Cell-free DNA Copy Number Research Articles

Multigene Copy Number Alteration Risk Score Biomarker-Based Enrichment Study Designs in Metastatic Castrate-Resistant Prostate Cancer.

A composite multigene risk score derived from tumor-biology alterations specific to metastatic castrate-resistant prostate cancer (mCRPC) state was evaluated as a classifier to design biomarker-based enrichment clinical trials. A plasma cell-free DNA copy number alteration risk score based on alterations in 24 genes was simulated to develop a biomarker classifier-based clinical trial design enriched for high-risk patients to detect a survival advantage of a novel treatment (hazard ratio of 0.70 with 80% power). We determined the design trade-offs between the number of patients screened and enrolled when varying the type of patients to enrich and the extent of enrichment needed. For a 2-year overall survival end point in mCRPC state, fully enriching patients with mCRPC having a high-risk score of 3 or more (the 95th percentile of a range of risk scores in patients with mCRPC) was determined to require screening to a maximum of 4,149 patients to enroll 259 patients for the targeted effect size. A nonenriched trial was determined to require enrolling 689 patients to be equivalently powered. We identified a pragmatic alternative, which is to enrich patients with mCRPC with a risk score of 1 or more (the 67th percentile) and an enrichment fraction of 0.25. This would require screening 658 patients to enroll 584 patients, and it maximizes the ability to detect a difference in treatment effect by risk score. A plasma multi-CNA risk score classifier can feasibly be leveraged to design an enrichment trial in mCRPC. Enriching 25% of patients screened with a risk score >1 was observed to be optimal for obtaining an adequately powered, biomarker-based mCRPC-enriched clinical trial.

Evaluating the effectiveness of pre-operative diagnosis of ovarian cancer using minimally invasive liquid biopsies by combining serum human epididymis protein 4 and cell-free DNA in patients with an ovarian mass

ObjectiveTo assess the feasibility of scalable, objective, and minimally invasive liquid biopsy-derived biomarkers such as cell-free DNA copy number profiles, human epididymis protein 4 (HE4), and cancer antigen 125 (CA125)...

Circulating tumour DNA (ctDNA) burden quantified through low-pass whole genome sequencing as an early pharmacodynamic biomarker of therapeutic response in patients enrolled on phase 1 trials.

23 Background: Early phase trials are conducted to determine the recommended dose and safety of an investigational therapy. Targeted therapies and immunotherapy may not always have dose-dependent toxicities. Therefore, dose finding is increasingly reliant on optimal biological dosing. Pharmacodynamic biomarkers are increasingly important to demonstrate proof of concept. Using ctDNA allows for dynamic and early tumour assessments compared to imaging. We sought to evaluate ctDNA as an early pharmacodynamic biomarker of therapeutic response. Methods: Patients (pts) enrolled on phase 1 trials at the National Cancer Centre Singapore were prospectively consented to collect serial blood samples at defined timepoints (pre-treatment, mid-cycle 1, end of cycle 1, and with subsequent imaging evaluations). ctDNA burden was quantified using low pass whole genome sequencing (lpWGS) with both established (ichorCNA) and novel (cell-free DNA degradation) bioinformatic analyses of ctDNA burden, and targeted panel sequencing. Early kinetics of ctDNA burden prior to imaging evaluation was evaluated as response (reduction in ctDNA burden) versus progression (increase in ctDNA burden). Results: Between May 2021 to Oct 2022, 68 plasma samples (median 3, range 2-5) were collected from 22 pts. Median age was 56 years (range 37-77) and 73% were male. Pts were enrolled on trials of targeted therapy (55%), immunotherapy (40%) or combinations of both (5%). Common tumour types were lung (36%), colorectal (23%) and pancreatic (9%) cancer. Best response on phase 1 trial as per imaging and RECIST v1.1 was partial response (PR) in 9%, stable disease (SD) in 45%, progressive disease (PD) in 27% and not evaluable (NE; taken off trial early due to toxicity) in 18%. Quantification of ctDNA burden using cell-free DNA fragment length pattern demonstrated enhanced limit of detection in detecting baseline ctDNA compared to cell-free DNA copy number profile based ichorCNA method (100% vs 59%). For pts on targeted therapies, early kinetics of ctDNA burden indicated ctDNA response in 9/13 pts, with subsequent imaging showing PR (n=2), SD (n=2), PD (n=2) or NE (n=3). In the remaining 4/13 pts on targeted therapies with early ctDNA progression, subsequent imaging showed SD (n=3) or NE (n=1). For pts on immunotherapy, early ctDNA progression was seen in 8/10 pts, with subsequent imaging showing SD (n=5) and PD (n=3). Patient enrolment and sample analysis is ongoing to further validate these initial findings and updated data will be presented. Conclusions: Early ctDNA evaluation via lpWGS may represent an early pharmacodynamic biomarker of therapeutic response. ctDNA has the potential to provide early proof of concept, particularly for targeted therapies, that may influence subsequent decisions for dose determination.

Cell-free DNA copy number variations predict efficacy of immune checkpoint inhibitor-based therapy in hepatobiliary cancers

BackgroundThis study was designed to screen potential biomarkers in plasma cell-free DNA (cfDNA) for predicting the clinical outcome of immune checkpoint inhibitor (ICI)-based therapy in advanced hepatobiliary cancers.MethodsThree cohorts including...

Identification of somatic copy number variations in plasma cell free DNA correlating with intrinsic resistances to EGFR targeted therapy in T790M negative non-small cell lung cancer.

BackgroundAbout 20–30% EGFR-mutant non-small lung cancer show intrinsic resistance to EGFR targeted therapies. Compared to T790M positive in acquired resistance patients, little is known about EGFR-TKI intrinsic resistance for T790M negative patients.MethodsThirty-one patients with advanced stage lung cancer, including 18 patients with intrinsic resistance (PFS <6 months) and 13 patients with acquired resistance (PFS >36 months) but are negative for plasma T790M were recruited in the study. Plasma cell free DNA was profiled by low coverage whole genome sequencing with median genome coverage of 1.86X by Illumina X10. Sequencing coverage across chromosomes was summarized by samtools, and normalized by segmentation analysis as provided by R package ‘DNACopy’.ResultsThe most frequent chromosomal changes were found on chr7, chr1 and chr8. Among them, chr7p gains were found in 12 (66.7%) intrinsic resistance and 4 (30.7%) acquired resistance patients. The gene EGFR was found located on the focal amplification peak of chr7p. The performance of 7p gain to predict intrinsic resistance reaches AUC =0.902. Similarly, focal amplifications were also found on chromosome 5, 16 and 22, where tumor related gene PCDHA@, ADAMTS18 and CRKL were located. Focal deletions were also found in chr1, 8, 10 and 16, where genes SFTPA1/2, DLC1, PTEN and CDH1 are located.ConclusionsThe results suggest cell free DNA copy number might be a useful peripheral blood tumor biomarker for predicting intrinsic resistance of EGFR targeted therapy and prognosis.

Persistently increased cell-free DNA concentrations only modestly contribute to outcome and host response in sepsis survivors with chronic critical illness

BackgroundAlthough early survival from sepsis has improved with timely resuscitation and source control, survivors frequently experience persistent inflammation and develop chronic critical illness. We examined whether increased copy number of endogenous alarmins, mitochondrial DNA, and nuclear DNA are associated with the early “genomic storm” in blood leukocytes and the development of chronic critical illness in hospitalized patients with surgical sepsis. MethodsA prospective, observational, cohort study of critically ill septic patients was performed at a United States tertiary health care center. Blood samples were obtained at multiple time points after the onset of sepsis. Droplet Digital polymerase chain reaction (Bio-Rad Laboratories, Hercules, CA) was performed to quantify RHO (nuclear DNA) and MT-CO2 (mitochondrial DNA) copies in plasma. Leukocyte transcriptomic expression of 63 genes was also measured in whole blood. ResultsWe enrolled 112 patients with surgical sepsis. Two experienced early death, 69 recovered rapidly, and 41 developed chronic critical illness. Both mitochondrial DNA and nuclear DNA copy number were increased in all sepsis survivors, but early nuclear DNA, and not mitochondrial DNA, copy number was further increased in patients who developed chronic critical illness. Cell-free DNA copy number was associated with in-hospital but not long-term (180-day and 365-day) mortality and were only weakly correlated with leukocyte transcriptomics. ConclusionIncreased cell-free DNA copy number persists in survivors of sepsis but is not strongly associated with leukocyte transcriptomics. Nuclear DNA but not mitochondrial DNA copy number is associated with adverse, short-term, clinical trajectories and outcomes.

Elevated plasma cell-free DNA copy number variations to predict survival for cancer patient with spinal metastasis.

e14522 Background: Approximately 70% of patients with cancer have evidence of spinal metastatic disease at the time of their deaths. The spinal column is the most common location among osseous sites for metastatic deposits. Expected overall survival is the major determinant of treatments for those ones suffering from intractable pain, neurologic deficits, and even paralysis. We sought to evaluate copy number variations (CNV) in spinal metastatic cancer via cfDNA and determine if CNV is associated with prognosis and treatment decision. Methods: In this study, 314 patients with pathologically confirmed spinal metastatic cancers were recruited since November 2015. 314 plasma samples were sent to low-coverage genome-wide sequencing of cfDNA from plasma followed by a customized bioinformatics workflow UCAD. Tokuhashi score was also evaluated for each patient before surgery. Statistical correlation with clinical index like prognosis was estimated. Results: 280 evaluable data were collected (34 samples failed Quality Control). The median follow-up time is 276 days. 114 (40.7%) patients died during follow up till December the 25th, 2018. Elevated CNVs was found in 109 (38.9%) patients, including 9(69.2%) head&amp;neck, 15(46.9%) liver and gallbladder, 27(44.3%) lung, 7(38.9%) breast, 2(28.6%) prostate, 5(19.2%) thyroid cancer, 4 (10.5%) kidney and 20(40.0%) cancer with unknown primary site. Further analyses showed that patients with elevated CNVs were found with worse survival. The median overall survival (OS) was 298 (95% CI: 258-422) days, as compared with those low-CNVs status with median OS 657 (95% CI:433-NA) days (Hazard ratio = 3.73 [95% CI: 2.22-6.27], P &lt; 0.01). Especially for patients with low Tokuhashi score (≤8) who have the predictive survival less than 6 months, CNVs score distinguish those well-prognosis patients with median OS 433 (95% CI: 308-NA) days from the worse survival group with median OS 285(95% CI:243-348) days (Hazard ratio = 2.42 [95% CI:1.38-4.25], P = 0.013). Conclusions: We present the largest cfDNA genomic characterization of spinal metastatic cancers. Specific CNVs features are enriched in spinal metastasis cancers with different primary sites. Elevated CNVs in plasma cfDNA is significantly associated with worse survival in a large spinal metastatic cancer cohort. This demonstrates that cfDNA CNVs could be a useful marker in estimating the survival time of spinal metastasis cancer patients for whom the outcome is mainly dependent on the selection of proper treatment.

Plasma cell-free DNA copy number analysis by low coverage whole genome sequencing to monitor breast cancer relapse in CA15-3 and CEA silent patients.

e13036Background: CA15-3 and CEA are the commonly used serum tumor biomarkers for monitoring breast cancer relapse. However, it is limited by sensitivity. Here we investigated copy number variation...

Tumor cell-free DNA copy number instability compared to CA19-9 as an early predictor of response to systemic therapy in pancreatic ductal adenocarcinoma (PDAC).

e14524 Background: Humoral tumor markers are used clinically for real-time assessment of therapeutic efficacy. In pancreatic ductal adenocarcinoma (PDAC) the predominant marker is CA19-9, which is not expressed by 10 to 30% of patients depending on race. We compared plasma cell-free DNA (cfDNA) copy number based assay with changes in serum CA19-9 levels and radiological responses to predict responses to systemic therapy. Methods: In a laboratory blinded, prospective multicenter pilot study, 40 non-resectable PDAC patients, treated with (m)FOLFIRINOX, CAPIRI, or gemcitabine +/- nab-paclitaxel) are currently enrolled. CA19-9 was determined in the local center’s laboratory. Tumor cfDNA was measured with a copy-number instability (CNI) scoring assay, determined by next generation sequencing in a centralized laboratory. The CNI score assesses the amount of cfDNA with somatic macro-alterations originating from malignant neoplasms. The difference of the values before commencing therapy (baseline) and prior to cycle 2 (either rising or falling) was calculated as a predictor of standardized radiological evaluation of chemotherapeutic efficacy. Results: 37 patients (3 drop-outs) had data for baseline and cycle 2, of which CA19-9 was elevated and evaluable in 29 patients. The direction from baseline to cycle 2 of CA19-9 and CNI scores were in agreement in 18/29 patients. 9 of 11 cases with discordant CNI score and CA19-9 had treatment response data, and CNI correlated with 7/9 (78%); in contrast 7/9 had rising CA19-9, when response was stable disease or better (22% concordance). In the 27 patients with available imaging, CNI predicted better (n = 18) than CA19-9 (n = 10) (p = 0.03 Fisher’s exact). Conclusions: This comparative study on cfDNA versus CA19-9 suggest that cfDNA CNI quantitation is a potentially more reliable blood based marker for early real-time assessment of efficacy in systemic PDAC therapy than CA19-9, compared to standard of care imaging. The better prediction after the first cycle might be due to the very short in vivo half-life of cfDNA ( &lt; 1hr) compared to about one week for CA19-9. These results justify a larger prospective validation trial.

Circulating cell-free DNA copy-number profiles as a biomarker in melanoma

Circulating cell-free DNA copy-number profiles as a biomarker in melanoma

Abstract 3138: Changes in tumor cell-free DNA copy number instability (CNI) predict therapeutic response in metastatic cancers

Abstract Background: Tumor cell-free DNA (cfDNA) has the potential to provide minimally invasive patient specific biomarkers to monitor tumor burden. Gains and losses of chromosomal regions - as a hallmark of cancer - have been detected in plasma as copy number aberrations (CNAs), and for several cancers a relation to tumor size has been reported. Longitudinal observations during anti-cancer therapy have been mostly anecdotal. We measured CNAs changes during treatment by computing a genomic copy number instability index (CNI) of cfDNA to evaluate its potential to predict cytotoxic chemotherapy (chemo) response. Methods: 24 patients (pts) with advanced esophageal cancer (EC; n = 2), colorectal cancer (CRC; n = 3), non-Hodgkin lymphoma (NHL; n = 3), pancreatic ductal adenocarcinomas (PDAC; n = 4), and non-small cell lung cancer (NSCLC; n = 12) were included and assessable for response. DNA was extracted from pre-treatment plasma samples at baseline and sequentially for up to six cycles of chemotherapy. Copy-numbers were called from shotgun sequencing (Illumina) after mapping and quality filtering reads were counted in 5.5MBp windows (sliding) yielding a read coverage of 24,000-fold per bin. The read counts were transformed into log2 ratios and converted into a score based on Gaussian transformations. Concentration of total cfDNA was determined by digital PCR. Treatment response by imaging was recorded by RECIST 1.1 or EORTC PET/CT criteria. For pts with baseline CNI&amp;gt;40 (representing the 99.99% level for non-cancer healthy controls), CNI change was considered predictive of response or stable disease when there was a reduction of CNI ≥50% relative to baseline. Disease progression was demonstrated when: 1) CNI was ≥50% over nadir and &amp;lt;80% reduction relative to baseline; OR, 2) CNI was ≥5 fold the nadir; OR, 3) CNI demonstrated an absolute increase by 1,000 above baseline CNI. For pts with baseline CNI≤40, CNI change was considered predictive of response or stable disease when CNI remained ≤40, and PD when CNI≥2X above the baseline CNI. CNI was an early predictor of response if these results were observed by ∼3-8 weeks prior to imaging or a major change in pt's clinical status. Results: CNI was measured in 124 samples. Median baseline cfDNA was 7,238 cp/mL (range 2,319-91,978) and CNI was 171 (range 26-10,170). In general, NHL and tumors with high lymph node disease burden had highest baseline cfDNA and CNI, whereas overall the CNI was not correlated to the cfDNA content. 22 of 24 pts (91.7%) had CNI changes that were predictive of treatment response. For at least 15 pts, CNI change predicted response ∼3-8 weeks prior to scan results demonstrating response or PD. Conclusions: CNI change may serve as predictor (potentially early predictor) of therapeutic response to standard chemo for the investigated cancer types. Highest baseline cfDNA and CNI appear to be present in lymph node predominant disease, suggesting more readily shed DNA into the circulation. Citation Format: Glen J. Weiss, Julia Beck, Donald P. Braun, Kirsten Bornemann-Kolatzki, Heather Barilla, Rhiannon Cubello, Ashish Sangal, Robert P. Whitehead, Madappa Kundranda, Vivek Khemka, Howard B. Urnovitz, Ekkehard Schutz. Changes in tumor cell-free DNA copy number instability (CNI) predict therapeutic response in metastatic cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3138.

Tumor cell-free DNA copy number instability (CNI) to predict therapeutic response to immunotherapy prior to cycle 2.

3027Background: Tumorcell-free DNA (cfDNA) provides minimally invasive patient specific biomarkers to monitor tumor burden. Gains and losses of chromosomal regions have been detected in plasma as c...

Cell-free DNA copy number variations as a marker for breast cancer in a large study cohort.

11013 Background: Massive parallel sequencing provides high numbers of cell-free nucleic acid serum DNA sequences (cfDNA) that can detect trace amounts of tumor derived chromosomal imbalances and copy number variations (CNVs) in patients with cancer. The aim of this study was to determine if there is a difference between the cfDNA CNVs from patients with breast cancer (BrCa) compared to healthy controls. Methods: DNA extracted from serum samples of 225 BrCa (Stage 1 to 4) and 205 gender and age-matched healthy controls (HC) was amplified using random primers, tagged with a unique molecular identifier per sample, sequenced on an Illumina HiSeq system and aligned to the human genome (Build 37). Hits were counted in sliding 1Mbp interval regions and normalized. Using a Random-Resampling procedure, a model was established to distinguish BrCa from HC using the copy number variations (CNV) and cross validated. Results: From 1,100 rounds of random resampling (50/50), a set of 31 regions was selected, based on the frequency of occurrence in the models. Using 20 random sets of a 10-fold cross validation, the selected regions were found to be highly significant discriminators between BrCa and HC (p&lt;10-5). When using a final linear model with 16 regions the AUC of a diagnostic ROC curve was found to be 0.895 for all samples, for Stage I and II the AUC was 0.86 compared to 0.93 for the higher stages. The final model included three regions from chromosome 8 and 1 and two regions from chromosome 15, the remaining regions were found as one per chromosome. Conclusions: Using comparative massive parallel sequencing of cfDNA from cancer patients vs. controls, we were able to show that a 16-region model based on CNV, is useful to distinguish patients with breast cancer from matched controls. Genomic instabilities that are shed into the circulation from breast cancer may play a role in screening, monitoring or as companion diagnostic tests in breast cancer.