The impact of diabetes mellitus type 2 on the steroidogenesis of male Zucker Diabetic Fatty rats.

The aim of this study was to evaluate the impact of diabetes mellitus type 2 (DM2) on the male endocrine system of Zucker Diabetic Fatty (ZDF) rats. Sexually mature ZDF rats were divided to a lean (control) and obese group, and had diabetes confirmed by blood tests. For the in vivo experiment, fasting blood was collected to obtain blood plasma. In case of the in vitro experiments, testicular fragments were cultured for 24 h, and the culture medium was collected. The concentrations of testosterone (T), androstenedione (A4), dehydroepiandrosterone (DHEA-S), estradiol (E2), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were quantified in the blood plasma and the medium by the ELISA method, while cholesterol (CHOL) was assessed spectrophotometrically. A significant decline of T (36.31 %), A4 (25.11 %) and FSH (26.99 %) as well as a significant increase of CHOL and E2 (36.17 %) was observed in the blood plasma of obese ZDF rats in comparison to the control. Under in vitro conditions, a significant decrease of FSH (23.35 %) accompanied by an increase of E2 was observed in the obese group compared to the control. In the case of CHOL, LH, T, DHEA and A4 no significant differences were observed. Our results suggest that except for FSH and E2 all steroid biomolecules were synthetized normally by the testicular tissue, however a dramatic endocrine disturbance was observed at the system level. We may conclude that DM2 has negative effects on systemic hormone secretion and these alterations are more pronounced in combination with obesity.

Circulating RNA as a tumor marker: detection of 5T4 mRNA in breast and lung cancer patient serum.

Cancer is commonly associated with the inappropriate mRNA expression of nonmutated genes. Recently, several tumor-associated RNA species, including tyrosinase mRNA and telomerase RNA, have been demonstrated in plasma and serum. The presence of tumor RNA in plasma and serum affords the opportunity to diagnose or stratify patients with cancer when tissue is not readily available. To exemplify the potential for pharmacogenomic and phenotypic stratification of the cancer patient, we evaluated serum for 5T4 mRNA. 5T4 is a trophoblast glycoprotein frequently overexpressed in epithelial malignancies that provides a potential target for cancer therapeutics. Serum was collected from 19 patients with advanced breast cancer (5 patients) or non-small-cell lung cancer (14 patients), and from 25 normal control volunteers having amplifiable RNA. RNA extracted from the serum was RT-PCR amplified using heminested, two-stage reactions, with products detected by gel electrophoresis. 5T4 mRNA was reproducibly detected in 8/19 (42%) cancer patient sera, including 2/5 breast cancer patient sera and 6/14 lung cancer patient sera, but in only 3/25 (12%) normal control sera (p = 0.035). The potential for circulating mRNA to identify patients who might benefit from a 5T4-directed therapy offers an example of the utility of circulating RNA as a tumor marker.

Serum BCL2/IGH DNA in follicular lymphoma patients: a minimal residual disease marker.

The majority of follicular lymphoma patients carry a t(14,18) juxtaposing the BCL2 oncogene to the immunoglobulin heavy chain joining region (IgH). Molecular analysis for follicular lymphoma-specific DNA translocations may permit evaluation of minimal residual disease (MRD). We identify extracellular BCL2/IGH transgene DNA in the serum of patients with follicular lymphoma, and evaluate its utility as a surrogate marker. DNA was harvested from both the sera and bone marrow of 5 stage IV follicular lymphoma patients prior to and after chemotherapy and following a novel vaccine-based regimen. Serial PCR amplifications were performed using heminested BCL2-specific major breakpoint cluster region (MBR) primers and the immunoglobulin heavy chain consensus primer. Amplification products were detected by agarose gel electrophoresis, and comparison was made to amplification products from the original tumor biopsy. Results show that four of the five lymphoma patients carried extracellular BCL2/IGH transgene DNA in their serum. The remaining patient did not have an amplification product from either the tumor or the serum, suggesting either the absence of a translocation or the presence of a variant translocation not detectable with this primer set. Transgene DNA was detectable in serum even in patients with MRD, comparing favorably with bone marrow results. In at least one patient, the presence of the transgene in serum at the conclusion of therapy preceded relapse. In conclusion, it seems that tumor-specific, extracellular DNA is present in the serum of follicular lymphoma patients, including those with MRD. Because extracellular DNA may be released into the bloodstream by tumor throughout the body it may be less subject to sampling error, and appears to be an ideal surrogate marker.

Somatic mutation screening: identification of individuals harboring K-ras mutations with the use of plasma DNA.

BACKGROUND: Many cancers are attributed to somatic mutation of DNA. We investigated whether it is feasible to detect cancer-associated somatic mutations in patients with neoplasms by using plasma DNA. METHODS: Plasma samples were prospectively collected from 240 patients undergoing colonoscopy. Colorectal biopsies were performed as clinically indicated in 135 patients, and risk factor information was available from 232 patients. DNA was extracted from plasma and colorectal tissue and was amplified by use of a polymerase chain reaction method that enriches for mutations in codon 12 of the K-ras oncogene. Molecular, histologic, and clinical data were compared by use of two-sided Fisher's exact test. RESULTS: Mutations in the K-ras gene detected in the plasma of 64 (28%) of 232 patients were statistically significantly associated with colorectal cancer risk factors (P =.0002). Of those patients having tissue available for comparison (n = 135), mutations in the K-ras gene were found in the tissues of 35 patients, and 29 (83%) of these 35 showed mutations in plasma samples. In contrast, the plasma assay was negative in 93 of the 100 patients whose tissue K-ras was wild-type. Among patients without biopsies (n = 105), 28 had mutated K-ras in their plasma DNA, despite the absence of remarkable colonoscopy findings; 24 of these 28 patients had risk factors for colorectal cancer. Overall, 25 (39%) of 64 patients showing mutations in plasma DNA had colorectal neoplasms with K-ras mutations compared with five (3%) of 176 patients without K-ras mutations in plasma DNA. CONCLUSION: Plasma DNA assays for the detection of mutations in K-ras codon 12 may provide a feasible method to screen populations for somatic mutations frequently found in neoplasms. The clinical utility of using this test in screening populations requires further study.

p53 and APC mutations are detectable in the plasma and serum of patients with colorectal cancer (CRC) or adenomas.

BACKGROUND: A variety of tumor-related DNA has been detected in the plasma and serum of cancer patients, including RAS, the BCL2-IgH transgene, and p53. It is not known whether the APC gene, which is frequently mutated in colorectal cancer (CRC), can be identified in the plasma of CRC patients. Similarly, it is unknown whether another tumor suppressor gene altered in CRC, p53, is detectable in people with precursor lesions to CRC. MATERIALS AND METHODS: The plasma of 240 colonoscopy patients was tested for mutations at two frequently altered sites (codons 175 and 248). A restriction enzyme-mediated enrichment assay was used to detect these mutants. We also tested tumor tissue from 8 patients with CRC for mutations in the mutation cluster region of the APC gene using direct DNA sequencing. Corresponding plasma from any positive patient was then similarly sequenced. RESULTS: Three plasma p53 mutations were identified. Two of these patients had adenomas at biopsy, and 1 had a hyperplastic polyp. All were tested for the same p53 mutations, and 1 of the adenomas was positive. One of the 8 CRC patients had a 5-base-pair deletion in the cancer and the same deletion was detectable in that patient's plasma, although at an amount that we estimate at 1-5% of the total APC DNA present. CONCLUSIONS: APC mutations are detectable in the plasma of CRC patients. p53 mutants are detectable in the plasma of colorectal adenoma patients. These may have important implications for cancer screening and diagnosis in the large intestine and suggest that all malignant and premalignant DNA alterations from the colon are identifiable in the blood.

Complete molecular remissions induced by patient-specific vaccination plus granulocyte-monocyte colony-stimulating factor against lymphoma.

Lymphomas express a tumor-specific antigen which can be targeted by cancer vaccination. We evaluated the ability of a new idiotype protein vaccine formulation to eradicate residual t(14;18)+ lymphoma cells in 20 patients in a homogeneous, chemotherapy-induced first clinical complete remission. All 11 patients with detectable translocations in their primary tumors had cells from the malignant clone detectable in their blood by PCR both at diagnosis and after chemotherapy, despite being in complete remission. However, 8 of 11 patients converted to lacking cells in their blood from the malignant clone detectable by PCR after vaccination and sustained their molecular remissions. Tumor-specific cytotoxic CD8+ and CD4+ T cells were uniformly found (19 of 20 patients), whereas antibodies were detected, but apparently were not required for molecular remission. Vaccination was thus associated with clearance of residual tumor cells from blood and long-term disease-free survival. The demonstration of molecular remissions, analysis of cytotoxic T lymphocytes against autologous tumor targets, and addition of granulocyte-monocyte colony-stimulating factor to the vaccine formulation provide principles relevant to the design of future clinical trials of other cancer vaccines administered in a minimal residual disease setting.

Detection of tumor messenger RNA in the serum of patients with malignant melanoma.

Serum RNases are known to be elevated in patients with cancer. Consequently, it is not clear whether human mRNA with sufficient integrity as to permit reverse transcription-PCR (RT-PCR) amplification is detectable in serum. We examined serum from six patients with malignant melanoma for human tyrosinase mRNA using RT-PCR. Serum from 20 normal volunteers served as controls. Tyrosinase mRNA could be demonstrated in serum from four of the six melanoma patients with detection by gel electrophoresis and confirmation by blotting amplified product to a tyrosinase-specific probe. The serum remained tyrosinase mRNA positive, even if passed through a 0.45 microm filter prior to RNA extraction, indicating that the mRNA was extracellular at the time of extraction. Tyrosinase mRNA could not be detected in any control serum (0 of 20 individuals). The presence and integrity of amplifiable RNA was confirmed in all serum specimens (patients and controls) by RT-PCR amplification of c-abl mRNA. Amplifiable RNA could be demonstrated regardless of whether serum was freshly drawn or stored frozen for several years. We conclude that human mRNA can be extracted and amplified from serum. The ability to amplify tumor mRNA from serum may have important utility in cancer diagnostics and monitoring.

Transmission of mitochondrial DNA heteroplasmy in normal pedigrees.

The presence of multiple mitochondrial genotypes (heteroplasmy) has been studied in normal individuals. Six multigenerational normal families were screened for heteroplasmy by PCR of the mitochondrial control region and the cytochrome c oxidase intergenic regions. Two individuals from different families exhibited multiple length polymorphisms in a homopolymeric tract at positions 16184-16193 and a grandmother in a third family was heteroplasmic for both cytosine and thymidine at position 15945. Although the 15945 T variant comprised 28% of the grandmother's mitochondrial DNA, this sequence was not present in any of her descendants. Heteroplasmy was detected in 2.5% of the 96 mother-offspring pairs, consistent with the possibility that it may not be rare.

KRAS oncogene mutations suggest a common histogenetic origin for pleomorphic giant cell tumor of the pancreas, osteoclastoma of the pancreas, and pancreatic duct adenocarcinoma.

Giant cell neoplasms of the pancreas are rare tumors of uncertain histogenesis. Mutation of the KRAS oncogene is common in typical pancreatic duct adenocarcinoma. We have analyzed DNA from five pancreatic tumors with giant cells for mutations in the KRAS oncogene and found alterations of the second position of codon 12 in each case (four G > A transitions and one G > C transversion). The common mutation pattern in tumors with giant cells and duct adenocarcinoma suggests a common route to malignant transformation and may indicate a shared histogenesis. We also tested 11 cases of malignant fibrous histiocytoma, a histological mimic of pleomorphic giant cell tumor, for mutations in the KRAS oncogene. The absence of KAS mutations in each of the malignant fibrous histiocytomas (MFHs) and in other histologically similar tumors may provide assistance in the differential diagnosis of pleomorphic pancreatic tumors.

Detection of mutant K-ras DNA in plasma or serum of patients with colorectal cancer.

Increased understanding of the molecular basis of colorectal cancer and recognition that extracellular DNA circulates in the plasma and serum of cancer patients enables new approaches to detection and monitoring. We used a polymerase chain reaction (PCR) assay to demonstrate mutant K-ras DNA in the plasma or serum of patients with colorectal cancer. Plasma or serum was fractionated from the blood of 31 patients with metastatic or unresected colorectal cancer and from 28 normal volunteers. DNA was extracted using either a sodium chloride or a gelatin precipitation method and then amplified in a two-stage PCR assay using selective restriction enzyme digestion to enrich for mutant K-ras DNA. Mutant K-ras DNA was detected in the plasma or serum of 12 (39%) patients, all confirmed by sequencing, but was not detected in any of the normal volunteers. K-ras mutations were detected in plasma or serum regardless of sex, primary tumour location, principal site of metastasis or proximity of chemotherapy and surgery to blood sampling. Tumour specimens available for 19 of the patients were additionally assayed for ras mutations and compared with blood specimens. Our results indicate mutant K-ras DNA is readily detectable by PCR in the plasma or serum of patients with advanced colorectal cancer. Thus, plasma- or serum-based nucleic acid amplification assays may provide a valuable method of monitoring and potentially detecting colorectal cancer.

Loss of heterozygosity and microsatellite instability at the retinoblastoma locus in osteosarcomas.

Studies of osteosarcoma cell lines or frozen tissue have detected loss of heterozygosity (LOH) at the retinoblastoma (RB) locus by Southern blot analysis or restriction fragment length polymorphism. Most archived clinical specimens cannot be analyzed by these techniques. We analyzed formalin-fixed, paraffin-embedded samples from 19 cases of osteosarcoma for molecular changes at the RB locus using polymerase chain reaction amplification of polymorphic short tandem repeat sequences (microsatellite repeats). Four repeat sequences, two within and two flanking the RB gene, were analyzed. Fourteen of 18 informative cases (78%) showed molecular changes at the RB locus. LOH was identified in 13 cases (72%). Unexpectedly, microsatellite instability (MI) was found in eight cases (44%). All of the cases of MI involved alterations of more than one repeat unit, and six of eight were associated with LOH. LOH was identified at three unlinked loci in one case and at a single locus in another Microsatellite analysis of archival tissue yields prevalence rates of LOH comparable to those found by other methods and has the added advantage of showing MI. The ability to use formalin-fixed, paraffin-embedded tissue extends genetic analysis to routinely processed surgical material and may permit molecular confirmation of challenging cases of osteosarcoma.