Schackert Hk

DHPLC mutation analysis of the hereditary nonpolyposis colon cancer (HNPCC) genes hMLH1 and hMSH2.

Denaturing high-performance liquid chromatography (DHPLC) is an efficient method for detection of mutations involving a single or few numbers of nucleotides, and it has been successfully used for mutation detection in disease-related genes. Colorectal cancer is one of the most common cancers, and mutations in the genes for hereditary nonpolyposis colon cancer (HNPCC), hMLH1 and hMSH2, also involve mainly point mutations. Sequence analysis is supposed to be a screening method with high sensitivity; however, it is time-consuming and expensive. We therefore decided to test sensitivity and reproducibility of DHPLC for 71 sequence variants in hMLH1 and hMSH2 initially found by sequence analysis in DNA samples of German HNPCC patients. DHPLC conditions of the PCR products were based on the melting pattern of the wild-type sequence of the corresponding PCR fragments. All but one of the 71 mutations was detected using DHPLC (sensitivity of 97%). Running time per sample averaged only 7 min, and the system is highly automated. Thus DHPLC is a rapid and sensitive method for the detection of hMLH1 and hMSH2 sequence variants.

Sequence analysis of the mismatch repair gene hMSH6 in the germline of patients with familial and sporadic colorectal cancer

To evaluate the involvement of hMSH6 in colorectal cancer, the complete coding sequence and flanking intron regions of the gene were analyzed by DNA sequencing in 10 patients fulfilling Bethesda Guidelines for colorectal tumors and 10 patients with sporadic colorectal carcinoma. In addition, 10 mono‐ and 10 dinucleotide repeat markers were analyzed for microsatellite instability. A protein‐truncating T insertion at codon 218 was identified in the index person of a hereditary non‐polyposis colorectal cancer (HNPCC)‐like kindred and was accompanied by a somatic T deletion in the tumor. The tumor of this patient was positive for mono‐ but negative for dinucleotide repeat instability and lacked allelic losses at loci frequently affected in colorectal carcinomas. A novel amino acid change, F340S, was found in a patient with sporadic colon and breast cancer and leukemia but was not detected in 246 chromosomes from healthy anonymous blood donors. In addition, we describe 2 silent and 15 intronic sequence variants not previously reported. Although the frequency is low, we present further evidence for hMSH6 germline mutations that predispose patients to HNPCC‐like phenotypes and suggest that mono‐ and dinucleotide repeat instability testing may be useful for distinguishing between individuals harboring an hMSH2 or hMLH1 mutation and a mutation of the hMSH6 gene. Int. J. Cancer 85:606–613, 2000.

Frequent loss of heterozygosity at the 19p13.3 locus without LKB1/STK11 mutations in human carcinoma metastases to the brain.

Inactivating germline mutations of the novel putative tumor-suppressor gene LKB1/STK11 at 19p13.3 have been shown to cause Peutz-Jeghers syndrome (PJS), an autosomal dominantly inherited disease characterized by a predisposition to mucocutaneous pigmentations, as well as various benign and malignant neoplasms. To elucidate the role of LKB1/STK11in the carcinogenesis of primary and secondary human brain tumors, a total of 309 tumors were analyzed for loss of heterozygosity (LOH) at microsatellite loci D19S886, DI9S878, and D19S565. Low LOH rates were observed for glioma (17.3%, n = 139), meningioma (5.3%, n = 57), schwannoma (0%, n = 21), pituitary adenoma (18.8%, n = 16), primary CNS lymphoma, neuroblastoma, plasmocytoma, medulloblastoma, germinoma, and papilloma of the choroid plexus (6.6%, n = 15). In contrast, brain metastases exhibited a mean LOH frequency of 42.6% (n = 61), with breast (56.3%) and lung cancer metastases (58.3%) being most frequently affected. Genomic DNA sequencing of the complete coding region of LKB1/STK11 was performed in all brain metastases exhibiting LOH (n = 26); no mutation was revealed, but we did find a germline mutation in a PJS patient. Despite high LOH fiequencies at the 19p13.3 locus in carcinoma metastases to the brain and occasional mutations reported for certain primary carcinomas, there are no mutations in LKB1/STK11. This fact suggests that alterations of LKB1/STK11 occur relatively early in tumorigenesis and are rarely involved in the development of carcinoma metastases. Based on these findings, the genes adjacent to LKB1/STK11 may be relevant for the development of metastases to the brain from certain carcinomas.

Clonal stimulation or inhibition of human colon carcinomas and human renal carcinomas mediated by transforming growth factor-beta 1.

We examined various human carcinomas and cells populating a single human neoplasm to determine whether they exhibit a heterogeneous response to the effects of transforming growth factor-beta 1 (TGF-beta). Using recently established human colon carcinoma and renal cell carcinoma under defined in vitro conditions, we observed intertumoral and intratumoral heterogeneity and polarity of responses to TGF-beta (growth inhibition or stimulation) that did not correlate with the metastatic phenotype of the cancer cells as assessed in athymic nude mice. TGF-beta mediated both cytostatic and cytolytic effects against sensitive tumor cells, and these responses were not related to the effects of TGF-beta on the cell-cycle traverse. The human colon carcinoma and renal cell carcinoma, however, exhibited differences in the expression of TGF-beta receptors.

Development of immunogenic colorectal cancer cell lines for vaccination: Expression of CD80 (B7.1) is not sufficient to restore impaired primary T cell activation in vitro

The capacity of colorectal carcinoma and melanoma cell lines to induce primary versus effector T lymphocyte activation in vitro was investigated. Established epithelial tumour cell lines derived from colorectal carcinoma and melanoma did not activate a primary proliferative response of resting T lymphocytes in allogeneic mixed lymphocyte tumour cell cultures (MLTCs). In contrast, the same tumour cells were effectively lysed by preactivated cytolytic T cell clones. This demonstrates that tumour cells are impaired in inducing a primary immune response but are susceptible to effector immune responses. Attempts at improving primary T cell activation revealed that exogenous cytokines, including interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and interleukin-2 (IL-2), were not effective. Expression of CD80 (B7.1), by transfecting a CD80 cDNA into the melanoma cell line SkMel63, improved T cell proliferation considerably. In contrast, CD80 expression in two colorectal carcinoma cell lines (SW480, SW707) did not result in T cell activation. This was not due to lack of class II MHC expression on SW480 since coexpression of a HLA-DR3 alloantigen and CD80 had no effect. Our data suggest that de novo CD80 expression is not, in general, sufficient to improve primary T cell activation by human tumour cells.

Clinical consequences of molecular diagnosis in families with mismatch repair gene germline mutations

Abstract. Hereditary nonpolyposis colorectal cancer (HNPCC), clinically defined by the Amsterdam criteria, is associated with mismatch repair gene germline mutations. This study was performed to evaluate the efficiency of combined clinical and molecular diagnostics in identifying carriers of a mutated gene in families meeting criteria of the Bethesda guidelines and to examine the influence of molecular diagnosis on clinical decision-making in carriers and noncarriers. Seventy-two patients meeting criteria of the Bethesda guidelines were tested for microsatellite instabilities (MSI). MSI-H tumors were found in 38 (52.8%) index patients. Complete sequencing of hMLH1 and hMSH2 in 38 MSI-H patients and of hMSH6 in one of these patients revealed 15 pathogenic germline mutations, including three novel mutations, and three novel unclassified germline variants. Twelve of the 15 pathogenic mutations were found in patients fulfilling the Amsterdam I/II criteria. Surgical and genetic counseling was offered to the affected families; as a result of molecular diagnosis in the 15 families, 26 index patients and affected carriers and 8 asymptomatic carriers of a mutated mismatch repair gene were included in the surveillance program, and 26 noncarriers were excluded from this program. Although germline mutations are detected in only 20.8% of patients fulfilling criteria of the Bethesda guidelines, family history and MSI-H tumor classification are both strong indicators for germline mutations in hMSH2, hMLH1, and hMSH6 genes, resulting in a 51.9% mutation detection rate. Identification of individual mutation status allows clear-cut decisions on whether or not inclusion in surveillance programs is indicated.

BRCA2 germline mutations among early onset breast cancer patients unselected for family history of the disease.

Editor—Germline mutations in the BRCA1 and BRCA2 genes are associated with an increased risk of breast and ovarian cancer.1-3 Different patient populations, predominantly with at least some familial aggregation of breast/ovarian cancer, have been screened to estimate the frequency of mutations and identify women for whom mutation analysis should be considered. The prevalence of BRCA1 and BRCA2 gene mutations has been studied in only a few population based series of patients.4-8 We tested for BRCA2 germline mutations in 40 patients diagnosed with breast cancer before the age of 40, unselected for family history of the disease. These patients were drawn from breast cancer patients recruited between 1992 and 1995 in a population based genetic epidemiological case-control study conducted in a region around Heidelberg in Germany.9 The mean age of onset of the disease in the tested population was 33.8 years (range 25-39 years). Written informed consent was obtained from all patients. The 26 coding exons and flanking intronic regions were analysed from genomic DNA using published10 11 or newly designed primers (available on request). Polymerase chain reactions contained 50 ng DNA, 200 mmol/l of each dinucleotide, 1.5-3.0 mmol/l MgCl, 200 nmol/l of each primer, and …

Pancreatic surgery gastric secretion and ulcers in the rat: increased ulcer development following pancreatic half resection or duct occlusion

In the rat, both partial resection of the pancreas and occlusion of the side branches of the biliodigestive duct were investigated with respect to their influence on gastric secretion (acid, pepsin, sodium), gastric mucosal blood flow (MBF), development of gastric ulcers, and gastrin and somatostatin in the blood. On the 14th postoperative day the exocrine pancreatic function is reduced and ulcer index and severity are significantly enhanced. There are no simultaneous changes in gastric secretion or MBF. Aortal gastrin was decreased and somatostatin was unchanged. We conclude that: in the rat, reduction of exocrine pancreatic function should be considered an ulcerogenic factor; factors others than gastric hypersecretion or reduced MBF are responsible for ulcer formation, and an etiological role of either circulating gastrin or somatostatin is doubtful.

618 Molecular diagnosis and surgical therapy of hereditary colerectal cancer

Familial adenomatous polyposis (FAP) which is caused by APC gene mutations is a paradigm for presyrnptomatic molecular diagnostics and preventive surgical treatment. Sigmoidoscopy identifies symptomatic probands and presymptomatic relatives presenting with hundreds of colorectal adenomas. If sigmoidoscopy does not confirm FAP in relatives, either due to late onset of adenoma development or noncarrier status, molecular testing may identify affected patients or exclude noncarriers. Since combined clinical/molecular diagnosis can identify affected relatives about twenty years earlier than symptomatic probands, sixty of our probands but none of the presymptomatic relatives presented with colorectal cancer. Restorative proctocolectomy fullowed by an ileoanal J-pouch procedure prevents colorectal cancer and preserves sphincter function in FAP patients. Although familial adenomatous polyposis accounts for less than I% of all colorectal cancers, hereditary nonpolyposis colorectal cancer (HNPCC) may account for up to 15%. Presymptomatic molecular diagnosis of FAP and HNPCC and preventive surgical treatment might be effective tools to further decrease mortality due to colorectal cancer.