Loss Of Heterozygosity Patterns Research Articles

High frequency of CDKN2A alterations in esophageal squamous cell carcinoma from a high-risk Chinese population.

Because previous studies have shown that loss of heterozygosity (LOH) is common on chromosome arm 9p in esophageal squamous cell carcinoma (ESCC) and that genetic alterations in CDKN2A and CDKN2B on 9p are also common, we sought to determine whether LOH and these genetic alterations are related. We performed LOH studies on chromosome bands 9p21-p22 and searched for genetic alterations of CDKN2A and CDKN2B in 56 ESCCs from a high-risk Chinese population. Seventy-three percent of patients were found to have LOH at one or more loci on chromosome bands 9p21-p22, and LOH occurred more frequently in patients with a family history of upper gastrointestinal cancer than in those with a negative family history (P = 0.01, global permutation test). CDKN2A mutations (point mutations, deletions, insertions) were observed in 25% (14 of 56) of cases, and the LOH pattern was significantly different for individuals with and without a CDKN2A mutation (P = 0.01, global test). Three new single nucleotide polymorphisms (SNPs) and 2 previously reported SNPs were identified in this group of patients. Intragenic allelic loss at polymorphic sites in CDKN2A was detected in 32% (18 of 56) of patients. Seven of the 56 (13%) cases exhibited what is considered classic evidence (n = 4) or showed potential evidence (n = 3) of biallelic inactivation. Only one alteration was observed in CDKN2B, G171A in the 5' untranslated region. Both mutation and intragenic allelic loss in CDKN2A appear to play a role in the development of ESCC.

Genetic evolution of α fetoprotein producing gastric cancer

Background: α Fetoprotein (AFP) producing gastric cancer is an unusual form of aggressive adenocarcinoma with a complex histological picture, including enteroblastic and hepatoid differentiation. Aims: To investigate the genetic events...

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

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

Flow Cytometric Sorting of Paraffin-Embedded Tumor Tissues Considerably Improves Molecular Genetic Analysis

The characterization of genetic aberrations in paraffin-embedded tumor material is impaired by contaminating normal cells. In the present study on the genetic causes of loss of HLA expression in diffuse large B-cell lymphoma (DLBCL), we compared the efficacy of microdissection with flow cytometric sorting of tumor cells. Single-cell suspensions from paraffin-embedded material of 5 DLBCL cases were stained for CD79a and DNA content (propidium iodide). Fluorescent in situ hybridization (FISH) using HLA class II and chromosome 6 centromeric probes and loss of heterozygosity (LOH) analysis with 5 HLA-specific microsatellite markers were performed on microdissected and flow cytometry– sorted fractions. FISH confirmed considerable enrichment of the samples after flow cytometric sorting and disclosed tumor heterogeneity in 4 cases. Moreover, lymphomas with a so-called zebra LOH pattern in the microdissected material showed unambiguous LOH after flow cytometric sorting, revealing in 1 case a biologically relevant hemizygous deletion in the HLA region.

Microallelotyping Defines the Monoclonal or the Polyclonal Origin of Mixed and Collision Endocrine-Exocrine Tumors of the Gut

Mixed endocrine-exocrine tumors of the gut are a heterogeneous group of neoplasms with uncertain histogenesis showing different morphologic and clinical features. The aim of this work is to clarify the histogenesis of these tumors by studying the genetic profile of both the endocrine and exocrine components. We performed an allelotyping analysis of five mixed endocrine-exocrine tumors (two gastric and three colonic) and one rectal collision tumor, using 35 polymorphic microsatellite markers covering a total of six chromosomes, including 3, 5q, 6, 11, 17, and 18. The loss of heterozygosity (LOH) analysis showed concurrent losses of the same allele in both the endocrine and exocrine components in all of the five mixed tumors composed by a poorly differentiated endocrine carcinoma or a well differentiated endocrine carcinoma associated with adenocarcinoma or adenoma. Among these tumors an identical LOH pattern was frequently found on chromosomes 17p, 18q, and 5q. Additional allelic losses restricted to the poorly differentiated endocrine carcinoma were often observed. On the contrary, in the only collision tumor composed by a well differentiated endocrine carcinoma associated with adenocarcinoma, completely different allelotypes between the two components were detected. These findings confirm a close genetic relationship between the two distinct histologic components within mixed endocrine-exocrine tumors, supporting the hypothesis that a monoclonal mechanism of tumorigenesis is the most frequent genetic event in mixed exocrine-endocrine tumors. The clonal divergence observed in the only collision tumor, composed by a well differentiated endocrine carcinoma associated with an adenocarcinoma, confirms the existence of double tumors growing next to each other coincidentally but showing different histogenesis and different tumorigenetic pathways.

Fractional allele loss is a valuable marker for human lung cancer detection in sputum

Using PCR-based microsatellite DNA analysis with 48 markers we examined sputum and bronchial washing for genetic alterations compared with lymphocyte extracted DNA from 124 lung cancer patients and 36 healthy donors as normal control. Microsatellite alterations (MA) in at least one locus were detected in all cancer patient-derived specimens but only in 22.2% of the healthy donors. Loss of heterozygosity (LOH) was detected in bronchial washings from 101 non-small cell lung cancer (NSCLC) predominantly on 17p13.1–p13.3 (69.7%), 9p13.3–p24.1 (63.3%), 1p34.2–p36.22 (48.5%), 13q12.1–q13.1 (47.7%) and 3p22.3–p23 (42.7%). In bronchial washings from 23 small cell lung cancer (SCLC) LOH was detected mostly on 3p22.3–p23 (88.6%), 17p13.1–p13.2 (82.3%), 5q32–q33.1 (66.6%), 13q12.2–q13.1 (65.6%) and 9q22.33–q31.3 (52.9%). The different LOH patterns indicate that different genetic background may be responsible for the different physiology of NSCLC and SCLC. The fractional allele loss (FAL) mean value of all cancer specimens was 0.243±0.021 compared with 0.007±0.008 of healthy donors with a confidence interval (CI) 99.5%. Only seven out of 124 lung cancer specimens (5.2%) exhibited FAL value less than 0.083, the highest was observed in the healthy donors group. FAL appears to be a likely indicator for lung cancer detection. Microsatellite instability (MIN) was detected in 8.7% of SCLC and 4.0% of NSCLC bronchial washings in at least three loci tested. LOH and MIN detection in sputum and bronchial washing from the same patient was 77.6%. Calculation of these indexes per marker exhibits significant variations that could be attributed to diffuse lung disorders or non-cancer specific genetic alterations.

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.

Deletion Mapping of Chromosome 1 in Early Onset and Late Onset Breast Tumors – AComparative Study in Eastern India

Younger women exhibit more aggressive pathologic features of breast cancer (BC) compared to their older counterparts. Young age has been shown to be an independent predictor of adverse prognosis. These findings have raised the question of whether these differences are also present at the genetic level. Twenty-five early onset (age < or = 40 years) tumors including 4 bilateral tumors, and 26 late onset (>40 years) breast tumors, including 2 bilateral tumors, were examined for loss of heterozygosity (LOH) at chromosome 1 using 11 polymorphic microsatellite markers. A comparative study revealed high frequencies of LOH in chr. 1p36 (61%), 1p31.3 (40%), 1p21.3 (50%) and 1q22-23.2 (56%) in a younger group, and chr. 1p36 (46%), 1p34.2 (48%), and 1q22-23.2 (52%) in an older group. These differences in LOH frequency in these two age groups were significant for chr. 1p21.3 (p = 0.025) only. These data suggest that the deletion pattern in early onset breast tumors is not fully identical to late onset breast tumors. Similar differential deletion patterns of LOH in the 5 highly deleted regions were seen in premenopausal and postmenopausal groups. An association was seen between LOH at chr. 1p34.2 and chr. 1q22-23.2 and higher grade of the tumors in older women. Among the highly deleted regions, the deletion at chr. 1p36 was found to occur early in both groups because of common allelic loss in the bilateral tumors.

Chemical carcinogens induce varying patterns of LOH in mouse T-lymphocytes.

We have shown previously that a wide range of mutagenic carcinogens are capable of inducing loss of heterozygosity (LOH) at the endogenous Aprt locus in mouse splenic lymphocytes. To investigate whether LOH might be caused by a single common mechanism, we set out to determine the extent of LOH by microsatellite analysis along (the Aprt gene containing) mouse chromosome 8. Aprt+/- hybrid B6C3F1 mice were treated with mutagens that induce different classes of DNA lesions, i.e. bulky DNA adducts, DNA methylation, DNA inter-strand crosslinks or DNA strand breaks. Aprt mutant frequencies (MF) in this C57Bl/6-C3H hybrid background were significantly reduced for mitomycin C (MMC) and methylmethanesulfonate (MMS) in comparison with MF in C57Bl/6 background, suggesting either enhanced repair or reduced formation of MMC- or MMS-induced mutagenic lesions in a hybrid B6C3F1 background. In contrast, Aprt MF after dimethylbenz[a]anthracene (DMBA), methylnitrosurea (MNU) and etoposide treatment were similar in both genetic backgrounds. Microsatellite analysis of Aprt mutant clones indicated a dominant role for mitotic recombination (MR) in generating spontaneous, DMBA- and etoposide-induced LOH at APRT: However, over 80% of the MMC-induced Aprt LOH mutants had lost heterozygosity for all markers tested, suggesting that either the crossover points were located close to the centromere or that these mutants arose by chromosome loss and duplication of the remaining chromosome 8. A substantial fraction (40%) of MNU-induced Aprt mutants had lost the wild-type Aprt allele, but had retained heterozygosity at all polymorphic markers tested at chromosome 8 indicating an important role for deletions in LOH formation by MNU. Patterns of MR differed quite dramatically for the various chemical mutagens tested, suggesting different mechanisms to be involved in inducing recombination between homologous chromosomes. In addition, non-random adduct formation and repair between chromosomal regions, i.e. heterochromatin versus euchromatin, may contribute to a non-random distribution of recombinational crossover points.

Evidence that superficial basal cell carcinoma is monoclonal from analysis of the Ptch1 gene locus.

Superficial basal cell carcinoma (BCC) arises as an apparently multifocal proliferation of tumour nests attached to the epidermis, which is at odds with a monoclonal origin. Computer-assisted reconstruction shows that these nests join in three dimensions, but it remains unknown whether this tumour is monoclonal. An early event in BCC formation, loss of heterozygosity (LOH) at the Patched 1 (Ptch1) locus, can be used as a tool to address whether this tumour is monoclonal. To determine whether superficial BCC is monoclonal by analysing individually microdissected superficial BCC nests and looking for the same pattern of LOH in each. Six cases of superficial BCC were analysed for LOH at the Ptch1 gene locus using the D9S287 microsatellite marker. Identical allelic patterns were sought in each nest from a given tumour. These patterns were no allelic loss, loss of the shorter allele or loss of the longer allele, each with a respective probability of occurrence, as estimated from published findings. All cases were informative. Four cases showed no LOH in each nest and two showed loss of the same allele. If these nests arose independently, then the probability of this result was between 4 x 10-11 and 2 x 10-14. The lack of LOH, seen in four cases, could be due to monoclonal expansion of a cell retaining both D9S287 alleles, or due to a polyclonal proliferation. Therefore, a separate analysis excluding these cases was done, giving a probability of between 2.2 x 10-4 and 1.0 x 10-7. These probabilities were so extreme that it was unlikely that the nests arose independently, thus providing the first molecular evidence that superficial BCC is monoclonal.

Molecular evidence for putative tumour suppressor genes on chromosome 13q specific to BRCA1 related ovarian and fallopian tube cancer

Background/Aims: Loss of heterozygosity (LOH) on chromosome 13q has been reported to occur frequently in human ovarian cancer, and indications have been found that chromosome 13 may also play a...

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.

Metachronous bilateral mammary metaplastic and infiltrating duct carcinomas: A molecular study for clonality

Mammary metaplastic carcinoma is uncommon. In this study, both carcinoma and sarcoma components of a metaplastic carcinoma and a subsequent metachronous contralateral infiltrating ductal carcinoma were analyzed by microsatellite analysis for the loss of heterozygosity (LOH) patterns at multiple sites on chromosome arms 3p, 6q, 8, 9p, 11, 13q, 14q, 16q, and 17p. The LOH patterns between the carcinoma and sarcoma components in the first tumor were similar, indicating clonality. The LOH patterns between the first and second tumors were different at all chromosome arms, indicating different clonality and a second primary. We demonstrated a second primary carcinoma in a patient with previous metaplastic carcinoma rather than a metastasis with carcinoma component only. HUM PATHOL 33:677-679. Copyright 2002, Elsevier Science (USA). All rights reserved.

Detection of genetic alterations induced by low-dose X rays: analysis of loss of heterozygosity for TK mutation in human lymphoblastoid cells.

To elucidate the genetic influence of low-dose ionizing radiation at the chromosome level, we exposed human lymphoblastoid TK6-20C cells to 10 cGy of X rays. The TK mutation frequency was 5.7 +/- 1.3 x 10(-6) at the background level and 6.9 +/- 2.8 x 10(-6) after X irradiation. Although this small increase was not statistically significant (P = 0.40), we applied multilocus analysis using 4 TK locus markers and 12 microsatellite loci spanning chromosome 17 for TK mutants exhibiting loss of heterozygosity (LOH). The analysis demonstrated a clear effect of low-dose ionizing radiation. We observed radiation-specific patterns in the extent of hemizygous LOH in 14 TK mutants among the 92 mutants analyzed. The deleted regions in these patterns were larger than they were in the control mutants, where those restricted to the TK locus. Surprisingly, the radiation-specific LOH patterns were not observed among the 110 nonirradiated TK mutants in this study. They were identified previously in TK6 cells exposed to 2 Gy of X rays. We consider these hemizygous LOH mutants to be a result of end-joining repair of X-ray-induced DNA double-strand breaks.

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.

Genetics of synchronous uterine and ovarian endometrioid carcinoma: Combined analyses of loss of heterozygosity, PTEN mutation, and microsatellite instability

Synchronous development of carcinomas in the endometrium and ovaries is a fairly common phenomenon, but distinction of a single clonal tumor with metastasis from 2 independent primary tumors may present diagnostic problems. To determine clonality and the occurrence of progression, we microdissected multiple foci from 17 cases of synchronous endometrioid carcinomas and studied loss of heterozygosity (LOH), microsatellite instability (MI), and PTEN mutations. In 14 of the 17 cases, genetic alterations were either homogeneous or found in only some of the foci. LOH was detected for 10q (4 cases), 17p (2 cases), and 2p, 5q, 6q, 9p, 11q, 13q, and 16q (1 case each). Four cases had the MI phenotype with discordant MI patterns between both tumor sites, thus indicating a biclonal or triple clonal process. In 3 of 6 cases with PTEN mutations, identical mutations in both tumor sites indicated a single clonal neoplasm. Altogether, 14 synchronous tumors were genetically diagnosed as follows: single clonal tumor, characterized by concordant genetic alterations in both tumor sites, including identical LOH, identical PTEN mutations, and/or identical sporadic allelic instability patterns (4 cases); single clonal tumor with genetic progression, homogeneous LOH or identical PTEN mutations in both tumor sites and progressive LOH in ovarian metastatic foci (2 cases); and double (7 cases) or triple clonal tumors (1 case), determined by discordant PTEN mutations, heterogeneous LOH, and/or discordant MI patterns. Thus, 35% of synchronous tumors were monoclonal, 47% were polyclonal, and 18% were undetermined. The favorable prognosis of synchronous endometrioid carcinomas may be due to the occurrence of PTEN mutations in both independent and metastatic tumors, the MI-positive independent primary tumors, and the low frequency of LOH. HUM PATHOL 33:421-428. Copyright 2002, Elsevier Science (USA). All rights reserved.

Loss of heterozygosity analysis of cutaneous melanoma and benign melanocytic nevi: Laser capture microdissection demonstrates clonal genetic changes in acquired nevocellular nevi

The molecular pathology of the common nevocellular nevus (NCN) and its relationship to the genetic model of malignant melanoma (MM) progression has not been fully characterized. We used laser capture microdissection of archival formalin-fixed material to study 34 melanocytic lesions (19 MM and 15 NCN). Twelve of the 15 NCN were acquired, 3 of which were congenital; none had dysplastic features. Ten polymorphic markers on five chromosomal regions (1p36, 6q22-23.3, 8p22-24, 10q23, and 11q23) were selected for loss of heterozygosity (LOH) analysis, based on previous studies demonstrating involvement in MM pathogenesis and progression. Loss of heterozygosity at any allelic locus was observed in 18 of 19 (95%) MM and in 9 of 15 (60%) NCN. Of the three congenital nevi analyzed, none showed LOH at any informative locus. The frequency of allelic loss was highest in the MM at 6q22-23.3 (64%), followed by 10q23 (62%), 8p22-24 (43%), 11q23 (43%), and 1p36 (13%). In the 15 NCN, the most frequent allelic losses were detected at 6q22-23.3 (29%), 1p36 (27%), and 10q23 (25%), with lower frequencies of LOH at 11q23 (10%) and 8p22-24 (7%). LOH at a single polymorphic marker, D6S1038, was detected in 70% of the MM and in 36% of the NCN, suggesting the presence of putative tumor-suppressor genes (TSGs) critical in melanocytic neoplasia at 6q22-23.3. The presence of clonal genetic alterations in acquired NCN justifies their classification as a benign neoplasm. The pattern of LOH in NCN is not random; rather, the relative frequencies of LOH at the chromosomal regions examined are consistent with a multistep model of MM progression that begins with NCN. Molecular analysis of NCN reiterates established epidemiologic and morphologic notions that NCN are precursor lesions for MM. HUM PATHOL 33:191-197. Copyright 2002, Elsevier Science (USA). All rights reserved.

Mapping common deleted regions on 5p15 in cervical carcinoma and their occurrence in precancerous lesions

BackgroundPrevious studies have shown that the short arm of chromosome 5 (5p) exhibit frequent genetic changes in invasive cervical carcinoma (CC), and that these changes arise early during the carcinogenesis, in precancerous lesions. These data therefore suggest that loss of candidate tumor suppressor genes located on 5p is associated with the development of CC. However, the precise location of 5p deletions is not known.ResultsWe performed a detailed deletion mapping of 5p in 60 cases of invasive CC. We found that 60% of the tumors exhibit a 5p loss of heterozygosity (LOH). The patterns of LOH allowed us to identify two minimal regions of deletions, one at 5p15.3 spanning a 5.5 cM genetic distance and a second site of 7 cM at 5p15.2-15.3. In addition, we also identified 5p deletions in 16% lesions of high-grade cervical intraepithelial neoplasia (CIN). 5p LOH was found in 63% of HPV 16 positive tumors, while only 33% tumors with other HPV-types had 5p LOH. The differences in frequency of 5p LOH between tumors harboring HPV16 in combination with other HPV types and tumors harboring HPV16 DNA alone were significantly higher, suggesting a synergistic effect of high-risk types in causing genomic instability.ConclusionThese findings implicate the presence of tumor suppressor gene(s) on 5p relevant to CC tumorigenesis.

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.

Loss of heterozygosity in tumour-adjacent normal tissue of breast and bladder cancer

Normal tumour-adjacent breast tissue samples from 12 breast cancer patients forming six monozygotic twin pairs were analysed for loss of heterozygosity (LOH) on chromosomes 1, 13 and 17. 7 patients showed LOH at one or more markers. Each of them had a different LOH pattern. Only one twin pair showed LOH at the same locus, but the twins had lost a different allele. Multiple (n=1–13), histologically normal samples were collected from 6 bladder cancer patients and analysed for LOH on chromosomes 3 and 9. On chromosome 9, all 6 patients analysed showed LOH in at least one sample and one marker. Four of them also showed LOH on chromosome 3. Samples surrounding different tumours of a given patient resembled each other. More heterogeneity was seen between the patients, even though they shared some similarities in LOH clustering. The results demonstrate that tumour-adjacent normal tissues already harbour genetic changes typical for tumours. These alterations can reveal the earliest changes leading to tumorigenesis.