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Background: Microsatellite instability (MSI) and allelic imbalance involving chromosome arms 5q, 8p, 17p, and 18q are genetic alterations commonly found in colorectal cancer. We investigated whether the presence or absence of these genetic alterations would allow stratification of patients with Astler-Coller stage B2 or C colorectal cancer into favorable and unfavorable prognostic groups., Methods: Tumors from 508 patients were evaluated for MSI and allelic imbalance by use of 11 microsatellite markers located on chromosome arms 5q, 8p, 15q, 17p, and 18q. Genetic alterations involving each of these markers were examined for associations with survival and disease recurrence. All P values are two-sided., Results: In univariate analyses, high MSI (MSI-H), i.e., MSI at 30% or more of the loci examined, was associated with improved survival (P =.02) and time to recurrence (P =.01). The group of patients whose tumors exhibited allelic imbalance at chromosome 8p had decreased survival (P =.02) and time to recurrence (P =.004). No statistically significant associations with survival or time to recurrence were observed for markers on chromosome arms 5q, 15q, 17p, or 18q. In multivariate analyses, MSI-H was an independent predictor of improved survival (hazard ratio [HR] = 0.51; 95% confidence interval [CI] = 0.31-0.82; P =.006) and time to recurrence (HR = 0.42; 95% CI = 0.24-0.74; P =.003), and 8p allelic imbalance was an independent predictor of decreased survival (HR = 1.89; 95% CI = 1.25-2.83; P =. 002) and time to recurrence (HR = 2.07; 95% CI = 1.32-3.25; P =.002)., Conclusions: Patients whose tumors exhibited MSI-H had a favorable prognosis, whereas those with 8p allelic imbalance had a poor prognosis; both alterations served as independent prognostic factors. To our knowledge, this is the first report of an association between 8p allelic imbalance and survival in patients with colorectal cancer.

Denaturing high performance liquid chromatography (DHPLC) has been described recently as a method for screening DNA samples for single nucleotide polymorphisms and inherited mutations. Thirty-eight DNAs, 22 of which were heterozygous for previously characterized rearranged transforming gene (RET) or cystic fibrosis transmembrane conductance regulator gene (CFTR) mutations or polymorphisms, were examined using DHPLC analysis to assess the accuracy of this scanning method. Ninety-one per cent (20/22) of the PCR amplicons from specimens with heterozygous RET or CFTR sequence showed elution profiles distinct from corresponding homozygous normal patterns; whether the profiles for two amplicons containing heterozygous RET sequence were distinct from homozygous cases was equivocal. To investigate the usefulness of this method for detecting mutations in tumor DNAs, each of the phosphatase and tensin homologue deleted on chromosome ten gene (PTEN) exons were examined for mutations in 63 malignant gliomas. Seventeen PTEN PCR products from this series of brain tumors showed elution profiles indicating sample heterozygosity and in each instance conventional sequencing confirmed the presence of a mutation. PTEN amplicons containing exons 1, 3 and 5 were sequenced for each of the 63 tumor DNAs to determine whether any mutations may have escaped DHPLC detection, and this analysis identified one such alteration in addition to the eight mutations that DHPLC had revealed. In total, DHPLC identified 37 of 40 (92.5%) PCR products containing defined sequence variation and no alterations were indicated among 196 amplicons containing homozygous normal sequence.

Germline mutations in exons 10, 11, and 16 of the RET protooncogene are associated with the heritable cancer syndromes multiple endocrine neoplasia (MEN) type 2A, familial medullary thyroid carcinoma (FMTC), and MEN type 2B. Nonradioactive mutation analysis with nondenaturing Phastgels and the Phast System was performed on DNA amplified by the polymerase chain reaction from exons 10, 11, and 16 of the RET protooncogene from patients with MEN 2A, MEN 2B, or FMTC. The analysis requires approximately 45-90 min for electrophoresis and 35 min for staining. This assay detected 20 of 21 different mutations that represented approximately 90% of all known mutations associated with these lesions. A rare silent polymorphism within exon 10 was also detected. This form of mutation analysis provides simple, rapid, and highly sensitive nonradioactive detection of mutations known to be associated with MEN 2A, FMTC, and MEN 2B.

Mutations in the RET protooncogene have recently been demonstrated in families with multiple endocrine neoplasia (MEN) types 2A and 2B. We have studied pheochromocytomas from 29 individuals who had no clinical evidence of MEN-2A or -2B to determine the frequency of germline and/or somatic mutations in exons 10, 11, and 16 of the RET protooncogene. Of the 29 tumors examined, 3 (10%) were found to have a mutation in 1 of the 3 exons. These mutations were not found in the DNA from the peripheral blood from these individuals, indicating that the mutations in the tumors were somatic in origin. Although we cannot exclude the possibility of mutations in other regions of the RET protooncogene, our data suggest that 1) individuals presenting with apparently sporadic pheochromocytomas are not likely to have undiagnosed MEN-2A or -2B; and 2) somatic mutations in exons 10, 11, and 16 in the RET protooncogene contribute to the process of tumorigenesis in a small percentage of sporadic pheochromocytomas.

A unique kindred manifesting medullary thyroid carcinoma and corneal nerve thickening without other aspects of the multiple endocrine neoplasia syndrome (MEN) was analyzed by linkage analysis using four highly polymorphic (CA)n repeat markers (sTCL-1, D10S141, ZNF22, and sJRH-1). Additionally, the RET protooncogene was examined for specific mutations by DNA sequence analyses in all affected family members. Screening of 11 family members spanning 4 generations revealed 7 subjects with corneal nerve thickening; of these subjects, 3 had abnormal pentagastrin-stimulated calcitonin studies, and these 3 subjects were each found to have C-cell hyperplasia or medullary thyroid carcinoma at surgery. Linkage analysis showed cosegregation of alleles (as defined by the above markers), with the presence of both corneal nerve thickening and medullary thyroid carcinoma/C-cell hyperplasia (maximum LOD score, 2.69; consistent with, but not proving linkage). DNA sequence analysis showed that none of the affected individuals had mutations in either exon 10 or 11, or in exon 16 of the RET protooncogene, regions where mutations have been described for MEN type 2A (MEN-2A) and MEN-2B families, respectively. Thus, compared to the defined syndromes of MEN-2A and MEN-2B, this kindred appears to represent a true clinical overlap syndrome whose genetic basis may be distinct from these two syndromes.

Ten kindreds (95 individuals) with multiple endocrine neoplasia, type 2 (MEN 2) were analyzed by linkage analysis using four highly polymorphic (CA)n-repeat markers (sTCL-1, D10S141, ZNF22, and sJRH-1). Additionally, we examined the RET proto-oncogene for specific mutations by DNA sequence analyses in these 10 plus 14 members of 3 additional kindred. Nine families had MEN 2A, two had MEN 2B, and two had medullary thyroid cancer alone (FMTC). Using these four markers, all 10 kindreds were informative, with 10 individuals predicted to be presymptomatic MEN 2 gene carriers and 23 individuals predicted not to be carriers. DNA sequence analysis of exons 10 and 11 of the RET proto-oncogene revealed a mutation in all nine MEN 2A kindreds. A missense mutation was found in each case, leading to a loss of a cysteine residue (codon 618 of exon 10 or codon 634 of exon 11). In the MEN 2A families, the linkage analysis and RET mutation analysis gave concordant results for prediction of gene carriers in 100% of the individuals tested. No mutations were found in the two kindreds with FMTC or the two MEN 2B kindreds. Two individuals from two different MEN 2A kindreds were identified who had abnormal calcitonin stimulation tests but were not MEN 2A gene carriers by both linkage analysis and RET mutation analysis. These individuals presumably represented the sporadic occurrence of abnormal calcitonin stimulation tests in the general population. These studies provide further support for a role of the RET proto-oncogene in the pathogenesis of MEN 2A. Additionally, in the absence of identifiable RET proto-oncogene mutations, linkage analysis using (CA)n-repeat markers is a highly accurate alternative for the identification of MEN 2 or FMTC gene carriers.

A case of lymphocyte-depletion Hodgkin's disease is described for the purpose of reviewing the criteria currently used to distinguish this disease from other pleomorphic large-cell malignancies. A 76-year-old man with a 3-month history of daily fevers underwent extensive evaluation and exploratory laparotomy, which revealed only two large, separate splenic tumor nodules. Postoperatively, the patient remained asymptomatic. Histologically, the tumor was composed of giant cells, including both typical Reed-Sternberg forms and mononuclear variants with inflammatory stromal response along its borders. Immunoperoxidase showed tumor cells to be strongly reactive for Leu-M1 (CD15), BER-H2 (CD30), Leu-3 (CD4), and T11 (CD2) and weakly reactive for Leu-4 (CD3) but nonreactive for EMA, LCA, lysozyme, Leu-9, Leu-M3, Leu-M5, and immunoglobulin light chains. Southern blot analysis revealed an isolated clonal band for kappa light chain only. Included in the discussion of this case of primary splenic lymphocyte-depletion Hodgkin's disease is a review of clinical, histologic, immunohistochemical, and gene-rearrangement characteristics of what can be defined as lymphocyte-depletion Hodgkin's disease.

We describe a dual-isotope assay for measuring the concentration of estrogen receptors and progesterone receptors in tumor cytosols. The concentrations of these receptors are derived from separate Scatchard-plot analyses of a single dextran-coated charcoal assay that incorporates both radioiodinated estradiol and tritiated R5020 as the labeled ligands. The two isotopes, radioiodine and tritium, are easily measured with a liquid-scintillation counter. The concentrations of estrogen and progesterone receptors derived from the dual-label assay are identical to values derived from the respective single-label assays. Moreover, values obtained from an assay with dextran-coated charcoal that incorporates radioiodinated estradiol are identical to those obtained from an assay in which tritiated estradiol is used as the labeled ligand. The dual-isotope assay requires both less time and less tissue.

Cytosolic estrogen receptors from human breast carcinoma (approximately 50 patients) and uterine myometrium tissue (approximately 20 patients) were analyzed by isoelectric focusing on agarose gels, by their ability to bind to isolated nuclei, and by ultracentrifugation, under diverse conditions. Through the use of diisopropylfluorophosphate (DFP) and sodium molybdate (Mo), two in vitro processes were examined by these techniques: receptor activation and receptor degradation. These agents are capable of inhibiting these processes selectively so that the resulting stabilized forms of the estrogen receptor can be characterized in more detail. The predominant molecular forms, as determined by the parameters stated above, vary dramatically depending on the presence of DFP or Mo. At least six distinct species of the estrogen receptor were resolved by isoelectric focusing. Of these, four appear to be products of proteolysis. These had isoelectric points of 6.9, 6.8, 6.4, and 6.3, with a combined sedimentation coefficient of approximately 3.2S. The nonactivated form of the receptor, prepared in the presence of both DFP and Mo, focused at pH 4.8 and had a sedimentation coefficient of approximately 9.5S. Finally, the activated form detected in the presence of DFP sedimented at 8.0S and had an apparent pI of 5.4.

Recombinant DNA technology promises to play an increasingly important role in the future of medicine. Application of this technology to the study of human disease will help us to define and clarify the molecular pathology of many clinical disorders, provide new diagnostic tools and approaches, and, finally, will provide new therapeutic agents including gene-replacement therapy. We have begun to exploit these powerful new techniques to aid in the laboratory diagnosis of several genetic disorders for which reliable assays are currently not available, such as "Fragile X" syndrome.