TSC22D1 and PSAP predict clinical outcome of tamoxifen treatment in patients with recurrent breast cancer.

Purpose Two genes, TSC22 domain family, member 1 (TSC22D1) and prosaposin (PSAP) were identified in an in vitro functional screen for genes having a causative role in tamoxifen resistance. These genes were also present in our previously established 81-gene signature for resistance to first-line tamoxifen therapy. The aim of this study was to investigate the predictive value of these genes for tamoxifen therapy failure in patients with recurrent breast cancer. Experimental Design The mRNA levels of TSC22D1 and PSAP were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) in 223 estrogen receptor-positive primary breast tumors of patients with recurrent disease treated with first-line tamoxifen therapy. The main objective of this study was the length of progression-free survival (PFS). Results High mRNA levels of TSC22D1 and PSAP were significantly associated with shorter PFS and both were independent of the traditional predictive factors (HR = 1.30, 95% CI = 1.04-1.64 P = 0.023; and HR = 1.40, 95% CI = 1.03-1.88, P = 0.029, respectively). In multivariate analysis, patients with high mRNA levels of both genes associated significantly with no clinical benefit (OR = 0.19, 95% CI = 0.06-0.62, P = 0.006) and had the shortest PFS (HR = 2.05, 95% CI = 1.29-3.25, P = 0.002). Conclusion These results confirm our previous in vitro and tumor-related findings and are indicative for the failure of tamoxifen treatment in breast-cancer patients. Both TSC22D1 and PSAP are associated with clinical outcome and may have a functional role in therapy resistance.

Downregulation of SIAH2, an ubiquitin E3 ligase, is associated with resistance to endocrine therapy in breast cancer.

PURPOSE: In our microarray analysis we observed that Seven-in-Absentia Homolog 2 (SIAH2) levels were low in estrogen receptor (ER) positive breast tumors of patients resistant to first-line tamoxifen therapy. The aim of this study was to evaluate SIAH2 for its (a) predictive/prognostic value, and (b) functional role in endocrine therapy resistance. PATIENTS AND METHODS: SIAH2 expression was measured with quantitative Real-Time-PCR (qRT-PCR) in 1205 primary breast tumor specimens and related to disease outcome. The functional role of SIAH2 was determined in human breast cancer cell lines ZR-75-1, ZR/HERc, and MCF7. Cell lines were treated with estrogen (E2), anti-estrogen ICI164.384 or epidermal growth factor (EGF). Moreover, MCF7 was treated with ICI164.384 after silencing SIAH2 expression. RESULTS: SIAH2 was not prognostic in 603 lymph node negative patients who had not received adjuvant systemic therapy. In multivariate analysis of ER-positive tumors of 235 patients with recurrent disease, SIAH2 as continuous variable, significantly predicted first-line tamoxifen treatment failure (OR = 1.48; P = 0.05) and progression-free survival (PFS) (HR = 0.79; P = 0.007). Furthermore, in primary breast cancer patients treated with adjuvant tamoxifen, SIAH2 predicted metastasis-free survival (MFS) (HR = 0.73; P = 0.005). In vitro experiments showed that SIAH2 silencing in MCF7 cells resulted in resistance to ICI164.384-treatment when compared with mock silenced cells (P = 0.008). Interestingly, in ZR cells transfected with EGFR (ZR/HERc), SIAH2 expression was induced by E2 but downregulated by EGF. CONCLUSION: In primary breast tumor specimens as well as in vitro low SIAH2 levels associated with resistance to endocrine therapy. Moreover, SIAH2 expression showed an opposite regulation by E2 and EGF.

Association of an extracellular matrix gene cluster with breast cancer prognosis and endocrine therapy response.

PURPOSE: We previously discovered an extracellular matrix (ECM) gene cluster associated with resistance to first-line tamoxifen therapy of patients with metastatic breast cancer. In this study, we determined whether the six individual ECM genes [collagen 1A1 (COL1A1), fibronectin 1 (FN1), lysyl oxidase (LOX), secreted protein acidic cysteine-rich (SPARC), tissue inhibitor of metalloproteinase 3 (TIMP3), and tenascin C (TNC)] were associated with treatment response, prognosis, or both. EXPERIMENTAL DESIGN: In 1,286 primary breast tumors, mRNA expression (quantitative real-time PCR) was related to clinicopathologic factors and disease outcome in univariate and multivariate analysis including traditional factors. RESULTS: TIMP3, FN1, LOX, and SPARC expression levels (continuous variables) were significantly associated with distant metastasis-free survival (MFS) in 680 lymph node-negative untreated patients (P<0.03). Using a calculated linear prognostic score, these patients were evenly divided into five prognostic groups with a significant difference in 10-year MFS of approximately 40% between the two extreme prognostic groups. Furthermore, high TNC expression as continuous variable was associated with (a) shorter MFS in 139 estrogen receptor-positive and lymph node-positive patients who received adjuvant tamoxifen therapy (hazard ratio, 1.53; P=0.001), and (b) no clinical benefit (odds ratio, 0.81; P=0.035) and shorter progression-free survival (hazard ratio, 1.19; P=0.002) in 240 patients in whom recurrence was treated with tamoxifen as first-line monotherapy. These results were also significant in multivariate analyses. CONCLUSION: FN1, LOX, SPARC, and TIMP3 expression levels are associated with the prognosis of patients with breast cancers, whereas TNC is associated with resistance to tamoxifen therapy. Further validation and functional studies are necessary to determine the use of these ECM genes in decisions regarding treatment and whether they can serve as targets for therapy.

HOXB13-to-IL17BR expression ratio is related with tumor aggressiveness and response to tamoxifen of recurrent breast cancer: a retrospective study.

PURPOSE: A HOXB13-to-IL17BR expression ratio was previously identified to predict clinical outcome of breast cancer patients treated with adjuvant tamoxifen. However, this ratio may predict a tumor's response to tamoxifen, its intrinsic aggressiveness, or both. PATIENTS AND METHODS: We have measured the HOXB13 and IL17BR expression levels by real-time polymerase chain reaction in 1,252 primary breast tumor specimens. Expression levels were normalized to housekeeper gene levels and related to clinicopathologic factors for all patients. The primary objective of this study was to determine the relationship of a HOXB13-to-IL17BR ratio with tumor aggressiveness and/or with response to tamoxifen therapy in estrogen receptor (ER) -positive disease. We selected ER-positive tumors, and clinical end points for the HOXB13-to-IL17BR ratio were disease-free survival (DFS) in patients with primary breast cancer (N = 619) and progression-free survival (PFS) in patients with recurrent breast cancer treated with first-line tamoxifen monotherapy (N = 193). The odds ratio (OR) and hazard ratio (HR) and their 95% CI were calculated, and all P values were two-sided. RESULTS: The HOXB13-to-IL17BR ratio was significantly associated with DFS and PFS. In multivariate analysis, HOXB13-to-IL17BR ratio expression levels were associated with a shorter DFS for node-negative patients only. Corrected for traditional predictive factors, the dichotomized HOXB13-to-IL17BR ratio was the strongest predictor in multivariate analysis for a poor response to tamoxifen therapy (OR = 0.16; 95% CI, 0.06 to 0.45; P < .001) and a shorter PFS (HR = 2.97; 95% CI, 1.82 to 4.86; P < .001). CONCLUSION: High HOXB13-to-IL17BR ratio expression levels associate with both tumor aggressiveness and tamoxifen therapy failure.

Mismatch repair and treatment resistance in ovarian cancer.

BACKGROUND: The treatment of ovarian cancer is hindered by intrinsic or acquired resistance to platinum-based chemotherapy. The aim of this study is to determine the frequency of mismatch repair (MMR) inactivation in ovarian cancer and its association with resistance to platinum-based chemotherapy. METHODS: We determined, microsatellite instability (MSI) as a marker for MMR inactivation (analysis of BAT25 and BAT26), MLH1 promoter methylation status (methylation specific PCR on bisulfite treated DNA) and mRNA expression of MLH1, MSH2, MSH3, MSH6 and PMS2 (quantitative RT-PCR) in 75 ovarian carcinomas and eight ovarian cancer cell lines RESULTS: MSI was detected in three of the eight cell lines i.e. A2780 (no MLH1 mRNA expression due to promoter methylation), SKOV3 (no MLH1 mRNA expression) and 2774 (no altered expression of MMR genes). Overall, there was no association between cisplatin response and MMR status in these eight cell lines. Seven of the 75 ovarian carcinomas showed MLH1 promoter methylation, however, none of these showed MSI. Forty-six of these patients received platinum-based chemotherapy (11 non-responders, 34 responders, one unknown response). The resistance seen in the eleven non-responders was not related to MSI and therefore also not to MMR inactivation. CONCLUSION: No MMR inactivation was detected in 75 ovarian carcinoma specimens and no association was seen between MMR inactivation and resistance in the ovarian cancer cell lines as well as the ovarian carcinomas. In the discussion, the results were compared to that of twenty similar studies in the literature including in total 1315 ovarian cancer patients. Although no association between response and MMR status was seen in the primary tumor the possible role of MMR inactivation in acquired resistance deserves further investigation.

Molecular profiling of platinum resistant ovarian cancer.

The aim of this study is to discover a gene set that can predict resistance to platinum-based chemotherapy in ovarian cancer. The study was performed on 96 primary ovarian adenocarcinoma specimens from 2 hospitals all treated with platinum-based chemotherapy. In our search for genes, 24 specimens of the discovery set (5 nonresponders and 19 responders) were profiled in duplicate with 18K cDNA microarrays. Confirmation was done using quantitative RT-PCR on 72 independent specimens (9 nonresponders and 63 responders). Sixty-nine genes were differentially expressed between the nonresponders (n=5) and the responders (n=19) in the discovery phase. An algorithm was constructed to identify predictive genes in this discovery set. This resulted in 9 genes (FN1, TOP2A, LBR, ASS, COL3A1, STK6, SGPP1, ITGAE, PCNA), which were confirmed with qRT-PCR. This gene set predicted platinum resistance in an independent validation set of 72 tumours with a sensitivity of 89% (95% CI: 0.68-1.09) and a specificity of 59% (95% CI: 0.47-0.71)(OR=0.09, p=0.026). Multivariable analysis including patient and tumour characteristics demonstrated that this set of 9 genes is independent for the prediction of resistance (p<0.01). The findings of this study are the discovery of a gene signature that classifies the tumours, according to their response, and a 9-gene set that determines resistance in an independent validation set that outperforms patient and tumour characteristics. A larger independent multicentre study should further confirm whether this 9-gene set can identify the patients who will not respond to platinum-based chemotherapy and could benefit from other therapies.

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.

Combined vascular endothelial growth factor and TP53 status predicts poor response to tamoxifen therapy in estrogen receptor-positive advanced breast cancer.

PURPOSE: In recent studies, we showed that TP53 gene mutation or high levels of cytosolic vascular endothelial growth factor (VEGF) in estrogen receptor (ER)-alpha-positive primary breast tumors predict a poor disease outcome for patients treated with first-line tamoxifen for advanced disease. Mutant TP53 may up-regulate VEGF, whereas, on the other hand, wild-type TP53 may decrease VEGF production. EXPERIMENTAL DESIGN: In the present study, we aimed to assess the combined predictive value of TP53 gene mutation and VEGF status of 160 advanced breast cancer patients with ER-positive tumors who were treated with tamoxifen (median follow-up from start of tamoxifen treatment, 64 months). To assess TP53 gene mutation status, the entire open reading frame was sequenced; for VEGF status, an ELISA was used. RESULTS: In univariate analysis, both TP53 gene mutation (28% of the tumors) and a VEGF level above the median value were significantly associated with a short progression-free survival, post-relapse overall survival, and a poor rate of response to tamoxifen. In Cox multivariate regression analysis including the traditional predictive factors, the addition of TP53 gene mutation and VEGF status, alone or in combination, significantly predicted a poor efficacy of tamoxifen treatment. When the two factors were combined, a significantly decreased odds ratio was seen for the rate of response (odds ratio, 0.27). Similarly, an increased hazard ratio (HR) was seen for progression-free survival (HR, 2.32) and post-relapse overall survival (HR, 1.68) in the group with mutant TP53 and high VEGF compared with the group with both risk factors absent. CONCLUSIONS: Combined TP53 gene mutation status and high VEGF levels of ER-positive primary breast tumors independently predict a poor course of the disease of patients with advanced breast cancer treated with tamoxifen. These patients, having unfavorable tumor characteristics, might benefit more from other types of (individualized) treatment protocols.

Associations between common polymorphisms in TP53 and p21WAF1/Cip1 and phenotypic features of breast cancer.

The tumour suppressor gene TP53 and its downstream effector p21 are thought to play major roles in the development of breast cancer. We investigated three common sequence variants in TP53 and p21 for possible associations with the risk of breast cancer and with various phenotypic features of this disease. A total of 351 cases were available for study. Germline DNA obtained from female subjects of similar age but without cancer was used to estimate the TP53 and p21 genotype frequencies in a control population. A single nucleotide polymorphism in intron 2 of p21 was associated with slightly increased breast cancer risk (RR = 1.4, P = 0.011) and with well/moderately differentiated tumour histology (P = 0.029). The 16 bp insertion polymorphism in intron 3 of TP53 was associated with poor histological grade (OR = 2.3, P = 0.013) independently of other pathological features. The codon 31 polymorphism in p21 was strongly linked to negative progesterone receptor status (OR = 3.4, P = 0.0001), suggesting this variant may have functional significance for the progesterone signalling pathway in breast cancer. These results add to the growing body of evidence that genetic variants can influence not only the risk of breast cancer but also the disease phenotype.