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Background Matrix metalloproteases (MMPs), enzymes with the ability to degrade the extracellular matrix, play an important role in tissue invasion by cutaneous malignant melanoma (CMM). One specific MMP, collagenase-3 (MMP-13), is thought to have a key function in the activation of MMP.Aims To evaluate the expression of MMP-13 in CMM and assess its possible relationship to clinical and pathological parameters.Methods MMP-13 expression was analyzed in 51 paraffin-embedded tumor samples from patients with invasive CMM, ten samples from in situ melanomas, and in eight samples from benign lesions (three dermal melanocytic nevi, three compound melanocytic nevi and two atypical melanocytic nevi) using immunohistochemical techniques. The median follow-up period in patients with invasive CMM was 50 months.Results Benign lesions were consistently negative for MMP-13, whereas three of the ten in situ melanomas (30%) and 23 of the 51 invasive CMMs (45%) showed positive immunostaining for MMP-13. The percentage of MMP-13-positive tumors correlated significantly and positively with the mitotic index (p=0.002) in invasive CMM. However, our results did not show any significant association between tumoral MMP-13 expression and relapse-free survival in patients with invasive CMM.Conclusions MMP-13 appears to be a factor associated with tumor aggressiveness in CMM. It seems to eliminate an important barrier not only against tumoral invasion but also against proliferation.

Background Cathepsin D is the proteolytic enzyme most frequently implicated as a prognostic factor in primary breast cancer. In the present study we evaluated by means of an immunoradiometric assay the tumor content of this protease in primary breast cancer, its relationship with tumor-related clinical and pathological parameters, and its prognostic significance in a large series of breast cancer patients.Method The study comprised 1033 women with histologically established invasive breast cancer. Cathepsin D was measured in cytosol samples by means of an immunoradiometric assay to determine the total amount of cathepsin D (52 kDa, 48 kDa and 34 kDa). Evaluation of relapse-free survival and cause-specific survival was performed in the group of 1003 patients without evidence of metastasis at the time of initial diagnosis. The median follow-up of the patients who were free of recurrence was 54 months.Results Cathepsin D levels showed a wide range among the studied tumors (n=1033; median (range) 41 (0.9–2504) pmol/mg protein). Statistical analysis showed that the median cathepsin D levels were considerably higher in large tumors (T2–4) than in smaller ones (T1) (p=0.017), as well as in node-positive than in node-negative tumors (p=0.004). Cathepsin D levels were also higher in ductal tumors than in the other histological types (p=0.001), as well as in moderately or poorly differentiated tumors (p<0.001). Likewise, the median value of the protease was significantly higher in ER or PgR-positive tumors than in hormone receptor-negative ones (p=0.011 and p=0.004, respectively), as well as in aneuploid tumors than in diploid tumors (p=0.029). Multivariate analysis demonstrated that elevated cathepsin D levels (>59 pmol/mg protein) were notably associated with a shorter cause-specific survival in the whole group of patients with breast cancer, as well as in the subgroup of node-positive patients (p<0.05).Conclusions This study suggests that elevated intratumoral cathepsin D levels may identify a subset of node-positive breast cancer patients showing a high probability of earlier death.

Background Epidermal growth factor receptor (EGFR) and c-erbB-2 are membrane receptors expressed in a variety of solid human cancers and directly correlated with poor prognosis. The objective of this work was to evaluate the EGFR and c-erbB-2 levels in non-resectable gastric carcinomas, their possible relationship with a variety of clinicopathological tumor parameters, and their prognostic significance.Methods This was a prospective analysis of 65 patients with unresectable gastric carcinomas (UICC R1 or R2), who underwent palliative surgery and were followed up for a median period of 13 months. Membranous EGFR levels were examined by radioligand binding assays and cytosolic c-erbB-2 levels by means of an immunoenzymatic assay.Results There was a wide variability in EGFR (80.3-2910 fmol/mg of protein) and c-erbB-2 (0.4-10071 NHU/mg of protein) levels in neoplastic tissues from patients with unresectable gastric carcinomas. Median c-erbB2 was significantly higher in tumors of the intestinal type than in tumors of the diffuse type (p=0.035) and in R2 than in R1 tumors (p=0.016). Statistical analysis showed that there was no relationship between tumor c-erbB-2 or EGFR content and any other patient or tumor characteristics. However, high levels of EGFR were significantly associated with a shorter overall survival (p=0.01).Conclusion Our data suggest a role of both transmembrane proteins in the progression of gastric cancer. EGFR and c-erbB-2 contents in unresectable gastric cancer could be utilized as appropriate biological markers for selecting candidates for treatment based on EGFR and/or c-erbB-2 inhibition.

Aims We analyzed the tPA content in primary gastric carcinomas and surrounding mucosa in order to assess the relationship between tPA content, clinicopathological tumor characteristics, and estrogen and progesterone receptor content. We evaluated the prognostic value of this serine protease in gastric cancer patients.Patients and methods 122 resected gastric neoplasms and 95 adjacent mucosa samples were studied. The tPA content was measured in cytosol by an ELISA method. Cytosolic ER and PgR were measured with a solid phase enzyme immunoassay.Results Cytosolic tPA levels in neoplastic tissues (median 1.0 ng/mg prot) were significantly lower (p=0.002) than those found in paired mucosa samples (median 2.3 ng/mg prot). There was no significant association between tPA levels and clinicopathological parameters or PgR content, but tPA levels were significantly correlated with ER content. The intermediate-tPA-content group, corresponding to samples with between 0.3 and 1.70 ng/mg protein, proved to have a significantly high risk of relapse.Conclusions We found a wide variability in tPA levels in gastric carcinoma and adjacent mucosa samples, with significantly decreased levels in tumors and a significantly positive relationship between tPA levels and ER status. There was a non-monotonic relationship between tPA levels and prognosis in patients with gastric cancer.

Pepsinogen C is an aspartyl protease mainly involved in the digestion of proteins in the stomach, and an androgen-inducible protein in breast cancer cells. The aims of this study were to evaluate the expression and clinical significance of this enzyme in the primary tumors of prostate cancer patients with bone metastasis who were scheduled to receive antiandrogenic therapy. This study was prospectively performed in 28 stage D2 prostate cancer patients who, after diagnosis, received maximum androgen blockade. Pepsinogen C tumor expression was analyzed in samples (24 from needle biopsy cylinders and four from transurethral resection specimens) from primary tumors using an immunohistochemical assay. Twelve prostate carcinomas (42.8%) were positive for pepsinogen C. Pepsinogen C was a significant prognostic factor to predict a longer overall survival in the patients of our study (p<0.01). Pepsinogen C can be a new prognostic factor and a useful biological marker of androgen dependency in prostate cancer.

In this prospective study we have quantified by means of ELISA-methods the cytosolic content of estrogen (ER) and progesterone receptors (PgR) in tumoral tissue and paired normal mucosa from 163 patients with resectable colorectal cancer. Survival analysis was performed in a subgroup of 120 patients and the mean follow-up period was 24.9 months. The cutoff for ER and PgR levels was set at 1 fmol/mg protein. On the basis of this cutoff 20.9% of the cancers were ER positive and 25.8% were PgR positive; normal adjacent tissue presented ER in 18.4% and PgR in 24.5%. Our results did not show any significant correlation between ER and PgR levels in neoplastic tissues. Howewer, a correlation was found in normal mucosa samples (p=0.02). Statistical analysis showed that there was no correlation between tumor ER and PgR content and patient age or sex, tumor location, Dukes’ stage, histological differentiation, DNA ploidy status and S-phase fraction. Furthermore, the results did not show any statistical differences in relapse-free and overall survival curves calculated for patients classified according to the hormone receptor content of their tumors.ER and PgR were detected at low levels in normal and neoplastic colorectal tissues without any significant relationship to either clinicopathological tumor characteristics or patient outcome. Their possible role in colorectal cancer remains to be elucidated.

pS2, a 60-amino-acid chain peptide which is the most widespread estrogen-induced RNA messenger in MCF-7 breast cancer cells, is normally detected in the epithelium of gastric mucosa. The aims of this work were to evaluate the cytosolic pS2 content and its clinical significance in gastric carcinomas. Cytosolic pS2 levels were examined by immunoradiometric methods in 108 patients with primary gastric adenocarcinomas. The mean follow-up period was 23.3 months. The cytosolic pS2 levels of the tumors ranged widely, i.e., from 0.1 to 3217 ng/mg protein. There were no significant differences in pS2 content between tumors (mean ± standard error: 137.2±31.4 ng/mg protein) and paired adjacent mucosa samples (n=84; mean ± standard error: 249.6±32.6 ng/mg protein), nor were there any significant differences in tumoral pS2 levels with respect to clinicopathologic parameters such as patient age and sex or tumor location, stage, histologic type or grade. However, the results indicated that high intratumoral pS2 levels were significantly and independently associated with an unfavorable outcome in the overall group of patients (p=0.0266) and in patients with resectable gastric cancer (p=0.003). In conclusion, pS2 may represent a useful biological marker in gastric cancer.

Pepsinogen C is an aspartyl-proteinase usually involved in the digestion of proteins in the stomach, and an androgen- inducible protein in breast cancer cells. In this study we evaluated its expression and clinical significance in patients with resectable pancreatic cancer. Pepsinogen C expression was examined by immunohistochemical methods in a series of 73 pancreatic carcinomas. The prognostic value of pepsinogen C was retrospectively evaluated by multivariate analysis. A total of 21 (28.8%) pancreatic carcinomas stained positively for pepsinogen C. The percentage of pepsinogen C-positive tumors was significantly higher in well-differentiated tumors (38.3%) than in moderately differentiated (15.8%) and poorly differentiated (0%) tumors (p<0.05). In addition, statistical analysis revealed that pepsinogen C expression was associated with clinical outcome. Thus, patients with pepsinogen C-negative tumors have a poorer overall survival than those with pepsinogen C-positive tumors. Our results led us to consider that the expression of pepsinogen C may represent a useful biological marker in pancreatic cancer. Expression of this protein may be a marker of gastric-type differentiation of the tumors and it might also reflect the existence of a complete hormone receptor pathway in a subset of pancreatic carcinomas.

The objective of this work was to evaluate the epidermal growth factor receptor (EGFR) content in gastric cancer, its possible relationship with clinicopathological parameters of tumors and its prognostic significance. Membranous EGFR levels were examined by radioligand binding assays in 110 patients with gastric cancer. The mean follow-up period was 30.7 months. EGFR levels of tumors ranged widely, from 0.3 to 510 fmol/mg protein. EGFR levels were significantly higher (p<0.0005) in neoplastic tissue than in paired adjacent mucosa samples (median) (n= 84; 8.7 vs. 3.9 fmol/mg protein). Intratumoral EGFR levels were significantly correlated with tumor stage (p<0.05), and were higher in patients with stage III tumors (median) (7.6, 6.4, 12.3 and 7.5 fmol/mg protein for stages I, II, III and IV, respectively). In addition, the tumor/mucosa ratios of the EGFR content were significantly higher (p<0.05) in patients with stage III tumors (1, 1.8, 3.9, and 0.92, respectively). Although there was no significant relationship between EGFR levels of tumors and overall survival, the results suggest a role for EGFR in tumor progression of gastric cancer.

The aim of this study was to evaluate, by means of an immunoenzymatic assay, the membranous and cytosolic c-erbB-2 oncoprotein contents in primary tumors and in adjacent mucosa from gastric cancer patients. Fifty-two patients with primary gastric adenocarcinomas were enrolled in this prospective study. c-erbB-2 protein levels were significantly higher in membranous than in cytosolic samples, both in neoplastic tissues (median: 3602 vs 525 NHU/mg protein; p<0.0001) and in adjacent mucosa samples (median: 3174 vs 509 NHU/mg protein; p<0.0001). Nevertheless, there was a significant positive relation between membranous and cytosolic c-erbB-2 protein contents in both neoplastic tissue (p<0.001) and adjacent mucosa (p<0.001) samples. There was no significant difference in the membranous c-erbB-2 protein content between neoplastic tissues and adjacent mucosa samples. However, the cytosolic c-erbB-2 content was significantly higher in neoplastic tissues than in adjacent mucosa (p<0.05). Finally, the results did not show any significant correlations of these oncoprotein contents with patient characteristics, clinicopathologic parameters and overall survival of the study population.

The aim of this work was to evaluate the cytosolic contents of hyaluronic acid (HA) and cathepsin D (CatD) in gastric carcinomas and their possible relationships with the clinicopathological parameters of the tumors. Our study demonstrated a wide variability in the cytosolic levels of HA (mean ± SEM: 3748 ± 411 ng/mg protein) and cathepsin D (52 ± 4 pmol/mg protein) in the tumors of 78 gastric cancer patients. In addition, the tumoral contents of HA and CatD were significantly higher (p<0.005) in diffuse type (HA: 6027 ± 1099 ng/mg protein; CatD: 75 ± 13 pmol/mg protein) than in intestinal type (HA: 2735 ± 242 ng/mg protein; CatD: 42±3 pmol/mg protein) carcinomas. These data suggest that both markers may contribute to the biological characterization of gastric carcinomas.

We have examined by immunohistochemistry the ability of human carcinomas of various origin to produce pepsinogen C, an aspartyl proteinase mainly involved in the digestion of proteins in the stomach and recently found to be associated with breast carcinomas. Of the 268 tumors analyzed 80 (29.8%) showed positive staining for pepsinogen C. These positive tumors included 12 gastric (38.7% of the 31 examined cases), nine pancreatic (42.8%), two renal (20%), 12 prostatic (40%), three bladder (27.3%), 14 endometrial (29.7%) and 18 ovarian (40%) carcinomas. We also detected 10 melanomas (50%) that were positive for pepsinogen C. By contrast, immunohistochemical staining for the proteinase was not detected in colorectal, cervical, lung and basal cell skin carcinomas. These results demonstrate that pepsinogen C, a proteolytic enzyme of highly restricted expression in human tissues, can also be expressed by a wide variety of human carcinomas. In addition, and similar to pepsinogen C expression in breast carcinomas, the production of this enzyme by different human tumors might be related to putative hormonal alterations associated with the development and progression of these tumors.

The aim of this study was to evaluate the cytosolic tissue-type plasminogen activator (tPA) content in colorectal cancer, its possible relationship with the clinicopathologic parameters of tumors, and its prognostic significance. We have therefore examined by immunoenzymatic assay the cytosolic tPA content in tumors and paired surrounding normal mucosa samples from 162 colorectal cancer patients. Cytosolic tPA levels were significantly higher in surrounding normal mucosa samples than in neoplastic tissues (4.01±5.07 vs 2.63±5.82 ng/mg protein; p<0.0001). By contrast, no significant correlation was found between tPA content and clinicopathologic tumor parameters such as location, Dukes’ stage, histologic grade, and DNA content or S-phase fraction. However, the results indicated that a high cytosolic tPA content (>0.75 ng/mg protein) in tumors predicted for a shorter relapse-free and overall survival (both p<0.05) in 123 resectable colorectal cancer patients who were prospectively evaluated during a mean follow-up period of 32.2 months. This suggests that tPA may give additional information to that provided by other biochemical markers currently used in colorectal cancer.

We evaluated in 214 patients with primary colorectal cancer the prognostic value of the preoperative serum levels of CEA and CA195. For CEA these levels were above the cutoff of 6 ng/ml in 31.3% of patients, whereas for CA195 they were higher than 12 U/ml in 35.9% of patients. The simultaneous use of both antigens increased the sensitivity to 49%, which was significantly higher than that of CEA (p<0.001) and CA195 (p<0.01) taken singly. The mean preoperative CEA levels were significantly (p<0.001) correlated with Dukes’ stage only, while there was a significant correlation between preoperative serum levels of CA195 and Dukes’ stage (p<0.001), grade of differentiation (p<0.01) and tumor location (p<0.05).The results indicated that high preoperative serum levels of CEA and CA195 were associated with a shorter overall survival (p<0.0001). In addition, separate Cox multivariate analysis showed that preoperative CA195 was, after Dukes’ stage, the strongest factor to predict overall survival (p<0.0001).

In a prospective study we evaluated in 48 patients with primary ovarian carcinoma the prognostic value of the preoperative circulating serum levels of CA 125 and TAG-72. Serum levels of CA 125 were above the cutoff level of 35 U/ml in 68% of patients, TAG-72 levels were higher than 6 U/ml in 50% of patients, while the simultaneous use of the two markers increased the sensitivity to 75%. Pretreatment CA 125 and TAG-72 levels were significantly lower (p < 0.05, for both) in patients with well differentiated tumors than in those with moderate or poor differentiation. Similarly, both marker levels were significantly higher (p < 0.001) in patients with residual disease after cytoreductive surgery than in those with no residual tumor. In addition, the CA 125 levels were also higher in initial stages (I-II) than in more advanced stages (III-IV) (p < 0.05), whereas TAG-72 levels were higher (p < 0.05) in patients with mucinous or endometrioid tumors than in those with serous carcinomas. The results further indicated that high preoperative serum levels of CA 125 and TAG-72 were associated with a shorter overall survival (p < 0.001 and p < 0.01, respectively). Finally, separate Cox multivariate analysis showed that preoperative CA 125 and TAG-72 serum levels were, after stage, the strongest factors to predict overall survival (p < 0.0001, p < 0.05 and p < 0.005, respectively) in patients with ovarian carcinoma.

We evaluated in 74 patients with resectable primary gastric carcinoma, the prognostic value of the preoperative circulating serum levels of CEA and TAG-72. Serum levels of CEA were above the cutoff level of 6 ng/ml in 18.9% of patients; TAG-72 levels were higher than 6 U/ml in 31% of patients. Pretreatment mean CEA levels were significantly lower (p<0.01) in patients with stage I tumors (2.9 ± 0.3 ng/ml) than in those with more advanced tumors (stage II: 14.5 ± 6.8 ng/ml; stage HI-TV: 6.8 ± 1.5 ng/ml). Similarly, significant differences in mean TAG-72 serum levels were found between stage I (3.5 ± 1.8 U/ml) and stage II and stage III-IV (30.4 ± 20.7 U/ml and 26.1 ± 9.7 U/ml, respectively) (p<0.05). In addition, TAG-72 levels were also higher in poorly differentiated and moderately differentiated tumors (38.5 ± 20.1 U/ml and 23.1 ± 9.4 U/ml, respectively) than in well differentiated tumors (4.4 ± 0.9 U/ml) (p<0.05). The results further indicated that high preoperative serum levels of CEA predicted shorter relapse-free survival duration (p<0.01), and that high TAG-72 levels were associated with shorter relapse-free and overall survival (p < 0.0001 and p < 0.0005, respectively). In addition, separate Cox multivariate analysis showed that preoperative TAG-72 was, after stage, the strongest factor to predict both relapse-free and overall survival (p < 0.0001 and p < 0.005, respectively) in patients with gastric cancer.

Serum prolactin (PRL) concentrations at baseline and after TRH stimulation were determined in 15 healthy women and in 51 premenopausal patients suffering from Gross Cystic Breast Disease. All women were in the luteal phase of the menstrual cycle and patients were divided into three groups according to cyst type at presentation. Basal hormone levels were within the normal range in the control group and in the three cystic breast disease groups. The maximum PRL response to TRH stimulation was significantly higher (p < 0.001) in patients with type I cysts (low Na+/K+intracystic ratio and apocrine epithelium) than in patients with type II cysts (high Na+/K+intracystic ratio and flattened epithelium), type III cysts (intermediate Na+/K+intracystic ratio and mixed epithelium) and in normal women. Serum PRL concentrations corresponding to samples obtained 60 and 90 minutes after stimulation remained higher in the first group of patients. These results led us to consider the existence of an altered central regulation of PRL secretion in patients with type I cysts at presentation.