17q12 microduplications: a challenge for clinicians.

In the recent years, some cases of 17q12 deletions and duplications have been reported, but the clinical impact of these imbalances is still to be fully elucidated. In particular, 17q12 duplications elude syndrome classification, since they are associated with a wide phenotypic spectrum, ranging from very mild to quite severe phenotypes. Here, two unrelated patients with the same 1.2 Mb microduplication of 17q12 are reported. Comparing these patients' phenotype with those previously published, it emerges that the more patients reported, the more difficult is finding common characteristics, even in presence of exactly the same genetic anomaly. The role of the genes duplicated in this region and the impact of this chromosomal imbalance are discussed.

Marine yeast Candida aquaetextoris S527 as a potential immunostimulant in black tiger shrimp Penaeus monodon.

A marine yeast Candida aquaetextoris S527 as a source of immunostimulant in Penaeus monodon was studied. Yeast diet was prepared by incorporating 10% C. aquaetextoris S527 biomass into a standard shrimp diet and administered in P. monodon at different frequencies (daily, once in three days, once in seven days and once in ten days) followed by challenge with white spot syndrome virus (WSSV). Immune parameters such as total protein, total hemocyte count, pro-phenoloxidase, nitroblue tetrazolium reduction, alkaline phosphatase activity and acid phosphatase activity were tested. Expression profile of antimicrobial peptide (AMP) genes viz., anti-lipopolysaccharide factor (ALF), crustin-1, crustin-2, crustin-3, penaeidin-3 and penaeidin-5; immune genes viz., alpha-2-macroglobulin (α-2-M), astakine, peroxinectin, prophenol oxidase (proPO) and transglutaminase, and WSSV genes viz., DNA polymerase, endonuclease, protein kinase, immediate early gene, latency related gene, ribonucleotide reductase, thymidine kinase and VP28 were analyzed. The study demonstrated that marine yeast diet administered once every seven days conferred better protection to P. monodon against WSSV infection, supported by the hematological and immune gene expression profiles analyzed.

Nasal nitric oxide and nitric oxide synthase expression in primary ciliary dyskinesia.

No study has evaluated the correlation between different expression of nitric oxide synthase (NOS) isoforms in nasal epithelial cells and nasal NO (nNO) level in primary ciliary dyskinesia (PCD). Gene expression of endothelial (NOS3) and inducible NOS (NOS2) and their correlation with nNO level, ciliary function and morphology were studied in patients with PCD or secondary ciliary dyskinesia (SCD). NOS3 gene polymorphisms were studied in blood leukocytes. A total of 212 subjects were studied (48 with PCD, 161 with SCD and three normal subjects). nNO level correlated with mean ciliary beat frequency (p = 0.044; r = 0.174). The lower the nNO level the higher was the percentage of immotile cilia (p<0.001; r = -0.375). A significant positive correlation between NOS2 gene expression and nNO levels was demonstrated in all children (p = 0.001; r = 0.428), and this correlation was confirmed in patients with PCD (p = 0.019; r = 0.484). NOS2 gene expression was lower in PCD than in SCD (p = 0.04). The NOS3 isoform correlated with missing central microtubules (p = 0.048; r = 0.447). nNO levels were higher in PCD subjects with the NOS3 thymidine 894 mutation, and this was associated with a higher ciliary beat frequency (p = 0.045). These results demonstrate a relationship between nNO level, NOS mRNA expression and ciliary beat frequency.

Clinical and genetic findings in a series of Italian children with pure hereditary spastic paraplegia.

BACKGROUND: hereditary spastic paraplegias (HSP) are a group of neurodegenerative disorders characterized by progressive lower extremity spastic weakness. SPG7, SPG4 and SPG3A are some of the autosomal genes recently found as mutated in recessive or dominant forms of HSP in childhood. SPG31 is more often associated with a pure spastic paraplegia phenotype, but genotype-phenotype correlation is still unclear. The aims of the current study was: (i) to verify the mutational frequency of SPG4, SPG3A, SPG31 and SPG7 genes in our very-well-selected childhood sample, and (ii) to improve our knowledge about the clinical and electrophysiological HSP phenotypes and their possible correlation with a specific mutation. METHODS: a sample of 14 Italian children affected by pure HSP (mean age at diagnosis 5.9 years) was extensively investigated with electrophysiological, neuroradiological and genetic tests. RESULTS: three SPG4 mutations were identified in three patients: two novel missense mutations, both sporadic, and one multiexonic deletion already reported. A novel large deletion in SPG31 gene involving exons 2-5 was also detected in one young patient. No mutations in the SPG7 and in the SPG3A genes were found. CONCLUSIONS: our data confirm that HSP represent a heterogeneous group of genetic neurodegenerative disorders, also in sporadic or autosomal recessive early onset forms. Multiplex Ligation-dependent Probe Amplification-based mutation screening for SPG4 and SPG31 genes would be added to sequencing-based screening of SPG4, SPG31 and SPG3A genes in the routine diagnosis of HSP children.

Increasing financial burden of revision total knee arthroplasty.

We reviewed the peri-operative and financial data of patients who underwent revision total knee arthroplasty in our institution between 1997 and 2006. The aims were to compare difference in cost between aseptic and septic cases and to identify the sources of preventable cost increase in revision knee procedure. The study group comprised 117 women (65%) and 62 men (35%). The median age of patients decreased from 73 years (37-83 years) in 1997-2001 to 70 years (15-91 years) in 2002-2006, a decline of 4% (P < 0.05). The mean ASA scores also dropped from 3 to 2 between the two periods. Despite this, the mean total cost of revision knee procedure continued to increase. Patients undergoing revision arthroplasty because of infection had much higher (P = 0.0001) cost compared to their aseptic counterpart. Increase in the costs of investigations (P < 0.05) and implant (P < 0.05) was the major contributing factors. The cost of implants increased by 32-35% (P < 0.05) depending on implant selection. Changing demographics will increase the requirement for this surgery and thus increase its overall cost to society. Cost increases associated with unnecessary investigations, prolonged hospital stay and use of expensive implants should be avoided.

PS-341 (Bortezomib) inhibits proliferation and induces apoptosis of megakaryoblastic MO7-e cells.

PS-341 (Bortezomib) is a dipeptide boronic acid proteasome inhibitor with antitumor activity that induces apoptosis in different human cancer cell lines. We investigated effects of PS-341 (Bortezomib) on cell proliferation, cell cycle progression, induction of apoptosis and differentiation in a megakaryoblastic (MO7-e) cell line. PS-341 was able to retain NF-kappaB in the cytoplasm and inhibit cell growth (IC(50)=22.5 nM), in a dose/time-dependent way. This anti-proliferative activity resulted to be lineage-specific, because other leukemic cell lines (KG1a, K562/R7, HL60/DNR) were unaffected by the PS-341 treatment. Moreover, PS-341 in MO7-e induced a significant pro-apoptotic effect from 10 nM concentration (40% versus 12% in the control, p<0.05). On the other hand, at lower concentration (5 nM), Bortezomib blocked cell cycle in the G2 phase. Finally, this compound was able to down-regulate WT1 expression. No significant effects on cell differentiation were found. Because a spontaneous NF-kappaB activation has been reported in megakaryocytes from patients affected by myeloproliferative disorders, Bortezomib would so be an attractive therapeutic tool for these malignancies, including essential thrombocythemia or idiopathic myelofibrosis. Preliminary data show an inhibiting activity of Bortezomib in the megakaryocytic colonies formation. Finally, also down-regulation of the WT1 gene Bortezomib-driven could be relevant, because of the role that this gene would play in the pathogenesis of acute and chronic myeloproliferative disorders.

Molecular characterization of 6 unrelated Italian patients with 5alpha-reductase type 2 deficiency.

Steroid 5alpha-reductase (5alphaR) deficiency (OMIM number #264600) is a rare 46,XY disorder of sex differentiation caused by mutations in the 5alphaR type 2 gene (SRD5A2) resulting in dihydrotestosterone deficiency during fetal development. We report on the analysis of the SRD5A2 gene in 6 unrelated 46,XY Italian patients with external genitalia morphology ranging from predominantly female to nearly completely male. Three subjects were seen and assessed at birth, 1 patient was referred to us before puberty, and 2 at postpubertal age. Six different causative mutations (5 missense and 1 nonsense) and a rare polymorphism were identified. Four patients presented homozygous single-base substitutions. These SRD5A2 mutations were located in exon 2 (variant Cys133Gly), exon 4 (Gly196Ser and Ala207Asp) and exon 5 (Tyr235Phe). A fifth subject was a compound heterozygote who carried a nonsense mutation in exon 1 (Trp53X) and a second SRD5A2 alteration in exon 5 (Tyr235Phe). The final patient presented a mutation in only 1 allele (Gly34Trp) together with the Ala49Thr variant. The molecular characterization of these patients made it possible to identify novel mutations and to confirm, before gender assignment or any surgical approach, the suspected 5alphaR deficiency in 2 newborns, 1 of whom had inconclusive hormonal data. 5alphaR deficiency in subjects without parental consanguinity and the presence of compound heterozygotic patients suggest that SRD5A2 mutations carrier frequency may be higher than previously thought.

Alterations of Fas (APO-1/CD 95) gene and its relationship with p53 in non small cell lung cancer.

The Fas (APO-1/CD95) system regulates a number of physiological and pathological processes of cell death. The ligand for Fas induces apoptosis by interacting with a transmembrane cell surface Fas receptor. The key role of the Fas system has been studied mostly in the immune system, but Fas mutations, one of the possible mechanisms for resistance to apoptosis signaling, may be involved in the pathogenesis of non-lymphoid malignancies as well. To better understand the potential involvement of Fas system in non-small cell lung cancer (NSCLC) we evaluated Fas and Fas-ligand mRNA expression by polymerase chain reaction in 102 tumor samples and in 44 normal surrounding tissues. Although over 60% of the human NSCLC analysed expressed both genes, they seem to be unable to induce apoptosis in vivo by autocrine suicide. In this regard, we investigated in 79 cases, the promoter and the entire coding region of the Fas gene by polymerase chain reaction, single strand conformation polymorphism and DNA sequencing for detecting putative alterations. Sixteen tumors (20.25 %) were found to have Fas alterations, in promoter and/or exon region. In all cases samples carried heterozygous alterations and mostly showed simultaneous mutations of p53 gene. Moreover, the quantitative analysis of Fas mRNA expression showed high levels of Fas messenger associated with p53 wild-type status alone. Taken together, these findings point to an involvement of Fas/Fas-ligand system in the development of NSCLC, suggesting that the loss of its apoptotic function might be linked to p53 alterations which contribute to the self-maintenance of cancer cells.

The effect of cytokines, including IL4, IL7, stem cell factor, insulin-like growth factor on childhood acute lymphoblastic leukemia.

We have investigated the effects of some interleukins, such as interleukin (IL) 4, IL7, stem cell factor (SCF) and insulin-like growth factor (IGF-1), known to be involved in human lymphopoiesis, on proliferation, clonal growth and differentiation of cells from two acute lymphoblastic leukemia (ALL) derived pre-B cell lines, that is, Nalm 1, Nalm 6 and purified blasts from 37 childhood ALL. IL4 did not display any promoting activity, an inhibitory effect being observed in two patients. IL7 showed an heterogeneous responsiveness, not related to immunophenotype or cytogenetic features, proliferation and clonal growth being observed in a minority of ALL. In other patients no or even inhibitory effects on proliferation were observed. In one case this inhibition of DNA synthesis was accompanied by maturation of the cells, as demonstrated by the induced expression of surface immunoglobulins (slg); other IL7 treated samples failed to express slg, but showed a decreased expression of terminal deoxynucleotidyl transferase and cALL antigen, suggesting that the cells have a potential of limited maturation by IL7. SCF, known to synergize with IL7 in the most primitive stages of normal B cell development, did not enhance the IL7 response in B cell precursor ALL. Finally IGF-1 failed to induce a proliferative response and clonal growth in BCP ALL either alone or in combination with IL7.

Identification of Fas (APO-1/CD95) and p53 gene mutations in non-small cell lung cancer.

Fas (APO-1/CD95) is a broadly expressed death receptor involved in a series of physiological and pathological apoptotic processes. One of the possible mechanisms for resistance to apoptosis signaling in the immune system as well as in the pathogenesis of non-lymphoid malignancies is the presence of Fas mutations within the entire gene. We investigated, in 79 non-small cell lung cancer (NSCLC) samples, the promoter and the entire coding region of the Fas gene by polymerase chain reaction, single strand conformation polymorphism and DNA sequencing in order to detect putative alterations. Sixteen of 79 tumor samples (20.2%) were found to have Fas alterations, either in promoter or exon region. Since the loss of Fas apoptotic function might be linked to p53 alterations, which are often involved in the development of NSCLC, we analyzed p53 status in 40 of the 79 NSCLC samples. p53 mutations were found to be more frequently present than Fas gene alterations (25 out of 40 cases, 62.5%). These data increase the knowledge regarding mutations of apoptosis-genes involved in the pathogenesis of NSCLC, and give benefits for the clinical management of this type of tumor.

Correlation between cytogenetic and histopathological findings in 65 human meningiomas.

The correlations between cytogenetic and histopathological findings were analyzed in 65 cases of human meningiomas. Clonal chromosome abnormalities were present in 28 cases (43%). The overall female/male ratio was 1.7, but it was 1.1 in the group of 28 cases with chromosomal abnormalities. Abnormalities of chromosome 22 as sole abnormality predominate in the female patients. The mean age of patients with normal karyotype was significantly lower (50.7 years) than that of patients with chromosome changes (57.3 years). The tumor origin was predominantly at the base in the patients with normal karyotype but different at the convexity, falx cerebri, and spinal cord. The five abnormal cases from the spinal cord all showed involvement of chromosome 22. The proportion of chromosome anomalies was different in the various histological types, and a significant difference was found between the meningotheliomatous (23%) and psammomatous (58%) types. The cytogenetically abnormal cases of the psammomatous type all showed involvement of chromosome 22. In three patients with multiple meningiomas, we found different karyotypes in the different tumors of the same patient, which may indicate a multifocal origin of the tumors.

Clonal chromosome changes in renal carcinoma do not correlate with clinical stages and histopathologic grades.

We analyzed the correlations between chromosome abnormalities and clinical and histopathologic characteristics in 77 cases of renal cell carcinoma (RCC). Chromosome changes such as +5,+7,+8,+10,+18,+X,+Y, and -Y have been excluded from the analysis because they also occur in nonneoplastic kidney tissue and cytogenetic analysis indicates that these anomalies are not involved in tumor progression. The most frequent specific chromosome abnormalities in this sample were 3p rearrangements, trisomy 17, and hyperdiploidy and were not related to tumor stage or grade or to development of distant metastases.

Graft-versus-leukemia effects after allogeneic bone marrow transplantation are active also in the presence of clones with chromosomal anomalies in addition to the Ph chromosome.

Two male patients with Philadelphia-chromosome (Ph+) chronic myelogenous leukemia (CML) underwent allogeneic bone marrow transplantation (ABMT) in the first chronic phase after busulfan treatment. In both cases, the donor was a sister, and engrafting was demonstrated by chromosome analyses which showed only donor cells in the BM. Cytogenetic relapse occurred 29 and 30 months after ABMT, respectively, when host cells reappeared: in both cases, the Ph and additional anomalies typical of the blastic phase of CML were evident. We then monitored the chromosome picture for 52 and 39 months, respectively: no striking evolution occurred, and cells with the Ph and additional anomalies persisted together with donor cells, which were a minority in the first patient and a great majority in the second throughout the observation period. A clinical relapse was observed in the first patient, but the disease never progressed to a blastic phase, whereas the second patient has not relapsed 7 years after ABMT. We reviewed data from the literature on cytogenetic relapse after ABMT in CML without clinical relapse, especially the 12 patients in whom cytogenetic relapse included chromosome anomalies in addition to the Ph, as in our patients. We suggest that graft-versus-leukemia (GVL) reactions in such patients are able to arrest progression of the leukemic blastic clone and prevent a possible relapse in blastic phase.

Recessive cancer genes in meningiomas? An analysis of 31 cases.

Cytogenetic studies on 31 human meningiomas revealed clonal abnormalities in 14 of them. Monosomy 22 was present in three cases as the only abnormality, and in five it was associated with monosomy 18, monosomy 14, loss of X, loss of Y, and trisomy 20, respectively. We found a number of rearrangements involving chromosome #22: an i psu dic(22)(pter----q11::q11----pter) in two cases and a t(18;22)(q12;q11) in another case. Two cases showed a complex translocation involving #7 and #14: t(2;7;14)(q23;q36;q22) and t(1;7;14)(q25;q32;q22), respectively. Other clonal chromosome abnormalities were del(1p) (present in two cases); der(9)t(9;?)(q34;?); der(7)t(7;?)(q31;?); der(22)t(22;?)(q11;?); and a 9p+ chromosome. The relevance for the pathogenesis of human meningiomas of these chromosome anomalies is also discussed with reference to the previous literature. The possible involvement of recessive cancer genes present on the long arm of chromosome #22 is also discussed.

Central precocious puberty in 48,XXYY Klinefelter syndrome variant.

We report the first case of central precocious puberty in a patient with 48,XXYY Klinefelter syndrome variant. We also report clinical characteristics, growth pattern, endocrine data and pathological testicular findings. The patient did not receive medical care for his precocious pubertal development, because of adequate height prognosis, and reached normal height for both his target height and Klinefelter patients. Since precocious puberty seems to occur in Klinefelter syndrome and its variants, we advise karyotype analysis in boys with mental retardation, gynecomastia, small testes and precocious onset of puberty.

Chronic lymphocytic leukemia (B-cell) in the course of polycythemia vera. Description of a case with an unusual chromosomic anomaly.

The present paper describes the case of a patient who developed a B-cell chronic lymphocytic leukemia (B-CLL) 15 months after the diagnosis of polycythemia vera, which had been treated only with phlebotomies. In spite of lymphocytosis and the clinical signs and symptoms of leukemia, the patient exhibited at the same time presumptive elements of polycythemia (high LAP index levels, a high number of neutrophils). Cytogenetic investigations, carried out after the appearance of B-CLL, revealed the presence of an unusual abnormality (18 p+) both in bone marrow not stimulated by mitogens and in PWM-stimulated circulating lymphocytes. This case, which is the ninth of its kind described in the literature, offers some interesting observations about the association between myeloproliferative and lymphoproliferative syndromes.

Myocardial stunning after resuscitation from cardiac arrest following spinal anaesthesia.

Cardiac arrest associated with spinal anaesthesia has been well researched. Myocardial stunning after successful resuscitation from cardiac arrest is seen in up to 2/3(rd) of in-hospital cardiac arrests. Myocardial stunning after resuscitation from cardiac arrest associated with spinal anaesthesia has probably not been reported earlier. Our case, an ASA physical status I lady, posted for tubal reanastomosis surgery developed bradycardia followed by asystole, approximately 5 minutes after giving subarachnoid block. Return of spontaneous circulation (ROSC) was achieved within 2 minutes with cardiopulmonary resuscitation (CPR) and defibrillation for pulseless ventricular tachycardia. Patient developed delayed pulmonary oedema, which was probably due to myocardial stunning. In the present case, inadequate preloading could have precipitated bradycardia progressing to cardiac arrest which, after resuscitation led to reversible myocardial dysfunction. We conclude that early vasopressor infusion, titrated fluids and echocardiography should be considered in immediate post cardiac arrest phase following spinal anaesthesia.

FOLFOXIRI plus bevacizumab as first-line treatment in BRAF mutant metastatic colorectal cancer.

BACKGROUND: BRAF V600E mutation plays a negative prognostic role in metastatic colorectal cancer (mCRC), leading to a median Progression Free Survival (PFS) of 4-6months with first-line conventional treatments. Our group recently reported in a retrospective exploratory analysis of a phase II trial that FOLFOXIRI (5-FU/LV+Oxaliplatin+Irinotecan) plus bevacizumab might allow to achieve remarkable results in terms of PFS and Overall Survival (OS) also in this poor-prognosis subgroup. The aim of this work was to prospectively validate our retrospective finding. PATIENTS AND METHODS: This phase II trial was designed to detect an increase in 6month-Progression Free Rate (6m-PFR) from 45% to 80% in a population of BRAF mutant mCRC patients treated with first-line FOLFOXIRI plus bevacizumab. Secondary end-points were PFS, OS, response rate (RR) and the analysis of outcome parameters in the pooled population consisting of both retrospectively and prospectively included patients. This trial is registered with ClinicalTrials.gov, number NCT01437618. RESULTS: Two-hundred-fourteen potentially eligible mCRC patients were screened for BRAF mutational status. Fifteen BRAF mutant patients (7%) were included in the validation cohort. At a median follow up of 25.7months, 6m-PFR was 73%. Median PFS and OS were 9.2 and 24.1months, respectively. In the pooled population, at a median follow up of 40.4months, 6m-PFR was 84%. Median PFS and OS were 11.8 and 24.1months, respectively. Overall RR and disease control rate were 72% and 88%, respectively. CONCLUSION: Lacking randomised trials in this specific molecular subgroup, FOLFOXIRI plus bevacizumab might be a reasonable option for the first-line treatment of BRAF mutant mCRC patients.