Henk Portengen

Molecular Classification of Tamoxifen-Resistant Breast Carcinomas by Gene Expression Profiling

PURPOSE To discover a set of markers predictive for the type of response to endocrine therapy with the antiestrogen tamoxifen using gene expression profiling. PATIENTS AND METHODS The study was performed on 112 estrogen receptor-positive primary breast carcinomas from patients with advanced disease and clearly defined types of response (ie, 52 patients with objective response v 60 patients with progressive disease) from start of first-line treatment with tamoxifen. Main clinical end points are the effects of therapy on tumor size and time until tumor progression (progression-free survival [PFS]). RNA isolated from tumor samples was amplified and hybridized to 18,000 human cDNA microarrays. RESULTS Using a training set of 46 breast tumors, 81 genes were found to be differentially expressed (P < or = .05) between tamoxifen-responsive and -resistant tumors. These genes were involved in estrogen action, apoptosis, extracellular matrix formation, and immune response. From the 81 genes, a predictive signature of 44 genes was extracted and validated on an independent set of 66 tumors. This 44-gene signature is significantly superior (odds ratio, 3.16; 95% CI, 1.10 to 9.11; P = .03) to traditional predictive factors in univariate analysis and also significantly related with a longer PFS in univariate (hazard ratio, 0.54; 95% CI, 0.31 to 0.94; P = .03) as well as in multivariate analyses (P = .03). CONCLUSION Our data show that gene expression profiling can be used to discriminate between breast cancer patients with progressive disease and objective response to tamoxifen. Additional studies are needed to confirm if the predictive signature might allow identification of individual patients who could benefit from other (adjuvant) endocrine therapies.

Oncogene amplification and prognosis in breast cancer: Relationship with systemic treatment ☆

In the present study, we aimed to clarify the potential of oncogene amplifications as markers for the prediction of (i) (relapse-free) survival, (ii) response to first-line endocrine therapy and (iii) subsequent chemotherapy in patients with recurrent breast cancer. To attain this goal, amplification of different oncogenes (HER-2/neu, c-MYC and INT-2) was studied in primary tumors of a series of 259 patients with breast cancer (median follow-up of 72 mo). Of these tumors, 49.8% did not contain an amplification of any of the oncogenes studied, whereas in the amplified subgroup, INT-2 was amplified in 13%, HER-2/neu in 24% and c-MYC in 20% of the tumors. In univariate analysis, INT-2 amplification was associated with an increased risk of relapse (p < 0.03), especially in the subgroups of 85 node-negative (p = 0.05) and 156 ER/PgR-positive patients (p = 0.01). Cox multivariate regression analysis showed that c-MYC was the only oncogene whose amplification was significantly related with the rate of relapse. With respect to amplification in patients developing metastatic disease, who received first-line hormonal therapy (n = 114), HER-2/neu amplification was associated with a less favorable response to endocrine therapy (objective response rate only 17% and a progression-free survival (PFS) of only 4% at 12 mo). Interestingly, distinct INT-2 amplification might predict a better response to endocrine therapy (objective response rate of 56%, and a PFS after relapse of 42% at 12 mo).(ABSTRACT TRUNCATED AT 250 WORDS)

The prognostic value of epidermal growth factor receptor (EGF-R) in primary breast cancer: Results of a 10 year follow-up study

SummaryIn a study on 214 patients with primary breast cancer (median follow-up 8.5 yr, maximum follow-up 15 yr), EGF-R was negatively correlated to estrogen receptor and progesterone receptor, whereas no association was found with age, lymph node status, and tumor size. Initially, after a follow-up of 5 yr, there was a tendency to a significant association between EGF-R levels and tumor recurrence rate (p=0.08). Patients with tumors containing intermediate levels of EGF-R experienced a longer relapse-free survival (RFS) than did patients with tumors possessing lower or higher levels of EGF-R. This effect was most pronounced in the subgroup of patients with positive axillary lymph nodes. However, after 10 yr follow-up, this association appears to be lost (p=0.28) as shown in this update. A similar phenomenon was observed for the ER. While at 5 yr follow-up ER status had significant prognostic value (p=0.01), at 10 yr follow-up this significance also appears to be lost (p=0.40). However, tumor size, lymph node status, grade, and PgR status maintained significant prognostic value by univariate analysis.Based on 40 separate studies comprising 5232 patients, the mean percentage of EGF-R positivity reported in breast cancer is 45% (range 14–91%). Nine out of 15 different studies showed in some way a significant negative association between EGF-R and RFS by univariate analysis, and 2 others showed a tendency to such a relationship. Of 7 studies applying multivariate analysis, two demonstrated an independent prognostic value of EGF-R for RFS and two others a tendency to a significant correlation, whereas three did not. It may be concluded that EGF-R status has more or less prognostic value in patients with primary breast cancer, but the prognostic power decreases with longer follow-up. Of great clinical significance is the association of EGF-R with hormone resistance. Therefore EGF-R status can be used for selection of type of treatment. Finally, EGF-R might be useful as a target for new treatment modalities.

Prevalence of amplification of the oncogenes c-myc, HER2/neu, and int-2 in one thousand human breast tumours : correlation with steroid receptors

The frequency of oncogene amplification described in the literature shows a large fluctuation, which could be attributed to the study of relatively small series of tumours, to selection of subgroups of patients, or, especially in retrospective studies, to selection of tumour material from the tumour-bank. To address this question, we have studied amplification of c-myc, HER2/neu and int-2/bcl-1 genes in a series of 1052 collected human breast tumours. The retrospective and prospective subgroups in this collected series of tumours were of equal size. c-myc was amplified in 17.1%, HER2/neu in 18.7% and int-2/bcl-1 in 14.1%, of all breast cancer specimens studied. In the retrospective subgroup the prevalence of amplification was 18.1% for c-myc; 22.6% for HER2/neu and 11.6% for int-2/bcl-1, whereas in the prospective subgroup an incidence of amplification of 16.1%, 15.1% and 16.3% for c-myc, HER2/neu and int-2/bcl-1, respectively was observed. HER2/neu amplification was negatively correlated with oestrogen receptor (ER) and progesterone receptor (PR) status (P less than 0.0001; for both), c-myc amplification was more prevalent in the PR-negative subpopulation (P less than 0.05) and int-2/bcl-1 amplification was positively correlated with ER status (P less than 0.001).

Association of DNA Methylation of Phosphoserine Aminotransferase with Response to Endocrine Therapy in Patients with Recurrent Breast Cancer

To understand the biological basis of resistance to endocrine therapy is of utmost importance in patients with steroid hormone receptor-positive breast cancer. Not only will this allow us prediction of therapy success, it may also lead to novel therapies for patients resistant to current endocrine therapy. DNA methylation in the promoter regions of genes is a prominent epigenetic gene silencing mechanism that contributes to breast cancer biology. In the current study, we investigated whether promoter DNA methylation could be associated with resistance to endocrine therapy in patients with recurrent breast cancer. Using a microarray-based technology, the promoter DNA methylation status of 117 candidate genes was studied in a cohort of 200 steroid hormone receptor-positive tumors of patients who received the antiestrogen tamoxifen as first-line treatment for recurrent breast cancer. Of the genes analyzed, the promoter DNA methylation status of 10 genes was significantly associated with clinical outcome of tamoxifen therapy. The association of the promoter hypermethylation of the strongest marker, phosphoserine aminotransferase (PSAT1) with favorable clinical outcome was confirmed by an independent quantitative DNA methylation detection method. Furthermore, the extent of DNA methylation of PSAT1 was inversely associated with its expression at the mRNA level. Finally, also at the mRNA level, PSAT1 was a predictor of tamoxifen therapy response. Concluding, our work indicates that promoter hypermethylation and mRNA expression of PSAT1 are indicators of response to tamoxifen-based endocrine therapy in steroid hormone receptor-positive patients with recurrent breast cancer.

Insulin-like growth factor-1 receptors and insulin-like growth factor-1-like activity in human primary breast cancer.

In the current study the authors have investigated whether human primary breast cancer specimens contain insulin‐like growth factor‐1 (IGF‐1) receptors (IGF‐1‐R) or IGF‐1‐like activities. Simultaneously, epidermal growth factor (EGF) receptors (EGF‐R) and cytosolic estrogen receptor (ER), progesterone receptor (PR), and EGF‐like activity were determined. All tumors assayed contained a single class of specific iodine 125 (125I)‐IGF‐1 binding sites (Kd: median 106, range 48–755 pM; n = 32) with limited capacity (Bmax: median 147, range 19–11,900 fmol/mg membrane protein). Seventy percent of 44 tumors (50% ER+, PR+), displayed specific 125I‐EGF binding with a wide range of values (median 13, range 2–215 fmol/mg protein, n = 31). A positive relationship was apparent between the amount of IGF‐1‐R with ER and PR (Spearman; 2P < 0.02 and 2P < 0.01, respectively; n = 32), whereas for EGF‐R a negative relationship was observed (for both 2P < 0.01; n = 44). All tumors contained endogeneous IGF‐1‐like and EGF‐like activities as measured by radioreceptor assay on acid‐ethanol extracted cytosols (median 15, range 3–131 ng/mg protein, n = 78 for IGF‐1; and median 86, range 26–517 ng/mg protein, n = 142 for EGF). Tumor contents of IGF‐1‐like and EGF‐like activities showed a negative relationship with ER (2P < 0.1 for IGF‐1, n = 78; and 2P < 0.001 for EGF, n = 142), and with PR (2P < 0.05, n = 78; and 2P < 0.001, n = 142). No relationship was observed between the tumor contents of IGF‐1‐like and EGF‐like activities. In conclusion, these data support the view that IGF‐1 and EGF can act as autocrine or paracrine growth factors in human breast cancer.

Prognostic value of PS2 and cathepsin D in 710 human primary breast tumors: multivariate analysis.

PURPOSE Evaluation of the prognostic value of cytosolic PS2 (pS2 protein) and cathepsin D in a large series of breast cancer patients by multivariate analysis taking into account steroid receptors and conventional prognostic factors. PATIENTS AND METHODS Prognostic factors were analyzed in 710 primary breast cancers (median follow-up, 4 years). PS2 and cathepsin D were measured by radiometric immunoassays. Estrogen receptor (ER) and progesterone receptor (PgR) status were assessed by radioligand binding assays and multiple-point Scatchard analysis. RESULTS The best cutoff point for PS2 to discriminate between positive (61% of the tumors) and negative was 2 ng/mg protein (univariate P value in 5-year relapse-free survival = .003). For cathepsin D, no sensible cutoff point could be chosen, since there was a continuous association between the level of cathepsin D and relapse rate (P = .001). In Cox multivariate analysis, relapse rate decreased with age of premenopausal/perimenopausal patients and with PS2 or steroid receptor positivity, and increased with the size of the tumor, the number of positive lymph nodes, and increasing levels of cathepsin D. In analysis for overall survival, age of both premenopausal/perimenopausal and postmenopausal patients, tumor size, the number of positive lymph nodes, ER/PgR, and PS2 were all independently associated with the rate of death. The level of cathepsin D was positively correlated with the rate of death, but this trend was not statistically significant. Separate Cox multivariate analyses for relapse-free survival in subgroups of patients as defined by nodal status showed that the contribution of PS2 and cathepsin D was the strongest in the node-negative subgroup. Node-negative patients with tumors containing PS2 values < or = 2 ng/mg protein and cathepsin D values more than 70 pmol/mg protein experienced a 4.5-fold increase in relapse rate as compared with those with PS2 levels greater than 2 ng/mg protein and cathepsin D levels < or = 30 pmol/mg protein. CONCLUSION PS2 and cathepsin D are independent prognostic factors in primary breast cancer and lymph node-negative patients.

Which Cyclin E Prevails as Prognostic Marker for Breast Cancer? Results from a Retrospective Study Involving 635 Lymph Node–Negative Breast Cancer Patients

Purpose: To evaluate the prognostic value of cyclin E with a quantitative method for lymph node–negative primary breast cancer patients. Patients and Methods: mRNA transcripts of full-length and splice variants of cyclin E1 (CCNE1) and cyclin E2 (CCNE2) were measured by real-time PCR in frozen tumor samples from 635 lymph node–negative breast cancer patients who had not received neoadjuvant or adjuvant systemic therapy. Results: None of the PCR assays designed for the specific splice variants of the cyclins gave additional prognosis-related information compared with the common assays able to detect all variants. In Cox multivariate analysis, corrected for the traditional prognostic factors, high levels of cyclin E were independently associated with a short distant metastasis-free survival [hazard ratio (HR), 3.40; P < 0.001 for CCNE1 and HR, 1.76; P < 0.001 for CCNE2, respectively]. After dichotomizing the tumors at the median level of 70% tumor cells, the multivariate analysis showed particularly strong results for CCNE1 in the group of 433 patients with stroma-enriched primary tumors (HR, 5.12; P < 0.001). In these tumors, the worst prognosis was found for patients with estrogen receptor–negative tumors expressing high CCNE1 (HR, 9.89; P < 0.001) and for patients with small (T1) tumors expressing high CCNE1 (HR, 8.47; P < 0.001). Conclusion: Our study shows that both CCNE1 and CCNE2 qualify as independent prognostic markers for lymph node–negative breast cancer patients, and that CCNE1 may provide additional information for specific subgroups of patients.

How ADAM-9 and ADAM-11 Differentially From Estrogen Receptor Predict Response to Tamoxifen Treatment in Patients with Recurrent Breast Cancer: a Retrospective Study

Purpose: To evaluate the predictive value of the disintegrin and metalloproteinases, ADAM-9, ADAM-10, ADAM-11, and ADAM-12, and of the matrix metalloproteinases, MMP-2 and MMP-9, in patients with recurrent breast cancer treated with tamoxifen. Experimental Design: A retrospective study was done on 259 frozen specimens of estrogen receptor–positive primary breast carcinomas from patients who developed recurrent disease and were treated with tamoxifen as the first line of therapy. The expression levels of the biological factors were assessed by real-time quantitative reverse transcriptase PCR. Results: Using log-transformed continuous variables, increasing levels of ADAM-9 [odds ratio (OR) = 1.41; P = 0.015] and decreasing levels of MMP-9 (OR, 0.81; P = 0.035) predicted favorable disease control independent from the traditional predictive factors. Furthermore, when tumors were dichotomized at the median level of 70% tumor cell nuclei, our univariate analysis showed particularly strong results for the group of 153 patients with primary tumors containing 30% or more stromal cells. Although estrogen receptor levels lost their predictive power for this group of patients, high levels of ADAM-9 (OR, 1.59; P = 0.007) and ADAM-11 (OR, 1.65; P = 0.001) were significantly associated with a higher efficacy of tamoxifen therapy. Conclusions: Our results show that especially for primary tumors containing stromal elements, the assessment of mRNA expression levels of ADAM-9 and ADAM-11 could be useful to identify patients with recurrent breast cancer who are likely to benefit or fail from tamoxifen therapy.

p53 protein accumulation predicts poor response to tamoxifen therapy of patients with recurrent breast cancer.

PURPOSE Mutations of the p53 gene are frequently observed in primary breast cancer and accumulation of p53 protein has been used as a surrogate marker of p53 inactivation. Previous studies have shown that p53 accumulation is related to poor prognosis in primary breast cancer. We studied whether p53 protein accumulation is a predictive factor for response to tamoxifen treatment in patients with recurrent breast cancer. PATIENTS AND METHODS Levels of p53, estrogen receptor (ER), progesterone receptor (PgR), and urokinase-type plasminogen activator (uPA) were assayed in cytosolic extracts derived from primary tumors of 401 tamoxifen-naive patients who developed recurrent disease. All patients in the study received tamoxifen therapy upon relapse (median follow-up, 69 months). Association of tested factors with response to tamoxifen treatment was studied by logistic regression analysis, and with survival after the start of treatment by Cox univariate and multivariate regression analysis. RESULTS p53 levels (median, 0.23 ng/mg protein) were not related to ER or PgR levels, but positively correlated with uPA (P < .0001). In a test for trend, we observed an association of p53 protein levels with response to tamoxifen therapy. When dichotomized (at the median value), 42% in the p53-high versus 56% in the p53-low group showed a response. In multivariate analysis, including patient and tumor characteristics, p53 accumulation retained significance with the rate of response (odds ratio [OR], 0.48; 95% confidence interval [CI], 0.31 to 0.74; P < .001). Also in multivariate analysis, reduced survival after the start of tamoxifen therapy was observed in the p53-high group (relative hazards rate [RHR], 1.56, 95% CI, 1.17 to 2.10; P = .002). A statistically significant association between p53 levels and decreased tamoxifen response was seen only in the subset of patients whose tumors expressed low levels of ER or PgR (<75 fmol/mg protein). CONCLUSION Measurement of primary tumor p53 levels may be effective in predicting response to tamoxifen therapy in recurrent breast disease. However, more confirming studies on the association between p53 protein accumulation and response to antiestrogen therapy are needed before tumor p53 levels can be used in routine clinical practice.