Supplementary Data from Genome-Wide Sequencing of Cell-Free DNA Identifies Copy-Number Alterations That Can Be Used for Monitoring Response to Immunotherapy in Cancer Patients
Authors: Jensen Taylor J. , Goodman Aaron M. , Kato Shumei , Ellison Christopher K. , Daniels Gregory A. , Kim Lisa , Nakashe Prachi , McCarthy Erin , Mazloom Amin R. , McLennan Graham , Grosu Daniel S. , Ehrich Mathias , Kurzrock Razelle 
Published: April 2023

<p>Supplementary Table 1. Patient clinical characteristics (N=44 patients). *PFS values with a (+) indicate response is ongoing at time of data censoring Abbreviations: cfDNA = cell-free DNA; CR = complete response; GE = gene equivalents per mL; GE junction = gastroesophageal junction; GIN = genome instability number; NSCLC = non-small cell lung cancer; PFS = Progression-free survival; PR = partial response; SBRT = stereotactic body radiation therapy; SCC = squamous cell cancer; SD = stable disease Supplementary Figure 1. GIN values for 6014 samples submitted for noninvasive prenatal testing for which no CNAs were detected using NIPT algorithms. Red dashed line represents the established threshold of a GIN value of 170 based on empirical distribution resulting in no GIN values higher than 170 in this training cohort. Supplementary Figure 2. Values for all baseline samples. Patients are divided based upon clinical response to immunotherapy treatment (response = stable disease for > 9 months or complete or partial response; No response (progressor) = progressive or stable disease {less than or equal to} 9 months; Unevaluable = ongoing stable disease not yet reaching 6 months. Top: GIN values for the baseline sample from each of 44 patients. Bottom: Total cfDNA per mL of plasma collected from each patient. Values represent total genome equivalents (GE) based on a single locus droplet digital PCR assay. There was no difference in GIN (p = 0.0.48) or in GE (p =0.49) between responders (N = 13 patients) and non-responders (N = 27 patients) (Supplementary Table 1). Abbreviations: cfDNA = cell-free DNA; GE = gene equivalents per mL; GIN = genome instability number; PCR = polymerase chain reaction. Supplementary Figure 3. Relationship between the total amount of cfDNA and the GIN in 44 baseline plasma samples. Spearman correlation coefficient = 0.49. Supplementary Figure 4. Overview of Patient 110 where the GIN prediction was discordant with RECIST criteria (stable disease for 4 months (hence labelled as progressor)). A) Genome-wide cfDNA profiles from each of the time points described above each plot. Sequencing reads were assigned to 50 kilobase (kb) non-overlapping segments of the human reference genome. The normalized read counts from each segment of the genome are shown with alternating colors delineating chromosomes. A LOESS regression was performed for each chromosome (white lines). Deviations above and below the median value (dashed black line) indicate amplifications and deletions, respectively. B) GIN values for each collected time point relative to treatment initiation. C) Relative change in CNA-specific z-scores over time, consisent with clonal evolution. Each line corresponds with the genomic locus described by like colored "*" in panel A. Note that the region on Chromosome 9 denoted with the purple * appears in all samples and reflects the GIN; however, the region on Chromosome 14 (green *) decreases over time while the region on Chromosome 12 (pink *) increases in the same samples. Collectively, these data are consistent with clonal selection in the tumor measured in the cfDNA. Supplementary Figure 5. Examples of GIN profiles of eleven patients predicted to be non-responders by GIN (responder defined as achieving SD>9 months, PR or CR). X axis = GIN value; Y axis = day since start of treatment. The data from patient 111 is colored green due to showing neither an increased or decreased GIN upon initial evaluation. Open circles represent measured samples with time relative to the treatment initiation (day 0; x-axis). Black dashed line represents the baseline GIN for each patient. Supplementary Figure 6. An independent cohort of 43 samples was processed through two independent sequencing runs and the GIN calculated for each. Cor=pearson correlation coefficient. Red dashed line represents the linear regression of these data.</p>