Rajeswari Avula

Epigenetic silencing of TCEAL7 (Bex4) in ovarian cancer

Epigenetic silencing by hypermethylation of CpGs represents a mechanism of inactivation of tumor suppressors. Here we report on the cloning of a novel candidate tumor suppressor gene TCEAL7 inactivated by methylation in ovarian cancer. TCEAL codes for a 1.35 kb transcript that was previously reported to be downregulated in ovarian cancer by cDNA microarray and suppression subtraction cDNA (SSH) analyses. This report focuses on the elucidation of mechanisms associated with TCEAL7 downregulation. Expression of TCEAL7 is downregulated in a majority of ovarian tumors and cancer cell lines but induced by 5-aza-2′-deoxycytidine treatment in a dose-dependant manner, implicating methylation as a mechanism of TCEAL7 inactivation. Sequence analyses of bisufite-modified genomic DNA from somatic cell hybrids with either the active or the inactive human X chromosome reveal that TCEAL7 is subjected to X chromosome inactivation. Loss of TCEAL7 expression in primary tumors and cell lines correlates with methylation of a CpG site within the promoter. In vitro methylation of the CpG site suppresses promoter activity whereas selective demethylation of the SmaI site attenuates the suppression. Finally, re-expression of TCEAL7 in cancer cell lines induces cell death and reduces colony formation efficiency. These data implicate TCEAL7 as a cell death regulatory protein that is frequently inactivated in ovarian cancers, and suggest that it may function as a tumor suppressor.

Studies of humoral immunity to preprohypocretin in human leukocyte antigen DQB1*0602-positive narcoleptic subjects with cataplexy

BACKGROUND Canine models for narcolepsy have mutations of the hypocretin receptor 2 gene, and preprohypocretin knockout murine lines exhibit narcoleptic-like behaviors. Human narcolepsy with cataplexy is associated with human leukocyte antigen DQB1*0602 and reduced hypocretin levels in cerebrospinal fluid, suggesting an autoimmune diathesis. We tested the hypothesis that DQB1*0602-positive narcoleptic subjects with cataplexy have immunoglobulin (Ig)G reactive to human preprohypocretin and its cleavage products. METHODS Serum samples of 41 DQB1*0602-positive narcoleptic subjects with cataplexy and 55 control subjects were studied, as were 19 narcoleptic and 13 control samples of cerebrospinal fluid. We tested for IgG reactive to preprohypocretin and its major cleavage products (including hypocretin 1 and 2), using immunoprecipitation assays (IP), immunofluorescence microscopy (IF) of Chinese hamster ovarian cells expressing preprohypocretin, and Western blots. RESULTS There was no evidence for IgG reactive to preprohypocretin or its cleavage products in CSF of subjects with narcolepsy as measured by IPs, Western blots, and IF. Although the IP with CSF and the C-terminal peptide showed significant differences by two methods of comparison, the control subjects had higher counts per minute than narcoleptic subjects, which was opposite to our hypothesis. CONCLUSIONS The hypothesis that DQB1*0602-positive narcoleptic subjects with cataplexy have IgG reactive to preprohypocretin or its cleavage products was not supported.

SULT1A1, CYP2C19 and disease-free survival in early breast cancer patients receiving tamoxifen

AIM Tamoxifen biotransformation to endoxifen, a potent antiestrogen, is catalyzed by CYP2D6. In addition, CYP2C19 and SULT1A1 have also been implicated in the metabolism of tamoxifen. We sought to evaluate the importance of SULT1A1 copy number and CYP2C19*17 on disease-free survival (DFS) in postmenopausal women randomized to tamoxifen monotherapy in North Central Cancer Treatment Group 89-30-52 from January 1991 to April 1995. MATERIALS & METHODS We extracted DNA from paraffin-embedded tumors and determined tumor SULT1A1 copy number and CYP2C19*17 genotype. The association of genotype with DFS was determined using the log-rank test. Multivariate cox modeling was performed using traditional prognostic factors, as well as CYP2D6 genotype. SULT1A1 copy number and CYP2C19*17 genotype was determined in 190 out of 256 patients (95% Caucasian). RESULTS The median follow-up for living patients was 14 years. DFS did not differ according to SULT1A1 copy number (p = 0.482) or CYP2C19*17 genotype (p = 0.667). Neither SULT1A1 copy number or CYP2C19*17 genotype was associated with disease recurrence in this cohort. CONCLUSION Future studies are needed to identify whether other genetic and environmental factors which affect tamoxifen metabolism are associated with tamoxifen clinical outcomes.

Correlation of CYP2B6, CYP2C19, ABCC4 and SOD2 genotype with outcomes in allogeneic blood and marrow transplant patients

CYP2B6, CYP2C19, ABCC4, and SOD2 have been implicated in adverse drug reactions and survival after cyclophosphamide (CPA) treatment. 110 BMT patients who received high dose CPA treatment were genotyped for variants in these genes and the results were correlated with toxicity and relapse. CYP2B6 genotype significantly influenced overall toxicity suggesting active CYP2B6 alleles led to higher rates of overall toxicity. The p.R487C deficiency allele was significantly associated with a lower rate of overall toxicity and a higher rate of relapse. SOD2 rs4880 V16A polymorphism was associated with significantly less CPA-related overall toxicity and significantly lower relapse rates by Kaplan-Meier analysis although the SOD2 finding regarding relapse was not significant when evaluated by the cumulative incidence function.

Tamoxifen pharmacogenetics of CYP2D6, CYP2C19, and SULT1A1: long term follow-up of the North Central Cancer Treatment Group 89-30-52 adjuvant trial.

CTRC-AACR San Antonio Breast Cancer Symposium: 2008 Abstracts Abstract #6037 Background: Tamoxifen (Tam) is biotransformed to the potent antiestrogen, endoxifen, by the CYP2D6 enzyme. We previously demonstrated that patients (pts) receiving adjuvant TAM with impaired CYP2D6 metabolism due to CYP2D6 (*4) and/or concurrent administration of a CYP2D6 inhibitor had a higher risk of recurrence. Other studies suggest CYP2C19*17 (Schroth JCO 2007) and SULT1A1 *2 (Nowell JNCI 2002) may be associated with treatment outcome. With extended follow-up, we sought to evaluate the importance of comprehensive CYP2D6 genotyping as well as the potential association between CYP2C19*17 and SULT1A1 copy number with clinical outcome in pts randomized to TAM in NCCTG 89-30-52. Methods: Using DNA derived from paraffin embedded sections, CYP2D6 and SULT1A1 genotype were determined using quantitative multiplex PCR and CYP2C19 by sequencing. Pts administered the following CYP2D6 inhibitors (fluoxetine, paroxetine, sertraline, cimetidine, amiodarone, doxepin, ticlopidine and haloperidol) were considered as intermediate (IM) or poor metabolizers (PM) based on the potency of CYP2D6 inhibition. CYP2D6 phenotype was defined as follows: extensive metabolizers (EM) were pts not administered an inhibitor who did not carry a null allele (*3, *4, *6) and who were not homozygous for an IM allele (*10, *17, *41). CYP2D6 IM were either heterozygous for a null allele or homozygous for an IM allele but not administered an inhibitor or CYP2D6 EM administered a weak inhibitor. PM were pts homozygous for a null allele, or any patient administered a potent inhibitor. The association between genotype or CYP2D6 phenotype and clinical outcome was determined using the log-rank test. Multivariate Cox modeling was performed using traditional prognostic factors . Results : The median follow-up of living pts is now 14.5 years. Of 256 pts. randomized to TAM, genotype was determined for CYP2D6 (n=210), CYP2C19*17 (n=170), and SULT1A1 (n=169) with the following allele frequencies: CYP2D6 *3 (.02), *4(.20), *6 (.01), *10 (.03), *17 (.00), *41(.08), and CYP2C19*17 (.22). The frequency of SULT1A1 copy number alleles (CNA) was 1 (4%), 2 (67%), 3 (20%), and 4+ (9%). 14/227 patients (6%) were administered an inhibitor. A multivariate analysis accounting for nodal status and tumor size demonstrated that compared to CYP2D6 EM, CYP2D6 PM had significantly shorter time to recurrence (TTR) (HR 4.0, p=0.001) and DFS (HR 2.0, p=0.02) and CYP2D6 IM tended to have shorter TTR (HR 1.8, p=0.08) and DFS (HR 1.4, p=0.10). DFS did not differ by SULT1A1 copy number (p=0.62) or CYP2C19 *17 (p=0.47) Conclusion: Long term follow-up of pts in NCCTG 89-30-52 confirms the importance of CYP2D6 metabolism and further demonstrates the importance of comprehensive genotyping and phenotyping with CYP2D6 inhibitors. We could not identify an association between CYP2C19*17 or SULT1A1 copy number with recurrence but further evaluation is needed in larger cohorts. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 6037.

Technical Advances for the Clinical Genomic Evaluation of Sudden Cardiac Death: Verification of Next-Generation Sequencing Panels for Hereditary Cardiovascular Conditions Using Formalin-Fixed Paraffin-Embedded Tissues and Dried Blood Spots

Background— Postmortem genetic testing for heritable cardiovascular (CV) disorders is often lacking because ideal specimens (ie, whole blood) are not retained routinely at autopsy. Formalin-fixed paraffin-embedded tissue (FFPET) is ubiquitously collected at autopsy, but DNA quality hampers its use with traditional sequencing methods. Targeted next-generation sequencing may offer the ability to circumvent such limitations, but a method has not been previously described. The primary aim of this study was to develop and evaluate the use of FFPET for heritable CV disorders via next-generation sequencing. Methods and Results— Nineteen FFPET (heart) and blood (whole blood or dried blood spot) specimens underwent targeted next-generation sequencing using a custom panel of 101 CV-associated genes. Nucleic acid yield and quality metrics were evaluated in relation to FFPET specimen age (6 months to 15 years; n=14) and specimen type (FFPET versus whole blood and dried blood spot; n=12). Four FFPET cases with a clinical phenotype of heritable CV disorder were analyzed. Accuracy and precision were 100% concordant between all sample types, with read depths >100× for most regions tested. Lower read depth, as low as 40×, was occasionally observed with FFPET and dried blood spot. High-quality DNA was obtained from FFPET samples as old as 15 years. Genomic analysis of FFPET from the 4 phenotype-positive/genotype unknown cases all revealed putative disease-causing variants. Conclusions— Similar performance characteristics were observed for next-generation sequencing of FFPET, whole blood, and dried blood spot in the evaluation of inherited CV disorders. Although blood is preferable for genetic analyses, this study offers an alternative when only FFPET is available.

Analysis of Cell-Free DNA to Assess Risk of Tumoremia Following Endoscopic Ultrasound Fine-Needle Aspiration of Pancreatic Adenocarcinomas

Background & Aims Cellular and nuclear material from tumors disseminates into the bloodstream (tumoremia), but it is not clear whether medical procedures cause release of this material or contribute to formation of metastases. We performed a prospective study of blood samples from patients with pancreatic adenocarcinoma (PDAC) to determine whether endoscopic ultrasound‐guided fine needle aspiration (EUS‐FNA) associates with markers of tumoremia. Methods We obtained peripheral blood from 104 patients (35 with PDAC) before and after EUS‐FNA of primary tumors; blood samples from 69 healthy individuals were used as controls. Plasma concentrations of cell‐free DNA (cfDNA) were measured, and cfDNA and primary tumor samples were analyzed to detect activating mutations in KRAS. Potential development of tumoremia was defined by an increase in cfDNA of 2‐fold or more, and/or detection of mutant KRAS in samples collected after FNA from patients whose blood samples did not contain detectable mutant KRAS before FNA. Results Peripheral blood concentrations of cfDNA were 1200 ng/ml (500–3300 ng/ml) before FNA vs 1400 ng/ml (900–4000 ng/ml) after FNA (P = .391). Tumoremia was detected in 10/35 patients (28.6%): 7 patients had a ≥2‐fold increase in cfDNA concentration (20.6%) and 3 patients had circulating tumor DNA with KRAS mutations after FNA that were not detected before FNA (8.8%). New distant metastases were detected in 1.3 ± 0.82 patients with tumoremia vs 0.64 ± 0.81 without (P = .0375). Overall mortality did not differ significantly between patients with tumoremia (10/10 deaths, 100%) vs those without (19/25 deaths, 76%) nor did survival times of deceased patients (13.3 months for patients with tumoremia; range, 5.8–14.9 months vs 11.1 months for patients without tumoremia; range, 5.5–14.5 months). However, 6 patients without tumoremia were alive at a mean 23.9 months after EUS‐FNA (range, 19.9–25 months after EUS‐FNA) vs none of the patients with tumoremia. Conclusion In patients with PDAC, EUS‐FNA associates with increased plasma concentration of cfDNA and increased detection of mutant KRAS after the procedure (markers of tumoremia and possible new distant metastasis). Although levels of cfDNA and activating mutations in KRAS are logical markers of tumoremia, they may not serve as the ideal biomarkers of this process. These findings are preliminary and do not indicate a need to modify current practice, yet further studies are needed.

Abstract 505: Optimized 7-Plex cell-free DNA assay for clinically relevant KRAS mutations in colorectal cancer

Background: KRAS gene mutations are present in approximately 40% of colorectal adenocarcinomas and predict for nonresponse to the anti-epidermal growth factor receptor antibodies used in routine clinical practice. The most common activating mutations in this malignancy occur in codons 12 and 13 of exon 2. Determination of KRAS mutational status from tumor samples is an essential tool for managing patients with colorectal cancer (CRC) but requires tissue obtained by invasive percutaneous or surgical biopsies. Recent advances allow for the isolation and analysis of cell-free DNA (cfDNA) from the peripheral blood. A highly sensitive multiplex technology that uses low amounts of input DNA is needed to establish cfDNA as a liquid biopsy to personalize care. Methods: Digital droplet PCR (ddPCR) is a particularly sensitive method for detecting rare and low copy targets. Related work led to 7 individual assays, and to companion 4-plex and 3-plex assays, to accommodate the 7 common KRAS mutations and internal controls. A single 7-plex assay is favored to maximize efficiency and minimize cost. We optimized the RainDrop Digital PCR System to collectively detect the G12/G13 KRAS mutations, given that documentation of a mutation (not the specific mutation) is the factor of clinical relevance. Commercially available cell lines and genomic DNA aliquots were used as reference standards and for determinations of sensitivity and limit of blank (LOB). Initial clinical validation samples included tumor DNA derived from FFPE tissue (n = 10), cfDNA from healthy donors (n = 10), and cfDNA from metastatic CRC patients with tumors of known KRAS mutation status (n = 4). All assays were run in triplicate to confirm reproducibility. Results: Custom Taqman MGB probes were selected for KRAS wild type (WT), G12A, G12C, G12D, G12R, G12S, G12V, and G13D to allow for testing in a single reaction. LOB was 9 per 1×10(6) droplets on the basis of WT controls, including cfDNA samples from healthy donors. Evaluation of cell line DNA, genomic DNA aliquots, and FFPE samples demonstrates that the assay detects mutant KRAS with 100% accuracy and 0.1% mutant DNA in a total input of 10 ng WT/mutant DNA. With the clinical samples, concordant KRAS findings between cfDNA and tissue were obtained for two patients with WT tumors and one patient with a KRAS G12S mutant tumor. cfDNA analysis did not detect the G12V mutation identified in the colon primary of the other patient. Evaluation of additional clinical samples is in progress. Conclusions: Our 7-plex G12/G13 KRAS assay has excellent accuracy and is able to detect the KRAS mutations of interest at low (0.1%) mutation frequencies. Additional studies are being performed on prospectively collected plasma samples from patients with metastatic CRC of known KRAS mutation status. Large scale correlation between tumor mutation status and cfDNA findings will be performed to validate our assay for clinical implementation. Citation Format: Rajeswari Avula, Benjamin R. Kipp, Jesse S. Voss, Keegan E. Haselkorn, W. Edward Highsmith, Kevin C. Halling, Jeremy C. Jones, Steven R. Alberts, Michael B. Campion, Cassandra J. Nelson, Jin Jen, Eric D. Wieben, Julie M. Cunningham, Minetta C. Liu. Optimized 7-Plex cell-free DNA assay for clinically relevant KRAS mutations in colorectal cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 505.