Nguyen, Phuong L.

Introduction Germline predisposition syndromes (GPS) are inherited disorders associated with germinal aberrations that increase the risk of malignancies. While aberrations in certain genes increase the risk for all types of malignancies (Tp53, ATM, CDKN2A, CHEK2), there is a growing list of genes associated specifically with hematological malignancies (GATA2, RUNX1, DDX41, ETV6, ANKRD26). At our institution, we have established a hematology GPS clinic to diagnose and manage GPS and with this report, detail our experience with 130 patients. Methods GPS were investigated in pediatric and adult patients with one or more first degree relatives with hematological/visceral malignancies or in those with antecedent thrombocytopenia (ANKRD26, RUNX1, ETV6), or with specific syndromic features (short telomere syndromes/STS, GATA2 haploinsufficiency, Fanconi anemia/FA, Shwachman-Diamond syndrome/SDS). Depending on the phenotype, specific functional assays such as flow-FISH for telomere length assessment and chromosomal breakage assays were ordered. After informed consent and genetic counselling, germline testing was carried out on peripheral blood mononuclear cell, skin fibroblast, or hair follicle-derived DNA. A custom-designed marrow failure NGS panel (200 genes) was used in most cases and interrogation of variants, in silico studies, and functional assays were carried out as previously described (Mangaonkar et al MC Proc 2019). Copy number variations were identified by aCGH. At the time of progression/worsening cytopenias, bone marrow/lymph node biopsies and NGS (next generation sequencing) were carried out where indicated. Results 130 patients with germline predisposition have been identified to date. The spectrum of disorders seen include STS 29 (22%), FA 17 (13%), GATA2 16 (12%), Diamond Blackfan anemia/DBA 13 (10%), RUNX1-FPD 12 (9%), ATM deletions/mutations 11 (8%), ANKRD26 6 (5%), SDS 5 (4%), DDX41 4 (3%), MPL 3 (2%), CHEK2, MECOM, Tp53 mutations 2 (2%) each, and CBL, CEPBA, ELANE, NF1, CDKN2A, CSF3R, ETV6, and GATA1 mutations, 1 (1%) each. Evidence for clonal evolution (CCUS) and hematological malignancies were seen in 51 (39%) patients, involving all the aforementioned genes/syndromes with the exception of DBA, CBL, ETV6, MPL, CSF3R, and GATA1. Seven (64%) of 11 patients with germline ATM deletions/mutations developed lymphoid malignancies; homozygous ATM (Follicular NHL-1, Burkitt lymphoma-1, T-ALL-1, T-LPD-1) and heterozygous ATM (T-PLL-1, DLBCL-1, CLL-1). Clonal evolution occurred in 11 (69%) of 16 GATA2 haploinsufficient patients (CCUS-2, MDS-3, CMML-1, AML-5) and in 7 (58%) of 12 RUNX1-FPD patients (CCUS-1, MDS-1, MDS/MPN-3, AML-2). Five of 29 (17%) STS patients had clonal progression (CCUS-2, MDS-2, AML-1), and 5 (29%) of 17 FA patients progressed to MDS-2 or AML-3. JMML was seen in one patient with a germline NF1 mutation, while 1 (20%) of 5 SDS patients progressed to AML. NGS data at progression was available in 24 (55%) of 44 myeloid/CCUS progressions, with somatic truncating ASXL1 mutations being most frequent (29%), followed by RAS pathway mutations (15%). AML/MDS progressions in STS, FA, and SDS were universally associated with complex/monosomal karyotypes, translating to refractory disease. Seventeen (39%) of 44 patients with myeloid predisposition underwent allogenic HCT (GATA2-7, FA-3, RUNX1-FPD-3, STS-2, NF1-1, Tp53-1), with 10 (59%) being alive at last follow up (Table 1). Conclusion We demonstrate the spectrum of germline aberrations associated with predisposition to hematological malignancies and outline the phenotypic heterogeneity of clonal transformation. The advent of NGS allows identification of clonal progression earlier than morphological changes, with mutations in ASXL1 and RAS pathway genes being commonly implicated. This study supports the universal development of dedicated germline predisposition clinics. Disclosures Pruthi: CSL Behring: Honoraria; Genentech Inc.: Honoraria; Bayer Healthcare: Honoraria; HEMA Biologics: Honoraria; Instrumentation Laboratory: Honoraria; Merck: Honoraria.

Abstract Objectives Distinguishing between T-cell and natural killer (NK)–cell neoplasms could be difficult given their overlapping immunophenotype. In this study, we investigated whether a flow cytometry assay with cytoplasmic staining for CD3 could be used for this purpose. Methods Flow cytometry immunophenotyping was performed on 19 surface CD3 (sCD3)–negative mature T-cell neoplasms, 10 sCD3-positive mature T-cell neoplasms, 13 mature NK-cell neoplasms, and 19 normal controls. In addition to routine antibody panels (CD2, sCD3, CD4, CD5, CD7, CD8, CD16, CD45, CD56, CD57, CD94, CD158a, CD158b, CD158e, NKG2A TCRγ/δ), cytoplasmic staining for a monoclonal CD3 antibody (clone SK7/Leu-4) was assessed in all cases. A molecular study for T-cell receptor (TCR) gene rearrangement and an immunohistochemical study for TCRβ were performed. Results Our data showed all T-cell neoplasms were uniformly positive for cytoplasmic CD3 (cCD3) regardless of sCD3 expression, whereas 85% of NK-cell neoplasms completely lacked cCD3 expression. The 2 cases with classic NK-cell immunophenotype but partial cCD3 expression showed no molecular genetic features of T-cell lineage by TCR gene rearrangement studies. Conclusions Uniform cCD3 positivity and homogeneous cCD3 negativity highly suggest T-cell and NK lineage, respectively. When partial cCD3 expression is encountered, additional confirmatory studies should be pursued for the most accurate lineage assignment.

8534 Purpose: Tumor necrosis factor (TNF) is a putative mediator of the cancer anorexia/weight loss syndrome. This study was designed to determine if etanercept (a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human 75 kilodalton TNF receptor linked to the Fc portion of human IgG1) could palliate this syndrome. Methods: 63 evaluable patients were randomly assigned to etanercept 25 mg SQ twice/week versus a comparably-administered placebo, both of which were to be given for at least 12 weeks. All patients had an incurable malignancy, acknowledged loss of weight and/or appetite as a concern, and reported weight loss of > 5 pounds over 2 months and/or a daily intake of < 20 calories/kg body weight. The above sample size provided 81% power to detect a 24% difference in the percentage of patients who gained >/= 10% of baseline weight. Results: At baseline, groups were comparable on age, gender, tumor type, and degree of weight loss. Over time, weight gain was minimal in both groups; no patient gained >/= 10% baseline weight. Appetite questionnaires (the NCCTG Anorexia/Cachexia Questionnaire and FACT-AN) revealed negligible improvements in both groups. Median survival was comparable: 175 days versus 148 in etanercept-treated and placebo-exposed patients, respectively (p=0.82). Finally, preliminary data on adverse events showed that etanercept-treated patients had higher rates of neurotoxicity (29% versus 0%) but lower rates of anemia (0% versus 19%) and thrombocytopenia (0% versus 14%). Infection rates were negligible in both groups. Clinical correlative data on TNF genotyping will be available at the time of the meeting. Conclusion: This TNF inhibitor does not appear to palliate the cancer anorexia/weight loss syndrome. The study was supported by CA37404, the American Institute for Cancer Research, and Amgen. No significant financial relationships to disclose.

Background With the exception of therapy related MDS due to treatment with alkylating agents and/or radiation or with a topoisomerase II inhibitor, little is known about the etiology of myelodysplastic syndromes (MDS). A few studies have evaluated pre-existing medical conditions as risk factors for MDS, with evidence suggesting that autoimmune disease is associated with an increased risk of MDS. Here, we evaluated associations between history of autoimmune disease, overall and by individual disease subtype, and risk of MDS in a population-based case control study. Methods MDS cases were identified by rapid case ascertainment through the population-based Minnesota Cancer Surveillance System (MCSS). Participants were recruited if they were diagnosed with MDS between April 1, 2010 and October 31, 2014. Eligibility criteria included residence in Minnesota, age at diagnosis between 20 and 85 years, and ability to understand English or Spanish. Centralized pathology and cytogenetics review were conducted to confirm diagnosis and classify by subtypes. Controls were identified through the Minnesota State driver's license/identification card list. History of autoimmune disease was reported using a self-administered questionnaire; proxy interviews were not conducted. Unconditional logistic regression with adjustment for age, sex and confounders (education, NSAID use, benzene exposure, BMI) was used to calculate adjusted odds ratios (ORs) and 95% confidence intervals (CI) for autoimmune disease and MDS. Results A total of 399 cases with a confirmed diagnosis of MDS and 1399 controls were included. Cases were significantly more likely to report a diagnosis of any autoimmune disease when compared with controls (OR=1.49, 95% CI 1.13-1.97). As illustrated in Figure 1, specific associations were observed for hypothyroidism (OR=2.07, 95% CI 1.39-3.08), ulcerative colitis (OR=2.26, 95% CI 1.09-4.69), Crohn's disease (OR=3.88, 95% CI 1.23-12.2), and systemic lupus erythematosus (SLE; OR=7.52, 95% CI 1.98-28.6). Autoimmune conditions were more commonly reported in both female cases and female controls when compared with males; however, associations for hypothyroidism and ulcerative colitis were significantly associated with MDS in males in an analysis stratified by sex. Associations between autoimmune disease and MDS remained statistically significant after exclusion of therapy-related MDS cases. Discussion Our results validate previous findings of an association between autoimmune disease and MDS risk from population-based registry studies and case-control studies. In addition, our results provide additional evidence to suggest that this association is largely driven by hypothyroidism, inflammatory bowel disease, and SLE. This increased MDS risk following a diagnosis for an autoimmune condition is hypothesized to be due to shared genetic or environmental risk factors, treatment for autoimmune diseases, altered immune surveillance or damage to the bone marrow caused by the autoimmune condition. Further studies are required to confirm which of these is the most plausible explanation. Disclosures No relevant conflicts of interest to declare.

Background MDS is a heterogeneous disease and the revised International Prognostic Scoring System (IPSS-R) is utilized in prognostication. The percentage (%) of blasts in the bone marrow is determined in the aspirate morphologically. Though the former is the standard of care the blast percentage is also reported by flow cytometry and biopsy which can many times be inconsistent. We previously presented the utilization of biopsy based blast percentage which showed meaningful prognostic groups compared to aspirate. In this study we compare the blasts as reported by the aspirate and flow cytometry in MDS-EB in calculating IPSS-R. Methods The MDS database was reviewed for cases of MDS-EB after due IRB approval at the Mayo clinic. We calculated IPSS-R scores based on the aspirate blast % (IPSS-RAsp) and flow blast% (IPSS-Rfl). The aspirate blast percentage was reported morphologically. Suboptimal aspirates were excluded from the study. The flow blast percentage was determined by immunophenotyping. The overall survival (OS) was determined by IPSS-RAsp and IPSS-RFl. OS estimates were calculated by Kaplan-Meier curves and log-rank testing using JMP v.13. Uno's concordance statistic was used to compare the 2 risk scoring systems. Results Of 1322 patients, 431 (33%) cases were identified with MDS-EB out of which 120 (29%) cases had blasts reported in the aspirate and flow. Based on aspirate MDS EB1: 54% (n=65), MDS EB2 46% (n=55). The hematological, cytogenetic and R-IPSS categories were compared between MDS-EB1 and MDS- EB 2. The blast percentage and hemoglobin levels was significantly different between MDS-EB1 and EB2 as seen in table 1, however the IPSS-R risk groups were not significantly different. The flow cytometry was concordant with aspirate in 66/120 (55%) cases. Out of the dis-concordant cases only 20% (11/54) was upstaged by flow cytometry with most of the patients being down staged as expected by the techniques used in processing the blood and hence not reliable when reported low (Figure 1). The OS outcomes based on the IPSS- R asp, IPSS-Rfl areshown in figure 2A,2B .The p value with aspirate based R-IPSS was more significant than flow cytometry based R-IPSS (p= 0.0007 vs 0.0174). We compared the two models for observed OS differences using the Uno model which was not statistically significant. (p= 0.6) Conclusions Both models did not show a difference which is likely due to the very small sample size. However flow cytometry did down stage more patients when disconcordant and may have less value in that setting. It would be ideal to compare all 3 models aspirate, biopsy and flow cytometry however we did not have enough number of patients to do the comparison. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Al-Kali:Astex Pharmaceuticals, Inc.: Research Funding.

Introduction: Whereas morphological assessment and cytogenetics have been the cornerstone for the diagnosis of myelodysplastic syndrome (MDS), flow cytometry and mutational analyses are novel, evolving techniques. These diagnostic procedures play a role in identifying pre-MDS cases including idiopathic cytopenia of undetermined significance (ICUS) and clonal cytopenia of undetermined significance (CCUS). Aim: To assess flow cytometric findings in ICUS and CCUS and their correlation with progression to MDS or myeloid neoplasm (MN), concurrent genetic aberrations, and underlying pathological evolution. Methods: Patients (Pts) who had undergone evaluation to rule out MDS and had retrospectively undergone flow cytometry, cytogenetic, mutational and morphological assessment were included. Flow cytometry results were classified as normal (no abnormality or 1 equivocal result), abnormal (2 abnormalities or at least 1 abnormality and 1 equivocal result), or atypical (1 abnormality or 2-3 equivocal results). Next generation sequencing included mutation analysis of: ASXL1, BCOR, BRAF, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, GATA1, GATA2, IDH1, IDH2, JAK2, KIT, KRAS, MPL, MYD88, NOTCH1, NPM1, NRAS, PHF6, PTPN11, RUNX1, SETBP1, SF3B1, TERT, TET2, TP53, U2AF1, WT1, ZRSR2. Results: A) Characteristics: 229 pts were assessed (median age 60 and 69% were males). Based on pathologist interpretation, 87 (38%) pts met diagnostic criteria of MDS, 106 (46%) pts had normal/reactive bone marrow (BM), and 36 (16%) pts had BM findings suspicious for MDS. Median time to follow-up was 24 months for MDS pts, and 32 months for pts with cytopenias. B) Morphology: Pts who had a BM biopsy that was suspicious for MDS by pathologist interpretation were more likely to develop MDS compared to pts with normal/reactive pathology report with an incidence rate (IR) of 16% versus 0.9% (p= .0005). Pts who were suspicious for MDS based on the BM biopsy were more likely to have underlying mutations compared to pts with normal/reactive BM (IR 83% versus 69%, p= .2). C) Flow cytometry: In the non-MDS cohort, 96 (67%) pts had normal flow results, 29 (20%) pts had atypical flow, and 17 (12%) pts had abnormal flow results compared to 16 (18%) pts, 18 (21%) pts, and 53 (61%) pts in the MDS cohort, respectively. Of the pts who had normal flow result (n=112), 16 were MDS, 16 were suspicious for MDS/MN, and 80 were normal/reactive. Pts who had biopsy interpretation suspicious for MDS/MN were more likely to have abnormal flow cytometry compared to normal/reactive BM biopsy (IR 55% versus 24%, p=.0003). IR of MDS was 4% in normal flow, 3% in atypical flow, and 12% in abnormal flow (p= .5). Likewise, IR of MN was 4% in normal flow, 20% in atypical flow, and 30% in abnormal flow (p= .1). D) Genetics (cytogenetics and mutation profiling): Cytogenetics was normal in 123 (86%) pts and abnormal in 19 (13%) pts. Pts who had abnormal cytogenetics were more likely to develop MN (IR 57% versus 5%, p= .002). Out of the 50 non-MDS pts tested for NGS, 38 (76%) were positive for mutations. 15 (39%) pts had 1 mutation, 8 (21%) pts had 2, 3 (8%) pts had 3, 6 (16%) pts had 4, 2 (5%) pts had 5, 1 (3%) pt had 7, and 1 (3%) pt had 8 mutations (61% had >1 mutation). Pts with positive mutations were more likely to develop MDS during follow-up compared to pts who did not have mutations (IR 16% versus 0%, p=.06). Additionally, flow cytometric aberrations significantly correlate with the number of mutations (p= .03). MN correlated significantly with BCOR mutation and RUNX1 (IR 60% (p= .008) and 50% (p=.02), respectively. E) Interaction between variables: In pts with positive mutations, flow cytometry did not correlate with progression to MDS with an IR of 17% of MDS in normal flow, 11% in atypical flow, and 17% in abnormal flow, (p= .9). There was no impact on overall survival neither in pts with abnormal flow results vs normal nor in pts with positive mutations vs wild type. Conclusion: Conventional diagnostic techniques remain the cornerstone of MDS diagnosis with flow cytometry and NGS arising as novel techniques that correlate significantly with progression of pre-MDS pts to MDS. Flow had an impact on pathology final interpretation but did not add value in pt with a positive mutation analysis. Larger studies are needed to confirm our findings. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Al-Kali:Astex Pharmaceuticals, Inc.: Research Funding.

Background: Genetic variants resulting in quantitative defects in the production of α-spectrin are associated with autosomal recessive hereditary spherocytosis (HS). Because erythroid cells produce α-spectrin in a 3-4:1 ratio in relation to β-spectrin, one defective allele is typically well-tolerated and a single null allele combined with a second partially defective allele may display a sub-clinical phenotype. Protein expression can be reduced by nonsense, frameshift, missense or splicing variants but reported large deletion events are extraordinarily rare (n = 1). Splicing variants include the low expression alleles αLELY - Low Expression LYon (SPTA1:c.6531-12C>T, p.?) and αLEPRA-Low Expression PRAgue (SPTA1:c.4339-99C>T, p.?) which reduce protein expression by 50% and 16%, respectively. Here, we present a family study with a whole gene SPTA1 deletion that characterizes the patient phenotype when a full gene mutation is co-inherited with the αLEPRA and αLELY low expression alleles. Methods: The 6 month-old male proband had a history of congenital Coombs negative hemolytic anemia requiring transfusion from 3 months of age and in whom a red cell membrane disorder was clinically suspected. Pretransfusion CBC and peripheral blood smear results were unknown. Transfused CBC indices were as follows: RBC 4 x 1012/L, HGB 11 g/dL, MCV 76 fL, MCH 27 pg, MCHC 36 g/dL and a reticulocyte count of 1.8%. The proband's mother, of Cuban descent, and father, of European descent, had no history of anemia or HS. Targeted high throughput sequencing of genes related to hereditary hemolytic anemia was performed for the detection of single nucleotide variants (SNVs) and small insertion/deletion variants (indels). Relative quantitative real time PCR (RT-PCR) was utilized to confirm large structural variants. Results: Sequencing identified a large heterozygous deletion that spanned the entire SPTA1 gene (NC_000001.11:g.(?_158656337)_(158581024_?) in the proband and his mother. In addition, the proband had inherited hemizygous αLEPRA from his father. The mother had hemizgous αLELY. The father showed heterozygous αLEPRA and no other SPTA1 variants (Table 1). Conclusion: To our knowledge, only Iolascon et al in 2011 has reported a SPTA1 gene large deletion (SpectrinExeter, exon 11-exon 12 deletion, loss of 83 amino acids) which resulted in a structurally abnormal protein and ellipto-poikilocytosis phenotype when in trans to αLELY. Our current case is the first reported SPTA1 full gene deletion and our family study allows comparison of the effects of coinheritance of the low expression alleles αLEPRA and αLELY. When in combination with complete absence of the SPTA1 gene in trans configuration, αLELY manifests in subclinical effects and αLEPRA causes a recessive HS phenotype, which resulted in severe anemia. This contrast further demonstrates that, unlike αLEPRA, αLELY does not decrease the α-spectrin concentration sufficiently to contribute to the baseline phenotype regardless of the severity of the second allele mutation for quantitative disorders such as HS. Disclosures No relevant conflicts of interest to declare.

Background: Voxelotor (GBT440) is a small molecule allosteric effector with high specificity for hemoglobin (Hb) that inhibits Hb S polymerization by increasing hemoglobin oxygen affinity. Developed as an oral, once-daily medication, it is undergoing phase 3 randomized clinical trials in sickle cell disease patients. Therapeutic monitoring of Hb quantitative fractions is routine in sickle cell disease patients to assess medication and transfusion effects and future treatment requirements. In the clinical laboratory, some therapies can alter the Hb fraction percentages or characteristic protein migration/elution patterns and can mimic complex hemoglobinopathy disorders causing misinterpretation or unnecessary additional testing. We present the effects of voxelotor on hemoglobin identification/monitoring testing in multiple testing platforms. Methods: A whole blood anticoagulated sample from a male patient who was undergoing treatment for sickle cell disease was received for routine hemoglobin testing. Five additional samples with similar features were also received from three male patients (age range of all patients was 18 to 22 years) with sickle cell disease. Hemoglobin protein analysis was performed using standard methods including cation-exchange high performance liquid chromatography (HPLC), capillary electrophoresis (CE), isoelectric focusing (IEF), intact globin chain mass spectrometry (MS), sickle solubility and hemoglobin stability studies. DNA sequencing of the alpha (HBA1/HBA2) and beta globin (HBB) genes was performed on one sample. Results: CE (initial and 50:50 mix, figures 1A and 1B, respectively) revealed a Hb S peak with a shoulder and additional small peaks at position 203 (D zone) and 234 (cusp of zones E/A2). In some cases the 234 peak interfered with instrument designation of the A2 peak (50:50 mix with normal) and shifted the Hb S peak into the E zone which risks variant misidentification (figure 1C). Our laboratory overcame this limitation by substituting the normal sample with beta thalassemia trait to enhance the Hb A2 peak after mixing. HPLC tracings (figure 1D) showed a duplex peak pattern in the D and S windows with separate peak quantification percentages (index case showed elution at 4.18 and 4.37 minutes with 14.9% and 66.4%, respectively). The dual peaks detected by HPLC partially mimicked compound Hb S/D-Punjab. IEF (figure 1E) showed additional bands at -4.7, and -11.5 (Schneider-Barwick ratios). The additional peaks identified by CE and IEF mimicked an alpha chain hemoglobin variant; however, the expected hybrid patterns were atypical prompting further investigation. Sickle solubility was positive, heat stability studies were negative and mass spectrometry did not definitively detect abnormal peaks other than βS. DNA sequencing of the alpha and beta globin genes did not reveal abnormalities other than homozygous Hb S. Similar hemoglobin patterns were identified in all samples tested. Conclusion: Voxelotor treatment in sickle cell patients results in a distinctive pattern alteration in hemoglobin testing. Methods may not give accurate variant percentage estimation and the treatment may result in shifts in variant mobility causing erroneous laboratory results. Novel therapies are increasing in the treatment of hemoglobin disorders and medication effect should be a consideration when atypical patterns are encountered in the laboratory. Knowledge of these artifacts by clinicians and laboratorians is paramount to avoid erroneous diagnosis, unnecessary additional testing and inaccurate Hb variant quantitation for monitoring purposes. Notification of current treatment regimens to testing facilities is highly recommended in these patients. Figure 1 Disclosures No relevant conflicts of interest to declare.