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[Graphic][1] Introduction . Availability of the 2 TPO-RAs Romiplostim (Rom) and Eltrombopag (El) offers an effective treatment option for primary ITP patients (pts). However, some pts are either not responsive or lose response - i.e. desired platelet (plt) count achieved but not sustained over time, or experience wide fluctuations in plt count with either TPO-RA. Adverse events may cause treatment discontinuation. Finally, ptOs preference may be an important issue considering the different route and timing of administration of the two agents. Availability of two TPO-RAs for clinical use, with different molecular structure and site of binding within the TPO receptor, makes it appealing to try switching with the aim of overcoming treatment limitations of either agent. The present survey offers insight into outcome of TPO-RA switching in a group of ITP pts treated at 8 Centers representative of the Italian territory. Patients. Charts of ITP pts on treatment who underwent TPO-RA switch were retrospectively reviewed. Results . Between Jan 2009 and Feb 2015, 57 of 249 pts on TPO-RA (22,9%) underwent switch: El ˆ Rom 26/57 (45.6%), Rom ˆEl 31/57 (54.4%). Median age at 1st TPO-RA administration was 55 yrs (range 16-81); M/F = 23/34. Median disease duration prior to 1st TPO: 58 mos (range 2-648). Median lines of previous therapy 3 (range 1-6; splenectomy: 23/57, 40.4%). Overall 42/57 pts (73,7%) had received maximum product dose as per prescribing information prior to switch. Table 1 summarizes reasons for TPO-RA switch and outcome. Overall, 32/57 pts (56.1%) achieved, regained or maintained a response upon switching. The majority of pts (39/57, 68.5%) were switched for efficacy issues, i.e. failure to respond to 1st TPO (27 pts) or response loss (12 pts); among these 39 pts, 48.7% responded to the 2nd TPO-RA. One pt lost response to Rom because of development of neutralizing antibodies; response was regained upon switching to El. In this subgroup of pts, disease duration and lines of previous therapy (but not splenectomy status) seem to have an impact on response to switching. Each one month increase in disease duration determines a 0.7% decrease in the odds of achieving a response (WaldOs test p=0.065). More than 2 lines of therapy determine a 72% decrease in the odds of achieving a response (WaldOs test p=0.077). Either TPO-RA switch sequence was equally effective in yielding response (FisherOs exact test p=0.752) and age at 1st TPO-RA had no impact on response. Of the 18 pts switched for reasons other than efficacy, 13 (72.2%) maintained a response on the 2nd TPO-RA: 5/6 switched for plt count instability (counts stabilized = 2/5 responding pts), 4/7 switched for ptOs preference, 4/5 switched for side effects. Four pts (1 plt count instability, 3 ptOs preference) underwent Odouble switchO (i.e. Rom ˆ El ˆ Rom): re-exposure to Rom was not associated with response loss. Discussion . Switching enables approximately 56% of pts to achieve, regain or maintain a plt response; switching for inefficacy yields lower response rates (48.7%) compared to switching for reasons other than efficacy (72.2%) Plt counts fluctuation stabilized in 40% of pts. Re-exposure to R in the 4 pts who underwent Odouble switchO was not associated with response loss, confirming absence of tachyphylaxis with this TPO-RA. Our results are in line with those reported by Khellaf (Haematologica 2013) and Gonzales-Porras (BJH 2014): TPO-RA switch can be a safe and appealing treatment option for ITP pts who experience suboptimal results with either agent. | REASON for SWITCHING | n (%) | NR (%) | R (%) | CR (%) | | | --------------------- | --------- | --------- | --------- | --------- | | | All | 57 (100) | 25 (43.9) | 14 (24.6) | 18 (31.5) | | | El->Rom | 26 (45.6) | 10 (38) | 8 (31) | 8 (31) | | | Rom->El | 31 (54.4) | 10 (48.4) | 6 (19.4) | 10 (32.3) | | | 1st TPO-RA failure | 27 (47.4) | 16 (59) | 4 (15) | 7 (26) | | | El->Rom | 15 | 7 (47) | 3 (20) | 5* (33) | | | Rom->El | 12 | 9 (75) | 1 (8) | 2 (17) | | | Loss of response | 12 (21.1) | 4 (33) | 2 (17) | 6 (50) | | | El->Rom | 4 | 2 (50) | 1 (25) | 1 (25) | | | Rom->El | 8 | 2 (25) | 1 (12.5) | 5 (62.5) | | | Plt count fluctuation | 6 (10.5) | 1 (17) | 3 (50) | 2 (33) | | | El->Rom | 2 | | 1 | 1 | | | Rom->El | 4 | 1 | 2 | 1 | | | PtOs preference | 7 (12.3) | 3 (42.9) | 2 (28.6) | 2 (28.6) | | | El->Rom | | | | | | | Rom->El | 7 | 3 | 2 | 2 | | | Adverse eventii | 5 (8.8) | 1 (20.0) | 3 (60.0) | 1 (20.0) | | | El->Rom | 5 | 1 | 3 | 1 | | | Rom->El | | | | | | * CR: complete response; R: response; NR: no response (Rodeghiero et al, Blood 2009) * *1 NR secondary to neutralizing antibodies development * ii1 hepatic enzyme increase, 1 CPK increase; 2 skin rash; 1 retinal thrombosis. Table 1. Disclosures De Stefano: Janssen Cilag: Research Funding; Shire: Speakers Bureau; Amgen: Speakers Bureau; Bruno Farmaceutici: Research Funding; Novartis: Research Funding, Speakers Bureau; Roche: Research Funding; Celgene: Speakers Bureau; GlaxoSmithKline: Speakers Bureau. [1]: /embed/inline-graphic-2.gif

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are represented by rare but life-threatening cutaneous adverse reactions to different drugs. Previous studies have found that in a Han Chinese population from Taiwan and other Asian Countries, a strong genetic association between HLA-class I alleles (B*15:02, B*58:01) and SJS and TEN was induced by carbamazepine and allopurinol, respectively. To identify genetic markers that covered the MHC region, we carried out a case-control association enrolling 20 Caucasian patients with SJS/TEN. Our patient series included 10 cases related to paracetamol, 7 to allopurinol and 3 to different drugs (plaquenil, itraconazol, nabumetone). Healthy controls were represented by 115 Caucasian bone marrow or stem cell donors. The HLA-A*, B*, C*, DRB1*, DQB1*, DQA1* and DPB1* genotyping were determined. The frequencies of HLA-A*33:03 as well as C*03:02 and C*08:01 were significantly higher in SJS/TEN patient subgroup showing allopurinol drug-induced severe cutaneous adverse reactions (SCAR) as compared to controls (28.6% vs 0%, P=0.00002, Pc=0.0011; 28.6% vs 0%, P=0.00002, Pc=0.001; 28.6% vs 0%, P=0.00002, Pc=0.001, respectively). In the same subgroup the frequencies of B*58:01, DRB1*15:02 and DRB1*13:02 alleles, although considerably higher than in control group (42.8% vs 5.2%, P=0.003; 28.6% vs 1.7%, P=0.005; 28.6% vs 3.5%, P=0.037, respectively), appeared no more statistically different after P correction (Pc=0.248; Pc=0.29; Pc=1.00, respectively). In addition, in 10 of the 20 SJS/TEN patient subgroup with paracetamol-induced SCAR no statistically significant association with HLA alleles could be found. However, in the same SJS/TEN patient subgroup showing allopurinol drug-induced SCAR, haplotype analysis indicated that B*58:01, DRB1*13:02 and DRB1*15:02 alleles, that in a single allele analysis lost statistical significance after P correction, may still confer susceptibility, because the B*58:01-DRB1*13:02 and DRB1*15:02-DQB1*05:02 are positively associated with the disease (14.2% vs 0.43%, P= 0.00001, Pc=0.00028; 14.2% vs 0.43%, P=0.00001, Pc=0.00028, respectively). Our results show that in contrast to SCAR-related to paracetamol, where HLA alleles do not appear to be involved, HLA molecules behave as a strong risk factor for SCAR-related to allopurinol even when a limited number of patients are considered.

An independent clinical assessment was compared with flow cytometry (FCM) and cytomorphology results obtained on 227 cerebrospinal fluids investigated for hematologic malignancy, in a retrospective longitudinal study with a median observation time of 11 months. A combined method assessment (CMA), defining "positive" a sample if at least one method gave "positive" results, was also tested. Eleven out of 55 screening samples and 53 out of 166 follow-up samples resulted positive at clinical evaluation. FCM and CM were concordant with positive clinical assessment in 68.5% and 51.5% of cases, respectively. According to CMA, 10.5% of samples (resulting false negative by either FCM or cytomorphology) were rescued as true positive. FCM retained significantly higher accuracy than cytomorphology (p = 0.0065) and 100% sensitivity when at least 220 leukocytes were acquired. CMA accuracy was higher than FCM accuracy and significantly higher than cytomorphology accuracy in the analysis of all samples (p < 0.0001), samples from mature B/T cell neoplasms (p = 0.0021), and samples drawn after intrathecal treatment (p = 0.0001). When acquiring ≥220 leukocytes, FCM accuracy was poor, and combining cytomorphology added statistically significant diagnostic advantage (p = 0.0043). Although FCM is the best diagnostic tool for evaluating CSF, morphology seems helpful especially when clinically positive follow-up samples are nearly acellular.

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.

Flow cytometry and cytomorphology results on 92 body cavity fluids [61 effusions and 31 bronchoalveolar lavage fluids (BALF)] from hematologic malignancy were compared with retrospective clinical outcome. We observed double true positive/negative results in 67 cases (73%), and double false negative results in 2 cases (2%). Immunophenotyping accounted for true positive/negative results in 22 out of 23 mismatched cases (25%), and retained significantly higher accuracy than that of cytomorphology especially in effusions and differentiated lymphoma. In BALF analysis, immunophenotyping and cytomorphology sensitivity was 75% and 0%, respectively. Flow cytometry retains the highest accuracy in detecting neoplastic cells in body cavity fluids.

Abstract 2351 Poster Board II-328 Background: although the clinical course of patients with B-cell chronic lymphocytic leukemia (CLL) is often complicated by autoimmune phenomena few data are available concerning the prevalence of autoimmune diseases in this lymphoprolipherative disorder as well as their clinical outcome. Autoimmune hemolytic anemia (AIHA) is the most common form, occurring in 10% to 25% of patients during CLL course. DAT may be positive at some time during the course of the disease in up to 35% of cases, but overt AIHA occurs less frequently. Although AIHA may occur in asymptomatic untreated CLL, it is more common in patients with advanced-stage disease. So far it is unknown if also the biological characteristics of CLL may have any influence on the risk of developing autoimmune phenomena. Recently, an exceedingly high prevalence of un-mutated IgVH, with an excess of VH1 and VH3 families has been described in patients with autoimmune thrombocytopenia and DAT status has been correlated with clinical and laboratory prognostic factors in CLL Chinese population. Aim: the current retrospective study was conducted to investigate the relation between incidence of positive DAT test and biological features of CLL patients. Patients and methods: in our institution 158 patients with CLL were studied with DAT at some time during the disease course. Characteristics of patients are summarized in the table. DAT resulted positive in 42 patients (26.6%), overt AIHA occurred in 28,6% of them. Univariate analysis by Fisher's exact test was performed to establish the association between DAT result and AIHA development with the following variables: age, sex, Binet stage, LDH and beta2 microglobulin (B2m) at diagnosis, IgVH status, VH families, ZAP70, CD38 and FISH analysis. Results: the incidence of positive DAT was significantly increased in presence of higher B2m (p 0.011) and highly correlated with IgVH un-mutated status (p 0.014). No prevalence or correlation for the IgVH families was recorded between DAT positive and negative patients. In the subgroup of patients with poor risk FISH chromosome aberration (deletion in 17p13, 11q22 or 6q21) we found a trend (p 0.064) for higher frequency of positive DAT compared to good risk (deletion in 13q14 or trisomy 12). A trend for increased positivity of DAT has been also found in patients with positive ZAP70 (p 0.083) and increased LDH value at diagnosis (p 0.051). Among DAT-positive patients none of biological and clinical characteristics listened above showed a significant correlation with the risk to develop AIHA. Conclusions: in our series DAT positivity correlated with poor risk biological features, however no difference was detected in terms of OS between patients with positive or negative test. Furthermore, we could speculate that patients with un-mutated IgVH status are characterized by a peculiar antibody reactivity, resulting in a major failure controlling the emergence of auto-antibodies from normal B cells. Although the results of this study warrant further confirmation from larger series, the documented association of poor biological features and DAT positivity in CLL patients, suggests that this laboratory test may improve prognostic evaluation at diagnosis. Disclosures: No relevant conflicts of interest to declare.

Human tryptase is a serine protease expressed in mast-cells. We previously observed that AML blast cells, cultured in vitro from a KIT D816Y patient, give rise to adherent cells with mast-cell like phenotype and tryptase was released in the serum-free medium. To correlate total serum tryptase (ts-try) levels with cytogenetic features and KIT mutational status, we analyzed serum samples from AML patients at diagnosis. In 70 out of 155 patients (45%) we detected elevated ts-try (>15 ng/mL), significantly linked to t(8;21) (P < .001) and inv(16) (P = .007). In patients that achieved complete remission the ts-try decreased to normal values. In 75 patients screened for KIT mutation, we found a clear relationship between elevated ts-try and mutated patients with t(8;21) (P < .001). In conclusion, we propose that checking for ts-try at diagnosis of AML may be a simple tool to select patients to be addressed to KIT mutation screening.

Background: To evaluate the clinical outcome of patients with relapsed or refractory follicular lymphoma treated with immunochemotherapy, in vivo purging and high-dose therapy with autotransplant. Patients and methods: Sixty-four patients were enrolled in the trial. Primary end point was progression-free survival (PFS). Secondary end points were the in vivo purging effect on stem-cell harvest and the impact of molecular response on the outcome. Results: At enrollment, 59% of patients were PCR+ for bcl-2 rearrangement in bone marrow (PCR-informative). After the immunochemotherapy, before mobilization, 97% obtained complete response or partial response and 87% of patients informative for bcl-2 were molecularly negative. Sixty-one patients proceeded to in vivo purging and peripheral blood stem cell (PBSC) mobilization with rituximab and high-dose AraC. The median number of CD34+ cells collected was 16.6 · 10 6 /kg. Of 33 PCR-informative patients, the harvests resulted in PCR2 in all. Fifty-eight patients received high-dose therapy and autotransplant of in vivo purged PBSC. After a median follow-up of 3.5 years, 41 patients are in complete remission. Five-year PFS is 59%. Conclusion: This study demonstrates that patients with advanced relapsed or refractory follicular lymphoma treated with immunochemotherapy, in vivo purging and autotransplant may obtain long-lasting PFS. In bcl-2-positive patients, in vivo purging allows the harvest of lymphoma-free PBSC. Absence of the bcl-2 rearrangement after autotransplant is associated with persistent clinical remission.

We studied circulating (C)CD34 + cells by flow cytometry in 96 patients with myelodisplastic syndromes (MDS) at diagnosis, and in a subset of 35 cases during follow-up. CCD34 + counts were stratified within both International Prognostic Scoring System (IPSS) and World Health Organization (WHO) categories. Counts >10/μl were associated with poorer leukemia-free survival, a prognostic value for evolution independent from that of WHO, and a higher progression probability within intermediate-risk IPSS and WHO classes. When serial measurements were performed, counts >10/μl more frequently correlated to evolution. Separating newly diagnosed patients on the basis of 10/μl cut-off of circulating CD34 + cells retains prognostic utility, especially in intermediate-risk MDS.

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 Conclusions: Both markers of myeloid cell committment (i.e., CD33 and CD34) and markers of myeloid cell immaturity (i.e., CD117) correlate with BM blast WHO category and IPSS score. Expression of CD33 and CD34 may have prognostic significance independently from blast percentage. The evaluation of CD33, CD34 and CD117 expression on not enriched BM samples may represent a simple tool for the prognostic assessment of MDS patients, in addition to previously established methods. Tab. 1: Correlation of CD33, CD34, CD117 expression with IPSS and percentage of BM blast in 50 MDS pts at diagnosis

Introduction: A phase II multi-center study was performed to investigate the efficacy of Bortezomib with high-dose dexamethasone (Vel-Dex) as induction therapy in multiple myeloma (MM) patients (pts) candidates to high-dose therapy. Methods: Patients were planned to receive 4 courses of Vel-Dex (Bortezomib 1.3 mg/m2 i.v. on days 1, 4, 8, 11; oral Dexamethasone 40 mg on days 1–4 and 8–11 every 3 weeks), followed by 2 courses of DCEP 4 weeks apart with stem cell collection, and a single autologous transplant with melphalan 200 mg/m2. Patients were untreated, aged ≤65 years, with Durie-Salmon stage III, II, or I in progression. Criteria of response were: CR: negative serum/urine immunofixation with Results: From March 2006 to June 2007, 52 out of the 54 planned pts entered the protocol. Patient characteristics at enrolment were: male/female 33/19; median age 57 years (37–65); IgG/IgA/light-chain 33/9/10 pts; stage III/II/I in progression 44/5/3 pts; ISS I/II/III 21/14/17 pts; cytogenetic analysis showed del 13 in 54%, t (4;14) in 15%. Thirty-nine of 52 enrolled pts are evaluable for efficacy and toxicity after 4 Vel-Dex courses. Six pts were withdrawn (3 for progression, 2 for toxicity, 1 patient withdrew informed consent). Overall response rate (ORR) was 85%, with 67% major responses (CR 33%, nCR 26%, VGPR 8%), PR 18%, stable disease 7%, progression 8%. No statistically significant correlation was found between response and either age, stage, ISS, or unfavorable cytogenetics. Friedman ANOVA (p=0.00001) and Wilcoxon Matched Pairs (p Discussion: This study shows that Vel-Dex as first line therapy produces high response rates in MM pts (ORR 85%, major response 67%). Toxicity was generally predictable and manageable. Stem cells were successful harvested in all patients. Vel-Dex appears an effective and safe pre-transplant treatment for younger MM patients.

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

Introduction: Flow cytometry is well known to detect malignant cells in peripheral blood and bone marrow of patients with hematologic malignancies. However, its role in evaluating non-hematic body fluid contamination by tumor cells is largely unexplored. Patients and Methods: Data detected by flow cytometry in non-hematic body fluid samples drawn between 2002 and 2007 from patients with hematologic neoplasms were retrospectively compared with morphological findings obtained from cytospin slides. Immunophenotyping was carried out by using disease-specific multicolor panels of quadruple monoclonal antibodies, conjugated with the fluorochromes FITC, PE, PerCP, and APC, respectively. Acquisition of information on 1x104 to 1x105 stained cells depending on the whole sample cellularity was assessed on a dual-laser FACSCalibur flow cytometer using the CellQUEST software (Becton Dickinson, San José, CA, USA). Results: Fourty-five samples (bronchoalveolar fluid, n=5; ascites, n=2; hydrocele, n=2; pleural effusion, n=8; and cerebrospinal fluid, n=28) from 32 patients were available for comparison. Diagnoses were as follows: chronic myelomonocitic leukemia (n=1), acute promyelocytic leukaemia (n=2), acute myelomonocytic leukaemia (n=1), B-chronic lymphocitic leukemia (n=4), follicular lymphoma (n=1), acute lymphoblastic leukaemia (n=6), lymphoblastic lymphoma (n=1), high grade non-Hodgkin’s lymphoma (NHL), Burkitt-like (n=3), diffuse large B-cell NHL (n=6), peripheral T-cell NHL (n=3), and NHL, unspecified (n=4). Flow cytometry detected neoplastic cells in 24 cases. Of these cases, only 17 were positive also by morphology. In 7 cases, in which tumor cells were detected by flow cytometry but not by morphology, clinical data confirmed the presence of the disease. Flow cytometry did not show neoplastic cells in 21 cases. Of these cases, only 18 were negative also by morphology. In the remaining 3, the suggestion of diffuse large B-cell NHL contamination by morphology was not confirmed by flow cytometry, demonstrating T-reactive lymphocytes that were clearly negative for disease-specific markers. Conclusions: Our data suggest that flow cytometry is a useful tool complementary to morphology for the screening of non-hematic body fluid contamination in patients with hematologic neoplasms.

Antifungal therapy may be unable to eradicate invasive mycosis in leukemia patients. The presence of persisting pulmonary nodules owing to mycosis seems to increase the risk of fungal relapse after chemotherapy and transplant procedures. Between 1997 and 2004, 10 acute leukemia patients underwent pulmonary surgery for invasive mycosis. The median time from diagnosis of mycosis to surgery was 135 days (range 21-147). Three patients underwent emergency surgery, owing to hemoptysis. In the other seven patients with nodule/cavitation remaining after antifungal treatment, surgery (three wedge resections, four lobectomies) was scheduled before transplant. Pathologic examination confirmed two aspergillosis and three zygomycosis. The only side effect was pneumothorax in one case. Nine patients were considered cured. Six patients underwent bone marrow transplantation (three allogeneic, three autologous) with antifungal prophylaxis without relapse during the transplant procedure. In selected patients scheduled for bone marrow transplantation, surgical resection of localized pulmonary fungus nodules combined with antifungal prophylaxis seem to be an effective treatment for preventing mycotic relapse.

Anna Maria Frustaci, Alessandra Tedeschi, Paola Picardi, Roberto Cairoli, Marco MontilloDepartment of Hematology, Niguarda Cancer Center, Niguarda Ca' Granda Hospital, Milan, Italy Abstract: Treatment aim for chronic lymphocytic leukemia has been radically changed over the past years from providing only a palliative approach to reaching disease eradication and improving survival. Ofatumumab is a monoclonal humanized antibody with peculiar in vitro and in vivo properties, at present approved for double fludarabine and alemtuzumab refractory chronic lymphocytic leukemia. Its efficacy in this subset of patients, who typically have an unfavorable prognosis, facilitated its use in different Phase II and III trials. Ofatumumab as single agent or combined with chemotherapeutic or biologic agents, led to sundry results in the setting of both previously treated or untreated patients. Its role in maintenance therapy is also under investigation. Further advances concerning ofatumumab administration as first line therapy in combination with chlorambucil, came recently from the COMPLEMENT 1 study. Results from this trial will open the door to new perspectives of its use in treatment-naïve patients. Ofatumumab was well tolerated in almost all the studies, with the main adverse events relating mostly to infusion reaction. Hematologic toxicity, especially neutropenia, was also common. A significant improvement in patients' quality of life was reported following ofatumumab treatment and this was mainly due to its effect on constitutional symptoms. Nevertheless, some concerns remain regarding the long-term efficacy of the drug in terms of response duration and survival. The real strength of this drug needs to be confirmed by further studies and direct comparative trials. Keywords: ofatumumab, chronic lymphocytic leukemia, refractory, alemtuzumab, fludarabine, high risk

Systematic data on the ability of pegfilgrastim to mobilize stem cells after chemotherapy are scarce. We evaluated the efficacy of a single 6 mg dose of pegfilgrastim for mobilizing peripheral blood stem cells (PBSC) in aggressive lymphoma patients. Between July 2004 and October 2005, 17 aggressive non-Hodgkin's lymphoma and 11 poor-risk Hodgkin's lymphoma were treated with cycles containing cisplatin-aracytin. At the end of chemotherapy, the patients received 6 mg of pegfilgrastim. Duration of grade 4 neutropenia, adverse events, time to neutrophil recovery, peak and harvest of CD34+ cells were recorded. Twenty-seven out of 28 patients harvested a median of 17.3 x 10(6)/CD34+ cells (range 2.5-28.9) after a median of 9 days (range 8-12 days), with a single apheresis procedure in 25 cases. All patients had grade 3-4 neutropenia, median duration 3 days. The only adverse event was mild bone pain. To date, 13 patients have been autografted with a median of 15.4 x 10(6) CD34+ pegfilgrastim-mobilized cells per kg (range 2.5-28.9) with rapid and sustained engraftment. Mobilization, harvesting and autografting of pegfilgrastim-mobilized PBC can be successfully achieved in pretreated patients with aggressive lymphoma.

Purpose Treatment with alemtuzumab has resulted in negative responses for minimal residual disease (MRD) in patients with chronic lymphocytic leukemia (CLL). In a prior analysis we demonstrated that it is possible to achieved MRD negativity, as assessed by polyclonality of immunoglobulin heavy chain after consolidation with alemtuzumab. This phase II study evaluated 34 patients with CLL who received alemtuzumab consolidation in an effort to improve the quality of their response to fludarabine-based induction. Subsequent peripheral blood stem-cell (PBSC) collection and transplantation, tolerability, and pharmacokinetics also were assessed. Patients and Methods Thirty-four patients younger than 65 years who had a clinical response to fludarabine-based induction therapy received alemtuzumab 10 mg subcutaneously three times per week for 6 weeks. PBSCs were collected after mobilization with cytarabine and granulocyte colony-stimulating factor. Blood samples for pharmacokinetics study were taken between days 1 and 31. Results The complete response rate improved from 35% after fludarabine induction to 79.4% after alemtuzumab consolidation, including 19 patients (56%) who achieved MRD negativity. The most common adverse events were injection-site reactions and fever. Cytomegalovirus reactivation occurred in 18 patients, all of whom were successfully treated with oral ganciclovir. PBSC collection was successful in 24 (92%) of 26 patients, and 18 patients underwent autologous PBSC transplantation. Alemtuzumab plasma concentrations increased gradually during the first 2 weeks and accumulated more rapidly thereafter. Conclusion Subcutaneously administered alemtuzumab was effective, safe, and well tolerated as consolidation therapy in patients with CLL who responded to fludarabine induction therapy. Subsequent PBSCT was feasible thereafter.

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 =

Distinct forms of tyrosine kinase domain (TKD), juxtamembrane domain, exon 8, and internal tandem duplication (ITD) mutations of c-KIT, were observed in about 46% of core binding factor leukemia (CBFL) patients. To evaluate their prognostic significance, 67 adult patients with CBFL were analyzed to ascertain the c-KIT mutation status. In acute myeloid leukemia (AML) with t(8;21), the presence of c-KIT TKD mutation at codon 816 (TKD(816)) was associated with a high white blood cell count at diagnosis (median, 29.60 x 10(9)/L) and a higher incidence (33%) of extramedullary leukemia (EML) during the course of the disease. Data also showed that the TKD(816) mutated patients (n = 12) had a significantly higher incidence of relapse and a lower overall survival (OS) at 24 months, compared with the 17 c-KIT unmutated (c-KIT(-)) patients (90% vs 35.3%, P = .002; 25% vs 76.5%, P = .006, respectively). No difference in relapse incidence (P = .126) and OS (P = .474) was observed between the c-KIT mutated other than TKD(816) (n = 7) and the c-KIT(-) patients. These findings indicate that c-KIT TKD(816) mutation has a negative impact on the outcome of AML with t(8;21).

Chronic myeloid leukemia (CML) is a disease of stemming from genetic damage to a hematopoietic stem cell. Despite nilotinib being a very effective drug for the treatment of CML, drug resistance can emerge. In the contest of the REL-PhilosoPhi34 study on behalf of the Rete Ematologica Lombarda, we performed an exploratory study aimed to identify the gene expression signature of bone marrow (BM) CD34+/lin- cells of CML patients at diagnosis as well as after 12 months of nilotinib to investigate the genes and pathways responsible for the response or resistance to nilotinib. Microarray experiments were performed using the latest generation Affymetrix GeneChip HTA 2.0 to further investigate genomic complexity. The study was developed in two steps as follows: In the first step we analyzed 30 CML patients and from the comparison between the BM CD34+/lin- cell counts from each patient at diagnosis and after 3 and 6 months of nilotinib, patients were divided into 2 classes: class 1 (n=24) showed highly reduced levels of CD34+/lin- cells while class 2 (n=6) demonstrated increasing levels of CD34+/lin- cells after 3 and 6 months of nilotinib, respectively (Fig.1). The 30 patients can be divided into 6 groups showing different patterns of CD34+/lin- cellularity (Figure 1). Data was preprocessed and normalized using Robust Multi-array Average (RMA) algorithm. The Significant Analysis of Microarrays (SAM) was used to identify genes with statistically significant changes in expression in CML patients. P-values were corrected for multiple testing using false discovery rate. No transcripts were selected as differentially expressed using this threshold. However, if a nominal significance level alpha equal to 0.05 is adopted together with a fold-change threshold equal to 2 (absolute value), 56 transcripts were selected in the comparison between the 2 groups of CML patients. Among them, we focused on NFKBIAgene which was over expressed in class 1 compared to class 2.NFKBIA is involved in 68 pathways regulating apoptosis (PI3K,NF-kB) and encodes IkBα protein belonging to the NF-kB pathway which is a potential downstream target of BCR-ABL1 due to its role in regulating cell survival and proliferation. Of note, 31/56 transcripts are located on chromosome 15 suggesting that this region could be crucial for transcriptional regulation of CML correlated to nilotinib response. The second step analyzed the GEP of CD34+/lin- cells of 8 patients at diagnosis (class 1) vs. the same 8 patients after 12 months of nilotinib (class 2) to investigate the gene expression changes and the pathways induced by nilotinib treatment. Data was preprocessed and analyzed as described above. 247 probes (corresponding to 51 genes and 180 non-annotated transcripts) were selected with a p-value lower than 0.05 after multiple testing correction and using a fold-change threshold equal to 3 (absolute value). Functional enrichment analysis was performed using DAVID and highlighted the following pathways and genes: ¯ Defense response and immune system: CAMP, CRISP3, CYBB, RGS1, CEACAM8, LTF (cell growth and differentiation), MNDA and HP (positive regulation of apoptosis) were over expressed in class 2 with high fold changes of 7.65, 9.95, 4.40, 3.47, 4.28, 3.61, 3.60, 3.95, respectively ¯ Transcriptional regulation and cell cycle: MMP8was under expressed in class 1 with the high fold change of 8,94 ¯ S100A12, S100A8, S100A9 (regulation of the cell cycle and differentiation) were over expressed in class 2 with the FC of 8.68, 3.96, 4.49, respectively ¯ RNA5S-ribosomal pseudogenes 310-311-312-313-314-315-316-317-320-363 were downregulated in class 2 ¯ 10 small-nuclear-RNAs and 11 small-nucleolar RNA were differently deregulated between the 2 classes ¯ APOC1, PLBD1 (lipid metabolism) were overexpressed and underexpressed in class 1, respectively In conclusion, this is the first study of GEP of CD34+/lin- cells with HTA 2.0 which demonstrated that 51 genes mostly involved in immune system, transcriptional regulation and cell cycle were significantly differently expressed from the comparison between CML patients at diagnosis and after 12 month of nilotinib. Ongoing studies are focused on examining the genetic and biologic mechanisms underlying nilotinib response or resistance to predict response or failure providing new insights into the molecular mechanisms in CML. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Aims and backgroundIn order to increase the prognostic significance of polymerase chain reaction (PCR) data it has been suggested that quantitative PCR can be used to measure tumor burden. However, this option has not yet been definitely supported or refuted in patients with follicular lymphoma (FL). We decided to evaluate whether knowledge of the quantitative level of minimal residual disease and its variations can be of use in the management of FL patients.MethodsWe used qualitative and competitive PCR to study 11 patients with refractory or relapsed FL harboring the t(14;18) translocation who underwent autologous (nine patients) or allogeneic (two patients) stem cell transplantation (SCT). Competitive PCR was performed with a multiple competitor carrying specific sequences including Bcl2/lgH MBR and mcr, and the β-globin gene.ResultsAfter a median post-SCT follow-up of 44 months (range, 12-62), overall survival was 91% and disease-free survival 82%. The quantitative PCR data showed that: 1) effective chemotherapy before SCT substantially (1-2 log) reduced the tumor burden in the bone marrow (BM); 2) the increase in rearranged DNA detected in BM was associated with disease progression and relapse; 3) a PCR-negative autograft seemed to lead to lasting molecular remission even when it was performed in patients with a low level of BM infiltration before transplant; and 4) allo-SCT made and maintained the BM PCR negative even in the presence of a greater tumor burden before SCT. Six of the nine patients having CR after SCT (four auto and two allo) are in continuous molecular remission.ConclusionsIn FL patients qualitative and quantitative PCR may provide data that can be helpful for the prognostic evaluation of tumor progression and the early detection of impending relapse by highlighting biological features such as the quality of the infused material, the tumor burden at transplant, and the behavior of tumor cells after transplant.

Background. Pegfilgrastim is a polyethylene glycol (PEG)-conjugated form of G-CSF in which filgrastim is bound covalently to a 20kDa PEG molecule; this formulation increases serum half-life of G-CSF due to decreased renal elimination. Following the subcutaneous injection of a 6 mg single dose of pegfilgrastim, serum levels of G-CSF are maintained over a period of 2 weeks. In patients with lymphoma, pegfilgrastim is as effective as filgrastim in shortening the time of neutropenia after cytotoxic chemotherapy; however, the ability of pegfilgrastim to mobilize stem cells after chemotherapy is poorly understood and the optimal mobilization strategy remains controversial. Aims. We evaluate the efficacy of a single fixed 6 mg dose of pegfilgrastim after salvage therapy with cisplatin-containing chemotherapy regimens in mobilizing peripheral blood stem cells in aggressive lymphoma patients. Moreover the possibility of a sufficient collection of CD34+ PBSC (cell dose > 2,5 x106/Kg) in a single procedure was tested. Methods. Between July 2004 and July 2005 twenty two pretreated patients with aggressive lymphoma (8 Hodgkin’s lymphoma, 11 diffuse large B cell lymphoma, 3 anaplastic lymphoma) received salvage chemotherapy with cisplatin-based regimens: (R)-DHAP, dexametasone, cisplatin, cytarabine, or (R)-IPAD idarubicin, dexametasone, cisplatin, cytarabine, with or without Rituximab. A 6 mg single dose of pegfilgrastim was given from day +1 or +2 after chemotherapy completion. Duration of grade 4 neutropenia, adverse events, time to neutrophil and CD34+ cell recovery were recorded. Stem cell collection was started when the absolute number of CD34+ was not less than 20/μL. Leukapheresis was daily performed until the minimum target cell dose of 2.5 x 10 6 CD34+ cells /kg was reached. Results. Following the administration of either (R)-DHAP or (R)-IPAD regimens, 21/ 22 pts (95%) were able to harvest a median of 14,2 x 10 6/Kg CD34+ cell (range 2,4– 45,8), after a median of 9 days from stimulation (range 8–12). A single apheresis procedure was sufficient to obtain a median of 14,8 x 10 6/kg CD34+ cell (range 5,2–45,8) in 18/21 pts (86%) Grade 4 neutropenia was present in all pts with a median duration of 3 days (range 1–7). Pegfilgrastim determined mild bone pain as only adverse event. Seven patients underwent autologous bone marrow transplantation and all of them showed a rapid and sustained engraftment after high-dose chemotherapy. Conclusions. In aggressive lymphoma patients a single dose of pegfilgrastim following chemotherapy completion was safe and highly effective in enhancing the mobilization of CD34+ PBSC. Stem cell collection was performed in a single procedure in most of patients (86%) obtaining a stem cell harvest of a median of 14,8 x 106/Kg CD34+ cells. In 7 autologous bone marrow transplanted patients these results determined early engraftment and sustained hematological reconstitution.

High dose therapy represents the gold standard therapy for newly diagnosed multiple myeloma (MM) patients (pts), with no definite agreement about the adoption of single or double transplant. From January 2000 to December 2004, 151 consecutive MM pts aged ≤65 years in stage II, III or I in progression according to Durie-Salmon were enrolled in a multicenter no randomised high dose program including a tandem transplant (Tx1; Tx2). The protocol was designed as follows: 2 pulse-VAD as induction, 2 DCEP to mobilise peripheral blood stem cells (PBSC), double auto-transplant 3-6 months apart each conditioned with high-dose Melphalan at the dose of 200 mg/m2. Patients characteristics at the enrolment: males 76 (51%), females 75 (49%), median age 55 (range: 35–65), stage I in progression 26 (17%), stage II 25 (16%), stage III 100 (67%). Response rates after each phase for the evaluable patients are reported in the table below VAD (151 pts) DCEP (146 pts) Tx1 (119 pts) Tx2 (63 pts) CR (%) 4 9 18 29 VGPR (%) 28 35 48 60 PR (%) 44 30 25 9 SD (%) 18 10 2 0 Progr (%) 6 16 7 0 Patients not addressed to transplant for mobilization failure were only 5%. Most of the patients (75%) collected ≥ 4x106CD34+cells/Kg after each DCEP-cycle which were considered adequate to rescue hemopoiesis after each transplant. The whole protocol was well-tolerated. In particular, no therapy related mortality was associated to pulse-VAD, or DCEP, and no difference between Tx1 and Tx2 as far the transplant related mortality was registered (1.5% after each transplant). Second transplant was not performed in 48 pts for the following reasons: 8 pts (7%) did not collect enough PBSC, 8 pts (7%) have had severe toxicity with the first transplant; 8 pts (7%) underwent allo-TMO; 7 pts (6%) had progressive disease and 15 pts (12%) refused Tx2. Finally only 76 pts (50% of the enrolled pts) completed the program with the second transplant. Analysing data on an intention-to-treat basis, median follow-up was 30 months, median Progression Free Survival (PFS) was 31 months, median overall survival (OS) was not reached. The median Event Free Survival (calculated from the completion of Tx1 to progression or any other event) was 20 months. No difference in terms of PFS and EFS was found comparing pts who finally received only Tx1, with those who completed the protocol (p=0.9; p=0.5). The EFS was not statistically different for patients receiving one or two transplant even when the analysis was performed according to the type of response achieved after Tx1. In conclusion, despite higher percentage of good quality responses (CR+VGPR) can be obtained with 2 transplants with respect to 1 (66% vs 89%) without additional toxicity, no difference in terms of PFS or EFS were observed between the patients who underwent 1 or 2 transplants. Thus, keeping into account the more complex management of patients in a tandem transplant program, it might be more advantageous to perform as initial therapeutic approach, high-dose protocol including only 1 transplant procedure.

Objective Alterations in growth factor signaling pathways may be a frequent collaborating event in AML1-ETO-mediated leukemogenesis. Gain-of-function KIT receptor mutations have been reported in adult AML patients, especially those with core binding factor leukemia (CBFL). We have previously reported a new gain-of-function KIT Asn822Lys mutation that is constitutively expressed in the Kasumi-1 CBFL cell line, and has recently been described in two childhood AML patients. To explore the molecular basis of the effects of this mutation in the appropriate context of hemopoietic dysregulation, we investigated KIT downstream signaling in the Kasumi-1 cell line by means of STI 571 (Imatinib, Gleevec) pharmacological inhibition. Materials and Methods We investigated KIT Asn822Lys mutant-initiated signaling in Kasumi-1 cell line, and characterized the inhibitory effect of the STI 571 protein tyrosine kinase inhibitor on downstream signaling. Results The use of STI 571-mediated inhibition impaired the tyrosine phosphorylation of KIT Asn822Lys and its association with the p85 subunit of phosphatidylinositol 3′-kinase (p85 PI3K ). The downstream constitutive phosphorylation of JNK1/2 and STAT3 was also significantly inhibited, but STI 571 had no effect on the constitutive activation of Akt, thus suggesting that it is due to other signaling in Kasumi-1 cells. STI 571 inhibited the KIT-mediated proliferation of Kasumi-1 cells in a dose-dependent manner. Conclusions These findings show the role of PI3K in KIT Asn822Lys -mediated constitutive activation through the Akt-independent downstream signaling pathway of JNK, and also demonstrate the mutant's susceptibility to STI 571, which may therefore have therapeutic potential in CBFL patients with susceptible KIT mutations.

We report a high incidence of neutropenia in patients treated with rituximab prior to and following autologous stem cell transplantation (ASCT). Fourteen patients with follicular or mantle-cell lymphoma were treated with high dose (HD) therapy followed by an in vivo-purged autologous graft. Ten of these patients received two additional courses of rituximab after the transplant. Seven experienced severe neutropenia after the second administration. Our data suggest that early administration of rituximab following a transplant may favor the onset of neutropenia.

Mutations of the c-kit gene have been reported in myeloproliferative disorders. We describe here a case of Ph þ (b2a2) chronic myelogenous leukemia that, during the course of disease, showed an unusual bone marrow mast-cell infiltration. A mutational screening for the c-kit gene, performed on DNA routinely cryopreserved during the follow-up, evidenced the D816Yactivating mutation as an additional genetic abnormality. Treatment with imatinib mesylate resulted in a substantial decrease of the BCR-ABL/ABL ratio and in the absence of c-kit mutation. It is likely that the superimposed c-kit mutation, in this case, may account for the transient bone marrow mastocytosis. The Hematology Journal (2004) 5, 273–275. doi:10.1038/sj.thj.6200348

Chronic lymphocytic leukemia (CLL) cells could be undetectable by flow cytometry or polymerase chain reaction after sequential treatment with fludarabine and Campath-1H. Concern has been raised regarding the ability to mobilize sufficient peripheral blood progenitor cells (PBPCs) for autografting after purine analogues, and there are few data about PBPC collection after Campath-1H. In all, 16 CLL patients responding to sequential chemo-immunotherapy entered the study. In 10, mobilization regimen consisted of granulocyte colony-stimulating factor (G-CSF) 5-10 microg/kg/die. Patients failing mobilization or not achieving the target of 2.5 x 10(6) CD34+ cells/kg underwent a second attempt using intermediate-dose (ID) Ara-C, 800 mg/m(2) every 12 h for six doses+G-CSF. PBPC collection after G-CSF alone was successful in two out of 10 patients. An adequate number of CD34+ cells were collected after ID Ara-C+G-CSF in eight patients failing the mobilization with G-CSF alone and in five out of six who did not receive G-CSF before. Greater yields of PBPCs were collected with Ara-C+G-CSF compared with G-CSF alone (13.8 vs 3.3). The extrahematologic toxicity was manageable. In conclusion, PBPC collection is feasible in CLL patients treated with sequential therapy including fludarabine and Campath-1H. Excellent yields were obtained in 92.8% of patients primed with ID Ara-C+G-CSF.

A 43 year-old woman presented with fever, abdominal pain, epato-splenomegaly, ascites, cholestasis, anemia, thrombocytopenia and previous diagnosis of sclerosing cholangitis based on liver biopsy and endoscopic retrograde cholangiopancreatography(ERCP). The bone marrow biopsy and the revision of liver biopsy using antitryptase stain diagnosed systemic mastocytosis. Because of the aggressive course of the disease the patient was treated with an acute myeloid leukaemia chemotherapy regimen without success.

Although an increasing number of patients with chronic lymphocytic leukemia (CLL) may achieve complete remission (CR) relapse is almost inevitable, due to re-emergence of the malignant clone. The eradication of minimal residual disease (MRD) is associated with improved remission durability and has great potential in offering the possibility of cure in many lymphoid malignancies. The monoclonal antibody alemtuzumab is the foundation of many eradication-based treatment approaches because of its ability to achieve clinical remissions and to successfully purge MRD. We investigate the use of subcutaneous (sc) alemtuzumab for the eradication of residual disease in the bone marrow of patients in clinical response after fludarabine treatment, as determined by NCI criteria. At least 8 weeks after fludarabine treatment, 35 B-CLL patients (13 female, 22 male; median age 55 [range 39–64] years) received sc alemtuzumab, three times weekly for 6 weeks, at escalating doses up to 10 mg. Residual disease was assessed using a consensus polymerase chain reaction methodology to detect the clonality of IgH sequences. Patients obtaining a successfull peripheral blood stem cell (PBSC) harvest (CD34+ 2.5 x 109/l) after either granulocyte colony-stimulating factor (G-CSF) or intermediate-dose Ara-C (800 mg/m2, every 12 h, for six doses) plus (G-CSF) were considered eligible for autologous PBSC transplant. Pharmacokinetic parameters were estimated in seven patients. Serum samples of alemtuzumab were assayed by indirect immunofluorescence with target cells (HUT-78, a human T cells line) and antibody concentration was calculated by comparison with a standard curve. Post fludarabine responses were: 10 CRs, 11 nodular partial responses (PRn) and 14 partial responses (PR). Post alemtuzumab responses were: 29 CRs, 4 PRn, 2 PR. Overall, 51% of patients converted to polyclonal IgH pattern (MR). Of the patients in CR before alemtuzumab, 7 achieved MR; nine of the patients in PRn before immunotherapy, improved to CR, five achieving MR. Of the fourteen patients in PR before alemtuzumab, twelve improved (two PRn, ten CR) and six achieved MR. Twenty-three out of 25 patients successfully mobilized PBSC. Fourteen patients have been transplanted so far. Subcutaneous alemtuzumab was generally well tolerated; fifteen patients (57%) developed cytomegalovirus (CMV) reactivation, but CMV disease was prevented by prompt treatment with oral ganciclovir. Alemtuzumab serum concentrations measured at second week after the start of therapy, just before the next dose (Ctrough), were relatively stable: Ctrough = 0.79±0.6 mg/ml. Sequential treatment with fludarabine and alemtuzumab is feasible, effective and safe. Overall 83% of patients reached CR according to NCI criteria. Eighty-four percent of patients improved to CR or PRn after alemtuzumab. Residual disease was not detected in 51% of patients. Sequential treatment did not compromise PBSC mobilization, allowing patients to proceed to autologous transplantation.

C-Kit point mutations in core binding factor leukemias: correlation with white blood cell count and the white blood cell index

Mucormycosis infections, caused by fungi of the families Rhizopus, Mucor or Absidia, are typically rapidly progressive and often fatal. We report a 27-year-old male with acute myeloid leukemia (AML) developing an invasive pulmonary-CNS mucormycosis during the neutropenic period after salvage induction chemotherapy; the infection was successfully controlled with surgery and antifungal therapy. The patient received two courses of consolidation chemotherapy and underwent autologous stem cells transplantation (ASCT) while receiving secondary antifungal systemic prophylaxis with liposomal Amphotericin B (L-AMB, Ambisome). There was no clinical, radiological or microbiological evidence of mycotic reactivation during the bone marrow transplantation (BMT) procedure.

Molecular remission and reconstitution of a full chimera with arsenic trioxide in a patient with acute promyelocytic leukemia relapsed after allogeneic bone marrow transplantation

The aim of our study was to evaluate the impact of an early intensification programme including chemotherapy (CHT), autologous stem cell transplantation (ASCT) and radiation therapy (RT) in patients with primary mediastinal large B cell lymphoma (MLCL) with sclerosis presenting with adverse prognostic factors. Between 1993 and 1999, 19 patients with MLCL were referred to our institution. Four patients were classified as low risk according to the age-adjusted International Prognostic Index (AA-IPI). Fifteen (79%) were categorised in the high–intermediate or high risk group and were considered eligible for ASCT. Induction therapy consisted of VACOP-B (etoposide, doxorubicin, cyclophosphamide, vincristine, prednisone and bleomycin) for 12 weeks. After induction therapy the four low risk patients achieved a complete remission (CR) and did not undergo ASCT. Of the 15 poor risk patients, five achieved CR, seven partial remission (PR), and three showed refractory disease (RD). All these patients received mobilising therapy consisting of high-dose cyclophosphamide. After peripheral stem cell (PSC) collection, to obtain a greater tumor mass reduction before transplantation, the seven patients in PR underwent further treatment with high-dose etoposide and those with RD received two cycles of DHAP (dexamethasone, cytarabine and cisplatin). At the time of ASCT, seven patients were in CR, six in PR and two had RD. After transplantation using BEAM as preparative regimen, all patients but one achieved a CR. Seven patients with minimal (

Summary. Options for relapsed/refractory indolent lymphoma include chemotherapy, immunotherapy and high-dose therapy with autologous support. The best combination of these approaches, however, is not defined. We treated 10 patients with relapsed/refractory follicular (n = 7) or mantle cell lymphoma (n = 3) using chemotherapy, immunotherapy, high-dose therapy and autotransplant in a sequence of four phases, each designed to play a specific role in tumour eradication. After the debulking with VACOP-B (doxorubicin, cyclophosphamide, etoposide, vincristine, prednisone, bleomycin) (phase 1), 9/10 patients responded but none achieved a molecular response. After the immuno-chemotherapy phase, which combined Rituximab with vincristine and cyclophosphamide, seven patients were in complete response (CR) and three in good partial response (PR), and all those with a molecular marker of disease showed a disappearance of the signal from marrow and blood. Phase 3, which coupled high-dose cytarabine with Rituximab, was effective in mobilizing an adequate number of progenitor cells that were polymerase chain reaction negative in all informative cases. Phase 4 consisted of high-dose therapy with autologous support followed by two doses of Rituximab. Autograft was performed in nine patients. The haematopoietic recovery was as expected. This sequence of chemotherapy, immuno-chemotherapy, stem cell mobilization with in vivo purging and autotransplant, organized in four blocks of treatment, was simple to administer and devoid of toxic effects. It permits rapid attainment of clinical and molecular response and enables the harvest of lymphoma-free peripheral blood progenitor cells even in heavily pretreated patients with relapsed or refractory disease.

DCEP (dexamethasone, cyclophosphamide, etoposide, and cisplatin) has proved to be an effective salvage therapy for refractory-relapsed MM patients. Little is known, however, about its potential as mobilizing therapy. The aim of this study was to evaluate the efficacy of DCEP in mobilizing PBSC and to define its toxicity. Fifty-five MM patients received DCEP followed by G-CSF as part of high-dose programs including autologous transplantation. At the time of mobilization, 40 patients had previously received VAD only, and 15 alkylating agents. Mobilization was successful (minimum number of CD34(+) cells 2 x 10(6)/kg) in 48/55 patients (87%), and 41/55 patients (75%) collected >4 x 10(6)/kg CD34(+) cells. Of the seven patients who did not mobilize stem cells, five (71%) had been previously exposed to alkylating agents. The median number of CD34(+) cells harvested was 5.8 x 10(6)/kg (range 2.1-22.4). There was no treatment-related mortality. The side-effects of DCEP were always tolerable. No neutropenia

A G→T transversion at nucleotide 2467 of the c-KIT gene leading to Asp816→Tyr (D816Y) substitution in the phosphotransferase domain has been previously identified in a patient with rapidly progressing AML-M2 and mast cell involvement; the patient's blasts had a 47,XY,+4,t(8;21)(q22;q22) karyotype. Herein we confirm the simultaneous presence of both major chromosomal changes by multicolor fluorescence in situ hybridization (FISH) on interphase CD34+ mononuclear cells. By setting up culture leukemic blasts, spontaneous differentiation of adherent cells with mast-cell like features was proved by histochemical and immunoenzymatic analyses. Fluorescence in situ hybridization evidence of trisomy 4 confirmed the origin of differentiated cells from the leukemic blasts. Semiquantitative polymerase chain reaction (PCR) and phosphoimage densitometry of wild-type and mutated KIT alleles on bone marrow blasts made it possible to demonstrate that chromosome 4 trisomy led to a double dosage of the mutated KIT allele. This finding, and that of trisomy 7 and MET mutation in hereditary renal carcinoma represent the only cases of human tumors in which an increased number of chromosomes carrying an oncogene activated by point mutation have been detected.

To the editor: C-kit mutations in core binding factor leukemias Positivity for CD117(c-kit) expression is present in 80% of acute myeloblastic leukemia (AML) cases,[1][1] and has been associated with the expression of CD33, a marker of myeloid precursors, and CD13, a myeloid-associated antigen.[2

The SH2 domain-containing tyrosine phosphatase PTPN6 (SHP-1, PTP1C, HCP) is a 68 kDa cytoplasmic protein primarily expressed in hematopoietic cell development, proliferation and receptor-mediated mitogenic signaling pathways. By means of direct dephosphorylation, it down-regulates a broad spectrum of growth-promoting receptors, including the Kit tyrosine kinase, activated to elicit a prominent cascade of intracellular events by stem cell factor binding. The pivotal contribution of PTPN6 in modulating myeloid cell signaling has been revealed by the finding that shp-1 mutation is responsible for the overexpansion and inappropriate activation of myelomonocytic populations in motheaten (me/me) and motheaten viable (me(v)/me(v)) mice. Association of PTPN6 with c-Kit and negative modulation of the myeloid leukocyte signal transduction pathways prompted us to examine the expression of the protein tyrosine phosphatase PTPN6 gene in CD34(+)/CD117(+) blasts from acute myeloid leukemia patients. We identified and cloned cDNAs representing novel PTPN6 mRNA species, derived from aberrant splicing within the N-SH2 domain leading to retention of intron 3. Sequence analysis of cDNA clones revealed multiple A-->G editing conversions. The editing of PTPN6 mRNA mainly occurred as an A-->G conversion of A(7866), which represents the putative branch site in IVS3 of PTPN6 mRNA. Evidence that editing of A(7866) abrogates splicing has been obtained in vitro by using an edited clone and its backward clone generated by site-directed mutagenesis. The level of the aberrant intron-retaining splice variant, evaluated by semi-quantitative RT-PCR, was lower in CD117(+)-AML bone marrow mononuclear cells at remission than at diagnosis, suggesting the involvement of post-transcriptional PTPN6 processing in leukemogenesis.

The primary role of protooncogene c-kit in mast cell differentiation is supported by the development of mast cells from CD34+/CD117+(c-kit) myeloid precursors. Growth factor independence, neoplastic transformation and differentiation of mast cells were found in association with c-kit activating mutations in both murine and human mastocytoma and mast cell diseases. We have identified a novel c-kit mutation (D816Y) in peripheral blood mononuclear cells from a patient with AML (M2), massive presence of mast cells in bone marrow and rapid progression of the disease. The mutation, a G-->T transversion at nt 2467 of the c-kit gene resulting in Asp816-->Tyr substitution, corresponds to the D814Y and D817Y mutations identified and characterized in the murine P815 mastocytoma and the rat RBL-2H3 mast cell leukemia cell lines. The absence of SCF transcripts that we found by RTPCR in the patient's blasts indicates that, also in humans, this activating mutation leads to SCF independent growth. The expression of the mutant allele on Kit signaling may be further enhanced by trisomy of chromosome 4 (carrying the c-kit gene) in the patient's blasts. From these findings it is concluded that mast cells could be generated from a leukemic CD34/CD117-positive clone, that combines the antigenic expression of mast cell precursor to the growth and differentiation factor-independence which was derived by the c-kit D816Y mutation.

The translocation t(15;17)(q24;q21), unique to acute promyelocytic leukemia (APL), gives rise to PML/RAR alpha fusion transcripts detected by the sensitive reverse transcriptase-polymerase chain reaction (PCR) technique. PCR may help in the diagnosis and in monitoring minimal residual disease. Reversion of PCR to negative is obtained by chemotherapy (CT) alone or in combination with all-trans retinoic acid (ATRA). Here we show a serial PCR study of 10 APL cases. Five cases were studied at the time of diagnosis, and all were PCR positive for the rearranged transcripts (three bcr1 type, two bcr3 type). Seven cases in complete remission (CR) after one cycle of induction CT were persistently PCR negative, one case in CR after ATRA rescue was persistently PCR positive (bcr1 type), one patient (bcr3 type) relapsed 15 months after the PCR-negative CR and one patient died early. Seven patients underwent bone marrow transplantation (BMT) (five allogeneic, two autologous). One of them died early after take of the allogeneic BMT, the other six cases studied by serial PCR were persistently negative. At a median follow-up of 31 months (range 9-39), none of these six cases had relapsed. PCR data characterize the CR at the molecular level and evaluate the efficacy of different treatments, including BMT. The data may help to define a standardized schedule for PCR follow-up, and are also potentially useful to establish the time required before judging patients with persistently negative PCR to be cured. BMT as post-induction treatment in first CR is also discussed.

The Philadelphia chromosome t(9;22)(q34;q11) is a cytogenetic marker for chronic myelogenous leukaemia (CML), and is also present in some acute leukaemias. The translocation in CML gives rise to two BCR/ABL chimeric transcripts (b3a2 and b2a2) encoding a 210-kD tyrosine kinase protein. These leukaemia-specific transcripts can be detected easily by the reverse transcriptase polymerase chain reaction (PCR). PCR has improved the possibility of detecting minimal residual leukaemia cells in Ph-positive patients, especially after bone marrow transplantation (BMT). With PCR, we looked for BCR/ABL transcripts in 30 patients with CML and 4 with essential thrombocythaemia at time of diagnosis, finding a significant difference in the platelet counts of CML patients carrying b3a2 or b2a2 transcripts. The BCR/ABL transcript was monitored by PCR in 6 CML patients after BMT. The usefulness of PCR in clinical practice at time of diagnosis, and the biological and clinical significance of positive/negative PCR results, in patients with transplants, are discussed.

Ninety-eight untunnelled central venous catheters were positioned in the subclavian or internal jugular vein in 42 patients with acute leukemia. The procedure was feasible at bedside and carried a risk of catheter-related infection of 21%, similar to that of tunnelled catheters. The persistence of exit-point infection is the main route of infection and requires catheter removal.

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 vitro, AML blasts are capable to produce and release the α-protryptase constitutively. Recently, elevated levels of serum tryptase have been detected in certain FAB subtypes of AML, particularly in AML-M4eo. In an attempt at correlating the levels of tryptase with FAB classification and cytogenetics, we have analyzed serum samples collected at diagnosis, from 103 AML and 57 ALL patients referred to our Institution. 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. We found elevated tryptase levels (more than 15 ng/ml) in 46 out of 103 AML-patients (44.6%) and in 1 out of 57 ALL-patients (1.75%) (p = 89.3 ng/ml) (p = 0.0001), in comparison with other cytogenetic groups (diploid, n= 43; t(15;17), n=10; other, n=29). Our data suggest that elevated serum tryptase levels at diagnosis may play a role as a marker for CBF AML subtypes.

Contamination of reinfused stem cells with leukemic cells is a major concern affecting outcomes after ASCT in patients with CLL. In fact, outcomes after ASCT strongly correlate with disease status before transplantation. Persistence of minimal residual disease (MRD), as detected by consensus primer PCR, or the switch from a negative to a positive MRD status during follow-up, are both highly predictive of clinical relapse after ASCT. In order to minimize the risk of contaminating the collection with leukemic cells, in our institution patients who undergo an ASCT receive alemtuzumab before peripheral blood stem cell (PBSC) mobilization, to purge in vivo any residual disease. We evaluated the outcome of ASCT in 20 CLL patients who were pretreated with fludarabine (FAMP) containing regimens and subsequently received alemtuzumab SC (10 mg x 3/w for 6 weeks) to purge MRD. A FAMP containing regimen had been administered as first-line treatment in 18 cases and as second-line in 2 cases; median number of FAMP cycles administered was 6 (range, 4–8). All but 1 PBSC patient was mobilized with Ara-C (800mg/m2/12h x 3 d) followed by G-CSF, while the last patient received only G-CSF. Median age at transplant was 56 y (range, 45–65); all patients (pts)were in CR based on the NCI-WG criteria. Of 20 pts who underwent PBSC harvest, polyclonal IgH rearrangement was evident in 13 pts (65%), as assessed by PCR. The conditioning regimen consisted of 12 Gy TBI plus cyclophosphamide 120 mg/kg in 14 pts < 60 yrs, and Melphalan 180 mg/sqm in 6 pts >60 years. Median number of CD34+ cells reinfused was 17.4 x106/kg (range, 3.1–30.4), and in 13 cases the reinfused product was polyclonal for IgH. The median time for PMN (>500/ml) and PLT (>20000/ml) recovery was 9 (range, 8–11) and 11 (range, 9–13) days respectively. During marrow aplasia 13 patients experienced an episode of fever >38°C with a median duration of 2 days (1–8); in 3 pts the fever was of unknown origin, in 8 cases sepsis was due to Staphilococcus epidermidis, and in 1 case it was due to P. aeruginosa. Intravenous antibiotics were administered in 11 cases, and only 1 patient required intravenous antifungal therapy. One patient died due to a pulmonary fungal infection sustained by Aspergillus Terreus. No incidence of grade 3–4 nonhematologic toxicity was observed. During the 3 months post-transplant 2 pts required hospitalization: 1 for a fever, and the other for acute polyneuropathy. No pathogens were isolated in either case. At 11 months post-transplant 1 patient developed thrombocytopenia. None of the pts developed CMV reactivation, even in the 9 cases, which became CMV positive during alemtuzumab treatment. Herpes Zoster was observed in 2 patients at 5 and 10 months after transplant. At a median of 28 months after receiving alemtuzumab (range, 15–48 months), and a median 17 months after PBSC transplantation (range, 1–41 months), 19 pts are in CR. At the 9- and 12-month post-transplant evaluation, performed on 16 and 12 pts respectively, all but 1 patient showed polyclonal IgH rearrangement. ASCT after sequential treatment with FAMP and Campath-1H is feasible with no significant increase in major infections; a substantial number of patients achieved a sustained polyclonal IgH rearrangement after transplant.

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