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The APL0406 study showed that arsenic trioxide (ATO) and all-trans retinoic acid (ATRA) are not inferior to standard ATRA and chemotherapy (CHT) in newly diagnosed, low-intermediaterisk acute promyelocytic leukaemia (APL). We analysed the kinetics of promyelocytic leukaemia-retinoic acid receptor-α (PML-RARα) transcripts by real-time quantitative PCR (RQ-PCR) in bone marrow samples from 184 patients and assessed the prognostic impact of fms-related tyrosine kinase 3-internal tandem duplication (FLT3-ITD) in 159 patients enrolled in this trial in Italy. After induction therapy, the reduction of PML-RARα transcripts was significantly greater in patients receiving ATRA-CHT as compared with those treated with ATRA-ATO (3.4 vs 2.9 logs; P=0.0182). Conversely, at the end of consolidation, a greater log reduction of PML-RARα transcripts was detected in the ATRA-ATO as compared with the ATRA-CHT group (6.3 vs 5.3 logs; P=0.0024). FLT3-ITD mutations had no significant impact on either event-free survival (EFS) or cumulative incidence of relapse in patients receiving ATRA-ATO, whereas a trend for inferior EFS was observed in FLT3-ITD-positive patients receiving ATRA-CHT. Our study shows at the molecular level that ATRA-ATO exerts at least equal and probably superior antileukaemic efficacy compared with ATRA-CHT in low-intermediaterisk APL. The data also suggest that ATRA-ATO may abrogate the negative prognostic impact of FLT3-ITD.

Health-related quality of life (HRQOL) is an important goal of therapy for chronic myeloid leukemia (CML) patients treated with current molecular-targeted therapies. The main objective of this study was to investigate factors associated with long-term HRQOL outcomes of CML patients receiving imatinib. Analysis was performed on 422 CML patients recruited in an observational multicenter study. HRQOL was assessed with the Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36). Key socio-demographic and clinical data were investigated for their association with HRQOL outcomes. Chronic fatigue and social support were also investigated. Univariate and multivariate linear regression analyses were used to identify independent factors associated with HRQOL outcomes. Fatigue was the only variable showing an independent and consistent association across all physical and mental HRQOL outcomes (P<0.01). Differences between patients reporting low versus high fatigue levels were more than eight and seven times the magnitude of a clinically meaningful difference, respectively, for the role physical (Δ=70 points) and emotional scale (Δ=63 points) of the SF-36. Fatigue did not occur as an isolated symptom and was most highly correlated with musculoskeletal pain (r=0.511; P≤0.001) and muscular cramps (r=0.448; P≤0.001). Chronic fatigue is the major factor limiting HRQOL of CML patients receiving imatinib.

Although the occurrence of thrombosis in acute promyelocytic leukemia (APL) has been reported during retinoic acid treatment, no studies carried out in large clinical cohorts have specifically addressed this issue. We analyzed 124 APL patients treated with the all-trans retinoic acid and idarubicin protocol and compared clinico-biologic characteristics of 11 patients who developed thrombosis with those of 113 patients who had no thrombosis. In seven patients, the events were recorded during induction, whereas in four patients deep vein thrombosis occurred in the post-induction phase. Comparison of clinico-biological characteristics of patients with and without thrombosis revealed in the former group higher median white blood cell (WBC) count (17 x 10(9)/l, range 1.2-56, P=0.002), prevalence of the bcr3 transcript type (72 vs 48%, P=0.01), of FLT3-ITD (64 vs 28%, P=0.02), CD2 (P=0.0001) and CD15 (P=0.01) expression. No correlation was found with sex, age, French-American-British subtype, all-trans-retinoic acid syndrome or with thrombophilic state that was investigated in 5/11 patients. Our findings suggest that, in APL patients consistent biologic features of leukemia cells may predict increased risk of developing thrombosis.

In this trial, acute myeloid leukemia patients (pts) aged 61-80 years received MICE (mitoxantrone, etoposide and cytarabine) induction chemotherapy in combination with different schedules of granulocyte colony-stimulating factor administration. Pts in complete remission were subsequently randomized for two cycles of consolidation therapy: mini-ICE regimen (idarubicin, etoposide and cytarabine) given according to either an intravenous (i.v.) or a 'non-infusional' schedule. Among the 346 pts randomized for the second step, 331 pts received consolidation-1 and 182 consolidation-2. A total of 290 events (255 relapses, 35 deaths in first CR) have been reported. The median follow-up was 4.4 years. No significant differences were detected in terms of disease-free survival (median 9 vs 10.4 months, P=0.15, hazard ratio (HR) =1.18, 95% confidence interval (CI) 0.94-1.49) - primary end point - and survival (median 15.7 vs 17.8 months, P=0.19, HR=1.17, 95% CI 0.92-1.50). In the 'non-infusional' arm grade 3-4 vomiting (10 vs 2%; P=0.001) and diarrhea (10 vs 4%; P=0.03) were higher than in the 'i.v.' arm, whereas time to platelet recovery >20 x 10(9)/l (median: 19 vs 23 days; P=0.02) and duration of hospitalization (mean: 15 vs 27 days; P<0.0001) was shorter. The 'non-infusional' consolidation regimen resulted in an antileukemic effect similar to the intravenous regimen, which was less myelosuppressive and associated with less hospitalization days.

The therapeutic activity and toxicity profile of gemtuzumab ozogamicin were assessed in 40 patients >60 years of age with acute myeloid leukemia (AML) who were not considered eligible for conventional chemotherapy because of advanced age or poor performance status. The drug was administered at the dose of 9 mg/m2 as a single 2-h i.v. infusion on days 1 and 15. Patients who achieved a complete remission (CR/CRp) were to receive a consolidation with two additional injections of the immunotoxin at the same dose. The overall CR/CRp rate was 17% (95% CI, 8-32%). The CR/CRp rate in patients 61-75 years old was 33% (6/18), and 5% (1/22) in patients older than 75 years. Induction death occurred in seven patients (17%), all aged above 75 years. Overall survival was significantly longer in patients aged 61-75 years than in older individuals (P=0.05), and in CD33+ cases than in CD33- cases (P=0.05). We conclude that the dose/schedule of gemtuzumab ozogamicin used in this trial is too toxic in the age group over 75 years. For these patients, additional studies with reduced doses of the immunotoxin are warranted.

Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) has a dismal prognosis. We prospectively evaluated minimal residual disease (MRD) by measuring BCR/ ABL levels with a quantitative real-time PCR procedure after induction and after consolidation in 45 adults with Ph+ ALL who obtained complete hematological remission after a high-dose daunorubicin induction schedule. At diagnosis, the mean BCR-ABL/GUS ratio was 1.55 +/- 1.78. A total of 42 patients evaluable for outcome analysis were operationally divided into two MRD groups: good molecular responders (GMRs; n = 28) with > 2 log reduction of residual disease after induction and > 3 log reduction after consolidation therapy, and poor molecular responders (PMRs; n = 14) who, despite complete hematological remission, had a higher MRD at both time points. In GMR, the actuarial probability of relapse-free, disease-free and overall survival at two years was 38, 27 and 48%, respectively, as compared to 0, 0 and 0% in PMR (P = 0.0035, 0.0076 and 0.0026, respectively). Salvage therapy induced a second sustained complete hematological remission in three GMR patients, but in no PMR patient. Our data indicate that, as already shown in children, adult Ph+ ALL patients have a heterogeneous sensitivity to treatment, and that early quantification of residual disease is a prognostic parameter in this disease.

In all, 17 consecutive patients in hematological complete remission (HCR) of acute promyelocytic leukemia (APL) received allogeneic stem cell transplantation (SCT) from an HLA-identical sibling and were monitored by reverse transcriptase polymerase chain reaction of PML/RARalpha prior and after transplant. Median age was 31 years (range 3-50 years). At 10 years, the actuarial probabilities of nonrelapse mortality, relapse and disease-free survival were 32% (95% CI: 8-56%), 33% (95% CI: 6-60%) and 46% (95% CI: 22-70%). Six patients tested PCR +ve (1st HCR n=2; 2nd HCR n=3; 3rd HCR n=1) and 11 PCR -ve (2nd HCR n=11) pre-SCT. Of the six patients PCR +ve, two showed early persistence of PCR positivity and converted to sustained PCR negativity after CSA withdrawal (one died of secondary tumor in molecular remission and one is alive in relapse), while four converted to PCR -ve rapidly (one died of the underlying disease and three are in molecular remission). Of the 11 patients PCR -ve pre-SCT, six died (four of transplant-related mortality, one of relapse and one after heart transplantation) and five are alive, four in molecular remission and one is in relapse. Allogeneic SCT seems a valid option for advanced APL, particularly for the poor prognostic group of patients with pre-SCT molecularly persistent disease.

In all, 134 elderly patients (median age 66 years, range 60-75 years) with newly diagnosed acute promyelocytic leukemia (APL) were enrolled in two successive protocols of the Italian multicenter group GIMEMA. All patients received an identical induction with all-trans retinoic acid and idarubicin; 116 (86%) entered complete remission (CR), two (2%) were resistant and 16 (12%) died during induction. After CR, 106 patients received further therapy whereas 10 did not, because of refusal (n=5) or toxicity (n=5). Consolidation consisted of three chemotherapy courses in the AIDA protocol (AIDA, 67 patients) or, since 1997, of an amended protocol including only the first cycle (amended AIDA, aAIDA, 39 patients). In the AIDA group, 43 patients (64%) completed consolidation, while seven (11%) and 17 (25%) patients were withdrawn after first and second courses, respectively; nine patients (13%) died in CR and 12 (18%) relapsed. In the aAIDA group, all patients received the assigned treatment; two patients (5%) died in CR and six (15%) relapsed. In the AIDA and aAIDA series, the 3-year overall and discase-free survival rates were 81 and 83% (P=NS), 73 and 72% (P=NS), respectively. We highlight here the frequency and severity of complications linked to intensive chemotherapy in this clinical setting and suggest that, in APL of the elderly, less intensive postremission therapy allows significant reduction of severe treatment-related toxicity and may be equally effective.

This report used the framework of a large European study to investigate the outcome of patients with and without an HLA-identical sibling donor on an intention-to-treat basis. After a common remission-induction and consolidation course, patients with an HLA-identical sibling donor were scheduled for allogeneic transplantation and patients lacking a donor for autologous transplantation. In all, 159 patients alive at 8 weeks from the start of treatment were included in the present analysis. In total, 52 patients had a donor, 65 patients did not have a donor and in 42 patients the availability of a donor was not assessed. Out of 52 patients, 36 (69%) with a donor underwent allogeneic transplantation (28 in CR1). Out of 65 patients, 33 (49%) received an autograft (27 in CR1). The actuarial survival rates at 4 years were 33.3% (s.e. = 6.7%) for patients with a donor and 39.0% (s.e. = 6.5%) for patients without a donor (P = 0.18). Event-free survival rates were 23.1% (s.e. = 6.2%) and 21.5% (s.e. = 5.3%), respectively (P = 0.66). Correction for alternative donor transplants did not substantially alter the survival of the group without a donor. Also, the survival in the various cytogenetic risk groups was not significantly different when comparing the donor vs the no-donor group. This analysis shows that patients with high-risk myelodysplastic syndrome and secondary acute myeloid leukemia may benefit from both allogeneic and autologous transplantation. We were unable to demonstrate a survival advantage for patients with a donor compared to patients without a donor.

Infections represent a frequent complication of chemotherapy used for acute myeloid leukaemia (AML) and are associated with important toxicity frequently leading to treatment discontinuation. Acute promyelocytic leukaemia (APL) is a unique AML subset requiring tailored therapy including all-trans retinoic acid and anthracycline-based chemotherapy. We analysed in this study the incidence and type of infections complicating the clinical course of 89 consecutive APL patients receiving the AIDA protocol at a single institution. A total of 179 febrile episodes were registered during induction and consolidation, 52% of which were of unknown origin. Infections were clinically and microbiologically documented in 10.6 and 37.4% of cases, respectively. Coagulase-negative staphylococci represented the major cause of septicaemia (28%) and were more frequently isolated during induction, whereas viridans group streptococci, the second pathogen most frequently isolated from blood (27%), represented the principal pathogen detected during consolidation and were significantly associated with mucositis. Gram-negative bacteria accounted for 33.3% of all blood isolates. Fungal infections were only occasionally observed. Bloodstream infections in APL patients were compared with those documented in 271 consecutive patients affected by other subtypes of AML. The incidence of total septicaemia episodes, of staphylococcal bacteraemias and of fungaemias was significantly higher in patients with other AMLs. Empirical antibiotic therapy with ceftriaxone plus amikacin was effective in 73% of APL cases, most of the remaining cases being successfully managed by the addition of teicoplanin. One single death apparently related to infectious complication was recorded. Overall, infections led to antileukaemic treatment withdrawal in six patients, five of whom currently remain in haematologic remission for 13-106 months. These results indicate that a particular pattern of infections is observed in APL patients receiving ATRA plus anthracycline-based chemotherapy and that these appear to be effectively counteracted by standard management.

Variable numbers of CD34+ cells can be harvested from the blood of AML patients in CR after G-CSF supported mobilization following consolidation chemotherapy. We hypothesized that a decreased ability to mobilize stem cells reflects a chemotherapy-induced reduction in the number of normal and leukemic stem cells. We therefore analyzed whether the mobilizing capacity of these patients was of prognostic significance. 342 AML-patients in first CR received daily G-CSF from day 20 of the consolidation course and underwent 1-6 aphereses to obtain a minimum dose of 2 x 10(6) CD34+ cells/kg. Afterwards they were randomized for autologous bone marrow (BM) or blood SCT. As a surrogate marker for the mobilizing capacity, the highest yield of CD34+ cells of a single apheresis was adopted. Patients could be categorized into four groups: no harvest (n = 76), low yield (<1 x 10(6) CD34+/kg; n = 50), intermediate yield (1-6.9 x 10(6) CD34+ cells/kg; n = 128) and high yield (> or = 7 x 10(6) CD34+ cells/kg; n = 88). The median follow-up was 3.4 years; 163 relapses and 16 deaths in CR were reported. Autologous blood or BM SCT was performed in 36%, 64%, 81% and 88%, respectively, of the patients assigned to the no harvest, low, intermediate and high CD34+ yield group. The 3-year disease-free survival rate was 46.7%, 65.0%, 50.4% and 26.9% (P = 0.0002) and the relapse incidence was 47.5%, 30.1%, 43.1% and 71.9% (P < 0.0001). Multivariate Cox's proportional hazards model showed that the CD34+ yield was the most important independent prognostic variable (P = 0.005) after cytogenetics. Patients with the highest mobilizing capacity have a poor prognosis due to an increased relapse incidence.

Alterations in the FLT3 gene, including internal tandem duplications (ITDs) and D835 mutations occur frequently in acute myelogenous leukemia. We investigated the prevalence and clinico-biological correlations of FLT3 ITDs and D835 mutations in 90 patients with acute promyelocytic leukemia (APL) receiving the AIDA protocol. Twenty patients in which both presentation and relapse material was available were analyzed sequentially. Thirty-three patients (37%) harbored the ITD, and seven (7.7%) the D835 mutation in blasts obtained at diagnosis. Presence of ITDs was strongly associated with high WBC count (P = 0.0001), M3 variant (P = 0.0004), and the short (BCR3) PML/RARalpha isoform (P = 0.003). There was no difference in response to induction in the two ITD+ve and ITD-ve groups, while a trend towards inferior outcome was observed for ITD+ve cases when analyzing disease-free survival (DFS) and relapse risk (RR). These differences, however, did not reach statistical significance. Sequential studies showed variable patterns in diagnostic and relapse material, ie ITD (-ve/-ve, +ve/+ve, +ve/-ve, -ve/+ve) and D835 (-ve/-ve, +ve/-ve, -ve/+ve). Our results indicate that FLT3 alterations are associated in APL with more aggressive clinical features and suggest that these lesions may not play a major role in leukemia progression.

We have treated 20 patients, affected by acute myelogenous leukemia in advanced phase of the disease, with intravenous high-dose recombinant interleukin-2 (IL2) as induction treatment, achieving a complete remission (CR) in 11/20 of patients (55%). All CR patients were planned to receive a maintenance program with lower subcutaneous doses of IL2 until relapse. Currently, 5/11 patients are alive in continuous complete remission with a minimum follow-up of 9 years from IL2 induction. In the aim to investigate the treatment's side-effects during or after prolonged IL2 therapy, we decided to submit these patients to a clinical and immunological evaluation. Four patients have been evaluated as one, who independently stopped IL2 after 6 years, refused the check-up. No organ-specific treatment sequelae that may decrease the quality of life or may be life-threatening were found, concerning renal, liver and cardiovascular function. Endocrine abnormalities were detected in three patients, the most serious being a severe hypothyroidism, which prompted cessation of IL2 maintenance after 6 years and required thyroid supplementation treatment. Immunological studies were carried out prior to the last IL2 cycle and showed high levels of CD3-positive T cells expressing the IL2 receptor alpha chain (CD25), both in the peripheral blood and in the bone marrow. Our study shows that low-dose IL2 can be given for a prolonged period of time without serious organ-specific late sequelae and with a good quality of life.

Within 285 adult acute lymphoblastic leukemias (ALL) included in the multicenter GIMEMA 0496 trial and prospectively studied by conventional cytogenetics, 18 cases (6%) with long arm deletion of chromosome 6 (6q) were identified. These cases were divided into: (i) del(6q) only (n = 6); (ii) del(6q) plus other numerical and/or structural abnormalities (n = 8); (iii) del(6q) and other 'specific' translocations (n = 4). The biologic and clinical features of the patients carrying this anomaly, as well as their outcome, were compared with those of 267 patients without del(6q). A T cell phenotype was more frequently associated with del(6q) cases in general (P = 0.001) and particularly with cases presenting del(6q) as the isolated abnormality (P = 0.0027). No significant difference with respect to multidrug resistance (MDR)/P glycoprotein expression was observed between the two groups of patients (21% vs 28% of MDR-positive cases, respectively). A BCR-ABL fusion transcript was less frequently detected in cases with del(6q) (11%) compared with those without the anomaly (29%). p15 and p16 deletions were identified by Southern blot analysis in 21% of cases with del(6q) and in 26% of cases without del(6q). In this latter group, a T cell phenotype was less frequently associated with p15 and/or p16 deletion than in the group carrying del(6q) (36% vs 100% of cases, P = 0.011). Overall, patients with ALL and del(6q) had a high complete remission (CR) rate (83%); however, they had a lower 18 month event-free survival (31% vs 41%) and a higher relapse rate (70% vs 37%, P = 0.02) compared with patients without del(6q). To date, this is the largest series of adult ALL cases reported with del(6q) homogeneously treated, which have also been prospectively studied for MDR expression and for the detection of known fusion genes. This anomaly, as an isolated change, identifies a subset of cases with hyperleukocytosis (median WBC count 52 x 10(9)/l) and a strict correlation with a T cell phenotype. Overall, del(6q) seems to be associated with an unfavorable clinical outcome, although this finding will need to be confirmed by extended FISH analysis.

Comparisons of the effectiveness of chemotherapy and transplantation in AML in first complete remission (CR) have focused almost exclusively on patients with de novo disease. Here we used Cox modelling to compare these strategies in patients with MDS and s-AML treated by the Leukemia Group of the EORTC or at the MD Anderson Cancer Center. All patients were aged 15-60. The 184 EORTC patients received conventional dose ara-C + idarubicin + etoposide for remission induction, and after one consolidation course, were scheduled to receive an allograft, or an autograft if a sibling donor was unavailable. The 215 MDA patients received various high-dose ara-C containing induction regimens, and in CR, continued to receive these regimens at reduced dose for 6-12 months. CR rates were 54% EORTC and 63% MDA (P = 0.09). Sixty-five of the 100 EORTC patients who entered CR received a transplant in first CR. Disease-free survival in patients achieving CR was superior in the EORTC cohort, the 4-years DFS rates were 28.9% (s.e. = 4.8%) EORTC vs 17.3% (s.e. = 3.7%) MDA (P = 0.017). Survival from CR was not significantly different in the EORTC and MDA groups, as was survival from start of treatment. After accounting for prognostic factors the conclusions were unchanged. Despite various problems with the analysis discussed below, the data suggest that neither transplantation nor chemotherapy, as currently practised, can be unequivocally recommended for these patients in first CR and that questions as to the superior modality may be less important than the need to improve results with both.

The objective of the study was to evaluate the incidence, characteristics, treatment and outcome of acute megakaryoblastic leukemia (AMeL) in patients enrolled in GIMEMA trials. Between 1982 and 1999, 3603 new consecutive cases of AML aged over 15 years were admitted to GIMEMA trials. Of them, 24 were AMeL. The incidence of AMeL among AML patients enrolled in GIMEMA trials was 0.6% (24/3603). Diagnosis was based on morphological criteria. Out of 11 cytogenetic studies performed two presented chromosome 3 abnormalities. Twelve patients (50%) reached a CR, five (21%) died in induction and seven (27%) were unresponsive. The median duration of CR was 35 weeks (range 10-441). Seven patients underwent transplantation procedures (1 BMT, 4 aBMT, 2 aPBSCT). Four patients died in CR due to chemotherapy-related complications. Comparing the CR rate between AMeL and the other cases of AML enrolled in GIMEMA trials, no differences were observed. These results were mirrored for different age groups. The median survival was 40 weeks. At present, after a follow-up of a minimum of 2 years, only two patients are alive in CR, all the others having died. A 5-year Kaplan-Meier curve shows a disease-free survival of 17% and an actuarial overall survival of 10%. AMeL is a rare form of AML. The CR duration and the overall survival in this group of patients are very poor, even if similar to those observed in other AML. Furthermore, a high number of deaths in CR were observed. On the basis of these data, a specific therapeutic approach, possibly with innovative treatments, should be evaluated.

Extramedullary (EM) involvement occurs infrequently in acute promyelocytic leukemia (APL) and usually involves skin and CNS. We describe seven patients (four observed at a single institution) who relapsed in various sites of the auditory apparatus, including the external canal and middle ear (temporal bone). Front-line treatment included ATRA and chemotherapy (six patients) or chemotherapy alone (one patient). Three patients had concomitant hematologic relapse, two had molecular relapse and two were in hematologic and molecular remission when ear localization was documented. Local symptoms that stimulated further diagnostic studies included ear bleeding/discharge in the first patient, but were mild in the others (hypoacusia, five patients; earache, two patients). Ear involvement by leukemia was documented by histological and/or molecular studies after local surgery in five cases, and by CT scan or NMR in the remaining patients. We suggest that the ear might represent a specific sanctuary for disease involvement in APL.

We studied the impact of cytogenetics and kind of induction/consolidation therapy on 848 adult acute myeloid leukemia (AML) patients (age 15-83). The patients received three types of induction/consolidation regimen: standard (daunorubicin and cytosine arabinoside (3/7); two cycles); intensive (idarubicin, cytosine arabinoside and etoposide (ICE), plus mitoxantrone and intermediate-dose Ara-C (NOVIA)); and low-dose (low-dose cytosine arabinoside). CR patients under 60 years of age, if an HLA-identical donor was available received allogeneic stem cell transplantation (allo-SCT); otherwise, as part of the program, they underwent autologous (auto)-SCT. CR rates significantly associated with 'favorable' (inv(16), t(8;21)), 'intermediate' ('no abnormality', abn(11q23), +8, del(7q)) and 'unfavorable' (del (5q), -7, abn(3)(q21q26), t(6;9), 'complex' (more than three unrelated cytogenetic abnormalities)) karyotypes (88% vs 65% vs 36%, respectively; P = 0.0001). These trends were confirmed in all age groups. On therapeutic grounds, intensive induction did not determine significant increases of CR rates in any of the considered groups, with respect to standard induction. Low-dose induction was associated with significantly lower CR rates. Considering disease-free survival (DFS), multivariate analysis of the factors examined (including karyotype grouping) showed that only age > 60 years significantly affected outcome. However, in cases where intensive induction was adopted, 'favorable' karyotype was significantly related to longer DFS (P = 0.04). This was mainly due to the favorable outcome of t(8;21) patients treated with intensive induction. Patients receiving allo-SCT had significantly longer DFS (P = 0.005); in particular, allo-SCT significantly improved DFS in the 'favorable' and 'intermediate' groups (P = 0.04 and P = 0.048, respectively). In conclusion our study could provide some guidelines for AML therapy: (1) patients in the 'favorable' karyotype group seem to have a longer DFS when treated with an intensive induction/consolidation regimen, adopted before auto-SCT instead of standard induction; this underlines the importance of reinforcement of chemotherapy, not necessarily based on repeated high-dose AraC cycles. Allo-SCT, independently of induction/consolidation therapy, should be considered an alternative treatment; (2) patients in the 'intermediate' karyotype group should receive allo-SCT; (3) patients in the 'unfavorable' karyotype group should be treated using investigational chemotherapy, considering that even allo-SCT cannot provide a significantly longer DFS, but only a trend to a better prognosis.

Twenty-nine consecutive patients with high-risk hematological malignancy aged from 3 to 58 years underwent an unmanipulated graft from an HLA-identical sibling after an irradiation-free preparative regimen consisting of idarubicin (IDA), 21 mg/m2/day administered by continuous infusion on days -12 and -11, followed by busulphan (BU), 4 mg/kg/day orally from day -7 to -4, and cyclophosphamide (CY), 60 mg/kg/day intravenously on days -3 and -2 (IDA-BUCY2). Most clinically relevant extra-hematological regimen-related toxicities consisted of stomatitis observed in all subjects and hemorrhagic cystitis occurred in five cases (17%) within 100 days after transplant. Six patients (21%) developed a grade 2 acute graft-versus-host disease (GVHD) and three (10%) a grade 3 or 4; extensive chronic GVHD was assessed in nine of 22 (41%) evaluable patients. So far, 12 patients have died and 17 are alive, 16 of whom disease-free, 5-41 months after transplant (median, 15 months). The causes of death were related to GVHD in three patients, to sepsis in one and to disease recurrence in the remaining eight. At present, only one of nine relapsed patients is alive. For all patients the actuarial probability of survival (OS) at 1 and 2 years +/- standard error (s.e.) was 63 +/- 9% and 52 +/- 10%, respectively. The actuarial probabilities of disease-free survival (DFS), relapse and transplant-related mortality (TRM) at both 1 and 2 years +/- s.e. were 53 +/- 9%, 35 +/- 9% and 16 +/- 7%, respectively. These results are encouraging but not substantially different from those obtained in 28 patients with malignancy in advanced phase transplanted after the standard BUCY2 regimen, who had an actuarial probability of OS, DFS, relapse and TRM projected at 10 years +/- s.e. of 54 +/- 10%, 57 +/- 9%, 36 +/- 9% and 11 +/- 6%, respectively. Although the retrospective comparison between the two groups does not seem to show any advantage in the use of the IDA intensified regimen, only a prospective randomized trial could answer this question.

The first multicentric approach to childhood acute lymphoblastic leukemia (ALL) treatment in Italy started in the early 1970s when the Associazione Italiana di Ematologia ed Oncologia Pediatrica (AIEOP) was founded. Since then the AIEOP has conducted nationwide chemotherapy protocols. Results obtained in three different periods (1982-1986, 1987-1990, 1991-1995) are reported here. Treatment schedules have been characterized by a progressive intensification of systemic therapy and by a progressive substitution of protracted intrathecal therapy for cranial irradiation as central nervous system (CNS) preventive therapy. In the third period cranial radiotherapy (CRT) has been administered only to patients at high risk of relapse or with CNS involvement at diagnosis (about 15% of the overall population). A progressive improvement of therapeutic results, with a steady reduction of isolated CNS relapse rates have been obtained in the three periods considered here. The AIEOP experience shows that CRT can be safely omitted in non-high risk patients, unless they are T-ALL patients with WBC count at the diagnosis > or =100,000/mm3, and that intensification of treatment allows the improvement of overall results with a reduction of the impact of NCI prognostic criteria. Over the years, AIEOP has also continued to foster active cooperation at an international level. In the ongoing AIEOP ALL 2000 study, conducted in cooperation with the BFM group, patients are stratified according to the presence of translocations t(9;22) and t(4;11) and to treatment response (either initial steroid therapy or induction) or minimal residual disease). This cooperation will allow an adequate recruitment of patients to answer relevant randomized questions in the context of a study in which patients are stratified according to minimal residual disease findings.

The feasibility and safety of outpatient management of acute promyelocytic leukemia (APL) during the aplastic phase after intensive consolidation chemotherapy, the incidence and types of complications requiring readmission to hospital, and the number of hospital days spared by this policy have been prospectively evaluated. After chemotherapy administration, patients were evaluated on an ambulatory basis. In the event of any complication they referred to the Emergency Unit (EU) of our Department dedicated to outpatients with hematologic diseases. Forty patients with APL observed over a 4 year period were eligible for intensive chemotherapy. After the achievement of complete remission they received a total of 104 consolidation courses and in 98 instances they were followed on an ambulatory basis. There were 41 cases (42%) of rehospitalization for fever (40 cases) or severe anemia (one case). Only one patient died due to a brain hemorrhage. Streptococcus viridans was the organism most frequently isolated from blood. Empiric once-a-day antibacterial therapy with ceftriaxone and amikacin was effective in 87% of the cases and made possible early discharge in 28% of the cases to continue the antibiotic therapy on an outpatient setting. Patients were managed out of the hospital for 76% of the post-consolidation neutropenia period. Thanks to the availability of an EU specifically dedicated to outpatients with hematologic diseases, out-hospital management of APL patients after consolidation therapy appeared to be safe, well accepted, potentially cost-saving, and contributed to saving the risk of developing severe nosocomial infections.

A home care service has been implemented at our center with the aim of offering domiciliary assistance to patients with hematologic malignancies in advanced phase. We report our experience concerning the home management of these patients in the setting of infective complications. Of 151 patients in home care, 70 (46%) developed a total of 109 febrile episodes, performance status and neutrophil count significantly affecting the incidence of infections. Fever was of unknown origin in 51% of cases and microbiologically and clinically documented infections accounted for 26 and 23% of the cases, respectively. Oral ciprofloxacin in patients not neutropenic and intravenous ceftriaxone plus amikacin in neutropenic patients was shown to be effective and suitable for empiric home antibacterial treatment; in fact, 65% of febrile episodes responded to the initial antibacterial therapy with a further 16% after modification. Overall, 19.3% of the infective episodes were fatal, the prognosis appearing to be similar to that usually observed in the same category of patients in an inpatient setting. Our experience appears to show that a home care program could be the option of choice for patients with advanced cancer even in the setting of infective complications. It could improve the quality of life of patients and of their families, and it could save these subjects the risk of developing infections by resistant nosocomial isolates.

The outcome of a cohort of 218 consecutive patients who failed to respond to a single course of standard daunorubicin plus ARAC (three + seven) induction regimen has been retrospectively evaluated to assess the characteristics of this group of AML patients and the effectiveness of second-line induction programs. Seventy-four of the 218 patients (33.9%) attained complete remission with salvage chemotherapies. The multivariate analysis of pretherapy characteristics of the patients showed that peroxidase positivity and age were the most important factors in determining whether or not the patient would have a favorable response to second-line induction regimen. In addition, comparison of marrow characteristics at diagnosis with those of marrow after the first-line therapy (marrow leukemic index, MLI) provided the greatest differences between second-line CR and resistant patients. Finally, peroxidase positivity and MLI predicted for remission duration and overall survival. Allogeneic BMT, however, appeared the most important factor for survival and event-free survival of remitting patients. These results are of importance when considering that better defined prognostic factors provide an objective rationale for selecting appropriate strategies for the treatment of patients who do not respond to a single course of induction regimen.

Acute promyelocytic leukemia (APL) is a medical emergency which requires rapid diagnosis and tailored treatment. Detection of the PML/RARalpha fusion gene in APL blasts is critical to start promptly the specific therapy with all-trans retinoic acid (ATRA). APL lacking this genetic lesion have been reported as being ATRA resistant. Reverse-transcription polymerase chain reaction (RT-PCR) has been extensively used to detect the PML/RARalpha cDNA. The reported PML/RARalpha amplification techniques are laborious and time consuming, and include conventional RNA extraction, cDNA synthesis and a two-round (nested) PCR. We hereby describe a few variations of the commonly adopted RNA extraction and PML/RARalpha RT-PCR protocols which allow a molecular diagnosis of APL to be carried out in less than 5 h. Processing of small volumes of leukemic cell lysate (0.5 ml) in a microfuge allows extraction of good quality RNA in 1 h. After reverse transcription to obtain cDNA, a 'hot start' PCR procedure was adopted which enabled us to amplify clearly visible and specific products after a single (not nested) amplification round. The PML/RARalpha fusion gene was detected in the blasts of six consecutive APL at diagnosis, and an APL-tailored protocol including ATRA was started in each case within 6 h of admission. On repeated experiments, the assay proved highly specific and sensitive for the rapid detection of all PML/RARalpha transcript types. Our data should encourage the use of this rapid procedure for the diagnosis of both typical APL and, particularly, less typical cases awaiting urgent therapeutic intervention.

Thirty-one patients with either advanced AML (18) or blastic CML (13) were treated with an intensive timed sequential combination of VP-16 (100 mg/m2/day i.v., days 1-3 and 8-10), intermediate-dose Ara-C (500 mg/m2 i.v. over 1 h q 12 h, days 1-3 and 8-10) and carboplatin (150 mg/m2/day i.v. continuous infusion, days 1-3 and 8-10). CR rates were 9/18 (50%) for patients with AML and 9/13 (69%) for those with blastic CML, for an overall CR rate of 58%. Among patients with AML, CR rates for specific subgroups were: primary resistant disease 2/6; resistant relapse 1/5; second relapse 6/7. Ten patients were refractory to VAC and three (10%) died of complications during marrow hypoplasia. Median overall survival was 7 months, and median DFS of the 18 responders 4 months. The major toxicity was myelosuppression and infection. The VAC regimen has significant activity and acceptable toxicity in myelogenous leukemias. The very high response rate observed in blastic CML warrants further testing of carboplatin-based regimens in this poor-risk form of leukemia.

We report 72 blastic crises (BC), occurring in 238 Ph+ chronic myeloid leukemia (CML) patients treated in chronic phase (CP) with alpha-interferon (IFN) for a median time of 51 months (range 7-96). The 238 patients were grouped by Sokal's risk at diagnosis in low- (LR), intermediate- (IR) and high-risk (HR), and by CP treatment. Group 1: 160 patients (57% LR, 31% IR, 12% HR) given IFN alone in early CP. Group 2: 31 patients (65% LR, 32% IR, 3% HR) given IFN alone in late CP. Group 3: 23 patients (78% LR, 22% IR) given IFN before and after autologous stem cell transplantation (ASCT). Group 4: 24 patients (83% LR, 17% IR) given IFN after ASCT. Of the 72 BC, 52 (72%) were myeloid (My), and 20 (28%) lymphoid (Ly). Overall BC incidence was similar in all CP treatment groups, although with a prevalence of Ly BC in groups 3 + 4 vs groups 1 + 2, (p = NS); the incidence of BC was higher in HR patients (P = NS), but on the whole it was lower than expected on the basis of historical controls. Lymphoid BC was more frequent in LR than in IR + HR patients (P < 0.05), and was more frequent in responders to IFN, than in non-responders (P < 0.05). In conclusion, a subset of patients with low risk at diagnosis, better response to IFN and proneness to evolve into Ly BC can be identified. The role played by IFN in this context remains to be defined.

This paper reported on a series of 49 elderly patients over 60 years of age affected by acute lymphoblastic leukemia (ALL), observed at our institution from 1969 to 1993. The biological characteristics considered, median WBC count, FAB classification, immunophenotype at onset of disease, were no different from those of our adult ALL series. Overall complete remission (CR) rate of these patients was 59%, 18% had resistant disease and 23% died during induction. Overall median survival was 9 months and overall median duration of CR was 7 months. All patients were divided according to treatment into two groups: group A (13 patients) received an intensive treatment including vincristine (VCR), prednisone (PDN), daunorubicin (DNR) and L-asparaginase (L-Asp), while in group B (36 patients) were included patients who received mild conventional induction with VCR and PDN. In group A, 77% of patients achieved CR and 23% died during induction. All patients were hospitalized during induction treatment. During follow-up, among 10 CRs, five (50%) died in CR because of hemorrhage (two), infections (two) and myocardial infarction (one); five (50%) relapsed. Median survival was 4 months and median duration of CR was 3.5 months. In group B, 53% of patients obtained CR, 25% had resistant disease and 22% died during induction. During induction, 44% of patients were hospitalized. During follow-up, among 19 CRs, 14 (74%) relapsed and three (15%) died in CR because of infection (two) and cardiorespiratory failure (one). Three patients (15%) are still alive: two in first CR and one in second CR. No statistical differences between the two groups in terms of CR rate or survival were noted. Standardized therapeutic trials are needed better to evaluate results in these patients, considering also the introduction of new therapeutic agents or supportive treatments, such as growth factors.

We report a case of therapy-related acute myeloid leukemia (t-AML), M4 FAB subtype, with t(10;11)(p14;q21) chromosome abnormality developed in a patient treated for acute promyelocytic leukemia (APL) after 4 years of continuous complete remission (CCR). Two distinct forms of t-AML have been described: the classical type and the second type. Our case has many characteristics in common with the second type of t-AML such as: exposure to topoisomerase II active agents (idarubicin (IDA), mitoxantrone (MITOX), etoposide (VP16)), M4 FAB subtype, a latency period of 39 months and absence of a preleukemic phase. However, it differs in the chromosome 11 breakpoint (band q21 instead of q23) and absence of ALL-1 (Hrx, MLL, Htrx) gene involvement. This can represent the second observation of t-AML occurring after treatment for APL.

We treated a patient with chronic myeloid leukaemia in accelerated phase with autologous bone marrow transplantation. Before reinfusion, cells were purged in vitro with a 26-mer phosphorothioate antisense oligodeoxynucleotide specific for the B2A2 junction. Incubation with antisense oligodeoxynucleotides produced a 24 and 41% reduction of CFU-GM and CD34+ cells, respectively. However, an in vitro test previously performed as a screening for patient inclusion in this procedure, revealed a 38 and 75% reduction of colony formation after 24-h and 168-h incubation, respectively. The patient showed bone marrow engraftment 15 days after reinfusion and haematological reconstitution after 17 and 25 days for platelets and neutrophils, respectively. Using fluorescence in situ hybridization in interphase nuclei, we demonstrated the presence of a proportion of Ph-negative cells in repeated controls after the autograft. The patient is now in unmaintained complete haematological remission 9 months after the autograft.

Scanty information is available on acute promyelocytic leukemia (APL) in children, and whether differences are present with respect to the adult form. The experience of the Italian Pediatric Hematology and Oncology Group (AIEOP) will be presented with respect to the following aspects: 1. Incidence of APL. The incidence of APL is generally considered to account for 3-9% of acute myelogenous leukemia (AML) in children and approximately 10-15% in adults. Recently a single Italian pediatric institution reported that APL constituted one third of observed acute nonlymphocytic leukemia (AnLL) cases. Data from the AIEOP cooperative study group have confirmed that APL in Italy is more frequently observed in children as compared to other countries. Environmental and/or genetic factors should be considered to explain such differences. 2. Diagnosis of M3v. The clinical and biological features of the largest series of childhood M3v will be presented and the problems encountered in the proper separation of 'classic' M3 and M3v in children will be discussed. 3. Clinical Aspects. The clinical features of the APL patients enrolled in the AIEOP study groups since 1989, will be presented with emphasis on the recent experience with the use of all-trans retinoic acid. 4. Analysis of PML/RAR alpha Fusion Transcripts. An RT-PCR analysis of 32 pediatric APL cases from cryopreserved bone marrow samples has been performed. It is concluded that APL in children did not differ significantly from the adult form, with the exception of a higher incidence of PML bcr3 breakpoint.

We investigated the occurrence of late events (beyond 2 years) in patients with acute leukaemia who received an allogeneic (BMT) (n = 1059), or an autologous bone marrow transplantation (ABMT) (n = 656) in Europe during the period from January 1979 to December 1990. Patients with no recurrence of leukaemia at 2 years had overall 82% chance of being alive in complete remission at 9 years following transplantation regardless of the nature of the leukaemia, the status at transplant, and the type of transplant. The incidence of late relapses continuously decreased with time. The latest relapses in acute myelogenous leukaemia (AML) were observed following BMT at 6.6 years in a patient transplanted in first remission (CR1) and at 3.7 years in a patient transplanted in second remission (CR2), and following ABMT at 6 years and 5.1 years respectively. The latest relapses in acute lymphoblastic leukaemia (ALL) were observed following BMT at 4 years in a patient transplanted in first remission (CR1) and at 6.8 years in a patient transplanted in second remission (CR2), and following ABMT at 5.3 years and 4.5 years respectively. Several factors predictive for late relapse or death were identified. Patients allografted experienced a lower frequency of late relapse than patients autografted. Of the numerous other prognostic factors studied, female sex in AML, the use of total body irradiation (TBI) in ALL and status in CR1, rather than CR2-3, for both ALL and AML allografted were correlated with a lower relapse incidence. The use of TBI in ALL was also associated with a better LFS and survival. The absence of acute graft-versus-host disease (GVHD) in allografted AML correlated with better LFS and better survival, but had no influence on the relapse incidence. This study indicates that patients alive and well at 2 years post transplant have a very high probability of being cured, but the possibility of late relapse still remains.

Adult Philadelphia-chromosome-positive acute lymphoblastic leukemia (Ph1-positive ALL) represents about 30% of all adult ALL, and is considered a poor prognosis disease, since complete remission (CR), which can be achieved in 50-70% of cases, is usually short in patients treated with conventional chemotherapy. Presently bone marrow transplantation, performed early in first CR is becoming the treatment of choice, as it has shown to be able to cure some cases. In a ten-year period, at our department, among 108 adult ALL patients, in which cytogenetics was successfully carried out at diagnosis, 24 (22%) resulted Ph1-positive. Molecular biology was performed in 13 out of these 24 patients: 10 patients showed a p210 rearrangement and three p190. All patients have been treated at induction with conventional drugs, with a CR rate of 96%. As post-remission therapy, the first 17 cases (group 1) followed a chemotherapeutic program, like the other adult ALL; while the remaining six patients (group 2) have been enrolled in a pilot study including early BM transplantation. In group 1, the median overall duration of first CR is 7 months; 50% of relapses were recorded within the first 6 months, although in this group five patients exhibited a first CR prolonged more than 30 months. In group 2, among the six patients, three were submitted to allogeneic bone marrow transplantation (BMT), and three to autologous bone marrow transplantation (ABMT). Overall median duration of first CR is 13 months. Three patients relapsed, three are in continuous CR for 11, 31 and 32 months.

Acute promyelocytic leukemia (M3) is, as one of the FAB subtypes of AML, included in the EORTC/GIMEMA AML-8A and 8B randomized trials. In these trials 1519 patients were included, 477 of them in non-Italian EORTC-LCG centers and 1042 in GIMEMA centers. A total of 80 patients were classified as M3 including 18 patients with M3-variant. Thirty-nine were male and 41 female. Ages ranged from 15 to 59 years; 25 (31.3%) of them were younger than 30, 34 (42.5%) between 30 and 45, and 21 (26.3%) older than 45 years of age. 56.3% of the patients had leukocytes less than 5 x 10(9)/l at the time of diagnosis vs. 24.9% of the patients belonging to the other FAB subtypes. Remission induction consisted of a standard protocol with 3 days daunorubicin and 7 days of cytosine arabinoside. Forty-three patients (53.8%) achieved a complete remission compared to 64.6% of the remaining AML patients. After salvage treatment this percentage increased to 70%, which is the same as for the other AML subtypes. Thirteen (16.3%) patients died during remission induction, mainly due to hemorrhagic complications. This percentage is significantly higher than the death rate (9.1%) in the other FAB subtypes of AML. All patients received one course of consolidation treatment. Post consolidation treatment could be either standard maintenance, intensive consolidation courses, autologous or allogeneic transplantation, according to the guidelines of the treatment protocols. At present, relapses almost all in the bone marrow, are seen in only 34.9% of the M3 patients, compared to 48.4% in the remaining AML patients. Disease-free survival for patients less than 45 years of age with the M2 and M3 subtypes was approximately 50% at 3 years compared to 30-40% for the other FAB subtypes. Despite the higher death rate during induction, the long-term survival results were better for M3 patients in comparison with the remaining AML patients. The projected survival at 3 years was 50% for M3 patients vs. 38% for remaining patients.

Scanty information is available on acute promyelocytic leukemia (APL) in children, and whether differences are present with respect to the adult form. The experience of the Italian Pediatric Hematology and Oncology Group (AIEOP) will be presented with respect to the following aspects: 1. Incidence of APL. The incidence of APL is generally considered to account for 3-9% of acute myelogenous leukemia (AML) in children and approximately 10-15% in adults. Recently a single Italian pediatric institution reported that APL constituted one third of observed acute nonlymphocytic leukemia (AnLL) cases. Data from the AIEOP cooperative study group have confirmed that APL in Italy is more frequently observed in children as compared to other countries. Environmental and/or genetic factors should be considered to explain such differences. 2. Diagnosis of M3v. The clinical and biological features of the largest series of childhood M3v will be presented and the problems encountered in the proper separation of 'classic' M3 and M3v in children will be discussed. 3. Clinical Aspects. The clinical features of the APL patients enrolled in the AIEOP study groups since 1989, will be presented with emphasis on the recent experience with the use of all-trans retinoic acid. 4. Analysis of PML/RAR alpha Fusion Transcripts. An RT-PCR analysis of 32 pediatric APL cases from cryopreserved bone marrow samples has been performed. It is concluded that APL in children did not differ significantly from the adult form, with the exception of a higher incidence of PML bcr3 breakpoint.

5-Aza-2'-deoxycytidine (Decitabine) is a new cytosine analog with potent antileukemic activity and able to induce in vitro gene activation and cellular differentiation by a mechanism probably involving DNA hypomethylation. The aim of this pilot study was to evaluate the efficacy and the toxicity of Decitabine, used as single induction agent, in the treatment of poor prognosis acute myeloid leukemia (AML) patients, and to explore its mechanism of action. A total of 12 patients were treated with Decitabine at 90-120 mg/m2 as a four hour intravenous infusion, three times daily for three consecutive days every four to six weeks. A minimum of two courses were required for response evaluation and to consider a patient as therapeutic failure. A total of 10/12 patients were fully evaluable for response; three patients achieved a complete remission (CR) and one a partial remission (PR). Extra-hematological toxicity was generally mild. As for the mechanism of action, both a differentiation induction effect and a cytotoxic mechanism have been observed. In particular, CRs and PRs were probably obtained through the induction of leukemia cell differentiation as shown by the kinetic of remission and immunotyping studies. The preliminary results of this ongoing study suggest that Decitabine may have a prominent role in the treatment of those AML patients with poor general conditions and/or advanced age.

We review the role of adhesion molecule expression on malignant lymphoid cells as delineated by experimental studies and clinical observation. Adhesion molecules of the Ig superfamily, integrins, selectins, and the lymphocyte homing receptor CD44 mediate cell-to-cell and cell-to-extracellular matrix interactions. These molecules have been investigated with the aim (i) of defining certain biological features of the malignant cells, (ii) of providing a rationale to understand tumor organization, metastasis and organ specificity, and (iii) of detecting disease subsets and prognostic groups.

Based on pre-clinical findings and on more recent preliminary in vivo data it has been suggested that immunotherapy with Interleukin 2 (IL2) may be employed in the treatment of acute leukemia patients. Here we shall discuss the biological and clinical observations which indicate that this innovative therapeutic approach may play a role in the management of minimal residual disease.

In an attempt to prolong disease-free survival in children with acute leukemia, we tested the feasibility of interleukin-2 (IL-2) administration after an autologous bone marrow transplantation (ABMT). We report the clinical and biological data obtained in three children with acute myelocytic leukemia (AML) in second complete remission (CR) and in seven children with acute lymphocytic leukemia (ALL) in second or subsequent CR, who received IL-2 at a median interval of 78 days (range 38-125) from ABMT. Patients were treated with 1-2 cycles of IL-2 given by continuous infusion over a 5-day period using a daily escalating protocol, from 100 micrograms/m2 per day to the maximum tolerated dose, followed after 3 weeks by low-dose IL-2 for 5 days monthly over a 6-h infusion on an out-patient basis. Side effects greater than grade 2 (WHO system), consisting of thrombocytopenia, fever, cutaneous rash, nausea and vomiting, diarrhoea were common during the high-dose IL-2 cycles, but resolved 24-48 h after stopping IL-2. Only one patient developed liver toxicity (grade 3, WHO) on day +3 of the first cycle which prompted us to stop the administration of IL-2. An increase in lymphocytes and eosinophils was also observed. IL-2 treatment was followed by a normalization of NK function and by the generation of a high proportion of endogenous LAK cells. All seven ALL patients relapsed at a median of 5 months (range 1-23). Two AML patients relapsed at 1 and 11 months, while the other is still in continuous CR at 23 months after IL-2 treatment. Our IL-2 schedule for treatment of leukemia in children after ABMT is thus feasible but its efficacy requires further investigation.

In this paper we review some of the preclinical findings which have led us to believe that immunotherapy with interleukin 2 (IL2)/lymphokine activated killer (LAK) cells may be a feasible approach in the management of acute myeloid leukemia. The main clinical and biological results so far obtained with IL2 treatment, and the currently ongoing protocols and strategies are discussed.