Hepatitis C Virus Positive Patients on the Waiting List for Liver Transplantation: Turnover and Characteristics of the Population on the Eve of the Therapeutic Revolution With Direct-Acting Antivirals.

INTRODUCTION: Antivirals direct acting (DAA) for hepatitis C virus (HCV) have brought a revolution in the field of transplantation. It is likely to think that in the future patients on the waiting list for liver transplantation (LT) will no longer be registered for HCV-related cirrhosis but for liver disease from other causes. On the eve of this change, we show a snapshot of the Italian waiting list for LT. METHODS: From October 1, 2012 to September 30, 2013, we estimated the total number of patients on the liver waiting list as intention to treat (ITT), the number of incident cases, and the delistings, particularly in the HCV positive (HCV+) population. Gender, median age, etiology and prognosis of liver disease, presence of hepatocellular carcinoma (HCC), reason for delisting, mean waiting time for LT, and rate of death on waiting list were evaluated. RESULTS: In the time period, there were 517 new patients who were HCV+ (median age, 53 years): 255 (49.3%) mono-infected with HCV, 236 (45.7%) co-infected with HCV and hepatitis B virus (HBV), 11 (2.1%) co-infected with HCV and human immunodeficiency virus (HIV), and 15 (2.9%) co-infected with HCV, HBV, and HIV. The median model for end-stage liver disease (MELD) score at listing was 17 and HCC was present in 206 (39.8%) cases. HCV+ patients delisted were 442 (61.9%), 355 (80.3%) for LT. The mean waiting time to transplantation was 1.9 months; the percentage of death was 7.6%. CONCLUSIONS: This snapshot of the waiting list for LT in the year before the advent of DAA drugs will allow us to assess whether and how they will change the waiting list for LT when we start to look at the impact of new therapies on the waiting list.

A novel oncogenic BTK isoform is overexpressed in colon cancers and required for RAS-mediated transformation.

Bruton's tyrosine kinase (BTK) is essential for B-cell proliferation/differentiation and it is generally believed that its expression and function are limited to bone marrow-derived cells. Here, we report the identification and characterization of p65BTK, a novel isoform abundantly expressed in colon carcinoma cell lines and tumour tissue samples. p65BTK protein is expressed, through heterogeneous nuclear ribonucleoprotein K (hnRNPK)-dependent and internal ribosome entry site-driven translation, from a transcript containing an alternative first exon in the 5'-untranslated region, and is post-transcriptionally regulated, via hnRNPK, by the mitogen-activated protein kinase (MAPK) pathway. p65BTK is endowed with strong transforming activity that depends on active signal-regulated protein kinases-1/2 (ERK1/2) and its inhibition abolishes RAS transforming activity. Accordingly, p65BTK overexpression in colon cancer tissues correlates with ERK1/2 activation. Moreover, p65BTK inhibition affects growth and survival of colon cancer cells. Our data reveal that BTK, via p65BTK expression, is a novel and powerful oncogene acting downstream of the RAS/MAPK pathway and suggest that its targeting may be a promising therapeutic approach.

Mg(++)-induced endothelial cell migration: substratum selectivity and receptor-involvement.

The activation of endothelial cells during angiogenesis requires cell spreading and migration. These processes are influenced by extracellular signals such as chemoattractants from the local microenvironment. We have shown previously that transmembrane Ca++ influx is necessary for motility and cell spreading, thus we hypothesized that the extracellular divalent cations Mg++ and Ca++ may regulate human umbilical vein endothelial cell (HUVEC) spreading and act as chemoattractants. Studies demonstrated that extracellular Mg++ induced a statistically better spread phenotype when cells were plated on multiple extracellular matrix substrata; Ca++ promoted cell spreading only on vitronectin. Mg++ but not Ca++ acted as a potent chemoattractant when HUVEC migrated on gelatin- and type IV collagen- but not on vitronectin-coated filters. A checkerboard analysis of migration showed that Mg++ induces both chemokinetic and chemotactic migration peaking at 0.1 and 10 mM, respectively. An equivalent effect of oligomycin was seen on motility to Mg++ or to vascular endothelial growth factor (VEGF) in extracellular Mg(++)-free conditions, ruling out an exclusive role for Mg++ as a migration energy producer. The Mg(++)-stimulated chemotaxis was inhibited > 60% by pertussis toxin, d-erythrosphingosine, and tyrphostin B48, but unaffected by cholera toxin exposure. These data suggest that Mg(++)-induced chemotaxis may be promoted through a Gi protein-coupled receptor pathway with a requirement for protein kinase C activity and protein tyrosine phosphorylation. Thus, Mg++ may be a newly recognized receptor-mediated chemoattractant for endothelial cells.

Quantitative multiparametric immunophenotyping in acute lymphoblastic leukemia: correlation with specific genotype. I. ETV6/AML1 ALLs identification.

The t(12;21)(p13;q22) fusion gene is the most frequent genetic lesion described in precursor B cell acute lymphoblastic leukemia (ALL) of childhood occurring in a quarter of cases. This gene rearrangement is associated with a good outcome presenting a high response rate to chemotherapy. In spite of its potential clinical relevance, the t(12;21) translocation usually goes undetected with conventional cytogenetic procedures. In the present study we utilized an objective flow cytometric approach (multiparametric quantitative analysis) for the phenotypic characterization of this type of ALL. We studied a total of 74 precursor B-ALL children, including 21 t(12;21)+ and 53 t(12;21)- cases. Our results show that the t(12;21)(p13;q22)+ ALLs display a higher intensity of CD10 (P = 0.0016) and HLADR (P = 0.005) expression together with lower levels of the CD20 (P = 0.01), CD45 (P = 0.01), CD135 (P = 0.003) and CD34 (P = 0.03) antigens as compared to the t(12;21) cases. Moreover, as regards CD34 expression, we observed a more heterogeneous antigen expression within individual patients with higher coefficients of variation (median of 202 vs 88, P = 0.0001). A multi-variate analysis disclosed that with the immunophenotypic approach used identification of t(12;21)+ cases can be achieved with a sensitivity of 86% and a specificity of 100%. We conclude that childhood precursor B-ALL carrying the t(12;21) translocation display characteristic phenotypic features which could provide a rapid, simple, sensitive and specific screening method to select for those cases that should undergo confirmatory molecular analysis.

Interferon treatment of chronic hepatitis C in patients cured of pediatric malignancies.

BACKGROUND AND OBJECTIVE: Chronic hepatitis C was a frequent complication in patients treated for malignancy until the introduction of anti-HCV screening tests for blood donors. The association between chronic hepatitis C and progression to cirrhosis and hepatocellular carcinoma has been reported in about 20% and 5% of patients, respectively, within 20-30 years of infection. In adult patients, interferon has proved to be effective in decreasing the abnormal values of transaminases and the level of HCV viremia. Our purpose was to assess efficacy of and tolerance to interferon in a group of young patients who had acquired HCV infection during a period of chemotherapy. DESIGN AND METHODS: Interferon-a (IFN) was administered to 26 adolescents and young adults (13 males, age range 17-36 years; median age 24) with chronic hepatitis C, including 4 with hepatitis B virus co-infection, who had been treated for leukemia or solid tumor 5 to 19 years before joining this trial. Patients were treated with natural IFN alpha at a dose of 4 MU/m(2) thrice weekly for 12 months and followed up for another 6 months thereafter. RESULTS: Nine patients stopped treatment during the first 6 months because of side effects (2 cases) or lack of response. At the end of the trial, 8 (31%) cases had responded, with alanine amino-transferase normalization and clearance of hepatitis C virus (HCV) RNA. A sustained response was only documented in 15% of cases, however, irrespective of any hepatitis B virus co-infection. The 2 patients with HCV genotype 2 were both responders, whereas only 8% of those with genotype 1 responded. INTERPRETATION AND CONCLUSIONS: These data show that the efficacy of IFN in this series of young patients is similar to that reported for otherwise healthy adults with hepatitis C. Patients with genotype 2 are strong candidates for IFN treatment while other therapeutic strategies should be designed for patients with HCV genotype 1.

High-dose melphalan with autologous stem-cell rescue in metastatic rhabdomyosarcoma.

PURPOSE: The European Collaborative MMT4-91 trial was conducted as a prospective nonrandomized study to evaluate the potential benefit of high-dose melphalan as consolidation of first complete remission in children with stage IV rhabdomyosarcoma. PATIENTS AND METHODS: Fifty-two patients in complete remission after six courses of chemotherapy received "megatherapy": 42 received melphalan alone, whereas 10 received melphalan in combination with etoposide, carboplatin/etoposide, or thiotepa/busulfan and etoposide. The outcome of this group of patients was compared with that observed in 44 patients who were also in complete remission after six courses of identical chemotherapy (plus surgery or radiotherapy) but went on to receive a total of up to 12 courses of conventional chemotherapy (four cycles). No differences were found between the two groups regarding clinical characteristics, chemotherapy received before complete remission, or response to chemotherapy. In particular, there was no significant difference between the groups for site of primary tumor, histologic subtype, age at presentation, presence of bone or bone marrow metastases, or number of metastases. RESULTS: The 3-year event-free survival (EFS) and overall survival (OS) rates were 29.7% and 40%, respectively, for those receiving high-dose melphalan or other multiagent high-dose regimens and 19.2% and 27.7%, respectively, for those receiving standard chemotherapy. The difference was not statistically significant (P =.3 and P =.2 for EFS and OS, respectively). There was a significant prolongation in the time from the last day of high-dose chemotherapy or the end of chemotherapy cycle 4 to the time of relapse in those receiving megatherapy (168 days for patients receiving megatherapy v 104 days for those receiving standard therapy; P =.05). CONCLUSION: The addition of a high-dose alkylating agent to consolidation therapy may have prolonged progression-free survival in this poor-risk patient group, but it did not significantly improve the ultimate outcome.

Regulation of the RhoA pathway in human endothelial cell spreading on type IV collagen: role of calcium influx.

We have shown that nonvoltage-operated Ca(2+) entry regulates human umbilical vein endothelial cell adhesion, migration, and proliferation on type IV collagen. We now demonstrate a requirement for Ca(2+) influx for activation of the RhoA pathway during endothelial cell spreading on type IV collagen. Reorganization of actin into stress fibers was complete when the cells where fully spread at 90 minutes. No actin organization into stress fibers was seen in endothelial cells plated on type I collagen, indicating a permissive effect of type IV collagen. CAI, a blocker of nonvoltage-operated Ca(2+) channels, prevented development of stress fiber formation in endothelial cells on type IV collagen. This permissive effect was augmented by Ca(2+) influx, as stimulated by 0. 5 microM thapsigargin or 0.1 microM ionomycin, yielding faster development of actin stress fibers. Ca(2+) influx and actin rearrangement in response to thapsigargin and ionomycin were abrogated by CAI. Activated, membrane-bound RhoA is a substrate for C3 exoenzyme which ADP-ribosylates and inactivates RhoA, preventing actin stress fiber formation. Pretreatment of endothelial cells with C3 exoenzyme prevented basal and thapsigargin-augmented stress fiber formation. While regulation of Ca(2+) influx did not alter RhoA translocation, it reduced in vitro ADP-ribosylation of RhoA (P(2)<0. 05), suggesting Ca(2+) influx is needed for RhoA activation during spreading on type IV collagen; no Ca(2+) regulated change in RhoA was seen in HUVECs spreading on type I collagen matrix. Blockade of Ca(2+) influx of HUVEC spread on type IV collagen also reduced tyrosine phosphorylation of p190Rho-GAP and blocked thapsigargin-enhanced binding of p190Rho-GAP to focal adhesion kinase. Thus, Ca(2+) influx is necessary for RhoA activation and for linkage of the RhoA/stress fiber cascade to the focal adhesion/focal adhesion kinase pathway during human umbilical vein endothelial cell spreading on type IV collagen.

Peripheral blood stem cell collection and transplantation in paediatric malignancies: a monocentric experience.

Thirty-seven patients underwent peripheral blood stem cell (PBSC) collection from May 1994 to May 1997. Twenty-five were males and 12 were females, the median age at collection was 11.5 years (range 1-27.4) and the median weight was 38 kg (range 9-80). As mobilising chemotherapy, cyclophosphamide, etoposide, doxorubicin and cytosine arabinoside were the drugs most frequently used in association with G-CSF for a total of 47 courses. Sixty-one aphereses were performed with a median collection of CD34+ and CFU-GM cells/kg of 3.6 x 10(6) (range 0.6-31.8) and 24.4 x 10(4) (range 0.1-1260), respectively. Minimal residual disease (MRD) was found in five of the 30 investigated aphereses. Twenty-one of the 37 patients underwent high-dose chemotherapy with autologous stem cell rescue: in seven the stem cell source was peripheral blood and bone marrow. The median duration of hospitalization was 18 days for the PBSC group and 23 days for the PBSC/ABMT group. Overall survival was 78.7% at a median follow-up of 18 months (range 2-31) and the DFS was 52% without difference depending on stem cell source. Compared to a historical group of ABMT patients, the PBSC group showed a statistical advantage in terms of neutrophils and platelet engraftment, blood and platelet requirements, and length of hospitalization. PBSC collection is a feasible procedure also in the paediatric setting providing that vascular access is adequate. As already reported, PBSC transplant results in faster engraftment and shorter hospitalization that could allow a better utilization of health financial resources. The question whether the source of stem cells could influence transplant outcome would require a prospective randomised study.

Veno-occlusive disease of the liver in children treated for Wilms tumor.

INTRODUCTION: Hepatotoxicity consistent with the clinical diagnosis of veno-occlusive disease (VOD) of the liver has been suspected after conventional anti-cancer chemotherapy in children. METHODS: To establish the incidence of hepatotoxicity and its relationship with VOD, we analyzed toxicity data obtained on 511 children affected by Wilms tumor and treated according to the SIOP-9 protocol. They all received pre- and postnephrectomy chemotherapy using dactinomycin (AD) and vincristine (VCR) +/- other drugs +/- radiotherapy according to surgical stage and histology. RESULTS: Sixty-four patients suffered at least one episode of hepatotoxicity and 41 satisfied the criteria for a clinical diagnosis of VOD. In this latter group, toxicity occurred during preoperative treatment in 15 patients and was confirmed histopathologically in 9 of the 16 liver biopsies obtained. There was a higher percentage of children aged less than 1 year at diagnosis in the VOD group than in the other patients (24% vs. 11.4%). The degree of liver damage in the younger patients seems important, as suggested by a higher increase in transaminases. VOD developed in 12% of the 68 irradiated children vs. 7% in the non-irradiated group. Statistical analysis showed an increased risk of VOD in younger patients (p < 0.001) and in those receiving radiotherapy (p < 0.001). All patients recovered after 6-180 days using supportive therapy only. CONCLUSIONS: (1) 8% of children treated according to the SIOP-9 protocol, developed hepatotoxicity consistent with VOD. Excluding patients who received radiotherapy, the incidence was 6%. These figures are much higher than in earlier reports, though different diagnostic criteria were used. (2) Chemotherapy with AD and VCR seems to be a major cause of VOD. (3) Risk factors are young age and concomitant radiotherapy. (4) VOD does not prejudice positive outcome for these patients.

Second malignant neoplasms in patients treated on SIOP Wilms tumour studies and trials 1, 2, 5, and 6.

The incidence of second malignant neoplasms (SMNs) was investigated among 1,988 patients with complete data, enrolled in the SIOP Wilms tumor trials and studies 1, 2, 5, and 6, treated between September 1971 and October 1987. By the end of 1992, eight SMNs were documented, whereas only 1.3 were expected (standardized incidence ratio [SIR] = 4.15; 95% CI = 1.79, 8.17). The risk increases in the first 10 years from diagnosis, while no apparent excess of risk is observed in the subsequent periods. This finding however is difficult to interpretdue to the low statistical power. The cumulative incidence of a second cancer observed at 15 years after Wilms tumor diagnosis was 0.65%. Six SMNs were registered in the cohort of patients treated in the SIOP studies 1, 2 and 5 (999 cases) compared to the two cases observed in the SIOP6 cohort (989 cases). If the suggested reduced incidence of second cancers between SIOP1-5 and SIOP6 patient cohorts is confirmed by longer follow-up, it might reflect changes in the treatment protocols.

Chronic hepatitis C virus infection after treatment for pediatric malignancy.

Sera of 658 patients who had completed treatment for pediatric malignancy were analyzed by a second-generation enzyme-linked immunosorbent assay and recombinant immunoblot assay test to assess the prevalence of hepatitis C virus (HCV)-seropositivity. All HCV-seropositive patients underwent detailed clinical, laboratory, virologic, and histologic study to analyze the course of HCV infection. One hundred seventeen of the 658 patients (17.8%) were positive for HCV infection markers. Among the 117 anti-HCV+ patients, 41 (35%) were also positive for markers of hepatitis B virus infection with or without delta virus infection markers, 91 (77.8%) had previously received blood product transfusions, and 25 (21.4%) showed a normal alanine aminotransferase (ALT) level during the last 5-year follow-up (11 of them never had abnormal ALT levels). The remaining 92 patients showed ALT levels higher than the upper limit of normal range. Eighty-one of 117 (70%) anti-HCV+ patients were HCV-RNA+, with genotype 1b being present in most patients (54%). In univariate analysis, no risk factor for chronic liver disease was statistically significant. In this study, the prevalence of HCV infection was high in patients who were treated for a childhood malignancy. In about 20% of anti-HCV+ patients, routes other than blood transfusions are to be considered in the epidemiology of HCV infection. After a 14-year median follow-up, chronic liver disease of anti-HCV+ positive patients did not show progression to liver failure.

Analyses of prognostic factors in a retrospective review of 92 children with ependymoma: Italian Pediatric Neuro-oncology Group.

The principal aim of this report is to present the results of multivariate analyses conducted to identify clinical prognostic factors in 92 children aged < 16 years with ependymoma (EPD) retrospectively collected in seven Italian centres. They were treated over a 16-year period (1977-1993). Treatment modalities varied. Surgery and radiotherapy (RT) was the "gold standard" management method for the majority of these children. Only in the late 1980s did some of them receive chemotherapy (CT), mainly with vincristine, lomustine (CCNU) and prednisone. The median follow-up of the entire study population is 36 months (average 43 months; range 12 to 214 months). The 10-year overall (OS) and the progression-free survival (PFS) of the study population were 55.5% (CI 41.4-69.4%) and 34.7% (CI 21.4-47.8%), respectively. Age (< 5 years; > 5 years), sex, site (infratentorial vs. supratentorial), histology (anaplastic/malignant vs. non-anaplastic/non-malignant), type of resection (complete vs. incomplete); use and fields of RT, and of CT employed were entered in a multivariate regression model to test their impact on OS and PFS. On univariate analysis, radical surgery, the use of RT and age more than 5 years at the time of diagnosis achieved statistically significant values for predicting long-term OS and PFS. Histology reached marginal statistical significance but only for PFS. When those variables were entered in a multivariate analysis only radical resection (P = 0.00142 and 0.0001) resulted a significant factor for predicting long-term OS and PFS, while the use of RT reached a marginal statistical significance, but only for PFS (P = 0.05). Children who had the tumour completely resected did significantly better than all the others who had less than a complete resection, with a 10-year OS and PFS for the two groups of patients of 69.8% (CI 53-86.5%) and 57.2% (CI 40.3-75%) and of 32.5% (CI 8.5-57.6%) and 11.1% (0-24.4%), respectively. These findings suggest that, for childhood EPD, radical resection should be pursued as much as reasonably possible. Thus, it seems justified proposing for future trials, patient stratification by entity of surgical resection.

Regulation of cell spreading during differentiation in the muscarinic M5 receptor tumor-suppressor model.

Activation of the muscarinic receptor in Chinese hamster ovary (CHO) cells results in a reversal of the malignant phenotype for which spreading into a bipolar, fibroblastic morphology is a marker. The process of morphologic change requires multiple events, including alterations in adhesions to substrates and cytoskeletal re-arrangement. In this report, we demonstrate the calcium-dependent involvement of p125FAK in this cellular shape change using an inhibitor of ligand-induced calcium influx, carboxyamido-triazole (CAI). p125FAK becomes tyrosine-phosphorylated after exposure to the agonist carbachol (CC), reaching maximal phosphorylation prior to initiation of cellular shape change at 1 hr into CC exposure (386 +/- 103%). Phosphorylation remained elevated through the shape change (4-12 hr). CHOm5 cell exposure to the Ca2+-mobilizing agents maitotoxin and ionomycin also resulted in p125FAK phosphorylation. Inhibition of Ca2+ influx with CAI, an inhibitor of ligand-induced Ca2+ influx, had little effect on CC-induced phosphorylation but partially inhibited ionomycin-mediated p125FAK phosphorylation. While the intracellular Ca2+ chelator BAPTA failed to prevent CC-induced p125FAK tyrosine phosphorylation, it inhibited phosphorylation due to ionomycin. CC induced Ca2+-independent binding of phosphorylated p125FAK selectively to the C-terminal SH2 domain of phosphatidylinositol-3'-kinase (PI3K). Further, CC, maitotoxin and ionomycin induced in vitro kinase activity of p125FAK for the exogenous substrate poly(Glu4Tyr1). Kinase activity stimulated by all 3 agonists was inhibited by preincubation with either CAI or BAPTA. Our results indicate that increasing intracellular Ca2+ can stimulate both p125FAK autophosphorylation and kinase activity; however, p125FAK phosphorylation in response to CC also may be induced through a Ca2+-independent pathway.

Control of type IV collagenase activity by components of the urokinase-plasmin system: a regulatory mechanism with cell-bound reactants.

The urokinase-type plasminogen activator (uPA) and the matrix-degrading metalloproteinases MMP-2 and MMP-9 (type IV collagenases/gelatinases) have been implicated in a variety of invasive processes, including tumor invasion, metastasis and angiogenesis. MMP-2 and MMP-9 are secreted in the form of inactive zymogens that are activated extracellularly, a fundamental process for the control of their activity. The physiological mechanism(s) of gelatinase activation are still poorly understood; their comprehension may provide tools to control cell invasion. The data reported in this paper show multiple roles of the uPA-plasmin system in the control of gelatinase activity: (i) both gelatinases are associated with the cell surface; binding of uPA and plasmin(ogen) to the cell surface results in gelatinase activation without the action of other metallo- or acid proteinases; (ii) inhibition of uPA or plasminogen binding to the cell surface blocks gelatinase activation; (iii) in soluble phase plasmin degrades both gelatinases; and (iv) gelatinase activation and degradation occur in a dose- and time-dependent manner in the presence of physiological plasminogen and uPA concentrations. Thus, the uPA-plasmin system may represent a physiological mechanism for the control of gelatinase activity.

Clinical relevance of DNA ploidy and proliferative activity in childhood rhabdomyosarcoma: a retrospective analysis of patients enrolled onto the Italian Cooperative Rhabdomyosarcoma Study RMS88.

PURPOSE: Evaluation of the possible clinical relevance of DNA ploidy and proliferative activity assessed as S-phase fraction (SPF) in childhood rhabdomyosarcoma (RMS). PATIENTS AND METHODS: We conducted a retrospective study on 59 RMS patients enrolled onto the ICS-RMS88 protocol (seven botryoid, 35 embryonal, and 17 alveolar RMS), for which formalin-fixed paraffin-embedded (FFPE) tissue was available. Nuclear suspensions for cytometric investigation were obtained using a mechanical disaggregation. Tumors were distinguished according to their DNA index (DI) value as follows: diploid (0.9 < DI < 1.1), hyperdiploid (1.1 < or = DI < 1.8 or DI > or = 2.2), and tetraploid (1.8 < or = DI < 2.2); for analysis of SPF, a cutoff value of 14% was used. RESULTS: DNA histograms were diploid in 19 (33%) cases, hyperdiploid in 29 (49%), and tetraploid in 10 (32%). One patient showed both a hyperdiploid and a tetraploid peak. The 5-year overall survival (OS) rate by ploidy status was 73% in hyperdiploid patients as compared with 33% and 25% in diploid and tetraploid patients, respectively (P = .0012). A striking difference emerged when the 5-year OS for the combined diploid and tetraploid RMS groups was compared with survival of the hyperdiploid RMS group: 30% versus 73%, respectively (P = .0006). In addition, the SPF was prognostically relevant: 5-year OS by SPF less than or greater than 14% was 70% and 36%, respectively (P = .009). Multivariate analysis confirmed the importance of DNA content (P = .0006) and SPF (P = .034) in predicting survival. CONCLUSION: These findings confirm that ploidy and SPF are important new prognostic factors that are able to identify selected groups of patients at high risk of treatment failure, even if the tumor's presentation is favorable according to standard criteria.

New anti-angiogenesis agents: review of the clinical experience with carboxyamido-triazole (CAI), thalidomide, TNP-470 and interleukin-12.

Angiogenesis was postulated to be a critical prognostic factor and therapeutic focus for malignancy more than two decades ago. Recent studies indicate quantitative assessments of microvessel count to be an independent prognostic variable for disease-free and overall survival in a wide variety of tumors, and that angiogenesis may be a feasible target against which to intervene pharmacologically. Several new and old agents have been found to have anti-angiogenic activity and have reached clinical trial. This review will focus on four agents under investigation in the US: carboxyamido-triazole (CAI), thalidomide, TNP-470 and interleukin (IL)-12. CAI, originally identified for its anti-invasive capacity, has been shown to inhibit tumor and endothelial cell proliferation by inhibition of calcium uptake. It is administered orally, is generally well tolerated, and has been shown to induce disease stabilization and occasional reductions in tumor mass. Thalidomide was shown to inhibit growth factor-induced neovessel formation, a process that can also explain its earlier devastating clinical toxicity. It is administered orally, and is currently in phase II clinical trials for prostate cancer, glioblastoma multiforme and breast cancer. TNP-470 is a fumagillin analog that has been shown in in vivo models to be a potent inhibitor of angiogenesis at concentrations that are cytostatic to endothelial cells and tumor cells. Lastly, IL-12 may exert its anti-angiogenic effects through activation of interferon-gamma to up-regulate interferon-inducible protein-10, an anti-angiogenic cytokine. Phase I clinical trials of IL-12 have shown disease stabilization in several tumor types in response to s.c. administration or using genetically engineered IL-12-expressing patient fibroblasts. These promising new agents join the matrix metalloproteinase inhibitors as important new drugs in the anti-cancer armamentarium.

Down-regulation of tumour gelatinase/inhibitor balance and preservation of tumour endothelium by an anti-metastatic ruthenium complex.

The anti-metastatic ruthenium complex Na[trans-RuCl4(DMSO)Im] was given i.p. at 22 and 44 mg/kg/day, on days 8-13 after tumour implantation, to mice carrying s.c. implants of MCa mammary carcinoma. The aim of the study was to compare the effects on lung metastasis formation with those on primary tumour cells. This investigation was based on flow cytometry analysis after propidium iodide and acridine orange staining, histology of tumour parenchyma and RT-PCR analysis for the type-IV collagenases MMP-9 and MMP-2 and their respective inhibitors TIMP-1 and TIMP-2 mRNAs. Na[trans-RuCl4(DMSO)Im] is not cytotoxic for tumour cells but has the capacity of interacting with nucleic acids, giving a general reduction of nucleic acid content as shown by a marked reduction of acridine orange staining and a tendency to a reduction of DNA polyploidy with marked reduction of 8n and 4n cell populations. Na[trans-RuCl4(DMSO)Im] also influences a proteolytic system which has the potential of degrading the basement membrane and has been related to metastatic aggressiveness: it markedly reduces, in a dose-dependent manner, MMP-2/TIMP-2 balance, but not that of MMP-9/TIMP-1. The different enzyme/inhibitor mRNA levels between untreated and treated tumours seem to be unaffected by tumour-infiltrating lymphocytes and are paralleled by the maintenance of connective tissue around blood vessels in the tumour mass. Correspondingly, lung metastasis formation is markedly reduced, to less than 10% of that seen in controls.

72-kilodalton type IV collagenase, type IV collagen, and Ki 67 antigen in serous tumors or the ovary: a clinicopathologic, immunohistochemical, and Serological study.

The immunohistochemical expression of 72-kDa type IV collagenase [matrix-metalloproteinase (MMP)-21], basement membrane component type IV collagen and proliferation-related antigen Ki 67 were investigated in 43 benign, borderline, and malignant serous tumors of the ovary. The results were compared with the histotypes of ovarian serous tumors and with their clinical behavior. Serum evaluation of MMP-2 was performed in 14 patients with cystadenocarcinoma and the data compared with that of a control group. The basement membrane (BM) was continuous in benign cystadenomas and in some borderline tumors, whereas it was discontinuous or absent in other borderline tumors, in borderline tumors with microinvasion, and cystadenocarcinomas. The percentage of MMP-2- and Ki 67-expressing cells increased from cystadenomas to borderline tumors, being the highest in malignant tumors; a frequent basal disposition of the MMP-2 cytoplasmic granules also was observed in cystadenocarcinomas. Statistical analysis demonstrated that MMP-2 expression was inversely related to BM integrity. Serum MMP-2 values did not differ from that of the control group. Cox regression analysis showed that tumor stage and grade were significant prognostic factors, whereas MMP-2 and Ki 67 immunohistochemical expression added no further significant information to the prognosis. The investigators conclude that the correlation between increasing MMP-2 expression and BM alteration gives support to the hypothesis of a direct role of the metalloproteinase in the process of destructive stromal invasion. MMP-2, type IV collagen, and Ki 67 immunodetection varied according to the histologic classification of ovarian serous tumors. However, neither these factors nor the serum evaluation of MMP-2 appear useful as prognostic predictors in this series.