ALBERT

Description: Normal peripheral blood mononuclear cells (PBMC) were cocultured with a human lung cancer cell line (LC89) transduced with the interleukin-2 (IL-2), IL-7, granulocyte-macrophage colony-stimulating factor (GM-CSF ), and tumor necrosis factor-α (TNF-α) genes to evaluate the capacity of the engineered cells to: allow survival of CD3+ and CD56+ cells, generate cytotoxic effectors with HLA class I restricted and unrestricted antitumor activity, and interfere in the molecular organization of the CD3/T-cell receptor associated signal transduction machinery. When PBMC were cultured up to 3 weeks with IL-2 releasing LC89 cells (LC89/IL-2), the number of viable CD3+ and CD56+ lymphocytes was much greater than in cultures with parental cells or with LC89 cells transduced with the other cytokine genes. After 1 week of coculture, a variable degree of restricted and unrestricted killing directed against different targets was observed. When the cultures were prolonged up to 3 weeks, LC89/IL-2 cells induced a marked increase in specific cytotoxic activity, which was coupled to a further enhancement of unrestricted lytic function. In the presence of LC89/IL-7 cells the degree of specific lysis remained unchanged, whereas unrestricted effectors were markedly decreased. No cytotoxic activity could be induced by LC89/GM-CSF and LC89/TNF-α cells in the few lymphocytes surviving after 3 weeks of culture. Coculture of parental LC89 cells with PBMC was consistently associated with a downmodulation in the expression of the CD3 ζ chain, as well as of the tyrosine kinases p56lck and ZAP-70. On the contrary, LC89/IL-2 cells, and not LC89 cells transduced with the IL-7, GM-CSF, or TNF-α gene, were capable of reverting the immunosuppressive effect exerted by the tumor cells. This protective effect could be maintained in cultures prolonged up to 4 weeks. When the same cultures were set up in Transwell, ie, with a membrane separation between cancer cells and PBMC, the expression of the CD3 ζ chain and of the p56lck and ZAP-70 tyrosine kinases remained unchanged under all culture conditions, indicating that the downmodulation of T-cell signal transduction molecules requires a direct cell to cell contact. These results show that transfer of the IL-2 gene into the DNA of human cancer cells promotes both restricted and unrestricted antitumor activity, and is capable of restoring and maintaining the expression of molecules involved in the process of T-cell mediated tumor cell recognition, thus underlining the potential role of the IL-2 gene in the design of vaccination protocols with cytokine gene transduced cancer cells.

Description: Normal peripheral blood mononuclear cells (PBMC) were cocultured with a human lung cancer cell line (LC89) transduced with the interleukin-2 (IL-2), IL-7, granulocyte-macrophage colony-stimulating factor (GM-CSF ), and tumor necrosis factor-α (TNF-α) genes to evaluate the capacity of the engineered cells to: allow survival of CD3+ and CD56+ cells, generate cytotoxic effectors with HLA class I restricted and unrestricted antitumor activity, and interfere in the molecular organization of the CD3/T-cell receptor associated signal transduction machinery. When PBMC were cultured up to 3 weeks with IL-2 releasing LC89 cells (LC89/IL-2), the number of viable CD3+ and CD56+ lymphocytes was much greater than in cultures with parental cells or with LC89 cells transduced with the other cytokine genes. After 1 week of coculture, a variable degree of restricted and unrestricted killing directed against different targets was observed. When the cultures were prolonged up to 3 weeks, LC89/IL-2 cells induced a marked increase in specific cytotoxic activity, which was coupled to a further enhancement of unrestricted lytic function. In the presence of LC89/IL-7 cells the degree of specific lysis remained unchanged, whereas unrestricted effectors were markedly decreased. No cytotoxic activity could be induced by LC89/GM-CSF and LC89/TNF-α cells in the few lymphocytes surviving after 3 weeks of culture. Coculture of parental LC89 cells with PBMC was consistently associated with a downmodulation in the expression of the CD3 ζ chain, as well as of the tyrosine kinases p56lck and ZAP-70. On the contrary, LC89/IL-2 cells, and not LC89 cells transduced with the IL-7, GM-CSF, or TNF-α gene, were capable of reverting the immunosuppressive effect exerted by the tumor cells. This protective effect could be maintained in cultures prolonged up to 4 weeks. When the same cultures were set up in Transwell, ie, with a membrane separation between cancer cells and PBMC, the expression of the CD3 ζ chain and of the p56lck and ZAP-70 tyrosine kinases remained unchanged under all culture conditions, indicating that the downmodulation of T-cell signal transduction molecules requires a direct cell to cell contact. These results show that transfer of the IL-2 gene into the DNA of human cancer cells promotes both restricted and unrestricted antitumor activity, and is capable of restoring and maintaining the expression of molecules involved in the process of T-cell mediated tumor cell recognition, thus underlining the potential role of the IL-2 gene in the design of vaccination protocols with cytokine gene transduced cancer cells.

Description: Background: Life expectancy in Essential Thrombocythemia (ET) patients is superimposed to normal population. The main causes of death are thrombotic events and evolution into myelofibrotic phase or secondary myelodisplasia/acute leukemia. Approximately 50% to 65% of patients with ET carry activating mutations in the Janus kinase 2 gene (JAK2), and an additional 5% in the thrombopoietin receptor gene (MPL) whereas 10 to 20% of patients have mutated calreticulin gene (CALR). Non-mutated JAK2, CALR and MPL ET (triple negative-TN) are about 10%. Patients and methods : In this study we analysed the prognostic value of JAK2V617F, CALR and MPL mutational status on outcome and thrombotic risk in a retrospective cohort of 138 ET patients defined according to WHO criteria, diagnosed from 1974 to 2013 in a single Italian centre (Turin). JAK2V617F mutation was assessed by Quantitative Real–Time PCR on DNA from peripheral blood or bone marrow samples. JAK2 negative cases were then analyzed for CALR mutations by Gene Scan Analysis in combination with direct sequencing or MPL W515L/K by Allelic Discrimination Real-Time PCR assay. Overall survival (OS), cumulative incidence of myelofibrotic transformations (CI-MT) and thrombosis (CI-TB) were calculated from the date of diagnosis. The between-group comparison for OS was performed with the log-rank test, whereas for CI-MT and CI-TB we using the Gray’s test considering death as competing event and adjusting for presence of cardiovascular risk factors . Results: Among 138 ET patients, 103 (74.6%) carried the JAK2V617F mutation, 3 (2.2%) carried activating mutations of MPL exon 10, 16 (11.6%) carried mutations of CALR exon 9, and only 16 patients (11.6%) had none of these markers (TN). An high incidence of elevated haemoglobin levels and/or haematocrit (males 〉16.5 g/dl or 49% and females 〉16.0 g/dl or 48%) was significantly associated with JAK2 positivity [13 pts JAK2+ (14.77%) vs 0 pts JAK2- (Fisher test p=0.019)]. Similarly, the CI-TB, analysed with a competing-risk approach, was higher in patients with a JAK2 mutation [(5-year CI-TB: 23.7% JAK2, 0% CALR, 0% MPL, 12.5% TN; P

Description: Translocations of the anaplastic lymphoma kinase (ALK) gene have been described in anaplastic large-cell lymphomas (ALCLs) and in stromal tumors. The most frequent translocation, t(2;5), generates the fusion protein nucleophosmin (NPM)–ALK with intrinsic tyrosine kinase activity. Along with transformation, NPM-ALK induces morphologic changes in fibroblasts and lymphoid cells, suggesting a direct role of ALK in cell shaping. In this study, we used a mass-spectrometry–based proteomic approach to search for proteins involved in cytoskeleton remodeling and identified p130Cas (p130 Crk-associated substrate) as a novel interactor of NPM-ALK. In 293 cells and in fibroblasts as well as in human ALK-positive lymphoma cell lines, NPM-ALK was able to bind p130Cas and to induce its phosphorylation. Both of the effects were dependent on ALK kinase activity and on the adaptor protein growth factor receptor–bound protein 2 (Grb2), since no binding or phosphorylation was found with the kinase-dead mutant NPM-ALKK210R or in the presence of a Grb2 dominant-negative protein. Phosphorylation of p130Cas by NPM-ALK was partially independent from Src (tyrosine kinase pp60c-src) kinase activity, as it was still detectable in Syf-/- cells. Finally, p130Cas-/- (also known as Bcar1-/-) fibroblasts expressing NPM-ALK showed impaired actin filament depolymerization and were no longer transformed compared with wild-type cells, indicating an essential role of p130Cas activation in ALK-mediated transformation.

Description: Background: Prognostic value of bone marrow (BM) fibrosis grading in myeloproliferative neoplasm (MPN) is still debated. Polycythemia Vera (PV) and Essential Thrombocythemia (ET) are long term outcome MPN; however, they could evolve to adverse secondary myelofibrosis-MF or acute leukemia-AL. Aims and Methods: We retrospectively analyzeda cohort of 579 World Health Organization-defined PV (n=180) and ET (n=399) patients, and examined the prevalence and prognostic relevance of BM reticulin fibrosis. All patients were diagnosed between 1990 and 2013 and were recruited in Turin (n= 436) and Bologna (n=143), Italy. BM biopsy sample were reviewed by local pathologist and fiber scoring was performed according to a 3-graded system. Eligibility criteria included the availability of BM samples at diagnosis. Patients with grade 2 or 3-fibrosis were excluded. Overall survival (OS) was evaluated from diagnosis to death using Kaplan Meyer method and Hazard Ratio were estimated with the Cox Model. Cumulative incidence of MF and AL evolution were estimated considering death from any cause as a competing event and compared between groups using the Gray's test. Results: Overall, we observed 115 (63%) grade 0 and 65 (36%) grade 1 fibrosis and 291 (72%) grade 0 and 108 (27%) grade 1 among PV and ET patients, respectively (p= 0.028) We analyzed effect on clinical outcome separately. PV With a median follow up of 110 months (IQR:70-170), 5 and 10-years OS were 96% and 87%, respectively. Stratifying patients based on fibrosis degree, we observed 15 (13%) and 16 (25%) deaths for grade 0 and 1 fibrosis respectively, with 5 and 10-years OS of 98% vs 90% and 92% vs 82% for grade 0 and grade 1, respectively (p 0.076). Neither clinical findings nor thrombosis were significantly different between fibrosis degree. JAK2 V617F or exon 12 mutation status and allele burden was similar into subgroup. Cumulative incidence of MF evolution at 5 and 10-years was 2,8% and 7,2% vs 3,8% and 18,7% for grade 0 and grade 1 respectively (p 0.123). Cumulative incidence of AL evolution at 10- year was 4,2 % for both grade whereas at 15-years was 4,2% vs 19% for grade 0 and 1, respectively. ET At a median follow up of 75 months (IQR:39-120), 5 and 10-years OS were 98% and 90%, respectively. We observed 22 (8%) and 16 (55%) deaths for grade 0 and 1 respectively, with 5 and 10-years OS of 98% and 90% for grade 0 vs 97% and 89% for grade 1, respectively (p 0.358). The mutation status was analyzed in 379 patients and showed: 62% JAK2V617F, 19% CALR (type-1/1-like 14% and type2/2-like 5%), 3% MPLW515, 63 patients were triple negative for the above mutations. During follow-up, patients with grade 0 fibrosis showed more thrombotic events, 41 cases (14%; the 71% JAK2V617F-positive) vs 17 (16%). At multivariate analysis MPL mutation showed a higher risk of MF evolution compared to triple negative with an HR of 5,8 (p 0.0014) and to JAK2V617 mutation with an HR of 9,5 (p 0.002). Cumulative incidence of MF evolution was at 5-years 0,5% and 9% and at 10-years 6,2% and 18% for grade 0 and 1, respectively (p 0.0001). Cumulative incidence of AL evolution at 5 and 10-years was 0% for grade 0 and 13% and 7,3% for grade 1, respectively (p 0.096). However grade 0 showed a higher cumulative risk of AL evolution at 15-years (6,9% vs 10% for grade 0 and 1, respectively) (p=0.096). Conclusion: According to data recently published, in ET patients grade 1 BM fibrosis seems to correlate with a higher cumulative risk of MF and AL evolution, whereas in PV patients seems to correlate to a trend of higher mortality, even if not statistically significantly. Data reaffirm the importance of BM examination as part of diagnostic criteria in all MPN. Disclosures Cavo: Janssen-Cilag: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Millennium: Consultancy, Honoraria. Vitolo:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria; Gilead: Honoraria; Celgene: Honoraria; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Description: Background: Myelofibrosis (MF) is a Ph-negative myeloproliferative neoplasm, classified as Primitive (PMF) or secondary to Polycythemia Vera (PPV-MF) or to Essential Thrombocythemia (PET-MF). Treatments for MF include hydroxycarbamide (HU) and JAK-inhibitors. PMF has a better overall survival than secondary MF, but data from literature are not sufficient to predict whether age, molecular biology and therapy may affect overall survival or incidence of post-diagnosis complications in these different subsets of patients. Methods: Retrospectively analyse a cohort of World Health Organization-defined MF patients, diagnosed from 1988 to 2015 by a single Italian haematological centre (Turin), and observe possible differences on overall survival and post-diagnosis infective, thrombotic or hemorrhagic complications based on age, therapy and molecular biology. Overall survival (OS) was estimated from diagnosis to death for any cause using Kaplan Meyer method and Hazard Ratio (HR) were estimated with the Cox Model. Dichotomous outcomes (post-diagnosis infective, thrombotic, hemorrhagic complications) were compared between groups using chi-squared test. Results: We analysed123 MF patients: 58 (47%) primary MF and 65 (53%) secondary MF (28 PPV-MF and 37 PET-MF). Shared by age, patients were assessed into three groups: under 60 years (40 pts, 33%), between 60-69 years (28 pts, 23%) and over 70 years (55 pts, 44%) Median follow-up was 40 months. Sixty-eight (55%) patients were positive for JAK2-mutation, 14 (11%) for CALR-mutation and 4 (3,2%) for MPL-mutation whereas 15 (12%) patients were triple-negative. Ninety-nine (80,5%) patients received a therapy with HU or JAK-inhibitors, while 24 patients (19,5%) did not receive any cytoreductive therapy. At a median follow-up of 36 months, OS rates were significantly different according to age cohorts: 100% in patients under 60 years, 96% in those between 60-69 years and 78% in patients over 70 years (p=0.001). 36-months OS was also significantly different between PMF and secondary MF: 96% (95% IC: 84-98) versus 86% (95% IC: 85-99) (p=0.01) (HR=0.35). Impact on OS seems to be stronger, even though not significant, in patients under 60 years than in those over 60 years [60ys HR=0.61 (p=0.363)] Shared patients by age (under 60ys, over 60ys), no significant difference was found in the rate of: infective post-diagnosis complications (34% vs 42%, p=0,792); thrombotic events (19% vs 21%, p=0,425) or hemorrhagic events (22 % vs 19%, p=0,855). Post-diagnosis infections, thrombotic or hemorrhagic complications were present in 38%, 15% and 22%, respectively in patients on treatment whereas in 42%, 8% and 12%, respectively in treatment-naïve patients. Therapy did not impact on age nor on primary or secondary nature of MF. The rate of post-diagnosis complications (infective, thrombotic or hemorrhagic) was similar among age cohorts: 51 % in under 60 year-old patients, 50% in 60-69 and 58% in over 70 patients (p=0,741); moreover post-diagnosis complications were observed in 50% of patients who did not receive treatment and in 55% of those on treatment (p=0,689). At last significant connections between JAK2/CALR/MPL positivity and different cohorts of age were not been observed. Conclusion: In patients with MF, our study showed a significant impact of age over 60ys on OS. We confirmed that PMF has a better survival than secondary MF, independently from age, even if the differences seem to be more important in under 60ys group. Moreover we observe a not significant difference between the therapy effect and molecular profile in PMF or secondary MF. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Evaluation of WT1 expression is becoming an attractive marker in acute myeloid leukaemia (AML) for minimal residual disease (MRD) detection. Recent studies correlate therapy response with WT1 copy levels in the bone marrow (BM) offering an additional tool beside multiparameter flow-cytometry (MFC). No well-known data are available regarding its impact on the outcome after hematopoietic cell transplantation (HCT). Patients/Methods One hundred and two consecutive clinically and molecularly well characterized AML patients (pts) were transplanted in a single hematology center from 2004 to 2013. Indication for allogeneic HCT included high cytogenetic and molecular risk according to WHO criteria, high leukocytosis, extramedullary manifestations or secondary AML at diagnosis. Further, primary induction failure was considered as an indication for allogeneic HCT. WT1 expression was analyzed by real time polymerase chain reaction (RT-PCR), using the standardized European LeukemiaNet method on BM samples before HCT and during each follow up control. Only pts in first CR before HCT were included in the analysis. Cumulative incidence of relapse (CIR) was estimated considering death for other causes than relapse as a competing event. Univariate and multivariate Fine & Gray Regression models were used to test the association between CIR and pre-HCT BM WT1 levels. Linearity of the relationship between CIR and the WT1 level was investigated using a mathematical transformation (restricted cubic spline). Aim of this retrospective analysis was to investigate the impact of pre-HCT BM WT1 expression in first CR pts on predicting relapse after HCT. Results A BM WT1 evaluation pre-HCT was available in 89 out of 102 pts. Among them, 62 achieved a CR after induction treatment. Relapsed pts in CR after reinduction chemotherapy (n=16) or with disease persistence pre-HCT (n=2) and pts refractory to treatment (n=9) were excluded from analysis. The patient subgroup displaying a first CR before HCT had a median age at diagnosis of 49 years (range: 20-65). Patient/donor relationship involved 26 (42%) sibling, 32 (52%) matched unrelated and 4 (6%) haplo-identical donors. On the basis of standard cytogenetics, molecular biology and clinical criteria (global disease risk) pts were classified according to the following risk groups: 54 (87%) high, 5 (8%) intermediate and 3 (5%) low risk. Acute GVHD occurred in 24 (39%) whereas chronic GVHD was documented in 18 (28%) pts. Twenty-six (42%) deaths were documented after HCT, 21 (34%) due to relapse and 5 (8%) because of treatment related mortality (TRM). Pre-HCT BM WT1 expression was correlated with CIR. A cut point of 150 WT1 copies was applied according to the slope change of relapse hazard, and subsequently used for CIR analysis. Pts displaying a pre-HCT BM WT1 level 〉 150 copies (n=18) had a higher CIR (2-year CIR 52.5%, 95% CI: 27.1-77.8) compared to pts with a BM WT1 level ≤ 150 copies (n=44, 2-year CIR 27.7% (95% CI: 13.4-42.1). WT1 copy level 〉 150 showed a significantly higher risk of relapse in univariate analysis (HR 2.9, 95% CI 1.2-6.7, p=0.014). In multivariate analysis pre-HCT BM WT1 expression was confirmed as a significant independent risk factor for CIR when adjusted for patient/donor relationship, presence of GVHD, global disease risk and competitive risk of mortality due to TRM (HR 3.4, 95% CI 1.3-8.5, p=0,010), Figure. Conclusions In the present study, pre-HCT BM WT1 levels discriminated significantly for CIR in a cohort of AML in first CR. A cut off level of 150 BM WT1 copies pre-HCT had a powerful statistically significant discriminating value to predict the risk of relapse, independently from already established risk factors. The prognostic value of WT1 was confirmed also when TRM as competitive risk for mortality was added in the multivariable model. Based on these results, WT1 is a promising candidate as MRD tool. Further prospective studies are required to confirm these results. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Wilms Tumor gene 1 (WT1) is overexpressed in the vast majority of patients (pts) in Acute Myeloid Leukemia.(1) Recently the decrease of WT1 expression was suggested to reflect cytoreduction after chemotherapy in AML.(2) However, despite its potential role in minimal residual disease (MRD) assessment, at the moment there is no consensus on its clinical application. Patients/Methods 233 clinically and molecularly well characterized consecutive AML pts were treated with intensive induction chemotherapy according to age, performance status and co-morbidities in a single hematology center. WT1 expression was analyzed by Real Time Polymerase Chain Reaction (RT-PCR), using the standardized European LeukemiaNet (ELN) method, at baseline and after induction chemotherapy. Further, WT1 expression was compared with several demographic (sex, age), clinical (organomegaly and leucocytosis), biological (genetic and genomic aberrations, immunophenotype) and therapy related variables. Aim of this retrospective analysis was to investigate the role of WT1 expression in risk stratification of de novo AML, its potentially added value to already established risk factors and its possible role for response evaluation. Results WT1 expression at diagnosis was determined in the bone marrow of all pts with a mean value of 9379 copies (range 1-83200 copies). There was no statistical significance in the difference of WT1 expression when pts were divided on the basis of demographic or clinical variables. Interestingly with increasing age of pts at diagnosis WT1 expression decreased significantly, for instance the patient group 19-30 years (yrs) showed higher copies than the 41-50 yrs- or the 51-60 yrs group, p=0.019 and p=0.004, respectively. Organomegaly or amount of blasts in peripheral blood didn’t show any correlation, whereas a high number of blasts in the bone marrow expressing AML specific antigens displayed significant correlation with high WT1 expression (p=0.024). There was no significant correlation between WT1 expression and cytogenetic abnormalities, whereas there was a significant correlation between WT1 expression and the presence or absence of FLT3. Patients with FLT3 mutations (n=38) had significantly higher WT1 expression levels than pts with FLT3 wild type (wt) (n=170) at baseline, 58709,4 vs 8710,3 copies (p=0.0008). Significant correlation was also obtained when subdividing the pts on the basis of NPM1 mutations. Patients presenting a NPM1 mutation showed higher WT1 expression than NPM1 wt pts (p=0.018). Even the combination of FLT3 and NPM1 yielded statistically significant correlation with WT1 expression. Patients with FLT3 and NPM1 mutations had higher WT1 copies than FLT3 and NPM1 wt pts (p=0.028). Bone marrow analysis of WT1 expression was repeated after induction chemotherapy in 133 pts (57%) with a mean value of 891 copies (range 0-23261 copies). The achievement of complete remission (CR) according to ELN criteria (3) in 110 pts was related to significant decrease of WT1 expression (p350 copies) after first induction chemotherapy showed reduced disease-free survival (DFS) compared to 89 pts in CR with low WT1 expression (