Characterization of breast cancer subtypes by quantitative assessment of biological parameters: relationship with clinicopathological characteristics, biological features and prognosis.

OBJECTIVES: Gene expression analysis has identified several breast cancer subtypes, including luminal, epidermal growth factor receptor-2 positive (HER2+), and basal-like. To determine if our proposed molecular taxonomy correlates with biological and clinical behavior. This is based on four biological markers: estrogen and progesterone receptors (ER and PR, respectively), HER2 and the epidermal growth factor receptor-1 (HER1), all of them being determined by quantitative assays. STUDY DESIGN: The biological parameters were examined by enzyme immunoassay, radioligand-binding assay or ELISA, in tumors from 787 patients with invasive breast cancer. Patients were prospectively evaluated over a median follow-up period of 50 months. Subtype definitions were as follows: luminal (ER+), HER2+ (HER2+, ER-, PgR-) and basal-like (HER2-, ER-, PgR-). In addition, we divided basal tumors into two groups based on their HER1 status. RESULTS: A 55.8% of tumors were of luminal type, 11.9% basal-like HER1+, 10.7 basal-like HER1-, and the remainder 21.6% HER2+. Both HER2+ and basal-like subtypes were more frequent in younger and premenopausal women, showing a higher percentage of cases of poorly differentiated tumors and higher S-phase fraction, when compared with those of luminal subtype. Multivariate analysis demonstrated that the subtype of tumor was related to both relapse and overall survival, being those of luminal subtype associated with the best prognosis. CONCLUSIONS: Through the classification of breast tumors in four groups, according to their ER, PgR, HER2 and HER1 status, it is possible to obtain a major division of breast tumors associated with significant differences in biological features and clinical behavior.

Overexpression of matrix metalloproteinases and their inhibitors in mononuclear inflammatory cells in breast cancer correlates with metastasis-relapse.

An immunohistochemical study was performed using tissue microarrays and specific antibodies against matrix metalloproteinase (MMP)-1, -2, -7, -9, -11, -13 and -14, tissular inhibitors of metalloproteinase (TIMP)-1, -2 and -3. More than 2600 determinations on cancer specimens from 131 patients with primary ductal invasive tumours of the breast were performed. To identify specific groups of tumours with distinct expression profiles the data were analysed by unsupervised hierarchical cluster analysis by each cellular type. We did not find well-defined cluster of cases for tumour cells or fibroblastic cells. However, for mononuclear inflammatory cells the dendogram shows a first-order division of the tumours into two distinct MMP/TIMP molecular profiles, designated group 1 (n=89) and group 2 (n=42). Matrix metalloproteinase-7, -9, -11, -13 and -14, and TIMP-1 and -2, were identified as showing significant high expression in group 2 compared with group 1. Multivariate analysis demonstrated that clustering for mononuclear inflammatory cells was the most potent independent factor associated with distant relapse-free survival (group 2: 5.6 (3.5-9.6), P<0.001). We identify a phenotype of mononuclear inflammatory cells infiltrating tumours, which is associated with the development of distant metastasis. Therefore, this finding suggests that these host inflammatory cells could be a possible target for inhibition of metastasis.

Study of matrix metalloproteinases and their inhibitors in breast cancer.

An immunohistochemical study was performed using tissue microarrays and specific antibodies against matrix metalloproteinases (MMPs) 1, 2, 7, 9, 11, 13, 14, and their tisullar inhibitors (TIMPs) 1, 2, and 3. More than 2600 determinations on cancer specimens from 131 patients with primary ductal invasive tumours of the breast (65 with and 66 without distant metastasis) and controls were performed. Staining results were categorised using a score based on the intensity of the staining and a specific software program calculated the percentage of immunostained cells automatically. We observed a broad variation of the total immunostaining scores and the cell type expressing each protein. There were multiple and significant associations between the expression of the different MMPs and TIMPs evaluated and some parameters indicative of tumour aggressiveness, such as large tumour size, advanced tumour grade, high Nottinham prognostic index, negative oestrogen receptor status, peritumoural inflammation, desmoplastic reaction, and infiltrating tumoural edge. Likewise, the detection of elevated immunohistochemical scores for MMP-9, 11, TIMP-1, and TIMP-2, was significantly associated with a higher rate of distant metastases. The expression of MMP-9 or TIMP-2 by tumour cells, MMP-1, 7, 9, 11, 13, or TIMP-3 by fibroblastic cells, and MMP-7, 9, 11, 13, 14, TIMP-1, or TIMP-2 by mononuclear inflammatory cells, was also significantly associated with a higher rate of distant metastases.

Cytosolic levels of TFF1/pS2 in breast cancer: Their relationship with clinical-pathological parameters and their prognostic significance.

BACKGROUND: The Trefoil Factor 1 (TFF1/pS2), a peptide consisting of 60 amino acids, is the most abundant estrogen-induced messenger RNA present in MCF-7 breast cancer cells. The objective of this work was to evaluate the cytosolic TFF1 content in breast carcinomas, its possible relationship with different clinical-pathological parameters, and its potential prognostic significance and predictive value. METHODS: Cytosolic TFF1 levels were examined by immunoradiometric assay in 1031 patients with invasive breast cancer. The median follow-up period was of 50 months. RESULTS: There was a wide variability of cytosolic TFF1 levels in tumors (0.9-743.2 ng/mg protein). Statistical analysis showed that TFF1 levels were significantly higher in premenopausal patients (p = 0.001), as well as in tumors showing any of the following characteristics: good differentiation (p = 0.0001), ER and PgR positivity (p = 0.0001 and p = 0.001, respectively), diploidy (p = 0.045) and a high S-phase fraction (p = 0.001). In addition, the presence of high intratumoral TFF1 levels (cut-off: 2 ng/mg protein) was independently associated with a shorter overall survival in the group of patients as a whole (p = 0.001) as well as in the subgroup with node-negative breast cancer (p = 0.0004). Likewise, high intratumoral TFF1 levels were associated with a more prolonged overall survival in patients who received adjuvant tamoxifen (p = 0.004). CONCLUSIONS: In breast cancer patients, intratumoral TFF1 levels are associated with a better clinical outcome, especially in those with node-negative tumors. In addition, TFF1 levels have a low but significant predictive value in regards to response to adjuvant therapy with tamoxifen.

Expression and prognostic value of EGFR in invasive breast cancer.

Epidermal growth factor receptor (EGFR) is a membrane receptor expressed in a variety of solid human cancers and directly related with poor prognosis. The objective of this work was to evaluate the EGFR content in breast carcinomas, its possible relationship with different clinical-pathological parameters, and its potential prognostic significance and predictive value. EGFR levels were examined by radioligand binding assays in 846 patients with invasive breast cancer. The median follow-up period was 50 months. There was a wide variability of EGFR levels among the studied tumors (0.01-403 fmol/mg protein). Statistical analysis showed that EGFR levels were significantly higher in younger patients (p=0.0001). EGFR were also notably higher in ER-negative or PgR-negative tumors than in ER-positive (p=0.0001) or PgR-positive tumors (p=0.001). In addition, the presence of high intratumoral EGFR levels (cut-off: 6 fmol/mg protein) was associated with both shorter relapse-free survival (p=0.04) and overall survival (p=0.01) in the group of patients as a whole, as well as with overall survival in the subgroup of patients without any type of systemic adjuvant treatment (p=0.02). However, EGFR levels did not achieve significance as independent prognostic factor in the multivariate analysis. There is a wide variability of intratumoral EGFR levels in breast carcinomas, and these protein levels correlated positively with a poor prognosis in the t univariate analysis. However, further studies are necessary in order to assess the possible clinical value of EGFR in combination with other essential components of the EGFR family network.

Significance of cytosolic hyaluronan levels in gastric cancer.

BACKGROUND: Hyaluronan a high-molecular weight glycosaminoglycan, is considered to be involved in the growth and progression of malignant tumours. The objective of this work was to evaluate the cytosolic hyaluronan content in gastric cancer cells, its possible relationship with clinicopathological tumour parameters and its potential prognostic significance. METHODS: Cytosolic hyaluronan levels were examined utilizing immunoenzymatic techniques in 129 patients with gastric cancer. The mean follow-up period for these patients was 28 months. RESULTS: Cytosolic hyaluronan levels ranged widely in tumours as well as in adjacent mucosal samples (median (range) 2822 (50-24,523) versus 3650 (507-20,782) ng/mg protein). Statistical analysis showed that tumour hyaluronan levels correlated significantly with patient's sex and the presence of lymphatic invasion. In addition, high tumour hyaluronan levels were significantly associated with shorter overall survival period (p<0.05). CONCLUSIONS: Our results suggest that high tumoral cytosolic hyaluronan levels are associated with lesions of unfavorable outcome in gastric cancer patients. Thus, hyaluronan may provide additional prognostic information to that given by other biochemical markers currently used in gastric cancer.

Membranous levels of c-erbB-2 oncoprotein in gastric cancer: their relationship with clinicopathological parameters and their prognostic significance.

BACKGROUND: The protein encoded by the c-erbB-2 gene is a membrane receptor expressed in a variety of solid human cancers and directly related to poor prognosis. The objective of this work was to evaluate the clinical value of the quantification of membranous oncoprotein levels in gastric cancer. MATERIALS AND METHODS: Membranous c-erbB-2 levels were examined by means of a sandwich immunoenzymatic assay in 82 patients with gastric cancer. The median follow-up period for these patients was 16 months. In addition, c-erbB-2 expression was analyzed by immunohistochemistry in 57 gastric carcinomas. RESULTS: Membranous c-erbB-2 levels ranged widely in the studied tumors (44-112,000 NHU/mg protein). Median c-erbB2 content was significantly higher in intestinal-type tumors than in diffuse-type tumors (p = 0.01). In addition, high levels of c-erbB-2 were significantly associated with shorter relapse-free survival and overall survival in patients with resectable gastric carcinomas (p = 0.01 and p = 0.04, respectively). However, the correlation between immunohistochemistry and ELISA determinations did not reach statistical significance. CONCLUSION: Our results suggest a potential prognostic value of membranous c-erbB-2 quantification by immunoenzymatic assay in gastric cancer. However, its possible role in the selection of patients with a view to the possible introduction of Herceptin as a novel drug against gastric cancer is at present uncertain.

Epidermal growth factor receptor and c-erbB-2 contents in unresectable (UICC R1 or R2) gastric cancer.

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.

Clinical significance of the epidermal growth factor receptor and HER2 receptor in resectable gastric cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR or HER1) and its homolog c-erbB-2 (HER2) are membrane receptors. Both EGFR and HER2 genes are overexpressed in a variety of solid human cancers and are related to poor prognosis of the patients. The objective of this work was to evaluate the EGFR and HER2 contents in resectable gastric cancer, their possible relationship with clinicopathologic parameters of tumors, and their prognostic significance. METHODS: This was a prospective analysis of 63 patients with resectable gastric carcinomas, with a mean follow-up period of 40.7 months. Membranous EGFR levels were examined by radioligand binding assays, and cytosolic HER2 levels were examined by means of an immunoenzymatic assay. RESULTS: There was a wide variability of EGFR (1-1,239 fmol/mg of protein) and HER2 (7-20,863 NHU/mg of protein) levels in tumors. There was no significant correlation between these levels and patient or tumor characteristics. However, high levels of EGFR and HER2 were significantly associated with a shorter overall survival period (P =.03 and P =.02, respectively). CONCLUSIONS: There is a wide variability in membranous EGFR levels and in cytosolic HER2 levels in gastric cancer, which seems to be related to the biological heterogeneity of these tumors. In addition, high tumor EGFR and HER2 levels were associated with an unfavorable outcome in patients with resectable gastric cancer.

Cytosolic levels of an estrogen-induced breast cancer-associated peptide (TFF1/pS2) in colorectal cancer: clinical significance and relationship with steroid receptors.

BACKGROUND: The Trefoil Factor 1 (TFF1/pS2), a peptide consisting of 60 amino acids, is the most abundant estrogen-induced messenger RNA in MCF-7 breast cancer cells and is also expressed by colorectal carcinomas. The objective of this work was to evaluate the cytosolic TFF1 content in colorectal carcinomas, its possible relationship with estrogen and progesterone receptors as well as with clinicopathological tumor parameters, and its potential prognostic significance. METHODS: Cytosolic TFF1 levels were examined by immunoradiometric assay in 178 patients with resectable colorectal cancer. The mean follow-up period was 32 months. RESULTS: There was a wide variability of cytosolic TFF1 levels in tumor-surrounding mucosa samples (0.09-42.5 ng/mg protein) as well as in tumors (0.01-270 ng/mg protein). Comparison of paired mucosa and carcinoma samples showed significantly higher TFF1 levels in tumors (mean: 17.1 ng/mg protein) than in mucosa samples (10 ng/mg protein) (p = 0.027). TFF1 levels were significantly higher in mucosa samples surrounding distal colon and rectal tumors (p = 0.0001) and in tumor samples obtained from older patients (p = 0.007). However, there were no significant differences in tumor TFF1 levels with respect to clinicopathological parameters such as the patient's sex, tumor location, stage, histological grade, ploidy, S-phase, or tumor estrogen and progesterone receptors. In addition, there was no significant relationship between tumor TFF1 levels and disease outcome. CONCLUSIONS: TFF1 may play an as yet undetermined role in the tumorigenesis of colorectal carcinomas. However, cytosolic levels of TFF1 do not seem to have any prognostic significance in colorectal carcinomas.

Prognostic significance of tissue-type plasminogen activator (tPA) content in gastric cancer and surrounding mucosa.

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.

Clinical significance of epidermal growth factor receptor content in gastric cancer.

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.

[Preoperative serum levels of the carcinoembryonic antigen (CEA) and prognosis in colorectal cancer]. / Niveles séricos preoperatorios del CEA y pronóstico en el cáncer colorrectal.

OBJECTIVE: To analyze the prognostic value of the preoperative serum levels of the carcinoembryonic antigen (CEA) in primary colorectal carcinoma. MATERIAL AND METHODS: Preoperative serum levels of CEA were analyzed in 275 colorectal cancer patients, who were followed up for a minimum of 5 years, or until death. RESULTS: The percentage of positivities for the preoperative serum levels of CEA (> 6 ng/ml) was positively and significantly associated with the tumoral stage (A: 10,5%; B: 38,8%; C: 32,2%; y D: 72%; p < 0,0001). In addition, the elevated serum values of the antigen were significantly associated, in the univariate analysis, with short survival in the overall group of patients (p < 0,0001). However, the multivariate analysis only showed an independent prognosis value of the CEA in the subgroup of patients with stage C tumors. CONCLUSIONS: Preoperative serum levels of CEA may be useful to predict tumoral extension, and also for the prognosis regarding stage C colorectal cancer patients.

Prognostic significance of cytosolic pS2 protein content in gastric cancer.

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.

Hyaluronic acid as prognostic marker in resectable colorectal cancer.

BACKGROUND: Hyaluronic acid (HA), an extracellular high molecular mass polysaccharide, is thought to be involved in the growth and progression of malignant tumours. The objective of this work was to evaluate the cytosolic HA content in resectable colorectal cancer, its possible relationship with clinicopathological parameters of tumours and its prognostic significance. METHODS: Cytosolic HA levels were examined by radiometric assay in 120 patients with resectable colorectal cancer. The mean follow-up period was 33.4 months. RESULTS: Cytosolic HA levels of tumours ranged widely, from 30 to 29 412 ng per mg protein. Intratumour HA levels were significantly correlated with Dukes stage (P < 0.005), and were higher in patients with advanced tumours (mean(s.e.m.) 2695(446), 2858(293) and 5274(967) ng per mg protein for stages A, B and C respectively). In addition, Cox multivariate analysis demonstrated that tumour HA levels higher than 2000 ng per mg protein predicted shorter relapse-free survival and overall survival periods (both P < 0.05). CONCLUSION: There is a wide variability in cytosolic HA levels in colorectal carcinomas, which seems to be related to the biological heterogeneity of these tumours. In addition, high tumour cytosolic HA levels were associated with an unfavourable outcome in patients with resectable colorectal cancer. HA may provide additional information to that given by other biochemical markers currently used in colorectal cancer.

Tissue-type plasminogen activator (tPA) content in colorectal cancer and in surrounding mucosa: relationship with clinicopathologic parameters and prognostic significance.

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.

C-erbB-2 oncoprotein content in colorectal cancer and in surrounding mucosa: relationship with clinicopathologic parameters and prognostic significance.

BACKGROUND: c-erbB-2 is a transmembrane signaling molecule closely related in structure to the epidermal-growth-factor receptor (EGFR) but biologically distinct from it. c-erbB-2 has been implicated in cell transformation and tumor pathogenesis, but very little is known about its content and clinical significance in colorectal cancer. AIMS: To evaluate the c-erbB-2 content in colorectal cancer and its possible relationship with clinicopathologic parameters from tumors and prognostic significance. METHODS: Membranous and cytologic c-erbB-2 oncoprotein contents were examined by an immunoenzymatic assay in tumors and paired normal surrounding mucosa samples from 131 colorectal cancer patients. In addition, survival analysis were prospectively performed in a subgroup of 69 consecutive patients with resectable colorectal carcinomas, who underwent a mean follow-up period of 28 months. RESULTS: In the overall group of patients, c-erbB-2 levels were significantly higher in membranous than in cytosolic samples, in neoplastic tissues (5,830.4 +/- 1085.3 vs. 934.2 +/- 107.5 NHU/mg protein; p < 0.0001) and in surrounding normal mucosa samples (5,257.8 +/- 646.3 vs. 837.4 +/- 187.4 NHU/mg protein; p < 0.0001). Nevertheless, a significant positive relation was found between membranous and cytosolic oncoprotein levels in these two paired sets (p < 0.0001, for both). There were no significant differences in membranous or cytosolic c-erbB-2 protein levels between neoplastic tissues and surrounding mucosa samples in this overall group of patients. In addition, the results did not show significant correlations of these oncoprotein contents with clinicopathologic parameters from tumors such as location, stage, histologic grade, and DNA content or S-phase fraction. However, the results indicated that low membranous c-erbB-2 content (< 4,500 NHU/mg protein) in tumors predict shorter relapse-free survival and overall survival (p < 0.05, for both) in resectable colorectal cancer patients. CONCLUSIONS: There are a wide variability of both membranous and cytologic c-erbB-2 contents in colorectal carcinomas, which seems to correspond to the biological heterogeneity of these tumors. In addition, our results also demonstrate that high membranous c-erbB-2 levels are associated with lesions of favorable evolution in resectable colorectal cancer patients.