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Abstract Background: Mechanisms of hereditary erythrocytosis have been elucidated recently. These include high oxygen affinity (HOA) hemoglobin (Hb) variants, bisphosphoglycerate mutase (BPGM) deficiency, abnormalities in the erythropoietin receptor (EPOR) and oxygen-sensing pathway (OSP) proteins prolyl hydroxylase domain-2 (PHD2), hypoxia-inducible factor-2 alpha subunit (HIF2A), and von Hippel-Lindau (VHL). We present our experience with these disorders. Methods: Evaluation of erythrocytosis patients' blood samples for HOA Hb variants by protein or DNA sequencing methods has been performed in our laboratory for over 30 years. Testing has included alkaline/acid electrophoresis, isoelectric focusing, capillary electrophoresis, high performance liquid chromatography, mass spectrometry and Sanger sequencing. BPGM deficiency was determined by enzyme activity assay and/or genetic testing. Since 2012 we have evaluated the EPOR, EGLN1 (PHD2), EPAS1(HIF2A), and VHL genes. Reflexive evaluations, which include testing for p50 and pertinent Sanger sequencing of HBB, HBA1, HBA2, EPOR (exon 8), EGLN1 (exons 1-5), EPAS1 (exon 12) and VHL, have been performed on a subset of cases. Genetic results were correlated with clinical and phenotypic data. Results: Retrospective database review identified 62 confirmed HOA Hb variants (48β, 14α) with clinical erythrocytosis (Table 1). Multiple Hb variants were silent on at least one protein method but showed decreased p50 values. One homozygous BPGM mutation case was identified. Of the 394 cases evaluated for EPOR and OSP abnormalities, 39 cases (10%) contained genetic alterations. Of these, 11 were known pathogenic mutations and 28 were novel alterations including 4 pathogenic, 12 likely pathogenic and 12 variants of unknown significance (VUS). Eighteen EGLN1, 10 EPAS1, and 7 EPOR alterations were detected, all heterozygous. Four VHL mutation cases were identified: 3 homozygous/compound heterozygous known mutations and one novel heterozygous VUS (p.R200Q). All EPOR mutations resulted in a premature stop codon. Most of the EPAS1 alterations were located near proline 531, although two predicted splice site disturbances were found. The EGLN1 alterations were novel, variable, and affect the enzyme's catalytic domain. All 4 VHL cases involved the R200 amino acid position in at least one allele. Serum Epo levels varied: decreased in EPOR (5/5); normal in PHD2 (11/11) and most HIF2A (6/7) and VHL (3/4) cases; increased in 1 HIF2A and 1 VHL case. p50 was normal in EPOR and OSP cases (31/31 tested). Conclusion: A spectrum of abnormalities was identified. Salient points include: 1) Diagnostically, a high index of suspicion for HOA Hb variants is required as they can give false negative results by routine protein detection methods. p50 and Epo levels were useful for triage. 2) Phenotypically, serum Epo levels were low in EPOR and normal in PHD2 and HIF2A cases. Unexpectedly, the two classic Chuvash polycythemia VHL cases were associated with normal Epo levels. 3) Mechanistically, the alterations were truncating in EPOR, variable and scattered in EGLN1 and clustered near proline 531 in EPAS1. VHL mutations were rare and consistently affected the R200 amino acid position in at least one allele. Novel alterations were frequent, especially in EGLN1. 4) Clinically, many patients were asymptomatic but a subset demonstrated recurrent headaches and one had chest pain. Clotting complications included cerebrovascular accident in one EPAS1 and EGLN1 case each and portal vein thrombosis in one EGLN1 case. Table 1. Clinically significant high oxygen affinity Hb variants at Mayo Clinic n Beta Alpha >30 Malmo Tarrant 10 - 20 Bethesda, San Diego, Andrew-Minneapolis, M-Saskatoon, Olympia, Syracuse, Abruzzo Dallas 5 - 10 Ty Gard, Ypsilanti, Alberta, Coimbra, North Chicago J-Cape Town 2 - 4 Brigham, Kempsey, Little Rock, Puttelange, Wood, Brisbane, Cowtown, Creteil, Linkoping, Osler, Potomac, Providence, Ranier, Sparta, Vanderbilt, Heathrow, Helsinki, Hiroshima, Homozygous HPFH, McKees-Rocks, Pierre Benite, Regina Chesapeake, Ethiopia, Chiapas, Columbia Missouri, Burlington, Legnano 1 Alcorn County*, Bologna-St. Orsola, Bunbury, Cambridge-MA, Cardarelli, Chandigarh, Johnstown, Nantes, Nebraska, Olomouc, Palmerston North, Pitie-Salpetriere, Saint-Jacques, South Milwaukee Linwood, Longview*, Milledgeville, Sarasota Springs, Voorhees * novel Disclosures Pardanani: Stemline: Research Funding.

Abstract Background: Myelodysplastic syndromes (MDS) are rare hematological neoplasms that are more typically seen in elderly patients. Young patients (< 50 years old) have been reported to comprise between 3-6% in the Surveillance, Epidemiology and End Results (SEER) (Ma X et al, Cancer 2007; Rollison R et al, Blood 2008) with a better overall survival (OS). We hereby report the characteristics of young MDS patients with long survival follow-up. Methods: A total of 1012 MDS patients' data from Oct 1993 to Dec 2015 at Mayo Clinic were reviewed after appropriate IRB approval was obtained. All cases had their bone marrow slides reviewed at our institution. Patients youngers than 50 years old (yMDS) were included as cohort 1, while the rest was included as control group (cohort 2). Prognostic factors were analyzed by univariate and multivariate analyses. 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: Characteristics: We identified 68 (7%) yMDS patients with a median age of 42 years (range, 18-49). Female gender was more common in yMDS (43% vs 31%, p= 0.05). Upon comparison between cohort 1 and 2, only platelets were significantly lower in yMDS (61 vs 102, p <0.0001), but not white blood cells, hemoglobin or degree of marrow fibrosis. MDS subtyping according to World Health Organization 2016 showed single lineage dysplasia in 15% vs 2%, multilineage dysplasia 24% vs 36% , ring sideroblasts 9% vs 15%, isolated del (5q) 1% vs 3%, excess blasts 44% vs 31%, and unclassifiable in 6% vs 5%. As expected, therapy related MDS (t-MDS) was more frequent in yMDS (33% vs 17%, p= 0.005). Transformation to acute myeloid leukemia (AML) was also more frequent in yMDS (28 % vs 11%, p= 0.0004). Compared to cohort 2, yMDS IPSS-R scores were very high, high, intermediate, low, and very low in 24% vs 13%, 29% vs 16%, 21% vs 20%, 19% vs 35%, and 7% vs 17%, respectively. 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 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 high risk groups, respectively (p= 0.09). Median OS was shorter in t-MDS vs de novo MDS in cohort 1 (13 vs 47 months, p = 0.04). Young patients who had transformed to AML had a 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. IPSS-R was less powerful in detecting differences between its risk groups for this patient population, although more than half of the patients with yMDS had either high or very high risk. Among this cohort of yMDS patients, there was a significantly higher proportion with therapy-related myeloid neoplasms compared to older patients (one third of patients), with subsequent higher rates of transformation to AML and higher allogeneic HCT. In our study, we did not find an improved OS for yMDS patients compared to older patients. Disclosures Al-Kali: Celgene: Research Funding; Onconova Therapeutics, Inc.: Research Funding.

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.