Loss of nuclear localized and tyrosine phosphorylated Stat5 in breast cancer predicts poor clinical outcome and increased risk of antiestrogen therapy failure

Abstract

Purpose: To investigate nuclear localized and tyrosine phosphorylated Stat5 (Nuc-pYStat5) as a marker of prognosis in node-negative breast cancer and as a predictor of response to antiestrogen therapy.

Patients and methods: Levels of Nuc-pYStat5 were analyzed in five archival cohorts of breast cancer by traditional diaminobenzidine-chromogen immunostaining and pathologist scoring of whole tissue sections or by immunofluorescence and automated quantitative analysis (AQUA) of tissue microarrays.

Results: Nuc-pYStat5 was an independent prognostic marker as measured by cancer-specific survival (CSS) in patients with node-negative breast cancer who did not receive systemic adjuvant therapy, when adjusted for common pathology parameters in multivariate analyses both by standard chromogen detection with pathologist scoring of whole tissue sections (cohort I; n = 233) and quantitative immunofluorescence of a tissue microarray (cohort II; n = 291). Two distinct monoclonal antibodies gave concordant results. A progression array (cohort III; n = 180) revealed frequent loss of Nuc-pYStat5 in invasive carcinoma compared to normal breast epithelia or ductal carcinoma in situ, and general loss of Nuc-pYStat5 in lymph node metastases. In cohort IV (n = 221), loss of Nuc-pYStat5 was associated with increased risk of antiestrogen therapy failure as measured by univariate CSS and time to recurrence (TTR). More sensitive AQUA quantification of Nuc-pYStat5 in antiestrogen-treated patients (cohort V; n = 97) identified by multivariate analysis patients with low Nuc-pYStat5 at elevated risk for therapy failure (CSS hazard ratio [HR], 21.55; 95% CI, 5.61 to 82.77; P < .001; TTR HR, 7.30; 95% CI, 2.34 to 22.78; P = .001). CONCLUSION Nuc-pYStat5 is an independent prognostic marker in node-negative breast cancer. If confirmed in prospective studies, Nuc-pYStat5 may become a useful predictive marker of response to adjuvant hormone therapy.

Fig 1.

Low levels of nuclear localized and tyrosine phosphorylated Stat5 (Nuc-pYStat5) predict unfavorable breast cancer prognosis. (A-B) Kaplan-Meier analysis of Nuc-pYStat5, detected by mouse monoclonal antipYStat5 AX1 antibody and standard diaminobenzidine (DAB) immunohistochemistry (IHC) with pathologist review of whole tissue sections, in cohort I revealed that low expression of Nuc-pYStat5 was prognostic of (A) reduced time to recurrence of breast cancer and (B) poor breast cancer-specific survival (CSS). (C-E) Comparative validation of mouse monoclonal AX1 with rabbit monoclonal antipYStat5 antibody E208. (C) Detection of nuclear localized and tyrosine phosphorylated Stat5 by DAB chromogen IHC in healthy human breast tissue surgical explants incubated ex vivo with (positive control) or without (negative control) human prolactin (100 nmol/L; 60 minutes) using antipYStat5 antibodies AX1 (16 hours' incubation with manual staining protocol; upper panels) or E208 (20 minutes' incubation with autostainer protocol; lower panels). (D) Representative immunofluorescent images from two serial sections of a breast tissue stained with AX1 or E208 antibodies indicating comparable detection of Nuc-pYStat5. (E) Correlation of Nuc-pYStat5 expression detected by AX1 or E208 and quantified by automated quantitative analysis (AQUA) in serial sections of an assorted breast tissue microarray. (F) Kaplan-Meier analysis of breast CSS in cohort II indicated that risk of death from breast cancer was significantly elevated in patients whose tumors expressed low Nuc-pYStat5 as quantified by immunofluorescence and AQUA analysis. Censored cases (+) and number of patients per group are indicated.

Fig 2.

Loss of nuclear localized and tyrosine phosphorylated Stat5 (Nuc-pYStat5) during breast cancer progression. Levels of Nuc-pYStat5 as detected by immunofluorescence and quantified by automated quantitative analysis (AQUA) were significantly reduced in invasive ductal carcinoma (IDC; n = 72) and lymph node metastases (LN Met; n = 17) when compared with normal breast tissue (n = 27) and ductal carcinoma in situ (DCIS; n = 14) in progression array cohort III. (*) P = .012; (***) P < .001.

Fig 3.

Low levels of nuclear localized and tyrosine phosphorylated Stat5 (Nuc-pYStat5) predict increased risk of failure of antiestrogen therapy. (A-B) Low levels of Nuc-pYStat5 detected by diaminobenzidine chromogen immunohistochemistry (IHC) and pathologist scoring in patients treated with antiestrogen therapy (cohort IV) predicted (A) poor breast cancer-specific survival (CSS) and (B) reduced time to recurrence (TTR) of breast cancer. (C-D) Immunofluorescence and quantitative automated quantitative analysis of Nuc-pYStat5 expression in breast cancer patients treated with antiestrogen monotherapy (cohort V) revealed that low expression of Nuc-pYStat5 was predictive of (C) poor CSS and (D) reduced TTR of breast cancer. Kaplan-Meier plots with censored cases (+) and number of patients per group indicated.