ALBERT

Description: Background: Refining risk stratification of acute myeloid leukemia (AML) using molecular profiling, especially those with intermediate cytogentic risk, is becoming standard of care. However, current WHO and ELN classifications are focused on few markers, mainly FLT3, NPM1, and CEBPA. While these abnormalities are relatively common, not all patients with AML and intermediate or normal cytogenetics will have abnormalities in these genes leaving large percentage of patients without refined risk stratification. We demonstrate that using 8 different AML-related genes are adequate to provide one or more molecular markers to further risk stratify patients with de novo AML. Method: Using direct sequencing we analyzed 211 samples referred from community practice with the diagnosis AML for molecular analysis. All samples were evaluated prospectively for mutations in FLT3, NPM1, IDH1, IDH2, CEBPA, WT1, RUNX1, and TP53 using direct sequencing. Fragment length analysis was used in addition to sequencing for FLT3 and NPM1. Available morphology, cytogenetics, and clinical data along with history were reviewed. Results: Of the 211 samples tested 103 (49%) had at least one or more molecular abnormality adequate for refining the risk classification. The mutations detected in these 103 patients were as follows: 27 (26%) FLT-ITD, 10 (10%) FLT3-TKD, 30 (29%) NPM1, 7 (7%) CEBPA, 14 (14%) IDH1, 13 (13%) IDH2, 10 (10%) WT1, 38 (37%) RUNX1, and 2 (2%) TP53. There was significant overlap and most patients had more than one mutation as illustrated in the graph below. However, if the testing was restricted to FLT3, NPM1, CEBPA and DNMT3A, only 56 (54%) would have had refined risk classification and 46% of patients would have remained without subclassification. The most striking finding was that all the remaining patients, who had no molecular abnormality detected in any of these 8 genes, had either history of MDS evolved to AML, therapy-related AML, or cytogenetic abnormalities other than intermediate (multiplex cytogenetic abnormalities or core-binding factor abnormality). Conclusion: Using FLT3, NPM1, CEBPA, and DNMT3A is inadequate for the molecular characterization of patients with AML. Patients with de novo AML and intermediate risk cytogenetics can be adequately prognostically subclassified and molecularly studied by testing only 8 genes. More importantly, this data confirms that the molecular biology driving de novo AML is significantly different from that driving MDS, AML with myelodysplasia-related changes, therapy-related AML, or AML with core binding factor or multiplex cytogenetics. Unlike de novo AML, these entities should be molecularly studied using MDS-specific driver genes. Furthermore, this data suggests that different therapeutic approaches should be developed for MDS and MDS-related AML. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: New world nonhuman primates of the genus Aotus (owl monkeys) can be categorized by 11 distinct karyotypes (K). It has been demonstrated that monkeys of K-VI persistently have one order of magnitude more eosinophils (EOS) in the peripheral blood than K-I monkeys. The purpose of this study was to investigate the basis for this difference and examine EOS recruitment using two cutaneous models of inflammation. Peripheral blood EOS were isolated on metrizamide gradients to 〉 or = 95% purity and then used for phenotypic studies. There were no significant differences when comparing karyotypes in the ratio of normodense (K-I, 6.4% +/- 3.8%; K-VI, 21.1% +/- 8.8%) EOS or their survival in culture (K-I, 5.3% +/- 2.9% at 72 hours; K-VI, 2.8% +/- 0.7% at 72 hours) (P 〉 .05). Examination of bone marrow revealed that K- VI monkeys had greater than fivefold more EOS and EOS precursors than K- I animals. To examine EOS function in recruitment, monkeys of each karyotype were given a single intradermal injection of Escherichia coli lipopolysaccharide (LPS) or human recombinant (PMN) and mononuclear cells occurred in response to LPS as early as 4 hours after injection; the severity of infiltration was similar in both karyotypes and at all time points up to 24 hours. In contrast, by 8 hours after intradermal injection of RANTES, leukocyte infiltration in K-I monkeys consisted mostly of PMN (94.8% +/- 0.7%) that were predominantly EOS. In comparison, there was essentially no infiltrate in K-VI animals at all time points. There was no difference in VCAM-1 expression in response to intradermal LPS or RANTES between the two karyotypes. These results suggest that the genetic basis of peripheralblood eosinophilia in K-VI owl monkeys is likely a function of heightened eosinophilopoiesis and depressed recruitment kinetics from the peripheral circulatory pool in response to RANTES.

Description: Acute myeloid leukemia (AML) is currently distinguished from myelodysplastic syndrome (MDS) based on the presence of 20% blasts in bone marrow, an arbitrary cut-off adopted by the WHO classification and replacing the 30% cut-off required by the older FAB (French, American and British) classification. Patients with t(15;17), t(8;21), or inversion 16 cytogenetic abnormalities are classified as having AML irrespective of the percentage of blasts. We explored the possibility that currently defined molecular abnormalities can distinguish AML from MDS without relying on an arbitrary percentage of blasts in the bone marrow. We compared the molecular profiles obtained by next generation sequencing (NGS) from consecutive patients with a clinical diagnosis of AML or MDS by WHO criteria. Methods: NGS data from 251 patients with the diagnosis of AML and 294 patients with the diagnosis of MDS was studied. All samples were analyzed using a panel of 25 genes including FLT3, NPM1 SF3B1, CBL, DNMT3A, ASXL1, BRAF, CEBPA, CSFR3, ETV6, EZH2, IDH1, IDH2, JAK2, c-KIT, KRAS, NRAS, PHF6, PTPN11, RUNX1, SETBP1, TET2, TP53, WT1, and ZRSR2. We compared the frequency of mutations in each gene between AML and MDS patients. Results: Mutations in FLT3 and NPM1 were uniquely and commonly detected in AML (27% and 22%, respectively). In contrast, mutations in SF3B1 gene were uniquely dominant (22%) in MDS and FLT3 and NPM1 mutations were rare (2% and 3%, respectively). SF3B1 mutations were extremely rare in AML (1%). Overall, 102 (41%) of all AML patients had mutations in either FLT3 or NPM1 and 8% of AML patients had mutations in both FLT3 and NPM1. In addition, WT1 gene was mutated in 8% of AML cases, but none of the MDS cases showed WT1 mutation. TET2 gene was commonly mutated in both AML and MDS (25% and 36%, respectively), but the frequency was significantly higher in MDS (P=0.003). IDH1, IDH2, NRAS, and PTPN11 were mutated slightly more often in AML than in MDS, while ASXL1, EZH2, and ZRSR2 were more frequently mutated in MDS than in AML. There was no statistically significant difference in mutation frequency between AML and MDS for the other genes analyzed. Conclusion: Mutations in FLT3, NPM1 and WT1 are molecular abnormalities characteristically detected in patients with AML and can be used as objective criteria for the classification of AML rather than blast count in bone marrow. These mutations are detected in 49% of AML patients. This suggests that approximately half of AML patients can be diagnosed based on the detection of molecular abnormalities, irrespective of bone marrow morphology. The presence of mutation in SF3B1 gene is also a characteristic molecular finding for MDS. Table. AML (No=251) MDS (No=294) P-Value No % No % FLT3 68 27 5 2 0.00001 NPM1 55 22 8 3 0.0001 SF3B1 3 1 66 22 0.00006 CBL 4 2 10 3 NS DNMT3A 51 20 51 17 0.07 ASXL1 44 18 75 26 0.01 BRAF 3 1 1 0 NS CEBPA 38 15 51 17 NS CSFR3 11 4 11 4 NS ETV6 3 1 6 2 NS EZH2 8 3 25 9 0.03 IDH1 20 8 7 2 0.03 IDH2 17 7 7 2 0.04 JAK2 4 2 10 3 NS KIT 2 1 0 0 NS KRAS 11 4 6 2 NS NRAS 34 14 18 6 0.01 PHF6 5 2 2 1 NS PTPN11 26 10 6 2 0.01 RUNX1 31 12 33 11 NS SETBP1 5 2 9 3 NS TET2 64 25 105 36 0.003 TP53 61 24 75 26 NS WT1 19 8 0 0 0.01 ZRSR2 7 3 30 10 0.02 Disclosures No relevant conflicts of interest to declare.

Description: In order to determine whether the tumor-promoting phorbol esters are capable of inducing normal human committed granulocytic-monocytic progenitor cells (CFUc) to proliferate and differentiate in the absence of granulocyte-monocyte colony-stimulating activity (CSA), we studied the effects of these compounds on human granulopoiesis in vitro. We found that when light-density human marrow cells or peripheral blood leukocytes were depleted of adherent cells and then incubated in semisolid tissue culture medium under conditions optimal for CFUc growth, phorbol myristate acetate (PMA) and its congeners produced no measurable stimulatory effect on the proliferation of CFUc in the absence of added CSA. Likewise, when light-density marrow cells that had not been depleted of adherent cells were plated in the cultures, no stimulation of CFUc colony growth resulted from the addition of PMA. However, when light-density peripheral blood leukocytes were used as a target source of CFUc without first subjecting them to adherence separation, enhanced proliferation and differentiation of CFUc were noted in cultures that contained PMA. To investigate the possibility that CSA production by monocytes in these cultures in response to activation by PMA might account for the enhanced colony formation that we observed, we incubated isolated peripheral blood monocytes in short- term liquid suspension cultures and found that in the presence of PMA, large quantities of CSA were secreted into the surrounding medium. Finally, we noted that when marrow cell suspensions were suboptimally stimulated by low concentrations of CSA added to the cultures, the effects of PMA on CFUc proliferation were unpredictable, enhancing colony formation in some cases and inhibiting it in others. Our data indicate that although the tumor-promoting phorbol esters do not appear capable of directly stimulating the proliferation or differentiation of human CFUc in the absence of CSA, they may do so indirectly by causing auxiliary cells such as monocytes to secrete CSA.

Description: Background: CALR gene is frequently mutated in patients with myeloproliferative neoplasmas (MPN). Almost all mutations are indel - some with large (〉50 bp) deletion. Detecting this type of mutations with acceptable sensitivity is difficult by sequencing. Fragment length analysis (FLA) is a reliable technique in detecting this type of mutations. Furthermore, FLA allows quantifying the mutant DNA and better evaluation of tumor load. Determining tumor load can be confusing and difficult when the mutation is biallelic. Distinguishing between patients with single allele mutation from those with biallelic mutations might add another dimension in predicting clinical behavior and determining the tumor load. We explored using cell free DNA in peripheral blood plasma to test for CALR mutations and determining tumor load. Methods: Using Direct bidirectional sequencing and FLA, we detected CALR indel mutations in 71 of 522 (14%) patients suspected of having MPN and referred for testing for CALR mutation. No sample showed point mutation. DNA from cells and cell free DNA in plasma was available from 31 of the 71 cases. The mutant DNA peak was quantified and the relative percentage of mutant DNA was calculated in both cellular DNA and cell free plasma DNA. FLA and Sanger sequencing data were compared between cellular DNA and cell free plasma DNA. Any ratio 〉55% was considered as evidence of biallelic mutation. Results: As expected all positive samples by cellular DNA testing also showed the indel mutation in cell free plasma by FLA. However, four of the 31 (13%) positive samples by FLA failed to show the mutation on Sanger sequencing. The most likely cause for failing to detect the mutation by Sanger is low level mutation load and the lower sensitivity of Sanger sequencing. When we compared ratios of mutant peak (tumor load) between cellular DNA and cell free DNA in plasma, mutant CALR DNA was significantly higher (P=0.0002, Wilcoxon matched pairs test) in cell free DNA in plasma than in cellular DNA. More importantly, we were able to determine the presence of homozygous mutation (〉55% mutant DNA) in 5 of 31 (16%) patients when cell free DNA in plasma is used. In contrast, only 1 of 31 (3%) patients showed evidence of biallelic mutation when cellular DNA is used. Most of the mutations (25 of 31, 81%) were deletions. As deletions results in smaller size amplicon on the FLA and better amplification efficiency, we set 55% as a cut-off for biallelic mutation to account for the more efficient amplification of the deleted peak. Conclusion: Cell free DNA in plasma is more reliable than cellular DNA for the detection of CALR mutations and for determining tumor load. Testing for CALR must include fragment length analysis. More importantly, biallelic CALR mutation is frequent and the clinical relevance of biallelic CALR mutation needs to be investigated. Disclosures No relevant conflicts of interest to declare.

Description: Background: In patients presenting with cytopenia, myelodysplastic syndrome (MDS) should be considered, but confirmation of diagnosis requires bone marrow biopsy and morphologic and cytogenetic evaluation. It is extremely difficult to rely on subjective morphologic features to confirm the diagnosis of MDS, when the karyotype is normal and blasts are not increased. Objective criteria for the diagnosis of MDS are needed in these cases. With recent advances in the characterization of molecular abnormalities in MDS, diagnosis of early MDS is becoming more objective by documenting the presence of MDS-specific molecular abnormalities in cases with appropriate clinical presentation. Since MDS is a disease of excessive apoptosis in bone marrow, DNA resulting from the apoptosis is abundant in circulation. We explored the potential of using cell free DNA in peripheral blood plasma using next generation sequencing (NGS) to confirm the diagnosis of early MDS without the need for marrow biopsy. Methods: Total nucleic acid was extracted from the plasma of 16 patients presenting with cytopenia and confirmed diagnosis of early MDS (blasts

Description: Key Points CD32a antibodies induce thrombocytopenia and hypersensitivity reactions in FCGR2A mice. Effector-deficient CD32a antibodies prevent IgG-induced thrombosis and shock in FCGR2A mice.

Description: Introduction: Diagnosis of myelodysplastic syndrome (MDS) can be very difficult when blast count in bone marrow is

Description: Introduction: Diagnosis of myelodysplastic syndrome (MDS) based on bone marrow morphology can be very difficult, when blasts are not increased. The demonstration of cytogenetic abnormalities in these cases can confirm the diagnosis, providing cytopenia is documented. Cytopenia is usually the major reason for initiating work-up for myelodysplasia and , in general, cases with unicytopenia are the most difficult to make the diagnosis. In principle the recent characterization of the molecular abnormalities underlying the biology of MDS should provide objective biomarkers that can be used to confirm the diagnosis of MDS in the absence of cytogenetic abnormalities. Toward this goal, we developed a 14-gene panel to detect molecular abnormalities in patients referred to rule out MDS with blast count