Abbott Molecular Research Articles

P1.02-025 Evaluation of NGS and RT-PCR Methods for ALK Assessment in European NSCLC Patients: Results from the ETOP Lungscape Project

The reported prevalence of ALK rearrangement in NSCLC ranges from 2%-7%, depending on population and detection method. The primary standard diagnostic method is fluorescence in situ hybridization (FISH). Recently, immunohistochemistry (IHC) has also proven to be a reproducible and sensitive technique. Reverse transcriptase-polymerase chain reaction (RT-PCR) has been advocated and most recently the advent of targeted Next-Generation Sequencing (NGS) for ALK and other fusions has become possible. This is one of the first studies comparing all 4 techniques in resected NSCLC from the large ETOP Lungscape cohort. 96 cases from the ETOP Lungscape iBiobank (N=2709) selected based on any degree of IHC staining (clone 5A4 antibody, Novocastra, UK) were examined by FISH (Abbott Molecular, Inc.; Blackhall, JCO 2014), central RT-PCR and NGS. H-score 120 is used as cutoff for IHC+. For both RT-PCR and NGS, RNA was extracted from the same formalin-fixed, paraffin-embedded tissues. For RT-PCR, primers were used covering the most frequent ALK translocations. For NGS, the Oncomine™ Solid Tumour Fusion Transcript Kit was used, allowing simultaneous sequencing of 70 ALK, RET and ROS1 specific fusion transcripts associated with NSCLC, as well as novel ALK translocations using 5’-3’ ALK gene expression ‘Imbalance Assay’. NGS provided results for 90 cases, while RT-PCR for 77. Overall, 70 cases have results for all 4 methods, with fully concordant 60 (85.7%) cases (49 ALK-, 11 ALK+). Before employing the ‘Imbalance Assay', in 5 of the remaining 10 cases, NGS differs from the other methods (3 NGS-, 2 NGS+), while in the other 5, NGS agrees with RT-PCR in all, IHC in 2, and FISH in 1. Using the concordant result of at least two of the three methods as true negative/positive, the specificity and sensitivity of the fourth is 96/94/100/96% and 94/94/89/72% for IHC/FISH/RT-PCR/NGS, respectively (incorporating imbalance: NGS sensitivity=83%). Imbalance scores are presented here for 18 NGS- cases: 9 ‘NGS-/FISH+/IHC+’, 9 ‘NGS-/FISH-/IHC-‘. Among the ‘NGS-/FISH+/IHC+’, there is strong evidence of imbalance in 4 cases (score’s range: 0.0144-0.0555), uncertain in 5 (range: 0.0030-0.0087), and no evidence (scores≤0.0004) in the 9 negative cases. NGS is a useful screening tool for ALK rearrangement status, superior to RT-PCR when RNA yield is limited. When using NGS, it is critically important to integrate the 5’-3’ imbalance assay and to confirm with one or more additional methods in the ‘imbalance’ cases. Data further highlight the possibility of missing actionable rearrangements when only one screening methodology is available.

P1.02-048 MET Exon 14 Skipping Mutations and Gene Amplifications Are Not Simultaneous Events in NSCLC

Mutations in the MET exon 14 RNA splice acceptor and donor sites, which lead to exon skipping, have been described with responsiveness to crizotinib. Until now, patient selection has been made in view of MET amplification/high polysomy and protein overexpression, with discrepancies in positivity criteria. We investigated the prevalence of abnormal MET mutational status and the subsequent gene copy number alterations (CNAs) in NSCLC. We routinely tested 190 lung adenocarcinomas and adenosquamous carcinomas for MET exon 14 and flanking introns mutations by PCR-direct sequencing, and for MET gene CNAs by FISH (Abbott Molecular). Amplifications were defined as mean gene by mean centromere ratio ≥2.2, and high gains as mean gene ≥5.0. RT-PCR was performed to validate mutations leading to exon 14 skipping. We collected clinical-pathological data together with EGFR and KRAS mutational status and ALK, ROS1 and RET rearrangements. MET alterations were found in 34 patients (17.9%): 11 mutated cases (5.8%), eight gene amplifications (4.2%), and 15 high gains (9.1%). Six out of 11 mutations were confirmed to lead to exon 14 skipping (3.2%). Remarkably, none of these exon 14 skipped cases had concurrent MET amplification nor high gains. Although, KRAS p.G12C and EGFR 19 exon mutations were found concomitant with MET mutation in two of these cases. Among the 14 confirmed MET altered cases (6 exon 14 skipped and 8 amplified): 13 patients were male (93%), with a median age of 65.7 years (range: 40-91), nine current smokers (64%) (40 pack-years, range: 20-60), and eight diagnosed in an advanced stage disease (III and IV) (57%). Correlation with c-MET protein expression by IHC is ongoing, and data will be presented at the meeting. As no concurrent MET mutated and amplified cases were found, our data support prospective identification of both, MET exon 14 skipping mutations and gene amplifications. These mutations define a new subset of NSCLC patients that should be analyzed independently of the status of MET gene copy number.

A comparative analysis of immunohistochemistry and fluorescent in situ hybridization assay to detect anaplastic lymphoma kinase status in lung adenocarcinoma cases: A search for a testing algorithm.

Testing for echinoderm microtubule-associated protein-like 4 (EML4) anaplastic lymphoma kinase (ALK) translocation by fluorescence in situ hybridization (FISH) is well established whereas the Food and Drug Administration (FDA) ALK immunohistochemical (IHC) test is relatively new. The aim of this study is to compare FDA-approved ALK IHC test (D5F3 clone) with the standard ALK FISH test. A validation and a test arm with 100 and 200 cases of Formalin-Fixed, Paraffin-embedded blocks of lung adenocarcinoma, respectively, comprised the material. All cases had ALK IHC test on automated Ventana Benchmark XT IHC slide stainer using anti-ALK D5F3 rabbit monoclonal primary antibody; when positive tumor cells (any percentage) showed strong granular cytoplasmic staining. For the FISH test, Vysis ALK Dual Color Break Apart Rearrangement Probe (Abbott Molecular Inc.,) was used to detect ALK gene 2p23 rearrangements; when positive the red and green signals were split two signal diameter apart and/or isolated 3'red signal were detected in more than 15% tumor cells. The ALK FISH results were available in all 100 validation cases and 64-test arm cases which formed the basis of this analysis. The ALK IHC test was positive in 16% cases; four discordant cases were ALK IHC positive but ALK FISH negative, but no case was ALK IHC negative and ALK FISH positive. There was 100% sensitivity, 90.5% specificity, and 93.75% accuracy. A negative ALK IHC result obviates the need for a FISH test barring those with a strong clinical profile, and a positive ALK IHC result is sufficient basis for the initiation of treatment.

P1.02-029 Infrequent Staining Patterns in ALK Immunohistochemistry: Correlation with Fish Analysis

AKL gene rearrangements are predictive alterations in non-small cell lung carcinomas (NSCLC). Immunohistochemistry (IHC) has become a valuable tool in assessing ALK status, however unusual staining patterns, such as heterogeneous diffuse moderate and focal intense stains, may occur and can make evaluation difficult. We correlated immunohistochemistry unusual staining patterns with ALK status by fluorescence in situ hybridization (FISH). Of 851 cases tested, we found 14 (1.6%) cases with inconclusive staining patterns that can be summarized in: a) diffuse granular cytoplasmic moderate stain, with or without background mucin stain (10 cases) b) focal intense granular cytoplasmic stain in overall negative or weakly positive tumors (4 cases). IHC was performed on an automatized Benchmark staining module using Ventana ALK (D5F3) CDx assay with Optiview amplification kit. FISH was performed using ALK break-apart probe set (Vysis LSI ALK Dual Color, Abbott Molecular). Cases were considered ALK-FISH positive if ≥15% tumor cells showed split red and green signals (separation of 2 diameters or more) and/or single red signals. Of 10 moderate granular cytoplasmic stain cases, 4 had also abundant mucin background stain 6 where markedly heterogeneous with areas of weak and moderate cytoplasmic granular stain. Nine were FISH negative, one yielded no signals (uninformative result) and one specimen corresponded to an acid decalcified specimen and was not evaluated by FISH. Focal intense stain was observed in 5 samples, 3 corresponded to surgical specimens and the rest to small needle biopsies, one of the surgical specimens was FISH positive and the rest, negative. Since FDA approval of Ventana ALK (D5F3) IHC CDx Assay, IHC has become a widely used tool for assessing ALK status. Guidelines suggest that weak granular stain should be interpreted as negative and focal intense granular stain in any number of cells, as positive. Even though our sample is small, moderate granular stain was consistently negative by FISH analysis, however, focal intense stain shows more discordant results between tests. To date, no suggestions are made on what should be the minimum amount of tumor in a sample to report an IHC assay. Even though some of these patients with IHC positive/FISH negative results have been reported as responders to Crizotinib, further studies are needed. One specimen with moderate cytoplasmic IHC stain was uninformative due to lack of signals. This raises the issue of the need to standardize pre-analytical variables, which can be difficult in some areas of Latin America.

PUB051 Comprehensive Analysis of Chromosomal Alterations Involving CDKN2A and 22q in Malignant Pleural Mesothelioma

The promise of personalized genomic medicine relies on the identification and clinical management of patients on the basis of specific genomic alterations that drive clinically heterogenous malignancies. While recent large cohort studies of malignant pleural mesothelioma (MPM) tumors have confirmed that CDKN2A and NF2 are among the most commonly mutated genes, the clinical and biological significance of these molecular alterations remains incompletely characterized. Herein, we explore whether loss of CDKN2A and/or 22q define molecular subclasses in a large cohort of MPM patients. FISH analysis was performed using the Vysis LSI9p21/CEP9 Dual Color Probe Set (Abbott Molecular, Des Plaines, IL) for the p16 locus at 9p21, CEP9 at the chromosome 9 centromeric region, and Vysis TUPLEI/LSI ARSA Dual color Probe set (Abbott) for TUPLEI at 22q11.2, and ARSA at 22q13. At least 50 nuclei were observed per tumor. Aberrations observed in ≥ 2% of nuclei was considered abnormal, based on normal mesothelial cell controls. Loss of CDKN2A or 22q was explored in relation to demographic, pathological and clinical factors using Fisher’s exact test. Overall survival (OS) was estimated using the Kaplan–Meier method and compared between groups using the logrank test. CDKN2A and 22q FISH data along with patient’s clinical information were collected from 592 MPM patients. Loss of CDKN2A was detected in 329 (56%), and of 22q in 326 (55%) patients. CDKN2A loss was significantly more common among men (p=0.0027) compared to women, and among men older than 60 compared to younger men (p = 0.0005). Loss of CDKN2A was also significantly correlated with self-reported asbestos exposure (p=0.0419), preoperative anemia (p=0.0007), and epitheliod tumor histology (p<0.0001). Loss of 22q was unrelated to most clinical factors explored, being only marginally associated with N2-N3 lymph node status compared to N0-N1 (p=0.0429). Survival analysis showed that patients with CDKN2A loss, with (n=228) or without (n=101) concurrent loss of 22q, demonstrated reduced OS compared to patients with either wild-type copy number of both markers (n=165; p<0.0001) or loss of 22q only (n=98; p < 0.0001). This study demonstrates a previously unappreciated relationship between demographic, clinical, pathological and outcome characteristics of MPM in association with CDKN2A versus 22q loss. The pattern of alteration involving these chromosomal regions may, therefore, suggest the possibility of distinct molecular subgroups. Further analyses are needed to demonstrate its potential role in precision medicine.

Superior Identification of Prognostic Relevant Copy Number Abnormalities By SNP-Based Genomic Arrays As Compared to Interphase FISH in Multiple Myeloma

Multiple myeloma (MM) is a neoplasm that exhibits a broad heterogeneity in both biological behavior and clinical presentation. Specific copy number abnormalities (CNAs) such as hyperdiploidy, 1p loss, 1q gain, 13q loss and 17p loss (including the TP53 gene), and IGH translocations, such as t(4;14)(p16;q32) and t(14;16)(q32;q23), provide important information regarding prognosis and treatment response.Interphase fluorescence in situ hybridization (FISH) on enriched plasma cells, currently used in clinical diagnostics of MM, is a targeted test aimed at specific genomic loci. However, it is laborious and provides only genetic information of the probe targets. Microarray-based genomic profiling is a high-resolution tool that enables genome-wide analyses for copy number alterations (CNA), including focal CNA (<5 Mb) and regions of copy neutral loss of heterozogosity (CNLOH) that cannot be identified by FISH. A limitation of SNP-based array is its inability to identify balanced translocations.The aim of this study was to compare FISH with SNP-based genomic arrays with respect to the detection yield for prognostic relevant genetic copy number abnormalities in enriched plasma cell samples from MM patients. In addition we have set up a diagnostic work flow in which on one sample of enriched plasma cells interphase FISH for (balanced) IGH translocations as well as SNP-based array for identification of CNA can be performed.SNP-based genomic array profiling and FISH were performed in 37 MM patients. After enrichment of CD138 plasma cells half of each sample was treated with 0.075M KCl and, subsequently, fixed with 3:1 methanol/acetic acid and transferred to a microscopic slide for subsequent FISH. From the remaining part of the CD138-enriched plasma cell fraction DNA was extracted to perform SNP-based genomic array. Interphase FISH was performed according to standard methods using the following probes D5S23/D5S721/CEP9/CEP15, LSI13 (13q14), LSI TP53 (17p13.1) (all from Abbott Molecular, USA), and CDKN2C/CKS1B (from Cytocell, UK). 200 nuclei were analyzed by two different investigators and the detection limit was set at 20% as proposed by the EMN (Ross et al 2012; Haematologica 97:1272-1277). SNP-based array was performed using the CytoScan HD array platform (Affymetrix, USA), using the interpretation criteria as proposed by Schoumans (Schoumans et al 2016; Genes Chromosomes Cancer 55:480-491). Data regarding FISH and SNP-based array were obtained in a fully blinded fashion.All prognostic relevant CNA as observed by FISH were also identified when only SNP-based genomic arrays would have been performed, including 4 cases with loss of 1p, 19 cases with gain of 1q, 14 cases with loss of 13q, 4 cases with loss of 17p, and 20 cases with a hyperdiploid karyotype. However, SNP-based arrays identified 20 additional prognostic relevant abnormalities which were not observed by interphase FISH for several reasons. Due to a higher detection limit of the applied SNP-based array platform, 2 cases with loss of 17p (abnormality present in 15-20% of the cells) and 1 case with loss of 13q and a hyperdiploid karyotype (present in 15% of the cells) were observed by SNP-array only. Four cases showed a 1p21 or 1p16 loss, which were not observed by FISH since these deleted regions were outside the 1p32 probe target region. In 3 cases tumor-associated regions of CNLOH were observed involving the regions 13q and 17p. Finally, in 4 cases in which FISH was suggestive for a hyperdiploid karyotype, the SNP-based array information regarding whole genome analysis and allele frequencies demonstrated that these 4 cases appeared to have a doubled up DNA content in their plasma cells, and therefore the losses of 1p, 13q and 17p were not observed by interphase FISH.In conclusion, we demonstrate that SNP-based arrays are superior in the identification of prognostic relevant CNA in MM. SNP-based array do identify all CNA as observed by FISH, and in addition, identifies additional prognostic relevant abnormalities, such as loss of 1p, 13q, and 17p, that escaped the detection by FISH. The prognostic relevance of the CNLOH and the loss of 1p21 and 1p16 regions requires further evaluation in prospective clinical trials. DisclosuresZweegman:Celgene: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria, Research Funding.

Rearrangements of Tyrosine Kinase (TK) Genes in Chronic Myeloproliferative Disorders (CMPD): A FISH Study

CMPDs are heterogeneous hematopoietic stem cell disorders caused by the constitutive activation of specific TK genes induced by mutations or, exceptionally, chromosomal translocations that often escape conventional cytogenetics (CC) identification as they often involve poorly stained regions. The demonstration of these TK gene rearrangements is twofold relevant: diagnostically, as these translocations identify specific entities within the 2016 WHO classification of CMPDs and clinically, as most TK can be targeted with specific TK inhibitors.Thus, this study employed FISH probes specific for TK genes to establish the incidence of these chromosomal translocations, to identify uncommon TK translocation partners, to establish whether eosinophils are part of the clonal cell population and to find any potential correlation with clinical parameters and outcome. From January 2005-December 2015 43 consecutive patients (pts) were analysed; 13 females and 31 males with a median age of 47 years (range 22-72). According to WHO classification, 20 pts were diagnosed as atypical chronic myeloid leukemia (aCML), 22 as chronic eosinophilic leukemia (CEL) and one as AML/T lymphoblastic lymphoma (T-LL). Median follow-up was 39 months (range 8-136). At the time of the study one pt died and one experienced disease progression. FISH probes were obtained from Kreatech (Amsterdam, NL), Abbot Molecular Inc. (Chicago, Il, USA) and from BACPAC Resources Center at C.H.O.R.I. (Oakland, USA) after determining their Mb position using UCSC genome browser on Human Feb. 2009 assembly. The commercial probes, applied according to manufacturer's guidelines were: ON FIP1L1-CHIC2-PDGFRA (4q12) Del, Break; ON PDGFRB (5q33) Break; ON FGFR1 (8p12) Break; ON JAK2 (9p24) Break; LSI BCRABL. The BAC probes RP11-484L21 and RP11-880I16 covering the PCM1 gene were labelled and applied as previously described. i-FISH, cut-off values were obtained from the analysis of 300 nuclei from ten normal samples and were fixed at 10%.An abnormal FISH pattern was revealed in 12 pts (27.9%): 5/20 (25%) with aCML, 6/22 (27.2%) with CEL and one with AML/T-LL. Two aCML pts presented a trisomy 8, one a t(9;13)(q34;q14) which involved the ABL gene and a not yet identified partner gene, one at(9;22)(p24;q11) which involved the ABL and BCR genes and one a t(8;22)(p11;q11) which involved the FGFR1 gene. Interestingly, after one month this last pt progressed to AML and on CC showed a duplication of the derivative chromosome 8. Two CEL pts showed a JAK2 rearrangement: one who carried a t(8;9)(p22;p24) on CC displayed the classical PCM1-JAK2 gene fusion, the other who carried a t(3;8)(?;p24) not revealed by CC harboured a fusion between the JAK2 gene and a not yet identified partner. Three additional CEL pts showed a PDGFRB rearrangement which escaped CC detection too. In these pts who on CC showed a t(1;5)(?;q33) with loss of the reciprocal translocation product, a t(5;8)(q33;?) and a t(5;12)(q33;?), the PDGFRB translocation partner has been not yet identified. The last chromosomally normal CEL pt showed a PDGFRA deletion. Thus, in 4 pts FISH with BAC probes is still on-going in order to search the unknown translocation partners of the JAK2 and PDGFRB genes. Noteworthy, despite the fact that all these pts presented a relevant peripheral eosinophilia (≈65%), FISH performed on peripheral blood smears always revealed a normal pattern. The sole AML/T-LL pt who carried a t(8;13)(p11;q12) which produced the classical FGFR1-ZNF198 gene fusion failed to respond to conventional chemotherapy and died of disease related complications. From a clinical point of view 3 aCML/CEL pts with TK rearrangements responded to TK inhibitors experiencing a haematological improvement including one complete remission (CR); the t(8;22) positive pt entered CR after induction chemotherapy.In conclusion, i) FISH effectively reveals cryptic TK translocations in about 36% of chromosomally normal CMPDs; ii) these rearrangements are more common in CELs than in aCML; iii) peripheral blood eosinophils may show a normal FISH pattern; iv) FISH can effectively be used to monitor the clonal cell population during disease outcome. DisclosuresNo relevant conflicts of interest to declare.

Screening for ROS1 gene rearrangements in non-small-cell lung cancers using immunohistochemistry with FISH confirmation is an effective method to identify this rare target.

To assess the prevalence of ROS1 rearrangements in a retrospective and prospective diagnostic Australian cohort and evaluate the effectiveness of immunohistochemical screening. A retrospective cohort of 278 early stage lung adenocarcinomas and an additional 104 prospective non-small-cell lung cancer (NSCLC) cases referred for routine molecular testing were evaluated. ROS1 immunohistochemistry (IHC) was performed (D4D6 clone, Cell Signaling Technology) on all cases as well as fluorescence in-situ hybridization (FISH) using the ZytoVision and Abbott Molecular ROS1 FISH probes, with ≥15% of cells with split signals considered positive for rearrangement. Eighty-eight cases (32%) from the retrospective cohort showed staining by ROS1 IHC, and one case (0.4%) showed ROS1 rearrangement by FISH. Nineteen of the prospective diagnostic cases showed ROS1 IHC staining, 12 (12%) cases of which were confirmed as ROS1 rearranged by FISH. There were no ROS1 rearranged cases that showed no expression of ROS1 with IHC. The ROS1 rearranged cases in the prospective cohort were all EGFR wild-type and anaplastic lymphoma kinase (ALK) rearrangement-negative. The sensitivity of ROS1 IHC in the retrospective cohort was 100% and specificity was 76%. ROS1 rearrangements are rare events in lung adenocarcinomas. Selection of cases for ROS1 FISH testing, by excluding EGFR/ALK-positive cases and use of IHC to screen for potentially positive cases, can be used to enrich for the likelihood of identifying a ROS1 rearranged lung cancer and prevent the need to undertake expensive and time-consuming FISH testing in all cases.

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.

Monitoring of cytomegalovirus (CMV) infection in solid organ transplant recipients: quantitation of CMV DNAemia by two real-time polymerase chain reaction assays

&lt;em&gt;Background and aim:&lt;/em&gt; Quantification of cytomegalovirus (CMV) DNAemia is essential in clinical management of post-transplant infection. We evaluated the performances of two quantitative real-time polymerase chain reaction (PCR) assays. &lt;br /&gt;&lt;em&gt;Materials and Methods&lt;/em&gt;: 114 serial whole blood samples collected from 14 actively infected transplant recipients were processed by Abbott RealTime CMV PCR kit (Abbott Molecular) and CMV ELITe MGB™ kit (ELITech Group). The Quality Control for Molecular Diagnostics human CMV panels was also tested. &lt;br /&gt;&lt;em&gt;Results&lt;/em&gt;: Sixteen (14%) samples resulted negative and 59 (51.7%) positive with a quantitative result for both assays. In the 59 samples, the coefficient of correlation was 0.856. Bland-Altman analysis showed a mean difference of &amp;lt;0.11 log10 copies/mL (standard deviation=0.38 log10 copies/mL). The assays gave CMV-DNA loads differing by 1 log10 DNA copies/mL in 57 samples (96.6%) and by &amp;lt;0.5 log10 DNA copies/mL in 48 samples (81.3%). Eleven (9.6%) samples were positive with a quantitative result with Abbott and negative with ELITech. Sixteen (14%) positive samples with a quantitative result for Abbott resulted positive but below the lower limit of quantification (LLQ) for ELITech. Twelve (10.5%) samples resulted negative with ELITech and positive but below the LLQ with Abbott. No samples were positive with ELITech and negative with Abbott. &lt;br /&gt;&lt;em&gt;Conclusions&lt;/em&gt;: The assays showed a good correlation between CMVDNA levels detected and variation in CMV-DNA &amp;lt;0.5 log10 was observed in the majority of the samples. The viral load kinetic profiles of the assays were overlapping in all patients, but Abbott showed higher sensitivity in samples containing lower amount of DNA. The clinical value of this greater sensitivity requires further investigation.

HLA-A, HLA-B and HLA-DRB1 allele and haplotype frequencies of 10918 Koreans from bone marrow donor registry in Korea.

The human leucocyte antigen (HLA) system is the most polymorphic genetic system in humans, and HLA matching is crucial in organ transplantation, especially in hematopoietic stem cell transplantation. We investigated HLA-A, HLA-B and HLA-DRB1 allele and haplotype frequencies at allelic level in 10918 Koreans from bone marrow donor registry in Korea. Intermediate resolution HLA typing was performed using Luminex technology (Wakunaga, Japan), and additional allelic level typing was performed using PCR-single-strand conformation polymorphism method and/or sequence-based typing (Abbott Molecular, USA). Allele and haplotype frequencies were calculated by direct counting and maximum likelihood methods, respectively. A total of 39 HLA-A, 66 HLA-B and 47 HLA-DRB1 alleles were identified. High-frequency alleles found at a frequency of ≥5% were 6 HLA-A (A*02:01, *02:06, *11:01, *24:02, *31:01 and *33:03), 6 HLA-B (B*15:01, *35:01, *44:03, *51:01, 54:01 and *58:01) and 8 HLA-DRB1 (DRB1*01:01, *04:05, *04:06, *07:01, *08:03, *09:01, *13:02 and *15:01) alleles. At each locus, A*02, B*15 and DRB1*14 generic groups were mostdiverse at allelic level, consisting of 9, 12 and 11 different alleles, respectively. A total of 366, 197 and21 different HLA-A-B-DRB1 haplotypes were estimated with frequencies of ≥0.05%, ≥0.1% and ≥0.5%, respectively. The five most common haplotypes with frequencies of ≥2.0% were A*33:03-B*44:03-DRB1*13:02 (4.97%), A*33:03-B*58:01-DRB1*13:02, A*33:03-B*44:03-DRB1*07:01, A*24:02-B*07:02-DRB1*01:01 and A*24:02-B*52:01-DRB1*15:02. Among 34 serologic HLA-A-B-DR haplotypes with frequencies of ≥0.5%, 17 haplotypes revealed allele-level diversity and majority of the allelic variation was arising from A2, A26, B61, B62, DR4 and DR14 specificities. Haplotype diversity obtained in this study is the most comprehensive data thus far reported in Koreans, and the information will be useful for unrelated stem cell transplantation as well as for disease association studies.

From clinical sample to complete genome: Comparing methods for the extraction of HIV-1 RNA for high-throughput deep sequencing

The BEEHIVE (Bridging the Evolution and Epidemiology of HIV in Europe) project aims to analyse nearly-complete viral genomes from >3000 HIV-1 infected Europeans using high-throughput deep sequencing techniques to investigate the virus genetic contribution to virulence. Following the development of a computational pipeline, including a new de novo assembler for RNA virus genomes, to generate larger contiguous sequences (contigs) from the abundance of short sequence reads that characterise the data, another area that determines genome sequencing success is the quality and quantity of the input RNA. A pilot experiment with 125 patient plasma samples was performed to investigate the optimal method for isolation of HIV-1 viral RNA for long amplicon genome sequencing. Manual isolation with the QIAamp Viral RNA Mini Kit (Qiagen) was superior over robotically extracted RNA using either the QIAcube robotic system, the mSample Preparation Systems RNA kit with automated extraction by the m2000sp system (Abbott Molecular), or the MagNA Pure 96 System in combination with the MagNA Pure 96 Instrument (Roche Diagnostics). We scored amplification of a set of four HIV-1 amplicons of ∼1.9, 3.6, 3.0 and 3.5kb, and subsequent recovery of near-complete viral genomes.Subsequently, 616 BEEHIVE patient samples were analysed to determine factors that influence successful amplification of the genome in four overlapping amplicons using the QIAamp Viral RNA Kit for viral RNA isolation. Both low plasma viral load and high sample age (stored before 1999) negatively influenced the amplification of viral amplicons >3kb. A plasma viral load of >100,000 copies/ml resulted in successful amplification of all four amplicons for 86% of the samples, this value dropped to only 46% for samples with viral loads of <20,000 copies/ml.

Rapid virological response assessment by Abbott RealTime hepatitis C virus assay for predicting sustained virological responses in patients with hepatitis C virus genotype 1 treated with pegylated-interferon and ribavirin

The lower limits of virus detection of hepatitis C virus (HCV) RNA detection assays are continuously improving. We aimed to assess the utility of more precise definition of 4th week viral load [rapid virological response (RVR)] in predicting sustained virological response (SVR) in HCV genotype 1 patients treated with pegylated-interferon (PEG-IFN) and ribavirin. Clinical data of treatment-naïve HCV genotype 1 patients were retrospectively collected from 2009 to 2014. Patients were grouped according to 4th week viral load as follows: undetectable (n = 90) and detectable but not quantifiable (< 12 IU/mL, n = 27). All patients received PEG-IFNα-2a or -2b and ribavirin for 24 weeks. Serum HCV RNA levels were measured by Abbott RealTime (ART; Abbott Molecular, Abbott Park, IL, USA) HCV assay. SVR was 95.5% and 63% in the undetectable group and < 12 IU/mL group of 4th week viral load, respectively. The between-group difference in SVR was significant (p < 0.001). We determined 4th week viral load was independently associated with SVR (odds ratio = 19.28; p = 0.002) and a good predictor of SVR [area under the curve (AUC) = 0.775; p = 0.001]. ART HCV assays had a stronger SVR predictive value in HCV genotype 1 patients, indicating that only the undetectable group of 4th week viral load patients measured by ART HCV assay should be considered for shorter treatment time (24 weeks) with PEG-IFN and ribavirin.

Frequency and Clinical Characteristics of Intrachromosomal Amplification of Chromosome 21 in Korean Childhood B-lineage Acute Lymphoblastic Leukemia.

BackgroundIntrachromosomal amplification of chromosome 21 (iAMP21) is known to be associated with poor prognosis in B-cell ALL (B-ALL). To determine the frequency and clinical characteristics of iAMP21 in Korean B-ALL patients, we performed FISH and multiplex ligation-dependent probe amplification (MLPA) analyses.MethodsA total of 102 childhood B-ALL patients were screened with ETV6-RUNX1 FISH probes (Abbott Molecular, USA). The presence of an iAMP21 was confirmed by using MLPA P327 iAMP21-ERG probemix (MRC Holland, The Netherlands).ResultsiAMP21 was detected in one of the screened B-ALL patients (1/102 patients, 1.0%) who presented the ALL immunophenotype and complex karyotype at initial diagnosis. The patient relapsed twice after bone marrow transplantation. MLPA showed 12.5-Mb and 4.28-Mb regions of amplification and deletion, respectively.ConclusionsThe frequency of iAMP21 is considerable in Korean pediatric patients. Our report suggests that iAMP21 in childhood B-ALL has very unfavorable impact on patient's prognosis. Additional methods such as MLPA analysis is essential to rule out patients with equivocal interphase FISH results.

Aneuploidies level in sperm nuclei in patients with infertility.

Male infertility is a relevant social and medical problem. Male infertility is mostly caused by genetic disorders. The purpose of the study was to analyze the correlation of chromosome aberrations, as well as DNA fragmentation and various manifestations of spermatogenesis disorder. Sperm samples of 58 males with infertility and 23 conditionally healthy males were studied. All patients diagnosed with asthenozoospermia, teratozoospermia, oligoasthenozoospermia and oligoteratozoospermia underwent subsequent analysis of sperm DNA fragmentation. Sperm DNA fragmentation was examined with sperm chromatin dispersion test (sperm chromatin dispersion, Spermprocessor, India) with an Axioscope 40 fluorescent microscope. Fluorescence in situ hybridization with fluorescent probes (Vysis Multi Vysion PGT, Abbot Molecular) was used to study chromosome abnormalities in sperm nuclei with regard to X and Y chromosomes, as well as to chromosomes 18 and 21. It was found that the development of pathospermia was characterized by genetic discontinuity, which manifests as DNA fragmentation and disjunction of chromosomes in meiosis with spermatogenesis. It was also found that the prevailing type of pathospermia in men with infertility was oligozoospermia. In addition, this group also had the highest rate of numerical chromosome abnormalities. This was caused by the degeneration of spermatozoids with aneuploidies in chromosomes.

12P Volatolomic signatures of anaplastic lymphoma kinase gene rearrangement in adenocarcinoma

Background: Mutations in the exon 14 of the MET gene affecting RNA splice acceptor and donor sites, which lead to exon skipping, have been recently associated to responsiveness to crizotinib. Up to date, patient selection for Met inhibitors has been made according to MET amplification/high polysomy and/or protein overexpression. We have investigated the prevalence of MET exon 14 mutations and their correlation with gene copy number alterations (CNAs) in NSCLC patients. Methods: We tested 132 lung adenocarcinomas and adenosquamous carcinoma samples for mutations in MET exon 14 by Sanger sequencing and for MET gene CNAs by FISH (Abbott Molecular). We collected clinical data together with EGFR and KRASmutational status and ALK, ROS1 and RET rearrangements. Results: MET alterations were found in 23 patients (17.5%): 3 exon 14 skipping mutations (2.3%), 8 gene amplifications (6.1%), and 12 high polysomy cases (9.1%). Among the 11 MET altered cases (3 mutated and 8 amplified): 8 patients were male, with a median age of 64.5 years (range: 46–91), current or former smokers (50 pack/years, range: 30–80), and all were diagnosed in an advanced stage disease (III and IV). None of the 3 mutated cases had concurrent MET CNAs. One MET mutated case presented also a KRAS p.G12C substitution while the other two were wild type for EGFR (exons 18 to 21) and KRAS (exon 2). Interestingly, patients with MET alterations have a shorter overall survival (p < 0.001). Conclusions: We have identified alterations of the MET gene (mutations in exon 14 and CNAs) in 8.4% of NSCLC patients. Our data support the usefulness of prospective screening of MET exon 14 mutations and gene amplifications due to their prognostic and predictive role. These mutations define a new subset of NSCLC patients that should be analyzed independently of the MET gene copy number status. Legal entity responsible for the study: Institut Hospital del Mar d’Investigacions Mediques (IMIM), Barcelona, Spain Funding: Institut Hospital del Mar d’Investigacions Mediques (IMIM), Barcelona, Spain Disclosure: All authors have declared no conflicts of interest.

11P Prognostic impact of MET mutations in exon 14 and copy number alterations in a series of NSCLC patients

Background: Mutations in the exon 14 of the MET gene affecting RNA splice acceptor and donor sites, which lead to exon skipping, have been recently associated to responsiveness to crizotinib. Up to date, patient selection for Met inhibitors has been made according to MET amplification/high polysomy and/or protein overexpression. We have investigated the prevalence of MET exon 14 mutations and their correlation with gene copy number alterations (CNAs) in NSCLC patients. Methods: We tested 132 lung adenocarcinomas and adenosquamous carcinoma samples for mutations in MET exon 14 by Sanger sequencing and for MET gene CNAs by FISH (Abbott Molecular). We collected clinical data together with EGFR and KRASmutational status and ALK, ROS1 and RET rearrangements. Results: MET alterations were found in 23 patients (17.5%): 3 exon 14 skipping mutations (2.3%), 8 gene amplifications (6.1%), and 12 high polysomy cases (9.1%). Among the 11 MET altered cases (3 mutated and 8 amplified): 8 patients were male, with a median age of 64.5 years (range: 46–91), current or former smokers (50 pack/years, range: 30–80), and all were diagnosed in an advanced stage disease (III and IV). None of the 3 mutated cases had concurrent MET CNAs. One MET mutated case presented also a KRAS p.G12C substitution while the other two were wild type for EGFR (exons 18 to 21) and KRAS (exon 2). Interestingly, patients with MET alterations have a shorter overall survival (p < 0.001). Conclusions: We have identified alterations of the MET gene (mutations in exon 14 and CNAs) in 8.4% of NSCLC patients. Our data support the usefulness of prospective screening of MET exon 14 mutations and gene amplifications due to their prognostic and predictive role. These mutations define a new subset of NSCLC patients that should be analyzed independently of the MET gene copy number status. Legal entity responsible for the study: Institut Hospital del Mar d’Investigacions Mediques (IMIM), Barcelona, Spain Funding: Institut Hospital del Mar d’Investigacions Mediques (IMIM), Barcelona, Spain Disclosure: All authors have declared no conflicts of interest.

PD13-05 FLUORESCENCE IN SITU HYBRIDIZATION (FISH) IN THE DIAGNOSIS OF BLADDER AND UPPER TRACT UROTHELIAL CARCINOMA: A SINGLE-INSTITUTION EXPERIENCE

You have accessJournal of UrologyBladder Cancer: Upper Tract Transitional Cell Carcinoma I1 Apr 2016PD13-05 FLUORESCENCE IN SITU HYBRIDIZATION (FISH) IN THE DIAGNOSIS OF BLADDER AND UPPER TRACT UROTHELIAL CARCINOMA: A SINGLE-INSTITUTION EXPERIENCE Mark Mann, Ryan Cleary, Leonard Gomella, Scott Hubosky, Peter McCue, Demetrius Bagley, Costas Lallas, and Edouard Trabulsi Mark MannMark Mann More articles by this author , Ryan ClearyRyan Cleary More articles by this author , Leonard GomellaLeonard Gomella More articles by this author , Scott HuboskyScott Hubosky More articles by this author , Peter McCuePeter McCue More articles by this author , Demetrius BagleyDemetrius Bagley More articles by this author , Costas LallasCostas Lallas More articles by this author , and Edouard TrabulsiEdouard Trabulsi More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.976AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder cancer (BC) is the second most common urological cancer while upper tract urothelial carcinoma (UTUC) is far rarer with approximately 7000 new cases per year. Urine cytology is the only widespread urinary marker is use but has many limitations including low sensitivity and wide interpreter variability. We evaluated the multi target UroVysion (Abbott Molecular, IL) fluorescence in situ hybridization (FISH) assay for the diagnosis of urothelial cancer (UC) in patients with BC only, UTUC only, and combined BC and UTUC. METHODS A single institution retrospective analysis was conducted comparing sensitivity, specificity, positive predictive value, and negative predictive values. FISH within 6 months of endoscopic evaluation were obtained from outpatient voided urine samples. Our institutional pathology department confirmed disease stage from specimens obtained during endoscopic evaluations for lower tract disease. For upper tract disease, disease was confirmed by retrograde ureteroscopy, biopsies of visual lesions, and site specific upper tract cytology. RESULTS A total of 415 patients submitted voided FISH specimens. Seventy-seven patients after evaluation for UC had no evidence of malignancy. One hundred and seventy-six patients had BC, 65 had UTUC, and 32 had both BC and UTUC. Overall FISH was more sensitive than cytology 54.9% in comparison with cytology 42.2% (p=0.01). However, specificity favored cytology 92.9% compared to 73.5% with FISH (p<0.01). After analyzing BC only patients, the same significant finding of increased sensitivity and decreased specificity was identified. For UTUC, FISH had a trend toward being more sensitive, but it was not significant. For both upper and lower tract disease there was no difference in the sensitivities. Cytology had improved positive predictive value (PPV) over FISH, 76.9% in comparison to 64.6% (p=0.02). Negative predictive value (NPV) also favored cytology 74.2% vs. 64.9% (p = 0.02). When analyzing individual cohorts, cytology had improved PPV for BC alone patients. UTUC showed no difference for PPV and NPV. For both UTUC and BC, NPV was slightly favored for FISH over cytology 93.2% vs. 91.2% (p= 0.03). CONCLUSIONS Voided urine FISH testing does offer a higher detection of UC for BC compared to voided cytology; however specificity was worse. In analysis of the separate cohorts, FISH does not appear to improve detection of UC in patients with either UTUC only or both BC and UTUC. FISH alone should not replace cytology as a standalone urinary tumor assay for detection of UC. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e299 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Mark Mann More articles by this author Ryan Cleary More articles by this author Leonard Gomella More articles by this author Scott Hubosky More articles by this author Peter McCue More articles by this author Demetrius Bagley More articles by this author Costas Lallas More articles by this author Edouard Trabulsi More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract P1-01-13: Does HER2 immunohistochemistry-guided targeted FISH analysis help identify intratumoral heterogeneity in breast cancer?

Abstract Introduction: The updated 2013 ASCO/CAP HER2 guidelines proposed revised scoring with the aim of capturing all patients eligible for targeted therapy. The current guidelines also address intratumoral heterogeneity using immunohistochemistry (IHC) as a platform to identify and target heterogeneously staining areas for FISH analysis. We wanted to determine if identifying the most intense areas of staining in IHC equivocal / 2+ cases would help identify genetic heterogeneity and potentially more FISH amplified patients. Materials and Methods: Our lab offers HER2 immunohistochemistry by HercepTest™ (Dako) or 4B5 (Ventana), and dual probe FISH (Abbot Molecular or Dako IQ). We offer HER2 testing as IHC or FISH only, and also as a reflex FISH following equivocal (2+) IHC. HER2 tests performed and interpreted after 10/2013, following the implementation of the ASCO/CAP 2013 guidelines, to 5/2015 were included in this study. Both IHC and FISH were manually read following ASCO/CAP 2013 guidelines by one and two observers respectively. Equivocal (2+) cases with heterogeneous staining showing areas with ≥ 10% weak or moderate circumferential membrane staining were circled by a pathologist and reflexed to HER2 FISH. In those cases where samples were received for FISH testing only, the pathologist circled the entire area of invasive tumor on H&amp;E slides. All circled tumor areas were evaluated before signal enumeration of the FISH probes. Results: 1805 HER2 FISH test requests received in our laboratory during the study period had interpretable FISH results. 1210 of these cases did not have prior HER2 IHC testing performed in our laboratory ("FISH only"), 595 had reflex FISH testing following equivocal (2+) IHC ("IHC2+/reflex FISH"). Amplification rates between the "IHC2+/reflex FISH" group of patients with targeted areas for FISH and the "FISH only" group where the entire invasive carcinoma was marked on H&amp;E slide and scored were similar (16.3% and 20% respectively). 10/595 (1.7%) "IHC2+/reflex FISH", and 15/1210 (1.4%) "FISH only" groups showed genetic heterogeneity by FISH with discrete population of amplified tumor cells respectively (p=0.4) FISH Testing Utilizing Entire Tumor Area * n=1210FISH Testing Utilizing Most Intense Staining Areas (following equivocal IHC) n=595Genetic Heterogeneity15 (1.4%)10 (1.7%)Amplified242 (20.0%)97 (16.3%)*includes equivocal IHC, and FISH as first line of testing Conclusion: The updated HER2 ASCO/CAP guidelines recommend that intratumoral heterogeneity for HER2 may be easier to detect with IHC which can be used to guide FISH enumeration. In our study, identifying areas of more intense staining on IHC slides for targeting FISH analysis did not result in a difference in identification of genetic heterogeneity. Citation Format: Gulbahce HE, Factor RE, Geiersbach KB, Downs-Kelly E. Does HER2 immunohistochemistry-guided targeted FISH analysis help identify intratumoral heterogeneity in breast cancer?. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-01-13.

The interpopulation differences between nucleotide sequence polymorphisms in alleles of the STR-loci of human chromosomal DNA

The objective of the present work was to study the phenomenon of nucleotide sequence polymorphism in alleles of the STR-loci of human chromosomal DNA and to estimate its interpopulation differences with a view to the search for the molecular-genetic markers to be used as an efficient tool for the determination of belonging of the subjects of interest to a given population. We undertook the comprehensive analysis of amplified DNA fragment sequence polymorphism (AFSP) and amplified DNA fragment length polymorphism (AFLP) with the use of the PLEX-ID-TM analytical mass-spectrometry platform (Abbott Molecular, USA). The interpopulation differences were estimated in terms of the presence or the absence of single nucleotide replacements (SNP) in the STR markers based on a few population samples. Some of the loci of interest were found to have allelic variants the occurrence of which was significantly different in individual samples. Such alleles are of importance for the further investigation since they can be regarded as potential ethno-geographical markers. Their application opens up the new promising prospects for the expert detection of the ethnic affiliation of individual subjects.