To cite this article: Glembotsky AC, Bluteau D, Espasandin YR, Goette NP, Marta RF, Marin Oyarzun CP, Korin L, Lev PR, Laguens RP, Molinas FC, Raslova H, Heller PG. Mechanisms underlying platelet function defect in a pedigree with familial platelet disorder with a predisposition to acute myelogenous leukemia: potential role for candidate RUNX1 targets. J Thromb Haemost 2014; 12: 761-72 Summary. Background: Familial platelet disorder with a predisposition to acute myelogenous leukemia (FPD/ AML) is an inherited platelet disorder caused by a germline RUNX1 mutation and characterized by thrombocytopenia, a platelet function defect, and leukemia predisposition. The mechanisms underlying FPD/AML platelet dysfunction remain incompletely clarified. We aimed to determine the contribution of platelet structural abnormalities and defective activation pathways to the platelet phenotype. In addition, by using a candidate gene approach, we sought to identify potential RUNX1-regulated genes involved in these defects. Methods: Lumiaggregometry, a-granule and dense granule content and release, platelet ultrastructure, a IIb b 3 integrin activation and outside-in signaling were assessed in members of one FPD/AML pedigree. Expression levels of candidate genes were measured and luciferase reporter assays and chromatin immunoprecipitation were performed to study NF-E2 regulation by RUNX1. Results: A severe decrease in platelet aggregation, defective a IIb b 3 integrin activation and combined ad storage pool deficiency were found.However, whereas the number of dense granules was markedly reduced, a-granule content was heterogeneous. A trend towards decreased platelet spreading was found, and b 3 integrin phosphorylation was impaired, reflecting altered outside-in signaling. A decrease in the level of transcription factor p45 NF-E2 was shown in platelet RNA and lysates, and other deregulated genes included RAB27B and MYL9. RUNX1 was shown to bind to the NF-E2 promoter in primary megakaryocytes, and wild-type RUNX1, but not FPD/AML mutants, was able to activate NF-E2 expression. Conclusions: The FPD/AML platelet function defect represents a complex trait, and RUNX1 orchestrates platelet function by regulating diverse aspects of this process. This study highlights the RUNX1 target NF-E2 as part of the molecular network by which RUNX1 regulates platelet biogenesis and function.

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Although CALR(+) patients were at higher risk of developing anemia, anagrelide proved effective among all molecular subsets, indicating that mutational status does not seem to represent a major determinant of choice of cytoreductive treatment among essential thrombocythemia therapies.

The low frequency of MPL mutations in this cohort is in agreement with previous studies. The finding of normal Mpl levels in MPLW515L-positive platelets indicates this mutation does not lead to dysregulated Mpl expression, as frequently shown for myeloproliferative neoplasms. The lack of spontaneous STAT3 and STAT5 activation and the normal response to TPO is unexpected as MPLW515L leads to constitutive receptor activation and hypersensitivity to TPO in experimental models.

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INTRODUCTION: Recent studies have suggested that tumor microenviroment (TME) may play an important role in lymphomagenesis and tumor progression in non-Hodgkin lymphoma. Tumor-infiltrating lymphocytes and myeloid-derived cells could provide valuable prognostic information independent from tumor characteristics alone. In diffuse large B-cell lymphoma (DLBCL) several attempts have been made to capture prognostic variables associated with TME. Peripheral blood monocytes, lymphocytes and natural killer (NK) cells have been shown to predict outcome in DLBCL patients (pts). Immunohistochemistry and gene-expression profile on tissue biopsies have also been studied as prognostic indicators. In this study we assessed the prognostic significance of the percentage of infiltrating T-lymphocytes (TL) and NK cells measured by flow cytometry (FC) in tissue biopsies of DLCBL. AIMS: -To determine the prognostic impact on survival of the percentage of infiltrating TL and NK cells measured by FC in tissue biopsies. -To evaluate whether this variables can provide additional information when superimposed on the R-IPI. METHODS: We selected pts with DLBCL and available tissue biopsy FC data at the time of diagnosis who received treatment at our institution between 2012 and 2018. Clinical information such as age, gender, stage, serum lactate dehydrogenase levels, R-IPI and cell of origin were collected from medical records. FC analysis was performed with 8-color FC panels according to international Euroflow protocols. Percentage of TL and NK-cells in lymph node biopsy by FC was compared to the normal values determined by Battaglia et al (Immunology 2003). Both parameters were analyzed as dichotomized variables: low vs. normal-high. The survival analysis was estimated with Kaplan-Meier method. The comparison between variables was performed through log-rank test and multivariate analysis with Cox regression. RESULTS: A total of 75 pts were included in this retrospective study. Pts characteristic are summarized in Table 1. All pts were treated with immunochemotherapy regimens. Complete remission rate was 82%. Median PFS and OS were 35.2 and 83.2 months respectively, with a median follow up time of 27.8 months (range: 4.3-177.5). In our cohort, the R-IPI was able to discriminate OS and PFS into poor and good-very good risk (median OS of 27.8 months vs. not reached, and median PFS of 12.8 vs. 82.6 months, p<0.001, respectively). A low percentage of TL in lymph node biopsy samples was associated with inferior OS (median OS 34.3 months vs. not reached, p:0.001). This subgroup also had inferior PFS, not statistically significant (median PFS 16 vs. 118 months, p:0.08). Regarding NK-cells, low values had significantly worse OS (median OS 57.4 months vs. not reached, p:0.03). Decreased PFS, not statistically significant, was also found in this subgroup (median PFS of 15.5 vs. 40.4 months, p:0.079). Both the TL and R-IPI remained independent predictors of survival on multivariate analysis with HR of 5.5 (CI 95% 1.88-16.07, p: 0.002), and 5.8 (CI 95% 2.20-15.33, p<0.001) respectively (Table 2). The percentage of TL in lymph nodes was able to further stratify clinical outcome in all R-IPI categories. In pts in the good-very good R-IPI and normal-high TL risk group no deaths occurred, compared to a median OS of only 22.2 months in pts with poor R-IPI and low TL values (p<0.001). Interestingly the outcomes of pts with only one of the adverse variants (low TL or poor R-IPI) were very similar (figure 4). Similarly, PFS in the good-very good R-IPI with normal-high TL was significantly higher than in the poor risk R-IPI with low percentage of TL in tissue samples (median PFS of 118.5 months vs. 9.3 months respectively, p<0.001), (Figure 3). CONCLUSIONS: Tumor infiltrating TL in lymph nodes of pts with DLBCL measured by FC showed prognostic impact in OS in our cohort. Data obtained from FC is easily acquired and should be taken into consideration when studying TME. Furthermore, the percentage of TL was able to provide additional prognostic information when superimposed on the R-IPI, identifying both a subgroup with an exceptionally good outcome and a very high-risk subset of pts. Early strategies aiming to improve TL in poor risk patients could constitute an appealing approach. Disclosures No relevant conflicts of interest to declare.

Introduction Estimated incidence of cHL in Argentina is 842 cases/year (Globocan 2018). There is no local data regarding response rates (RR) to FL. GATLA (Grupo Argentino de Tratamiento de Leucemia Aguda) reported 3 years progression free survival (PFS) rates of 90% and overall survival (OS) of 98% regardless of stage. HL has a high cure rate; 10% are primary refractory and 30% relapse after achieving complete remission (CR). In stage I-IIa, 5 years OS is estimated around 90% and 60% in stage IV (Ann Hematol 2019). Objectives Primary: To learn the RR, PFS and associated variables after FL of cHL in public (PuI) and private institutions (PrI) in Argentina. Secondary: To learn the OS rates. To study epidemiological characteristics of the patients (Pts) in participating institutions and reveal differences which may affect the response to treatment. Materials and methods Retrospective analysis of consecutive Pts with diagnosis of cHL from 1/1/2008 to 2/1/2019 with available follow up data. Descriptive statistics was performed in clinical variables and histopathological findings. Quantitative variables were expressed as median an interquartile range (IQR) and qualitative variables as total number and percentage (%). Survival rates were estimated by the Kaplan-Meier method and compared by the log-rank test. OS was measured from the date of diagnosis to date of death or last follow-up visit. Results 520 Pts from 7 PuI and PrI in Buenos Aires and Rosario were examined. 22 Pts had nodular lymphocyte predominant HL. Data on the 498 Pts with cHL is presented. Median follow up: 37.4 months (CI95% 17.7-63.5). Pts characteristics: Table 1. The median time from diagnosis to FL was 22 days (IQR 14-42), significantly shorter in PrI (32.5 (IC95% 27-38) vs. 49.3 (IC95% 38.5-60.2); p=0.0027). 96.5% of Pts received ABVD as FL, dose modifications or transitory suspension were required in 17.1%, and 82.1% received all cycles properly. CR was achieved in 83.4% of Pts and partial remission (PR) in 6.3%. The % achieving CR was higher in PrI; more PR were achieved in PuI. 10.3% had progressive disease (PD) at the end-of FL. 85.4% (n=373) had negative end-of-treatment FDG-PET results (DS1-3). Interim PET scan was performed in 70% of Pts (n=357), with 83.8% achieving metabolic CR but only 15.5% (n=70) being treated with response-adapted strategies (6.5% deescalated to AVD). Regarding hematologic toxicity, anemia, neutropenia and thrombocytopenia were found in 28.5%, 56.4% and 7.2% of Pts, respectively. Febrile neutropenia was reported in 9 Pts. 28.6% developed non-hematologic toxicities (41/144 pulmonary toxicity). 51 Pts had primary refractory disease and 69 (14%) relapsed during follow-up (median time to relapse 4.4 months (CI95% 0-13)). 65 Pts died (12.5%), 34 due to lymphoma progression and the remaining 31 due to toxicity. 2 years OS rate was 91% (CI95% 88% - 94%) and 85% at 5 years (CI95% 80% - 89%). There was no difference in OS between PrI and PuI (p=0.27); every day of delay in the beginning of FL increased 0.89 (IC95% 0.6-1.8) the risk of achieving PR or PD at the end of FL. 5 years PFS rate was 76% (CI95% 70-81) (figure 1-2: OS according to risk group and PFS). Outcomes were statistically better in women, age younger than 60, non-bulky disease, absence of extranodal disease or risk factors such as leukocytosis, lymphopenia and hipoalbuminemia. Pts with normal ESD, stage I-III, early favorable and advanced favorable stages and Charlson score <3 also showed survival advantage (p<0.01). On multivariate analysis Charlsons score and end-of-treatment FDG-PET scan remained independent predictors of OS with HR of 1.2 (CI95%1.1-1.7; p=0.001) and 2.3 (CI95% 1.7-3.2; p<0.0001), respectively. Conclusions This is one of the largest retrospective cohorts reported in cHL. Epidemiology characteristics, RR, PFS, and associated variables are similar to the ones reported in literature. Five years OS proved to be higher than previously reported. ABVD is the chemotherapy regimen of choice in our country and as our study shows, is well tolerated but not exempt from toxicity. Early FL initiation improves outcome. PET scan was widely used but only 15.5% of the Pts were treated with response adapted strategies. Taking into account that in 47.6% of the Pts toxicity was the main cause of death, the use of PET-guided treatment in our population should be strongly considered. * The first four authors have equal contribution figure 1 Disclosures No relevant conflicts of interest to declare.

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