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We studied the prevalence and degree of tumor cell infiltration (TCI) in bone marrow (BM) aspirates of 89 infants with stage 4/4 S neuroblastoma and correlated them with MYCN gene status and patient outcome. TCI was scored 0, +, ++, and +++, the last corresponding to an infiltration greater than 10%. TCI 0 was more frequent in stage 4 than in stage 4 S. TCI + and ++ were equally represented. TCI +++ was found only in 9 patients, all with typical stage 4 features (bone or lung involvement). Overall survival was not significantly influenced by the presence and degree of TCI.

Summary. Factor VIII gene inversion of intron 1 has recently been reported to be the mutation responsible for haemophilia A in about 5% of severe cases. In our series of patients, which is made up of 77 Italian cases negative for intron 22 inversion, the mutation was found in three sporadic and in one familial patients, with an overall frequency of 5.2%. The carrier status of the patients’ female relatives was assessed by mutation analysis and showed that only two-thirds of cases could be considered truly sporadic. The germ-line origin of the mutation was investigated in the two sporadic families by haplotype analysis on genomic DNA of the patients’ maternal grandparents. These studies indicated that both mutation events had occurred in the germ cell lines of the patients’ healthy grandfather, suggesting that, as already demonstrated for the inversion of intron 22, the male germ cell line is more susceptible to the intrachromosome recombination which leads to the inversion of intron 1.

Summary. We describe 18 novel mutations, unreported in the Haemophilia A mutation Databases, that have been identified in a cohort of unrelated, Italian patients affected with haemophilia A (HA). Screening of the factor VIII gene (FVIII) was performed using denaturing high-performance liquid chromatography (DHPLC) and direct sequencing. Eight mutations were characterized as non-missense alterations, and the remaining 10 were missense mutations. Heterozygosity for the identified mutations was observed in the female relatives of patients belonging to eight families with sporadic cases. In an attempt to understand better the causative effect of the mutations and the clinical variability of the patients, missense mutation consequences were investigated for: (1) the nature of the new amino acid; (2) the location of the substituted amino acid within crystallographic and theoretical models; and (3) the degree of conservation of the native residue in factor VIII (FVIII) protein and FVIII-related protein family aligned sequences. These research tools have provided evidence that the mutations we describe involve residues that were conserved, at least in FVIII proteins, in all the species we compared.

AC133+ cells may represent an alternative source of transplantable haemopoietic progenitor cells to CD34+ cells. Here, we have addressed the characterization of umbilical cord blood (UCB) AC133+ cells and compared their immunophenotypic and functional features with those of UCB CD34+ cells. UCB AC133+ and CD34+ cell fractions were purified by magnetic cell sorting, analysed by flow cytometry, tested for their content in blast cell colony-forming units (CFU-Bl), erythroid and granulocyte-macrophage colony-forming units before and after expansion in the presence of various haemopoietic growth factor combinations. Median AC133+ cell yield was 62.3%, and median AC133+ population purity was 97.9%. AC133+ cells were found to contain significantly more CFU-Bl than CD34+ cells; furthermore, the replating efficiency, i.e. the number of CFU-Bl capable of generating secondary colonies, was higher in the former than in the latter cells. Both AC133+ and CD34+ cells displayed an increased ability to give rise to committed progenitors after 7-day expansion in liquid cultures. These data suggest that the AC133+ cell subset is a heterogeneous pool of immature and more differentiated cells that can be maintained and expanded in well-defined culture conditions. In comparison with CD34+ cells, UCB AC133+ cells appear to contain a higher number of early haemopoietic progenitors.

Ectopic mRNA was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) in patients with duplication of F8 gene exon 13, a mutation which has been demonstrated to be a cause of mild hemophilia A in 32% of Northern Italian subjects. Two different transcripts originate from mutated genomic DNA, due to alternative splice processes. The larger-sized transcript contains both duplicated exons 13, the smaller one contains only one exon 13. The residual FVIII:C activity which accounts for the mild hemophilia A phenotype derives from the latter transcript.

A basic tenet of the Lyon hypothesis is that X inactivation occurs randomly with respect to parental origin of the X chromosome. Yet, nonrandom patterns of X inactivation are common - often ascertained in women who manifest recessive X-linked disorders despite being heterozygous for the mutation. Usually, the cause of skewing is cell selection disfavouring one of the cell lineages created by random X inactivation. We have identified a three generation kindred, with three females who have haemophilia A because of extreme skewing of X inactivation. Although they have both normal and mutant factor VIII (FVIII) alleles, only the mutant one is transcribed; and, they share an XIST allele that is never transcribed. The skewing in this case seems to result from an abnormality in the initial choice process, which prevents the chromosome bearing the mutant FVIII allele from being an inactive X.

A rearrangement of exon 13 in the factor VIII gene has been identified as the causative mutation in 32% of Northern Italian patients with mild hemophilia A. We have demonstrated that all share a common haplotype, thus suggesting that the mutation likely occurred in a single ancestor. To date, no predominant mutation has been identified in mild hemophilia A, therefore it would be extremely useful to carry out more extensive studies to ascertain whether the mutation is confined to northern Italy.

Previous studies have shown that hemophilia B (HB) is the result of several different mutations, mostly single nucleotide substitutions, in the factor IX (FIX) gene. In order to evaluate the impact of mutation analysis on genetic counseling in sporadic and uninformative HB familial pedigrees, we re-analyzed by the conformation sensitive gel electrophoresis (CSGE) technique 14 patients, previously studied by restriction fragment length polymorphisms (RFLPs). A single mutation was present within the FIX gene of each patient: 12 mutations were single base substitutions, 1 was a base insertion, and 1 was a four nucleotide deletion; 4/12 mutations have not been described so far. By identifying the detrimental mutations in affected males, carrier status was correctly diagnosed in all the women we studied; 3/12 de novo events were found in maternal meioses with a 25% mutation rate. Identification of the genetic defect was also successfully applied to three prenatal diagnoses.

We report on a girl with congenital hypoplastic anaemia, "coarse" face, generalized hypertrichosis with scalp hypotrichosis, short fifth finger, hypoplastic toenails, and mental retardation. A sister of the proposita, who died at the age of 1 year, had severe congenital anaemia, hypoplastic fingernails, low birth weight, failure to thrive, and repeated upper respiratory tract infections. Based on family history, we suspect that hypoplastic anaemia and the same multiple congenital anomalies-mental retardation syndrome (MCA/MR) were also present in this sister. To the best of our knowledge, this patient represents the first report of congenital hypoplastic anaemia and such a complex MCA/MR syndrome, probably inherited as an autosomal recessive trait.

Ectopic mRNAanalysis andmolecular modelling substantiate severe haemophilia in a patient with a FVIII gene splice mutation

Advanced neuroblastoma is one of the most lethal pediatric malignancies. Several antitumor compounds are able to induce tumor regression, and a dose-response relationship has been demonstrated for some of them. A significant improvement in both response rate and duration of survival has thus been obtained by treatment intensification [1–4]. In the Italian experience, the median survival time of children treated with aggressive chemotherapy has been doubled compared with historical controls [5]. However, the higher initial response rate and the prolonged remission time has resulted in only a marginal improvement in cure rate, presently not exceeding 25% [6, 7]. Hematopoietic growth factors (colony-stimulating factors, CSFs), by mitigating the myelotoxic effect of chemotherapy, may reduce the treatment-related morbidity and thus permit the delivery of higher dosages and the shortening of intervals between chemotherapy courses [8, 9].

10056 Background: The INSS definition of stage 4S neuroblastoma allows a bone marrow infiltration (BMI) no greater than 10%. More extensive involvement is to be consider stage 4. However, the degree of BMI may be difficult to establish, since tumor cells in the marrow commonly aggregate in clumps. So far, no studies have specifically investigated this issue. In this study we have reviewed the bone marrow slides of infants with disseminated neuroblastoma enrolled between January 1998 and July 2008 in the Italian Neuroblastoma Registry. Methods: BMI was arbitrarily considered No significant financial relationships to disclose.

The translocation (8;21) is a typical marker of the M2 subtype of acute nonlymphocytic leukemia, whereas the Philadelphia (Ph) chromosome is predominantly associated with chronic myelogenous leukemia, and seldom with immature acute leukemias, either lymphoblastic or nonlymphoblastic. Furthermore, the association between t(8;21) and a Ph in the same cell is extremely rare. We present a case of secondary acute promyelocytic leukemia with a karyotype 46,XY,t(8;21), t(9;22) at onset. At relapse the patient lost the Ph, while maintaining the t(8;21). This apparently paradoxical pattern of cytogenetic features and evolution is discussed.

Only two cases of Noonan Syndrome (NS) associated with tumor have previously been reported. The authors describe two new cases of NS with acute lymphoblastic leukemia (ALL), which were part of a series of 370 consecutive ALL untreated patients.

Immunological characterization of lymphoid subpopulations has assumed an important role in the study of lymphoproliferative disorders, in the attempt to detect the nature of malignant cells and discover the possible relationship between malignancies and immunological status.

Several studies document the importance of immunity in the host-tumor relationship in neuroblastoma patients, evidentiating a correlation between clinical compromission and immune impairment. In this study we evaluated some aspects of cellular and humoral immune capacity in 50 neuroblastoma patients at onset. The aim of this investigation was to define, if any, the common immune pattern of these patients, and to evidentiate a possible correlation with prognosis. Immune tests performed were serum immunoglobulin quantitation, absolute value of total T and B lymphocytes, and lymphocyte reactivity to phytohemagglutinin (PHA), Concanavalin A (ConA), and Pokeweed mitogen (PWM). In patients with localized or regional disease, diminished values of lymphocytes were observed in 4/16 cases, a datum highly correlated to a poor final outcome (P

Studies on CSF lymphocyte markers demonstrated normally a high percentage of E-rosette forming cells ( 90 %). By a microtechnique the relative proportion of T, B and null CSF cells was evaluated in two groups of children with ALL. As CNS prophylaxis patients of the first group received i.t. MTX during induction, cytosine arabinoside during consolidation and MTX every two months for three years as maintenance. Children of the second group received X-ray during induction and consolidation (total dose 2400 rads). Patients were in complete remission from 24 to 32 months for the first group and from 11 to 17 months for the second one. Results showed that the lymphocytes from CSF of the first group had a much lower percentage of T and T gamma cells in comparison to the second group; B cells were virtually absent, null cells were abnormally high in the first group. CSF cells of patients immediately after treatment with TCT demonstrated a high percentage of T cells. The lack of T cells in the group of children treated three years with i.t. MTX seems to cause such an inununological damage to discourage from this type of CNS prophylaxis.