ALBERT

Description: 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

Description: Introduction: Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are myeloproliferative neoplasms (MPN) with similar driver mutations. The three hallmark molecular alterations in these diseases are JAK2, MPL and CALR mutations. Patients with myelodysplastic/myeloproliferative (MPN/ MDS) neoplasms such as refractory anemia with ring sideroblasts and thrombocytosis (RARS-T) also present with the same hallmark genetic changes. Nevertheless, roughly 10% of these patients do not present mutations in neither of these genes. Recent data suggest that triple negative patients have a more aggressive clinical course. While the molecular alterations present in patients with MPN have been extensively studies, the genomic profile of triple negative TE/PMF/RARS-T has not been described. To better characterize these patients we performed whole exome / genome sequencing of paired granulocytes and skin from 15 triple negative MPN patients Methods: A total of 15 patients with triple negative MPN or MPN/ MDS [PMF (N=6)/TE (N=8)/RARS-T (N=1)] were analyzed. DNA was extracted from CD66b+ magnetic bead selected granulocytes (EasySep, Stem Cell Technologies) and matched skin biopsies with QiaAmp DNA Mini kit (Qiagen). Whole-exome targeted capture was carried out on 3 μg of genomic DNA, using the SureSelect Human Exome Kit 51Mb version 4 (Agilent Technologies, Inc., Santa Clara, CA, USA). The exome library was sequenced with 100 bp paired-end reads on an Illumina HiSeq2000. Somatic variants calls were generated by combining the output of Somatic Sniper (Washington University), Mutect (Broad Institute) and Pindel (Washington University). Tumor coverage was 150x and germline coverage was 60x. The combined output of these 3 softwares 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 ≥5% in tumor and ≤10% in germline; ratio of allele fraction tumor:germline 〉2). All JAK2 and CALR mutations were validated through Sanger sequencing. Validations of other somatic mutations are under way at this point. Results: First we asked whether other hematopoietic related genes could be responsible for the pathogenesis of the triple negative cases. With that goal we searched for high confidence mutations in genes that are mutated in at least 1% of patients with hematopoietic tumors on COSMIC (catalog of somatic mutations in cancer) database and also genes known to be recurrently mutated in myeloid malignancies. Only 6 out of 15 patients presented mutations in other myeloid related genes. The diagnosis of these patients were PMF=4, TE=2. The hematopoietic related genes mutated in these patients were: ASXL1 (n=4), CUX1 (n=3), NRAS (n=2) and ATM, CBL, CSFR3, CREBBP, DNMT3A, ETV6, EZH2, JARID2, MLL2, PHF6, SRSF2, STAG2, TET2, GNAS, U2AF1 (n=1). Noteworthy, we have found one known oncogenic mutation in CSFR3, an alteration supposed to be specific for chronic neutrophilic leukemia and atypical CML, in a patient with ET, and an oncogenic mutation in GNAS in a patient with PMF. In addition, the patient with RSRA-T had a putative oncogenic mutation in PTPN11 Remarkably, the average number of hematopoietic related mutations in these patients was 5, significantly higher than the total number of mutations found in another cohort of patients with either JAK2 (average = 1.7) or CALR mutations (average = 1.9). Although our numbers are small, we may speculate that the high incidence of ASXL1 mutations (28%) associated with a high number of prognostically detrimental mutations can partially explain the worse outcomes associated with triple negative MPN. Noteworthy is also the high prevalence of CUX1 mutations in this subset of patients (21%) when compared to other myeloid malignancies in general. Regarding the other 9 patients for whom no hematopoietic mutations could be identified, 6 patients had ET, 2 patients PMF and one patient had RARS-T. Conclusion: We have shown that: i-patients with triple negative MPN are molecularly heterogeneous, with one group presenting a high number of hematopoietic related mutations, ii-the most common mutations present in these patients are ASXL1, CUX1 and NRAS, iii-The majority of these patients do not present mutations in hematopoietic related genes, what suggests that non-described molecular mechanisms are operating in these patients. Disclosures No relevant conflicts of interest to declare.

Description: Background:Treatment options for patients with relapsed/refractory chronic lymphocytic leukemia (CLL) or mantle cell lymphoma (MCL) are limited. Ibrutinib is a first-in-class small-molecule inhibitor of Bruton's tyrosine kinase. These extended access programs (EAP) provided patient access to ibrutinib in Brazil and real-world safety data was collected, at a time when the medication was not commercially available in Brazil. Methods:These two prospective, multicenter, open-label EAPs of single-agent ibrutinib were conducted between April 2013 and August 2017, and enrolled Brazilian patients with relapsed/refractory CLL or MCL. Eligible patients must have progressive disease after at least one prior therapy and not suitable for retreatment with purine analogue therapy. After a 30-day screening, eligible patients received once-daily oral ibrutinib 420 mg (CLL) or 560 mg (MCL) continuously until disease progression, unacceptable toxicity, absence of clinical benefit, or end of EAP, whichever occurred first. Doses could be withheld or reduced based on toxicity. Patients were monitored for safety and disease evaluations were conducted per routine local standard of care practices. Results:Of 33 CLL patients enrolled, 32 received one dose of drug or more and were included in the safety analysis. Median age was 62.5 years, and most patients were male (n=24; 75%) and white (n=27; 84.4%). The median time from CLL diagnosis to study inclusion was 83.8 months and from diagnosis to relapsed/refractory state, 42.0 months. The median number of ibrutinib cycles was 12.0 (1.0-16.0) with a median treatment duration of 11.1 (0.9-11.6) months. Eight patients discontinued due to adverse event (AE; n = 4; 12.5%), consent withdrawal (n = 2; 6.3%), death (n = 1; 3.1%), or disease progression (n = 1; 3.1%). AEs leading to treatment discontinuation were intestinal bleeding, neutropenia, infection, and gastric tumor (one patient each). Three (9.4%) patients had dose reductions: one (3.1%) for neutropenia, febrile neutropenia with pneumonia, or worsening fatigue. 21 patients (65.6%) had at least one Grade ≥3 (G3) AE or serious AE (SAE). The most frequent G3 or SAEs included neutropenia in 8 (25.0%), fatigue (1), leukocytosis (1), and pneumonia (3). No atrial fibrillation or bleeding AEs were reported. Among the 47 G3 or SAEs, 17 (36.2%) were serious, 38 (80.9%) were suspected to be related to ibrutinib, and 39 (83.0%) were resolved without sequelae. All 13 MCL patients enrolled in the study were included in the safety analysis. The median age was 60.0 years, and most patients were male (n=9; 69.2%) and white (n=9; 69.2%). The median number of prior treatment regimens were 3. The median time from diagnosis to the first dose of ibrutinib was 20.4 months. The median number of ibrutinib cycles was 19 (4.0-34.0) with a median treatment duration of 16.8 (3.6-30.5) months. Eight patients discontinued because of either death (n=3; 23.1%) or disease progression (n=5; 38.5%). The three patients died with treatment-emergent G4 or higher AEs, including pneumonia (G5; probably treatment-related [TR]), sepsis (G5; not TR), and dyspnea (G4; doubtful TR); 8 patients (61.5%) had at least one G3 or higher treatment-emergent AE. The most frequent AE was diarrhea (n=3; 23.1%), and other AEs were reported in one patient each (i.e. abdominal hernia, anemia, appendicitis, dyspnea, febrile neutropenia, influenza, leukocytosis, neutropenia, pneumonia, productive cough, renal failure/insufficiency, retroperitoneal abscess, thrombocytopenia). Three (23.1%) patients had ibrutinib dose modifications: one (7.7%) each because of appendicitis/tuberculosis, thrombocytopenia, and diarrhea/retroperitoneal abscess/dyspnea. No atrial fibrillation or bleeding AEs were reported. Among the 20 G3 or higher treatment-emergent AEs, 14 (70%) were suspected to be related to ibrutinib and 15 (75%) were resolved. Conclusions: This is the first real-world experience with ibrutinib monotherapy for CLL and MCL in Brazil. Overall, treatment was well tolerated with no unexpected toxicities. No atrial fibrillation or bleeding AEs were reported. Of 32 patients with relapsed/refractory CLL, 24 (80%) remained on therapy, 4 (12.5%) discontinued due to AEs, 1 (3.1%) each died or experienced disease progression. Among 13 patients with relapsed/refractory MCL, 5 (38.5%) remained on the therapy, 3 (23.1%) died and 5 (38.5%) experienced disease progression. Disclosures Chiattone: Janssen: Honoraria, Research Funding. Fogliatto:Novartis: Consultancy; Janssen: Honoraria, Research Funding; Roche: Consultancy, Speakers Bureau. Scheinberg:Novartis: Consultancy, Speakers Bureau; Janssen: Honoraria, Research Funding; Pfizer: Speakers Bureau. Bigni:Janssen: Honoraria, Research Funding. Rodrigues:Janssen: Honoraria, Research Funding. Garicochea:Janssen: Honoraria, Research Funding. Pimenta:Janssen: Honoraria, Research Funding. Boechat:Janssen: Honoraria, Research Funding. Musacchio:Janssen: Honoraria, Research Funding. Goncalves:Janssen: Honoraria, Research Funding. Vieira:Janssen: Honoraria, Research Funding. Santos:Janssen: Employment. Grings:Janssen: Employment. Parisi:Janssen: Employment. Barreyro:Janssen: Employment.

Description: Introduction: Primary Myelofibrosis (PMF) and Essential Thrombocythemia (ET) are myeloproliferative neoplasms with similar genetic backgrounds. Both diseases are characterized, at the molecular level, by mutations in the genes JAK2, MPL and CALR. In addition recurring mutations is several other genes have been described in myeloid malignancies in general. Although the differential diagnosis between PMF and ET may be straight forward in most cases, there is a significant clinical and pathologic overlap between these two conditions, making the differential diagnosis difficult sometimes, mostly between early PMF and ET. With the goal of utilizing genomic information to better differentiate ET from PMF we decided to identify and compare all genomic alterations present in patients with ET and PMF, through whole exome / genome sequencing of paired granulocytes and skin. Methods: A total of 84 patients with either PMF (N=48) or ET (N=36) were analyzed. DNA was extracted from CD66b+ magnetic bead selected granulocytes (EasySep, Stem Cell Technologies) and matched skin biopsies with QiaAmp DNA Mini kit (Qiagen). Whole-exome targeted capture was carried out on 3 μg of genomic DNA, using the SureSelect Human Exome Kit 51Mb version 4 (Agilent Technologies, Inc., Santa Clara, CA, USA). The exome library was sequenced with 100 bp paired-end reads on an Illumina HiSeq2000. Somatic variants calls were generated by combining the output of Somatic Sniper (Washington University), Mutect (Broad Institute) and Pindel (Washington University). Tumor coverage was 150x and germline was 60x. The combined output of these 3 softwares 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 ≥5% in tumor and ≤10% in germline; ratio of allele fraction tumor:germline 〉2). All JAK2 and CALR mutations were validated through Sanger sequencing. Validations of other somatic mutations are under way at this point. For this work, other myeloid driver mutations were defined as mutations occurring recurrently in myeloid malignancies in the medical literature, and in this cohort of patients these mutations were present in the following genes: ASXL1, ATM, CALR, CBL, CUX1, DNMT3A, EZH2, GATA2, GNAS, IDH1, IDH2, JAK2, MPL, NRAS, SH2B3, SF3B1, STAG2, TET2, NFE2, SMC3, SUZ12, PRPF8, SRSF2, U2AF1, TP53. Fisherxs exact test was used for statistical comparisons. Results: The most common mutated genes after JAK2 and CALR were ASXL1 (n=16), TET2 (n=9) and DNMT3A (n=9). After data analysis, the patients could be divided in 7 groups based on the genomic profile: A – JAK2 mutation as the single genetic abnormality (JAK2_Single) (N=24), B – JAK2 plus other myeloid driver mutations (JAK2_Plus) (N=25), C - CALR mutation as the single genetic abnormality (CALR_Single) (N=11), D – CALR plus other myeloid driver mutations (CALR_Plus) (N=9), E – MPL mutation (N=1), F – Triple negative without other myeloid driver mutations (TN_Single) (N=8), G – No JAK2, CALR or MPL (triple negative) but with other myeloid driver mutations (TN_plus) (N=6) 1 – The presence of 3 or more total myeloid driver mutations was strongly associated with a diagnosis of PMF Table 1mut2TE282PMF2521 P= 0.0002 2 – The presence of ASXL1 mutations was strongly associated with a diagnosis of PMF Table 2ASXL1+ASXL1-TE135PMF1533 P=0.0007 In order to validate our findings in an independent cohort of patients, we performed the same analysis using data from 2 published studies that evaluated myeloid multi-gene panels in ET and PMF (Nangalia J, NEJM 2013) (Lundberg P, Blood, 2014). We pooled together all patients with ET (N=117) and PMF (N=56) from both studies and repeated the two previous analyses, that confirmed the previous results: Table 3mut2TE1106PMF4214P=0.0005ASXL1+ASXL1-TE4113PMF1442P=3.9E-05 Conclusions: We have demonstrated that ASXL1 mutations as well as a number of myeloid driver mutations higher than two is strongly associated with PMF. This information may be useful in the near future to improve the differential diagnosis between ET and PMF. Disclosures No relevant conflicts of interest to declare.

Description: 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