# Monitoring Immunotherapy Response Using Cell-Free DNA Fragmentomes

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

## Affiliations  
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 cell-free DNA (cfDNA) assays are costly and rely on prior knowledge of tumor-specific mutations.
- Here, we demonstrate the utility of DELFI Tumor Fraction (DELFI-TF), a tumor- and mutation-independent 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 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 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 dynamics were consistent with treatment response assessment using imaging.
- Baseline ctDNA levels predicted clinical outcome.
- 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).

**(Table 1: Patient Characteristics)**  
| Characteristic                        | Participants (Samples) | Median Age |  
|---------------------------------------|------------------------|------------|  
| Sex, n(%)                            |                        |            |  
| Male                                  | 59 (54%)               |            |  
| Female                                | 50 (46%)               |            |  
| Stage, n (%)                         |                        |            |  
| Stage III                             |                        |            |  
| Histology, n(%)                      |                        |            |  
| Adenocarcinoma                       | 84(77%)                |            |  
| Squamous cell                        | 23(21%)                |            |  
| Large cell carcinoma                  | 1(1%)                  |            |  
| Unknown histology                     | 1(1%)                  |            |  
| Prior treatment, n (%)               |                        |            |  
| Yes                                   | 72 (66%)               |            |  
| No                                    | 37 (34%)               |            |  
| Treatment, n (%)                     |                        |            |  
| Immunotherapy only                    | 80(73%)                |            |  
| Immunotherapy+ chemotherapy           | 29(27%)                |            |  
| Censored                              | 29 (27%)               |            |  
| Progressive disease                   | 80 (73%)               |            |

## RESULTS
- 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 time points were analyzed.
- 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 longer PFS and OS (log-rank p<0.001) compared to those in the molecular disease progression (mPD) group.

## CONCLUSIONS
- 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.
- Landmark molecular response early after ICI initiation identifies patients with differential clinical outcomes, which opens a window of opportunity for therapeutic intervention.