Citations

Abstract INTRODUCTION: Our group has demonstrated that MASS-FIX is a quick, inexpensive, and accurate means to diagnose and monitor the serum and urine of patients with plasma cell disorders. Screening can be done with a MALDI-TOF MS and samples reflexed to microflow liquid chromatography coupled with electrospray ionization (ESI) and Q-TOF MS (microLC-ESI-Q-TOF MS). Because this technique provides a mass/charge (m/z) for a given patient's monoclonal protein, this method can provide greater sensitivity and specificity to monitor for complete response (CR), especially in patients receiving therapeutic monoclonal antibodies. Our goal was to assess the performance of miRAMM in patients with AL who have been classified as complete response using conventional means. METHODS: We identified 77 patients with AL who had both documented CR by immunofixation (Seibia) of the serum (SIFE), urine IFE (UIFE), and serum free light chain (FLC; The Binding Site) and paired serum samples to test by MALDI-TOF and ESI-TOF. No urine samples were available to test. Paired serum samples from baseline and approximately one year post-therapy were immunoaffinity purified using nanobodies targeting kappa, lambda, alpha, gamma and mu as previously described. For the MALDI-TOF (Bruker Microflex, LT), a range of 9,000 to 32,000 m/z was acquired. The m/z distribution was then visually inspected for the presence of a peak that was distinct from the polyclonal background in both the M+1 and M+2 light chain mass ranges. For the ESI-TOF, spectra were also collected on an TripleTOF 5600 quadrupole time-of-flight mass spectrometer (ABSciex, Vaughan ON, CA) in ESI positive mode with a Turbo V dual ion source with an automated calibrant delivery system. TOF MS scans were acquired from m/z 600−2500 with an acquisition time of 100 ms. RESULTS: Median age of the cohort was 58 (range 42, 81). Fifty-eight percent were male. No test was 100% sensitive at baseline with positive results as follows: abnormal FLC ratio, 82%; positive SIFE, 70%; 73% positive UIFE; positive serum MASS-FIX, 71%; and ESI-TOF, 79% . There was light chain isotype agreement in all cases, except for 2 patients for whom the SIFE and the FLCr disagreed. Of the 56 patients with baseline positive MASS-FIX, there was evidence of the original monoclonal protein in 5 patients (see figure for example of spectra illustrating same m/z before and after therapy). Of the 63 patients who had baseline positive ESI-TOF, there was evidence of the original monoclonal protein in 8 patients. Small unrelated monoclonal proteins were seen both by SIFE and by mass spectrometry techniques. With the spectrometry techniques, however, these transient oligoclonal bands could be distinguished from the original monoclonal protein when they shared the same isotype based on the difference in m/z. DISCUSSION: At baseline, the MALDI was able to identify the baseline monoclonal protein in a comparable number of patients as SIFE, UIFE, and FLC. Approximately 10% of patients thought to be in CR using routine screening tests were found to have persistence of their original clone by MALDI and by ESI-TOF. Small post-therapy unrelated monoclonal proteins were also seen both by IFE and by MALDI, but either the presence of a different isotype or in the case of MALDI, a different m/z, made it clear that the monoclonal protein was not related to the original clone. The sensitivity of the assay will improve significantly when we formalize the introduction of free light chain magnetic beads to capture the serum FLCs. Routine use of MASS-FIX of the urine will also increase performance characteristics. Figure. Figure. Disclosures Dispenzieri: Celgene, Takeda, Prothena, Jannsen, Pfizer, Alnylam, GSK: Research Funding. Gertz:Prothena: Honoraria; celgene: Consultancy; spectrum: Consultancy, Honoraria; Teva: Consultancy; Physicians Education Resource: Consultancy; annexon: Consultancy; Apellis: Consultancy; Alnylam: Honoraria; Medscape: Consultancy; Amgen: Consultancy; janssen: Consultancy; Research to Practice: Consultancy; Abbvie: Consultancy; Ionis: Honoraria. Kumar:Novartis: Research Funding; KITE: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; KITE: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncopeptides: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding. Dingli:Alexion Pharmaceuticals, Inc.: Other: Participates in the International PNH Registry (for Mayo Clinic, Rochester) for Alexion Pharmaceuticals, Inc.; Alexion Pharmaceuticals, Inc.: Other: Participates in the International PNH Registry (for Mayo Clinic, Rochester) for Alexion Pharmaceuticals, Inc.; Millennium Takeda: Research Funding; Millennium Takeda: Research Funding. Kapoor:Takeda: Research Funding; Celgene: Research Funding. Russell:Vyriad: Equity Ownership.

Abstract In a 20-year period, 223 patients (median age, 64.8 years) with myelofibrosis with myeloid metaplasia (MMM) had therapeutic splenectomy at our institution. Primary indications for surgery were transfusion-dependent anemia (45.3%), symptomatic splenomegaly (39.0%), portal hypertension (10.8%), and severe thrombocytopenia (4.9%). Operative mortality and morbidity rates were 9% and 31%, respectively. The 203 survivors of surgery had a median postsplenectomy survival time (PSS) of 27 months (range, 0-155). Among preoperative variables, thrombocytopenia (platelet count less than 100 × 109/L) and nonhypercellular bone marrow were identified as independent risk factors for decreased PSS. Durable remissions in constitutional symptoms, transfusion-dependent anemia, portal hypertension, and severe thrombocytopenia were achieved in 67%, 23%, 50%, and 0% of the patients, respectively. Histologic or cytogenetic features of bone marrow obtained before splenectomy did not predict a response in cytopenias. After splenectomy, substantial enlargement of the liver and marked thrombocytosis occurred in 16.1% and 22.0% of the patients, respectively. The thrombocytosis was associated with an increased risk of perioperative thrombosis and decreased PSS. The rate of blast transformation (BT) was 16.3%, and the risk of BT was higher in the presence of increased spleen mass and preoperative thrombocytopenia. However, the PSS of patients with BT was not significantly different from that of patients without BT. We conclude that presplenectomy thrombocytopenia in MMM may be a surrogate for advanced disease and is associated with an increased risk of BT and inferior PSS. However, the development of BT after splenectomy may not affect overall survival and does not undermine the palliative role of the procedure for the other indications.

Abstract Proteomics with great sensitivity and specificity effectively analyzes proteins by prior tryptic digestion and subsequent analysis by LC-MS/MS of the tryptic digest. We have utilized this approach in the development of a LC-MS/MS method to characterize minimum residual disease in multiple myeloma. The abundant antibodies produced by multiple myeloma plasma cells are identical and appear as a spike (M-spike) upon protein electrophoresis. The M-spike and histological examination of the bone marrow constitute part of the myeloma diagnostic repertoire. Upon treatment, myeloma cells and the antibodies they produce are reduced in number and amount and become increasingly hard to detect. The bone marrow biopsy serves as the “gold standard” test for clinical remission. We have developed a sensitive test for the presence of the monoclonal antibody produced by the plasma cells which may serve as a substitute for invasive bone marrow biopsy. We have focused on tryptic peptides comprising the variable CDR regions of the Ig light chains that are unique to each patients antibody clone. Utilizing 2-5 µL of patient plasma/serum from the initial M-spike sample we have utilized SDS-PAGE to yield a crudely purified immunoglobulin light chain. The light chain band is isolated, reduced, alkylated and trypsin digested with subsequent LC-MS/MS analysis to identify CDR specific tryptic peptide(s). These can be identified as variable peaks (elution time and mass) in a base peak ion chromatogram where constant region peptides of either lambda or kappa light chains (clone dependent) serve as “internal standards” for identifying the CDR tryptic peptides. The peptide’s mass and its corresponding MS/MS spectra are unique to this CDR tryptic peptide and this patient’s clone. The unique diagnostic peptide is isolated in subsequent samples by immunoaffinity purification of the target kappa/lambda clone, SDS-PAGE separation and LC-MS/MS analysis of the light chain gel band. An extracted ion chromatogram is generated based upon the CDR peptides identified in the initial analysis of the M-spike sample. In patients in clinical remission the presence of a significant signal from the targeted peptides indicates that targeting a CDR peptide from the M-spike protein is more sensitive than currently available diagnostic tools including immunofixation. Comparison of this test to the gold standard bone marrow biopsy will be examined. Disclosures: Barnidge: Mayo Foundation: provisional patent application for technology, provisional patent application for technology Patents & Royalties.

Abstract Abstract 4048 Poster Board III-983 Introduction Iron deficiency is the most common nutritional disorder in the world with an estimated two billion affected persons. Although commonly considered environmental in origin, the existence of multiple genetic disorders of iron metabolism in man, rodents and other vertebrates suggest a genetic contribution to iron deficiency. Methods: The Hemochromatosis and Iron Overload Screening (HEIRS) Study is a multi-center, multi-ethnic study in which transferrin saturation (TS), serum ferritin (SF), and HFE mutations were determined in 101,168 adults. To identify genomic locations associated with iron deficiency, we performed a genome-wide association study (GWAS) using DNA collected from white HEIRS Study participants who had SF ≤ 12 μg/L (cases) and an equal number of white controls (SF > 100 μg/L in men, SF > 50 μg/L in women) frequency-matched to cases by sex and geographic location. Men aged ≥ 25 y and women ≥ 50 y were included in both groups. Tissue body iron, an index of iron deficiency, was estimated from serum transferrin receptor (sTfR) and SF. Genotyping was performed with the Illumina HumanCNV370K Beadchip platform. Quality control filters excluded single nucleotide polymorphisms (SNPs) or samples with > 5% missing genotypes, SNPs showing heterozygosity or Hardy-Weinberg deviations (P<10−7), and SNPs with minor allele frequency < 0.02. Population admixture/structure was assessed using principal component analysis. Regression analysis was used to examine the association between outcomes (case-control status, tissue body iron, serum ferritin, transferrin receptor, serum iron, total iron-binding capacity [TIBC], and unsaturated iron-binding capacity [UIBC]) and each SNP genotype variable; covariates included age, sex, and geographic location. Replication for 56 SNPs was conducted in a population attending primary care clinics at a Veterans Affairs (VA) medical center using the iPlex platform. Eligibility within the VA replication population was restricted to age and self-reported white ethnicity as for the HEIRS subset from a total of 2559 people (138 women). VA participants with SF ≤ 20 μg/L were classified as iron-deficient cases and frequency matched 1:2 with controls (men with SF > 100 μg/L and women with SF > 50 μg/L). Results The GWAS genomic control parameter was not significantly different from 1.0. There were 392 cases (96 men) and 390 controls (96 men) in the HEIRS subset GWAS with average age (SD) of 59 (10) y and 61 (11) y, respectively. Geometric mean SF (minimum, maximum), and mean (SD) for sTfR and tissue body iron in the HEIRS subset were 7.5 (1.2, 12) μg/L, 6.4 (3.77) mg/kg and -2.0 (2.50) for cases and 141 (51, 881) μg/L, 3.0 (0.98) mg/kg and 10.8 (2.5) for controls. After quality control tests, GWAS analysis included genotype data for 331,060 SNPs in 734 individuals (364 cases, 370 controls). For the VA replication population there were 67 male and 11 female cases, and 136 male and 27 female controls for whom DNA was successfully prepared; the average age (SD) was 68 (12) y for cases and 65 (11) y for controls. Regression analysis identified seven SNPs within four independent regions that replicated associations found in the GWAS (GWAS P<1×10-4 and VA P<0.05).The SNP rs6735681 on chromosome 2p24 was associated with serum iron (GWAS P<3.9×10-5, VA P=0.038). Three SNPs on chromosome 2p14 (rs6750096, rs2698541 and rs2698530) significantly influenced both TIBC and UIBC (GWAS P<2.9×10-5, VA P< 0.04 for all). Two SNPs in the TF gene region on chromosome 3q22 also showed significant effects on TIBC and UIBC (GWAS P<4.7×10-6, VA P<0.03 for all). The SNP rs10512064 on chromosome 9q21 was associated with serum ferritin concentration and tissue body iron (GWAS P<2.5×10-5, VA P<0.05 for both). Conclusion: From these GWAS and replication studies, we have identified three new genetic loci and one known iron gene, TF, associated with iron phenotype variability. These results point to specific loci as targets for gene identification and TF polymorphisms as determinants of iron metabolism, which in turn may play a role in regulation of body iron status. Disclosures: No relevant conflicts of interest to declare.

In a 20-year period, 223 patients (median age, 64.8 years) with myelofibrosis with myeloid metaplasia (MMM) had therapeutic splenectomy at our institution. Primary indications for surgery were transfusion-dependent anemia (45.3%), symptomatic splenomegaly (39.0%), portal hypertension (10.8%), and severe thrombocytopenia (4.9%). Operative mortality and morbidity rates were 9% and 31%, respectively. The 203 survivors of surgery had a median postsplenectomy survival time (PSS) of 27 months (range, 0-155). Among preoperative variables, thrombocytopenia (platelet count less than 100 × 109/L) and nonhypercellular bone marrow were identified as independent risk factors for decreased PSS. Durable remissions in constitutional symptoms, transfusion-dependent anemia, portal hypertension, and severe thrombocytopenia were achieved in 67%, 23%, 50%, and 0% of the patients, respectively. Histologic or cytogenetic features of bone marrow obtained before splenectomy did not predict a response in cytopenias. After splenectomy, substantial enlargement of the liver and marked thrombocytosis occurred in 16.1% and 22.0% of the patients, respectively. The thrombocytosis was associated with an increased risk of perioperative thrombosis and decreased PSS. The rate of blast transformation (BT) was 16.3%, and the risk of BT was higher in the presence of increased spleen mass and preoperative thrombocytopenia. However, the PSS of patients with BT was not significantly different from that of patients without BT. We conclude that presplenectomy thrombocytopenia in MMM may be a surrogate for advanced disease and is associated with an increased risk of BT and inferior PSS. However, the development of BT after splenectomy may not affect overall survival and does not undermine the palliative role of the procedure for the other indications.

Abstract Background: Myeloablative hematopoietic cell transplantation (HCT) provides a cure for children with hemoglobinopathies, but transplant related early and late morbidity remains a challenge. Toxicities associated with myeloablative chemotherapy include early complications such as mucositis, hemorrhagic cystitis, seizures, and hepatic injury. Late effects such as infertility and compromised linear growth impair long-term quality of life, often decreasing enthusiasm for the procedure. Reduced intensity preparative regimens are especially attractive in children with non-malignant disorders to mitigate these toxicities but have been associated with increased risk of graft rejection. The primary objective of this study was to determine the toxicity and efficacy of a non-myeloablative preparative regimen using alemtuzumab, fludarabine, and melphalan followed by HCT from HLA matched related donors (MRD) in children with hemoglobinopathies. Methods: Following institutional review board approval, and parent and/or patient consent, participants were enrolled at 18 centers. Children < 21 years of age with severe sickle cell disease (SCD) manifestations, or transfusion dependent (> 8 red blood cell transfusions per year) thalassemia with a MRD and a performance status > 40 were eligible for inclusion. The preparative regimen included alemtuzumab (total dose 48 mg) IV (between days –22 and –19), fludarabine (30 mg/m2/day) (days –8 to –4) and melphalan (140 mg/m2) on day -3. Graft versus host disease (GVHD) prophylaxis included a calcineurin inhibitor (tapered after day 100, and methotrexate (7.5 mg/m2 on days 1, 3 and 6) or mycophenolate mofetil. Five patients also received methylprednisone (1 mg/kg/day) between days 1 and 28; this practice was discontinued in 2007. Results: A total of 52 children (43 with SCD and 9 with thalassemia), median age 11 years (range, 10m - 20y) underwent HCT between March 2003 and July 2014. Of these, 46 received bone marrow, 5 received marrow and cord blood (CB), and 1 received CB alone. Median follow up was 35.5 months (range, 3 – 136). Forty-nine children were alive at last follow up (Figure 1); 48 were symptom-free; one CB recipient had disease recurrence following graft rejection and successfully underwent a 2nd HCT. No hepatic veno-occlusive disease was noted. Three deaths 6, 11 and 21 months post HCT were from GVHD related causes [bronchiolitis obliterans (n=1); infection with GVHD (n=2)]. The cumulative incidence of graft failure and transplant related mortality was 1.9% and 5.7% respectively. The mean time to engraftment of neutrophils (ANC >500/cu mm) and platelets (>50,000/cu mm) was 13.0 (range 5-21) and 25.9 (range 8-120) days respectively. Three patients had neurologic toxicity (seizures/PRES) post HCT. At the time of last follow up, 35 patients (67%) were complete donor chimera (>90% donor) and 16 (31%) were mixed chimera (28-89% donor). Acute GVHD (grade 1-3) was noted in 30.8%. Four had grade I, 6 had grade II, and 6 had grade III GVHD. No patient developed grade IV aGVHD. Chronic GVHD was noted in 13%. Of 43 patients that were alive without disease and >6 months post HSCT, 38 had successfully discontinued all immune suppression and maintained donor chimerism post withdrawal. Immune reconstitution was robust by the end of the first year post transplant; infectious complications (23 of 30 CMV+ recipients had reactivation) were noted primarily in the first 3 months post HSCT. Conclusions: Children with hemoglobinopathies undergoing MRD HCT tolerated this non-myeloablative preparative regimen well, with minimal early toxicities. The event-free survival rate was comparable to those achieved with myeloablative regimens. Withdrawing immunosuppression was not associated with graft loss or recurrent GVHD. GVHD complications resulted in a low mortality rate. Alternative GVHD prophylaxis may further improve event-free survival. No gonadal toxicity (determined by hormone levels) has been identified to date in 15 HCT recipients of pubertal age (>13 years). Follow up however is early and in progress to determine long-term toxicities. These results support consideration of reduced intensity conditioning regimens for HCT in children with hemoglobinopathies as an alternative to standard myeloablative regimens. Figure 1: Overall/Event-free survival following non-myeloablative conditioning andMRD transplants for hemoglobinopathy Figure 1:. Overall/Event-free survival following non-myeloablative conditioning andMRD transplants for hemoglobinopathy Disclosures No relevant conflicts of interest to declare.

Abstract Abstract 2105 The existence of multiple inherited disorders of iron metabolism in man, rodents and other vertebrates suggests genetic contributions to iron deficiency. We hypothesized that common variants in genes involved in iron metabolism may modulate susceptibility or resistance to the development of iron deficiency in humans. To examine the association between single nucleotide polymorphisms (SNPs) in key genes involved in iron metabolism pathways, we previously performed a genome-wide association study using DNA collected from white men aged ≥25 y and women ≥50 y in the Hemochromatosis and Iron Overload Screening (HEIRS) Study with serum ferritin (SF) ≤12 μg/L (cases) and controls (SF >100 μg/L in men, SF >50 μg/L in women). We now report on a multiethnic follow-up association study of HEIRS participants. Candidate SNPs were identified from our GWAS and the scientific literature. Population samples of whites, African Americans, Hispanics, and Asians from the U.S. and Canada were analyzed separately for association between SNPs and case-control status and each of seven quantitative outcomes including serum iron, total iron-binding capacity (TIBC), unsaturated iron-binding capacity (UIBC), transferrin saturation, SF, serum transferrin receptor, and body iron. There were 1084 white (357 cases, 727 controls), 153 Asian (51 cases, 102 controls), 221 African American (77 cases, 144 controls) and 233 of 239 Hispanic individuals (79 cases, 160 controls) that passed quality control. For the African-American and Hispanic samples, ancestry proportions were estimated based on genotypes of ancestry informative markers. Regression analysis was used to examine the association between case-control status and quantitative serum iron measures and 1134, 1115, 1113 and 1134 SNP genotypes in the white, African-American, Hispanic, and Asian population samples, respectively. Model predictors included age, sex, the estimated ancestry proportion (for African American and Hispanic only), genotype, and measured covariates that showed nominally significant associations with the outcome. Three chromosomal regions showed evidence of association across multiple populations, including SNPs in the TF gene on chromosome 3q22, the TMPRSS6 gene on chromosome 22q12, and loci on chromosome 18q21. SNP rs1421312 in TMPRSS6 was associated with serum iron in whites (p=4.7×10−7) and was replicated in African Americans (p=0.0012).Twenty SNPs in the TF gene region were significantly associated with TIBC in the white sample (p<4.4×10−5); six SNPs were replicated in other ethnicities (p< 0.01). SNP rs10904850 in the CUBN gene on 10p13 was significantly associated with serum iron in the African-American sample (P=1.0×10−5). Mutations in the TMPRSS6 gene have been implicated in iron-refractory iron deficiency anemia through linkage studies. We found a novel SNP in TMPRSS6 that was associated with serum iron in whites and replicated in African Americans, suggesting a role for this SNP in increasing the risk of iron deficiency in affected persons. Our results confirm known associations with iron measures and give evidence of their role in different ethnic groups, a unique aspect of this study, suggesting origins in a common founder. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: Current laboratory methods for detection of monoclonal gammopathies include a panel of serum protein electrophoresis (SPEP), immunofixation electrophoresis (IFE) and serum free light chain quantitation (FLC). Our group has recently described a new assay based on matrix assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) which is capable of detecting, isotyping and quantitating M-proteins in a single assay. The basic principle of the method involves leveraging the unique mass resulting from light chain (LC) Ig gene rearrangement in B-cells. In a similar fashion to SPEP, the mass distribution of LCs after dissociation from their heavy chains can be examined for the presence of an over expressed light chain. In this study we retrospectively compared the MALDI-TOF MS method to SPEP, IFE and Hevylite ratios for monitoring patients with monoclonal gammopathies. Methods: Serum from patients who had at least 1 diagnostic and 4 serial follow-up samples was enriched for IgG, IgA, IgM, kappa and lambda using nanobodies. After disassociating the heavy and light chains by reduction, the five purified samples were spotted onto a Bruker Microflex MALDI plate. Automated acquisition (~10 seconds/sample) was performed and the five LC mass spectra from each enrichment were overlaid. M-proteins were detected, quantitated and isotyped by the presence of distinct peaks (m-spike) within LC mass to charge regions. The serial patient samples were then measured by MALDI-TOF and a comparison of results was made to previous aquired data. The results were then analyze in light of clinical and lab data in order to evaluate the ability of the MALDI-TOF MS method to monitor patients with monoclonal gammopathies. Results: In M-protein positive patient samples, serial dilution revealed MALDI-TOF MS to have ~10-times lower limit of detection than IFE. M-protein isotype in the cohort by MALDI-TOF MS was 100 percent concordant with the isotype from IFE. The MALDI TOF M-protein quantitation was linear over a clinically acceptable range (0.1 to 6 g/dL) and had improved linearity over SPEP at lower M-protein levels. M-proteins quantitation by MALDI-TOF MS compared well with SPEP with a slope of 1.16 (R2 -0.93). Hevylite Ig ratios for each isotype were statistically similar to those from MALDI-TOF MS demonstrating the ability of the MALDI to measure isotype suppression. For some patients, the MALDI method was able to detect M-proteins in patients with normal Hevylite ratios. Finally, the results for each patient were then compared over the course of monitoring. The time evolution change in M-protein concentration was similar among the three methods with the exception of a few samples in which the MALDI-TOF detected residual M-proteins in treated patients not detected by the other methods. Conclusion: This preliminary study demonstrates that MALDI-TOF MS method can provide detection, quantitation and isotyping data suitable for monitoring patients. This is a significant finding since the MALDI-TOF method is amendable to automation, is rapid and in its current format is cost-competitive with current clinical assays. Disclosures Murray: Mayo Clinic: Patents & Royalties: Patent Application Filed. Mills:Mayo Clinci: Patents & Royalties: Patent Application Filed. Barnidge:Mayo Clinic: Patents & Royalties: Patent Application Filed.