Mutations in codons 12 and 13 of the KRAS oncogene are relatively common in colorectal and lung adenocarcinomas. Recent data indicate that these mutations result in resistance to anti-epidermal growth factor receptor therapy. Therefore, we assessed Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS codon 12/13 mutations in formalin-fixed paraffin-embedded samples, including 58 primary and 42 metastatic colorectal adenocarcinomas, 63 primary and 17 metastatic lung adenocarcinomas, and 20 normal colon samples. Of 180 tumor samples, 62.2% were KRAS mutant positive, and 37.8% were negative. Melting curve analysis yielded no false positive or false negative results, but had 10% equivocal calls. Melting curve analysis also resulted in 4 cases with melting curves inconsistent with either wild-type or codon 12/13 mutations. These patterns were generated from samples with double mutants in codons 12/13 and with mutations outside of codons 12/13. Pyrosequencing yielded no false positive or false negative results as well. However, two samples from one patient yielded a pyrogram that was flagged as abnormal, but the mutation subtype could not be determined. Finally, using an electronic cutoff of 10%, Sanger sequencing showed 11.1% false positives and 6.1% false negatives. In our hands, the limit of detection for Sanger sequencing, pyrosequencing, and melting curve analysis was approximately 15 to 20%, 5%, and 10% mutant alleles, respectively.
Distinction of hydatidiform moles from nonmolar specimens and their subclassification as complete hydatidiform mole (CHM) versus partial hydatidiform mole (PHM) are important for clinical practice and investigational studies to refine ascertainment of risk of persistent gestational trophoblastic disease which differs among these entities. Immunohistochemical analysis of p57 expression, a paternally imprinted maternally expressed gene on 11p15.5, and molecular genotyping are useful for improving diagnosis. CHMs are characterized by androgenetic diploidy, with the loss of p57 expression owing to lack of maternal DNA. Loss of p57 expression distinguishes CHMs from both PHMs (diandric triploidy) and nonmolar specimens (biparental diploidy) which retain expression. In the process of evaluating molar specimens in our laboratory with p57 immunohistochemistry and molecular genotyping, we identified a morphologically typical androgenetic diploid CHM with aberrant diffuse p57 expression. Molecular genotyping by short tandem repeat markers and genome-wide copy number analysis by single nucleotide polymorphism array established androgenetic diploidy with retained maternal copies of chromosomes 6 and 11, with aberrant p57 expression attributable to the latter. This case, only the second reported to date, illustrates the value of combined traditional pathologic and ancillary molecular techniques for refined diagnosis of molar specimens. Specimens with morphologic features suggestive of CHM yet retaining p57 expression should be subjected to molecular genotyping to establish a definitive diagnosis because misclassification as PHM underestimates the risk of persistent gestational trophoblastic disease. We recommend use of p57 immunohistochemistry and molecular genotyping to evaluate all products of conception specimens for which there is any consideration of a diagnosis of hydatidiform mole. Genome-wide analysis has the potential to assist in localizing imprinted genes critical for determining the morphologic and behavioral phenotypes of hydatidiform moles.
Chronic myelogenous leukemia (CML) is the first human cancer causally linked to a specific chromosomal abnormality, the Philadelphia chromosome (Ph), which is a product of a reciprocal translocation between chromosome 9 and 22 [t(9;22)(q34;q11.2)]. This particular translocation results in the BCR-ABL gene fusion that was subsequently shown to have transforming activity due to the deregulated tyrosine kinase activity of ABL.1 In CML, Ͼ95% of the breakpoints involve the M-bcr region consisting of BCR introns downstream of either exon 13 (e13, previously known as b2) or 14 (e14, previously known as b3) and introns upstream of ABL exon 2 (a2). These BCR-ABL e13a2 and e14a2 fusions result in a 210-kd fusion protein.2 There are two less common breakpoints in the intronic region between the alternative BCR exon 2 known as m-bcr, and between BCR exons 19 and 20, known as -bcr, which encode a 190-kd (e1a2) and 230-kd fusion protein (e19a2), respectively.3,4 Rare atypical breakpoints have also been sporadically reported and can be grouped into four categories: BCR breakpoints originating within introns that lie outside M-bcr, m-bcr, or -bcr fused to ABL a2; BCR breakpoints occurring within exons fused to ABL a2; typical BCR breakpoints (M-bcr, m-bcr, or -bcr) fused to ABL breakpoints located downstream of a2; and transcripts containing intervening sequences between BCR and ABL a2. There are multiple methods for detecting the BCR-ABL translocation including cytogenetics, Southern blot, fluorescence in situ hybridization (FISH), and reverse transcription polymerase chain reaction (RT-PCR) (including quantitative qRT-PCR). Each method has advantages and disadvantages. Cytogenetics, Southern blot, and some FISH-based assays should be able to detect essentially all BCR-ABL translocations regardless of the breakpoints, while RT-PCR assays are limited in the breakpoints detected based on the location of the primers and probes. However, cytogenetics, Southern blot, and FISH all have limits of detection of approximately
Background Intraductal papillary mucinous neoplasms (IPMNs) are one of the 3 known curable precursor lesions of invasive pancreatic ductal adenocarcinoma, an almost uniformly fatal disease. Cell lines from IPMNs and their invasive counterparts should be valuable to identify gene mutations critical to IPMN carcinogenesis, and permit high-throughput screening to identify drugs that cause regression of these lesions. Methods To advance the study of the biological features of IPMNs, we attempted in vivo and in vitro growth of selected IPMNs based on the hypothesis that IPMNs could be grown in the most severely immunodeficient mice. We examined fourteen cases by implanting them into nude, severe combined immunodeficient (SCID), and NOD/SCID/IL2Rγnull (NOG) mice, in addition to direct culture, to generate tumor xenografts and cell lines. One sample was directly cultured only. Results Thirteen tumors were implanted into the 3 types of mice, including 10 tumors implanted into the triple immunodeficient NOG mice, where the majority (8 of 10) grew. This included 5 IPMNs lacking an invasive component. One of the explanted IPMNs, with an associated invasive carcinoma, was successfully established as a cell line. Tumorigenicity was confirmed by growth in soft agar, growth in immunodeficient mice, and the homozygous deletion of p16/cdkn2a. Epithelial differentiation of the cell line was documented by cytokeratin expression. Patient origin was confirmed using DNA fingerprinting. Conclusions Most non-invasive IPMNs grow in NOG mice. We successfully established one IPMN cell line, and plan to use it to clarify the molecular pathogenesis of IPMNs.
The vast majority of trisomies in spontaneous abortions (SAB) are single and of maternal origin , most frequently due to meiosis I errors. Triple trisomies are exceedingly rare (ϳ0.05% of spontaneous abortions) , most often of maternal origin , and associated with increased maternal age. Some trisomic SAB specimens can exhibit abnormal villous morphology simulating a partial hydatidiform mole , a distinct form of hydatidiform mole characterized by diandric triploidy. A SAB specimen from a 27-year-old woman, G1P0 at 8 weeks gestational age , was reviewed in consultation to address the finding of morphological features suggestive of a partial hydatidiform mole but DNA ploidy analysis yielding a diploid result. The villi were irregularly shaped and hydropic but lacked trophoblastic hyperplasia; p57 expression was retained. Since fully developed features of a partial hydatidiform mole were lacking , additional analysis was performed. Molecular genotyping and single nucleotide polymorphism array analysis demonstrated biparental diploidy with trisomy of chromosomes 7 , 13, and 20 , all of paternal origin. The three trisomies may have originated from paternal meiosis II errors, or from mitotic nondisjunction. We believe this to be the first report of triple trisomy in a SAB confirmed to be of paternal origin.
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