Abnormal ECM1 expression in B-ALL patients
The ECM1 transcript levels in ND (median 101.3%, range 0-5411.7%) and RR B-ALL patients (median 39.1%, range 0-6365.5%) were significantly increased than it in normal control (median2.8%, range 0-4.97%) (p<0.01). While the ECM1 transcript level was significantly decreased in CR patients (median 3.3%, range 0-196.9%) compared with the ND and RR B-ALL patients (p<0.01). Besides, there was no statistically significant difference in ECM1 transcript levels between children(median71.8%, range 0-5411.7%) and adult patients(median106.3%, range 0-2431.6%) (p＞0.05)(Fig.1).
Then the data of 71 adult patients (age 14 years or older) were collected for further analysis. The baseline characteristics are summarized in Table 2. The median age was 37 years old. And 62.9% (n=44) patients were male. The median WBC count, hemoglobin, and plate count at diagnosis were 17.5 (range 1.6–586.2) ×109 /L, 82(range28.9-158) g/L and 56(range 4-338) ×109 /L respectively. Among them, 51 patients were common-B-ALL, 10 patients were progenitor B-ALL and 10 patients were precursor-B-ALL. Regarding the molecular biological features, 32 patients had BCR-ABL fusion (Ph+), 4 patients had MLL rearrangement. According to the cytogenetic risk, 57.5%(n=41) patients were poor risk. 24 patients were MRD positive at the end of the induction therapy. 53.5% (n=38) patients only received chemotherapy and the other patients received allo-HSCT.
High expression of ECM1 at diagnosis predicted poor outcomes in adult B-ALL patients
To assess the clinical significance of ECM1 in adult B-ALL patients, we next examined the relationship between ECM1 expression level and the clinical characteristics of 71 patients. Patients were grouped into ECM1-Low or ECM1-High according to the median baseline ECM1 expression levels. As shown in Table 2, compared with ECM1-Low group, the patients in ECM1-High group were more likely to have higher platelet number (p =0.004), BCR-ABL fusion (p=0.004), and cytogenetic poor risk (p=0.021). There were no statistically significant differences in age, gender, WBC count, hemoglobin, immunophenotype, WT1, IKZF1, MLL, MRD status, HSCT between two groups.
The median follow-up time was 18 months (range 1–78 months). The CR rates after first cycle of induction therapy were similar between ECM1-Low and ECM1-High group (80.6% vs.77.1%; p=0.725). Kaplan–Meier survival analysis was then performed to compare the outcomes of patients dichotomized by ECM1 expression. Patients with high ECM1 expression had a significantly worse 3-year OS (11.4% vs. 36.1%, p=0.015) and 3-year RFS (9.7% vs. 29.4%, p=0.047) than those with low ECM1 expression (Fig.2).
High expression of ECM1 at diagnosis independently predicts poor prognosis of adult B-ALL patients
Furthermore, a Cox proportional hazard regression model was set up to assess the effect of ECM1 on the prognosis of adult B-ALL patients. Age (≥ vs. < 35 years), WBC count (≥ vs. < 30×109/L), BCR-ABL fusion status (negative/positive), treatment (chemotherapy alone vs. chemotherapy/allo-HSCT), MRD at the end of induction therapy (negative/positive), risk group (poor risk vs. standard risk) and ECM1 expression levels (low/high) were included. The univariate cox regression analysis indicated age≥35 years, MRD positive at the end of induction therapy，chemotherapy alone, high ECM1 expression as strong prognostic predictors for poor OS. Besides, MRD positive at the end of induction therapy and chemotherapy alone were also significantly related to poorer RFS. WBC count, BCR-ABL fusion status and risk group had no effects on OS or RFS. To further verify the robustness value of ECM1 expression, multivariate analysis was performed to determine risk assessment related to survival and relapse. Most remarkably, MRD positive at the end of induction therapy, chemotherapy alone and high ECM1 expression were independent adverse prognostic factors for OS. Moreover, MRD positive at the end of induction therapy and chemotherapy alone were independent adverse prognostic factors for RFS. Together, our data revealed that the expression level of ECM1 at diagnosis could serve as an independent prognostic biomarker of clinical outcomes in adult B-ALL patients (Table 3).
Time course analysis of ECM1 expression
To determine the utility of ECM1 as MRD markers, expression of the ECM1 was evaluated at the diagnosis, CR, and relapse. ECM1 and BCR-ABL expression in three continuous CR Ph+ patients during follow-up generally changed in parallel (Fig.3). Expression of the two markers was high at the primary diagnosis and decreased when achieved CR. Furthermore, an increase in expression of ECM1 and BCR-ABL was observed in four relapse Ph+ patients. In addition, among eight continuous CR Ph- patients, decreased expression of ECM1 was observed in four patients who had a high baseline ECM1 expression and achieved CR after induction therapy. Expression of ECM1 was continuous low both at the primary diagnosis and CR for the other four patients. Similarly, an increase in expression of ECM1 was observed in four relapse Ph- patients. In particular, for a patient with negative molecular biomarkers, expression of ECM1 during follow up generally changed in parallel with MRD quantified by analyzing LAIPs using flow cytometry (Fig.4). Taken together, ECM1 could be a marker of MRD in adult B-ALL patients with negative molecular biomarkers at diagnosis.