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Identification of a putative protein profile associated with tamoxifen therapy resistance in breast cancer.

Umar A, Kang H, Timmermans AM, Look MP, Meijer-van Gelder ME, den Bakker MA, Jaitly N, Martens JW, Luider TM, Foekens JA, Pasa-Tolić L - Mol. Cell Proteomics (2009)

Bottom Line: Identification of proteins that are associated with tamoxifen resistance is a first step toward better response prediction and tailored treatment of patients.ENPP1, EIF3E, and GNB4 were significantly associated with progression-free survival upon tamoxifen treatment for recurrent disease.Extracellular matrix metalloproteinase inducer levels were higher in therapy-resistant tumors and significantly associated with an earlier tumor progression following first line tamoxifen treatment (hazard ratio, 1.87; 95% confidence interval, 1.25-2.80; p = 0.002).

View Article: PubMed Central - PubMed

Affiliation: Erasmus Medical Center Rotterdam, Josephine Nefkens Inst., Dept. of Medical Oncology, Laboratory of Genomics and Proteomics of Breast Cancer, Dr. Molewaterplein 50, Be 430c, P. O. Box 2040, 3000 CA Rotterdam, The Netherlands. a.umar@erasmusmc.nl

ABSTRACT
Tamoxifen resistance is a major cause of death in patients with recurrent breast cancer. Current clinical factors can correctly predict therapy response in only half of the treated patients. Identification of proteins that are associated with tamoxifen resistance is a first step toward better response prediction and tailored treatment of patients. In the present study we intended to identify putative protein biomarkers indicative of tamoxifen therapy resistance in breast cancer using nano-LC coupled with FTICR MS. Comparative proteome analysis was performed on approximately 5,500 pooled tumor cells (corresponding to approximately 550 ng of protein lysate/analysis) obtained through laser capture microdissection (LCM) from two independently processed data sets (n = 24 and n = 27) containing both tamoxifen therapy-sensitive and therapy-resistant tumors. Peptides and proteins were identified by matching mass and elution time of newly acquired LC-MS features to information in previously generated accurate mass and time tag reference databases. A total of 17,263 unique peptides were identified that corresponded to 2,556 non-redundant proteins identified with > or = 2 peptides. 1,713 overlapping proteins between the two data sets were used for further analysis. Comparative proteome analysis revealed 100 putatively differentially abundant proteins between tamoxifen-sensitive and tamoxifen-resistant tumors. The presence and relative abundance for 47 differentially abundant proteins were verified by targeted nano-LC-MS/MS in a selection of unpooled, non-microdissected discovery set tumor tissue extracts. ENPP1, EIF3E, and GNB4 were significantly associated with progression-free survival upon tamoxifen treatment for recurrent disease. Differential abundance of our top discriminating protein, extracellular matrix metalloproteinase inducer, was validated by tissue microarray in an independent patient cohort (n = 156). Extracellular matrix metalloproteinase inducer levels were higher in therapy-resistant tumors and significantly associated with an earlier tumor progression following first line tamoxifen treatment (hazard ratio, 1.87; 95% confidence interval, 1.25-2.80; p = 0.002). In summary, comparative proteomics performed on laser capture microdissection-derived breast tumor cells using nano-LC-FTICR MS technology revealed a set of putative biomarkers associated with tamoxifen therapy resistance in recurrent breast cancer.

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Immunohistochemical staining of EMMPRIN. EMMPRIN immunohistochemical staining was performed on an independent sample set of 156 tissues using TMA. A, overview of TMA; B, negatively stained tissue; C, 1+ membrane stain; D, 2+ stain; E, 3+ stain. Overview picture was taken at 5× magnification; other pictures were taken at 100× magnification.
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f7: Immunohistochemical staining of EMMPRIN. EMMPRIN immunohistochemical staining was performed on an independent sample set of 156 tissues using TMA. A, overview of TMA; B, negatively stained tissue; C, 1+ membrane stain; D, 2+ stain; E, 3+ stain. Overview picture was taken at 5× magnification; other pictures were taken at 100× magnification.

Mentions: To independently validate differential EMMPRIN protein abundance between OR and PD patients, IHC was performed using our primary breast cancer TMA. Among the different tissues, there were 156 breast tumors of patients who received first line tamoxifen therapy after recurrence. This set of tumors had no overlap with the discovery set tumors. In total, 130 tumors showed reproducible IHC staining on the TMA when assays were performed in triplicate. Patient and tumor characteristics are described in Table IV. Different staining outcomes were categorized as undetectable, weak, medium, and strong membrane staining. Weak membrane staining, present in <10% of tumor cells, was scored as 1+. Medium membrane staining, present in 10–50% of tumor cells, was scored as 2+. Strong membrane staining, observed in >50% of tumor cells, was assigned score 3+ (Fig. 7). These scoring outcomes were subsequently related to clinical endpoints. We observed that none of the CR tumors displayed EMMPRIN staining, whereas highest EMMPRIN staining (3+) was observed in PD tumors (Table V). This finding, originally indicated using LC-MS-based technology, was thus confirmed by IHC. For comparison, we defined a “clinical benefit” group composed of tumors showing NC for >6 months, CR, and PR and a “no clinical benefit” group representing NC for ≤6 months and PD tumors. Absence of detectable EMMPRIN levels showed a significant clinical benefit with an odds ratio of 2.98 (95% CI, 1.32–6.73; p = 0.009). The presence of detectable EMMPRIN levels was more frequently observed in premenopausal women (X2 = 11.7; p < 0.001) and in patients with a shorter disease-free interval (X2 = 11.2; p = 0.004) defined as the time from primary diagnosis to recurrence (Table VI). In addition, Cox regression analysis showed that presence of EMMPRIN significantly correlated with shorter progression-free survival from the start of tamoxifen treatment (HR, 1.87; 95% CI, 1.25–2.80; p = 0.002) (Fig. 8). Thus, high EMMPRIN levels correlate with poor outcome on first line tamoxifen treatment.


Identification of a putative protein profile associated with tamoxifen therapy resistance in breast cancer.

Umar A, Kang H, Timmermans AM, Look MP, Meijer-van Gelder ME, den Bakker MA, Jaitly N, Martens JW, Luider TM, Foekens JA, Pasa-Tolić L - Mol. Cell Proteomics (2009)

Immunohistochemical staining of EMMPRIN. EMMPRIN immunohistochemical staining was performed on an independent sample set of 156 tissues using TMA. A, overview of TMA; B, negatively stained tissue; C, 1+ membrane stain; D, 2+ stain; E, 3+ stain. Overview picture was taken at 5× magnification; other pictures were taken at 100× magnification.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2690491&req=5

f7: Immunohistochemical staining of EMMPRIN. EMMPRIN immunohistochemical staining was performed on an independent sample set of 156 tissues using TMA. A, overview of TMA; B, negatively stained tissue; C, 1+ membrane stain; D, 2+ stain; E, 3+ stain. Overview picture was taken at 5× magnification; other pictures were taken at 100× magnification.
Mentions: To independently validate differential EMMPRIN protein abundance between OR and PD patients, IHC was performed using our primary breast cancer TMA. Among the different tissues, there were 156 breast tumors of patients who received first line tamoxifen therapy after recurrence. This set of tumors had no overlap with the discovery set tumors. In total, 130 tumors showed reproducible IHC staining on the TMA when assays were performed in triplicate. Patient and tumor characteristics are described in Table IV. Different staining outcomes were categorized as undetectable, weak, medium, and strong membrane staining. Weak membrane staining, present in <10% of tumor cells, was scored as 1+. Medium membrane staining, present in 10–50% of tumor cells, was scored as 2+. Strong membrane staining, observed in >50% of tumor cells, was assigned score 3+ (Fig. 7). These scoring outcomes were subsequently related to clinical endpoints. We observed that none of the CR tumors displayed EMMPRIN staining, whereas highest EMMPRIN staining (3+) was observed in PD tumors (Table V). This finding, originally indicated using LC-MS-based technology, was thus confirmed by IHC. For comparison, we defined a “clinical benefit” group composed of tumors showing NC for >6 months, CR, and PR and a “no clinical benefit” group representing NC for ≤6 months and PD tumors. Absence of detectable EMMPRIN levels showed a significant clinical benefit with an odds ratio of 2.98 (95% CI, 1.32–6.73; p = 0.009). The presence of detectable EMMPRIN levels was more frequently observed in premenopausal women (X2 = 11.7; p < 0.001) and in patients with a shorter disease-free interval (X2 = 11.2; p = 0.004) defined as the time from primary diagnosis to recurrence (Table VI). In addition, Cox regression analysis showed that presence of EMMPRIN significantly correlated with shorter progression-free survival from the start of tamoxifen treatment (HR, 1.87; 95% CI, 1.25–2.80; p = 0.002) (Fig. 8). Thus, high EMMPRIN levels correlate with poor outcome on first line tamoxifen treatment.

Bottom Line: Identification of proteins that are associated with tamoxifen resistance is a first step toward better response prediction and tailored treatment of patients.ENPP1, EIF3E, and GNB4 were significantly associated with progression-free survival upon tamoxifen treatment for recurrent disease.Extracellular matrix metalloproteinase inducer levels were higher in therapy-resistant tumors and significantly associated with an earlier tumor progression following first line tamoxifen treatment (hazard ratio, 1.87; 95% confidence interval, 1.25-2.80; p = 0.002).

View Article: PubMed Central - PubMed

Affiliation: Erasmus Medical Center Rotterdam, Josephine Nefkens Inst., Dept. of Medical Oncology, Laboratory of Genomics and Proteomics of Breast Cancer, Dr. Molewaterplein 50, Be 430c, P. O. Box 2040, 3000 CA Rotterdam, The Netherlands. a.umar@erasmusmc.nl

ABSTRACT
Tamoxifen resistance is a major cause of death in patients with recurrent breast cancer. Current clinical factors can correctly predict therapy response in only half of the treated patients. Identification of proteins that are associated with tamoxifen resistance is a first step toward better response prediction and tailored treatment of patients. In the present study we intended to identify putative protein biomarkers indicative of tamoxifen therapy resistance in breast cancer using nano-LC coupled with FTICR MS. Comparative proteome analysis was performed on approximately 5,500 pooled tumor cells (corresponding to approximately 550 ng of protein lysate/analysis) obtained through laser capture microdissection (LCM) from two independently processed data sets (n = 24 and n = 27) containing both tamoxifen therapy-sensitive and therapy-resistant tumors. Peptides and proteins were identified by matching mass and elution time of newly acquired LC-MS features to information in previously generated accurate mass and time tag reference databases. A total of 17,263 unique peptides were identified that corresponded to 2,556 non-redundant proteins identified with > or = 2 peptides. 1,713 overlapping proteins between the two data sets were used for further analysis. Comparative proteome analysis revealed 100 putatively differentially abundant proteins between tamoxifen-sensitive and tamoxifen-resistant tumors. The presence and relative abundance for 47 differentially abundant proteins were verified by targeted nano-LC-MS/MS in a selection of unpooled, non-microdissected discovery set tumor tissue extracts. ENPP1, EIF3E, and GNB4 were significantly associated with progression-free survival upon tamoxifen treatment for recurrent disease. Differential abundance of our top discriminating protein, extracellular matrix metalloproteinase inducer, was validated by tissue microarray in an independent patient cohort (n = 156). Extracellular matrix metalloproteinase inducer levels were higher in therapy-resistant tumors and significantly associated with an earlier tumor progression following first line tamoxifen treatment (hazard ratio, 1.87; 95% confidence interval, 1.25-2.80; p = 0.002). In summary, comparative proteomics performed on laser capture microdissection-derived breast tumor cells using nano-LC-FTICR MS technology revealed a set of putative biomarkers associated with tamoxifen therapy resistance in recurrent breast cancer.

Show MeSH
Related in: MedlinePlus