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The College of American Pathologists offers biannual proficiency testing for molecular analysis of fragile X syndrome. The purpose of this study was to analyze laboratory performance on the fragile X proficiency surveys from 2001 to 2009.Individual laboratory responses were analyzed for accuracy of genotype determination (normal, gray zone, premutation, or full mutation) and size analysis of the FMR1 trinucleotide repeat region. The analytical sensitivity and specificity of testing for fragile X were calculated, and laboratory performance for trinucleotide repeat sizing was evaluated.Overall, laboratories demonstrated analytical sensitivity of 99% and 96% for detection of full mutations associated with fragile X syndrome in males and females, respectively; analytical sensitivity of 98% for detection of premutations; and analytical specificity of 99.9%. Size measurements of the CGG repeat region were acceptable from most laboratories, with an increase in the range of reported sizes observed for larger repeat expansions.Molecular genetic testing for fragile X syndrome demonstrated excellent sensitivity and specificity by laboratories participating in the College of American Pathologists (CAP) surveys. Allele sizing demonstrated good performance overall with improved accuracy over the study period. Participation in proficiency testing can aid laboratories in assessing individual performance and need for calibration of assays.

Purpose: A multicenter, open-label, phase II trial was conducted to evaluate the efficacy, safety, and tolerability of selumetinib in iodine-refractory papillary thyroid cancer (IRPTC). Experimental Design: Patients with advanced IRPTC with or without follicular elements and documented disease progression within the preceding 12 months were eligible to receive selumetinib at a dose of 100 mg twice daily. The primary endpoint was objective response rate using Response Evaluation Criteria in Solid Tumors. Secondary endpoints were safety, overall survival, and progression-free survival (PFS). Tumor genotype including mutations in BRAF, NRAS, and HRAS was assessed. Results: Best responses in 32 evaluable patients out of 39 enrolled were 1 partial response (3%), 21 stable disease (54%), and 11 progressive disease (28%). Disease stability maintenance occurred for 16 weeks in 49%, 24 weeks in 36%. Median PFS was 32 weeks. BRAF V600E mutants (12 of 26 evaluated, 46%) had a longer median PFS compared with patients with BRAF wild-type (WT) tumors (33 versus 11 weeks, respectively, HR = 0.6, not significant, P = 0.3). The most common adverse events and grades 3 to 4 toxicities included rash, fatigue, diarrhea, and peripheral edema. Two pulmonary deaths occurred in the study and were judged unlikely to be related to the study drug. Conclusions: Selumetinib was well tolerated but the study was negative with regard to the primary outcome. Secondary analyses suggest that future studies of selumetinib and other mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK; MEK) inhibitors in IRPTC should consider BRAF V600E mutation status in the trial design based on differential trends in outcome. Clin Cancer Res; 18(7); 2056–65. ©2012 AACR.

The past 25 years have witnessed the field of molecular pathology evolving from an imprecisely defined discipline to a firmly established medical subspecialty that plays an essential role in patient care. During this time, the training, certification, and licensure requirements for directing and performing testing in a molecular pathology or molecular diagnostics laboratory have become better defined. The purpose of this document is to describe the various board certifications available to individuals seeking certification in molecular diagnostics at the level of laboratory director, supervisor, or technologist. Several national organizations offer certification in molecular pathology or molecular diagnostics for doctoral-level clinical scientists to function as the director of a molecular diagnostics laboratory. Furthermore, 12 states and Puerto Rico require licensing of medical technologists, including those working in molecular diagnostic laboratories. The information provided here updates a 2002 document by the Training and Education Committee of the Association for Molecular Pathology and has been expanded to include certification and licensing requirements for laboratory technologists.

Purpose We examined the feasibility of using CYP2D6 genotyping to determine optimal tamoxifen dose and investigated whether the key active tamoxifen metabolite, endoxifen, could be increased by genotype-guided tamoxifen dosing in patients with intermediate CYP2D6 metabolism. Patients and Methods One hundred nineteen patients on tamoxifen 20 mg daily ≥ 4 months and not on any strong CYP2D6 inhibiting medications were assayed for CYP2D6 genotype and plasma tamoxifen metabolite concentrations. Patients found to be CYP2D6 extensive metabolizers (EM) remained on 20 mg and those found to be intermediate (IM) or poor (PM) metabolizers were increased to 40 mg daily. Eighty-nine evaluable patients had tamoxifen metabolite measurements repeated 4 months later. Results As expected, the median baseline endoxifen concentration was higher in EM (34.3 ng/mL) compared with either IM (18.5 ng/mL; P = .0045) or PM (4.2 ng/mL; P < .001). When the dose was increased from 20 mg to 40 mg in IM and PM patients, the endoxifen concentration rose significantly; in IM there was a median intrapatient change from baseline of +7.6 ng/mL (−0.6 to 23.9; P < .001), and in PM there was a change of +6.1 ng/mL (2.6 to 12.5; P = .020). After the dose increase, there was no longer a significant difference in endoxifen concentrations between EM and IM patients (P = .84); however, the PM endoxifen concentration was still significantly lower. Conclusion This study demonstrates the feasibility of genotype-driven tamoxifen dosing and demonstrates that doubling the tamoxifen dose can increase endoxifen concentrations in IM and PM patients.

Purpose: Advances in genetic sequencing technology have the potential to enhance testing for genes associated with genetically heterogeneous clinical syndromes, such as primary ciliary dyskinesia. The objective of this study was to investigate the performance characteristics of exon-capture technology coupled with massively parallel sequencing for clinical diagnostic evaluation. Methods: We performed a pilot study of four individuals with a variety of previously identified primary ciliary dyskinesia mutations. We designed a custom array (NimbleGen) to capture 2089 exons from 79 genes associated with primary ciliary dyskinesia or ciliary function and sequenced the enriched material using the GS FLX Titanium (Roche 454) platform. Bioinformatics analysis was performed in a blinded fashion in an attempt to detect the previously identified mutations and validate the process. Results: Three of three substitution mutations and one of three small insertion/deletion mutations were readily identified using this methodology. One small insertion mutation was clearly observed after adjusting the bioinformatics handling of previously described SNPs. This process failed to detect two known mutations: one single-nucleotide insertion and a whole-exon deletion. Additional retrospective bioinformatics analysis revealed strong sequence-based evidence for the insertion but failed to detect the whole-exon deletion. Numerous other variants were also detected, which may represent potential genetic modifiers of the primary ciliary dyskinesia phenotype. Conclusions: We conclude that massively parallel sequencing has considerable potential for both research and clinical diagnostics, but further development is required before widespread adoption in a clinical setting.

To examine the incidence of congenital cytomegalovirus (CMV) infection relative to common genetic etiologies of hearing loss in a pediatric population with sensorineural hearing loss (SNHL), and to characterize intracranial radiological abnormalities in patients with CMV-associated hearing loss.Retrospective study.Academic tertiary care center.A total of 112 pediatric patients with confirmed SNHL.The association of congenital CMV infection status with abnormal brain magnetic resonance imaging (MRI) scans and the frequencies of congenital CMV infection, gap junction β-2 (GJB2) mutations, and the mitochondrial DNA (mtDNA) 1555AG mutation in children with SNHL.Of 109 patients, 11 (10%) had positive results for CMV DNA; 10 of the 11 had normal GJB2 sequence and had negative test results for the mtDNA 1555AG mutation. Brain MRI scans for 97 patients demonstrated a higher proportion of abnormalities in patients with positive CMV test results (80%) compared with those with no detectable CMV DNA (33%) (P = .006). GJB2 mutations and the mtDNA 1555AG mutation were seen in 10 of 88 patients (11%) and 1 of 97 patients (1%) with SNHL, respectively.The presence of brain abnormalities in most patients with congenital CMV infection suggests that neurological damage in otherwise asymptomatic patients may not be limited to SNHL. Congenital CMV infection accounted for a significant proportion of patients with SNHL, with an incidence rate comparable with that of GJB2-related SNHL.

Cardiomyopathies are an important and heterogeneous group of common cardiac diseases. An increasing number of cardiomyopathies are now recognized to have familial forms, which result from single-gene mutations that render a Mendelian inheritance pattern, including hypertrophic cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy and left ventricular noncompaction cardiomyopathy. Recently, clinical genetic tests for familial cardiomyopathies have become available for clinicians evaluating and treating patients with these diseases, making it necessary to understand the current progress and challenges in cardiomyopathy genetics and diagnostics. In this review, we summarize the genetic basis of selected cardiomyopathies, describe the clinical utility of genetic testing for cardiomyopathies and outline the current challenges and emerging developments.

Pharmacogenetic testing is becoming more common; however, very few quality control and other reference materials that cover alleles commonly included in such assays are currently available. To address these needs, the Centers for Disease Control and Prevention's Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing community and the Coriell Cell Repositories, have characterized a panel of 107 genomic DNA reference materials for five loci (CYP2D6, CYP2C19, CYP2C9, VKORC1, and UGT1A1) that are commonly included in pharmacogenetic testing panels and proficiency testing surveys. Genomic DNA from publicly available cell lines was sent to volunteer laboratories for genotyping. Each sample was tested in three to six laboratories using a variety of commercially available or laboratory-developed platforms. The results were consistent among laboratories, with differences in allele assignments largely related to the manufacturer's assay design and variable nomenclature, especially for CYP2D6. The alleles included in the assay platforms varied, but most were identified in the set of 107 DNA samples. Nine additional pharmacogenetic loci (CYP4F2, EPHX1, ABCB1, HLAB, KIF6, CYP3A4, CYP3A5, TPMT, and DPD) were also tested. These samples are publicly available from Coriell and will be useful for quality assurance, proficiency testing, test development, and research.

In the past several decades, treatment of hematolymphoid disorders has made dramatic strides. However, the presence or development of resistance to various chemotherapeutic agents is an important problem that affects response to therapy. Drug resistance can be either intrinsic to cancer cells or acquired while on therapy. In this chapter, the major molecular mechanisms of resistance to therapy in hematolymphoid disorders are discussed and techniques that are used to detect resistance to therapy are described. General markers of resistance to therapy, such as cytogenetic markers of resistance or functional assays to measure response to therapy, are not described in this chapter. Rather, this chapter focuses on the detection of specific molecular mechanisms of drug resistance due to the presence or alteration of a specific gene product.

There are multiple techniques to detect defining chromosomal translocations and other abnormalities, including conventional cytogenetic analysis, fluorescence in situ hybridization (FISH), spectral karyotyping (SKY), DNA microarray analysis, polymerase chain reaction (PCR) analysis, and immunohistochemical (IHC) analysis. These various techniques with their advantages and limitations will be discussed in this chapter.

The Institute for Pharmacogenomics and Individualized Therapy (IPIT) at the University of North Carolina at Chapel Hill (NC, USA) is a collaborative, multidisciplinary unit that brings together faculty from different disciplines and crosses the traditional departmental/school structure to perform pharmacogenomics research. IPIT investigators work together towards the goal of developing therapies to enable the delivery of individualized medical care. The NIH-supported Comprehensive Research on Expressed Alleles in Therapeutic Evaluation (CREATE) group leads the field in the evaluation of pathways regulating drug activity, and also provides a foundation for future IPIT research. IPIT members perform bench research, clinical cohort analysis and prospective clinical intervention studies, research on the integration of pharmacogenomic therapy into practice and research to foster global health pharmacogenomics application through the Pharmacogenetics for Every Nation Initiative. IPIT Investigators are actively incorporating a pharmacogenomics curriculum into existing teaching programs at all levels.

Well-characterized reference materials (RMs) are integral in maintaining clinical laboratory quality assurance for genetic testing. These RMs can be used for quality control, monitoring of test performance, test validation, and proficiency testing of DNA-based genetic tests. To address the need for such materials, the Centers for Disease Control and Prevention established the Genetic Testing Reference Material Coordination Program (GeT-RM), which works with the genetics community to improve public availability of characterized RMs for genetic testing. To date, the GeT-RM program has coordinated the characterization of publicly available genomic DNA RMs for a number of disorders, including cystic fibrosis, Huntington disease, fragile X, and several genetic conditions with relatively high prevalence in the Ashkenazi Jewish population. Genotypic information about a number of other cell lines has been collected and is also available. The present study includes the development and commutability/genotype characterization of 10 DNA samples for clinically relevant mutations or sequence variants in the following genes: MTHFR; SERPINA1; RET; BRCA1; and BRCA2. DNA samples were analyzed by 19 clinical genetic laboratories using a variety of assays and technology platforms. Concordance was 100% for all samples, with no differences observed between laboratories using different methods. All DNA samples are available from Coriell Cell Repositories and characterization information can be found on the GeT-RM website.

Responses to warfarin (Coumadin) anticoagulation therapy are affected by genetic variability in both the CYP2C9 and VKORC1 genes. Validation of pharmacogenetic testing for warfarin responses includes demonstration of analytical validity of testing platforms and of the clinical validity of testing. We compared four platforms for determining the relevant single nucleotide polymorphisms (SNPs) in both CYP2C9 and VKORC1 that are associated with warfarin sensitivity (Third Wave Invader Plus, ParagonDx/Cepheid Smart Cycler, Idaho Technology LightCycler, and AutoGenomics Infiniti). Each method was examined for accuracy, cost, and turnaround time. All genotyping methods demonstrated greater than 95% accuracy for identifying the relevant SNPs (CYP2C9 *2 and *3; VKORC1 −1639 or 1173). The ParagonDx and Idaho Technology assays had the shortest turnaround and hands-on times. The Third Wave assay was readily scalable to higher test volumes but had the longest hands-on time. The AutoGenomics assay interrogated the largest number of SNPs but had the longest turnaround time. Four published warfarin-dosing algorithms (Washington University, UCSF, Louisville, and Newcastle) were compared for accuracy for predicting warfarin dose in a retrospective analysis of a local patient population on long-term, stable warfarin therapy. The predicted doses from both the Washington University and UCSF algorithms demonstrated the best correlation with actual warfarin doses.

Introduction: Analysis of tumors for somatic mutations of individual cancer-associated genes has proven valuable in defined clinical scenarios, but the incorporation of multi-gene panels into routine clinical use has proven complex. Here, we describe UNCseq™, a single-institution experience evaluating how care providers use testing of a 247 gene panel in a study of >1400 patients with cancer. Approach: Somatic and germline DNA was captured and sequenced in the context of an IRB-approved clinical trial, with somatic events (point mutations (PM) and copy number alterations (CNA)) determined using an institution-designed bioinformatic pipeline. Mutations deemed ‘actionable’ by a molecular tumor board (MTB) were confirmed in a CLIA-compliant manner, and reported to the patient's caregiver. The clinical use of sequencing information by caregivers was determined through follow-up questionnaire. Results: Somatic events were noted in 444 of 718 (62%) patients as of 11/30/2014 (79% PM/21% CNA). Although 247 genes were analyzed, reports were only made regarding a minority (77) of genes. PMs of PIK3CA/PTEN/KRAS/BRAF/PIK3R1 and CNAs of CCDN1/EGFR/ERBB2/FGFR1 were the most commonly reported events. Non-canonical (71%) events were observed more frequently than canonical events (29%, p Conclusions: An analysis of 247 genes for somatic mutations in patients with advanced cancer is cost-effective and feasible, and can lead to significant changes in clinical care in a minority of patients. Non-canonical events are common, and determination of events for reporting requires pathological review by an MTB. Patients with advanced disease and certain tumor types benefit most from cancer panel sequencing. A majority of patients harbor actionable events, although changes in therapeutic care are less frequent largely because of practical considerations related to care delivery. These data suggest a need to re-structure clinical trials in the era of modern genomic testing. Citation Format: David Neil Hayes, Juneko E. Grilley-Olson, David A. Eberhard, Nirali M. Patel, Joel S. Parker, Karen E. Weck, William Y. Kim, Michele C. Hayward, H. Shelton Earp, Norman E. Sharpless. The impact of gene panel sequencing on clinical care in patients with cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT133. doi:10.1158/1538-7445.AM2015-CT133

Hepatitis C virus is an RNA virus that is associated with chronic infection in the majority of people infected. Chronic infection with hepatitis C virus is the cause of significant morbidity and mortality worldwide and is associated with a large spectrum of liver disease including cirrhosis and hepatocellular carcinoma. End-stage liver disease due to chronic hepatitis C virus infection is currently the leading indication for liver transplantation in the USA. Hepatitis C virus genotyping of viral isolates circulating in the blood during chronic infection has become an important part of hepatitis C virus monitoring in chronically infected patients, and is useful as a prognostic indicator and to direct duration of therapy. This review will summarize information on hepatitis C genotyping, describe the limitations of current commercially available methods, give information on more recently developed methods, and provide a look to the future in terms of where advances in hepatitis C virus genotyping assays need to be made.

BK virus-allograft nephropathy (BKVAN) is an increasingly recognized complication after kidney transplantation. Quantitative tests have been advocated to monitor patients, but data demonstrating their efficacy are relatively limited. We developed a real-time PCR assay to quantitate BK virus loads in the setting of renal transplantation, and we correlated the BK virus load with clinical course and with the presence of BK virus in renal biopsy specimens. BK virus loads were measured in urine, plasma, and kidney biopsy samples in three clinical settings: (i) patients with asymptomatic BK viruria, (ii) patients with active BKVAN, and (iii) patients with resolved BKVAN. Active BKVAN was associated with BK viremia greater than 5 × 103 copies/ml and with BK viruria greater than 107 copies/ml in all cases. Resolution of nephropathy led to resolution of viremia, decreased viruria levels, and disappearance of viral inclusions, but low-level viral DNA persisted in biopsy specimens even for patients whose viruria was cleared. All but one patient in the resolved BKVAN group carried a urinary viral load below 107 copies/ml. Viral loads in patients with asymptomatic viruria were generally lower but in some cases overlapped with levels more typical of BKVAN. One patient with asymptomatic viruria and with a viral load overlapping values seen in BKVAN had developed nephropathy by the time of follow-up. In conclusion, serial measurement of viral loads by quantitative PCR is a useful tool in monitoring the course of BK virus infection. The results should be interpreted in conjunction with the clinical picture and biopsy findings.

Training in molecular pathology in the United States is undergoing development toward a more structured format involving accreditation of training programs and the availability of recognized professional credentials. The traditional apprenticeship for molecular pathology, composed of experience in a molecular biology laboratory with a strong research focus is being replaced by formal training in residency, fellowship, and other postdoctoral training programs that have a clinical focus. This is a reflection of increasing importance of this field to clinical practice, and to a growing desire of persons interested in molecular pathology to undertake formal training programs. These developments are bringing molecular pathology in line with other clinical laboratory specialties for which structured training and certification have long been the norm. An important measure of educational achievement is success in professional examinations leading to recognized credentials. These credentials should attest to the holder’s professional competence as a practitioner in the field and are frequently used for this purpose by licensing and other regulatory agencies. In the past decade, and especially in the last 5 years, a number of routes for certification in molecular pathology by examination have been offered by nationally recognized credentialing agencies. This paper provides an update on these certification routes reflective of the growing interest in molecular pathology education. The principal Boards that offer certification in the United States are the American Board of Pathology, the American Board of Medical Genetics, the American Board for Clinical Chemistry, the National Credentialing Agency for Laboratory Personnel, and the American Board of Bioanalysis. The American Board of Medical Genetics offered a combined examination in Clinical Molecular Genetics and Clinical Biochemical Genetics in 1990 and has offered an examination in Clinical Molecular Genetics alone since 1993. Because of the overlap between molecular genetics and molecular pathology, we have included this examination in this listing. The information provided here is intended to summarize the requirements for these exams. Complete requirements are available from the boards listed here.

Extracellular superoxide dismutase (EC-SOD) is highly expressed in lung tissue. EC-SOD contains a heparin-binding domain that is sensitive to proteolysis. This heparin-binding domain is important in allowing EC-SOD to exist in relatively high concentrations in specific regions of the extracellular matrix and on cell surfaces. EC-SOD has been shown to protect the lung against hyperoxia in transgenic and knockout studies. This study tests the hypothesis that proteolytic clearance of EC-SOD from the lung during hyperoxia contributes to the oxidant-antioxidant imbalance that is associated with this injury. Exposure to 100% oxygen for 72 h resulted in a significant decrease in EC-SOD levels in the lungs and bronchoalveolar lavage fluid of mice. This correlated with a significant depletion of EC-SOD from the alveolar parenchyma as determined by immunofluorescence and immunohistochemistry. EC-SOD mRNA was unaffected by hyperoxia; however, there was an increase in the ratio of proteolyzed to uncut EC-SOD after hyperoxia, which suggests that hyperoxia depletes EC-SOD from the alveolar parenchyma by cutting the heparin-binding domain. This may enhance hyperoxic pulmonary injury by altering the oxidant-antioxidant balance in alveolar spaces.

Although the 5′ untranslated region (5′ UTR) is the most conserved region of the hepatitis C virus (HCV) genome, it has been suggested that interrogation of this region is sufficient for determination of the HCV genotype. We compared two methods of determination of the HCV genotype: (i) direct sequencing of the DNA of the NS-5b region and (ii) reverse line probe assay (LiPA; INNO-LiPA HCV II; Innogenetics N.V.) of the 5′ UTR. There was 100% concordance between the two methods for genotype but only 80% concordance for subtype. A significant percentage of genotype 1a isolates were misclassified by LiPA as genotype 1b. Sequence analysis revealed that the only consistent difference in the 5′ UTR for these genotype 1a isolates misclassified as genotype 1b was a single nucleotide (A/G) at position −99 of the HCV genome. All isolates with discordant results analyzed had a G at this position, consistent with LiPA determination of these samples as subtype 1b. However, sequence analysis of 222 nucleotides in the NS-5b region clearly identified all of these isolates as subtype 1a. Population distribution data from the University of Pittsburgh Medical Center of over 200 samples analyzed by sequencing of the NS-5b region and over 1,000 samples analyzed by LiPA also indicated that INNO-LiPA HCV II cannot accurately differentiate HCV genotype 1a isolates from HCV genotype 1b isolates. We provide evidence that the A/G at position −99 represents a sequence polymorphism in the HCV genome that cannot differentiate subtype 1a from subtype 1b isolates. In conclusion, the 5′ UTR is not heterogeneous enough for use in determination of the HCV subtype and cannot be used for differentiation of HCV genotypes 1a and 1b.

We present the case of a 15-year-old female with acute promyelocytic leukemia and a new variant chromosome rearrangement identified as ins(15;17)(q22;q12q21) by conventional cytogenetic analysis. This finding was confirmed by fluorescence in situ hybridization using the PML-RARA DNA probe and whole chromosome paints 15 and 17. A typical PML-RARA fusion transcript consistent with a breakpoint in intron 3 of the PML gene and intron 2 of the RARA gene was identified by reverse transcription polymerase chain reaction.

A role of tumor necrosis factor-alpha (TNF-alpha) In the immunopathogenesis of hepatitis C virus (HCV) infection has been proposed. The novel herpes virus, human herpes virus-6 (HHV-6), is amongst the most potent inducers of cytokines, including TNF-alpha. The impact of HHV-6 viremia on the progression of recurrent HCV hepatitis was assessed in 51 HCV-positive liver transplant recipients. The frequency of recurrent HCV hepatitis did not differ between patients with HCV viremia (47.6%, 10/21) as compared with those without HCV viremia (46.7%, 14/30, p = 0.9). However, the patients with HHV-6 viremia had a significantly higher fibrosis score upon HCV recurrence than those without HHV-6 viremia (mean 1.5 vs. 0.3, p = 0.01). An association between cytomegalovirus (CMV) viremia and HCV recurrence was not documented; 50% (15/30) of the patients with CMV viremia and 42.8% (9/21) of those without CMV viremia had recurrent HCV hepatitis (p0.5). Receipt of ganciclovir (administered upon the detection of CMV viremia) was associated with lower total Knodell score (mean 5.2 vs. 6.9, p = 0.05) and a trend towards lower fibrosis score (mean 0.44 vs. 1.00, p = 0.12) in patients with recurrent HCV hepatitis. Thus, HHV-6 viremia in HCV-positive liver transplant recipients identified a subgroup of patients at increased risk for early fibrosis upon HCV recurrence.

Background: Clinical cancer samples frequently have significant contamination by stromal cells that increase the difficulty of mutation calling. Histopathologic light microscopy (LM) estimation of purity is commonly used to qualify samples for molecular testing. With large datasets generated by current technologies, more accurate estimations of purity may be facilitated by use of SNP chip (SNP) or sequencing (seq) data. We present a computational framework that considers a variety of technical factors, including percent (%) tumor, seq quality, and mutation type that impact the detection and evaluation of somatic mutations reported to patients. Methods: LCCC1108 (“The development of a tumor molecular analyses program and its uses to support treatment decisions”) is a registry study for cancer patients that performs targeted sequencing with a panel of >200 clinically actionable genes for clinical use. A subset of patients have additional analyses performed including DNA SNP and RNA seq. Any clinically actionable variant is orthogonally confirmed in a CLIA-certified lab. Results: As of 12/1/2013, 816 patients have enrolled. 350 cases have both molecular and LM estimates of tumor purity. Agreement between molecular estimates of % tumor and LM review was low (Pearson's correlation coefficient 0.16 for the most favorable comparison). No clear etiology could explain the low agreement, although examination of extreme cases in which LM approached 100% tumor and molecular estimates approached zero generally included tumors with low rates of molecular alterations (e.g. adenoid cystic carcinoma, chronic myelogenous leukemia). Additionally, independent assessment of % tumor by molecular analysis such as SNP vs. seq also had low agreement (0.13), although a clear pattern could be seen in the disagreement for certain cases. Methods that rely heavily on copy number(CN) alterations often under-predicted estimates of % tumor relative to methods that relied on the mutant allele fraction (MAF) in cases where there were few CN alterations but where point mutations predominated. Excluding these outliers, the agreement was high and molecular estimates of % tumor more reproducible. Most molecular tumor estimates performed more poorly or failed as the % stromal infiltration rose above 50%. Seq estimates of % tumor tended to be noisier for coverage Conclusion: In clinical practice, accurate estimates of % tumor spanning the clinical spectrum are critical for somatic mutation calling. Pure analytic approaches to estimates of % tumor may fail in tumors with low rates of somatic mutation and CN alteration. Ultimately, a combination of methods will be required to describe samples for optimal variant detection. Citation Format: Heejoon Jo, Nirali M. Patel, Karen E. Weck, David A. Eberhard, D. Neil Hayes, Joel S. Parker, Michele Hayward, Ashley Salazar, Juneko E. Grilley-Olson. Correlating molecular and histopathologic tumor purity: An analysis of 816 patients. [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 5598. doi:10.1158/1538-7445.AM2014-5598

561 Background: Tamoxifen treated breast cancer patients with diminished CYP2D6 activity have lower endoxifen exposure, which may lead to inferior treatment outcomes. Increasing the daily tamoxifen...

Fundamental issues remain unresolved regarding the possible contribution of viruses to vascular pathology, as well as the role of the immune system in regulating these processes. Here we demonstrate that infection of mice with gamma-herpesvirus 68 (gammaHV68) provides a novel model for addressing these issues. Interferon-gamma receptor-deficient (IFNgammaR-/-) mice died weeks to months after gammaHV68 infection from a severe large-vessel panarteritis. GammaHV68-infected B cell-deficient and normal weanling mice exhibited milder large-vessel arteritis. Immunohistochemical analyses demonstrated gammaHV68 antigen in arteritic lesions and revealed a striking tropism of gammaHV68 for smooth muscle cells. These studies demonstrate that IFN-gamma is essential for control of chronic vascular pathology induced by gammaHV68 and suggest gamma-herpesviruses as candidate etiologic agents for human vasculitis.

Murine gammaherpesvirus 68 (gammaHV68) infects mice, thus providing a tractable small-animal model for analysis of the acute and chronic pathogenesis of gammaherpesviruses. To facilitate molecular analysis of gammaHV68 pathogenesis, we have sequenced the gammaHV68 genome. The genome contains 118,237 bp of unique sequence flanked by multiple copies of a 1,213-bp terminal repeat. The GC content of the unique portion of the genome is 46%, while the GC content of the terminal repeat is 78%. The unique portion of the genome is estimated to encode at least 80 genes and is largely colinear with the genomes of Kaposi's sarcoma herpesvirus (KSHV; also known as human herpesvirus 8), herpesvirus saimiri (HVS), and Epstein-Barr virus (EBV). We detected 63 open reading frames (ORFs) homologous to HVS and KSHV ORFs and used the HVS/KSHV numbering system to designate these ORFs. gammaHV68 shares with HVS and KSHV ORFs homologous to a complement regulatory protein (ORF 4), a D-type cyclin (ORF 72), and a G-protein-coupled receptor with close homology to the interleukin-8 receptor (ORF 74). One ORF (K3) was identified in gammaHV68 as homologous to both ORFs K3 and K5 of KSHV and contains a domain found in a bovine herpesvirus 4 major immediate-early protein. We also detected 16 methionine-initiated ORFs predicted to encode proteins at least 100 amino acids in length that are unique to gammaHV68 (ORFs M1 to 14). ORF M1 has striking homology to poxvirus serpins, while ORF M11 encodes a potential homolog of Bcl-2-like molecules encoded by other gammaherpesviruses (gene 16 of HVS and KSHV and the BHRF1 gene of EBV). In addition, clustered at the left end of the unique region are eight sequences with significant homology to bacterial tRNAs. The unique region of the genome contains two internal repeats: a 40-bp repeat located between bp 26778 and 28191 in the genome and a 100-bp repeat located between bp 98981 and 101170. Analysis of the gammaHV68, HVS, EBV, and KSHV genomes demonstrated that each of these viruses have large colinear gene blocks interspersed by regions containing virus-specific ORFs. Interestingly, genes associated with EBV cell tropism, latency, and transformation are all contained within these regions encoding virus-specific genes. This finding suggests that pathogenesis-associated genes of gammaherpesviruses, including gammaHV68, may be contained in similarly positioned genome regions. The availability of the gammaHV68 genomic sequence will facilitate analysis of critical issues in gammaherpesvirus biology via integration of molecular and pathogenetic studies in a small-animal model.

e14522 Background: KRAS mutation status is an important clinical variable for targeting treatment in metastatic colon cancer. Many community practices send out mutational analysis to laboratories which utilize allele specific primer extension (ASPE) to identify mutations in only codons 12 or 13 of the KRAS gene. Our institutional molecular pathology laboratory performs pyrosequencing (PS) of KRAS codons 12, 13, and 61 which can potentially identify more KRAS mutations. We undertook this study to determine whether there were analytical differences in the results for specific mutations between these techniques. Methods: We obtained 77 sets of paraffin slides from a local practice from patients with metastatic adenocarcinoma of the colon whose KRAS mutation status had previously been analyzed by ASPE. After macrodissection for tumor enrichment, DNA was extracted from unstained paraffin slides and analyzed for KRAS codon 12, 13, and 61 mutations using the PyroMark KRAS v2.0 test. The results of KRAS testing by PS were then compared to those by ASPE. Exact 95% confidence intervals are reported. Results: None of the patients with KRAS codon 12 or 13 mutations called by ASPE (0/77, 0% [0%-4.7%]) changed to wild-type (WT) upon retesting by PS methodology. However, six patients had a discordant result between their ASPE-based testing and PS-based testing (6/77, 7.8% [2.9%-16.2%]). Two patients had differences in the amino acid (AA) at codon 12 (2/77, 2.6% [0.3%-9.1%]). Three patients were found to have a codon 61 mutation (3/77, 3.9% [0.8%-11.0%]), including two patients who were called KRAS WT by ASPE (2/30, 7% [0.8%-22%]). Finally, one patient with a call of a codon 13 mutation by ASPE changed to WT by PS (1/77, 1.3% [0.0% - 7.0%]). Conclusions: Almost 8% of patients who had KRAS testing by PS had a discrepancy in results compared with commercial ASPE testing alone. Although ASPE appears to be very accurate in detecting KRAS mutations, it missed KRAS codon 61 mutations in about 4% of the samples tested. This may have some clinical impact as we learn more about this subgroup of patients in the future.

e11095 Background: CYP2D6 genotyping has been investigated to avoid suboptimal response to tamoxifen, and although current data do not support routine genotyping in postmenopausal women, there is a...

Background: Pharmacogenomics is an emerging area for breast cancer research. Little is known about how well patients understand pharmacogenomics or the rationale for research in this area. The objective of this study was to analyze patient understanding of a clinical trial involving CYP2D6 genotyping to guide tamoxifen (T) therapy for breast cancer.Methods: We conducted a survey of understanding of pharmacogenomics and the purposes of a clinical trial among patients (pts) eligible for LCCC0801, a prospective Phase 2 study of CYP2D6 genotype-guided therapy for pts on tamoxifen for breast cancer. In this trial, we evaluated baseline endoxifen (E) levels and the impact of increased T dose to 40 mg/day among pts with any dysfunctional CYP2D6 alleles. The primary endpoint of change in E levels is not yet reported. All trial participants and those who declined participation were eligible for this survey. The research nurse administered 11 written questions at time of consent related to the purpose of this study and the nature of pharmacogenomic research. Pts had unlimited time to complete the survey written in a 5 point scale (strongly agree, agree, not sure, disagree, strongly disagree). For pts declining to enroll in the parent study, we offered an identical companion survey to which they could separately give consent.Results: Of 118 pts in the parent study, 117 completed the survey. Following informed consent, all respondents expressed confidence that they understood the purpose of the trial, 75% strongly agreed that they understood the purpose of the study. 98% of participants understood that this was a study of how different people respond to T, but 42% also incorrectly felt that this was a study of how different types of breast cancer respond to T, and 30% incorrectly felt that this study evaluated genetic risk for developing breast cancer. Though the consent form clearly stated that there may be no direct benefit to participants and that the purpose of the study was to help future pts, 68% reported that they would benefit directly, and only 22% felt the study was designed only to help future pts. When asked if the study involved genetics, 14% of pts disagreed, or were unsure. 45% of participants were uncomfortable or unsure with “having your doctor determine your T dose from the results of a genetic test.” Among a small sample of pts who declined trial participation but consented to the survey (13/30 decliners, 43%), compared to trial participants, fewer reported strong confidence in understanding the purpose of the trial (38% vs. 75%, p=0.0034), and a greater percentage identified an inaccurate purpose of the trial (69% vs. 42%, p = 0.043).Conclusions: After informed consent, a high percentage of participants in a pharmacogenomic clinical trial are able to correctly identify the primary purpose of the research, but a substantial minority hold false views about what the trial is designed to investigate. The majority of participants believe that they will directly benefit from trial participation, and few may understand that the primary purpose of the study is to improve care for future patients. Opportunities exist for improved understanding and communication of pharmacogenomic research and further evaluation of this area is needed. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 6082.

Background: Tamoxifen is a selective estrogen receptor modulator that is the most commonly used and cost effective agent for hormone sensitive breast cancer with documented efficacy in prevention, treatment of preneoplasia, and treatment in both the adjuvant and metastatic settings. However, tamoxifen is a prodrug, and up to half of those taking it may not receive the full benefit because of genetic differences in CYP2D6 that affect metabolism to its active form, endoxifen. Tamoxifen is FDA approved for use at both 20mg/day and 40 mg/day, however by convention is dosed at 20mg. We aimed to determine if endoxifen levels can be manipulated by genotype-guided dosing of tamoxifen.Methods: 118 patients on tamoxifen ≥ 4 months and not on potent CYP2D6 inhibiting medications enrolled in Lineberger Comprehensive Cancer Center (LCCC) trial 0801. Genotyping was performed using the CYP450 Amplichip® (Roche Diagnostics) for 2D6 alleles: *1-11, *15, *17, *19, *20, *29, *35, *36, *40, *41, *1XN, *2XN, *4XN, *10XN, *17XN, *35XN and *41 XN. Tamoxifen dose was increased from 20mg to 40mg in patients with any intermediate or poor metabolizing (IM or PM) alleles [but not in patients homozygous for extensive metabolizing (EM) alleles]. Endoxifen levels were drawn at baseline and 4 months later. Assuming that endoxifen levels in IM pts are 40% lower than EM at baseline (Jin et al., 2005) and with a one-sided significance level of 0.025 and a sample size of 40 patients with intermediate metabolizing CYP2D6 genotypes, this study would have 84% power to detect a 40% increase in endoxifen.Results: Of the 118 patients, 25 withdrew or were removed from study, leaving 93 who have completed this study and who are evaluable for the primary analysis. Genotyping results were presented at 2009 ASCO meeting (Irvin et al.). For this analysis, 19 (20%) are African-American, 69 (74%) are non-Hispanic white, 2 are Hispanic, and 3 are Asian. For the 93 evauable patients, genotyping revealed 31 (33%) EM/EM, 1 EM/UM (ultra-rapid), 20 (22%) EM/IM, 19 (20%) EM/PM, 4 (4%) IM/IM, 9 (10%) IM/PM, 9 (10%) PM/PM and 1 unknown. The primary outcome, the tamoxifen metabolite levels, including endoxifen, will be available in July.(Supported by NC University Cancer Research Fund, NCI SPORE, Laboratory Corporation of America, Roche Diagnostics) Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 410.

Viral regulatory region sequence variations in kidney tissue obtained from patients with BK virus nephropathy. Background The polyomavirus genome contains a noncoding control region (NCCR), which is believed to contain the enhancer elements that are important activators of viral transcription. We hypothesized that DNA sequence variations in this region play a role in the pathogenesis of BK virus allograft nephropathy (BKVAN). Methods Tissue sections were prepared from 26 paraffin-embedded biopsies with BKVAN obtained from 15 patients. The sections were lysed in a proteinase K buffer and subjected to DNA sequencing using Big Dye cycle sequencing reactions run on an automated DNA sequencer (ABI 310). NCCR anatomy was compared to the genomic sequence of archetype BKV strain WW . Results Twelve putative transcription factor binding sites showed nucleotide substitutions, deletions, or duplications in at least one of the biopsies studied. Changes were most commonly found in binding sites for granulocyte/macrophage stimulating factor promoter (24/26 biopsies) and nuclear factor-I (NF-I) transcription factor (21/22 biopsies). Less frequent changes were seen at other binding sites, including T antigen (3/22 biopsies) and the cytomegalovirus immediate early (CMV IE-1) promoter (3/22 biopsies). Five biopsies showed complex rearrangements reminiscent of those described in JC virus–associated progressive multifocal leukoencephalopathy. Conclusion The regulatory region of BKV shows sequence heterogeneity in biopsy tissue with viral nephropathy. Sequence variations frequently affect putative transcription factor binding sites, with potential implications for promoter activity related to genes important in viral replication. Architectural rearrangements were found in some cases but did not appear to be a prerequisite for the pathogenesis of BKVAN.

Abstract 2102 Poster Board II-79 BACKGROUND: Two enzymatic complexes are related to therapeutic response to coumarin: 1)cytocrome P-450 2C9 (CYP2C9), that metabolizes the drug; and 2)vitamin K epoxide reductase complex subunit 1 (VKORC1), targeted by coumarin. Haplotypes CYP2C9 *2 and *3 and VKORC1*2, more frequent in Caucasians, are related to coumarin sensitivity. VKORC1*3 and *4 confer resistance to the anticoagulant and are found mainly in Africandescents. MATERIAL AND METHODS: To determine genetic variability associated with altered warfarin response among Brazilian patients, sixty-two adult patients showing resistance or sensitivity to warfarin were genotyped for variants in CYP2C9 and VKORC1 associated with alteredwarfarin response. Fifty-one were users of low daily doses (¶ 2.5mg) and 11 users of high daily doses (>10mg) of warfarin.RESULTS: Among the 51 patients on low doses of warfarin, VKORC1 1639 (3673) G>A polymorphism, which defines VKORC1*2, was present in 48 (94.1%). Of these, thirty-two patients (66.7%) were Caucasians, 9 (18.7%) were of African descent, 4 (8.4%) were of both African and Brazilian-Indian descent and 3 (6.2%) were of Brazilian-Indian descent. Additionally, 27(52.9%) patients had at least one CYP2C9*2 or CYP2C9*3 allele, 21(63.6%) of Caucasians and 6 (54%) of African-descents. For the 11 patients on high doses of warfarin, 6 (54.5%) patients had at least one variant allele of VKORC1. Four patients were found with a non-coding VKORC1 polymorphism: VKOR3482(8773)C>T or VKOR 1173(6484)C>T. The VKOR 3482(8773)C>T, which defines the haplotype VKORC1*3F, was present in 2 African-descentpatients and VKOR 1173(6484)C>T, associated with VKORC1*2, was present in 3 patients. Two African-descent patients had a coding mutation Val66Met(V66M). CONCLUSION: The presence of the rare V66M mutation in two high dose outlier patients implies that this variant may be more frequent among African Brazilians and has implications for future warfarin studies in other individuals of African descent. Disclosures: No relevant conflicts of interest to declare.

Background KRAS mutations may predict poor response to radiotherapy. Downstream events from KRAS, such as activation of BRAF, AKT and ERK, may also confer prognostic information but have not been tested in rectal cancer (RC). Our objective was to explore the relationships of KRAS and BRAF mutation status with p-AKT and p-ERK and outcomes in RC. Methods Pre-radiotherapy RC tumor biopsies were evaluated. KRAS and BRAF mutations were assessed by pyrosequencing; p-AKT and p-ERK expression by immunohistochemistry. Results Of 70 patients, mean age was 58; 36% stage II, 56% stage III, and 9% stage IV. Responses to neoadjuvant chemoradiotherapy: 64% limited, 19% major, and 17% pathologic complete response. 64% were KRAS WT, 95% were BRAF WT. High p-ERK levels were associated with improved OS but not for p-AKT. High levels of p-AKT and p-ERK expression were associated with better responses. KRAS WT correlated with lower p-AKT expression but not p-ERK expression. No differences in OS, residual disease, or tumor downstaging were detected by KRAS status. Conclusions KRAS mutation was not associated with lesser response to chemoradiotherapy or worse OS. High p-ERK expression was associated with better OS and response. Higher p-AKT expression was correlated with better response but not OS.

Background Metastatic melanoma involving the esophagus is rare; the occurrence of metastatic melanoma in a background of Barrett esophagus is rarer still. We report a case of an 80 year-old male who presented to our institution for workup of Barrett esophagus with high-grade dysplasia and who proved to have metastatic melanoma occurring in the background of Barrett esophagus, the first report of this kind, to our knowledge, in the English literature. Case presentation An 80 year-old Caucasian male was diagnosed at an outside institution with Barrett’s esophagus with high grade dysplasia and presented to our institution for therapy. The patient underwent endoscopic mucosal resection using a band ligation technique of an area of nodularity within the Barrett esophagus. Microscopic examination demonstrated extensive Barrett esophagus with high-grade dysplasia as well as a second tumor which was morphologically different from the surrounding high-grade dysplasia and which was positive for S-100, HMB 45 and Melan-A on immunohistochemistry, consistent with melanoma. Further workup of the patient demonstrated multiple radiologic lesions consistent with metastases. Molecular studies demonstrated that the melanoma was positive for the 1799T>A (V600E) mutation in the BRAF gene. The overall features of the tumor were most consistent with metastatic melanoma occurring in a background of Barrett esophagus with high-grade dysplasia. Conclusion This case demonstrates a unique intersection between a premalignant condition (Barrett esophagus with high grade dysplasia) and a separate malignancy (melanoma). This report also shows the utility of molecular testing to support the hypothesis of primary versus metastatic disease in melanoma.