Objective: To analyze the outcome of patients treated with gemtuzumab ozogamycin combined with co... more Objective: To analyze the outcome of patients treated with gemtuzumab ozogamycin combined with conventional therapy treated at Hospital Israelita Albert Einstein. Methods: 14 patients who had high risk features (secondary leukemia, unfavorable cytogenetics, and refractory disease) were treated with gemtuzumab ozogamycin combined with conventional therapy and their outcome was analysed by reviewing their medical records. results: Overall response rate was 58%, with 43% achieving complete response, with a median followup of 11 months, event-free survival was 3 months. Eleven patients died, 6 of them due to refractory acute myeloid leukemia. Only four patients presented with grade 3 to 4 toxicities and only one patient had sinusoidal obstruction syndrome after bone marrow transplant. conclusion: gemtuzumab ozogamycin combined with chemotherapy is a feasible treatment regimen in acute myeloid leukemia patients. However, further studies are necessary to clarify which subgroup of patients may benefit from this treatment.
Objective: To analyze the outcome of patients treated with gemtuzumab ozogamycin combined with co... more Objective: To analyze the outcome of patients treated with gemtuzumab ozogamycin combined with conventional therapy treated at Hospital Israelita Albert Einstein. Methods: 14 patients who had high risk features (secondary leukemia, unfavorable cytogenetics, and refractory disease) were treated with gemtuzumab ozogamycin combined with conventional therapy and their outcome was analysed by reviewing their medical records. results: Overall response rate was 58%, with 43% achieving complete response, with a median followup of 11 months, event-free survival was 3 months. Eleven patients died, 6 of them due to refractory acute myeloid leukemia. Only four patients presented with grade 3 to 4 toxicities and only one patient had sinusoidal obstruction syndrome after bone marrow transplant. conclusion: gemtuzumab ozogamycin combined with chemotherapy is a feasible treatment regimen in acute myeloid leukemia patients. However, further studies are necessary to clarify which subgroup of patients may benefit from this treatment.
Abstract 4531 Introduction: Cytomegalovirus (CMV) is one of the most common viral pathogens post-... more Abstract 4531 Introduction: Cytomegalovirus (CMV) is one of the most common viral pathogens post-allogeneic stem cell transplantation (SCT). However, CMV reactivation and disease are relatively uncommon events after autologous stem cell transplantation (ASCT), with a reported incidence of 2–9%. There are few studies describing risk factors for development of CMV reactivation post-ASCT. Objective: To describe the incidence and risk factors for CMV reactivation post-ASCT in our institution and its impact on survival. Methods: The charts of patients who underwent ASCT at our institution from January 2005 until July 2009 were manually reviewed. CMV reactivation was detected by antigenemia assay and/or real-time PCR. Variables associated with CMV in a univariate analysis with a…
Introduction: Busulfan (Bu) is used in the conditioning regimen for hematopoietic stem cell trans... more Introduction: Busulfan (Bu) is used in the conditioning regimen for hematopoietic stem cell transplantation (HSCT). Therapeutic drug monitoring (TDM) of Bu with subsequents adjustments doses based on a “target” therapeutic concentration may reduce toxicity after HSCT. Objectives: To evaluatethe impact of TDM of Bu and clinical outcomes in patients with acute leukemia that underwent to allogeneic matched related donor (MRD) and allogeneic matched unrelated donor (MUD) HSCT. Patients and methods: From January 2009 to January 2014, we prospectively analyzed 42 patients with diagnosis of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) who underwent TDM of Bu (IV or oral) before transplantation (test dose) and TDM on 1st day of conditioning regimen. Samples were collected at 0, 30’, 60’ and subsequently every hour until 6 hours after administration of busulfan. The plasma was extracted by HPLC (High Performance Liquid Chromatography). All the patients that were submitted to TDM had a test dose 15 days to 48 hours before transplantion. The dose of Bu was adjusted during the first day of the conditioning regimen based on test dose. At the same time we analyzed 21 patients in the retrospective group who did not underwent TDM (from 2004 to 2010). Results: In the retrospective group (n=21), all of them underwent to MRD transplantation. Six (46.2%) were in first complete remission (CR1), 18(85.7%) patients received Bu and cyclophosphamide (BuCy) and the mean of age was 38 years (18-55 Yo). The median of CD34+ cells was 5.4 x 106/kg. The second group consisted by patients that received oral Bu (n= 21): 7 (33.3%) underwent to MUD transplantation, 14 (66.6%) to MRD transplantation, 8 (44.4%) patients were second complete remission (CR2), 4 (22.2%) had active disease status with a mean age of 32.7 years (14-58 Yo). Fifteen (71.4%) received BuCy and 16 (76.2%) received cells from peripheral blood. The median of CD34+ cells was 5.8 x106/kg. The median area under the curve (AUC) in 24 hours was 4950 μMol.min (3196.6- 8212 μMol.min). The third group was IV Bu (n= 21): 7 (33.3%) patients underwent MRD and 14 (66.6%) MUD transplantation, 7 (33.3%) patients were CR1, 7 (33.3%) had active disease or prior HSCT with a mean of age 52.7 years (20-74 Yo). The majority of patients received fludarabine and Bu (n=18; 85.7%) as conditioning and bone marrow was the main source. Immunosuppression was based on FK-506 and methotrexate (90.5% of patients). The median of AUC in 24 hours was 5690 μMol.min (3539.6- 8881.8 μMol.min). The cumulative incidence (CI) of sinusoidal obstructive syndrome (SOS) in the retrospective group and IV Bu were 9.5% for both, while in the group oral Bu it was at 19% (p = 0.566). The median AUC of Bu received during conditioning for those who died of SOS was lower in oral Bu than IV Bu (4872 uMol.min vs 5732 uMol.min respectively). The CI of acute graft-versus-host disease (aGVHD) at D+100 was 38.1% in the retrospective group, 40.6% in oral Bu and 42.9% in IV Bu. Chronic GVHD was 13.6% in oral Bu, 34% in IV Bu and 42.9% in the retrospective group (p = 0.142). The CI of relapse at D+100 was 19% in IV Bu, 4.8% in the retrospective group and oral Bu did not have this event. The IC of death at D+100 was 34.9% in group oral Bu, 9.5% in IV Bu and 14.3% in the retrospective (p = 0.102). The CI of relapse at 1.5 years was 35.8% in the IV Bu, 34.8% in oral Bu and 14.3% in the retrospective group. The CI of death at 1.5 years was 9.5% in group IV Bu, 53.5% in oral Bu and 34.3% in the retrospective (p = 0.015). Among patients who died until D+100, the median of AUC was 5732 μMol.min (5578.5-6818.5 μMol.min) during the conditioning for IV Bu and 4872 μMol.min (3448-8212 μMol.min) for oral Bu. The range between the AUC was large and there was no correlation with patients who died. Conclusion: In acute leukemia SOS had an impact on mortality at D+100 after HSCT (p…
Introduction: Hematopoietic stem cell transplantation (HSCT) was offered primarily as a therapeut... more Introduction: Hematopoietic stem cell transplantation (HSCT) was offered primarily as a therapeutic option for severe sickle cell disease in the context of myeloablative matched sibling donor transplants over the last two decades and helped to establish the benefits of transplantation for this disorder. In recent years, the transplant community has set out to explore ways to make stem-cell transplantation more available to patients with the disease, define indications and better timing, and offset toxicities with novel approaches to conditioning and better supportive care. In this context, neurological complication such as stroke disease and blood flow alteration in medial cerebral artery constitute the main indications of HSCT but neurological complications are the main causes of TRM in D+100 and is important to find ways to prevent this problem. Objective: Describe early neurological complications in sickle patients undergoing related HSCT on single specialized center and its following after the early diagnosis. Material and Methods: Seven patient were undergone related HSCT for sickle disease from 2011 to 2013. All patients filled inclusion criteria in the study and signed agreement term. Results: Seven HSCT were developed in the period, being four males. The average age was 13 years old (7-24). The HSCT indications were previous stroke, cerebral flow alteration on Doppler, acute chest syndrome and alloimunization. All patient were on blood transfusion therapy. The conditioning regimen was BuCy + ATG and the GVHD prophylaxis was MTX and CSA. Related donors were chosen with 10/10 HLA match and graft source marrow. The median of the neutrophil engraftment was D+ 25 and the platelets engraftment was in D+60. Two patient died, one by intestinal and liver GVHD on D+120, and another with sagittal sinus thrombosis and hemorrhagic stroke on D+3. Other two patients showed PRES syndrome related to cyclosporine use. The patients showed generalized seizures with tomographic neurological alteration. After the imunossupressor change to tacrolimus and the change of Phenytoin to Lamotrigine, the patient had total resolution of neurological complications without development of neurological sequelae. Patients who used Lamotrigine since the beginning of the conditioning have not shown neurological alterations. Conclusion: The PRES Syndrome and Stroke are two of the main causes of mortality related to the use of calcineurin inhibitors. Patients with sickle disease have shown endothelial and cerebral microcirculation changes, which made them highly susceptible to neurological complication. The control of blood pressure, maintenance of 50.000 platelets level and the use of Lamotrigine as prophylaxis of seizures seems to decrease the risk of neurological complications. Prospective studies with lamotrigine as primary prevention of PRES Syndrome must be performed. Patients with severe neurological alteration as vessels stenosis more than 90% and Moya-Moya Syndrome must be better evaluated before the conditioning because of high TRM risk. Disclosures No relevant conflicts of interest to declare.
Introduction: Multiple myeloma is an incurable hematologic malignancy. However, many novel drugs ... more Introduction: Multiple myeloma is an incurable hematologic malignancy. However, many novel drugs (lenalidomide, carfilzomib, daratumumab, elotuzumab and ixazomib) have recently been approved in Brazil for this disease. Proving that the availability of these drugs increases survival is not straightforward, since pivotal trials with these drugs were designed to show benefit in progression-free survival and not in overall survival. Hypothesis: We have hypothesized that the availability of novel drugs would reduce the risk of death. Methods: This is a real-world, unicenter analysis that included patients with multiple myeloma treated between 1993 and 2017. The availability of novel drugs (lenalidomide, carfilzomib, daratumumab, elotuzumab and ixazomib) was treated as a time-dependent covariate and analyzed by Cox model. The analysis was controlled for the International Staging System (ISS) and for age. Results: With a median follow-up of 6 years, we included 151 patients with multiple myeloma. Fourty percent had ISS-I, 34% ISS-II and 26% ISS-III. Mean age was 60.5 y/o (SD: 12.2). There has been 51 deaths, and 5-y overall survival was 90% for ISS-I, 80% for ISS-II and 54% for ISS-III (p=0.0004). In multivariable analysis, age (HR=1.06, p<0.0001) and ISS-III (HR=4.51, p<0.0001) were risk factors for death, while the availability of two or more novel drugs was protective (HR=0.31, p=0.006). The availability of a single novel drug was also protective (HR=0.58), although it did not reach statistical significance (p=0.13). Adjusted Simon-Makuch survival plot is in figure 1 (adjusted for ISS III and 60 y/o). Discussion: Our results show that the availability of two or more novel drugs increases overall survival in multiple myeloma. This is an important finding, since the pivotal trials were designed to show advantages in progression-free survival and, once achieved, the experimental drug was offered to the control group, hampering overall survival analyses. Overall survival benefit, therefore, are more likely to be seen in real-world studies. We included the availability of novel drugs as a time-dependent, and the interpretation of our results is that patients alive when two novel drugs became available had benefit in overall survival. Our study has two weaknesses. Two or more novel drugs were more likely to be available for patients from a more recent time. This difference could be due to changes in supportive care, but there has been no major change in supportive care for multiple myeloma patients. Another weakness is that patient alive in the more recent years may have not received novel drug-based treatment. Considering that multiple myeloma is an incurable disease, this is unlikely for the majority of patients. In conclusion, the availability of two or more novel drugs for multiple myeloma improves survival. Figure Disclosures No relevant conflicts of interest to declare.
Introduction The role of the endothelial cell in the pathogenesis of Ph-negative MPNs is still no... more Introduction The role of the endothelial cell in the pathogenesis of Ph-negative MPNs is still not elucidated. Some have reported the presence of the JAK2V617F mutation in endothelial colony forming cells (ECFC) isolated from peripheral blood in patients with Ph-negative MPNs (Teofili L et all, Blood 2011). Others, however, did not find such an association (Piaggio G et al, Blood 2009). In patients with Budd-Chiari Syndrome (BCS), the JAK2V617F mutation has been found in endothelial cell (ECs) isolated by micro dissection from liver biopsies in patients both with and without MPN (Sozer S et al, Blood 2009). Besides the JAK2V617F mutation, other mutations (e.g. ASXL1, TET2, DNMT3A, SRSF2) have been described in patients with Ph-negative MPNs, but their presence has not been evaluated in patients with BCS who carried the JAK2V617F mutation. Objectives 1. To evaluate the presence of the JAK2V617F mutation in CECs from patients with BCS both with and without concomitant Ph-negative MPNs; 2. To determine the mutational landscape of granulocytes in patients with BCS who harbored the JAK2V617F mutation but did not have the clinical diagnosis of a Ph-negative MPN. Methods We identified 10 patients from our institution who had a diagnosis of BCS and harbored the JAK2V617F mutation in granulocytes. Three patients died from hepatic failure before they could be evaluated by bone marrow biopsy, so 7 patients remain for the analysis. All patients were investigated for the presence of Ph-negative MPNs with bone marrow trephine biopsy. ECs assays were performed according to the method of Hill. Briefly, Ficoll-Paque density gradient–isolated mononuclear cells were plated on fibronectin coated 6-well dishes with EndoCult medium (Stem Cell Technologies) for 48 hours, when non adherent cells were recovered and re-plated in a new dish at 106/mL concentration. After an additional 5 days, adherent cells were plucked and analyzed by flow cytometry. The ECs population was sorted using a FACS Aria BD Biosciences sorter according to the following phenotype: CD45-PerCP-negative, CD31-FITC-positve, VEGFR2-PE-positive, CD34-PECy7-positive, CD133-APC-negative. The presence of the JAK2V617F mutation was investigated by allele-specific PCR. Paired DNA (sorted CD66b-granulocytes/skin biopsy) from 3 patients with JAK2V617F-positive BCS without a clinical diagnosis of Ph-negative MPN was subjected to whole exome sequencing on a Illumina HiSeq 2000 platform using Agilent SureSelect kit. 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), followed by in-house filters to reduce false positive calls. Results We were able to obtain CECs from all 7 patients. The purity of the CECs populations obtained was over 96% in all cases. Among the 7 patients with BCS, five did not have any clinical feature of a Ph-negative MPN, with a normal bone marrow biopsy. Results are summarized in the table. The JAK2V617F mutation was positive in the CECs from 5 cases, including 3 patients who only had BCS. In one patient with BCS solely the reaction did not work, and in another the JAK2 was wild-type in the ECs. The mutation was positive in CECs from both patients with myelofibrosis and BCS. Three patients with BCS solely were evaluated by whole exome sequencing. The only known pathogenic abnormality found was the JAK2V617F mutation, albeit at a low allele fraction (5%, 6% and 12.6%). Conclusion The presence of the JAK2V617F mutation in CECs from patients with BCS who did and did not have a diagnosis of Ph-negative MPN suggest that the mutation plays an important role in the development of vascular complications in these patients. Further studies with a larger number of patients are needed to precisely define the importance of CECs in the pathogenesis of MPNs. The sole presence of the JAK2V617F mutation in circulating granulocytes at a very low allele fraction in patients with BCS without Ph-negative MPNs suggest that these patients have a pre-malignant clone that would probably remain undiagnosed had it been not for the development of hepatic venous thrombosis. Disclosures No relevant conflicts of interest to declare.
Introduction: Development of massive sequencing of whole exomes (WES; whole exome sequencing) has... more Introduction: Development of massive sequencing of whole exomes (WES; whole exome sequencing) has revolutionized DNA sequencing, leading to several studies detailing the genetic profile of patients with several types of neoplasms, including MPNs and MDS/MPNs. One important challenge is to identify recurrently mutated genes that may play a role in disease pathogenesis. It is important to consider that cohort size is critical in the analysis to determine putative oncogenic driver genes, and most cohorts published in MPNs and MDS/MPNs have a limited sample size. One possibility to circumvent this limitations is to combine datasets of different studies. Herein we report the combined analysis of WES data of 403 patients with these disorders, including 124 patients from our center and 279 cases that were previously reported in the literature. Patients and Methods: We analyzed a combined cohort of 403 patients in this study with a diagnosis of Ph-negative MPN or MDS/MPN. For the 124 patients included from our center, reads were aligned and processed following Genome Analysis Toolkit best practices, and somatic variants were called combining the output of SomaticSniper, Mutect and Pindel. Regarding public data, we selected 6 studies that evaluated patients with MPNs and MDS/MPNs through WES and where sample-level data on all individual mutations that were identified were available. Data on identified mutations was extracted from supplementary material of the articles. Combined data on genomic mutations from the whole cohort was used to predict driver genes using softwares MutSigCV and IntOGen (www.intogen.org). Genes considered as being significantly mutated were genes with q-value Results: Diagnosis of the cohort included essential thrombocythemia (ET; N=110), polycythemia vera (PV; N=93), myelofibrosis (MF; N=98), chronic myelomonocytic leukemia (CMML; N=82), atypical CML (aCML; N=15) and more rare disorders (MPN-unclassified [N=2], MDS/MPN-unclassified [N=2] and refractory anemia with ring sideroblasts and thrombocytosis [N=1]). There were a total of 7519 identified mutations, with a median of 9 nonsilent coding mutations per sample (range 0-311 mutations). There were 2 patients (both with a diagnosis of ET) who had no somatic mutations identifed. Analysis of the mutational data using both MutSigCV and IntOGen algorithms revealed a total of 26 genes considered as putative driver genes in these neoplasms: JAK2, TET2, ASXL1, SRSF2, CALR, DNMT3A, SF3B1, CBL, NRAS, EZH2, U2AF1, RUNX1, KRAS, CUX1, MPL, ZRSR2, TP53, IDH2, PHF6, SH2B3, BCL11A, GATA2, ZBTB33, ETNK1, IDH1 and PPM1D (Figure) . A mutation in at least one these genes was found in 92.5% of samples. Among these 26 genes, 2 genes were not previously reported to be mutated in these diseases ( ZBTB33 and BCL11A ), and one gene was previously reported to be associated with other types of neoplastic diseases ( PPM1D ). ZBTB33 encodes the transcriptional regulator Kaiso and was found to be mutated in 6 samples (MF=2, ET=2, PV=1, CMML=1). Three mutations were frameshift indels, and three mutations were missense mutations, one of them (p.I37T) located in the POZ/BTB domain that regulates protein homodimerization. The second gene that was found to be recurrently mutated is BCL11A , which encodes a zinc-finger protein. There were 6 cases of BCL11A mutations, most of them in MDS/MPNs (CMML=3, aCML=2, MF=1). Mutations were all missense and clustered in two asparagine residues located at positions 391 and 756. Finally, four patients (CMML=2, PV=1, MF=1) presented with truncating mutations in the last exon of the PPM1D gene. These mutations have been reported to be activating, leading to an increase in activity of the Wip1 phosphatase and suppression of p53 activity. PPM1D mutations have been reported to be associated with clonal hematopoiesis, MDS and solid tumors. To the best of our knowledge this is one of the first reports detailing their presence in MPNs. Conclusions: Combining datasets across different studies has the potential to increase power and saturate discovery of recurrently mutated genes. This permits prioritization of genes for functional analysis to confirm their role in disease pathogenesis. Larger cohorts than the ones analyzed here may provide even more meaningful data on putative driver genes in MPNs and MDS/MPNs. Disclosures No relevant conflicts of interest to declare.
Introduction Busulfan (BU) is one of the most used alkylating agent in mieloablative conditioning... more Introduction Busulfan (BU) is one of the most used alkylating agent in mieloablative conditioning regimens (CR) for patients submited to Hematopoietic Stem Cell Transplantation (HSCT). Its therapeutic effect is correlated to area under the curve (AUC) having a wide variability among patients. Ideal AUC is not been well stablished yet but it is known that higher levels can impact survival by increasing extra medular toxicity. In the other way, lower levels can be associated to a higher incidence of relapse. Two BU formulations are available, oral (PO) or Intrvenous (IV). Although IV BU can developed more reliable AUC, there are few data comparing both formulations. Objectives To compare the pharmacokinetics (PK) of PO and IV BU based conditioning regimens in patients submited to HSCT and to determinate the best target BU AUC in order to reduce acute toxicity after HSCT. Methods and Results 149 patients were prospectively evaluated. All recieved myeloablative (MA) BU based conditioning regimen (BU associated to Cyclophosphamide(Cy), Melphalan(Mel), Cy/VP-16, Fludarabine(Flu)/Thiotepa, Flu/Mel,Mel/Gencitabine, Mel/Cy, Flu/clofarabine, Clofarabine/Thiotepa). 56 (37,5%) received a MA reduced intensity CR (Bu associated to Flu). 56 (37.5%) patients recieved a stem cells from a matched related donor, 45(30.2%) from matched unrelated donor, 9(6%) from related haploidentical donor and 17 (11.4%) from cord blood. The median age was 30,2 years (range 1,3-73). 86 (57.7%) had acute leukemias and myelodisplastic syndrome, 28(18.8%) had lymphomas, multiple myeloma and chronic leucemias, 32(21.4%) had diferente benign diseases. 83 (55.7%) patients recieved PO and 63(44.3%) IV BU. All patients had BU PK monitoring at least one time during CR. 81 (54.4%) patients also performed a pre transplant BU test dose (32mg/m2 for IV and 1mg/kg for PO BU). There were 3 BU targeted AUC: 4000, 5000 and 6000 µMol.min, according to institutional protocols based on disease risk and patient age. Historical control of 53 patients who received myeloablative conditioning regimen with no BU PK monitoring were used. Mucositis, Sinusoidal obstruction Syndrome (SOS) Overall survival (OS), Transplant related mortality (TRM) and relapse were analysed according to AUC targeted dose. 184 (91%) patients developed mucositis at 5 (range:0-11) days after HSCT. There were no diference in either incidence (p=0,15) or severity (p=0,19) of mucositis in patients recieving PO(n=66, 32.6%) or IV (n=83, 41%) BU and historical control (n=53, 26.2%) (p=0,15). Pre transplant test dose had also no impact on incidence and severity of mucositis (p=0,75). More importantly, AUC targeted dose had no impact in either mucositis incidence or severity (p=0.75). Among all 202 patients, 23 (11,4%) developed SOS until day 30 post HSCT. The use of pre transplant Bu test dose had no impact in SOS incidence: Gray-subhazard ratio (SHR)SHR 0.70 - CI95% 025-1.91; (p=0.48). BU formulations (PO or IV) had no impact in SOS (p=0.73) either. Patients recieving higher BU AUC targeted dose had an incresed risk to develop SOS after HSCT: Incidence of SOS in patients who recieved AUC =5000 was 5% and 16% respectively (SHR 3.39, p=0.034, CI 95% 1.09-10.52). In contrast, this finding had no impact in day Transplant related TRM and 1 year OS: patients who revieved AUC =5000, had 1 year OS of 61% and 66% respectivelly (p=0.52). Patients who revieved AUC =5000, had 100 day TRM of 12% and 12% respectivelly (p=0.42). We also found that patients who recieved AUC from 4000 to 5000 had a lower OS, showing a protective effect of lower AUC: 1 year OS for patients who recieved AUC 4000t to 5000 was 65% (IC95% 55-74%) HR: 0.65, (p=0.33). In the other hand, patients who recieved lower AUC targeted dose (4000) had higher incidence of relapse: SHR: 0.42, p=0.08, CI 95% 0.15-1.1 Conclusion In this cohort of patients, toxicity and survival were not influenced by BU formulation (oral or IV). More importantly, patients recieving higher Bu AUC targeted dose had higher incidence of SOS and targeting AUC between 4000 to 5000 had a positive impact in 1 year OS but are more likely to relapse (AUC 4000). We also, shown that using Bu test dose, does not increase toxicities or mortality in patients reciving Bu condicioning regimen. Therefore, we can conclude that PK Bu monitoring is important in either PO or IV BU formulation, in order to reduce toxicities such as SOS and mortality. Disclosures: Santos:Novartis: Consultancy, Research Funding, Speakers Bureau.
Clinical Lymphoma, Myeloma & Leukemia, Jun 1, 2015
Background: The median survival of patients with primary myelofibrosis (PMF) is 5 to 7 years afte... more Background: The median survival of patients with primary myelofibrosis (PMF) is 5 to 7 years after diagnosis. In the majority of patients with PMF somatic mutations were detected either in Janus Kinase 2 (JAK2; in 60% of patients), Calreticulin (CALR; in 25% of patients) or MPL (in 5% of patients) genes. Neither mutation was detected 5% to 10% of PMF patients. Patients with mutated JAK2 are known to have a more aggressive disease compared to patients with mutated CALR. However patients with mutated JAK2 and high allele burden have a favorable outcome compared to patients with a low mutated JAK2 burden. Aim: To develop a model that uses genetic information to predict survival outcome of patients with PMF. Patients and Methods: Bone marrow samples were collected from 344 patients with PMF that were followed at MD Anderson Cancer Center between 2000 and 2013 (157 months). All samples were screened for JAK2 and for mutations in CALR. Patients who did not have a mutation in either gene were also screened for mutations in MPL. Results: In 226 patients (66%) JAK2 was detected and in 43 (13%) CALR was mutated. Of the 75 patients who did not have JAK2 or CALR mutations, 16 (21%) had mutated MPL. In 59 patients (17%), none of those mutations was detected. In the 226 patients who harbored the JAK2 mutation, a cut-point of 50% dichotomized patients into those with a high JAK2 burden and a favorable overall survival (OS; median OS: 80 months) and those with a low JAK2 burden and an adverse OS (median OS: 50 months). Age (above 65 years) and mutation status (low JAK2 burden or triple-negative) were independent risk factors. Patients with a favorable mutation status and age below 65 had a median survival of 126 months (n 1⁄4 82). Patients with either one risk factor, age above 65 (n 1⁄4 88) or adverse mutation status (n 1⁄4 87) had intermediate survival expectancy. The two risk factors were additive and patients age > 65 years and adverse mutation status (n 1⁄4 87) had a median survival of 35 months. Conclusions: Age and mutation status are independent predictors of survival in patients with PMF and stratify patients into 4 groups of equal size with very different survival outcome. 703 Mutational Profiling of JAK2V617F vs. CALR mutated Primary Myelofibrosis Fabio Santos, Renato Puga, Bianca Lisboa, Welbert Pereira, Mariana Miyagi, Evelyn Mata, Tarcila Datoguia, Isabel Bello, Michelli Diniz, Sandra Nakashima, Guilherme Perini, Ricardo Helman, Nelson Hamerschlak, Paulo Campregher Hematology/Oncology, Hospital Israelita Albert Einstein
Mucosa-associated lymphoid tissue (MALT) is the most common extranodal lymphoma, with one third o... more Mucosa-associated lymphoid tissue (MALT) is the most common extranodal lymphoma, with one third of the cases occurring in the stomach. Surgical treatment is a possible option. This case report describes an obese woman (body mass index 46 kg/m2) seeking bariatric surgery, with elevated serum cholesterol and uric acid, negative for Helicobacter pylori but with a positive biopsy for MALT (immunohistochemistry), stage IA. She was submitted to a Roux-en-Y gastric bypass and gastric resection. Two months later, she had lost 20 kg and the MALT lymphoma was in complete remission.
5127 NOTCH1 is a proto-oncogene with activating mutations described in a variety of malignancies,... more 5127 NOTCH1 is a proto-oncogene with activating mutations described in a variety of malignancies, including acute lymphoblastic leukemia (ALL), mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). While the prognostic significance of NOTCH1 mutations remains controversial in ALL, recent data suggest that NOTCH1 PEST domain mutations are associated with adverse prognosis in patients with CLL. NOTCH1 mutations are found in around 8% of CLL patients at diagnosis and more than 30% of patients with advanced disease. Since this disease has a heterogeneous clinical course and few prognostic markers, we aimed at designing a fast, cost effective and robust assay to detect NOTCH1 PEST domain mutations in patients with CLL for the clinical laboratory. While 92% of the mutations in NOTCH1 PEST domain found in CLL are insertions or deletions, only 8% are represented by point mutations. Therefore we decided to use a fragment analysis approach in our assay. Given that a single mutati...
Introduction The role of the endothelial cell in the pathogenesis of Ph-negative MPNs is still no... more Introduction The role of the endothelial cell in the pathogenesis of Ph-negative MPNs is still not elucidated. Some have reported the presence of the JAK2V617F mutation in endothelial colony forming cells (ECFC) isolated from peripheral blood in patients with Ph-negative MPNs (Teofili L et all, Blood 2011). Others, however, did not find such an association (Piaggio G et al, Blood 2009). In patients with Budd-Chiari Syndrome (BCS), the JAK2V617F mutation has been found in endothelial cell (ECs) isolated by micro dissection from liver biopsies in patients both with and without MPN (Sozer S et al, Blood 2009). Besides the JAK2V617F mutation, other mutations (e.g. ASXL1, TET2, DNMT3A, SRSF2) have been described in patients with Ph-negative MPNs, but their presence has not been evaluated in patients with BCS who carried the JAK2V617F mutation. Objectives 1. To evaluate the presence of the JAK2V617F mutation in CECs from patients with BCS both with and without concomitant Ph-negative MPNs...
Introduction: Development of massive sequencing of whole exomes (WES; whole exome sequencing) has... more Introduction: Development of massive sequencing of whole exomes (WES; whole exome sequencing) has revolutionized DNA sequencing, leading to several studies detailing the genetic profile of patients with several types of neoplasms, including MPNs and MDS/MPNs. One important challenge is to identify recurrently mutated genes that may play a role in disease pathogenesis. It is important to consider that cohort size is critical in the analysis to determine putative oncogenic driver genes, and most cohorts published in MPNs and MDS/MPNs have a limited sample size. One possibility to circumvent this limitations is to combine datasets of different studies. Herein we report the combined analysis of WES data of 403 patients with these disorders, including 124 patients from our center and 279 cases that were previously reported in the literature. Patients and Methods: We analyzed a combined cohort of 403 patients in this study with a diagnosis of Ph-negative MPN or MDS/MPN. For the 124 patients included from our center, reads were aligned and processed following Genome Analysis Toolkit best practices, and somatic variants were called combining the output of SomaticSniper, Mutect and Pindel. Regarding public data, we selected 6 studies that evaluated patients with MPNs and MDS/MPNs through WES and where sample-level data on all individual mutations that were identified were available. Data on identified mutations was extracted from supplementary material of the articles. Combined data on genomic mutations from the whole cohort was used to predict driver genes using softwares MutSigCV and IntOGen (www.intogen.org). Genes considered as being significantly mutated were genes with q-value Results: Diagnosis of the cohort included essential thrombocythemia (ET; N=110), polycythemia vera (PV; N=93), myelofibrosis (MF; N=98), chronic myelomonocytic leukemia (CMML; N=82), atypical CML (aCML; N=15) and more rare disorders (MPN-unclassified [N=2], MDS/MPN-unclassified [N=2] and refractory anemia with ring sideroblasts and thrombocytosis [N=1]). There were a total of 7519 identified mutations, with a median of 9 nonsilent coding mutations per sample (range 0-311 mutations). There were 2 patients (both with a diagnosis of ET) who had no somatic mutations identifed. Analysis of the mutational data using both MutSigCV and IntOGen algorithms revealed a total of 26 genes considered as putative driver genes in these neoplasms: JAK2, TET2, ASXL1, SRSF2, CALR, DNMT3A, SF3B1, CBL, NRAS, EZH2, U2AF1, RUNX1, KRAS, CUX1, MPL, ZRSR2, TP53, IDH2, PHF6, SH2B3, BCL11A, GATA2, ZBTB33, ETNK1, IDH1 and PPM1D (Figure) . A mutation in at least one these genes was found in 92.5% of samples. Among these 26 genes, 2 genes were not previously reported to be mutated in these diseases ( ZBTB33 and BCL11A ), and one gene was previously reported to be associated with other types of neoplastic diseases ( PPM1D ). ZBTB33 encodes the transcriptional regulator Kaiso and was found to be mutated in 6 samples (MF=2, ET=2, PV=1, CMML=1). Three mutations were frameshift indels, and three mutations were missense mutations, one of them (p.I37T) located in the POZ/BTB domain that regulates protein homodimerization. The second gene that was found to be recurrently mutated is BCL11A , which encodes a zinc-finger protein. There were 6 cases of BCL11A mutations, most of them in MDS/MPNs (CMML=3, aCML=2, MF=1). Mutations were all missense and clustered in two asparagine residues located at positions 391 and 756. Finally, four patients (CMML=2, PV=1, MF=1) presented with truncating mutations in the last exon of the PPM1D gene. These mutations have been reported to be activating, leading to an increase in activity of the Wip1 phosphatase and suppression of p53 activity. PPM1D mutations have been reported to be associated with clonal hematopoiesis, MDS and solid tumors. To the best of our knowledge this is one of the first reports detailing their presence in MPNs. Conclusions: Combining datasets across different studies has the potential to increase power and saturate discovery of recurrently mutated genes. This permits prioritization of genes for functional analysis to confirm their role in disease pathogenesis. Larger cohorts than the ones analyzed here may provide even more meaningful data on putative driver genes in MPNs and MDS/MPNs. Disclosures No relevant conflicts of interest to declare.
Objective: To analyze the outcome of patients treated with gemtuzumab ozogamycin combined with co... more Objective: To analyze the outcome of patients treated with gemtuzumab ozogamycin combined with conventional therapy treated at Hospital Israelita Albert Einstein. Methods: 14 patients who had high risk features (secondary leukemia, unfavorable cytogenetics, and refractory disease) were treated with gemtuzumab ozogamycin combined with conventional therapy and their outcome was analysed by reviewing their medical records. results: Overall response rate was 58%, with 43% achieving complete response, with a median followup of 11 months, event-free survival was 3 months. Eleven patients died, 6 of them due to refractory acute myeloid leukemia. Only four patients presented with grade 3 to 4 toxicities and only one patient had sinusoidal obstruction syndrome after bone marrow transplant. conclusion: gemtuzumab ozogamycin combined with chemotherapy is a feasible treatment regimen in acute myeloid leukemia patients. However, further studies are necessary to clarify which subgroup of patients may benefit from this treatment.
Objective: To analyze the outcome of patients treated with gemtuzumab ozogamycin combined with co... more Objective: To analyze the outcome of patients treated with gemtuzumab ozogamycin combined with conventional therapy treated at Hospital Israelita Albert Einstein. Methods: 14 patients who had high risk features (secondary leukemia, unfavorable cytogenetics, and refractory disease) were treated with gemtuzumab ozogamycin combined with conventional therapy and their outcome was analysed by reviewing their medical records. results: Overall response rate was 58%, with 43% achieving complete response, with a median followup of 11 months, event-free survival was 3 months. Eleven patients died, 6 of them due to refractory acute myeloid leukemia. Only four patients presented with grade 3 to 4 toxicities and only one patient had sinusoidal obstruction syndrome after bone marrow transplant. conclusion: gemtuzumab ozogamycin combined with chemotherapy is a feasible treatment regimen in acute myeloid leukemia patients. However, further studies are necessary to clarify which subgroup of patients may benefit from this treatment.
Abstract 4531 Introduction: Cytomegalovirus (CMV) is one of the most common viral pathogens post-... more Abstract 4531 Introduction: Cytomegalovirus (CMV) is one of the most common viral pathogens post-allogeneic stem cell transplantation (SCT). However, CMV reactivation and disease are relatively uncommon events after autologous stem cell transplantation (ASCT), with a reported incidence of 2–9%. There are few studies describing risk factors for development of CMV reactivation post-ASCT. Objective: To describe the incidence and risk factors for CMV reactivation post-ASCT in our institution and its impact on survival. Methods: The charts of patients who underwent ASCT at our institution from January 2005 until July 2009 were manually reviewed. CMV reactivation was detected by antigenemia assay and/or real-time PCR. Variables associated with CMV in a univariate analysis with a…
Introduction: Busulfan (Bu) is used in the conditioning regimen for hematopoietic stem cell trans... more Introduction: Busulfan (Bu) is used in the conditioning regimen for hematopoietic stem cell transplantation (HSCT). Therapeutic drug monitoring (TDM) of Bu with subsequents adjustments doses based on a “target” therapeutic concentration may reduce toxicity after HSCT. Objectives: To evaluatethe impact of TDM of Bu and clinical outcomes in patients with acute leukemia that underwent to allogeneic matched related donor (MRD) and allogeneic matched unrelated donor (MUD) HSCT. Patients and methods: From January 2009 to January 2014, we prospectively analyzed 42 patients with diagnosis of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) who underwent TDM of Bu (IV or oral) before transplantation (test dose) and TDM on 1st day of conditioning regimen. Samples were collected at 0, 30’, 60’ and subsequently every hour until 6 hours after administration of busulfan. The plasma was extracted by HPLC (High Performance Liquid Chromatography). All the patients that were submitted to TDM had a test dose 15 days to 48 hours before transplantion. The dose of Bu was adjusted during the first day of the conditioning regimen based on test dose. At the same time we analyzed 21 patients in the retrospective group who did not underwent TDM (from 2004 to 2010). Results: In the retrospective group (n=21), all of them underwent to MRD transplantation. Six (46.2%) were in first complete remission (CR1), 18(85.7%) patients received Bu and cyclophosphamide (BuCy) and the mean of age was 38 years (18-55 Yo). The median of CD34+ cells was 5.4 x 106/kg. The second group consisted by patients that received oral Bu (n= 21): 7 (33.3%) underwent to MUD transplantation, 14 (66.6%) to MRD transplantation, 8 (44.4%) patients were second complete remission (CR2), 4 (22.2%) had active disease status with a mean age of 32.7 years (14-58 Yo). Fifteen (71.4%) received BuCy and 16 (76.2%) received cells from peripheral blood. The median of CD34+ cells was 5.8 x106/kg. The median area under the curve (AUC) in 24 hours was 4950 μMol.min (3196.6- 8212 μMol.min). The third group was IV Bu (n= 21): 7 (33.3%) patients underwent MRD and 14 (66.6%) MUD transplantation, 7 (33.3%) patients were CR1, 7 (33.3%) had active disease or prior HSCT with a mean of age 52.7 years (20-74 Yo). The majority of patients received fludarabine and Bu (n=18; 85.7%) as conditioning and bone marrow was the main source. Immunosuppression was based on FK-506 and methotrexate (90.5% of patients). The median of AUC in 24 hours was 5690 μMol.min (3539.6- 8881.8 μMol.min). The cumulative incidence (CI) of sinusoidal obstructive syndrome (SOS) in the retrospective group and IV Bu were 9.5% for both, while in the group oral Bu it was at 19% (p = 0.566). The median AUC of Bu received during conditioning for those who died of SOS was lower in oral Bu than IV Bu (4872 uMol.min vs 5732 uMol.min respectively). The CI of acute graft-versus-host disease (aGVHD) at D+100 was 38.1% in the retrospective group, 40.6% in oral Bu and 42.9% in IV Bu. Chronic GVHD was 13.6% in oral Bu, 34% in IV Bu and 42.9% in the retrospective group (p = 0.142). The CI of relapse at D+100 was 19% in IV Bu, 4.8% in the retrospective group and oral Bu did not have this event. The IC of death at D+100 was 34.9% in group oral Bu, 9.5% in IV Bu and 14.3% in the retrospective (p = 0.102). The CI of relapse at 1.5 years was 35.8% in the IV Bu, 34.8% in oral Bu and 14.3% in the retrospective group. The CI of death at 1.5 years was 9.5% in group IV Bu, 53.5% in oral Bu and 34.3% in the retrospective (p = 0.015). Among patients who died until D+100, the median of AUC was 5732 μMol.min (5578.5-6818.5 μMol.min) during the conditioning for IV Bu and 4872 μMol.min (3448-8212 μMol.min) for oral Bu. The range between the AUC was large and there was no correlation with patients who died. Conclusion: In acute leukemia SOS had an impact on mortality at D+100 after HSCT (p…
Introduction: Hematopoietic stem cell transplantation (HSCT) was offered primarily as a therapeut... more Introduction: Hematopoietic stem cell transplantation (HSCT) was offered primarily as a therapeutic option for severe sickle cell disease in the context of myeloablative matched sibling donor transplants over the last two decades and helped to establish the benefits of transplantation for this disorder. In recent years, the transplant community has set out to explore ways to make stem-cell transplantation more available to patients with the disease, define indications and better timing, and offset toxicities with novel approaches to conditioning and better supportive care. In this context, neurological complication such as stroke disease and blood flow alteration in medial cerebral artery constitute the main indications of HSCT but neurological complications are the main causes of TRM in D+100 and is important to find ways to prevent this problem. Objective: Describe early neurological complications in sickle patients undergoing related HSCT on single specialized center and its following after the early diagnosis. Material and Methods: Seven patient were undergone related HSCT for sickle disease from 2011 to 2013. All patients filled inclusion criteria in the study and signed agreement term. Results: Seven HSCT were developed in the period, being four males. The average age was 13 years old (7-24). The HSCT indications were previous stroke, cerebral flow alteration on Doppler, acute chest syndrome and alloimunization. All patient were on blood transfusion therapy. The conditioning regimen was BuCy + ATG and the GVHD prophylaxis was MTX and CSA. Related donors were chosen with 10/10 HLA match and graft source marrow. The median of the neutrophil engraftment was D+ 25 and the platelets engraftment was in D+60. Two patient died, one by intestinal and liver GVHD on D+120, and another with sagittal sinus thrombosis and hemorrhagic stroke on D+3. Other two patients showed PRES syndrome related to cyclosporine use. The patients showed generalized seizures with tomographic neurological alteration. After the imunossupressor change to tacrolimus and the change of Phenytoin to Lamotrigine, the patient had total resolution of neurological complications without development of neurological sequelae. Patients who used Lamotrigine since the beginning of the conditioning have not shown neurological alterations. Conclusion: The PRES Syndrome and Stroke are two of the main causes of mortality related to the use of calcineurin inhibitors. Patients with sickle disease have shown endothelial and cerebral microcirculation changes, which made them highly susceptible to neurological complication. The control of blood pressure, maintenance of 50.000 platelets level and the use of Lamotrigine as prophylaxis of seizures seems to decrease the risk of neurological complications. Prospective studies with lamotrigine as primary prevention of PRES Syndrome must be performed. Patients with severe neurological alteration as vessels stenosis more than 90% and Moya-Moya Syndrome must be better evaluated before the conditioning because of high TRM risk. Disclosures No relevant conflicts of interest to declare.
Introduction: Multiple myeloma is an incurable hematologic malignancy. However, many novel drugs ... more Introduction: Multiple myeloma is an incurable hematologic malignancy. However, many novel drugs (lenalidomide, carfilzomib, daratumumab, elotuzumab and ixazomib) have recently been approved in Brazil for this disease. Proving that the availability of these drugs increases survival is not straightforward, since pivotal trials with these drugs were designed to show benefit in progression-free survival and not in overall survival. Hypothesis: We have hypothesized that the availability of novel drugs would reduce the risk of death. Methods: This is a real-world, unicenter analysis that included patients with multiple myeloma treated between 1993 and 2017. The availability of novel drugs (lenalidomide, carfilzomib, daratumumab, elotuzumab and ixazomib) was treated as a time-dependent covariate and analyzed by Cox model. The analysis was controlled for the International Staging System (ISS) and for age. Results: With a median follow-up of 6 years, we included 151 patients with multiple myeloma. Fourty percent had ISS-I, 34% ISS-II and 26% ISS-III. Mean age was 60.5 y/o (SD: 12.2). There has been 51 deaths, and 5-y overall survival was 90% for ISS-I, 80% for ISS-II and 54% for ISS-III (p=0.0004). In multivariable analysis, age (HR=1.06, p<0.0001) and ISS-III (HR=4.51, p<0.0001) were risk factors for death, while the availability of two or more novel drugs was protective (HR=0.31, p=0.006). The availability of a single novel drug was also protective (HR=0.58), although it did not reach statistical significance (p=0.13). Adjusted Simon-Makuch survival plot is in figure 1 (adjusted for ISS III and 60 y/o). Discussion: Our results show that the availability of two or more novel drugs increases overall survival in multiple myeloma. This is an important finding, since the pivotal trials were designed to show advantages in progression-free survival and, once achieved, the experimental drug was offered to the control group, hampering overall survival analyses. Overall survival benefit, therefore, are more likely to be seen in real-world studies. We included the availability of novel drugs as a time-dependent, and the interpretation of our results is that patients alive when two novel drugs became available had benefit in overall survival. Our study has two weaknesses. Two or more novel drugs were more likely to be available for patients from a more recent time. This difference could be due to changes in supportive care, but there has been no major change in supportive care for multiple myeloma patients. Another weakness is that patient alive in the more recent years may have not received novel drug-based treatment. Considering that multiple myeloma is an incurable disease, this is unlikely for the majority of patients. In conclusion, the availability of two or more novel drugs for multiple myeloma improves survival. Figure Disclosures No relevant conflicts of interest to declare.
Introduction The role of the endothelial cell in the pathogenesis of Ph-negative MPNs is still no... more Introduction The role of the endothelial cell in the pathogenesis of Ph-negative MPNs is still not elucidated. Some have reported the presence of the JAK2V617F mutation in endothelial colony forming cells (ECFC) isolated from peripheral blood in patients with Ph-negative MPNs (Teofili L et all, Blood 2011). Others, however, did not find such an association (Piaggio G et al, Blood 2009). In patients with Budd-Chiari Syndrome (BCS), the JAK2V617F mutation has been found in endothelial cell (ECs) isolated by micro dissection from liver biopsies in patients both with and without MPN (Sozer S et al, Blood 2009). Besides the JAK2V617F mutation, other mutations (e.g. ASXL1, TET2, DNMT3A, SRSF2) have been described in patients with Ph-negative MPNs, but their presence has not been evaluated in patients with BCS who carried the JAK2V617F mutation. Objectives 1. To evaluate the presence of the JAK2V617F mutation in CECs from patients with BCS both with and without concomitant Ph-negative MPNs; 2. To determine the mutational landscape of granulocytes in patients with BCS who harbored the JAK2V617F mutation but did not have the clinical diagnosis of a Ph-negative MPN. Methods We identified 10 patients from our institution who had a diagnosis of BCS and harbored the JAK2V617F mutation in granulocytes. Three patients died from hepatic failure before they could be evaluated by bone marrow biopsy, so 7 patients remain for the analysis. All patients were investigated for the presence of Ph-negative MPNs with bone marrow trephine biopsy. ECs assays were performed according to the method of Hill. Briefly, Ficoll-Paque density gradient–isolated mononuclear cells were plated on fibronectin coated 6-well dishes with EndoCult medium (Stem Cell Technologies) for 48 hours, when non adherent cells were recovered and re-plated in a new dish at 106/mL concentration. After an additional 5 days, adherent cells were plucked and analyzed by flow cytometry. The ECs population was sorted using a FACS Aria BD Biosciences sorter according to the following phenotype: CD45-PerCP-negative, CD31-FITC-positve, VEGFR2-PE-positive, CD34-PECy7-positive, CD133-APC-negative. The presence of the JAK2V617F mutation was investigated by allele-specific PCR. Paired DNA (sorted CD66b-granulocytes/skin biopsy) from 3 patients with JAK2V617F-positive BCS without a clinical diagnosis of Ph-negative MPN was subjected to whole exome sequencing on a Illumina HiSeq 2000 platform using Agilent SureSelect kit. 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), followed by in-house filters to reduce false positive calls. Results We were able to obtain CECs from all 7 patients. The purity of the CECs populations obtained was over 96% in all cases. Among the 7 patients with BCS, five did not have any clinical feature of a Ph-negative MPN, with a normal bone marrow biopsy. Results are summarized in the table. The JAK2V617F mutation was positive in the CECs from 5 cases, including 3 patients who only had BCS. In one patient with BCS solely the reaction did not work, and in another the JAK2 was wild-type in the ECs. The mutation was positive in CECs from both patients with myelofibrosis and BCS. Three patients with BCS solely were evaluated by whole exome sequencing. The only known pathogenic abnormality found was the JAK2V617F mutation, albeit at a low allele fraction (5%, 6% and 12.6%). Conclusion The presence of the JAK2V617F mutation in CECs from patients with BCS who did and did not have a diagnosis of Ph-negative MPN suggest that the mutation plays an important role in the development of vascular complications in these patients. Further studies with a larger number of patients are needed to precisely define the importance of CECs in the pathogenesis of MPNs. The sole presence of the JAK2V617F mutation in circulating granulocytes at a very low allele fraction in patients with BCS without Ph-negative MPNs suggest that these patients have a pre-malignant clone that would probably remain undiagnosed had it been not for the development of hepatic venous thrombosis. Disclosures No relevant conflicts of interest to declare.
Introduction: Development of massive sequencing of whole exomes (WES; whole exome sequencing) has... more Introduction: Development of massive sequencing of whole exomes (WES; whole exome sequencing) has revolutionized DNA sequencing, leading to several studies detailing the genetic profile of patients with several types of neoplasms, including MPNs and MDS/MPNs. One important challenge is to identify recurrently mutated genes that may play a role in disease pathogenesis. It is important to consider that cohort size is critical in the analysis to determine putative oncogenic driver genes, and most cohorts published in MPNs and MDS/MPNs have a limited sample size. One possibility to circumvent this limitations is to combine datasets of different studies. Herein we report the combined analysis of WES data of 403 patients with these disorders, including 124 patients from our center and 279 cases that were previously reported in the literature. Patients and Methods: We analyzed a combined cohort of 403 patients in this study with a diagnosis of Ph-negative MPN or MDS/MPN. For the 124 patients included from our center, reads were aligned and processed following Genome Analysis Toolkit best practices, and somatic variants were called combining the output of SomaticSniper, Mutect and Pindel. Regarding public data, we selected 6 studies that evaluated patients with MPNs and MDS/MPNs through WES and where sample-level data on all individual mutations that were identified were available. Data on identified mutations was extracted from supplementary material of the articles. Combined data on genomic mutations from the whole cohort was used to predict driver genes using softwares MutSigCV and IntOGen (www.intogen.org). Genes considered as being significantly mutated were genes with q-value Results: Diagnosis of the cohort included essential thrombocythemia (ET; N=110), polycythemia vera (PV; N=93), myelofibrosis (MF; N=98), chronic myelomonocytic leukemia (CMML; N=82), atypical CML (aCML; N=15) and more rare disorders (MPN-unclassified [N=2], MDS/MPN-unclassified [N=2] and refractory anemia with ring sideroblasts and thrombocytosis [N=1]). There were a total of 7519 identified mutations, with a median of 9 nonsilent coding mutations per sample (range 0-311 mutations). There were 2 patients (both with a diagnosis of ET) who had no somatic mutations identifed. Analysis of the mutational data using both MutSigCV and IntOGen algorithms revealed a total of 26 genes considered as putative driver genes in these neoplasms: JAK2, TET2, ASXL1, SRSF2, CALR, DNMT3A, SF3B1, CBL, NRAS, EZH2, U2AF1, RUNX1, KRAS, CUX1, MPL, ZRSR2, TP53, IDH2, PHF6, SH2B3, BCL11A, GATA2, ZBTB33, ETNK1, IDH1 and PPM1D (Figure) . A mutation in at least one these genes was found in 92.5% of samples. Among these 26 genes, 2 genes were not previously reported to be mutated in these diseases ( ZBTB33 and BCL11A ), and one gene was previously reported to be associated with other types of neoplastic diseases ( PPM1D ). ZBTB33 encodes the transcriptional regulator Kaiso and was found to be mutated in 6 samples (MF=2, ET=2, PV=1, CMML=1). Three mutations were frameshift indels, and three mutations were missense mutations, one of them (p.I37T) located in the POZ/BTB domain that regulates protein homodimerization. The second gene that was found to be recurrently mutated is BCL11A , which encodes a zinc-finger protein. There were 6 cases of BCL11A mutations, most of them in MDS/MPNs (CMML=3, aCML=2, MF=1). Mutations were all missense and clustered in two asparagine residues located at positions 391 and 756. Finally, four patients (CMML=2, PV=1, MF=1) presented with truncating mutations in the last exon of the PPM1D gene. These mutations have been reported to be activating, leading to an increase in activity of the Wip1 phosphatase and suppression of p53 activity. PPM1D mutations have been reported to be associated with clonal hematopoiesis, MDS and solid tumors. To the best of our knowledge this is one of the first reports detailing their presence in MPNs. Conclusions: Combining datasets across different studies has the potential to increase power and saturate discovery of recurrently mutated genes. This permits prioritization of genes for functional analysis to confirm their role in disease pathogenesis. Larger cohorts than the ones analyzed here may provide even more meaningful data on putative driver genes in MPNs and MDS/MPNs. Disclosures No relevant conflicts of interest to declare.
Introduction Busulfan (BU) is one of the most used alkylating agent in mieloablative conditioning... more Introduction Busulfan (BU) is one of the most used alkylating agent in mieloablative conditioning regimens (CR) for patients submited to Hematopoietic Stem Cell Transplantation (HSCT). Its therapeutic effect is correlated to area under the curve (AUC) having a wide variability among patients. Ideal AUC is not been well stablished yet but it is known that higher levels can impact survival by increasing extra medular toxicity. In the other way, lower levels can be associated to a higher incidence of relapse. Two BU formulations are available, oral (PO) or Intrvenous (IV). Although IV BU can developed more reliable AUC, there are few data comparing both formulations. Objectives To compare the pharmacokinetics (PK) of PO and IV BU based conditioning regimens in patients submited to HSCT and to determinate the best target BU AUC in order to reduce acute toxicity after HSCT. Methods and Results 149 patients were prospectively evaluated. All recieved myeloablative (MA) BU based conditioning regimen (BU associated to Cyclophosphamide(Cy), Melphalan(Mel), Cy/VP-16, Fludarabine(Flu)/Thiotepa, Flu/Mel,Mel/Gencitabine, Mel/Cy, Flu/clofarabine, Clofarabine/Thiotepa). 56 (37,5%) received a MA reduced intensity CR (Bu associated to Flu). 56 (37.5%) patients recieved a stem cells from a matched related donor, 45(30.2%) from matched unrelated donor, 9(6%) from related haploidentical donor and 17 (11.4%) from cord blood. The median age was 30,2 years (range 1,3-73). 86 (57.7%) had acute leukemias and myelodisplastic syndrome, 28(18.8%) had lymphomas, multiple myeloma and chronic leucemias, 32(21.4%) had diferente benign diseases. 83 (55.7%) patients recieved PO and 63(44.3%) IV BU. All patients had BU PK monitoring at least one time during CR. 81 (54.4%) patients also performed a pre transplant BU test dose (32mg/m2 for IV and 1mg/kg for PO BU). There were 3 BU targeted AUC: 4000, 5000 and 6000 µMol.min, according to institutional protocols based on disease risk and patient age. Historical control of 53 patients who received myeloablative conditioning regimen with no BU PK monitoring were used. Mucositis, Sinusoidal obstruction Syndrome (SOS) Overall survival (OS), Transplant related mortality (TRM) and relapse were analysed according to AUC targeted dose. 184 (91%) patients developed mucositis at 5 (range:0-11) days after HSCT. There were no diference in either incidence (p=0,15) or severity (p=0,19) of mucositis in patients recieving PO(n=66, 32.6%) or IV (n=83, 41%) BU and historical control (n=53, 26.2%) (p=0,15). Pre transplant test dose had also no impact on incidence and severity of mucositis (p=0,75). More importantly, AUC targeted dose had no impact in either mucositis incidence or severity (p=0.75). Among all 202 patients, 23 (11,4%) developed SOS until day 30 post HSCT. The use of pre transplant Bu test dose had no impact in SOS incidence: Gray-subhazard ratio (SHR)SHR 0.70 - CI95% 025-1.91; (p=0.48). BU formulations (PO or IV) had no impact in SOS (p=0.73) either. Patients recieving higher BU AUC targeted dose had an incresed risk to develop SOS after HSCT: Incidence of SOS in patients who recieved AUC =5000 was 5% and 16% respectively (SHR 3.39, p=0.034, CI 95% 1.09-10.52). In contrast, this finding had no impact in day Transplant related TRM and 1 year OS: patients who revieved AUC =5000, had 1 year OS of 61% and 66% respectivelly (p=0.52). Patients who revieved AUC =5000, had 100 day TRM of 12% and 12% respectivelly (p=0.42). We also found that patients who recieved AUC from 4000 to 5000 had a lower OS, showing a protective effect of lower AUC: 1 year OS for patients who recieved AUC 4000t to 5000 was 65% (IC95% 55-74%) HR: 0.65, (p=0.33). In the other hand, patients who recieved lower AUC targeted dose (4000) had higher incidence of relapse: SHR: 0.42, p=0.08, CI 95% 0.15-1.1 Conclusion In this cohort of patients, toxicity and survival were not influenced by BU formulation (oral or IV). More importantly, patients recieving higher Bu AUC targeted dose had higher incidence of SOS and targeting AUC between 4000 to 5000 had a positive impact in 1 year OS but are more likely to relapse (AUC 4000). We also, shown that using Bu test dose, does not increase toxicities or mortality in patients reciving Bu condicioning regimen. Therefore, we can conclude that PK Bu monitoring is important in either PO or IV BU formulation, in order to reduce toxicities such as SOS and mortality. Disclosures: Santos:Novartis: Consultancy, Research Funding, Speakers Bureau.
Clinical Lymphoma, Myeloma & Leukemia, Jun 1, 2015
Background: The median survival of patients with primary myelofibrosis (PMF) is 5 to 7 years afte... more Background: The median survival of patients with primary myelofibrosis (PMF) is 5 to 7 years after diagnosis. In the majority of patients with PMF somatic mutations were detected either in Janus Kinase 2 (JAK2; in 60% of patients), Calreticulin (CALR; in 25% of patients) or MPL (in 5% of patients) genes. Neither mutation was detected 5% to 10% of PMF patients. Patients with mutated JAK2 are known to have a more aggressive disease compared to patients with mutated CALR. However patients with mutated JAK2 and high allele burden have a favorable outcome compared to patients with a low mutated JAK2 burden. Aim: To develop a model that uses genetic information to predict survival outcome of patients with PMF. Patients and Methods: Bone marrow samples were collected from 344 patients with PMF that were followed at MD Anderson Cancer Center between 2000 and 2013 (157 months). All samples were screened for JAK2 and for mutations in CALR. Patients who did not have a mutation in either gene were also screened for mutations in MPL. Results: In 226 patients (66%) JAK2 was detected and in 43 (13%) CALR was mutated. Of the 75 patients who did not have JAK2 or CALR mutations, 16 (21%) had mutated MPL. In 59 patients (17%), none of those mutations was detected. In the 226 patients who harbored the JAK2 mutation, a cut-point of 50% dichotomized patients into those with a high JAK2 burden and a favorable overall survival (OS; median OS: 80 months) and those with a low JAK2 burden and an adverse OS (median OS: 50 months). Age (above 65 years) and mutation status (low JAK2 burden or triple-negative) were independent risk factors. Patients with a favorable mutation status and age below 65 had a median survival of 126 months (n 1⁄4 82). Patients with either one risk factor, age above 65 (n 1⁄4 88) or adverse mutation status (n 1⁄4 87) had intermediate survival expectancy. The two risk factors were additive and patients age > 65 years and adverse mutation status (n 1⁄4 87) had a median survival of 35 months. Conclusions: Age and mutation status are independent predictors of survival in patients with PMF and stratify patients into 4 groups of equal size with very different survival outcome. 703 Mutational Profiling of JAK2V617F vs. CALR mutated Primary Myelofibrosis Fabio Santos, Renato Puga, Bianca Lisboa, Welbert Pereira, Mariana Miyagi, Evelyn Mata, Tarcila Datoguia, Isabel Bello, Michelli Diniz, Sandra Nakashima, Guilherme Perini, Ricardo Helman, Nelson Hamerschlak, Paulo Campregher Hematology/Oncology, Hospital Israelita Albert Einstein
Mucosa-associated lymphoid tissue (MALT) is the most common extranodal lymphoma, with one third o... more Mucosa-associated lymphoid tissue (MALT) is the most common extranodal lymphoma, with one third of the cases occurring in the stomach. Surgical treatment is a possible option. This case report describes an obese woman (body mass index 46 kg/m2) seeking bariatric surgery, with elevated serum cholesterol and uric acid, negative for Helicobacter pylori but with a positive biopsy for MALT (immunohistochemistry), stage IA. She was submitted to a Roux-en-Y gastric bypass and gastric resection. Two months later, she had lost 20 kg and the MALT lymphoma was in complete remission.
5127 NOTCH1 is a proto-oncogene with activating mutations described in a variety of malignancies,... more 5127 NOTCH1 is a proto-oncogene with activating mutations described in a variety of malignancies, including acute lymphoblastic leukemia (ALL), mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL). While the prognostic significance of NOTCH1 mutations remains controversial in ALL, recent data suggest that NOTCH1 PEST domain mutations are associated with adverse prognosis in patients with CLL. NOTCH1 mutations are found in around 8% of CLL patients at diagnosis and more than 30% of patients with advanced disease. Since this disease has a heterogeneous clinical course and few prognostic markers, we aimed at designing a fast, cost effective and robust assay to detect NOTCH1 PEST domain mutations in patients with CLL for the clinical laboratory. While 92% of the mutations in NOTCH1 PEST domain found in CLL are insertions or deletions, only 8% are represented by point mutations. Therefore we decided to use a fragment analysis approach in our assay. Given that a single mutati...
Introduction The role of the endothelial cell in the pathogenesis of Ph-negative MPNs is still no... more Introduction The role of the endothelial cell in the pathogenesis of Ph-negative MPNs is still not elucidated. Some have reported the presence of the JAK2V617F mutation in endothelial colony forming cells (ECFC) isolated from peripheral blood in patients with Ph-negative MPNs (Teofili L et all, Blood 2011). Others, however, did not find such an association (Piaggio G et al, Blood 2009). In patients with Budd-Chiari Syndrome (BCS), the JAK2V617F mutation has been found in endothelial cell (ECs) isolated by micro dissection from liver biopsies in patients both with and without MPN (Sozer S et al, Blood 2009). Besides the JAK2V617F mutation, other mutations (e.g. ASXL1, TET2, DNMT3A, SRSF2) have been described in patients with Ph-negative MPNs, but their presence has not been evaluated in patients with BCS who carried the JAK2V617F mutation. Objectives 1. To evaluate the presence of the JAK2V617F mutation in CECs from patients with BCS both with and without concomitant Ph-negative MPNs...
Introduction: Development of massive sequencing of whole exomes (WES; whole exome sequencing) has... more Introduction: Development of massive sequencing of whole exomes (WES; whole exome sequencing) has revolutionized DNA sequencing, leading to several studies detailing the genetic profile of patients with several types of neoplasms, including MPNs and MDS/MPNs. One important challenge is to identify recurrently mutated genes that may play a role in disease pathogenesis. It is important to consider that cohort size is critical in the analysis to determine putative oncogenic driver genes, and most cohorts published in MPNs and MDS/MPNs have a limited sample size. One possibility to circumvent this limitations is to combine datasets of different studies. Herein we report the combined analysis of WES data of 403 patients with these disorders, including 124 patients from our center and 279 cases that were previously reported in the literature. Patients and Methods: We analyzed a combined cohort of 403 patients in this study with a diagnosis of Ph-negative MPN or MDS/MPN. For the 124 patients included from our center, reads were aligned and processed following Genome Analysis Toolkit best practices, and somatic variants were called combining the output of SomaticSniper, Mutect and Pindel. Regarding public data, we selected 6 studies that evaluated patients with MPNs and MDS/MPNs through WES and where sample-level data on all individual mutations that were identified were available. Data on identified mutations was extracted from supplementary material of the articles. Combined data on genomic mutations from the whole cohort was used to predict driver genes using softwares MutSigCV and IntOGen (www.intogen.org). Genes considered as being significantly mutated were genes with q-value Results: Diagnosis of the cohort included essential thrombocythemia (ET; N=110), polycythemia vera (PV; N=93), myelofibrosis (MF; N=98), chronic myelomonocytic leukemia (CMML; N=82), atypical CML (aCML; N=15) and more rare disorders (MPN-unclassified [N=2], MDS/MPN-unclassified [N=2] and refractory anemia with ring sideroblasts and thrombocytosis [N=1]). There were a total of 7519 identified mutations, with a median of 9 nonsilent coding mutations per sample (range 0-311 mutations). There were 2 patients (both with a diagnosis of ET) who had no somatic mutations identifed. Analysis of the mutational data using both MutSigCV and IntOGen algorithms revealed a total of 26 genes considered as putative driver genes in these neoplasms: JAK2, TET2, ASXL1, SRSF2, CALR, DNMT3A, SF3B1, CBL, NRAS, EZH2, U2AF1, RUNX1, KRAS, CUX1, MPL, ZRSR2, TP53, IDH2, PHF6, SH2B3, BCL11A, GATA2, ZBTB33, ETNK1, IDH1 and PPM1D (Figure) . A mutation in at least one these genes was found in 92.5% of samples. Among these 26 genes, 2 genes were not previously reported to be mutated in these diseases ( ZBTB33 and BCL11A ), and one gene was previously reported to be associated with other types of neoplastic diseases ( PPM1D ). ZBTB33 encodes the transcriptional regulator Kaiso and was found to be mutated in 6 samples (MF=2, ET=2, PV=1, CMML=1). Three mutations were frameshift indels, and three mutations were missense mutations, one of them (p.I37T) located in the POZ/BTB domain that regulates protein homodimerization. The second gene that was found to be recurrently mutated is BCL11A , which encodes a zinc-finger protein. There were 6 cases of BCL11A mutations, most of them in MDS/MPNs (CMML=3, aCML=2, MF=1). Mutations were all missense and clustered in two asparagine residues located at positions 391 and 756. Finally, four patients (CMML=2, PV=1, MF=1) presented with truncating mutations in the last exon of the PPM1D gene. These mutations have been reported to be activating, leading to an increase in activity of the Wip1 phosphatase and suppression of p53 activity. PPM1D mutations have been reported to be associated with clonal hematopoiesis, MDS and solid tumors. To the best of our knowledge this is one of the first reports detailing their presence in MPNs. Conclusions: Combining datasets across different studies has the potential to increase power and saturate discovery of recurrently mutated genes. This permits prioritization of genes for functional analysis to confirm their role in disease pathogenesis. Larger cohorts than the ones analyzed here may provide even more meaningful data on putative driver genes in MPNs and MDS/MPNs. Disclosures No relevant conflicts of interest to declare.
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Papers by Ricardo Helman