Nicola Stefano Fracchiolla

Central Venous Catheter-related Complications in Patients with Hematological Malignancies: A Retrospective Analysis of Risk Factors and Prophylactic Measures

Abstract We retrospectively analyzed the incidence of thrombotic and infectious complications in relation with the use of central venous catheters (CVCs), in a series of patients with hematological malignancies and low platelet and leucocyte counts. Patients and Methods: 126 patients with hematological malignancies were analyzed. A total of 207 CVCs were implanted: 137 centrally (CICCs) and 70 peripherally (PICCs). The median duration of the CVCs was 19 days for a total of 4051 catheter-days. Antithrombotic prophylaxis was unfractionated heparin (UFH), 2,500 IU daily by 24 h continuous infusion in 169 CVCs, low molecular weight heparin (LMWH), 3,800 IU daily by single bolus intravenous injection (i.v.) in 21 and warfarin in one. No prophylaxis was given in 16 CVCs. Thrombotic complications developed in 15.5% of the CVCs (7.9 events/1000 catheter days), and the frequency of infectious complications was 10.6% (5.2 events/1000 catheter days). On multivariate analysis thromboses were more frequent and earlier with PICCs than CICCs (p = 0.0001). and in patients on UFH (16.6%) than in LMWH prophylaxis (4.7%), but the last difference was not statistically significant. In conclusions the incidence of thrombotic complications in our series was comparable to that observed in non-thrombocytopenic patients and was significantly higher in those carrying PICC than CICC (p = 0.0001). There were fewer thrombotic events in the patients receiving i.v. LMWH prophylaxis than in those receiving i.v. UFH. The use of anticoagulants was safe and not associated with hemorrhages.

Molecular and immunohistochemical analysis of the bcl-1/cyclin D1 gene in laryngeal squamous cell carcinomas: Correlation of protein expression with lymph node metastases and advanced clinical stage

The molecular pathogenesis of laryngeal squamous cell carcinomas (LSCCs) is still only partially understood, although genetic alterations affecting various protooncogenes or tumor suppressor genes have often been detected.

The European Consensus on grading of bone marrow fibrosis allows a better prognostication of patients with primary myelofibrosis

We investigated the relationship between the International Prognostic Scoring System of the International Working Group for Myelofibrosis Research and Treatment and the European Consensus on grading of bone marrow fibrosis (MF) in patients with primary myelofibrosis. We compared them in 196 consecutive primary myelofibrosis patients (median follow-up 45.7 months; range 7.4–159). International Prognostic Scoring System classified 42 cases as low risk, 73 as intermediate risk-1, 69 as intermediate risk-2, and 12 as high risk; European Consensus on grading of bone marrow fibrosis classified 83 cases as MF-0, 58 as MF-1, 41 as MF-2, and 14 as MF-3. By the time of the analysis, 30 patients (15.3%) had died. Overall median survival was 3.8 years (95% confidence interval: 3.3–4.3). Multivariate analysis confirmed that both scoring systems independently predicted survival, with hazard ratios similar to those provided by univariate analysis (respectively, 2.40 (95% confidence interval: 1.47–3.91) and 2.58 (95% confidence interval: 1.72–3.89) but the likelihood ratio increased from 19.6 of the International Prognostic Scoring System or 29.0 of the European Consensus on grading of bone MF to 42.3 when both measures were considered together. Analysis of the overall survival curves documented that patients classified as having the most favourable rate with both prognostic scores (ie low risk and MF-0) survive longer than those with only one favourable score (ie low risk but MF >0 or MF-0, but International Prognostic Scoring System >low risk). In contrast, those patients classified as having the most unfavourable rate for both scores (high risk and MF-3) have a shorter survival than those with only one unfavourable score (ie high risk but MF<3 or MF-3, but International Prognostic Scoring System <high risk). In conclusion, our analysis suggests that better prognostication can be achieved in primary myelofibrosis patients when both systems are used together.

Analysis of p53 and ras Gene Mutations in Endometriosis

No activating mutations in codons 12, 13 and 61 of ras genes nor inactivating mutations in exons 5-9 of the p53 tumor suppressor gene were detected by polymerase chain reaction and single-strand conformation polymorphism methods in either eutopic or ectopic endometrium from 10 women with severe endometriosis.

Endothelial precursors and mature endothelial cells are increased in the peripheral blood of myelodysplastic syndromes

Increased angiogenesis has been demonstrated to be a significant prognostic factor in many solid tumors. In the oncohematological setting, it has been associated with myelodysplastic syndromes (MDS), chronic myeloid leukemia, acute lymphoid, and myeloid leukemias. Recently, increased circulating endothelial cells (CECs) have been associated with breast cancer and non-Hodgkin lymphoma (NHL). Based on these premises we analysed total and activated CECs, and endothelial precursors (CEPs) in 50 MDS patients and 20 healthy donors. CECs and CEPs were quantified by flow cytometry. CEC levels were compared with bone marrow (BM) microvessel density (MVD). In addition, some angiogenic factors, namely vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and soluble VEGF-Receptor2 (VEGFR2), were tested in the sera from 25 MDS patients. Total, activated CECs and CEPs were significantly increased in MDS when compared to control group (p<0.0001); whereas in the MDS cases no association was found with French--American--British (FAB), International Prognostic Scoring System (IPSS) subtypes or survival. Patients with higher CECs also showed higher MVD. Among the cytokines analysed, sVEGFR2 was significantly higher in the lower IPSS risk classes, while the levels of bFGF directly correlated with total and activated CECs. Taken together these data strengthen the hypothesis of a possible role of angiogenesis in MDS pathogenesis.

Prothrombotic Gene Mutations in Patients with Sudden Sensorineural Hearing Loss and Cardiovascular Thrombotic Disease

Objectives: Impaired cochlear perfusion seems to be an important event in sudden sensorineural hearing loss. Prothrombotic gene mutations have been related to vascular disorders and sudden hearing loss. We assessed the prothrombotic risk in 10 patients with sudden sensorineural hearing loss who had previously experienced cardiovascular events to support its vascular pathogenesis. Methods: Ten patients underwent hematologic tests (MTHFR C677T/A1298C, prothrombin G20210A, platelet GlyIIIaA1/A2, and V Leiden G1691A genotyping; fibrinogenemia; cholesterolemia; homocysteinemia; folatemia). The results were compared with those of 100 previously investigated patients with sudden hearing loss alone and those of 200 healthy controls. DNA was isolated from peripheral blood leukocytes, and the gene mutations were investigated by polymerase chain reaction and a LightCycler DNA analyzer. Results: Two patients had 2 mutant alleles, 6 had 3, and 2 had 4. The mean homocysteine, cholesterol, and fibrinogen levels were above the upper limit of normal; the mean folate levels were slightly above the lower limit of normal. Multiple mutations were more frequent in the patient group than in the previously analyzed patients and healthy controls. Conclusions: The association between inherited and acquired prothrombotic factors in patients with sudden sensorineural hearing loss and thrombotic diseases in other sites suggests that a multifactorial mechanism may underlie microvascular cochlear impairment. Hematologic investigation, including MTHFR, prothrombin, platelet, and V Leiden genotyping, may help to detect patients at potential risk of recurrent hearing loss and multiple microvascular diseases, and could be usefully performed in otherwise idiopathic sudden sensorineural hearing loss.

The Involvement of the Candidate Proto-Oncogene NFKB2/lyt-10 in Lymphoid Malignancies

NF-kappa B transcription factors regulate the expression of a variety of genes involved in immune responses and cell growth. In higher vertebrates, the NF-kappa B family encompasses five distinct members. Three NF-kappa B proteins, p65/RelA, RelB, and c-rel/Rel, have high transactivating potential in addition to their DNA binding activity. Two subunits, NF-kappa B1p50 and NF-kappa B2p52, coded respectively by the NFKB1 and NFKB2 genes, may only have DNA binding activity. Moreover, p50 and p52 subunits are translated as precursors, respectively p105 and p100, which can be processed into the mature active forms by the removal of their carboxy-terminal ankyrin domain. The five proteins share a homologous amino-terminal domain (rel domain) involved in DNA binding, dimerization, nuclear transport, and binding of regulatory subunits. All members form homo- and heterodimeric complexes with different DNA binding specificity and transactivating potential. Structural alterations of some members of the NF-kappa B gene family have been observed in lymphoid malignancies. In particular, the NFKB2 gene, localized on chromosome 10q24, represents a candidate proto-oncogene, since it has been found rearranged in certain types of lymphoma and more commonly in cutaneous lymphoma. Molecular analysis indicated that these rearrangements may occur as a consequence of chromosomal translocations or small internal chromosomal deletions. Rearrangements cluster within the carboxy-terminal ankyrin domain of the NFKB2 gene leading to the production of carboxy-terminally truncated proteins which, in some cases, are fused to heterologous protein domains. Experimental data showed that these abnormal proteins are constitutively localized in the nucleus, have lost the transcriptional repressor functions typical of normal NF-kappa B2p52 and may be capable of transactivation activity. These findings suggest that abnormal NFKB2 proteins may contribute to lymphomagenesis by altering the NF-kappa B system, both quantitatively and qualitatively, and leading to the activation of specific subsets of kappa B-controlled genes.

Effect of inositol hexaphosphate (IP 6 ) on human normal and leukaemic haematopoietic cells

Summary. Inositol hexaphosphate (IP6), a naturally polyphosphorylated carbohydrate, has been reported to have significant in vivo and in vitro anticancer activity against numerous tumours, such as colon, prostate, breast, liver and rhabdomyosarcomas. To confirm this activity in haematological malignancies and to characterize some of the mechanisms of IP6 action, we analysed its effects on human leukaemic cell lines and fresh chronic myelogenous leukaemia (CML) progenitor cells using a combined cellular and molecular approach. IP6 had a dose‐dependent cytotoxic effect on all of the evaluated cell lines, with accumulation in the G2M phase in two out of five cell lines tested. At the molecular level, cDNA microarray analysis after IP6 exposure showed an extensive downmodulation of genes involved in transcription and cell cycle regulation and a coherent upregulation of cell cycle inhibitors. Furthermore, IP6 treatment of fresh leukaemic samples of bone marrow CD34+ CML progenitor cells significantly inhibited granulocyte–macrophage colony‐forming unit (CFU‐GM) formation (P = 0·0062) in comparison to normal bone marrow specimens, which were not affected. No differentiating effect on HL60 cells was observed. Taken together, our results confirm the antiproliferative activity of IP6 and suggest that it may have a specific antitumour effect also in chronic myeloid leukaemias, via active gene modulation.

Identification of a tumor-associated mutant form of the NF-κB RelA gene with reduced DNA-binding and transactivating activities

Alterations of NF-κB family members have been found to be associated with various forms of lymphoid malignancies. In order to determine whether alterations of the RelA gene are involved in lymphomagenesis, we analysed a large and representative panel (200 cases) of such tumors. Southern blot analysis did not reveal any rearrangements or locus amplification, suggesting that structural alterations of the RelA gene may represent rare events in lymphoid neoplasia. By means of PCR-SSCP analysis, we were able to identify a single point mutation leading to amino acid substitution (codon 494, Glu-Asp) in the transactivating (TA) domain in one case of multiple myeloma. The mutated allele was expressed in the pathological bone marrow sample but not in the peripheral blood cells of the patient. We demonstrate that the RelA protein carrying this specific mutation (called RelA494D) has less transactivating ability than the normal RelA protein. Interestingly, the mutated protein has a lower affinity for κB binding sites both as a homodimer or in association with the NFKB1/p50 subunit. Transfection experiments using a Gal4-RelA494D fusion protein indicated that the mutation does not alter the intrinsic transactivating ability of the TA domain of RelA. Furthermore, in vitro translated RelA494D is able to dimerize efficiently with other NF-κB members, such as p50, cREL and IκBα. Our data therefore suggest that this mutation may alter the specific structural conformation needed for the DNA interaction of RelA, and provide insights into the amino acid sequences involved in mediating the biological activities of RelA.

Poor prognosis in non-villous splenic marginal zone cell lymphoma is associated with p53 mutations

We have recently reported a series of 15 non‐villous splenic marginal zone lymphoma patients, six of whom showed p53 mutations (40%). This molecular alteration did not correlate with any particular clinico‐pathologic feature at diagnosis. After a median follow‐up of 56 months, four cases evolved into aggressive fatal non‐Hodgkins lymphoma (NHL) and two had refractory progressive disease; interestingly, p53 mutations were demonstrated in five of these patients at diagnosis. As the patients with wild‐type p53 presented responsive or indolent disease, this genetic alteration may be an early marker of aggressive transformation or refractoriness. p53 evaluation at diagnosis could be advisable in this particular subset of NHL.