2002; 01

DLI is feasible with rapid engraftment and minimizes the risk of severe GVHD. Large prospective trials are required to prove that it preserves the GVL effect fully. ©2002, Ferrata Storti Foundation Key words: allogeneic transplantation, GVHD, GVL, CD34 selection, donor lymphocyte infusions. plication of DLI is GVHD, which is often associated with response to DLI. © F e r r a t a S t o r t i F o u n d a t i o n Design and Methods Patients and donors Twenty-four patients with hematologic malignancies, 18 males and 6 females, aged 14 to 56 years (median 46 years) were included. Their clinical characteristics are summarized in © F e r r a t a S t o r t i F o u n d a t i o n obtained from patients and donors and our institution's Ethical Committee approved the protocol. Clinical management The conditioning regimen depended on the clinical diagnosis. The following regimens were used: 1) Ara-C (12 g/m 2 ), cyclophosphamide (120 mg/m 2 ) and single dose total body irradiation-TBI (8 Gy) (AML and MDS patients); 2) Ara-C (18 g/m 2 ), melphalan (140 mg/m 2 ) and fractionated TBI (12 Gy) (ALL patients); 3) busulfan (16 mg/kg) and cyclophosphamide (120 mg/kg) (CML and 2 nd transplant patients); 4) cyclophosphamide (120 mg/kg) and fractionated TBI (12 Gy) (NHL and CLL patients). All patients were treated with 5 µg/kg/d lenograstim (Granocyte®) from day +1 until the granulocyte count was > 10 9 /L for three consecutive days or > 10 10 /L for one day. Six early patients (4 not HLA-identical to their donor and 2 AML in CR1) received a short course of methotrexate in addition to cyclosporine (CyA). Because of the low incidence of acute GVHD observed in the first 9 patients, GVHD prophylaxis was carried out with CyA alone for patients 10 to 24. The diagnosis and grading of acute and chronic GVHD was established as previously reported. Stem cell mobilization, collection and selection Donors received human granulocyte colonystimulating factor (G-CSF) (Granocyte®, kindly provided by Rhône-Poulenc-Rorer, Brussels, Belgium) at a dose of 10-15 µg/kg from day -5 through day -1 before transplant. Collection of PBSC was carried out on days -1 and 0, using a continuous flow blood cell separator (CS3000+, Baxter-Fenwall Laboratories, Deerfield, IL, or Cobe Spectra, Lakewood, CO, USA). The volume of blood processed was 12-16 liters. The PBSC from the first day of harvest were stored overnight in the patient's own plasma. Immediately after the second harvest, PBSC from the first and the second days of harvest were pooled. CD34 + cell selection was carried out using the Isolex 300i® magnetic cell separator (Nexell International, Wemmel, Belgium), according to the manufacturer's recommendations. Donor lymphocyte infusions Around day 60 post-transplantation, donors underwent 12-16 liter leukophereses on 2 consecutive days to collect lymphocytes. The collection from the first day of harvest was stored overnight in the patient's own plasma and pooled with the second harvest before processing by CD8 + selection using the Nexell Isolex 300i®. The CD8-negative fraction was recovered and divided into 3 aliquots containing 2×10 6 , 1×10 7 and 5×10 7 CD3 + cells/kg recipient for patients from #1 to #13. The 2 nd and 3 rd aliquots were cryopreserved in 10% DMSO in a controlled-rate freezer. After an interim analysis showed little acute or chronic GVHD associated with DLI, the schedule of DLI was changed to 2 aliquots of 1×10 7 and 5×10 7 CD3 + cells/kg recipient for patients 14 to 21. Aliquot 1 was injected fresh immediately after the CD8 depletion procedure (around day 60). Around day 100 (and 140 for patients 1 to 13), aliquot 2 (and 3) were thawed and infused into the patient. CD8-depleted DLI were not to be infused in case of an antecedent grade III or IV acute GVHD, or an antecedent extensive chronic GVHD, or active GVHD at the time of the scheduled infusion. Laboratory analyses Aliquots of the pooled PBSC as well as the CD34 + selected fraction were incubated with phycoerythrin-conjugated anti-CD34 monoclonal antibody (HPCA2; Becton-Dickinson, Palo-Alto, CA, USA) for 20 minutes at 20°C, washed and fixed. A total of 1×10 5 cells was analyzed using a FACS-scan analyzer (Becton-Dickinson). The percentage of CD34 + cells was defined with dot plot analysis using the whole nucleated cell population. The percentage of positive cells in the isotype control was subtracted from the CD34 + percentage to give the final percentage of CD34 + cells. Data acquisition was performed with the Cellquest software (BectonDickinson). Donor lymphocytes (before and after CD8 depletion) were similarly examined using double labeling with FITC-and PE-conjugated antibodies after treatment with a lysing solution. Complete blood counts were determined using a Technicon H2 cell counter (Bayer Diagnostics, Diegem, Belgium). Percentages of reticulocytes were obtained by an automated cytofluorometric method using the thiazole orange analog DEQTC. Statistical analyses Student's t-tests were used to compare cell subsets before and after CD8 depletion. The probability of GVHD, relapse, and survival as well as the speed of engraftment were studied by life-table analyses and Wilcoxon rank tests were used for comparisons between groups. Statistical analyses were carried out with Graphpad Prism (Graphpad Software, San Diego, CA, USA). Results Collection of PBSC, CD34 selection and engraftment kinetics PBSC were collected by leukophereses on two consecutive days, except in one 70-year old donor (patient #6) who had to undergo 4 consecutive leukophereses (and two CD34 selection procedures) because of poor yields. Most donors experienced bone pain and/or cephalalgia that were easily controlled with paracetamol, but no other complication was noted. A median of 10.15 (5.59 to 21.02) ×10 6 CD34 + cells/kg and 375 (127 to 656)×10 6 CD3 + cells/kg were collected CD8-depleted DLI after CD34-selected allo-PBSCT © F e r r a t a S t o r t i F o u n d a t i o n Collection of donor lymphocytes, CD8 depletion and CD8-depleted DLI Donor lymphocytes were collected on day 60 from all but 5 donors whose recipients died before or experienced serious complications Clinical data (Figure 1 and Table 4) Five patients died before receiving DLI. Patients #6 (second transplant) and #4 developed many early serious complications and died on day 81 of influenza pneumonia and on day 99 of a polymicrobial infection, respectively. Patient #19 developed severe renal and respiratory failure and died of infection at day 195. Patient #16 died of CLL and veno-occlusive disease of the liver on day 44 and patient #17 died of leukemia on day 38. The other 19 patients were in CR on day 60 and received the first DLI. Two ALL patients (patients #7 and 10) relapsed between days 60 and 100 and received the pooled 2 nd and 3 rd aliquots. Patient #7 achieved a CR with chemotherapy and DLI but relapsed later and died on day 313. Patient #10 died of leukemia on day 100. In addition, patient #12 died suddenly at home of unknown reason while enjoying continuous CR and presenting no complication other than depression. The other 16 received the scheduled 2 nd (and the 3 rd for patients #1, 2, 3, 5, 8, 9, 10, 13) DLI on days 100 (and 140). Fourteen of the 16 patients remain in CR 126 to 1344 (median 400) days post-transplantation. Patient #15 developed a central nervous system relapse on day 135, was reinduced into remission with chemotherapy and radiotherapy but relapsed again and died on day 309. Patient #3 experienced a biopsyproven massive relapse in the thymus on day 315 that completely regressed after cyclosporine discontinuation, but later relapsed again and she died on day 950. Finally, 3 patients died in CR after day 100: patient #13 died of pulmonary aspergillosis on day 351, patient #18 died suddenly at home of unknown reason on day 288 and patient #21 of acute grade IV GVHD on day 150. The remaining 11 patients were alive and disease-free 126 to 1344 (median 550) days after transplantation. Bone marrow chimerism Evaluation of bone marrow chimerism was performed in 6/7 patients with a donor of the opposite sex (n=7) by FISH with X and Y probes ( Acute and chronic GVHD (Figure 3) Acute GVHD occurred in 15 patients. It was of grade I in 9 patients, of grade II in 5 patients (3 with 1 HLA mismatch) and of grade IV in 1 patient (HLA mismatch). Thus, grade II-IV acute GVHD occurred more frequently in HLA-mismatched (4/8 or 50%) than HLA-matched (2/16 or 13%) transplants (p<0.05). Before DLI, the 60-day actuarial incidence of grade II-IV GVHD was 17% (4 patients) but 2 additional patients developed grade II and IV GVHD after DLI to produce a 150-day (after DLI) incidence of 28%. For HLA-identical sibling transplants, the 60-and 150-day actuarial incidences of grade II-IV acute GVHD were 0 and 13%, respectively ( CMV reactivation Eleven of the 24 patients experienced CMV reactivation (PCR positivity) before day 60 that was successfully reversed by ganciclovir treatment and none of them presented a clinical CMV infection. Relapse and survival (Figure 4) There was no relapse in the 12 standard risk patients but 6 of the high-risk patients relapsed (p<0.005) ( Discussion In agreement with previous studies, 17,19,21 CD34-selection resulted in our study in a 3.3 log elimination of T-cells while preserving hematologic reconstitution. Engraftment of neutrophils and platelets was prompt and significantly faster in patients who did not receive methotrexate. However, within the range studied, the number of © F e r r a t a S t o r t i F o u n d a t i o n CD34 + cells did not influence the speed of engraftment significantly. Prompt engraftment occurred with CD34 + cell doses as low as 1.46×10 6 /kg. Therefore, CD34 -selection preserved the engraftment capability of allogeneic PBSC. As previously suggested by others, 17,22 our results evidenced that CD34 selection reduces the risk of acute GVHD. The actuarial 60-day (before DLI) probability of grade II-IV acute GVHD was 17% in our study. This rate compares very favorably with results from studies of HLA-identical siblings receiving unmanipulated PBSC or BM. Moreover, our results also compared favorably with those of previous studies of pre-emptive DLI after T-cell-depleted BMT or PBSC transplantation. A few other studies have investigated the feasibility of adding T-cells back, also to heterogeneous groups of patients, a few weeks to a few months after T-cell-depleted (TCD) transplantation. Barrett gave either 2×10 6 /kg on day 30 and 5×10 7 /kg on day 45 or 1×10 7 /kg on day 30 to HLA-identical siblings after TCD BMT. Contributions and Acknowledgments YB designed the study and wrote the paper. FB analyzed the data and wrote the paper. JS © F e r r a t a S t o r t i F o u n d a t i o n PEER REVIEW OUTCOMES What is already known on this topic Transplantation of CD34 + selected cells from peripheral blood of allogeneic donors associates with a lower risk of acute and chronic graft-versus-host disease than unmanipulated transplants. Relapse risk may be increased in this setting, as a consequence of T-cell depletion, which leads to a decreased graft-versusleukemia effect. What this study adds CD8-depleted lymphocyte infusions starting on day 60 may be safely administered without triggering acute or chronic graft-versus-host disease. The relapse rate after this approach was similar to that observed in a control group of non-T-cell depleted transplants. Manuscript processing Potential implications for clinical practice This report demonstrates the feasibility of allogeneic CD34 + selected stem cell transplantation followed by the infusion of engineered cell-depleted fractions. Jordi Sierra Gil, Deputy Editor © F e r r a t a S t o r t i F o u n d a t i o

2001; 02

Differential diagnostic values of serum transferrin receptor, serum ferritin and related parameters in patients with various causes of anemia This study involved 147 subjects, divided into 4 groups: 50 healthy controls, 54 patients with iron deficiency anemia (IDA); 34 with anemia of chronic disorders (ACD) and 9 with thalassemia. Serum transferrin receptor (sTfR), ferritin, total ironbinding capacity (TIBC), unbound iron-binding capacity (UIBC) and complete blood count were measured in all subjects. In 17 patients with IDA, serum ferritin, sTfR, and hemoglobin were rechecked after iron therapy. sTfR could differentiate patients with IDA from healthy subjects and those with ACD. Serum ferritin was better at distinguishing IDA from other causes of anemia, and was more sensitive than sTfR in indicating the iron repletion in patients with IDA after iron therapy. Soluble serum transferritin receptor (sTfR) has been introduced as a new tool for diagnosing iron depletion. Serum ferritin was significantly lower in patients with IDA than in patients without, but was not different among the non-IDA patients (Groups A, C, D, Seventeen patients with IDA treated with oral iron were consecutively selected in order to monitor the changes of hemoglobin, sTfR and serum ferritin after iron therapy. The correlation between the change of serum ferritin and sTfR (r=-0.371, p =0.129) was not significant. Positive and negative correlations were found between serum ferritin and hemoglobin as well as between sTfR and hemoglobin, respectively (r=0.673 and -0.554; p =0.002 and 0.017, respectively). Serum ferritin changed more and faster than sTfR after iron repletion. In the present study, mean sTfR was highest in the patients with IDA, and was significantly higher in patients with IDA than in those with ACD and healthy subjects. Thus, it could be used to distinguish IDA from ACD as reported by others. 5,7 However, sTfR was not significantly different between IDA and thalassemia, and thus could not distinguish IDA from anemias other than ACD. Serum ferritin could distinguish IDA from other non-IDA anemic patients, but it made no significant discrimination among the patients without IDA. In 17 patients with IDA treated with oral iron, there was no correlation between the change of ferritin and sTfR (r=-0.371, p =0.129) after iron therapy. The change of serum ferritin was more significant than the change of sTfR after iron repletion. Thus, the decrease of sTfR did not occur so rapidly as the increase of serum ferritin after iron repletion. This is compatible with the results of another study. 8 Serum ferritin should be better than sTfR in indicating iron repletion after iron therapy in patients with IDA. Chao-Hung Ho Division of Hematology, Taipei Veterans General Hospital, Taipei, Taiwan, R.O.C. Groups Ferritin STfR T.S. Hb MCV sTfR/Fer sTfR/log Fe

2002; 06.mk

© F e r r a t a S t o r t i F o u n d a t i o

2001; 10

scientific correspondence osteoclasts, the presence of which agreed with our own findings. Zvaifler et al. Despite © F e r r a t a S t o r t i F o u n d a t i o

2001; 07

Background and Objectives. Although several studies have determined quality of life (QOL) in patients with hemophilia, generic questionnaires have rarely been used. The objectives of our study were; 1) to measure QOL and utility in patients with hemophilia using the Short Form 36 (SF-36) and the Euro-QOL questionnaires; 2) to evaluate the influence of some clinical variables on QOL and utility; 3) to assess the correlation between the two questionnaires. Design and Methods. All consecutive patients with hemophilia were asked to complete the SF-36 and the EuroQOL questionnaires. The following information was recorded from each patient: age, type of hemophilia, severity of disease, HCV and HIV infection, number of bleeding episodes and cumulative dose of coagulation factors over the previous year. These items were entered into a multivariate analysis to assess their effect on QOL. Correlation analyses were conducted to evaluate the relationship between the EuroQOL and SF-36. Results. Fifty-six patients completed the SF-36 and the EuroQOL questionnaires. The mean scores of the SF-36 ranged from 55.2 (general health) to 74.7 (social functioning). The EuroQOLself-classifier and the EuroQOLvas showed a mean score of 0.67 (SD=0.26) and 0.66 (SD=0.17), respectively. Among the clinical variables, age significantly influenced both the EuroQOL and the SF-36 scores. The EuroQOL indices showed a statistically significant correlation with each dimension of the SF-36. Interpretation and Conclusions. Our study quantified the degree to which QOL is impaired in patients with hemophilia by using both a generic questionnaire and a utility-based approach. ©2001, Ferrata Storti Foundation Key words: hemophilia, quality of life, utility T he research on quality of life (QOL) in hemophilia is still at an early stage of advancement. We used the SF-36 and the EuroQOL questionnaires to study QOL in the hemophilia patients referred to our regional Hemophilia Center in Tuscany. Our study had three objectives: 1) to measure QOL and utility in patients with hemophilia using a generic questionnaire (SF-36) and a utility questionnaire (EuroQOL); 2) to analyze the QOL and utility scores in the framework of a multivariate analysis for determining the influence of some clinical variables on these measurements; 3) to assess the correlation between the QOL and the utility measurements produced by the two questionnaires. Design and Methods Study design and data collection We studied all consecutive patients with hemophilia who were referred to our regional Hemophilia Center in Tuscany from 1 April, 2000 to 30 June, 2000. The study had a cross-sectional design; © F e r r a t a S t o r t i F o u n d a t i o n accordingly, patients were included in the study once only (i.e. on the occasion of their first contact with our Center during the study period). The eligibility criteria for patients' enrollment were the following: a) diagnosis of either hemophilia A or hemophilia B; b) age ≥ 16 years; c) referral as an in-patient or out-patient to the units of Hematology of the Careggi Hospital or to the Regional Hemophilia Center of Careggi; d) no measurable titer of inhibitors at the last visit (determined over the previous 12 months using the Bethesda assay); e) no regular administration of factor VIII or IX for prophylaxis (defined as at least two weekly prophylactic infusions of the deficient factor over the previous six months). The ineligibility of patients with inhibitors and those receiving a regular prophylaxis [criteria (d) and (e)] had the purpose of increasing the homogeneity of our QOL measurements. When the eligibility criteria were met, the inclusion criterion was simply written consent from the patient to participate in the study. All patients enrolled in the QOL study were asked to complete the SF-36 and the EuroQOL questionnaires. For the purpose of our study, the following information was obtained from each patient: or mild depending on whether the level of the deficient factor was less than 1%, between 1% and 5%, or greater than 5%, respectively); 6. presence of HCV infection determined by two criteria: a) anti-HCV antibody assayed by EIA (assay manufactured by Roche, Italy); and b) ALT more than 2.5 higher than upper limit of normal in at least two independent samples over a period of 6 months; 7. presence of HIV infection (determined by ELISA assay, Organon, Italy); 8. number of bleeding episodes over the previous year; 9. dosage of coagulation factors over the previous year. The information of items from 3 to 9 was used as clinical variables for our statistical analysis, and their influence was assessed on the results of both the SF-36 and the EuroQOL questionnaires. Questionnaires and scoring methods The SF-36 questionnaire measures 2 main health concepts (physical and mental health) with 36 items and 8 multi-item scales called dimensions or domains (physical functioning, role physical, bodily pain, general health, vitality, social functioning, role emotional, mental health). An additional 1-item measure assesses self-evaluated change in health status. Scores are assembled using the method of summated ratings; 10,13,14 the raw scores are then transformed to a 0 to 100 scale (with 0 and 100 assigned to the lowest and highest possible value, respectively). Highest transformed scores indicate better health. Two component summary scores, one concerning the physical dimension (physical component score or PCS) and the other concerning the mental dimension (mental component score or MCS), are also calculated as a result of a weighted combination of the 8 dimensions. The EuroQOL questionnaire 11,12 consists of 5 questions and contains a visual analog scale (VAS). Two separate utility estimates are calculated, the first (EuroQOLself-classifier) from the 5 questions [which are processed through a simple computation algorithm, In our study, the two questionnaires were used in the Italian version 9,11 and were self-administered in accordance with current recommendations. Normative data To assess the influence of hemophilia on QOL, the results obtained using the SF-36 in our patients were compared with those of 2,031 Italian normal subjects (normative data) reported previously by Apolone et al. 9 Since normative data from Italian subjects are lacking for the EuroQOL, no comparison with normative information is presented for this questionnaire. Statistical analysis The parameters estimated from the two questionnaires (namely: scores for each of the 8 dimensions of SF-36, summary physical score and summary mental score of SF-36, EuroQOL utility according to the self-classifier algorithm, EuroQOL utility according to the VAS) were presented as means with standard deviation (SD) inclusive of subgroup analyses where appropriate. To determine the influence of the clinical variables on the SF-36 and the EuroQOL measurements, a multivariate regression analysis (SPSS computer program for Windows, Version 8.0, SPSS Inc., 1997) was conducted to test the statistical association between each of the SF-36 and EuroQOL parameters and the clinical variables; this analysis estimated the significance level for each variable and calculated partial correlations and regression coefficients. Categorical variables stratified on more than two levels (e.g. disease severity stratified as severe, moderate, or mild) were handled as dummy variables. A backward stepwise method of variable elimination was used, wherein the variables with p<0.10 were eventually retained; the choice of using this p=0.10 limit was made to explore not only the associations with clear-cut statistical significance (p<0.05), but also those suggested in terms of statistical trends (0.05<p<0.10). A separate analysis tested the inter-relations between the SF-36 and the EuroQOL results (using Spearman's correlation); this analysis, too, was carried out using the SPSS computer program mentioned above. Results Our study recruited a total of 67 patients at their first contact with our Center over the study period. After excluding patients with inhibitors (n=7) and patients receiving prophylaxis (n=4), a total of 56 cases were enrolled in the QOL study; none of these patients refused to participate in the study. The main characteristics of this patient cohort are summarized in The administration of the SF-36 questionnaire to these patients gave the results shown in The results obtained with the EuroQOL questionnaire were the following: EuroQOLself-classifier = 0.68 (SD=0.26, valid cases=56), EuroQOLvas = 0.66 (SD=0.17, valid cases=55). Our comparison of the SF-36 results between hemophilia patients and normal subjects ( indicates that the 4 physical domains (physical functioning, role physical, bodily pain, general health) are all consistently affected by hemophilia. Among the mental-related domains, the role emotional dimension was worsened while the other 3 were unaffected. Among the clinical variables that influenced QOL and utility (multivariate regression analysis, HIV infection negatively influenced the mental health domain of the SF-36 and both EuroQOL measurements. Subgroup means (with SD) of all QOL and utility parameters are presented in In our multivariate analysis Discussion Two studies 6,7 have thus far utilized the SF-36 and/or the EuroQOL in patients with hemophilia ( *The cut-off for retaining a variable in the analysis was set at p=0.10. °A negative correlation coefficient indicates that the clinical variable worsens the QOL or the utility parameter and vice versa. † This symbol identifies paradoxical results in that the effect of the variable on QOL was opposite to that expected. © F e r r a t a S t o r t i F o u n d a t i o n In comparison with our results, Molho et al. 6 reported higher scores in the domains of physical functioning, role physical, general health, social functioning, and role emotional. On the other hand, the results of the two summary scores of the SF-36 were very similar to ours. In the case of Miners' investigation, 7 the average scores of the SF-36 (with the exception of the domain of physical functioning) and of the EuroQOL were higher than ours Quality of life in patients with hemophilia One limitation of our analysis is that its statistical power was probably insufficient to explore the effect on QOL of disease severity (split into 3 subgroups of 32 vs 15 vs 9 patients). For example, there was a paradoxical effect for moderate disease which had some scores lower than those found for severe disease. There is plenty of data in the literature on HCV in individuals without hemophilia that suggest HCV decreases QOL. Since in our study all individuals with severe hemophilia were HCV positive, it was impossible to rule this out. Because many advanced treatments for hemophilia have become available in the last 15 years, the impaired QOL level of our older patients might reflect the negative long-term effect of inadequate treatments received in young age; however, the decline that QOL indices generally show with age in normal subjects and in a variety of disease conditions might also have contributed to this finding. HIV infection ranked second in our multivariate analysis. As shown in The strong agreement between the two utility estimates and each of the 8 domains of the SF-36 In conclusion, the section of our study based on the SF-36 substantially confirmed the results of previous investigations and provided new information on the critical issue of which variables affect QOL in hemophilia. The section of our study based on the EuroQOL provided original data in terms of utility that could be useful for future cost-effectiveness studies evaluating this disease condition. Complete information about utility is the prerequisite to translate clinical outcome and cost of hemophilia patients into a cost/utility ratio (e.g. cost per QALY gained) and to place this latter parameter in the framework of those previously calculated for other medical interventions in other disease conditions. Finally, because relevant results were obtained Potential implications for clinical practice This study sheds light on the main factors that influence quality of life in hemophilia. This information can be of benefit to clinicians in order that they interact adequately with individual patients. © F e r r a t a S t o r t i F o u n d a t i o

2002; 01

F e r r a t a S t o r t i F o u n d a t i o n account diverse parameters such as peripheral cell counts, enzyme levels, histopathologic features, in addition to the percentage of BM blasts. Design and Methods Patients This is a multicenter retrospective analysis of 234 MDS patients evaluated from 1984 to 2000 proceeding from Argentinian hematologic centers: Instituto de Investigaciones Hematológicas (IIHE-MA); Hospital Privado de Córdoba, Hospital General de Agudos Dr. Carlos G. Durand, Hospital General de Agudos José María Ramos Mejía and others. A diagnosis of MDS was made after inspection of peripheral blood (PB) and BM to document the requisite dysplastic cytologic features. Trephine biopsy, cytogenetic studies, cytochemical and iron stains, immunophenotyping and, in some cases, ferrokinetics studies were performed to provide confirmatory diagnosis. All patients were categorized according to FAB criteria; 4,13 all had primary MDS without documented preceding radio-or chemotherapy. Most patients received supportive care, such as transfusion or polyvitamin therapy; only a minority received varying amounts of chemotherapy. Infections, bleeding, BM failure and LE were considered as MDS-related causes of death. The IPSS was applied according to Greenberg et al. 9 Table 1 provides a summary of the patients' clinical data. BM blasts, cytopenias, cytogenetic groups, FAB and IPSS classifications are presented in percentages and in absolute numbers, at the time of diagnosis. For each of these variables, the estimated median SV and the time to 25% of patients evolving to LE are also given. Cytogenetic pattern Chromosome analyses of 198 patients with MDS were performed in BM samples, according to standard procedures of our laboratory. Inclusion in the study required the analysis of ≥ 11 metaphase cells per patient. Chromosome identification and karyotype designation were made taking into account the International System for Human Cytogenetic Nomenclature. Statistics The Kaplan-Meier method was used for the univariate estimation of SV time and LE calculated from the day of diagnosis. Each variable was analyzed using the log-rank test. The level of statistical significance was fixed at 0.05. Results Patients The clinical, demographic and cytogenetic findings are summarized in Percentage of BM myeloblasts SV and LE curves for groups divided according to IPSS cut-off of blast percentage were significantly different (p<0.001). The median SV was 80 months and the LE (25%) was not achieved in the group with <5% blasts; while, for the groups with 5-10%, 11-20% and >20% blasts the median SV and LE (25%) were 31 and 17; 36 and 9; 9 and 3 months, respectively. However, no differences were observed between the group with 5-10% blasts and that with 11-20% regarding SV (p=0.225) and LE (p=0.085). Number of cytopenias SV and LE curves drawn according to the number of cytopenias defined by the IPSS were significantly different (p<0.001). The median SV was 77 months and the LE (25%) was not achieved in patients with 1 or no cytopenia; while, for those patients having 2 or 3 cytopenias the median SV was 31 months and LE had occurred in 25% by 9 months ( Detection of risk groups in MDS Cytogenetic findings A total of 198 cases could be successfully analyzed since no metaphases were available from 36 (15%). An abnormal karyotype was observed in 82 (41%) patients at diagnosis. The most common cytogenetic abnormalities were: -7 or del(7q) [ Twenty-two patients showed complex karyotypes Cytogenetic findings were subdivided according to IPSS into 126 (64%) good, 41 (21%) intermediate and 31 (15%) poor and the SV and LE curves according to these subdivisions showed significant differences (p=0.013 and p<0.001). The median SV and the times to 25% of patients undergoing LE were 60 and 46, 34 and 19, 28 and 5 months in the groups with good, intermediate and poor cytogenetic findings, respectively ( We observed that the proportion of normal karyotypes decreased according to worsening prognostic IPSS subgroup, since 48% of cases with normal karyotypes were present in the IPSS low risk group while only 4% of them were in the IPSS high risk group. The correlation between cytogenetic subgroups and IPSS showed that 84% of patients belonging to the good risk cytogenetic group were in low (48%) and Int-1 (36%) IPSS risk groups; 61% of patients with an intermediate cytogenetic risk group were in the Int-1 IPSS group; whereas, 84% of patients with poor cytogenetic findings were in Int-2 (42%) and high (42%) IPSS risk groups. These data show the importance of cytogenetic parameters IPSS SV and LE curves plotted according to IPSS stratification were significantly different (p<0.001). The median SV and the LE (25%) were not achieved in the low risk group but were 42 and 45; 33 and 25; 14 and 5 months for the Int-1, Int-2 and high risk groups, respectively ( IPSS vs. FAB subtyped patients The correlation between IPSS and FAB classifications identified patients with RA (5%) and RARS (15%) within the Int-2 group and those with RAEB (19%) within the high risk group as having worse prognosis The IPSS was not informative about RAEBt because the patients had an uniformly poor outcome in relation to their high blast count. With respect to CMML, it was too favorable because all patients (92%) were included in the low and Int-1 risk groups. Discussion In this study we evaluated the IPSS and its prognostic variables in a large group of patients with FAB-classified primary MDS over the past sixteen years. The median SV for our patients according to their FAB subtype was similar to those previously reported, From our studies and others, The karyotype of BM at diagnosis coincided strongly with chromosome alterations reported in other large series. The percentage of blasts in BM aspirates is one of the parameters with strong prognostic value. Several scoring systems have proposed different cut-offs 1,6,7 for this percentage besides the generally accepted one in the FAB criteria. Considering that there were great variations in SV and LE among patients belonging to the same FAB subtype, this integration of IPSS and FAB criteria allowed identification of worse prognostic subgroups within patients with RA, RARS and RAEB. The score value for blast percentage was null in RA and RARS, but adding the increased number of cytopenias and a poor cytogenetic pattern demonstrated the worse prognosis for these cases. These variables also improved the subdivision of RAEB patients in addition to their division according to blast percentage. FAB classification IPSS RA (%) RARS (%) RAEB (%) RAEBt (%) CMML (%) Total(%) Low 50 Our univariate analyses showed that FAB classification, IPSS and its variables, percentage of blasts, cytogenetic pattern and number of cytopenias, were predictive for SV and LE. These results showed that Argentine MDS patients have a similar behavior to other MDS populations and that the stratification of patients according to IPSS taking into account FAB subtypes will be helpful in developing risk-adapted therapeutic strategies for MDS. Contributions and Acknowledgments CB and IL were responsible for the conception and design of the study, interpretation of its results PEER REVIEW OUTCOMES What is already known on this topic Prognostication is particularly relevant in myelodysplastic syndromes (MDS). The major challenge for a prognostic score is to demonstrate its prognostic accuracy in an independent population. What this study adds This study shows the International Prognostic Scoring System (IPSS) for MDS is also valuable in an Argentinian cohort of MDS patients. This is of interest as the IPSS did not include South American patients. These data reinforce the argument that the IPSS should be used for planning therapy in MDS. Manuscript processing Potential implications for clinical practice Identification of differents groups of risk is very important for clinical practice in myelodysplastic patients. Guillermo F. Sanz, Associate Editor © F e r r a t a S t o r t i F o u n d a t i o

2000; 09

Background and Objectives. To assess the efficacy and the toxic profile of gemcitabine, a novel pyrimidine antimetabolite active against several solid tumors, we carried out a study in heavily pretreated Hodgkin's disease (HD) patients. Design and Methods. From May 1997 to January 1999, 14 pretreated patients (10 relapsed and 4 refractory to previous treatments) were enrolled in a phase II trial and treated with gemcitabine. The drug was given on days 1, 8 and 15 of a 28-day schedule at a dose of 1,200 mg/m 2 intravenously for a total of 6 cycles. Results. Two (14%) patients achieved complete remission (CR) and 4 (29%) had partial responses (PR) , giving an overall response rate of 43%. In the relapsed subset there was an overall response rate of 50% with 2 CR and 3 PR. Among the refractory patients there was only 1 PR (25%). Both patients who had relapsed after autologous bone marrow transplant achieved a response (1 CR and 1 PR). No major toxic effects were recorded. Interpretation and Conclusions. These data suggest that gemcitabine is an effective drug with a low toxicity profile in patients with heavily pretreated HD. Further trials using gemcitabine in combination with other conventional drugs are needed. Correspondence: Pier Luigi Zinzani, M.D., Istituto di Ematologia e Oncologia Medica "Seràgnoli", Policlinico S.Orsola, via Massarenti 9, 40121 Bologna, Italy. Phone: international +39-051390413 -Fax: international +39-051398973 -E-mail: [email protected] mens to treat HD patients who are refractory to firstline treatment or who have relapsed after two or more different lines of treatment. ©2000, Ferrata Storti Foundation Design and Methods From May 1997 to January 1999, 14 previously treated HD patients (10 relapsed and 4 refractory to previous chemotherapy regimens) completed therapy with gemcitabine. Criteria for entry into the study included: histologic diagnosis of HD; stage II-IV as outlined by the Costwolds Meeting; 23 disease resistant to primary and secondary treatment or relapsed after second or third complete remission (CR). At the time of recurrent or progressive disease before gemcitabine, all patients were restaged by chest Xray, hematologic and chemical profiles, bone marrow biopsy, measurement of all tumor masses, computerized tomography of the chest and abdomen, and biopsy of tumor masses when possible. Other studies included lymphography and liver biopsy when appropriate. Informed consent was obtained from all patients in accordance with the ethics policy of the institute, and the study was performed in line with the Helsinki declaration. The patients' characteristics are summarized in I n recent decades very effective polychemotherapy regimens have been developed for the treatment of Hodgkin's disease (HD). As a result, more than 70% of HD patients can now be cured with chemotherapy administered either alone or in combination with radiotherapy. 1 However, for those patients who relapse after initial treatment, salvage therapy remains a difficult challenge. The choice of therapy must be individualized to fit the clinical circumstances of the relapse. In recent years several attempts have been made to develop salvage regi- Ten of the 14 patients had relapsed. Of these, 8 patients had initially been treated with alternating or sequential MOPP-ABVD and then with the CEP regimen and 2 patients were treated first with one combination, the second being utilized as first salvage treatment (ABVD⇒MOPP) and then the IEV 7 regimen and ABMT. The remaining 4 patients were resistant to prior ABVD, MOPP and ABMT. Among the 10 relapsed patients, 6 had experienced a complete remission (CR) lasting more than 1 year with prior chemotherapy, and the other 4 patients had failed within 1 year of the initial chemotherapy. Treatment protocol Gemcitabine hydrochloride (Gemzar, Eli-Lilly, Italy) was supplied as a freeze-dried powder. The drug was diluted in normal saline and administered intravenously over 30 minutes. Gemcitabine was given on days 1, 8 and 15 of a 28-day schedule at a dose of 1,200 mg/m 2 for a total of six cycles. All cycles were delivered in an outpatient setting. No antiemetic prophylaxis was given, but nausea and vomiting could be treated routinely if necessary. Evaluation of response CR was defined as the complete disappearance of signs and symptoms due to lymphoma for at least 6 weeks; partial response (PR) was defined as a reduction of at least 50% in the product of the largest perpendicular diameters of all measurable lesions for a duration of at least 6 weeks. Disease progression was considered to be present when there was clear evidence of advancing disease, despite continuation of the treatment. Patients were evaluated by weekly history and physical examination, complete blood counts, and chemistry profiles. All signs, symptoms or laboratory abnormalities were assessed using ECOG criteria 24 for toxicities. One month after completion of the last course of therapy, clinical and pathologic evaluation was undertaken by repeating radiographic investigations, and bone marrow and/or liver biopsies which had been positive before treatment. Results Response The therapeutic results are shown in Side effects Gemcitabine treatment was generally well tolerated, and all the patients who responded completed Gemcitabine in pretreated HD Discussion The therapeutic approaches developed for HD patients who have failed first-line regimens or relapsed after a first or second CR include the use of conventional salvage chemotherapy regimens and, in recent years, high-dose chemotherapy with ABMT. It is apparent from the literature that an important variable affecting outcome is the ability of conventionaldose programs to reduce tumor volume before transplantation: thus, most of the long-term survivors in ABMT programs are patients who relapsed after chemotherapy but responded to subsequent chemotherapy. At the same time, although 90% of adults with advanced HD can achieve a CR with new polychemotherapy regimens such as BEACOPP, 25 it is too early to assess how many of them can ultimately be cured. With the previous generation of combination treatments, remission rates of 80% have been reported, but 30-50% of these patients still relapse and less than 25% of those in first relapse can be cured. In this study, mostly involving heavily pretreated relapsed patients, we obtained 2 (14%) CR with an overall response rate of 43%. Furthermore, 2 patients who initially obtained a PR went on to achieve a CR after ABMT. In addition, both patients who had had prior ABMT showed a response (1 PR and 1 CR). The patients who had been resistant to first-line treatment had the worst outcome, with only 1 obtaining a brief PR. In terms of side effects, we observed moderate myelosuppression but no organ toxicity. Standard antiemetics were not called for, and no cumulative toxicity patterns were observed. While confirming the preliminary data reported by other authors on the activity and modest toxicity profile of gemcitabine, 28,29 the present study shows that the drug can also be useful for heavily pretreated patients, including ones who have already undergone ABMT. One interesting therapeutic option is first to reduce the tumor burden by administration of gemcitabine prior to ABMT, with the aim of an eventual cure. Our data from the use of gemcitabine alone in heavily pretreated HD patients are very promising and, together with those reported by others, These findings, even with the limitation of a relatively short follow-up and the small size of the patient cohort, lead us to conclude that gemcitabine seems to be the most active of the new conventional drugs for HD. It has a substantial activity and, at the same time, an acceptable toxicity, as has already been evidenced in patients with other lymphomas such as pretreated aggressive and peripheral T-cell lymphomas. Contributions and Acknowledgments PLZ was the principal investigator involved in the conception of the study, its design, and the writing of the paper. MB and FG helped the principal investigator (PLZ) with data analysis and interpretation. PA, MT, VS, and PPP collected the study data. ST critically revised the paper and gave final approval for its submission. Disclosures Conflict of interest: none. Redundant publications: no substantial overlapping with previous papers. Manuscript processing Manuscript received May 15, 2000; accepted July 20, 2000. Potential implications for clinical practice ♦ On the basis of our data gemcitabine might be considered an interesting therapeutic option for relapsed/refractory HD patients. © F e r r a t a S t o r t i F o u n d a t i o

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F e r r a t a S t o r t i F o u n d a t i o n potentially useful in emergencies, 5 but few clinical trials have assessed their performance in this setting. Moreover, D-dimer assays have not been sufficiently investigated in patients with thrombosis of the great saphenous vein (GSV); this thrombosis should be considered separately from other superficial venous thromboses because of the possible progression of the thrombus to the deep system 14 and the potential risk of pulmonary embolisation. Design and Methods Patients Patients presenting spontaneously or referred by a general practitioner (GP) with symptoms of swelling and/or pain and/or inflammation in the lower limbs, and for whom the physician in charge at the ER suspected acute DVT or GSV thrombosis, were considered eligible for the study. Exclusion criteria were 1) signs and/or symptoms of acute pulmonary embolism (PE), 2) previous episode of VT in the same leg and/or objectively documented PE and 3) ongoing oral anticoagulant therapy. Taking into account the signs and symptoms at presentation, 2 eligible patients were classified as suspected as having 1) DVT, or 2) thrombosis of the GSV, involving at least one vein proximal to the deep system (at the cross or in the popliteal fossa), or 3) both of the previous. After the physical examination, all patients underwent compression ultrasonography (C-US) and blood sampling for D-dimer assay. Methods Compression ultrasonography was performed by operators (in charge at the ER or the Vascular Surgery Department) unaware of D-dimer results, using a high-resolution, electronically focused linear array transducer (7.5 MHz probe). The entire deep venous system between the proximal common femoral vein at the cross and the distal veins of the legs was evaluated as was the entire saphenous vein between its junction to the proximal deep venous system and the distal saphenous segment. Ultrasonography was performed using the currently accepted criteria for diagnosing VT; 3 briefly, C-US results were considered abnormal if a vein or venous segment was not fully compressible. Blood for D-dimer assay was drawn at presentation and tested by technicians unaware of C-US test results. Venous blood (9 vol.), anticoagulated with tri-sodium citrate (1 vol.), was taken in the ER from a forearm vein and sent to the central laboratory; results were available within 30 min. The Dimertest® (Dade Behring) was performed as previously described. The Dimertest® was compared to another agglutination reference method; the correlation was r=0.94 and the regression equation was y=1.19×. Intra-assay (within run) reproducibility was determined for 10 replications of 3 plasma samples that contained different levels of XDP. The results were equivalent for all replicates. Inter-assay (run to run) reproducibility was determined using 10 plasma samples with XDP titers ranging from 1 to 16. In 10 runs, the replications of these specimens did not vary by more than one titer. Statistical analysis and ethics Sensitivity, specificity, positive and negative predictive values for D-dimer were calculated using standard methods (2×2 tables); C-US was considered as the reference test. When indicated, 95% confidence intervals were calculated. Paired t-test and Pearson's χ 2 test were applied when indicated; a p value < 5% (two-tailed) was considered statistically significant. Patients gave oral consent to the study. Results Four hundred and seventy-eight consecutive patients were considered during the period February 1999-December 2000. Sixty-four patients were excluded (31 because evaluated in a setting other than the emergency unit, 4 because on oral anticoagulation, 20 because were lacking the D-dimer and 5 the C-US test results, 4 because of a recurrent DVT episode). Thus, 414 patients were considered eligible and included in the analysis. Among these, DVT was clinically suspected in 298 patients (of whom 177 were female [59.4%] and 121 males) and GSV thrombosis in 116 patients (66 female [56.8%] and 50 males). Among patients on heparin or low molecular weight heparin at the time of referral to the ER, 34 (11.4%) belonged to the DVT group and 20 (17.2%) to the GSV group Among patients clinically suspected of having a DVT, C-US was positive in 93 cases (31.8%, 7 of whom had distal DT [7.5%]), while in those suspected of having GSV thrombosis, C-US was positive in 52 (44.8%); four patients (7.6%) had concomitant involvement of the common femoral vein (CFV) at the cross The sensitivity, specificity, the positive and negative predictive values of the D-dimer assay in patients with suspected DVT and suspected GSV thrombosis are reported in The D-dimer assay is a relatively new tool. In combination with other diagnostic tests (such as C-US) or standardized pre-test clinical probability, this assay can be effectively used for refuting acute VTE at referral. 6-8 Several D-dimer assays have become available including classical ELISAs, latex agglutination tests, immunofiltration assays and whole blood Ddimer tests. These assays are useful if the results are rapidly available and the test has a high sensitivity with a reasonable specificity. These two characteristics vary widely in the published reports. 11 This heterogeneity can be explained by methodologic differences between studies (study design, selected cut-off value and diagnostic reference standard used) as well as clinical differences (study population and indication for D-dimer testing). Therefore, it is essential that each D-dimer assay is evaluated in the local setting and the specific population for which the test is proposed. In this respect, it appeared appropriate to us to set up diagnostic strategies suitable for the particular setting of an emergency unit. The results of our investigation permit us to make some considerations; first of all, although the overall accuracy of the Dimertest® is almost equivalent to that of other latex assays, 11 it appears slightly lower that that reported in recent investigations. Regarding the first issue, we used the D-dimer assay currently employed in our Institution, a semiquantitative latex assay, without an automated method for measurement; specifically, we did not evaluate the diagnostic accuracy of the test at cutoffs different from those suggested by the manufacturer. © F e r r a t a S t o r t i F o u n d a t i o n dimer assay. The situation is clearly different in patients with DVT, when the exclusion of the aforementioned variables improved the diagnostic accuracy of the assay, thus increasing the negative predictive value to 92.8%. Although anticoagulant therapy has been proven to be associated with low D-dimer concentration, 13 no data are currently available on the effect of a short-course of heparin on D-dimer accuracy. The role of a few days of heparin (2.5 days, median in our DVT patients) in reducing D-dimer levels can be debated; moreover, dosage and type of heparin widely varied among our patients. Nevertheless, taking into account these limits, our study demonstrated that even this anticoagulant approach may affect Ddimer results. Because the common practice of initiating heparin treatment if diagnostic imaging for acute venous thromboembolism is not immediately available, this information may have considerable clinical impact both on the first diagnosis of the diseases and, particularly, on the prospective of reducing the need for C-US in patients with a low suspicion of DVT and a negative D-dimer assay. Therefore, although the D-dimer assay may help ER physicians to make a diagnosis in patients with recent DVT, as a sole and simple laboratory assay, the test cannot absolutely exclude VT. At the present, the Ddimer test must be part of a combined approach that requires standardized evaluation of clinical probability along with objective tests (such as C-US) for confirming or refuting the clinical suspicion of VT. Contributions and Acknowledgments SS was responsible for the conception and th