The International Journal of Biological Markers

The metallothionein (MT) family is a class of low molecular weight, intracellular and cysteine-rich proteins presenting high affinity for metal ions. Although the members of this family were discovered nearly 40 years ago, their functional significance remains obscure. Four major MT isoforms, MT-1, MT-2, MT-3 and MT-4, have been identified in mammals. MTs are involved in many pathophysiological processes such as metal ion homeostasis and detoxification, protection against oxidative damage, cell proliferation and apoptosis, chemoresistance and radiotherapy resistance. MT isoforms have been shown to be involved in several aspects of the carcinogenic process, cancer development and progression. MT expression has been implicated as a transient response to any form of stress or injury providing cytoprotective action. Although MT participates in the carcinogenic process, its use as a potential marker of tumor differentiation or cell proliferation, or as a predictor of poor prognosis remains unclear. In the present review the involvement of MT in defense mechanisms to toxicity and in carcinogenicity is discussed.

The aim of this study was to evaluate the clinical value of CEA and CA 19-9 in a potential high-risk population of subjects with gastrointestinal complaints. The basic question was whether the determination of these markers, in addition to some other clinical features in this high risk population, could be helpful in diagnosing intraabdominal cancer. Two hundred and two patients with gastrointestinal complaints underwent standard diagnostic procedures and were followed for at least one year. For every patient, CEA and CA 19-9 levels were obtained at the first examination; the evaluating physician was blinded to the marker levels. The determinants of the likelihood of cancer were evaluated by multivariate analysis. Seventeen patients were diagnosed as having intraabdominal cancers. With the presence of melena (RR=101.63, p=0.007), nonspecific gastrointestinal symptoms (RR=12.54, p=0.026), increasing age (RR=1.09, p=0.028) and abnormal CEA (RR=240.79, p=0.000), the risk of having cancer increased significantly and independently. The presence of a primary gastric complaint was associated with a lower risk of cancer in this cohort (RR=0.01, p=0.04). Markers were not used in the diagnostic workup. In conclusion, in patients presenting with gastrointestinal complaints, the finding of elevated CEA levels may help in the diagnosis of cancer by prompting a more extensive search for intraabdominal cancer.

To study the behavior and possible correlations of neuron-specific enolase (NSE) with other clinicobiological parameters, we measured the cytosolic levels of this marker by means of an immunoradiometric assay (IRMA) in 95 squamous cell lung carcinoma samples. We also analyzed the levels of pS2, tissue-type plasminogen activator (t-PA), hyaluronic acid (HA), free beta subunit of human chorionic gonadotropin (β-HCG), CYFRA 21.1 and CA 125 in cytosol. On the cell surface we analyzed the concentrations of epidermal growth factor receptor (EGFR), HA, erbB-2 oncoprotein, CD44s, CD44v5 and CD44v6. Other parameters considered were clinical stage, lymph node involvement, histological grade (HG), ploidy and the cellular S-phase fraction measured by flow cytometry on nuclei obtained from fresh tissues.

In the 95 squamous cell carcinomas the cytosolic levels of NSE varied from 4.5 to 2235 ng/mg protein (median: 267) and were significantly higher (p<0.001) than those observed in 38 samples of normal pulmonary tissue obtained from the same patients (range: 56–657; median: 141.5). When classifying tumors according to the different parameters analyzed, we observed that the levels of NSE were higher in aneuploid than in diploid cases (p=0.046) and in those that were HG3 than in those that were HG2 (p<0.001). Tumors with high NSE levels (>422 ng/mg protein; 75th percentile) were more likely to have high S-phase values (p=0.012) and were more frequently aneuploid (p=0.038) and HG3 (p<0.001) than those with low levels of NSE (<180 ng/mg protein; 25th percentile).

These results lead us to the following conclusions: 1) the cytosolic concentrations of NSE are significantly higher in squamous cell carcinomas than in healthy pulmonary tissue, and 2) the cytosolic concentrations of NSE are not correlated with clinical stage or nodal involvement. However, in our study higher levels of the enzyme were statistically correlated with aneuploidy, histological grade 3 and S-phase. This may explain its association with poorer outcome and progression, but also the more favorable response of tumors with elevated NSE to chemotherapy, as suggested by other groups.

H-ras is a member of the ras superfamily of genes. This gene encodes for a 21 kDa protein (p21) which is located on the inner surface of the plasma membrane. Ras genes are involved in a wide variety of human tumors, and there is a known correlation between H-ras activation and breast carcinogenesis. H-ras contains a polymorphic region, a repeated hexanucleotide –GGGCCT - located in intron 1 close to the 5′ of the gene (HRM region). Three alleles of this region, P1, P2 and P3, have been identified that contain two, three and four repeats of the hexanucleotide, respectively. H-ras possesses a minisatellite DNA of the variable tandem repeat (VTR) which is located 1000 bp downstream of the gene displaying linkage disequilibrium with HRM. The purpose of this study was to estimate the frequency of P1, P2 and P3 in the normal population and in patients with breast cancer. We studied 56 biopsy specimens from patients with breast cancer, 61 normal blood samples, and 30 pairs of normal and tumoral breast tissues for VTR analysis. There was a difference in the distribution of P1, P2 and P3 alleles between normal and breast cancer samples. The frequency of P1 homozygosity was shown to be almost twice as high in women with breast cancer compared to healthy women (72% versus 39%). These results suggest that P1 homozygosity may be considered as a potential risk factor in breast carcinogenesis. In VTR analysis one sample presented a shift in mobility, but no polymorphism in the BstN I pattern of the 28 bp repetition core was observed.

In a retrospective study of 488 women with primary breast cancer, after a median follow-up of 10 years, we sought interactions between disease-free survival (DFS) and overall survival (OS) and tumor antigen levels of two components of the plasminogen system, urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1, and the transmembrane growth factor receptor c-erbB-2. We used ELISAs (American Diagnostica, Greenwich, CT, USA) to quantify uPA and PAI-1 antigen levels in cytosols, and a double monoclonal antibody-based assay (EIA) (Ciba Corning Diagnostics, Alameda, CA, USA) to quantify c-erbB-2 in membrane extracts of the same tissues. Weak positive correlations were found between uPA and c-erbB-2 (r_s=0.146; p=0.001) and between PAI-1 and c-erbB-2 (r_s=0.154; p<0.001). In the overall population, using univariate analyses, c-erbB-2 overexpression and high uPA and PAI-1 antigen levels (>300 IU/mg, >1.40 ng/mg and >5.53 ng/mg, respectively) were significantly associated with shorter DFS (p=0.003, p<0.001 and p<0.001, respectively) and OS (p<0.001 in all cases). Using multivariate analyses, PAI-1, node status and tumor size were independent predictors of DFS and c-erbB-2 was retained in the model only for OS. In the node-negative subgroup, PAI-1 was the strongest significant survival predictor both for OS (p=0.003; HR 2.52) and DFS (p<0.001; HR 2.39). This study shows that in primary breast cancer c-erbB-2 offers no additional prognostic information when uPA and/or PAI-1 are candidates in the multivariate analyses.

C677T and A1298C methylenetetrahydrofolate reductase (MTHFR) polymorphisms have been suggested to affect susceptibility to malignant lymphoma, possibly by altering DNA methylation. The DNA repair gene O⁶-methylguanine DNA methyltransferase (MGMT) is transcriptionally silenced by promoter hypermethylation in diffuse large B-cell lymphomas (DLBCL). We analyzed the MTHFR677 and MTHFR1298 genotypes in 111 DLBCL patients and 465 controls. No significant difference in the frequency of MTHFR polymorphisms between patients and controls and no significant association between MTHFR677 or MTHFR1298 genotypes and methylation of MGMT promoter were observed. These results indicate that MTHFR variants are not related to DLBCL development and MGMT hypermethylation.

In this study we assessed the prognostic significance of 90K/MAC-2BP serum levels in a group of 40 hepatocellular carcinoma patients. This glycoprotein is a new, interesting serum marker that reflects the immune reaction of the host against certain viral infections and tumors such as breast, ovarian and pancreatic cancer. Hepatocellular carcinoma (HCC) is one of the most widespread tumors in the world. AFP is currently the most useful marker for HCC, in spite of its poor diagnostic sensitivity. In this study 40 cirrhotic HCC patients were enrolled. The prevalence of viral hepatic infections in this group was 73% for HCV, 8% for HBV, and 8% for both viruses. Thirteen percent of the patients showed non-virus-related liver damage. 90K serum levels were assayed by an ELISA kit and AFP levels by a chemiluminescent enzyme immunometric system. The overall survival curves were estimated by the Kaplan-Meier method, taking into account age, sex, 90K and AFP serum levels. Statistical analysis showed a highly significant influence on overall survival of age below 70 years and 90K serum levels below the cutoff of 14 ng/mL. Serum AFP (≤20 ng/mL) had positive prognostic value only when it was associated with 90K levels (p<0.02, log-rank).