ALBERT

Description: Background Venous thrombosis (VT) frequently complicates the clinical course of cancer. Reported incidence of VT in many hematological neoplasms is up to 10%, a value comparable to that of solid tumors. Available data on the incidence and management of VT in Acute Leukemia (AL) are scanty and quite discordant. We have performed a multicenter retrospective study with the primary objective to evaluate the incidence of venous thrombotic complications in a population of patients with AL. Secondary objective was to evaluate the management of these complications in patients with AL. Materials and Methods Available clinical records of out and in-patients diagnosed with AL from January 2008 to June 2013 in 4 Regional Reference Hospitals were analyzed. Cases of venous thrombosis (VT), including thrombosis in atypical sites [Retinal occlusion (RO) and Cerebral Sinus Thrombosis (CST)], were reported in the current study. All data were recorded in a dedicated database. Available laboratory tests at diagnosis of VT included complete blood cells count (CBC), basal coagulation tests (PT, aPTT, fibrinogen), Antithrombin, anticoagulant Protein S and C and D-dimer. Instrumental Diagnosis of deep vein thrombosis (DVT), pulmonary embolism (PE) and RO and CST was performed according to ACCP guidelines. In the statistical analysis, logistic regression model was applied. Fisher’s exact test was used to determine relationships between categorical variables. All P-values represented were two-sided, and statistical significance was declared when P〈 0.05. Results Over a population of 831 patients with AL, 37 cases of VT were recorded, mainly (34/37 cases) in hospitalized patients: 24 cases were associated with Acute Myeloid Leukemia (AML) and 13 with Acute Lymphoblastic Leukemia (ALL). In the cohort of patients with VT, 23 were males (14 with AML, 9 with ALL) and 14 females (4 with AML, 10 with ALL), with a mean age of 46 ± 13,1 years; mean age of patients with AML and VT was 49 ± 12,8 years; mean age of patients with ALL and VT was 40,2 ± 12,2 years. Twelve patients presented at least a concomitant chronic disease; no one was receiving anticoagulant prophylactic treatment with low molecular weight heparin (LMWH) during hospitalization. There were 23 cases of DVT of upper arms, 9 cases of proximal DVT of limbs (one complicated with PE), 2 cases of RO, 1 of CST and 1 case of intracardiac clot. In 28/37 (75,6%) cases of recorded VT, a central venous catheter (CVC) was placed (Figure 1); moreover, 21/23 events of DVT of upper arms were significantly associated with a CVC insertion (p〈 0.01). In the other 2 cases, one patient had a bulky mediastinal disease and 1 was diagnosed with promyelocytic AML. VT occurred during chemotherapy (CHT) in 32/37 (86.4%) cases, the remaining 5 cases were diagnosed in concomitance with leukemia: in 20 cases, VT occurred at induction, in 7 at consolidation and in 5 during salvage CHT. In both subgroups with VT, there was no statistical significant difference between time at diagnosis of VT and time at diagnosis of AL. At CBC, thrombocytopenia was the most frequently observed laboratory abnormality. Basal coagulation tests and anticoagulant levels were normal in all cases. Inherited prothrombotic mutations were available only for 9/37 cases, 1 case was heterozygous for Factor V Leiden and 1 for Factor II (G20210A) mutation. Most VT episodes (32/37) were treated with LMWH at therapeutic doses for the first month after diagnosis, a dose reduction was recorded in the following months, mainly related to severe thrombocytopenia after CHT ; 1 case was treated with unfractioned heparin; four cases did not receive any treatment due to severe thrombocytopenia. No cases of VT–related deaths nor fatal complications during treatment for VT were recorded. Treatments with LMWH lasted from 3 to 6 months. All patients clinically recovered from VT, only 2 late recurrences (PEs) were observed. Conclusions The incidence (4.5%) of VT in the analyzed cohort of patients with AL is almost similar to only one previous report, even if the involved sites distribution appears quite different. In particular, RO has never been reported. Atypical sites VT must be suspected to be correctly diagnosed and treated. Anticoagulant treatment schedules and duration in patients with AL is influenced by many factors, mainly related to CHT and severe thrombocytopenia. The optimal management of VT in patients with AL requires further, prospective studies. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3097 Poster Board III-34 Backgound Adolescent and young (

Description: Abstract 1037 Background Among all the AML subtypes, APL has the distinction of being the most curable. The median age at diagnosis is 40 years, which is younger than with other AML subtypes. The fact that APL is more common in younger patients increases the likelihood that it may occur during fertile age. The introduction of ATRA and ATO has substantially modified the outcome of APL: in two successive studies 94% of patients achieved CR and the 6-yr OS rates (PETHEMA and GIMEMA) were about 80%. ATRA is highly effective in APL patients, but adverse effects such as retinoic acid syndrome, arrhythmias, headache, rash, dizziness have been reported. Moreover, retinoids are known to be teratogens and increased rates of spontaneous abortion and major fetal abnormalities have been reported. Most of the cases reported suggest that ATRA is relatively safe for both mother and fetus when used in the second and third trimesters. By contrast, when it was used in the first trimester, a negative foetal outcome was reported. No data have yet been reported on the outcomes of pregnancies in young patients with APL, occurring during CCR following ATRA-including chemotherapy regimens. Methods Herein we report 20 female patients who successfully conceived 21 healthy fetuses (two twins) and the outcomes in the patients and newborns. Clinical and demographic features of the 20 patients were as follows: median age at onset 25 yr. (range 18–35); according to the FAB classification 18 were M3 and 2 M3v, 8 at low, 10 intermediate and 2 high risk, with 13 bcr1, 1 bcr2 and 6 bcr3 PML/RARA fusion gene type. All the patients were treated according to the AIDA protocol, all achieved CR after induction and Complete Molecular Remission (CMR) after the 3rd consolidation cycle. Results Twenty successful pregnancies occurred, all in patients in CR (2 in second CR), off therapy for a median of 57 months (range 6–120). Nine pregnancies ended with spontaneous vaginal deliveries and 11 by cesarian section; resulting in the birth of 21 live, healthy newborns, without evidence of congenital anomalies. All 21 children showed normal development and growth during their respective follow-up periods; to date they are all in good health, median age 24 months (range 8–54). Conclusions The inclusion of ATRA in chemotherapy regimens for the treatment of APL had significantly improved the prognosis, changing the natural history of the disease, by increasing the incidence of CR, resistant disease being virtually absent, prolonging OS and DFS, and improving the quality of the CR, in terms of the hematologic and molecular response. The resulting prolongation of life expectancy allows these patients, mainly those off therapy, to resume their normal life style, including fulfilling a natural desire for children. The low median age at onset increases the probability of pregnancy post-chemotherapy. The long-term effects of ATRA-including regimens on fertility and on the newborn are not yet known. In our experience, pregnancy is feasible and does not pose additional risks. Disclosures: No relevant conflicts of interest to declare.

Description: Epigenetic silencing of tumor suppressor (TS) genes is a hallmark in human leukemias, particularly through DNA methylation. Cyclin-dependent kinase inhibitors (CKI) are, among other genes, frequently found methylated in their promoter region. This epigenetic modification has been described also in acute lymphoblastic leukemia (ALL). However, the relationship between aberrant DNA methylation and protein expression of TS genes has not yet been extensively evaluated in adult ALL series. The aim of this study was to analyze in primary cells from newly diagnosed adult ALL patients, uniformly treated according to the LAL2000 GIMEMA protocol, the promoter methylation status of p73, p21, p15 and p16, evaluating in addition the p21, p15 and p16 protein expression. The DNA methylation status of promoter regions was investigated, according to cell availability, using a widely accepted method based on bisulfite modification of DNA, followed by methylation-specific PCR assay (MSP). Protein expression was evaluated by Western blot. Normal peripheral blood lymphocytes, as already described, resulted unmethylated for p73, p21, p15 and p16, and did not express the p21, p15 and p16 proteins. In ALL patients, in contrast, only the p21 promoter region was found constantly unmethylated. The p15, p16 and p73 promoter genes were found methylated in 15/37 (40.5%), 8/43 (18.6%) and 9/36 (25%) patients, respectively. Only 2/23 cases (8.6%) resulted simultaneously methylated for p15, p16 and p73. The p21 and p15 protein expression was found in 28/85 (32.9%) and 44/85 cases (51.8%), respectively. The p16 protein, in contrast, was never expressed. The p16 methylation was associated with the T-ALL (P=0.005) phenotype and with higher white blood cell (WBC) counts (P=0.027). Resistance to spontaneous induction of apoptosis was significantly associated with p21 protein expression (P=0.019) and its co-expression with p15 (P=0.049). Achievement of CR was not influenced by gene methylation status, nor by single protein expression. Interestingly, the co-expression of p15 and p21 was associated with failure to induction treatment: only 6/63 (9.5%) patients co-expressing p15 and p21 obtained a CR (P=0.027). Multivariate analysis confirmed the unfavorable role of this protein co-expression (P=0.059) on CR achievement. In contrast, once patients achieved remission, p21 protein expression was associated with a prolonged DFS, as confirmed by multivariate analysis for DFS (P=0.039). In conclusion, p15 and p21 protein expression plays an unfavorable prognostic role in adult ALL patients independently of the p73, p21, p15 and p16 gene promoter methylation status.

Description: In 2001, the GIMEMA Group started a phase II study to evaluate Imatinib both in adult (study A, 18–60 years) and elderly (Study B, 〉61 years) patients with Philadelphia positive (Ph+) acute lymphoblastic leukemia (ALL). In study A, adult patients with Ph+ ALL received the standard GIMEMA induction (VCR, DNR, PDN, L-ASP) and consolidation (MTZ and high-dose Ara-C) treatments, without imatinib. All patients in hematological complete remission (HCR) after the consolidation, either with or withouth a donor, received imatinib p.o. at the dosage of 800 mg/day for 6 months. After completing the 6-months therapy, in the absence of safety concerns, if the investigator considered the patient eligible for treatment, the therapy with imatinib was continued. A total of 22 patients have been enrolled [7 in molecular complete remission (MCR) and 15 still BCR/ABL+]: 18 are still in HCR after a median follow up of 20 months (2 – 26). In particular, all the 7 patients PCR- when started imatinib are still in HCR (6 PCR- and 1 becoming PCR+), after a median follow-up of 19 months (7.6 – 24) and eleven of the 15 PCR+ patients are in CHR after a median follow-up of 19 months (1.2 – 26). Of the remaining 4 PCR+ patients, 1 died in CR after an alloSCT and 3 patients relapsed at 4, 9 and 11 months, respectively. The probability of disease-free survival at 2 years is 79% (C.I. 95%: 58,4 – 100,3). Figure Figure In study B, planned for elderly patients usually not eligible for intensive treatments, the induction therapy consisted of imatinib 800 mg/day p.o. associated to prednisone 40 mg/sqm/day for 30 days, withouth any other chemotherapy. Up to date only 12 patients have been enrolled. Median age was 67.5 yrs (61–78), 9 were females. Eleven (92%) obtained a CR with the prednisone-Imatinib association induction treatment, 1 patient discontinued the treatment for toxicity. The post-remissional treatment mainly consisted of imatinib alone: 8 patients are still in CR, after a median follow up of 7 months (4 – 15), 2 relapsed at 4 and 4 months and 1 died in CR due to a 2nd neoplasia. In conclusion, these studies, planned 3 years ago to verify the feasibility and toxicity of imatinib in adult and elderly Ph+ ALL patients, clearly suggest a strong activity of Imatinib also in this disease, not only giving the possibility of inducing a CR as monotherapy, but even of maintaining long-lasting hematological and molecular CR withouth allogeneic stem cell transplantation. Further studies are mandatory to finally identify the best way to integrate imatinib in the entire treatment strategy of this disease.

Description: In the GIMEMA LAL 0496 trial, which has been conducted in Italy between 1996 and 2000, patients with adult ALL received an induction therapy with VCR, DNR and ASP until they reached complete remission (CR). Consolidation therapy with Ara-C and CTX was then administered and, if still in CR, patients received a 3-year maintenance therapy. Ph+ ALL patients were considered at high risk and after the induction phase a stem cell transplant (SCT) was planned, allogeneic for patients with an HLA identical sibling or autologous. A total of 498 patients have been enrolled by 50 GIMEMA Centers. Of these, 29 patients (5.8%) with either t(4;11) or t(1;19), who received the standard post-remissional approach, showed a statistically significant worse prognosis after CR: the projected probabilities of DFS and OS at 2 years were 19% (C.I. 95%: 4.1–34.4) and 31% (C.I. 95%: 14.2–47.9), respectively. Thus, in the new protocol - GIMEMA LAL 2000 - these patients received an intensive post-remissional treatment including SCT. Between 2000 and 2003, a total of 383 patients have been enrolled in this new protocol. Of these, 23 (6.0%) were positive either for t(4;11) or for t(1;19). The clinical characteristics of these patients are comparable to those of the patients enrolled in the previous study and the overall CR rate is also super imposable. GIMEMA LAL 0496 vs GIMEMA LAL 2000 Fig. 1 shows the comparison in DFS at 2 years between LAL 0496 and LAL 2000; though not statistically significant, a trend towards a better prognosis is clearly seen. The DFS probability projected at 2 years is 52% (95% C.I. 26–78) compared to 19% (95% C.I. 4–35). The OS, projected at 2 years, is 42% (95% C.I. 14–70) compared to 31% (C.I. 95%: 14.2–47.9). Despite the relatively limited case series, these results suggest that for t(4;11)+ and t(1;19)+ adult ALL intensification of the post-remissional treatment with SCT is a feasible approach that may improve the outcome in this subset of patients at high risk of relapse with standard post-remissional therapies. Figure Figure GIMEMA LAL 0496 GIMEMA LAL 2000 p Patients t(4;11) or t(1;19) 29 23 0.7626 Sex (M/F) 14/15 8/15 0.3280 Age yrs, median (min-max) 37.8 (14.9–59.4) 39.4 (19.6–59.2) 0.1481 WBC x 109/L median (min-max) 41.2 (2.2–663.0) 50.3 (2.4–300.0) 0.9193 Phenotype (B/T) 29/0 23/0 - CR rate 92.8% 85.7% 0.6392

Description: Abstract 4848 Objectives: the prognosis of patients with cytogenetically normal acute myeloid leukemia (CN-AML) is highly variable and can be influenced by several clinical and biological variables. Nevertheless, some biological data may be conflicting and difficult to combine with the clinical ones. Methods: in order to propose a simple scoring system, we retrospectively analysed the clinical data of 337 patients newly diagnosed with CN-AMLs, aged less than 65 years, consecutively treated in eleven hematological Italian Centres from 1990 to 2005. Two hundred nineteen patients (65%) received a fludarabine-based induction regimen. All the other patients received a conventional induction regimen, including cytarabine, one anthracycline with or without etoposide. Univariate and multivariate analysis on event free survival and overall survival (EFS and OS) were performed. Patients addressed to allogeneic stem cell transplantation were censored at the time of transplant. Factors found to be significant in univariate analysis were tested in multivariate analysis. A numerical score was derived from the regression coefficients of each independent prognostic variable. The Prognostic Index Score (PIS) for each patient was then calculated by totalling up the score of each independent variable. Patients could thus be stratified into low-risk (score = 0–1), intermediate-risk (score = 2) and high-risk group (score grater than 3). The score obtained in this group of patients (training set) was then tested on 193 patients with newly diagnosed with CN-AMLs, aged less than 65 years, enrolled in the GIMEMA LAM99p clinical trial (validation set). Results: the clinical variables that were independent prognostic factors on EFS in the training set of patients were: age 〉 50 yrs (regression coefficient: 0.39, HR 1.5, score = 1), secondary AML (regression coefficient: 0.90, HR 2.5, score = 2) and WBC 〉 20 × 10^9/L (regression coefficient: 0.83, HR 2.3, score = 2). For what concerns the OS, the same variables showed the followings statistical data: age 〉 50 yrs (regression coefficient: 0.48, HR 1.6, score = 1), secondary AML (regression coefficient: 0.99, HR 2.7, score = 2) and WBC 〉 20 × 10^ 9/L (regression coefficient: 0.87, HR 2.4, score = 2). In the training set of patients, the median EFS was 22, 12 and 8 months in the low, intermediate and high-risk group (p

Description: Abstract 3327 Background: Critically ill patients are at high risk of developing venous thromboembolism (VTE) during their stay in the intensive care unit (ICU) because of premorbid medical and surgical conditions. The clinical consequences of Deep Vein Thrombosis (DVT) have the potential to be serious yet are frequently unrecognized in the Intensive Care Unit (ICU). In contrast to the extensive documentation on the short and long–term outcomes of patients with DVT evaluated in other clinical settings, little is known about the clinical course of this disease in the ICU setting. We hypothesized that both undetected and clinically evident VTE would affect the prognosis of critically ill patients. Purpose: To systematically review whether a diagnosis of DVT in critically ill patients affects clinically important outcomes including length of stay, duration of mechanical ventilation and mortality. Material and Methods: MEDLINE and EMBASE databases were searched up to June 2010. Two reviewers performed study selection independently. Studies were selected if evaluate one or more of the following outcomes: hospital and ICU mortality, duration of patient stay in hospital and in ICU, and duration of mechanical ventilation. Two investigators independently extracted and reviewed data from each study; including study and patient characteristics and outcomes. Association between DVT and hospital and ICU mortality, and the mean difference of duration of patient stay in hospital and in ICU, and duration of mechanical ventilation in patients with and without DVT were calculated using a random-effects model (DerSimionan and Laird method). Pooled results are reported as relative risk (RR) and mean difference and are presented with 95% confidence interval (CI) and with 2-sided P values. A P value of .05 or less was considered statistically significant. Statistical heterogeneity was evaluated using the I2 statistic, which assesses the appropriateness of pooling the individual study results [22]. The I2 value provides an estimate of the amount of variance across studies due to heterogeneity rather than chance. Cohen's Kappa for inter-rater agreement was used to assess inter-rater reliability. Results: Six studies for a total of 1518 patients were included in the systematic review. Patients diagnosed with DVT compared to those without DVT had increased ICU and hospital stay (7.3 days (95% CI 1.4 to 13.2; P= 0.02) and 16.5 days (95% CI 1.51 to 30.59; P= 0.03), respectively. Duration of mechanical ventilation appeared to be increased in patients with DVT although this difference was not statistically significant (weighted mean difference: 3.41 days 95 % CI –1.12 to 7.94; P=0.14). Patients diagnosed with DVT also had a marginally significant increase in the RR of hospital mortality (RR 1.31 95%CI,0.99 to 1.74,P=0.06), and a non statistically significant increase in the RR of ICU mortality (RR 1.96; 95% CI 0.74 to 5.19; P = 0.17). Conclusions: A diagnosis of DVT upon ICU admission appears to affect clinically important outcomes including length of ICU and hospital stay and hospital mortality. Further research involving larger prospective study designs are warranted. Disclosures: No relevant conflicts of interest to declare.

Description: Cyclin-dependent kinase inhibitors (CKI) regulate cell division resulting aberrantly expressed in many types of cancer. Alterations of CKI have been reported in acute leukemia, as the result of gene promoter methylation. Despite the common frequency of these alterations, little has been reported on the role of CKI aberrant protein expression and results are less clear, especially in acute lymphoblastic leukemia (ALL). The aim of this study was to analyze p21, p15 and p16 protein expression and their gene methylation status in primary cells from adult ALL cases enrolled in the LAL2000 GIMEMA protocol. Normal peripheral blood lymphocytes (PBL) and 91 primary samples from untreated ALL patients were evaluated in this study. The p21, p15 and p16 protein expression was analyzed by Western blot using the specifically MoAbs. The CKI gene methylation status was investigated using a widely accepted method based on bisulfite modification of DNA, followed by the use of the methylation-specific PCR assay (MSP). This assay was further validated in vitro by SSI methylase. Normal PBL from 10 healthy donors, as described, did not expressed all CKIs and resulted unmethylated. The p21 expression was found in 28/91 cases (30.8%); in contrast, samples were found constantly unmethylated. The p15 expression was found in 44/85 cases (51.8%) and its gene methylated in 41.7%; a significant correlation was found between absence of protein expression and gene methylation (P=0.040). The p16 resulted never expressed in adult ALL, while its promoter was found methylated in 8/42 cases (19.1%). A significant association (P=0.037) was observed between p21 expression and immunophenotype; in fact, 3/24 (12.5%) T-ALL and 24/65 (36.9%) B-lineage ALL expressed this protein. The p16 methylation was associated with T-ALL (P=0.082). Achievement of CR was not influenced by single protein expression, nor by gene methylation status. However, the co-expression of p15 and p21 was associated with failure to induction treatment; in fact, only 6/67 (9%) of patients co-expressing p15 and p21 achieved CR (P=0.021). In summary, in adult ALL p21 is not methylated and p16 is never found expressed, and CR achievement is adversely affected by the co-expression of p21 and p15. In conclusion, we report that in addition to CKI methylation, aberrant expression of CKI, namely p21 and p15, is associated with poor outcome in adult ALL, suggesting that chemotherapy resistance may be promoted in these cases by cell cycle arrest and/or abnormal survival.

Description: The GSI 103 AMLE was a randomized phase III clinical trial to explore the efficacy of DaunoXome (DNX) versus Daunorubicin (DNR) standard treatment in AML elderly patients. From 10/2001 to 2/2004, 301 patients aged 61 to 75 years with a diagnosis of AML according to WHO classification and eligible for intensive chemotherapy were enrolled in this trial by 39 GIMEMA Centers. Patients were randomized to receive induction treatment with DNR (45 mg/sqm day 1 to 3) or DNX (80 mg/sqm day 1 to 3) plus AraC (100 mg/sqm day 1 to 7 by continuous infusion): as consolidation therapy, patients in CR received a further course of the assigned induction treatment. After the consolidation course, patients in CR underwent a 2nd randomization independently from the 1st random, to receive as maintenance therapy either AraC (20 mg twice daily day 1 to 10) plus all-trans retinoic acid (ATRA) (45 mg/sqm day 1 to 10) every 28 days for a maximum of 12 cycles (Arm A) or no further treatment (Arm B). 153 patients were enrolled in the DNR arm and 148 in the DNX arm: the two arms were comparable as to relevant patient characteristics, including karyotype. In the DNR arm, 77 patients (50.3%) achieved CR, 56 (36.6%) were resistant and 20 (13.1%) died during induction (ID). In the DNX arm, 73 patients (49.3%) achieved CR, 47 (31.8%) were resistant and 28 (18.9%) died during induction. Differences between the two arms at univariate analysis were not statistically significant either considering the 3 different types of response or considering ID versus other (Chi-square 0.34 and 0.17 respectively). After CR, the effect of DNX seems to be time-related, due to higher incidence of early deaths in CR (11% vs 3%, p=0.053) and lower incidence of relapse beyond 6 months (at 24 months 60% vs 80%, p=0.064). This translates in a cross of the OS and DFS curves between the two arms, with initial advantage for the DNR arm followed by an advantage for DNX arm after 12 months from diagnosis. A time-dependent covariate Cox model and a landmark analysis on patients in CR after 6 months revealed a better outcome of DNX patients (p= 0.029 and p=0.092, respectively) in the long term DFS follow-up. No difference has been observed in the DFS curves according to maintenance randomization. In conclusion, DNX is as effective as DNR in the induction phase of AML elderly patients, with a trend of increased early toxicity (ID + deaths in CR): however, DNX seems to improve the OS and DFS in the long-term follow-up of these patients, due to a reduction of late relapses. Further trials in more selected elderly patients or in younger patients will be helpful to define the exact role of DNX in AML.