Trop2 is a cell-surface glycoprotein reported to be overexpressed in various types of adenocarcinomas with minimal expression in normal tissues. Recent findings that Trop2 expression correlates with tumor aggressiveness have increased interest in Trop2 as a potential target for cancer immunotherapy. The goal of this study was to extensively evaluate Trop2 expression at the transcript and protein levels in normal and tumor tissues. It was determined that Trop2 is overexpressed on some carcinomas relative to the corresponding normal tissue. However, in human and mouse, Trop2 is highly expressed at both the transcript and protein levels on several essential normal tissues. The findings suggest that the development of therapeutic agents to target Trop2 may require strategies that target Trop2 on malignant tissues in order to minimize potential toxicities to essential normal tissues that also express high levels of Trop2.

BackgroundScreening for colorectal cancer (CRC) has shown to reduce cancer-related mortality, however, acceptance and compliance to current programmes are poor. Developing new, more acceptable non-invasive tests for the detection of cancerous and precancerous colorectal lesions would not only allow preselection of individuals for colonoscopy, but may also prevent cancer by removal of precancerous lesions.MethodsPlasma from 128 individuals (cohort I – exploratory study: 73 cases / 55 controls ) was used to test the performance of a single marker, SEPT9, using a real-time quantitative PCR assay. To validate performance of SEPT9, plasma of 76 individuals (cohort II – validation study: 54 cases / 22 controls) was assessed. Additionally, improvement of predictive capability considering SEPT9 and additionally ALX4 methylation was investigated within these patients.ResultsIn both cohorts combined, methylation of SEPT9 was observed in 9% of controls (3/33), 29% of patients with colorectal precancerous lesions (27/94) and 73% of colorectal cancer patients (24/33). The presence of both SEPT9 and ALX4 markers was analysed in cohort II and was observed in 5% of controls (1/22) and 37% of patients with polyps (18/49). Interestingly, also 3/5 (60%) patients with colorectal cancer were tested positive by the two marker panel in plasma.ConclusionsWhile these data confirm the detection rate of SEPT9 as a biomarker for colorectal cancer, they also show that methylated DNA from advanced precancerous colorectal lesions can be detected using a panel of two DNA methylation markers, ALX4 and SEPT9. If confirmed in larger studies these data indicate that screening for colorectal precancerous lesions with a blood-based test may be as feasible as screening for invasive cancer.

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Aberrant DNA methylation occurs early in oncogenesis, is stable, and can be assayed in tissues and body fluids. Therefore, genes with aberrant methylation can provide clues for understanding tumor pathways and are attractive candidates for detection of early neoplastic events. Identification of sequences that optimally discriminate cancer from other diseased and healthy tissues is needed to advance both approaches. Using well-characterized specimens, genome-wide methylation techniques were used to identify candidate markers specific for colorectal neoplasia. To further validate 30 of these candidates from genome-wide analysis and 13 literature-derived genes, including genes involved in cancer and others with unknown functions, a high-throughput methylation-specific oligonucleotide microarray was used. The arrays were probed with bisulfite-converted DNA from 89 colorectal adenocarcinomas, 55 colorectal polyps, 31 inflammatory bowel disease, 115 extracolonic cancers, and 67 healthy tissues. The 20 most discriminating markers were highly methylated in colorectal neoplasia (area under the receiver operating characteristic curve > 0.8; P < 0.0001). Normal epithelium and extracolonic cancers revealed significantly lower methylation. Real-time PCR assays developed for 11 markers were tested on an independent set of 149 samples from colorectal adenocarcinomas, other diseases, and healthy tissues. Microarray results could be reproduced for 10 of 11 marker assays, including eight of the most discriminating markers (area under the receiver operating characteristic curve > 0.72; P < 0.009). The markers with high specificity for colorectal cancer have potential as blood-based screening markers whereas markers that are specific for multiple cancers could potentially be used as prognostic indicators, as biomarkers for therapeutic response monitoring or other diagnostic applications, compelling further investigation into their use in clinical testing and overall roles in tumorigenesis. (Mol Cancer Res 2007;5(2):153 -63)

Following adeno-associated virus (AAV)-mediated transduction, cellular RNA preparations can be contaminated with AAV single-stranded DNA. The single-stranded DNA genome of recombinant AAV vectors can serve as an efficient, but undesirable, template for traditional reverse transcriptase-polymerase chain reaction (RT-PCR) methods. Consequently, recombinant AAV gene therapy presents a unique challenge to the design of sensitive and reliable methods to detect vector-derived mRNA. Several methods have been proposed to reduce the presence of single-and doublestranded vector DNA without compromising RNA specificity. For example, DNase I, although widely used, can be ineffective at completely removing the AAV single-stranded DNA genome. We have developed a sensitive real-time RNA-Specific reverse transcriptase PCR (RS-PCR) method that is independent of DNase I treatment. The RS-PCR method relies on the generation of a first-strand cDNA template using a primer with a linker sequence, X, at the 5 0 -end such that synthesis of second-strand cDNA incorporates the X-linker sequence into the cDNA template. The RS-PCR then utilizes forward and reverse primers targeting AAV vector sequence and the X-primer site, respectively, while a vector-specific Taqman probe makes sensitive real-time detection possible. We present data to validate the sensitivity and RNA specificity of the RS-PCR method and propose two unique endogenous control strategies by monitoring expression of both b-glucuronidase and endogenous cystic fibrosis transmembrane conductance regulator (CFTR). Finally, we demonstrate the utility of this new RS-PCR method in detecting recombinant AAV-CFTR expression, including, an in vitro transduction assay and methods to support both preclinical and clinical trials.