposition gene (FWT2) at 19q13.3-q13.4. In addition, we observed loss of heterozygosity at 19q in tumors from individuals from two families in which 19q can be ruled out as the site of the inherited predisposing mutation. From these data, we hypothesize that alterations at two distinct loci are critical rate-limiting steps in the etiology of familial WTs.     

Waist to hip ratio and psychosocial factors in adults with insulin-dependent diabetes mellitus: the Pittsburgh Epidemiology of Diabetes Complications study

Loss of heterozygosity (LOH) at several chromosomal loci is a common feature of the malignant progression of human tumors. In the case of chromosome 11, LOH has been well documented in several types of solid neoplasms, including gastric carcinoma, suggesting the presence of suppressor gene(s) at 11p15 and 11q22-23. Little is currently known about the molecular events occurring during the development of gastric cancer. To define the regions of chromosome 11 involved in gastric cancer progression, we used high-density polymorphic markers to screen for LOH in matched normal and tumor tissue DNA from 60 primary gastric carcinomas. We found that 21% of the tumors showed LOH simultaneously at 11p15 and 11q22-23, 41% had LOH at 11p15, and 30% had LOH at 11q22-23. We confirm that the minimal critical area of LOH for 11p15.5 is the approximately 2-Mb region between loci D11S1318 and D11S988. However, when we analyzed the pattern of LOH according to the country of origin of the patient, LOH for 11q22-23 alone was found only in cases from Italy. The minimal critical region of LOH at 11q22-23 is identical to that identified for other solid tumors, suggesting that the same putative tumor suppressor gene(s) contained within this region is involved in the pathogenesis of several common human tumors.     

Allelotype and replication error phenotype of small cell lung carcinoma

Allelotype and replication error (RER) phenotype analyses were performed to clarify the pathogenetic significance of inactivation of tumor suppressor genes and genomic instability in the genesis and progression of small cell lung carcinoma (SCLC). We examined 37 cases of SCLC for loss of heterozygosity (LOH) and microsatellite instability at 49 loci on all 39 nonacrocentric chromosomal arms. LOH was frequently (>70%) detected on chromosomes 3p (29/32, 90.6%), 5q (15/21, 71.4%), 13q (25/26, 96.2%), 17p (22/25, 88.0%), and 22q (24/33, 72.7%). Frequent LOH (>70%) on these loci was observed even among seven cases of stage I tumors. The incidence of LOH on all 39 nonacrocentric chromosomal arms was not significantly different between primary tumors and metastases. These results suggest that inactivation of multiple tumor suppressor genes accumulates relatively early during progression of SCLC and it may be responsible for clinically and biologically aggressive phenotype of SCLC. RER was observed in 6/37 (16.2%) of SCLC, however, RER at multiple loci was observed only in two cases. Therefore, it was indicated that genomic instability is uncommon, but might play a role in the genesis of a small subset of SCLC.     

Loss of heterozygosity at 5q21-22 (adenomatous polyposis coli gene region) in oral squamous cell carcinoma is common and correlated with advanced disease

We determined the frequency of loss of heterozygosity (LOH) at chromosome 5q21-22 (adenomatous polyposis gene region) in oral SCC from 49 patients using PCR-based assays. Of 43 informative (heterozygous) tumors, 41.9% [95% confidence interval (CI)=27.0, 57.9] contained LOH at 5q21-22. LOH at 5q21-22 was strongly associated with stage at diagnosis: 100%, (3/3), 50% (13/26), and 14% (2/14) of tumors from patients with distant metastases, regional spread, and localized disease, respectively, contained this genetic alteration (P=0.01). There were no statistically significant associations between LOH at 5q21-22 and other patient or tumor characteristics, but LOH was more commonly found in the tumors of heavy smokers, infrequent alcohol consumers, and in tumors containing either p53 mutations or HPV-DNA. In univariate analyses, LOH at 5q21-22 was associated with poor prognosis (hazard ratio=1.8, 95%, CI 0.8, 4.5); this relationship did not persist after adjustment for stage of disease (hazard ratio=1.1, 95% CI=0.4, 3.1). These data provide further evidence that inactivation of the APC gene and/or other genes at 5q21-22 is common and may be involved in the development and/or progression of oral SCC. Larger studies are needed to determine whether LOH at 5q21-22 is linked to known oral SCC etiologic factors and/or the prognosis of oral SCC patients, as well as to genetic instability at other loci involved in these malignancies.     

New phototherapies

BACKGROUND: Ovarian epithelial tumors can be divided into subcategories often regarded as different stages of neoplastic transformation. Cystadenomas belong to the least aggressive subgroup and are noninvasive and nonmetastatic. Ovarian tumors of low malignant potential (LMP) are intermediate between cystadenomas and carcinomas and show markedly reduced invasive and metastatic abilities. Invasion and metastasis are the hallmarks of carcinomas, which constitute the most aggressive subgroup and can be further subdivided into different grades. PURPOSE: We performed comparative allelotype analyses of ovarian cystadenomas, LMP tumors, and carcinomas, reasoning that such analyses could provide clues about the molecular determinants of their phenotypic differences. Because we realized that allelic losses involving the X chromosome might be associated with LMP tumor development, we determined whether such losses were interstitial and whether they involved the active or the inactive X chromosome. METHODS: Frequencies of loss of heterozygosity (LOH) at specific loci in every chromosomal arm were determined in 16 ovarian cystadenomas, 23 ovarian LMP tumors, 15 low-grade ovarian carcinomas, and 35 high-grade ovarian carcinomas by use of either the polymerase chain reaction (PCR) or Southern blot analyses. We took advantage of the fact that DNA methylation is an important mechanism of X-chromosome inactivation to determine whether losses involving the X chromosome were in the active or the inactive copy. We analyzed the methylation status of retained alleles on the X chromosome by determining whether they could be amplified by PCR after digestion with the methylation-sensitive restriction endonuclease Hpa II. RESULTS: High-grade carcinomas contained frequent(>50%) LOH in four autosomal chromosome arms, i.e., 6q, 13q, 17p, and 17q. Except for 13q, these same chromosomal arms showed frequent LOH in low-grade carcinomas. LOH in autosomal chromosomes was comparatively rare in LMP tumors and was absent in cystadenomas. In contrast, half (eight of 16) of LMP tumors informative for a locus in the proximal portion of chromosome Xq showed LOH at that locus. These losses were the result of interstitial deletions in six of the eight cases and involved the inactive copy of the X chromosome exclusively. Similar losses in the X chromosome were not seen in either cystadenomas or low-grade carcinomas. CONCLUSIONS AND IMPLICATIONS: LOH at multiple loci is associated with the development of ovarian carcinomas but not with the development of cystadenomas and LMP tumors. However, the integrity of a locus in chromosome Xq that possibly escapes X-chromosome inactivation is important for the control of LMP tumor development. The fact that this locus does not appear to be involved in the genesis of low-grade carcinomas suggests that LMP tumors are not precursors of such carcinomas.     

Loss of heterozygosity and mutational analysis of the PTEN/MMAC1 gene in synchronous endometrial and ovarian carcinomas

Mutations of the human putative protein tyrosine phosphatase (PTEN/MMAC1) gene at chromosome 10q23 have been found frequently in type I endometrial carcinomas. Endometrioid adenocarcinoma is the most frequent histology seen in patients with clinically determined synchronous endometrial and ovarian carcinomas. We report a high incidence of PTEN/MMAC1 mutations and 10q23 loss of heterozygosity (LOH) in patients with synchronous endometrial and ovarian carcinomas. Paraffin-embedded precision microdissected tumors were analyzed for 10 matched synchronous endometrial and ovarian cancers and 11 matched control metastatic endometrial cancers. Single-stranded conformation polymorphism analysis was used to screen for mutations in all tumors and corresponding normal lymphocyte DNA. LOH was determined using a panel of four microsatellite markers within the PTEN/MMAC1 locus. PTEN/MMAC1 mutations were found in 43% (9 of 21) of the endometrial cancers studied, similarly represented in the clinically synchronous group (5 of 10 or 50%) and the advanced metastatic group (4 of 11; 36%; P = 0.53). In two of the five cases of clinically synchronous cancers, identical or progressive PTEN mutations were found in both the endometrial and ovarian cancers, suggesting that the ovarian tumor is a metastasis from the endometrial primary. PTEN/MMAC1 mutations in the advanced endometrial cancers were similar in the corresponding metastases. In one case, the mutation was seen in only one of two metastatic lymph nodes. The LOH analysis demonstrated 55% LOH in at least one PTEN/MMAC1 marker. These findings suggest that the putative tumor suppressor gene PTEN/MMAC1 may be a viable molecular marker to differentiate synchronous versus metastatic disease in a subset of clinically synchronous endometrial and ovarian carcinomas.