Signatera for detecting molecular residual disease from solid tumour cancers

Study size, design and location

Prospective cohort study evaluating the clinical utility of ctDNA assessment in 168 people with stage 3 colorectal cancer at 7 centres in Denmark and Spain.

Intervention

Signatera in addition to standard care. Samples for ctDNA analysis were collected at diagnosis, after surgery, during adjuvant therapy and at follow up.

Key outcomes

Recurrence occurred in 25% of people. Before surgery, 139 of 153 samples (91%) were ctDNA-positive. This reduced to 20 of 140 samples (14%) after surgery before adjuvant chemotherapy. The recurrence rate was 80% for people who tested ctDNA-positive and 18% for people who tested negative.

People with recurrence who tested ctDNA-negative had higher cfDNA than people who tested positive. Authors suggested surgical trauma could increase cfDNA release, which could potentially dilute ctDNA levels below the detection level. For this group, samples taken more than 2 months after surgery had lower levels of cfDNA than those taken at 2 weeks, with the ctDNA detection rate increasing from 0% to 80%.

People who tested ctDNA-positive postoperatively had shorter recurrence-free survival than those who tested negative (hazard ratio [HR] 7.0, 95% confidence interval [CI] 3.7 to 13.5; p<0.001). A multivariable cox regression showed ctDNA was the strongest predictor of recurrence-free survival (HR 30.97, 95% CI 10.6 to 90.2; p<0.001). Overall, 93 people had adjuvant therapy, with persistence of ctDNA after adjuvant therapy associated with shorter recurrence free survival (HR 50.8, 95% CI 15.4 to 167; p<0.001). Serial ctDNA analysis every 3 months detected recurrence with a median lead time of 9.8 months compared with standard care CT scans.

Strengths and limitations

Patients and healthcare professionals were blinded to the ctDNA result, so the study does not show how results may have affected treatment decisions on adjuvant chemotherapy.

Study size, design and location

Secondary analysis in 581 people who had surgery for muscle-invasive urothelial carcinoma. This study evaluated outcomes from a subgroup of 809 people in a global randomised phase 3 trial comparing adjuvant atezolizumab (n=406) with observation (n=403) for operable urothelial cancer (Bellmunt et al. 2021 [IMvigor010]). This study included centres from the UK.

Intervention

Signatera in addition to standard care in people having adjuvant atezolizumab or observation. Plasma samples were collected after surgery at baseline and 6 weeks after randomisation to treatment.

Key outcomes

At baseline, 214 people (37%) tested ctDNA-positive. People in the observation arm who tested ctDNA-positive (n=98) had significantly higher risk of disease recurrence than those who tested negative (disease-free survival HR 6.3, 95% CI 4.45 to 8.92; p<0.0001).

People who tested ctDNA-positive at baseline who went on to have adjuvant atezolizumab had better overall survival than those having observation (HR 0.59, 95% CI 0.41 to 0.86, median 15.8 months compared with 25.8 months). The benefit from adjuvant atezolizumab was not seen in people who tested ctDNA-negative (HR 1.31, 95% CI 0.77 to 2.23).

Change from ctDNA-positive to negative across the 2 timepoints was reported in 18.2% of people (18 of 99) who had adjuvant atezolizumab compared with 3.8% (3 of 79) in the observation arm. People who were no longer ctDNA‑positive after having adjuvant atezolizumab had better disease-free survival (HR 0.26, 95% CI 0.12 to 0.56; p=0.001) than those who still tested positive.

Strengths and limitations

This study had a reasonably large sample size which included 214 people who tested ctDNA-positive. It suggests that ctDNA-positive results may predict benefit from adjuvant atezolizumab. But ctDNA results were not used to guide treatment decisions and therefore did not affect clinical outcomes. Authors reported 2 timepoints which is less than other studies reporting serial ctDNA analysis. The manuscript was written with some co-authors from the company, but the company said the study was done independently.

Study size, design and location

Phase 2 trial in 106 people with solid tumour cancers who had pembrolizumab in Canada. The study included 5 cohorts: squamous cell cancer of head and neck, triple-negative breast cancer, high-grade serous ovarian cancer, malignant melanoma and mixed solid tumours.

Intervention

Signatera in addition to standard care in people having pembrolizumab. Samples were collected at baseline and before every 3 cycles of treatment.

Key outcomes

Overall, 94 people had enough tumour tissue for ctDNA analysis. Baseline ctDNA was found in 92 samples (98%). People with breast cancer and mixed solid tumours had the highest ctDNA levels while people with malignant melanoma had the lowest. Lower than median ctDNA levels at baseline were associated with better overall survival (adjusted HR 0.49, 95% CI 0.29 to 0.83), progression-free survival (adjusted HR 0.54, 95% CI 0.34 to 0.85) and clinical benefit rate (odds ratio [OR] 3.24, 95% CI 1.19 to 8.8).

At the start of cycle 3 of pembrolizumab, 45% of people (33 of 74) had lower ctDNA levels compared with baseline. Of these, 14 people (42%) had an objective response compared with only 1 (2%) person whose ctDNA level increased from baseline (OR 28.7, 95% CI 3.51 to 253). Change in ctDNA levels was also associated with better clinical benefit rate, overall survival (adjusted HR 0.36, 95% CI 0.18 to 0.71) and progression-free survival (adjusted HR 0.33, 95% CI 0.19 to 0.58). Authors reported that using change in ctDNA along with response evaluation criteria in solid tumours (RECIST) improved risk classification.

Strengths and limitations

The overall sample size was 94 people, with relatively small numbers in each of the 5 cohorts. The authors acknowledged that some cohort analysis was limited by the small sample sizes. Sample sizes also varied across timepoints depending on the number of viable samples available. Samples were collected prospectively but analysis was not done at the same time and did not impact treatment. Some authors were employed by the company.

Study size, design and location

Prospective cohort study in 68 people with localised muscle-invasive bladder cancer in Denmark. Everyone had transurethral resection of bladder tumour (TURBT) followed by neoadjuvant or first-line chemotherapy and cystectomy.

Intervention

Signatera in addition to standard care. Blood samples for ctDNA analysis were collected at scheduled clinical visits and before chemotherapy.

Key outcomes

Recurrence was found in 13 people (20%) with data (n=64). After resection before chemotherapy, 24 people were ctDNA-positive. Of these, 46% (11 of 24) had recurrence during the study compared with 3% of people (1 of 35) who were ctDNA-negative (HR 29.1, p=0.001).

Detecting ctDNA after chemotherapy before cystectomy was also prognostic. At this timepoint, 8 people were ctDNA-positive with a 75% recurrence rate. Comparatively, 55 people were ctDNA-negative with an overall recurrence rate of 11% (6 of 55; HR 12.0, p<0.001). Everyone who tested ctDNA-positive had residual disease or lymph node metastases at cystectomy. Overall, 35 people had no histologically proven residual cancer at cystectomy, of whom all were ctDNA-negative.

After cystectomy, 17 people were ctDNA-positive of whom 76% (13 of 17) had recurrence. No one who was ctDNA-negative at this timepoint (n=47) had recurrence. Multivariable Cox proportional hazards regression analysis found that ctDNA status was the strongest predictor of recurrence-free survival after cystectomy (HR 129.6, p<0.001). ctDNA was found a median of 96 days (range ˗83 to 245 days) before imaging found metastatic relapse. Serial analysis of ctDNA during surveillance after cystectomy found metastatic relapse with 100% sensitivity and 98% specificity.

Strengths and limitations

This was a prospective study with serial ctDNA analysis throughout treatment. The study does not report on the use of ctDNA analysis in treatment decisions or its impact on clinical practice. It is unclear if healthcare professionals saw ctDNA results. The company said it provided funding for the testing used in the study, but samples and methods were provided by the collaborators.

Study size, design and location

Prospective multicentre cohort study in 49 adults with breast cancer in the UK. People were recruited after surgery and adjuvant therapy and had no signs of metastatic disease.

Intervention

Signatera in addition to standard care. Samples for ctDNA analysis were collected every 6 months for up to 4 years.

Key outcomes

At the time of interim reporting of results, 18 people had recurrence of disease, of whom 16 (89%) tested ctDNA-positive. Everyone who did not relapse (31 of 49) tested ctDNA-negative with an assay specificity of 100%. Recurrence occurred within 50 months after surgery with ctDNA detected a median of 8.9 months before clinical relapse (range 14 to 721 days). ctDNA was also detected before positive results on CT scans and other standard care tests such as liver function tests and CA 15‑3.

Strengths and limitations

This study reports interim results of a UK multicentre study. Target sample size for the main study was 194 assuming a 20% dropout rate and a 20% rate of recurrence in 2 years. Authors reported that serial plasma samples were analysed blinded but no details of this was provided. It is unclear if healthcare professionals and patients were told ctDNA results or if this impacted treatment decisions. Some results were presented across breast cancer subtype, but sample sizes in these groups were very small which limits the certainty of findings. Authors reported that in addition to presence or absence of ctDNA, the levels of ctDNA can be used to track disease burden over time. But this was based on data from 12 people with only 1 person who did not have relapse. Some authors were affiliated with the company.

Study size, design and location

Multicentre cohort study in 130 people with stage 1 to 3 colorectal cancer in Denmark. Samples were collected prospectively. ctDNA analysis was retrospective and not shared with healthcare professionals or patients.

Intervention

Signatera in addition to standard care. Samples for ctDNA analysis were collected before surgery, after surgery, and every 3 months until death, withdrawal from the study or month 36.

Key outcomes

Five people were excluded because they were lost to follow-up or progressed to stage 4 disease. Overall, 24 people (19.2%) had radiologic recurrence. Before surgery, ctDNA was found in 88.5% of samples (108 of 122) while carcinoembryonic antigen (CEA) was found in 43.3% (53 of 122). After surgery before adjuvant therapy, 89.4% of people (84 of 94) tested ctDNA‑negative and 10.6% (10 of 94) positive. Recurrence was higher in people who tested ctDNA-positive (70%) compared with ctDNA-negative (11.9%).

Overall, 58 people had adjuvant chemotherapy. Recurrence was found in everyone who tested ctDNA-positive after adjuvant therapy and in 13.7% (7 of 51) who tested negative (HR 17.5, 95% CI 5.4 to 56.5; p<0.001). Serial ctDNA analysis after definitive treatment (n=75) identified recurrence with 88% sensitivity and 98% specificity. In this group, recurrence occurred in 14 of 15 people (93.3%) who tested ctDNA-positive compared with 2 of 60 (3.3%) who tested negative. Comparatively, CEA analysis found recurrence with 69% sensitivity and 64% specificity. ctDNA analysis detected recurrence a mean 8.7 months before CT scan, but this lead time was not found for CEA.

Strengths and limitations

Treatment and follow-up were clearly outlined and followed Danish Colorectal Cancer Group guidelines that were similar to NICE's guideline on colorectal cancer. The study had an overall sample size of 130 people of which 24 had recurrent disease. ctDNA analysis was retrospective and did not impact treatment decisions or clinical outcomes. Length of follow up varied as did the number of samples analysed at differing timepoints. This made it difficult to interpret changes in ctDNA status across the study. There was also some discrepancy in the reporting of group sizes which affected clarity of the reported findings. Some authors were affiliated with the company.