Improving the international prognostic index score using peripheral blood counts: Results of a large multicenter study involving 520 patients with diffuse large B cell lymphoma

The main purpose of this study was to assess whether it is possible to improve the prognostic impact of international prognostic index (IPI) score by combining it with peripheral blood counts. Thus, we evaluated the prognostic power of lymphocyte, neutrophil, and monocyte counts in 520 patients with diffuse large B cell lymphoma treated with R-CHOP, confirming that these parameters have a strong impact on overall survival (OS). Using revised IPI (R-IPI), 44% of patients were categorized as poor-risk and showed an OS at 5 years of 46%. As OS at 5 years of the 520 patients is 67%, it is clearly evident that R-IPI tends to overestimate the proportion of patients with poor prognosis. Accordingly, in an attempt to improve the discriminating power of R-IPI, we evaluated and compared three different scores by combining the neutrophil lymphocyte ratio (NLR) and absolute monocyte count (AMC) with the following values: (a) IPI score 3-5, (b) age > 60 years and performance status, (c) age ≥ 65 years and LDH > ULN. The three indexes studied, had a similar 5 years OS for the high-risk group (46%-52%), but the proportion of patients classified as poor-risk were 37%, 20%, and 32%, respectively, which are lower than 44% identified with R-IPI. Thus, while R-IPI overestimates the number of high-risk patients, after applying our models, it is possible to recognize patients who are truly at high-risk. Of the three scores, the most accurate appears to be that based on NLR, AMC, LDH > ULN and age ≥ 65 years, which identifies 32% of high-risk patients, correlating well with what is seen in clinical practice.     

Effects of androgen treatment in impotent men with normal and low levels of free testosterone

The relation between sexual function and serum free testosterone (fT) levels, which represent the active fraction of circulating testosterone, was evaluated. Two groups of impotent male subjects with mild hypogonadism were treated with oral testosterone undecanoate (TU); these men presented with tT/luteinizing hormone (LH) ratio and tT levels at the lower limits of normal. The first group had serum fT below 6.6 ng/ml, considered the lower normal value, according to our laboratory method, whereas the second group had normal fT limits. Administration of TU improved sexual function only in impotent men with low fT levels, but not in subjects with normal fT levels, even though the tT levels and the tT/LH ratio of the two groups were not significantly different. The results of our study suggest the presence of a minimun serum fT threshold, lying near the lower normal range, which determines the male sexual function. Moreover, serum fT levels were a more sensitive index than tT for identifying impotent men who can be successfully treated with androgens.     

Partial resection of distal humerus and reconstruction using autogenous iliac bone graft

Summary Seven patients with eccentrically placed tumours of the distal end of the humerus treated with partial resection and autogenous iliac bone grafting are reported. The functional results are discussed.

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Résumé Présentation de sept cas de tumeurs situées à l'extrémité distale de l'humérus et traitées par résection limitée et reconstruction par autogreffe lilaque. Discussion des résultats fonctionnels.

     

Analysis of alterations of oncogenes and tumor suppressor genes in chronic lymphocytic leukemia.

B cell chronic lymphocytic leukemia (B-CLL) represents the most frequent adult leukemia in the Western world. The molecular pathogenesis of B-CLL is largely unknown. Although initial reports on small panels of cases had suggested a role for Bcl-1 and Bcl-2 oncogene activation in B-CLL, later investigations failed to confirm these data. Among tumor suppressor genes, p53 mutations have been reported in a fraction of cases. In this study, we have attempted a conclusive definition of the involvement of dominantly acting oncogenes (Bcl-1 and Bcl-2) and tumor suppressor loci (p53, 6q-) in 100 cases of B-CLL selected for their CD5 positivity and Rai's stage (0 to IV). Rearrangements of Bcl-1 and Bcl-2 and deletions of 6q and 17p were analyzed by Southern blot using multiple probes. Mutational analysis (single strand conformation polymorphism and polymerase chain reaction direct sequencing) was used to assay p53 inactivation. No alterations of Bcl-1 or Bcl-2 were detected in the 100 cases tested. Mutations of p53 were found in 10/100 cases without any significant association with clinical stage. Deletions of 6q were present in 4/100 cases. Overall, our data indicate that: 1) contrary to previous reports, Bcl-1 and Bcl-2 rearrangements are not involved in CD5+ B-CLL pathogenesis and 2) p53 mutations are present in 10% of cases at all stages of the disease.     

Effects of antisense mediated inhibition of cathepsin K on human osteoclasts obtained from peripheral blood.

Cathepsin K is a cystein protease that displays a proteolytic activity against Type I collagen and is abundantly and selectively expressed in osteoclasts where it plays a critical role in bone degradation. Its direct role in bone tissue has been defined by knock-out mice studies and inhibiting strategies in animals models. However, direct proof of cathepsin K function in human osteoclast model in vitro is lacking. The aim of this study is to analyze cathepsin K expression and localization in human osteoclasts obtained from peripheral blood and to examine cathepsin K function in these cells by antisense oligodeoxynucleotide (AS-ODN) strategy. AS-ODN was added to the culture of osteoclast precursors induced to differentiate by RANKL and M-CSF. AS-ODN treatment produced a significant down-regulation of cathepsin K mRNA (>80%) and protein expression, as verified respectively by Real-time PCR and by immunocytochemistry or Western blot. The cathepsin K inhibition caused an impairment of resorption activity as evaluated by a pit formation assay ( p = 0.045) and by electron microscopy, while the acidification process was unaffected. We demonstrated that antisense strategies against cathepsin K are selectively effective to inhibit resorption activity in human osteoclasts, like in animal models.     

Characterization of estrogen receptor from human liver

Characterization of the estrogen receptor in cytosol from human male liver was undertaken to further understanding of the molecular basis of estrogen action in this tissue. By analysis of estrogen binding data of crude cytosol, saturable estrogen binding showed a Kd = 4.7 X 10(-10) M. High levels of nonsaturable binding were also detected. The estrogen-binding activities detected could be distinguished by their steroid specificity, hydrodynamic parameters, ionic properties, and sensitivity to proteolytic attack. Our findings also confirmed that the moderate-affinity estrogen binders found in rodent liver cannot be detected in human tissue. We concluded that the properties of estrogen receptor of human liver cytosol allow its separation from nonsaturable estrogen-binding components.     

Simultaneous visualization of seven different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy.

Combinatorial labeling of probes (i.e., with two or more different reporters) increases the number of target sequences that can be detected simultaneously by fluorescence in situ hybridization. We have used an epifluorescence microscope equipped with a digital imaging camera and computer software for pseudocoloring and merging images to distinguish up to seven different probes using only three fluorochromes. Chromosome-specific centromere repeat clones and chromosome-specific "composite" probe sets were generated by PCR in which different mixtures of modified nucleotides, including fluorescein-conjugated dUTP, were incorporated. Cosmid clones were labeled similarly by nick-translation. The technique has been used to delineate the centromeres of seven different human chromosomes, on both 4',6-diamidino-2-phenylindole-stained metaphase spreads and interphase nuclei, to map six cosmid clones in a single hybridization experiment and to detect chromosome translocations by chromosome painting. Multiparameter hybridization analysis should facilitate molecular cytogenetics, probe-based pathogen diagnosis, and gene mapping studies.     

Phenotypic and functional characteristics of tumor-associated lymphocytes in patients with malignant ascites receiving intraperitoneal infusions of recombinant interleukin-2 (rIL-2)

In the course of a phase I trial, in which recombinant IL-2 (rIL-2) was infused intraperitoneally (i.p.) in patients with peritoneal carcinomatosis, we evaluated the effect on "tumor-associated lymphocytes" (TAL) isolated from the ascitic fluid. No major changes in the percentages of cells expressing the CD3, CD4, CD8, Leu-7, OKM1 and WT-31 antigens were detected either in TAL or in peripheral blood lymphocytes (PBL) after 7 days of rIL-2 infusion. In contrast the percentages of TAL (but not PBL) expressing surface IL-2 receptor (Tac), or LAK-1 antigen were sharply increased. Analysis of cytolytic functions showed a potentiation of the lytic activity against natural-killer (NK) sensitive K562 target cells and the de novo appearance of lytic activity against fresh melanoma cells. In one patient IFN-gamma was detected in the ascitic fluid following rIL-2 infusion. T-cell clones derived from the patient were analyzed for the IFN-gamma production. While only approximately 40% of PB-derived control clones produced medium to low amounts of IFN-gamma, all of the TAL-derived clones produced medium to high amounts of the lymphokine.     

Normal human IgG prevents endothelial cell activation induced by TNFalpha and oxidized low-density lipoprotein atherogenic stimuli

Normal human immunoglobulin G (IgG) has anti-inflammatory and immuno-regulatory properties, which are exploited in the therapy of selected diseases. A putative mechanisms of action is the direct regulation of endothelial cell function by natural antiendothelial cell antibodies. Endothelium activation is a critical event in atherosclerosis. We have verified the ability of normal human IgG to modulate endothelial responses to the atherogenic stimuli tumour necrosis factor-alpha (TNFalpha) and oxidized low-density lipoproteins (oxLDL) in vitro. Confocal microscopy was used to visualize vascular cell adhesion molecule-1 (CD106) expression on endothelial cells, cytoplasmic free calcium ([Ca++]i) modifications and fluorescein-coupled oxLDL internalization. Cytokine secretion was measured by ELISA on cell supernatants. IgG prevented TNFalpha induced CD106 membrane expression and an increase in [Ca++]i, and inhibited the secretion of interleukin-6 (IL-6) and macrophage-colony-stimulating factor (M-CSF). IgG also inhibited CD106 expression induced by oxLDL and one pathway of their internalization, but were ineffective on oxLDL induced [Ca++]i rise and apoptosis. F(ab)'2 fragments from IgG, but not monoclonal IgG, reproduce IgG effects. These findings point to a regulatory role for specific antibodies included in circulating normal IgG towards proinflammatory responses of endothelial cells in atherogenesis and suggest possible development of new therapeutic strategies.