ALBERT

Description: Abstract 3350 Introduction: In the absence of serum hepatitis B (HB) surface antigen (HBsAg) and clinical evidence of HB, an occult HB virus (HBV) infection (OBI) is defined as the existence of HBV DNA in the serum, in the peripheral blood mononuclear cells (PBMC), and/or in the liver. Most frequently, OBI follows the resolution of acute hepatitis and continues indefinitely after HBsAg clearance and liver function improvement. Usually, this form of infection, termed secondary OBI (sOBI), is associated with the presence of serum anti-HB core antibodies (anti-HBc). Up to 20% of individuals with OBI do not react for either anti-HBc or any other serological indicator of HBV exposure. Consistent with the woodchuck model, this form of infection, termed primary OBI (pOBI), is characterized by the presence of DNA in both serum and PBMC, but not in the liver. Although reactivation of sOBI has been reported in a high proportion of hemopoietic stem cell transplant (HSCT) recipients, pOBI reactivation risk has not yet been fully investigated in the transplant setting. We followed up 9 long term surviving HSCT recipients with pOBI, in order to define its clinical significance in immune suppressed patients. Patients and Methods: A total of 75 consecutive patients with hematological disease, receiving autologous or allogeneic HSCT between April 2006 and March 2007, were included in a database and retrospectively examined. As a prerequisite for inclusion in the study, each of the following was needed: (i) negative serological indicators of HBV exposure and absence of clinical signs/symptoms of hepatitis at the time of HSCT, (ii) positive HBV DNA, as detected by polymerase chain reaction (PCR), in both serum and PBMC samples drawn within 6 months before transplant, and (iii) availability of stocked frozen serum/PBMC samples collected after HSCT from each patient. Following these criteria, 9 patients (7 males, 2 females) were selected for the analysis. Five patients (1 Hodgkin disease, 3 non-Hodgkin lymphomas, and 1 B-cell chronic lymphocytic leukemia) received autologous HSCT, and 4 patients (1 acute myeloid leukemia, 1 multiple myeloma, 1 myelodysplastic syndrome, and 1 acute lymphoblastic leukemia) received allogeneic HSCT. Median age at transplant was 49 (range: 33–63) years. No patient received lamivudine prophylaxis. Serum and PBMC samples collected after HSCT were analyzed for HBV serology and DNA detection. The latter was performed by in-house PCR amplification by nested primers (detection limit 50 copies/ml) from highly conserved S region encompassing a-determinant of HBV. Direct sequencing of all HBV DNA amplified products was used to confirm the specificity of the reaction. Results: In 8 cases, one serum/PBMC sample per patient was available, at a median of 27 (range: 21–34) months after HSCT; in 1 case, three serum/PBMC samples were available, at 17, 22, and 28 months after HSCT, respectively. During follow-up, positive HBV DNA was found, both in the serum and in the PBMC. All patients were confirmed to be infected with HBV subgenotype D1, with mutations known to impair HBsAg antigenicity. Three patients were confirmed to be further infected with HBV subgenotype A2. So far, 41 to 52 months after transplant (median: 46 months), neither patients showed seroconversion nor developed clinical hepatitis, suggesting pOBI persistence. Conclusions: Although the virus persisting in pOBI remains biologically competent, it is likely neither to reach liver-pathogenic doses nor to induce “classical” HBV reactivation during PBMC-restricted replication. However, prospective studies with large sample size and long-term follow-up are warranted, to define the actual clinical relevance of HBV occult infection. Indeed, OBI with low viremia could induce DNA damage, cellular demise, immune hypo-responsiveness, and oxidative stress in peripheral blood lymphocytes. Disclosures: No relevant conflicts of interest to declare.

Description: Conventional cytogenetics and molecular analyses allow to stratify acute myeloid leukemia (AML) patients into subgroups with different clinical and prognostic relevance. AMLs with unsuccessful cytogenetics and no known recurrent mutations are a subgroup of cases for which information on possible underlying genetic lesions of the leukemic cells is lacking and with a poorly defined outcome. Previous studies have quantified the rate of unsuccessful karyotyping in approximately 10% of the analyzed AML samples and it is conceivable that if cases with molecular rearrangements were to be included this figure could be lower. With the aim of investigating the prevalence and impact of cases with an undefined genetic profile (UGP), we studied 437 AML patients - 228 males and 209 females, with a median age of 50 years (range 1-81) - treated on successive intensive chemotherapy protocols at our Institution. Conventional cytogenetic and molecular analyses - RUNX1-RUNX1T1, CBFB-MY11, DEK-NUP214, FLT3-ITD, NPM1, BCR-ABL, MLL-PTD - were performed at diagnosis. According to the genetic alterations, patients were comprehensively stratified into three subgroups: a favorable risk group - t(8;21) RUNX1-RUNX1T1, inv(16) CBFB-MY11, normal karyotype with mutated NPM1 without FLT3-ITD - with a 5-year overall survival (OS) of 65%, an intermediate risk group - normal karyotype with mutated or wild type NPM1 and FLT3 ITD or wild-type NPM1 without FLT3-ITD, t(9;11)(p22;q23), cytogenetic abnormalities not classified as favorable or adverse - with a 5-year OS of 27% and an unfavorable risk group - inv(3)(q21q26) or t(3;3)(q21;q26), t(6;9)(p23;q34), DEK-NUP214, t(v;11)(v;q23), -5 or del(5q), -7, complex karyotype - with a 5-year OS of 11%. Thirty-three patients (7.5%) were identified as having an UGP and their baseline characteristics, as well as clinical outcome, were compared to those of patients with defined molecular and cytogenetic features. Patients with an UGP were older at the onset of the disease than those with a delineated genetic profile (median 55 vs 49 years). In addition, the proportion of UGP cases increased with age, being 3% in patients 50 years. The complete remission (CR) rate for UGP patients (69.6%) was similar to that of intermediate risk patients (71.1%), but inferior to that of patients with a favorable risk profile (90.5%) (p=0.0046) and better than that of unfavorable genetic risk patients (63.7%). After adjusting for age, gender, WBC and platelet count, Hb, marrow blast percentage at diagnosis and treatment, UGP remained an independent factor for lower CR rate with respect to patients with a favorable genetic risk profile. The frequency of relapse was significantly higher in patients with UGP compared to the favorable risk group (60.8% vs 32%) (p=0.011). In multivariate analysis, the 5-year OS of patients with UGP was significantly worse than that of patients with a favorable genetic risk profile (p

Description: AML patients early relapsing within 12 months, still represents an extremely poor prognosis setting. Cytogenetic and molecular data are not diagnostic in 20-25% of AML patients and intermediate 2 ELN risk category has still an undefined prognosis. Post induction and consolidation MRD might represent a new prognostic factor besides patients and disease characteristics. We have evaluated post induction and consolidation bone marrow mimimal residual disease (MRD) in 126 AML patients (median age: 61.5 years, range: 17-89) with 20 months median follow-up (range 2-79.9). We analysed abnormal leukemia immunophenotype (ALIP) by multiparameter flow cytometry (MPFC) and WT1 by RT-PCR as described by Buccisano et al and Cilloni et al. Cytogenetic, NPM and FLT3 status were performed in 111, 97 and 119 patients respectively, defining the molecular cytogenetic risk in 96 patients. WT1 was +ve in 91/114 patients (70%) at diagnosis (median 1,231.5; range: 2-268,784), in 11/71 (15.5%) post induction (median 1216; range:260-134,633) and in 8/66 (12,1%) post consolidation (median 627.8; range:258-45,338). MPFC MRD was +ve in 33/66 (50%) patients after induction and in 18/48 (37.5%) after consolidation. We analysed 12 month Cumulative Incidence of Relapse (CIR) adjusted by MRD status, patients and disease characteristics. 82/99 patients achieved CR, 40 relapsed in a median of 8 months (1-52 months) 29 within 12 months with 37.5% 1 yr CIR. Patients receiving chemotherapy (38), Autologous (14) and Allogeneic Transplant (39) as post consolidation treatment had 45%, 35% and 30% 1 yr CIR respectively. NPM+FLT3- patients had 25% 1 yr CIR, compared to 25% in NPM-FLT3-, 50% in NPM+FLT3+ and 47% in NPM-FLT3+. Patients with WT1 positive post induction and consolidation had 1 year CIR of 90% and 72.5% respectively. Patients with MPFC positive post induction and consolidation had a 1 year CIR of 48.6% and 44.5% respectively. Multivariate analysis identified post induction WT1 positive status as the main predictor of 1 year CIR. Patients with WT1 positive after induction had a a 15.8 RR of 1 year CIR(p50,000/ml at diagnosis) and age〉60 yrs also significantly predicted 1 year CIR with a RR of 7.22 (p=0.002) and 9.1 (p=0.001) respectively. In conclusion WT1 post induction status confirmed its prognostic significance in our series targeting a subset of early relapsing patients with an extremely poor outcome deserving experimental approaches. Disclosures No relevant conflicts of interest to declare.

Description: Deletion of the chromosome 20 long arm (del20q) has been reported in 3-7% of patients with Myelodysplastic Syndromes (MDS). In particular, isolated del20q seems to be associated with good prognosis, low risk of progression to AML and prolonged survival: however, very few reports addressed this subset of MDS patients up to now. To highlight this issue, we collected data from all patients with MDS and isolated del20q diagnosed and followed by Centers of the Gruppo Romano-Laziale Sindromi Mielodisplastiche (GROM-L). On the whole, 53 patients were analysed: the main features at diagnosis of these patients are reported in the Table. Platelet (PLT) count was 〈 100 x 109/l in 28 patients (52.8%), Hb level was 〈 10.0 g/dl in 23 patients (43.3%) and neutrophil count was 〈 1.0 x 109/l in 12 patients (22.6%). As to risk prognostication, according to IPSS score 51 patients (96.2%) had low/intermediate-1 risk and 2 (3.8%) had intermediate-2/high risk: according to r-IPSS, 8 patients (15.1%) had very low risk, 20 (37.7%) low risk, 19 (35.9%) intermediate risk and 6 (11.3%) high risk. During follow-up, 14 patients (26.4%) did not need any therapy and were only observed, 36 (67.9%) were treated with Erythropoietin (EPO), 1 (1.9%) with hypomethylating agents, 2 (3.8%) with other treatments (TNF-α inhibitor, interferon). Among the 36 patients treated with ESA after a median interval from diagnosis of 5.0 months [interquartile range (IR) 1.2 - 27.6], 24 (66.6%) received α-EPO, 11 (30.5%) β-EPO and 1 (2.9%) darbopoietin. Two patients were too early (〈 3 months of treatment) for response evaluation: among the 34 evaluable patients, 21 (61.7%) achieved stable erythroid response according IWG criteria (Hb increase 〉 1.5 g/dl in 18 patients and reduction 〉 50% of basal transfusion requirement in 3 patients) while 13 (38.2%) were resistant to EPO. Nine patients (17%) progressed to Acute Myeloid Leukemia (AML) after a median time from diagnosis of 16.8 months (IR 4.1 - 51.7). At the last follow-up, 21 patients (39.6%) died (13 from MDS/AML related causes and 8 from unrelated causes), 12 (22.6%) were lost to follow-up and 20(37.8%) were alive. Median Overall Survival (OS) of the entire cohort was 61.6 months (95%CI 42.2 - 81.0): the 10-year cumulative OS was 38.6% (95%CI 18.6 - 58.6) (Figure 1). In conclusion, as MDS represent a heterogeneous group of pathologies, many efforts are ongoing to identify patients with similar biological, clinical and prognostic features (eg 5q- syndrome, MDS with ring sideroblasts). From the scarce data in the literature and from our results, it is reasonable that also patients with isolated del20q may represent a distinct clinical and biological entity, with specific clinical and prognostic features (low PLT count, low number of marrow blasts, low IPSS and r-IPSS risk score, good response to EPO, long OS). The mechanisms underlying del20q are still unclear and warrant future molecular analysis. Disclosures Breccia: Novartis: Honoraria; BMS: Honoraria; Pfizer: Honoraria; Incyte: Honoraria.

Description: The CD15 antigen is an adhesion molecule normally expressed on neutrophils that mediates phagocytosis and chemiotaxis: it is also expressed on blasts of patients with acute myeloid leukemia (AML). Its prognostic role has been tested in different studies, including or not acute promyelocytic leukemia (APL), with conflicting results and its significance still remains unclear. To address this issue, a cohort of 460 AML patients of all ages with, the exclusion of APL, [M/F 243/217, median age 50.6 years (range 0.9 – 81.2)] intensively treated at our Institute between 1/1999 and 12/2010 was retrospectively evaluated. Overall, 61 patients (13.3%) evolved from a documented myelodysplastic phase (MDS): AML-ETO, CBFβ-MYH11, FLT3-ITD and NPM were positive in 35/438 (8.2%), 30/427 (7.0%), 55/409 (13.4%) and 67/200 (14.6%) evaluable patients, respectively. A favorable karyotype was found in 90/436 patients (20.6%) while an unfavorable profile was documented in 60/436 cases (13.8%). CD15 positivity was found in 171/406 evaluable patients (42.1%): in particular, CD15 was positive in 13/42 evaluable patients evolved from MDS (31.0%) compared with 158/364 evaluable patients without previous MDS (43.4%) (p=0.123). Induction treatments consisted of anthracycline (ACY) + cytarabine (Ara-C) +/- etoposide in 448 patients and of a fludarabine-based regimen in 12 patients. A complete remission (CR) was achieved by 334 patients (72.6%), while 82 (17.8%) were resistant and 44 (9.6%) died during induction: the median CR duration was 15.5 months (range 0.6 – 176.0), with a 2-year disease-free survival (DFS) rate of 45.1% (95% CI 39.6 – 50.6). The median overall survival (OS) was 14.4 months (range 0.3 – 177.0), with a 2-year OS rate of 42.2% (95% CI 37.5 – 46.9). Among the several variables tested at univariate analysis for CR achievement, age

Description: Conflicting results have been reported regarding the correlation between CD133, a surface marker of immature progenitors, and outcome in acute myeloid leukemia (AML). The expression of this antigen has also been reported in myelodysplastic syndromes (MDS), in particular in high-risk diseases, but always in small cohorts of patients and without a focus on the prognostic role of this antigen. Aim of our study was to establish a clinico-biologic correlation between CD133 expression and disease features at baseline in a large series of AML patients of different ages, with particular regard to the older age.Seven hundred AML patients consecutively diagnosed at a single Institution were retrospectively analyzed and enrolled in this study. There were 395 males and 305 females, with a median age of 54 years (range 1.1-90.4). A previous MDS phase was recognized in 124 patients. Several clinical and biologic features were recorded at baseline and retrospectively collected, such as age, gender, FAB and WHO morphologic classification, cytogenetic analysis, molecular alterations, hematologic parameters (Hb, platelet and WBC count), response to treatment and outcome. Overall, 157 patients expressed CD133. This first analysis was carried out on the older patient population (≥65 years) on the basis of the CD117 positivity. Seventy-three older patients expressed CD133 at baseline, whereas 36 patients were CD117+CD133-. Comparison between the two groups showed a significant prevalence of a previously recognized MDS phase in CD133+ patients (27% vs 13%, p=0.01), higher incidence of a complex karyotype or typical MDS cytogenetic aberrations (trisomy 8, del20q, del5q) (30% vs 8%, p=0.001) and of dysplastic morphologic features detectable in patients without a previous dysplastic identification (63% vs 27%, p=0.002). Forty-three patients in the CD133+ group and 21 patients in the CD133- group received intensive chemotherapy: the remission rate was 52% and 64%, respectively (p=0.06). The relapse rate was 25.5% in the CD133+ and 19% in the CD133- group, respectively (p=0.08). No differences were observed with regard to the hematologic parameters at baseline or in overall survival between the two groups. We then assessed the characteristics of cases negative for CD117, but CD133+ (13 patients) that were compared to the entire cohort of cases that were CD117+CD133+ (144 patients): again we found that, independently from the positivity for CD117, CD133 identified patients with a previously reported MDS phase (61% of patients CD117-CD133+), with a higher median age (69 years) and with dysplastic morphologic changes (100% CD117-CD133+). Taken together, our findings strongly suggest that CD133 can identify at diagnosis a previous MDS phase. In particular, the presence of this antigen in the setting of older de novo AML patients should be used to recognize early a subset of patients who, for the associated biologic features, could benefit from the use of hypomethylating agents as first line treatment. Further analyses, aimed at identifying the prognostic role of this antigen in a large cohort of patients treated with azacitidine, are warranted. Disclosures No relevant conflicts of interest to declare.

Description: Different biologic features have been associated with a more or less aggressive clinical course in chronic lymphocytic leukemia (CLL). In the present study, 20 patients with highly stable CLL observed at a single institution over a period of 10 to 23 years and who never required treatment were extensively characterized. The aim was to identify a distinct and reproducible biologic profile associated with disease stability that may be used to recognize at presentation CLL patients who are likely to have a very benign clinical course and for whom treatment is not indicated. The results obtained indicate that numerous parameters are closely associated with disease stability: a typical CLL morphology and immunophenotype, the lack of expression of the CD38 antigen, the mutated immunoglobulin (Ig) heavy (H) chain variable (V) pattern, the absence of p53 mutations, a CD4/CD8 ratio more than 1, the lack of 17p and 11q deletions and of complex karyotypic aberrations, and the occurrence of the 13q14 deletion. No case displayed the VH3-21 gene, linked in mutated CLL with a poor outcome. In addition, the VH1-69 gene associated with unmutated CLL cases was never detected. These biologic features were coupled with an indolent clinical course characterized by an unmodified clinical stage over time, and by lack of autoimmune phenomena and of major infections requiring parental antibiotics. At a time when aggressive therapeutic strategies are always more frequently used in the management of CLL, the distinctive features of patients with long-lived stable disease should be prospectively identified at presentation.

Description: Background Acute promyelocytic leukemia (APL) in the elderly has a favourable outcome in a significant proportion of patients (pts). Elderly and frail pts are usually treated with attenuated treatment schedules, mostly "according to medical judgment". The combination of all-trans retinoic (ATRA) and anthracycline-based chemotherapy (CT) has been the mainstay of treatment for many years. Alternative approaches, such as arsenic trioxide (ATO) and gentuzumab ozogamicin (GO) have recently been tested with success in this setting, even though no large series of elderly pts treated with CT-free first-line treatment have been published yet. Moreover, neither a "standard of care approach" nor a specific prognostic score system have been developed to guide physician in choosing the most adequate treatment schedule in this setting. Objective Aim of the present preliminary survey was to assess genetic and clinical features of APL in pts over 60 yrs. This cut off reflects the definition of "elderly patient" in most Italian APL GIMEMA trials. Moreover, both the real life outcome and safety data after either conventional anthracycline-based CT or alternative CT-free approaches were analysed. OS, response rate to either regimens and adverse event occurrence were collected. Methods This retrospective multicenter REL (Rete Ematologica Lombarda) survey, enrolled a total of 101 consecutive APL pts aged ≥60, treated between 2000-2018. Demographics, clinical data and therapy outcome data were recorded in a dedicated patient's report form. Statistical analysis was performed using the Kaplan Maier method, Log-rank test and Cox regression. Results For analysis, pts were grouped into different categories according to age and fitness. Tables 1 and 2 summarise clinical charcteristics and treatment administered. Performance status (PS) and number of comorbidities increase with age. Twentynine of 102 (28,4%) and 9/102 (8,8%) pts had a history of or subsequently developed a solid tumor respectively. High frequency of additive cytogenetic abnormalities was observed as well. CT+ATRA was preferentially administered, mostly with attenuated intensity, to younger pts with better PS, while ATO+ATRA was preferred in pts with reduced cardiac function and ATRA monotherapy was reserved to frail or over 80 yrs old pts. Rates of differentiation syndrome or infections or cardiac events were similar in the different treatment groups. Overall, complete remission (CR) rate after induction therapy was 94.16% [CI95%: 87,5%-97,8% ]. At a median follow-up of 40 mos (range 0-199), the overall relapse rate was 24%. Median time to relapse was 7 mos in 1/8 (12,5%) ATRA monotherapy treated pts and 13 mos (range 5-66) in the 23/68 (33,8%) pts receiving CT+ATRA. No relapse occurred among the 22 pts treated with ATO+ATRA (p 0.005). OS and EFS were significantly associated with pts' age (HR 9% and 7.6%; p

Description: The authors treated a total of 82 patients with Ph′-positive chronic myelogenous leukemia (CML) with recombinant interferon alpha-2b (IFN alpha-2b). Sixty-five patients in chronic phase (CP), 28 of whom were untreated and 37 pretreated, and nine patients in accelerated phase (AP), were started on IFN three times a week. Patients in CP were randomized to receive 2 or 5 X 10(6) IU/m2, while patients in AP were all given the dose of 5 X 10(6) IU/m2, in addition to concomitant chemotherapy. Patients in CP who were unresponsive to the lower dose were crossed to the higher dose. Of 63 evaluable patients in CP, 43 (68%) responded, 29 (46%) achieved complete hematologic response (CHR), and 14 (22%) achieved partial hematologic response (PHR). The response rate appeared to be significantly influenced by the IFN dose in pretreated patients. Of the nine patients in AP, two attained PHR and one CHR. More recently, eight previously untreated CP cases were submitted to daily IFN administration at doses from 2 to 5 X 10(6) IU/m2. This daily schedule was also applied to patients who had obtained, with the intermittent treatment, a PHR persisting unmodified for six months (nine patients) or an unstable CHR (five patients). Seven of the eight previously untreated patients, and five of the nine PHR patients crossed to daily IFN reached CHR. In the total series of previously untreated patients, the response rate proved to be significantly influenced by the initial risk status. Cytogenetic improvement was seen in 37 of 53 responders (70%) treated for more than 3 months, the median of Ph′-positive cells declining from 100% to 65% (range 0% to 95%). Complete suppression of Ph′ chromosome was observed in one case. The cytogenetic response was persistent for over 6 months in 21 patients, but the lowest value of Ph′ positivity was usually unstable. At a median follow-up of 56 weeks, 23 of 36 (64%) CHR patients remain in continued disease control with IFN. A blastic transformation (BT) occurred in seven of 21 unresponsive patients and in one of the 36 CHR patients. The authors' data confirm that IFN alpha- 2b, especially at daily doses, is effective in inducing clinical and cytogenetic response in a good proportion of patients with CML in the benign phase. Longer follow-ups will define the exact influence of this agent on the natural course of the disease.

Description: Recently, we showed that during the acute phase of immune-mediated thrombotic thrombocytopenic purpura (iTTP), ADAMTS13 circulates in an open conformation. Although the cause of this conformational change in acute iTTP remains elusive, ADAMTS13 is mainly closed in iTTP patients (i) in remission with an ADAMTS13 activity 〉50% and undetectable anti-ADAMTS13 autoantibodies, and (ii) after rituximab treatment, suggesting a role for anti-ADAMTS13 autoantibodies. Therefore, IgGs from 18 acute iTTP patients were purified and added to closed ADAMTS13 in healthy donor plasma. This resulted in open ADAMTS13 in 14/18 (78%) samples, proving that indeed anti-ADAMTS13 autoantibodies can induce an open ADAMTS13 conformation. To further elucidate the conformation of ADAMTS13 in iTTP patients, we studied a novel iTTP patient cohort (n=197) that also included plasma samples of iTTP patients in remission where ADAMTS13 activity was