Thymidylate synthase (TYMS) enhancer region genotype-directed phase II trial of oral capecitabine for 2nd line treatment of advanced pancreatic cancer.

PURPOSE: The primary aim of this study was to characterize the 6-month overall survival and toxicity associated with second-line capecitabine treatment of advanced pancreatic cancer patients harboring the TYMS *2/*2 allele. The secondary aim was to analyze the response rate and pharmacokinetics of capecitabine-based therapy in this patient population. Lastly, TYMS, ATM and RecQ1 single nucleotide polymorphism were analyzed relative to overall survival in patients screened for study participation. METHODS: Eighty patients with stage IV pancreatic cancer were screened for the *2/*2 TYMS allele. Patients with the *2/*2 TYMS polymorphism were treated with capecitabine, 1,000 mg/m2 twice daily for 14 consecutive days of a 21 day cycle. Screened patients not possessing TYMS *2/*2 were monitored for survival. Pharmacokinetic analysis was done during Cycle 1 of the therapy. RESULTS: Sixteen of the 80 screened patients tested positive for *2/*2 TYMS variant. Four out of the 16 eligible patients were treated on study. The study was terminated early due to poor accrual and increased toxicity. Three patients experienced grade 3 non-hematologic toxicities of palmer-plantar erythrodysesthesia, diarrhea, nausea and vomiting. Grade 2 toxicities were similar and occurred in all patients. Only one patient was evaluable for response after completion of three cycles of therapy. The presence of the *2/*2 TYMS genotype in all of the screened patients trended toward a decreased overall survival. CONCLUSION: To our knowledge, this study represents the first genotype-directed clinical trial for patients with pancreatic adenocarcinoma. Although the study was closed early, it appears capecitabine therapy in pancreatic cancer patients harboring the TYMS *2/*2 variant may be associated with increased non-hematologic toxicity. This study also demonstrates the challenges performing a genotype-directed study in the second-line setting for patients with advanced pancreatic cancer.

In vivo and in vitro propagation of intraductal papillary mucinous neoplasms.

Intraductal papillary mucinous neoplasms (IPMNs) are one of the three 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. 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 14 cases by implanting them into nude, severe combined immunodeficient (SCID), and NOD/SCID/IL2Rgamma(null) (NOG) mice, in addition to direct culture, to generate tumor xenografts and cell lines. One sample was directly cultured only. Thirteen tumors were implanted into the three types of mice, including 10 tumors implanted into the triple immunodeficient NOG mice, in which the majority (8 of 10) grew. This included five 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. 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.

Complex rearrangement of chromosomes 1, 7, 21, 22 in Ewing sarcoma.

The Ewing sarcoma (ES) family of tumors is characterized by nonrandom chromosomal translocations involving the EWSR1 gene on chromosome 22 with one of the members of the ETS family of transcription factors. The majority of ES tumors are characterized by a balanced translocation t(11;22)(q24;q12), which results in the fusion of the 5' portion of EWSR1 gene with the 3'end of the FLI1 gene. Fusions with ERG, another member of the ETS family, occur in less than 10% of ES tumors, and can arise through complex chromosomal rearrangements. Here, we report a case of a 5-year-old female with an ES tumor in the thoracic region. G-banding and spectral karyotyping analysis demonstrated 46,XX,t(1;21;7)(q25;q22.3;q22). Metaphase fluorescence in situ hybridization (FISH) using the EWSR1 break-apart probe demonstrated a normal signal on both apparently normal chromosomes 22, and an additional EWSR1-5' signal on the derivative chromosome 21. Reverse-transcriptase polymerase chain reaction analysis of RNA isolated from the tumor demonstrated a EWSR1-ERG fusion transcript, fusing exon 7 of EWSR1 and exon 11 of ERG. These results are consistent with an additional copy of the 5' portion of EWSR1, which inverted and then inserted on chromosome 21 and fused to the 3' end of ERG. To our knowledge, this is the first report of a EWSR1-ERG fusion in an ES tumor with an apparently duplicated 5' portion of EWSR1, and with a complex translocation involving chromosomes 1, 7, and 21. This case adds to the spectrum of genetic rearrangements identified in ES tumors.

Abnormal villous morphology associated with triple trisomy of paternal origin.

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 (approximately 0.05% 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.

Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications.

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.

Complete hydatidiform mole with retained maternal chromosomes 6 and 11.

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.

A rare e14a3 (b3a3) BCR-ABL fusion transcript in chronic myeloid leukemia: diagnostic challenges in clinical laboratory practice.

Patients with chronic myelogenous leukemia have a t(9;22)(q34;q11.2) or variant translocation that results in a BCR-ABL fusion gene. BCR-ABL detection by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) is the standard practice for monitoring residual disease in patients with chronic myelogenous leukemia who receive tyrosine kinase inhibitor therapies. In this study, we describe a patient who tested positive for the BCR-ABL translocation by fluorescence in situ hybridization and cytogenetic analysis but tested negative by qRT-PCR molecular analysis at the time of diagnosis. Further PCR analysis and DNA sequencing with alternative primer sets demonstrated the presence of an e14a3 (also known as b3a3) BCR-ABL fusion. The e14a3 fusion is rare, but may be underreported as a result of many commercially available and laboratory-developed primer sets that fail to detect breakpoints in the ABL gene that are downstream of intron 1. For this patient, if the qRT-PCR assay had been used to monitor disease response/progression after treatment and not in conjunction with fluorescence in situ hybridization or cytogenetics at the time of diagnosis, the negative result would have been misinterpreted as molecular remission.