Laboratory-developed Assays Research Articles

Brief comparative evaluation of six open one-step RT-qPCR mastermixes for the detection of SARS-CoV-2 RNA using a Taqman probe

Facing the emergence of a new RNA virus, clinical laboratories are often helpless in the case of a shortage of reagents recommended by Reference Centres. To compare five open one step RT-qPCR reagents to the SuperScript™ III Platinum™ One-Step qRT-PCR kit (Invitrogen) considered as the reference one in France at the beginning of the pandemic for detection of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in respiratory specimens by using a laboratory-developed assay targeting the viral RNA dependant RNA polymerase (RdRp) gene. A total of 51 NUCLISENS easyMAG extracts from respiratory specimens was tested on ABI 7500 thermocycler with TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems), Luna® Universal Probe One-Step RT-qPCR Kit (New England Biolabs), GoTaq® Probe 1- Step RT-qPCR System (Promega), LightCycler® Multiplex RNA Virus Master (Roche) and One-step PrimeScript RT-PCR kit (Takara). The CT values obtained using the 5 challenged reagents were compared to those obtained using the reference assay. The percentages of concordance were all above 95 %. When comparing the CT values of the 48 extracts exhibiting CT values < 35 obtained with the reference reagent, the results were similar between the reagents although the differences of CT values were quite dispersed. All five reagents can be considered as alternative reagents to the reference for detecting SARS-CoV-2 RNA.

Empiricism in Microsampling: Utilizing a Novel Lateral Flow Device and Intrinsic Normalization to Provide Accurate and Precise Clinical Analysis from a Finger Stick.

Phlebotomy plays a key role in clinical laboratory medicine but poses certain challenges for the patient and the laboratory. Dried blood spots simplify collection and stabilize specimens effectively, but clinical reference intervals are based primarily on serum or plasma. We evaluated use of dried separated blood plasma specimens to simplify plasma sample collection via finger stick; however, this sampling technique posed substantial analytical challenges. We discuss herein our efforts to overcome these challenges and provide accurate and precise clinical measurements. Microsamples of whole blood were collected via finger stick using a collection device employing laminar-flow separation of cellular blood and plasma fractions with subsequent desiccation. Samples were analyzed on modern autoanalyzers with FDA-approved reagent and calibration systems, as well as commercially available reagents with laboratory-developed assay parameters. Measured analyte concentrations from extracted dried plasma samples were normalized to a coextracted endogenous analyte, chloride. Chloride normalization reduced variability incurred through extraction and undefined plasma volume. Excellent correlation of normalized measurements from dried finger-stick samples (whole blood and plasma) versus matched venous samples facilitated developing mathematical transformations to provide concordance between specimen types. Independent end-to-end performance verification yielded mean biases <3% for the 5 analytes evaluated relative to venous drawn samples analyzed on FDA-approved measurement systems. Challenges inherent with this microsampling technique and alternate sample matrix were obviated through capabilities of modern autoanalyzers and implementation of chloride normalization. These results demonstrate that self-collected microsamples from a finger stick can give results concordant with those of venous samples.

Evaluation of a quantitative RT-PCR assay for the detection of the emerging coronavirus SARS-CoV-2 using a high throughput system.

Facing the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), high-volume respiratory testing is demanded in laboratories worldwide. We evaluated the performance of a molecular assay for the detection of SARS-CoV-2 on a high-throughput platform, the cobas 6800, using the ‘open channel’ for integration of a laboratory-developed assay. We observed good analytical performance in clinical specimens. The fully automated workflow enables high-throughput testing with minimal hands-on time, while offering fast and reliable results.

Tumour mutational burden ring trial: Evaluation of targeted next-generation sequencing platforms for implementation in clinical practice

Abstract Background Tumour mutational burden (TMB) is a measurement of DNA variants in a tumour and is a potential biomarker of response to Immune Checkpoint Inhibitor (ICI) therapy. Patients with non-small cell lung cancer (NSCLC) whose tumours have a high TMB (≥10 mutations/Mb) might benefit from upfront ICI combination therapy. Clinical trials have measured TMB using Whole Exome Sequencing (WES) and/or the FoundationOne CDx (F1CDx) assay. In parallel, several commercial next-generation sequencing (NGS) assays have become available. Before implementation of TMB testing in clinical practice, technical and clinical validation are needed. Hence, a multi-center study was organised to establish concordance between several TMB assays. Methods Fifteen resection NSCLC formalin-fixed paraffin-embedded (FFPE) samples with broad TMB range were selected. Each participant received extracted DNA and was asked to report TMB values using their own protocol. In parallel, FFPE slides were analysed with F1CDx. Eight labs participated using five different methods: Oncomine TML assay (Thermofisher; n = 4), TSO500 assay (Illumina; n = 1), NEOplus assay (NEO New Oncology; n = 1), a 0,4 Mb targeted resequencing lab-developed assay (LDT; n = 1) and WES using three different TMB calculation methods (n = 1). Correlations between each platform and F1CDx were calculated. Also, the reported TMB category (high vs low) of the different platforms in comparison to F1CDx was evaluated for the fifteen samples. Results Assessment of TMB values obtained by the platforms and F1CDx demonstrated a high correlation (R2 between 0.81 and 0.94), except for the smaller LDT (R2 = 0.53). The TMB category (high vs low) reported by each platform showed concordance with the F1CDx category for eleven (73%) to thirteen (93%) of the fifteen samples. From the fifteen samples, the same category was reported by all different platforms for seven (47%) samples. Conclusion Our data show that assays from different providers can be used to predict TMB. However, samples with a TMB value around the cut-off of 10 mut/Mb are challenging and interpretation should occur with caution. Further studies are required before implementing these assays in routine clinical diagnosis. Legal entity responsible for the study The authors. Funding Bristol-Myers Squibb. Disclosure S. Lambin: Research grant / Funding (institution): Bristol-Myers Squibb. All other authors have declared no conflicts of interest.

BK Polyomavirus-specific T cell immune responses in kidney transplant recipients diagnosed with BK Polyomavirus-associated nephropathy

BackgroundAdjustment of immunosuppression is the main therapy for BK polyomavirus (BKPyV)-associated nephropathy (BKPyVAN) after kidney transplantation (KT). Studies of BKPyV-specific T cell immune response are scarce. Here, we investigated BKPyV-specific T cell immunity in KT recipients diagnosed with BKPyVAN.MethodsAll adult KT recipients with BKPyVAN diagnosed at our institution from January 2017 to April 2018 were included. Laboratory-developed intracellular cytokine assays measuring the percentage of IFN-γ-producing CD4+ and CD8+ T cells, after stimulation with large-T antigen (LT) and viral capsid protein 1 (VP1), were performed both at the time of diagnosis and after adjustment of immunosuppression.ResultsWe included 12 KT recipients diagnosed with BKPyVAN (7 proven, 4 presumptive, and 1 possible). Those with presumptive BKPyVAN had a median plasma BKPyV DNA load of 5.9 log10 copies/ml (interquartile range [IQR]: 4.9–6.1). Adjusted dosing of mycophenolic acid and tacrolimus with (86%) or without (14%) adjunctive therapies were implemented after diagnosis. There was a significantly higher median percentage of IFN-γ-producing CD4+ T cells to LT at a median of 3 (IQR: 1–4) months after adjustment of immunosuppression compared with at the time of diagnosis (0.004 vs. 0.015; p = 0.047). However, the difference between the median percentage of IFN-γ-producing CD4+ T cells to VP1 and CD8+ T cells to LT and VP1 did not reach statistical significance. Four (33%) patients achieved plasma BKPyV DNA clearance, and the remaining eight (67%) patients had persistent BKPyV DNAemia. Although eight (67%) patients developed allograft dysfunction, none required hemodialysis.ConclusionsWe observed a marginal trend of BKPyV-specific CD4+ T cell recovery after adjustment of immunosuppression in KT recipients diagnosed with BKPyVAN. A further study would be benefited to confirm and better assess BKPyV-specific immune response after KT.

Automated Extraction of Microbial DNA From Formalin-Fixed, Paraffin-Embedded Tissue Using the Siemens Tissue Preparation System

We describe the adoption of an automated nucleic acid extraction platform, Siemens Tissue Preparation System (TPS), for use with formalin-fixed, paraffin-embedded (FFPE) tissues intended for PCR microbial testing. The TPS uses a liquid handling robot to automate deparaffinization, followed by extraction and purification of nucleic acids using magnetic particle-based isolation. While it has been used previously for human DNA extraction, this is the first incorporation of the TPS into clinical microbiology practice for microorganism detection to our knowledge. Our laboratory transitioned to use of the TPS from manual deparaffinization of FFPE tissue with subsequent DNA extraction using Roche MagNA Pure (MP) instruments (manual/MP method). Unpublished data from our group demonstrated that the TPS performed better than the manual/MP method for FFPE tissue extraction (27% higher positivity in 86 FFPE specimens tested by both methods). We tracked total testing volumes, laboratory efficiency, TPS errors, and workflow. Additionally, we added a microtome instrument to our clinical microbiology laboratory to section FFPE blocks in order to increase efficiency by avoiding reliance on the histopathology laboratory. Since implementation, 710 FFPE specimens were processed on the TPS, with 724 downstream PCR tests run on extracts for eight laboratory-developed assays: Mycobacterium tuberculosis (MTB), Bartonella spp, Tropheryma whipplei, Coxiella burnetii, Kingella kingae, Coccidioides spp, Acanthamoeba spp, and Balamuthia mandrillaris/Naegleria fowleri. The most common tests run after TPS extraction included 468 (64.6%) MTB, 156 (21.5%) Bartonella, and 70 (9.7%) T whipplei; positivity rates were 4.9%, 6.4%, and 4.3%, respectively. Regarding workflow, use of the TPS decreased total processing time by approximately 70%. The TPS workflow from tissue block sectioning to DNA extraction averaged a total of 3.2 hours, of which 0.7 hours (21.8%) were hands-on time (microtome sectioning and TPS specimen loading/unloading). Comparatively, our manual/MP method averaged 10.6 hours, of which 1.1 hours (10.5%) involved hands-on time (microtome sectioning, manual deparaffinization using xylene and 95% ethanol, and MP loading/unloading). Approximately 7 hours of the manual/MP method were devoted to proteinase K lysis. The TPS accommodates 48 specimens per run, with each run lasting 2.5 hours. Our laboratory currently performs one run daily with an average of four specimens per run. Eleven (6.25%) failure events were noted in 176 runs over the past 9 months. Nine (82% of the errors) were attributed to robotic mechanical problems and two (18%) to human errors (reagent misplacement). The system software alerts the user when errors occur so the problem can be fixed and/or the run repeated. In conclusion, the automated TPS extraction system for FFPE is rapid and limits exposure to hazardous chemicals with minimal run failures. Transition of FFPE sectioning to the microbiology laboratory from the histology laboratory also increased efficiency.

Evaluation of clinical application of two molecular assays for detection of toxigenic Clostridium difficile

Objective In comparison with Xpert C. difficile/Epi through detection of Clostridium difficile toxin genes from clinical stool, the performance of a laboratory-developed (LD) assay was evaluated in detail. Methods A total of 176 stool specimens collected from patients with diarrhea in the First People′s Hospital of Yuhang District and the People′s Hospital of Yingzhou, Ningbo from August 1 to December 30 were detected by the two assays in parallel, and meanwhile the C. difficile strains will be isolated and identified for C. difficile toxin genes by a conventional PCR assay. The Cross-tabs Analysis was used for the results by using SPSS20.0 software. Results In comparison with the results of Xpert C. difficile/Epi as the standard, the LD assay had a sensitivity of 91.7% (22/24), a specificity of 100% (152/152), a positive predictive value (PPV) of 100% (22/22), and negative predictive value (NPV) 98.7% (152/154). The results of two assays were statistically coherent (Kappa=0.950, P<0.001). In comparison with culture and detection of toxin genes results, the LD assay had a sensitivity of 90.0% (18/20), a specificity of 97.0% (152/156), a PPV of 81.8% (18/22), and NPV of 98.7% (152/154)(Kappa=0.838, P<0.001), and the Xpert C. difficile/Epi assay had a sensitivity of 90.0% (18/20), a specificity of 96.0% (150/156), a PPV of 75.0% (18/24), and NPV of 98.7% (150/152)(Kappa=0.792, P<0.001). Conclusions The performance of the LD assay was similar to that of the Xpert C. difficile/Epi kit in detection of toxigenic C. difficile. The LD assay could be directly applied to detection of toxigenic C. difficile from clinical stool samples. The clinical application of this LD assay will also provide a domestic and promising diagnostic assay for diagnosis of C. difficile infection in China.(Chin J Lab Med, 2018, 41: 35-40) Key words: Clostridium difficile; Molecular Diagnostics; Clinical evaluation

Evaluation of a real-time PCR assay for the qualitative detection of Clostridium difficile toxin gene and its clinical application

Objective In comparison of the performances for the detection of Clostridium difficile toxin B genes from stool between BD MAX Cdiff assay and a laboratory-developed (LD) assay.The LD assay was evaluated in clinical application. Methods This study was a clinical application research. A total of 147 stool specimens from patients with diarrhea in Hangzhou First Hospital affiliated with Zhejiang Chinese Medical University were detected by the two assays from 1 July to 30 September 2014. DNA extraction and amplification of the tcdB gene were performed automatically on the BD MAX platform. Meanwhile, the tcdA and tcdB gene were detected by the LD real-time PCR assay after DNA extraction. Then, the results were analyzed by use of SPSS 10.0. Results A total of 147 stool samples were collected. There were 33 C. difficile positive cases and 114 negative cases detected by both of two assays. However, there were four stool samples had incongruent results. In comparison with BD MAX, the LD assay had a sensitivity of 93.94% (31/33), a specificity of 98.25% (112/114), a positive predictive value of 93.94% (31/33), and negative predictive value 98.25% (112/114). Furthermore, the results of the LD assay were statistically coherent with that of the BD assay (Kappa=0.922, P<0.01). Conclusions The LD assay was highly sensitive and accurate as BD MAX Cdiff assay in the detection of toxigenic Clostridium difficile. Furthermore, this LD assay could be also applied to detection of clinical stool samples directly with low cost. The assay will be more promising in diagnosis of toxigenic C. difficile in clinical application in China due to no additional instrument needed.(Chin J Lab Med, 2017, 40: 511-514) Key words: Clostridium difficile; Bacterial toxins; Molecular diagnostic techniques

Mycoplasma pneumoniae Macrolide Resistance in Children in Central Ohio Detected by Sequencing

Background Mycoplasma pneumoniae(Mp) is one of the most common causes of LRTI in school aged children, with estimates of up to 20% of cases of community-acquired pneumonia (CAP). Macrolides are the drug of choice for treating Mp infections, but in the last 2 decades there has been emergence of resistance worldwide. Reported rates in USA vary from 3.5 up to 10.8%, but rates in Ohio are unknown. Currently there is no well-established, standardized method for detecting macrolide resistance and antibiotic treatment is empiric because of these limitations. Our goal was to determine the frequency of macrolide resistance in children with Mp respiratory infections in central Ohio.MethodsA convenience sample of Mp positive PCR identified by our laboratory-developed assay, obtained from both inpatient and outpatient settings, were selected. These were then cultured using Remel’s SP4 glucose broth and then incubated at 35°C until isolates grew, or for a maximum of 4 weeks. All samples that yield positive cultures were then sequenced for the domain V of the 23S rRNA (nucleotide 1937–2154, accession no. X68422) using Sanger methods and sequences were compared with corresponding region of wildtype reference strain (ATCC 15322).ResultsFor the period of October 2015-March 2017, culture was attempted in 433 PCR-positive samples, and 347 (80%) yield an isolate. Sequencing was performed in those 347 samples and was successful in 334 (96%). A macrolide resistance mutation was detected in 10 samples (3%). From the 10 mutations detected, 7 (70%) were the A2063G mutation, and 3 (30%) were A2064G. We also detected a deletion in A2065 of unknown significance in 3 samples (0.9%).ConclusionIn this pediatric population, culture was successful in 80% of PCR positive samples, thus providing isolates for sequencing to be able to assess our local resistance. Our overall Mp resistance was 3%, suggesting that empiric treatment with macrolides is still appropriate in Central Ohio. Additional studies are needed to correlate the presence of these resistance mutations with phenotypic susceptibility testing and clinical outcomes.Disclosures O. Ramilo, Abbvie: Board Member, Consulting fee; Regeneron: Board Member, Consulting fee; Janssen: Board Member and Investigator, Consulting fee and Research grant; NIH: Grant Investigator, Research grant; A. Leber, BioFIre Dia gnostics: Research Contractor and Scientific Advisor, Research support, Speaker honorarium and Travel expenses

Analytical and clinical evaluation of the Abbott RealTime hepatitis B sequencing assay

BackgroundLong-term nucleoside analogue (NA) treatment leads to selection for drug-resistant mutations in patients undergoing hepatitis B virus (HBV) therapy. The Abbott RealTime HBV Sequencing assay (Abbott assay; Abbott Molecular Inc., Des Plaines, IL, USA) targets the reverse transcriptase region of the polymerase gene and as such has the ability to detect NA resistance-associated mutations in HBV. ObjectivesWe evaluated the analytical performance of the Abbott assay and compared its diagnostic performance to that of a laboratory-developed nested-PCR and sequencing method. Study designThe analytical sensitivity of the Abbott assay was determined using a serially-diluted WHO International Standard. To validate the clinical performances of the Abbott assay and the laboratory-developed assay, 89 clinical plasma samples with various levels of HBV DNA were tested using both assays. ResultsThe limit of detection of the Abbott assay, was 210IU/ml and it successfully detected mutations when the mutant types were present at levels ≥20%. Among 89 clinical specimens, 43 and 42 were amplification positive in the Abbott and laboratory-developed assays, respectively, with 87.6% overall agreement (78/89; 95% confidence interval [CI], 78.6–93.4). The Abbott assay failed to detect the minor mutant populations in two specimens, and therefore overall concordance was 85.3% (76/89), and the kappa value was 0.79 (95% CI, 0.67–0.90). ConclusionsThe Abbott assay showed comparable diagnostic performance to laboratory-developed nested PCR followed by direct sequencing, and may be useful as a routine method for detecting HBV NA resistance-associated mutations in clinical laboratory settings.

P021 Development of laboratory-developed chimerism analysis assay targeting t lymphocytes in hematopoietic stem cell transplantation

Aim Hematopoietic stem cells transplantation (HSCT) is one of the gold standard treatment for the patients with hematologic malignancies. After the transplantations, the engraftment of the stem cells is monitored using various methods. Short tandem repeats (STR) are one of the sensitive markers for chimerism. We designed laboratory-developed chimerism analysis assay using 8 short tandem repeat regions. Methods Total 38 patients who received allogeneic HSCT were involved in this study. The diagnosis of the patients included acute myeloid leukemia, precursor lymphoblastic leukemia, myelodysplastic syndrome, hemophagocytic lymphohistiocytosis, chronic eosinophilic leukemia, paroxysmal nocturnal hemoglobinuria, juvenile myelomonocytic leukemia, plasma cell myeloma, and congenital neutropenia. DNA was extracted from both ACD anticoagulated blood and T lymphocytes isolated by EasySep (STEMCELL Technologies, Vancouver, Canada). We designed the assays, named LDT 8, targeting D3S1358, D5S818, TH01, D7S820, D12S391, FGA, D18S51, and Penta E. The amplicons were analyzed using Performance Optimized Polymer 7 (Applied Biosystems Norwalk, USA) by 3130 xl genetic analyzer (Applied Biosystems). The results LDT8 were compared with commercially available PowerPlex 21 (Promega, Madison, USA) and Mentype 13 (Biotype, Dresden, Germany). In addition, we also compared the chimerism status of whole blood to that of T lymphocyte. Results Compared with PowerPlex 21 and Mentype 13 respectively, 90.1% and 84.9% of specimens showed less than 5%p in overall discrepancies. In the comparison between whole blood and T lymphocytes, most of specimens showed the higher recipient proportion of DNA in T lymphocytes. Conclusions We developed the economical and effective chimerism analysis assay using 8 short tandem repeat regions. Also, we identified discrepancies among two different cell sources. Further study for long-term monitoring of chimerism status in different cell subsets would be helpful in patients who received hematopoietic stem cell transplantation.

Validation of a panel of ADAMTS13 assays for diagnosis of thrombotic thrombocytopenic purpura: activity, functional inhibitor, and autoantibody test.

Quantitation of ADAMTS13 activity, functional inhibitors, and autoantibodies is crucial in diagnosis and management of thrombotic thrombocytopenic purpura. We compared and optimized commercial assay kits and validated a testing panel. Citrated plasma specimens from healthy volunteers and residual samples submitted for clinical testing were used in the study. Commercially available ADAMTS13 activity assays including ACTIFLUOR(™) ADAMTS13 (Sekisui Diagnostics, Stamford, CT, USA), LIFECODES ATS-13 (Gen-Probe Inc., San Diego, CA, USA), and TECHNOZYM(®) ADAMTS-13 (Technoclone, Vienna, Austria) were evaluated. Functional inhibitor assays were performed using internally developed mixing protocols. Two autoantibody assays were also evaluated: IMUBIND(®) (Sekisui Diagnostics) and TECHNOZYM(®) ADAMTS-13 INH ELISA kits (Technoclone). A laboratory-developed assay using ACTIFLUOR(™) reagents showed best agreement with the reference method, and full validation showed a reportable range of 5% (LLOQ) to 114% with a reference interval of ≥68%. Both intra- and interassay coefficients of variation were <10%. Inhibitor assays performed with the kits showed 95% overall agreement with the reference method. A modification of the TECHNOZYM(®) autoantibody assay showed 85% overall agreement with the reference method with imprecision approximately 20%. ADAMTS13 activity and inhibitor tests using ACTIFLUOR(™) reagents and modified TECHNOZYM(®) autoantibody ELISA showed superior performance compared to the other kits for clinical use in this study.

Pharmacogenetics of Cytochrome P450 Enzymes in American Indian and Caucasian Children Admitted to a Psychiatric Hospital

The cytochrome P450 (CYP450) enzyme system metabolizes many psychiatric medications. We compare frequencies of alleles and phenotypes for CYP2D6, CYP2C9, and CYP2C19 in American Indian (AI) and Caucasian youth treated at a psychiatric hospital in the Northwestern United States. A retrospective chart review evaluated CYP450 pharmacogenetic (PGx) data from the Shodair Children's Hospital Clinical Genetic Laboratory between 2006 and 2014. CYP2D6 genotyping was performed using the xTAG® CYP2D6 Kit (Luminex, Austin, TX). CYP2C9 and CYP2C19 genotyping was performed by laboratory-developed assays using allele-specific quantitative polymerase chain reaction (qPCR) and/or melt-curve analysis. A total of 123 AIs and 688 Caucasians met criteria for inclusion. The overall prevalence of CYP2D6 poor metabolizers was 8.3% (95% Confidence Interval [CI] 6.1%, 10.4%), 9.3% in Caucasians (95% CI 7.1%, 11.5%), and 2.4% in AIs (95% CI 0%, 5.2%). The overall prevalence of CYP2D6 ultrarapid metabolizers was 1.6% (95% CI 0.7%, 2.5%), 1.6% in the Caucasians (95% CI 0.7%, 2.5%), and 1.6% in AIs (95% CI 0%, 3.9%). The overall prevalence of CYP2C9 poor metabolizers was 3% (95% CI 1.7%, 4.2%), 3.2% in Caucasians (95% CI 1.8%, 4.6%), and 1.8% in AIs (95% CI 0%, 4.2%). The overall prevalence of CYP2C19 poor metabolizers was 2.5% (95% CI 1.3%, 3.6%), 2.9% in Caucasians (95% CI 1.6%, 4.2%), and 0% in AIs. The overall prevalence of CYP2C19 ultrarapid metabolizers was 1.5% (95% CI 0.6%, 2.4%), 1.6% in Caucasians (95% CI 0.6%, 2.6%), and 0.9% in AIs (95% CI 0%, 2.6%). This study is the first to identify differences in polymorphism frequencies of the CYP450 system in AIs and Caucasian youth admitted to a psychiatric hospital. Our findings warrant further study of these populations to determine if these differences are generalizable to the larger population of Caucasian and AI/Alaska Native youth in the Northwestern United States.

Requisite Analytic and Diagnostic Performance Characteristics for the Clinical Detection of BRAF V600E in Hairy Cell Leukemia

Detection of high-frequency BRAF V600E mutations in hairy cell leukemia (HCL) has important diagnostic utility. However, the requisite analytic performance for a clinical assay to routinely detect BRAF V600E mutations in HCL has not been clearly defined. In this study, we sought to determine the level of analytic sensitivity needed for formalin-fixed, paraffin-embedded (FFPE) and frozen samples and to compare the performance of 2 allele-specific polymerase chain reaction (PCR) assays. Twenty-nine cases of classic HCL, including 22 FFPE bone marrow aspirates and 7 frozen specimens from blood or bone marrow were evaluated using a laboratory-developed allele-specific PCR assay and a commercially available allele-specific quantitative PCR assay-myT BRAF Ultra. Also included were 6 HCL variant and 40 non-HCL B-cell lymphomas. Two cases of classic HCL, 1 showing CD5 expression, were truly BRAF V600E-negative based on negative results by PCR and sequencing despite high-level leukemic involvement. Among the remaining 27 specimens, V600E mutations were detected in 88.9% (17/20 FFPE; 7/7 frozen) and 81.5% (15/20 FFPE; 7/7 frozen), for the laboratory-developed and commercial assays, respectively. No mutations were detected among the 46 non-HCL lymphomas. Both assays showed an analytic sensitivity of 0.3% involvement in frozen specimens and 5% in FFPE tissue. On the basis of these results, an assay with high analytic sensitivity is required for the clinical detection of V600E mutations in HCL specimens. Two allele-specific PCR assays performed well in both frozen and FFPE bone marrow aspirates, although detection in FFPE tissue required 5% or more involvement.

Detection of enterovirus D68 in Canadian laboratories.

The recent emergence of a severe respiratory disease caused by enterovirus D68 prompted investigation into whether Canadian hospital and provincial laboratories can detect this virus using commercial and laboratory-developed assays. This study demonstrated analytical sensitivity differences between commercial and laboratory-developed assays for the detection of enterovirus D68.

Report of New Haplotype for ABCC2 Gene: rs17222723 and rs8187718 in cis

The ATP-binding cassette, subfamily C [CFTR/MRP], member 2 (ABCC2) gene is a member of the ATP-binding cassette transporters and is involved in the transport of molecules across cellular membranes. Substrates transported by ABCC2 include antiepileptics, statins, tenofovir, cisplatin, irinotecan, and carbamazepine. Because of the pharmacogenomics implications, we developed a clinical laboratory-developed assay to test for seven variants in the ABCC2 gene: c.3563T>A (p.V1188E, rs17222723), c.1249G>A (p.V417I, rs2273697), c.3972C>T (p.I1324I, rs3740066), c.2302C>T (p.R768W, rs56199535), c.2366C>T (p.S789F, rs56220353), c.-24C>T (5'UTR, rs717620), and c.4544G>A (p.C1515Y, rs8187710). During the validation process, we noted several DNA samples, obtained from the Coriell Cell Repository, that contained both c.3563T>A, c.4544G>A, and a third variant, suggesting that c.3563T>A and c.4544G>A are in cis on the chromosome in some individuals. We obtained DNA samples from a trio (father, mother, and child), tested their ABCC2 variants, and confirmed that c.3563T>A and c.4544G>A were in cis on the same chromosome. Here, we report a new haplotype in ABCC2.

Evaluation of four molecular methods for the diagnosis of tuberculosis in pulmonary and blood samples from immunocompromised patients

The present study analysed the concordance among four different molecular diagnostic methods for tuberculosis (TB) in pulmonary and blood samples from immunocompromised patients. A total of 165 blood and 194 sputum samples were collected from 181 human immunodeficiency virus (HIV)-infected patients with upper respiratory complaints, regardless of suspicious for TB. The samples were submitted for smear microscopy, culture and molecular tests: a laboratory-developed conventional polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) and the Gen-Probe and Detect-TB Ampligenix kits. The samples were handled blindly by all the technicians involved, from sample processing to results analysis. For sputum, the sensitivity and specificity were 100% and 96.7% for qPCR, 81.8% and 94.5% for Gen-Probe and 100% and 66.3% for Detect-TB, respectively. qPCR presented the best concordance with sputum culture [kappa (k) = 0.864)], followed by Gen-Probe (k = 0.682). For blood samples, qPCR showed 100% sensitivity and 92.3% specificity, with a substantial correlation with sputum culture (k = 0.754) and with the qPCR results obtained from sputum of the corresponding patient (k = 0.630). Conventional PCR demonstrated the worst results for sputa and blood, with a sensitivity of 100% vs. 88.9% and a specificity of 46.3% vs. 32%, respectively. Commercial or laboratory-developed molecular assays can overcome the difficulties in the diagnosis of TB in paucibacillary patients using conventional methods available in most laboratories.

Epidermal growth factor receptor signaling pathway is frequently altered in ampullary carcinoma at protein and genetic levels

Our objective was to explore alteration of the epidermal growth factor receptor (EGFR) signaling pathway in ampullary carcinoma. Immunohistochemical studies were employed to evaluate expression of amphiregulin as well as expression and activation of EGFR. A lab-developed assay was used to identify mutations in the EGFR pathway genes, including KRAS, BRAF, PIK3CA, PTEN, and AKT1. A total of 52 ampullary carcinomas were identified, including 25 intestinal-type and 24 pancreatobiliary-type tumors, with the intestinal type being associated with a younger age at diagnosis (P=0.03) and a better prognosis (P<0.01). Expression of amphiregulin correlated with better differentiation (P<0.01), but no difference was observed between two major histologic types. Expression and activation of EGFR was more commonly seen in the pancreatobiliary type (P<0.01). Mutations were detected in 50% of the pancreatobiliary type and 60% of the intestinal type. KRAS was the most common gene mutated in the pancreatobiliary type (42%) as well as the intestinal type (52%). Other mutations detected included PIK3CA, SMAD4 and BRAF. KRAS mutations at codons 12 and 13 did not adversely affect overall survival. In conclusion, EGFR expression and activation were different between intestinal- and pancreatobiliary-type ampullary carcinoma. KRAS mutation was common in both histologic types; however, the incidence appeared to be lower in the pancreatobiliary type compared with its pancreatic counterpart, pancreatic ductal adenocarcinoma. Mutational analysis of the EGFR pathway genes may provide important insights into personalized treatment for patients with ampullary carcinoma.

Identifying a Subpopulation for a Tailored Therapy: Bridging Clinical Efficacy From a Laboratory-Developed Assay to a Validated In Vitro Diagnostic Test Kit

In the United States, regulatory approval of a therapy that is tailored to a subpopulation may require the coapproval of a companion in vitro diagnostic (IVD) tool for identifying that subpopulation. Unfortunately, for many reasons, development of the companion IVD may lag such that it is unavailable during a pivotal clinical trial of the therapy. Instead, a laboratory-developed test (LDT) may be used on clinical trial specimens to identify the subpopulation on whom to evaluate the therapy. However, remaining specimen material is saved so that when the companion IVD is ready for market, the specimens can be retested, in an effort to “bridge” from the LDT to the IVD. Unfortunately, retest results can be missing or invalid because some subjects lack remaining specimen material or because what remains is unevaluable (e.g., due to insufficient specimen material, inadequate specimen quality). We frame the bridging analysis problem as one of estimating drug efficacy in the IVD-defined subpopulation. We develop a closed-form approach, as well as approaches based on multiple imputation and bootstrapping to address the missing data problem. We discuss this in the context of a case study involving a recent submission and approval in the United States of a drug and IVD in oncology.

Abstract P4-05-14: Molecular and immunohistochemical profile of invasive micropapillary breast carcinoma

Abstract Background: Invasive micropapillary carcinoma (IMPC) of the breast is an uncommon disease subtype that is generally considered aggressive. A variety of carcinomas can exhibit micropapillary histology including lung, bladder, and ovary. Immunohistochemical (IHC) and genomic studies at these other organ sites have identified potential therapeutic targets including BRAF mutations. The purpose of this study was to characterize the IHC and molecular profile of a cohort of IMPC that could suggest potentially useful targeted therapies. Design: Eight tumor blocks (from seven patients) of IMPC were identified from the archives at the University of Iowa Hospitals and Clinics from 1997-2012. IHC stains for ER, PR, HER2, PTEN (Dako; clone 6H2.1, 1:100) and NM23H1 (Santa Cruz Biotechnology, clone: Nm301, 1:50) were performed. ER, PR, and HER2 were score according to ASCO/CAP guidelines. PTEN and NM23H1 were scored using a 4-tier system: P: strong homogenous; H: heterogeneous (positive with negative foci); R: reduced in more than 50%; and L: lost in all or majority. Genomic DNA was isolated from the formalin fixed paraffin embedded tumor samples used for IHC. BRAF V600 mutational analysis was analyzed using a laboratory-developed primer extension assay. Massive parallel sequencing (next generation sequencing, NGS) was performed using the Ion AmpliSeq™ Cancer Hotspot Panel v2 (Life Technologies, Carlsbad, CA). Results: The patients were 50-83 years of age. Six had Elston-Ellis Grade II tumors and one was Elston-Ellis grade I. Four patients had axillary lymph node metastases. All cases examined showed strong and diffuse estrogen and progesterone receptor expression with no over-expression of HER2. Complete PTEN loss was observed in 4 cases including 3 with lymph node positive disease, three of these four cases had concurrent reduced expression of NM23H1. No mutations in exon 15 of the BRAF gene were detected. By NGS, one sample showed a KITc.A1621C (pM541) polymorphism, three had no mutations, and sequencing from the other three was unsatisfactory. Molecular and IHC Profile of Seven Patients with IMPC of the BreastHistologic GradeAxillary Lymph Node MetastasisER/PRHER2PTENNM23H1NGS2NoPositive/PositiveNegativePPUnsatisfactory1NoPositive/PositiveNegativeLRNegative2NoPositive/PositiveNegativeHPIntronic2YesPositive/PositiveNegativeHPUnsatisfactory2YesPositive/PositiveNegativeLRKIT c.A1621C (pM541)2YesPositive/PositiveNegativeLPNegative2YesPositive/PositiveNegativeLRUnsatisfactory Conclusions: Loss of PTEN expression was observed in 57% of cases (4/7) and was often associated with lymph node metastasis. A direct correlation between PTEN and NM23H1 expression was observed in six cases. No BRAF or other mutations were detected. PTEN was included in the NGS panel. Reduced expression observed in our study is most likely due to posttranscriptional modifications. Concurrent reduced expression of the metastatic suppressor NM23H1 may play a role in the clinical behavior of IMPC. The high frequency of PTEN loss may have implications for targeted therapy towards the PI3K pathway in this subgroup of patients. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-05-14.