Lawrence B. Faulkner

Mutations in the thrombopoietin receptor, Mpl, in children with congenital amegakaryocytic thrombocytopenia

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare disorder of undefined aetiology. The disease presents with severe thrombocytopenia and absence of megakaryocytes in the bone marrow. Furthermore, CAMT patients may develop bone marrow aplasia. To obtain more insight into the mechanism underlying CAMT, five children were analysed. All patients had increased plasma thrombopoietin (Tpo) levels, indicating a platelet production defect. Bone marrow‐derived CD34+ stem cells from three patients were cultured in an in vitro liquid culture system to study megakaryocytopoiesis. CD34+ cells from two of the three patients failed to differentiate into megakaryocytes. The lack of megakaryocyte formation could imply that a defect in the c‐mpl gene, encoding the Tpo receptor, exists. Sequencing of c‐mpl revealed mutations in four of five patients. Three patients had point mutations and/or a deletion in the coding regions of c‐mpl. All point mutations led to an amino acid substitution or to a premature stop codon. In one patient, a homozygous mutation in the last base of intron 10 was found that resulted in loss of a splice site. This study showed that mutations in c‐mpl could be the cause of thrombocytopenia in CAMT in the majority of patients. Furthermore, Tpo has been shown to have an anti‐apoptotic effect on stem cells. Therefore, mutations in c‐mpl might not only affect megakaryocyte formation but may also impair stem cell survival, which could explain the occurrence of bone marrow failure as final outcome in patients with CAMT.

Pediatric peripheral blood progenitor cell collection: haemonetics MCS 3P versus COBE Spectra versus Fresenius AS104

BACKGROUND: An increasing number of apheresis machines are becoming available for peripheral blood progenitor cell (PBPC) collection in children. STUDY DESIGN AND METHODS: At the Childrens Hospital of Florence (Italy), three apheresis machines were evaluated: MCS 3P (Haemonetics) (10 procedures in 4 patients, aged 10–12 years, weight 23.5‐64 kg), Spectra, (COBE) (8 procedures in 3 patients, aged 4–17 years, weight 19–59 kg), and AS104 (Fresenius) (24 procedures in 9 patients, aged 2–16 years, weight 13.6‐60 kg). For PBPC quantitative analysis, CD34 cytofluorimetry was employed. Relevant variables analyzed included efficiency of CD34+ cell extraction and enrichment, mononuclear cell purity and red cell contamination of the apheresis components, and platelet count decreases after leukapheresis. RESULTS: No significant differences in CD34+ cell‐extraction abilities were found. However, the AS104 provided consistently purer leukapheresis components in terms of mononuclear cell and CD34+ cell enrichment (441 +/− 59%, vs. 240 +/− 35% and 290 +/− 42% for MCS 3P and Spectra, respectively). Postapheresis platelet counts dropped the least with the AS104. The smallest patient who underwent apheresis with MCS 3P (the only machine working on discontinuous flow and hence with greater volume shifts) weighed 23.5 kg and tolerated the procedure well, with no signs of hemodynamic instability. No significant complications were observed. CONCLUSION: All machines seem to have comparable PBPC extraction efficiency, but the AS104 seems to give the component with the greatest PBPC enrichment. This feature might be relevant for further ex vivo cell processing (CD34+ cell selection, expansion, and so on).

Detection of Neuroblastoma Cells in Bone Marrow and Peripheral Blood by Different Techniques Accuracy and Relationship with Clinical Features of Patients

Purpose: Detection of metastatic tumor cells in bone marrow (BM) and peripheral blood (PB) of children with neuroblastoma is crucial for prognosis and planning of therapy. Aims of this large descriptive repeated survey were to evaluate the diagnostic accuracy of different techniques in diagnostic samples obtained at several disease course time points and to correlate positive results with patient clinical features and outcome. Experimental Design: BM aspirates, trephine biopsies, PB, and peripheral blood stem cell (PBSC) samples from Italian children with neuroblastoma were analyzed by morphological and histologic techniques, as well as by immunocytochemistry (IC) for disialoganglioside GD2 and reverse transcription-PCRs (RT-PCRs) for tyrosine hydroxylase (TH) and pgp9.5 genes. The diagnostic odd ratio (DOR) was used to measure the accuracy of the different techniques. Results: A total of 2,247 evaluations were done on 561 BM, 265 PB, and 69 PBSC samples from 247 patients. IC showed the best accuracy. Whereas TH RT-PCR accuracy was satisfactory, that of pgp9.5 was very low. Positive results obtained by IC in BM and PB samples at diagnosis from stage 1, 2, and 3 patients correlated with unfavourable outcome. No correlation was found between positive results obtained by IC or TH RT-PCR in BM, PB, and PBSC samples from stage 4 patients and their outcome. Conclusions: Because of its elevated diagnostic accuracy, IC may represent a useful adjunct to conventional morphological techniques, especially in view of its potential prognostic role in patients with localized disease. Longitudinal multicenter studies are warranted to definitely establish the clinical usefulness of TH RT-PCR.

G-CSF serum pharmacokinetics during peripheral blood progenitor cell mobilization: neutrophil count-adjusted dosage might potentially improve mobilization and be more cost-effective

The optimal dosing schedule of G-CSF for peripheral blood progenitor cell (PBPC) mobilization is still under investigation although many centers use 10 μg/kg/day in a single subcutaneous dose. However, G-CSF clearance increases with increasing absolute neutrophil count (ANC). Hence a G-CSF dosage adjusted to ANC might be a reasonable approach. We measured G-CSF trough serum levels by sandwich ELISA assay at different ANCs in eight patients undergoing treatment with filgrastim at 10 μg/kg/day in a single subcutaneous dose. A total of 26 samples were analyzed, and a strong correlation between increasing ANC and decreasing G-CSF levels was found by linear regression analysis (P < 0.0003, r2 = 0.4199). For ANC values above 5000/μl the trough serum levels, ie 24 h after administration, were consistently below the level that provides maximal clonogenic precursor stimulation in vitro (10 ng/ml). Serial serum G-CSF measurements performed in three patients at 0, 3, 6, 9 and 24 h after G-CSF administration, showed a reduction of the area under the curve (AUC) with increasing ANC. For an ANC of 20000/μl or greater, the G-CSF serum level fell under the maximal in vitro stimulation threshold of 10 ng/ml within 12 h. This preliminary pharmacokinetic data seems to suggest that an ANC-adjusted G-CSF dosing schedule might improve the design of PBPC mobilization regimens.

Detection procedures for neuroblastoma cells metastatic to blood and bone marrow: blinded comparison of chromogranin A heminested reverse transcription polymerase chain reaction to tyrosine hydroxylase nested reverse transcription polymerase chain reaction and to anti-GD2 immunocytology.

Specific and sensitive tumor cell detection is becoming increasingly important for diagnosing and staging as well as for the therapeutic management of neuroblastoma patients. We propose a chromogranin A heminested reverse transcription polymerase chain reaction (CgA hn RT-PCR) procedure for the detection of neuroblastoma minimal residual disease in peripheral blood and bone marrow samples. The results were checked in comparison with the presently available procedures (i.e., with the tyrosine hydroxylase nested RT-PCR [TH n RT-PCR] and with the immunocytochemical approach using anti-GD2 antibodies). Controls from healthy patients or from people with unrelated disease (12 samples of bone marrow and 23 samples of peripheral blood) and serial dilution experiments using neuroblastoma cell lines (SKNLP, SKNFI, STA6, STA8) showed CgA hn RT-PCR full specificity and sensitivity ranging from 103 to 106 (depending on the cell line). The results compared favorably with those obtained using TH n RT-PCR. Preliminary data obtained analyzing bone marrow and peripheral blood specimens from stage IV neuroblastomas showed substantially overlapping results between CgA and TH n RT-PCR procedures. Our data support the potential usefulness of CgA heminested RT-PCR as a specific and sensitive procedure for minimal disease detection in neuroblastoma. A prospective evaluation of this tool in clinical studies might be warranted.

In vivo cytoreduction studies and cell sorting-enhanced tumor-cell detection in high-risk neuroblastoma patients: Implications for leukapheresis strategies

PURPOSE To improve autologous leukapheresis strategies in high-risk neuroblastoma (NB) patients with extensive bone marrow involvement at diagnosis. PATIENTS AND METHODS Anti-G(D2) immunocytochemistry (sensitivity, 1 in 10(5) to 10(6) leukocytes) was used to evaluate blood and bone marrow disease at diagnosis and during the recovery phase of the first six chemotherapy cycles in 57 patients with stage 4 NB and bone marrow disease at diagnosis. A total of 42 leukapheresis samples from the same patients were evaluated with immunocytology, and in 24 of these patients, an anti-G(D2) immunomagnetic enrichment step was used to enhance tumor-cell detection. RESULTS Tumor cytoreduction was much faster in blood compared with bone marrow (3.2 logs after the first cycle and 2.1 logs after the first two cycles, respectively). Bone marrow disease was often detectable throughout induction, with a trend to plateau after the fourth cycle. By direct anti-G(D2) immunocytology, a positive leukapheresis sample was obtained in 7% of patients after either the fifth or sixth cycle; when NB cell immunomagnetic enrichment was applied, 25% of patients had a positive leukapheresis sample (sensitivity, 1 in 10(7) to 10(8) leukocytes). CONCLUSION Standard chemotherapy seems to deliver most of its in vivo purging effect within the first four cycles. In patients with overt marrow disease at diagnosis, postponing hematopoietic stem-cell collection beyond this point may not be justified. Tumor-cell clearance in blood seems to be quite rapid, and earlier collections via peripheral-blood leukapheresis might be feasible. Immunomagnetically enhanced NB cell detection can be highly sensitive and can indicate whether ex vivo purging should be considered.

Use of percutaneous radial artery catheter for peripheral blood progenitor cell collection in pediatric patients.

BACKGROUND : Leukapheresis procedures require adequate flow rates, which in children may frequently involve invasive vascular access placement.

Peripheral blood progenitor uncontrolled‐rate freezing: a single pediatric center experience

BACKGROUND: Controlled‐rate freezing (CRF) followed by storage in liquid nitrogen is employed by most centers as the standard procedure for peripheral blood progenitor cell (PBPC) cryopreservation. Uncontrolled‐rate freezing (URF) at −80°C is more simple, time‐saving, less expensive, and, possibly, as effective as CRF. The aim of this retrospective analysis was to compare CRF and URF in childhood transplantation.