Detection of PICALM-MLLT10 ( CALM-AF10 ) and outcome in children with T-lineage acute lymphoblastic leukemia

Death early (DE) during the induction phase of therapy is a rare but dramatic event in children with acute myeloid leukemia (AML). Recent reports emphasize that DE in patients with leukemia is related to aggressive disease, signaled by hyperleukocytosis, and to the early administration of intensive chemotherapy. Many efforts have been made to better characterize subgroups of patients with AML by defining biologic markers associated with aggressive disease. Even if little is known about the molecular mechanisms that regulate blast migration into tissues, adhesion receptors are likely to play an important role in this process. We have attempted to identify the molecular characteristics of an aggressive subset of pediatric patients with AML through a prospective evaluation of the CD56+ neural cell adhesion molecule (NCAM) and CD94 expression. For this purpose we developed an RT-PCR semi-quantitative methodology, using specific primers from NCAM, CD94 and β-actin as an internal quality and quantity control. We analyzed a total population of 44 children with AML, distributed as follow: 5 with M3v (group A), 9 with APL (group B) and 30 with non-APL AML (group C). We found a dramatic correlation in group A between high expression of NK-related genes and occurrence of early death. In groups B and C, primarily used as controls, we found a strong correlations between a high level of NCAM-CD94 expression and two cases with APL, three with FAB M5, two with FAB M2 AML, one case with FAB M4 and a child with dendritic cell leukemia (DCL). We primarily found that 9 out of 13 cases presenting with a high level of NCAM-CD94 expression died of disease-related events during the early days of induction. Our data strongly suggest that doses and timing of chemotherapy induction in children with AML need to be modulated because of the high risk of hemorrhagic events. These appear to be related to the presence of high concentration of adhesion molecules secondary to acute blast lysis and to the associated severe coagulopathy. We know of no pharmacological correctives for this phenomenon. It would be important to develop specifically designed molecules in order to disrupt the interactions with adhesion molecules, and thus to improve the clinical course of both children and adults with AML during the early days of induction.

Background: Myelomonocytic precursors from acute or chronic leukemias can differentiate to dendritic cells in vitro , but leukemias with a dendritic cell immunophenotype are rare, have been reported mainly in adults, and their molecular pathogenesis is unknown. Dendritic cells are classified as Langherans, myeloid and lymphoid/plasmacytoid cells, but leukemias arising from dendritic cells are unclassified in the FAB system. We identified a new entity of pediatric acute myeloid leukemia (AML) presenting with morphologic and immunophenotypic features of mature dendritic cells, which is characterized by MLL gene translocation. Methods and Results: Standard methods were used to characterize the morphology, immunophenotype, karyotype and MLL translocations in 3 cases of pediatric AML. The patients included two boys and one girl diagnosed with AML between 1–6 years old. Their clinical histories and findings included fever, pallor, abdominal and joint pain, adenopathy, hepatosplenomegaly, normal WBC counts but anemia and thrombocytopenia. and no evidence of CNS disease. The bone marrow aspirates were hypocellular and replaced completely by large blasts with irregular nuclei, slightly basophilic cytoplasm, and prominent cytoplasmic projections. There were no cytoplasmatic granules or phagocytosis. Myeloperoxidase and alpha napthyl esterase reactions were negative, excluding FAB M5 AML, and the morphology was not consistent with any standard FAB morphologic diagnosis. The leukemic blasts in all three cases were CD83+, CD86+, CD116+, consistent with differentiated myeloid dendritic cells, and did not express CD34, CD56 or CD117. MLL translocations were identified in all 3 cases. In the first case FISH analysis showed t(10;11)(p12;q23) and RT-PCR identified and a ‘5-MLL-AF10-3’ fusion transcript. In the second case FISH analysis showed t(9;11)(p22;q23) and RT-PCR identified and a ‘5-MLL-AF9-3’ fusion transcript. In the remaining case, the MLL gene rearrangement was identified by Southern blot analysis and RT-PCR showed an MLL - AF 9 fusion transcript. The fusion transcripts in all 3 cases were in-frame. Remission induction was achieved with intensive chemotherapy, and all three patients have remained in durable remission for 30–60 months after hematopoietic stem cell transplantation. Conclusions. We have characterized a new pediatric AML entity with features of mature dendritic cells, MLL translocation and an apparently favorable prognosis. The in-frame MLL fusion transcripts suggest that chimeric MLL oncoproteins underlie its pathogenesis. The partner genes in all 3 cases were known partner genes of MLL that encode transcription factors. This study increases the spectrum of leukemias with MLL translocations. Comprehensive morphological, immunophenotypic, cytogenetic and molecular analyses are critical for this diagnosis, and will reveal its frequency and spectrum as additional cases are uncovered.

Natural killer (NK) cell lymphomas are rare in the USA and Europe but more common in Asia and Central America although very rare among children. We report a case of Epstein-Barr virus-positive NK lymphoma/leukemia, that showed peculiar features represented by a very long clinical course with a significant interval between the first clinical signs and the diagnosis, detection of neoplastic cells in the peripheral blood but not in the bone marrow, and good response to treatment and clinical outcome.

Pediatric Blood & CancerVolume 43, Issue 2 p. 185-185 Letter to the Editor Use of PEG-interferon alfa-2a plus ribavirin as treatment for chronic HCV hepatitis in a child cured of ALL Luca Lo Nigro MD, Corresponding Author Luca Lo Nigro MD lucaln@yahoo.com Center of Pediatric Hematology and Oncology, University of Catania, Catania, ItalyCenter of Pediatric Hematology Oncology, University of Catania, via S. Sofia 78, 95123 Catania, Italy.===Search for more papers by this authorPaola Guardo BD, Paola Guardo BD Clinical Pathology Service, M. Ascoli-Tomaselli Hospital, Catania, ItalySearch for more papers by this authorMilena La Spina MD, Milena La Spina MD Center of Pediatric Hematology and Oncology, University of Catania, Catania, ItalySearch for more papers by this authorElena Mirabile PhD, Elena Mirabile PhD Center of Pediatric Hematology and Oncology, University of Catania, Catania, ItalySearch for more papers by this authorPaola Pisana MD, Paola Pisana MD Center of Pediatric Hematology and Oncology, University of Catania, Catania, ItalySearch for more papers by this authorGino Schilirò MD, Gino Schilirò MD Center of Pediatric Hematology and Oncology, University of Catania, Catania, ItalySearch for more papers by this authorPaola Guardo BD, Paola Guardo BD Clinical Pathology Service, M. Ascoli-Tomaselli Hospital, Catania, ItalySearch for more papers by this author Luca Lo Nigro MD, Corresponding Author Luca Lo Nigro MD lucaln@yahoo.com Center of Pediatric Hematology and Oncology, University of Catania, Catania, ItalyCenter of Pediatric Hematology Oncology, University of Catania, via S. Sofia 78, 95123 Catania, Italy.===Search for more papers by this authorPaola Guardo BD, Paola Guardo BD Clinical Pathology Service, M. Ascoli-Tomaselli Hospital, Catania, ItalySearch for more papers by this authorMilena La Spina MD, Milena La Spina MD Center of Pediatric Hematology and Oncology, University of Catania, Catania, ItalySearch for more papers by this authorElena Mirabile PhD, Elena Mirabile PhD Center of Pediatric Hematology and Oncology, University of Catania, Catania, ItalySearch for more papers by this authorPaola Pisana MD, Paola Pisana MD Center of Pediatric Hematology and Oncology, University of Catania, Catania, ItalySearch for more papers by this authorGino Schilirò MD, Gino Schilirò MD Center of Pediatric Hematology and Oncology, University of Catania, Catania, ItalySearch for more papers by this authorPaola Guardo BD, Paola Guardo BD Clinical Pathology Service, M. Ascoli-Tomaselli Hospital, Catania, ItalySearch for more papers by this author First published: 17 May 2004 https://doi.org/10.1002/pbc.20084Citations: 7Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article.Citing Literature Volume43, Issue2August 2004Pages 185-185 RelatedInformation

Detection of Philadelphia chromosome (Ph) in childhood T‐lineage acute lymphoproliferative disorders is a rare event. Additional cytogenetic abnormalities are particularly uncommon in ALL. We here report two cases with T lineage acute lymphoproliferative disorders (T‐ALL and T‐NHL) presenting with both cytogenetic alterations and BCR‐ABL fusion transcripts, associated with an aggressive presentation and a poor outcome. We point out firstly on the cytogenetic aberrations, supporting the hypothesis of multi‐lineage involvement of ALL expressing Ph chromosome; secondly, on the persistence of T‐cell leukemic clone detected by minimal residual disease (MRD) analysis, despite of the early disappearance of BCR‐ABL fusion transcript. © 2003 Wiley‐Liss, Inc.

Analysis of minimal residual disease (MRD) can predict outcome in childhood acute lymphoblastic leukemia (ALL). We applied a chemiluminescent methodology in 20 children with ALL. We detected MRD at different time-points throughout the follow-up of our patients, concluding that chemilumi-nescent detection of MRD is a reliable, safe and sensitive method. Recent prospective studies clearly demonstrate the prognos-tic value of MRD in children with ALL. 1,2 Several methodologies are available for MRD analyses in ALL patients. 1-5 In order to test a safe, sensitive and reliable method for those laboratories in which neither radioactive analysis nor the TaqMan strategy can be carried out, 3 we present here a report on application of digox-igenin (DIG)-labeled patient-specific probes for a chemilumi-nescent detection of MRD in children with ALL. Twenty children with ALL diagnosed at our institution and treated according to the ongoing protocol of the Associazione Italiana di Ematologia ed Oncologia Pediatrica (AIEOP-ALL 95) were included in this study. The childrens' characteristics are listed in Table 1. We collected samples at 5 time-points (TP): after 43 days (TP1), after three months (TP2), five (TP3), seven (TP4) and 24 months (TP5) of therapy. T-cell receptor (TCR) γ and TCR δ gene rearrangements were identified and characterized by performing diagnostic polymerase chain reactions (PCR), het-eroduplex and sequencing analyses using standardized techniques. 6,7 PCR of follow-up were performed using different protocols in order to amplify TCRγ and TCRδ rearrangements. 7 Seven microliters of the products were spotted onto positively charged nylon membranes (NYTRAN N+, Roche Boehringer). Next, 200 pmol of each oligonucleotide were 3'-end labeled with DIG-ddUTP using a DIG oligonucleotide 3'-end labeling kit (Roche Molecular Biochemicals, Mannheim, Germany) according to manufacturer's instructions. The membranes were prehy-bridized at 68°C for 2 hours using 25 mL hybridization solution (Roche Molecular Biochemicals) and then hybridized in a sealed plastic bag with 3 mL hybridization solution and 20 µL of the labeling probe (200 pmol). The membranes were incubated overnight at 54°C and then washed twice in 2 × SSC, 0.1% SDS at room temperature for 5 min. The membranes were soaked with blocking solution (Roche Molecular Biochemicals) for 30 min at room temperature. Twenty-four milliliters of blocking solution containing 2.4 µL of anti-DIG-alkaline phosphatase Fab fragments (Roche Molecular Biochemicals) were added, followed by incubation for 30 min. Finally, the membranes were washed twice with washing buffer (Roche Molecular Biochemicals) for 15 min and incubated with detection buffer (Roche …

We studied 15 Sicilian subjects with Hb H disease correlating clinical examinations with hematological and molecular data. Seven different alpha-tha1 mutations were identified: four deletion types (--MED --CAL, -alpha3.7, -alpha4.2) and three nondeletion types (alpha(Ncol)alpha, alpha(Hph)alpha, alphaCSalpha). All the patients had a zero-gene chromosome (--MED or --CAL), while the third alpha gene was deleted (-alpha3.7, -alpha4.2) or inactive (alpha(Ncol)alpha, alpha(Hph)alpha, alphaCSalpha). In patients with the nondeletion genotype the analysis of hematological values revealed lower levels of RBC and Hb A2 and significantly higher levels of Hb H. The clinical variability was remarkable, ranging from totally asymptomatic conditions, casually diagnosed, to severe thalassemia intermedia with marked hemolytic crises, liver and spleen enlargement and the necessity for frequent transfusions. The genotype did not justify the gravity of the phenotype in every case, and the differences in clinical manifestations, also notable, are not easily explainable in subjects who apparently have the same genotype.

Sicily, at the center of the Mediterranean, has been the meeting place of Eastern and Western civilizations, and in the Sicilian population the presence of many different alterations in the globin gene clusters can surely be considered testimony of past colonizations. From 1975 to 1994, 100,000 Sicilian subjects were screened by us to monitor the presence of hemoglobin (Hb) structural variants. In this paper we present the data gathered, emphasizing the high incidence (2.5%) of carriers of at least one abnormal Hb, and the great heterogeneity of globin molecular defects on the island. Twenty-six different mutations were identified: the most common occur in the beta-globin gene (beta(S), beta(C), deltabeta(Lepore), beta(G-San José), beta(O-Arab), but also quite frequent is the mutated allele alpha(J-Oxford). The chromosome haplotypes associated with some of them were characterized. Two uncommon Hbs, Copenhagen and D Punjab, and some 18 rare variants complete the wide spectrum of structural alterations of globin genes in Sicily. We think they are de novo mutations prevalently. It is not possible to exclude that the presence of a few of them is related to migratory phenomena, particularly from North Africa and East Asia. Numerous thalassemic alleles complete the picture of globin gene mutations in Silicy.