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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.

Background: Circulating tumor DNA (ctDNA) is an emerging biomarker in non-Hodgkin lymphomas (NHLs). Current methods for ctDNA minimal residual disease (MRD) are limited by two factors - low input DNA amounts and high background error rates. VDJ sequencing (i.e., IgHTS) has low background but is limited by low cell-free DNA (cfDNA). Tracking multiple mutations via CAPP-Seq improves sensitivity, but detection is limited by background errors. Clustered mutations have been described in multiple cancers including NHLs and potentially have lower error rates. We explored clustered mutations from whole-genome sequencing (WGS) to identify 'phased variants' (PVs), defined as multiple mutations on a single DNA molecule (Fig 1A). We designed a method to capture PVs for improved ctDNA detection and explored its utility for MRD in DLBCL. Methods: We reanalyzed WGS from 1455 tumors across 11 cancer types. We identified genomic regions recurrently containing PVs and designed an assay for deep cfDNA sequencing. We applied this assay to 171 patients with large B-cell lymphomas. We compared the performance of PVs for disease detection to current ctDNA techniques, including CAPP-Seq and duplex sequencing. Results: To utilize PVs, mutations must occur within a typical cfDNA strand (~170bp). We measured the frequency of putative PVs in WGS, focusing on pairs of mutations occurring within &lt;170bp. PVs were more frequent in NHLs than any other histology (median: DLBCL, 642; FL, 307; Burkitt, 89.5; CLL, 34; breast, 46; lung, colorectal, melanoma, bladder, cervical, head & neck &lt; 10 per case; P &lt; 0.001 for NHLs vs others). PVs in NHLs were enriched in single base substitution mutational signatures associated with activation-induced cytidine deaminase (AID) (SBS84 & 85). PVs in NHLs occurred in stereotyped regions, including canonical AID targets such as IGH, IGK, and IGL, as well as 44 other AID targets (Schmitz, NEJM 2018) (Fig 1B). We additionally identified novel regions not previously implicated as targets of AID, including LPP, XBP1, BZRAP1, and HLA-DQ. We designed an approach for enriching PVs from ~115kb (Phased variant Enrichment Sequencing, PhasE-Seq) and other regions recurrently mutated in B-NHLs. We compared PhasE-Seq and CAPP-Seq using tumor and plasma samples from 16 patients. Compared to CAPP-Seq, PhasE-Seq yielded more SNVs and PVs per case (median SNVs: 331 vs 114, P&lt;0.001; PVs: 729 vs 222.5, P &lt;0.001). We next applied PhasE-Seq to 171 patients with untreated lymphomas (DLBCL, 148; primary mediastinal B-cell lymphoma, PMBCL, 23) profiling 58 tumor and 171 plasma samples with matched germline. We observed significant differences in the distribution of PVs between subtypes - for example, GCB-DLBCL had more PVs in BCL2, MYC, and SGK1, while ABC-DLBCL had more in PIM1 and IGHV4-34 (Fig 1C). Similarly, we noted enrichment in PVs in PMBCL in CIITA, SOCS1, CD83, and ITPKB. We then compared PhasE-Seq to alternative methods for MRD detection. We used limiting dilutions of patient ctDNA down to 1:1,000,000 to establish the detection limit (LOD, Fig 1D). PhasE-Seq outperformed CAPP-Seq and duplex sequencing for recovery of expected tumor content, with a high degree of linearity down to ~1:1,000,000. We applied standard CAPP-Seq and PhasE-Seq to patient cfDNA samples after two cycles of front-line therapy (n=92). We previously reported a 2.5-log reduction in ctDNA as prognostic at this time-point (Kurtz, JCO 2018). Using CAPP-Seq, 58% (53/92) of samples were undetectable. Using PhasE-Seq, 30% (16/53) of samples not detected by CAPP-Seq had evidence of MRD, with levels as low as 2:1,000,000. In patients with ctDNA undetected by CAPP-Seq, detection by PhasE-Seq significantly stratified outcomes (Fig 1E). Conclusions: PVs are frequent in NHLs, likely due to AID, and correlate with disease biology. PhasE-Seq allows for superior detection of ctDNA, including MRD detection in the majority of patients after 2 cycles. Targeted sequencing of ctDNA should consider PVs to maximize detection and guide precision approaches. Figure 1: A) Structure of phased variants B) Distribution of putative PVs from WGS data C) Genomic enrichment in PVs in lymphoma subtypes D) Dilution series comparing PhasE-Seq, CAPP-Seq, and duplex sequencing E) Waterfall plot showing ctDNA level vs outcome; undetectable ctDNA by CAPP-Seq is highlighted F) EFS of patients with undetectable ctDNA by CAPP-Seq after 2 cycles, stratified by PhasE-Seq Disclosures Kurtz: Roche: Consultancy. Diehn:Roche: Consultancy; AstraZeneca: Consultancy; Novartis: Consultancy; BioNTech: Consultancy; Quanticell: Consultancy. Alizadeh:Pharmacyclics: Consultancy; Janssen: Consultancy; Genentech: Consultancy; Roche: Consultancy; Gilead: Consultancy; Celgene: Consultancy; Chugai: Consultancy; Pfizer: Research Funding.

7565 Background: Detection of circulating tumor DNA (ctDNA) has prognostic value in diverse tumors, including DLBCL. Despite uses for assessing molecular response to therapy, current methods using immunoglobulin or hybrid-capture sequencing have suboptimal sensitivity, particularly when disease-burden is low. This contributes to a high false negative rate at key milestones such as at the end of therapy (EOT; Kumar A, ASH 2020). We explored the utility of detecting multiple mutations (phased variants, PVs) on individual cell-free DNA (cfDNA) strands to improve MRD in DLBCL. Methods: We applied Phased Variant Enrichment and Detection Sequencing to track PVs from 485 specimens from 117 DLBCL patients undergoing first-line therapy. We sequenced cfDNA prior to, during, and after therapy to assess the prognostic value of MRD. We compared the performance of PhasED-Seq to current techniques, including SNV-based CAPP-Seq and duplex sequencing. Results: To establish its detection limit for ctDNA, we compared the background error-profile of of PVs and SNVs in cfDNA sequencing from healthy subjects. PV-detection by PhasED-Seq demonstrated a lower background profile than SNVs, even when considering duplex molecules (n = 12; 8.0e-7 vs 3.3e-5 and 1.2e-5; P < 0.0001). We also assessed analytical sensitivity within a ctDNA limiting dilution series from 3 patients, simulating tumor fractions from 0.1% to 0.00005% (1:2,000,000). PhasED-Seq outperformed SNV-based methods and duplex sequencing for recovery of expected tumor content below 0.01% (P < 0.0001 and P = 0.005 respectively by paired t-test). We then explored disease detection in clinical samples. We identified SNVs and PVs from pretreatment tumor or plasma and followed these variants in serial cfDNA. Using SNV-based methods, 40% and 59% of patients had undetectable ctDNA after 1 or 2 cycles (n = 82 and 88). However, 24% and 25% of these cases had detectable ctDNA by PhasED-Seq. Importantly, MRD detection by PhasED-Seq was prognostic for event-free survival even in patients with undetectable ctDNA by SNVs. We next explored the utility of PhasED-Seq at the EOT in 19 subjects, 5 of whom experienced eventual disease progression. While only 2/5 cases with progression had detectable disease at EOT using SNVs, PhasED-Seq detected all 5/5 cases. PhasED-Seq also correctly identified all patients (14/14) without clinical relapse as having no residual disease, including one patient who discontinued therapy after 1 cycle due to toxicity, but remains in remission > 5 years after this single treatment. This resulted in superior classification of patients for EFS using PVs compared with SNVs (C-statistic: 0.98 vs 0.60, P = 0.02). Conclusions: Tracking PVs results in significantly lower background rates than SNV-based approaches, enabling detection to parts per million range. PhasED-Seq improves on disease detection in DLBCL at the EOT, allowing possible MRD-driven consolidative approaches.