Allelic Loss Of Heterozygosity Research Articles

Secretin-induced Duodenal Aspirate of Pancreatic Juice (SIDA): Utility of Commercial Genetic Analysis.

Secretin-induced duodenal aspiration (SIDA) of pancreatic duct fluid has been proposed for pancreatic neoplasm screening in very high-risk patients. We sought to determine the clinical yield and safety of commercially-analyzed SIDA samples in patients at moderately elevated risk. A prospectively maintained institutional database of pancreatic fluid DNA profiles was retrospectively reviewed. Fifty-seven patients underwent SIDA testing, most commonly for intraductal papillary mucinous neoplasms (n=43) and not otherwise specified solitary cysts (n=9). SIDA mutation yield was low compared to 37 concomitant endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) samples of pancreatic fluid: KRAS (2.5% vs. 40.0%), GNAS (2.6% vs. 11.1%) and allelic loss of heterozygosity (3.1% vs. 0%). Patients undergoing SIDA alone experienced no complications while 3 patients with concomitant EUS-FNA had post-procedural pancreatitis. The genetic yield of commercially-analyzed SIDA samples was relatively low in a moderately elevated risk cohort. SIDA testing may have a better safety profile than EUS-FNA.

Association of TIMP3 expression with vessel density, macrophage infiltration and prognosis in human malignant melanoma

AimsSeveral anti-tumour properties have been ascribed to the tissue inhibitor of matrix metalloproteinases-3 (TIMP3) gene, including inhibition of neovascularisation in tumour xenografts. Reduced protein expression has been linked to promoter hypermethylation and allelic loss of heterozygosity in various human malignancies. In melanoma-positive lymph nodes from patients, we evaluated the association between TIMP3 expression, vessel density, macrophage infiltration and potential correlations with disease-free survival (DFS) and overall survival (OS). Patients and methodsTIMP3 expression was analysed by immunohistochemistry (IHC) in melanoma lymph node biopsies of stage III melanoma patients (n = 43). Blood vessel density and macrophage infiltration were quantitatively assessed and correlation with TIMP3 expression was investigated. Methylation status of the gene promoter was determined using methylation-specific polymerase chain reaction (MSP). Protein expression and promoter methylation status were investigated for associations with DFS and OS. ResultsReduced expression of TIMP3, as determined by IHC, was observed in 74% of the cases (32 in 43). A significant inverse correlation was observed between TIMP3 expression and vessel density (p = 0.031). Correlation between TIMP3 expression and macrophage infiltration was not statistically significant (p = 0.369). MSP analysis revealed methylation of the gene promoter in 18% (7 in 38) of the analysed cases. No differences in OS and DFS were observed between cases with high and low TIMP3 expression. Gene promoter methylation was significantly associated with both poor 5-year DFS (p = 0.024) and OS (p = 0.034). ConclusionsOur data indicate that TIMP3 is a dominant negative regulator of angiogenesis in cutaneous melanoma and gene silencing by promoter methylation is associated with poor outcome.

Loss of heterozygosity of TRIM3 in malignant gliomas

BackgroundMalignant gliomas are frequent primary brain tumors associated with poor prognosis and very limited response to conventional chemo- and radio-therapies. Besides sharing common growth features with other types of solid tumors, gliomas are highly invasive into adjacent brain tissue, which renders them particularly aggressive and their surgical resection inefficient. Therefore, insights into glioma formation are of fundamental interest in order to provide novel molecular targets for diagnostic purposes and potential anti-cancer drugs. Human Tripartite motif protein 3 (TRIM3) encodes a structural homolog of Drosophila brain tumor (brat) implicated in progenitor cell proliferation control and cancer stem cell suppression. TRIM3 is located within the loss of allelic heterozygosity (LOH) hotspot of chromosome segment 11p15.5, indicating a potential role in tumor suppression. ...MethodsHere we analyze 70 primary human gliomas of all types and grades and report somatic deletion mapping as well as single nucleotide polymorphism analysis together with quantitative real-time PCR of chromosome segment 11p15.5.ResultsOur analysis identifies LOH in 17 cases (24%) of primary human glioma which defines a common 130 kb-wide interval within the TRIM3 locus as a minimal area of loss. We further detect altered genomic dosage of TRIM3 in two glioma cases with LOH at 11p15.5, indicating homozygous deletions of TRIM3.ConclusionLoss of heterozygosity of chromosome segment 11p15.5 in malignant gliomas suggests TRIM3 as a candidate brain tumor suppressor gene.

Combined genome‐wide allelotyping and copy number analysis identify frequent genetic losses without copy number reduction in medulloblastoma

Detailed analysis of mechanisms of genetic loss for specific tumor suppressor genes (TSGs; e.g., RB1, APC and NF1) indicates that TSG inactivation can occur by allelic loss of heterozygosity (LOH), without any alteration in DNA copy number. However, the role and prevalence of such events in the pathogenesis of specific malignancies remains to be established on a genome-wide basis. We undertook a detailed molecular assessment of chromosomal defects in a panel of nine cell lines derived from primary medulloblastomas, the most common malignant brain tumors of childhood, by parallel genome-wide assessment of LOH (allelotyping) and copy number aberrations (comparative genomic hybridization and fluorescence in situ hybridization). The majority of genetic losses observed were detected by both copy number and LOH methods, indicating they arise through the physical deletion of chromosomal material. However, a significant proportion of losses (17/42, 40%) represented regions of allelic LOH without any associated copy number reduction; these events involved both whole chromosomes (10/17) and sub-chromosomal regions (7/17). Using this approach, we identified medulloblastoma-characteristic alterations, e.g., isochromosome for 17q, MYC amplification and losses on chromosomes 10, 11, and 16, alongside novel regions of genetic loss (e.g., 10q21.1-26.3, 11q24.1-qter). This detailed genetic characterization of the majority of medulloblastoma cell lines provides important precedent for the widespread involvement of copy number-neutral genetic losses in medulloblastoma and demonstrates that combined assessment of copy number aberrations and LOH will be necessary to accurately determine the contribution of chromosomal defects to tumor development.

Detection and molecular characterisation of thyroid cancer precursor lesions in a specific subset of Hashimoto's thyroiditis.

Hashimoto's thyroiditis (HT) represents the most common cause of hypothyroidism and nonendemic goiter, but its clinical and pathological heterogeneity opens the question if this disease should be more properly considered as a spectrum of different thyroid conditions rather than as a single nosological entity. In this study, we analysed 133 cases of HT for the expression of galectin-3, a lectin molecule involved in malignant transformation, apoptosis and cell cycle control. An unexpected expression of galectin-3 was demonstrated in a subset of HT together with the presence of HBME-1, c-met and cyclin-D1 that are also involved in malignant transformation and deregulated cell growth. Furthermore, a loss of allelic heterozygosity in a specific cancer-related chromosomal region was demonstrated in some HT harbouring galectin-3-positive follicular cells, by using laser capture microdissection. On the basis of the morphological and molecular findings we identified four subsets of HT: (a) HT with classic features of chronic autoimmune thyroiditis; (b) HT associated to hyperplastic/adenomatous lesions; (c) HT harbouring thyroid cancer precursors; (d) HT associated to unequivocal thyroid microcarcinomas. Our findings provide a well-substantiated morphological and molecular demonstration that HT may include a spectrum of different thyroid conditions ranging from chronic autoimmune thyroiditis to thyroiditis triggered by specific immune-response to cancer-related antigens.

Genetic alterations in bronchial mucosa and plasma DNA from individuals at high risk of lung cancer.

Evidence suggests that the majority of lung cancer patients have tumour-derived genetic alterations in circulating plasma DNA, and that this may be developed as a diagnostic tool. To this end, we have studied 60 individuals attending bronchoscopy clinic, with symptoms suspicious of lung cancer, for genetic alterations in bronchial mucosa biopsy (n = 47) and plasma (n = 40) DNA. Thirteen of 47 individuals from whom biopsies were taken displayed allelic loss of heterozygosity (LOH) in biopsy DNA for at least 1 of 4 markers. All 13 of these individuals had neoplastic tumour cells in their biopsies and were subsequently diagnosed with cancer. Thirteen of 40 individuals from whom plasma was taken displayed a plasma DNA LOH, and 12 of these 13 individuals were subsequently diagnosed with cancer. LOH in plasma was generally representative of LOH in the corresponding biopsy. In terms of sensitivity, using just 4 markers, biopsy LOH and plasma LOH were found in 13 of 44 (30%) and 12 of 29 (41%), respectively, of those patients subsequently diagnosed with cancer. Two patients were positive for LOH in plasma samples that pre-dated a diagnosis of cancer by several months. These data suggest that assay of genetic alterations in circulating plasma DNA may be developed as a useful addition to conventional techniques for the diagnosis of lung cancer.

Expression of Apoptosis-Regulating Proteins in Chronic Lymphocytic Leukemia: Correlations With In Vitro and In Vivo Chemoresponses

B-cell chronic lymphocytic leukemia (B-CLL) represents a neoplastic disorder caused primarily by defective programmed cell death (PCD), as opposed to increased cell proliferation. Defects in the PCD pathway also contribute to chemoresistance. The expression of several apoptosis-regulating proteins, including the Bcl-2 family proteins Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, and BAD; the Bcl-2–binding protein BAG-1; and the cell death protease Caspase-3 (CPP32), was evaluated by immunoblotting using 58 peripheral blood B-CLL specimens from previously untreated patients. Expression of Bcl-2, Mcl-1, BAG-1, Bax, Bak, and Caspase-3 was commonly found in circulating B-CLL cells, whereas the Bcl-XL and BAD proteins were not present. Higher levels of the anti-apoptotic protein Mcl-1 were strongly correlated with failure to achieve complete remission (CR) after single-agent therapy (fludarabine or chlorambucil) (P = .001), but the presence of only seven CRs among the 42 patients for whom follow-up data were available necessitates cautious interpretation of these observations. Higher levels of the anti-apoptotic protein BAG-1 were also marginally associated with failure to achieve CR (P = .04). Apoptosis-regulating proteins were not associated with patient age, sex, Rai stage, platelet count, hemoglobin (Hb) concentration, or lymph node involvement, although higher levels of Bcl-2 and a high Bcl-2:Bax ratio were correlated with high numbers (>105/μL) of white blood cells (WBC) (P = .01; .007) and higher levels of Bak were weakly associated with loss of allelic heterozygosity at 13q14 (P = .04). On the basis of measurements of apoptosis induction by fludarabine using cultured B-CLL specimens, in vitro chemosensitivity data failed to correlate with in vivo clinical response rates (n = 42) and expression of the various apoptosis-regulating proteins. Although larger prospective studies are required before firm conclusions can be reached, these studies show the expression in B-CLLs of multiple apoptosis-regulating proteins and suggest that the relative levels of some of these, such as Mcl-1, may provide information about in vivo responses to chemotherapy. In vitro chemosensitivity data, however, do not appear to be particularly useful in predicting responses in B-CLL.

Expression of Apoptosis-Regulating Proteins in Chronic Lymphocytic Leukemia: Correlations With In Vitro and In Vivo Chemoresponses

AbstractB-cell chronic lymphocytic leukemia (B-CLL) represents a neoplastic disorder caused primarily by defective programmed cell death (PCD), as opposed to increased cell proliferation. Defects in the PCD pathway also contribute to chemoresistance. The expression of several apoptosis-regulating proteins, including the Bcl-2 family proteins Bcl-2, Bcl-XL, Mcl-1, Bax, Bak, and BAD; the Bcl-2–binding protein BAG-1; and the cell death protease Caspase-3 (CPP32), was evaluated by immunoblotting using 58 peripheral blood B-CLL specimens from previously untreated patients. Expression of Bcl-2, Mcl-1, BAG-1, Bax, Bak, and Caspase-3 was commonly found in circulating B-CLL cells, whereas the Bcl-XLand BAD proteins were not present. Higher levels of the anti-apoptotic protein Mcl-1 were strongly correlated with failure to achieve complete remission (CR) after single-agent therapy (fludarabine or chlorambucil) (P= .001), but the presence of only seven CRs among the 42 patients for whom follow-up data were available necessitates cautious interpretation of these observations. Higher levels of the anti-apoptotic protein BAG-1 were also marginally associated with failure to achieve CR (P= .04). Apoptosis-regulating proteins were not associated with patient age, sex, Rai stage, platelet count, hemoglobin (Hb) concentration, or lymph node involvement, although higher levels of Bcl-2 and a high Bcl-2:Bax ratio were correlated with high numbers (>105/μL) of white blood cells (WBC) (P= .01; .007) and higher levels of Bak were weakly associated with loss of allelic heterozygosity at 13q14 (P= .04). On the basis of measurements of apoptosis induction by fludarabine using cultured B-CLL specimens, in vitro chemosensitivity data failed to correlate with in vivo clinical response rates (n = 42) and expression of the various apoptosis-regulating proteins. Although larger prospective studies are required before firm conclusions can be reached, these studies show the expression in B-CLLs of multiple apoptosis-regulating proteins and suggest that the relative levels of some of these, such as Mcl-1, may provide information about in vivo responses to chemotherapy. In vitro chemosensitivity data, however, do not appear to be particularly useful in predicting responses in B-CLL.

DNA Mapping in Growth and Developmental Disorders

DNA mapping techniques are being increasingly applied to familial and acquired disorders affecting growth and development. To understand the potential applications of these techniques, one must first have a good understanding of the components and basic structure of DNA, the genetic code, the basic structure of genes and the roles that various components of gene structure play in regulating gene expression. Following review of this basic information, a variety of DNA mapping techniques including in situ hybridization, Southern blotting, polymerase chain reaction amplification, DNA sequencing and linkage analysis will be covered. Applications of these techniques can enable mapping of unknown genes by detection of loss of allelic heterozygosity or use of linkage analysis and genetic maps. With increasing ease, mutations such as deletions, expansions, rearrangements and point mutations can be detected in diseases such as congenital adrenal hyperplasia, cystic fibrosis, diabetes insipidus, growth hormone deficiency, fragile X syndrome, Laron dwarfism and Turner syndrome. From this discussion, a better understanding of methods of gene localization, uses of genetic maps and rapid, convenient methods to detect a variety of molecular derangements causing familial disorders affecting growth and development will be gained.

A rapid and non-radioactive PCR based assay for the detection of allelic loss in human gliomas.

Studies of the loss of allelic heterozygosity (LOH) in tumour tissues have evolved as an important tool for the identification of chromosomal regions which are likely to harbour tumour suppressor genes. The classical procedure to determine LOH has been restriction fragment length polymorphism (RFLP) analysis and Southern blotting, a time consuming method requiring radioisotopes and several micrograms of DNA. Recently, the use of highly polymorphic microsatellites of the CA-dinucleotide repeat class and polymerase chain reaction (PCR) has considerably advanced and facilitated the detection of LOH in tumour tissues. We here describe a strategy to identify LOH based on PCR amplification of CA-dinucleotide repeats, denaturing polyacrylamide gel electrophoresis (PAGE) and nucleic acid detection with a sensitive silver staining protocol. In a comparative study of 20 astrocytomas, this rapid technique was able to identify all cases of LOH on chromosome 17 p that had previously been found in these tumours by RFLP analysis and Southern blotting. This non-radioactive PCR based assay has a great potential for LOH studies in human tumours.

Loss of heterozygosity for alleles on chromosome 10 in human brain tumours.

We analysed for loss of alleles on chromosome 10, 25 astrocytomas, 3 ependymomas, 2 medulloblastomas, 2 juvenile pilocytic astrocytomas, 2 gangliogliomas, 1 subependymal giant cell astrocytoma and 1 anaplastic oligoastrocytoma. A battery of 12 DNA markers spanning chromosome 10 was employed. Loss of heterozygosity on chromosome 10 was seen in 16 tumours (13 glioblastoma multiforme, 2 anaplastic astrocytomas, and 1 anaplastic oligoastrocytoma), but not in any of the low-grade astrocytomas examined. High-resolution restriction fragment length polymorphism (RFLP) analysis showed that the loss of alleles in a number of tumours involved two separate large regions of chromosome 10 (10p-proximal 10q and distal 10q). However, a small common region of deletion overlap could not be identified. Our data indicate that the loss of alleles on chromosome 10 is a common finding, seen in over two-thirds of malignant astrocytomas, and may be suggestive of the presence of two or more chromosome 10 tumour suppressor genes involved in astrocytoma formation. Nevertheless, the possibility of these genetic changes being secondary and not causative of the deregulated cell growth cannot be excluded. Regardless of the mechanisms involved, however, chromosome 10 deletions may be a genetic marker for malignant astrocytomas.

Loss of allelic heterozygosity at the harveyras locus in human oral carcinomas

The Harvey ras locus was examined for restriction fragment polymorphism and loss of allelic heterozygosity in 62 oral cancer patients. Southern blot analysis on BamHI digests of the tumour tissue DNA, revealed 23 patients with H-ras-1 heterozygosity. The probes used to study the polymorphism were the BamHI 6.6-kb fragment encoding the complete H-ras-1 sequence plus the variable tandem repeat (VTR) region, and the 1-kb MspI fragment encoding the VTR region. The allelic heterozygosity was better resolved by PvuII and further confirmed by TaqI. In addition, TaqI digestion demonstrated a unique VTR rearrangement indicated by 2.1-kb, 0.9-kb and 0.6-kb fragments, implying additional TaqI sites, in three of the patients. Further analysis of matched tumor tissue and peripheral blood cell DNA from the same patient demonstrated tumor-associated loss of one of the allelic fragments in 7/23 (30%) of the patients with H-ras-1 heterozygosity. However, the loss was not significantly correlated to clinicopathological parameters staging the disease. Thus, our data showing loss of H-ras-1 alleles and VTR rearrangement, with relatively high incidence (9/23; 39%) in the oral cancer patients at various stages of the disease, implies H-ras-1 involvement as an early event in the process of oral carcinogenesis.

Loss of allelic heterozygosity on distal chromosome 1p in Merkel cell carcinoma: A marker of neural crest origins?

The location of genes involved in tumor evolution has been inferred from experiments in which loss of constitutional heterozygosity has been detected in tumor DNA at high frequency in specific chromosome regions. For example, cytogenetic and molecular abnormalities on chromosome 1p have been reported in tumors such as malignant melanoma and neuroblastoma which arise in cells derived from embryonic neural crest tissue. To extend these observations, we have examined tumor DNA from three cases of Merkel cell carcinoma for evidence of loss of constitutional heterozygosity on the short arm of chromosome 1. In all three cases, heterozygous allelic deletions of varying extent on distal chromosome 1p were detected in tumor DNA. Comparisons with neural crest tumors suggest that loss of heterozygosity on distal chromosome 1p in Merkel cell tumors may be a marker of neural crest origin.