Levels of circulating cell-free serum DNA in benign and malignant breast lesions

PURPOSES OF THE STUDY: We analyzed circulating cell-free DNA in the serum of patients with benign and malignant breast disease and in healthy individuals to determine its diagnostic value. BASIC PROCEDURES: Serum samples were obtained from 50 healthy individuals, 33 patients with malignant breast disease and 32 patients with benign breast disease. Circulatory DNA was extracted from serum samples. Cell-free DNA was quantified by real-time quantitative PCR for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Tissue samples from patients with malignant and benign breast lesions were histopathologically examined. MAIN FINDINGS: The mean levels of circulating cell-free DNA in serum samples were 41,149 genome equivalents (GE)/mL in patients with malignant disease, 30,826 GE/mL in patients with benign disease, and 13,267 GE/mL in healthy individuals. Healthy individuals had significantly lower levels of cell-free DNA than patients with malignant or benign breast disease (p=0.001, p=0.031). No significant difference was observed between malignant and benign disease. There was a correlation between cell-free DNA levels and tumor size but not with other tumor characteristics. PRINCIPAL CONCLUSION: Our results suggest that levels of circulating cell-free DNA in serum could have diagnostic value to discriminate between healthy individuals and patients with breast lesions but not between patients with malignant and benign breast lesions.     

Transforming growth factor beta 1 serum levels in patients with preinvasive and invasive lesions of the breast

Transforming growth factor beta (TGF-beta)1 is thought to be involved in breast carcinogenesis. TGF-beta1 acts in an antiproliferative manner in the early stages of breast carcinogenesis, but promotes tumor progression and metastases in the advanced stages of the disease. No data have been published on serum TGF-beta1 in breast cancer. We investigated TGF-beta1 serum levels in patients with breast cancer (n=135), ductal carcinoma in situ (DCIS) I to III (n=67) or fibroadenoma (n=35), and in healthy women (n=40) to determine its value as a differentiation marker between malignant, pre-invasive and benign diseases and as a predictive marker for metastatic spread. Median (range) TGF-beta1 serum levels in patients with breast cancer, DCIS I-III or benign breast lesions and in healthy women were 48.8 (18-82.4) pg/mL, 45.3 (26.9-58.3) pg/mL, 47.2 (17.2-80.5) pg/mL and 51.6 (30.9-65.1) pg/mL, respectively (p=0.2). In breast cancer patients TGF-beta1 serum levels showed no statistically significant correlation with tumor stage, lymph node involvement, histological grade, estrogen receptor status and progesterone receptor status. Our data fail to indicate any correlation between serum TGF-beta1 levels and clinicopathological parameters of breast diseases. Serum TGF-beta1 levels do not provide clinical information in addition to established tumor markers.     

Evaluation of cell-free tumour DNA and RNA in patients with breast cancer and benign breast disease

High levels of DNA and RNA released by apoptotic and necrotic cells circulate in the blood of cancer patients. In the present study we determined the applicability of the quantification of nucleic acids and their genetic alterations as minimally invasive tool for breast cancer screening. The relative concentrations of DNA and RNA were determined in preoperative serum of 102 breast cancer patients, 32 patients with benign breast disease and 53 healthy women. The mean follow-up time of the cancer patients was 6.2 years. Loss of heterozygosity (LOH) at four polymorphic markers (D13S159, D13S280, D13S282 at region 13q31-33 and D10S1765 at PTEN region 10q23.31) was analyzed by PCR-based fluorescence microsatellite analyses using cell-free DNA. The serum levels of DNA (p = 0.016) and RNA (p = 0.001) could differentiate between healthy women and cancer patients, but could not discriminate malignant from benign breast lesions. A significant correlation of serum DNA with RNA levels was observed in all groups (p = 0.018). Increased serum DNA levels (but not RNA levels) in cancer patients were associated with a poorer overall (p = 0.021) and disease-free survival (p = 0.025). The occurrence of LOH at all markers significantly correlated with lymph node status (p = 0.026). In addition, the LOH frequency at D13S280 (p = 0.047) and D13S159 (p = 0.046) associated with overall and disease-free survival, respectively. In conclusion, the quantification of cell-free tumour DNA had diagnostic and prognostic values in breast cancer patients, and DNA loss at the region 13q31-33 may be an indication of lymphatic tumour cell spread.     

Breast cancer diagnostics based on extracellular DNA and RNA circulating in blood

Extracellular DNA and RNA were extracted from blood plasma and cell surface-bound fractions of healthy women and patients with fibroadenoma and breast cancer. Frequency of methylation of RASSF1A, Cyclin D2, and RARβ2 genes was detected in the extracellular DNA using methylation-specific PCR. Methylation of at least one of these genes was found in plasma of 13% patients with nonmalignant breast fibroadenoma and in 60% of breast cancer patients. Employment cell-surface bound DNA as the substrate for PCR increased the detection frequency of gene methylation up to 87% in patients with fibroadenoma and 95% in breast cancer patients. In clinically healthy women the methylation markers have not been found in extracellular DNA. GAPDH, RASSF8, Ki-67 mRNAs, and 18S rRNA copies were quantified using RT-qPCR of extracellular RNA circulating in blood of patients with breast tumors and healthy controls. The major part of blood extracellular RNA is associated with cell surface. ROC analysis has shown that differences in concentrations 18S RNA, RASSF8, and Ki-67 mRNAs in blood plasma are highly sensitive and specific in discrimination of benign and malignant breast tumors. Thus, analysis of methylated forms of tumor suppressor genes in blood extracellular and quantification of specific extracellular RNA circulating in blood plasma may detect mammary gland tumors and discriminate malignant and benign neoplasms.     

A serological marker of the N-terminal neoepitope generated during LOXL2 maturation is elevated in patients with cancer or idiopathic pulmonary fibrosis

ObjectivesLysyl oxidase like 2 (LOXL2) is associated with poor prognosis in idiopathic pulmonary disease (IPF) and cancer. We developed an Enzyme-linked immunosorbent assay (ELISA) targeting the LOXL2 neo-epitope generated through the release of the signal peptide during LOXL2 maturation.Design and methodsAn ELISA targeting the N-terminal site of the human LOXL2 was developed including technical optimization and validation steps. Serum LOXL2 was measured in patients with breast, colorectal, lung, ovarian, pancreatic and prostate cancer, melanoma, IPF and in healthy controls (n = 16).ResultsA technically robust and specific assay was developed. LOXL2 was detectable in serum from healthy controls and showed reactivity towards recombinant LOXL2. Compared to controls, LOXL2 levels were significantly (p < 0.001–0.05) elevated in serum from patients with breast, colerectal, lung, ovarian and pancreatic cancer (mean range: 49–84 ng/mL), but not in prostate cancer (mean: 36 ng/mL) and malignant melanoma patients (41 ng/mL). Serum LOXL2 was elevated in IPF patients compared to healthy controls (mean: 76.5 vs 46.8 ng/mL; p > 0.001)ConclusionsA specific ELISA towards the N-terminal neo-epitope site in LOXL2 was developed which detected significantly elevated serum levels from patients with above-mentioned cancer types or IPF compared to healthy controls.     

Serum levels of tumor necrosis factor alpha correlate with response to neoadjuvant chemotherapy in locally advanced breast cancer

It has been shown that serum levels of tumor necrosis factor alpha (TNF-alpha) are increased in breast cancer patients. There are few data available on the reduction of serum levels of this cytokine following chemotherapy. The aim of this study was to determine the effect of neoadjuvant chemotherapy on serum concentrations of TNF-alpha and the relation to response rates in locally advanced breast cancer. Twenty consecutive patients with non-inflammatory stage III-B breast cancer achieving a partial or complete clinical response to three courses of neoadjuvant chemotherapy followed by modified radical mastectomy were prospectively included in the study and evaluated. Sera were collected before the start and after the termination of chemotherapy. Serum concentrations of TNF-alpha were measured by an ELISA method. The pathological response rates were also evaluated and recorded. The control group consisted of 12 healthy age-matched women. The mean pre-treatment TNF-alpha value of breast cancer patients was 15.9 +/- 0.9 pg/mL while it was 5.8 +/- 1.7 pg/mL in the control group; the difference was statistically significant (p < 0.0001). The serum levels of TNF-alpha were markedly decreased in patients with partial and complete responses compared to pre-treatment values (p < 0.0001). There was also a difference in TNF-alpha levels in patients with partial vs complete responses (p < 0.0001). The relative change between pre- and post-treatment values correlated significantly with the type of response (p = 0.004). These results suggest that the serum concentration of TNF-alpha can be an indicator of response and could be used in clinical decision-making for patients with locally advanced breast cancer.     

乳腺癌患者血清内IL-6 和CCL-18表达及其与临床病理因素的关系

目的:探究乳腺癌患者血清内白细胞介素-6(IL-6)和趋化因子配体-18(CCL-18)表达水平及其与临床病理因素的关系。方法:本研究于2014年4月~2015年4月期间,选择我院收治31例乳腺癌患者(乳腺癌组)、29例良性肿瘤患者(良性肿瘤组)与30例健康体检者(对照组)为研究对象,采用酶联免疫吸附试验(ELISA)法测定所有研究对象的血清IL-6与CCL-18水平,采用免疫组化法检测患者的临床病理参数。结果:乳腺癌组患者血清IL-6和CCL-18水平均显著高于良性肿瘤组和对照组,良性肿瘤组血清IL-6和CCL-18水平高于对照组,差异均有统计学意义(P0.05);血清IL-6S水平与雌激素受体(ER)、肿瘤增殖抗原(Ki67)、肿瘤TNM分期及淋巴转移存在关联(P0.05),血清CCL-18水平与Ki67与肿瘤TNM分期存在关联(P0.05)。结论:IL-6和CCL-18在乳腺癌患者内出现高表达,且均可预示患者肿瘤的发展恶化,影响预后。     

Are 90K/MAC-2BP serum levels correlated with poor prognosis in HCC patients? Preliminary results

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 (< or = 20 ng/mL) had positive prognostic value only when it was associated with 90K levels (p < 0.02, log-rank).     

Lipid peroxidation and antioxidant status in blood and tissue of malignant breast tumor and benign breast disease

Changes in the levels of malondialdehyde (MDA), nitrate and nitrite (as an index of nitric oxide production), lipid hydroperoxide (LOH), total antioxidant capacity (TAC), lipids (total cholesterol and triglycerides) and lipoproteins (HDL- and LDL-cholesterol) were estimated in breast cancer patients (n = 15) and benign breast disease (n = 15). Serum and tissue MDA levels were found to be decreased in breast cancer patients compared to the benign group (p < 0.05). In contrast, nitrate and nitrite levels were increased in serum and tissue of the cancer group compared to benign breast disease patients (p < 0.05). Compared to the benign group, tissue TAC levels were elevated in the breast cancer patient group (p < 0.05). Total cholesterol and HDL-cholesterol were elevated in the benign group compared with cancer patients (p < 0.05). These findings support the hypothesis that lipid peroxidation in serum and tissue of benign breast disease is greater than in breast cancer. However, the enhanced levels of nitric oxide may be in response to inflammation in patients with breast cancer. Total antioxidant status is lower in benign tissue than in cancerous tissue, probably to compensate for this elevated free radical production.     

Association between estradiol, estrogen receptors, total lipids, triglycerides, and cholesterol in patients with benign and malignant breast tumors

This study addresses the correlation between the levels of estradiol (E₂), total lipids, triglycerides, and cholesterol in serum and tissue samples of age-matched patients with benign (40 cases; 16 were premenopausal and 24 were postmenopausal) and malignant (50 cases; 17 were premenopausal and 33 were postmenopausal) breast tumors. Estradiol levels were determined in serum and cytosol, estrogen receptors (ER) were assayed in cytosol, and total lipids, triglycerides and cholesterol were determined in serum and membrane fractions of all benign and malignant breast disease patients. Serum E₂ was significantly higher in malignant cases than benign ones (P<0.05) with a significant reduction (40%) in postmenopausal than premenopausal women. ER-positive tumors were significantly higher in postmenopausal women with malignant breast tumors than benign cases (P<0.05). Tissue levels of total lipids, triglycerides, and cholesterol were highly significantly increased in breast cancer women than women with benign breast diseases (P<0.05, P<0.005 and P<0.05 respectively) and they were also significantly correlated with estradiol levels. It could be concluded that the uptake of lipids from plasma by the tumor tissue is greatly correlated to estradiol and it may confirm the possible role of lipids as risk factor in breast cancer.     

Circulating CA 15.3 levels in breast cancer. Our present experience

CA 15.3 is an antigen expressed by human breast carcinoma cells, and defined by two monoclonal antibodies, 115D8 and DF3. We used IRMA to determine the circulating serum levels of CA 15.3 in 1178 subjects with breast cancer, non-breast malignancies, benign diseases and controls. A threshold level of 40 U/ml was established with 140 healthy controls and 650 patients with benign diseases (respectively 0% subjects and 1.5% patients had abnormal antigen levels). Elevated CA 15.3 was found in 12 of 184 patients with malignancies different from breast cancer (6.5%), either epithelial carcinomas with distant metastases, mainly in the liver, or primary liver tumors. Breast cancer patients (n = 204) were analysed by prior therapy, UICC stage and WHO response to therapy. Eight of 134 (5.9%) patients with stage II or III breast cancer at presentation and no evidence of disease (NED) had elevated CA 15.3. All of 22 patients with stage IV breast cancer not responding to therapy (SD and PD) had antigen levels greater than 40 U/ml, as did 10 of 34 (29.4%) stage IV patients in objective response (CR + PR). Three of 14 pretreatment patients had abnormal marker levels, and they later proved to have distant metastases. Serum CA 15.3 values were statistically different (p less than 0.01) in NED (20.6 +/- 11.2 U/ml), CR + PR (33.5 +/- 24.0 U/ml), stable disease (98.8 +/- 50.4 U/ml) and progressive disease (greater than 200 U/ml) breast cancer patients. Our results suggest that circulating CA 15.3 antigen levels agree with the stage of breast cancer and with the response to therapy.     

Serum HER-2 extracellular domain: relationship with clinicobiological presentation and prognostic value before and after primary treatment in 701 breast cancer patients

PURPOSE: To determine the clinical correlations and prognostic value of serum HER-2 (sHER-2) before and after primary breast cancer treatment. METHODS: sHER-2 from 701 consecutive patients with stage I-III tumors (median follow-up 7.7 years) was assayed by an enzyme-linked immunosorbent assay (Immuno 1, Bayer Diagnostics). RESULTS: The median pretreatment sHER-2 concentration was 8.30 ng/mL (range 3.15-82.00 ng/mL). Forty-seven patients (6.7%) had sHER-2 concentrations >12 ng/mL (cutoff level). Pretreatment sHER-2 correlated positively with CA 15.3 (p=0.0169), pathological tumor size (p=0.0082), number of invaded lymph nodes (pN, p=0.0160) and histological grading (p=0.0086). Kaplan-Meier analyses indicated that pretreatment sHER-2 was of prognostic value for contralateral breast cancer (p=0.0018), metastasis-free survival (MFS) (p=0.0008) - particularly lung (p=0.0082) and liver metastases (p=0.0035) - and overall disease-specific survival (DSS) (p=0.0020). According to pN status, pretreatment sHER-2 was of prognostic value only for pN-positive patients (p=0.0017). When combined with estradiol or progesterone receptor status, patients with elevated sHER-2 and receptor-negative tumors had a significantly shorter DSS (p<0.0001 for both receptors). Post-treatment sHER-2 also had individual prognostic value for MFS (p=0.0144) and DSS (p=0.0212). In multivariate analysis, only sHER-2 after primary treatment was an independent prognostic variable for MFS and DSS (p=0.0078 and p=0.0058, respectively). CONCLUSION: sHER-2 elevation in early breast cancer correlates with the principal criteria of tumor aggressiveness, thus permitting selection of patients with a high risk of visceral metastases and contralateral breast tumors. Post-treatment sHER-2 is an independent prognostic factor enabling to identify patients likely to benefit from aggressive adjuvant treatments.     

Monitoring therapy by serum HER-2/neu

We have evaluated the performance of the Bayer Immuno 1 serum HER-2/neu assay. The precision is excellent and varied between 1.7% and 2.1% at values of HER-2/neu ranging from 16.8 ng/mL to 108.6 ng/mL. In normal women who were followed on a monthly basis the average deviation was 6%. The concentration (mean +/- SD) in normal women was 8.7 +/- 3.2 ng/mL. There was no difference between pre and postmenopausal women. The normal/abnormal cutoff was defined as greater than 15 ng/mL. In normal women and those with benign disease the specificity varied between 95% and 100%. In women with breast cancer the sensitivity was 1.7% in stage I disease, 3.0% in stage II, 16.7 in stage III and 35.5% in stage IV. There were 56 elevations (14.7%) in the total group of 285 women with breast cancer. There was an excellent correlation with a microtitre plate method (r=0.9944). Longitudinal studies showed clearly that women who expressed HER-2/neu in their tissue had elevated serum levels and these levels reflected the clinical course of the patient. More extensive control studies are required to establish the role of HER-2/neu assays in the management of women with breast cancer.     

Assessment of Ki-67 as a potential biomarker in patients with breast cancer

Breast cancer is the most common cancer in females, it accounts for one third of all malignancies affecting women. Appropriate biomarkers play significant role in predicting the prognosis and decide the specific therapy to each patient. In this study we aimed at evaluating the value of Ki-67 as a prognostic marker in breast cancer patients and to analyze the associations between Ki-67 and their clinicopathological parameters. This study included 92 patients with developed non metastatic breast cancer and 10 women had benign breast tumor served as controls. We measured the serum level by ELISA technique and tissue expression of Ki-67 by immunohistochemical technique. Our results showed that there were no statistically significant differences in serum Ki-67 levels between the two studied groups. As for Ki-67expression in breast cancer cells, the score increases with increase of tumor size, grade, premenopausal, Ki-67 expression in estrogen and progesterone receptor positive tumors showed lower values than estrogen and progesterone negative tumors, while higher Ki-67 expression was more frequently associated with HER2-positive. In conclusion; our study supports the finding that tissue Ki-67 expression may add prognostic information to that obtained from classical prognostic factors and can provide data of significant value to other important prognostic indicators such as pathological grading, and axillary lymph node involvement.     

Telomerase activity in breast cancer in Western India (Gujarat)

The purpose of this study was to examine the association of telomerase activity with clinical and histopathological prognostic variables in primary breast cancer (n=64). Telomerase activity in breast cancer was also compared with that in benign (n=10) and non-malignant tissues (n=8; post-lumpectomy tissues histopathologically defined as containing no residual tumor). The parameter was assessed using the Telomerase PCR ELISA kit. Values above OD 0.120 were considered positive. Estrogen and progesterone receptors (ER and PgR) were assayed by the dextran-coated charcoal method and levels >10 fmol/mg cytosol protein were taken as positive. Telomerase activity was detected in 20% and 50% of the patients with benign lesions and primary breast cancer, respectively, and in 50% of post-lumpectomy breast tissues histopathologically defined as containing no residual tumor. Telomerase activity was present in all stages of breast carcinoma and showed a significant inverse correlation with lymph node status (p=0.006), lymphatic invasion (p=0.035) and necrosis (p=0.033). Moreover, when stage II patients were grouped according to nodal involvement, a trend towards significance was observed (p=0.055). No correlation was observed with ER and PgR. The results of our study suggest that telomerase activity might be associated with the presence of cancer cells. Furthermore, telomerase activation may occur early in breast cancer and may be periodically downregulated during subsequent tumor progression.     

Reliability and discriminant validity of HER2 gene quantification and chromosome 17 aneusomy analysis by real-time PCR in primary breast cancer

There is an increasing demand for the evaluation of HER2 status in breast cancer. In this study, sections from fixed tissues and triton extracts of tissue homogenates were obtained from 163 malignant breast tumors and analyzed in parallel using immunohistochemistry combined with fluorescence in situ hybridization, as gold standard tests, and an ELISA test (c-erbB2/c-neu Rapid Format ELISA, Oncogene Research Products, USA). Tumor DNA was employed to evaluate two quantitative PCR methods: the HER2/neu DNA Quantification Kit (Roche Diagnostics GmbH, Germany), which uses the gastrin chromosome 17 reference gene, and our recently developed Oncolab qPCR assay, where both a chromosome 17 gene (somatostatin receptor type II (SSTR2)) and a non-chromosome 17 reference gene (glyceraldehyde-3-phosphate deshydrogenase (GAPDH)) were used to detect an increase in HER2 gene copy number and to evaluate the aneusomy of chromosome 17, respectively. By IHC/FISH and ELISA, HER2 was overexpressed in 27 (16.6%) and 24 (14.7%) samples, respectively. With the Roche and Oncolab qPCR assays, 29 (17.8%) samples showed a ratio of HER2/gastrin > or = 2.0 and 26 (16.0%) showed a ratio of HER2/SSTR2 > or = 2.0, respectively. In samples presenting HER2/SSTR2 <2.0 and HER2/GAPDH > or = 2.0, which was indicative of a chromosome 17 polysomy, we observed a modest increase in HER2 protein expression. Complete agreement between the four methods for HER2 status determination was obtained for 154 (94.5%) samples. Overall, these results demonstrate that quantitative PCR is a reliable method for analyzing HER2 status and chromosome 17 polysomy.     

Clinical significance of plasma MMP‐2 and MMP‐9 levels as biomarkers for tumor expression in breast cancer patients in Egypt

Matrix metallopeptidase 2 (MMP-2) and matrix metallopeptidase 9 (MMP-9) are involved in the breakdown of extracellular matrix in normal physiological processes as well as in disease processes, such as cancer metastasis. We conducted this work to study the role of MMP-2 and MMP-9 in breast cancer by measuring their plasma concentrations before and after surgery. Also, to examine if their levels can reflect the stage of disease and prognosis. Forty-eight breast cancer patients and 13 patients with benign breast diseases were included in the study. MMP-2 and MMP-9 levels were measured by ELISA and semi-quantitative real-time PCR. MMP-2 and MMP-9 levels in plasma were determined by ELISA immediately before surgery and during 6 to 12 months after curative surgery. We observed a significant increase in the level of MMP-9 mRNA expression in breast cancer patients in comparison to their normal breast tissues and to tissues of benign breast disease. In all TNM tumor stages, the plasma levels of MMP-2 and MMP-9 were increased significantly before curative surgery in the studied patients with breast carcinoma and decreased significantly after surgery. Both MMP-2 and MMP-9 may be used as a possible marker for follow-up or as a marker that reflects the response of the disease to treatment.

     

The clinical significance of thymidine kinase 1 measurement in serum of breast cancer patients using anti-TK1 antibody

The activity of total thymidine kinase in serum (S-TK) has been used as a tumor maker for decades. To date such activity has been determined using [125]I-iodo-deoxyuridine as a substrate. The aim of this study was to develop a new, antibody-based technique for the measurement of cytoplasmic thymidine kinase (TK1) in serum. Both mono- and polyclonal antibodies against S-TK1 were used in dot blot assay. S-TK1 was characterized by SDS and IEF techniques. Sixty-five breast cancer patients were studied, including 17 preoperative and 38 postoperative tumor-free patients and 10 patients with metastases to the lymph nodes (N1-2). They were compared to patients with benign tumors (n=21) and healthy volunteers (n=11). S-TK1 was low (0-1.0 pM) in healthy volunteers, while in preoperative patients the level was increased 6-110-fold. Significant differences were observed between preoperative patients and healthy volunteers (p=0.005), preoperative patients and patients with benign tumors (p<0.001), and preoperative patients and postoperative patients without metastases (p<0.001). No significant difference was observed between preoperative patients and postoperative patients with metastases (p=0.191). The S-TK activity in preoperative patients was also high in serum, but no decrease was observed following surgery. In conclusion, the anti-TK1 antibody could be a good marker for monitoring the response of breast cancer patients to therapy.     

Clinical significance of cathepsin D concentration in tumor cytosol of primary breast cancer

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.     

Surrogate markers of tumoral angiogenesis

BACKGROUND: Angiogenesis is a prerequisite for tumor growth and metastasis. Vascular cell adhesion molecule1 (VCAM-1) is expressed on endothelial cells as a result of vascular endothelial growth factor (VEGF) stimulation. PURPOSE: To determine if measurement in serum of VEGF or VCAM-1 provides an accurate measure of tumor angiogenesis. METHODS: VCAM-1 and VEGF were measured in the serum of women with early and advanced breast cancer by ELISA. Levels were compared to levels of VCAM-1 and VEGF in women with normal breasts and levels of the endothelial glycoprotein von Willebrand factor. Levels of VEGF and VCAM-1 in women with early breast cancer were correlated with established clinicopathological prognostic markers and intratumoral microvessel density (IMD). RESULTS: In early breast cancer serum VCAM-1 correlated closely with the microvessel density in tumors (r=0.61, p<0.001). Women with lymph node-positive and high-grade tumors had higher levels of serum VCAM-1 than women with lymph node-negative and low-grade tumors. Serum VEGF demonstrated no correlation with established prognostic features or IMD. Levels of VCAM-1 and VEGF were raised in women with advanced breast cancer. CONCLUSION: Serum VCAM-1 is a surrogate marker of angiogenesis in breast cancer and its measurement may help in the assessment of antiangiogenic drugs currently in phase II trials.