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Circulating cell-free DNA (ccfDNA) is used increasingly as a cancer biomarker for prognostication, as a correlate for tumor volume, or as input for downstream molecular analysis. Determining optimal blood processing and ccfDNA quantification are crucial for ccfDNA to serve as an accurate biomarker as it moves into the clinical realm. Whole blood was collected from 50 subjects, processed to plasma, and used immediately or frozen at -80°C. Plasma ccfDNA was extracted and concentration was assessed by real-time quantitative PCR (qPCR), fluorimetry, and droplet digital PCR (ddPCR). For the 24 plasma samples from metastatic pancreatic cancer patients, the variant allele fractions (VAF) of KRAS G12/13 pathogenic variants in circulating tumor DNA (ctDNA) were measured by ddPCR. Using a high-speed (16,000 × g) or slower-speed (4100 × g) second centrifugation step showed no difference in ccfDNA yield or ctDNA VAF. A two- versus three-spin centrifugation protocol also showed no difference in ccfDNA yield or ctDNA VAF. A higher yield was observed from fresh versus frozen plasma by qPCR and fluorimetry, whereas a higher yield was observed for frozen versus fresh plasma by ddPCR, however, no difference was observed in ctDNA VAF. Overall, our findings suggest factors to consider when implementing a ccfDNA extraction and quantification workflow in a research or clinical setting.

We have previously demonstrated an independent association between high levels of plasma circulating cell-free DNA (ccfDNA) concentration and poor survival outcomes in patients with newly diagnosed glioblastoma. Given that somatic mutations are rarely detected in glioblastoma patient plasma, we reasoned that DNA shed by tumor (circulating tumor DNA, ctDNA) was not likely to be a driver of prognostic increases in ccfDNA. To investigate the tissue of origin for this prognostic ccfDNA, we analyzed the plasma ccfDNA methylation signatures of 23 patients with newly diagnosed glioblastoma using the Illumina Infinium EPIC array, a genome-wide DNA methylation technique covering over 850,000 CpG sites. Deconvolution of the ccfDNA methylation signatures based on published reference methylomes revealed an increased proportion of ccfDNA derived from granulocytes in glioblastoma specimens compared to healthy controls (p < 0.0001). Further, this granulocyte proportion was increased in patients with high ccfDNA levels (above median value) compared to patients with low ccfDNA (p = 0.0001). Granulocyte proportion correlated with ccfDNA concentration (Spearman r = 0.64, p = 0.001). The top two gene sets identified by differential methylation analysis followed by gene set enrichment analysis (methylGSA) between high- and low-ccfDNA specimens were “neutrophil activation involved in immune response” (GO:0002283, p = 3.4E-23) and “neutrophil degranulation” (GO:0043312, p = 3.4E-23). Additional analysis of ccfDNA fragment size identified larger fragments ( > 700 bp) as the major source of the prognostic signal (log-rank p = 0.002 for progression-free survival) compared to traditionally sized ccfDNA fragments (50-700 bp, log-rank p = 0.12). Taken together, these data suggest that granulocytes are the primary contributing source of prognostic ccfDNA in glioblastoma. Future studies are needed to determine the mechanism through which granulocyte-derived ccfDNA is associated with clinical outcomes in glioblastoma and to explore related therapeutic opportunities.

Up to 85% of patients with resectable pancreatic ductal adenocarcinoma (PDAC) experience metastatic relapse after curative intent surgery with many recurring early. Detection of such occult metastases (those thought to be below the level of detection of standard of care imaging at the time of resection) could steer patients to a different treatment course rather than delaying systemic therapy until after recovery from inappropriate surgery. Here we investigate the potential of pancreas-specific circulating cell-free DNA (cfDNA) as a biomarker for the detection of occult metastatic disease and as a prognostic biomarker. Blood specimens were collected from 53 patients (15 negative control healthy subjects, 11 positive control metastatic PDAC patients, and 27 resectable PDAC patients prior to resection), processed to plasma, and banked. Plasma cfDNA was extracted, quantified, treated with bisulfite, and used as template for PCR amplification of 9 marker loci that are uniquely unmethylated in DNA of pancreatic acinar or duct cells. Following deep sequencing of PCR products, the fraction of cfDNA molecules derived from the pancreas was determined and multiplied by the total cfDNA concentration to yield pancreas-specific cfDNA. Recurrence and survival data were abstracted from the medical record and receiver operator curve analysis was utilized to determine statistical significance. Metastases were categorized as overt (present at diagnosis), occult (discovered during or within 4 months of curative intent surgery), or two-year (discovered during or within two years of curative intent surgery). Pancreas-derived cfDNA was significant for the detection of occult or overt metastases in our full cohort (18 of 52 evaluable subjects) with an area under the curve (AUC) of 0.86 (95% Confidence Interval, 0.74-0.80) and 0.91 (0.83-1.00) for liver-specific occult or overt metastases (15 of 52). It was borderline significant for the detection of occult metastases in the resectable sub-cohort (7 of 27) with an AUC of 0.71 (0.47-0.96) but significant for the detection of occult liver-specific metastases (5 of 27) with an AUC of 0.79 (0.62-0.96). Further, detection of overt metastases or two-year metastases (28 of 50) was significant with an AUC of 0.85 (0.74-0.96). In the resectable sub-cohort, it was also significant for the detection of two-year metastases (17 of 25) with an AUC of 0.79 (0.60-0.97) and prognostic for 2-year overall survival (12 of 24) with an AUC of 0.81 (0.62-1.00) in the resectable sub-cohort. Liver-derived cfDNA was also analyzed and was always outperformed by pancreas-specific cfDNA. In this pilot cohort, enumeration of pancreas-specific cfDNA shows promise as biomarker of occult metastatic disease, two-year metastatic progression, and two-year overall survival in resectable PDAC. Further investigation of a larger cohort and potential combination with other known markers like CA19-9 and tumor size is underway; results for an additional ~40 patients will be available by the time of the meeting. Citation Format: Jacob E. Till, Roni Ben-Ami, Ruth Shemer, Kristine Kim, Aseel Abdalla, Samuele Cannas, Charles M. Vollmer, Mark H. O'Hara, Ben Z. Stanger, Yuval Dor, Erica L. Carpenter. Pancreas-specific circulating cell-free DNA for detection of occult metastases and prognosis in resectable pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A030.

BACKGROUND GBM genomic profiling relies on sequencing a limited tumor tissue sample, which is invasive and may underrepresent molecular heterogeneity. CSF, which can be obtained less invasively, is in intimate contact with tumor lesions and may better capture the full genomic profile of the tumor. However, datasets with contemporaneously collected CSF and tissue to support this claim have been lacking. To evaluate the performance of CSF NGS, we conducted a pilot study in patients with GBM undergoing a resection for suspected recurrence following first-line chemoradiotherapy. METHODS Paired CSF and tissue samples were sequenced using a hybrid capture-based NGS assay. Clinically meaningful variants were defined as those that are potentially targetable using off-label or clinical trial options, exhibit prognostic value, or may predict response or resistance to specific treatments. RESULTS Eighteen patients were enrolled, and 13 of 18 CSF samples (72.2%) were sequenced successfully. At least one variant was detected in all CSF samples analyzed. A median of 7 variants (range 1—67) was detected per sample across 54 genes. The median variant allele fraction was 0.6% (range 0.2—72.4%). Among 38 clinically meaningful genes, 102 variants were detected; 25 (24.5%) were detected in both tissue and CSF, while 60 (58.8%) were detectable solely in CSF. Hypermutation was detected by CSF in one patient. Of the 82 variants detected in this patient’s tumor, 15 (18.3%) were identified in both tissue and CSF, 15 (18.3%) were identified only in the tissue, and 52 (63.2%) were identified only in the CSF. CONCLUSIONS CSF NGS detects clinically meaningful variants at a substantial rate and frequently identifies mutations not detected by matched tissue NGS. These results suggest that CSF may be a suitable source material for tumor profiling, overcoming the limitations of tissue, and may also provide a more comprehensive tumor profile.

BACKGROUND We have previously demonstrated that high baseline plasma cfDNA concentration is associated with poor survival in patients with newly diagnosed GBM. The mechanism of this association remains unknown. To explore whether differences in the immune landscape between high- vs. low-cfDNA patients may play a role in their divergent clinical outcomes, we phenotyped tumors from patients with high vs. low cfDNA using mass cytometry by time of flight (CyTOF). METHODS We performed CyTOF on frozen tumor infiltrate suspension from a pilot cohort of patients with previously untreated GBM with known baseline plasma cfDNA concentration (Bagley, Clin Cancer Res 2020). CyTOF was used to simultaneously measure expression of 39 molecules related to immune cell lineage, differentiation state, and function. Differences in immune cell infiltrates between high- and low-cfDNA patients were assessed using Mann-Whitney U tests. RESULTS Four patients with high cfDNA (median 57, range 33-90 ng/mL) were compared to six patients with low cfDNA (median 12, range 7-16 ng/mL). Immune cell infiltrates with increased adaptive cells (high monocytes and T cells, p=0.05) were present in high-cfDNA compared to low-cfDNA patients. While > 70% of the infiltrating T cells were exhausted in both groups, the pattern of exhaustion was significantly different in high- vs. low-cfDNA patients, with less CXCR5+CD69+ and more CXCR5-CD69- (p=0.008) progenitor exhausted T cells in cfDNA-high patients. CONCLUSIONS In this GBM pilot study, we demonstrated differences in the tumor immune infiltrate in patients with high vs. low baseline plasma cfDNA concentration. Preclinical studies will be needed to determine if this explains the association between high plasma cfDNA and poor outcomes previously observed in patients. Our results may have implications for the use of cfDNA concentration as a predictive biomarker for immunotherapy, as tumors with more intermediate progenitor (CXCR5-CD69-) exhausted T cells may respond better to PD-1 checkpoint blockade.

Background We aimed to determine whether plasma cell-free DNA (cfDNA) concentration is associated with survival in patients with isocitrate dehydrogenase (IDH) wild-type glioblastoma (GBM). Methods Pre-operative and post-chemoradiotherapy blood samples were prospectively collected from patients with newly diagnosed IDH wild-type GBM. Patients underwent surgical resection or biopsy and received adjuvant radiotherapy with concomitant temozolomide. Cell-free DNA (cfDNA) was isolated from plasma and quantified using SYBR Green-based q polymerase chain reaction (qPCR). Results Sixty-two patients were enrolled and categorized into high vs. low cfDNA groups relative to the pre-operative median value (25.2 ng/mL, range 5.7–153.0 ng/mL). High pre-operative cfDNA concentration was associated with inferior PFS (median progression-free survival (PFS), 3.4 vs. 7.7 months; log-rank P = .004; hazard ratio [HR], 2.19; 95% CI, 1.26–3.81) and overall survival (OS) (median OS, 8.0 vs. 13.9 months; log-rank P = .01; HR, 2.43; 95% CI, 1.19–4.95). After adjusting for risk factors, including O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status, pre-operative cfDNA remained independently associated with PFS (HR, 2.70; 95% CI, 1.50–4.83; P = .001) and OS (HR, 2.65; 95% CI, 1.25–5.59; P = .01). Post-hoc analysis of change in cfDNA post-chemoradiotherapy compared to pre-surgery (n = 24) showed increasing cfDNA concentration was associated with worse PFS (median, 2.7 vs. 6.0 months; log-rank P = .003; HR, 4.92; 95% CI, 1.53–15.84) and OS (median, 3.9 vs. 19.4 months; log-rank P < .001; HR, 7.77; 95% CI, 2.17–27.76). Conclusions cfDNA concentration is a promising prognostic biomarker for patients with IDH wild-type GBM. Plasma cfDNA can be obtained noninvasively and may enable more accurate estimates of survival and effective clinical trial stratification.

Background Plasma cell-free DNA (cfDNA) concentration is lower in glioblastoma (GBM) compared to other solid tumors, which can lead to low circulating tumor DNA (ctDNA) detection. In this study, we investigated the relationship between multimodality magnetic resonance imaging (MRI) and histopathologic features with plasma cfDNA concentration and ctDNA detection in patients with treatment-naive GBM. Methods We analyzed plasma cfDNA concentration, MRI scans, and tumor histopathology from 42 adult patients with newly diagnosed GBM. Linear regression analysis was used to examine the relationship of plasma cfDNA concentration before surgery to imaging and histopathologic characteristics. In a subset of patients, imaging and histopathologic metrics were also compared between patients with and without a detected tumor somatic mutation. Results Tumor volume with elevated (>1.5 times contralateral white matter) rate transfer constant (Kep, a surrogate of blood–brain barrier [BBB] permeability) was independently associated with plasma cfDNA concentration (P = .001). Histopathologic characteristics independently associated with plasma cfDNA concentration included CD68+ macrophage density (P = .01) and size of tumor vessels (P = .01). Patients with higher (grade ≥3) perivascular CD68+ macrophage density had lower volume transfer constant (Ktrans, P = .01) compared to those with lower perivascular CD68+ macrophage density. Detection of at least 1 somatic mutation in plasma cfDNA was associated with significantly lower perivascular CD68+ macrophages (P = .01). Conclusions Metrics of BBB disruption and quantity and distribution of tumor-associated macrophages are associated with plasma cfDNA concentration and ctDNA detection in GBM patients. These findings represent an important step in understanding the factors that determine plasma cfDNA concentration and ctDNA detection.

BACKGROUND Plasma circulating tumor DNA (ctDNA) is rarely detectable by traditional methods in patients with GBM. As a result, unlike in lung and other cancers, serial next generation sequencing of ctDNA for monitoring GBM tumor burden has been challenging. In light of the low tumor fraction (TF) of DNA fragments in GBM patient plasma and the urgent need to improve upon MRI for tracking GBM tumor burden, we conducted a pilot study in patients with newly diagnosed GBM using the C2 intelligence platform (C2i Genomics), which leverages genome-wide mutational integration for highly sensitive ctDNA detection. METHODS Plasma was collected pre- and post-operatively in patients with newly diagnosed GBM undergoing surgical resection/biopsy. cfDNA was extracted, quantified, and analyzed for fragment size. Genomic DNA (gDNA) was extracted from matched tumor tissue. Whole genome sequencing (WGS) was performed on both gDNA and cfDNA. A specific copy number alteration (CNA) compendium was created for each patient to generate a readout of TF (Zviran, Nat Medicine 2020). We assessed the association between TF at post-operative day 1 (a surrogate for residual disease) and OS, adjusting for other prognostic factors using Cox regression. RESULTS 37 patients were enrolled. For samples with high tumor fraction (n=5), a statistically significant (p< 1e-4) correlation between CNA profiles of tumor tissue and plasma samples was observed. Post-operative TF above the median value was associated with inferior OS (median 7.7 vs. 19.3 months, p=0.019). This association persisted after adjusting for age, O6-methylguanine-DNA methyltransferase methylation status, extent of resection, and performance status (adjusted HR 2.5, 95% CI 1.1-5.6, p=0.03). CONCLUSION Genome-wide mutational integration enables ultra-sensitive detection of ctDNA in GBM patient plasma. Post-operative TF measured by the C2i test is independently associated with OS in newly diagnosed GBM, providing the foundation to evaluate this technology for personalized prognostication and disease monitoring.

PURPOSE We have previously demonstrated the potential role of liquid biopsy, specifically plasma cell-free DNA (cfDNA), as a non-invasive biomarker for prognostication in patients with glioblastoma. In separate prior studies, we have also developed MRI-based radiomic signatures to predict survival outcomes in glioblastoma. In this study, for the first time, we evaluated the potential of combining radiomic signatures, epidemiological and clinical variables, and plasma cfDNA quantification for upfront prediction of overall survival (OS) in patients with newly diagnosed glioblastoma. METHODS Quantitative radiomic features were extracted from multiparametric MRI (T1, T1Gd, T2, T2-FLAIR) scans of a discovery cohort of 505 and an independent replication cohort of 50 IDH-wildtype glioblastoma patients. For the independent replication cohort, pre-surgical plasma cfDNA was extracted and quantified. In the first stage, a radiomic signature was created for stratification of patients into categories of short (OS ≤ 6 months) and long (OS ≥ 18 months) survivors using a cross-validated XGBoost method based on the discovery cohort, which was tested independently on the replication cohort. In the second stage, the radiomic signature and clinical variables were integrated to build a second-stage signature using a cross-validated support vector machine (SVM) classifier to stratify the patients into short and long survivor categories. In the third stage, the value of the second-stage signature integrated with cfDNA concentration was assessed through a cross-validated SVM regression method. RESULTS The combination of radiomic, clinical, and cfDNA variables resulted in the best overall predictive accuracy, with Pearson’s correlation coefficient of 0.59 (p< 0.0001) between actual and predicted OS. CONCLUSION In this study, we evaluated the value of combining plasma cfDNA, radiomic, and clinical variables for predicting OS, and showed that it could act as an effective non-invasive prognostic and patient stratification tool in patients with newly diagnosed glioblastoma.

4122 Background: Traditional imaging-guided therapeutic decision-making for patients with metastatic pancreatic ductal adenocarcinoma (mPDAC) may lag and, on occasion, be misleading. The concept of liquid biopsy-based molecular response holds promise for proximate and accurate therapy monitoring and assessment of emerging resistance to therapy. Here we investigate the association between baseline (pre-treatment) level and early, on-treatment changes in plasma circulating cell-free DNA (ccfDNA) mutant KRAS (ctKRAS) with progression-free survival (PFS) and overall survival (OS) in mPDAC. Methods: 189 plasma samples were analyzed from 123 total patients with mPDAC. An initial cohort included 54 patients treated at the University of Pennsylvania who received first-line standard of care (SOC) regimens and had a baseline plasma sample. Of these, 21 also had an on-therapy sample collected at ̃8 weeks. We also analyzed an independent cohort of 69 patients enrolled in the PRINCE trial (NCT03214250) who had a baseline sample, of which 45 also had an on-treatment sample at ̃8 weeks. PRINCE trial patients received gemcitabine/nab-paclitaxel with immunotherapy (I/O) agents (APX005M and/or nivolumab). ctKRAS variant allele fraction (VAF) was quantified by droplet digital PCR on pre-amplified ccfDNA. Baseline ctKRAS was dichotomized at 5% VAF. ctKRAS clearance was defined as detectable ctKRAS at baseline followed by ctKRAS becoming undetectable in the on-treatment sample. Results: Baseline ctKRAS (above/below 5% VAF) and ctKRAS clearance were associated with PFS and OS in both cohorts (Table). Further, in a multivariate cox regression model, ctKRAS clearance associated with improved PFS (HR 3.8, 1.4-10.9 or 3.6, 1.8-7.2) in both the SOC and I/O cohorts, respectively, and OS in the SOC cohort (HR 5.5, 1.5-20.8) after adjusting for baseline VAF. Conclusions: Baseline ctKRAS is significantly associated with OS and PFS in mPDAC in both independent cohorts. Further, early on-treatment ctKRAS clearance is strongly associated with improved PFS and OS, independent of baseline ctKRAS VAF. These data strongly support further investigation of ccfDNA as a biomarker of response and resistance to therapy.[Table: see text]

2508 Background: Due to significant interpatient heterogeneity, survival outcomes vary widely in patients with GBM. Novel prognostic biomarkers are needed. We aimed to determine the prognostic impact of baseline plasma cfDNA concentration in patients with GBM. Methods: We analyzed 84 patients with newly diagnosed GBM and at least 7 months of follow-up time. The first 41 patients comprised a previously published derivation cohort (Bagley, Clin Cancer Res 2020). The subsequent 43 patients served as an independent validation cohort. cfDNA was extracted from plasma collected prior to initial surgical resection and quantified by qPCR for a 115 bp amplicon of the human ALU repeat element. Receiver operating characteristic (ROC) curve analysis was used in the derivation cohort to (1) assess the accuracy of plasma cfDNA concentration for predicting progression-free survival status at 7 months (PFS-7), a landmark based on the median PFS for newly diagnosed GBM (Stupp, N Engl J Med 2005), and (2) derive the optimal cutoff for dichotomizing patients into high- and low-cfDNA groups. In the validation cohort, logistic regression was used to measure the association of plasma cfDNA concentration (high vs. low) with PFS-7, adjusted for age, isocitrate dehydrogenase ( IDH) 1/2 mutational status, 0-6-methylguanine-methyltransferase ( MGMT) methylation, extent of resection, and performance status. Multivariate Cox regression was used for overall survival (OS) analysis. Results: In the derivation cohort, the optimal cutoff for plasma cfDNA was 25.0 ng/mL (area under the curve [AUC] = 0.663), with inferior PFS and OS in patients with cfDNA above this cutoff (PFS, median 4.9 vs. 9.5 months, log-rank p = 0.001; OS, median 8.5 vs. 15.5 months, log-rank p = 0.03). In the validation cohort, baseline plasma cfDNA concentration over the cutoff was independently associated with a lower likelihood of being alive and progression-free at 7 months (adjusted OR 0.13, 95% CI 0.02 – 0.75, p = 0.02). OS was also worse in in the validation cohort in patients with high plasma cfDNA (adjusted HR 3.0, 95% CI 1.1 – 8.0, p = 0.03). Conclusions: In patients with newly diagnosed GBM, high baseline plasma cfDNA concentration is associated with worse survival outcomes independent of other prognostic factors. Further validation in a larger, multicenter study is warranted.