Citations

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 Maintenance of telomere length has long been established to play a role in the biology of cancer, and several lines of evidence suggest that it may be especially important in myeloid malignancy. Measuring leukocyte telomere length (LTL) directly in case control studies is problematic because telomere length is likely to be influenced by the disease process. Recent studies have led to the identification of a robust genetic predictor of LTL, which we have used to compare differences in predicted telomere length in case control studies of myeloid leukemia and myelodysplastic syndromes (MDS) using Mendelian randomization (MR). MR is a method used to measure the association between a trait (e.g. telomere length) and an outcome (e.g. myeloid malignancy) that can't be measured directly by utilizing genetic variants known to be associated with the trait as instrumental variables. The use of genetic predictors of telomere length reduces the possibility that the association is explained by reverse causation and confounding likely to be relevant in case-control studies where telomere length is measured directly because genotypes in the general population should be randomly distributed. Additionally, predicted telomere length has the added advantage of representing telomere length over the lifetime of the individual while measuring LTL directly will represent only one time point in an individual's life. Methods Myeloid leukemia (AML and CML) and MDS cases were identified by rapid case ascertainment through the Minnesota Cancer Surveillance System (MCSS). Centralized pathology and cytogenetics reviews were conducted to confirm diagnosis and classify by subtypes. Controls were identified through the Minnesota State driver's license/identification card list. We used the Sequenom platform to genotype seven SNPs that were validated predictors of LTL in a recently published meta-analysis. We estimated associations between individual SNPs and myeloid malignancy using multivariable logistic regression. Mendelian randomization was used to evaluate the association between predicted LTL and risk of myeloid malignancy. Results We included 253 AML cases, 353 MDS cases, 145 CML cases, and 1,042 controls. We observed a significant association between longer predicted telomere length and AML in analyses adjusted for sex and age (OR 4.82 per additional kilobase of average telomere length, 95% CI 1.32-17.63). The individual SNP analyses suggest that the association is largely driven by rs10936599, located in TERC (OR 1.47 per allele, 95% CI 1.14-1.90; Figure). We did not observe statistically significant associations between predicted LTL and MDS or CML. Discussion In this analysis of predicted telomere length and myeloid malignancy, we identified a significant association between longer predicted leukocyte telomere length and AML. In contrast, no significant association was observed for MDS or CML although we did observe an elevated OR for longer predicted telomere length and MDS that did not reach statistical significance. Figure 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.

Abstract PURPOSE/OBJECTIVE Dose constraints for re-irradiation of recurrent primary brain tumors are not well-established, especially for treatment volumes too large for stereotactic radiotherapy. This prospective trial was performed to test dose constraints for conventionally-fractionated re-irradiation of recurrent primary brain tumors MATERIALS/METHODS A single-institution, prospective trial of 21 adults with recurrent brain tumors was performed. Electronic dosimetry records from the first course of radiation (RT1) were obtained and deformed onto the simulation CT for the second course of radiation (RT2). Treatment plans for RT2 were developed that met protocol-assigned dose constraints for RT2 alone and the composite dose of RT1+RT2. Dose constraints were also based on histology and interval since RT1. The primary endpoint was the rate of symptomatic brain necrosis after RT2. RESULTS Twenty one adults enrolled from March 2017 to May 2018. Twelve had glioblastoma, four had oligodendroglioma, two had anaplastic astrocytoma, and one each had choroid plexus papilloma, hemangiopericytoma, and pleomorphic xanthroastrocytoma (PXA). Twenty patients were treated with VMAT and one was treated with proton CSI. Median RT1-RT2 interval was 45 months (range, 9–141 months). Median RT2 dose was 42.8 Gy (range, 17.5–60 Gy). Median PTV volume was 208 cc (range, 7–1537 cc). Median imaging followup was 9 months (range, 1–20 months). Two months after RT2, the patient with PXA developed a trapped temporal horn adjacent to the RT2 treatment volume; pathology from emergent resection revealed necrotic brain tissue. The patient recovered fully and lived another 18 months until dying of disease progression. No other patient developed symptomatic radionecrosis. Median overall survival from RT2 for all patients was 11 months (range, 3–20 months). CONCLUSION Re-irradiation can be performed with conventionally fractionated schemes. Given the low rate of symptomatic radionecrosis, the dose constraints described here are a starting point for future studies of conventionally fractionated re-irradiation.

Abstract INTRODUCTION Glioblastoma has a poor prognosis, further complicated by postoperative seizures. We analyzed various factors associated with postoperative seizures in glioblastoma. METHODS Retrospective chart review of 932 patients who underwent craniotomy for glioblastoma (777=83.4% newly diagnosed; 155=16.6% recurrent) at our institution (2002–2014). Postoperative seizures were defined as those occurring within 14 days of surgery. RESULTS Our cohort consisted of 49.9% (n=465) males with mean age of 55.8 (range 9–91) years. In total, 69 (7.4%) had postoperative seizures with mean time from surgery to seizure of 3.1 days (IQR=1–4). Of 536 patients (57.5%) receiving seizure prophylaxis, those treated with Keppra were more likely to have postoperative seizures compared to those on Dilantin (10.9% vs 5.0%; p=0.022). Primary tumor patients were less likely to seize postoperatively compared to recurrent tumor patients (9.7% vs 19%; p=0.016). Patients presenting with seizure as chief complaint (12.1% vs 5.1%; p=0.025) and those with >20% of Chromosome 10 deletion were more likely to have seizures postoperatively (12.4% vs 6.1%; p=0.0029). Risk factors for postop seizures for newly-diagnosed patients included subtotal resection versus gross total resection (28.0% vs 7.1%; p=0.022). Risk factors for postop seizures for recurrent patients were >5% EGFR amplification (23.5% vs 5.8%; p=0.0021) and hyponatremia within mean 2.5 days (IQR 0–1) after surgery (20.8% vs 7.1%; p=0.035). CONCLUSION We identified several risk factors for postoperative seizures in glioblastoma patients, most of which were clinical (seizure as chief complaint, the antiepileptic chosen for prophylaxis, and extent of resection), while some were molecular and linked to genes whose products have demonstrated links to epileptogeneicity (Chromosome 10, EGFR). These findings may assist physicians in detecting glioblastoma patients that are at higher risk for postoperative seizures and in providing prophylaxis to reduce morbidity.

Background Implant-based breast reconstruction (IBR) accounts for 70% of postmastectomy reconstructions in the United States. Improving the quality of surgical care in IBR patients through accurate measurements of outcomes is necessary. The purpose of this study is to compare the American College of Surgeons National Surgical Quality Improvement Program (NSQIP) data from our institution to our complete institutional health records database. Methods Data were collected and recorded for all patients undergoing IBR at our institution from 2015 to 2017. The data were completely identified and compared with our institutional NSQIP database for demographics and complications. Results The electronic health records data search identified 768 IBR patients in 3 years and NSQIP reported on 229 (30%) patients. Demographics were reported similarly among the 2 databases. Rates of tissue expander/implant infections (5.9% vs 1.8%; P = 0.003) and wound dehiscence (3.5% vs 0.4%; P = 0.003) were not reported similarly between our database and NSQIP. However, the rates of hematoma (2.7% vs 1.8%) and skin flap necrosis (2.5% vs 1.8%) were comparable between the two databases. In our database, 43% of all complications presented after 30 days of surgery, beyond NSQIP's capture period. Conclusions Databases built on partial sampling, such as the NSQIP, may be useful for demographic analyses, but fall short of providing data for complications after IBR, such as infections and wound dehiscence. These results highlight the utility and importance of complete databases. National comparisons of clinical outcomes for IBR should be interpreted with caution when using partial databases.

OBJECTIVES: Diabetic gastroparesis (Gp) occurs more often in type 1 diabetes mellitus (T1DM) than in type 2 diabetes mellitus (T2DM). Other diabetic end-organ complications include peripheral neuropathy, nephropathy, and retinopathy (together termed triopathy ). This study determines the prevalence of diabetic complications (retinopathy, nephropathy, and peripheral neuropathy) in diabetic patients with symptoms of Gp, assessing the differences between T1DM and T2DM and delayed and normal gastric emptying (GE). METHODS: Diabetic patients with symptoms of Gp underwent history and physical examination, GE scintigraphy, electrogastrography with water load, autonomic function testing, and questionnaires assessing symptoms and peripheral neuropathy. RESULTS: One hundred thirty-three diabetic patients with symptoms of Gp were studied: 59 with T1DM and 74 with T2DM and 103 with delayed GE and 30 without delayed GE. The presence of retinopathy (37% vs 24%; P = 0.13), nephropathy (19% vs 11%; P = 0.22), and peripheral neuropathy (53% vs 39%; P = 0.16) was not significantly higher in T1DM than in T2DM; however, triopathies (all 3 complications together) were seen in 10% of T1DM and 3% of T2DM ( P = 0.04). Diabetic patients with delayed GE had increased prevalence of retinopathy (36% vs 10%; P = 0.006) and number of diabetic complications (1.0 vs 0.5; P = 0.009); however, 39% of diabetic patients with delayed GE did not have any diabetic complications. DISCUSSION: In diabetic patients with symptoms of Gp, delayed GE was associated with the presence of retinopathy and the total number of diabetic complications. Only 10% of patients with T1DM and 3% of those with T2DM had triopathy of complications, and 39% of diabetic patients with Gp did not have any diabetic complications. Thus, the presence of diabetic complications should raise awareness for Gp in either T1DM or T2DM; however, diabetic Gp frequently occurs without other diabetic complications.

Abstract Background CDI is an important cause of morbidity and mortality in SOT and HCT patients (pts). In retrospective single-center analyses, severe disease and relapse were common. We undertook a multicenter prospective observational study to evaluate outcomes of CDI among both SOT and HCT patients. Methods Adults with a first episode of CDI, defined as 3 liquid stools/24 h with the detection of C. difficile toxin in stool, within the first 2 years of SOT or HCT were recruited from 12 centers internationally in the INSIGHT network. At enrollment, demographics, comorbidities, medication histories and outcomes were collected prospectively over 90 days to assess clinical cure, recurrences and complications and to define baseline risk factors for clinical cure and recurrent CDI. Results 132 patients (81 SOT, 51 HCT (32 allogeneic)) were enrolled: median age 56 years, 62.1% were males, 97% were hospitalized. 80.3% were diagnosed by DNA assay. CDI occurred a median of 20 days post transplant (IQR: 6–133). 108 patients were on PPIs. 98.5% were on antibiotics before CDI. 1st line treatment regimen was oral vancomycin in 66 patients (40 SOT, 26 HCT), metronidazole in 48 patients (27 SOT, 21 HCT), both drugs in 14 (10 SOT, 4 HCT), fidaxomicin (3) and linezolid (1). Rejection within 60 days before CDI was uncommon (6.2% SOT) as was GVHD (27.5%). 110 patients (83%, 95% CI: 46–89)) (65 SOT, 45 HCT) had clinical cure; 18% (95% CI: 11–27) had recurrent CDI, 2 were admitted to the ICU due to CDI, 11 (8.3%) died (2 HCT related to CDI). Among baselines variables, only first-line regimen was associated with a higher rate of clinical cure (P = 0.003), most notably for SOT. Factors that did not have a statistically significant negative impact on clinical cure included sex, age > 60, race, country, transplant type, steroids, diabetes, CMV viremia/disease, WBC > 15,000, creatinine > 1.5 mg/dL, or specific antibiotic given prior to CDI. Higher recurrence rates were associated with metronidazole-only regimen (OR: 4.6, 95% CI: 1.6–12.8; P = 0.004) and a history of CMV after transplant (OR: 5.2, 95% CI: 1.7–15.7; P = 0.003). Conclusion Despite their immunosuppressed state, recurrence, ICU admission and mortality occurred in a minority of SOT and HCT with CDI. Initial use of metronidazole and CMV viremia/disease were associated with higher recurrence rates. Disclosures All authors: No reported disclosures.