Demographic information for patients included in the study are presented in Table 1. The cohort was predominantly post-menopausal (median age 62.2 ± 11.7) and included racially and ethnically diverse women (50% non-white race, 32% Hispanic ethnicity). A broad range of uterine histologies was represented including all grades of endometrioid adenocarcinoma (EAC, N = 47), uterine papillary serous carcinoma (UPSC, N = 15), carcinosarcoma (CS, N = 15), leiomyosarcoma (LMS, N = 8), and clear cell carcinoma (CCC, N = 6). These patients had predominantly International Federation of Gynecology and Obstetrics (FIGO) stage I disease (45%) but over half of the cohort had more advanced disease (stage II- 18%, stage III- 24%, stage IV- 13%) at the time of diagnosis. Patients with more aggressive tumor subtypes such as UPSC, CS, LMS and CCC generally had larger tumors, deeper myometrial invasion, and more frequent LVSI or lymph node involvement than those with EAC.
The median total serum cfDNA concentration (prior to magnetic bead purification) was 69.2 ng/mL (IQR 37.4, 132.3). There was no significant association between total serum cfDNA concentration and body mass index (p = 0.17). There were no significant differences in total serum cfDNA concentration between control subjects (60.8 ng/mL IQR 5.3, 108.0) and women with cancer (70.7 ng/mL IQR 37.7, 138.2) (p = 0.33). There were also no significant differences in total serum cfDNA concentration across histologic subtypes (p = 0.06), grade of cancer (p = 0.46), stage of cancer (p = 0.33), percent myometrial invasion (p = 0.29), tumor size (p = 0.11), presence of LVSI (p = 0.12), or nodal involvement (p= 0.81).
The bioanalyzer profiles of fragment size distribution revealed a predominant peak of ~ 150-200 bp with a small additional peak around 350 bp and contamination of higher molecular weight bands around 10,380 bp (Figure 1A-C). After purification with SPRI AMPure beads as described in the “Methods” section, the high-molecular weight DNA was removed (Figure 1 D-F) and the corresponding bands were no longer visible, leaving only the desired LMW cfDNA with peaks at 150 base pairs and additional peaks corresponding to nucleosomal fragments (Figure 1G-I).
The median serum LMW concentration (after bead purification) was 23.8 ng/mL (IQR 14.9, 44.4) for the entire cohort (Table 2). There was no significant association between LMW cfDNA and body mass index (p = 0.53). LMW cfDNA concentration was significantly higher in women with cancer (25.8 ng/mL IQR 16.0, 49.6) relative to controls (15.5 ng/mL, IQR 9.3, 25.8) (p < 0.01) (Figure 2). Moreover, it was significantly higher in women with early stage (stages I and II) cancer compared to the controls (p < 0.01). Among cancer patients, there were also significant differences in LMW cfDNA concentration among histologic subtypes (p= 0.02). LMS (65.4 ng/mL IQR 45.7, 164.3) and CS (32.2 ng/mL IQR 17.9, 79.0) had the highest concentrations and EAC (25.2 ng/mL IQR 17.6, 44.4) had the lowest (Figure 3). Within cancer patients, LMW cfDNA concentration was also significantly associated with cancer stage. Median LMW cfDNA concentration for patients with stage IV disease was significantly higher (57.2 ng/mL IQR 16.4, 246.1) than those with stage I -III disease (24.2 ng/mL IQR 16.0, 39.3) (p < 0.01) (Figure 4). We also found marginally significant associations between LMW cfDNA concentration and the presence of LVSI (p=0.06), and tumor size (p=0.05), and no statistical significant association with the percent of myometrial invasion (p = 0.37), presence of nodal metastases (p = 0.39) or tumour grade (p=0.49).
Over a median follow-up of 51.9 months, a total of 91 subjects contributed a total of 4574 person-months at risk. Thirty-three people within the cohort (36.3%) died of cancer, and the 24-month survival rate was 75%. Cox-proportional hazard modelling showed that the LMW cfDNA concentration is strongly associated with overall survival (p < 0.01), with hazard ratio at 1.53 as the concentration doubles. When analysing survival outcomes by quartile of LMW cfDNA concentration, patients with concentrations above the 75th percentile (> 49.6 ng/mL) had significantly worse survival than the remainder of subjects within the cohort (14.5 vs 41.8 months respectively, logrank p-value = 0.04) (Figure 5). Subjects with high-risk uterine histologies had worse overall survival (12.9 months) than those with endometrioid histologies (57.6 months) (p < 0.01) and subjects with leiomyosarcoma had the worst outcomes (4.8 months) (Figure 5). When analysing survival outcomes by quartile of cfDNA concentration, those with concentrations above the 75th percentile (>49.6 ng/mL) had significantly worse survival than the remainder of subjects within the cohort (14.5 vs 41.8 months respectively, logrank p-value = 0.04) (Figure 6). This high-risk group included 22 women with various histologies including those with low grade EAC (N = 5), grade 2 EAC (N = 2), grade 3 EAC (N = 3), CS (N=5), UPSC (N= 1) and LMS (N = 6). It also included women with both early stage (FIGO I-II, 54.5%) and late stage (FIGO III-IV, 45.5%) disease.