Microsatellite instability (MSI) analysis of colorectal cancers is clinically useful to identify patients with hereditary nonpolyposis colorectal cancer (HNPCC)caused by germline mutations of mismatch repair genes. MSI status may also predict cancer response/ resistance to certain chemotherapies. We evaluated the MSI Analysis System (Promega Corp.; five mononucleotide and two pentanucleotide repeats) and compared the results to the Bethesda panel, which interrogates five microsatellite loci recommended by the 1997 National Cancer Institute-sponsored MSI workshop (three dinucleotide and two mononucleotide repeats). Thirty-four colorectal cancers were analyzed by both assays. The overall concordance between the two assays was 85% (29 of 34). There was complete concordance between the two assays for all of the MSI-high (11 of 11) and microsatellite stable (MSS; 18 of 18) cases. In the 11 MSI-high cases, all 5 of the mononucleotide loci in the MSI Analysis System demonstrated shifted alleles (100% sensitivity), and each shift resulted in products that were smaller in size than the germline alleles. All (5 of 5) of the cases interpreted as MSI-low by the Bethesda assay were interpreted as MSS by the MSI Analysis System. Our results suggest that the MSI Analysis System is generally superior and may help resolve cases of MSI-low into either MSI-high or MSS.

FLT3 is a receptor tyrosine kinase that is expressed on early hematopoietic progenitor cells and plays an important role in stem cell survival and differentiation. Two different types of functionally important FLT3 mutations have been identified. Internal tandem duplication mutations arise from duplications of the juxtamembrane portion of the gene and result in constitutive activation of the FLT3 protein. This alteration has been identified in ϳ20% to 30% of patients with acute myelogenous leukemia and appears to be associated with a worse prognosis. The second type of FLT3 mutation, missense mutations at aspartic acid residue 835, occurs in ϳ7.0% of acute myelogenous leukemia cases. These mutations also appear to be activating and to portend a worse prognosis. Identification of FLT3 mutations is important because it provides prognostic information and may play a pivotal role in determining appropriate treatment options. We have developed an assay to identify both internal tandem duplication and D835 FLT3 mutations in a single multiplex polymerase chain reaction. After amplification, the polymerase chain reaction products are analyzed by capillary electrophoresis for length mutations and resistance to EcoRV digestion. Here we describe the performance characteristics of the assay, FMS-like tyrosine kinase 3 (FLT3 also known as STK1 and flk2) is a member of the class III receptor tyrosine kinase family that also includes PDGF-R, KIT, and FMS. 1 The FLT3 protein is normally expressed on hematopoietic stem progenitor cells and appears to play an important role in stem cell survival, and the development of dendritic and natural killer cells. 2,3 FLT3 is overexpressed in most cases of acute myeloid leukemia (AML). 4,5 In addition, analysis of leukemic blasts from AML patients has identified two specific somatic mutations of the FLT3 gene. 6,7 Identification of these mutations in AML patients provides independent prognostic information that may also prove important for treatment optimization.The first and best-studied FLT3 mutation is an internal tandem duplication (ITD) mutation. ITD mutations typically result from the duplication and tandem insertion of a portion of the juxtamembrane (JM) region (exons 11 to 12) of the FLT3 wild-type gene. 6 The lengths of the duplicated segments have been reported to range in size from 6 to 180 bases and are always in frame. 8,9 ITD mutations result in the constitutive autophosphorylation of the FLT3 receptor and are thus gain-of-function mutations of the FLT3 proto-oncogene. 10 FLT3 ITD mutations have been reported to occur in 20 to 30% of patients with AML and have been associated with an increased relapse risk, decreased disease-free survival, decreased event-free survival, and decreased overall survival. 8,9,11 In a multivariate analysis of FLT3 ITD mutations, cytogenetic risk group, presentation white blood cell count, percentage BM blasts at diagnosis, age, gender, and FAB type in 854 AML patients, the presence of a FLT3 ITD mutation was the most significant factor adversely a...

Adenomatous polyposis coli (APC) is a tumor suppressor gene important in colorectal tumorigenesis. A genetic variant of APC, I1307K, results from a T-to-A transversion at nucleotide 3920 which converts the wild-type sequence to a homopolymer tract (A(8)). The I1307K alteration is not itself oncogenic, but creates a hypermutable region (A(8)) that is prone to frame-shift mutations. The APC I1307K variant occurs in approximately 6% of the Ashkenazi Jewish population and is reported to approximately double an individual's risk for colorectal cancer. Here we describe a single nucleotide primer extension assay for the detection of the APC I1307K mutation. Following PCR amplification, nucleotide 3920 of the APC gene is directly sequenced using single nucleotide primer extension technology. The assay is in a multiplex format allowing simultaneous forward and reverse sequencing of the I1307K variant, which provides an internal, independent confirmation of each testing result. The assay was validated against 60 samples previously characterized by an allele-specific oligonucleotide (ASO) hybridization assay, with 100% concordance of results. Compared to the ASO assay, this single nucleotide primer extension assay requires significantly less technical time to perform, and has a greatly increased throughput capacity. The single nucleotide extension assay provides a highly sensitive and specific assay to identify individuals with the APC I1307K gene variant who may benefit from increased colorectal screening.