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Introduction Diagnosis of myelodysplastic syndromes (MDS) is usually challenging. In this context, we have attempted to employ data derived from automated analysis of bone marrow (BM) samples as an ancillary tool for the discrimination between reactive marrow and MDS. Methods A total of 101 BM anticoagulated samples referred for flow cytometry (FCM) analysis on the clinical suspicion of MDS had been previously counted in a Mindray BC‐6800 hematology analyzer (testing set). Among them, 22/101 randomly selected BM samples (comparison set) had been also simultaneously counted by an Advia 2120 and a CELL‐DYN Sapphire hematology analyzer. Selected parameters obtained by Mindray BC‐6800 were retrospectively evaluated with ROC and regression analysis in an attempt to formulate a discriminative scoring system (SS) for MDS. This system was further evaluated in the comparison set. Results The diagnosis of MDS was established in 37/101 patients assessed (“MDS” group). Three patients were diagnosed with myelodysplastic/myeloproliferative neoplasm (MDS/MPN), while 61 revealed a “reactive” bone marrow (“RBM” group). Statistical analysis revealed significant differences in Hb, RDW‐CV%, NRBC%, and RET% values between the “MDS” and the “RBM” group. Specific cutoff values were then indicated and employed for the formulation of a SS of high sensitivity (86.84%) and specificity (86.89%). The encouraging performance characteristics of the proposed SS were also confirmed in the BM comparison set. Conclusion Automated BM counts on hematology analyzers contributed to the formulation of a SS for the screening discrimination between reactive and MDS BM fluids, which seems to be applicable and informative, regardless of the analyzer used.

Amifostine (AMF) promotes in vitro growth and survival of hematopoietic progenitors. In this study we evaluated the efficacy of AMF in the treatment of anemia in patients with low-risk myelodysplastic syndromes (MDS) and the possible predicting value for response to AMF therapy of two types of in vitro clonogenic assays. Two different doses of AMF, 300 mg/m2 (group A, 11 patients) or 400 mg/m2 (group B, 16 patients), were studied. AMF was given three times weekly for 3 weeks, i.v., followed by 2 weeks off therapy. Patients were evaluated after two cycles of treatment. Partially or nonresponding patients of group A received 400 mg/m2 AMF and were reevaluated. An increase of hemoglobin (Hb) values of more than 2 g/dl and a 100% decrease in transfusion requirements for at least 6 weeks were defined as a complete response (CR) while an increase of Hb values of 1-2 g/dl or a 50% decrease in transfusion requirements was considered as a partial response (PR). In group A, two out of 11 (18.1%) patients achieved a CR with the initial dose and one of the nine that received 400 mg/m2 AMF achieved a PR. In group B, three out of 16 (18.7%) patients achieved a PR; the overall response rate in both groups was 22.2%. In group A, bone marrow progenitor assay was performed pre- and post-amifostine treatment. Erythroid burst-forming units (BFU-E) were increased in six out of 11 (54.5%) patients, and this increase preceded the rise in Hb levels in three of them. In group B, a clonogenic assay was performed in 11 out of 16 patients before AMF treatment. In vitro results after pretreatment with 500 microM amifostine confirmed the response of two MDS patients that achieved a PR. No response in vitro was observed in all eight nonresponding patients and in one PR patient. The lack of response in the clonogenic assays predicted for nonresponse to treatment with a predictive power of 91.8%. We conclude that 300 mg/m2 is an adequate initial treatment for low-risk MDS patients and both clonogenic assays have a strong predicting value for response to treatment.

The FCR combination in a phase III study (Blood 102, abstract #373, p110a,2003) has shown promising results in the treatment of chronic lymphocytic leukemia (CLL) patients leading in increase of proportion of complete remissions and improved survival. Based on these encouraging results we tried to study the efficacy and safety of the FCR combination in patients of our Haematologic Unit. Seventeen patients, 8 males and 9 females with a median age of 69,5 years old, with relapsed/refractory or de novo CLPD ( 9 CLL and 8 NHL patients) were enrolled in this study between February 2002 and August 2004. Fifty percent of CLL patients had Rai stage I/II and the rest 50% had Rai stage III/IV disease. Four NHL patients had also Ann Arbor stage I/II and the rest four Ann Arbor stage III/IV disease. They were treated with Rituximab 375 mg/m2 on day1 in combination with Fludarabine and Cyclophosphamide ( 25 mg/m2 and 250 mg/m2 respectively) for days 2 to 4, every 4 weeks, for 6 consecutive cycles. Nine patients had a history of a prior unsuccessful treatment. Overall, 14 out of 17 evaluable patients were responsive to the treatment [12 patients complete response (CR) and 2 patients partial response (PR), overall response rate (ORR) 82%]. The remaining 3 patients did not show any response (NR), 2 progressive and 1 stable disease. Hematological toxicity was acceptable ( grade 2–3 neutropenia in 6/17 patients, grade 2–3 anemia and thrombocytopenia in 2/17 patients). There were no septic episodes except one case with neutropenic fever. There were no adverse events like nausea or vomiting except one patient with a serious anaphylactic reaction due to Rituximab administration. Three CLL patients died because of stable or progressive disease. In conclusion, this preliminary report suggests that the FCR regimen is an effective and safe treatment for CLPD patients, achieving higher CR rates than previous treatments. A longer follow up of a larger number of patients is required to confirm an improved survival in these patients.

BACKGROUND: Current evidence supports the existence of circulating clonal B cells in Multiple Myeloma (MM). However, attempts to enumerate and phenotypically characterise them have so far provided inconsistent results. Most investigators have studied unselected peripheral blood (PB) mononuclear cells, among which the clonal ones make only a small minority. Moreover, in most cases, numerical chromosomal changes were employed as a clonal marker, but aneuploidy is considered a late event in myelomagenesis. To overcome these difficulties, we have followed an alternative approach, by studying purified PB B cells and focusing on chromosomal translocations involving the immunoglobulin heavy chain gene (IGH) on region 14q32, a frequent, early and possibly crucial pathogenetic event in MM. METHODS: The study included 33 MM patients with 14q32 rearrangements, detected by conventional cytogenetics or florescence in-situ hybridisation (FISH) in the bone marrow at diagnosis. PB CD19+ cells were immunomagnetically isolated (>99% purity) and cytocentrifuged on slides. The slides were studied with a FICTION technique, ie a combination of FISH using a “break-apart” IGH probe set (Vysis Inc, Downers Grove, Il, USA) and indirect immunofluorescence for CD34, CD5, CD10, CD23 and CD38. To avoid the possibility of contaminating plasma cells, isolates with >1% CD19+CD38+++ cells on flow cytometry were excluded from FICTION study. RESULTS: “Positive” cells above the cutoff level of false positivity (4%) were detected in 25 cases (75.7%), ranging from 4% to 33% (median 9%) among the total CD19+ population. These cells were found to consistently express CD10 (83% to 100%, median 96%) and CD38 (79% to 100%, median 89%). They less commonly expressed CD23 (39% to 67%, median 46%) and very rarely CD34 (0% to 5%, median 0%) and CD5 (0% to 3%, median 0%). CONCLUSIONS: Our data suggest that circulating cells bearing IGH rearrangements are the rule in MM, making a small but detectable fraction of CD19+ cells. There mmunophenotypic profile supports the concept that clonal B cells represent advanced ontogenetic rather than early stages in B lineage differentiation. Finally, the virtual absence of CD5+ clonal cells is in accord with the view that the high number of PB CD5+ B cells in MM reflects an immunoregulatory network and does not result from clonal expansion.

BACKGROUND: Plasmacytoid dendritic cell (DC2) acute leukemia is a recently described hematological malignancy with distinct clinical and phenotypic features. Yet, with fewer than 40 cases studied so far, the cytogenetic profile of this entity is still elusive. Previous studies (Leroux et al, 2002) have revealed recurrent chromosomal aberrations affecting certain genomic targets and the analysis of additional cases is expected to clarify whether these aberrations - alone or in combination - may constitute specific markers for the disease. METHODS: Seven patients with a well-established diagnosis of DC2AL (based on clinical, morphological and phenotypic features) were included in the study. A conventional cytogenetic analysis (CCA) was performed on unstimulated bone marrow cultures. Additionally, the diagnostic marrow smears were studied with interphase fluorescent in-situ hybridization (FISH), using commercially available probes for 5q31, 6q21, 7q31, 9p21 (p16 gene), 9q34, 11q23 (MLL gene), 12p13 (TEL gene), 13q14, 17p13 (p53 gene) and 21q22 (AML1 gene) regions. In case of any abnormal finding, further study was performed with the use of centromeric or other appropriate arm/chromosome enumeration probe to distinguish between region deletion or overrepresentation and numerical aberrations of the carrier arm/chromosome. RESULTS: An abnormal karyotype (by either CCA or FISH) was documented in all seven cases studied, with most lesions indicating the loss of genomic material. The most common finding was a rearrangement of the 21q22 region (involving the AML1 gene). Deletions of 6q21, 9q34, 11q23, 12p13 and 13q14, trisomy 12 and trisomy of 13q were detected in only one case each. Interestingly, some aberrations, including the three cases of AML1 rearrangement, were missed on CCA, suggesting a submicroscopic abnormality. CONCLUSIONS: The study failed to define recurrent chromosomal lesions in DC2AL, other than 21q22 rearrangements. Our results point to a possible pathogenetic role of the AML1 gene. Since AML1 is involved in both myeloid and lymphoid malignancies, it makes a reasonable candidate for leukemogenesis also in the case of DC2AL. Additional investigation at the cytogenetic and molecular level is needed to verify this assumption.