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Abstract Abstract 278 Background: Overactive JAK-STAT signaling as a result of gain-of-function mutations (eg, JAK2V617F) and/or high circulating levels of inflammatory cytokines is considered to play a key role in the pathogenesis of myeloproliferative neoplasms. Ruxolitinib, a selective oral inhibitor of JAK1 and JAK2, demonstrated a significant reduction in spleen volume (SV) and improvements in myelofibrosis (MF)-related symptoms in a double-blind placebo-controlled trial (COMFORT-I). The objective of this analysis was to evaluate the efficacy of ruxolitinib across patient (pt) subgroups in COMFORT-I. Methods: Pts with MF were randomized to start placebo or ruxolitinib at a dose of 15 mg or 20 mg PO BID depending on baseline platelet count (100–200 ×109/L or >200 ×109/L, respectively). The dose was optimized for efficacy and safety during treatment. SV change was measured by MRI; MF symptoms were assessed using a daily diary (modified Myelofibrosis Symptom Assessment Form [MFSAF] v2.0) over 1 wk prior to dosing and throughout the 24 wks of dosing. The percent changes from baseline to wk 24 in SV and MFSAF Total Symptom Score (TSS, a measure of combined scores for abdominal discomfort, pain under ribs on left side, early satiety, itching, night sweats, and bone/muscle pain) were compared for ruxolitinib and placebo pts across the following subgroups: MF disease subtype, age, International Prognosis Scoring System (IPSS) risk group, presence/absence of JAK2V617F mutation, baseline hemoglobin, baseline spleen size (palpable spleen length), and baseline TSS. Survival was estimated by Kaplan-Meier method. Changes in SV and TSS Across Subgroups: 309 pts were randomized: 155 to ruxolitinib and 154 to placebo. Ruxolitinib demonstrated consistent benefit compared with placebo in both SV and TSS across all subgroups evaluated (Table). The impact of symptom severity on response was evaluated by baseline TSS quartiles (maximum score for TSS = 60). Ruxolitinib pts with baseline TSS of <8.5, 8.5-<16.5, 16.5-<25.5 and ≥25.5 had mean percent changes in SV of −28.0, −31.4, −31.7 and −34.8, respectively, vs +8.1 for all placebo pts combined. The mean percent change in TSS for these same subgroups was −40.5, −47.2, −48.1 and −48.2 vs +41.8 for all placebo pts combined. These data indicate that pts with modest to marked symptoms all benefit from ruxolitinib therapy in terms of both SV and TSS. Survival Analysis: 13 ruxolitinib and 24 placebo pts died during the study or during extended follow-up (median follow-up of 52 and 51 wks, respectively), representing a hazard ratio (95% CI) of 0.499 (0.254, 0.98) (p=0.0395). For ruxolitinib- and placebo-treated pts, respectively, the probability of survival (95% CI) >48 wks was 0.98 (0.92, 0.99) and 0.90 (0.81, 0.95) for pts with baseline hemoglobin values ≥10 g/dL and 0.84 (0.72, 0.91) and 0.77 (0.63, 0.86) for pts with baseline hemoglobin <10 g/dL. Conclusions: Pts receiving ruxolitinib had higher response rates than placebo based on reductions in SV and improvements in TSS at wk 24 regardless of baseline subgroup: MF disease subtype, age (≤65 or >65 y), IPSS risk group (intermediate-2 or high-risk), presence or absence of JAK2V617F mutation, hemoglobin level (≥10 g/dL or <10 g/dL), palpable spleen length (≤10 cm or >10 cm), and symptom severity (TSS quartile). In addition, the overall survival analysis suggested a benefit with ruxolitinib therapy over placebo. Disclosures: Verstovsek: Incyte: Research Funding. Mesa:Incyte: Research Funding; Lilly: Research Funding; SBio: Research Funding; Astra Zeneca: Research Funding; NS Pharma: Research Funding; Celgene: Research Funding. Gotlib:Incyte: Consultancy, Research Funding. Levy:Incyte: Employment, Equity Ownership. Gupta:Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Catalano:Incyte: Honoraria; Novartis: Honoraria. Deininger:BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genzyme: Research Funding. Miller:Incyte: Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau. Winton:Incyte: Consultancy. Arcasoy:Incyte: Research Funding. Lyons:Alexion: Consultancy, Honoraria; Telik: Research Funding; Incyte: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Novartis: Research Funding. Vaddi:Incyte: Employment. Erickson-Viitanen:Incyte: Employment. Sun:Incyte: Employment. Sandor:Incyte: Employment. Kantarjian:Incyte: Research Funding; Novartis: Consultancy, Research Funding.

Abstract The amyloidoses are heterogeneous diseases associated with the deposition of insoluble proteins or peptides with a characteristic beta-diffraction pattern extracellularly. At the current time, over 27 different extracellular fibril proteins are known to cause disease in humans. Iatrogenic amyloidosis is a rare, often not sought diagnosis. On occasion, patients with drug induced amyloidosis can present with other systemic features reminiscent of systemic immunoglobulin-derived (AL) amyloidosis, and may present a diagnostic challenge. Treatment of the latter often involves chemotherapy and/or stem cell transplantation, while the former tends to remain localized and needs local therapy; thus accurate diagnosis is critical. To this end, proteomic analysis of amyloid tissue has proven to be an invaluable tool in amyloid typing. We have analyzed the biochemical composition of iatrogenic amyloid using laser capture/tandem mass spectrometry (LC-MS/MS)-based proteomic analysis in 52 cases of insulin and enfuvirtide (Fuzeon¨) associated amyloidosis. In brief, 10-μm-thick sections of formalin-fixed paraffin-embedded tissues were stained with Congo red. Congo red positively staining tissue as viewed with a fluorescent light source appeared bright red. Positive areas were dissected using laser microdissection to a volume of at least 60,000 μm2; three microdissections were analyzed for each case. The microdissected material was collected into 0.5-ml microcentrifuge tube caps containing 35 μL Tris/EDTA/0.002% Zwittergent buffer. Microdissected fragments were subjected to a heat-mediated antigen retrieval method (98C for 90minutes) before being denatured via sonication and subsequently digested into tryptic peptides overnight using 0.5ug of trypsin. The resulting digests were then analyzed with nanoflow LC-MS/MS. The MS/MS spectra of each case were matched against a composite protein sequence database using three different search algorithms (Sequest, X!Tandem, and Mascot). The composite database contained the human SwissProt entries but was also augmented with known immunoglobulin variant domains, known amyloidogenic mutations from literature, the enfuvirtide amino acid sequence, and common contaminants. Reversed protein sequences were appended to the database for estimating the false discovery rates of the identifications. The peptide identification results were filtered using Scaffold software (Proteome Software, Portland, OR) and then filtered peptides were assembled into protein identifications. Candidate proteins with at least one high-confident (probability of identification &gt;90%) unique peptide identification and at least four MS/MS spectral matches were considered for clinical interpretation. For each case, we created a personalized proteomic profile that lists all the confident protein identifications in each of the microdissection along with their respective MS/MS spectral counts. The number of MS/MS spectra matching to a protein is considered as a semi-quantitative measure of its abundance. The most abundant amyloidogenic protein detected across all microdissections and as interpreted in the context of the clinical history is considered to be the amyloid subtype. Figure 1 shows the results of insulin amyloidosis. Figure 2 shows the results of enfuvirtide amyloidosis. Amyloid deposits are shown to be composed of the recombinant drug in addition to amyloid precursor proteins such as apolipoprotein A-I, A-IV, E and serum amyloid P (SAP).Figure 1. Insulin-associated amyloidosisFigure 1. Insulin-associated amyloidosisFigure 2Enfuvirtide (Fuzeon¨)-associated amyloidosisFigure 2. Enfuvirtide (Fuzeon¨)-associated amyloidosis In conclusion, we show the biochemical composition of all known drug-induced iatrogenic amyloidosis and provide the utility of proteomic analysis in elucidating amyloid subtyping for accurate diagnosis and management. Legend: A spectral count number of greater than 4 is significant. The green boxes denote protein identification at a probability of over 95%, and yellow 80-94%. In figure 1, insulin (# 2) and in figure 2, enfuvirtide (#5) are shown in abundance, additionally other amyloid precursors such as apolipoproteins A-IV, E, A-I and SAP are also seen. Disclosures: No relevant conflicts of interest to declare.

Abstract Myeloproliferative disorders (MPDs), diagnosed in 15,000 individuals annually, are characterized by overproduction of lineage-committed blood cells, thrombotic events, occasionally marrow fibrosis or progression to acute myelogenous leukemia and frequently a point mutation (V617F) in the JAK2 kinase. Mutant JAK2 expression results in erythrocyte overproduction in almost all cases of polycythemia Vera (PV). We investigated the molecular mechanisms driving erythroid skewed differentiation and the capacity of a selective JAK2 inhibitor (TG101348) to normalize PV progenitor differentiation. Both JAK2 V617F+ transduced cord blood and PV progenitor erythroid colony formation was potently inhibited by TG101348 (300 nM). Bioluminescent immunocompromised mouse transplantation studies revealed that TG101348 (150 mg/kg) treatment significantly reduced human erythroid engraftment by both PV and JAK2 V617F+ cord blood progenitors. Moreover, the imbalance between GATA-1 and PU.1 transcripts typical of JAK2 V617F+ progenitors normalized after treatment with TG101348. In addition, TG101348 inhibited both JAK2-mediated STAT5 phosphorylation and AKT-regulated GATA-1 phosphorylation in an erythropoietin responsive cell line suggesting a dual mechanism for the erythroid inhibitory effects of TG101348. Because of selective inhibition of JAK2 V617F+ progenitors both in vitro and in vivo, TG101348 may be an effective molecularly targeted inhibitor of JAK2 V617F+ driven MPDs in clinical trials.

Abstract Background Immunoglobulin light chain-associated amyloidosis (AL) is caused by deposition of immunoglobulin light chain molecules with unique, clonotypic variable (V) regions. Detection and identification of the V region, however, has been challenging due heterogeneity inherent in V regions. We developed a new method for detecting IGKV and IGLV region fragments in AL deposits using protein mass spectrometry and novel bioinformatics approach. We report distinct IGKV and IGLV usage in localized versus systemic AL. Methods Shotgun protein mass spectrometry on amyloid plaques was performed as previously described (Blood. 2009; 114: 4957-9). Peptide tandem mass spectra (MS/MS) were searched against a composite sequence database containing SwissProt's human complete proteome augmented with 1764 IG V region sequences obtained from ImMunoGeneTics database and a Mayo Clinic internal database. Reversed sequences were appended to the database for estimating identification false discovery rates (FDRs). Peptide identifications were processed with Scaffold software. Confident protein identifications (probability > 0.9) with at least one unique peptide identifications and five MS/MS matches were considered. Detected variable gene family (if any) with most number of peptide and spectral matches is considered to be present in the deposit. The method was first validated in 8 cases of AL amyloidosis with known IGKV and IGLV region sequences and then applied on 1238 systemic and 393 localized AL cases. Differences between groups were measured by generating hypergeometric p-values. Results In all 8 AL cases with known IGKV and IGLV region usage, gene family identified in the AL deposit via proteomics matched the gene family inferred from bone marrow plasma cells by Sanger sequencing of IGLC genes. Armed with this method, we analyzed the amyloid proteomics data from 1238 patients with systemic AL amyloidosis and 393 patients with localized AL amyloidosis. The anatomical site distribution in systemic cohort was 296 GI tract, 364 heart, 225 kidney, 81 liver and 272 fat aspirate; Localized cohort has 78 bladder, 158 lung and 157 skin cases. Figure 1 shows the normalized frequency of the variable gene families detected between the systemic and localized AL amyloidosis. For AL-kappa, KV1 was more prevalent in systemic cases when compared to localized cases (p=7.2E-12) suggesting KV1 clones as a hallmark for systemic AL-kappa amyloidosis. KV3 was more frequently seen in localized AL-kappa cases when compared to systemic AL-kappa cases (p =2.8E-11). For AL-lambda, LV1 and LV2 gene families were more prevalent in localized cases when compared to systemic cases (p= 0.039 and 2.7E-05). LV6 was more prevalent in systemic AL-lambda cases (p=1.4E-07). We also detected a higher incidence of mixed AL/AH type in localized AL (18%) when compared to systemic AL (5%; Odds Ratio=4.2673, p<0.0001). We next turned to the organ specific IGKV and IGLV gene family usage patterns in patients with localized amyloidosis. For 147 localized AL-kappa cases, KV3 was most dominant in lung (64%) and KV1 was dominant in skin (38%). For 246 localized AL-lambda cases, LV2 was most prevalent in lung (34%) and LV3 was most prevalent in skin (36%). For bladder, both LV1 and LV2 had comparable prevalence (42% and 32%). For 361 systemic AL-kappa cases, KV1 consistently ranked at the top of gene families used in GI tract (55%), heart (71%), kidney (59%), liver (75%) and fat aspirate (50%). For 877 systemic AL-lambda cases, LV2 was predominant in GI tract (30%), LV3 in heart (35%) and LV6 in kidney (33%). The prevalence of LV1, LV2 and LV3 in liver is comparable (36%, 22% and 31%). LV6 and LV3 had comparable prevalence (22% and 24%) in AL-lambda fat aspirates. Conclusion The novel proteomics method detects IGLC V family usage in large cohorts of AL patients. It identifies unique profiles in systemic and localized cases, and in different organ sites. This information will be helpful in determining systemic versus localized nature of AL amyloidosis at diagnosis and to assess risk of specific end organ involvement. We found also a strong association between IGKV and IGLV gene usage and organ involvement. Disclosures: No relevant conflicts of interest to declare.

Abstract Amyloidosis is a group of hereditary and acquired disorders of diverse pathogenesis united by end organ damage due to tissue deposition of misfolded proteins. The diagnosis is typically based on demonstrating Congo red positive amyloid deposits in tissue biopsies of clinically involved sites. Because treatment options for amyloidosis patients are highly varied and intricately tied to the identity of the protein that constitutes the amyloid deposits, adequate diagnostic evaluation now requires identification of the amyloidogenic protein. Traditionally, immunohistochemistry with antibodies to amyloid-associated proteins was the clinical technique of choice for subtyping amyloid deposits in tissue sections. This technique suffers from poor analytical sensitivity and specificity and is a suboptimal technique on which to base treatment decisions for amyloidosis patients. We developed a highly accurate and reproducible liquid chromatography electrospray tandem mass spectrometery (LC-MS/MS)-based shotgun proteomics assay for subtyping amyloid deposits (Blood. 2009; 114: 4957-9) from microdissected paraffin embedded tissue samples. Implementation of this assay in a CAP/CLIA clinical laboratory has subsequently transformed the standard of amyloidosis care at Mayo Clinic. Since 2008, we utilized the assay in routine practice to identify 20 different amyloid types in tissue samples from diverse anatomic sites from 4139 patients (Table 1). Although AL and ATTR were most common, the cases include a large variety of unusual, and in many cases clinically unsuspected amyloid types. In addition, the amyloid protein subtypes had different frequencies in different anatomic sites (A partial list of tissue sites and their amyloid types analyzed in this study includes: bone marrow: AL 82%, ATTR 15%, AA 2%, other 1%; kidney: AL 51%, ALect2 18%, AA 17%, AH 5%, AFib 4%, other 5%; liver: AL 64%, ALect2 23%, AApoA1 6%, AA 3%, ATTR 2%, other 2%; heart: ATTR 60%, AL 37%, AANF1%, other 2%; GI tract: AL 78%, ATTR 13%, AA 6% AApoA4 1%, other 2%; eye [primarily adnexae] AL 63%; eye [primarily cornea] TGFB induced protein ig-h3 34%, other 3%). One caveat is that these distributions of amyloid types may not accurately reflect population prevalence. More likely, the findings reflected a composite prevalence that included challenging amyloidosis cases.Table 1Frequency of amyloid subtypes observed in the Mayo Amyloid Cohort (n=4139). Asterisk denotes non-canonical subtypesAmyloid SubtypeNumber (%) of casesAL2553 (61.68)ATTR1015 (24.52)AA151 (3.65)ALect2148 (3.58)AIns48 (1.09)Keratin*36 (0.87)AApoA130 (0.72)AH27 (0.65)AFib26 (0.63)TGFB1-IP*22 (0.53)AApoA420 (0.48)AANF14 (0.34)Ab2M12 (0.29)AGel12 (0.29)ASem112 (0.29)APro7 (0.17)ALys3 (0.07)ACal2 (0.05)Enfuvirtide*2 (0.05)AIAPP2 (0.05) The prevalence of diverse and unexpected amyloid types in many tissues suggest that shotgun proteomic-based analysis may be the only way to accurately assign amyloid subtype in tissue samples. The technique avoids the analytical problems of immunohistochemistry and the bias introduced by assigning probable amyloid type based on clinical presentation and disease distribution. It provides the clinician with an amyloid subtype that allows for confident individualized patient management. Accurate amyloid subtyping by LC-MS/MS is not only possible, but has proven to be a robust routine clinical tool for the evaluation of numerous amyloidosis patients in our practice. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 5146 Background: Myelofibrosis (MF) is characterized by splenomegaly, debilitating symptoms, hematologic abnormalities, and shortened survival. COMFORT-I is a Phase III randomized, double-blind, placebo-controlled study of the JAK1 and JAK2 inhibitor ruxolitinib in patients (pts) with MF. The placebo arm of this ongoing study provides a unique population in which to assess the clinical burden of MF in a controlled setting. Methods: 151 pts with intermediate-2 or high-risk primary-MF (PMF), post-polycythemia vera-MF (PPV-MF), or post-essential thombocythemia-MF (PET-MF) who were not candidates for other therapies were randomized to receive placebo in COMFORT-I after a 28-day washout of previous MF therapies. Assessments included MF symptoms measured daily using an electronic diary (modified Myelofibrosis Symptom Assessment Form [MFSAF] v2.0), spleen volume measured every 12 wks by blinded abdominal MRI or CT scans, and laboratory measures at least monthly through wk 24. The Patient-Reported Outcomes Measurement System (PROMISE) Fatigue and Patient Global Impression of Change (PGIC) questionnaires were administered at each visit. Pts with missing data (baseline or post-baseline assessment measured), or pts who withdrew or crossed over to active treatment before a study visit were not included in analyses. Results: 151 pts received 1 or more doses of placebo (median exposure 204 days). The study included 65% high-risk and 35% intermediate-2 risk pts, with a median of 2.5 years since diagnosis. 55% had PMF, 31% PPV-MF, and 14% PET-MF. Regardless of prior MF therapy (57% were on hydroxyurea), there was continual worsening of symptoms over time as assessed by the MFSAF Total Symptom Score (TSS, comprised of scores for night sweats, itching, abdominal pain and discomfort, early satiety and bone/muscle pain) (see Figure). At wk 24, mean/median spleen volume increased by +8.1%/+8.5%, and both intermediate-2 and high-risk pts demonstrated progressive disease over the course of the study. Mean/median PROMIS Fatigue score worsened by 9.1%/6.5%. Mean/median score for the PGIC (a 7-point scale ranging from 1 = very much improved to 7 = very much worse, with 4 = no change) was 4.2/4.0 at wk 24; 19.6% of pts reported feeling much worse or very much worse. Mean/median total body weight changed from 72.0 kg/72.2 kg to 70.1 kg/69.7 kg at wk 24. Hematology assessments (median, baseline vs wk 24) were as follows: hemoglobin, 105 vs 106 g/L; platelets, 233 vs 219 x109/L; neutrophils, 14.8 vs 16.0 x109/L; white blood cell count, 18.4 vs 20.1 x109/L. Two of 151 pts had new-onset (new or worsening) grade 3 or grade 4 thrombocytopenia, and 23/151 pts had new-onset grade 3 or grade 4 anemia. 16 pts met protocol-specified criteria for unblinding and crossover to active treatment before wk 24, which was a 25% increase in MRI-based spleen volume either with or without increased pain or weight loss; thus, mean and median values reported at wk 24 may represent an underestimate of the degree of worsening in disease parameters. Conclusion: The signs and symptoms of MF continued to worsen over a 6-month time frame in this placebo population. The data illustrate the progressive nature of MF and support the need for effective therapeutic interventions. Moreover, treatment benefit must be benchmarked against continual evolution of the disease in the absence of treatment. Early recognition of the debilitating symptoms of MF and intervention with effective therapy to prevent continual worsening of the disease should be strongly considered. Disclosures: Verstovsek: Incyte: Research Funding. Mesa:Incyte: Research Funding; Lilly: Research Funding; SBio: Research Funding; Astra Zeneca: Research Funding; NS Pharma: Research Funding; Celgene: Research Funding. Gotlib:Incyte: Consultancy, Research Funding. Levy:Incyte: Employment, Equity Ownership. Gupta:Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Catalano:Incyte: Honoraria; Novartis: Honoraria. Deininger:BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genzyme: Research Funding. Miller:Incyte: Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau. Winton:Incyte: Consultancy. Arcasoy:Incyte: Research Funding. Lyons:Amgen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Incyte: Research Funding; Telik: Research Funding; Alexion: Consultancy, Honoraria; Novartis: Research Funding. Vaddi:Incyte: Employment. Erickson-Viitanen:Incyte: Employment. Sun:Incyte: Employment. Sandor:Incyte: Employment. Kantarjian:Incyte: Research Funding; Novartis: Consultancy, Research Funding.

Abstract In CN-AML, mutations of specific genes have prognostic significance and are used for treatment guidance. However, these mutations are in general not mutually exclusive and CN-AML patients (pts) frequently harbor mutational combinations with unclear prognostic significance because of the concurrent presence of mutations with favorable (CEBPA, NPM1) and unfavorable (ASXL1, DNMT3A, IDH1, IDH2, FLT3-ITD, MLL-PTD, RUNX1, TET2, WT1) impact on outcome. Here, we report the frequency and clinical significance of isolated mutations and their combinations in 364 CN-AML pts. Younger [<60 years (y); n=163] and older (≥60 y; n=201) pts were intensively treated on frontline CALGB/Alliance protocols and, per protocol, did not undergo allogeneic stem cell transplantation in first complete remission (CR). Median follow-up for pts alive was 8.7 y. Pts were analyzed centrally for mutations in the ASXL1, CEBPA, DNMT3A, IDH1, IDH2, NPM1, RUNX1, TET2, and WT1 genes and the presence of FLT3-ITD, FLT3-TKD and MLL-PTD. We found that 99% of pts had ≥1 and 88% ≥2 of the 12 mutations. Specifically, 11% of pts had 1 gene mutated (n1), 40% had 2 (n2), 31% 3 (n3), and 18% harbored ≥4 mutated genes (n4). The distribution of the different n groups in younger and older pts was similar (Fig 1). The expected frequency of each mutated gene in every numerical (n) group was calculated as follows: [no. of pts with the mutated gene] x [total no. of mutations in the n group] / [total no. of mutations in all pts], and compared with the observed frequency. CEBPA, IDH2, NPM1, and RUNX1 mutations were overrepresented in the n1 and n2 groups (P<.001, P<.001, P=.002, P=.02, respectively) and thus likely represent “early” events in leukemogenesis. In contrast, DNMT3A, FLT3-ITD, and TET2 mutations were more frequent in the n3 and n4 groups (P=.008, P<.001, P=.02, respectively) suggesting that they are likely “late” events. The distribution among age groups and clinical impact of the “early” mutations (i.e., CEBPA, NPM1, IDH2) presenting as isolated mutations or in combination with other mutations were then investigated. Isolated RUNX1 mutations could not be evaluated due to small sample size. Younger pts were more likely to harbor an isolated “early” mutated gene than older pts (21% v 12%, P=.03). Specifically, isolated CEBPA and NPM1 mutations were more frequent in younger pts compared with the older (7% v 2%, P=.007; 7% v 3%, P=.07, respectively). In pts with isolated CEBPA mutations, these were always bi-allelic. Single “early” mutated genes varied in their impact on CR achievement (CEBPA: 100%, NPM1: 94%, IDH2: 67%), and overall survival (OS; 3-y OS: CEBPA: 87%, NPM1: 69%, IDH2: 22%). Acquisition of additional mutations adversely modified the prognostic significance of favorable “early” mutations in the CEBPA (Fig 2A) and NPM1 (Fig 2B) genes, but left unchanged the unfavorable effect of IDH2 mutations (Fig 2C). Two mutational combinations were exceptions to this: younger pts with both NPM1 and DNMT3A mutations (8 pts) had an excellent prognosis with 3-y OS of 100%; and older pts with concurrent NPM1 and IDH2 mutations (9 pts; all IDH2 mutations were in codon R140) had a high 3-y OS rate of 67%. We conclude that CEBPA and NPM1 mutations mainly define pts with favorable prognosis when they present as single markers, whereas mutated IDH2 predominantly associates with adverse outcome. The NPM1+DNMT3A and NPM1+IDH2 mutational patterns may define novel favorable molecular subtypes in younger and older CN-AML pts, respectively, but these results require corroboration in larger pt cohorts. Disclosures: Mrózek: AMERICAN SOCIETY OF HEMATOLOGY: I will review abstracls submitted for presentation at the 55th ASH Annual Meeting in the 611 category Other. Eisfeld:AMERICAN SOCIETY OF HEMATOLOGY: I will review abstracls submitted for presentation at the 55th ASH Annual Meeting in the 608 category Other.

Abstract Background: Neutropenia is the most common predisposing factor that increases the susceptibility of cancer patients (pts) to infection. The use of fluoroquinolones (FQs) as prophylaxis during chemotherapy-induced neutropenia has been shown to decrease the frequency of gram-negative (GN) infections (Cullen et al. NEJM 2005) with the benefit of reduced infection-related mortality in neutropenic pts with cancer (Bucaneve et al. NEJM 2005) and was instituted as part of our institution-wide standard clinical practice in January 2006. The widespread use of FQs for prophylaxis of pts with neutropenia and hematologic malignancies raises concerns about the evolving resistance of organisms. Methods: This retrospective observational study compared acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) pts, ages 18 and older who were admitted to the hematology service and received levofloxacin prophylaxis (LEVP) within 60 days of admission with those who were admitted but did not receive LEVP during the period of chemotherapy-induced neutropenia. Pts had to have a positive sterile site culture up to 96 hours prior to admission excluding Staphylococcus coagulase negative, Propionibacterium, Corynebacterium, and other common colonizing organisms. Analysis also compared the rates of Methicillin-resistant Staphylococcus aureus (MRSA) infections, positive Vancomycin-resistant Enterococci (VRE) swabs, and positive Clostridium difficile cultures and evaluated them for a correlation LEVP. Results: A total of 213 pts were evaluated between September 2004 and May 2007. Ninety-four pts received LEVP and 119 did not. Patient characteristics were similar between groups. There was a shift from almost equal incidence of gram-positive (GP) and GN cultures in the group without prophylaxis (51% vs 49%, respectively) to a predominance of GP cultures with LEVP (68% vs 32%, respectively). There was a significant decrease in the number of sterile site cultures of GN organisms (n = 94 vs 37, respectively), while cultures of GP organisms remained similar (n = 98 vs. 77, respectively). GN organisms in sterile site cultures occurred less often in the LEVP group, however; the incidence of resistance to levofloxacin in GN was higher (32% vs 15%), particularly the susceptibility profiles of Pseudomonas sp., Escherichia coli, and Stenotrophomonas maltophilia. Both groups had equal cultures of Staphylococcus aureus, but the group receiving LEVP had a greater incidence of MRSA (55% vs 30%). Rates of positive VRE swabs declined and had no apparent correlation with the use of levofloxacin as prophylaxis. There were more documented positive Clostridium difficile cultures in the pts receiving LEVP (29% vs 22%, respectively), but rates of Clostridium difficile have shown a constant increase throughout the institution; therefore, LEVP should not be considered the sole contributing factor to an increased rate of positive cultures. Conclusions: The epidemiology and microbial susceptibility profile of positive sterile site cultures of acute leukemia pts receiving LEVP during chemotherapy-induced neutropenia differed from those that did not. The LEVP group had a decrease in the absolute number of sterile site cultures, number and percent of GN sterile site cultures. There was an increase in GN organism resistance to levofloxacin and the incidence of several GP organisms increased with the number of GP organisms remaining constant.

Abstract ECOG, CALGB and SWOG (coordinating group) enrolled 899 patients with newly diagnosed MM to receive VAD x 4 (813 were considered eligible), followed by randomization to PBSC-supported MEL-TBI (n=261) versus VBMCP (n=255), using CTX 4.5g/m2 + G-CSF for PBSC mobilization in all patients. Responders to VBMCP or MEL-TBI were randomized to IFN (n=121) or no maintenance (n=121). With a median follow-up of alive patients of 60 mos, median EFS and OS from 1st randomization were 25 and 62 mos for MEL-TBI and 21 and 53 mos for VBMCP (p=.14 for EFS, p=.87 for OS). EFS and OS from IFN randomization were also identical at 23/59 mos for IFN versus 18/74 mos for observation (p=.2/.6). 87 VBMCP failures received salvage autotransplants, with a post-relapse median survival of 24 mos which was similar to the 27 mos observed among the remaining 83 VBMCP failures managed with standard salvage therapies. When examined in the context of 2 other randomized trials (IFM 90, MRC VII), OS from baseline of S932, limited to patients <60 yr (as in IFM 90 + MRC VII), is intermediate between the high dose arms of IFM 90/MRC VII and the standard dose arms of these two trials (randomization at baseline) (Figure 1). OS from the time of randomization to high or standard dose therapy on S9321 (again restricted to age <60 yr) was similar to OS timed from transplant for the high dose arms of IFM 90 and MRC VII. (Figure 2). Figure Figure The comparable outcome after high- versus standard-dose therapies on S9321, interpreted in the context of 2 other published randomized clinical trials, may relate to (1) higher dose intensity with VBMCP than with other standard regimens or (2) a detrimental effect of TBI in S9321. We conclude that with the current follow-up MEL-TBI is no better than VAD/VBMCP with the option of salvage transplant and that IFN maintenance is of no benefit in this maintenance setting. Data from this trial will be provided to MRC to be included in their on-going meta-analyses of high-dose therapies and IFN maintenance.

Abstract Previous work has suggested a relationship between disease activity and in vivo birth rates of chronic lynphocytic leukemia (CLL) cells, as measured by incorporation of deuterium into DNA during heavy water (2H2O) consumption. By extension, CLL-cell kinetics may be a useful prognostic marker in early stage disease, with an increased birth rate predicting shorter time to disease progression and poor prognosis. We are conducting a multi-center study to evaluate the relevance of proliferation rates to both known prognostic markers and to disease progression during 2.5 years of clinical follow up in previously untreated patients with early stage disease. Patients are given a daily dose of 2H2O for 6 weeks and CLL-cell birth rates are measured. We examined the leukemia cells of 36 patients for known prognostic markers (ZAP 70 expression, CD38 expression, IgVH mutation status) and CLL birth rates. Subjects were Rai Stage 0 (n=15), 1 (n=17), or 2 (n=4); mean age 57 +/− 9 (range 41–78); mean time from diagnosis to enrollment 2.1 +/− 2.2 y (range 0.1–10 y). ZAP-70 expression (20% cutoff) was positive in 44%, CD38 expression (30% cutoff) was positive in 35%, and use of unmutated IgVH genes was found in 45% of the subjects. CLL-cell birth rates ranged from 0.09 to 0.97% per day. Utilizing the previously published cut-off value of 0.35% newly divided cells per day, 33% of subjects had a high proliferation rate. Data on all biomarkers were available for 33 subjects. Nine subjects had 4/4 favorable prognostic indicators and 5 subjects had 4/4 unfavorable prognostic indicators. Agreement between prognostic markers, as assessed by kappa coefficient, was strongest for the ZAP-70 v. IgVH mutational status comparison. Comparisons were also significant for ZAP-70 v. CD38 and IgVH mutational status v. CD38. Birth rate was not significantly correlated with any of the other prognostic markers, suggesting it may be a useful independent predictor of disease progression. Isolation of CLL-cells from the bone marrow at the end of the 6 wk labeling period in 5 subjects revealed matching proliferation rates in the marrow and blood in 3 subjects. In 2 subjects, marrow enrichment was lower, suggesting a more slowly proliferating pool in the marrow, the pathophysiologic significance of which is unclear. In 5 subjects, kinetic analysis revealed a very delayed entry of newly divided cells into the blood compartment, presumably from tissue sites of proliferation. This parameter may reflect pool size of CLL-cells in tissues and correlation with lymph node burden is being evaluated. In summary, analysis of CLL-cell birth rates in vivo in these early stage patients provides unique information about the biology of CLL. Correlations with disease progression during the follow-up period will help determine the utility of this test as a prognostic biomarker.