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Lung cancer is a common disease with a poor prognosis. Genomic alterations involving the KRAS gene are common in lung carcinomas, although much is unknown about how different mutations, deletions, and expressions influence the disease course. The first approval of a KRAS-directed inhibitor was recently approved by the FDA. Mutations in the KRAS gene have been associated with poor prognosis for lung adenocarcinomas, but implications of the loss of heterozygosity (LOH) of KRAS have not been investigated. In this study, we have assessed the LOH of KRAS in early-stage lung adenocarcinoma by analyzing DNA copy number profiles and have investigated the effect on patient outcome in association with mRNA expression and somatic hotspot mutations. KRAS mutation was present in 36% of cases and was associated with elevated mRNA expression. LOH in KRAS was associated with a favorable prognosis, more prominently in KRAS mutated than in wild-type patients. The presence of both LOH and mutation in KRAS conferred a better prognosis than KRAS mutation alone. For wild-type tumors, no difference in prognosis was observed between patients with and without LOH in KRAS. Our study indicates that LOH in KRAS is an independent prognostic factor that may refine the existing prognostic groups of lung adenocarcinomas.

Primary mediastinal B-cell lymphoma (PMBCL) is the only non-Hodgkin’s lymphoma variant responding to immune checkpoint inhibitor (ICI) therapy, approximately in half of the cases; however, no molecular markers predicting a response to ICI therapy in PMBCL have been described so far. In this study, we assessed the incidence of the loss of heterozygosity (LOH), elevated microsatellite alteration at selected tetranucleotides (EMAST), and microsatellite instability (MSI) in the tumor genomes of 72 patients with PMBCL undergoing high-dose chemotherapy treatment at the National Research Center for Hematology (Moscow, Russia). Tumor DNA was isolated from biopsy samples taken at diagnosis. Control DNA was isolated from the blood of patients in complete remission or from buccal epithelium. STR-profiles for LOH and EMAST were assessed by PCR with COrDIS Plus multiplex kit (Gordiz Ltd., Moscow, Russia). LOH was detected in 37 of 72 patients (51.4%). EMAST was found in 40 patients (55.5%); 24 had a combination of EMAST with LOH. MSI-high was not found, while MSI-low was detected only in one patient. The association of certain genetic lesions with the clinical outcome in patients receiving treatment according to the standard clinical protocol R-Da-EPOCH-21 has been estimated (58 patients out of 72) and no associations with the worst overall or event-free survival were found.

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.

Most sporadic colorectal cancer reflects acquired mutations in the adenomatous polyposis coli (APC) tumor suppressor gene, while germline heterozygosity for mutant APC produces the autosomal dominant disorder Familial Adenomatous Polyposis (FAP) with a predisposition to colorectal cancer. In these syndromes, loss of heterozygosity (LOH) silences the remaining normal allele of APC, through an unknown mechanism, as the initiating step in transformation. Guanylyl cyclase C receptor (GUCY2C) and its hormones, uroguanylin and guanylin, have emerged as a key signaling axis opposing mutations driving intestinal tumorigenesis. Indeed, uroguanylin and guanylin are among the most commonly repressed genes in colorectal cancer. Here, we explored the role of APC heterozygosity in mechanisms repressing hormone expression which could contribute to LOH. In genetic mouse models of APC loss, uroguanylin and guanylin expression were quantified following monoallelic or biallelic deletion of the Apc gene. Induced biallelic loss of APC repressed uroguanylin and guanylin expression. However, monoallelic APC loss in Apcmin/+ mice did not alter hormone expression. Similarly, in FAP patients, normal colonic mucosa (monoallelic APC loss) expressed guanylin while adenomas and an invasive carcinoma (biallelic APC loss) were devoid of hormone expression. Thus, uroguanylin and guanylin expression by normal intestinal epithelial cells persists in the context of APC heterozygosity and is lost only after tumor initiation by APC LOH. These observations reveal a role for loss of the hormones silencing the GUCY2C axis in tumor progression following biallelic APC loss, but not in mechanisms creating the genetic vulnerability in epithelial cells underlying APC LOH initiating tumorigenesis.

This paper presents design and practical implementation of single-phase inverter based on selective harmonic elimination-pulse width modulation (SHE-PWM) technique. Microcontroller mega type Arduino used as a controller for producing the gate pulses. The optimized switching angles determination results in wide range of output voltage. Depending on number of switching angles, the lower order harmonics (LOHs) can be eliminated to improve the output voltage waveform. A comparison study using MATLAB/Simulink for sinusoidal-PWM and SHE-PWM techniques, which shows for the same LOH in the output voltage waveform, the SHE-PWM has less number of pulses per half cycle than sinusoidal-PWM strategy. The reduction in number of pulses results less switching losses. The simulation done using ten switching angles to drive R-L load. A prototype of SHE-PWM inverter with R-L load is used to validate the simulation results.

Mutations in BRCA1 result in predisposal to breast and ovarian cancers, but many variants exist with unknown clinical significance (VUS). One is BRCA1 c.4096+3A>G, which affects production of the full-length BRCA1 transcript, while augmenting transcripts lacking most or all of exon 11. Nonetheless, homozygosity of this variant has been reported in a healthy woman. We saw this variant cosegregate with breast and ovarian cancer in several family branches of four Icelandic pedigrees, with instances of phenocopies and a homozygous woman with lung cancer. We found eight heterozygous carriers (0.44%) in 1820 unselected breast cancer cases, and three (0.15%) in 1968 controls (p = 0.13). Seeking conclusive evidence, we studied tumors from carriers in the pedigrees for wild-type-loss of heterozygosity (wtLOH) and BRCA1-characteristic prevalence of estrogen receptor (ER) negativity. Of 15 breast and six ovarian tumors, wtLOH occurred in nine breast and all six ovarian tumours, and six of the nine breast tumors with wtLOH were ER-negative. These data accord with a pathogenic BRCA1-mutation. Our findings add to the current knowledge of BRCA1, and the role of its exon 11 in cancer pathogenicity, and will be of use in clinical genetic counselling.

Loss of heterozygosity (LOH) on chromosome 18q23 is associated with significantly decreased survival in head and neck cancer. In agreement with such tumor suppressive roles, the loss of function of genes located in this region can be achieved through LOH and promotor hypermethylation. In this study, the methylation status of promoters of 18q23 genes in 243 head and neck cancer patients was assessed by quantitative methylation-specific PCR. Promoter methylation was then compared to various clinical characteristics and patient survival. GALR1 and SALL3 promoter methylation correlated with reduced disease-free survival (log-rank test, p = 0.018 and p = 0.013, respectively). Furthermore, based on multivariate Cox proportional hazards analysis, these methylation events were associated with poor disease-free survival, with hazard ratios of 1.600 (95% confidence interval: CI, 1.027&ndash, 2.493, p = 0.038) and 1.911 (95% CI, 1.155&ndash, 3.162, p = 0.012), respectively. By comparison, GALR1 and SALL3 methylation were not prognostic for overall survival in The Cancer Genome Atlas (TCGA) cohort. Our findings suggest that the methylation status of 18q23 genes could serve as important biomarkers for the prediction of clinical outcomes in well-annotated head and neck squamous cell carcinoma cohorts. GALR1 and SALL3 methylation could thus help to facilitate risk stratification for individualized treatment.

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.

To investigate a possible relation between penile Doppler ultrasound examination (PDUE) parameters and efficacy of chronic therapy with tadalafil (TAD) combined with a protocol of aerobic physical activity (PA) in patients with late onset hypogonadism (LOH).The study evaluated 30 patients consecutively enrolled with LOH and erectile dysfunction which present contraindication to hormonal replacement therapy for concomitant prostate disease. These patients were subjected to a combined protocol with phosphodiesterase V selective inhibitors (TAD 5 mg daily) and aerobic PA.After three months, we observed significant improvements in erectile function [IIEF-5, median (IQR) = 13.0 (7.0-18.0) versus 6.0 (5.0-6.75); p 0.01] and of the main metabolic [homeostatic model assessment index, median (IQR) = 2.5 (1.62-3.37) versus 3.0 (2.0-3.75); p 0.01; body mass index, median (IQR) = 27.0 (24.0-28.75) versus 27.5 (24.0-29.5)] and vascular parameters [peak systolic velocity, median (IQR) = 29.5 (24.25-31.0) versus 28.0 (23.0-24.25); acceleration time, median (IQR) = 114 (105.25-134.0) versus 115.0 (106.5-134.0)], assessed by PDUE.PA in association with phosphodiesterase V inhibitors could compensate the effects of hypogonadism on erectile function and facilitate the clinical response to these drugs even in the absence of adequate serum concentrations of total testosterone.

Publisher's version (útgefin grein)., Mutations in BRCA1 result in predisposal to breast and ovarian cancers, but many variants exist with unknown clinical significance (VUS). One is BRCA1 c.4096+3A>G, which affects production of the full-length BRCA1 transcript, while augmenting transcripts lacking most or all of exon 11. Nonetheless, homozygosity of this variant has been reported in a healthy woman. We saw this variant cosegregate with breast and ovarian cancer in several family branches of four Icelandic pedigrees, with instances of phenocopies and a homozygous woman with lung cancer. We found eight heterozygous carriers (0.44%) in 1820 unselected breast cancer cases, and three (0.15%) in 1968 controls (p = 0.13). Seeking conclusive evidence, we studied tumors from carriers in the pedigrees for wild-type-loss of heterozygosity (wtLOH) and BRCA1-characteristic prevalence of estrogen receptor (ER) negativity. Of 15 breast and six ovarian tumors, wtLOH occurred in nine breast and all six ovarian tumours, and six of the nine breast tumors with wtLOH were ER-negative. These data accord with a pathogenic BRCA1-mutation. Our findings add to the current knowledge of BRCA1, and the role of its exon 11 in cancer pathogenicity, and will be of use in clinical genetic counselling., This research was funded by the Landspitali University Hospital Research Fund, grant A-2019-001, and by the Icelandic association "Walking for Breast Cancer Research" (Göngum saman).

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.

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.

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.

Neuroendokrine Tumoren des gastroenteropankreatischen Systems (GEP-NET) sind charakterisiert durch die Expression neuroendokriner Marker sowie die Synthese, Speicherung und Sekretion von Peptidhormonen und/ oder biogenen Aminen. Es handelt sich um mehr als 50 unterschiedliche Tumorentitäten mit klinisch sehr unterschiedlichem Verlauf. Sie können sporadisch oder familiär auftreten. Die hohe biologische und klinische Vielfalt äußert sich in unterschiedlichen genetischen Profilen auf DNA- und mRNA-Ebene, welche wie folgt zusammengefasst werden können: 1. NET unterscheiden sich genetisch von den häufigen nichtneuroendokrinen Karzinomen des Verdauungstraktes. 2. Die klinisch-pathologische Heterogenität der GEP-NET spiegelt sich auch auf genetischer Ebene wider. 3. Bei pankreatischen NET finden sich deutliche genetische Unterschiede zwischen Insulinomen und anderen Entitäten. 4. Die Tumorprogression geht bei pankreatischen NET mit einer Zunahme von genetischen Veränderungen einher. 5. Pankreatische NET unterscheiden sich genetisch von gastrointestinalen NET. 6. Sporadische GEP-NET unterscheiden sich genetisch von hereditären (familiären) GEP-NET.

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.

Esophageal adenocarcinoma (EAC) develops from Barrett's esophagus (BE), wherein normal squamous epithelia is replaced by specialized intestinal metaplasia in response to chronic gastroesophageal acid reflux. BE can progress to low- and high-grade dysplasia, intramucosal, and invasive carcinoma. Both BE and EAC are characterized by loss of heterozygosity, aneuploidy, specific genetic mutations, and clonal diversity. Given the limitations of histopathology, genomic and epigenomic analyses may improve the precision of risk stratification. Assays to detect molecular alterations associated with neoplastic progression could be used to improve the pathologic assessment of BE/EAC and to select high-risk patients for more intensive surveillance.

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.

We investigated whether genetic lesions such as loss of heterozygosity (LOH) are detected in prostatic cells obtained by prostatic massage during early diagnosis of prostate cancer (CaP) and discussed their clinical relevance. Blood and first urine voided after prostatic massage were collected in 99 patients with total prostate-specific antigen (PSA) between 4 and 10 ng ml−1, prior to prostate biopsies. Presence of prostatic cells was confirmed by quantitative RT–PCR analysis of PSA mRNA. Genomic DNA was analysed for LOH on six chromosomal regions. One or more allelic deletions were found in prostatic fluid from 57 patients analysed, of whom 33 (58%) had CaP. Sensitivity and specificity of LOH detection and PSA free to total ratio

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.

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

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.

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 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 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.

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.

Background Vasoactive intestinal polypeptide secreting tumors(VIPomas) are rare endocrine tumors of the pancreas with an estimated incidence of 0.1 per million per year. The molecular mechanisms that mediate development of VIPomas are poorly investigated and require definition. Methods A genome- and gene expression analysis of specimens of a primary pancreatic VIPoma with hepatic metastases was performed. The primary tumor, the metastases, the corresponding healthy tissue of the liver, and the pancreas were compared with each other using oligonucleotide microarrays and loss of heterozygosity (LOH). Results The results revealed multiple LOH events and several differentially expressed genes. Our finding of LOH and downregulation was conspicuous in the microarray analysis for the mismatch repair gene MSH2 in the primary pancreatic VIPoma tumor, the hepatic metastasis but not in the corresponding healthy tissue. Further a strong overexpression of the chemokine CXCR4 was detected in the hepatic metastases compared to its pancreatic primary. With a review of the literature we describe the molecular insights of metastatic development in VIPoma. Conclusion In VIPoma, defects in the mismatch repair system especially in MSH2 may contribute to carcinogenesis, and increased CXCR4 may be associated with liver metastasis.

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.

Duisburg, Essen, Univ., Diss., 2010 Ziel der vorliegenden Dissertation war die Eingrenzung von Kandidatengenen, die zur Progression von Retinoblastomen mit einem Verlust von genomischem Material bzw. einem LOH auf Chromosom 16q und Metastasierung in den Glaskörper führen. Ein Teilaspekt dieser Zielsetzung war es, ein Testsystem zu etablieren, das zur Untersuchung der eingegrenzten Kandidatengene genutzt werden konnte. Zur Einengung der Kandidatengene wurden die in einer Vorstudie (Gratias et al., 2007) definierten LOH-Bereiche mit Hilfe einer hochauflösenden SNP-Array-Analyse weiter eingegrenzt. Die eingegrenzte LOH-Region hatte eine Größe von ungefähr 6,6 MB, beinhaltete 86 Gene und erstreckte sich innerhalb des Chromosoms 16q von 81,4 Mb bis 88,0 Mb. Unter Verwendung eines eigenständig geschriebenen Programms konnte innerhalb des gesamten Genoms zwei partielle LOHs in 8 von 8 Tumoren im kodierenden Bereich von Cadherin 13 definiert werden. Bei einer Expressionsstudie zur Retinaentwicklung der Maus mittels Expressionsmikroarrays konnte gezeigt werden, dass über die Hälfte von ca. 45000 Genen signifikant reguliert sind. Eine Korrelation der Ergebnisse der SNP-Array und der Mikroarrayanalyse identifizierte 35 Gene, die innerhalb der 6,6 Mb großen LOH-Region liegen und deren orthologe Gegenparts in der Maus signifikant in der Retinaentwicklung reguliert sind. Eine weitere Eingrenzung mit Hilfe von Mikroarraydaten von Retinoblastomen mit und ohne 16q LOH konnte die zuvor identifizierten Gene auf 26 Gene eingrenzen, unter denen sich das Gen Cadherin 13 befand. Zusätzliche Studien mittels qPCR zeigten, das Cadherin 13 im Vergleich zur humanen gesunden Retina in Retinoblastomzelllinien über das 60-fach herunterreguliert ist. Im Kontext der aktuellen Literatur lassen die in der vorliegenden Dissertation erzielten Ergebnisse den Schluss zu, dass Cadherin 13 eine Schlüsselrolle in der Progression von Retinoblastomen mit Metastasierung in den Glaskörpern zukommt. Mit dem ex ovo CAM-Modell wurde zudem ein in vivo Testsystem für die Untersuchung der 26 eingegrenzten Kandidatengene und speziell für Cadherin 13 etabliert sowie für zukünftige Untersuchungen an primären Retinoblastomen erweitert.

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.

Prostate cancer (PC) development reflects a complex sequence of biologic and molecular events. Several inheritable and somatic genetic changes have been identified. The knowledge of the molecular basis of PC can improve our understanding of the causes of this common cancer and provide information on prognosis and treatment. To date, however, no molecular studies have yet yielded consistent information that is ready to be incorporated into clinical practice. We reviewed the current literature on the molecular biology of prostate cancer and analyzed different potential tumor markers according to the classical concepts of oncogenes, suppressor genes, and the more modern concepts of genes involved in detoxification or inflammatory pathways of cancer progression. This review aims to identify trends in PC research and suggests potential clinical applications for diagnosis, prognosis, prevention and treatment.

Gesamthabilitationsschrift, Gliome und Meningeome sind die häufigsten primären intrakraniellen Tumore. Während letztere oft heilbar sind, so gibt es keine befriedigende Therapie für Gliome. Erst ein Verständnis genetischer Ursachen der Tumore bietet die Möglichkeit einer differenzierteren Diagnostik und verbesserten Therapie. In den vorliegenden Studien wurde gezeigt, dass Hirntumore genetische Veränderungen aufweisen, die z.T. mit spez. klinischen Verläufen oder charakteristischer Morphe assoziiert sind. Ausgehend von diesen chromosomalen Veränderungen wurde versucht, spezifische molekulargenetische Veränderungen zu identifizieren. Dieses erforderte neuartige Methoden der genetischen Annotation. So konnten in Astrozytomen (AZ) Kandidatenregionen näher eingegrenzt werden. Es konnte gezeigt werden, dass in AZ häufig CDKN2A und CDKN2B deletiert sind und dass Zellinien eine weitgehend gleiche Häufigkeit dieser Mutationen (Mut) im Vergleich zum Primärtumor aufweisen. Es wurde eine deutlich niedrigere Mut-häufigkeit in PIK3CA nachgewiesen, als initiale Studien implizierten. Es wurde erstmalig ein Tumor dokumentiert, welcher sowohl eine PIK3CA als auch eine PTEN Mut aufwies. Die Analyse von Microarray- Expressionsdifferenzen zwischen Glioblastomen (GBM) und anaplastischen Oligodendrogliomen (OG) erlaubte eine bessere klinische Prädiktion, als es die Histologie ermöglichte. In GBM wurden LOH 9p und 10q als Prediktionsmarker für eine Temozolomid Therapie identifiziert. 19q spielt eine wichtige Rolle in der Pathogenese glialer Tumore. Die Identifikation des Tumorsuppressorgens auf 19q13.3 wurde zum einen durch die erste detaillierte Annotation der Kandidatenregion, zum anderen durch den Ausschluss eines parentalen Imprintings weiter geführt. Ein diagnostisch verwendbarer Polymorphismus in PLA2G4C wurde gefunden. Aktivierte Onkogene sind in OG mit Wahrscheinlichkeit anzunehmen. PDGFRA ist solch ein Kandidat, doch gelang kein Nachweis von Mut. Mut wurden jedoch in PIK3CA in anaplastischen OG in geringer Zahl gefunden. In der histologischen Diagnostik wird eine Vielzahl an Varianten von Meningeomen (MG) beschrieben. Es konnte gezeigt werden, dass die seltenen Varianten unterschiedliche Frequenzen an NF2 Mut aufweisen. Weiterhin wurde eine Bedeutung von DAL-1 für multiple MG ausgeschlossen., Gliomas and meningiomas are the most frequent primary intracranial tumors. In many cases the therapeutic options are limited. Understanding the genetic background of these tumors might help to improve diagnosis and therapy of these tumors. In the presented studies it was shown that these tumors exhibit certain genetic alterations that are linked to a specific clinical course or to a characteristic phenotype. Based on these chromosomal alterations different genetic mutations were analyzed. This approach was supported by newly available data of the human genome project that offered new option of chromosomal annotation. By this, certain astrocytomas (AC) candidate regions were narrow down in their size. It was demonstrated that there are frequent deletion of CDKN2A and CDKN2B in AC and that cell lines exhibit a similar mutation frequency than the primary tumor. The frequency of PIK3CA mutations was found to be much lower than initially reported in AC. Microarray-based expression differences between glioblastomas (GBM) and oligodendrogliomas (OG) allowed a better survival prediction than histology. LOH 9p and 10q was identified to be a marker for a better clinical outcome in temozolamide- treated GBM patients. Chromosomal alterations on 19q are important for AZ and OZ as well. The candidate region 19q13.3 was annotated with a high resolution and parental 19q imprinting was excluded to be a mechanism of 19q alterations for OZ. A polymorphism in PLA2G4C was observed that is helpful in genetic diagnosis. Presumably, there are activated oncogenes on OG. However, no mutations were seen in the candidate gene PDGFRA that was analyzed in detail. But a small number of OZ exhibited mutations in an other oncogene named PIK3CA. There are multiple different histological variants of meningiomas. The genetic background of these variants remains unclear in many instances. However, it was observed that different rare meningiomas variants demonstrate varying mutation frequencies in the NF2 tumor suppressor gene. Furthermore, it was found that DAL-1 is not important for multiple meningiomas.

Background and aim: Colon cancer tumorigenesis is a multistep process of mutation accumulation in a number of oncogenes and tumour suppressor genes. NF1 gene protein, neurofibromin, acts as a tumour suppressor by turning the active form of Ras into an inactive form. This molecular switch has an important role in the control of the cell cycle and differentiation, and changes in Ras activity are present in many different cancers. This is the first study to investigate the role of NF1 in sporadic colon cancer. Methods: We investigated loss of heterozygosity (LOH) at the NF1 locus. Real time reverse transcription-polymerase chain reaction was used to determine NF1 mRNA expression in tumours and corresponding normal tissue. Expression of neurofibromin was analysed by immunohistochemistry. Relative ratio of NF1 mRNA type I and II isoform expression was also examined. Results: LOH of the NF1 gene was detected in 20.7% of heterozygous samples. NF1 mRNA expression was significantly increased in tumour tissue compared with corresponding normal tissue (p = 0.04291). There was a statistically significant increase in NF1 type I isoform expression (p = 0.0005) in tumour tissue compared with corresponding normal colon tissue. NF1 isoform type II was predominantly expressed in normal tissue while the NF1 isoform type I prevailed in tumour samples. The transition from dominant expression of isoform type II in normal mucous tissue 15 cm away from the tumour to dominant expression of isoform type I in tumour tissue itself was detected. Total neurofibromin expression increased as tumours were more advanced but expression of wild-type neurofibromin remained the same. Conclusions: Our findings suggest that the NF1 gene may play a role in the development and progression of colon cancer and the NF1 gene may be a potential tumour marker and a new potential target for colon cancer therapy.

Background: The discovery that genetic alterations in oncogenes and tumour suppressor genes accompany tumour formation in many human tumours has encouraged the search for genes that promote or suppress tumour spread and metastasis; nm23 is a promising candidate for a metastasis suppressing gene. Aims: To evaluate whether expression of nm23-H1 protein or loss of heterozygosity (LOH) of the nm23-H1 gene is associated with colon cancer progression. Materials/Methods: Paraffin wax embedded tissue sections were analysed immunohistochemically. DNA isolated from normal and tumour tissue was used for LOH analysis using a variable nucleotide tandem repeat (VNTR) marker located in the untranslated 5′ region of the nm23-H1 gene. RNA isolated from tumour and normal tissue was used for “real time” RT-PCR. Results: Of 102 adenocarcinomas examined, 58.8% stained weakly for nm23-H1 protein. There was a negative correlation between nm23-H1 positivity and tumour histological grade. In VNTR analysis, 70.2% of patients were informative and 27.4% of tumours had nm23-H1 LOH. There was a positive correlation between nm23-H1 LOH and both tumour histological grade and Dukes’s stage. Expression of nm23-H1 mRNA was increased in 22 of 30 colon tumours compared with normal tissue. No significant correlation was found between nm23-H1 mRNA expression and histological grade or Dukes’s stage of tumours. Conclusions: These findings suggest that nm23-H1 protein expression in early stages may have a role in suppressing metastasis in sporadic colon cancer, whereas at a later stage both reduced nm23-H1 protein expression and LOH of the nm23-H1 gene may play role in colon cancer progression and metastasis.

Aim: The objective of this thesis was to find genes and chromosomal regions involved in neuroblastoma (NB) tumor progression. NB is a childhood tumor of the sympathetic nervous system that generally occurs spontaneously. Biologically, NB has a complex heterogeneity from tumor progression to tumor regression, dependent on clinical stage and age at diagnosis. The main genetic markers, which are also of prognostic value in NB, are amplification of the oncogene MYCN, deletion of chromosome arm 1p and gain of chromosome arm 17q. Results: We have been shown using fluorescence in situ hybridization (FISH) on a Scandinavian tumor material, that 17q gain is present in approximately 65% of all NB stages, is significantly associated with poor prognosis and predicts survival. The gene encoding somatostatin receptor 2 (SSTR2), localized in chromosome region 17q24, was not found to be mutated in any NB, when analyzed with PCR-based single stranded conformation polymorphism/heteroduplex (SSCP/HD) and DNA sequencing. In a tentative effort of defining of the location of a general embryonal tumor suppressor gene (TSG) on 1p, we combined the smallest region of overlap (SRO) of 1p deletions in NB tumors and germ cell tumors (GCTs). We thus delimited the NB/GCT SRO to approximately 5 cM between markers D1S508 and D1S244, and fine-mapped this region by radiation hybrid mapping and construction of a bacterial artificial chromosome (BAC) contig. A homozygously deleted region in an NB cell line was found to partially overlap the proximal part of the 5 cM-SRO defined by us, which further focused our search for a TSG to a 500 kb candidate region in 1p36.22. Two attractive candidate NB TSGs, DFFA and CASP9, are both located in 1p36.2 and encode key apoptotic mediators. In fact, DFFA resides in the 500 kb TSG candidate region. Via sequence analysis of the entire tumor material, we found three different coding alterations in DFFA which all affect the highly conserved N-terminal regulatory domain of DFF45. Using RT-PCR and real-time RT-PCR (TaqMan) studies, we were able to show that both DFFA and CASP9 are preferably expressed in NB tumors with favorable outcome. It has been proposed that lack of apoptosis plays an important role in tumor progression. We therefore screened an array with cDNAs involved in the apoptotic process, to find genes differentially expressed in NB tumors with unfavorable versus favorable biology. Using real-time RT-PCR analysis, we verified the differential expression of several transcripts encoding mitochondrial apoptotic mediators. Conclusions: We have shown that 17q gain is the most frequently detected alteration in NB and that it is associated with established prognostic factors. We narrowed down the TSG candidate region on 1p and found mutations in a gene localized in the region possessing fundamental functions in apoptosis. Our results also suggest that the mitochondrial apoptotic pathway is suppressed at multiple steps in advanced stages of NB tumors, due to imbalance between anti-apoptotic and pro-apoptotic mediators.

The most frequent genetic alterations described in neuroblastoma (NB) are amplification of MYCN oncogene and deletion of chromosome 1p, although somatic deletions have been demonstrated at other chromosomal intervals. Since loss of heterozygosity (LOH) at distal 4p has been observed in about 20-29% of neuroblastomas, we have evaluated deletions in 41 Italian NB samples by LOH analysis at loci mapping to 4p as follows: pter-D4S2936-D4S412-D4S2957-D4S432-D4S3023-D4S431-cen. Our analysis showed allele losses in eight out of 41 samples (19.5%) and allowed the identification of a smallest region of overlapping deletion (SRO) of 3.0 cM, delimited by D4S412 and D4S3023. Two of these tumors with 4p LOH are from patients belonging to a family with recurrent NB. Interestingly the genotyping of this family revealed an identical haplotype that includes the nonrecombinant loci D4S412, D4S2957 and D4S432 shared by all affected children and demonstrated that this haplotype is retained in the two tumors carrying somatic deletions from patients of this family. Furthermore linkage analysis was performed in two NB families and yielded an overall lod-score of 3.0 in the interval including the haplotype. This provides a confirmatory indication that the region delimited by D4S2936 and D4S3023, which also includes the new defined SRO, may harbor NB predisposing gene/s.

Approximately 2/3 of patients diagnosed with superficial transitional cell carcinoma of the urinary bladder (TCC) will recur within 2 years. Loss of chromosome 9 and loss of heterozygosity (LOH) at 9q34 in index TCCs identify a subset of patients at high risk of recurrence. This study explores genetic alterations on chromosomes 4, 8, 11 and 17 as predictors of recurrence. A total of 109 carcinomas were investigated at 26 loci. DNA was extracted from microdissected archival normal/tumour tissue and was analysed for loss of heterozygosity (LOH). Fluorescent PCR was performed and genotyping carried out on a Perkin Elmer ABI377 sequencer. LOH of D11S490 or D17S928 was significantly more frequent in index carcinomas of patients who experienced recurrence compared to those with no recurrence (P = 0.004 and 0.019 respectively). These results suggest that loss of these regions is associated with recurrence of TCC. Further investigation is required to identify genes in these regions, which might be responsible for driving recurrence in TCC of the urinary bladder. © 2001 Cancer Research Campaign http://www.bjcancer.com

Carcinogenesis results from an accumulation of several genetic alterations. Mutations in the p53 gene are frequent and occur at an early stage of lung carcinogenesis. Loss of multiple chromosomal regions is another genetic alteration frequently found in lung tumours. We have examined the association between p53 mutations, loss of heterozygosity (LOH) at frequently deleted loci in lung cancer, and tobacco exposure in 165 tumours from non-small cell lung cancer (NSCLC) patients. A highly significant association between p53 mutations and deletions on 3p, 5q, 9p, 11p and 17p was found. There was also a significant correlation between deletions at these loci. 86% of the tumours with concordant deletion in the 4 most involved loci (3p21, 5q11–13, 9p21 and 17p13) had p53 mutations as compared to only 8% of the tumours without deletions at the corresponding loci (P< 0.0001). Data were also examined in relation to smoking status of the patients and histology of the tumours. The frequency of deletions was significantly higher among smokers as compared to non-smokers. This difference was significant for the 3p21.3 (hMLH1 locus), 3p14.2 (FHIT locus), 5q11–13 (hMSH3 locus) and 9p21 (D9S157 locus). Tumours with deletions at the hMLH1 locus had higher levels of hydrophobic DNA adducts. Deletions were more common in squamous cell carcinomas than in adenocarcinomas. Covariate analysis revealed that histological type and p53 mutations were significant and independent parameters for predicting LOH status at several loci. In the pathogenesis of NSCLC exposure to tobacco carcinogens in addition to clonal selection may be the driving force in these alterations. © 2001 Cancer Research Campaign http://www.bjcancer.com

Studies of Li-Fraumeni syndrome fibroblasts heterozygous for germline TP53 mutations have shown that loss of heterozygosity (LOH) occurs during passaging and is associated with genomic instability, such as chromosomal aberrations and aneuploidy. to investigate the genomic changes associated with LOH in Li-Fraumeni (LF) fibroblasts, we have analysed cell strains at increasing population doublings (PD) using Comparative Genomic Hybridization (CGH). We have looked at three groups of cell strains: LF mutation-carrying strains which showed LOH for TP53, LF mutation-carrying strains which did not show LOH, and strains from normal individuals. Using CGH, we have detected loss of distinct chromosomal regions associated with LOH in 4 out of 5 mutation-carrying strains. In particular we have found loss of chromosomal regions containing genes involved in cell cycle control or senescence, including loss of 9p and 17p in these strains. Other recurrent changes included loss of chromosomes 4q and 6q, regions shown to contain one or more tumour suppressor genes. No genomic alterations were detected at cumulative PD in the normal strains or in the LF mutation-carrying strains which did not show LOH for TP53. We have also analysed the three groups of strains for microsatellite instability and somatic TP53 mutations, and have found genetic alterations in only one strain. © 2000 Cancer Research Campaign