Citations

AbstractPrevious studies have shown that K562 chronic myelogenous leukemia cells are resistant to induction of apoptosis by a variety of agents, including the topoisomerase II (topo II) poison etoposide, when examined 4 to 24 hours after treatment with an initiating stimulus. In the present study, the responses of K562 cells and apoptosis-proficient HL-60 acute myelomonocytic leukemia cells to etoposide were compared, with particular emphasis on determining the long-term fate of the cells. When cells were treated with varying concentrations of etoposide for 1 hour and subsequently plated in soft agar, the two cell lines displayed similar sensitivities, with a 90% reduction in colony formation at 5 to 10 μmol/L etoposide. After treatment with 17 μmol/L etoposide for 1 hour, cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP), DNA fragmentation, and apoptotic morphological changes were evident in HL-60 cells in less than 6 hours. After the same treatment, K562 cells arrested in G2 phase of the cell cycle but otherwise appeared normal for 3 to 4 days before developing similar apoptotic changes. When the etoposide dose was increased to 68 μmol/L, apoptotic changes were evident in HL-60 cells after 2 to 3 hours, whereas the same changes were observed in K562 cells after 24 to 48 hours. This delay in the development of apoptotic changes in K562 cells was accompanied by delayed release of cytochrome c to the cytosol and delayed appearance of peptidase activity that cleaved the fluorogenic substrates Asp-Glu-Val-Asp-aminotrifluoromethylcoumarin (DEVD-AFC) and Val-Glu-Ile-Asp-aminomethylcoumarin (VEID-AMC) as well as an altered spectrum of active caspases that were affinity labeled with N-(Nα-benzyloxycarbonylglutamyl-Nε-biotinyllysyl) aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone [z-EK(bio)D-aomk]. On the other hand, the activation of caspase-3 under cell-free conditions occurred with indistinguishable kinetics in cytosol prepared from the two cell lines. Collectively, these results suggest that a delay in the signaling cascade upstream of cytochrome c release and caspase activation leads to a long latent period before the active phase of apoptosis is initiated in etoposide-treated K562 cells. Once the active phase of apoptosis is initiated, the spectrum and subcellular distribution of active caspase species differ between HL-60 and K562 cells, but a similar proportion of cells are ultimately killed in both cell lines.

Abstract Abstract 4886 Detection of high molecular weight light chain oligomers in urinary exosomes of patients with AL amyloidosis. Background Exosomes are microvesicles that are part of the multivesicular body (MVB) pathway. They are created by the inward budding of the cell surface membrane and contain both surface bound membrane proteins and cytosolic proteins which can be used to identify the cell of origin. Immunoglobulin light chain amyloidosis (AL) occurs as the result of amyloid formation by the misfolding of monoclonal light chains (LC) and deposition of these amyloid fibrils in various soft tissues. This reaction requires the organization of the monoclonal LC's into protofibrils which are then weave into amyloid fibrils. This study was undertaken to determine whether urinary exosomes of glomerular origin contain intermediate species of amyloid formation. Method Urine samples from patients with AL, light chain deposition disease (LCDD), multiple myeloma (MM) and monoclonal clonal gammopathy of undetermined significance (MGUS) were collected. Urinary exosomes were isolated and separated into fractions by gradient centrifugation. Western blots were performed on the urinary exosome fractions using anti-kappa or anti-lambda antibodies. Glomerular fractions were identified using antibodies directed toward podocin. Results Urine samples were collected from 5 patients with AL, 2 from LCDD, 1 from MM and 1 MGUS. On the Western blot, immunoglobulin LC were seen in all exosomal fractions in patients with AL amyloidosis, LCDD, MM but not MGUS which is similar to normal controls (not shown). In patients with AL, oligomeric species were found in the highest concentrations in fraction 4 and 5 (Figure 1). Fraction 4 and 5 were also stained for podocin, a glomerular protein (not shown). The highest molecular weight species was ∼250 kd which corresponds to a LC decamer. High molecular weight species were also identified in 1 of 2 LCDD patients corresponding to a tetramer. The band was identified in fraction 10 which had polycystin-1 expression suggesting a tubular origin. No high molecular weight LC species was found in patients with MM or MGUS. Conclusion Our study found high molecular weight LC species corresponding to the intermediates involved in protofibril formation in urinary exosomes of patients with AL. Smaller (tetramer) high molecular weight LC species were also found in a patient with LCDD but not in patients with MM and MGUS. Not only were the high molecular weight LC species found exclusively in the diseases characterized by deposition of LC aggregates, they were also found in the segments of the nephron where the deposits were expected: glomerulus for AL and tubular epithelium for LCDD. This is consistent with our current understanding of the pathogenic mechanisms of these diseases. We believe urinary exosomes are a powerful tool in the study of diseases involving self-aggregation of monoclonal proteins. It has tremendous potential in both diagnostic and scientific research in this area. Disclosures Gertz: celgene: Honoraria; millenium: Honoraria, Membership on an entity's Board of Directors or advisory committees.

The cDNA for eosinophil granule major basic protein (MBP) encodes a prepromolecule with a total length of 222 amino acids (preproMBP). PreproMBP includes a secretory leader of 15 amino acids, an acidic propiece of 90 amino acids, and a basic MBP portion of 117 amino acids. The function of the propiece, which has a predicted pI of 3.9, is unknown, but it gives proMBP an overall acidic charge. Because proMBP is not found in mature eosinophils, we analyzed eosinophil differentiation in interleukin-5 (IL-5)–stimulated umbilical cord stem cells cultured for 24 days. By immunofluorescence, proMBP appeared by day 6 and peaked on day 18, whereas MBP was prominent at days 12 to 24. By day 6, Western blots detected heterogeneous glycosylated 33-kD proMBP; its peak expression occurred on day 12. Western blots showed sequential processing of 33-kD proMBP to an 18-kD intermediate form and finally to 14-kD MBP. By dual label immunoelectron microscopy, proMBP was localized primarily to large uncondensed eosinophil granules, whereas MBP was localized to granules containing a condensed central area. Thus, proMBP is likely expressed and processed as the granule condenses in a multistep process to 14-kD MBP in differentiating eosinophils.

Previous studies have shown that K562 chronic myelogenous leukemia cells are resistant to induction of apoptosis by a variety of agents, including the topoisomerase II (topo II) poison etoposide, when examined 4 to 24 hours after treatment with an initiating stimulus. In the present study, the responses of K562 cells and apoptosis-proficient HL-60 acute myelomonocytic leukemia cells to etoposide were compared, with particular emphasis on determining the long-term fate of the cells. When cells were treated with varying concentrations of etoposide for 1 hour and subsequently plated in soft agar, the two cell lines displayed similar sensitivities, with a 90% reduction in colony formation at 5 to 10 μmol/L etoposide. After treatment with 17 μmol/L etoposide for 1 hour, cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP), DNA fragmentation, and apoptotic morphological changes were evident in HL-60 cells in less than 6 hours. After the same treatment, K562 cells arrested in G2 phase of the cell cycle but otherwise appeared normal for 3 to 4 days before developing similar apoptotic changes. When the etoposide dose was increased to 68 μmol/L, apoptotic changes were evident in HL-60 cells after 2 to 3 hours, whereas the same changes were observed in K562 cells after 24 to 48 hours. This delay in the development of apoptotic changes in K562 cells was accompanied by delayed release of cytochrome c to the cytosol and delayed appearance of peptidase activity that cleaved the fluorogenic substrates Asp-Glu-Val-Asp-aminotrifluoromethylcoumarin (DEVD-AFC) and Val-Glu-Ile-Asp-aminomethylcoumarin (VEID-AMC) as well as an altered spectrum of active caspases that were affinity labeled with N-(Nα-benzyloxycarbonylglutamyl-Nε-biotinyllysyl) aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone [z-EK(bio)D-aomk]. On the other hand, the activation of caspase-3 under cell-free conditions occurred with indistinguishable kinetics in cytosol prepared from the two cell lines. Collectively, these results suggest that a delay in the signaling cascade upstream of cytochrome c release and caspase activation leads to a long latent period before the active phase of apoptosis is initiated in etoposide-treated K562 cells. Once the active phase of apoptosis is initiated, the spectrum and subcellular distribution of active caspase species differ between HL-60 and K562 cells, but a similar proportion of cells are ultimately killed in both cell lines.

Abstract We recently reported that the induction of polyploidization of malignant megakaryocytes shows great promise as a new therapy for acute leukemia. Polyploidization inducers such as dimethylfasudil (diMF) and MLN8237, both of which target Aurora A kinase (AURKA), induce proliferation arrest, polyploidization, expression of megakaryocyte differentiation markers and apoptosis of leukemic megakaryocytes in vitro and in vivo. Since megakaryocytes in primary myelofibrosis (PMF) show impaired polyploidization and maturation, and likely directly contribute to the disease, we predicted that polyploidization inducers would provide a new therapeutic strategy. To determine the effect of these compounds on the growth of MPN cells, we first treated the JAK2 V617F mutant megakaryocytic SET2 cell line with varying doses of MLN8237 and diMF. Both compounds effectively and dose dependently inhibited proliferation, induced polyploidization and upregulation of lineage specific markers CD41 and CD42, and increased apoptosis. Furthermore, MLN8237 synergized with ruxolitinib to induce apoptosis of the SET2 cells and also potently induced growth arrest of JAK2 inhibitor persistent SET2 cells. We observed a similar polyploidization and differentiating activity of MLN8237 and diMF on megakaryocytes derived from primary human PMF progenitors. The ability of these agents to induce polyploidization was specific, as the non-megakaryocyte fractions of the cultures were not affected. Next, we assayed the activity of polyploidization inducers on progression of MPNs in two mouse models: JAK2V617F conditional knockin mice and mice engrafted with MPLW515L expressing bone marrow progenitors. Of note, spleens from both mouse models displayed a robust increase in both total and phosphorylated forms of AURKA relative to control animals, further suggesting that AURKA is a rational target in this disease. We first assayed the activities of MLN8237 and diMF in the MPLW515L bone marrow transplantation model. Recipient mice develop a rapid MPN characterized by leukocytosis, thrombocytosis and bone marrow fibrosis. Both MLN8237 and diMF reduced the disease burden, as evidenced by significant reductions in the liver and spleen weights, white cell counts and platelet counts. Both compounds also led to a significant decrease of fibrosis in the bone marrow, diminished infiltration of megakaryocytes and granulocytes in the liver, and a profound reduction in the numbers of megakaryocytes within the spleen. Moreover, plasma levels of TGF-β a known myelofibrogenic cytokine, were decreased by more than 3-fold by the drug treatment. Both diMF and MLN8237 led to selective polyploidization of megakaryocytes in the spleen as well as marked reductions in the levels of p-AURKA. Of note, neither agent affected the extent of phosphorylation of STAT3 or STAT5. Therefore, we tested whether the combined use of a JAK inhibitor and a polyploidy inducer would show enhanced activity in vivo. Indeed, the combination of MLN8237 and ruxolitinib led to greater reductions in tumor burden in the MPLW515L mouse model than either agent alone. Similar results were obtained using the JAK2V617F knock-in model. To further validate our conclusion that AURKA is a target in PMF, we infected Aurkafl/fl floxed bone marrow progenitors with MPLW515L and transplanted the cells to irradiated recipients. Excision of both alleles of Aurka by Cre mediated recombination completely resolved the disease, while heterozygous deletion of Aurka significantly reduced the disease burden, in a manner similar to treatment with MLN8237. Given that heterozygous deletion of Aurka does not alter normal hematopoiesis in mice, the fact that a 50% reduction in kinase expression was associated with a significant decrease in disease burden suggests that there is an effective therapeutic window in which AURKA inhibitors will be effective against MPN while sparing normal hematopoiesis. Although JAK inhibitors provide symptomatic relief, it is becoming clear that they are not curative. Thus, there is an urgent need to develop new agents to use in combination with JAK inhibitors. Our data reveal that inducing polyploidization and differentiation of dysplastic megakaryocytes in PMF ameliorates features of the disease both in vitro and in vivo. These results support the initiation of clinical studies that combine a JAK inhibitor with an AURKA inhibitor. Disclosures: Crispino: Sanofi: Research Funding.

Abstract The effect of 6-MP therapy on the duration of remissions induced by adrenal corticosteroids has been studied as a model for testing of new agents. Ninetytwo patients under age 20 entered the study and were accepted for analysis. Sixty-two (67 per cent) had complete or partial remissions induced by corticosteroids. Patients in remission were randomly assigned to maintenance therapy with either 6-MP or placebo. The median duration of 6-MP-maintained complete remissions was 33 weeks and for placebo, 9 weeks. A sequential experimental design was used to analyze remission times while the study was in progress. This resulted in the study being stopped after analysis of the remission times of 21 pairs of patients (42 patients). Overall survival was not significantly different for the two treatment programs, since patients maintained on placebo were treated with 6-MP when relapse occurred. The activity of the known active antileukemic compound 6-MP was readily detected by this experimental design without compromise of optimal survival. Such a design should prove useful for the evaluation of new agents and also permit study of the remission maintenance activity of a compound separately from its remission inducing activity.

Abstract The efficacy of three therapeutic programs for acute leukemia were compared. These programs included (1) Methotrexate (Phase I) followed by 6-mercaptopurine (Phase II); (2) 6-mercaptopurine (Phase I) followed by Methotrexate (Phase II); and (3) Combination Therapy, i.e., 6-mercaptopurine given in combination with Methotrexate. In children with acute lymphocytic leukemia the remission rate was 59 per cent for combination therapy, 47 per cent for 6-mercaptopurine, and 29 per cent for Methotrexate. The better remission rate for combination therapy is consistent with that predicted if it is assumed that 6-mercaptopurine and Methotrexate act independently. The median duration of complete remissions for the three treatments was not different (4 to 5 months). However, long lasting remissions were more frequent in patients receiving combination therapy. The median survival from the onset of therapy to death was 9 months. There were no differences between the three treatment programs as regards survival. In adults the remission rate was 15 per cent for combination therapy, 21 per cent for 6-mercaptopurine and 7 per cent for Methotrexate. As regards survival in adults, early deaths were more common in patients who received MTX as initial therapy, whereas after 5 months survival was somewhat better in those patients receiving combination therapy. In both children and adults there was no evidence that prior treatment with one of the antimetabolites altered response to the other antimetabolite. This result differs from those in animal models, and its effect on our concept of the mechanism of resistance is discussed. Responsiveness to the second course of antimetabolite therapy (Phase II) was as good as that to the first course of treatment. This was true for the remission rate, remission duration, and even for survival when appropriate corrections were made. Thus, responsiveness to drug therapy is maintained as the disease progresses temporally. It may be concluded therefore that new agents can be effectively studied in patients with "late" disease. Responsiveness to Phase II therapy was independent of responsiveness to Phase I. The most common and severe toxic manifestations related to the bone marrow and gastrointestinal tract. There were no major differences quantitatively in the toxicity for the three treatment programs in children in spite of the fact that the drugs were given in full dosage in the combination program. Oral ulcers and a generalized erythematous rash occurred significantly more frequently in patients receiving Methotrexate. Jaundice was significantly more frequent in adults and in patients receiving 6-MP.

Abstract Among mechanisms underlying cytotoxic drug resistance is activation of diverse DNA damage response (DDR) pathways. Poly(ADP-ribose) polymerases (PARP)-1/2 facilitate both single- and double-strand break (DSB) repair and play a key role in the base excision repair (BER) of chemotherapy-damaged DNA. The PARP inhibitor veliparib (V) potentiates the cytotoxicity of different chemotherapeutics, including temozolomide (TEM). TEM induces distinct alkylating events in neoplastic cells whose ultimate outcome depends on the interaction of BER, mismatch repair (MMR), O(6)-methylguanine-DNA methyltransferase (MGMT), and DSB repair. While clinical activity of TEM has been mainly observed in MGMT-deficient AML, V potentiated cytotoxicity of TEM in leukemia cells in vitro in the setting of MGMT overexpression or deficient MMR pathway (Mol Cancer Ther, 2009). Methods: We conducted a Phase I study to determine maximum tolerated dose (MTD) and recommended Phase II dose (RP2D) of V+TEM, using a 3+3 dose escalation. Patients (pts) ≥60 years (yrs) with newly diagnosed poor cytogenetic-risk AML/ALL who were not candidates for intensive therapy, or ≥18 yrs with relapsed/refractory AML/ALL, secondary AML (therapy-related or arising from MDS or MPN), and CMMoL-2 were eligible. Any number of prior regimens, including allogeneic transplant (alloSCT), were allowed. V was given orally day (d)1 once, then twice a day on d4-12 at one of 6 dose levels (DL) (DL1A-B 20mg; DL2-DL3-DL4-DL5-DL6: 40-80-120-150-200 mg). TEM was given orally once a day on d3-9 (DL1A 150 mg/m2/d; DL1B-DL6 200 mg/m2/d). 28-day cycles (cy) were repeated depending on response/tolerability (4-6 weeks delay allowed) with V on d1-8 and TEM d1-5. TEM was taken on empty stomach with antiemetics and V was taken irrespective of meals. Results: Forty-nine pts with median age 69 yrs (range, 22-88; 47% ≥70) were treated. Of 47 AML pts, 29 (62%) had secondary AML and 27 (57%) adverse karyotype. Median number of prior treatments for AML was 1 (range, 0-6): 18 (38%) had median 1 prior therapy (range, 1-3) for MDS; 30 (64%), 9 (18%), 34 (69%) received hypomethylating agents, alloSCT and intensive chemotherapy, respectively. Overall 42 (85%) pts were refractory to their last treatment. Pts received a median of 1 (range, 1-7) cy of therapy. Two did not complete cy 1, pt withdrawal d5 and progressive fungal pneumonia d9 with death d15 of progressive disease (PD). The MTD/RP2D was defined at V 150 mg and TEM 200 mg/m2; 2 of 4 pts treated at V 200 mg and TEM 200 mg/m2 developed dose-limiting toxicity of grade (gr) 3 oral mucositis/esophagitis. The most frequent drug-related toxicities (NCI CTC v4) were gr 1/2 nausea/vomiting (39%), fatigue (26%), oropharyngeal mucositis (26%), constipation (12%), and diarrhea (10%). Other common toxicities were infectious, including febrile neutropenia (29%), pneumonia (20%), bacteremia (18%). One (2%) pt died ≤d30 and 12 (24%) ≤d60 mainly of PD (1 pt fungal pneumonia before count recovery d31). Overall response rate was 33% (complete remission (CR), hematologic improvement (HI)/stable disease) with 8 (16%) pts achieving CR (1 CRi). Median overall survival was 5.03 months, for all responders 11.58 months, and for CR pts 19.89 months (Fig 1). Responses occurred at all DLs. Three CR pts underwent alloSCT; 2 remain in CR at ~3 yrs. Pharmacokinetics (PK): V or TEM PKs were not altered with co-administration. There was a correlation between the DLT of mucositis and V single (Cmax P=0.005; AUC P=0.009) and multiple dose exposure (Cmax P = 0.02; AUC P=0.03). Pharmacodynamics and pharmacoepigenetics: Four of 39 pts examined had MGMT methylation (3 CR; 75%) and 2 had BRCA-1 methylation (1 HI) in peripheral blood (PB) or bone marrow (BM) mononuclear cells (MC). Defective FancD2 pathway was observed in the BMMC of 19/19 pts using FancD2 ubiquitylation assays but did not correlate with response. V reduced PAR levels by >75% in PBMC of most pts and in the presence of TEM. Induction of γ-H2AX in CD34+ cells was seen upon V/V+TEM treatment. Conclusion: V plus TEM demonstrated safety and activity in this resistant and elderly leukemia population. Response rate was higher in MGMT methylated pts, but responses were also seen in pts who had no MGMT methylation, had failed multiple therapies, had secondary AML and/or adverse karyotype. Future clinical study should aim to identify pts with defective DDR pathways who are most likely to respond to this therapeutic approach. Figure 1. Figure 1. Disclosures Off Label Use: Temozolomide is not approved for AML. Beumer:Millenium: Other: Research support. Gore:Celgene: Consultancy, Honoraria, Research Funding.

Background: Local, intrawound use of antibiotic powder, such as vancomycin and tobramycin, in spinal fusion surgery has become an increasingly common prophylactic measure in an attempt to reduce rates of postsurgical infection. However, the effects of localized antibiotic delivery on fusion remain unclear. The objective of this study was to examine the in vivo effects of intraoperative local delivery of 2 antibiotics commonly used in bone-grafting surgery on spinal fusion outcomes in a rat model. Methods: Single-level (L4-L5), bilateral posterolateral intertransverse process lumbar fusion surgery was performed on 60 female Lewis rats (6 to 8 weeks of age) using syngeneic iliac crest allograft mixed with clinical bone-graft substitute and varying concentrations of antibiotics (n = 12 each): (1) control without any antibiotics, (2) low-dose vancomycin (14.3 mg/kg), (3) high-dose vancomycin (71.5 mg/kg), (4) low-dose tobramycin (28.6 mg/kg), and (5) high-dose tobramycin (143 mg/kg). Eight weeks postoperatively, fusion was evaluated via micro-computed tomography (µCT), manual palpation, and histological analysis, with blinding to treatment group. In the µCT analysis, fusion-mass volumes were measured for each rat. Each spine specimen (L4-L5) was rated (manual palpation score) on a scale of 2 to 0 (2 = fused, 1 = partially fused, and 0 = non-fused). Results: The mean fusion-mass volume on µCT (mm 3 ) was as follows: control, 29.3 ± 6.2; low-dose vancomycin, 26.3 ± 8.9; high-dose vancomycin, 18.8 ± 7.9; low-dose tobramycin, 32.7 ± 9.0; and high-dose tobramycin, 43.8 ± 11.9 (control versus high-dose vancomycin, p < 0.05; and control versus high-dose tobramycin, p < 0.05). The mean manual palpation score for each group was as follows: control, 1.46 ± 0.58; low-dose vancomycin, 0.86 ± 0.87; high-dose vancomycin, 0.68 ± 0.62; low-dose tobramycin, 1.25 ± 0.71; and high-dose tobramycin, 1.32 ± 0.72 (control versus high-dose vancomycin, p < 0.05). The histological analyses demonstrated a similar trend with regard to spinal fusion volume. Conclusions: Intraoperative local application of vancomycin, particularly at a supraphysiological dosage, may have detrimental effects on fusion-mass formation. No inhibitory effect of tobramycin on fusion-mass formation was observed. Clinical Relevance: When spine surgeons decide to use intraoperative intrawound antibiotics in spinal fusion surgery, they should weigh the reduction in surgical site infection against a possible inhibitory effect on fusion.

Abstract Abstract 2893 Introduction: Monoclonal B-Cell Lymphocytosis (MBL) is a common asymptomatic condition characterized by a clonal B-cell population detected in the blood or bone marrow. The prevalence of MBL is approximately 3–5% in adults over age 40 but rises with age. MBL is a precursor state to CLL and other types of indolent non-Hodgkin lymphoma (NHL). Only a small subset of patients with MBL comes to clinical attention, typically during evaluation for low grade lymphocytosis (designated now as clinical MBL). The rate of progression to CLL requiring treatment among individuals with clinical MBL is ∼1%/yr, and the vast majority of patients with MBL will never develop a B-cell malignancy. Recent data indicate that individuals with MBL are at increased risk of infection similar to CLL. It is unknown whether individuals with MBL are at risk for other adverse outcomes associated with CLL, such as increased risk of non-hematologic cancers (NHC). We investigated the incidence of NHC among patients with clinical MBL and compared this to a cohort of general medicine patients and a cohort of CLL patients. Methods: Mayo Clinic Rochester (MCR) is the primary center for hematologic care in a region including southeastern Minnesota, northern Iowa, and western Wisconsin. No other hematology specialty centers are available within a 50 mile radius of MCR. To explore the risk of NHC in a local, community dwelling cohort of individuals with clinically-identified, CLL-phenotype MBL, we used the MCR CLL database to identify all individuals with newly diagnosed MBL seen in the MCR Hematology Division between January 1, 1999 and December 31, 2009. Analysis was limited to 154 MBL cases residing within 50 miles of MCR. The comparison cohort consisted of 596 adult general medicine patients, with no prior cancer, residing within 50 miles of MCR, who were seen for a general medical exam and enrolled as controls in a case-control study for NHL between April 25, 2004 and December 31, 2009. Rates of NHC in the MBL cohort were also compared to a cohort of 174 patients with newly diagnosed CLL seen in the same time interval as the MBL cohort and also living within 50 miles of MCR. NHC were identified using the Mayo Clinic Cancer Tumor Registry as well as review of medical records in all 3 cohorts. Patients with a NHC diagnosis >3 months prior to the diagnosis date of MBL/CLL were excluded from analysis while those with MBL/CLL and a NHC within 3 months of one another were considered to have a concurrent diagnosis. Results: There were 125 patients in the MBL cohort, 153 in the CLL cohort, and 596 in the control cohort after excluding patients with prior cancers. The MBL and CLL patients tended to be older (median age MBL=69, CLL=68, Control=58; p<0.0001) and more likely to be male (% male MBL=61, CLL=67, Control=46; p<0.0001) than the controls. After a median follow-up of 4.2 years (range 0–11), 20 (16%) individuals with MBL developed NHC as compared to 22 (4%) of the controls (Hazard Ratio (HR) MBL relative to control=5.0; 95% confidence interval (CI)=(2.6, 9.4; p<0.0001). Twenty CLL patients (13%) developed NHC, supporting a similar rate to that of MBL patients (HR MBL relative to CLL=1.3; 95% CI=(0.6, 2.5; p=0.49;See Figure). Specifically, the MBL cohort had a higher incidence of cancer of the breast, lung, and nervous system than that of the control cohort. The higher risk of NHC among individuals with CLL and MBL persisted even when patients with “concurrent diagnosis” of CLL/MBL and NHC were excluded (p=0.006) and when patients treated for progressive CLL were censored at the date of treatment (p=0.001) Finally, we performed a pooled multivariable analysis of all patients (n=874) adjusting for age, sex, major co-morbidities, presence of MBL, and diagnosis of CLL. Age (HR each year older=1.03; 95% CI 1.01–1.06; p=0.003), major comorbidities (HR=1.81; 95% CI 1.07–3.00; p=0.02), CLL (HR=2.10; 95% CI 1.01–4.01; p=0.04), and MBL (2.47; 95% CI 1.30–4.69; p=0.02) were independent risk factors for NHC. The results were similar when the effects of age was modeled in a non-linear (e.g. logarithmic) manner. Conclusions: In this cohort study, patients with newly diagnosed clinical MBL had a 2.5-fold increased risk of developing NHC relative to the control cohort. MBL was an independent risk factor for development of NHC after adjusting for age, sex, and other comorbidities. This finding adds to the growing evidence that clinical MBL has a distinct clinical phenotype. Disclosures: Shanafelt: Genentech: Research Funding; Glaxo-Smith-Kline: Research Funding; Cephalon: Research Funding; Hospira: Research Funding; Celgene: Research Funding; Polyphenon E International: Research Funding.