Monitoring Immunotherapy Response Using Cell-Free DNA Fragmentomes

Monitoring immunotherapy response using cell-free DNA fragmentomes

1 2 1 1 1 2 2 2 2 1 Bahar Alipanahi, Lavanya Sivapalan, Jamie Medina, Bridget Tripp, Zachary Skidmore, Paola Ghanem, Gavin Pereira, Nisha Rao, Kavya Velliangiri, Caitlin Shonewolf, Noushin 2 1 1 1 1 1 2 2 1 2 Niknafs, Bryan Chesnick, Jennifer Tom, Stephen Cristiano, Peter Bach, Nicholas C. Dracopoli, Robert Scharpf, Victor Velculescu, Lorenzo Rinaldi, Valsamo Anagnostou

1 2 Delfi Diagnostics, Inc., Baltimore, MD, USA; The Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA

BACKGROUND

● Accurate and timely detection of disease progression in patients receiving immune checkpoint inhibitors (ICIs) is challenging given the lack of reliable biomarkers of clinical response. ● Current targeted next-generation sequencing

● Current targeted next-generation sequencing cell free DNA (cfDNA) assays are costly and rely on prior knowledge of tumor-specific mutations. ● Here, we demonstrate the utility of DELFI Tumor

● Here, we demonstrate the utility of DELFI Tumor Fraction (DELFI-TF), a tumor- and mutationindependent cfDNA fragmentome approach to monitor immunotherapy response in patients with metastatic non-small cell lung cancer (mNSCLC).

METHODS

● A cohort of 324 longitudinal blood samples were collected from 109 mNSCLC patients treated with ICI-containing regimens (ICI cohort); (Table 1). ● An independent cohort consisting of 47

● An independent cohort consisting of 47 longitudinal samples obtained from 15 mNSCLC patients undergoing treatment with both immunotherapy and chemotherapy was utilized to show the concordance with MAF and RECIST (Multi-Treatment cohort). ● Plasma-derived cfDNA was processed with whole

● Plasma-derived cfDNA was processed with whole genome sequencing (WGS) at low coverage (~4x). ● Circulating tumor burden was quantified as the

● Matched white blood cells were used to filter out germline and clonal hematopoietic variants. ● DELFI-TF, a random forest regression model

● DELFI-TF dynamics were consistent with treatment response assessment using imaging. ● Baseline ctDNA levels predicted clinical outcome.

● Baseline ctDNA levels predicted clinical outcome. ● Landmark molecular response assessment,

● Landmark molecular response assessment, defined as ctDNA detection above the limit of blank (LOB) at 3 to 9 weeks of ICI initiation, strongly predicted progression-free (PFS) and overall survival (OS). ● LOB estimated as the maximum of 95th

overall survival (OS). ● LOB estimated as the maximum of 95th percentile of DELFI-TF in 3 non-cancer cohorts. ● Clinical assessment of landmark PFS at 6 months

● In both the ICI and Multi-Treatment cohorts, DELFI-TF scores were strongly correlated with maxMAF (n=324, r=0.94, p<0.001, Pearson and n=47, r=0.94, p<0.001, Pearson respectively). ● Changes in DELFI-TF and maxMAF at all

● Clinical assessment of landmark PFS at 6 months revealed a significant association between ctDNA molecular response and durable clinical benefit (DCB; p < 0.001, Fisher’s exact test). ● Patients with molecular response (mR) attained

● Patients with molecular response (mR) attained longer PFS and OS (log-rank p<0.001) compared to those in the molecular disease progression (mPD) group.

Characteristic

Participants (samples) 109 (324)

Median Age 68 (36-91)

Stage, n (%) Stage III

DELFI-TF scores strongly correlate with maxMAF DELFI-TF was applied to all longitudinal samples of the ICI and Multi-Treatment cohorts.

Prior treatment, n (%) Yes

● DELFI-TF was applied to all longitudinal samples of the ICI and Multi-Treatment cohorts.

Clinical Status Censored

Table 1. Patient characteristics.

● Figure 2 and Figure 3 illustrate the correlation between DELFI-TF and maxMAF in the ICI cohort (n=324, r = 0.94, p < 0.001, Pearson correlation) and the Multi-Treatment cohort (n=47, r = 0.94, p < 0.001, Pearson correlation). Axis are scaled using a square root transformation.

DELFI-TF dynamics have strong correlation with maxMAF dynamics and are consistent with the clinical outcomes DELFI-TF change between serial timepoints were computed in the ICI cohort. Figure 4

Figure 2. Correlation between DELFI-TF and max MAF in the ICI cohort.

● DELFI-TF change between serial timepoints were computed in the ICI cohort. Figure 4 illustrates a strong correlation between DELFI-TF and maxMAF changes. ● DELFI-TF changes captured clinical outcomes. Figure 5 displays DELFI-TF in longitudinal

may be informative for risk classification and subsequent intervention. • DELFI-TF dynamics closely recapitulate radiographic responses by RECIST, which represent the current golden standard for therapy response assessment in patients with solid tumors. • Landmark molecular response early after ICI initiation identifies patients with differential clinical outcomes, which opens a window

• DELFI-TF is a cfDNA fragmentome–based tumor- and mutation- independent liquid biopsy approach that reliably and rapidly captures circulating tumor burden. • Patients with higher fragmentome-TF levels are at higher risk for disease progression and identification of such patients prior to ICI

• Landmark molecular response early after ICI initiation identifies patients with differential clinical outcomes, which opens a window of opportunity for therapeutic intervention. anagnostoulab.org | @ValsamoA | @HopkinsThoracic | @MolecularOncLab

• Murray et al. “Elucidating the Heterogeneity of Immunotherapy Response by Longitudinal ctDNA Tracking in Lung Cancer” Clinical Cancer Research, 2024 • Anagnostou et al. “ctDNA response after pembrolizumab in non-small cell lung cancer: phase 2 adaptive trial results” Nature Medicine, 2023.

● DELFI-TF changes captured clinical outcomes. Figure 5 displays DELFI-TF in longitudinal samples of representative patients with radiographic progressive disease (PD), stable disease (SD) and partial/complete response (PR/CR).

Figure 1. Liquid biopsy analyses performed at baseline and during treatment with ICI evaluated for cell-free DNA fragmentation patterns and mutant allele fraction (MAF).

Conclusions DELFI-TF is a cfDNA fragmentome–based tumor- and mutation- independent liquid biopsy approach that reliably and rapidly

• Anagnostou, V. et al. “Dynamics of tumor and immune responses during immune checkpoint blockade in non-small cell lung cancer” Cancer Research, 2019.

Figure 4. Correlation between DELFI-TF and maxMAF changes (n=215, r = 0.9, p < 0.001).

Figure 5. DELFI-TF dynamics for patients with different radiographic responses.

Figure 6. Kaplan Meier curves for progression-free survival by baseline DELFI-TF absolute scores.

DELFI-TF baseline values and molecular response predict clinical outcomes with ICI Baseline DELFI-TF scores were dichotomized by the median value in the ICI cohort; low

● Baseline DELFI-TF scores were dichotomized by the median value in the ICI cohort; low baseline DELFI-TF scores were associated with longer PFS (4.6 vs 14.7 months for DELFI-TF low and DELFI-TF high subgroups respectively, log rank p=0.0046) Absence of detectable DELFI-TF (below LOB) at any sampled timepoint within 3 to 9

DELFI-TF low and DELFI-TF high subgroups respectively, log rank p=0.0046) ● Absence of detectable DELFI-TF (below LOB) at any sampled timepoint within 3 to 9 weeks of ICI treatment initiation was defined as molecular response. ● Clinical assessment of landmark PFS at 6 months revealed a significant association

weeks of ICI treatment initiation was defined as molecular response. ● Clinical assessment of landmark PFS at 6 months revealed a significant association between DELFI-TF molecular response and durable clinical benefit (DCB; p < 0.001, Fisher’s exact test). ● Patients with mR attained longer progression free survival (16.04 vs 2.70 months, log-

Fisher’s exact test). ● Patients with mR attained longer progression free survival (16.04 vs 2.70 months, logrank p<0.001) compared to those in the mPD group. ● Patients with mR attained longer overall survival (40.64 vs 6.28 months, log-rank