BACKGROUND:The authors hypothesized that low doses of the hypomethylating agent 5‐azacitidine may maximize the graft‐versus‐leukemia effect and may be tolerated well after allogeneic transplantation (HSCT).METHODS:The drug was given to 17 patients with acute leukemia as salvage for disease recurrence after HSCT (n = 9 patients) or as maintenance therapy (n = 8 patients). 5‐Azacitidine was given subcutaneously daily for 5 days and was repeated every 4 weeks at doses of 16 mg/m2 (n = 4 patients), 24 mg/m2 (n = 9 patients), and 40 mg/m2 (n = 4 patients). A median of 8 cycles was delivered. The median follow‐up was 16 months and 11 months after HSCT and 5‐azacitidine treatment, respectively.RESULTS:Five of 9 patients with recurrent disease responded. Four of 13 responding patients developed disease recurrence while they were receiving 5‐azacitidine after a median of 10 months. The actuarial 1‐year event‐free and overall survival rates were 55% and 90%, respectively. There were no extramedullary toxicities, and no graft‐versus‐host disease exacerbation was observed.CONCLUSIONS:Low‐dose 5‐azacitidine may induce durable remissions for patients who develop disease recurrence after HSCT. Further follow‐up and a larger group of patients will be necessary to confirm these observations. Cancer 2009. © 2009 American Cancer Society.

BackgroundBecause hepatitis C virus infection causes hepatic and immunological dysfunction, we hypothesized that seropositivity for this virus could be associated with increased nonrelapse mortality after allogeneic hematopoietic stem cell transplantation. Design and MethodsWe performed a case-control study of the outcomes of patients who were hepatitis C virus seropositive at the time of allogeneic hematopoietic stem cell transplantation (N=31). Patients positive for hepatitis C virus were considered candidates for stem cell transplantation only if they had no significant evidence of hepatic dysfunction. Matched controls (N=31) were seronegative for viral hepatitides and were paired according to age, diagnosis, disease stage, conditioning regimen and donor type. We also compared the hepatitis C virus seropositive patients to all seronegative patients (all controls, N=1800) transplanted during the same period, to adjust for other confounding effects. ResultsThe median age of the seropositive patients was 49 (range 26-72); 15 had acute myeloid leukemia/myelodysplastic syndrome, 6 had chronic myeloid leukemia/myeloproliferative disease, 6 non-Hodgkin's lymphoma, 2 myeloma, 1 acute lymphocytic leukemia and 1 Hodgkin's lymphoma; 61% had poor risk disease; 68% had related donors; 68% received reduced intensity conditioning; 7 patients had mildly abnormal alanine transaminase levels (all less than three times the upper limit of normal) and 1 patient had minimally elevated bilirubin. These characteristics were similar to those of the matched control group. Median overall survival was 3, 18 and 20 months, and 1-year survival was 29%, 56% and 56%, in the hepatitis C virus, matched and all controls groups, respectively (hazard ratio for death 3.1, 95% confidence interval 1.9 -5.6, p<0.001 in multivariate analysis). Non-relapse mortality at 1 year was 43%, 24% and 23%, respectively (hazard ratio 3.3, 95% confidence interval 1.8 -7.1, p<0.01). Disease progression and graft-versus-host disease rates were comparable. ConclusionsHepatitis C virus seropositivity is a significant risk factor for non-relapse mortality after allogeneic hematopoietic stem cell transplantation even in patients with normal or minimally abnormal liver function tests.Key words: hematopoietic stem cell transplantation, hepatitis C virus, transplant-related mortality.Citation: Ramos CA, Saliba RM, de Pádua L, Khorshid O, Shpall EJ, Giralt S, Patah PA, Hosing CM, Popat UR, Rondon G, Khouri IF, Nieto YL, Champlin RE, and de Lima M. Impact of hepatitis C virus seropositivity on survival after allogeneic hematopoietic stem cell transplantation for hematologic malignancies. Haematologica 2009; 94:249-257. doi:10.3324/haematol.13756 ©2009 Ferrata Storti Foundation. This is an open-access paper. Impact of hepatitis C virus seropositivity on survival after allogeneic hematopoietic stem cell transplantation for hematologic malignancies

The use of CB for stem cell transplantation is increasing. A major disadvantage of CB is delayed and failed engraftment when compared to marrow or blood transplantation. Double CBT and ex-vivo expansion are both strategies being investigated in order to improve hematopoietic reconstitution. We compared these approaches with the aim of improving the clinical outcomes of CBT patients. 48 patients were randomized to receive either two unmanipulated (UNM) CB units (N=24) or one UNM unit and one unit from which all the cells were EXP ex-vivo (N=24). Most CB units were HLA 4/6 matches. Diagnoses were AML (N=16), ALL(N=13), NHL(N= 5), HD(N=7), CML (N=5), and CLL(N=2). Patients (Table 1) were heavily pre-treated, with a median of 3 (1–8) prior regimens including autotransplants in 18%. Preparative regimens included ATG and either fludara plus dose-adjusted busulfan(N=13; myeloid diseases), or melphalan and thiotepa (N=21; lymphoid malignancies/HD); patients not eligible for high-dose therapy received non-myeloablative fludara plus cyclophosphamide and 200 GyTBI (=11) or melphalan(n=3). GVHD prophylaxis was tacrolimus plus either 3 doses of 5 mg/m2 methotrexate or MMF (table 1). Ex-vivo expansion: the smallest unit was CD133-selected using the CliniMACS device (day -14). The T cell-containing CD133-negative fraction was frozen. The CD133+ fraction was cultured for 14 days in media containing SCF, G-CSF and TPO. On day 0, the 2nd UNM unit was infused, followed by the CD133-negative and the EXP fractions. Results. Infused median total nucleated cells (TNC)×108/Kg was 0.36 and 0.36, and median CD34×106/Kg was 0.16 and 0.13, respectively for EXP and UNM pts. Median TNC fold-expansion was 26 (0.44–275) and for CD34+ cells, 2.2 (0–18). There was a nonsignificant trend to more rapid engraftment in patients receiving EXP cells. Patients with &gt;50-fold versus &lt;50-fold TNC expansion engrafted in a median of 19 days(11–21) versus 23 (16–31), and achieved platelets&gt;20K/ul at a median of 29 days(17–44) versus 42(29–56). 25 patients are alive [median follow up is 11.1 mo (2–33.6)]. 100-day and 1-year non-relapse mortality rates are 10% and 22%, respectively (table 2). Chimerism showed that 1 CB unit dominated in all patients. Among 19 evaluable EXP patients, in 63% the UNM unit provided 100% of the hematopoiesis from day +30; the EXP unit was predominant in 3 cases (for 12, 2 and 2 mo.) and present but not predominant in 4 (5–25%, for 12, 7, 3 and 3 mo). Conclusion: EXP CBT was safe. The range of fold-expansion was highly variable. Accrual continues focusing on strategies which improve CB expansion. Outcomes Expanded Unmanipulated P C.Incid.: cumulative incidence Time to ANC500 (median/95%CI) 20 days (15, 31) 23.5 (21, 34) 0.4 Proportion engrafting ANC500 82% (n=19) 79% (n=19) 1 Time to PLT20K (median/95%CI) 41 (32, NA) 54 (40, NA) 0.4 Proportion engrafting PLT 67% (n=16) 58% (n=14) 0.38 1-year survival 60% 40% 0.45 aGVHD gd II–IV/ III–IV 32% / 5% 41% / 9% NS C.Incid. cGVHD 47% (28–78) 31% (15–65) NS Expanded Unmanipulated P Complete remission at UCBT 42% 67% 0.15 Median weight 82 (20-113) 76 (51-144) 0.6 Median age 40.4 (4-66) 38.1 (9-65) 0.8 Ablative preparative regimen 67% 75% 0.8 GVHD prophylaxis with methotrexate 48% 48% NS

Disease relapse is the most frequent cause of treatment failure after HSCT in patients (pts) with refractory AML/MDS. AZA inhibits the enzyme DNA methyltransferase and leads to DNA hypomethylation. It may induce leukemic cell differentiation and increased immunogenicity, therefore potentially magnifying the graft-versus-leukemia effect. Lower doses are likely to be better tolerated after HSCT and to be as effective as larger doses in inducing hypomethylation. We hypothesized that AZA maintenance will result in lower relapse rates, and designed a phase I clinical trial to determine the safest dose and schedule combination. We also monitored global methylation post HSCT as a surrogate marker for changes in methylation status during AZA. Methods: Pts with AML or high-risk MDS not in 1st complete remission (CR), not candidates for ablative regimens were eligible. Conditioning regimen was gemtuzumab ozogamicin 2 mg/m2 (day -12), fludarabine 120mg/m2, and melphalan 140mg/m2. GVHD prophylaxis was tacrolimus/mini-methotrexate. ATG was administered to recipients of unrelated donor (UD) HSCT. We investigated 3 AZA doses: 8, 16, and 24 mg/m2 daily × 5 starting on day +42, and given for 1–4 28-day cycles (schedule). An outcome-adaptive method was used in order to determine both dose and schedule (number of cycles): pts were assigned to a dose/schedule combination chosen on the basis of the data (toxicity) from all pts treated previously in the trial. Patients in CR on transplant day +30, without gd III/IV GVHD, platelet >10,000/mm3 and ANC >500/mm3 were eligible to receive SQ AZA. The methylation status of long interspersed nuclear elements (LINE) was analyzed by pyrosequencing and used as a surrogate marker of global DNA methylation in mononuclear cells of 22 pts(55%) that received AZA for at least 1 cycle. Results: 40 patients were enrolled; median age was 57 years (22–72). Diagnoses were MDS with high IPSS(n=6) and AML (n=34). Disease status at HSCT: complete remission (CR), 10% (n=4); chemo-naive MDS, 2% (n=1); induction failure, 35% (n=14), 1st/2nd relapse, 42.5% (n=21). Patients had received a median of 5 courses of chemotherapy (0–23) prior to HSCT, and the median comorbidity Charlson score was 3 (0–8). Donors were related (n=23) or UD(n=17), and stem cell source was bone marrow(n=8) or peripheral blood(n=32). Median follow-up of alive pts is 11 mo (2.5–20; n=21). 49 cycles of AZA were delivered at 8 (n=7 pts), 16 (n=4) and 24 mg/m2 (n=12 pts). AZA-associated toxicities were grade I/II hematologic, nausea and fatigue. There was no increase in GVHD: gd II-IV aGVHD, and chronic GVHD rates were 34% and 30%. 11 pts have relapsed, 2 while on AZA (16 and 24 mg/m2). Day +30 and +100 non-relapse mortality was 5% and 12%. Mean LINE methylation results were as follows: baseline: 43.51% (±5; n=22); on cycle 1, 5th day of AZA: 24 mg/m2=43.58% (±5; n=9); 16 mg/m2: 36.93% (±6.5; n=4); 8 mg/m2: 42.86% (±5; n=3). There were trends towards lower levels of global methylation at 16mg/m2 in subsequent cycles. The trial design has reached the higher dose and the maximum number of cycles given the absence of AZA-related major toxicities. Conclusion: AZA at 24 mg/m2 is safe and can be administered for at least 4 cycles. The lack of toxicity and the methylation studies indicate that higher doses and longer periods of administration should be investigated.

Introduction: The development of next-generation sequencing has made it feasible to interrogate the entire genome or exome (coding genome) in a single experiment. Accordingly, our knowledge of the somatic mutations that cause cancer has increased exponentially in the last years. MPNs and MDS/MPD are chronic myeloid neoplasms characterized by an increased proliferation of one or more hematopoietic cell lineages, and an increased risk of transformation to acute myeloid leukemia (AML). MPNs and MDS/MPDs are heterogenous disorders, both in clinical presentation and in prognosis. We sought to determine the genetic landscape of Ph-negative MPNs and MDS/MPD through next-generation sequencing. Methods: Paired DNA (sorted CD66b-granulocytes/skin biopsy) from 102 patients with MPNs or MDS/MPD was subjected to whole exome sequencing on a Illumina HiSeq 2000 platform using Agilent SureSelect kit. Diagnosis included primary myelofibrosis (MF; N=42), essential thrombocythemia (ET; N=28), polycythemia vera (PV; N=12), chronic myelomonocytic leukemia (CMML; N=10), systemic mastocytosis (MS; N=6), MDS/MPD-Unclassified (N=2) and post-MPN AML (N=2). Tumor coverage was 150x and germline coverage was 60x. Somatic variants calls were generated by combining the output of Somatic Sniper (Washington University), Mutect (Broad Institute) and Pindel (Washington University). The combined output of these 3 tools was further filtered by in-house criteria in order to reduce false-positive calls (minimum coverage at both tumor/germline ≥8 reads; fraction of reads supporting alternate allele ≥10% in tumor and ≤10% in germline; ratio of allele fraction tumor:germline >2; excluding mutations seen in SNP databases). All JAK2 and CALR mutations were validated through Sanger sequencing. Validation of other somatic mutations is currently underway. Analysis of driver mutations was made with the Intogen web-based software, using the Oncodrive-FM and Oncodrive-cluster algorithms (www.intogen.org). Significantly mutated genes were considered as those with a q-value of <0.10. Results: We identified a total of 309 somatic mutations in all patients, with each patient having an average of 3 somatic abnormalities, fewer than most solid tumors that have been sequenced so far. Mutations occurred in 166 genes, and 40 of these were recurrently somatically mutated in Ph-negative MPNs. By the Oncodrive-FM algorithm, the following genes were identified as the most significantly mutated driver genes in Ph-negative MPNs and MDS/MPDs (in order of significance): CALR, ASXL1, JAK2, CBL, DNMT3A, U2AF1, TET2, TP53, RUNX1, EZH2, SH2B3 and KIT. By the Oncodrive-cluster algorithm, which considers clustering of mutations at a hotspot, the following genes were significantly mutated: KIT, JAK2, SRSF2 and U2AF1. Somatic mutations were seen in genes that are mutated at a low frequency in Ph-negative MPNs, including ATRX, BCL11A, BCORL1, BIRC5, BRCC3, CSF2RB, CUX1, IRF1, KDM2B, ROS1 and SUZ12. Consistent with the clinical phenotype, 96 patients (94%) had mutations that lead to increased cellular proliferation, either through activation of the JAK-STAT pathway (e.g. JAK2, CALR) or mutations that activated directly or indirectly signaling by receptor tyrosine kinases (e.g. FLT3, KIT, CBL). Besides biological pathways regulating cell proliferation, the most commonly implicated pathways included regulation of DNA methylation (e.g. DNMT3A, TET2), mRNA splicing (e.g. U2AF1, SRSF2) and histone modifications (e.g. ASXL1, EZH2), seen in 27%, 25% and 22% of patients, respectively. Abnormalities in these 3 pathways were more often seen in MF, MDS/MPD and CMML, as compared to PV and ET (65% vs. 20%; p<0.0001). Conclusions: Our study represents one of the largest series of patients with these neoplasms evaluated by whole exome sequencing, and together with the published data helps to delineate the genomic landscape of Ph-negative MPNs and MDS/MPDs. The majority of the most frequent mutations seen in Ph-negative MPNs have already been reported. Nevertheless, there are several low frequency mutations that need to be further studied and functionally validated in vitro and in vivo for a deeper knowledge of the pathophysiology of MPNs. Besides activation of cellular proliferation, abnormalities of DNA methylation, histone modification and mRNA splicing emerge as the most important biological pathways in these disorders. Disclosures No relevant conflicts of interest to declare.