Leveraging phased variants for personalized minimal residual disease detection in localized non-small cell lung cancer.

Abstract

8518 Background: Detection of circulating tumor DNA (ctDNA) has prognostic value in lung cancer and could facilitate minimal residual disease (MRD) driven approaches. However, the sensitivity of ctDNA detection is suboptimal due to the background error rates of existing assays. We developed a novel method leveraging multiple mutations on a single cell-free DNA molecule (“phased variants” or PVs) resulting in an ultra-low error profile. Here we develop and apply this approach to improve MRD in localized NSCLC. Methods: To identify the prevalence of PVs, we reanalyzed whole genome sequencing (WGS) from 2,538 tumors and 24 cancer types from the pan-cancer analysis of whole genomes (PCAWG). We applied Phased Variant Enrichment and Detection Sequencing (PhasED-Seq) to track personalized PVs in localized NSCLC. We compared PhasED-Seq to a single nucleotide variant (SNV)-based ctDNA method. Results: In the PCAWG dataset, we found that PVs were common in both lung squamous cell carcinomas (LUSC, median 1,268/tumor; rank 2nd) and adenocarcinomas (LUAD, median 655.5/tumor; rank 3rd). However, PVs did not occur in stereotyped genomic regions. Thus, to leverage PhasED-Seq, we performed tumor/normal WGS to identify PVs, followed by design of personalized panels targeting PVs to allow deep cfDNA sequencing. We performed personalized PhasED-Seq for 5 patients with localized NSCLC. PVs were identified from WGS of tumor FFPE and validated by targeted resequencing in all cases (median 248/case). The background rate of PVs was lower than that of SNVs, even when considering duplex molecules (background: SNVs, 3.8e-5; duplex SNVs, 1.0e-5; PVs, 1.2e-6; P < 0.0001). We next assessed PhasED-Seq for MRD detection in 14 patient plasma samples. Both SNVs and PhasED-Seq had high specificity in healthy control cfDNA (95% and 97% respectively). Using SNVs, ctDNA was detected in 5/14 samples; PhasED-Seq detected all of these with nearly identical tumor fractions (Spearman rho = 0.97). However, PhasED-Seq also detected MRD in an additional 5 samples containing tumor fractions as low as 0.000094% (median 0.0004%). We analyzed serial samples from a patient with stage III LUAD treated with chemoradiotherapy (CRT) and durvalumab. SNV-based ctDNA and PhasED-Seq detected similar MRD levels (0.8%) prior to therapy. However, 3 samples collected during CRT, as well as before and during immunotherapy, were undetectable by SNVs. SNV-based ctDNA then re-emerged at disease recurrence. PhasED-Seq detected MRD in all 3 samples not detected by SNVs with tumor fractions as low as 0.00016%, including prior to immunotherapy (8 months prior to progression). Similar improvements were seen in samples not detected by SNVs from 2 additional patients. Conclusions: Personalized ctDNA monitoring via PVs is feasible and improves MRD detection in localized NSCLC. PhasED-Seq allows clinical studies testing personalized treatment based on MRD.

Making the Rounds: Exploring the Role of Circulating Tumor DNA (ctDNA) in Non-Small Cell Lung Cancer

“…In 2021, Kurtz et al developed a new technology, termed Phased Variant Enrichment and Detection Sequencing (PhasED-Seq) (Foresight Diagnostics Inc., Aurora, CO, USA), to improve the sensitivity of ctDNA MRD detection [ 120 , 123 ]. Assays that utilize molecular barcoding, such as CAPP-Seq, rely on duplex sequencing to improve the limit of detection, but duplex strand recovery is often suboptimal in the MRD setting [ 41 , 124 ].…”

“…Using this novel strategy, the authors showed that PhasED-Seq outperforms duplex sequencing with CAPP-Seq, yielding a limit of detection below one part per million [ 123 ]. PhasED-Seq was then applied to 14 plasma samples, collected from five patients with localized NSCLC, for MRD detection [ 120 ]. PhasED-Seq detected ctDNA MRD in 10 of the 14 samples, while a SNV-based ctDNA method only detected MRD in 5 of the 14 samples.…”

“…PhasED-Seq detected ctDNA MRD in 10 of the 14 samples, while a SNV-based ctDNA method only detected MRD in 5 of the 14 samples. Furthermore, the authors compared both methods to detect MRD in three longitudinal samples from a patient with unresectable stage III NSCLC who underwent curative-intent treatment with CRT and consolidation ICI [ 120 ]. While the SNV-based method failed to detect ctDNA MRD in all of the samples, PhasED-Seq detected ctDNA MRD at each of these timepoints [ 120 ].…”

“…Furthermore, the authors compared both methods to detect MRD in three longitudinal samples from a patient with unresectable stage III NSCLC who underwent curative-intent treatment with CRT and consolidation ICI [ 120 ]. While the SNV-based method failed to detect ctDNA MRD in all of the samples, PhasED-Seq detected ctDNA MRD at each of these timepoints [ 120 ]. These findings suggest the potential of PhasED-Seq as an ultrasensitive method for ctDNA MRD detection in patients with NSCLC.…”

“… Timeline of ctDNA MRD studies in patients with NSCLC. A timeline of studies that have advanced our understanding of ctDNA MRD as a prognostic and potentially predictive biomarker in patients with NSCLC, who were treated with a curative-intent [ 39 , 115 , 116 , 117 , 118 , 119 , 120 , 121 , 122 ]. …”