Search

JC virus (JCV) is a double-stranded DNA virus belonging to family Polyomaviridae. It causes progressive multifocal leukoencephalopathy (PML), mainly in immunosuppressed people. JCV had been shown to require the serotonin 2A receptor for host cell entry. We report a case of clinical, neuroradiological and virological response of biopsy-proven PML in a 33-year-old comatose woman after treatment with the anti-psychotic drug risperidone. Since risperidone is the tightest binding of current drugs to this receptor we think this may have blocked JCV entry in our patient, allowing her immune recovery and viral clearance.

PS-341 (Bortezomib) is a novel dipeptide boronic acid proteasome inhibitor with in vitro and in vivo antitumor activity that induces apoptosis in different human cancer cell lines. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, has been reported to induce apoptosis of leukemic cells by increasing the cyclin-dependent kinase inhibitor p21 or generating reactive oxygen species (ROS). Co-exposure of BCR/ABL-positive cells both sensitive and resistant to imatinib to these compounds has been previously reported resulting in an increased apoptotic rate. To extend this observation, we examined the effect of treatment with bortezomib or/and SAHA of a megakaryoblastic cell line (MO7-e). Cell proliferation, ROS production, cell cycle progression, induction of apoptosis and differentiation has been investigated. Bortezomib was shown to retain NF-kB in the cytoplasm and inhibit cell growth (IC50=20nM), in a dose/time-dependent way. This anti-proliferative activity resulted to be lineage-specific, because other leukemic cell lines were unaffected by the bortezomib treatment. Moreover, bortezomib in MO7-e cells increased ROS production and induced a significant pro-apoptotic effect (50% vs 5% in control). Finally, cell cycle was blocked in the G2 phase and bortezomib was able to down-regulate WT1 expression, gene that could play a relevant prognostic role in myeloproliferative disorders. Moreover, any significant effect on cell differentiation was found. SAHA also resulted able to inactivate NF-kB and to inhibit cell proliferation, at 1.5 uM. It did not increase significantly ROS production, blocked cell cycle in the G1 phase and down-regulated WT1 expression (10 fold minus than bortezomib). Neverthelles, SAHA also did not induce differentiation of megakaryoblatic cells. Co-exposure of this cell line to minimally toxic concentrations of bortezomib (5 nM) and SAHA (0.5 uM), resulted in a significant increase of anti-proliferative (50% of growth inhibition vs 15% with bortezomib and 10% with SAHA alone) and pro-apoptotic effect (45% vs 20% of bortezomib and 15% of SAHA alone). Interestingly, immunocitochemistry assays detecting the NF-kB p65 subunit showed that the co-exposure to bortezomib and SAHA resulted in a minor NF-kB inactivation than that achieved with single compounds. This finding was confirmed by the pre-incubation of MO7-e cells with SAHA in respect of the pre-incubation with bortezomib or the simultaneously addition of the two drugs. In fact, pre-incubating megakaryoblasts with SAHA, the anti-proliferative effect of bortezomib significantly decreased. In conclusion, this study supports the association of a proteasome with a histone deacetylase inhibitor, in a time-sequence-related way, especially in chronic myeloproliferative disorders where a spontaneous NF-kB activation and a WT1 over-expression have been reported.

TCR gamma/delta profiles were analyzed in 13 multiple myeloma patients after allogeneic non-myeloablative transplantation. Results show that both aGVHD and minimal residual disease (MRD) eradication did significantly affect TCR gamma/delta profile. During follow-up, six patients developed an aGVHD episode; in five of them, this event fitted with a modification of the TCR profile. Eleven patients achieved PCR-negativity during follow-up. In the 90% of them, the appearance of a new predominant TCR peak was concomitant to the disappearance of the IgH clone. These results suggest that different T gamma/delta populations would sustain GVM and GVH effects after non-myeloablative allogeneic transplant.

In this study we serially evaluated the chimerism status in 20 multiple myeloma patients allotransplanted with a reduced intensity regimen. All patients engrafted, with total 75% overall responses and 35% of CRs. After a median follow-up of 35 months, seven patients (35%) died, three of them due to disease progression. Four patients died before day +100, with a TRM of 20%. Nine patients (45%) developed aGVHD and six (40%) had cGVHD. Twenty-five percent of patients achieved full donor chimerism (FDC) before day +100, 42% before day +200 and 75% 24 months after graft. In our series, level of chimerism did not correlate with either the quality of response or aGVHD. No significant differences were found between bone marrow and peripheral blood samples. Analogously, even if donor DNA percentage often resulted higher in the PMN fraction than in the mononuclear one, these differences were not significant after statistical analysis. On the other hand, cGVHD was associated with increased rates of FDC, with 6/6 cases showing a full donor pattern in concomitance of the cGVHD versus 5/9 cases presenting a FDC in the group of patients without cGVHD (p=0.057). The Kaplan-Meier estimates of OS and PFS at 2 years were 59% and 58%, respectively; chimerism pattern did not impact in the predicting clinical outcome. In summary, our study shows that a stable engraftment and high frequency of donor chimerism are achievable after a reduced intensity conditioning regimen. Moreover, even as result of a single center experience, we suggest that chimerism, graft-versus-myeloma and GVHD would represent distinct entities that require larger immunological studies for further clarification.

Different mechanisms could sustain Imatinib resistance, including overexpression of MDR1, a gene already known to be responsible for multidrug resistance in other hematologic malignancies. In search for a possible correlation, BCR-ABL and MDR1 expression were measured in 115 serial bone marrow samples from 33 CML patients during Imatinib treatment. All patients achieved complete hematologic responses, and 22 patients also achieved complete cytogenetic responses, with median BCR-ABL mRNA values significantly lower than those observed in the group of cases that were persistently Philadelphia positive. All three cases treated during the accelerated phase showed disease progression after an initial period of remission; all presented either increased levels of BCR-ABL or MDR1 3 months before clinical progression. In the subgroup of cases treated during the chronic phase, BCR-ABL and MDR1 levels were significantly correlated after 3 and 6 months (88 and 80%, respectively) but not after 12 months of treatment (32%). Reported data maintain that MDR1 expression would play an important role in Imatinib resistance when the disease is not fully controlled (e.g., progressive disease or during the first months of treatment).

Granulocyte function may be altered after in vivo G-CSF administration and this has been related to both an immaturity of mobilized cells and to a defect in F-actin polymerization. In this paper we show that in resting Filgrastim (non-glycosylated G-CSF)-pulsed cells, F-actin polymerization, membrane-linked RhoA and cell polarization are enhanced compared to those found in resting Lenograstim (glycosylated G-CSF)-cells. The basal hyper-activation of RhoA could be responsible for the morphological and functional modifications of Filgrastim-mobilized cells. Moreover, Filgrastim-mobilized cells, but not Lenograstim-mobilized cells, are unable to correctly respond to LPS stimulation, as demonstrated by minor further RhoA activation and cell elongation.

We report the case of the development of two different stages of the same clonal disorder in two patients sharing the same bone marrow due to a previous bone marrow allotransplant. The transplanted patient developed severe aplasia with myeloid blasts, different from those of the previously cured leukemia. Chimerism evaluated by microsatellite analyses confirmed a full donor phenotype. At the same time, the donor of the bone marrow transplantation developed a refractory anemia with excess blasts. We speculate on the presence of an undetectable pre-existing pathological clone in the transplanted bone marrow, which have evolved in the two patients.

Purine analogues have dramatically improved the outcome of patients affected by hairy cell leukemia (HCL), although complete eradication of disease was achieved in few cases. The purpose of this study was to evaluate the role of Rituximab in eradicating minimal residual disease (MRD) in HCL patients after a pre-treatment with 2-chloro-deoxy-adenosine (2-CdA). Ten patients received four cycles of Rituximab after administration of Cladribrine. Before starting anti-CD20 antibody, two patients were in complete remission, six in partial remission and two showed no significant response to Cladribrine. All cases resulted IgH-positive. Median time from the last 2-CdA infusion was 5.7 months. Eight of 10 patients [four in partial remission (PR), two in complete remission (CR) and two unresponsive after 2-CdA] were evaluable for response. Two months after the end of anti-CD20 therapy, all evaluated patients presented a complete haematological remission. Moreover, Rituximab increased percentage of molecular remission up to 100% 1 yr after the end of treatment. Interestingly, in all cases but one, including those persistently polymerase chain reaction (PCR)-positive, semi-quantitative molecular analyses showed MRD levels lower than those found before Rituximab administration. Toxicity was very mild. The present results not only confirm the therapeutic effect of Rituximab, but also show its relevance in eradicating MRD in HCL.

Multidrug resistance (MDR) is a complex phenomenon that includes the expression of many different genes regulating drug transport or metabolism, cellular repair or detoxification mechanisms. The co-expression of several genes could be at the basis of the resistant phenotype in vivo. In order to test a possible prognostic role of the expression and co-expression of several MDR-related genes (MDR1, topoisomerase IIalpha, topoisomerase IIbeta, MRP, GSTpi, LRP), 35 patients affected by acute myeloid leukemia (AML) were tested by RT-PCR assays. In our series, topoisomerase IIbeta was significantly co-expressed with MRP (p = 0.05), GSTpi (p = 0.017) and LRP (p = 0.005). GSTpi was co-expressed with LRP (p = 0.03) and MRP (p = 0.007); on the other hand, 53.8% of patients were LRP and MRP-positive (p = 0.02). The PCR-positivity did not differ according to biological/clinical characteristics of patients, including age; this latter was the only parameter conditioning the response and overall survival. Neither the expression nor the co-expression of the tested genes was significantly correlated with the response to the induction treatment and long-term outcome.

Idiopathic myelofibrosis (IMF) is a clonal stem cell disorder characterized by reactive fibrosis of bone marrow sustained by a complex cytokine network. At present, no efficacious therapy for this disease exists. Synthetic carboxy-terminal pentapeptide of osteogenic growth factor (sOGP10-14) can increase bone marrow cellularity and the number of haematopoietic colonies; this study evaluated the activity of sOGP10-14 in IMF. Fragments of bone marrow biopsies from patients affected by IMF were cultured with or without the addition of sOGP10-14. Cellular density was evaluated by image analysis, and transforming growth factor-beta1 (TGF-beta1) concentration was immunologically assayed in the supernatant of cultured bone marrow biopsies. The proliferation rate of the megakaryoblastic M07-e cell line, cultured in the presence of either granulocyte-macrophage colony stimulating factor or thrombopoietin (TPO), and with or without sOGP10-14, was evaluated. Megakaryocyte colony forming unit (CFU-Mk) assay was performed on bone marrow samples of IMF patients with or without sOGP10-14. After 14 d, bone marrow cellularity was significantly increased in samples cultured with the pentapeptide. Moreover, sOGP10-14 induced a significant increase of TGF-beta in culture supernatants. TPO-primed proliferation of M07-e was reduced by sOGP10-14, and the pentapeptide significantly reduced CFU-Mk on IMF bone-marrow-derived cells. sOGP10-14 increased ex vivo bone marrow cellularity in IMF. This action could be related to the megakaryocyte inhibition induced by the interference of this pentapeptide with growth factor activities. These findings suggest that a deficiency of osteoblast-related factors may play a role in bone marrow failure in IMF.

Osteogenic growth peptide (OGP) is a peptide exerting regulatory effects on the bone and on bone marrow. The carboxy-terminal pentapeptide (OGP10-14) is the biologically active portion of OGP. We evaluated OGP10-14 hematopoietic activity performing colony-forming tests on human stem cells derived by bone marrow, peripheral blood and cord blood. Granulocyte-macrophage colony-forming unit (CFU) were significantly increased in OGP10-14-treated samples, while granulocyte-erythrocyte-monocyte-megakaryocyte CFU and burst-forming unit (BFU) erythroid were increased only in the cord blood test.Moreover, OGP10-14 preserves stem cells self renewal potential in long-term culture (LTC) initiating cells and acts directly on CD34+ enriched cells or by increasing activity of stem cell factor (SCF) and granulocyte-megakaryocyte colony-stimulating factor.

Osteogenic growth peptide (OGP) increases blood and bone marrow cellularity in mice, and enhances engraftment of bone marrow transplant. Carboxy-terminal pentapeptide (OGP10-14) holds several properties of full-length polypeptide. We evaluated whether synthetic OGP-derived pentapeptide (sOGP10-14) has some activity on peripheral blood cell recovery after cyclophosphamide-induced aplasia, and on stem cell mobilization. Peripheral blood stem cell (PBSC) mobilization was evaluated by administering granulocyte-colony stimulating factor (G-CSF) or sOGP10-14 after cyclophosphamide (CTX) injection. Haematological parameters and CD34/Sca-1 positive cells were sequentially evaluated. Colony-forming tests were performed in bone marrow cells from CTX-, G-CSF- and sOGP10-14-treated mice. sOGP10-14 was able to enhance band cells and monocyte recovery after cyclophosphamide administration. White blood cell (WBC) counts reached the maximum peak by day +10 but, on day +7, a significant recovery was already detected in sOGP10-14 treated mice. On day +10 the WBC increase in sOGP10-14-treated mice was comparable to that found in G-CSF treated ones. Moreover, CD34/Sca-1 positive early precursors were significantly mobilized by sOGP10-14 compared to the control group. In sOGP10-14-treated mice, the colony-forming unit-granulocyte-macrophage-megakaryocyte (GEMM-CFU) and burst-forming unit-erythroid (BFU-E) were significantly increased in bone marrow cells in comparison to mice treated with CTX only. These results suggest a central role of sOGP10-14 in bone and bone marrow interaction, and a possible role of sOGP10-14 as a mobilizing agent.

Seventy-two patients with non-Hodgkin's lymphoma were evaluated for the presence of molecular markers (IgH, bcl-1, bcl-2 rearrangement) on bone marrow, at diagnosis and after PBSCT, and on harvests in order to find a possible predictive role of minimal residual disease on treatment outcome. At diagnosis, 41 (59%) out of 69 available bone marrows showed molecular involvement. Fifty-six percent of leukaphereses were involved, mainly indolent lymphoma (P = 0.001) or advanced disease (P = 0.01). Ex vivo purging cleared only one stem collection out of 31 PCR-positive leukaphereses. Aggressive lymphomas showed both a longer overall survival (OS) (P = 0.03) and relapse-free survival RFS (P = 0.02) when transplanted with unpurged stem cells, whereas indolent NHL survival was not influenced by ex vivo purging. Twenty out of 26 samples taken during follow-up had bone marrow involvement at diagnosis. Of these, 15 cleared their bone marrow; both OS and RFS were significantly longer in the PCR-negative cases (P = 0.05 and P = 0.005). At 1 year after PBSCT, 75% of patients were PCR negative, with 50% molecular remissions; the relapse rate was 55% for patients still PCR positive vs 29% for those who were PCR negative. Thus, after high-dose chemotherapy, close molecular monitoring of MRD using qualitative PCR techniques seems to represent a reliable prognostic indicator.

Granulocytic sarcoma and subsequent acute leukemia recurrence with different biologic characteristics 5 years after allogeneic bone marrow transplantation for acute myeloid leukemia

A 55-year-old female with standard risk AML in second CR received an allogenic transplant from an HLA-matched sibling, using a nonmyeloablative conditioning regimen (NMST). On day +139, she rejected her graft with autologous reconstitution. She received a second NMST from a different HLA-matched sibling with an identical conditioning regimen and immunosuppression. On day +110, she rejected the second graft, with autologous reconstitution with blasts. She received a third allograft from the first sibling with a myeloablative busulfan-based conditioning regimen. She is now day +270, in CR, with full donor chimerism.

Abstract 1371 Background: IGHV1-69 gene identifies the most frequent IGHV gene in chronic lymphocytic leukemia (CLL) and identifies the paradigm of unmutated CLL (U-CLL), being used in roughly 1/3 U-CLL. It is often rearranged to form subsets of stereotyped HCDR3 patterns, likely selected and transformed from the natural naïve B-cell repertoire (Blood. 2010; 115:71-7). Being unmutated, IGHV1-69 CLL are hypothetically expected to have competent tumor B-cell receptors (BCR) and to progress more rapidly. However, it has not been investigated if progression occurs similarly in all the subsets. Aims: we aimed to investigate the prognostic significance of mutational status and of stereotypic B-cell receptors in IGHV1-69+ CLL. Methods: Nucleotide sequences of the tumor IGHV1-69/D/J rearrangement, clinical and molecular prognostic parameters at diagnosis and clinical status at follow-up of 294 IGHV1-69+ CLL patients were obtained from 22 hematological Institutions in Italy. CLL B-cell derived IGHV1-69 rearrangements were scanned for HCDR3 stereotypic patterns and assigned to subsets according to the criteria by Murray et al (Blood. 2008; 111: 1524–1533). Enpoint of outcome was time to progression requiring first treatment according to NCI criteria (TTFT) in Rai stage 0 CLL. Results: Of 294 IGHV1-69+ CLL, 264 (89,8%) were unmutated, 168 (57,1%) were assigned to subsets, of which subsets 7 (n=23, 7,8%), 6 (17, 5,8%), 3 (13, 4,4%), 5 (10, 3,4%) and 9 (10, 3,4%) were the most frequent. CD38, ZAP70, normal or sole del13, +12, del11 and del17p scored positive in 109/264 (58,7%), 139/245 (56,7%), 128/248 (50,5%), 51/248 (20,6%), 43/248 (17,3%) and 34/248 (13,7%). CLLs were reclassified as 18 (6,1%) clinical MBL, 101 (34,4%) Rai 0, 155 (52,7%) Rai I-II and 20 (6,8%) Rai III-IV CLL. Subset 6 was also UM in 16/17 (94,1%) cases. Prevalence of CD38 (p Conclusions: our analysis documents and confirms that unmutated status of IGHV, and not stereotypy, is a relevant prognosticator of outcome (TTFT) in CLL. In the IGHV1-69 CLL it exclude a role of IGHV gene use for CLL progression. However, the good prognosis of Rai 0 U-CLL assigned to subset 6 suggests a differential clinical benign course of this particular subset, irrelevant of unmutated status. One possibility is that the IGHV1-69/D3-16/J3 rearrangements of subset 6 produce a tumor-specific BCR with stereotypic HCDR3 patterns that are anergized by antigen while circulating in the peripheral blood in early stage (Rai 0) CLL. Disclosures: No relevant conflicts of interest to declare.

Although novel therapies for chronic lymphocytic leukemia have resulted in higher hematologic response rates, the complete eradication of disease rarely occurs. Alemtuzumab (Campath-1H) seems to be extremely effective in this role in pretreated patients. The authors used a molecular semiquantitative polymerase chain reaction (PCR) method to assess the ability of alemtuzumab to induce PCR negativity in eight patients pretreated with fludarabine. IgH rearrangement was coamplified with a housekeeping gene and fluorescent PCR products were analyzed on a DNA automatic sequencer. Each patient was evaluated at diagnosis, after fludarabine, and after Campath-1H. The median interval between the last therapy course with fludarabine and the start of Campath-1H was 14 weeks. Patients received subcutaneous doses up to 10 mg, three times a week, for 12 weeks, with a median dose of 190 mg. After six cycles with fludarabine, only one patient (12.5%) achieved molecular remission, and in three other patients IgH levels decreased by 0.5 to 1 log. At the beginning of Campath-1H administration, all patients were PCR positive, including the one previously found to be negative. At the end of treatment, five patients achieved molecular remission (62.5%), four of them within 1 month after the end of therapy. Seventy-two percent of responses, with 43% of complete responses, were documented on bone marrow smears. A significant reduction of lymph node and spleen diameters was noted in 50% and 33% of patients, respectively. Four patients showed grade 2 skin reaction at the site of the subcutaneous injection and grade 1 or 2 fever. Two patients developed neutropenia (grade 2 and 3) and two hemolytic episodes. Three patients showed cytomegalovirus and one herpes zoster and Epstein-Barr virus reactivation. These results show that Campath-1H represents an efficacious in vivo purging tool with a safe profile.