Kevin Qu

Circulating microRNAs as biomarkers for hepatocellular carcinoma.

Goals We investigated whether measurement of serum levels of the microRNAs (miRNAs) miR-16, miR-195, and miR-199a, alone or in combination with conventional serum markers, can help to differentiate hepatocellular carcinoma (HCC) from chronic liver diseases (CLDs). Background Recent reports suggest a link between aberrant expression of miRNA, and HCC. Study This retrospective analysis was conducted using sera from 105 HCC patients, 107 CLD patients, and 71 normal control subjects. The miRNAs were measured using real-time reverse transcription-polymerase chain reaction. The conventional HCC markers &agr;-fetoprotein (AFP), lens culinaris agglutinin-reactive AFP (AFP-L3%), and des-&ggr;-carboxyprothrombin (DCP) were measured with commercial kits. Results Serum levels of miR-16 and miR-199a were significantly lower in HCC than in CLD patients or control subjects (P<0.01). As a single marker, miR-16 had the highest sensitivity for HCC, followed by miR-199a, AFP, DCP, AFP-L3%, and miR-195. The combination of miR-16, AFP, AFP-L3%, and DCP yielded the optimal combination of sensitivity (92.4%) and specificity (78.5%) for HCC, overall and when analysis was restricted to patients with tumors size smaller than 3 cm. As a second-line HCC marker, miR-16 yielded positive HCC predictions in 18 of the 26 (69.2%) HCC patients with negative results on all 3 conventional markers, most of whom had tumors size smaller than 3 cm; miR-16 was falsely positive in only 12 of 96 (12.5%) CLD patients. Conclusions The addition of miR-16 to conventional serum markers improved sensitivity and specificity for HCC. Use of miR-16 for second-line testing in cases considered negative on the basis of conventional HCC markers should be explored in larger, prospective studies.

Distribution of the ugt1a1*28 polymorphism in Caucasian and Asian populations in the Us: a genomic analysis of 138 healthy individuals

The hepatic isoform 1A1 of uridine diphosphate glucuronosyltransferase is responsible for glucuronidation and detoxification of SN-38, the active metabolite of irinotecan. The presence of an additional TA repeat in the TATA sequence of the UGT1A1 promoter leads to a significant decrease in SN-38 glucuronidation. Patients with the UGT1A1 (TA)7 allele are more likely to experience severe neutropenia and diarrhea following irinotecan chemotherapy. We assessed the distribution of the UGT1A1 (TA)n polymorphism in healthy male and female US residents of European and Asian descent. We used a fluorescent polymerase chain reaction-based assay to detect UGT1A1 (TA)n polymorphisms in 138 healthy volunteers (56 Caucasians, 37 Chinese, 37 Filipino and eight Japanese) between the ages of 18 and 65 years. The χ2-test was used to assess between-group differences in the distribution of UGT1A1 (TA)n genotypes. The UGT1A1 (TA)6/6 genotype was significantly more common in Asians than in Caucasians (76 vs. 46%), whereas the (TA)6/7 (39 vs. 20%) and (TA)7/7 (13 vs. 5%) genotypes were more common in Caucasians than in Asians. Genotype distributions did not differ significantly between men and women in either group. The UGT1A1 (TA)5/5 genotype was detected in one Caucasian woman. In conclusion, consistent with previous reports, the UGT1A1 (TA)7/7 genotype was significantly more common in Caucasians than in Asians. UGT1A1 (TA)n/n genotype distribution did not vary with sex in individuals of European or Asian descent.

Ubiquitin‐proteasome profiling for enhanced detection of hepatocellular carcinoma in patients with chronic liver disease

Background and Aim:  A reliable test for the detection of hepatocellular carcinoma (HCC) could improve disease management. Recent reports suggested a link between abnormalities in the ubiquitin‐proteasome system (UPS) and HCC. We investigated the potential of using UPS markers, along with HCC markers, to differentiate HCC from chronic liver disease (CLD).

Abstract 4675: Detection of ALK, ROS1, and RET translocations in non-small cell lung cancer (NSCLC) patients by intragenic differential expression analysis

BACKGROUND: ALK, ROS1, and RET translocations are frequently detected in NSCLC patients. Crizotinib, a tyrosine kinase inhibitor (TKI), was approved by the FDA in 2011 to treat NSCLC in patients harboring ALK translocations as detected by an FDA-approved assay. However, the FDA-approved ALK FISH assay is technically challenging, with failures due to pre-analytic variables. Another approach, intragenic differential expression (IDE), detects translocations by comparing expression levels of the 5′ end with the 3′ end of target gene transcripts. In this study we developed and evaluated a rapid IDE assay to screen for ALK, ROS1, and RET translocations, independent of the fusion partner. METHODS: A total of 419 samples (408 randomly-selected NSCLC clinical samples, ALK positive and ROS1 positive cell lines (2 each), and 7 previously-tested RET-positive clinical samples) were used to develop and evaluate performance characteristics of the IDE assays. To determine IDE scores, levels of ALK, ROS1, and RET expression were first determined by quantitative RT-PCR measurement of the 5′- and 3′- ends of the respective transcripts. The differences in expression levels were calculated as ΔCt (Ct5′ - Ct3′). High ΔCt values indicate presumptive presence of gene translocations. 212/408 NSCLC samples were analyzed by ALK FISH and EML4-ALK RT-PCR, and 196/408 samples were analyzed by EML4-ALK RT-PCR. RESULTS: Thirty-one of the 408 (7.6%) clinical samples tested positive for ALK rearrangements by IDE. Among them, 20 were confirmed by FISH and/or EML4-ALK (true positive, 64.5%), while 11 were negative by FISH and/or EML4-ALK (false positive, 35.5%). One of 10 ALK FISH positive samples tested negative by both ALK IDE and EML4-ALK RT-PCR analysis (false negative), while one of 202 FISH-negative sample tested positive by both EML4-ALK and ALK IDE. ALK IDE exhibited 94.5% (189/200) concordance with ALK FISH and 96.0% (356/371) concordance with the EML4-ALK assay. For ROS1, both ROS1-positive cell lines and 4/408 (1.0%) NSCLC samples tested positive for ROS1 by IDE. Among the 4 IDE-positive NSCLC samples, 1 was confirmed by ROS1 FISH. For RET, all 7 known positives and 10/408 (2.5%) NSCLC samples tested positive by IDE. Three of six RET IDE positive NSCLC samples were confirmed by RET FISH. Overall, ALK, ROS1, and RET translocations were mutually exclusive in NSCLC patients. The lung IDE assay had a failure rate of 3.7%. CONCLUSION: These findings demonstrate the feasibility of using IDE to detect ALK, ROS1, and RET gene translocations. These assays may have potential as a screening tool to select patients for further confirmation by FISH for TKI-targeted therapy. The IDE concept can be applied to a wide range of somatic translocations. Citation Format: Shih-Min Cheng, Cindy Barlan, Feras Hantash, Heather R. Sanders, Patricia H. Chan, Vladimira Sulcova, Marc A. Sanidad, Kevin Qu, Joann C. Kelly, Fatih Z. Boyar, Anthony D. Sferruzza, Frederic M. Waldman. Detection of ALK, ROS1, and RET translocations in non-small cell lung cancer (NSCLC) patients by intragenic differential expression analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4675. doi:10.1158/1538-7445.AM2014-4675

Abstract 3156: MicroRNA profiling for the detection of melanoma

Background: Numerous reports have suggested a link between aberrant expression of microRNAs (miRNAs) and various forms of cancer, including melanoma. We examined miRNA expression patterns in melanoma and nevi to identify candidate biomarkers for differentiating between these melanocytic lesions. Method: The study used formalin-fixed paraffin-embedded (FFPE) tissues from 78 patients with melanoma and 98 with nevi (18 compound nevi, 20 intradermal nevi, 20 junctional nevi, 20 blue nevi, and 20 spitz nevi). All lesions were confirmed by at least 2 pathologists. Total RNA was extracted from the FFPE tissue and hybridized onto the Affymetrix GeneChip miRNA array (Affymetrix, Santa Clara, CA), which measure 850 human microRNAs. After determining an optimal set of miRNAs for differentiation using random forest algorithm, we retested specimens for this set using quantitative real-time PCR (qPCR) on a TaqMan miRNA system (Applied Biosystems, Foster City, CA). Results: Expression levels of 257 miRNAs differed significantly between melanomas and nevi (p values 0.75 for distinguishing melanomas from nevi, and 74 exhibited more than a two-fold difference. An optimized set of 7 miRNAs provided good differentiation of melanomas from nevi, with an AUORC of 0.94 and 87.7% concordance with pathology-determined tissue type. miRNA array data were further validated by qPCR: for 5 of the 7 miRNAs identified above, qPCR results showed significant correlation with the miRNA array results (average Pearson correlation coefficient = 0.95). Conclusions: These data suggest that the miRNA expression pattern from FFPE tissue holds promise for distinguishing melanomas from nevi. Use of this miRNA set as a diagnostic marker requires further validation and exploration in a large-scale study. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3156. doi:1538-7445.AM2012-3156

Gene Expression Analysis for Tumor Profiling

Publisher Summary This chapter reviews technologies required to identify and separate tumor tissue from normal tissue and then extract genomic material (i.e., RNA or DNA) for analysis, which requires purification, amplification, and expression analysis of the gene or gene products. The common technology used to develop these genomic signatures involves tumor libraries using primary and metastatic cancers whose provenance is clear. These specimens may be frozen or more commonly are FFPE. Analysis requires extracting RNA from the tumor tissue, using it as a template for cDNA, and building a gene expression profile using a multigene microarray as commercially available. This approach requires a training set of samples used to develop the expression signature and a second set of samples used to validate the signature. Gene silencing through methylation has promulgated a new area of research known as epigenetics where post-translational changes are evaluated as they effect gene expression. It summarizes the important observations on tumor profiling in the context of clinical care. Most neoplastic disease can be traced to its site of origin by clinical or pathological features, thereby allowing the treating physician to choose appropriate therapy. Tumor genotypes and the resulting RNA or protein phenotypes are becoming increasingly important in our understanding of malignancy.

Abstract 2723: Circulating microRNA as a biomarker for screening for hepatocellular carcinoma

Background: Recent reports suggest a link between aberrant expression of microRNAs (miRNAs, miR) and various cancers, including hepatocellular carcinoma (HCC). We examined whether measurement of serum levels of 3 miRNAs (miR16, miR195, and miR199a) could be used to differentiate HCC from HCV-related chronic liver disease (CLD). Method: The 3 miRNAs were measured using real-time PCR-based assays in sera from 104 patients with HCC, 59 patients with CLDs, and 67 normal control subjects. Small nuclear RNA U6 was used as an internal control. Cutoff values were determined by visual inspection of miRNA distribution curves. Serum levels of the conventional HCC markers alpha-fetoprotein (AFP), AFP-L3, and des-gamma-carboxyprothrombin (DCP) were also measured, with cutoffs based on established reference ranges. Results: Median serum levels of miR16, miR195, and miR199a were significantly lower in patients with HCC than in CLD and control subjects (all p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2723.