Tumor DNA In Plasma Research Articles

PROGNOSTIC VALUE OF LIQUID BIOPSY IN CRC: A LITERATURE REVIEW

Relevance: Liquid biopsy is a modern, quite appropriate, and promising method for diagnosing malignant neoplasms for oncology. The method allows us to determine the level of freely circulating tumor cells – micrometastases, tumor DNA, microRNA, and exosomes in blood plasma- and detect various genetic changes. A literature review of current scientific publications on liquid biopsy techniques, indexed in Medline, PubMed, and Medscape, was carried out as part of the work.
 The study aimed to review is to assess the prognostic significance of liquid biopsy, to determine the place of the method in current recommendations, and its expediency from the point of view of the practice.
 Methods: The information search was conducted in the Medline, PubMed, and Medscape databases, with a search depth of 8 years. Data from randomized controlled trials, clinical trials, reviews, systematic reviews, and meta-analyses were analyzed. The review includes both full-fledged articles in the public domain and abstracts to obtain complete information on the problem.
 Results: Liquid biopsy surpasses tissue biopsy in simplicity and speed of research, easy repeatability, and minimal invasiveness, as well as the possibility of dynamic monitoring of progression – the overall clonal transformation of the tumor and the emergence of resistance to treatment.
 The disadvantages of this method are low sensitivity, difficulty in correctly interpreting biomarkers and determining their specificity, and high risk of false positive and false negative results due to dormant tumor cells.
 Conclusion: At present, the Liquid biopsy method is relevant and in demand, but it needs to be tested on a validated sample of the main population, and in order to achieve effective clinical use, important work needs to be done to standardize both preanalytical and analytical procedures and generalize them for all components of liquid biopsy.

Circulating tumor DNA in plasma as an early predictor for pathological lymph node involvement and (neo-)adjuvant chemotherapy in patients with colon cancer.

3532 Background: Postoperative chemotherapy is standard for colon cancer patients with pathological lymph node involvement (pN+). If the presence of pN+ could be accurately predicted prior to surgery, the initiation of neoadjuvant chemotherapy could potentially reduce the presence of micrometastasis and enhance survival. The objective of this study was to assess the utility of preoperative methylated circulating tumor DNA (meth-ctDNA) as a predictive marker for pN+ and its clinical value as a marker for the initiation of neoadjuvant chemotherapy. Methods: The clinical data of 203 colon cancer patients (stage I-III) was collected from the Colorectal Cancer Database established at Danish Colorectal Cancer Center South, Vejle Hospital, Denmark. Patients were randomized into a discovery and a validation cohort. Plasma collected preoperatively was analyzed for three different meth-ctDNAs (Neuropeptide Y, Galactose-3-O-Sulfotransferase 3, and KN Motif and Ankyrin Repeat Domains 1) by droplet digital PCR (ddPCR). Results: Based on analysis of the discovery cohort, samples were considered positive if two or more of the three methylation markers had at least three positive droplets in the ddPCR analysis. 40% of patients in the discovery cohort and 46% of patients in the validation cohort were considered positive. When using meth-ctDNA as a diagnostic tool to predict pN+, the sensitivity and specificity in the discovery cohort were 42% and 61% respectively. In the validation cohort the values were 54% and 59%, respectively. AUC was < 60% in both cohorts. Meth-ctDNA was associated to clinical tumor (T)- and node (N)-category in both cohorts (p≤0.01). Four-year disease free survival (DFS) in patients with and without detectable meth-ctDNA was 60% and 90% in the discovery cohort (p = 0.01), and 62% and 78% in the validation cohort (p = 0.04). Meth-ctDNA remained the strongest prognostic factor of DFS in the multivariate analysis compared to clinical T- or N-category, gender and age (p = 0.065, HR 2.46, 95% CI 1.03-5.88), however not statistically significant. Four-year overall survival (OS) in patients with and without detectable meth-ctDNA was 75% and 95% in the discovery cohort (p = 0.006), and 78% and 91% in the validation cohort (p = 0.02). In the multivariate analysis, meth-ctDNA was the strongest prognostic factor of OS compared to clinical T- or N-category, gender and age (p = 0.016, HR 4.54, 95% CI 1.32-15.6). Conclusions: The preoperative measurement of meth-ctDNA was not found to be a clinically significant predictor of pN+ or a reliable indicator for the initiation of neoadjuvant chemotherapy. Nonetheless, the results showed that preoperative ctDNA is a powerful prognostic indicator, suggesting that randomized controlled trials should be conducted to determine the efficacy of neoadjuvant chemotherapy in colon cancer patients with preoperative detectable ctDNA.

Development of a high-performance multi-probe droplet digital PCR assay for high-sensitivity detection of human papillomavirus circulating tumor DNA from plasma

ObjectivesTo develop a high-performance droplet digital PCR (ddPCR) assay capable of enhancing the detection of human papillomavirus (HPV) circulating tumor DNA (ctDNA) in plasma from patients with HPV-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC). Materials and MethodsPlasma samples from subjects with HPV+ OPSCC were collected. We developed a high-performance ddPCR assay designed to simultaneously target nine regions of the HPV16 genome. ResultsThe new assay termed ‘ctDNA HPV16 Assessment using Multiple Probes’ (CHAMP- 16) yielded significantly higher HPV16 counts compared to our previously validated ‘Single-Probe’ (SP) assay and a commercially available NavDx® assay. Analytical validation demonstrated that the CHAMP-16 assay had a limit of detection (LoD) of 4.1 copies per reaction, corresponding to < 1 genome equivalent (GE) of HPV16. When tested on plasma ctDNA from 21 patients with early-stage HPV+ OPSCC and known HPV16 ctDNA using the SP assay, all patients were positive for HPV16 ctDNA in both assays and the CHAMP-16 assay displayed 6.6-fold higher HPV16 signal on average. Finally, in a longitudinal analysis of samples from a patient with recurrent disease, the CHAMP-16 assay detected HPV16 ctDNA signal ∼ 20 months prior to the conventional SP assay. ConclusionIncreased HPV16 signal detection using the CHAMP-16 assay suggests the potential for detection of recurrences significantly earlier than with conventional ddPCR assays in patients with HPV16+ OPSCC. Critically, this multi-probe approach maintains the cost-benefit advantage of ddPCR over next generation sequencing (NGS) approaches, supporting the cost-effectiveness of this assay for both large population screening and routine post-treatment surveillance.

Genomic profiling of NGS-based ctDNA from Chinese non-small cell lung cancer patients.

Cell-free circulating tumor DNA (ctDNA) in plasma enables rapid and repeat testing of actionable mutations. Next-generation sequencing (NGS) is an attractive platform for multiplex sequencing capabilities compared to traditional methods such as PCR. The purpose of this study is to evaluate the value of the NGS-based ctDNA assay and to identify the genomic alteration profile of ctDNA in real-world Chinese non-small cell lung (NSCLC) patients. In total, 294 Chinese patients with pathological diagnosis of Phase III-IV NSCLC were enrolled. 3-4mL peripheral blood was collected and NGS-based analysis was carried out using a 20-gene panel. The analytical sensitivity and specificity of ctDNA NGS-based assay was validated using droplet digital PCR (ddPCR). We have tested 570 sites from 286 samples using ddPCR, which included 108 positive sites and 462 negative sites from NGS results, and the concordance rate was 99.8% (418/419) for single-nucleotide variants (SNVs) and 96.7% (146/151) for insertions and deletions (InDels). The most frequent genes were TP53 (32%), EGFR (31.97%), KRAS (6.46%), PIK3CA (4.76%), and MET (4.08%). Exon 19 deletion (19del) was the most common alteration in EGFR and G12C was the most common alteration in KRAS. Furthermore, the detection rate of TP53 was higher in the male and patients with squamous cell carcinoma. We also found the prevalence of TP53 in L858R was higher than in 19del (61.29% vs. 40%; p = 0.1115). The results indicate that the results of NGS-based ctDNA assay are highly consistent with ddPCR. In Chinese NSCLC patients, TP53 mutation was more frequently associated with male and squamous cell carcinoma. The prevalence of concomitant mutations in L858R may be different from that in 19del.

Abstract CT216: Response to first-line (1L) pembrolizumab (pembro) + chemotherapy (chemo) in non-small cell lung cancer (NSCLC) by blood tumor mutational burden (bTMB): the phase 2 KEYNOTE-782 trial

Abstract Background: 1L pembro + platinum-based chemo has shown clinical activity in metastatic NSCLC regardless of tissue tumor mutational burden (tTMB) status. bTMB assessed using circulating tumor DNA in plasma may be a surrogate for tTMB. The single-arm, phase 2 KEYNOTE-782 study (NCT03664024) evaluated the correlation of bTMB with efficacy of 1L pembro + chemo in nonsquamous NSCLC. Methods: Eligible patients (pts) had histologically or cytologically confirmed stage IV nonsquamous NSCLC with measurable disease per RECIST v1.1 and were not eligible for EGFR-, BRAF-, ROS1-, or ALK-directed therapy. Pts were not previously treated for advanced or metastatic disease, had an ECOG PS of 0 or 1, and had an evaluable biopsy sample for biomarker analysis. All pts received intravenous pembro 200 mg Q3W + platinum chemo doublet Q3W (pemetrexed 500 mg/m2 + 4 cycles of carboplatin AUC 5 mg/mL/min or cisplatin 75 mg/m2). The primary objective was to evaluate the association of baseline bTMB with ORR per RECIST v1.1 by investigator assessment. Secondary objectives were to determine safety and the association of baseline bTMB with PFS per RECIST v1.1 by investigator assessment and OS. A study-specific next-generation sequencing-based assay using a 1.9 Mb/654-gene cancer panel, which includes specific lung cancer-associated gene targets, was used to measure bTMB (continuous scale) in cell-free DNA extracted from baseline plasma samples. Paired white blood cell DNA sequencing was also performed to eliminate potential clonal hematopoiesis-derived somatic mutations. Database cutoff: November 5, 2021. Results: 117 pts were enrolled; median age was 64.0 years (range, 37-85), and the majority were male (60.7%), had an ECOG PS of 1 (69.2%), and no brain metastases at baseline (92.3%). Median time from first dose to data cutoff was 19.3 months (range, 1.0-35.5). ORR was 40.2% (95% CI, 31.2-49.6; 6 CRs, 41 PRs), median PFS was 7.2 months (95% CI, 5.6-9.8), and median OS was 18.1 months (95% CI, 13.5-25.6). Treatment-related adverse events (TRAEs) occurred in 113 pts (96.6%) and grade 3-5 TRAEs occurred in 56 (47.9%). Eight pts (6.8%) died due to a TRAE (febrile neutropenia and pneumonitis [n = 2 each], and septic shock, pulmonary sepsis, general physical health deterioration, and neutropenia [n = 1 each]). bTMB data were available for 101 pts. The area under the receiver operating curve for bTMB as a continuous variable to discriminate response was 0.47 (95% CI, 0.36-0.59). The posterior probabilities for a positive association of bTMB with PFS and OS were 16.8% and 7.8%, respectively. Conclusions: Baseline bTMB showed no evidence of an association with ORR, PFS, or OS in pts with nonsquamous NSCLC treated with 1L pembro + chemo. These findings indicate no clinical utility of bTMB in this patient population and treatment setting. No new safety signals were observed. Citation Format: Jair Bar, Emilio Esteban, Delvys Rodríguez-Abreu, Santiago Ponce Aix, Zsuzsanna Szalai, Enriqueta Felip, Maya Gottfried, Mariano Provencio Pulla, Andrew Robinson, Andrea Fülöp, Suman B. Rao, D. Ross Camidge, Giovanna Speranza, Steven M. Townson, Julie Kobie, Mark Ayers, Elisha J. Dettman, Robert McDaniel, Byoungsok Jung, David Burkhardt, Ruth Mauntz, Tibor Csőszi. Response to first-line (1L) pembrolizumab (pembro) + chemotherapy (chemo) in non-small cell lung cancer (NSCLC) by blood tumor mutational burden (bTMB): the phase 2 KEYNOTE-782 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr CT216.

Investigate the application of postoperative ctDNA-based molecular residual disease detection in monitoring tumor recurrence in patients with non-small cell lung cancer--A retrospective study of ctDNA.

To evaluate whether postoperative circulating tumor DNA (ctDNA) in plasma of patients with non-small cell lung cancer (NSCLC) can be used as a biomarker for early detection of molecular residual disease (MRD) and prediction of postoperative recurrence. This study subjects were evaluated patients with surgical resected non-small cell lung cancer. All eligible patients underwent radical surgery operation followed by adjuvant therapy. Tumor tissue samples collected during operation were used to detect tumor mutation genes, and blood samples collected from peripheral veins after operation were used to collect ctDNA. Molecular residue disease (MRD) positive was defined as at least 1 true shared mutation identified in both the tumor sample and a plasma sample from the same patient was. Positive postoperatively ctDNA was associated with lower recurrence-free survival (RFS).The presence of MRD was a strong predictor of disease recurrence. The relative contribution of ctDNA-based MRD to the prediction of RFS is higher than all other clinicopathological variables, even higher than traditional TNM staging. In addition, MRD-positive patients who received adjuvant therapy had improved RFS compared to those who did not, the RFS of MRD-negative patients receiving adjuvant therapy was lower than that of patients not receiving adjuvant therapy. Post-operative ctDNA analysis is an effective method for recurrence risk stratification of NSCLC, which is beneficial to the management of patients with NSCLC.

Abstract 2290: DNA methylation markers for sensitive detection of circulating tumor DNA in plasma from patients with gastroesophageal adenocarcinomas

Abstract Background: We have previously shown that detection of methylated ctDNA in the three genes c9orf50, KCNQ5, and CLIP4 is a highly sensitive method (named the TriMeth assay) to discriminate patients with colorectal cancer from healthy individuals (1). The publicly available Infinium HumanMethylation450K BreadChip DNA methylation array indicates that these three markers are also highly methylated in gastric and esophageal cancer. In this study, we investigate the ability of the TriMeth assay to detect ctDNA in patients with gastric and gastroesophageal junction (GEJ) adenocarcinomas. Methods: Purified DNA was extracted from tumor tissue and plasma from 63 patients with gastric/GEJ adenocarcinomas. Sodium bisulfite conversion was performed to distinguish methylated from unmethylated cytosines. Droplet digital PCR (ddPCR) with methylation-specific primers and probes was performed to detect hypermethylated sites in the three genes c9orf50, KCNQ5, and CLIP4. The TriMeth assay was considered positive if at least two out of three markers were methylated according to a predefined cut-off. Eleven plasma samples from healthy individuals were used as negative controls.Results: In tumor tissue, methylated copies were detected in all 29 surgical samples (100% sensitivity), with varying allele frequencies. In plasma, methylated copies were detected in 14 out of 18 samples (78% sensitivity) from patients with advanced disease, with varying sensitivity amongst the three markers (AUC for the three markers CLIP4, C9orf50, and KCNQ5 was 0.61, 0.86, and 0.94, respectively). In plasma from patients with resectable disease, methylated copies were detected in 10 of 17 samples (59% sensitivity), although only 7/17 (41% sensitivity) were positive according to the predefined cut-off. All 11 controls were negative for methylation signals (100% specificity). Conclusion and Further Perspectives: Using a pre-defined cut-off value, the TriMeth assay detected methylated DNA in all tumor samples. The sensitivity of the TriMeth assay in plasma was 78% in advanced disease and 41% in resectable disease. Further investigations of the TriMeth assay in patients with resectable gastric and GEJ adenocarcinomas are needed. We plan to validate the sensitivity of the TriMeth assay for treatment monitoring and detection of residual disease.

Abstract 1036: Detection of circulating tumor DNA by whole genome sequencing enables prediction of recurrence in stage III colorectal cancer patients with great inter-lab reproducibility

Abstract Background: Detection of circulating tumor DNA (ctDNA) in plasma indicates the presence of cancer. In patients with very few copies of tumor DNA circulating in the blood, the blood sampling process becomes a potentially limiting factor. For ctDNA methods based on a single or a few genomic targets, it becomes stochastic whether the collected sample contains any tumor DNA fragments from targeted regions. To overcome this sampling limitation, we developed C2inform; a whole genome sequencing (WGS) approach, which detects ctDNA using a cumulative patient-specific signal from thousands of mutations throughout the entire genome. Aim: Here, we aim to I) evaluate the performance of C2inform in patients with UICC stage III colorectal cancer (CRC) using samples collected after the end of curatively intended treatment and serially during surveillance, II) assess the inter-lab reproducibility of C2inform and, III) investigate the potential for using the serial WGS data to track tumor genomic evolution in recurrence patients. Methods: From a cohort of 146 stage III CRC patients, including 37 patients with recurrence, 2 mL plasma samples were collected serially for up to three years (n = 1309, median 10 samples per patient). By WGS of tumor and blood-derived normal DNA, a mutational signature was established for each patient. Enhanced by an AI-based error suppression model, this signature was used to screen 20x WGS plasma cfDNA profiles for the presence of ctDNA. To evaluate the reproducibility, paired samples (n = 2 x 187 samples) were processed and sequenced at two independent laboratories while the bioinformatics processing of samples was identical. Using coverage, split-read, and discordant read-pair information, genomic structural alterations were identified in serial plasma samples from 19 recurrence patients and compared to tissue WGS from subsequent recurrence metastasis. Results: Preliminary results showed that detection of ctDNA after the end of treatment was associated with poor prognosis (HR = 7.1, 95% CI: 3.2-15.9). The presence of ctDNA during surveillance was a predictor of recurrence (HR = 9.5, 95% CI: 3.6-25.5) and enabled the detection of recurrence up to 21 months (median: 9.5 months) before detection by radiological imaging. We found indication of tumor evolution by searching serially collected plasma samples for novel genomic changes, which were confirmed by WGS of the metastatic tissue. Analysis of paired samples showed great reproducibility of C2inform with a high agreement between both ctDNA detection (Cohens Kappa = 0.9) and the estimated ctDNA level (r² = 0.99). Conclusion: C2inform showed great inter-lab reproducibility and enabled prediction of recurrence after treatment and during surveillance. Thus, C2inform has the potential to guide clinical decision-making during the postoperative management of CRC patients. Citation Format: Amanda Frydendahl, Jesper Nors, Mads Heilskov, Ester Ellegaard Sørensen, Thomas Reinert, Jesper Bramsen, Danielle Afterman, Tomer Lauterman, Maja Kuzman, Santiago Gonzalez, Dunja Glavas, James Samdbeck, Dillon Maloney, Jurica Levatic, Sunil Deochand, Michael Yahalom, Ryan Ptashkin, Iman Tavassoly, Zohar Donenhirsh, Eric White, Ravi Kandasamy, Ury Alon, Paz Polak, Anders Husted Madsen, Uffe Schou Løve, Per Vadgaard Andersen, Ole Thorlacius-Ussing, Lene Hjerrild Iversen, Kåre Andersson Gotschalck, Boris Oklander, Asaf Zviran, Claus Lindbjerg Andersen. Detection of circulating tumor DNA by whole genome sequencing enables prediction of recurrence in stage III colorectal cancer patients with great inter-lab reproducibility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1036.

Abstract 2137: Clinical utility of circulating tumor DNA in patients with advanced KRAS G12C-mutated NSCLC treated with sotorasib

Abstract Introduction: The CodeBreaK 200 trial showed that in patients (pts) with advanced KRAS G12C mutated (KRAS+) NSCLC sotorasib, a KRAS G12C-specific inhibitor, is superior to docetaxel for progression free survival (PFS) (HR 0.66) with a one-year PFS of 25%. We hypothesized that the detection of circulating tumor DNA (ctDNA) in plasma could allow for treatment response prediction and longitudinal monitoring. We analyzed serial plasma samples at baseline and within 3 months of start of sotorasib to evaluate ctDNA changes and correlation with clinical response. Methods: Pts with sotorasib treated KRAS+ NSCLC were prospectively enrolled in our biomarker START-TKI study (NCT05221372) after written informed consent. Plasma samples were collected prior to treatment (T0) and at first response evaluation (T1). The TruSight Oncology 500 ctDNA panel was used for mutation detection in cell-free DNA (cfDNA). cfDNA KRAS/TP53/STK11/KEAP1 status was determined and compared to tumor tissue pathology reports to filter out false positives. Radiological response and PFS was assessed per RECIST 1.1. Results: Between May 2021 and August 2022, 35 pts were included (table 1). Of these, 29 (83%) had detectable ctDNA KRAS G12C at T0, and 24 had both T0 and T1 samples. A decrease in variant allele frequency (VAF) at T1 compared to T0 was observed in 88% (n=21); 42% (n=10) showed complete clearance of ctDNA KRAS G12C. Six-months PFS was 83% in T0 negative pts (n=6) versus 43% in T0 positive pts (n=29); and 65% in pts with complete clearance (n=10) versus 14% in pts with incomplete clearance (n=11). VAF increase was seen in 3 pts, of which 1 had progression at T1. Conclusions: In pts with KRAS+ NSCLC treated with sotorasib, baseline ctDNA and ctDNA clearance within 3 months of treatment correlated with treatment response. Pts with undetectable KRAS ctDNA at baseline, or with complete clearance at first evaluation, had superior PFS. PFS will be updated as follow-up duration extends. Table 1. Patient cohort Liver metastasis KRAS p.G12C ctDNA at T0, median VAF % (range) KRAS p.G12C ctDNA at T1, median VAF % (range) Median change in VAF % (range) TP53/STK11/KEAP1 ctDNA T0 T1 response Reason EOT ꝉ Time on treatment (months) * Negative at T0 (n=6) No = 6 0 NE NE NE 1x PR; 4x SD; 1x PD 2x PD; 4x ongoing &amp;gt;8 months 6 (1 - 11) Positive at T0 (n=29) No = 20; Yes = 9 2.6 (0.1 - 46.7) 0.6 (0.1 - 38.9) -95% (-100 - +39) 4x TP53; 2x STK11; 1x STK11●; 2x KEAP1; 2x KEAP1●; 2x TP53 + KEAP1; 1x TP53 + STK11● 4x PR; 21x SD; 2x PD; 2x NE 19x PD; 3x toxicity; 6x ongoing &amp;gt;4 months; 1x loss to follow up 3 (1 - 15) Complete clearance at T1 (n=10) No = 6; Yes = 4 1.4 (0.1 - 8.4) 0 100% 2x TP53; 1x TP53 + KEAP1; 1x KEAP1; 1x KEAP1● 1x PR; 9x SD 6x PD; 1x toxicity; 2x ongoing &amp;gt;6 months; 1x loss to follow up 5 (1 - 11) Incomplete clearance at T1 (n=11) No = 7; Yes = 4 3.6 (1.3 - 46.7) 0.4 (0.1 - 29.4) -89% (-23 - -99) 2x TP53; 1x STK11; 1x STK11●; 2x TP53 + STK11●; 1x TP53 + STK11 + KEAP1; 1x STK11 + KEAP1 3x PR; 7x SD; 1x PD 8x PD; 2x toxicity; 1x ongoing &amp;gt;4 months 3 (1 - 8) Increase in VAF at T1 (n=3) No = 2; Yes = 1 1.0 (0.5 - 35.2) 1.1 (0.6 - 38.9) +11% (+10 - +39) 1x TP53●; 1x TP53 + KEAP1; 1x STK11● + KEAP1● 1x PD; 2x SD 3x PD 3 (1 - 15) Positive T0, no T1 available (n=5) No = 5 6.2 (0.3 - 24.6) NE NE 1x TP53; 1x TP53●; 1x KEAP1; 1x STK11 + KEAP1 3x SD; 2x NE 2x PD; 3x ongoing &amp;gt;3 months 2 (1 - 3) NE = Not evaluated; ● = Positive cell-free DNA (cfDNA), not tested in tumor tissue; PR = Partial response; SD = Stable disease; PD = Progressive disease; EOT = End of treatment; ꝉ for PFS analysis patients who discontinued treatment due to toxicity were censored at treatment discontinuation, and patients with ongoing sotorasib treatment were censored at data cut off (16-Nov-2022); * = excluding ongoing patients. Citation Format: Sophie M. Ernst, Ronald van Marion, Peggy N. Atmodimedjo, Evert de Jonge, Ron H. Mathijssen, Marthe S. Paats, Peter de Bruijn, Ron H.N. van Schaik, Hendrikus J. Dubbink, Anne-Marie C. Dingemans. Clinical utility of circulating tumor DNA in patients with advanced KRAS G12C-mutated NSCLC treated with sotorasib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2137.

Abstract 3387: ctDNA dynamics in early stage node negative lung cancers

Abstract Introduction: Patients with early-stage lung cancers have a high risk of relapse and death even after curative surgery. Detection of circulating tumour DNA (ctDNA) in plasma perioperatively is associated with shorter recurrence free survival (RFS). ctDNA-Lung-Detect is an investigator-initiated prospective study of ctDNA detection and association with RFS in patients with early stage non-small cell lung cancer (NSCLC). Methods: Patients with clinically staged T&amp;lt;4cm N0 NSCLC planned for surgical resection at the Princess Margaret/University Health Network underwent ctDNA assessment before and after surgery (~1 month and 1 year). ctDNA minimal residual disease (MRD) was detected using the highly sensitive and specific tumor-informed Residual Disease and Recurrence (RaDaR®, Inivata, Cambridge, UK) assay, which can track up to 48 tumor-specific variants in plasma. Results: Since August 2021, 64 eligible patients were enrolled (Table 1). One had isolated metastasis from a remote non-lung primary and subsequently excluded; another had their preoperative sample missed but was included. Preoperative ctDNA was detected in 15/62 (23.8%) patients and 1/63 (1.6%) post-operatively at the 1-month landmark timepoint (occult N2 disease found at surgery). All but one (93.3%) had ctDNA clearance with surgical resection. Two patients have recurred radiographically and in plasma at 1 year post-operatively, 1/15 with preoperative ctDNA detected (6.7%) and 1/47 without preoperative ctDNA detected (2.1%). Patients with higher T stage, tumor size, squamous histology, and without actionable driver alterations were more likely to have ctDNA detected at any timepoint. Conclusions: In patients with early stage NSCLC (small node negative tumors), 24% have detectable ctDNA pre-operatively. Surgical resection led to ctDNA clearance in 93% of cases. Our results support ongoing testing beyond the initial MRD landmark. Assessment of the impact on RFS post resection is ongoing. Citation Format: Jamie Feng, Tom Waddell, Kazuhiro Yasufuku, Andrew Pierre, Shaf Keshavjee, Jonathan Yeung, Marcelo Cypel, Laura Donahoe, Elliot Wakeam, Marc de Perrot, Jennifer Law, Alexandra Salvarrey, Lisa W. Le, Jennifer Lister, Michael Cabanero, Ming Tsao, Christodoulos Pipinikas, Karen Howarth, Natasha Leighl. ctDNA dynamics in early stage node negative lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3387.

Increasing the Capture Rate of Circulating Tumor DNA in Unaltered Plasma Using Passive Microfluidic Mixer Flow Cells.

A limiting factor in using blood-based liquid biopsies for cancer detection is the volume of extracted blood required to capture a measurable number of circulating tumor DNA (ctDNA). To overcome this limitation, we developed a technology named the dCas9 capture system to capture ctDNA from unaltered flowing plasma, removing the need to extract the plasma from the body. This technology has provided the first opportunity to investigate whether microfluidic flow cell design can affect the capture of ctDNA in unaltered plasma. With inspiration from microfluidic mixer flow cells designed to capture circulating tumor cells and exosomes, we constructed four microfluidic mixer flow cells. Next, we investigated the effects of these flow cell designs and the flow rate on the rate of captured spiked-in BRAF T1799A (BRAFMut) ctDNA in unaltered flowing plasma using surface-immobilized dCas9. Once the optimal mass transfer rate of ctDNA, identified by the optimal ctDNA capture rate, was determined, we investigated whether the design of the microfluidic device, flow rate, flow time, and the number of spiked-in mutant DNA copies affected the rate of capture by the dCas9 capture system. We found that size modifications to the flow channel had no effect on the flow rate required to achieve the optimal capture rate of ctDNA. However, decreasing the size of the capture chamber decreased the flow rate required to achieve the optimal capture rate. Finally, we showed that, at the optimal capture rate, different microfluidic designs using different flow rates could capture DNA copies at a similar rate over time. In this study, the optimal capture rate of ctDNA in unaltered plasma was identified by adjusting the flow rate in each of the passive microfluidic mixer flow cells. However, further validation and optimization of the dCas9 capture system are required before it is ready to be used clinically.

Cell-Free Urine and Plasma DNA Mutational Analysis Predicts Neoadjuvant Chemotherapy Response and Outcome in Patients with Muscle-Invasive Bladder Cancer.

To investigate the use of plasma and urine DNA mutation analysis for predicting neoadjuvant chemotherapy (NAC) response and oncological outcome in patients with muscle-invasive bladder cancer. Whole-exome sequencing of tumor and germline DNA was performed for 92 patients treated with NAC followed by radical cystectomy (RC). A custom NGS-panel capturing approximately 50 mutations per patient was designed and used to track mutated tumor DNA in plasma and urine. A total of 447 plasma samples, 281 urine supernatants, and 123 urine pellets collected before, during, and after treatment were analyzed. Patients were enrolled from 2013 to 2019, with a median follow-up time of 41.3 months after RC. We identified tumor DNA before NAC in 89% of urine supernatants, 85% of urine pellets, and 43% of plasma samples. Tumor DNA levels were higher in urine supernatants and urine pellets compared with plasma samples (P < 0.001). In plasma, detection of circulating tumor DNA (ctDNA) before NAC was associated with a lower NAC response rate (P < 0.001). Detection of tumor DNA after NAC was associated with lower response rates in plasma, urine supernatant, and urine pellet (P < 0.001, P = 0.03, P = 0.002). Tumor DNA dynamics during NAC was predictive of NAC response and outcome in urine supernatant and plasma (P = 0.006 and P = 0.002). A combined measure from plasma and urine supernatant tumor DNA dynamics stratified patients by outcome (P = 0.003). Analysis of tumor DNA in plasma and urine samples both separately and combined has a potential to predict treatment response and outcome.

Plasma circulating tumor DNA profiling in patients with chemo-refractory germ cell tumors.

Molecular analysis of tumor tissues has been extensively analyzed in germ cell tumors. However, genetic analysis of plasma circulating tumor DNA has been limited. Our objective was to analyze genetic alterations in circulating tumor DNA as well as its impact on prognosis in patients with chemo-refractory germ cell tumors. We included 13 patients with chemo-refractory germ cell tumors who relapsed after second-line or higher previous chemotherapy and performed targeted sequencing of plasma cell-free DNA using an AVENIO Expanded kit. Tumor-specific genetic alterations were identified in all patients. The most frequently mutated gene was TP53 (53.4%), followed by PTEN (23.1%), GNAS (15.4%) and MTOR (15.4%). Moreover, EGFR amplification (38.5%) and MET amplification (15.4%) were also identified. We defined two or more single nucleotide variants detected in plasma cell-free DNA as circulating tumor DNA-positive. Kaplan-Meier analysis revealed that overall survival was significantly shorter in circulating tumor DNA-positive patients than circulating tumor DNA negative-patients (median overall survival 3.13 vs. 8.73 months; p=0.042). Analysis of plasma circulating tumor DNA could detect genetic alterations in patients with chemo-refractory GCT. Moreover, detectable circulating tumor DNA in plasma was associated with poor prognosis in those patients. These results suggest that liquid biopsy using analysis of plasma circulating tumor DNA may be clinically useful for germ cell tumor patients.

Detection of Circulating Tumor DNA in Plasma Using Targeted Sequencing.

Cell-free DNA (cfDNA) is the degradation product of extracellular DNA. Circulating tumor DNA (ctDNA), as a fraction of cfDNA, comes from tumor cells and contains variations, including mutation, deletion, insertion, rearrangement, copy number variation, and methylation. Therefore, biomarkers identified in ctDNA show promising clinical applications in early diagnosis, recurrence monitoring, and conducting individualized treatment. In this chapter, we introduce experimental workflow and bioinformatic pipeline of targeted sequencing of cfDNA.

Tumor genomic profiling and personalized tracking of circulating tumor DNA in Vietnamese colorectal cancer patients.

Colorectal cancer (CRC) is the fifth most common cancer with rising prevalence in Vietnam. However, there is no data about the mutational landscape and actionable alterations in the Vietnamese patients. During post-operative surveillance, clinical tools are limited to stratify risk of recurrence and detect residual disease. In this prospective multi-center study, 103 CRC patients eligible for curative-intent surgery were recruited. Genomic DNA from tumor tissue and paired white blood cells were sequenced to profile all tumor-derived somatic mutations in 95 cancer-associated genes. Our bioinformatic algorithm identified top mutations unique for individual patient, which were then used to monitor the presence of circulating tumor DNA (ctDNA) in serial plasma samples. The top mutated genes in our cohort were APC, TP53 and KRAS. 41.7% of the patients harbored KRAS and NRAS mutations predictive of resistance to Cetuximab and Panitumumab respectively; 41.7% had mutations targeted by either approved or experimental drugs. Using a personalized subset of top ranked mutations, we detected ctDNA in 90.5% of the pre-operative plasma samples, whereas carcinoembryonic antigen (CEA) was elevated in only 41.3% of them. Interim analysis after 16-month follow-up revealed post-operative detection of ctDNA in two patients that had recurrence, with the lead time of 4-10.5 months ahead of clinical diagnosis. CEA failed to predict recurrence in both cases. Our assay showed promising dual clinical utilities in residual cancer surveillance and actionable mutation profiling for targeted therapies in CRC patients. This could lay foundation to empower precision cancer medicine in Vietnam and other developing countries.

Current Role and Future Potential of CSF ctDNA for the Diagnosis and Clinical Management of Pediatric Central Nervous System Tumors.

Most pediatric central nervous system (CNS) tumors are located in eloquent anatomic areas, making surgical resection and, in some cases, even biopsy risky or impossible. This diagnostic predicament coupled with the move toward molecular classification for diagnosis has exposed an urgent need to develop a minimally invasive means to obtain diagnostic information. In non-CNS solid tumors, the detection of circulating tumor DNA (ctDNA) in plasma and other bodily fluids has been incorporated into routine practice and clinical trial design for selection of molecular targeted therapy and longitudinal monitoring. For primary CNS tumors, however, detection of ctDNA in plasma has been challenging. This is likely related at least in part to anatomic factors such as the blood-brain barrier. Due to the proximity of primary CNS tumors to the cerebrospinal fluid (CSF) space, our group and others have turned to CSF as a rich alternative source of ctDNA. Although multiple studies at this time have demonstrated the feasibility of CSF ctDNA detection across multiple types of pediatric CNS tumors, the optimal role and utility of CSF ctDNA in the clinical setting has not been established. This review discusses the work-to-date on CSF ctDNA liquid biopsy in pediatric CNS tumors and the associated technical challenges, and reviews the promising opportunities that lie ahead for integration of CSF ctDNA liquid biopsy into clinical care and clinical trial design.

Detection of Cell Free Tumor DNA in Plasma of Patients with Large B-Cell Lymphoma of the Sanctuary Sites By Digital Droplet PCR

Background: Primary central nervous system lymphoma (PCNSL) and primary testicular lymphoma (PTL) are rare mature B-cell lymphoproliferative diseases that arise at immunological sanctuary sites. Cytomorphology (CM) and flow cytometry (FCM) analysis of spinal fluid is diagnostic in about 15% of PCNSL. In the remaining patients, a tumor biopsy is necessary, which carries the risk of complications. Response assessment in PCNSL and PTL relies on imaging studies. However, MRI scans and 18F-FDG PET-scans often cannot differentiate between active tumor and scar tissue. Hence, there is a need for non-invasive tools to aid in diagnosis and help in response assessment. PCNSL and PTL are characterized by a high incidence of hotspot mutations in MYD88 and CD79B. Detection of these mutations in cell free DNA (cfDNA) might aid in diagnosis and follow-up of disease activity in patients with PCNSL and PTL. Digital droplet PCR (ddPCR) offers a robust inexpensive tool with a fast turnaround time allowing sensitive detection of such hotspot mutations. Methods: Plasma was available for a total of 26 patients (20 PCNSL and 6 PTL) in the OncoLifes biobank of the University Medical Center Groningen. All patients gave informed consent. Corresponding tumor samples were available for 17 cases (14 PCNSL and 3 PTL). A ddPCR was performed for MYD88 L265P and in case of sufficient cfDNA also for CD79B Y196X (Bio-Rad Laboratories, Hercules, USA). The ddPCR Assay for CD79B Y196X targets the mutation Y196H c.586T>C, Y196D c.586T>G, Y196S c.587A>C, Y196C c.587A>G, and Y196F c.587A>T. The cut off value for the allele frequency (AF) used was ≥0.5%. Results: The MYD88 L265P and CD79B Y196X mutations were detected in 9/14 (64.2%) and 2/13 (15.4%) of the PCNSL tissue samples. The two CD79B Y196X positive tissue cases had a concomitant MYD88 L256P. The MYD88 L265P mutation was detected in 2/20 (10%) cfDNA samples. The CD79B Y196X mutation was detected in 1/19 (5.2%) cfDNA samples; remarkably, this mutation was not detected in the corresponding tissue sample. In PTL, MYD88 L265P mutations were detected in 3/3 of tumor tissues and a CD79B Y196X mutation was detected in 1/2 of tumor tissues, which co-occurred with the MYD88 L265P. In PTL, 2/6 (33.3%) and 1/6 (16.6%) cfDNA samples were positive for MYD88 L265P and CD79B Y196X. Overall, hotspot MYD88 and/or CD79B mutations were detected in cfDNA in 5 /26 cases (18.4%) (Figure 1). Figure 1. ddPCR results of MYD88 and CD79BConclusion: We explored the feasibility of using a combination of two common hotspot mutations in MYD88 and CD79B in PCNSL and PTL plasma samples to aid in diagnosis and disease monitoring. The data presented show that the value of ddPCR for hotspot mutations in plasma of PCNSL and PTL patients is limited. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prospective Study of the Usefulness of Liquid Biopsy in Patients with Unknown Fever Suspected of Malignant Lymphoma

Background: Malignant lymphoma, represented by intravascular large B-cell lymphoma (IVLBCL), is an important differential disease in classical fever of unknown origin (FUO). Bone marrow aspiration (BMA) and random skin biopsy (RSB) are useful in IVLBCL diagnosis, but their positivity rates are clinically insufficient and the procedures are invasive. The molecular genetic analysis of lymphomas using cell-free tumor DNA (cfDNA) in plasma (liquid biopsy) may be effective for IVLBCL (Shimada et al., Blood. 2021 Mar 18;137(11):1491). We conducted a multicenter prospective clinical trial to investigate the usefulness of liquid biopsy for early lymphoma diagnosis among patients with classical FUO. Methods: Patients with the following criteria were enrolled: (1) clinically suspected malignant lymphoma, (2) persistent fever for >3 weeks, refractory to antibiotics, (3) higher than standard lactate dehydrogenase, (4) unbiopsiable lesions, and (5) age ≥20 years. Liquid biopsies were performed as follows: (1) MYD88 L265P/CD79 Y196 mutation analysis using droplet digital PCR (ddPCR), (2) B-cell receptor (BCR)/T-cell receptor (TCR) reconstruction analysis using next generation sequencing (NGS), and (3) comprehensive genetic analysis using the hematopoietic oncogene panel (426 genes) and NGS. Pathological malignant lymphoma was diagnosed at each institution; later, a central diagnosis was made by two additional lymphoma pathology specialists. Results: Fifty patients were enrolled from 15 centers from October 2019 to September 2021; one was excluded. The median age was 73.5 (21-89) years; 28 patients (57.1%) were male. There were 29 cases (59%), 28 (57.1%), and one (2%) of hematologic malignancy, malignant lymphoma, and MDS/AML, respectively (Fig. 1A). Twenty (71%) malignant lymphomas were B-cell lymphomas (17 of which were IVLBCL [61%]), and 8 (29%) were mature T/NK-cell lymphomas. Nonhematologic diseases were present in 20 (41%) patients (7 collagen diseases [4 adult-onset Still's disease, 3 others], 2 solid tumors, 2 infections [1 syphilis, 1 tuberculosis], and 9 FUO). FUO pathological investigations were performed in 47 (97.9%) and 43 (87.6%) patients with BMA and RSB, respectively. IVLBCL had a 53% positivity rate for initial BMA (9/17) and RSB (8/15; Fig. 1B). Only 65% patients tested positive for either initial BMA or RSB and a majority (15/17) of them were diagnosed based on repeated evaluations during their lifetime. The BMA positive rate in patients with mature T/NK-cell lymphoma (8 cases) was high (75%). The mean cfDNA concentration was 215 (5.6-1320) ng/mL, with no significant difference between patients with lymphoma and those with other diseases (279 vs. 130 ng/mL, p=0.073). In ddPCR analysis, 85% of B-cell lymphoma cases (N=20) had either MYD88 or CD79B mutations; the mutation detection rate in IVLBCL (N=17) was 94% (Fig.1B, 2). All patients with non-B-cell lymphoma tested negative. The positive rate of patients with B-cell lymphoma in the NGS-based BCR reconstruction analysis was 90% and negative in two patients with IVLBCL. BCR reconstitution was also observed in two patients with T/NK-cell lymphoma. Comprehensive panel analysis demonstrated that 19/20 (95%) B-cell lymphoma cases showed MYD88 or CD79B mutations; all IVLBCL cases showed mutations. LymphGen algorithm analysis showed that 15, 1, and 1 IVLBCL cases were categorized as MCD (with MYD88 or CD79b mutations), composite (including MCD), and other, respectively (Fig. 2). In T/NK-cell lymphomas, TCR reconstitution was positive in 3/8 T/NK-cell lymphomas. Panel analysis could hardly predict T/NK-cell lymphoma. DdPCR results were returned to the physician within a median time of 14 days. Of the six patients who were not diagnosed with initial BMA and RSB, five were positive for the MYD88/CD79B mutation, according to the ddPCR results. Three of them were diagnosed through additional liquid biopsy testing. Two patients were diagnosed with IVLBCL based on central diagnosis after death. Conclusions: In the classical FUO cases with suspected malignant lymphoma, 57% and 35% had malignant lymphoma and IVLBCL, respectively. Compared to the classical diagnostic methods, liquid biopsy can be used to detect lymphoma-specific genetic mutations at an earlier stage, being clinically useful for the early diagnosis of difficult-to-diagnose malignant B-cell lymphomas. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

185 (PB-098) Poster - Cost-effectiveness of abemaciclib in early breast cancer patients: one size fits all or tailoring to patients’ needs

Background: The addition of two years of abemaciclib treatment to standard adjuvant endocrine therapy in all patients with high risk ER+, HER2- early-breast cancer (EBC) has been approved by the European Medicines Agency (EMA). Implementation of this treatment in this setting creates additional burden on patients and will increase health care costs. Pre-selection of high-risk patients using detection of minimal residual disease (MRD) by determination of circulating tumor DNA (ctDNA) in plasma during follow-up is highly promising and could select the patients who will benefit from abemaciclib treatment.

Early cancer detection by a targeted methylation assay of circulating tumor DNA in plasma

Early cancer detection by a targeted methylation assay of circulating tumor DNA in plasma