P53 Loss Of Heterozygosity Research Articles

Frequent chromosome 9p losses in histologically normal nasopharyngeal epithelia from southern Chinese.

Nasopharyngeal carcinoma (NPC) is a common cancer among the Chinese population in the southern part of China. The incidence of this cancer drops markedly in northern China. A 6- to 24-fold difference exists between southern and northern Chinese. To investigate the early genetic events involved in the development of this cancer, we have examined loss of heterozygosity (LOH) on chromosome 9p, being one of the most frequent genetic alterations in NPC, in nasopharyngeal tissues including normal epithelia (NP), dysplastic lesions (DNP) and invasive carcinoma (NPC) from high-risk and low-risk regions. We found similar frequencies of 9p LOH in NPC from high-risk (77.8%) and low-risk (63.6%) regions (p = 0.43). In contrast, 45% of normal nasopharyngeal epithelia from the high-risk region showed 9p LOH, while none of the NP from the low-risk region showed such abnormalities (p = 0.002). Deletions of chromosome 9p were found in 66.7% dysplastic nasopharyngeal lesions. These findings suggest that LOH of chromosome 9p is an early event in the tumorigenesis of NPC. The increased risk of NPC in southern Chinese may be related to early loss of genetic materials as indicated by 9p LOH in the NP from high- and low-risk regions. We also reported previously that Epstein-Barr virus (EBV) latent infection was present in all high-grade DNP and NPC but not in any NP from fetuses or normal adults. Thus, early genetic alterations such as 9p LOH may take place prior to EBV latent infection and expand clonally thereafter.

Infrequent loss of heterozygosity of the major tumour suppressor genes in Indian oral cancers

Abstract.The loss of heterozygosity (LOH) in tumour suppressor gene loci such as p53, retinoblastoma (rb) and adenomatous polyposis coli (apc) were analyzed in oral cancer tissues with matched controls by employing polymerase chain reaction based/restriction fragment length polymorphism (PCR-RFLP), variable number of tandem repeats (PCR-VNTR) analysis and microsatellite assay. The PCR-RFLP analysis showed an infrequent LOH in rb (17%), p53 (11%) and apc (10%) loci in these cases. The microsatellite assay also revealed only a low frequency of LOH in the microsatellite markers such as TP53 (25%), D5S505 (10%) and D3S1067 (0%) in the same samples. In contrast to the present study, similar studies from Western countries have reported a high frequency of LOH in p53, rb and apc genes in oral cancer tissues. The present preliminary study indicates that the gene aberration by LOH may be an insignificant mechanism in Indian oral cancers with respect to the tumour suppressor genes examined.

Gene therapy for pancreatic cancer targeting the genomic alterations of tumor suppressor genes using replication-selective oncolytic adenovirus.

In order to develop an effective therapeutic intervention for patients with pancreatic cancer, we examined the genetic alternations of pancreatic cancer. Based on these results, we are developing a new gene therapy targeting the genetic character of pancreatic cancer using mutant adenoviruses selectively replication-competent in tumor cells. Loss of heterozygosity (LOH) of 30% or more were observed on chromosome arms 17p (47%), 9p (45%), 18q (43%), 12q (34%), and 6q (30%). LOH of 12q, 17p, and 18q showed the significant association with poor prognosis. These data strongly suggest that mutation of the putative suppressor genes, TP53 and SMAD4 play significant roles in the disease progression. Based on this rationale, we are developing a new gene therapy targeting tumors without normal TP53 function. E1B-55kDa-deleted adenovirus (AxE1AdB) can selectively replicate in TP53-deficient human tumor cells but not cells with functional TP53. We evaluated the therapeutic effect of this AxE1AdB on pancreatic cancer without normal TP53 function. The growth of human pancreatic tumor in SCID mice model was markedly inhibited by the consecutive injection of AxE1AdB. Furthermore, AxE1AdB is not only the strong weapon but also useful carrier of genes possessing anti-tumor activities as a virus vector specific to tumors without normal TP53 function. It was reported that uracil phosphoribosyl transferase (UPRT) overcomes 5FU resistance. UPRT catalyzes the synthesis of 5-fluorouridine monophosphate (FUMP) from Uracil and phosphoribosylpyrophosphate (PRPP). The antitumor effect of 5FU is enhanced by augmenting 5-fluorodeoxyuridine monophosphate (FdUMP) converted from FUMP, which inhibits Thymidylate Synthetase (TS). The therapeutic advantage of restricted replication competent adenovirus that expresses UPRT (AxE1AdB-UPRT) was evaluatedin an intra-peritoneal disseminated tumor model. To study the anti-tumor effect of AxE1AdB-UPRT/5FU, mice with disseminated AsPC-1 tumors were administered the adenovirus, followed by the 5FU treatment. It was shown that the treatment with AxE1AdB-UPRT/5FU caused a dramatic reduction of the disseminated tumor burden without toxicity in normal tissues. These results revealed thatthe AxE1AdB-UPRT/5FU system is a promising tool for intraperitoneal disseminated pancreatic cancer.

Microsatellite instability at tetranucleotide repeats in skin and bladder cancer.

Recently, a novel form of MSI has been described that occurs only at tetranucleotide repeat markers. This has been termed elevated microsatellite instability at selected tetranucleotide repeats (EMAST). EMAST has been related to alterations of the p53 gene, and to the nature of the repeat sequence. We initially tested whether loss of heterozygosity (LOH) at the p53 and the patched (ptch) genes was related to EMAST in a series of 61 non-melanoma skin cancer (NMSC) tumors. We then analysed a series of 57 primary bladder cancers for the presence of EMAST, testing whether this was related to mutation or expression of the p53 gene. In both NMSC and bladder tumors we found a high prevalence of EMAST (75.4 and 43.9%). In NMSC the prevalence of EMAST was higher in tumors that had either p53 or ptch LOH, although the difference was not statistically significant. There was a significant association of extensive EMAST (three or more loci) with mutations in p53 among the bladder cancer tumors, but no indication of elevated EMAST in tumors with abnormal p53 staining without mutation. The association of EMAST with p53 mutation was confined to non-invasive disease. Hence, EMAST likely reflects a particular pattern of somatic events that are interactive with p53 mutation, particularly common in skin cancer and limited to non-invasive disease in bladder cancer.

Prognostic significance of p16INK4a alterations and 9p21 loss of heterozygosity in locally advanced laryngeal squamous cell carcinoma.

The p16INK4a gene, localized within chromosome 9p21, has been identified as a cyclin-dependent kinase inhibitor and may negatively regulate the cell cycle acting as a tumor suppressor. Genetic alterations involving the 9p21 region are common in human cancers. A consecutive series of 64 untreated patients (median of follow up 53 months) undergoing surgical resection for locally advanced laryngeal squamous-cell carcinomas (LSCCs) has been studied prospectively. Our purpose was to investigate p16 alterations (9p21 allelic loss, hypermethylation and point mutations) and their possible association with clinico-pathological data and flow cytometric variables (DNA-ploidy and S-phase fraction (SPF)), and to determine the possible prognostic role of this gene in these tumors. PCR-based techniques were used for investigating 9p21 loss of heterozygosity (LOH) and methylation promoter status of the p16 gene. p16 mutations were detected by PCR-SSCP (single strand conformation polymorphism) and sequencing. 9p21 LOH was detected in 16/62 (26%) informative tumors, point mutations in 5% (3/64) and hypermethylation in 9% (6/64) of the cases. p16 alterations were significantly associated with high SPF and DNA-aneuploidy. By univariate analysis, poor histologic differentiation, stage IV, DNA-aneuploidy and p16 point mutations proved to be significantly related to quicker relapse, whereas these same factors, and in addition high SPF, 9p21 LOH and any p16 alterations were significantly related to shorter overall survival. By Cox proportional hazards analysis only histologic grade (G3) and p16 point mutations were independently related to both disease relapse and death. Our study has identified p16 point mutations as important biomolecular indicators in LSCCs.

Loss of heterozygosity in the clonal evolution of flat colorectal neoplasms.

In contrast to invasive colorectal carcinomas that develop in typical exophytic adenoma-carcinoma sequences, some invasive cancers may evolve from flat mucosal dysplastic lesions. Despite their relatively small size, these flat colorectal lesions are often associated with high-grade dysplasia and may show an aggressive clinical course. To delineate the genetic pathways in the clonal evolution of these tumors, multiple foci were microdissected from 13 cases and the allelic deletions of 15 chromosomal arms were analysed. Loss of heterozygosity (LOH) was detected most frequently on 17p (77%), followed by 18q (69%), and 5q (54%). In five cases with concomitant low-grade adenomas, only one case showed LOH in low-grade adenoma foci. In high-grade dysplasia with/without submucosal invasion, early and homogeneous LOH of one to several chromosomal arms was detected. Overall, homogeneous and thus early LOH were most frequently detected on 17p (seven of 10 cases with 17p LOH), followed by 3p (two of three cases with 3p LOH), and 5q (four of seven cases with 5q LOH). In addition to homogeneous LOH, the LOH patterns observed in different portions of dysplasias and invasive cancers in individual cases identified several different genetic patterns of tumour progression, either with linear or branching (divergent) trees. Positive immunostaining for p53 was detected in 10 of the 13 cases; of these, five cases were concomitant with 17p LOH in all of the microdissected foci, four cases were concomitant with 17p LOH in a majority of foci and, one case showed retention of 17p. Except for the flat configuration and early 17p LOH, genetic heterogeneity in the flat high-grade dysplastic foci was found to be similar to genetic chaos in the late dysplastic and preinvasive stages of exophytic adenoma. These findings suggest a potentially aggressive course for these neoplasms.

Identification of the small interstitial deletion at chromosome band 1p34-p35 and its association with poor outcome in oligodendroglial tumors.

To narrow down the putative tumor-suppressor gene locus and to assess the predictability of clinical courses by genomic alterations, we analyzed 46 oligodendroglial tumors for loss of heterozygosity (LOH) in the distal region of the short arm of chromosome 1. LOH at 1p was found in 43 tumors (93.5%), including all 28 oligodendrogliomas, all eight oligo-astrocytomas, six of eight anaplastic oligodendrogliomas, and in one of two anaplastic oligo-astrocytomas. Thirty-seven tumors showed LOH patterns consistent with a large terminal deletion, whereas six tumors showed LOH suggesting interstitial deletions. Our data also showed two small regions of overlap at 1p34-p35 (approximately 5.7 Mb) and at 1p36.1-p36.2 ( approximately 12 Mb). Among the six tumors with interstitial deletion, the proximal region was deleted in five tumors, whereas the distal region was deleted in only half of them. Overall, 91% of tumors showed deletion including this proximal region. To examine the clinical significance of the LOH pattern, the samples were classified into three groups: tumors without 1p LOH (Group 1, n = 3), tumors with an interstitial deletion (Group 2, n = 6), and tumors with a large terminal deletion (Group 3, n = 37). Both overall and progression-free survival of patients in Group 2 was extremely poor compared with those included in Group 3 (P = 0.0006 and P = 0.003, respectively). As to the clinical response to chemotherapy, nimustine prevented tumor recurrence in Group 3 (P = 0.034) but not in Group 2. Our results demonstrate that a putative tumor-suppressor gene(s) in oligodendroglial tumors is localized at 1p34-p35 and that small interstitial deletions, in contrast to large terminal deletions, are strongly predictive of both chemoresistance and aggressive characteristics of these tumors.

P53 gene mutation and genetic instability in superficial multifocal esophageal squamous cell carcinoma

Tumor multicentricity is occasionally observed in esophageal squamous cell carcinoma (SCC). We studied five surgically resected superficial multifocal esophageal SCCs for p53 gene mutation and genetic instability, using DNA extracted from microdissected areas. A total of 38 target areas (TAs) were analyzed in SCC, dysplasia, basal cell hyperplasia (BCH) and normal squamous epithelium. Analysis of the replication error (RER) at 10 microsatellite loci showed microsatellite instability in all TAs, as well as in normal squamous epithelium. p53 gene mutation was identified in 28.9% (11/38 TAs). All cases showed a common missense mutation in exon 8 at codon 273 (CGT-->CAT, Arg-->His), which was DNA contact mutation in the S10 beta strand. In association with microsatellite alterations, 7 of 9 TAs with p53 mutation in exon 8 at codon 273 also showed loss of heterozygosity (LOH) of p53 gene. LOH of p53 gene was detected in 83.8% (31/37 TAs). LOH at D2S123 on 2p16 near MSH2 gene and at D3S1611 on 3p22 near MLH1 gene was detected in 65.4% (17/26) and 71.4% (10/14) TAs, respectively. Frequencies of LOH at p53 and D2S123 were similar in non-cancerous areas and SCCs. LOH of p53 and D2S123 were found in 50% (5/10 TAs) of non-cancerous areas and 60% (9/15 TAs) of SCCs. Our results suggest that genetic instability induces esophageal tumor multicentricity, and that p53 gene contact mutation together with LOH are early events of the multistage carcinogenesis of multifocal primary esophageal SCC.

Fluorescence in situ hybridization analyses of chromosome band 1p36 in neuroblastoma detect two classes of alterations.

Chromosomal alterations in 1p36 were investigated in 196 neuroblastoma tumors using fluorescence in situ hybridization. Additionally, by using the same technique, it was determined whether MYCN was amplified in 149 of these. The most frequent finding was a deletion in 1p36, leading to monosomy of this region (29 cases, 15%). Furthermore, we found tumors with at least two intact copies of chromosome 1 and additional 1p36-deleted copies. Altogether, 21 tumors (11%) displayed this imbalance of 1p36. Similar to the cases with deletion, imbalances were predominantly found in stage 4 tumors (81%), and they were significantly associated with an increased patient age (P = 0.01). Nearly all 1p-deleted tumors showed amplification of MYCN (24/27 analyzed samples, 89%), whereas only 8 of 21 (38%) with imbalance did. Eight cases with imbalance were investigated for loss of heterozygosity (LOH) using microsatellite markers in 1p35-36. Only 4 displayed 1p36 LOH, whereas the remaining 4 were heterozygous. Both patients with deletion of 1p and with imbalance had a poor outcome [3-year rate of event-free-survival (EFS): 33 +/- 15% and 41 +/- 15%], which was significantly worse compared to the outcome of patients without 1p alterations (3-year EFS: 70 +/- 5%; P = 0.01 and P = 0.0059). We conclude that besides monosomic short arm deletions, imbalance of 1p36 is a strong marker of a poor prognosis in neuroblastoma and not necessarily associated with MYCN amplification and LOH.

Caspase-9 and Apaf-1 are expressed and functionally active in human neuroblastoma tumor cell lines with 1p36 LOH and amplified MYCN.

Important roles have been suggested for caspase-8, caspase-9 and Apaf-1 in controlling tumor development and their sensitivity to chemotherapeutic agents. Methylation and deletion of Apaf-1 and CASP8 results in the loss of their expression in melanoma and neuroblastoma, respectively, while CASP9 localization to 1p36.1 suggests it is a good candidate tumor suppressor. The status of CASP9 and Apaf-1 expression in numerous neuroblastoma cell lines with/without amplified MYCN and chromosome 1p36 loss-of-heterozygosity (LOH) was therefore examined to test the hypothesis that one or both of these genes are tumor suppressors in neuroblastoma. Although CASP9 is included in the region encompassing 1p36 LOH in all neuroblastoma cell lines examined, the remaining CASP9 allele(s) express a functional caspase-9 enzyme. Apaf-1 is also expressed in all neuroblastoma tumor cell lines examined. Thus, the CASP9 or Apaf-1 genes do not appear to function as tumor suppressors in MYCN amplified neuroblastomas. However, approximately 20% of the neuroblastoma cell lines with methylated CASP8 alleles are also highly resistant to staurosporine (STS)- and radiation-induced cell death, presumably because cytochrome c is not released from mitochondria. This suggests that a second, smaller sub-group of MYCN amplified neuroblastoma tumors exists with defect(s) in apoptotic signaling components upstream of caspase-9 and Apaf-1. Since no consistent differences in Bcl-2, Bcl-x(L) or Bax expression were seen in the STS- and radiation-resistant neuroblastomas, it suggests that a unique mitochondrial signaling factor(s) is responsible for the defect in cytochrome c release in this sub-group of tumors.

Reciprocal relationship between methylation status and loss of heterozygosity at the p14(ARF) locus in Australian and South African hepatocellular carcinomas.

Chromosome 9p21, a locus comprising the tumor suppressor genes (TSG) p16(INK4a) and p14(ARF), is a common region of loss of heterozygosity (LOH) in hepatocellular carcinoma (HCC). p14(ARF) shares exon 2 with p16 in a different reading frame. p14 binds to MDM2 resulting in a stabilization of functional p53. This study examined the roles of p14, p16 and p53 in hepatocarcinogenesis, in 37 Australian and 24 South African patients. LOH at 9p21 and 17p13.1, p14 and p16 mutation analysis, p14 and p16 promoter methylation and p14, p16 and p53 protein expression was examined. LOH at 9p21 was detected more frequently in South African HCC (P = 0.04). Comparable rates of p53 LOH were observed in Australian and South African HCC (10/22, 45%vs 13/22, 59%, respectively). Hypermethylation of the p14 promoter was more prevalent in Australian HCC than in South African HCC (17/37, 46%vs 7/24, 29%, respectively). In Australian HCC the prevalence of p14 methylation increased with age (P = 0.03). p16 promoter methylation was observed in 12/37 (32%) and 6/24 (25%) in Australian and South African HCC, respectively. Loss of p16 protein expression was detected in 14/36 Australian HCC whereas p53 protein expression was detected in 9/36. Significantly, a reciprocal relationship between 9p21 LOH and p14 promoter hypermethylation was observed (P < or = 0.05). No significant association between p14 and p53 was seen in this study. The reciprocal relationship identified indicates different pathways of tumorigenesis and likely reflects different etiologies of HCC in the two countries.

Prognostic impact of molecular genetic alterations in hepatoblastoma

Background During recent years, we identified characteristic genetic alterations in sporadic hepatoblastoma (HB). These include loss of heterozygosity (LOH) at the chromosomal region 11p15.5, LOH on chromosome arms 1p and 1q, activating mutations in exon 3 of the β-catenin gene, as well as overexpression of the c-met oncogene, which encodes the hepatocyte growth factor receptor. We now wanted to evaluate the prognostic relevance of these abnormalities concerning the outcome in a large group of patients. Procedure All but 7 of 56 patients with HB were treated either with primary resection for small tumors, or neo-adjuvant chemotherapy with ifosfamide, cisplatin and doxorubicin (IPA) before delayed surgery in case of extended disease and additional adjuvant IPA therapy in all cases. Seven tumors were primarily resected and adjuvantly treated with different cytotoxic drugs. LOH 11p15.5, LOH 1p, and LOH 1q were detected in tumor tissue in comparison to normal liver and/or peripheral blood leukocytes by PCR based microsatellite analysis. Beta-catenin mutations were analysed with SSCP, deletion screening and direct sequencing. RT-PCR was used for identification of c-met mRNA overexpression. The results were correlated with the tumors' stage, histological type and epithelial differentiation, as well as with the patients' outcome. Results Overall disease-free survival after median follow-up of 5 years was 43/56 patients (77%). LOH 11p15.5 was found in 15/56, LOH1p in 11/53, LOH1q in 7/53 and β-catenin mutations in 25/55 HB. 13/23 HB had a c-met overexpression. LOH 11p15.5 and LOH 1p were significantly more often found in embryonal HB, while there was no correlation of other genetic alterations with histological type or differentiation. Furthermore, statistical analysis revealed no correlation of any of these disorders with initial tumor stage, nor with the patients' outcome. Conclusion None of the investigated molecular genetic alterations are suited to serve as a prognostic indicator in HB. Further investigations, especially genetic screening of tumor tissue may reveal prognostic markers for this neoplasm. Med Pediatr Oncol 2002;38:104–108. © 2002 Wiley-Liss, Inc.

Gene therapy for pancreatic cancer based on genetic characterization of the disease.

In order to develop an effective therapeutic intervention for patients with pancreatic cancer, we analyzed the association of loss of heterozygosity (LOH) with the clinicopathological features of the disease. Based on these results, we are developing a new gene therapy that targets the genetic character of pancreatic cancer, using mutant adenoviruses that are selectively replication-competent in tumor cells. LOH of 30% or more was observed on chromosome arms 17p (47%), 9p (45%), 18q (43%), 12q (34%), and 6q (30%). LOH of 12q, 17p, and 18q showed significant association with poor prognosis. These data strongly suggest that mutations of the putative tumor suppressor genes, TP53 and SMAD4, play significant roles in the disease progression. Based on this rationale, we are developing a new gene therapy that targets tumors without normal TP53 function. The E1B-55kDa-deleted adenovirus can selectively replicate in TP53-deficient human tumor cells, but not in cells with functional TP53. We evaluated the therapeutic effect of this E1B-55kDa-deleted mutant adenovirus on pancreatic cancer without normal TP53 function. The growth of a human pancreatic tumor in a severe combined immunodeficiency (SCID) mouse model was markedly inhibited by consecutive injections of the E1B-55kDa-deleted adenovirus. Furthermore, the replication-competent adenovirus is not only a strong weapon itself but it is also a useful carrier of genes that possess antitumor activities, as a virus vector specific to tumors without normal TP53 function.

Prognostic impact of molecular genetic alterations in hepatoblastoma

During recent years, we identified characteristic genetic alterations in sporadic hepatoblastoma (HB). These include loss of heterozygosity (LOH) at the chromosomal region 11p15.5, LOH on chromosome arms 1p and 1q, activating mutations in exon 3 of the beta-catenin gene, as well as overexpression of the c-met oncogene, which encodes the hepatocyte growth factor receptor. We now wanted to evaluate the prognostic relevance of these abnormalities concerning the outcome in a large group of patients. All but 7 of 56 patients with HB were treated either with primary resection for small tumors, or neo-adjuvant chemotherapy with ifosfamide, cisplatin and doxorubicin (IPA) before delayed surgery in case of extended disease and additional adjuvant IPA therapy in all cases. Seven tumors were primarily resected and adjuvantly treated with different cytotoxic drugs. LOH 11p15.5, LOH 1p, and LOH 1q were detected in tumor tissue in comparison to normal liver and/or peripheral blood leukocytes by PCR based microsatellite analysis. Beta-catenin mutations were analysed with SSCP, deletion screening and direct sequencing. RT-PCR was used for identification of c-met mRNA overexpression. The results were correlated with the tumors' stage, histological type and epithelial differentiation, as well as with the patients' outcome. Overall disease-free survival after median follow-up of 5 years was 43/56 patients (77%). LOH 11p15.5 was found in 15/56, LOH1p in 11/53, LOH1q in 7/53 and beta-catenin mutations in 25/55 HB. 13/23 HB had a c-met overexpression. LOH 11p15.5 and LOH 1p were significantly more often found in embryonal HB, while there was no correlation of other genetic alterations with histological type or differentiation. Furthermore, statistical analysis revealed no correlation of any of these disorders with initial tumor stage, nor with the patients' outcome. None of the investigated molecular genetic alterations are suited to serve as a prognostic indicator in HB. Further investigations, especially genetic screening of tumor tissue may reveal prognostic markers for this neoplasm.

Loss of heterozygosity of chromosome 3p in patients with carcinoma of the cervix

Loss of heterozygosity (LOH) at chromosome 3p frequently occurs in squamous cell carcinomas of the uterine cervix and indicates the probable sites of tumor suppressor genes that play a role in the development of this tumor. To study such recessive mutations involved in multistep cervical carcinoma, we separated tumors and normal DNA pairs from 43 invasive squamous cell carcinomas of the cervix on archived slides. We studied LOH using polymerase chain reaction (PCR) with 10 polymorphic DNA marker on the 3p12-26.1 region. HPV type 16 and 18 were preformed by PCR on tumor specimens. LOH at one or more loci was observed in 15 cases(35%). HPV DNA of any types was detected in 35 cases of all tumors (81%). HPV 16 was detected in 32 cases(74%) and HPV 18 in 9 cases (21%). In the 32 cases with positive HPV 16 infection, 9 (28%) had LOH, while in the 11 with negative HPV 16 infection, 6(55%) had LOH. Three cases in the 9 positive HPV 18 infection(33%) had LOH, while 12 in the 34 negative HPV 18 infection (35%) had LOH. Our results confirm that no relationship was found between 3p LOH and HPV.

Genetic alterations in serrated adenomas: Comparison to conventional adenomas and hyperplastic polyps

Serrated adenoma is a recently described entity characterized by the presence of a hyperplastic (serrated) growth pattern combined with cytologic features of dysplasia. In contrast to conventional (nonserrated) adenomas, the molecular features of serrated adenomas have been poorly studied. Thus, it remains unclear if serrated adenomas are simply a morphologic variant of conventional adenomas or represent a different biologic entity. In this study, 46 serrated adenomas from 39 patients, 32 conventional (nonserrated) adenomas from 31 patients, and 18 hyperplastic polyps from 16 patients were evaluated for loss of heterozygosity (LOH) of APC, p53, p16, and 3p and for K-ras mutations of codons 12, 13, and 61 by polymerase chain reaction (PCR) analysis. Serrated adenomas demonstrated LOH of at least one genetic locus in 32.6% of cases. LOH of the APC gene, 3p, p53, and p16 was seen in 19.4%, 14.2%, 9.3%, and 13.8% of cases, respectively. K-ras mutations were observed in 18% of cases. Similar to serrated adenomas, conventional adenomas demonstrated at least one LOH event in 37.5% of cases and K-ras mutations in another 19% of cases. LOH of APC, 3p, p53, and p16 was observed in 22%, 33%, 5.8%, and 13.4% of cases, respectively. There were no significant differences in either the total number of genetic events or the presence of LOH of any of the individual markers between serrated adenomas and conventional adenomas. However, hyperplastic polyps showed LOH in 22% of cases and a single K-ras mutation (11%). The prevalence of LOH in hyperplastic polyps was lower than both serrated adenomas and conventional adenomas (P <.05). These results support the hypothesis that serrated adenomas represent a biologically similar morphologic variant of conventional adenomas. HUM PATHOL 33:87-91. Copyright © 2002 by W.B. Saunders Company

Studying the heterogeneity of brain tumors using medium throughput LOH analysis.

The theory of tumor progression by clonal evolutionposits that a succession of increasingly malignant clonesarise, in turn, driven by the step-wise accumulation ofmutations (1). At each point in this process it is thoughtthat one cell in the currently dominant clone acquires anew mutation, allowing it to outcompete its siblings. As aresult, its descendants enjoy a higher rate of net growth,making them the new dominant clone whose growtheclipses that of preceding clones, until it too is left behindin the wake of the next mutation. Some direct evidencefor this model exists, for example, from analyzing the roleof TP53 in brain tumors. In an elegant study of primaryand recurrent glioma, it was possible to find the mutationprevalent in recurrent tumors represented in some cells ofthe primary lesion, suggesting that this molecular defectmarked the dominant clone about to emerge (2).In the strictest interpretation of the clonal evolutionmodel, a single clone makes up the bulk of the tumor at agiven stage (Fig. 1a). Any observed tumor heterogeneity isconsidered to be due to the remains of ancestral clones,whose sidelined descendants may persist but do not de-fine the clinical course of the disease. This view fits thedata from many studies where tumors are considered tobe, to a first approximation, homogeneous entities, and allof a tumor’s cells are combined in one sample and ana-lyzed using molecular markers of progression. This inter-pretation also is reassuring in its implication that effectivetreatment will result from targeting the dominant clone.The validity of the inherent assumptions that a tumor canbe treated as a single entity is an increasingly importantconcern, particularly as new therapies based on the per-ceived biological characteristics of an individual patient’stumor, become available.Although what may be termed the dominant cloneinterpretation of the theory of clonal evolution may verywell describe many tumor types, particularly those char-acterized by extreme changes in growth rate and invasive-ness with concomitant well-defined histologic stages, itmay not explain all cancers. Here we discuss data fromanalyses of loss of heterozygosity (LOH), which measuresclonally inherited genetic damage associated with the in-activation of tumor suppressor genes (3). Therefore LOHrelates directly to cancer progression and also serves as atool for following clonal patterns. Our data suggest thatother views of the theory of clonal evolution besides thedominant clone interpretation may need to be consideredin some tumor types.

Deletion mapping of 6q21-26 and frequency of 1p36 deletion in childhood endodermal sinus tumors by microsatellite analysis.

The most common malignant germ cell tumor of early childhood is the endodermal sinus tumor (CEST), also known as yolk sac tumor. Previous cytogenetic studies of CEST have demonstrated recurrent deletion of distal regions of chromosomes 1p and 6q. Studies utilizing comparative genomic hybridization have likewise demonstrated loss of distal 6q, however these studies show discrepant data concerning chromosome 1 abnormalities. This study analyses 18 CESTs for loss of heterozygosity (LOH) of distal chromosome 6q utilizing 17 microsatellite markers and 13 tumors were analysed for LOH of distal 1p using two microsatellite markers. LOH of 6q was found in 13/18 tumors (72 %). This data confirms that loss of genetic material on 6q is one of the most common abnormalities in CESTs and narrows the region of loss, enabling candidate tumor suppressor genes to be identified and analysed. In addition, LOH of 1p36 was identified in five of 11 informative tumors, clarifying prior conflicting data and confirming that 1p deletion is a common event in CESTs.

Cancer risk assessment in long-standing pouchitis

In a small subgroup of patients with ulcerative colitis (UC) undergoing proctocolectomy and restorative ileal pouch-anal anastomosis (IPAA), a colonic-like pouch mucosa with severe and persistent villous atrophy (type C pattern) develops. Neoplastic transformation of the mucosa in the neorectum may occur in these patients. We hypothesized that genetic alterations associated with colorectal carcinoma (CRC) could be an early finding in this transformational process and thus potentially useful as clinical monitors in carcinoma risk assessment. In six patients with long-standing severe pouchitis and a type C-pattern mucosa, biopsies were obtained from five different locations of the pouches. DNA was PCR-amplified and analyzed by automated fragment analysis for loss of heterozygosity (LOH) at chromosome 5q14-22, 17p12-13, and 18q12-22. Point mutations of the K-ras and adenomatous polyposis coli (APC) genes were studied by sequencing. The patients had varying degrees of dysplasia and one displayed DNA aneuploidy. Loss of heterozygosity at 5q15-22 was detected in three of five biopsies in one patient. This particular patient had no signs of dysplasia or DNA aneuploidy and a normal exon 15 sequence of the APC gene. No alterations of either the K-ras or the APC genes or LOH of 5q, 17p, or 18q were seen in any of the other patients. Dysplasia, aneuploidy, and LOH in 5q may all reflect different parts of an atrophic mucosa-dysplasia-carcinoma sequence, in line with current concepts of carcinogenesis for CRC in long-standing pouchitis. Further studies of histological and molecular events in IPAA patients with severe atrophy are warranted.

Molecular heterogeneity of oligodendrogliomas suggests alternative pathways in tumor progression.

To identify different genetic molecular profiles in oligodendrogliomas and to evaluate their prognostic significance. The main genetic alterations reported in glial tumors were investigated in 26 oligodendrogliomas (10 World Health Organization grade II and 16 World Health Organization grade III). Correlation between identified molecular changes and pathologic grade or clinical course was subsequently analyzed using univariate and multivariate statistical analyses. Loss of heterozygosity (LOH) on chromosome 1p, 19q, and 10; P16/CDKN2A homozygous deletion; EGFR (epidermal growth factor receptor) amplification; and TP53 and PTEN mutations were observed in 14 (54%), 15 (58%), 9 (35%), 7 (27%), 5 (19%), 1 (4%), and 0 cases. LOH 1p and 19q were tightly associated (p < 0.0001). A mutual exclusion was found between LOH 1p/19q and EGFR amplification (p = 0.01), P16/CDKN2A deletions (p = 0.001), or LOH on 10q (p = 0.03), suggesting the existence of distinct genetic subsets in oligodendrogliomas. On univariate analysis, age <50 years (p = 0.002) and LOH 1p (p = 0.01) were associated with a longer progression-free survival (PFS) whereas LOH 10q (p = 0.03) and EGFR amplification (p = 0.007) were associated with a worse PFS. In multivariate analyses, age <50 years (p = 0.001) and LOH 1p (p = 0.006) remained independent predictive factors for PFS. These results provide evidence for two alternative molecular pathways of progression in oligodendrogliomas. The first one is associated with loss of 1p and 19q and the second one with P16/CDKN2A deletion, 10q loss, and EGFR amplification. The findings confirm the value of loss of 1p as predictor of longer progression-free survival; in addition, the study demonstrates the unfavorable impact of 10q loss and EGFR amplification on the prognosis.