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1.  KRAS and CREBBP mutations: a relapse-linked malicious liaison in childhood high hyperdiploid acute lymphoblastic leukemia 
Leukemia  2015;29(8):1656-1667.
High hyperdiploidy defines the largest genetic entity of childhood acute lymphoblastic leukemia (ALL). Despite its relatively low recurrence risk, this subgroup generates a high proportion of relapses. The cause and origin of these relapses remains obscure. We therefore explored the mutational landscape in high hyperdiploid (HD) ALL with whole-exome (n=19) and subsequent targeted deep sequencing of 60 genes in 100 relapsing and 51 non-relapsing cases. We identified multiple clones at diagnosis that were primarily defined by a variety of mutations in receptor tyrosine kinase (RTK)/Ras pathway and chromatin-modifying genes. The relapse clones consisted of reappearing as well as new mutations, and overall contained more mutations. Although RTK/Ras pathway mutations were similarly frequent between diagnosis and relapse, both intergenic and intragenic heterogeneity was essentially lost at relapse. CREBBP mutations, however, increased from initially 18–30% at relapse, then commonly co-occurred with KRAS mutations (P<0.001) and these relapses appeared primarily early (P=0.012). Our results confirm the exceptional susceptibility of HD ALL to RTK/Ras pathway and CREBBP mutations, but, more importantly, suggest that mutant KRAS and CREBBP might cooperate and equip cells with the necessary capacity to evolve into a relapse-generating clone.
PMCID: PMC4530204  PMID: 25917266
2.  T-cell receptor Vβ skewing frequently occurs in refractory cytopenia of childhood and is associated with an expansion of effector cytotoxic T cells: a prospective study by EWOG-MDS 
Blood Cancer Journal  2014;4(5):e209-.
Immunosuppressive therapy (IST), consisting of antithymocyte globulin and cyclosporine A, is effective in refractory cytopenia of childhood (RCC), suggesting that, similar to low-grade myelodysplastic syndromes in adult patients, T lymphocytes are involved in suppressing hematopoiesis in a subset of RCC patients. However, the potential role of a T-cell-mediated pathophysiology in RCC remains poorly explored. In a cohort of 92 RCC patients, we prospectively assessed the frequency of T-cell receptor (TCR) β-chain variable (Vβ) domain skewing in bone marrow and peripheral blood by heteroduplex PCR, and analyzed T-cell subsets in peripheral blood by flow cytometry. TCRVβ skewing was present in 40% of RCC patients. TCRVβ skewing did not correlate with bone marrow cellularity, karyotype, transfusion history, HLA-DR15 or the presence of a PNH clone. In 28 patients treated with IST, TCRVβ skewing was not clearly related with treatment response. However, TCRVβ skewing did correlate with a disturbed CD4+/CD8+ T-cell ratio, a reduction in naive CD8+ T cells, an expansion of effector CD8+ T cells and an increase in activated CD8+ T cells (defined as HLA-DR+, CD57+ or CD56+). These data suggest that T lymphocytes contribute to RCC pathogenesis in a proportion of patients, and provide a rationale for treatment with IST in selected patients with RCC.
PMCID: PMC4042298  PMID: 24786393
3.  Validation of flow cytometric phospho-STAT5 as a diagnostic tool for juvenile myelomonocytic leukemia 
Blood Cancer Journal  2013;3(11):e160-.
To diagnose juvenile myelomonocytic leukemia (JMML) is sometimes challenging, because around 10% of patients lack molecular abnormalities affecting Ras-MAPK (mitogen-activated protein kinase) pathway and other diseases such as cytomegalovirus infection can mimic clinical signs of JMML. In order to validate a phospho-specific flow cytometry assay assessing phospho-signal transducer and activator of transcription factor 5 (p-STAT5) as a new diagnostic tool for JMML, we examined 22 samples from children with JMML and 47 controls. CD33+/CD34+ cells from 22 patients with JMML showed hyperphosphorylation of STAT5 induced by sub-saturating doses of granulocyte-macrophage colony-stimulating factor (GM-CSF). Using a training set of samples (11 JMML and 23 controls), we identified a threshold for p-STAT5-positive after stimulation with 0.1 ng/ml GM-CSF (17.17%) that discriminates JMML from controls. This threshold was validated in an independent series (11 JMML, 24 controls and 7 cases with diseases other than JMML) where we demonstrated that patients with JMML could be distinguished from other subjects with a sensitivity of 91% (confidence interval (CI) 59–100%) and a specificity of 87% (CI 70–96%). Positive and negative predictive values were 71% (CI 42–92%) and 96% (CI 82–100%), respectively. In conclusion, flow cytometric p-STAT5 profiling is a reliable diagnostic tool for identifying patients with JMML and can contribute to consistency of current diagnostic criteria.
PMCID: PMC3880439  PMID: 24241400
juvenile myelomonocytic leukemia; phospho-specific flow cytometry; phospho-STAT5; GM-CSF
4.  Inhibition of Notch3 signalling induces rhabdomyosarcoma cell differentiation promoting p38 phosphorylation and p21Cip1 expression and hampers tumour cell growth in vitro and in vivo 
Cell Death and Differentiation  2011;19(5):871-881.
Rhabdomyosarcoma (RMS) is a paediatric soft-tissue sarcoma arising from skeletal muscle precursors coexpressing markers of proliferation and differentiation. Inducers of myogenic differentiation suppress RMS tumourigenic phenotype. The Notch target gene HES1 is upregulated in RMS and prevents tumour cell differentiation in a Notch-dependent manner. However, Notch receptors regulating this phenomenon are unknown. In agreement with data in RMS primary tumours, we show here that the Notch3 receptor is overexpressed in RMS cell lines versus normal myoblasts. Notch3-targeted downregulation in RMS cells induces hyper-phosphorylation of p38 and Akt essential for myogenesis, resulting in the differentiation of tumour cells into multinucleated myotubes expressing Myosin Heavy Chain. These phenomena are associated to a marked decrease in HES1 expression, an increase in p21Cip1 level and the accumulation of RMS cells in the G1 phase. HES1-forced overexpression in RMS cells reverses, at least in part, the pro-differentiative effects of Notch3 downregulation. Notch3 depletion also reduces the tumourigenic potential of RMS cells both in vitro and in vivo. These results indicate that downregulation of Notch3 is sufficient to force RMS cells into completing a correct full myogenic program providing evidence that it contributes, partially through HES1 sustained expression, to their malignant phenotype. Moreover, they suggest Notch3 as a novel potential target in human RMS.
PMCID: PMC3321627  PMID: 22117196
Notch; rhabdomyosarcoma; differentiation; muscle cells; sarcoma
5.  Growth factors in ischemic stroke 
Data from pre-clinical and clinical studies provide evidence that colony-stimulating factors (CSFs) and other growth factors (GFs) can improve stroke outcome by reducing stroke damage through their anti-apoptotic and anti-inflammatory effects, and by promoting angiogenesis and neurogenesis. This review provides a critical and up-to-date literature review on CSF use in stroke. We searched for experimental and clinical studies on haemopoietic GFs such as granulocyte CSF, erythropoietin, granulocyte-macrophage colony-stimulating factor, stem cell factor (SCF), vascular endothelial GF, stromal cell-derived factor-1α and SCF in ischemic stroke. We also considered studies on insulin-like growth factor-1 and neurotrophins. Despite promising results from animal models, the lack of data in human beings hampers efficacy assessments of GFs on stroke outcome. We provide a comprehensive and critical view of the present knowledge about GFs and stroke, and an overview of ongoing and future prospects.
PMCID: PMC4373358  PMID: 20015202
stroke; growth factors; G-CSF; EPO; GM-CSF; SCF; VEGF; IGF-1; SDF-1α
6.  Autologous stem cell transplantation in the treatment of systemic sclerosis: report from the EBMT/EULAR Registry 
Annals of the Rheumatic Diseases  2004;63(8):974-981.
Objective: To analyse the durability of the responses after haematopoietic stem cell transplantation (HSCT) for severe systemic sclerosis (SSc) and determine whether the high transplant related mortality (TRM) improved with experience. This EBMT/EULAR report describes the longer outcome of patients originally described in addition to newly recruited cases.
Methods: Only patients with SSc, treated by HSCT in European phase I–II studies from 1996 up to 2002, with more than 6 months of follow up were included. Transplant regimens were according to the international consensus statements. Repeated evaluations analysed complete, partial, or non-response and the probability of disease progression and survival after HSCT (Kaplan-Meier).
Results: Given as median (range). Among 57 patients aged 40 (9.1–68.7) years the skin scores improved at 6 (n = 37 patients), 12 (n = 30), 24 (n = 19), and 36 (n = 10) months after HSCT (p<0.005). After 22.9 (4.5–81.1) months, partial (n = 32) or complete response (n = 14) was seen in 92% and non-response in 8% (n = 4) of 50 observed cases. 35% of the patients with initial partial (n = 13/32) or complete response (n = 3/14) relapsed within 10 (2.2–48.7) months after HSCT. The TRM was 8.7% (n = 5/57). Deaths related to progression accounted for 14% (n = 8/57) of the 23% (n = 13/57) total mortality rate. At 5 years, progression probability was 48% (95% CI 28 to 68) and the projected survival was 72% (95% CI 59 to 75).
Conclusion: This EBMT/EULAR report showed that response in two thirds of the patients after HSCT was durable with an acceptable TRM. Based on these results prospective, randomised trials are proceeding.
PMCID: PMC1755096  PMID: 15249325
7.  Phase I/II trial of autologous stem cell transplantation in systemic sclerosis: procedure related mortality and impact on skin disease 
Annals of the Rheumatic Diseases  2001;60(6):577-584.
BACKGROUND—Systemic sclerosis (SSc, scleroderma) in either its diffuse or limited skin forms has a high mortality when vital organs are affected. No treatment has been shown to influence the outcome or significantly affect the skin score, though many forms of immunosuppression have been tried. Recent developments in haemopoietic stem cell transplantation (HSCT) have allowed the application of profound immunosuppression followed by HSCT, or rescue, to autoimmune diseases such as SSc.
METHODS—Results for 41 patients included in continuing multicentre open phase I/II studies using HSCT in the treatment of poor prognosis SSc are reported. Thirty seven patients had a predominantly diffuse skin form of the disease and four the limited form, with some clinical overlap. Median age was 41 years with a 5:1 female to male ratio. The skin score was >50% of maximum in 20/33 (61%) patients, with some lung disease attributable to SSc in 28/37 (76%), the forced vital capacity being <70% of the predicted value in 18/36 (50%). Pulmonary hypertension was described in 7/37 (19%) patients and renal disease in 5/37 (14%). The Scl-70 antibody was positive in 18/32 (56%) and the anticentromere antibody in 10% of evaluable patients. Peripheral blood stem cell mobilisation was performed with cyclophosphamide or granulocyte colony stimulating factor, alone or in combination. Thirty eight patients had ex vivo CD34 stem cell selection, with additional T cell depletion in seven. Seven conditioning regimens were used, but six of these used haemoimmunoablative doses of cyclophosphamide +/- anti-thymocyte globulin +/- total body irradiation. The median duration of follow up was 12 months (3-55).
RESULTS—An improvement in skin score of >25% after transplantation occurred in 20/29 (69%) evaluable patients, and deterioration in 2/29 (7%). Lung function did not change significantly after transplantation. One of five renal cases deteriorated but with no new occurrences of renal disease after HSCT, and the pulmonary hypertension did not progress in the evaluable cases. Disease progression was seen in 7/37 (19%) patients after HSCT with a median period of 67 (range 49-255) days. Eleven (27%) patients had died at census and seven (17%) deaths were considered to be related to the procedure (direct organ toxicity in four, haemorrhage in two, and infection/neutropenic fever in one). The cumulative probability of survival at one year was 73% (95% CI 58 to 88) by Kaplan-Meier analysis.
CONCLUSION—Despite a higher procedure related mortality rate from HSCT in SSc compared with patients with breast cancer and non-Hodgkin's lymphoma, the marked impact on skin score, a surrogate marker of mortality, the trend towards stabilisation of lung involvement, and lack of other treatment alternatives justify further carefully designed studies. If future trials incorporate inclusion and exclusion criteria based on this preliminary experience, the predicted procedure related mortality should be around 10%.

PMCID: PMC1753658  PMID: 11350846
8.  Late pulmonary sequelae after childhood bone marrow transplantation 
Thorax  1999;54(2):131-135.
BACKGROUND—Respiratory function in transplanted children is important because of the long life expectancy of bone marrow transplant recipients, particularly children. Attention is now being focused on the late sequelae of treatment on organ system function. A few papers have been published but available data are somewhat conflicting.
METHODS—A cross sectional study aimed at evaluating the late effects of transplantation on lung function was performed in a group of 52 young patients who were given autologous or allogeneic bone marrow transplants during childhood for haematological malignancies.
RESULTS—No patients reported chronic respiratory symptoms. The distribution of respiratory function patterns showed that only 62% of patients had respiratory function within the normal limits; 23% had a restrictive pattern and 15% had isolated transfer factor impairment. The percentage of patients with lung function abnormalities was higher in those who (1) received a bone marrow transplant after two or three complete remissions compared with those who were transplanted immediately after the first remission (54% vs 21%; p<0.02), (2) underwent allogeneic bone marrow transplantation rather than an autologous transplantation (45% vs 26%; p = 0.06), and (3) had a pulmonary infection compared with those without (56% vs 26%; p = 0.07).
CONCLUSIONS—In spite of the absence of chronic respiratory symptoms there is a high prevalence of children with late pulmonary sequelae after bone marrow transplantation. Regular testing is recommended after transplantation, in particular in subjects at higher risk of lung injuries, such as those receiving transplants after more than one remission, those receiving allogeneic transplants, and those having suffered from pulmonary infections. When lung function abnormalities become apparent, long term follow up is necessary to see whether they become clinically relevant. All patients should remain non-smokers after transplantation and should have active early and aggressive treatment for respiratory illnesses.

PMCID: PMC1745418  PMID: 10325917
9.  Immunotherapy with low-dose recombinant interleukin 2 after high-dose chemotherapy and autologous stem cell transplantation in neuroblastoma. 
British Journal of Cancer  1998;78(4):528-533.
The purpose of this study was to evaluate in a phase I-II trial whether low doses of recombinant human interleukin 2 (rHuIL-2) over a prolonged period of time are safe and effective in eradicating or controlling minimal residual disease in children with neuroblastoma given high-dose chemotherapy (HDCT) and autologous stem cell transplantation (ASCT). From January 1992 to July 1996, 17 consecutive patients, with either stage IV or relapsed neuroblastoma, were enrolled. Patients received rHuIL-2 after a median time interval (min-max) of 105 days (56-153) after HDCT and ASCT. The protocol consisted of 2 'priming' courses of rHuIL-2 at escalating doses administered intravenously at 72-h intervals, followed by 'maintenance' with 11 monthly and six bimonthly boosting 5-day courses administered subcutaneously on an outpatient basis. At April 1997, 7 out of the 17 patients had completed the treatment schedule, four had discontinued treatment because of toxicity and four because of relapse; the remaining two patients are still on treatment, having completed 15 courses. Expansion of T lymphocytes, together with an increase in both natural killer cells and in activated T lymphocytes was evidenced. After a median (min-max) follow-up time of 30 (16-64) months, 12 out of 17 patients are alive and well. Two patients relapsed and died 14 and 35 months after transplant. Three patients are alive after having relapsed at 41, 21 and 13 months. The actuarial 2-year event-free survival and overall survival are 67% and 92% respectively. Intermittent administration of low doses of rHuIL-2 given for a long period of time is well tolerated and seems capable of controlling minimal residual disease after HDCT and ASCT in children with high-risk neuroblastoma.
PMCID: PMC2063104  PMID: 9716039
10.  The role of occupation and a past history of malaria in the etiology of classic Kaposi's sarcoma: a case-control study in north-east Sardinia. 
British Journal of Cancer  1997;76(11):1518-1520.
A case-control study was performed to determine the role of rural factors including occupation and previous malaria exposure in the development of classic Kaposi's sarcoma (CKS) in a high incidence area of Europe. The occurrence of CKS association with other malignancies was also examined. The results showed that the risk of having CKS was significantly increased in subjects farming cereals, while a previous history of malaria did not influence the risk of developing CKS. A near-significant increase in associated tumours was found.
PMCID: PMC2228163  PMID: 9400951
11.  Antimicrobial activity of human pancreatic juice and its interaction with antibiotics. 
Pancreatic juice (PJ) should be a factor of variability in the antimicrobial activity of antibiotics eliminated by the pancreas during pancreatic infections. We studied its effects on the activity of antimicrobial drugs with different mechanisms of action. Samples of pure PJ were collected from 16 patients with stabilized external pancreatic fistulas. The antimicrobial activity of the juice at different concentrations (from 1.25 to 100%) alone and in combination with mezlocillin, imipenem, ceftriaxone, gentamicin, ofloxacin, and ciprofloxacin was studied by a microbiological method (continuous turbidimetric recording of bacterial growth). The human PJ showed dose-dependent antimicrobial activity that increased directly with the concentration. The activity of the antibiotics at bactericidal concentrations were not modified by the PJ, while the combination with subinhibitory concentrations produced the following variable and different effects: (i) additivity with mezlocillin, ceftriaxone, gentamicin, and ciprofloxacin and autonomy (no interaction) with imipenem and ofloxacin against Providencia rettgeri and (ii) additivity with ceftriaxone, ofloxacin, gentamicin, imipenem, and mezlocillin and autonomy with ciprofloxacin against Escherichia coli. In the presence of PJ, fluoroquinolones showed constant positive effects, while beta-lactams showed more variable antimicrobial activity. Antibiotic concentrations and PJ pharmacodynamics are the main factors determining the final effect of the interaction in vitro. These results may be useful in choosing antibiotics for the treatment of pancreatic infections when they are supplemented with the pharmacokinetic data for each drug.
PMCID: PMC163480  PMID: 8878588
12.  Toxicity in man due to stain removers containing 1,2-dichloropropane. 
There are reports that 1,2-dichloropropane, a constituent of many commercial solvents and stain removers in Italy, has caused severe liver damage and, sometimes, acute renal failure. Between 1980 and 1983 three cases of 1,2-dichloropropane intoxication (1 by ingestion, 2 by inhalation) were observed. Clinical features included severe liver damage, acute renal failure (2 patients), haemolytic anaemia, and disseminated intravascular coagulation. The most surprising features were haemolytic anaemia and disseminated intravascular coagulation which have not been reported before. The clinical picture was similar despite different modes of exposure.
PMCID: PMC1007575  PMID: 4063220
13.  ADAR2-editing activity inhibits glioblastoma growth through the modulation of the CDC14B/Skp2/p21/p27 axis 
Oncogene  2012;32(8):998-1009.
Grade IV astrocytoma or glioblastoma multiforme (GBM) is one of the most aggressive and lethal tumors affecting humans. ADAR2-mediated A-to-I RNA editing, an essential post-transcriptional modification event in brain, is impaired in GBMs and astrocytoma cell lines. However, the role of ADAR2 editing in astrocytomas remains to be defined. Here, we show that ADAR2 editing rescue in astrocytomas prevents tumor growth in vivo and modulates an important cell cycle pathway involving the Skp2/p21/p27 proteins, often altered in glioblastoma. We demonstrate that ADAR2 deaminase activity is essential to inhibit tumor growth. Indeed, we identify the phosphatase CDC14B, which acts upstream of the Skp2/p21/p27 pathway, as a novel and critical ADAR2 target gene involved in glioblastoma growth. Specifically, ADAR2-mediated editing on CDC14B pre-mRNA increases its expression with a consequent reduction of the Skp2 target protein, as shown both in vitro and in vivo. We found that, compared to normal brain, both CDC14B editing and expression are progressively impaired in astrocytomas from grade I to IV, being very low in GBMs. These findings (1) demonstrate that post-transcriptional A-to-I RNA editing might be crucial for glioblastoma pathogenesis, (2) identify ADAR2-editing enzyme as a novel candidate tumor suppressor gene and (3) provide proof of principle that ADAR2 or its substrates may represent a suitable target(s) for possible novel, more effective and less toxic approaches to the treatment of GBMs.
PMCID: PMC3579159  PMID: 22525274
RNA editing; ADAR2; GBM; cell cycle; CDC14B

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