Abstract Abstract 3653 Background. The rearrangement Bcl2/IgH is detectable by PCR in more than half of patients with follicular lymphoma. Different authors reported on the long-term prognostic impact of the persistence of this rearrangement (minimal residual disease or MRD). In this study, we report about MRD significance in 534 patients randomized to receive 8 doses of Rituximab associated with 8 cycles of CVP, or 6 cycles of CHOP or FM, and no maintenance in the context of the Fondazione Italiana Linfomi (FIL) multicenter phase-III “FOLL05” trial (NCT00774826). Patients and methods. For qualitative PCR analysis, samples were centralized. Quantitative assays were performed by the 4 laboratories of the “FIL MRD NETWORK”. Qualitative and quantitative PCR were performed according to the Euro-MRD guidelines. Results. At baseline, 424 out of the 504 patients enrolled were assessed for Bcl2/IgH and 223 (52,6%) were Bcl2/IgH positive (molecular marker=MM). No significant differences were detected between cases with and without molecular assessment at the enrollment or after therapy, and those with positive or negative MM for the main clinical and prognostic features, and for treatment allocation. At the first time-point (within 6 weeks after the end of therapy), 153 of the 223 cases PCR-positive at enrollment were re-assessed: 110 (69,6%) achieved PCR negativity. MRD was not significantly correlated to clinical features or quality of clinical response. Three-year PFS was better for patients achieving PCR negativity at the sixth week than those retained MM (69% vs 50%), but this difference was not statistically significant. On the contrary, PFS was significantly conditioned by the PCR status at 12 and 24 months (87 and 66 cases evaluable), with 3-year PFS of 66% for cases PCR-negative versus 41% for those PCR-positive at 12 months (p=0.015, see Figure 1), and 84% vs 50% (p=0.014) at 24 months. Moreover, the probability of being PCR-negative by 24 months was significantly lower for cases receiving R-CVP (18% vs 41% in the R-CHOP and 41% in the R-FM, p=0.035). PCR-negativity resulted in better PFS both in CR and in PR patients (3-year PSF=72% for cases CR/PCR- vs 32% for those CR/PCR+ vs 62% for those PR/PCR- and 25% for patients in PR/PCR+; p=0.001). When PCR negativity in patients with or without complete response and PCR negativity by 12 months were considered in a multivariate analysis together with performance status, FLIPI, response, and age, the PCR negativity retained its favorable impact on PFS (HR=-1.2, 95% CI:0.32–0.19, p=0.001). Finally, in 105 cases tumor burden at the diagnosis was assessed by quantitative PCR. The median value was 3 × 10−3 copies; cases displaying values <1 × 10−4 showed a clear advantage in terms of PFS (3-year PFS of 80% vs 59% for those with >1 × 10−4 copies p=0.015). A significant MRD clearance was observed at the end of therapy: the mean value of Bcl2/IgH was 2 × 10−1 copies before therapy versus 5 × 10−3 at the first time-point. No differences in the entity of the MRD clearance were observed according to the arm of treatment. Conclusions. In conclusion, this study shows that R-CHOP and R-FM are more effective to clear MRD in follicular lymphoma compared to R-CVP. Moreover, the most prominent impact on PFS is exerted by the molecular status detected at least 6 months after the end of therapy. This observation is in line with the hypothesis of a quite slow, but long-term sustained, MRD clearance exerted by rituximab-based therapies. Finally, this study shows that patients with <1 × 10−4 copies of Bcl2/IgH at diagnosis would have longer PFS, thus supporting the usefulness of a quantitative PCR monitoring at diagnosis. Disclosures: No relevant conflicts of interest to declare.

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).

A 55-year-old woman presented with staphylococcal toxic shock syndrome (TSS). During the course of the disease a significant lymphocytosis appeared, and a high number of TcRalphabeta+CD3+CD4-CD8- (double-negative, DN) lymphocytes was observed both in bone marrow and in peripheral blood samples. Correction of the altered lymphocyte immunophenotype was observed only 6 weeks after recovery from TSS. The immunophenotype of circulating and bone marrow lymphocytes was also studied during a phase of an aspecific febrile episode observed 2 months after recovery, but no subset of DN lymphocytes was found. A small subset of DN lymphocytes can be found in normal bone marrow, liver, thymus, and skin. These cells show peculiar immune regulatory properties and can increase in certain autoimmune diseases. Our findings may represent a specific effect of lymphocyte stimulation by the staphylococcal exotoxin, which is the effector agent of TSS.

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-beta(1) (TGF-beta(1) ) 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.

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.

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.

Large granular lymphocyte proliferative status represents a group of clonal and nonclonal lymphoproliferative disorders of natural killer (NK) or T-cell lineages with common morphological features. Cellular differences may sustain the clinical polymorphism observed in these disorders. Here we report a case of large granular lymphocyte disease unusually expressing CD4+CD8+ clonal T cells and atypical cell morphology in bone marrow.

Heparin, heparan sulfate and chondroitin sulfate were evaluated for their possible role on proliferation and differentiation of hematological precursor cells from cord blood. For these purposes, different concentrations of glycosaminoglycans were added to methyl-cellulose in colony assay performed with human cord blood derived cells. A volume of 10 μg/ml heparin induces a significant increase of both granulocyte–monocyte and granulocyte colonies, and a decrease of erythroid-colonies, more evident in the presence of 100 μg/ml. Heparan sulfate-treatment induces a significant increase of all granulocyte-monocyte colonies derived from CFU-granulocyte-monocyte, CFU-granulocyte and CFU-monocyte precursors. A significant decrease of multipotent cells was also observed. On the other hand, chondroitin sulfate induces an increase of granulocyte-colonies and a decrease of erythroid-colonies. Glycosaminoglycans with different structure may be useful to increase the number of specific colonies. The selective and differential binding of glycosaminoglycans with several growth factors and the regulation of their activities is discussed.

Arsenic trioxide has recently been reported to be successful in the treatment of promyelocytic leukemia. Several concerns about the use of this toxic agent are currently reducing its potential clinical use even in severely ill patients. In this report we describe the results achieved by As2O3 with all-trans retinoic acid in a patient suffering from secondary, relapsed, resistant promyelocytic leukemia. Several complications, including sepsis and an extensive area of skin necrosis, did not allow us to treat the patient further with chemotherapy. With As2O3 and ATRA therapy, the patient obtained a complete molecular remission without any significant toxicity and, subsequently, it was possible to perform a bone marrow autograft in a state of complete remission.

Multidrug resistance is frequently detected in haematological malignancies and in acute leukaemias with a poor prognosis. In the last few years, several reports seem to suggest that the new anthracycline derivative idarubicin and the anthraquinone mitoxantrone have some advantages in the management of untreated or relapsed acute leukaemias compared with older anthracyclines. This could be due to a different interaction of these drugs with multidrug resistance. To evaluate this possibility, we compared the activity of doxorubicin (DOXO), epirubicin (EPI), idarubicin (IDA) and mitoxantrone (MITO) on a murine, multidrug resistant, leukaemic cell line (P-388/Dx) cultured in vitro. id50 of IDA and MITO was in the ng range whereas that of DOXO and EPI was in the μg range. Moreover, IDA has a resistance index of 50 whereas DOXO has one of 250. Verapamil is able to almost completely abolish the resistance to IDA. Efflux experiments confirm that verapamil increases IDA intracellular concentration. IDA and MITO appear to be less involved in multidrug resistance than older anthracyclines.

BACKGROUND Lymphocytes from patients affected by B-cell chronic lymphocytic leukemia (B-CLL) have frequently been shown to be positive for the multidrug resistance (MDR) phenotype. However, this phenotype does not seem to be responsible for the resistance to alkylating agents usually employed in the management of CLL. METHODS Lymphocytes from 42 patients were evaluated by flow cytometry for P-170 expression and by spectrophotometry for glutathione-S-transferase (GST) activity. RESULTS Our findings show that GST is not related to any clinical parameter but is increased in treated patients. Conversely, 85% of patients were positive for P-170 and this was related to the percentage of CD5/CD19-positive lymphocytes. CD5/CD19-negative patients were also negative for P-170. MDR was not related to any clinical parameter evaluated nor to GST activity in lymphocytes. CONCLUSIONS MDR is constitutively expressed in B-cell chronic lymphocytic leukemia and seems to be related to a CD5/CD19 B-CLL phenotype. The increase of GST activity in treated patients is statistically significant (p < 0.005).

Vitamin D binding protein (Gc) is present on the surface of several blood cells and may interfere with the activity of 1,25(OH)2D3. It has previously been reported that Gc may bind to the U-937 line which is known to differentiate upon exposition to 1,25(OH)2D3. In the present paper, we evaluate the expression of Gc on the surface of U-937 and HL-60 lines. Both cell lines did not express Gc on their surface but U-937 cells were able to bind human purified Gc added to the medium whereas HL-60 were not. After culturing with 1,25(OH)2D3, HL-60 became able to bind Gc. This property seems to be related to the monocytic differentiation induced by 1,25(OH)2D3. Conversely, when present together, 1,25(OH)2D3 reduces binding on U-937.