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.

FIP1L1-PDGFRA in chronic eosinophilic leukemia and BCR-ABL1 in chronic myeloid leukemia affect different leukemic cells.

We investigated genetically affected leukemic cells in FIP1L1-PDGFRA+ chronic eosinophilic leukemia (CEL) and in BCR-ABL1+ chronic myeloid leukemia (CML), two myeloproliferative disorders responsive to imatinib. Fluorescence in situ hybridization specific for BCR-ABL1 and for FIP1L1-PDGFRA was combined with cytomorphology or with lineage-restricted monoclonal antibodies and applied in CML and CEL, respectively. In CEL the amount of FIP1L1-PDGFRA+ cells among CD34+ and CD133+ cells, B and T lymphocytes, and megakaryocytes were within normal ranges. Positivity was found in eosinophils, granulo-monocytes and varying percentages of erythrocytes. In vitro assays with imatinib showed reduced survival of peripheral blood mononuclear cells but no reduction in colony-forming unit growth medium (CFU-GM) growth. In CML the BCR-ABL1 fusion gene was detected in CD34+/CD133+ cells, granulo-monocytes, eosinophils, erythrocytes, megakaryocytes and B-lymphocytes. Growth of both peripheral blood mononuclear cells and CFU-GM was inhibited by imatinib. This study provided evidence for marked differences in the leukemic masses which are targeted by imatinib in CEL or CML, as harboring FIP1L1-PDGFRA or BCR-ABL1.

Genomic gain at 6p21: a new cryptic molecular rearrangement in secondary myelodysplastic syndrome and acute myeloid leukemia.

Fluorescence in situ hybridization and comparative genomic hybridization characterized 6p rearrangements in eight primary and in 10 secondary myeloid disorders (including one patient with Fanconi anemia) and found different molecular lesions in each group. In primary disorders, 6p abnormalities, isolated in six patients, were highly heterogeneous with different breakpoints along the 6p arm. Reciprocal translocations were found in seven. In the 10 patients with secondary acute myeloid leukemia/myelodysplastic syndrome (AML/MDS), the short arm of chromosome 6 was involved in unbalanced translocations in 7. The other three patients showed full or partial trisomy of the 6p arm, that is, i(6)(p10) (one patient) and dup(6)(p) (two patients). In 5/7 patients with unbalanced translocations, DNA sequences were overrepresented at band 6p21 as either cryptic duplications (three patients) or cryptic low-copy gains (two patients). In the eight patients with cytogenetic or cryptic 6p gains, we identified a common overrepresented region extending for 5-6 megabases from the TNF gene to the ETV-7 gene. 6p abnormalities were isolated karyotype changes in four patients. Consequently, in secondary AML/MDS, we hypothesize that 6p gains are major pathogenetic events arising from acquired and/or congenital genomic instability.

Incidence and significance of cryptic chromosome aberrations detected by fluorescence in situ hybridization in acute myeloid leukemia with normal karyotype.

To better define the incidence and significance of cryptic chromosome lesions in acute myeloid leukemia (AML), fluorescence in situ hybridization (FISH) studies were performed in interphase cells and, when appropriate, in metaphase cells and in morphologically intact BM smears. Fifty-five adult de novo AML (group A) and 27 elderly AML or AML after myelodysplastic syndrome (AML-MDS) (group B) were tested using probes detecting the following anomalies: -5, -7, +8, deletions of 5q31, 7q31, 12p13/ETV6, 17p13/p53, 20q11. All the patients had a normal karyotype in more than 20 cells and tested negative for the common AML-associated fusion genes. No patient in group A was found to carry occult chromosome anomalies, whereas 8/27 patients in group B (P < 0.0001) showed 5q31 or 7q31 deletion (three cases each), a 17p13/p53deletion or trisomy 8 (one case each) in 33-60% interphase cells. Metaphase cells showed only one hybridization signal at 5q31 (three cases) and 7q31 (one case), whereas two normal signals at 7q31 and chromosome 8 centromeres were seen in two patients with 7q deletion and trisomy 8 in interphase cells. The majority of blast cells (76-94%) carried the chromosome anomaly in all cases; erythroid involvement in a minority of cells was seen in three patients. In group B, the presence of occult chromosome anomalies was associated with exposure to myelotoxic agents in the workplace (5/8 cases vs 3/19, P = 0.026) and with a lower complete remission rate (0/6 patients vs 7/12, P = 0.024). We arrived at the following conclusions: (1) cryptic chromosome deletions in the order of a few hundred kb magnitude may be found in a fraction of elderly AML or MDS-related AML and not in de novo adult AML with normal karyotype; (2) these chromosome lesions are usually represented by submicroscopic rearrangements; (3) they display a specific pattern of cell-lineage involvement arguing in favor of their role in the outgrowth of the leukemic blast cells; (4) they are associated with a history of exposure to myelotoxic agents in the workplace and, possibly, with resistance to induction treatment.

H4(D10S170), a gene frequently rearranged in papillary thyroid carcinoma, is fused to the platelet-derived growth factor receptor beta gene in atypical chronic myeloid leukemia with t(5;10)(q33;q22).

The molecular cloning of the t(5;10)(q33;q22) associated with atypical chronic myeloid leukemia (CML) is reported. Fluorescence in situ hybridization (FISH), Southern blot, and reverse transcriptase- polymerase chain reaction analysis demonstrated that the translocation resulted in an H4/platelet-derived growth factor receptor betaR (PDGFbetaR) fusion transcript that incorporated 5' sequences from H4 fused in frame to 3' PDGFbetaR sequences encoding the transmembrane, WW-like, and tyrosine kinase domains. FISH combined with immunophenotype analysis showed that t(5;10)(q33;q22) was present in CD13(+) and CD14(+) cells but was not observed in CD3(+) or CD19(+) cells. H4 has previously been implicated in pathogenesis of papillary thyroid carcinoma as a fusion partner of RET. The H4/RET fusion incorporates 101 amino acids of H4, predicted to encode a leucine zipper dimerization domain, whereas the H4/PDGFbetaR fusion incorporated an additional 267 amino acids of H4. Retroviral transduction of H4/PDGFbetaR, but not a kinase-inactive mutant, conferred factor-independent growth to Ba/F3 cells and caused a T-cell lymphoblastic lymphoma in a murine bone marrow transplantation assay of transformation. Mutational analysis showed that the amino-terminal H4 leucine zipper domain (amino acids 55-93), as well as H4 amino acids 101 to 386, was required for efficient induction of factor-independent growth of Ba/F3 cells. Tryptophan-to-alanine substitutions in the PDGFbetaR WW-like domain at positions 566/593, or tyrosine-to-phenylalanine substitutions at PDGFbetaR positions 579/581 impaired factor-independent growth of Ba/F3 cells. H4/PDGFbetaR is an oncoprotein expressed in t(5;10)(q33;q22) atypical CML and requires dimerization motifs in the H4 moiety, as well as residues implicated in signal transduction by PDGFbetaR, for efficient induction of factor-independent growth of Ba/F3 cells. (Blood. 2001;97:3910-3918)

Associated vascular lesions in patients undergoing coronary artery bypass grafting.

Atherosclerotic involvement of extracoronary arteries in patients undergoing myocardial revascularization can cause severe postoperative complications and increase postoperative mortality. Between January and November 1998, routine preoperative echo-Doppler study of carotid vessels, abdominal aorta and iliac-femoral arteries was performed in all patients undergoing coronary artery bypass grafting (CABG) at our institution, in order to assess the prevalence and the degree of associated vascular lesions. Correlations between echo-Doppler findings, angiographic patterns of coronary lesions and atherosclerotic risk factors were analyzed in all cases. Among 302 patients undergoing CABG, 186 (61.6%) had carotid disease, with a haemodynamically significant stenosis (>70%) of internal carotid in 31 (10.2%). Twenty-three patients had asymptomatic severe carotid disease. A significant correlation between severity of coronary disease and prevalence of severe carotid disease was found (p = 0.02). An abdominal aortic dilatation (diameter > 25 mm) was found in 20 cases (6.6%), with a diameter >35 mm in 7 patients (2.3%), 6 with triple-vessel coronary disease, and 1 with double-vessel disease. Atherosclerotic lesions of iliac-femoro-popliteal axis were found in 165 (54.6%) patients, with a strong correlation to the severity of coronary disease (p = 0.02); lesions were haemodynamically significant (> 70%) in 48 (15.8%) cases. Symptoms of carotid and peripheral vascular disease are no reliable predictors of perioperative risk in patients undergoing CABG. Non-invasive complete arterial investigation should be routinely performed in these patients, in order to plan the most suitable operative approach and to prevent perioperative vascular complications.

[Prevalence of vascular disease in candidates for myocardial revascularization with aortocoronary bypass: review of the literature and practical implications]. / Prevalenza di patologia vascolare in soggetti candidati a rivascolarizzazione miocardica mediante bypass aortocoronarico: revisione della letteratura ed implicazioni pratiche.

BACKGROUND: Cardiovascular disease remains the main cause of death and morbidity in the industrialized world. Atherosclerosis is a slowly progressive disease; coronary artery disease may be the first presentation of a systemic pathology. The association between coronary artery disease and peripheral vascular disease has often been confirmed by multicenter trials; nevertheless it still remains a subject of debate. METHODS: In order to assess the incidence of coronary artery disease and the degree of associated vascular lesions, between January 1997 and September 1999, in the Department of Cardiothoracic Surgery of the Second University of Naples (Italy), all candidates to coronary artery bypass grafting (CABG) were submitted to routine preoperative echo color Doppler study of the carotid vessels, abdominal aorta and iliac-femoral arteries. The correlation between the echo color Doppler findings, the angiographic patterns of coronary lesions and atherosclerotic risk factors was analyzed in all cases. RESULTS: Among 540 patients undergoing CABG, 418 (77.4%) had carotid disease, with a stenosis > 70% in 62 (11.3%). Forty-nine (79%) patients had asymptomatic severe carotid disease. A significant correlation between the severity of coronary disease and the incidence of severe carotid disease was found (p = 0.02). An abdominal aortic dilation was found in 37 cases (6.7%). Its diameter exceeded 35 mm in 14 patients (2.5%) and in 8 it was associated with triple vessel coronary disease. Atherosclerotic lesions of the iliac-femoro-popliteal axis were found in 394 (72.9%) patients and strongly correlated with the severity of coronary artery disease (p = 0.02); lesions were hemodynamically significant in 91 (16.8%) cases. CONCLUSIONS: Our study emphasizes the association between coronary artery disease and vascular disease. Non-invasive complete arterial investigation should be routinely performed in patients undergoing CABG.

Cytogenetic characterization of acute myeloid leukemia in Shwachman's syndrome. A case report.

We report on a case of acute myeloid leukemia in a 17-year old boy affected by Shwachman Diamond syndrome (SDS). Conventional cytogenetics at diagnosis revealed an abnormal clone with complex karyotypic changes including typical myeloid aberrations, such as monosomy 5, tetrasomy of chromosome 8, trisomy 9, and deletion of the short arm of chromosome 12. The boy was treated with conventional chemotherapy and reached complete remission of leukemia, confirmed by cytogenetics and fluorescence in situ hybridization. Nevertheless he failed to regenerate normal marrow cellularity and blood cell count. Cytogenetic information on hematologic malignancies in SDS patients are discussed.

t(4;11)(q21;p15) translocation involving NUP98 and RAP1GDS1 genes: characterization of a new subset of T acute lymphoblastic leukaemia.

Two cases of T acute lymphoblastic leukaemia (T-ALL) with an identical t(4;11)(q21;p15) translocation were identified within a prospective study on the biological and clinical features of adult ALL patients enrolled into the therapeutic protocol ALL0496 of the GIMEMA Italian Group. In both cases, the molecular characterization showed an involvement of the NUP98 gene on 11p15 which rearranges with the RAP1GDS1 gene on 4q21. The morphological and immunological features of the leukaemic cells, as well as the clinical behaviour and response to induction therapy, were the same in both patients. Based on the available data, the t(4;11)(q21;p15) translocation involving the NUP98-RAP1GDS1 fusion gene emerges as a new highly specific genetic abnormality that characterizes a subset of T-ALL.

Interphase FISH for Y chromosome, VNTR polymorphisms, and RT-PCR for BCR-ABL in the monitoring of HLA-matched and mismatched transplants.

Thirty-six sex-mismatched transplants were studied using fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) methods. Molecular cytogenetics was performed using interphase FISH with a centromeric probe for chromosome Y, and PCR amplification was performed with a set of VNTR microsatellite loci. In addition, reverse transcriptase-PCR (RT-PCR) for BCR-ABL fusion was used to investigate cases of Philadelphia chromosome (Ph)-positive chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). Our integrated approach of post-transplant monitoring was helpful in documenting successful transplants and in controlling the size of Ph-positive clones in CML. A striking overlap was found between results from FISH analysis and PCR for polymorphic loci.

Genomic instability and recurrent breakpoints are main cytogenetic findings in Hodgkin's disease.

BACKGROUND AND OBJECTIVE: Successful cytogenetic studies in Hodgkin's disease (HD) are rare, and, except for hyperdiploidy, no chromosome changes typical for this disorder have been described. The purpose of this study was to collect cytogenetic information from a new series of lymphoid neoplasms diagnosed either as classical HD or as Hodgkin's-like anaplastic large cell lymphoma (HD-like ALCL), according to the REAL Classification. DESIGN AND METHODS: We studied 27 cases of HD and 10 cases of HD-like ALCL. Cytogenetic investigations were performed on lymph nodes (35 cases), bone marrow or pleural effusion. A large screening of slides was performed to detect abnormal metaphases despite the low mitotic index of Reed-Sternberg cells. In addition to ours, available published data were analyzed in detail to identify recurring cytogenetic events. RESULTS: Metaphases which could be analyzed were obtained in 86.5% of cases, with 59.4% showing abnormal clones. We found a peculiar kind of cytogenetic instability in which, despite variations in the type of structural rearrangements, chromosome breakpoints were non-randomly distributed. Moreover, from our data plus those collected from literature on HD (total 177 cases), the number of breakpoints was higher in patients in a more advanced clinical stage. INTERPRETATION AND CONCLUSIONS: Cytogenetic studies in HD are highly informative regarding clonality, provided large numbers of metaphases are examined. Based on karyotype, genetic changes in HD and HD-like ALCL are similar. Results are consistent with a high degree of chromosomal instability and predominance of hyperdiploid complex karyotypes. Chromosome breakpoints are non-randomly distributed and more numerous in advanced clinical stages.

Trisomy 6 is the hallmark of a dysplastic clone in bone marrow aplasia.

Clonal hematopoiesis with trisomy 6 as the sole karyotypic change was revealed by cytogenetics in two cases of aplastic anemia. In both patients, dyserythropoiesis was characterized by asynchrony of maturation between nucleus and cytoplasm, binucleated elements, and intercytoplasmic connections. In addition to conventional cytogenetics, the size of the trisomic clone was evaluated by fluorescence in situ hybridization on fixed cells at diagnosis and in the course of the disease by using an alpha-satellite centromeric probe for chromosome 6. Moreover, in situ hybridization on bone marrow smears showed that dysplastic erythrocytes as well as myeloid cells belonged to the trisomic clone. Trisomy 6 identifies a subgroup of hematologic disorders with bone marrow hypo-aplasia and dyserythropoiesis.