Gaetano Vitelli

Increased serum levels of vascular endothelial growth factor predict risk of progression in early B-cell chronic lymphocytic leukaemia.

The present study is the first to evaluate serum levels of vascular endothelial growth factor (VEGF) in B‐cell chronic lymphocytic leukaemia (CLL). All 68 B‐cell CLL patients and 31 control subjects analysed had detectable serum levels of VEGF, with no statistically significant difference between two proups. An aberrant increase of circulating levels of VEGF was found in only 17.6% of cases. B‐cell CLL patients whose serum VEGF levels were higher than the median (i.e. 194.8 pg/ml) or 75th percentile (i.e. 288.5 pg/ml) values were more frequently at an advanced clinical stage. In contrast, no correlation with other clinico‐biological features representative of either tumour mass [bone marrow (BM) histology, peripheral blood (PB) lymphocytosis, beta‐2 microglobulin (β‐2m), LDH, interleukin‐6 (IL‐6)] or disease‐progression (DP) [lymphocyte doubling time (LDT)] was found.  Serum levels of VEGF predicted the risk of DP in early CLL. Among 41 patients in Binet stage A, progression‐free survival (PFS) was significantly shorter in those patients whose VEGF serum concentrations were above the median value. Interestingly, characteristics of stage A patients stratified according to the median value of VEGF were similar with respect to many clinico‐biological features, thus suggesting a possible independent prognostic role for such a marker. Finally, when added to the Rai subclassification, VEGF serum levels identified two groups with different PFS within stages I–II.  We conclude that increased serum levels of VEGF can be considered useful for predicting the risk of DP and add prognostic information to the Rai subclassification of stage A CLL.

Increased serum levels of matrix metalloproteinase-9 predict clinical outcome of patients with early B-cell chronic lymphocytic leukaemia.

Background and Methods: Serum levels of matrix metalloproteinase‐9 (MMP‐9) which agree with progression in solid and haematological tumours were correlated to the risk of disease progression in 62 patients with early (Binet stage A) B‐cell chronic lymphocytic leukaemia (CLL). Sera were taken at diagnosis and tested by an enzyme‐linked immunosorbent assay.

Serum angiogenin is not elevated in patients with early B‐cell chronic lymphocytic leukemia but is prognostic factor for disease progression

Abstract:  The association between angiogenin and cancer progression and poor outcome in solid tumors has been documented, but its significance in leukemias has not been evaluated. Using an ELISA technique (Quantikine Human Angiogenin Immunoassay; R&D Systems), we measured serum angiogenin levels in 77 previously untreated Binet stage A B‐cell chronic lymphocytic leukemia (CLL) patients. No difference in angiogenin serum levels could be found between patients (median: 295 ng/mL; range: 74–1700) and 15 age‐ and sex‐matched healthy controls (median: 264 ng/mL; range: 29–1835) (P = NS; Mann–Whitney test). Increased angiogenin serum level was associated with higher LDH (P = 0.03) and β2‐m (P = 0.007) concentrations. However, angiogenin did not reflect the extent of bone marrow (BM) angiogenesis as evaluated by microvessel area (P = 0.611), circulating levels of vascular endothelial growth factor (VEGF) (P = 0.873) and basic fibroblastic growth factor (FGF‐2) (P = 0.421). When the 25 patients with available data were stratified into the four major cytogenetic categories (normal karyotype, 13q as a sole aberration, 12q trisomy, 11q or 17p deletion) and aberrations were compared with angiogenin serum levels, no correlation was found (P = 0.651; Kruskall–Wallis test). A cut‐off of angiogenin serum level corresponding to median (i.e. 330 ng/mL) or higher identified later upstaging and longer progression‐free survival (PFS). The 5‐yr PFS was 51.5% for patients with angiogenin levels lower than median and 85% for patients with higher values [P = 0.03; hazard ratio (HR) = 2.86; 95% CI: 1.08–6.72]. Although in multivariate analysis only Rai substages (P = 0.00001) and peripheral blood lymphocytosis (P = 0.009) retained their prognostic significance, angiogenin could be incorporated into the Rai substages thus leading to the identification of the following risk categories: (i) stage 0 (angionenin >330 ng/mL); (ii) stage 0 (angiogenin <330 ng/mL) + stage I–II (angiogenin >330 ng/mL); and (iii) stage I–II (angiogenin <330 ng/mL). The 40‐month PFS were as follows: 85%, 65%, 25% (χ2 for trend = 6.33; d.f. = 1; P = 0.01). In conclusion, serum angiogenin levels although not increased in comparison with healthy controls, may predict clinical outcome of patients with early CLL and help to refine Rais stratification.

Increased serum BAFF (B-cell activating factor of the TNF family) level is a peculiar feature associated with familial chronic lymphocytic leukemia

In a series of 84 chronic lymphocytic leukemia (CLL) patients we sought to establish whether BAFF (B-cell activating factor of the TNF family) circulating levels correlated with clinical characteristics of disease. BAFF serum levels were significantly higher in 20 healthy controls (i.e., median 695 ng/mL, range 389-1040) in comparison to the whole population of CLL patients (median 376, range 93-8914; P<0.0001). After setting a cut-off at the median value observed in healthy controls (i.e., 695 ng/mL) we found that 6 out of 15 (40%) patients with familial CLL had increased BAFF levels while the same occurred only in 5 out of 64 (7.2%) patients with sporadic CLL (P=0.0007). No significant difference in age (P=0.82), sex (P=0.97), Binet clinical stage (P=0.20), incidence of autoimmune hemolytic anemia (AIHA) or immune thrombocytopenia (ITP) (P=0.47), mutational status of IgVH (P=1.00), CD38 (P=0.34) and ZAP-70 expression (P=0.16) could be detected between patients with sporadic and familial CLL, respectively. The only feature characterizing familial CLL patients was a higher serum BAFF level (sporadic CLL 336 ng/mL, range 93-925; familial CLL 601 ng/mL, range 138-8914; P=0.002). Our data suggest that BAFF levels are elevated in patients with familial CLL. The small cohort of patients used implies that a larger study is needed to reinforce the observation.

Prevalencia de las infecciones de los virus A, B, C y E de la hepatitis en dos grupos de niños de nivel socioeconómico distinto de Santa Cruz, Bolivia

Fundamento y objetivos: Se ha examinado la epidemiologia de las hepatitis A y E, que se transmiten por via gastroenterica, y de las hepatitis B y C, que se transmiten por via parenteral o sexual, en ninos de diferente condicion social residentes en la ciudad de Santa Cruz de la Sierra, Bolivia. Material y metodo: Se selecciono a 1.393 ninos de dos escuelas, una frecuentada por ninos que pertenecen a la mejor clase social de la ciudad (grupo A), y la otra, por ninos de clase social mas pobre (grupo B). Las muestras de sangre fueron obtenidas por medicos de la Universidad de Roma La Sapienza. Los anticuerpos sericos contra los virus de las hepatitis A, B, C y E, y el antigeno de superficie del virus B, se determinaron con metodos inmunoenzimaticos. La significacion se valoro con la prueba de la *2. Resultados: Los anticuerpos contra el virus A estaban presentes en el 82% de los ninos examinados, con una diferencia estadisticamente significativa entre ambos grupos (el 56,3 frente al 94,8%). La prevalencia de los anticuerpos anti-HBc y anti-HBs aumento con la edad, de modo que la infeccion se adquirio prevalentemente en la adolescencia, con una diferencia estadisticamente significativa entre los grupos A y B (el 1,1 frente al 3,8%). El mismo fenomeno se observo en la evaluacion de los anticuerpos anti-VHC (4,7% de positividad solo en el grupo B). Por ultimo, la presencia de anticuerpos contra el virus de la hepatitis E se observo solo en el 1,7% de la poblacion estudiada. Conclusiones: En Bolivia, como en otros paises en vias de desarrollo, las hepatitis viricas representan un grave problema de salud publica. La difusion de la hepatitis virica puede controlarse mejorando las condiciones higienicas y las costumbres de vida. Ademas, un plan de vacunacion contra los virus A y B de la hepatitis es indispensable para la poblacion que vive en un area endemica.

Prevalence of infections by hepatitis A, B, C and E viruses in two different socioeconomic groups of children from Santa Cruz, Bolivia

BACKGROUND AND OBJECTIVES The epidemiology of hepatitis A, E, B and C was analyzed in 1,393 children living in Santa Cruz de la Sierra, Bolivia. They were distributed in two groups according to the social condition. MATERIALS AND METHOD 1,393 children were selected from two different schools: one attended by children belonging to a high social class of the town (group A), and the other school attended by children belonging to the poorest social class (group B). Blood samples were drawn by a team of physicians from Rome University La Sapienza. Serum antibodies against hepatitis A, B, C and E virus, and the hepatitis B surface antigen were evaluated by immunometric methods. The significance was evaluated using the *2 test. RESULTS Antibodies against hepatitis A virus were detected in 82% of examined children, with a significant difference between the two groups (56.3% vs 94.8%). The incidence of anti-HBc antibodies increased with age, so the infection is acquired prevalently in adolescence with a significant difference between both groups (1.1% vs 3.8%). The same phenomenon was observed with anti-HCV antibodies (4.7% positivity only in group B). Serum antibodies against hepatitis E virus were observed in 1.7% cases. CONCLUSIONS In Bolivia, as in other developing countries, viral hepatitis represents a serious burden for public health. Spreading of viral hepatitis can be controlled upon improving hygienic conditions and customs. Moreover, a vaccination plan against hepatitis A and B virus is necessary for the population living in endemic areas.

Serum levels of syndecan-1 in B-cell chronic lymphocytic leukemia: Correlation with the extent of angiogenesis and disease-progression risk in early disease

Syndecan-1 is a transmembrane proteoglycan generally not expressed in mature B-cell neoplasias like chronic lymphocytic leukemia (CLL). Moreover, information dealing with the evaluation of soluble syndecan-1 in CLL are lacking. We measured syndecan-1 concentrations in serum drawn at the time of diagnosis from 67 B-cell CLL patients (Binet stage A, 46; stage B, 7; stage C, 14). For this purpose a syndecan-1 enzyme-linked immunosorbent assay (ELISA, Diaclone, Besancon, France) was used. Detectable levels of syndecan-1 were found in all patients, although serum concentrations were significantly lower in CLL patients in comparison to age- and sex-matched controls (P = 0.02; Mann-Whitney test). No correlation was found with Binet clinical stages (P = 0.796), β2-microglobulin (P = 0.923), hemoglobin level (P = 0.605), platelet count (P = 0.992) and lymphocyte doubling time (P = 0.709). Only an association with absolute peripheral blood lymphocytosis (PBL) (P = 0.01) and LDH (P = 0.05) could be detected. Serum levels of syndecan-1 did not parallel those of several angiogenic cytokines such as vascular endothelial growth factor (VEGF) (P = 0.963), basic fibroblastic growth factor (FGF-2) (P = 0.216), angiogenin (P = 0.478), metalloproteinase-9 (MMP-9) (P = 0.125) as well as bone marrow (BM) microvessel density (P = 0.110). The same applied with adhesion molecules such as CD54 (P = 0.233), CD108 (P = 0.799), CD44 (P = 0.816) and CD31 (P = 0.508). Interestingly, the inverse correlation (r = −0.4967; P = 0.03) between serum concentrations of syndecan-1 and plasma levels of stromal derived growth factor-1 (SDF-1) is in keeping with the different function, respectively, pro- and anti-apoptotic, of these molecules. In 46 Binet stage A patients, serum levels of syndecan-1 were further evaluated as a dichotomous variable with respect to progression-free survival (PFS), an end-point surrogate for overall survival in early B-cell CLL. The best separation of curves was seen with a cut-off point at the median value of syndecan-1 (i.e. 36.5 pg/ml). Median PFS was not reached in the patient group with low syndecan-1, compared to a median of 34 months observed in the remaining patients (P = 0.018; HR = 0.208; 95% CI = 0.115 – 0.816). At the multivariate analysis performed including variables significant in the univariate analysis [i.e. PBL (P = 0.03) and syndecan-1 (P = 0.01)], only syndecan-1 retained a trend of significance (P = 0.08). Despite the pro-angiogenic activity of syndecan-1 which mediates FGF-2 binding and activity, no correlation with either angiogenic cytokines or the extent of BM angiogenesis was found in CLL. The inverse correlation with plasma levels of SDF-1 suggests an involvement in the processes leading to apoptosis. Finally, our results highlight the involvement of syndecan-1 in the mechanisms of disease-progression of early CLL.

Serum insulin-like growth factor is not elevated in patients with early B-cell chronic lymphocytic leukemia but is still a prognostic factor for disease progression.

Abstract:  Objectives: Insulin‐like growth factor 1 (IGF‐1) is an important growth and anti‐apoptotic factor for the cancer cells in several malignancies and in multiple myeloma recent studies support the hypothesis of a role for IGF‐1 in disease progression; however, clinico‐biological relevance of IGF‐1 was never studied in B‐cell chronic lymphocytic leukemia (CLL). Patients and methods: Using a quantitative sandwich immunoassay technique (ELISA) (Quantikine®, Human IGF‐1 and IGFBP‐3, R&D Systems), we measured the concentration of IGF‐1 and its major binding protein IGF‐binding protein 3 (IGFBP‐3) in serum drawn at the time of diagnosis from 77 Binet stage A CLL patients. Results: Either IGF‐1 or IGFBP‐3 were significantly decreased compared with healthy age‐ and sex‐matched controls (P < 0.0001 for both; Mann–Whitney test). Serum levels of IGF‐1 and IGFBP‐3 paralleled each other (P = 0.002); in contrast, no significant correlation was found between serum levels of IGF‐1 and clinico‐hematological variables including age (P = 0.253), sex (P = 0.270), Rai clinical substages (P = 0.140), lactate dehydrogenase (P =0.956), β2‐microglobulin (P = 0.368), lymphocyte count (P = 0.703) and lymphocyte doubling time (LDT, P = 0.233). When correlation were attempted with circulating levels of angiogenic cytokines such as vascular endothelial growth factor (P = 0.971), basic fibroblastic growth factor (P = 0.695), angiogenin (P = 0.282) or adhesion molecules such as vascular cell adhesion molecule‐1 (P = 0.318), intercellular adhesion molecule‐1 (P = 0.883) and platelet endothelial cell adhesion molecule‐1 (P = 0.772) similar results were found. Serum levels of IGF‐1 were further evaluated as a dichotomous variable with respect to progression‐free survival (PFS), an endpoint surrogate for overall survival in early B‐cell CLL. The best separation of curves was seen with the cutoff point at the 75th percentile of IGF‐1 levels (i.e., 93 pg/mL). Median PFS was 63 months in the patient group with low IGF‐1, compared with a median PFS of 40 months in the remaining patients (P = 0.03). In the multivariate analysis performed including variables significant at univariate analysis [i.e. Rai substage (P = 0.002); LDT (P = 0.004), IGF‐1 (P = 0.01)], only Rai substage retained prognostic significance (P = 0.006). However, after removing from analysis LDT (only six of 77 had an LDT < 12 months), either IGF‐1 or Rai substage entered the model at a significant level (P = 0.03 and P = 0.01, respectively). Conclusions: IGF‐1 did not correlate with markers of tumor burden or clinical status in CLL thus suggesting that levels of this cytokine do not reflect the intrinsic malignancy of disease. Results of the present study highlight, however, its involvement in mechanisms of disease progression in early CLL.

Reduction of serum IGF-I levels in patients affected with Monoclonal Gammopathies of undetermined significance or Multiple Myeloma. Comparison with bFGF, VEGF and K-ras gene mutation

BackgroundSerum levels of IGF-I in patients affected with multiple myeloma (MM) have been scarcely studied. The present study is aimed to explore this point comparing 55 healthy subjects, 71 monoclonal gammopaties of uncertain significance (MGUS) and 77 overt MM patients. In the same subjects, basic FGF and VEGF, have been detected. All three mediators were analyzed in function of K-ras mutation and melphalan response. Concerning IGF-I, two representative monitoring examples have also been added.MethodsCytokine determinations were performed by commercially available ELISA kits, while K12-ras mutation was investigated on genomic DNA isolated from bone marrow cell specimens by RFLP-PCR assay.ResultsSignificant reductions of IGF-I levels were observed in MGUS and MM as compared with healthy controls. In addition, MM subjects showed significantly decreased serum IGF-I levels than MGUS. Conversely, increasing levels were observed for bFGF and VEGF, molecules significantly correlated. A multivariate analysis corrected for age and gender confirmed the significant difference only for IGF-I values (P = 0.01). K12-ras mutation was significantly associated with malignancy, response to therapy and with significantly increased serum bFGF levels.ConclusionIGF-I reduction in the transition: Controls→MGUS→MM and changes observed over time suggest that IGF-I should be furtherly studied in future clinical trials as a possible monitoring marker for MM.

Selection of Patients with Monoclonal Grammopathy of Undetermined Significance is Mandatory for a Reliable Use of lnterleukin-6 and Other Nonspecific Multiple Myeloma Serum Markers

Serum levels of various immunochemical markers of clinical interest, as interleukin-6 (IL-6), C-reactive protein (CRP) and beta 2-microglobulin (beta 2M), were measured in sera from 98 subjects affected with monoclonal gammopathy of undetermined significance (MGUS; 80% of which bearing cancer too) and from 39 patients with multiple myeloma (MM). In addition, the ratio between serum IgG/IgA amounts (GAR) was also calculated in monoclonal gammopathies of IgG type. Consistent with our previous investigations, we found that tumor presence significantly influenced the serum levels of the various markers (except GAR) in MGUS patients; in fact, only when comparing MGUS without tumor and MM patients, was a clear difference observed for all markers considered. The data presented discourage the use of IL-6, CRP and beta 2M as discriminant indices between MGUS and MM patients, unless a careful selection of MGUS subjects is performed. Further investigations on these potential markers are therefore needed for a more rational clinical application.