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Humoral and cell-mediated immune responses, specific for lipopolysaccharide (LPS), were evaluated before and after oral immunization with the Ty 21a strain of Salmonella typhi in a group of healthy volunteers. No rise in seric and foecal antibody titres, detected by the ELISA technique, was seen after vaccination. On the contrary, we were able to demonstrate the development of specific cell-mediated immunity, as assayed by the leukocyte migration inhibition test, 21 days after vaccination. This conversion was still present 40 days after the last dose of vaccine. We believe that the Ty 21a strain of S. typhi, recently proposed for immunization against typhoid fever and based on the use of live micro-organisms, is effective due to its ability to induce specific cell-mediated immunity.

Objective The medullar blast count is a milestone in the prognostic assessment in myelodysplastic syndromes (MDS). The optical microscopy (OM) may sometimes be inaccurate in this disease. The aim of this work is to test the flow immunocytometric (FCM) determinations of medullar immature cells (CD45±) and the expression, among them, of CD33, CD34, and CD117 markers, for their prognostic relevance. Methods In a retrospective analysis of 98 patients affected by MDS, the IPSS was re-calculated by means of the FCM determination of blasts. Survival of patients at low or intermediate-1 IPSS risk was compared with the survival of patients at intermediate-2 or high IPSS risk. In the 64 cases with OM blast count lower than 5%, the survival of patients with the FCM count of medullar blasts ≤2% was compared with that of patients with FCM count >2%. Results Each single marker had a prognostic weight comparable to the optical blast count. The FCM blast count was particularly efficient in distinguishing the risk of having up to 2% or more than 2% of blasts in patients without OM excess of blasts. Conclusion This method is interesting as prognostic tool, especially in patients without excess of blast.

BACKGROUND Extracorporeal photopheresis (ECP) is a recognized second-line treatment for steroid-refractory chronic graft-versus-host disease (cGVHD). Treatment course is usually long, expensive, and demanding for patients, so predictors for response are needed. We carried out a retrospective study on cGVHD patients treated at our institution with the aim to identify a possible correlation between apheretic yields composition and probability of response. STUDY DESIGN Patients treated for at least 6 months were eligible for the study. Flow cytometry data, including absolute counts of lymphocytes and their subpopulations in ECP products from cGVHD patients, were collected. For each cell population 1) the median dose per procedure harvested during the first 3 months of treatment and 2) the cumulative dose collected in the same period were compared with clinical response. RESULTS A total of 726 ECP procedures were performed in 15 patients. Overall response, defined as either a complete response (CR) or a partial response according to National Institutes of Health criteria, was obtained in 10 of 15 patients (66.7%), and CR, in eight of 15 (53.3%). According to Cox regression analysis, the probability of achieving an overall response is significantly correlated with the median number of CD3+, CD3+CD4+, and CD3+CD8+ lymphocytes collected during the early treatment phase (first 3 months). CONCLUSION Our data suggest that CD3+ cell evaluation in ECP during the early phase of treatment course could predict response and help identify patients who deserve further treatment.

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.

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.

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: 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.

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.

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.

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.

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.