ALBERT

Description: Introduction: Although blast-rich specimens immunophenotype studies in myelodysplastc syndromes (MDS) could associate bone marrow (BM) blast expression of CD7 and/or CD117 antigens with poor outcome (Ogata et al., Blood 2002), the prognostic role of markers of myeloid cell immaturity and committment in not enriched BM samples is largely unexplored. Patients and Methods: The expression of CD33, CD34 and CD117 antigens in not enriched BM samples of 50 newly diagnosed MDS was compared with both BM blast WHO category and IPSS score. Immunophenotyping was carried out by using the panel of quadruple monoclonal antibodies CD34/CD117/CD45/CD33, conjugated with the fluorochromes FITC, PE, PerCP, APC, respectively. Acquisition of information on 1x105 stained cells corresponding to the whole BM cellularity was assessed on a dual-laser FACSCalibur flow cytometer using the CellQUEST software (Becton Dickinson, San José CA USA). Multiple group comparisons were made using non parametric ANOVA for BM blasts; general linear model with Wald’s test and Kruskal-Wallis (KW) test to confirm significance was used for IPSS. Results: According to IPSS, 5 (10%) low risk, 27 (54%) intermediate risk-1, 14 (28%) intermediate risk −2 and 4 (8%) high risk pts were identified, respectively. The expression of CD33, CD34 and CD117 significantly correlated with both blast WHO category and IPSS, as shown in the Table 1. Interestingly, by analyzing the subset of 30 pts with BM blasts

Description: α and β-tryptase genes cluster on the short arm of human chromosome 16 and encode lineage-associated serine proteases that are abundantly expressed in mast-cells and, in trace amounts, in basophils. Under physiologic conditions no other myeloid cells express tryptases. However, in several myeloid leukemia cell lines and in AML blasts, the level of tryptase is elevated. In an attempt at correlating the levels of tryptase with cytogenetic features and the KIT and FLT3 mutational status, we analyzed serum samples collected at diagnosis from 150 AML and 57 ALL adult patients. The total serum concentration was determined by UniCAP 100 and UniCAP Tryptase Fluorenzyme Immunoassay Kit (Pharmacia-Upjohn, Uppsala, Sweden). The median value of tryptase level in the control group (50 healthy people; mean age 35 y, range 20–50; M/F= 26/24) amounted to less than 5 ng/ml, ranging from 1 to 15 ng/ml. We detected elevated tryptase levels (more than 15 ng/ml) in 66 out of 150 AML-patients (44%) and in 1 out of 57 ALL-patients (1.75%; median value 1.2 ng/ml) (p = 〈 0.0005, Fisher’s exact test ). In AMLs data showed that elevated tryptase values are significantly bound to patients with t(8;21) (n = 26, p =

Description: Introduction Several studies have recently pointed out the adverse impact of KIT mutations (mutKIT) on relapse incidence (RI) and overall survival (OS) in AML pts with t(8;21). By contrast, the prognostic significance of mutKIT in pts with inv(16) remains unclear. Purpose of this study is to evaluate the prevalence and the prognostic impact of mutKIT in inv(16)(p13q22). Patients and Methods Fifty adults with inv(16) AML at diagnosis (median age 46.6 yrs, range: 17–88; M/F: 30 /20), were centrally analyzed for mutKIT in exon 2, 8, 10, 11 and 17. Mutations were detected using sequencing and other sensitive assays such as ARMS (amplification refractory mutation system) PCR for D816Y and D816H and enzymatic digestion with HINFI for D816V and with Tsp509I for N822K. Results Data showed a prevalence of KIT mutation of 34% (17/50 pts). Among the mutKIT cases, we detected mutations in exon 17 (n=12), exon 8 (n= 4) and exon 10 (n=1). There was no difference between the mutKIT vs the unmutated (KIT-) patients in the median of WBC count at presentation (WBC 13.9 x109/L, range 4.4 to 277.5 vs 19.4 x109/L, range 2.5 to 130; Mann-Whitney U test: p = 0.649). Of the 42 patients (age 〈 60 years) who received intensive chemotherapy, 13 resulted mutKIT upon mutational screening. Complete remission (CR) was achieved in 13/13 (100%) mutKIT vs 27/29 (93%) KIT- patients (Fisher’s exact test: p 〉 0.999). At a median follow-up of 26 months (range: 2–144), 9/13 (69%) mutKIT and 8/27 (29%)KIT- pts relapsed;the Kaplan-Meier plots revealed KIT mutations to be a significant factor adversely affecting RI (log-rank test: p = 0.017) but not OS (61% in mutKIT vs 75% in KIT-;log-rank test: p = 0.331). Conclusion KIT mutations are associated with a greater probability of relapse following CR, without affecting OS, in AML pts with inv(16) aged

Description: C-KIT gene mutations are not a rare event in Core Binding Factor Leukemia (CBFL). To investigate their prognostic impact in this setting , we attempted at correlating the KIT mutational status with the WBC count, the level of the WBC-index, the presence of extramedullary disease (EMD) and the outcome, in t(8;21)(n=30) and inv(16) (n=20) AML patients. On the basis of genomic DNA analysis of c-KIT gene exons 2, 8, 10, 11, 17 for mutation detection, we categorized 21 patients (42%) in the “KIT mutated group” (KIT+) and 29 patients (58%) in the “KIT unmutated group” (KIT−). The median age at diagnosis was 46.4 and 45.6 years for the KIT(+) and KIT(−) groups, respectively (P=0.872). Among the KIT(+) patients, we found mutations in exon 8 (n = 4), exon 10 (n =1), exon 11 (n = 1) and exon 17 (n = 15). We recorded mean WBC counts of 28.6 x 109/L vs 34.5 x 109/L (P=0.051), and a mean WBC-index, expressed as WBC x (% Bone Marrow blasts/100), of 37.2 vs 15.5 (P=0.0395), for KIT(+) and KIT(−), respectively. Seven out of 50 patients experienced an EMD: in 6 cases (28.57%) this event occurred in the KIT(+) group. 40 patients (age 〈 60 years) were evaluable for clinical response; 18 out of them were KIT mutated. At a median follow-up of 24 months (range 10–107), we recorded for KIT(+) and KIT(−), respectively: CR incidence of 88.8 % (16/18) vs 100% (22/22) (P=0.38); Relapse Incidence 81.3% (13/16) vs 31.81% (7/22), P=0.0072; OS 27.7% (5/18) vs 77.3% (17/22), P=0.0049; DFS 16.7% (3/18) vs 77.3% (17/22), P=0.0005. Using a log-it linear model for univariate and multivariate regression analysis, we found a significant contribution to death (P=0.048) and relapse (P= 0.045) exerted by mutation in the whole group of patients; this effect was more evident when the analysis was restricted to patients

Description: Portal vein thrombosis (PVT) is a potentially fatal complication of splenectomy; the prevalence of PVT remains up till now controversial, such as the related-risk factors. The hematologic patients who underwent splenectomy in Niguarda Hospital, Milan, Italy, between January 1995 and November 2004 were retrospectively reviewed to identify the prevalence of post-splenectomy PVT and risk factors associated with its development. Splenectomy was performed because of malignant (n = 41) or non-malignant (n = 44) hematologic diseases. Indications for splenectomy in the groups with and without PVT were compared with Fisher’s exact test. We tested the reliability of platelet count as a marker of PVT, analysing the receiver operator characteristic (ROC) curve. Cut-off level was chosen for best prediction in term of sensitivity and specificity. Among 85 patients who underwent splenectomy, the associated non-malignant diseases were immune thrombocytopenic purpura (n = 40), hemolytic anemia (n = 2), Evan’s syndrome (n = 1), cryoglobulinemia (n = 1); malignant diseases were lymphoproliferative (n = 39) and myeloproliferative disorders (n = 2). Four cases of PVT (4,71%) were diagnosed. One of 2 patients with a myeloproliferative disorder had PVT. PVT was also present in 3/39 (7.69%) patients with lymphoproliferative malignancies, all with splenic marginal zone B-cell variety non-Hodgkin’s lymphoma (3/16) . The median splenic weight in patients who developed PVT was 1800 g (range 1500–1980 ) and the median post-surgery platelet count was 661 x 109/liter (range 211–1250). No patient with non-malignant diseases had portal vein thrombosis. The incidence of the event was of 0.0000523 new cases for day-patient, with a 0.005% new incidental case probability. Splenic weight, with a cut-off value of 500 grams for the risk analysis (p=0.029), was significantly correlated to thrombosis. PVT was also significantly correlated to the entity of the post-surgery thrombocytosis (p=0.024), setting the threshold value of thrombosis risk at 850.000/uL (Sensibility 0.50 - Specificity 0.95). Patients with myeloproliferative and lymphoproliferative disorders seem at higher risk for the development of PVT than those with non-malignant diseases. A splenic weight over 500 grams and a post-surgery thrombocytosis over 850.000/uL are significantly associated with the occurrence of PVT. The presence of these two risk factors should require the prophylactic use of adequate doses of low molecolar weight heparin.

Description: Introduction. Fc-mediated clearance of platelet (plt) by splenic macrophages is a characteristic feature of immune thrombocytopenia (ITP) and has so far been addressed as the main mechanism of plt destruction. Recently (Li J et al. ASH 2014 abs#467), an Fc-independent mechanism has been proposed which involves mainly the liver, mostly mediated by anti-GPIb antibodies (Abs). These Abs were shown to induce plt activation leading to GPIb desialylation. In turn, plt lacking sialic acid are removed by the hepatic Ashwell-Morell receptor (AMR).In order to test the clinical relevance of this novel mechanism of plt clearance, plt kinetic studies (PKS) and Ab type data were retrospectively reviewed in adult ITP patients (pts). Patients. Charts of ITP pts who had data on both PKS and Ab testing were retrospectively reviewed. Pts are enrolled in a local Italian data base and informed consent to clinical data use is given at enrollment. Results. A total of 82 pts (M 34) were identified; median age at ITP diagnosis was 46.3 yrs (range 7.5-78.9 yrs); median disease duration at PKS was 12.5 mos (range 0.7-278.9 mos). Of the 82 pts included: 59 (71.9%) had already undergone splenectomy between year 2000 and 2015; 23 were studied but splenectomy has not been performed yet. All pts underwent or are candidate to splenectomy because of steroid-dependent ITP; 1 pt only was primarily refractory to steroids and IVIG treatment. Results of PKS and autoAb testing are summarized in Figure 1. Antibody testing: 45/82 (55.9%) of pts tested positive to one or more than one antiGP Abs (direct test); 10/82 pts tested positive to both direct and indirect test (not shown in Fig). PKS results: Ab negative pts: spleen 25/37, liver 0; mixed 12/37; Ab positive pts: spleen 25/45, liver 4/45, mixed 16/45. A trend toward an association between number of Ab positivity and site of clearance was observed: every single unit increase in the number of Ab positivity (i.e.: none vs 1 vs 2 vs 3 vs both direct and indirect Ab testing) is associated with a 33% increase in the odds of liver involvement (Wald's test after logistic regression p= 0.104). No association was found between either isolated or combined anti-GPIb-lX Ab positivity and site of plt clearance (Fisher's exact test p=0.235). Splenectomy outcome: CR 50/59; R 4/59; NR 5/59. (Fig. 2) Conclusions. Recent studies have proposed that Ab type may determine pltÕs fate in ITP by activating either Fc- or non Fc-mediated mechanisms of plt clearance resulting in splenic or hepatic uptake respectively. However, our data on PKS and Ab type do not support such a mechanism being clinically relevant in ITP pts. Although an increase in the number of Ab positivity increases the probability of liver involvement in plt clearance, no association was found between type/number (single vs multiple antiGP) of Ab positive test and site of plt clearance. Moreover, response to splenectomy doesnÕt seem to be influenced by either Ab type and/or site of plt clearance. However, it may well be that AMR-mediated hepatic plt clearance rather represents a physiological mechanism involved in plt and hemostasis homeostasis. Plt desialylate as they circulate, thereby becoming the primary AMR ligand and this interaction is involved in the regulation of TPO production by hepatocytes (Grozovsky R et al. ASH 2014, abs #2). Desialylation also occurs when plt are activated by several physiological stimuli (S¿rensen AL et al. Curr Opin Hematol, 2008) and AMR clearance of activated plt may be relevant in attenuating the coagulopathy associated with sepsis (Ellies LG et al. Proc Natl Acad Sci USA 2002; Grewal PK et al. Nat Medicine 2008)- Table 1. Ab pattern n (%) site of plt clearance (111-Indium labeled plt) splenic hepatic mixed All patients 82 (M34/F48) Ab negative 37 (45.1) 25 (67.6%) 0 (0%) 12 (32.4.%) Ab positive 45 (54.9) 25 (55.5) 4 (9%) 16 (35.5%)  one positive Ab 17/45 GpIIb-IIIa 11 7 2 2 GpIb-IX 4 2 1 1 GpIa-IIa 2 1 0 1  two positive Abs 9/45 GpIIb-IIIa & GpIb-IX 2 1 0 1 GpIIb-IIIa & GpIa-IIa 7 5 0 2  three positive Abs 16/38 GpIIb-IIIa & GpIb-IX & GpIa-IIa 19 9 1 9 Table 2. Ab pattern Outcome after splenectomy site of plt clearance (111-indium labeled plt) spleen liver mixed Ab negative (27/59) CR + R 17 0 9 NR 1 one positive Ab (13/59) IIb-IIIa* CR 4 2 1 NR 0 0 0 Ib-IX CR 2 0 1 NR 0 1 0 Ia-IIa CR 1 0 0 NR 0 0 1 two positive Abs (8/59) IIb-IIIa; Ib-IX CR 1 0 1 NR 0 0 0 IIb-IIIa; Ia-IIa CR+R 4 0 2 NR 0 0 0 three positive Abs (11/59) IIb-IIIa; Ia-IIa; Ib-IX CR 6 0 3 NR 1 1 0 *4 pts had both direct and indirect positive ab testing Disclosures No relevant conflicts of interest to declare.

Description: Abstract 2934 Poster Board II-910 Background: hepatitis C virus (HCV) infection has been associated with increased risk of developing B-cell lymphoprolipherative disorders through chronic immune stimulation. Discordant data have been reported about the prevalence of HCV infection in patients with Waldenström's Macroglobulinemia (WM) and the putative role of HCV in lymphomagenesis of this non-Hodgkin Lymphoma subtype remains controversial. Aim: the current retrospective study was conducted to assess the impact of this infection itself in the clinical and biological features and outcome among WM patients as compared with those with HCV negative WM. Patients and methods: we analyzed 140 WM patients with tested anti-HCV antibody (HCV-Ab). HCV-Ab positivity was detected in 21 cases (15%). Clinical characteristics of patients at diagnosis of WM are reported in the table below. Results: HCV positivity was correlated to lower counts of platelets, neutrophil granulocytes, haemoglobin and with presence of cryoglobulins or autoantibodies and splenomegaly. Interestingly we even found a strong link between the presence of HCV and serum parameters of tumor burden such as beta-2 microglobulin (B2-M) and lactate dehydrogenase (LDH). Furthermore we did not reveal any outcome implications in terms of disease progression needing treatment, time from diagnosis to first therapy, overall survival between HCV- and HCV+ patients. Overall, 88 patients (63%) received treatment for disease progression with schedules including Rituximab in 46 cases (52% of treated patients). Rituximab was administered even in HCV-RNA positive patients associated to Cyclophosphamide and Fludarabine and this did not translate in hepatitis development. HCV-RNA was strictly monitored during immunotherapy and we did not observe any significant flair. Conclusions: in our series of patients HCV infection does not seem to affect clinical outcome of disease. Moreover, in our experience patients with documented infection can receive the same schedule of treatment including intensive chemotherapy even with monoclonal antibodies without development of further toxicity. Disclosures: No relevant conflicts of interest to declare.

Description: Background The p53 protein is an onco-suppressor protein encoded by the TP53 gene, which is mutated in 5-10% of cases of de novo myelodysplastic syndromes (MDS). In 75% of cases TP53 mutations lead to store the p53 protein within the nucleus of the neoplastic cells. TP53 mutations were shown to have an unfavorable prognostic impact in patients with MDS. The immunohistochemical (IHC) expression of p53 in bone marrow (BM) biopsy has in itself negative impact on prognosis in low risk MDS, especially in MDS with isolated del(5q) category. However, the p53 cut-off value related to prognosis has not been established with accuracy, ranging between 1 and 5% in different reports. Moreover, no data are available on the possible prognostic impact of p53 BM expression and cut-off levels in patients with higher risk MDS. Aim To evaluate the prognostic value of IHC expression of p53in BM biopsies from patients with intermediate, high and very high R-IPSS risk MDS Methods BM biopsies performed at diagnosis in patients with intermediate, high and very high R-IPSS risk MDS with a follow up of at least three years were revised and screened for IHC p53 expression. Percentage of p53 expression was evaluated by two independent pathologists (L.B.; M.T.), and related to patient survival. Only cells with strong p53 staining were counted as positive. The statistical evaluations were carried out with the logistic analysis and the influence of p53 expression on survival was analyzed by Cox regression. A ROC analysis was carried out using theYouden method to analyze the optimal cut-off value influencing the survival. The verification was performed with the positive and negative predictive values (respectively PPV and NPV), the sensibility and the specificity with their respective 95% confidence intervals (95%CI). Survivorships were estimated with the Kaplan-Meier product limit method, followed by the logrank test, and by the Cox proportional-hazard regression. The association among categorical variables was evaluated by Fisher exact test. Results A total of 60 BM biopsies performed at MDS diagnosis were screened for p53 expression. Themedian age of these 60 patients was 67 years (range 19 - 82). Diagnoses, according to WHO, were RCMD in 26/60 (43.3%) cases; RAEB1 in 21/60 (35%) cases; RAEB2 in 13/60 (21.7%) cases. The IPSS-R was intermediate in 43 (71.7%) cases; high in 9 (15%) cases and very high in 8 (13.3%) cases. Cytogenetic risk according to the IPSS-R stratification was: very low in 1(1.6%) case; low in 30 (50%) cases; intermediate in 10 (16.7%) cases; high in 12 (20%) cases; very high in 7 (11.7%) cases. Median overall survival was 41 months. The p53 expression was: 〈 1% in 39 cases (65.0%), 1% in 5 cases (8.3%), 2% in 6 cases (10.0%), 3% in 2 cases (3.3%), 5% in 3 cases (5.0%),at least 10% in 5 cases (8.3%). Upon analysis, a significant association between percentage of p53 expression and patient survival was found (p=0.013; Hazard Ratio 1.067; 95%CI: 1.014 - 1.124). A cut-off value of 10% p53 expression was associated with outcome (specificity 100%; sensibility 13.5%;PPV 100%; NPV 41.8%). Specifically, as shown in figure 1, a significantly better overall survival was observed in the 55 (91.7%) patients whose BM p53 expression was 〈 10% compared to the 5 (8.3 %) patients with a BM p53 expression at least 10% (p=0.0038). No association was found between either BM blast countor BM grade of fibrosis and p53 expression.A significant association between the cytogenetic risk according to R-IPSS stratification and the expression of p53was instead found: any single unitary arbitrary increase in the cytogenetic risk score rises by 1600% the odds of a BM p53 expression at least 10% (p=0.015). Conclusion In our study population we confirm the unfavorable prognostic significance of BM p53 expression in higher risk MDS patients. Contrary to the reported cut-off values of p53 expression in low risk MDS, in our cohort of higher risk MDS the levels related to prognosis were greater (10% compared with 1 to 5% according to different reports). A tentative explanation for this difference may be that factors other than p53 expression strongly impact on survival in patients with higher risk MDS. Thus the negative prognostic value of p53 only emerges at higher levels of expression. The association between p53 expression and the IPSS-R cytogenetic risk score, if confirmed on a larger cohort, should be evaluated in specific biologic investigations. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Diffuse Large B-cell lymphoma (DLBCL) is an heterogeneous disease in terms of biology, clinical presentation and prognosis. According to the putative Cell-Of-Origin (COO), DLBCL of the general HIV negative population can be classified into three molecular subtypes by gene expression profiling (GEP): the germinal center B-cell type (GCB), the activated B-cell type (ABC) and the unclassifiable type (UC). Immuno-histochemistry (IHC) models routinely surrogate GEP in the COO assignment, identifying GCB and non-GCB type. Hans algorithm is one of the most widely used IHC models and its concordance with GEP varies from 70 to 90% (Meyer PN et al, JCO 2011). NanoString (NS) 20-genes assay has emerged as a feasible technique for DLBCL molecular subgroups identification and shows a high concordance with GEP (Scott DW et al, Blood 2014). DLBCL classification according to COO categories has shown prognostic impact in the HIV negative population, since clinical outcome of non-GCB type is inferior if compared to GCB type in patients (pts) receiving CHOP or Rituximab-CHOP. DLBCL in HIV positive population have shown different biological features. COO subtypes distribution and its prognostic impact has not been extensively studied in HIV setting and still needs to be defined. Aim of the study: To evaluate the proportion of COO subtypes in HIV-associated DLBCL according to IHC and NS assay, the concordance of these two different diagnostic methods, and the prognostic impact of COO assignment; to analyze BCL-2 and MYC protein expression and their correlation with outcome; to determine the prognostic value of baseline clinical characteristics, such as stage, LDH value and CD4+ lymphocyte count. Methods: We retrospectively evaluated 66 cases of HIV positive pts with newly diagnosed DLBCL from 2000 to 2016, in 5 Italian Institutions. Histological samples were centrally reviewed by a panel of expert Pathologists for DLBCL diagnosis confirmation, BCL-2 and MYC protein expression and COO assignment according to Hans algorithm (Hans CP et al, Blood 2004). The cut off considered for BCL-2 and MYC overexpression was 70% and 40% respectively. NS Lymph2Cx assay for COO was performed in all pts. Clinical data were gathered from pts medical records. Results: Pts were mostly male (73%) and median age at DLBCL onset was 45 years (range: 28-83). 80% of pts had advanced stage lymphoma and 34% showed a high-intermediate or high IPI score. HIV first detection was concomitant to DLBCL diagnosis in 30% of pts. At DLBCL diagnosis median CD4+ cell count was 188/microL (range: 8-1172) and 34/66 pts (52%) had detectable HIV viral load. COO assigned by IHC was GCB in 31/66 pts (47%) and non-GCB in 35/66 (53%); COO allocation by NS assay was 57% GCB (n=34/60 pts) and 43% non-GCB (ABC n=11, 18%; UC n=15, 25%). IHC algorithm and NS assay concordantly assigned COO subtypes in 80% of pts with a Cohen's kappa=0,604 (p 200/microL has a protective role both for progression and death (2-years PFS: 62% vs 42%, p=0.02, HR: 0.41; 2-years OS: 72% vs 42%, p=0.002, HR: 0.30). Stage IV disease and elevated baseline LDH were associated with inferior PFS (p=0.0158 and p=0.02) and OS (p=0.0076 and p=0.034). Conclusions: This analysis of 66 HIV-associated DLBCL confirmed that GCB is the more frequent subtype identified by NS, as previously reported (Baptista MJ et al, Hemat Oncol 2017). We found an high concordance between Hans algorithm and NS in the COO subtypes allocation. However, COO classification showed no impact on pts outcome. FISH assays for detection of MYC, BCL-2 and BCL-6 rearrangements are ongoing in the entire study population. Disclosures Rusconi: Celgene: Research Funding.