Background: BK polyomavirus reactivation can occur following allogeneic hematopoietic stem cell transplantation (allo-HSCT) and may lead to hemorrhagic cystitis (BKPyV-HC). We hypothesized that development of BKPyV-HC is associated with increased mortality post allo-HSCT. Methods: We retrospectively reviewed data on 133 adult patients (≥18 years old) who underwent allo-HSCT from 2007 until 2014 at Hospital Israelita Albert Einstein in São Paulo, Brazil. Results: Thirty-six patients presented with BKPyV-HC after a median time of 42 days, with a 1-year cumulative incidence probability of 28.9% (95% CI 21.5%-36.7%). In a multivariate Cox model, risk factors for development of BKPyV-HC included younger age, male sex, development of grade 2-4 acute graft-versus-host disease and recipients of umbilical cord blood grafts. Development of grade 3-4 BKPyV-HC (but not grade 1-2) was associated with a decreased overall survival (OS) in a multivariate Cox model (hazard ratio [HR] 7.51, P < 0.0001) and an increased risk of TRM (HR 3.66, P < 0.0001). Grade 3-4 BKPyV-HC was also associated with an increased risk of relapse that did not reach statistical significance (HR 3.01, P = 0.07). Median overall survival (OS) post-BKPyV-HC was 4.7 months, and cidofovir had no impact on survival.Conclusion: Development of BKPyV-HC appears to be associated with decreased survival following allo-HSCT. K E Y W O R D Sallogeneic stem cell transplantation, BK virus reactivation, hemorrhagic cystitis, mortality

This content is licensed under a Creative Commons Attribution 4.0 International License.Successful treatment of post-transplant relapsed acute myeloid leukemia with FLT3 internal tandem duplication using the combination of induction chemotherapy, donor lymphocyte infusion, sorafenib and azacitidine. Report of three cases ABSTRACTAcute myeloid leukemia is a hematopoietic stem cell neoplastic disease associated with high morbidity and mortality. The presence of FLT3 internal tandem duplication mutations leads to high rates of relapse and decreased overall survival. Patients with FLT3 internal tandem duplication are normally treated with hematopoietic stem cell transplantation in first complete remission. Nevertheless, the incidence of post-transplant relapse is considerable in this group of patients, and the management of this clinical condition is challenging. The report describes the outcomes of patients with FLT3 internal tandem duplication positive acute myeloid leukemia who relapsed after allogeneic hematopoietic stem cell transplantation and were treated with the combination of re-induction chemotherapy, donor lymphocyte infusion, sorafenib and azacitidine. Three cases are described and all patients achieved prolonged complete remission with the combined therapy. The combination of induction chemotherapy followed by donor lymphocyte infusion, and the maintenance with azacitidine and sorafenib can be effective approaches in the treatment of post-hematopoietic stem cell transplant and relapsed FLT3 internal tandem duplication positive acute myeloid leukemia patients. This strategy should be further explored in the context of clinical trials.

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.

Introduction: Mutations that activate the RAS-RAF-MEK-ERK pathway have long been known to occur in patients with solid tumors and hematological malignancies. The most common mutations occur in the Ras family of GTPases (HRAS, NRAS, KRAS) and the Raf family of serine-threonine kinases (ARAF, BRAF, CRAF). In myeloid malignancies, RAS mutations have mainly been described in patients with acute myeloid leukemia, chronic myelomonocytic leukemia (CMML) and myelodysplastic syndrome. There are few studies describing the incidence of mutations of the RAS-RAF-MEK-ERK pathway in patients with MPNs other than CMML. Objective: To describe the incidence, clinical features and prognostic impact of Ras and Raf mutations in patients with Ph-negative MPNs and MPN/MDS-U Methods: Paired DNA (sorted CD66b-granulocytes/skin biopsy) from patients with MPNs or MPN/MDS was subjected to whole exome sequencing on a Illumina HiSeq 2000 platform using Agilent SureSelect kit (see our abstract “Whole Exome Sequencing of Myeloproliferative Neoplasms and Myelodysplastic/Myeloproliferative Disorders”). 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. Statistical calculations were done in Stata, v11.0. Results: We found clonal activating mutations of the RAS-RAF-MEK-ERK pathway in 8 patients (6.7% of cases). Diagnosis included primary myelofibrosis (PMF; N=5), MDS/MPD-U (N=2) and essential thrombocythemia (ET; N=1). Their clinical features are summarized in Table 1 (three of these patients [UPIs #11, #13, #99] are also described in the abstract “Genomic Profile of Patients with Triple Negative (JAK2, CALR and MPL) Essential Thrombocythemia and Primary Myelofibrosis”). There were 7 NRAS mutations and 1 BRAF mutation. In 5 cases the variant allele fraction (VAF) of reads in the tumor sample indicated that the mutation was present in a subclone at the time of sequencing. We next compared the clinical features of these 8 patients with 79 patients (MF=43, ET=35, MDS/MPD=1) who did not harbor these mutations. Patients with NRAS/BRAF mutations had lower hemoglobin (8.3 vs. 11.8 g/dL, p=0.001), higher white blood cell counts (28.37 vs. 7.7 x109/L, p=0.008) and had higher lactate dehydrogenase (1041 vs. 685 IU/L, p=0.02). They also had worse overall survival compared to unmutated cases (Hazard ratio [HR]=11.57; p=0.001). Most patients with NRAS/BRAF mutations had a high number of concomitant driver mutatons (median 5 vs. 1; p<0.0001). When the number of driver mutations was analyzed together with NRAS/BRAF mutations in a Cox model, NRAS/BRAF mutations were no longer independent predictors of survival (HR=1.48; p=0.61). Conclusions: Activating mutations of the RAS-RAF-MEK-ERK pathway occur in 6-7% of patients with Ph-negative MPNs, and they tend to co-occur with a high number of concomitant driver mutations. In most cases the mutation was present in a subclone, suggesting that they are late occurring. Patients with NRAS/BRAF mutations had a trend for worse outcome, but that was mainly dependent on the total number of driver mutations. The activity of MEK and BRAF inhibitors needs to be explored in patients with Ph-negative MPNs who harbor activating mutations of the RAS-RAF-MEK-ERK pathway. Table 1. Clinical features of patients with NRAS/BRAF mutations UPI Diagnosis Mutation VAF Concomitant driver genes and Chromosomal abnormalities Outcomes 7 MF NRAS p.G12S 47% ASXL1, CALR, STAG2, U2AF1 Died from disease progression 11 MF NRAS p.G12R 5% ASXL1, CBL, CUX1 (double mutant), EZH2 Died from disease progression 13 MF NRAS p.G12D 48% ASXL1, DNMT3A, ETV6 (double mutant) JARID2, U2AF1 Died from disease progression 18 MF NRAS p.G13D 25% JAK2, Del(5q) Underwent allogeneic transplantation; disease relapsed day+80; alive 29 MDS/MPD-U BRAF p.D594G 25% JAK2, Del(5q) Transformed to AML; entered CR with induction chemotherapy; underwent allogeneic transplantation; disease relapsed day+35; alive 99 ET NRAS p.G12D 43% ASXL1, CSF3R, STAG2 Alive 109 MF NRAS p.Q61R 19% CALR, DNMT3A, ZRSR2 Alive 122 MDS/MPD-U NRAS p.G12S 7% ASXL1, EZH2 (double mutant), PTPN11, TET2 (double mutant) Transformed to AML; underwent allogeneic transplantation; died on day+58 Disclosures No relevant conflicts of interest to declare.

Introduction: The treatment of human hematologic malignancies has rapidly advanced through the application of genomic platforms that have identified drug-able targets and companion diagnostics (e.g. BCR-ABL, IDH-1) and added new classes of targeted agents to the established compendium of cytotoxics. Despite these advances, the complexity, redundancy and promiscuity of cellular transformation remain incompletely understood at the molecular level. This has led to a renewed interest in whole cell experimental models for drug discovery. Laboratory platforms that measure cellular response to cytotoxic insult at the phenotypic level have been shown to correlate significantly with clinical response, and have the capacity to provide insights into chemotherapy selection and drug development. The Ex Vivo Analysis of Programmed Cell Death (EVA/PCD) uses metabolic and morphologic features of drug induced cell death to measure both cytotoxic and targeted drug effects in human primary cultures. We applied EVA/PCD in 20 heavily pre-treated, drug refractory patients from the Hospital Israelita Albert Einstein (HIAE) in Sao Paulo - Brazil. Methods: Peripheral blood, node biopsy or bone marrow aspirates were submitted by overnight courier. Cells isolated by density centrifugation were evaluated by dose response curves that were interpolated to provide LC50 values for comparison with our databases by Z-score. Patients with Acute Lymphoblastic Leukemia (ALL, N=5) , Acute Myeloid Leukemia (AML, N=6), Non-Hodgkin Lymphoma (NHL, N=4) or Multiple Myeloma (MM, N=4) had received a mean of 5, median of 4 (range 1-8) prior therapies, 7 with prior bone marrow transplantation (BMT). Results: of 20 specimens, 16 (80%) provided viable tumor for EVA/PCD. A mean of 8, median of 7(range 3-22) cytotoxics and a mean 7, median of 5(range 1-20) targeted agents were evaluated. Findings were reported by day 7. Nine of 16 patients were treatment candidates, with 5 lost to follow up, 3 dying of sepsis before evaluation and 1 achieving complete remission (CR) with radiation plus Rituximab. Of 7 patients who received assay directed therapy there were 3 CR (43%), 2 partial responses (PR: 28%) and 2 progressive disease (PD: 29%) for an overall response rate of 71%. Conclusion: These results establish the feasibility of laboratory directed therapy in heavily pre-treated patients, with 80% of submitted samples providing actionable results. Although the extremely advanced state of these patients limited the capacity to undergo treatment in some cases, the achievement of CR's and PR's in this drug refractory cohort is of interest. Clinical responses by disease, treatment history and drugs received will be reported. Studies correlating molecular profiles with phenotypic analyses are currently under development. Disclosures Evans: Rational Therapeutics: Employment. Nagourney:Rational Therapeutics: Employment.