3.1 ALK pathway signature score is associated with de-differentiation of BC
The association between ALK pathway signature score and seven clinical characteristics of BC were analyzed in 42 affymatrix datasets individually by univariate regression analysis. Figure 1 showed that in most of the datasets, ALK pathway signature score was a significant unfavorable factor for ER-positive (overall OR 0.30, 95% CI, 0.26-0.35, p=3.02E-58) and PR-positive (overall OR, 0.45, 95% CI 0.39-0.51, p=5.43E-32) tumors, while it was a significant favorable factor for grade 3 tumors (overall OR 2.35, 95% CI 2.01-2.75, p=9.69E-27). By contrast, no apparent associations of ALK pathway scores with age, HER2 status, lymph node status and tumor size were observed in most of the datasets (Fig. 2).
The 42 datasets were further merged into four cohorts, and similar associations of ALK pathway scores with the seven clinical characteristics were observed in the four merged cohorts (Supplementary Fig. 1). Multivariate logistic regression was also performed in these merged cohorts to assess whether the association of ALK pathway scores with estrogen receptor (ER) status, progesterone receptor (PR) status and tumor grade remain significant after adjustment for other covariates. Overall, the ORs for association of ALK pathway score with ER status stay constant when the pathway score was tested separately with correction for age, tumor grade, and tumor size, status of PR, HER2 or lymph node (Fig. 3A). Similar trend was also observed for the associations of ALK pathway scores with tumor grade or PR status, except that the association with PR status was almost abolished when adjusted for ER status (Fig. 3B and C). These results indicate that ALK pathway score is an independent risk factor for the presence of ER-negative and high grade BC, or in the other words, ALK pathway score is an independent factor associated with de-differentiation of BC since loss of ER expression and high pathological grade are the features of de-differentiated BC .
3.2 ALK pathway signature score is associated with pathological complete response (pCR) of BC
Logistic regression analysis showed that ALK pathway score was positively associated with pCR rate of BC in 15 individual datasets that contain patient’s neoadjuvant response information (overall OR 1.67, 95% CI 1.45-1.93, p=2.00E-12) (Fig. 4A). This association reached statistical significance (p<0.05) in 8 of the 15 datasets, and approached the borderline of significance (p=0.08) in 2 of the datasets (Fig. 4A). As to the remaining 5 datasets with p > 0.08, two (GSE16446, and GSE18864) contain only ER-negative BC samples, and another two (GSE37946 and GSE50948) are mainly composed of HER2-positive samples (Supplementary Table 1), suggesting that ER and HER2 status affect the association between ALK pathway score and pCR rate. This effect was also observed in the cohort merged from the 15 datasets. As shown in Fig. 4B, an increase of one standard deviation (SD) of ALK pathway score is associated with 62% and 85% increase of pCR rate in ER-positive (95% CI 1.28-2.04, p=5.31E-5) and HER2-negative (95% CI 1.63-2.10, p=1.80E-21) BC respectively, while only with 17% and 28% increase in ER-negative (95% CI 1.02-1.34, p=0.03) and HER-positive (95% CI 1.03-1.58，p=0.02) BC respectively. In addition, ALK pathway score is also more strongly associated with pCR rate in grade 1&2 tumors (OR 2.16, 95% CI 1.63-2.87, p=8.09E-08) than in grade 3 tumors (OR 1.36, 95% CI 1.17-1.59, p=9.59E-05).
Multivariate logistic regression analysis on the merged cohort showed that the association of ALK pathway scores with pCR was hardly affected after adjustment for other clinical variables, except that the OR values decreased moderately when adjusted by ER or PR status (Fig. 4C).
3.3 ALK pathway signature score is associated with recurrence risk of BC
Logistic regression analysis showed that ALK pathway score was positively associated with recurrence risk of BC in 30 individual datasets in which patient’s survival information is available (overall HR 1.21, 95% CI 1.13-1.29, p=3.31E-08) (Fig. 5A). Among the 30 datasets, GSE16446, GSE31519, GSE58812 contain only ER-negative samples and GSE17907 is mainly composed of Her2-positive samples (Supplementary Table 1). ALK pathway score failed to achieve positive association significantly with recurrence risk in all of these 4 datasets. Four additional datasets (GSE 20711, GSE2603, GSE2990 and GSE7378) have sample size <100. Among the remaining 22 datasets, the association of ALK pathway with recurrence risk reached statistical significance in 11 datasets (Fig. 5A).
The positive association of ALK pathway score with recurrence risk remain statistically significant in three cohorts that were merged from the 30 datasets in Cox regression analysis (p=7.29E-07, 8.98E-05, 2.54E-03, respectively) (Fig. 5B-D) and Kaplan-Meier survival curve analysis (p=1.29E-06, 1.79E-03, 2.50E-03, respectively) (Fig. 6A-C).
Like the association with pCR, the association of ALK pathway score with recurrence risk was also more significant in HER2 negative (p=9.00E-03, 1.25E-03, 7.85E-05, respectively) and grade 1&2 (p=2.42E-03, 9.81E-03, 0.07, respectively) tumors than in HER2 positive (p=0.14, 0.11, 0.10, respectively) and grade 3 (p=0.36, 0.34, 0.38, respectively) tumors in the three cohorts (Fig. 5B-D). In addition, the ALK pathway score was also more significantly associated with cancer recurrence in patient with age>50 (p=2.31E-06, 0.03, 5.96E-03, respectively) than with age ≤50 years old (p=0.49, 0.24, 0.10, respectively) and in patient with positive lymph node (p=9.52E-06, 3.42E-04, 7.52E-03, respectively) than with negative lymph node (p=0.17, 0.18, 0.21, respectively) in the three cohorts (Fig. 5B-D).
3.4 ALK pathway signature score is associated with recurrence risk only in patients with residual disease (RD) following neoadjuvant chemotherapy
Our data showed that ALK pathway score is positively associated with both pCR rate and recurrence risk of BC. These results seem to be controversial since BC patients with pCR generally have better prognosis than patients with RD . A potential explanation is that ALK pathway signature score is associated with recurrence risk only in RD but not in pCR patients. To test this hypothesis, samples in cohort 4 that were annotated with both pCR and survival information were tested. Kaplan-Meier analysis showed that patients with pCR have much lower relapse rate than patients with RD (black vs grey, 5-year DFS rate: 0.93 vs 0.69, p=1.96E-05) (Fig. 6D). When RD patients were further stratified into two subgroups based on ALK pathway scores, the subgroup with low ALK pathway score had better DFS rate than that with high ALK score (blue vs violet, 5-year DFS rate: 0.60 vs 0.77, p=1.83E-04) (Fig. 6D). By contrast, the two subgroups formed by further stratifying the pCR patients based on ALK pathway scores showed similar DFS rates (green vs red, p = 0.67) (Fig. 6D).