Diagnosis and management of acquired aplastic anemia in childhood. Guidelines from the Marrow Failure Study Group of the Pediatric Haemato-Oncology Italian Association (AIEOP).

Acquired aplastic anemia (AA) is a rare heterogeneous disorder characterized by pancytopenia and hypoplastic bone marrow. The incidence is 2-3 per million population per year in the Western world, but 3 times higher in East Asia. Survival in severe aplastic anemia (SAA) has improved significantly due to advances in hematopoietic stem cell transplantation (HSCT), immunosuppressive therapy, biologic agents, and supportive care. In SAA, HSCT from a matched sibling donor (MSD) is the first-line treatment. If a MSD is not available, options include immunosuppressive therapy (IST), matched unrelated donor, or haploidentical HSCT. The purpose of this guideline is to provide health care professionals with clear guidance on the diagnosis and management of pediatric patients with AA. A preliminary evidence-based document prepared by a group of pediatric hematologists of the Bone Marrow Failure Study Group of the Italian Association of Pediatric Hemato-Oncology (AIEOP) was discussed, modified and approved during a series of consensus conferences that started online during COVID 19 and continued in the following years, according to procedures previously validated by the AIEOP Board of Directors.

Acute megakaryoblastic leukemia: experience of GIMEMA trials.

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.

Treatment of "poor risk" acute myeloid leukemia with fludarabine, cytarabine and G-CSF (flag regimen): a single center study.

We describe a single center experience of 41 consecutive patients with poor prognosis acute myeloid leukemia (AML) who received a single course of FLAG regimen consisting of Fludarabine 30 mg/m2/day plus Cytarabine 2 gr/m2/day (days 1-5) and G-CSF 5 mg/Kg/day (from day 0 to polymorphonuclear recovery) as salvage therapy. Eleven patients were primarily refractory to previous chemotherapy, 10 patients were in first relapse, 2 patients in second relapse and 7 patients in relapse after transplants. Eleven cases were defined as secondary AML (diagnosis of AML made after a preexisting diagnosis of myelodysplastic syndrome). The median age was 52.6 years (range 16-72); 29 patients were males and 12 females. Overall, 23 (56%) patients reached complete remission (CR), 3 patients died of infection (2) or hemorrhage (1) during induction, and 15 (36%) patients had resistant disease. The highest CR rates (80%) were obtained in relapsed cases; de novo and secondary AML registered 60% and 45% of CR rates, respectively. Patients achieving CR received a second FLAG course as consolidation and were submitted to an individualized program post-remission therapy, depending on the age and performance status. Hematological and non hematological toxicities were acceptable. In conclusion, our data confirm that FLAG is a an high effective treatment for poor prognosis AML and in young patients allows intensive post remissional therapy including allogeneic BMT.

A dysfunctional factor X (factor X San Giovanni Rotondo) present at homozygous and double heterozygous level: identification of a novel microdeletion (delC556) and missense mutation (Lys(408)-->Asn) in the factor X gene. A study of an Italian family.

Low levels of factor X (F.X) were detected in a 4-year-old boy who experienced acute lymphoblastic leukemia and bleeding manifestations. Laboratory data suggested the presence of a dysfunctional F.X molecule. Two novel F.X gene mutations were identified in the proband that was double heterozygous for both: a microdeletion (delC556) in exon VI resulting in a frameshift leading to a termination codon at position 226. This deletion was found in six family members with reduced F.X antigen and activity levels. A second mutation characterised by a G(1344)-->C transversion in exon VIII was detected in the proband resulting in a Lys(408)-->Asn substitution. This latter mutation was present in several asymptomatic family members from the paternal and the maternal side. The proband's sister was homozygous for the Lys(408)-->Asn substitution and exhibited low F.X activity with a normal antigen level. The naturally occurring F.X Lys(408)-->Asn (F.X(K408N)) variant was isolated from plasma of either homozygous or double heterozygous individuals. NH(2)-terminal sequencing of the heavy chain of F.X(K408N) failed to show any sequence abnormality in patients who were also carriers of the delC556, suggesting that this latter lesion accounted for the lack of F.X synthesis. Purified F.X Lys(408)-->Asn had an identical behaviour to normal F.X as judged by SDS-PAGE and immunoblotting. Clotting assay using purified F.X(K408N) and F.X-deficient plasma resulted in a laboratory phenotype similar to that observed in a homozygous subject for F.X Lys(408)-->Asn substitution. This is the first characterisation of a naturally occurring F.X variant with a mutation at the COOH-terminal end of the molecule.

Treatment of adults with acute lymphoblastic leukaemia in first bone marrow relapse: results of the ALL R-87 protocol.

Sixty-one adults aged <55 years with acute lymphoblastic leukaemia (ALL) in first bone marrow relapse were enrolled in an Italian cooperative study (ALL R-87 protocol) from 12 GIMEMA Institutions. The treatment programme consisted of: (1) an induction phase with intermediate-dose cytarabine (IDARA-C 1 g/m2, 6 h daily infusion x 6 d) plus idarubicin (IDA; 5 mg/m2/d x 6 d) and prednisone (40 mg/m2/d x 21 d), (2) a consolidation phase followed by (3) bone marrow transplant (BMT). Median first complete remission (CR) duration was 8.5 months (range 1-54 months). 34/61 patients achieved CR (56%); 24 (39%) failed to respond and three (5%) died during induction. Most responders (24 patients) could not enter the BMT programme; 15 relapsed early (median time to relapse 2 months); nine were withdrawn due to toxicity and one died in CR of infection. Nine of the 34 CRs underwent BMT (five autologous and four allogeneic). Three of the four allotransplanted patients are alive in continuous CR at 22, 43 and 63 months; only one of the five who underwent an autologous BMT is alive in CR at 46 months. The estimated disease-free survival (DFS +/- SE) at 36 months was 0.16 +/- 0.08 for all responders. Univariate analysis showed that previous therapy was the only prognostic factor influencing DFS. The estimated probabilities of event-free survival (EFS +/- SE) and survival +/- SE at 37 months were 0.09 +/- 0.04 and 0.10 +/- 0.04, respectively. The EFS was significantly better in patients with a preceding CR > or = 24 months, compared to those with a shorter first remission. Our results confirm the tolerance and efficacy of IDARA-C plus IDA in inducing CR in poor-risk adult ALL. Even though the number of transplanted patients was small, allogeneic BMT seems to give a real opportunity of cure in this category of patients.

Adult and childhood acute lymphoblastic leukemia: clinico-biological differences based on CD34 antigen expression.

BACKGROUND AND OBJECTIVE: The prognostic significance of CD34 antigen expression in acute lymphoblastic leukemias (ALL), especially in adult patients, is still not well established. In the present report we analyzed a series of biological and clinical findings from 128 ALL patients in order to evaluate the possible clinical significance of this marker. METHODS: The clinical and biological significance of CD34 expression, an early marker of hemopoietic cells, was analyzed by flow cytometry in a series of 128 patients affected by ALL, including 78 adults and 50 children under 15 years old. RESULTS: Overall, 68.7% of patients showed significant ( > 10%) CD34 expression. There was no difference between CD34+ and CD34- ALL with respect to age, sex, FAB morphology, hepatosplenomegaly, Plt count, Hb level, DNA index, P-170 expression. CD34+ ALL displayed a significantly lower frequency of extramedullary involvement, a lower LDH level and lower WBC count, lower proliferative activity (as evaluated by the Ki67 monoclonal antibody) than CD34- ALL. CD34 expression was also associated with early phenotypes in both B- and T-ALL, co-expression of myeloid antigens, and the presence of the Ph1 chromosome. Due to a different distribution of prognostic factors investigated, DFS and OS were both significantly better in CD34+ than in CD34- childhood ALL, whereas no statistical difference was found in adults. Multivariate analyses confirmed these data in children. INTERPRETATION AND CONCLUSIONS: Expression of the CD34 antigen is a positive prognostic factor in childhood ALL. In adult ALL the presence of this marker on leukemic cell does not seem to influence the clinical outcome of these patients.

Are "early" and "late" T-acute lymphoblastic leukemias different diseases? A single center study of 34 patients.

Clinical and biological parameters were retrospectively reviewed in 34 cases of T-lineage acute lymphoblastic leukemia (T-ALL), classified as "early" (20 cases) or "late" (14 cases) subgroups, according to the degree of blast cell differentiation, assessed by immunophenotyping. In "early" T-ALL, age, co-expression of "immature" (CD34 and HLA-Dr) or myeloid (My+) antigens, proliferative activity (as evaluated by Ki67 monoclonal antibody), and expression of the "multidrug-resistance" (MDR) phenotype (as determined by C-219 monoclonal antibody) were significantly higher, while WBC count and expression of CDl0 were significantly lower, than in "late" T-ALL. Furthermore, although no statistically significant difference was found between the two groups, "late" T-ALL more frequently displayed a greater extramedullary tumor mass ("lymphoma-like" syndrome), LI FAB morphology and a normal karyotype. A single patient, with "late" T-ALL, also showed positivity for TCR gamma/delta chains, specific monoclonal antibodies. On the whole, 30 patients (88.2%) achieved complete remission: 16(80%) were "early" and 14(100%) "late" T-ALL. No statistical difference was found between the two groups with respect to disease free survival (42% vs 54% at six years), whereas median overall survival was significantly shorter in "early" T-ALL (23 months vs median not yet reached at six years for "late" T-ALL, p < 0.05). We conclude that "early" and "late" T-ALL show clinical and biological differences, that could perhaps justify differential therapeutic approaches.

Increased CD10/TdT positive cells in the bone marrow of an infant with immune thrombocytopenia.

We describe the case of an infant with immune thrombocytopenia whose bone marrow showed an increased percentage of CD10/TdT-positive lymphoid cells that resembled the onset of an acute lymphoproliferative disorder. Genotypic analysis of bone marrow, however, failed to reveal the malignant origin of these B cell precursors. After 8 months of follow-up, the child is alive and well, and shows a chronic form of ITP. Although a relation between this B cell proliferation and the onset of ITP cannot be excluded, it is important to consider this atypical pattern as a benign hematologic condition.

Treatment of primary refractory and relapsed acute lymphoblastic leukaemia in children and adults: the GIMEMA/AIEOP experience. Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto. Associazione Italiana Ematologìa ed Ocologia Pediatrica.

One hundred and forty-seven patients aged < 55 years with advanced acute lymphoblastic leukaemia (ALL) were enrolled in an Italian cooperative study (ALL R-87). This protocol consists of an induction phase with idarubicin (IDA) plus intermediate-dose cytarabine (IDARA-C), followed by a consolidation phase and bone marrow transplant (BMT). Complete remission (CR) was achieved in 97/147 patients (66%) with a CR rate of 77% in children versus 51% in adults (P < 0.01). 48 responders (50%) underwent BMT. Probability of event-free survival (EFS +/- SE) was 10.2 +/- 3.1% at 56 months. EFS was 14.3 +/- 4.51% at 56 months for children versus 3.8 +/- 3.41% at 37 months for adults (P < 0.0001). Among patients treated in first relapse, EFS was 14.2 +/-7.79% for patients with CR > 18 months verus 6.6 +/- 3.17% for those with CR < 18 months (P < 0.0001). Projected disease-free survival (DFS +/- SE) was 15.4 +/- 4.61% at 55 months for all responders and 43.3 +/- 14.34% at 52 months for allografted patients. Projected overall probability of survival +/- SE for all patients was 18.8 +/- 4.13% at 56 months. This study confirms the efficacy of IDA plus IDARA-C in poor-risk. ALL patients. A more intensive post-remission therapy or alternative approach must be designed to improve long-term results.

Increased risk of neurological relapse in acute lymphoblastic leukemias with high levels of cerebrospinal fluid thymidine kinase at diagnosis.

Cerebrospinal fluid thymidine kinase (CSF-TK) was measured at diagnosis in 62 patients with acute lymphoblastic leukemia (ALL) without initial neurological manifestations, who achieved a complete remission after chemotherapy. During the follow-up period, 10 patients developed central nervous system (CNS) involvement. At the onset of the disease mean CSF-TK levels in these subjects were found to be significantly higher than those observed in patients without subsequent CNS complications. In particular, 7/10 (70%) of these patients who presented CSF-TK levels above the upper limit of normal (1.4 U/microliters) had evidence of a neurological relapse, while 49/52 (94.2%) of subjects with presenting CSF-TK levels of up to 1.4 U/microliters did not develop a neurological leukemic disease (p < 0.00001). The white blood cell count at diagnosis was significantly increased, but not directly correlated to CSF-TK levels, in the group with CNS involvement, while age, serum thymidine kinase levels and lactic dehydrogenase, FAB classification or immunophenotype were not different in patients with or without neurological relapse. In conclusion, increased levels of CSF-TK at presentation correlate with a high risk of subsequent CNS involvement in patients with responsive ALL.

A randomised clinical trial comparing idarubicin and cytarabine to daunorubicin and cytarabine in the treatment of acute non-lymphoid leukaemia. A multicentric study from the Italian Co-operative Group GIMEMA.

255 patients with acute non-lymphoid leukaemia (ANLL), observed between October 1984 and June 1987, entered a chemotherapy regimen consisting of induction therapy with cytarabine in combination with idarubicin (IDA/ARA) or daunorubicin (DNR/ARA), followed by consolidation with four courses of IDA + ARA plus 6-thioguanine (6-TG) or DNR + ARA + 6-TG and a 6 month maintenance therapy with 6-TG and ARA. The median age was 62 years (range 55-78 years) and 33 were aged more than 70 years. The treatment groups were comparable for median age, FAB type, performance status and initial blood counts. 249 patients were randomised, 124 to the IDA/ARA arm and 125 to the DNR/ARA arm. Complete remission was achieved in 50 patients (40%) on the IDA/ARA treatment program and 49 patients (39%) on DNR/ARA. No definite differences were found between patients receiving IDA/ARA and those treated with DNR/ARA as far as complete response (CR), overall survival, failure free and relapse free survival are concerned. 74% of the complete responders in the IDA/ARA arm and 51% in the DNR/ARA arm achieved CR after a single course of treatment. Resistant leukaemia was observed in 13.7% of the patients in the IDA/ARA arm and in 31.2% in the DNR/ARA one, whereas hypoplastic death occurred in 29% and 14.4%, respectively. In conclusion, our data failed to show any advantage of idarubicin over daunorubicin even though there is some evidence that IDA, despite the higher toxicity, is more rapid in eradicating leukaemia as proved by the higher CR rate obtained after one course of induction.

Clinical relevance of immunocytochemical detection of multidrug-resistance-associated P-glycoprotein in hematologic malignancies.

P-glycoprotein (P-170) is the phenotypic marker of tumoral cells that show the phenomenon of multidrug resistance (MDR). Using an immunocytochemical approach, we employed the monoclonal antibody C219 (which recognizes an epitope of such a glycoprotein) to evaluate in cytologic samples the expression of P-170 on neoplastic cells from 52 patients affected by different hematologic malignancies and its eventual correlation to clinical outcome. Longitudinal studies were also performed in 14 patients. Results obtained demonstrated that a) the so-called "MDR phenotype" may be heterogeneously represented (from less than 1 to 100% of positive cells) in hemopoietic tumors at diagnosis (without exposure to pharmacologic agents), as well as during the course of the disease, although a more substantial presence of P-170 occurred in treated patients. There was no correlation between neoplastic kinetic activity (such as expression of Ki 67 recognized nuclear proliferation-associated antigen) and P-170-positive cells. b) Percentage of positive cells as well as intensity of staining seemed to be important in determining MDR; in general, there was a strong correlation between expression of P-170 in more than 20% of neoplastic cells and a lack of response to chemotherapy. However, some false-positive and false-negative cases were observed. c) The detection of scattered P-170-positive cells may predict a pharmacologic selection of intrinsic or mutant-resistant clones.

[Determination of thymidine kinase in the cerebrospinal fluid as a marker of leukemic meningosis]. / Determinazione della Timidina Kinasi nel liquor cerebrospinale quale marker della meningosi leucemica.

Levels of Thymidine Kinase (TK) in cerebrospinal fluid (CSF) were longitudinally evaluated in 68 patients with acute leukemia. Values significantly above the normal range were found in all cases with meningeal involvement (in two subjects before the cytological evidence of leukemic cells in CSF). Such high levels decreased until normal values after an effective treatment. Two patients with multiple cerebral neoplastic nodules without infiltration of meninges showed normal CSF-TK values. No false positive cases were observed. Both kinetic activity and mass of leukemic cells appeared to influence CSF-TK levels. Thus, CSF-TK appears to be a promising marker in diagnosing and monitoring meningeal disease in the course of acute leukemia.

Significance and limits of cerebrospinal fluid beta-2-microglobulin measurement in course of acute lymphoblastic leukemia.

Cerebrospinal fluid beta-2-microglobulin (CSF-beta 2m) was measured longitudinally in 48 patients affected by acute lymphoblastic leukemia (ALL). Thirteen developed a central nervous system (CNS) involvement during the course of the disease; although moderately higher mean CSF-beta 2m levels were found in these subjects, no significant statistical differences were observed in comparison with patients without this complication and compared with the control group. No correlations were found between beta 2m and other biochemical parameters in CSF. Furthermore, CSF-beta 2m levels appeared to be influenced by previous combined chemoradiotherapeutic treatment for CNS prophylaxis, presence of meningeal non-neoplastic infiltrates, patients' ages, amount of CSF blasts, and their immunological phenotype. In particular, only clearly B-committed leukemic cells, when tested, showed a strong surface expression of beta 2m, as demonstrated by immunocytochemical detection of this protein on cell membrane. However, in specific cases, CSF beta 2m measurement and CSF/serum beta 2m ratio were helpful in diagnosing and monitoring isolated CNS disease. Such findings suggest that CSF-beta 2m assay may be a useful tool in the management of CNS involvement in the course of ALL in only selected patients, as several factors can modify the outcome.