Prognostic value of estrogen receptor-α and progesterone receptor in curatively resected colorectal cancer: a retrospective analysis with independent validations

The tissue samples from training and internal validation cohorts were obtained from resected specimens. The tissues were fixed in 10% buffered formalin (PH7.0) and embedded in paraffin. The paraffin-embedded tumor samples were sectioned continuously into 4-μm-thick sections. Then the sections were dewaxed in xylene and rehydrated in graded alcohols. A negative control was performed by replacing the primary antibody with a normal rabbit IgG antibody. Following antigen retrieval by microwave heating (95 °C for 20 min), sections were then incubated with primary monoclonal rabbit anti-human PR (Clone 1E2, Ventana Medical Systems. Inc) (for training set only) or ER (Clone SP1, Ventana Medical Systems. Inc) at 4 °C overnight. After washing, the sections were incubated with a horseradish peroxidase-labeled goat antibody against a mouse/rabbit secondary antibody (Envision; Dako, Glostrup, Denmark) at room temperature for 30 min. Then the signal was developed with 3, 3′-diaminobenzidine tetrahydrochloride (DAB). Section was counterstained with hematoxylin. The stain was examined by two pathologists independently. The data were obtained by calculating the mean number of positively stained cells in five to ten separate 400 × per high power field(HPF) and evaluated as negative or positive. Two independent observers blinded to the clinicopathological information scored the ER-α and PR expression levels in tumor cells by assessing (a) the proportion of positively stained cells: (0, < 5%; 1, 6 to 25%; 2, 26 to 50%; 3, 51 to 75%; 4, > 75%) and (b) the signal intensity: (0, no signal; 1, weak; 2, moderate; 3, strong). The score was the product of a × b. Considering that the number of patients with score > 0 in ER-α and PR expression were 19 (12.8%) and 32 (21.6%), we used dichotomic classification (positive/negative). Therefore, the patients were divided into subgroups: a high group (a × b > 0) and a low group (a × b = 0). The score a and b were the averages of scores of two independent observers. Immunohistochemical staining was showed in Additional file 1: Figure S1.

For training and internal validation cohorts, the final decisions with regard to treatment strategy and use of chemotherapy or radiotherapy were based on TNM stage, the multidisciplinary team’s (MDT) decision and patient choice. All patients were treated with definitive-intent surgery. Most of the patients with stage II-III rectal cancer received radiotherapy. 5-FU based chemotherapy was delivered concurrently with radiation in forms of three-dimensional conformal radiotherapy (3D-CRT), intensity-modulated radiotherapy (IMRT) or Volumetric Modulated Arc Therapy (VMAT). Neoadjuvant or adjuvant chemotherapy consisted of oxaliplatin (130 mg/m², day 1) with capecitabine (1000 mg/m², bid, days 1–14) every 3 weeks to a total of 6 months’ perioperative therapy or Leucovorin (400 mg/m² or 200 mg/m², day1), with 5-fluorouracil (5-FU) (bolus 400 mg/m² and then 1200 mg/m²/day over 46-48 h) every 2 weeks to a total of 6 months’ perioperative therapy. Of 148 patients, 12 (8.1%) patients received neoadjuvant chemotherapy and 86 (58.1%) patients received adjuvant chemotherapy. All patients from external validation cohort (TCGA dataset) underwent surgery.

Follow-up for training and internal validation cohorts was measured from the first day of treatment until the day of last examination or the day of death. Patients were evaluated every 3 months for the first 2 years, and then every 6 months for the next 3 years and finally annually. Relapse local disease was diagnosed pathologically by surgical resection, biopsy or cytology and/or by the detection of radiologically obvious lesions that increased in size over time. Additional tests were ordered when indicated to identify distant failure.

Multivariate analyses using the Cox proportional hazards model were used to estimate the hazard ratios (HR) and test independent significance by backward elimination of insignificant explanatory variables. Covariates included host factors (i.e., sex, age,), and tumor factors (i.e., tumor localization, stage), the criterion for statistical significance was set at p = 0.05 and p values were based on 2-sided tests.

The median follow-up duration was 46.8 months (3.1–73.5 months) for training cohort, 64.7 months (0.2–150.1 months) for internal validation cohort and 23.8 months (0.2–105.1 months) for external validation cohort, respectively. The patients’ baseline characteristics of three cohorts are presented in Table 1.

To investigate the effect of tumor expression of ER-α, PR on the outcomes of patients with CRC, the 5-year actuarial OS, DMFS and LRFS rates in training cohort were analyzed. On univariate analysis, low and high ER-α expression demonstrated significant differences in the 5-year OS (89% vs. 47%, p < 0.001), LRFS (95% vs. 71%, p < 0.001) and DMFS (95% vs. 70%, p < 0.001) (Fig. 1, Tables 2 and 3) rates of CRC patients, while PR were not found to be significantly associated with improved 5-year OS, LRFS and DMFS rates (Additional file 1: Table S1).

In the COX multivariate analysis, the following parameters were included: age (< 60 years,≥60 years), sex, location of tumor, stage (I, II-III) tumor expression of ER-α. Stage maintained statistical significance in OS. In addition, ER-α expression was an independent prognostic factor for OS in CRC patients with surgery (HR,5.061; p = 0.002) (Table 2), LRFS (HR, 8. 655; p = 0.002) and DMFS (HR, 6.610; p = 0.004) (Table 3).

To validate the prognostic value of ER-α, the 5-year actuarial OS, DMFS and LRFS rates in internal validation cohort and the 5-year actuarial OS rates in external validation cohort were analyzed. On univariate analysis, tumor expression of ER-α demonstrated significant differences in the 5-year OS rates in internal and external validation cohorts, which are 74% vs. 61% with p = 0.039 and 53% vs. 38% with p = 0.02 (Fig. 2), respectively. Whereas, univariate analyses indicated that ER- α expression had no significant association with DMFS and LRFS in internal validation cohort (Table 3). Since there is no data about local recurrence and distant metastasis in external validation cohort (TCGA dataset), DMFS and LRFS were not validated in this set.

In the COX multivariate analysis, ER-α expression was an independent prognostic factor for OS in both validation cohorts, with HR = 1.572, 95%CI (1.001–2.467), p = 0.049 and HR = 1.624, 95%CI (1.047–2.520), p = 0.031 for internal and external validation sets (Table 2).