Loss Of Heterozygosity Mutations Research Articles

Underlying mechanisms for LTF inactivation and its functional analysis in nasopharyngeal carcinoma cell lines

The lactoferrin (LTF) gene, located at 3p21.3, behaves like a tumor suppressor gene in diverse tumors. To elucidate the exact role of LTF in NPC, we first detected its expression level in seven NPC cell lines by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The results showed the mRNA level of LTF was nearly undetectable in all the seven NPC cell lines, while it could be detected in chronic nasopharyngitis tissues. Subsequently, we used methylation-specific PCR (MSP), microsatellite assay, PCR-single-strand conformation polymorphism (PCR-SSCP) and sequencing methods to examine the promoter methylation, loss of heterozygosity (LOH) and gene mutation of LTF in NPC cell lines respectively. Consequently, we found that 100% (7 of 7) of NPC cell lines were methylated in LTF promoter, only one cell line (14%, 1 of 7) had LOH and gene mutation of LTF, respectively, while LTF exhibited re-expression in all cell lines after 5-aza-dC treatment, indicating promoter methylation should be the key mechanism causing LTF downregulation in NPC cell lines. Furthermore, patched methylation assay confirmed that promoter methylation could down-regulate LTF gene expression in NPC cells. Finally, we investigated the function of LTF in NPC cell lines by gene transfection. Restoration of LTF expression in NPC cells resulted in blockage of cell cycle progression, significant inhibition of cell growth and a reduced colony-formation capacity in vitro and obviously weaker tumor formation potential in vivo. In conclusion, our data indicate LTF may participate in NPC carcinogenesis as a negative effector, that is, a tumor suppressor gene.

K-ras Mutations Correlate with Atypical Cytology and Elevated CEA Levels in Pancreatic Cystic Neoplasms

Benign pancreatic cystic neoplasms are important precursors to pancreatic adenocarcinoma, and offer the opportunity to prevent cancer. Conversely, prevention only occurs with surgical resection associated with significant morbidity and mortality, while the natural history of small cystic neoplasms is a slow and uncertain progression to malignancy. Markers that predict progression to malignancy are needed. Cyst fluid DNA analysis including K-ras mutations may predict more aggressive natural history of pancreatic cystic neoplasms. Sixty patients with pancreatic cysts measuring less than 3cm without solid component or pancreatic ductal dilation underwent EUS with fine needle aspiration. Nine had surgical resection. Cyst fluid was tested for cytology, CEA levels, and DNA analysis including K-ras mutations, and eight loss of heterozygosity mutations. Mutations were correlated with findings of atypia and CEA levels. Cyst fluid K-ras mutation was found in 30% of patients. Patients with mutated K-ras were more likely to have atypia on cytology or pathology (39 vs. 14%) and higher CEA (median 591 vs. 42) compared to wild-type K-ras. K-ras mutants were more likely to have two or more loss of heterozygosity mutations. Loss of heterozygosity mutations did not correlate with atypia or CEA levels. Cyst fluid K-ras mutation correlates with other markers of aggressive cyst behavior. EUS with cyst DNA analysis may alter management of smaller pancreatic cysts when surgery might otherwise be deferred. Further studies of cyst fluid DNA and long-term outcomes are needed.

Intraductal Papillary Mucinous Neoplasm Arising in a Heterotopic Pancreas: A Case Report

To the Editor : A 44-year-old man was referred to our outpatient gastroenterology clinic for a 3-year history of abdominal bloating and epigastric pain, which were relieved by belching. Th e past medical history was notable for HIV diagnosed 11 years ago, with a preserved CD4 count and no history of opportunistic infections. His medication regimen included lamivudine, zidovudine, and nevirapine, which he had started in the last year. Th ere was no family history of gastroenterologic disease. Physical exam was unremarkable. Laboratory studies including liver function tests and serum amylase / lipase were normal. Because of his persistent unexplained symptoms, a computer tomography scan was obtained, which revealed a 3 × 4 cm complex cystic lesion in the greater curvature of stomach ( Figure 1 ). Subsequent endoscopic ultrasound revealed a subepithelial complex cystic mass at the junction of stomach body and antrum. Th e wall layer could not be clearly identifi ed. A fi ne-needle aspirate of the cyst revealed serous-appearing brown-tinged fl uid. Cytology revealed only very scant infl ammatory cells, and chemistries were notable for amylase 0 IU/L and carcinoembryonic antigen 319 ng / ml. Molecular analysis of the fl uid was also performed; the sample was negative for the K-ras mutation, but there were loss-of-heterozygosity (LOH) mutations of 10q and 17q. Th e LOH mutations were low amplitude in clonal extent, and interpreted as “ consistent with an indolent, low grade, benign neoplastic processes. ” Th e patient subsequently underwent a limited partial gastrectomy for excision of the mass. Pathology of the surgical specimen revealed a 3 cm heterotopic pancreas with a 0.9 cm intraductal papillary mucinous neoplasm ( Figure 2 ), with low-grade adenomatous cytologic features. Th e prevalence of heterotopic pancreas is not well established, but surgical and autopsy studies in the 1940 – 1950s suggest a prevalence ranging from 0.6 to 15 % ( 1,2 ). Th e most common locations of heterotopic pancreas are the stomach, duodenum, and jejunum, but other locations including the esophagus, common bile duct, mediastinum, and fallopian tubes have also been described ( 3 ). Heterotopic pancreas is most commonly found incidentally at endoscopy, surgery, or during other investigations. Although most patients are asymptomatic, heterotopic pancreas can occasionally lead to gastrointestinal obstruction, upper gastrointestinal bleeding, obstructive jaundice, and other presentations depending on the location of the abnormal tissue. In addition to eff ects on adjacent organs, heterotopic pancreatic tissue can itself undergo pathological changes including pancreatitis and rarely malignant transformation ( 4,5 ). To our knowledge, this is only the second reported case of intraductal papillary mucinous neoplasm in heterotopic pancreas, providing another example of the fact that heterotopic pancreas can develop pathological changes similar to the native pancreas ( 6 ).

T1502: Performance Characteristics of K-RAS and LOH Mutation Patterns in Predicting Mucinous Pancreatic Cyst Histology

Commercially available pancreatic cyst fluid DNA analysis (RedPath) maybe helpful in the evaluation of pancreatic cysts (PCs). Early studies have demonstrated that a k-ras mutation has a high predictive value for mucinous histology and that multiple high amplitude loss of heterozygosity (LOH) mutations may be predictive of malignancy. However, few data exist regarding the prevalence of specific LOH mutations and the predictive value of specific LOH mutations for mucinous histology.

Molecular Evidence for the Neoplastic Potential of Hepatic Von-Meyenburg Complexes

Von-Meyenburg complexes (VMCs) have been shown to progress to cholangiocarcinoma (CC) in some cases. Histologic examination in such cases reveals a gradual transition of VMCs to intermediate lesions and finally to CC. The goal of this study was to determine if this histologic progression was also accompanied by sequential genetic alterations. Two cases that showed many VMCs with a transition to cholangiocarcinoma through intermediate lesions were analyzed. Multiple VMCs (away from the tumor), intermediate lesions and areas of frank CC were microdissected under stereoscopic guidance and were analyzed for allelic imbalance [loss of heterozygosity (LOH)] using a panel of 20 polymorphic microsatellite markers by polymerase chain reaction/electrophoresis. The 2 cases of CCs revealed LOH at 5 and 7 different genomic loci specific for each patient, respectively. Coexisting VMCs also exhibited LOH ranging from 0 to 3 loci in each case. Intermediate lesions showed LOH at a single locus in case 1, whereas the assay could not be performed in case 2 due to inadequate DNA yield. In case 2, the earliest acquired mutations were present in the VMCs supporting a causal relationship for neoplastic progression. Discordant LOH mutations were also present in the VMCs and the adenomatous lesions providing support that LOH detected at these sites did not passively migrate or reflect contamination. The results of the present study show that the histologic progression observed through these stages is also accompanied by LOH at loci harboring key oncogenes. The findings also support that VMCs are preneoplastic lesions that could progress to CC with time.

Loss of heterozygosity at 1p-19q induces a global change in oligodendroglial tumor gene expression

Oligodendroglial tumors presenting loss of heterozygosity (LOH) at 1p and 19q have been shown to be sensitive to chemotherapy, thus making 1p-19q status testing a key aspect in oligodendroglioma diagnosis and prognosis. Twenty-nine tumor samples (19 oligodendrogliomas, 10 oligoastrocytomas) were analyzed in order to obtain a molecular profile identifying those bearing 1p-19q LOH. Other genomic anomalies usually present in gliomas, such as EGFR amplification, CDKN2A/ARF deletion, 10q LOH and TP53 mutation, were also studied. Tumors with 1p-19q LOH overexpressed genes related to neurogenesis. Genes linked to immune response, proliferation and inflammation were overexpressed in the group with intact 1p-19q; this group could in turn be further divided in two subgroups: one overexpressing genes involved in immune response and inflammation that did not show major genetic aberrations other than the TP53 mutation and EGFR trisomy in a few cases, and another overexpressing genes related to immune response and proliferation that had a predominance of samples carrying several anomalies and presenting worse outcomes. This molecular signature was validated by analyzing a set of ten tumor samples (three oligodendrogliomas, seven oligoastrocytomas); all ten samples were correctly assigned. LOH at 1p-19q results in haploinsufficiency and copy number reduction of several genes, including NOTCH 2; this phenomenon produces a global change in gene expression inducing a pro-neural status that results in restrictions to cell migration and proliferation. Tumors without LOH at 1p-19q exhibit the opposite characteristics, explaining their more aggressive behavior.

A Tumor Sorting Protocol that Enables Enrichment of Pancreatic Adenocarcinoma Cells and Facilitation of Genetic Analyses

Molecular profiling of human cancer is complicated by both stromal contamination and cellular heterogeneity within samples from tumor biopsies. In this study, we developed a tissue-processing protocol using mechanical dissociation and flow cytometric sorting that resulted in the respective enrichment of stromal and tumor fractions from frozen pancreatic adenocarcinoma samples. Molecular profiling of DNA from the sorted populations using high-density single nucleotide polymorphism arrays revealed widespread chromosomal loss of heterozygosity in tumor fractions but not in either the stromal fraction or unsorted tissue specimens from the same sample. Similarly, a combination of KRAS mutations and chromosomal copy number changes at key pancreatic cancer loci, such as CDK2NA and TP53, was detected in a substantial proportion of the tumor fractions but not in matched stromal fractions from the same sample. This approach to tissue processing could greatly expand the amount of archived tissue that is available for molecular profiling of human cancer and enable a more accurate diagnosis of genetic alterations in patient samples.

Restoration of CAPAN-1 cells with functional BRCA2 provides insight into the DNA repair activity of individuals who are heterozygous for BRCA2 mutations

Mutations in the BRCA2 gene are associated with inherited, early-onset breast cancer. CAPAN-1 cells have been useful for studying how BRCA2 mutations contribute to malignant transformation. They exhibit loss of heterozygosity (LOH), and the remaining copy of BRCA2 has a 6174delT mutation, which causes a premature C-terminal truncation that removes the domains for DNA repair and the nuclear localization signals. The DNA repair protein RAD51, which interacts with BRCA2, exhibits impaired nuclear translocation in CAPAN-1. It has been speculated that RAD51 may require BRCA2 for nuclear entry and that C-terminally truncated BRCA2 may retain RAD51 in the cytoplasm. This may cause heterozygous individuals to exhibit deficient DNA repair and cell viability comparable to individuals with LOH or biallelic BRCA2 mutations. We simulated a heterozygous condition by using stably transfected CAPAN-1 cells with wild-type BRCA2. Fusion of a nuclear localization signal to RAD51 did not increase its ability to independently enter the nuclei of CAPAN-1 cells. Furthermore, restoration of functional BRCA2 did not significantly improve DNA repair, nor did it reestablish cell viability in CAPAN-1 cells. The results imply that C-terminally truncated BRCA2 hinders RAD51 nuclear translocation, possibly contributing to genetic instabilities in homozygous as well as heterozygous individuals.

Loss of Heterozygosity Predicts Poor Survival After Resection of Pancreatic Adenocarcinoma

BackgroundAmerican Joint Committee on Cancer (AJCC) staging for pancreatic adenocarcinoma is a validated predictor of prognosis but insufficiently discriminates postresection survival. We hypothesized that genetic analysis of resected cancers would correlate with tumor biology and postoperative survival. MethodsResected pancreatic ductal and ampullary adenocarcinomas (n = 50) were analyzed for loss of heterozygosity (LOH) at 15 markers including 5q(APC), 6q(TBSP2), 9p(p16), 10q(PTEN), 12q(MDM2), 17p(TP53), and 18q(DCC/SMAD4). KRAS exon 1 mutations were detected by sequencing. The primary endpoint of this interim data analysis was survival at 18 month median follow-up. ResultsNegative margins were achieved in 43 (86%) cases. AJCC stage was: Ia/b (3), IIa (16), IIb (31). KRAS mutations were detected in 31 cases (62%) and LOH in 26 (52%) with mean fractional allelic loss score 23 ± 16%. Median survival was significantly shorter with LOH (15.2 months versus not reached; p = 0.021) and KRAS mutations (19.6 months versus not reached; p = 0.038). Combining KRAS mutation with LOH was a powerful negative predictor in Cox regression (HR = 10.6, p = 0.006). Stage, nodal and margin status were not predictive of survival. ConclusionLOH and KRAS mutations indicate aggressive tumor biology and correlate strongly with survival in resected pancreatic ductal and ampullary carcinomas. Genetic analysis may improve risk stratification in future clinical trials.

407 Comparison of K-RAS-2 and LOH Mutational Analysis with Pancreas Cyst Fluid CEA Levels and Histology

BACKGROUND: The main objective of pancreatic cyst fluid analysis is to differentiate cysts of mucinous or malignant variety from other cysts which have a benign course. Previous investigators have reported that K-ras-2 point mutation and at least two mutations of allelic imbalance or loss of heterozygosity (LOH) with good quality DNA is characteristic of mucinous cystic neoplasm (MCN). OBJECTIVE: Identify the clinical impact of DNA analysis of pancreatic cyst fluid with its correlation to cyst fluid chemistry and histologic analysis. METHODS: This is a retrospective analysis of all consecutive patients with pancreatic cysts who presented for endoscopic ultrasound (EUS) examination with fine-needle aspiration (FNA) biopsy over an 18 month period until October 2007. DNA analysis was performed by RedPath Integrated Pathology (Pittsburg, PA). Fluid analysis included carcinogenic embryonic antigen (CEA) with values >192ng/dL suggestive of MCN. The standard for comparison was surgical histopathology when available. RESULTS: 27 consecutive patients with cysts and samples submitted for DNA analysis were examined including 15 men and 12 women (mean age 62.8 and 61.3 years, respectively). In 20 patients, all parameters including cyst fluid, DNA analysis, and histology were available for comparison. Consistent findings were seen in 7/20 (35%) in which all parameters suggested negative benign findings. 3/20 (15%) had positive K-ras-2 mutations of which only 2/3 had true histologic MCN suggesting a 33% false positivity. 7/20 (35%) of patients had at least 2 LOH mutations; however only 2/7 were actually positive on histology for MCN suggesting 71% false positivity. In the same group, CEA levels >192ng/dL were seen in 7/20 of which 4/7 had histologic malignancy demonstrating 42.9% false positivity. In a group of 9 malignant cysts (MCN, side branch intraductal papillary mucinous neoplasm, and cystic degeneration of adenocarcinoma), 7 (78%) had CEA levels >192ng/dL. In this group, 7 had DNA analysis of which 4/7 (57.1%) were falsely negative for K-ras-2 and 3/7 (42.9%) falsely negative for LOH. CONCLUSIONS: Consistency in histology, CEA levels, and K-ras-2 and LOH mutations was seen in only 35% of cases, all of which were benign cysts. In the detection of malignant cysts, elevated CEA levels were more predictive of histology in comparison to K-ras-2 or LOH mutations. Additionally, false positivity of LOH mutations was noted to be considerably higher than K-ras-2mutations or even fluid CEA levels. These findings suggest that DNAmutation analysis should not be used routinely but rather selectively in the evaluation of pancreatic cysts.

Comprehensive Analysis of Copy Number and Allele Status Identifies Multiple Chromosome Defects Underlying Follicular Lymphoma Pathogenesis

Follicular lymphoma (FL) constitutes the second most common non-Hodgkin's lymphoma in the Western world. The clinical course is variable and only in part explained by known tumor-intrinsic or -extrinsic factors. FL carries the hallmark chromosomal translocation t(14;18), deregulating the expression of Bcl-2, but this is not sufficient to explain either FL biology or clinical behavior. We have employed high-density genomic profiling technology using the Affymetrix 50K-XbaI oligonucleotide single nucleotide polymorphism-chip platform to interrogate the genomes of 58 fluorescence-activated cell-sorted (FACS) FL specimens for chromosomal copy number changes and 46 specimens for loss of heterozygosity (LOH). We report (a) previously unknown high-frequency copy-neutral LOH (uniparental disomy) in FL on chromosomes 1p (approximately 50%) and 6p (approximately 30%); (b) that del6q is complex, as reported, with at least two regions of minimal common loss at 6q13-15 and 6q23-24, and that in addition, approximately 8% of FL specimens contain a homozygous deletion at 6q23.3-24.1 that spans the negative NFkappaB regulator A20 and the p53 apoptosis effector PERP; (c) that combined analysis of chromosome 17p for LOH, copy number, and p53 mutations shows that most p53 mutations in FL do not involve del17p. Finally, we map high-frequency LOH with and without copy loss on chromosomes 9p, 10q, and 16p and genomic gains on 2p15-16 and 8q24.22-24.3. This comprehensive description of the pathologic anatomy of the FL genome uncovers novel genetic lesions and should aid with identification of genes relevant to FL biology and clinical behavior.

Adequacy of Colonoscopic Biopsy Specimens for Molecular Analysis: A Comparative Study With Colectomy Tissue

Molecular analyses of tumors are increasingly useful for prognosis and for guiding therapy. Colonoscopic biopsy provides the first source of tissue for most cases of colorectal carcinoma and therefore might become an important source for molecular analyses. We have addressed the question whether molecular analyses of colonoscopic biopsy yield results similar to the findings from the surgical specimen. Further, we analyzed 2 separate areas of the colectomy specimen to assess tumor heterogeneity. We evaluated 3 samples from each of 67 patients for point mutations in the KRAS gene, loss of heterozygosity (LOH) at the Adenomatous Polyposis Coli (APC) and Deleted in Colon Cancer (DCC) genes and for microsatellite instability (MSI) using polymerase chain reaction based techniques. The average time interval between biopsy and surgery was 2.2+/-0.15 weeks. Lesions were from all colon segments and all surgical stages. The degree of agreement between the biopsy and surgical sites was high for APC LOH, MSI, and KRAS mutations (kappa=0.85, 1.00, and 0.93, respectively) but less so for DCC LOH (kappa=0.62). Colonoscopic biopsies are an acceptable source of neoplastic DNA for studies of KRAS, APC LOH, and MSI, but less so for DCC LOH, primarily resulting from technical considerations.

K‐Ras and microsatellite marker analysis of fine‐needle aspirates from intraductal papillary mucinous neoplasms of the pancreas

Preoperative diagnosis of pancreatic cystic lesions is difficult despite the combination of cytomorphology, radiographic imaging characteristics, and fluid tumor markers such as carcinoembryonic antigen. Intraductal papillary mucinous neoplasms (IPMNs) represent a subset of preinvasive pancreatic cystic neoplasms and are associated with accumulated genetic mutations, especially K-ras and tumor suppressor genes such as p53. Application of molecular techniques to cyst fluid obtained by endoscopic ultrasound guided fine-needle aspiration (EUSFNA) may contribute to preoperative assessment. Sixteen patients with pancreatic cystic lesions had cyst fluid obtained by preoperative pancreatic EUSFNA or intraoperative aspiration. All patients subsequently underwent surgical resection of the pancreas and IPMN was documented in all (6 adenomas, 6 borderline tumors, and 4 carcinomas). DNA was extracted from the cyst fluids and mutational analysis for K-ras point mutations and loss of heterozygosity (LOH) analysis using a preselected panel of genomic loci were performed. LOH was observed in 3 of 4 carcinomas as compared to 4 of 11 adenomas and borderline lesions (1 was QNS). LOH and K-ras mutations were both acquired in 2 of 4 carcinomas and in 1 of 12 adenoma/borderline lesions. Although the study is small, molecular analysis for LOH and K-ras mutations is useful in the preoperative evaluation of cystic pancreatic lesions. Increasing degree of neoplasia appears to correlate with increased genetic abnormality using a panel of selected genomic markers.

Familial Paragangliomas: Genetic Grounds

About 10 to 50% of paragangliomas are familial and they may present as a solitary tumor or as multiple tumors, as occurs in 30 to 40% of familial paragangliomas. These tumors are generally confined to the head and neck region, but occasionally they are accompanied by adrenal or extra-adrenal paragangliomas. Paragangliomas also may be part of different syndromes, such as multiple endocrine neoplasia type 2 (over-representation of mutant RET), von Hippel-Lindau disease (VHL germline mutation and wildtype allelic deletion), and neurofibromatosis type 1. It now appears that germline inactivating heterozygous mutations in SDHB, SDHC, and SDHD, which encode subunits of mitochondrial complex II (succinate dehydrogenase, succinate-ubiquinone oxidoreductase), are responsible for 70% of familial cases and approximately 8% of nonfamilial cases. On the other hand, mutations in the subunit SDHA display a phenotype resembling other mitochondrial and Krebs cycle gene defects, which are often characterized by hypotonia, growth retardation, cardiomyopathy, myopathy, neuropathy, and metabolic derangement. In SDHB (1p36)- and SDHC (1q21)-linked families, disease inheritance is autosomal dominant. In SDHD (11q23)-linked families, the disease phenotype is expressed only upon paternal transmission of the mutation, consistent with maternal imprinting or with a somatic genetic mechanism targeting both the SDHD gene on 11q23 and a paternally imprinted gene on 11p15.5. In addition, loss of heterozygosity (LOH) on chromosome 11, mainly in 11q23 (PGL1), has been observed. A two-hit model comprising both LOH and point mutation appears to be the pathophysiologic basis for sporadic tumor development. Loss of chromosome 11 regions, including the deletion of PGL1 and PGL2 loci, may result in a more severe phenotype, as exemplified by the development of multiple tumors. In contrast with SDHD mutation carriers who have more frequent multifocal paragangliomas, SDHB mutation carriers are more likely to develop malignant disease and possibly extraparaganglial neoplasias, including renal cell and thyroid carcinomas.

Mutation induction after low-dose carbon-ion beam irradiation of frozen human cultured cells

To study the genetic influence of low-dose ionizing radiation at the chromosomal level, frozen human lymphoblastoid TK6 cells were exposed to a 10 cGy dose delivered by a carbon-ion (24.5 ± 2.0 keV/μm) beam. Mutation assays were performed within a few days or after about one month of preservation at -80°C following irradiation. The results showed an increase in mutation frequencies at the thymidine kinase (TK) gene locus of 1.6-fold (2.5 × 10-6 to 3.9 × 10-6) and 2.1-fold (2.5 × 10-6 to 5.3 × 10-6), respectively. The relative distributions of the observed mutation classes were not changed by the radiation exposure in either assay. Multilocus analysis using two TK locus markers and eleven microsatellite loci spanning chromosome 17 were used for the analysis of TK mutants exhibiting a loss of heterozygosity (LOH). An interesting characteristic was detected using this system; interstitial deletion patterns were observed in hemizygous LOH mutants, which were specific for radiation exposure, and considered to be the result of end-joining repair of carbon-ion-induced DNA double-strand breaks. The relative increase in TK mutation frequency of the irradiated cells after the longer preservation at -80°C is probably due to the lower cell-viability compared to the unirradiated level. These results clearly demonstrate that this type of analysis can be used for the detection of low-dose ionizing radiation effects in frozen cells.

Chromosome 11 monosomy in conjunction with a mutated SDHD initiation codon in nonfamilial paraganglioma cases

Paragangliomas of the head and neck region are a group of rare, usually benign, slow-growing tumors developing from paraganglionic chemoreceptors in most patients. Mutations in a subunit of the mitochondrial enzyme II complex (succinate dehydrogenase [SDHD]) were shown to be responsible for the formation of paragangliomas. In addition, loss of heterozygosity (LOH) on chromosome 11, mainly in 11q23 (PGL1), was observed recently. We analyzed DNA derived from tumor sections of three unrelated paraganglioma patients (one case with multiple paragangliomas, two cases with single tumors; all of them sporadic cases) for mutations in the SDHD gene by direct sequencing. Microsatellite-based LOH was performed, and events of chromosomal loss were validated by fluorescence in situ hybridization (FISH) on paraffin-embedded tumor and normal tissue by using centromeric satellite DNA. Sequence analysis revealed mutations in SDHD exon 1 in all patients, affecting the initiation codon (M1V). Another alteration was detected in exon 2 but was lacking in tumor DNA and therefore classified as polymorphism (H50R). LOH and FISH analyses demonstrated partial/total monosomy for chromosome 11 in the tumor samples tested. A common genetic mechanism appears to be the pathophysiologic basis for sporadic tumor development because the proposed two-hit model comprising both LOH and point mutation is manifest in our patients. Loss of chromosome 11 regions, including the deletion of PGL1 and PGL2 loci, may result in a more severe phenotype, as exemplified by the development of multiple tumors in one of the patients.

Pms2 deficiency results in increased mutation in the Hprt gene but not the Tk gene of Tk+/- transgenic mice

The effects of deficiency in the DNA mismatch repair (MMR) protein Pms2 were investigated using the endogenous mouse Hprt and Tk genes as reporters of intragenic mutation and loss of heterozygosity (LOH). Pms2(-/-)Tk(+/-), Pms2(+/+)Tk(+/-), Pms2(+/-)Tk(+/-) and Pms2(-/-)Tk(-/-) mice were bred from Pms2(+/-)Tk(+/-) mice. At 2 months of age, the body weight and splenic T lymphocyte yields were significantly lower in Pms2(-/-)Tk(-/-) mice than in littermates of the other genotypes. The mice were evaluated for their spontaneous mutant frequencies in the Hprt and Tk genes of splenic lymphocytes and their frequency of micronuclei in polychromatic erythrocytes from bone marrow. The cloning efficiency of lymphocytes derived from Pms2(-/-)Tk(-/-) animals was 12-fold lower than that of animals of the other genotypes. Compared with Pms2(+/+) and Pms2(+/-) mice, Pms2(-/-) mice had a 21- to 69-fold increase in the Hprt mutant frequency. The Hprt mutant frequency was equally high in Pms2-deficient, Tk(+/-) and Tk(-/-) mice. No significant Pms2-dependent change in mutant frequency was detected using the Tk mutational target. When individual Tk mutants were analyzed for LOH mutation by allele-specific genotyping, the fraction of LOH mutants was lower in Pms2-deficient than in Pms2-proficient mice (29.2 and 43.6%, respectively). The frequency of bone marrow micronuclei was significantly higher in Pms2(-/-)Tk(-/-) mice than in Pms2(-/-)Tk(+/-) mice. These observations suggest that the simultaneous occurrence of Tk and Pms2 deficiencies may cause a decrease in cell viability that diminishes the Tk mutational response, making it impossible to discern clearly the effect of Pms2 deficiency on LOH-type mutation using the Tk reporter system. The interaction between Tk deficiency and a component of the MMR system suggests that Tk-deficient cells may have higher levels of DNA polymerase misincorporation or endogenous DNA damage than Tk-proficient cells.

Distribution of genetic variants in preneoplastic areas of colorectal tumours

Aims: Loss of heterozygosity (LOH) may vary almost randomly within a colorectal tumour due to the heterogeneous morphologic character of these tumours. Despite this, as a rule, single biopsies are the source of genetic material used in studies of markers important for prognosis, clinical behaviour of the disease, or susceptibility of specific tumours to different treatment modalities.Methods: To evaluate the importance of intratumoural variation for the results of analysis of LOH and point mutations in colorectal cancer and to determine the frequency of genetic alterations in different types of pre-neoplastic areas of the tumours, 36 consecutively operated patients with colorectal cancer were studied. After fixation, specimens were mounted on large slides containing the whole tumour. The specimens were sub classified into different areas defined as normal tissue, normal tissue closely adjacent the tumour mass, adenoma, dysplasia and invasive cancer cells. These areas were dissected and subjected to DNA extraction.Results: The extracted genomic DNA was studied for LOH at chromosome 5q, 17p, and 18q and for k-ras mutations. Overall, a correlation between the intratumoural degree of neoplastic progression and the frequency of LOH and k-ras mutations was seen. These correlations were significant (p<0.008) except for dysplasia/adenomatous tissue versus invasive cancer. Microsatellite instability was found in 9% of the tumours, all except one in invasive parts of the tumours.Conclusions: This study demonstrates a statistical correlation between intratumoural differences in neoplastic degree of dedifferentiation and genetic instability in terms of LOH and point mutations of the k-ras gene in colorectal carcinoma. The importance of a careful dissection in order to localise the region with the highest probability of genetic aberrations and multiple biopsing must not be neglected. The observation that the prevalence of k-ras mutations and LOH are correlated to the degree of dedifferentiation within a colorectal tumour is in line with the concept that selected cell clones are responsible for the neoplastic progression of the tumour.

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

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

MEN1 gene alterations do not correlate with the phenotype of sporadic primary hyperparathyroidism.

Loss of heterozygosity (LOH) in the MEN1 region on chromosome 11q13 and MEN1 gene mutations have been found in a subset of sporadic parathyroid tumors. The question of whether these genetic abnormalities in the parathyroid tumors might influence the clinical and biochemical characteristics of primary hyperparathyroidism (PHPT) remains to be elucidated. The aim of the present study was to correlate the presence of MEN1 gene alterations in PHPT tumors with the clinical phenotype. Using microsatellite analysis for LOH at 11q13 and DNA sequencing of the coding exons, the MEN1 gene was studied in 38 parathyroid tumors of patients with sporadic PHPT. Fourteen tumors showed LOH at 11q13, and mutations of MEN1 gene were detected in 7 cases. The clinical and biochemical characteristics of patients were unrelated to the presence or absence of LOH and/or MEN1 gene mutations. In conclusion, MEN1 gene alterations are rather common in sporadic PHPT and their presence does not correlate with the clinical manifestations of the disease.