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The worldwide approval of the combination maintenance therapy of olaparib and bevacizumab in advanced high-grade serous ovarian cancer requires complex molecular diagnostic assays that are sufficiently robust for the routine detection of driver mutations in homologous recombination repair (HRR) genes and genomic instability (GI), employing formalin-fixed (FFPE) paraffin-embedded tumor samples without matched normal tissue. We therefore established a DNA-based hybrid capture NGS assay and an associated bioinformatic pipeline that fulfils our institution’s specific needs. The assay ́s target regions cover the full exonic territory of relevant cancer-related genes and HRR genes and more than 20,000 evenly distributed single nucleotide polymorphism (SNP) loci to allow for the detection of genome-wide allele specific copy number alterations (CNA). To determine GI status, we implemented an %CNA score that is robust across a broad range of tumor cell content (25–85%) often found in routine FFPE samples. The assay was established using high-grade serous ovarian cancer samples for which BRCA1 and BRCA2 mutation status as well as Myriad MyChoice homologous repair deficiency (HRD) status was known. The NOGGO (Northeastern German Society for Gynecologic Oncology) GIS (GI-Score) v1 assay was clinically validated on more than 400 samples of the ENGOT PAOLA-1 clinical trial as part of the European Network for Gynaecological Oncological Trial groups (ENGOT) HRD European Initiative. The “NOGGO GIS v1 assay” performed using highly robust hazard ratios for progression-free survival (PFS) and overall survival (OS), as well a significantly lower dropout rate than the Myriad MyChoice clinical trial assay supporting the clinical utility of the assay. We also provide proof of a modular and scalable routine diagnostic method, that can be flexibly adapted and adjusted to meet future clinical needs, emerging biomarkers, and further tumor entities.

The worldwide approval of the combination maintenance therapy of olaparib and bevacizumab in advanced high-grade serous ovarian cancer requires complex molecular diagnostic assays that are sufficiently robust for the routine detection of driver mutations in homologous recombination repair (HRR) genes and genomic instability (GI), employing formalin-fixed (FFPE) paraffin-embedded tumor samples without matched normal tissue. We therefore established a DNA-based hybrid capture NGS assay and an associated bioinformatic pipeline that fulfils our institution’s specific needs. The assay´s target regions cover the full exonic territory of relevant cancer-related genes and HRR genes and more than 20,000 evenly distributed single nucleotide polymorphism (SNP) loci to allow for the detection of genome-wide allele specific copy number alterations (CNA). To determine GI status, we implemented an %CNA score that is robust across a broad range of tumor cell content (25–85%) often found in routine FFPE samples. The assay was established using high-grade serous ovarian cancer samples for which BRCA1 and BRCA2 mutation status as well as Myriad MyChoice homologous repair deficiency (HRD) status was known. The NOGGO (Northeastern German Society for Gynecologic Oncology) GIS (GI-Score) v1 assay was clinically validated on more than 400 samples of the ENGOT PAOLA-1 clinical trial as part of the European Network for Gynaecological Oncological Trial groups (ENGOT) HRD European Initiative. The “NOGGO GIS v1 assay” performed using highly robust hazard ratios for progression-free survival (PFS) and overall survival (OS), as well a significantly lower dropout rate than the Myriad MyChoice clinical trial assay supporting the clinical utility of the assay. We also provide proof of a modular and scalable routine diagnostic method, that can be flexibly adapted and adjusted to meet future clinical needs, emerging biomarkers, and further tumor entities.

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.

Loss of heterozygosity (LOH) happens when a heterozygous genome loses one of the two alleles at a locus. This may have an evolutionary advantage in highly unstable genomes such as those of hybrids (Smukowski Heil et al., 2017). By extracting LOH from a hybrid we understand which alleles were beneficial in its evolution. This is important in wild, clinical, and industrial settings (Gabaldón, 2020). The genomic properties of hybrids are still, however, poorly understood. LOH are studied with reliable short-read sequencing data (Mixão et al., 2019), but the downstream analysis is often done with custom scripts that reduce reproducibility and do not to leverage the power of a computing cluster. Here we present a program called “JLOH” that streamlines LOH extraction from sequencing data maximizing parallel computing.

The layer-ordered heap (LOH) is a simple data structure used in algorithms that perform optimal top-$k$ on $X+Y$, algorithms with the best known runtime for top-$k$ on $X_1+X_2+\cdots+X_m$, and the fastest method in practice for computing the most abundant isotopologue peaks in a chemical compound. In the analysis of these algorithms, the rank, $\alpha$, has been treated as a constant and $n$, the size of the array, has been treated as the sole parameter. Here, we explore the algorithmic complexity of LOH construction with $\alpha$ as a parameter, introduce a few algorithms for constructing LOHs, analyze their complexity in both $n$ and $\alpha$, and demonstrate that one algorithm is optimal in both $n$ and $\alpha$ for building a LOH of any rank. We then apply this to improve performance in applications where they are employed, find an estimate for the optimal $\alpha$ given an $n$ and $k$ for top-$k$ on $X+Y$, and derive a novel algorithm for top-$k$ on a multinomial distribution. Finally, we show that the results of our LOH analysis correspond with empirical experiments of runtimes when applying the LOH construction algorithms to both a common task in machine learning and top-$k$ on $X_1+X_2+\cdots+X_m$ and that our estimate of the optimal $\alpha$ for top-$k$ on $X+Y$ corresponds well with empirical data.

We show that N-acetyltransferase 2 (NAT2) loss of heterozygosity can be targeted in >4% of colorectal cancers with the use of a small molecule. We identify and describe the effect of a compound that impairs the growth of colorectal tumors with slow NAT2 activity by half when compared to wild-type.

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.

Regulated erroneous protein translation (adaptive mistranslation) increases proteome diversity and produces advantageous phenotypic variability in the human pathogen Candida albicans. It also increases fitness in the presence of fluconazole, but the underlying molecular mechanism is not understood. To address this question, we evolved hypermistranslating and wild-type strains in the absence and presence of fluconazole and compared their fluconazole tolerance and resistance trajectories during evolution. The data show that mistranslation increases tolerance and accelerates the acquisition of resistance to fluconazole. Genome sequencing, array-based comparative genome analysis, and gene expression profiling revealed that during the course of evolution in fluconazole, the range of mutational and gene deregulation differences was distinctively different and broader in the hypermistranslating strain, including multiple chromosome duplications, partial chromosome deletions, and polyploidy. Especially, the increased accumulation of loss-ofheterozygosity events, aneuploidy, translational and cell surface modifications, and differences in drug efflux seem to mediate more rapid drug resistance acquisition under mistranslation. Our observations support a pivotal role for adaptive mistranslation in the evolution of drug resistance in C. albicans. IMPORTANCE Infectious diseases caused by drug-resistant fungi are an increasing threat to public health because of the high mortality rates and high costs associated with treatment. Thus, understanding of the molecular mechanisms of drug resistance is of crucial interest for the medical community. Here we investigated the role of regulated protein mistranslation, a characteristic mechanism used by C. albicans to diversify its proteome, in the evolution of fluconazole resistance. Such codon ambiguity is usually considered highly deleterious, yet recent studies found that mistranslation can boost adaptation in stressful environments. Our

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 and aim: Recent studies focusing on the discovery of common alterations across cases suffering from follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) have reported loss of heterozygosity (LOH) and deletions events targeting the 15q21 region, indicating the relevance of this region in the lymphomagenesis of FL and DLBCL. Herein, we investigated the genetic structure of this region by studying identified LOH and copy-loss events and examined this region in the lymphomagenesis of FL and DLBCL. Methods: Fine mapping of the genomic region between the 15q15.1 and 15q21.1 loci was performed using data from copy number variation (CNV) and high resolution LOH analyses of FL (n=21) and DLBCL (n=21) cases. Validation of LOH of this region was performed using microsatellites followed by quantitative-PCR (qPCR) to measure the transcriptional abundance of TP53BP1 and B2M. Also, direct sequencing of exons 1 and 2 of B2M was performed on tumor DNA from 24 FL and 23 DLBCL samples. Results: The integration of LOH and CNV data identified copy-loss alterations at the 15q21 loci spanning a 7.5 Mb region, covering two LOH regions, termed LOH-1 and LOH-2. The LOH-1 region spans 3.4 Mb and contains 53 genes, from which TP53BP1 (tumor-protein-p53-binding-protein-1) and B2M (Beta-2-Microglobulin) were identified as the most likely target genes due to their roles in DNA double strand break (DSB) repair and immune recognition, respectively. Expression analyses revealed a significant up-regulation of TP53BP1 in NHL with LOH, but no significant changes in B2M expression were observed. Direct sequencing of exons 1 and 2 in B2M in FL and DLBCL identified two monoallelic microdeletions associated with DLBCL. Conclusion: This study identified that deletion mapping to the 15q21 locus cover two LOH regions. LOH of the TP53BP1 and B2M genes appear to be common alterations in FL and DLBCL tumorigenesis.

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 e

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

Our previous studies of chromosome 14 transfer into tumorigenic esophageal squamous cell carcinoma (ESCC) cell line, SLMT, suggested the existence of tumor suppressor genes on chromosome 14. Gene expression profiling of microcell hybrids and the tumor segregants identified an interesting gene, LTBP-2 (latent transforming growth factor beta binding protein 2), which has been analyzed here for its role in ESCC. LTBP-2 maps to 14q24 and encodes a secreted protein, which is a component of the extracellular matrix microfibrils. LTBP-2 expression was downregulated in ESCC cell lines and tumor tissues. Promoter hypermethylation was found to be involved in LTBP-2 inactivation. Functional studies indicated its tumor-suppressive roles in ESCC. In the in vitro colony formation and Matrigel three-dimensional culture assays, LTBP-2 decreased the colony-forming abilities of ESCC cell lines. LTBP-2 expression was associated with reduction of cell migrating and invasive abilities. LTBP-2 could also reduce the tube-forming ability of endothelial cells. Moreover, LTBP-2 induced tumor suppression in in vivo nude mouse assays. Tissue microarray immunohistochemical staining analysis indicated that LTBP-2 expression is reduced in tumor tissues when compared to normal tissues, and LTBP-2 expression correlated significantly with the survival of ESCC patients. Thus, LTBP-2 appears to play an important role in ESCC.

Recent studies indicate that soluble tumor DNA and microsatellite alterations have been identified in the peripheral blood of small cell lung cancer patients. There is a possibility that these might be useful for preoperative diagnosis, deciding on treatment strategy and postoperative evaluation as a new biomarker to detect genetic alterations originating from primary cancerous lesions in the peripheral blood of non-small cell lung cancer patients. The purpose of this study was to determine whether genetic alterations in resected cancer tissue could be identified in the preoperative peripheral serum by microsatellite DNA analysis. Our preliminary data, however, showed that no microsatellite alterations in tumor DNA could be found in the preoperative serum DNA of non-small cell lung cancer patients.

Pancreatic endocrine tumors (PETs) may cause typical syndromes of hormone excess, or appear clinically non-functioning without hormonal symptoms. PETs occur sporadically, in association with the multiple endocrine neoplasia type 1 (MEN1) syndrome, or rarely the von Hippel-Lindau syndrome. Molecular genetic investigations may reveal pathways important for tumor development, and be of clinical use. The aim of this thesis was to investigate regulation of different genes involved in cell proliferation, and relate findings to signs of malignancy in PETs. The MEN1 gene on chromosome 11q13 was mutated in three out of eleven sporadic malignant PETs. Two nonsense mutations, causing truncation of the protein, and one missense mutation were found. Relation of allelic loss at 11q13 and 3p25 to malignant behavior was observed in sporadic PETs. Allelic loss at 18q21 was found in a subset of sporadic and MEN1-associated PETs, and mutation analysis of Smad4 excluded a tumor suppressor gene function. In PETs with allelic loss on chromosome 3p25, mutation analysis of WNT7A and HDAC11 excluded function as tumor suppressor genes. Menin, encoded by the MEN1 gene, was reported to regulate expression of the cyclin-dependent kinase inhibitors CDKN2C/p18, CDKN1B/p27, and CDKN2B/p15 in mouse pancreatic islet tumor models. Here, the mRNA expression of these genes was not related to MEN1 gene mutations in human PETs. Cyclin-dependent kinase 4 (CDK4) and the protooncogene c-Myc were found to be overexpressed regardless of MEN1 gene mutational status of the PETs. The CDK4 gene was neither amplified nor mutated. Targeting of CDK4 may present an alternative to traditional chemotherapy of PETs in the future.

We examined 21 ovarian and 24 breastovarian cancer families with BRCA1 mutations among a Japanese population. Twenty-four kinds of mutations were observed in these families. The DNAs of the mutation carriers (including the cancer patients) with these mutations were genotyped with microsatellite markers (D17S855,D17S1322,D17S1323 and D17S1325) located around the BRCA1 gene. The microsatellite genotyping showed that the alleles with the same mutations had the same haplotypes. This suggests that patients with the same mutation may have a common ancestor. On the other hand, a loss of heterozygosity (LOH) in the 4 microsatellite markers was observed in 48 (96.0%) of 50 tumor tissues examined. A combination of microsatellite genotyping and LOH analysis revealed that DNAs of the tumors retained the alleles with haplotypes common to the patients with the same mutation. Based on these results, we speculated on which mutations would occur in ovarian cancer families, working inversely from the haplotypes determined by this combination of methods. Consequently, T307A mutation in 2 families and C2919T in 4 families could be easily found. Applying these methods, we were able to detect haplotypes of 22 mutation alleles common to persons with the same mutations. Moreover, we employed LOH analysis and discovered two patients with a germline mutation of BRCA1 among 57 ovarian cancer patients without a family history of ovarian cancer. Determination of the retained haplotype by LOH analysis was proven to be an efficient tool to detect BRCA1 mutations from both familial and non-familial ovarian cancer patients., departmental bulletin paper

A previous model of morphological pathogenesis assumed that cervical carcinoma is of monoclonal origin and progresses through multiple steps from normal epithelium via CINS into invasive carcinomas. The aim of this study was to investigate the molecular mechanism of pathogenesis of cervical neoplasia. In the clonality study, we found that 75% (6/8) of informative cases of cervical carcinoma had identical patterns of loss of heterozygosity (LOH) in the multiple synchronous lesions, while the remaining cases had different LOU patterns. In an extensively studied "golden case", the multiple carcinoma and cervical intraepithelial neoplasia (CIN) lesions could be divided into several different clonal groups by the X-chromosome inactivation patterns, HPV 16 mutations and LOH patterns. The biggest clonal family included one CIN II, one CIN III and four carcinoma samples, while four other monoclonal families of carcinoma did not include CIN lesions. These results suggested that cervical carcinoma can be either monoclonal or polygonal and contains clones developing either directly or via multiple steps. In the study of HPV types and HPV16 variations, the results confirmed that specific HPV types are the cause of cervical carcinoma but failed to support the previous opinion that HPV16 E6 variants are more malignant than the prototype. We established a novel classification called oncogene lineage of HPV16, and found that additional variations of HPV 16 oncogenes might be a weak further risk factor for cervical carcinoma. In the study of LOH, we found that interstitial deletion of two common regions of chromosome 3p, i.e., 3p2l.1-3p2l.3, and 3p22, was an early event in the development of cervical carcinoma. The results showed that the hMLH1 gene, located in 3p22 and showing LOH in 43% of the studied cases, was not involved in the development of cervical carcinoma because neither the expression level of protein nor the gene sequence was altered in these cases. In summary, a sugge

Detecting mutations and genomic variations is fundamental indiagnosis, isolating disease genes, association studies,functional genomics and pharmacogenomics. The objective hasbeen to use and further develop a variety of tools andtechnologies to analyze these genetic alterations andvariations. The p53 tumor suppressor gene and short arm of chromosome 9have been used as genetic markers to investigate fundamentalquestions concerning early events preceding non-melanoma skincancers, clonal progression and timing of different mutationsand deletions. Conventional gel based DNA sequencing andfragment analysis of microsatellite markers were utilized forthis purpose. In addition, a sequence-specific PCR-mediatedartifact is discussed. Pyrosequencing, a bioluminometric technique based onsequencing-by-synthesis, has been utilized to determinemutation ratios in the p53 gene. In addition, in the case ofmultiple mutations, pyrosequencing was adopted to determineallelic distribution of mutations without the use of cloningprocedures. Exons 5 to 8 of the p53 gene were also sequenced bythis method. The possibility of typing single base variations bypyrosequencing has been evaluated. Two different nucleotidedispensation orders were investigated and data were comparedwith the predicted pattern for each alternative of the variableposition. Analysis of loss of heterozygosity was possible byutilizing single nucleotide polymorphisms. A modified allele-specific extension strategy for genotypingof single nucleotide polymorphisms has been developed. Throughthe use of a real-time bioluminometric assay, it has beendemonstrated that reaction kinetics for a mismatchedprimer-template is slower than the matched configuration,butthe end-point signals are comparable. By introduction ofapyrase, the problems associated with mismatch extensions havebeen circumvented and accurate data has been obtained. Keywords:fragment analysis, microsatellite, loss ofheterozygosity, DNA sequencing, pyrosequencing, cance, QC 20100415 NR 20140805

Pancreatic endocrine tumors (PETs) occur sporadically or in the familial multiple endocrine neoplasia type 1 (MEN1) syndrome, whereas midgut carcinoids are nonfamilial, malignant endocrine tumors of the intestine. For these tumor entities morphological criteria are of limited use for prognostic prediction and selection of treatment. Genetic characterization may give additional information of clinical use and reveal pathways involved in tumor development. Molecular genetic alterations in sporadic and MEN1-associated PETs and midgut carcinoids were studied with LOH and mutational analysis. In addition, immunohistochemistry was used to clarify gene expression. Detected genetic aberrations were correlated to the disease course of individual patients. Somatic mutations of the MEN1 gene at chromosome 11q13 were detected in 1/3 of sporadic PETs. Moreover, LOH was found in 70% of the lesions. All tumors with somatic MEN1 mutations displayed loss of the remaining allele showing that the MEN1 gene is involved in development of sporadic PETs. Sporadic and MEN1 PETs were analyzed for LOH at 3p, 11q13 and 18q. A relation of LOH at 11q13 and 3p to malignancy was found for the sporadic tumors. None of the benign tumors (all of them insulinomas) had allelic loss at 3p or 11q13, versus 92 % (p<0.01) of the malignant tumors (including malignant insulinomas). 1/4 of both sporadic and MEN1 lesions displayed LOH at 18q, without altered Smad4/DPC4. Genome-wide LOH screening of MEN1 PETs revealed multiple allelic deletions without general correlation to tumor size or malignancy. All tumors displayed LOH at the MEN1 locus, and 30% on chromosomes 3, 6, 8, 10, 18 and 21. Intratumoral heterogeneity was revealed, with chromosome 6 and 11 deletions in most tumor cells. Chromosome 6 deletions were mainly found in lesions from patients with malignant features. A similar genome-wide LOH screening was performed on midgut carcinoids. Deletions at chromosome 18q were found in 88% of the tumors ind

Vulval intraepithelial neoplasia (VIN) is thought to be the premalignant phase of human papillomavirus (HPV)-associated vulval squamous cell carcinoma (VSCC). Various molecular events have been suggested as markers for progression from VIN to VSCC, but loss of heterozygosity (LOH) in vulval neoplasia has rarely been studied in this context. We performed LOH analysis by polymerase chain reaction (PCR) amplification of polymorphic microsatellite markers at 6 chromosomal loci (17p13-p53, 9p21-p16, 3p25, 4q21, 5p14 and 11p15). The presence of HPV was assessed using consensus PCR primers and DNA sequencing. To examine any association between LOH and the presence of invasive disease, we analyzed 43 cases of lone VIN III, 42 cases of lone VSCC and 21 cases of VIN with concurrent VSCC. HPV DNA was detected in 95% of lone VIN III samples and 71% of lone VSCC samples. Fractional regional allelic loss (FRL) in VIN associated with VSCC was higher than in lone VIN (mean FRL 0.43 vs. 0.21, p < 0.005). LOH at 3p25 occurred significantly more frequently in HPV-negative VSCC than in HPV-positive VSCC (58% vs. 22%, p < 0.04). These data suggest that genetic instability in VIN, reflected by LOH, may increase the risk of invasion. In addition, molecular events differ in HPV-positive and -negative VSCC and 3p25 may be the site of a tumor suppressor gene involved in HPV-independent vulval carcinogenesis.

Frequent allelic deletions at chromosome 11q24-q25 have been described in both early and late onset breast cancers, suggesting the existence of a gene locus implicated in the initiation and/or progression of the disease. In the present study we fine mapped this region further by loss of heterozygosity (LOH) analysis in a population of early onset breast cancer cases (n = 102, 22 to 36 years old). Loss of chromosomal material was assessed for possible association with patient survival as well as Nottingham histologic grade (NHG). Additionally, we investigated the involvement of the 11q24-q25 locus in a group of familial breast cancer cases with no detectable BRCA1 or BRCA2 gene alterations (n = 32, ages 28 to 40 years). Among the consecutive patients, extensive LOH was observed for all markers at 11q24-q25, with frequencies ranging from 42% to 54%. Deletion at the D11S4125 marker was found to be associated with reduced survival (p = 0.026), whereas the adjacent D11S387 marker correlated with higher histologic grade (p = 0.042). In the familial cases, the most telomeric markers showed substantially lower proportions of LOH, ranging from 10% to 21%. Comparison of the two patient groups demonstrated that this difference in LOH frequency was statistically significant for the D11S4098, D11S968, D11S387 and D11S4125 markers (p = 0.020, p = 0.029, p = 0.0070 and p = 0.0030, respectively). We conclude that 11q25 may harbor a gene implicated in early onset breast cancer. Our data suggest that the most probable position for this locus is defined by the markers D11S387 and D11S4125 and furthermore that it may play a less significant role in familial breast cancer cases not linked to either of the BRCA genes.

Cervical cancer derives from a series of pre-invasive cervical neoplastic lesions termed CIN I-III (Cervical Intraepithelial Neoplasia) via typical multiple-step processes. In contrast to premalignant disorders in other organs, different degrees of CIN possess a distinct biological behaviour where a proportion spontaneously regress or persist, whereas some progress to invasive cancer. A meticulous examination of pathological, genetical and viological aspects of the mechanisms for this dynamic event was performed. Clonality and clonal lineages of CIN and invasive cancer were studied on microdissected single or multiple synchronous lesions. Independent CIN lesions were both monoclonal and polyclonal, whereas invasive cancer and adjacent CIN inevitably were monoclonal in origin. Mostly when multiple lesions were analyzed, the invasive cancer shared an identical clonality pattern with the co-existing CIN but one third of multiple CIN lesions were multifocal and their clonal origin was not identical to synchronous invasive cancer. The data suggest that CIN lesions vary in their biological behaviour although they are morphologically similar. Polyclonal CIN may represent a benign proliferation,whereas monoclonal CIN more likely progresses to an invasive stage. Multifocal origin of CIN suggests that multiple cells from the cervix undergo transformation simultaneously, implying an essential role of a "field defect" (HPV?) in cervical carcinogenesis. Genetic deletions (LOH) of chromosome 3p, 6p and 6q were investigated in CIN and SCC. The detection of allelic losses and the deletion mapping suggested that the alterations of 3p22-21.3, 3p21.1, HLA region on 6p and 6q16.1-21 were frequent and early genetic events in cervical cancer and imply that these chromosome regions harbour potential tumor suppressor genes important in thecervical carcinogenesis. HPV 16 E6 and L1 gene variations were analyzed in CIN lesions independent or synchronous with invasive cancer. Distribution of H

Mechanism of multiplicity in the glial tumor is still unknown. The p53 gene has been impliceted as a tumor suppressor gene and abnormality of this gene has been reported in many human glial tumors. Codon 72 polymorphism in the p53 gene can be detected by digestion of polymerase chain reaction-amplified DNA using allele-specific restriction endonuclease (PCR-RFLP). We analyzed the DNA from the tissue in the two separated enhanced lesions of Gd-MRI in the same patient using this PCR-RFLP method and detected loss of the same allele of the p53 gene in each enhanced area. This result suggests there is a possibility that the two separated lesions originated from same progenitor cell and the multiplicity of this case resulted from metastatic mechanism.