Marina Lanciotti

Homozygosis for (12) CA repeats in the first intron of the human IFN‐γ gene is significantly associated with the risk of aplastic anaemia in Caucasian population

Interferon‐γ (IFN‐γ) mediates the final damage of the stem cell compartment in Aplastic Anaemia (AA). Normal subjects homozygous for 12 (CA) repeats of polymorphism variable number of dinucleotide (CA) repeat (VNDR) in position 1349 of the IFN‐γ gene (IFNG) were shown to overproduce IFN‐γin vitro. We studied the distribution of polymorphism VNDR 1349 of IFNG in 67 Caucasian AA patients and in normal controls. Genotype (CA)12‐12, (homozygosis for allele 2) and the single allele 12 were significantly more frequent (Pu2003=u20030·005 and 0·004 respectively) in patients versus controls. The polymorphism was equally distributed in AA patients regardless of their response to immunosuppression. Homozygosity for 12 (CA) repeats of polymorphism VNDR 1349 of IFNG is strongly associated with the risk of AA in Caucasian subjects.

Congenital and acquired neutropenias consensus guidelines on therapy and follow‐up in childhood from the Neutropenia Committee of the Marrow Failure Syndrome Group of the AIEOP (Associazione Italiana Emato‐Oncologia Pediatrica)

The management of congenital and acquired neutropenias presents some differences according to the type of the disease. Treatment with recombinant human granulocyte-colony stimulating factor (G-CSF) is not standardized and scanty data are available on the best schedule to apply. The frequency and the type of longitudinal controls in patients affected with neutropenias are not usually discussed in the literature. The Neutropenia Committee of the Marrow Failure Syndrome Group (MFSG) of the Associazione Italiana di Emato-Oncologia Pediatrica (AIEOP) elaborated this document following design and methodology formerly approved by the AIEOP board. The panel of experts reviewed the literature on the topic and participated in a conference producing a document that includes recommendations on neutropenia treatment and timing of follow-up.

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 disease characterized by pancytopenia and hypoplastic bone marrow. The incidence is 2-3/million inhabitants/year, in Europe, but higher in East Asia. Survival in severe aplastic anemia (SAA) has markedly improved in the past 2 decades because of advances in hematopoietic stem cell transplantation, immunosuppressive and biologic drugs, and supportive care. In SAA hematopoietic stem cell transplant (HSCT) from a matched sibling donor (MSD) is the treatment of choice. If a MSD is not available, the options include immunosuppressive therapy (IST) or unrelated donor HSCT. The objective of this guideline is to provide healthcare professionals with clear guidance on the diagnosis and management of pediatric patients with AA. A preliminary, evidence-based document issued by a group of pediatric hematologists was discussed, modified and approved during a series of Consensus Conferences according to procedures previously validated by the AIEOP Board. The guidelines highlight the importance of referring pediatric patients with AA to pediatric centers with long experience in diagnosis, differential diagnosis, management, supportive care and follow-up of AA.

Possible role of toll‐like receptor 9 polymorphism in chemotherapy‐related invasive mold infections in children with hematological malignancies

with Beckwith–Wiedemann syndrome previously treated for hepatoblastomawithmulti-agent chemotherapy. J Pediatr Hematol Oncol 1996;18:419–420. 6. Roebuck DJ, Perilongo G. Hepatoblastoma: An oncological review. Pediatr Radiol 2006;36:183–186. 7. JamesGG,MelissaLB. Epidemiology of childhood cancer. In: Philip AP, David GP, editors. Principles and practice of pediatric oncology, 5th edition. Lippincott:Wlilliams &Wilkins; 2005. pp. 1–13. 8. Ikeda H, Matsuyama S, Tanimura M. Association between hepatoblastoma and very low birth weight: A trend or a chance? [see comment]. J Pediatr 1997;130:557–560. 9. Perentesis JP. Genetic predisposition and treatment-related leukemia. Med Pediatr Oncol 2001;36:541–548. 10. Inoue K, Asao T, Shimada T. Ethnic-related differences in the frequency distribution of genetic polymorphisms in the CYP1A1 and CYP1B1 genes in Japanese and Caucasian populations. Xenobiotica 2000;30:285–295.

Compound heterozygosity for two new TERT mutations in a patient with aplastic anemia

Dyskeratosis congenita (DC) is a genetically heterogeneous syndrome characterized by reticular skin pigmentation, nail dystrophy, mucosal leukoplakia, short telomeres, and a predisposition to bone marrow failure and malignancy. Patients carrying mutations in TERT show a wide clinical spectrum of abnormalities, including classical DC, isolated bone marrow failure and lung fibrosis. Here, we report the clinical description and biological analysis of a patient with compound heterozygosity for two new missense mutations in TERT (V96L and V119L). Both mutations segregate with a short telomere phenotype, though only V96L segregates with clinical signs of DC. Pediatr Blood Cancer. 2010;55:550–553.

Etiology, clinical outcome, and laboratory features in children with neutropenia: Analysis of 104 cases

Neutropenia is not uncommon in childhood. The aim of our study was to analyze the underlying causes of neutropenia and to evaluate its clinical significance in a series of children referred to our center.

NOLA1 gene mutations in acquired aplastic anemia

Telomerase complex genes mutations (DKC1, TERC, TERT, and NOP10) lead to premature telomere shortening and are responsible for different forms of dyskeratosis congenita. TERC and TERT mutations were also found in patients with aplastic anemia. The aim of this work is to analyze the possible involvement of the telomerase complex gene NOLA1, in a population of Italian AA patients.

Lethal sepsis and malignant transformation in severe congenital neutropenia: Report from the Italian Neutropenia Registry

To the Editor: The occurrence of lethal sepsis and myelodysplasia/acute myeloid leukemia (MDS/AML) may affect patients diagnosed with severe congenital neutropenia (SCN) at variable rates depending upon the cohorts studied [1]. The Severe Chronic Neutropenia International Registry (SCNIR) reported a cumulative incidence (CI) of sepsis/death of 10% after 15 years from the start of therapy with granulocyte colony-stimulating factor (G-CSF) [2]. The French Severe Chronic Neutropenia Registry (SCNFR) reported 6 septic deaths out of 101 SCN cases, while in the Swedish cohort nobody died of sepsis [3,4]. As for MDS/AML, the CIs after 15 years from the beginning of G-CSF therapy were 22%, 11% and 31%, respectively, in the SCNIR, SCNFR and Swedish cohorts [2,3,4]. In the present study, we evaluated the incidence of lethal sepsis and MDS/AML in patients registered in the Italian Neutropenia Registry (INR). Starting from 2003, the INR identified 350 patients affected with various type of neutropenia from 35 centers belonging to Associazione Italiana Ematologia-Oncologia Pediatrica (AIEOP). Among them, 23 had SCN and considered eligible for the study. Median age at diagnosis of SCN was 8.17 months (IQR: 2.83– 32.15) and median duration of follow up was 6.44 years (IQR; 2.99–12.05) (Table I). During the course of the study, two patients died of non-transplant related sepsis, after voluntary decision by the family to stop G-CSF treatment. The lethal sepsis CI was 10% after three years from the beginning of G-CSF treatment. One patient out of 23 developed MDS, while no leukemia cases were reported. The MDS CI was 6% (95% IC 1–35) at four years after the start of G-CSF. In our study, the incidence of MDS was far lower than that described in the Swedish cohort and in SCNIR patients treated with G-CSF doses greater than 8mcg/kg. On the contrary, it is closer to the French cohort and to the SCNIR group treated with G-CSF dosage lower than 8mcg/kg (15%). The reason for the low MDS incidence, could be the small size and the young age of the cohort; also, in some cases, the choice of “early” transplantion might have prevented the malignant transformation. Moreover, SCN cases (i.e., GATA2 gene defects) with a high transforming potential were not included in our cohort, possibly reducing the incidence of malignant events. On the other hand, the lethal sepsis risk in our cohort was similar to those described in SCNIR and in the French cohort. Deaths were mainly related to voluntary interruption G-CSF treatment, probably because underestimation of its vital importance and general lack of adherence to daily subcutaneous infusions. Even with the limitations of the small sample size and relatively short follow up, due to the young age of patients in the Registry and to the ongoing SCN cases registration, we emphasize that the management and follow up of patients with SCN remains a matter of concern. The education of patients and their families and strict follow up could minimize the risk of improper administration and help to perform early diagnosis of possible malignant evolution.

Chromosomal locus 19p13 as potential hotspot for aberrant gene expression in relapsed paediatric acute lymphoblastic leukaemia

We read with interest the article by Beesley et al (2005), which reported the possible gene expression signature of relapse in paediatric pre-B acute lymphoblastic leukaemia (ALL). The authors analysed RNA extracted from 11 pairs of cryopreserved pre-B ALL bone marrow specimens taken from the same patients at diagnosis and relapse using Affymetrix HGU133A high-density oligonucleotide microarray. They showed that the relapse specimens overexpressed genes involved in cell growth and proliferation when compared with their diagnostic counterparts. Moreover, several chromosomal loci, including 19p13, were identified as potential hotspots for aberrant gene expression in relapsed ALL. To further explore the possibility of relapse prediction in paediatric pre-B ALL, we used a different approach, based on the hypothesis that ALL patients that will relapse already show a different expression profile in their leukaemic cells at the time of diagnosis from. To test this hypothesis, we compared leukaemic gene expression profiles in a set of 10 bone marrow samples obtained from ALL paediatric patients at the time of diagnosis. Four patients relapsed during therapy or early after stopping therapy and six are in continuous complete remission at 10 years from diagnosis. All of them share the following features: therapeutic protocol, prednisone good responder status, pre-B lineage, absence of chromosomal translocation and presence of >85% of blasts in the marrow. RNA was analysed by Affymetrix high-density oligonucleotide microarrays HG-U95Av2. Comparative analysis was performed using Affymetrix Microarray Suite 5Æ0 software algorithms (mas 5). Functional class and chromosomal loci were assigned using the NetAffx GO Mining Tool. We found that 39 genes were upregulated in relapsed patients. Analyses of their biological functions showed a predominance of genes involved in signal transduction, cell-cycle progression and regulation of transcription, suggesting impaired cell proliferation and apoptosis in B cell precursors of ALL relapsed patients. Moreover, among the 39 upregulated genes, six (15%) (NDUFB7, CD97, NDUFS7, BSG, ZNF91 and KCNN1) were localised in the chromosomal locus 19p13, which was found to be the most prevalent. A common finding in our study and that of Beesley et al (2005) was the recurrent upregulation of genes located in chromosomal locus 19p13 in diagnostic specimens of relapsed patients or in relapse specimens. This finding was also confirmed by Beesley et al (2005) in a separate analysis of the relapse data from Yeoh et al (2002). In particular, BSG, mapped on 19p13 locus, was a perfect marker for diagnosis and relapse specimens. It was significantly upregulated in the relapse specimen with respect to the diagnosis specimen as also reported by Yeoh et al (2002). In our samples, BSG was already overexpressed at diagnosis in the leukaemic cells of relapsed patients. This finding suggests that it could be a good predictor of relapse in paediatric pre-B ALL, and therefore the role of BSG expression in the mechanism of leukaemia relapse should be investigated further. A large amount of literature is available on the relationship between chromosomal locus 19p13 and cancer; in particular, translocations involving this locus are common in pre-B paediatric ALL (Pui et al, 2004) and an association with poor prognosis and response to therapy has also been reported (Khalidi et al, 1999). The diverse biological features of ALL patients analysed in these studies probably accounts for the variability of the data, but the common findings could be an interesting starting point to further investigate the potential role of 19p13 locus in the pathogenesis and prognosis of childhood ALL.

Severe congenital neutropenia: A negative synergistic effect of multiple mutations of ELANE (ELA2) gene

Heterozygous mutations of the ELANE gene (previously termed ELA2) encoding neutrophil elastase are responsible for most cases of severe congenital neutropenia (SCN) and cyclic neutropenia (CN) (Dale et al, 2000; Horwitz et al, 2007). The presence of more than one mutation in ELANE is a rare phenomenon, described in only two cases to date (Salipante et al, 2007). In both cases, the presence of two mutations seems have mitigated the disease, possibly by moderating the effects on subcellular mislocalization. We report the presence of three point mutations in the ELANE gene of a SCN patient, a female of unaffected, non-consanguineous Italian parents. SCN was diagnosed at 4 years of age; the haematological data were white blood cell count 10Æ3 · 10/l (neutrophils 7%; eosinophils 2%; lymphocytes 72%; monocytes 19%); Hb 86 g/l, platelet count 74 · 10/l, the bone marrow analysis showed hypocellularity with a maturation block at the promyelocyte/ myelocyte stage. The patient had frequent febrile episodes, mainly caused by skin abscesses and bronco-pneumonia. Treatment with granulocyte colony-stimulating factor (G-CSF) was started at a dose of 5 lg/kg per d, to maintain the absolute neutrophil count between 1Æ5 and 3Æ0 · 10/l. She did well until reaching 8 years of age when she was admitted to our centre, where a diagnosis of myelodysplastic syndrome (MDS) and long-lasting invasive pulmonary mycosis was made. Bone marrow karyotype was 45, XX, )7. Subsequent bone marrow karyotype analysis also showed some clones with chromosome 21 trisomy. Three months later, the patient underwent malignant transformation into acute myeloid leukaemia (AML). Unrelated donor haemopoietic stem cell transplantation (HSCT) was performed and at present, 8 years after HSCT, she is alive and well in complete remission. Written informed consent to the study was obtained from the parents. This study was conducted in accordance with the Declaration of Helsinki. Sequencing of ELANE in the patient’s genomic DNA identified three different heterozygous single-base substitutions (Fig 1): a de novo missense mutation (2192G > A) within exon 3, generating a non-conservative amino acid substitution V53M and two previously undescribed variations. One variation is a single base missense change (2240G > A), also in exon 3, leading to an amino acid substitution V69M and the other is an intronic mutation (IVS322G > A). The automated splice site analysis of this latter variation showed that it does not affect ELANE gene splicing and thereafter is not likely to be directly involved in the pathogenesis of the disease. HAX1 and CSF3R mutation analyses were negative. We also sequenced the ELANE gene of the parents. The father had only wild type alleles, while the mother showed the single base change 2240G > A (V69M), but she has never had any symptoms of the disease and all her haematological data were in normal range, suggesting a non pathogenic mutation or a very rare polymorphic variation. In order to exclude the possibility of a polymorphic change, the ELANE exon 3 and the flanking intronic regions were screened in 95 healthy controls. Neither of the novel mutations (2240G > A and IVS3-22G > A) were detected in controls nor in 95 unrelated neutropenic patients previously screened for ELANE mutations. A single genomic DNA fragment of the patient, spanning the three mutations, was amplified by polymerase chain reaction and subcloned in Escherichia coli; 20 clones were genotyped by DNA sequencing, revealing that all three mutations always occurred in cis, determining the maternal origin of the mutated allele. In our patient the presence of multiple mutations seems to have a synergistic negative effect on the disease. In fact, the de novo V53M mutation has been previously described as specifically associated with CN phenotype (Bellanné-Chantelot et al, 2004) which is a mild syndrome, characterized by intermittent and less severe neutropenia and no increased propensity to develop AML/ MDS. The valine with methionine substitution in position 69 (V69M) has never been documented. This novel mutation was not pathogenic in the mother of our patient, while it might have had a detrimental synergistic interaction with the other mutations resulting in a faulty protein in the patient. To shed some light on the consequences of the two ELANE mutations at the protein level, we modelled the amino acid changes into the three-dimensional structure of the full-length Human Neutrophil Elastase 2 (Bode et al, 1986). This model shows (Fig 2) that both of the V53M and V69M substitutions occur in a region far from the catalytic triad of the enzyme making a direct interaction with the active site an unlikely occurrence. Both mutations give rise to a substitution of a valine with a methionine, which is a larger amino acid with a branched residue. The V53 residue is inside the protein structure and this substitution could destabilize the local fold of the protein, without profoundly altering the protein structure and it seems consistent with the mild syndrome (CN) associated with this amino acid substitution. On the other hand, V69M substitution occurs in a superficial area of the protein structure and it does not seem to perturb the local stability of the protein, in agreement with our case in which the patient’s mother, carrying the V69M substitution, is