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Candida metapsilosis is a member of the Candida parapsilosis species complex, a group of opportunistic human pathogens. Of all the members of this complex, C. metapsilosis is the least virulent, and accounts for a small proportion of invasive Candida infections. Previous studies established that all C. metapsilosis isolates are hybrids, originating from a single hybridization event between two lineages, parent A and parent B. Here, we use MinION and Illumina sequencing to characterize a C. metapsilosis isolate that originated from a separate hybridization. One of the parents of the new isolate is very closely related to parent A. However, the other parent (parent C) is not the same as parent B. Unlike C. metapsilosis AB isolates, the C. metapsilosis AC isolate has not undergone introgression at the mating type-like locus. In addition, the A and C haplotypes are not fully collinear. The C. metapsilosis AC isolate has undergone loss of heterozygosity with a preference for haplotype A, indicating that this isolate is in the early stages of genome stabilization.

Background: Breast cancer is a heterogeneous disease that can be subdivided on the basis of histopathological features, genetic alterations, and gene-expression profiles. PTEN gene is considered an established tumor suppressor gene in different types of cancer including breast cancer. However, the role of PTEN alterations in north Indian breast cancer has not been explored especially in defining a group with distinct histological factors. Methodology: 181 sporadic breast cancer and their adjacent normal tissues were included in the present study. We analyzed methylation and LOH through MS-PCR and microsatellite markers respectively. While, for PTEN protein expression, we used immunohistochemistry. All the molecular findings were correlated with the clinicopathological parameters of the patients to underline clinical relevance. Results: We found that LOH and methylation of the PTEN promoter were significantly associated with loss of PTEN protein expression, while, PTEN mutation was a rare event. Furthermore, out of 46 double hit cases (i.e., having both methylation and LOH), 70% (32/46) cases showed complete loss of PTEN expression (P= 0.0249). Both LOH and PTEN promoter methylation were associated significantly with age and clinical stage, while, methylation and loss of PTEN expression were associated with high grade and Her-2 negativity. In addition, a quadruple (ER/PR/ Her-2 and PTEN) negative group with distinct features was found. Conclusion: The pattern of PTEN expression and its correlation with the clinical parameters indicates that loss of PTEN expression defines a clinical group with distinct features. Hence, PTEN expression provides differential therapeutic strategies for north Indian breast cancer.

SUMMARY Although aneuploidy is found in the majority of tumors, the degree of aneuploidy varies widely. It is unclear how cancer cells become aneuploid or how highly aneuploid tumors are different from those of more normal ploidy. We developed a simple computational method that measures the degree of aneuploidy or structural rearrangements of large chromosome regions of 522 human breast tumors from The Cancer Genome Atlas (TCGA). Highly aneuploid tumors overexpress activators of mitotic transcription and the genes encoding proteins that segregate chromosomes. Overexpression of three mitotic transcriptional regulators, E2F1, MYBL2, and FOXM1, is sufficient to increase the rate of lagging anaphase chromosomes in a non-transformed vertebrate tissue, demonstrating that this event can initiate aneuploidy. Highly aneuploid human breast tumors are also enriched in TP53 mutations. TP53 mutations co-associate with the overexpression of mitotic transcriptional activators, suggesting that these events work together to provide fitness to breast tumors., In Brief Pfister et al. analyzed TCGA sequence data to identify drivers of aneuploidy in breast tumors. TP53 is mutated in most aneuploid tumors, and a large number of genes that control mitosis are overexpressed. The oncogenes E2F1, MYBL2, and FOXM1 that regulate mitotic transcription drive the overexpression of mitotic proteins to lower the fidelity of chromosome segregation.

Candida krusei is a diploid, heterozygous yeast that is an opportunistic fungal pathogen in immunocompromised patients. This species also is utilized for fermenting cocoa beans during chocolate production. One major concern in the clinical setting is the innate resistance of this species to the most commonly used antifungal drug fluconazole. Here, we report a high-quality genome sequence and assembly for the first clinical isolate of C. krusei, strain 81-B-5, into 11 scaffolds generated with PacBio sequencing technology. Gene annotation and comparative analysis revealed a unique profile of transporters that could play a role in drug resistance or adaptation to different environments. In addition, we show that, while 82% of the genome is highly heterozygous, a 2.0 Mb region of the largest scaffold has undergone loss of heterozygosity. This genome will serve as a reference for further genetic studies of this pathogen.

Background: Diffuse gastric cancer (DGC) is associated with the reduction or absence of the expression of the cell adhesion protein E-cadherin (encoded by the CDH1 gene). Molecular characteristics are less well described for mixed gastric cancer (MGC). The main somatic alterations that have been described in the CDH1 gene are mutations, loss of heterozygosity (LOH) and promoter methylation. The aim was to analyze CDH1 somatic alterations in Mexican patients with diffuse and mixed gastric cancer. Methods: We searched for mutations in the CDH1 gene in tumor DNA from DGC (n = 13) and MGC (n = 7) patients by next generation sequencing (NGS). Validation of findings was performed using Sanger sequencing. LOH was analyzed using dinucleotide repeat markers surrounding the CDH1 gene, and methylation was investigated by DNA bisulfite conversion and sequencing. E-cadherin protein deficiency was analyzed by immunohistochemistry. Results: Seventeen point variants were identified by NGS, 13 of them were validated by Sanger sequencing. Only 1/13 had not been previously reported (c.-137C > A), and 12/13 were already reported as polymorphisms. Two DGC cases presented LOH at the locus 16q22.1 (13.3%). CDH1 promoter methylation was positive in (7/11) 63.6% and (4/6) 66.6% of the cases with DGC and MGC, respectively. E-cadherin protein deficiency was observed in 58.3% of DGC cases while 100% in MGC cases. Conclusions: While no pathogenic somatic mutations were found that could explain the diffuse histology of gastric cancer in DGC and MGC, methylation was the most common somatic inactivation event of the CDH1 gene, and LOH was rare. The previously unreported c.-137C > A variant modify the CDH1 gene expression since it alters the binding sites for transcription factors. This work was supported by the “Consejo Nacional de Ciencia y Tecnología (CONACYT)” (grant Ciencia Básica 2013–1-222972) and by the “Fondo de Investigación en Salud (FIS), Instituto Mexicano del Seguro Social (IMSS)” (grant FIS/IMSS/PROT/G13/1189), that contributed to the design of the study, collection, analysis, interpretation of data and writing the manuscript. We thank the following institutions: 1) Coordinación de Investigación en Salud (CIS), Instituto Mexicano del Seguro Social (IMSS) for the support given to ARBC, through the Professional Development in the International Research of Graduate Students (PRODESI) program; 2) Fundación IMSS, A.C. for the research grant awarded to JYSL and MTMT; 3) FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operational Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT/ Ministério da Ciência, Tecnologia e Inovação in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274); 4) The project NORTE-01-0145-FEDER-000029, supported by Norte Portugal Regional Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); 5) FCT fellowships (SFRH/BPD/86543/2012 to JC; SFRH/BPD/89764/2012 to PO; PD/ BD/128007/2016 to AS). IPATIMUP integrates the i3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology.

We conducted the largest investigation of predisposition variants in cancer to date, discovering 853 pathogenic or likely pathogenic variants in 8% of 10,389 cases from 33 cancer types. Twenty-one genes showed single or cross-cancer associations, including novel associations of SDHA in melanoma and PALB2 in stomach adenocarcinoma. The 659 predisposition variants and 18 additional large deletions in tumor suppressors, including ATM, BRCA1, and NF1, showed low gene expression and frequent (43%) loss of heterozygosity or biallelic two-hit events. We also discovered 33 such variants in oncogenes, including missenses in MET, RET, and PTPN11 associated with high gene expression. We nominated 47 additional predisposition variants from prioritized VUSs supported by multiple evidences involving case-control frequency, loss of heterozygosity, expression effect, and co-localization with mutations and modified residues. Our integrative approach links rare predisposition variants to functional consequences, informing future guidelines of variant classification and germline genetic testing in cancer. A pan-cancer analysis identifies hundreds of predisposing germline variants.

Background: mutS homolog 2 (MSH2) deficiency may be involved in the development of microsatellite instability found in certain sporadic colorectal tumors. In addition to mutations or loss of heterozygosity resulting in complete loss of MSH2 function, polymorphisms affecting MSH2 expression have been also identified. Therefore, the aim of this study was to examine MSH2 status in sporadic colon cancer. Materials and Methods: MSH2 status was examined at the DNA, RNA and protein levels through loss of heterozygosity (LOH) analysis, quantitative real-time PCR and immunohistochemistry. MSH2 IVS10+12A>G polymorphism was examined by real-time single nucleotide polymorphism genotyping. Results: MSH2 LOH was more frequent in tumors larger than 5 cm (p=0.032), mRNA expression was also significantly lower and the same expression pattern was present in the corresponding normal mucosa of the same patient (p=0.013 and p=0.008, respectively). No association was found between IVS10+12A>G polymorphism and susceptibility to sporadic colon cancer. Conclusion: Altered MSH2 expression detected in sporadic colon tumors pointing to its role in colorectal tumorigenesis without a hereditary component.

Background: Germline BRCA2 mutations are associated with poorer outcome prostate cancer ( PCa) compared with sporadic tumours but this association remains to be characterised. In this study, we aim to assess if there is a signature set of copy number alterations ( CNA) that could aid to the identification of BRCA2- mutated tumours and would assist us to understand their aggressive clinical behaviour. Methods: High- resolution array comparative genomic hybridisation profiling of DNA from PCa and matched morphologically normal prostate samples from 9 BRCA2 germline mutation carriers and 16 non- carriers in combination with unsupervised analysis was used to define copy number features. Results: PCa from BRCA2 germline mutation carriers ( B2T) harbour significantly more CNA than non- carrier tumours ( NCTs) ( P = 14 x 10(-6)). A hundred and sixteen regions had a significantly different distribution with both false discovery rate ( FDR) and P value

Evolution of intratumoral genetic heterogeneity during colorectal tumor progression has not been investigated so far. Multiple sample areas in colorectal adenocarcinoma at early and advanced stages and in metastases were studied for the well-known genetic alterations: K-ras and p53 point mutations and loss of heterozygosity (LOH) on chromosomes 5q and 18q. In primary colorectal cancers (CRCs), intratumoral genetic heterogeneity was more often observed in early than in advanced stages, at 90 and 67%, respectively. All but one of the advanced CRCs were composed of one predominant clone and other minor clones, whereas no predominant clone has been identified in half of the early cancers. At the early stage, the last events that were produced, the p53 mutation and LOH of 18q, were also the most heterogeneous. At the advanced stage, the LOH of 5q and 18q were the most frequent heterogeneous events (67 and 58%, respectively). The intratumoral heterogeneity for mutations was significantly reduced, from the early to the advanced stages (from 60 to 20% for K-ras and from 70 to 20% for p53). On the other hand, a quasi absence of intratumoral genetic heterogeneity was observed for K-ras and p53 in distant metastasis. In conclusion, colorectal adenocarcinomas are characterized by marked intratumoral genetic heterogeneity. A reduction of the intratumoral genetic heterogeneity for point mutations and a relative stability of the heterogeneity for allelic losses indicate that, during the progression of CRC, clonal selection and chromosome instability continue, while an increase cannot be proven.

Purpose Tumor supressor gene FHIT was identified at chromosome 3p14.2 spanning the FRA3B fragile site and is very often inactivated in different types of cancer. The aim of this study was to examine the frequency of FHIT gene LOH as well as FHIT mRNA and protein expression in sporadic colon adenocarcinoma. Methods The results of LOH, real-time qRT-PCR and imunohistochemical analyses were correlated with clinico-pathological characteristics of patients and their tumors in order to evaluate the role of FHIT gene/protein in sporadic colon adenocarcinoma tumorigenesis. Results One hundred and thirty one (96.3%) samples were informative for both markers and 33/131 (25.2%) demonstrated LOH. Expression of FHIT mRNA was significantly decreased in colon tumors relative to that in corresponding normal tissue (p = 7.2 × 10 − 6 ). Most of the samples (54.0%) were negative for FHIT protein, 26.4% adenocarcinomas showed a weak to moderate immunostaining and 19.6% adenocarcinomas showed strong FHIT immunostaining. No correlation was found between FHIT gene LOH status, mRNA expression or FHIT protein immunostaining and clinico-pathological characteristics. Expression of FHIT mRNA was significantly decreased in FHIT LOH positive tumors (p = 0.027). Patients with LOH negative tumors or FHIT protein positive tumors had longer survival but this findings were not statistically significant. Conclusions Our overall results suggest that reduced expression of FHIT gene may be associated with the progression of these malignant tumors.

Background Epigenetic regulators play a critical role in the maintenance of specific chromatin domains in an active or repressed state. Disruption of epigenetic regulatory mechanisms is widespread in cancer cells and largely contributes to the transformation process through active repression of tumor suppressor genes. While mutations of epigenetic regulators have been reported in various lymphoid malignancies and solid cancers, mutation of these genes in HTLV-I-associated T-cell leukemia has not been investigated. Method Here we used whole genome next generation sequencing (NGS) of uncultured freshly isolated ATL samples and identified the presence of mutations in SUZ12, DNMT1, DNMT3A, DNMT3B, TET1, TET2, IDH1, IDH2, MLL, MLL2, MLL3 and MLL4. Results TET2 was the most frequently mutated gene, occurring in 32 % (10/31) of ATL samples analyzed. Interestingly, NGS revealed nonsense mutations accompanied by loss of heterozygosity (LOH) in TET2 and MLL3, which was further confirmed by cloning and direct sequencing of DNA from uncultured cells. Finally, direct sequencing of matched control and tumor samples revealed that TET2 mutation was present only in ATL tumor cells. Conclusions Our results suggest that inactivation of MLL3 and TET2 may play an important role in the tumorigenesis process of HTLV-I-induced ATL. Electronic supplementary material The online version of this article (doi:10.1186/s12943-016-0500-z) contains supplementary material, which is available to authorized users.

Colorectal cancer with its frequency, high mortality rate as well as many etiological unknowns is a challenge to contemporary science. Finally, genetic information could be used in near future for prevention of colorectal cancer, its early diagnosis and selection for the most suitable hospital treatment. In this study, we analysed genetic alterations of tumor suppressor genes and the possibility of quick and efficient screening method for identification of colorectal cancer. The study consisted of 54 samples of tumor and surrounding healthy tissue of patients with colorectal cancer, which is clasificated according to Bethesda and Amsterdams criterias. The investigation showed that genetic alterations of tumor suppressor gene NM 23 were present in 19/35 (54,29%) samples, and tumor suppressor gene p53 in 18/35 (51,43%), APC in 18/35 (51,43%), DCC2 tumor suppressor gene in 12/35 (34,29%), tumor suppressor gene RB1 in 8 /35 (22, 86%) and DCC 1 in 10/35 ( 28,57%) tumor tissue.

Introduction. Loss of heterozygosity (LOH) is frequently observed in urinary bladder neoplasms. In the reported study, an attempt was undertaken to determine the loss of heterozygosity of TP53(17p13), RB1(13q14), CDKN2A/ ARF(9p21) genes in DNA from neoplastic tissue, collected from patients with diagnosed urinary bladder carcinoma, and to compare the results with those of LOH evaluation in DNA isolated from urine sediment cells. Material and methods. After isolation, DNA was amplified (PCR) by means of primers to five polymorphic microsatellite markers, the products being then separated on agarose gel. Following the method, a total of 125 DNA samples were obtained, isolated from neoplastic cells, together with 125 corresponding DNA samples, isolated from urine sediment cells. results. The loss of heterozygosity in at least one marker was identified in 39.2%. (49/125) of DNA from studied tumors and in 34.3% (43/125) of DNA samples, isolated from urine sediment cells. An analysis of LOH from the DNA, isolated from urine sediment cells, allowed for identification of 81.8% of neoplastic tumors with 99.7% specificity. Conclusions. Our observations have demonstrated that LOH within 13q14, 17p13 and 9p21 loci is more often observed in clinically more advanced neoplasms. LOH in 17p13 locus is more frequently found in tumors at high histopathological stage, while in low-stage neoplasms, LOH is most often observed on chromosome 9. The high sensitivity (81.8%) and specificity (99.7%) of LOH studies in DNA, isolated from urine sediment cells, make this technique an advantageous, non-invasive method for detection and screening of bladder cancer.

Dermal neurofibromas (dNFs) are benign tumors of the peripheral nervous system typically associated with Neurofibromatosis type 1 (NF1) patients. Genes controlling the integrity of the DNA are likely to influence the number of neurofibromas developed because dNFs are caused by somatic mutational inactivation of the NF1 gene, frequently evidenced by loss of heterozygosity (LOH). We performed a comprehensive analysis of the prevalence and mechanisms of LOH in dNFs. Our study included 518 dNFs from 113 patients. LOH was detected in 25% of the dNFs (N = 129). The most frequent mechanism causing LOH was mitotic recombination, which was observed in 62% of LOH-tumors (N = 80), and which does not reduce the number of NF1 gene copies. All events were generated by a single crossover located between the centromere and the NF1 gene, resulting in isodisomy of 17q. LOH due to the loss of the NF1 gene accounted for a 38% of dNFs with LOH (N = 49), with deletions ranging in size from ∼80 kb to ∼8 Mb within 17q. In one tumor we identified the first example of a neurofibroma-associated second-hit type-2 NF1 deletion. Analysis of the prevalence of mechanisms causing LOH in dNFs in individual patients (possibly under genetic control) will elucidate whether there exist interindividual variation. Hum Mutat 32:78–90, 2011. © 2010 Wiley-Liss, Inc.

One function ascribed to apoptosis is the suicidal destruction of potentially harmful cells, such as cancerous cells. Hence, their growth depends on evasion of apoptosis, which is considered as one of the hallmarks of cancer. Apoptosis is ultimately carried out by the sequential activation of initiator and executioner caspases, which constitute a family of intracellular proteases involved in dismantling the cell in an ordered fashion. In cancer, therefore, one would anticipate caspases to be frequently rendered inactive, either by gene silencing or by somatic mutations. From clinical data, however, there is little evidence that caspase genes are impaired in cancer. Executioner caspases have only rarely been found mutated or silenced, and also initiator caspases are only affected in particular types of cancer. There is experimental evidence from transgenic mice that certain initiator caspases, such as caspase-8 and -2, might act as tumor suppressors. Loss of the initiator caspase of the intrinsic apoptotic pathway, caspase-9, however, did not promote cellular transformation. These data seem to question a general tumor-suppressive role of caspases. We discuss several possible ways how tumor cells might evade the need for alterations of caspase genes. First, alternative splicing in tumor cells might generate caspase variants that counteract apoptosis. Second, in tumor cells caspases might be kept in check by cellular caspase inhibitors such as c-FLIP or XIAP. Third, pathways upstream of caspase activation might be disrupted in tumor cells. Finally, caspase-independent cell death mechanisms might abrogate the selection pressure for caspase inactivation during tumor development. These scenarios, however, are hardly compatible with the considerable frequency of spontaneous apoptosis occurring in several cancer types. Therefore, alternative concepts might come into play, such as compensatory proliferation. Herein, apoptosis and/or non-apoptotic functions of caspases may even promote tumor development. Moreover, experimental evidence suggests that caspases might play non-apoptotic roles in processes that are crucial for tumorigenesis, such as cell proliferation, migration, or invasion. We thus propose a model wherein caspases are preserved in tumor cells due to their functional contributions to development and progression of tumors.

Glioblastoma multiforme is the most common type of primary brain tumor in adults. WWOX is a tumor suppressor gene involved in carcinogenesis and cancer progression in many different neoplasms. Reduced WWOX expression is associated with more aggressive phenotype and poor patient outcome in several cancers. We investigated alternations of WWOX expression and its correlation with proliferation, apoptosis and signal trafficking in 67 glioblastoma multiforme specimens. Moreover, we examined the level of WWOX LOH and methylation status in WWOX promoter region. Our results suggest that loss of heterozygosity (relatively frequent in glioblastoma multiforme) along with promoter methylation may decrease the expression of this tumor suppressor gene. Our experiment revealed positive correlations between WWOX and Bcl2 and between WWOX and Ki67. We also confirmed that WWOX is positively correlated with ErbB4 signaling pathway in glioblastoma multiforme.

Prostate cancer (PCa) is the most commonly diagnosed non-cutaneous cancer in male subjects in Western countries. The widespread use of prostate-specific antigen (PSA) has increased the detection of this cancer form in earlier stages. Moreover, it has increased the need for new diagnostic procedures to be developed for patient stratification based on risk of progression. We analysed laser-microdissected prostate tumour tissue from 43 patients with histologically verified PCa, using the new high-resolution Affymetrix Mapping 50K single-nucleotide polymorphism array. The results showed six major loss of heterozygosity regions at chromosomes 6q14-16, 8p23-11, 10q23, 13q13-21 and 16q21-24 and a novel region at chromosome 21q22.2, all of which reveal concomitant copy number loss. Tumour development was further characterised by numerous novel genomic regions almost exclusively showing copy number loss. However, tumour progression towards a metastatic stage, as well as poor differentiation, was identified by specific patterns of copy number gains of genomic regions located at chromosomes 8q, 1q, 3q and 7q. Androgen ablation therapy was further characterised by copy gain at chromosomes 2p and 10q. In conclusion, patterns of allelic imbalance were discovered in PCa, consisting allelic loss as an early event in tumour development, and distinct patterns of allelic amplification related to tumour progression and poor differentiation.

Background Adoption of new technology in both basic research and clinical settings requires rigorous validation of analytical performance. The OncoScan® FFPE Assay is a multiplexing tool that offers genome-wide copy number and loss of heterozygosity detection, as well as identification of frequently tested somatic mutations. Methods In this study, 162 formalin fixed paraffin embedded samples, representing six different tumour types, were profiled in triplicate across three independent laboratories. OncoScan® formalin fixed paraffin embedded assay data was then analysed for reproducibility of genome-wide copy number, loss of heterozygosity and somatic mutations. Where available, somatic mutation data was compared to data from orthogonal technologies (pyro/sanger sequencing). Results Cross site comparisons of genome-wide copy number and loss of heterozygosity profiles showed greater than 95% average agreement between sites. Somatic mutations pre-validated by orthogonal technologies showed greater than 90% agreement with OncoScan® somatic mutation calls and somatic mutation concordance between sites averaged 97%. Conclusions Reproducibility of whole-genome copy number, loss of heterozygosity and somatic mutation data using the OncoScan® assay has been demonstrated with comparatively low DNA inputs from a range of highly degraded formalin fixed paraffin embedded samples. In addition, our data shows examples of clinically-relevant aberrations that demonstrate the potential utility of the OncoScan® assay as a robust clinical tool for guiding tumour therapy. Electronic supplementary material The online version of this article (doi:10.1186/s12920-015-0079-z) contains supplementary material, which is available to authorized users.

Smad4 protein, whose gene is coded at chromosome 18q21.1, is an important tumour suppressor that mediates transforming growth factor-beta. It has been reported that inactivation of the Smad4 gene and allelic loss of chromosome 18q correlate with liver metastasis and poorer prognosis in colorectal cancers. Utilising a recently developed method of immunohistochemical staining for Smad4 protein, we focused on the specific impact of Smad4 protein expression on liver metastasis in colorectal cancer. We also evaluated the association between chromosome18q deletion and liver metastasis. We selected 20 colorectal cancers with liver metastasis for the experimental group, and 20 cases without liver metastasis for the control. In order to exclude the influence of lymph node metastasis, all cases were lymph node negative. In addition, the two groups were matched for tumour depth, tumour differentiation and tumour location. We compared the expression level of Smad4 protein immunohistochemically in these 20 matched pairs. We also compared the loss of heterozygosity status at chromosome 18q in these 20 matched pairs. Immunohistochemical staining revealed a significant difference (P = 0.024) in the level of Smad4 protein between the two groups. We also observed a significantly different (P=0.0054) ratio of allelic deletion at chromosome 18q21. Smad4 protein expression level and allelic loss at 18q21 are associated with the process of liver metastasis in colorectal cancers evaluated when excluding clinical and pathological features except for liver metastasis.

Background The pattern of allelic loss of heterozygosity (LOH) and PTEN mutations appear to be associated with the progression of gliomas leading to a decrement in the survival rate of patients. This present study was carried out to determine the LOH and PTEN mutational status in glioma patients and its association with patients' survival. Methods Thirty-seven Malaysian glioma patients of the Malay race were subject to PTEN mutational analysis and the presence of LOH using the cold single-strand conformation polymorphism method, and their clinical and paraclinical response were correlated. Results Among analysed glioma patients, seven (21.6%) cases with PTEN mutations were detected and 12 (32.4%) of 37 patients showed presence of LOH. Univariate analysis showed that tumour grade, vascularization, PTEN mutation, LOH and combination of both PTEN mutation and LOH were significantly associated with glioma patients' survival. Multivariate analysis revealed that no factors contributed to survival time. Conclusion The results show that PTEN mutation and LOH are quite frequent in Malaysian glioma patients. However, they have no impact on the survival outcome of patients.

Surgical cure of glioblastomas is virtually impossible and their clinical course is mainly determined by the biologic behavior of the tumor cells and their response to radiation and chemotherapy. We investigated whether response to temozolomide (TMZ) chemotherapy differs in subsets of malignant glioblastomas defined by genetic lesions. Eighty patients with newly diagnosed glioblastoma were analyzed with comparative genomic hybridization and loss of heterozygosity. All patients underwent radical resection. Fifty patients received TMZ after radiotherapy (TMZ group) and 30 patients received radiotherapy alone (RT group). The most common aberrations detected were gains of parts of chromosome 7 and losses of 10% 9p, or 13q. The spectrum of genetic aberrations did not differ between the TMZ and RT groups. Patients treated with TMZ showed significantly better survival than patients treated with radiotherapy alone (19.5 vs 9.3 months). Genomic deletions on chromosomes 9 and 10 are typical for glioblastoma and associated with poor prognosis. However, patients with these aberrations benefited significantly from TMZ in univariate analysis. In multivariate analysis, this effect was pronounced for 9p deletion and for elderly patients with 10q deletions, respectively. This study demonstrates that molecular genetic and cytogenetic analyses potentially predict responses to chemotherapy in patients with newly diagnosed glioblastomas.

Neurofibromatosis type 1 (NF1) patients have an 8-13% lifetime risk of developing malignant peripheral nerve sheath tumors (MPNST) which have a very poor prognosis. In this study, cells from eight MPNSTs (six primary and two recurrences) of six clinically and genetically well-characterized NF1 patients were taken into culture. Tracing of loss of heterozygosity (LOH) of the NF1, p53, and p16 gene regions or of abnormal karyotypes enabled identification of tumor cells from five MPNSTs. In two other MPNST-derived cell cultures, LOH of the relevant regions in the original tumors could not be detected, indicating that the obtained cells were nonneoplastic cells. Cells from most MPNSTs grew only under standard culture conditions but not under conditions optimized for Schwann cells. These cells were S100-negative and did not exhibit spindle shape which is a characteristic of Schwann cells. Drastically increased proliferation rates were found for most of the MPNST cells in comparison to Schwann cells derived from benign neurofibromas. Our study demonstrates that genetic analysis is effective and essential for verification of MPNST tumor cells in culture. These verified MPNST cells are valuable for further investigations of the biology and pathogenesis of this malignancy as well as for in vitro pharmacologic studies essential for the development of new therapies.

Changes in DNA copy number are one of the hallmarks of the genetic instability common to most human cancers. Previous micro-array-based methods have been used to identify chromosomal gains and losses; however, they are unable to genotype alleles at the level of single nucleotide polymorphisms (SNPs). Here we describe a novel algorithm that uses a recently developed high-density oligonucleotide array-based SNP genotyping method, whole genome sampling analysis (WGSA), to identify genome-wide chromosomal gains and losses at high resolution. WGSA simultaneously genotypes over 10,000 SNPs by allele-specific hybridisation to perfect match (PM) and mismatch (MM) probes synthesised on a single array. The copy number algorithm jointly uses PM intensity and discrimination ratios between paired PM and MM intensity values to identify and estimate genetic copy number changes. Values from an experimental sample are compared with SNP-specific distributions derived from a reference set containing over 100 normal individuals to gain statistical power. Genomic regions with statistically significant copy number changes can be identified using both single point analysis and contiguous point analysis of SNP intensities. We identified multiple regions of amplification and deletion using a panel of human breast cancer cell lines. We verified these results using an independent method based on quantitative polymerase chain reaction and found that our approach is both sensitive and specific and can tolerate samples which contain a mixture of both tumour and normal DNA. In addition, by using known allele frequencies from the reference set, statistically significant genomic intervals can be identified containing contiguous stretches of homozygous markers, potentially allowing the detection of regions undergoing loss of heterozygosity (LOH) without the need for a matched normal control sample. The coupling of LOH analysis, via SNP genotyping, with copy number estimations using a single array provides additional insight into the structure of genomic alterations. With mean and median inter-SNP euchromatin distances of 244 kilobases (kb) and 119 kb, respectively, this method affords a resolution that is not easily achievable with non-oligonucleotide-based experimental approaches.

Colorectal carcinoma is one of the most common cancers in Western countries. Most deaths related to colorectal cancer are caused by metastasis. Little is known about the genetic alterations associated with the metastatic phenotype. Deletions of the long arm of chromosome 10 have been reported in many types of tumour, including colorectal carcinomas (Frayling et al, 1997), and are correlated with tumour progression and/or metastasis formation in several of these cancers, such as glial tumours (Balesaria et al, 1999), lung cancer (Petersen et al, 1998), head and neck squamous cell carcinomas (Bockmuhl et al, 2002), bladder (Cappellen et al, 1997), prostate (Komiya et al, 1996) and breast carcinomas (Bose et al, 1998). Several putative or known tumour-suppressor genes have been mapped to 10q, including BMPR1A on 10q23.2 and PTEN/MMAC1/TEP1 on 10q23.3. Mutations in PTEN are associated with hereditary cancer predisposition syndromes (Liaw et al, 1997; Marsh et al, 1997) and, to a greater or lesser extent, with a wide variety of sporadic cancers (Ali et al, 1999; Bonneau and Longy, 2000). With the exception of endometrial cancer (Mutter et al, 2000), alterations to PTEN in cancer are almost exclusively detected in advanced stages of disease. Mutations in PTEN have been studied only in primary colorectal tumours, and this gene appears to be involved only in tumours with microsatellite instability (MSI+) (Guanti et al, 2000; Shin et al, 2001; Zhou et al, 2002). The presence of germ-line-inactivating mutations in the BMPR1A gene has been found to be responsible for a significant proportion of cases of juvenile polyposis syndrome, an inherited hamartomatous polyposis syndrome with a risk of colon cancer (Howe et al, 2001; Zhou et al, 2001). Although BMPR1A was a good candidate for involvement in the pathogenesis of sporadic colon cancer, no mutations have yet been identified in primary colorectal tumours displaying LOH at the BMPR1A locus (Howe et al, 2001).

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.

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

Human papillomavirus (HPV) is thought to cause some vulval squamous cell carcinomas (VSCC) by degrading p53 product. Evidence on whether HPV-negative VSCC results from p53 mutation is conflicting. We performed immunohistochemistry for p53 product on 52 cases of lone vulval intraepithelial neoplasia (VIN), 21 cases of VIN with concurrent VSCC and 67 cases of VSCC. We had previously performed HPV detection and loss of heterozygosity (LOH) analyses on these samples. Abnormal p53 immunoreactivity (p53-positive) rates in HPV-positive VSCC and HPV-negative VSCC were 22% (12/54) and 31% (4/13), respectively (P

One of the most common forms of carcinoma in women, cervical invasive squamous cell carcinoma (CIC), often coexists with multiple lesions of cervical intraepithelial neoplasia (CIN). CIC and CIN show heterogeneity with respect to both histopathology and biology. To understand the causes, origin, and model of progression of cervical carcinoma, we assessed the clonality of a case with multiple synchronous lesions by analyzing X chromosome inactivation polymorphism, human papillomavirus type 16 (HPV16) sequence variation/mutations, and loss of heterozygosity (LOH). Microdissection was performed on 24 samples from this case, representing the entire lesional situation. The combination of different X chromosome inactivation patterns, two HPV16 point mutations, and LOH at three genomic microsatellite loci, led to the identification of five different “monoclonal” lesions (CIN II, CIN III, and invasive carcinoma nests) and five different “polyclonal” areas (CIN II and normal squamous epithelium). This finding indicated that CIC can originate from multiple precursor cells, from which some clones might progress via multiple steps, namely via CIN II and CIN III, whereas others might develop independently and possibly directly from the carcinoma precursor cells. Our results also supported the view that HPV16 as a “field factor” causes cervical carcinoma, which is probably promoted by the loss of chromosomal material as indicated by the LOH.

Skin tumors were induced by gamma-irradiation in F(1) mice between C3H/He or BALB/c and MSM carrying a p53-deficient allele. The incidence was 39.1% (34/87) in p53(KO/+) mice of the C3H/MSM genetic background and 14.3% (19/133) in those of the BALB/MSM background. Interestingly, most of the tumors (82%) lost the wild-type p53 allele and no skin tumor was found in p53(+ / +) F(1) mice. This suggests a requirement of p53 loss for the skin cancer development. Genome scan localized a chromosomal locus showing frequent allelic losses near D12Mit2, which may harbor a tumor suppressor gene. In addition, 23 loci distributed on 13 chromosomes exhibited allelic losses at frequencies of more than 20%. The genome-wide occurrence of allelic losses suggests that genomic instability of the skin tumors may be implicated in radiation-induced carcinogenesis. The present study is the first to report a mouse model system useful for the analysis of radiation induction of skin cancer in man.

Background The analysis of cellular networks and pathways involved in oncogenesis has increased our knowledge about the pathogenic mechanisms that underlie tumour biology and has unmasked new molecular targets that may lead to the design of better anti-cancer therapies. Recently, using a high resolution loss of heterozygosity (LOH) analysis, we identified a number of potential tumour suppressor genes (TSGs) within common LOH regions across cases suffering from two of the most common forms of Non-Hodgkin’s lymphoma (NHL), Follicular Lymphoma (FL) and Diffuse Large B-cell Lymphoma (DLBCL). From these studies LOH of the protein tyrosine phosphatase receptor type J (PTPRJ) gene was identified as a common event in the lymphomagenesis of these B-cell lymphomas. The present study aimed to determine the cellular pathways affected by the inactivation of these TSGs including PTPRJ in FL and DLBCL tumourigenesis. Results Pathway analytical approaches identified that candidate TSGs located within common LOH regions participate within cellular pathways, which may play a crucial role in FL and DLBCL lymphomagenesis (i.e., metabolic pathways). These analyses also identified genes within the interactome of PTPRJ (i.e. PTPN11 and B2M) that when inactivated in NHL may play an important role in tumourigenesis. We also detected genes that are differentially expressed in cases with and without LOH of PTPRJ, such as NFATC3 (nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 3). Moreover, upregulation of the VEGF, MAPK and ERBB signalling pathways was also observed in NHL cases with LOH of PTPRJ, indicating that LOH-driving events causing inactivation of PTPRJ, apart from possibly inducing a constitutive activation of these pathways by reduction or abrogation of its dephosphorylation activity, may also induce upregulation of these pathways when inactivated. This finding implicates these pathways in the lymphomagenesis and progression of FL and DLBCL. Conclusions The evidence obtained in this research supports findings suggesting that FL and DLBCL share common pathogenic mechanisms. Also, it indicates that PTPRJ can play a crucial role in the pathogenesis of these B-cell tumours and suggests that activation of PTPRJ might be an interesting novel chemotherapeutic target for the treatment of these B-cell tumours. Electronic supplementary material The online version of this article (doi: 10.1186/1471-2164-15-390) contains supplementary material, which is available to authorized users.

Breast cancer susceptibility genes BRCA1 and BRCA2 are tumour suppressor genes the alleles of which have to be inactivated before tumour development occurs. Hereditary breast cancers linked to germ-line mutations of BRCA1 and BRCA2 genes almost invariably show allelic imbalance (AI) at the respective loci. BRCA1 and BRCA2 are believed to take part in a common pathway in maintenance of genomic integrity in cells. We carried out AI and fluorescence in situ hybridization (FISH) analyses of BRCA2 in breast tumours from germ-line BRCA1 mutation carriers and vice versa. For comparison, 14 sporadic breast tumours were also studied. 8 of the 11 (73%) informative BRCA1 mutation tumours showed AI at the BRCA2 locus. 53% of these tumours showed a copy number loss of the BRCA2 gene by FISH. 5 of the 6 (83%) informative BRCA2 mutation tumours showed AI at the BRCA1 locus. Half of the tumours (4/8) showed a physical deletion of the BRCA1 gene by FISH. Combined allelic loss of both BRCA1 and BRCA2 gene was seen in 12 of the 17 (71%) informative hereditary tumours, whereas copy number losses of both BRCA genes was seen in only 4/14 (29%) sporadic control tumours studied by FISH. In conclusion, the high prevalence of AI at BRCA1 in BRCA2 mutation tumours and vice versa suggests that somatic events occurring at the other breast cancer susceptibility gene locus may be selected in the cancer development. The mechanism resulting in AI at these loci seems more complex than a physical deletion. http://www.bjcancer.com © 2001 Cancer Research Campaign

Neuroblastoma is a heterogeneous tumour and its effective clinical management is dependent on accurate prognostic evaluation. In approximately 25% of patients amplification of the MYCN oncogene is known to be associated with a poor outcome. In order to identify additional molecular markers with prognostic potential in non-MYCN-amplified neuroblastomas, we looked for a correlation between clinical outcome and loss of heterozygosity (LOH) on four chromosomes that frequently show alteration in neuroblastoma (chromosomes 3, 4, 11 and 14). Chromosome 11q loss (with frequent parallel loss of chromosomes 3p, 4p and/or 14q) was found exclusively in tumours without MYCN amplification and was significantly associated with poor event-free survival. The 2-year event-free survival rate for 11q LOH cases was 30%, compared to 34% for MYCN-amplified cases and 100% for cases without these abnormalities. While 11q LOH was associated predominantly with advanced-stage disease, 2 cases with low-stage disease and 11q LOH both suffered relapses. We conclude that chromosome 11q loss defines a biologically distinct group of tumours without MYCN amplification that appear to have potential for aggressive metastatic growth. Thus this genetic alteration may be an important new prognostic marker in neuroblastoma. © 2001 Cancer Research Campaign http://www.bjcancer.com

The human tumour suppressor gene PTEN located at 10q23 is mutated in a variety of tumour types particularly metastatic cases and in the germline of some individuals with Cowdens cancer predisposition syndrome. We have assessed the status of PTEN and associated pathways in cell lines derived from 19 squamous cell carcinomas of the head and neck. Loss of heterozygosity is evident at, or close to the PTEN gene in 5 cases, however there were no mutations in the remaining alleles. Furthermore by Western analysis PTEN protein levels are normal in all of these SCC-HN tumours and cell lines. To assess the possibility that PTEN may be inactivated by another mechanism, we characterized lipid phosphatase levels and from a specific PIP3 biochemical assay it is clear that PTEN is functionally active in all 19 human SCCs. Our data strongly suggest the possibility that a tumour suppressor gene associated with development of SCC-HN, other than PTEN, is located in this chromosomal region. This gene does not appear to be MXI-1, which has been implicated in some other human tumour types. PTEN is an important negative regulator of PI3Kinase, of which subunit alpha is frequently amplified in SCC-HN. To examine the possibility that PI3K is upregulated by amplification in this tumour set we assessed the phosphorylation status of Akt, a downstream target of PI3K. In all cases there is no detectable increase in Akt phosphorylation. Therefore there is no detectable defect in the PI3K pathway in SCC-HN suggesting that the reason for 3q26.3 over-representation may be due to genes other than PI3K110α. © 2001 Cancer Research Campaign http://www.bjcancer.com

The genetic alterations responsible for the development of cutaneous lymphoma are largely unknown. Chromosome region 9p21 contains a gene locus encoding an inhibitor of cyclin-dependent kinase 4, and heterozygous deletions of this tumor suppressor gene (p16) have been shown in a variety of malignant tumors. We studied 11 randomly selected cutaneous CD30-positive large cell lymphomas. Several areas containing 20-50 CD30-positive lymphocytes were microdissected in each case and subjected to single-step DNA extraction. Loss of heterozygosity analysis was performed using polymorphic markers at 9p21 (IFNA, D9S171, D9S169) and 17p13 (TP53). Samples from normal cells apart from CD30-positive lymphocytes, e.g., CD30-negative lymphohistiocytic infiltrates and normal epidermal layer, were also obtained in all cases from the same slide for comparison with the tumor samples. Expression of CD30 and T-lineage antigens (CD3, CD45Ro) was confirmed in all cases. Immunohistochemical staining for p16 and p53 was performed using the monoclonal antibodies sc-1661 and DO-7, respectively. Of the 11 informative cases, seven (64%) exhibited loss of heterozygosity at least for one marker at 9p21 (p16), whereas no allelic deletions were found for the polymorphic marker at 17p13 (p53). On immunohistochemistry loss of the p16 protein was detected in two of 11 cases. Nuclear staining for p53 protein was found in four of 11 cases. Here, we provide the first evidence of the involvement of the tumor suppressor gene p16 in primary cutaneous large cell lymphoma. Whether p16 deletion in these lymphomas is associated with disease progression and whether this method could serve as an early marker to detect lymphomas at an early stage needs to be addressed in future studies. J Invest Dermatol 115:1104-1107 2000

Endocrine tumours of the pancreas, anterior pituitary or parathyroids arise either sporadically in the general population, or as a part of inherited syndromes such as multiple endocrine neoplasia type 1 (MEN 1). The mechanisms responsible for the development of sporadic endocrine lesions are not well understood, although loss of heterozygosity (LOH) of the MEN1 locus on chromosome 11q13 and somatic mutation of the MEN1 gene have been frequently associated with the development of MEN 1-type sporadic endocrine lesions. To further investigate the role of the MEN1 gene in sporadic endocrine tumorigenesis, we analysed DNA from 14 primary parathyroid lesions, 8 anterior pituitary tumours and 3 pancreatic tumours for the presence of somatic MEN1 gene mutations and LOH of seven microsatellite markers flanking the MEN1 locus. In addition, we similarly analysed 8 secondary parathyroid lesions which arose in patients with chronic renal failure. None of the patients studied had a family history of MEN 1. Three primary parathyroid lesions and one pancreatic tumour (glucagonoma) were found to have lost one allele at the MEN1 locus. Somatic mutations were identified by SSCP and sequence analysis in one of these parathyroid lesions (P320L) and in the glucagonoma (E179V). These results support previous findings that inactivation of the MEN1 tumour suppressor gene contributes to the development of sporadic MEN 1-type endocrine lesions but is not associated with the development of parathyroid hyperplasia seen in some renal failure patients. © 2000 Cancer Research Campaign

The purpose of this study was to determine whether point mutations and loss of the p53 gene take place in ulcerative colitis which is histologically negative for dysplasia. DNA was extracted from 13 frozen rectal or colon biopsies and blood samples. Ulcerative colitis was classified histologically as active (10 cases) and inactive (3 cases). Exons 5-8 were amplified by PCR, treated with exonuclease and shrimp alkaline phosphatase and sequenced by the dideoxy chain termination method with the Sequenase Version 2.0 DNA sequencing kit. PCR products of intron 6 and exon 4 were digested with MspI and AccII, respectively, for RFLP analysis. No p53 gene mutation was detected in these cases. The number of informative patients for loss of heterozygosity (LOH) at the p53 intron 6 was high, 11 out of 12 (92%), whereas no LOH was observed. LOH affecting p53 exon 4 was not detected in lesions from 5 of 12 patients (42%). In ulcerative colitis, tumor progression is similar to that in sporadic colon cancer, and other oncogenes and tumor suppressor genes are likely to be mutated before the p53 gene.

The tumour suppressor gene CDKN2A, located on chromosome 9p21, encodes the cell cycle regulatory protein p16. Inactivation of the CDKN2A gene could lead to uncontrolled cell growth. In order to determine the role of CDKN2A in the development of sporadic ovarian cancer, loss of heterozygosity at 9p21–22, homozygous deletion, mutation and methylation status of the CDKN2A gene as well as CDKN2A expression were examined in a panel of serous papillary ovarian cancer. The frequency of loss of heterozygosity (LOH) for one or more informative markers at 9p21–22 was 65% (15/23). The most common deleted region was located between interferon (IFN)-α and D9S171. Homozygous deletions and mutations of the CDKN2A gene were not found. There was no evidence of methylation in exon 1, but methylation in exon 2 of CDKN2A gene was found in 26% (6/23). Absence of CDKN2A gene expression was shown in 27% (6/22) at mRNA level and 21% (4/19) at protein level. These data suggest that the CDKN2A gene is involved in the tumorigenesis of ovarian cancer, but the mechanisms of CDKN2A gene inactivation in serous papillary ovarian cancer remains unclear. © 1999 Cancer Research Campaign

We have analysed the loss of heterozygosity (LOH) on chromosome bands 16q22–q24 in 24 primary gastric cancer tissues and found three regions of frequent allelic loss (16q22, 16q24.1–q24.3 and 16q24.3). The region for the most frequent allelic loss (63%) was in 16q24.1–q24.3. LOH of this region had no relationship with histological subtype, but a significant association between LOH and microscopic lymphangial invasion was observed. Although not significant, vascular and gastric wall invasions are also associated with LOH. The region includes the locus for the H-cadherin gene. Therefore we examined the genetic and epigenetic alterations of this gene. Markedly reduced expression was observed in gastric cancer cell lines compared with that of normal gastric mucosa. However, no mutation was found in this gene in any of the gastric cancer tissues or the gastric cancer cell lines. Furthermore, we analysed the methylation status of the 5'-flanking region of the gene, but no significant association was found. We suggest that some other tumour suppresser gene(s) in 16q24.1–q24.3 may be responsible for gastric carcinogenesis. © 1999 Cancer Research Campaign

We have studied a set of 40 human lobular breast cancers for loss of heterozygosity (LOH) at various chromosome locations and for mutations in the coding region plus flanking intron sequences of the E-cadherin gene. We found a high frequency of LOH (100%, 31/31) at 16q21–q22.1. A significantly higher level of LOH was detected in ductal breast tumours at chromosome arms 1p, 3p, 9p, 11q, 13q and 18q compared to lobular breast tumours. Furthermore, we found a significant association between LOH at 16 q containing the E-cadherin locus and lobular histological type. Six different somatic mutations were detected in the E-cadherin gene, of which three were insertions, two deletions and one splice site mutation. Mutations were found in combination with LOH of the wild type E-cadherin locus and loss of or reduced E-cadherin expression detected by immunohistochemistry. The mutations described here have not previously been reported. We compared LOH at different chromosome regions with E-cadherin gene mutations and found a significant association between LOH at 13 q and E-cadherin gene mutations. A significant association was also detected between LOH at 13q and LOH at 7q and 11q. Moreover, we found a significant association between LOH at 3 p and high S phase, LOH at 9p and low ER and PgR content, LOH at 17p and aneuploidy. We conclude that LOH at 16q is the most frequent chromosome alteration and E-cadherin is a typical tumour suppressor gene in lobular breast cancer. © 1999 Cancer Research Campaign

Loss of heterozygosity (LOH) in loci of the 15q15.1, 12p13, 1p32, 17q21 and 13q12–13 regions may collaborate in the inactivation of RAD51, RAD52, RAD54, BRCA1, BRCA2 and possibly other genes implicated in the repair of double-stranded DNA and in DNA recombination. We investigate allelic losses in microsatellites of the RAD51, RAD52, RAD54, BRCA1 and BRCA2 regions, and their correlations with nine pathologic parameters in 127 breast carcinomas. The LOH analysis was performed by amplifying DNA by PCR, using 15 markers of the 15q15.1, 12p13.3, 1p32, 17q21 and 13q12–13 regions. LOH was found in the RAD51 region in 32% of tumours, in the RAD52 region in 16%, in RAD54 in 20% and in the BRCA1 and BRCA2 regions in 49% and 44% respectively. Significant correlations between one or more regions with concomitant LOH and pathologic parameters were observed with respect to age (P = 0.008), oestrogen receptor content (P = 0.03), progesterone receptors (P = 0.003), higher grade (P = 0.001), more advanced stage (P = 0.004) and peritumoural vessel involvement (P < 0.0001). The number of cases in which LOH was observed simultaneously in two or more regions was always higher than expected on the basis of their statistical probability, and curiously, the three patients with LOH at five regions concomitantly were under the age of 30 years. These results suggest that LOH at these regions could be related to breast cancer, and probably to a poor tumour prognosis. © 1999 Cancer Research Campaign

Mutations of the p53 tumor suppressor gene are the most prevalent genetic alteration observed in a wide variety of human cancers. In this study we examined 63 methylcholanthrene (MCA)-induced sarcomas from C57BL/6N x C3H/HeN F1 (BCF1) or C3H/HeN x C57BL/6N F1 (CBF1) mice for p53 gene mutations and loss of heterozygosity (LOH) of chromosome 11. Mutation analysis was done on exons 5 to 8 of the p53 gene by polymerase chain reaction-single strand conformation polymorphism analysis. This identified 53 potential mutations in 45 sarcomas. Mutations were further confirmed by direct sequencing of the region. Forty-nine of the 53 cases (94%) were missense mutations, while the rest included two nonsense mutations, one silent mutation and one insertional mutation. Spectra of base substitutions were: 25 cases (47%) of G:C-->T:A transversion, 13 cases (25%) of G:C-->A:T transition (CpG site 15%), 13 cases (24%) of G:C-->C:G transversion, a case (2%) of A:T-->T:A transversion and a case (2%) of insertion. In addition, analysis of 5 polymorphic markers of mouse chromosome 11 revealed LOH in ten cases (22%) among those carrying p53 mutations. In nine of these 10 cases, the loss involved all 5 markers. In addition, the loss was biased toward the C57BL allele (9 cases). The present study establishes the pattern of mutation of the p53 gene in MCA-induced mouse sarcomas.

NF2/Merlin was first identified through its association with neurofibromatosis type 2 (NF2). However, accumulating evidence suggests a more general involvement in tumorigenesis and, in particular, a broader role in tumor suppression. The aim of this study was to examine NF2/Merlin involvement in sporadic colorectal cancer. This study is the first to examine the role of NF2/Merlin in sporadic colorectal cancer through LOH analysis at the NF2 locus and mRNA expression analysis via quantitative RT-PCR of total NF2, NF2 isoform I and II. In addition, Merlin protein expression was assessed by immunohistochemistry and Western blotting. NF2 LOH was detected in 20.0 % of heterozygous cases and was found to be more frequent in tumors larger than 5 cm in diameter (p = 0.041) and in tumors with a less differentiated phenotype (p = 0.027). No differences were observed in total NF2 and NF2 isoform I/isoform II mRNA expression between the tumors and their corresponding normal mucous tissues. NF2 isoform II was the most predominant isoform in all samples analyzed. mRNA expression levels of total NF2 and isoforms I and II were significantly lower in poorly differentiated tumors (p = 0.033, p = 0.036 and p = 0.044, respectively). Weak Merlin immunostaining was more frequent in poorly differentiated tumors (p = 0.034) and tumors classified as Dukes’ C (p = 0.023). A distinct pattern of Merin phosphorylation was observed in tumors compared to normal mucous tissues. Our data indicate that NF2/Merlin may serve as a potential target in the management of colorectal cancer.

We compared transcript expression, and chromosomal changes on a series of tumors and surrounding tissues to determine if there is evidence of field cancerization in colorectal cancer. Epithelial cells were isolated from tumors and areas adjacent to the tumors ranging from 1 to 10 cm. Tumor abnormalities mirrored those previously reported for colon cancer and while the number and size of the chromosomal abnormalities were greatly reduced in cells from surrounding regions, many chromosome abnormalities were discernable. Interestingly, these abnormalities were not consistent across the field in the same patient samples suggesting a field of chromosomal instability surrounding the tumor. A mutator phenotype has been proposed to account for this instability which states that the genotypes of cells within a tumor would not be identical, but would share at least a single mutation in any number of genes, or a selection of genes affecting a specific pathway which provide a proliferative advantage.

In this study we investigated the types and role of different genetic changes of PTCH1 gene in three different types of ovarian tumors: carcinomas, fibromas and dermoids. LOH of the PTCH1 region was detected in 27.3% ovarian carcinoma samples, 18.18% ovarian fibroma samples and 55.56% ovarian dermoid samples. No point mutations were detected in any of the three types of ovarian tumors. SNP c.3944C>T showed significant differences between ovarian carcinoma and control samples with the minor T allele being significantly higher in controls compared to ovarian carcinomas. Interestingly, a new polymorphism c.−1184G>A was found only in tumor samples and further analyses should be performed in order to elucidate its potential role in ovarian tumors.

We investigated mutations of the epithelial (E)‐cadherin gene and loss of heterozygosity (LOH) at flanking loci using three microsatellite markers on the long arm of chromosome 16 in 25 ductal carcinomas of the breast. Expression of E‐cadherin was also investigated immunohistochemically. No mutations were detected in exons 6 through 9 of the E‐cadherin gene. LOH was observed more frequently (42%) at D16S402 (16q‐ter) than at D16S421 (16q22.3‐q23.1) (17%), which is located near the E‐cadherin gene. Expression of E‐cadherin was observed at the cell borders in 92% (11/12) of the tumors examined. The absence of mutations in the E‐cadherin gene and its conserved expression suggest that inactivation of E‐cadherin does not contribute significantly to the invasion or metastatic potential of ductal carcinomas of the breast. Furthermore, the high frequency of LOH at 16q‐ter suggests the existence of another tumor suppressor gene which may play a crucial role in the genesis of ductal carcinomas of the breast.

OBJECTIVE: Genomic instability is a hallmark of malignant tissues. In this work, we aimed to characterize nuclear and mitochondrial instabilities by determining short tandem repeats and somatic mitochondrial mutations, respectively, in a cohort of Brazilian sporadic breast cancer cases. Furthermore, we performed an association analysis of the molecular findings and the clinical pathological data. METHODS: We analyzed 64 matched pairs of breast cancer and adjacent non-cancerous breast samples by genotyping 13 nuclear short tandem repeat loci (namely, D2S123, TPOX, D3S1358, D3S1611, FGA, D7S820, TH01, D13S317, D13S790, D16S539, D17S796, intron 12 BRCA1 and intron 1 TP53) that were amplified with the fluorescent AmpFlSTR Identifiler Genotyping system (Applied Biosystems, USA) and by silver nitrate staining following 6% denaturing polyacrylamide gel electrophoresis. Somatic mtDNA mutations in the D-loop site were assessed with direct sequencing of the hypervariable HVI and HVII mitochondrial regions. RESULTS: Half of the cancer tissues presented some nuclear instability. Interestingly, the D13S790 locus was the most frequently affected (36%), while the D2S123 locus presented no alterations. Forty-two percent of the cases showed somatic mitochondrial mutations, the majority at region 303-315 poly-C. We identified associations between Elston grade III, instabilities at 13q31 region (p = 0.0264) and mtDNA mutations (p = 0.0041). Furthermore, instabilities at 13q31 region were also associated with TP53 mutations in the invasive ductal carcinoma cases (p = 0.0207). CONCLUSION: Instabilities at 13q31 region and the presence of somatic mtDNA mutations in a D-loop site correlated with tumor aggressiveness.

Heterozygous Patched1 (Ptc1(+/-)) mice are prone to medulloblastoma (MB), and exposure of newborn mice to ionizing radiation dramatically increases the frequency and shortens the latency of MB. In Ptc1(+/-) mice, MB is characterized by loss of the normal remaining Ptc1 allele, suggesting that genome rearrangements may be key events in MB development. Recent evidence indicates that brain tumors may be linked to defects in DNA-damage repair processes, as various combinations of targeted deletions in genes controlling cell-cycle checkpoints, apoptosis and DNA repair result in MB in mice. Non-homologous end joining (NHEJ) and homologous recombination (HR) contribute to genome stability, and deficiencies in either pathway predispose to genome rearrangements. To test the role of defective HR or NHEJ in tumorigenesis, control and irradiated Ptc1(+/-) mice with two, one or no functional Rad54 or DNA-protein kinase catalytic subunit (DNA-PKcs) alleles were monitored for MB development. We also examined the effect of Rad54 or DNA-PKcs deletion on the processing of endogenous and radiation-induced double-strand breaks (DSBs) in neural precursors of the developing cerebellum, the cells of origin of MB. We found that, although HR and NHEJ collaborate in protecting cells from DNA damage and apoptosis, they have opposite roles in MB tumorigenesis. In fact, although Rad54 deficiency increased both spontaneous and radiation-induced MB development, DNA-PKcs disruption suppressed MB tumorigenesis. Together, our data provide the first evidence that Rad54-mediated HR in vivo is important for suppressing tumorigenesis by maintaining genomic stability. Oncogene (2011) 30, 4740-4749; doi:10.1038/onc.2011.178; published online 23 May 2011

Loss of heterozygosity (LOH) on chromosomes 1p, 4q, 5q, 8p, 13q, 16q, 17p, and 22q, and mutation of the p53 gene were simultaneously analyzed in 63 hepatocellular carcinomas (HCCs) with distinct histopathological grades, 80% of the tumors being from patients who had been exposed to hepatitis B virus (HBV) or hepatitis C virus (HCV). The frequencies of LOH on 8 chromosomes were 0–25% in 10 well differentiated HCCs, LOH being observed on 4q, 5q and 17p, 21–53% in 26 moderately differentiated HCCs, LOH on 8p and 17p being high, and 29–75% in 27 poorly differentiated HCCs, LOH on 17p, 4q and 8p heing the most frequent. p53 gene mutation was detected in moderately and poorly differentiated HCCs at 15% and 52%, respectively, but not at all in well differentiated HCCs. Of the mutations detected, 42% were transition mutation and only 5% were CpG transition, in contrast to the high frequencies of these types of mutations in colon tumors (78% and 54%, respectively). LOH on every chromosome and p53 mutation were more frequent in more advanced tumors, and accumulation of genetic changes increased with increase of the histopathological grade. Frequency of genetic changes in HCCs from HBV‐positive patients was comparable to that from HCV‐positive patients. The present results suggest that accumulation of genetic changes in multiple tumor suppressor genes, especially LOH on 17p, 4q and 8p, and mutation in p53 gene, are involved in the progression of liver cancer, and LOH on 17p and 4q precedes other genetic changes. Differences in the direction of p53 mutation between HCC and colon carcinoma suggest that liver carcinogens are distinct from colon carcinogens. Furthermore, mechanisms affecting the frequency of LOH in HCCs in HBV‐infected patients may be similar to those in HCV‐infected patients.

OBJECTIVE: Primary pigmented nodular adrenocortical disease (PPNAD) is the main endocrine manifestation of Carney complex, a multiple neoplasia syndrome caused by PRKAR1A gene mutations. The presence of PRKAR1A loss of heterozygosity (LOH) in adrenocortical tumorigenesis remains controversial. The aim of the present study is to investigate the presence of PRKAR1A LOH in adrenocortical cells in a patient with Carney complex. METHODS: The LOH was investigated using a PRKAR1A informative intragenic marker by GeneScan software analysis in DNA obtained from laser-captured microdissected cells of several adrenal nodules. Patients: A young adult male patient with Carney complex and his family were studied. RESULTS: A novel heterozygous mutation (p. Y21X) was identified at PRKAR1A in blood DNA of the male proband and his relatives. No PRKAR1A LOH was evidenced in the laser-captured microdissected cells from PPNAD tissue by different methodologies. CONCLUSION: We identified a new PRKAR1A nonsense mutation and in addition we did not evidence PRKAR1A LOH in laser-captured nodules cells, suggesting that adrenocortical tumorigenesis in PPNAD may occurs apart from the second hit. OBJETIVO: A doença adrenocortical nodular pigmentosa primária (PPNAD) é uma das manifestações do complexo de Carney, uma neoplasia endócrina múltipla causada por mutações no PRKAR1A. A perda de heterozigose (LOH) do PRKAR1A na tumorigenese adrenal permanece controversa dada à possibilidade de contaminação com o tecido normal. Nosso objetivo foi investigar a presença de LOH no PRKAR1A a partir de células do nódulo adrenal de um paciente com complexo de Carney. MÉTODOS: A pesquisa da LOH do PRKAR1A foi realizada através do estudo de um marcador intragênico em DNA de células do nódulo adrenal microdissecadas a laser, evitando contaminação com o tecido normal. Pacientes: Um paciente com PPNAD e cinco familiares foram estudados. RESULTADOS: A nova mutação (p. Y21X) foi identificada no PRKAR1A sem evidência de LOH no tecido adrenal. CONCLUSÃO: Identificamos uma nova mutação no PRKAR1A e não evidenciamos LOH nas células dos nódulos adrenocorticais, sugerindo que a PPNAD possa ocorrer na ausência de um segundo evento molecular.

International audience; This study assessed the prognostic value of several markers involved in gliomagenesis, and compared it with that of other clinical and imaging markers already used. Four-hundred and sixteen adult patients with newly diagnosed glioma were included over a 3-year period and tumour suppressor genes, oncogenes, MGMT and hTERT expressions, losses of heterozygosity, as well as relevant clinical and imaging information were recorded. This prospective study was based on all adult gliomas. Analyses were performed on patient groups selected according to World Health Organization histoprognostic criteria and on the entire cohort. The endpoint was overall survival, estimated by the Kaplan-Meier method. Univariate analysis was followed by multivariate analysis according to a Cox model. p14(ARF), p16(INK4A) and PTEN expressions, and 10p 10q23, 10q26 and 13q LOH for the entire cohort, hTERT expression for high-grade tumours, EGFR for glioblastomas, 10q26 LOH for grade III tumours and anaplastic oligodendrogliomas were found to be correlated with overall survival on univariate analysis and age and grade on multivariate analysis only. This study confirms the prognostic value of several markers. However, the scattering of the values explained by tumour heterogeneity prevents their use in individual decision-making.