ctDNA Identifies Adjuvant MRD Positivity, Relapse in Lung Cancer

Chris Abbosh, MD

In the adjuvant setting, circulating tumor DNA (ctDNA) may be a biomarker for the detection of post-surgical minimal residual disease (MRD) and for determining the clonality of relapsing disease. Results from the Lung TRACERx study, in which the biomarker was found, has the potential to set the stage for future clinical trials that are structured to increase the standard-of-care dosage in patients with non—small cell lung cancer (NSCLC) who become MRD-positive after surgery.

Findings from the prospective TRACERx (NCT01888601) lung study were presented by Chris Abbosh, MD, of University College London in the United Kingdom, during the 2020 American Association for Cancer Research (AACR) Virtual Annual Meeting.

There were 78 patients for whom patient-specific anchored-multiplex polymerase chain reaction (AMP) enrichment panels were developed for analysis. These patients had undergone surgery for stage I-III NSCLC and from these participants, 608 plasma samples were tested.

The process was to perform primary tumor excision and then complete multi-region sampling and submit them to deep whole-exome sequencing. Variants including 100 clonal single nucleotide variants (SNVs), 50 neoantigens, and 50 subclonal variants were then prioritized based on clonality/subclonality, high copy number status, and low background sequencing noise. This process is carried out in order to optimize MRD sensitivity and allow for phylogenetic tracking. An AMP was then constructed against the variant positions being tracked and was applied to cell-free DNA (cfDNA) in the pre-operative and post-operative settings.

Conventional adjuvant trials are considered inferior to MRD-driven adjuvant trials because conventional trials have a heterogenous patient population with a low relapse-event rate, study recruitment numbers are high and take more than 10 years to complete, and the escalation of standard-of-care therapy creates a risk for toxicity in patients who are cured by surgery.

"The traditional adjuvant trials are challenging because you need hundreds, if not thousands, of patients to adequately power these studies, and you need to follow-up patients for many years. These challenges largely arise from the fact that you’re dealing with a heterogeneous patient population, which includes some patients who are cured by surgery and some patients who have residual or metastatic disease, and you can’t differentiate between these populations," said Abbosh. "The promise of an MRD-driven adjuvant trial is that you can differentiate between these populations and you can conduct adjuvant trials in smaller, more relevant populations when you are only escalating treatment in patients who are destined to relapse from their disease."

Characteristics in MRD-driven trials that overcome the challenges of conventional adjuvant trials include low recruitment numbers, rapid read-out of study data, therapy escalation only in patients who are destined to relapse, and an opportunity to establish disease-free survival surrogates who are predicated on MRD clearance. This is achieved with the use of a novel MRD-caller which can calculate interlibrary aberrants to inform the MRD pool. Tri-nucleotide error rates were established from the library and the error rates were then used to answer the question of whether the MRD signal at the position of interest was higher than expected based on the background error collected in the library. When MRD signals are higher than expected, an MRD-caller is created.

According to preclinical data from 2017, MRD was detected during pre-adjuvant radiotherapy with possible clearance of residual disease in patients with NSCLC who had no evidence of clinical relapse. False positives were seen in patients with a molecularly proven second primary. Overall, 26 patients with a median of 8 post-operative samples per patient (range, 3-13) were included in the non-recurrence cohort and followed-up for a median of 1173 days (3.2 years). In the second primary cohort, 11 patients were included with a median of 7 post-operative samples per patient (range, 1-10). This cohort had a median follow-up of 840 days of disease-free survival. The second primaries identified were 5 second primary lung cancers and 6 non-lung cancers.

Out of 96 early-stage pre-operative samples, ≥2 SNVs were detected in lung adenocarcinomas, ≥2 SNVs were detected in lung squamous cell carcinomas, and there was a low detection of other NSCLCs. Altogether, 11 out of 58 (19%) of lung adenocarcinomas were detected, 30 out of 31 (97%) lung squamous cell carcinomas were detected, including 16 out of 17 in stage I, and 5 out of 7 (71%) other NSCLCs were detected. This research provided information that sensitivity of the assay scales with DNA input. At a high input variant fractions can be detected down to 0.003%, Abbosh noted.

Based on the 2020 TRACERx lung study data, non-adenocarcinoma histology continues to show a correlation with pre-operative ctDNA shedding in NSCLC. Of 88 early-stage pre-operative samples, 49% of lung adenocarcinomas were detected, 100% of lung squamous cell carcinomas were detected, and 75% of other NSCLCs were detected.

The 50-variant AMP-MRD assay in the study was validated and showed an 89% sensitivity for mutant DNA at a MAF of 0.008% when 25 ng of DNA was entered into the assay. The specificity was 100% (95% CI, 92%-100%). The sensitivity scaled according to DNA input and the number of variants tracked.

Out of 42 patients who suffered a relapse in the TRACERx lung study, ctDNA was detected at or before clinical relapse in 38 of them. Median ctDNA lead-time in these relapsed patients was 164 days (range, 6-1022) and the median time to relapse from surgery was 362 days (range, 41-1143). No ctDNA was detected for 100% of the patients with a second primary, which indicates that the specificity of the MRD assay informs better on primary tumors. The median disease-free survival in relapsed patients with no detectable ctDNA pre-operatively was 640 days (range, 404-1242).

Using large-scale personalized enrichment panels, clonal evolution from therapy to relapse can be monitored. In the study, this was executed using 132 tracked clonal SNVs with knowledge of the copy-number macro-evolution at relapse in up to 483 variants per patient. Relapse can also be categorized as monoclonal or polyclonal and find clear subclonal dynamics during systemic intervention for disease recurrence.

Data from the TRACERx lung study show that AMP personalized cfDNA detect low-frequency variant DNA accurately and at low assay DNA inputs that are consistent with an MRD setting. The study also shows that MRD lead times are impacted by pre-operative shedding dynamics. Overall, surveilling MRD can expose relapse ahead of standard-of-care imaging surveillance.

Abbosh stated that based on the study results, the field of NSCLC is ready for MRD-driven adjuvant trials.

Reference

Abbosh C, Frankell A, Garnett A, et al. Phylogenetic tracking and minimal residual disease detection using ctDNA in early-stage NSCLC: A lung TRACERx study. Presented at: 2020 American Association for Cancer Research Virtual Annual Meeting; April 27-28, 2020. Abstract CT023.