National Cancer Institute, Research Contracts Branch, PSCS, 6120 Executive Blvd, EPS/Room 638, Bethesda, MD 20892-7228

B -- REPLICATION ERROR (RER) TESTING AND DNA SEQUENCING SOL N02-CM-66200-82 POC Contact Michelle Scala, Contract Specialist or Janet Mattson, Contracting Officer on (301) 402-4509. The National Cancer Institute requires the services of a laboratory to provide microsatellite instability (RER testing) and DNA sequencing of various human tumor tissue samples. DNA extracted from tumor tissue, adenoma, normal colonic epithelia, other tissue, or peripheral blood will be provided for testing. Arrangements must be made for the expeditious and secure transfer of material from this source to the testing facility. Pick-up of the sample(s) must occur within 48 hours (Monday-Friday, 0900-1700, excluding holidays) of notification of the contractor via phone or fax. For the retrospective study, to the extent possible, the samples will be supplied in batches of greater than or equal to ten. For the prospective study, samples from individual patients are likely to be supplied. We currently anticipate providing material from 200-400 patients (or relatives) per year. Multiple tests may be performed on a given sample. The tests to be performed will include the following: 1. DETERMINATION OF MICROSATELLITE INSTABILITY (RER testing). Sets of primers individually flanking at least five and not more than ten regions of microsatellite DNA will be formulated to amplify the microsatellite regions by polymerase chain reaction. The specific microsatellite regions in the assay panel will have been chosen to provide the greatest sensitivity while at the same time offering the clearest distinction between ''true'' RER positive and negative malignancies. In any case the regions will include mono, di, tri, or tetra nucleotide repeats and be relatively dispersed throughout the human genome. The reactions will be carried out with one of each primer pair being labeled so as to allow for subsequent identification of the amplified product by electrophoresis of the amplified DNA products on an acrylamide denaturing gel. Based on the electrophoretic pattern (which, at times, will also require comparison with normal tissue from the same patient) a determination will be made as to whether the tumor shows notable microsatellite instability for each primer pair tested. The tumors will be caegorized as RER+ (those showing significant microsatellite instability in a relevant proportion of the microsatellite regions tested) or RER-. In the case of equivocal results additional primer sets will be tested until a determination of RER status can be made. TURN-AROUND TIME: For retrospective batch analysis a turnaround time of 8 weeks would be adequate. For the prospective studies, batch analysis may not be possible in most cases. Here, a turnaround time of 1 week from receipt of the sample would be required. 2. DETERMINATION OF THE NUCLEOTIDE SEQUENCE of MSH2, MLH1, PMS1, and PMS2. These determinations will be performed on RER+ tumors. Analysis will be performed on DNA extracted from tumor tissue for determination of the nucleotide sequence of the above-named four genes involved in nucleotide mismatch recognition and repair. Initially, the assay used will be bidirectional dideoxy nucleotide sequencing. As more rapid techniques become available they will be incorporated into the work. The sequencing will be carried out across the coding sequence of the entire gene (including intron-exon borders) using overlapping sets of sequencing primers. The contractor must have access to GenBank and EMBL nucleotide sequence databases. Confirmed, reproducible variance from sequences of the ''wild type'' versions of these genes available in the GenBank and EMBL databases will be noted. In addition notations will be made as to whether the changes defined are consistent with amino acid substitution, polypeptide chain termination, or neutral polymorphic variation. For the subset of tumors that show gene mutation, non-tumor tissue may be made available to determine germline status of the affected gene. For those individuals in whom a germline mutation is identified, additional sequencing may be requested on DNA provided from normal tissue of certain first degree relatives. The sequencing of the non-tumor tissue will be focused only on the particular segment of the particular gene that showed mutation in the tumor tissue. A database must be established for the storage and retrieval of sample numbers and gene specific sequences. The sequence(s) so generated will be incorporated into a data bank to provide a basis for individual and population comparisons and interpretation of sequence relevance and meaning. The provider of the samples has no objection to the integration of such sequence with those obtained rom other sources but, if so, the entire data bank must be made available to the requester of this service without charge. All responsible sources may submit a proposal which shall be considered by the agency. It is anticipated that the proposed contract will be for a basic year with two option years. Solicitation will be issued o/a November 20, 1995. NO collect calls will be accepted See Numbered Note(s): 1. (0304)

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