br Statistical analysis br The end points analysed in this
2.4. Statistical analysis
The end-points analysed in this L-Glutamine study were progression-free survival (PFS) and overall survival (OS). PFS was defined as the elapsed time between the date of baseline blood draw and either the date of clinical progression, death, or the last follow-up. OS was defined as the time from baseline blood collection to death or the last follow-up. Patients without an end-point event at the last follow-up were censored. We also evaluated treat-ment responses on the basis of longitudinally collected samples. Complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD) were assessed by imaging-based methods following the Response Evaluation Criteria in Solid Tumours (RECIST) guideline . For CTC-related analyses, a widely used cut-off of 5 CTCs per 7.5 mL whole blood was applied to stratify patients into high- and low-risk groups ; for ccfDNA-related analyses, the best cut-off point of ccfDNA amount, calculated based on ALU115 and ALU81 levels, was determined using receiver operating characteristic curve analysis. Com-parisons of demographic and clinical variables were conducted using a c2 test for categorical variables and an unpaired Student’s t test for continuous variables. The correlation between CTC and ccfDNA levels was evaluated by the Spearman’s rank-order correlation method. Survival curves were estimated using the KaplaneMeier method and compared using a log-rank test. Associations of CTC or ccfDNA levels with PFS or OS were evaluated using hazard ratios (HRs) with 95% confidence intervals (CIs) by a univariate and a multi-variate Cox proportional hazards model adjusting for age, ethnicity, body mass index (BMI), tumour grade, menopause status, breast cancer subtypes, previous therapies, treatments after baseline blood draw and ccfDNA/CTC, where appropriate. The proportional hazards assumption was validated using the test based on Schoenfeld residuals. Possible interaction effect be-tween CTC and ccfDNA on survivals was tested by including a product interaction term into the Cox pro-portional hazards model. Changes in CTC and ccfDNA levels over time with treatment responses and clinical outcomes were analysed and plotted using data of longitudinally collected samples. CTC or ccfDNA levels before and after the initiations of new therapies were
compared using a paired t test. SAS (version 9.4, SAS Institute) and STATA (version 11.0, STATA Corp.) software packages were used for the analyses in this study. All P values were 2-sided, with a P < 0.05 considered the threshold for statistical significance.
3.1. Patient characteristics
3.2. Associations of circulating tumour cell and total circulating cell-free DNA levels at baseline with patient outcomes
Among the 117 patients, 32 (27.4%) had elevated CTCs ( 5 CTCs) at baseline. This percentage was slightly lower than previous reports , possibly due to the larger number of TNBC patients in our cohort and to the fact that some patients in our cohort had undergone previous therapies that might have reduced the number of CTCs [19,39]. As expected, patients with elevated CTCs exhibited significantly unfavourable PFS (Plog-
with 5 CTCs had 2.58-fold (95% CI 1.63e4.07) increased risk for disease progression and 3.63-fold (95% CI 1.58e8.34) increased risk for death (Table 2). The associations of CTCs with patient outcomes remained significant after adjusting for age, ethnicity, BMI, tumour grade, menopause status, breast cancer sub-types, previous therapies and treatments after baseline blood draw (HR 2.93, 95% CI 1.78e4.81 for PFS; HR 6.76, 95% CI 2.55e17.92 for OS, Table 2). The survival differences between patients with high- and low-level ccfDNAs were also statistically significant (Plog-
Characteristics of metastatic breast cancer patients (N Z 117).
Variables N (%)
Family history of breast cancer
Breast cancer subtypes
Baseline cancer antigen 15.3
Number of previous chemotherapy lines
Number of previous hormone therapy lines
SD, standard deviation; BMI, body mass index; HER2, human epidermal growth factor receptor 2; TNBC, triple-negative breast cancer.
when analysing CTC, and we adjusted CTC when ana-lysing ccfDNA. These analyses yielded similar results with moderately reduced HRs, indicating that CTC or ccfDNA affected patient outcomes in a way that depended partly on one another but mostly did not overlap (Table 2).
3.3. Joint effects of baseline circulating tumour cell and circulating cell-free DNA levels on patient outcomes