Viswanatha, David S.

Abstract Introduction: The BCR-ABL1 genomic fusion event in chronic myelogenous and B-lymphoblastic leukemias (CML, ALL) commonly produces the major chimeric transcript variants e13 (or e14)-a2 or e1-a2, which are translated respectively to p210 and p190 bcr-abl1 oncoproteins. However, substantial transcript heterogeneity is present including alternative splice forms in a given leukemia, as well as rare alternative chimeric transcript types. Relatively little is known regarding the molecular response of alternative transcript variants of BCR-ABL1 during tyrosine kinase inhibition (TKI) therapy, in part because of the lack of effective disease burden monitoring methods. We present an approach to detection of the various BCR-ABL1 mRNA types using a multiplex quantitative RT-PCR (QRT-PCR) methodology. This technique enables diagnosis and molecular tracking of unusual BCR-ABL1 variants and can improve our understanding of how these uncommon leukemias behave in response to TKI therapies. Furthermore, the method is more generally applicable for simultaneous QRT-PCR detection and measurement of different mRNA species. Methods: Patient samples for diagnostic evaluation were obtained and total RNA extraction was performed using the Qiagen RNeasy Mini Kit (Qiagen USA, Valencia, CA). Reverse transcription was done using Multiscribe Reverse Transcriptase (Life Technologies, Grand Island, NY). Eleven fluorescent primer and probe mixes were designed (e1a2-FAM, e6a2-Cyc 5, e13a2-FAM, e14a2-FAM, e19a2-Cyc 5, e1a3-FAM, e12a3-Cyc 5, e13a3-FAM, e14a3-FAM, e19a3-Cyc 5, and Gusb reference control-HEX). 10 uL reactions were performed (95 C X 10 min, 45 cycles of 95 C X 10 sec and 60 C X 30 sec) using four multiplex wells per sample. Double strand oligonucleotides (oligos) for each target type were synthesized (IDT, Coralville IA) and diluted in a range of 106, 105, 104, 103, 100, 10, 1 and 0.1 copies/uL. Results of the new multiplex QRT-PCR assay were compared against our standard method of qualitative multiplex RT-PCR and fluorescent probe hybridization. Results: Results for the target BCR-ABL1 oligo controls are summarized in the table. The data show a wide dynamic range of detection with reproducible copy number detection at very low level. All PCR targets had excellent correlation coefficients (R2) and good to excellent PCR efficiency. Specificity was excellent, except for some cross-reactivity of A3 templates. This minor interference did not detract from assay performance or interpretation. Thirty-two patient samples were evaluated using the multiplex QRT-PCR assay and the current lab method. For 13 known BCR-ABL1 negative and 19 positive patient samples, concordance/accuracy and specificity were 100%. Furthermore, relative quantification of transcript abundance was obtained by the multiplex QRT-PCR approach, enabling distinction between diagnostic and post-treatment mRNA levels. Alternative splicing events producing additional transcripts (e1-a2) were resolved in the same specimen and always quantified well below the primary transcript level. Table 1.Transcript TypeDynamic Range(log)Sensitivity(copies/uL)Specificity(%)R2Efficiencye1-a2 (p190)6101000.971.74e6-a2 (p195)711000.951.76e13-a2/ e14-a2 (p210)61010011.99e19-a2 (p230)610E19A3 also detected0.991.89e1-a3 (p190)6101000.981.90e13-a3/ e14-a3 (p210)610E12A3 also detected11.99e12-a3410001000.991.78e19-a3 (p230)61010011.95 Conclusions: We demonstrate a powerful and relatively simple method for concurrent detection and quantitative measurement of multiple common and unusual alternative BCR-ABL1 mRNA transcripts. The results of this study provide a basis for more detailed monitoring of molecular disease levels during TKI therapy in leukemic patients with rare BCR-ABL1 types, and in turn, potentially enhance the ability to more optimally individualize clinical management for these patients. The platform is flexible and novel BCR-ABL1 transcript types can also be incorporated relatively easily. A similar strategy is applicable to the detection of chimeric mRNA species in other types of leukemias. Our approach can also be used to evaluate targeted RNA expression profiles of other prognostic or theranostic tumor markers in individual patients. Disclosures No relevant conflicts of interest to declare.

Key Points This article characterizes a cryptic IGH/CCND1 rearrangement in MCL by NGS. Mate-pair sequencing can help in accurately diagnosing MCL in cases of cyclin-D1–positive B-cell lymphoma with negative CCND1 FISH studies.

e18560 Background: Myelodysplastic syndromes (MDS) are rare hematological neoplasms typically seen in the elderly. Young MDS (yMDS) patients ( < 50 years old) have been reported to comprise between 3-6% in the Surveillance, Epidemiology and End Results (Ma X et al, Cancer 2007; Rollison R et al, Blood 2008) with a better overall survival (OS). Methods: 1012 MDS patients from 1993 to 2015 were found after IRB approval was obtained. All cases had their bone marrow slides reviewed at our institution. yMDS Patients were included as cohort 1, while the rest as cohort 2. Survival estimates were calculated using Kaplan-Meier curves and univariate and multivariate analyses was based on log-rank testing using JMP software version 10. Results: We found 68 (7%) yMDS patients with a median age of 42 years (range, 18-49). Female gender was more common (43% vs 31%, p = 0.05) while platelets were lower in yMDS (61 vs 102, p < 0.0001). Therapy related MDS (t-MDS) was more frequent (33% vs 17%, p = 0.005), as was transformation to acute myeloid leukemia (AML) in yMDS (28 % vs 11%, p = 0.0004). Allogenic hematopoietic cell transplantation (HCT) was more frequent in yMDS (42% vs 4%, p < 0.0001). Survival outcome: Median OS was longer for cohort 1 vs cohort 2 but did not reach statistical significance (43 vs 21 months, p = 0.1). Median progression free survival (PFS) was shorter for cohort 1 vs cohort 2 but also did not reach statistical significance (8 vs 12 months, p = 0.3). Median OS for cohort 1 based on R-IPSS was 44, 105, 40, 18, and 12 months for very low, low, intermediate, high and very high risk groups, respectively (p = 0.09). Median OS was shorter in t-MDS vs de novo MDS in yMDS (13 vs 47 months, p = 0.04). yMDS patients who transformed to AML had worse median OS (18 vs 93 months, p = 0.001). On multivariate analysis neither t-MDS nor R-IPSS had a statistically significant impact on OS. Conclusions: MDS is rarely diagnosed under the age of 50 with IPSS-R being less powerful in detecting mOS. Among yMDS patients, there was a higher proportion of t-MDS compared to older patients with subsequent higher rates of AML transformation and higher rates of allogeneic HCT. In our study, we did not find an improved OS for yMDS patients compared to older patients.

Hereditary erythrocytosis is associated with high oxygen affinity hemoglobin variants (HOAs), 2,3‐bisphosphoglycerate deficiency and abnormalities in EPOR and the oxygen‐sensing pathway proteins PHD, HIF2α, and VHL. Our laboratory has 40 years of experience with hemoglobin disorder testing and we have characterized HOAs using varied protein and molecular techniques including functional assessment by p50 analysis. In addition, we have more recently commenced adding the assessment of clinically relevant regions of the VHL , BPGM, EPOR , EGLN1 (PHD2), and EPAS1 (HIF2A) genes in a more comprehensive hereditary erythrocytosis panel of tests. Review of our experience confirms a wide spectrum of alterations associated with erythrocytosis which we have correlated with phenotypic and clinical features. Through generic hemoglobinopathy testing, we have identified 762 patients with 81 distinct HOA Hb variants (61 β, 20 α), including 12 that were first identified by our laboratory. Of the 1192 cases received for an evaluation specific for hereditary erythrocytosis, approximately 12% had reportable alterations: 85 pathogenic/likely pathogenic mutations and 58 variants of unknown significance. Many have not been previously reported. Correlation with clinical and phenotypic data supports an algorithmic approach to guide economical evaluation; although, testing is expanded if the suspected causes are negative or of uncertain significance. Clinical features are similar and range from asymptomatic to recurrent headaches, fatigue, restless legs, chest pain, exertional dyspnea and thrombotic episodes. Many patients were chronically phlebotomized with reported relief of symptoms.