Effect of myo-inositol and alpha-lipoic acid on oocyte quality in polycystic ovary syndrome non-obese women undergoing in vitro fertilization: a pilot study.

The aim of the present study was to evaluate the effectiveness of the combined administration of myo-inositol and α-lipoic acid in polycystic ovary syndrome (PCOS) patients with normal body mass index (BMI), who had previously undergone intracytoplasmic sperm injection (ICSI) and received myo-inositol alone. Thirty-six of 65 normal-weight patients affected by PCOS who did not achieve pregnancy and one patient who had a spontaneous abortion were re-enrolled and given a cycle of treatment with myo-inositol and α-lipoic acid. For all female partners of the treated couples, the endocrine-metabolic and ultrasound parameters, ovarian volume, oocyte and embryo quality, and pregnancy rates were assessed before and after three months of treatment and compared with those of previous in vitro fertilization (IVF) cycle(s). After supplementation of myo-inositol with α-lipoic acid, insulin levels, BMI and ovarian volume were significantly reduced compared with myo-inositol alone. No differences were found in the fertilization and cleavage rate or in the mean number of transferred embryos between the two different treatments, whereas the number of grade 1 embryos was significantly increased, with a significant reduction in the number of grade 2 embryos treated with myo-inositol plus α-lipoic acid. Clinical pregnancy was not significantly different with a trend for a higher percentage for of myo-inositol and α-lipoic acid compared to the myo-inositol alone group. Our preliminary data suggest that the supplementation of myo-inositol and α-lipoic acid in PCOS patients undergoing an IVF cycle can help to improve their reproductive outcome and also their metabolic profiles, opening potential for their use in long-term prevention of PCOS.

A new variation in the promoter region, the -604 C>T, and the Leu72Met polymorphism of the ghrelin gene are associated with protection to insulin resistance.

OBJECTIVE: Previous studies suggested that polymorphisms in the coding region of the preproghrelin were involved in the etiology of obesity and might modulate glucose-induced insulin secretion. We evaluated the association of a new variation, -604C>T, in the promoter region of the ghrelin gene, of Leu72Met (247C>A) and of Gln90Leu (265A>T), all haplotype-tagging single nucleotide polymorphisms (SNPs), with measures of insulin sensitivity in 1420 adult individuals. RESEARCH METHODS: The three SNPs were genotyped using ABI PRISM 7900 HT Sequence Detection System. We used multiple linear regression analysis for quantitative traits and THESIAS software for haplotype analysis. RESULTS: We observed a protective effect exerted by Met72 variant of Leu72Met SNP on insulin resistance parameters; a significant decreasing trend from Leu/Leu to Leu/Met and to Met/Met homozygous subjects in triglycerides, fasting insulin levels and HOMA-IR index (P=0.02, 0.01 and 0.003, respectively), and, consistently, an increase in ghrelin levels (P=0.003) was found. A significant decrease from CC to TC and to TT genotypes in insulin levels and HOMA-IR index was also detected (P=0.00l for both), but only in subjects homozygous for Leu72, where the protective effect of Met72 was not present. The haplotype analysis results supported the data obtained by the evaluation of each single SNP, showing the highest value of insulin levels and HOMA-IR index in the -604(c)247(c) haplotype intermediate value in -604(T)247(C) and lowest value in -604(C)247(A). CONCLUSION: Our observations suggest a protective role of the Met72 variant and of -604 T allele in modulating insulin resistance. These SNPs or an unknown functional variant in linkage disequilibrium could increase ghrelin levels and probably insulin sensitivity.

The glucose clamp reveals an association between adiponectin gene polymorphisms and insulin sensitivity in obese subjects.

Results concerning the association of adiponectin gene polymorphisms (single-nucleotide polymorphisms, SNPs) with obesity, type 2 diabetes (T2DM), metabolic disorders and insulin resistance have not lead to definite conclusions. The aim of our study was to investigate a possible association between the -11391G>A and -11377C>G SNPs of adiponectin gene and measure of insulin sensitivity evaluated by the hyperinsulinemic-euglycemic clamp in a group of 'uncomplicated' obese subjects (with no associated comorbidities) (n=99, mean age 35 years) with a history of obesity lasting at least 10 years. The study of uncomplicated obese subjects, free of possible confounding factors that could interfere with insulin sensitivity, such as pharmacological treatment, provides a good model to assess insulin sensitivity per se. We observed that subjects homozygous for the G allele at locus -11391 had lower M (mg/kg min)/fat-free mass (FFM) index and adiponectin levels compared to subjects with GA+AA genotypes (P=0.002 and P=0.03, respectively) and subjects carrying the -11377G variant had lower M (mg/kg min)/FFM index and adiponectin levels compared to noncarriers (P=0.003 and P=0.03, respectively). Our results imply that the two promoter SNPs, -11391G>A and -11377C>G, of the adiponectin gene are associated with a reduced insulin sensitivity evaluated by hyperinsulinemic-euglycemic clamp in obese subjects.

The use of zoledronic acid in patients with bone metastases from prostate carcinoma: effect on analgesic response and bone metabolism biomarkers.

Zoledronic acid is a bisphosphonate that is effective in the treatment of complications of metastatic bone disease. We have carried out a perspective study on 24 consecutive patients with prostate cancer metastatic to bone to verify the effect of zoledronic acid on analgesic response and a possible relationship with the levels of bone metabolism biomarkers. Eligibility for this study required prostate cancer patients with metastatic bone disease and pain not controlled by analgesics. Patients were excluded from the study if they were receiving cytotoxic chemotherapy or radiation therapy within three months. Eighteen patients (75%) were considered responder to acid zoledronic, only 6 patients did not respond. Before starting treatment (T0) mean Visual Analogue Scale was 7.8 (SE +/- 0.29), after 1 month therapy (T1) was 3.6 (SE +/- 0.3) and after three months (T2) was 3.1 (SE +/- 0.4) with a significant difference between T0 and T1 (p<0.0005) and between T0 and T2 (p<0.0005). Visual Analogue Scale improvement was positively correlated with decrease of C-telopeptide and bone phosphatase alkaline (p<0.05) serum levels.

Production of oxidants by ion irradiation of water/carbon dioxide frozen mixtures.

We present new experimental results on the formation of oxidants, such as hydrogen peroxide, ozone, and carbonic acid, under ion irradiation of icy mixtures of water/carbon dioxide at different ratios and temperatures (16 and 80 K). Pure water ice layers and mixtures with carbon dioxide were irradiated by 200 keV He+ ions. We found that the CO(2)/H(2)O ratio progressively decreased to a value of about 0.1, the H(2)O(2) production increased with increasing CO(2) abundance at both 16 and 80 K, and the CO and H(2)CO(3) production increased with increasing CO(2) abundance at 16 K. At 80 K, the synthesis of CO was less efficient because of the high volatility of the molecule that partially sublimed from the target. The production of carbonic acid was connected with the production of CO(3). O(3) was detected only after ion irradiation of CO(2)-rich mixtures. The experimental results are discussed with regard to the relevance they may have in the production of an energy source for a europan or a martian biosphere.

Accelerated diabetic glomerulopathy in galectin-3/AGE receptor 3 knockout mice.

Several molecules were shown to bind advanced glycation end products (AGEs) in vitro, but it is not known whether they all serve as AGE receptors and which functional role they play in vivo. We investigated the role of galectin-3, a multifunctional lectin with (anti)adhesive and growth-regulating properties, as an AGE receptor and its contribution to the development of diabetic glomerular disease, using a knockout mouse model. Galectin-3 knockout mice obtained by gene ablation and the corresponding wild-type mice were rendered diabetic with streptozotocin and killed 4 months later, together with age-matched nondiabetic controls. Despite a comparable degree of metabolic derangement, galectin-3-deficient mice developed accelerated glomerulopathy vs. the wild-type animals, as evidenced by the more pronounced increase in proteinuria, extracellular matrix gene expression, and mesangial expansion. This was associated with a more marked renal/glomerular AGE accumulation, indicating it was attributable to the lack of galectin-3 AGE receptor function. The galectin-3-deficient genotype was associated with reduced expression of receptors implicated in AGE removal (macrophage scavenger receptor A and AGE-R1) and increased expression of those mediating cell activation (RAGE and AGE-R2). These results show that the galectin-3-regulated AGE receptor pathway is operating in vivo and protects toward AGE-induced tissue injury in contrast to that through RAGE.

Lispro insulin in type 1 diabetic patients on a Mediterranean or normal diet: a randomized, cross-over comparative study with regular insulin.

Lispro (LP) and regular human (HR) insulins were compared in Type 1 diabetic (T1DM) patients on either a Mediterranean diet or normal diet. Twelve T1DM patients were recruited and randomized into two groups of 6, groups A and B. They were treated in different sequences (in 3-month intervals for 1 year). Group A: LP insulin and normal diet, LP insulin and Mediterranean diet, regular insulin and Mediterranean diet, regular insulin and normal diet. Group B: regular insulin and normal diet, regular insulin and Mediterranean diet, LP insulin and Mediterranean diet, LP insulin and normal diet. Each patient was treated with rapid acting insulin, either LP insulin or HR insulin, before each main meal and a dose of slow acting insulin at bedtime. Every 15 days the glycemic control, the incidence and frequency of hypoglycemic episodes, and any adverse events were evaluated. Every 3 months, hematology and a chemistry panel, pre- and post-prandial glycemic and insulinemic profiles were evaluated in all patients. HbA1c levels significantly decreased in LP patients on normal diet, post-prandial glycemic levels were significantly lower in LP than in HR patients from 30 min onwards, 15-min post-prandial insulin levels higher in LP- than in HR-treated patients, and hypoglycemic episodes were significantly less in LP- than in HR-treated patients. LP insulin, irrespective of the type of diet, results in more effective glycemic control, significantly reduces hypoglycemic episodes as opposed to traditional insulin therapy and seems to be more effective with a normal diet than with a Mediterranean diet.

Increased retinal endothelial cell monolayer permeability induced by the diabetic milieu: role of advanced non-enzymatic glycation and polyol pathway activation.

BACKGROUND: Increased vascular permeability could be involved in the pathogenesis of diabetic retinopathy. The present study was aimed at assessing whether high glucose concentrations can impair retinal endothelial cell barrier function directly, irrespective of changes in other determinants of permeability, and the role of non-enzymatic glycation and polyol pathway activation in these alterations. METHODS: Bovine retinal endothelial cells (BREC) were exposed for various periods to high glucose vs iso-osmolar mannitol and normal glucose containing media+/-agents mimicking or inhibiting advanced glycation end product (AGE) formation and polyol pathway activation. Monolayer permeability was assessed by measuring the transendothelial passage of (125)I-labeled proteins. RESULTS: Permeability increased significantly (up to +70%) in BREC exposed to high glucose, but not to mannitol, for 1-30 days, vs normal glucose control cells. Exposure to AGE-modified bovine serum albumin (BSA) (> or = 90%) and, to a lesser extent, sorbitol (+28%) mimicked the high glucose effect. The AGE formation and nitric oxide synthase (NOS) inhibitor aminoguanidine significantly reduced (by 60%) changes induced by 30-day exposure to high glucose, whereas methylguanidine, which inhibits only NOS activity, did not affect permeability. Aldose reductase or sorbitol dehydrogenase inhibitors decreased (by approximately 40%) the enhanced leakage produced by 1-day, but not 30-day, incubation in high glucose. CONCLUSIONS: The present results indicate that high glucose is capable of impairing retinal endothelial cell barrier function directly and that non-enzymatic glycation and polyol pathway activation may mediate these changes, with AGEs participating in the long-term alterations and increased flux through the sorbitol pathway in the short-term effect.

Differential expression levels of urokinase-type plasminogen activator and cathepsin D in locally advanced laryngeal squamous cell carcinoma: clinical implications.

The expression levels and the prognostic impact of urokinase-type plasminogen activator (uPA) and cathepsin D (CD) were evaluated in patients with locally advanced laryngeal squamous cell carcinoma (LSCC). uPA and CD protein levels were determined by immunoluminometric or immunoenzymatic assays in the cytosol of paired sets of tumor tissues and corresponding adjacent normal mucosa (NLM) from 57 patients with stage III/IV LSCC and were correlated with a number of clinicobiological parameters of this tumor including anatomical site, tumor grade, nodal status, clinical stage, DNA ploidy, proliferation rate, and patient outcome. Median uPA levels were significantly higher in LSCC than in NLM (1.8 ng/mg of protein vs 0.3 ng/mg; p<0.001) whereas median CD levels were not significantly increased in tumor tissue compared to NLM (24 pmol/mg vs 19 pmol/mg, p=0.063). No significant correlation was observed between uPA and CD concentrations in tumor tissues (r=-0.1; p=0.4). Furthermore, the distribution analysis of uPA and CD in tumors showed no correlation between expression levels of these proteinases and the parameters mentioned above including patient outcome. However, when data were matched according to each parameter examined it was observed that the differences in uPA content between LSCC and NLM, expressed as uPA tumor/normal tissue ratio (T/M), were more marked in clinically advanced and/or aggressive forms of LSCC (i.e., node positive, stage IV, poorly and moderately differentated, aneuploid multiclonal, low S-phase, subglottis tumors). These data suggest that in such tumors altered regulation of uPA may occur to a greater extent than in less aggressive and less advanced forms of LSCC. This phenomenon was not observed for CD. However, in tumors with a high proliferation rate, in stage IV tumors as well as in those located in the supraglottis, CD levels were significantly higher than those found in the corresponding NLM (p=0.008, p=0.02 and p=0.03, respectively). In conclusion, uPA is highly expressed in locally advanced LSCC and appears to be implicated in some key events of progression of this tumor such as local invasion and/or nodal involvement, whereas CD does not seem to have a role in promoting these processes. Nevetheless, neither of these proteinases seem to be prognostically useful in patients with stage III/IV tumors.

Role of galectin-3 as a receptor for advanced glycosylation end products.

The advanced glycosylation end product (AGE)-binding proteins identified so far include the components of the AGE-receptor complex p60, p90 and galectin-3, receptor for advanced glycosylation end products (RAGE), and the macrophage scavenger receptor types I and II. Galectin-3 interacts with beta-galactoside residues of several cell surface and matrix glycoproteins through the carbohydrate recognition domain and is also capable of peptide-peptide associations mediated by its N-terminus domain. These structural properties enable galectin-3 to exert multiple functions, including the modulation of cell adhesion, the control of cell cycle, and the mRNA splicing activity. Moreover, in macrophages, astrocytes, and endothelial cells, galectin-3 has been shown to exhibit a high-affinity binding for AGEs; the lack of a transmembrane anchor sequence or signal peptide suggests that it associates with other AGE-receptor components rather than playing an independent role as AGE-receptor. In tissues that are targets of diabetic vascular complications, such as the mesangium and the endothelium, galectin-3 is not expressed or only weakly expressed under basal conditions, at variance with p90 and p60 but becomes detectable with aging and is induced or up-regulated by the diabetic milieu, which only slightly affects the expression of p90 or p60. This (over)expression of galectin-3 may in turn modulate AGE-receptor-mediated events by modifying the function of the AGE-receptor complex, which could play a role in the pathogenesis of target tissue injury. Up-regulated galectin-3 expression may also exert direct effects on tissue remodeling, independently of AGE ligands, by virtue of its adhesive and growth regulating properties.

The diabetic milieu modulates the advanced glycation end product-receptor complex in the mesangium by inducing or upregulating galectin-3 expression.

Nonenzymatic glycation has been implicated in the pathogenesis of the dysregulated tissue remodeling that characterizes diabetic glomerulopathy, via the formation of advanced glycation end products (AGEs) and their binding to cell surface receptors. Several AGE-binding proteins have been identified so far, including p60, p90, and the adhesive and growth-regulating lectin galectin-3 (Gal-3), the components of the so-called AGE-receptor complex. This study aimed to evaluate the mesangial expression of the AGE-receptor complex and its modulation by the diabetic milieu, both in vivo, in non-diabetic versus streptozotocin-induced diabetic rats, and in vitro, in mesangial cells exposed to either normal glucose (NG) levels (5.5 mmol/l), as compared with high glucose (HG) levels (30 mmol/l) and iso-osmolar mannitol (M), or to native bovine serum albumin (BSA), as compared with glycated BSA with AGE formation (BSA-AGE) and glycated BSA in which AGE formation was prevented by aminoguanidine (BSA-AM). In vivo, Gal-3 protein and mRNA were not detectable in glomeruli from nondiabetic rats until 12 months after initiating the study. On the contrary, in diabetic rats, Gal-3 expression was observed at 2 months of disease duration, and it increased thereafter. Both p60 and p90 immunoreactivities were observed at the glomerular level with slightly increased expression of p90, but not p60, in diabetic versus nondiabetic animals. In vitro, Gal-3 was not detectable in mesangial cells cultured in NG (although it became evident after a certain number of passages in culture), whereas Gal-3 was detectable in cells grown on BSA. Prolonged exposure (2-4 weeks) of mesangial cells to HG but not to M, as well as growing cells on BSA-AGE and, to a lesser extent, BSA-AM, induced or significantly increased the expression of Gal-3, both protein (up to 2.65-fold) and mRNA (up to 3.10-fold) and its secretion in the medium (by approximately 50%). Both p60 and p90 were demonstrated in mesangial cells under NG conditions, and the expression of p90, but not p60, was upregulated by approximately 20% by HG or BSA-AGE. These results indicate that 1) under basal conditions, Gal-3, unlike p90 and p60, is not detectable in the mesangium but becomes expressed with aging and 2) the diabetic milieu induces or upregulates Gal-3 production, whereas it increases only slightly the expression of p90, but not p60. Gal-3 expression or overexpression may modulate the AGE-receptor-mediated events by modifying the function of the AGE-receptor complex. Additionally, it may exert direct effects on tissue remodeling by virtue of its adhesive and growth-regulating properties.

Prognostic significance of DNA ploidy, S-phase fraction, and tissue levels of aspartic, cysteine, and serine proteases in operable gastric carcinoma.

A consecutive series of 63 untreated patients undergoing surgical resection for stage I-IV gastric adenocarcinomas (GCs) has been prospectively studied. Our purpose was to analyze the predictive relevance of DNA ploidy, S-phase fraction (SPF), and tissue levels of lysosomal proteinases cathepsin D (CD), cathepsin B (CB), cathepsin L (CL), and urokinase-type plasminogen activator (uPA) and that of the intracellular cysteine proteinase inhibitor stefin A on clinical outcome. All of the patients taking part in this study were followed up for a median of 73 months. DNA aneuploidy was present in 71% of the cases (45/63), whereas 9% of these (4/45) showed multiclonality. Both DNA ploidy and SPF were associated with tumor-node-metastasis (TNM) stage and node status, whereas only DNA ploidy was related to depth of invasion. CB, CL, uPA, but not CD, levels were significantly higher in GC as compared to paired normal mucosa, whereas stefin A levels were lower in tumor tissues. CB levels were significantly associated with TNM stage, nodal status, histological grade, and DNA ploidy. At univariate analysis, only node involvement, advanced TNM stage, DNA aneuploidy, and high SPF proved to be significantly related to quicker relapse and to shorter overall survival, whereas depth of invasion was related only to survival. With multivariate analysis, only high SPF (>15.2%) was related to risk of relapse (RR = 8.50), whereas high SPF and DNA aneuploidy were independently related to risk of death (RR = 1.88 and 2.09, respectively). Our preliminary prospective study has identified SPF and DNA ploidy as important biological indicators for predicting the outcome of patients with GC.

Lysosomal cathepsins B and L and Stefin A blood levels in patients with hepatocellular carcinoma and/or liver cirrhosis: potential clinical implications.

The serum levels of lysosomal cathepsin B and L and Stefin A, an intracellular inhibitor of these proteolytic enzymes, were determined in patients with hepatocellular carcinoma (HCC) and/or liver cirrhosis (LC) and correlated with some clinical and biochemical parameters of these diseases. Cathepsin B serum levels were increased in HCC and in LC patients as compared to normal subjects (p < 0.001). However no difference was observed between HCC and LC groups. Interestingly, a significant relationship was evidenced between cathepsin B serum content and the grade of severity of cirrhosis (r = 0.41; p < 0.001). Cathepsin L was significantly elevated only in sera of cancer patients as compared to normal subjects or LC patients (p < 0.001) and significantly correlated with the number of malignant lesions (r = 0.49; p = 0.001). Stefin A serum levels were increased in HCC and LC patients as compared to healthy subjects (p < 0.02). However, these levels were significantly higher in the LC group as compared to the HCC group (p < 0.05). In cancer patients, a significant relationship was observed between Stefin A serum content and tumor size (r = 0.35; p < 0.05), number of neoplastic lesions (r = 0.556; p < 0.001) and serum alpha-fetoprotein (r = 0.38; p < 0.01). These data suggest that cathepsin B and L and Stefin A may be potentially useful as additional biochemical parameters to monitor the therapeutic response of these diseases to clinical treatments and to identify patients with cirrhosis developing precancerous lesions.

Lysosomal aspartic and cysteine proteinases serum levels in patients with pancreatic cancer or pancreatitis.

Lysosomal cathepsins D (CD), B (CB), and L (CL) serum levels were determined by immunoassays in patients with chronic (CHP) or acute (AP) pancreatitis and in patients with ductal pancreatic carcinoma (DPC) and correlated with some biological and clinical parameters of this tumor. CB serum concentrations significantly higher than those measured in healthy subjects (NS) were observed in CHP, AP, and DPC patients (p < 0.01). However, no significant difference was noted among these groups. Increased CL serum levels were evident only in cancer patients compared to NS, AP, or CHP groups (p < 0.05), while no difference was observed among these groups. Elevated CD serum values were observed in CHP and AP patients compared to healthy subjects or cancer patients (p < 0.01). In cancer patients no correlation between CD, CB, and CL and clinical stage or tumor size was found. However, significant correlations were observed only between serum CD and CA50 (p < 0.02) and between CD and CL (p < 0.05). No further relationship among the biochemical parameters examined was observed. The present data suggest that the different serum patterns of CD, CB, and CL in patients with pancreatitis and pancreatic cancer may be of clinical interest as additional biochemical parameters for the differential diagnosis of these diseases. However, further prospective clinical studies are needed to assess better their potential value as prognostic parameters to identify patients with pancreatitis at increased risk to develop pancreatic cancer.

Cathepsin D serum mass concentrations in patients with hepatocellular carcinoma and/or liver cirrhosis.

Cathepsin D serum mass concentrations were determined by enzyme immunoassay in patients with hepatocellular carcinoma (n = 51) and/or liver cirrhosis (n = 92) or benign steatosis (n = 16) and correlated with some biochemical and clinical properties of these diseases. Increased cathepsin D serum mass concentrations (P < 0.001) were observed in all these groups of patients as compared to normal subjects (n = 98). However, patients with steatosis had serum mass concentrations of this enzyme significantly lower (mean 2-3 fold) than those measured in cancer patients (P < 0.05) or cirrhotic patients (P < 0.001). Interestingly, significantly higher cathepsin D serum mass concentrations (mean + 62%) (P < 0.006) were determined in the cirrhosis group as compared to cancer patients. No correlation between cathepsin D and a number of clinical and biochemical properties examined, namely, alpha-foetoprotein, number of neoplastic lesions and tumour size in cancer patients or, Child-Pugh grade of severity of cirrhosis and other enzymes of liver function tests in the cirrhotic group was found. The present data and those from other studies which indicate that cathepsin D may be involved in carcinogenesis suggest that this enzyme may be potentially useful as an additional biochemical marker to identify cirrhotic patients who may develop precancerous hepatic nodules.

Cathepsin D, B and L circulating levels as prognostic markers of malignant progression.

Growing evidence indicates that lysosomal Cathepsins D (CD), B (CB) and L (CL) may promote carcinogenesis and tumor progression. Therefore, we evaluated their potential value as biochemical parameters of malignant progression in patients with benign diseases which may undergo malignant transformation, such as liver cirrhosis (LC) and chronic pancreatitis (CHP) as well as in hepatocellular carcinoma (HCC) and pancreatic cancer (DPC). CD, CB and CL serum levels were determined by immunoenzymatic assays in LC, CHP, HCC or DPC patients and correlated with a number of biochemical and clinical parameters of these diseases. CD serum levels were increased in LC, CHP and HCC, but not in the DPC group as compared to normal subjects (NS) (P < 0.01). Interestingly, higher levels of this enzyme were observed in LC patients compared to HCC patients ( P < 0.01). CB serum concentrations were increased in all patient groups (P < 0.01). However no difference was evidenced between benign and malignant diseases. CL serum levels were significantly increased only in DPC as compared to NS (P < 0.01) or CHP patients (P < 0.02) and in HCC as compared to NS (P < 0.01). The evaluation of CD, CB and CL serum pattern in LC, CHP, HCC and DPC patients may be useful as additional biochemical parameters in the differential diagnosis and therapeutic monitoring of these diseases. Prospective clinical investigations to assess the potential value of these enzymes as biochemical markers of malignant progression of LC or CHP are warranted by the present data.

Biological characterization of laryngeal squamous-cell carcinoma.

The traditional prognostic factors, including stage of disease and tumour grade, have shown a limited prognostic significance and an inability to predict clinical response to specific treatment in patients with laryngeal squamous-cell carcinoma. More recent data suggest that cell kinetics indices, DNA-ploidy, lysosomal cysteine proteinase expression and genetic changes of both tumour suppressor genes and protooncogenes may be considered as reliable and reproducible indicators of biological aggressiveness in these patients. Moreover, the frequency of different genetic alterations suggests that several pathways are involved in the genesis of these neoplasias and, in particular, it is very probable that p-53 expression and PCNA indices (increased in normal mucosa and preinvasive lesions) may constitute more important biomarkers for the early steps of laryngeal carcinogenesis.

Flow cytometric DNA analysis and lysosomal cathepsins B and L in locally advanced laryngeal cancer. Relationship with clinicopathologic parameters and prognostic significance.

BACKGROUND: The traditional factors of locally advanced laryngeal squamous cell carcinoma (LSCC) have limited predictive value for the identification of high risk patients. Therefore, it is extremely important to define prognostic factors that identify the more aggressive types. Reliable and reproducible prognostic indicators are being investigated to help clinicians identify high risk groups and address more rational treatment. METHODS: Flow cytometric DNA ploidy and S-phase fraction (SPF) measurements were performed on frozen tumor tissues from a consecutive series of 71 patients with Stage III and IV LSCC: Lysosomal cathepsin B and L activity levels were determined biochemically in matched paired sets of tumor tissue and normal mucosa samples. RESULTS: By univariate analysis, lymph node positivity, poor histologic differentiation, DNA aneuploidy, high SPF, and high tumor/mucosa ratio of cathepsin B activity were significantly related to risk of relapse, whereas only DNA aneuploidy and high SPF proved to be significantly related to risk of death. Multivariate analysis showed that high histologic grade and high SPF values (> 15.1%) were independent prognostic factors related to risk of relapse (relative risk [RR] = 3.54; 95% confidence limits [CL] = 1.05-12.0; and RR = 4.22; CL = 1.54-11.6, respectively), whereas only high SPF was related to risk of death (RR = 3.63; CL = 1.17-11.3). CONCLUSIONS: S-phase fraction is an independent predictor of relapse free and overall survival in patients with locally advanced LSCC. On the basis of these findings, SPF should be used in addition to other established prognostic factors to refine the prognostic assessment of these patients further. More studies are needed for a better evaluation of the prognostic significance of DNA ploidy and that of lysosomal cysteine proteinases in these tumors.