ctDNA Sequencing Research Articles

Detection of Tumor Recurrence via Circulating Tumor DNA Profiling in Patients with Localized Lung Cancer: Clinical Considerations and Challenges.

Simple SummaryCirculating tumor DNA is a novel biomarker with emerging uses in the clinical care of patients with cancer, including non-small-cell lung cancer. Already approved for use in various clinical settings in patients with metastatic non-small-cell lung cancer, recent research has focused on the ability of circulating tumor DNA to predict relapse of patients with localized disease after treatment with curative intent. Identifying patients at increased risk of relapse after treatment with curative intent remains challenging, but several groups have identified circulating tumor DNA kinetics as a potential means of aiding our risk stratification. Herein, we discuss current research that identifies longitudinal circulating tumor DNA kinetics as a highly sensitive and specific marker for relapse. Then, we identify important clinical considerations and challenges for moving forward with further studying and eventually using this biomarker for patients with localized disease in clinic.Approximately 30% of patients with non-small-cell lung cancer (NSCLC) present with localized/non-metastatic disease and are eligible for surgical resection or other “treatment with curative intent”. Due to the high prevalence of recurrence after treatment, adjuvant therapy is standard care for most patients. The effect of adjuvant chemotherapy is, however, modest, and new tools are needed to identify candidates for adjuvant treatments (chemotherapy, immunotherapy, or targeted therapies), especially since expanded lung cancer screening programs will increase the rate of patients detected with localized NSCLC. Circulating tumor DNA (ctDNA) has shown strong potential to detect minimal residual disease (MRD) and to guide adjuvant therapies. In this manuscript, we review the technical aspects and performances of the main ctDNA sequencing platforms (TRACERx, CAPP-seq) investigated in this purpose, and discuss the potential of this approach to guide or spare adjuvant therapies after definitive treatment of NSCLC.

Complementary Sequential Circulating Tumor Cell (CTC) and Cell-Free Tumor DNA (ctDNA) Profiling Reveals Metastatic Heterogeneity and Genomic Changes in Lung Cancer and Breast Cancer.

IntroductionCirculating tumor cells (CTCs) and cell-free tumor DNA (ctDNA) are tumor components present in circulation. Due to the limited access to both CTC enrichment platforms and ctDNA sequencing in most laboratories, they are rarely analyzed together.MethodsConcurrent isolation of ctDNA and single CTCs were isolated from lung cancer and breast cancer patients using the combination of size-based and CD45-negative selection method via DropCell platform. We performed targeted amplicon sequencing to evaluate the genomic heterogeneity of CTCs and ctDNA in lung cancer and breast cancer patients.ResultsHigher degrees of genomic heterogeneity were observed in CTCs as compared to ctDNA. Several shared alterations present in CTCs and ctDNA were undetected in the primary tumor, highlighting the intra-tumoral heterogeneity of tumor components that were shed into systemic circulation. Accordingly, CTCs and ctDNA displayed higher degree of concordance with the metastatic tumor than the primary tumor. The alterations detected in circulation correlated with worse survival outcome for both lung and breast cancer patients emphasizing the impact of the metastatic phenotype. Notably, evolving genetic signatures were detected in the CTCs and ctDNA samples during the course of treatment and disease progression.ConclusionsA standardized sample processing and data analysis workflow for concurrent analysis of CTCs and ctDNA successfully dissected the heterogeneity of metastatic tumor in circulation as well as the progressive genomic changes that may potentially guide the selection of appropriate therapy against evolving tumor clonality.

AJS-5 Forefront challenge in the SCRUM-Japan utilizing Artificial Intelligence (AI) – Virtual Sequencing (VSQ) and QA Commons (QAC) projects and beyond

Recent progress in artificial intelligence (AI) technologies make big data analyses possible, which may offer more precise, rapid, valuable information in terms of clinical implementation of precision oncology. Numerous genetic and epigenetic abnormalities may lead to various morphologies of cancer. However, exactly which gene abnormality causes which morphology is unknown. The SCRUM-Japan Genesis Virtual Sequencing (VSQ) Project aims to investigate a novel algorithm by synergistically fusing deep learning (DL) technology and pathological diagnostics for the prediction of cancer genome abnormalities. This was achieved by elucidating the association between the morphological findings and genetic abnormalities, including BRAF V600E mutations and MSI status directly linked to the therapeutic strategies for advanced colorectal cancer (CRC) patients, indicating that VSQ can appropriately predict these alterations in advanced CRC, potentially without performing NGS tests and may also enable a prompt initiation of systemic treatments in CRC patients. Clinical sequencing of tumor tissue or circulating tumor DNA (ctDNA) enables the identification of genomic alterations that can be targeted therapeutically, and the importance of knowledge-based curation systems is on the rise. We assessed the utility of the AI-based curation system, QA Commons (QAC) for clinical sequencing. It demonstrated the utility of QAC in treatment decision making by tissue or ctDNA sequencing, especially in information of clinical trials. We will initiate the 4th stage SCRUM-Japan MONSTAR-SCREEN project as -from genomics to multi-omics with AI- since April 2021, to assess clinical utility for analyzing tissue-based WES, WTS, and Protein analyses as well as circulating tumor NA (nucleic acid) analyses in all solid tumors, utilizing the DL technology. In this ASCO-JSMO joint symposium, I would like to present the current status and perspective in the SCRUM-Japan Project.

Abstract 535: Molecular profiling of brainstem gliomas by liquid biopsy

Abstract Background: Brainstem gliomas are heterogeneous diseases, many of which are difficult to safely resect and have limited treatment options due to their location. These constraints pose challenges to biopsy, which limits the use of routine molecular profiling and identification of personalized therapies. Here, we explored the potential of sequencing of circulating tumor DNA (ctDNA) isolated from the cerebrospinal fluid (CSF) of brainstem glioma patients as a less invasive approach for tumor molecular profiling. Cohorts and Methods:A retrospective analysis of brainstem glioma patients with CSF and tumor tissue available was completed. CSF was obtained from patients either intraoperatively (91.2%, 52/57), from ventricular-peritoneal shunt (3.5%, 2/57), or by lumbar puncture (5.3%, 3/57), all prior to surgical manipulation of the tumor. Deep panel sequencing of glioma-associated genes was performed on CSF-derived ctDNA and, where available, matched blood and tumor DNA from 57 patients, including nine medullary and 23 diffuse intrinsic pontine gliomas (DIPG). In addition, we have now completed a prospective study, in which 12 preoperative CSF samples were obtained from lumbar puncture and used to isolate and characterize ctDNA. Results: In the retrospective analysis, at least one tumor-specific mutation was detected in over 82.5% of CSF ctDNA samples (47/57). In cases with primary tumors harboring at least one mutation, alterations were identified in the CSF ctDNA of 97.3% of cases (36/37). In over 83% (31/37) of cases, all primary tumor alterations were detected in the CSF, and in 91.9% (34/37) of cases, at least half of the alterations were identified. Among ten patients found to have primary tumors negative for mutations, 30% (3/10) had detectable somatic alterations in the CSF. Finally, mutation detection using plasma ctDNA was less sensitive than sequencing the CSF ctDNA (38% vs. 100%, respectively). In the prospective study, at least one tumor-specific mutation was detected in over 83.3% of CSF ctDNA samples (10/12), which is consistent with the data from the retrospective cohort. Conclusion: Our study indicates that deep sequencing of CSF ctDNA is a reliable technique for detecting tumor-specific alterations in brainstem tumors. This approach may offer an alternative approach to stereotactic biopsy for molecular profiling of brainstem tumors. Citation Format: Changcun Pan, Tian Li, Liping Jiang, Hai Yan, Liwei Zhang. Molecular profiling of brainstem gliomas by liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 535.

Abstract 540: Molecular response evaluation of patients with metastatic colorectal cancer using circulating tumor DNA

Abstract Introduction: Patients with colorectal cancer with unresectable and isolated liver metastases (CRLM) are mostly treated with chemotherapy and targeted treatments like anti-epidermal growth factor receptor (EGFR) monoclonal antibody therapy. Clinical response evaluation of systemic treatment is performed by radiological CT imaging, which can detect disease progression. However, CT imaging does not always give information about the viability of the tumor tissue nor does it constitute the genomic changes of the tumor, i.e. development of sub-clones following the pressure of treatment. Cell-free circulating tumor DNA (ctDNA) derived from liquid biopsies is a minimally invasive biomarker that has great potential for tumor detection and is present in relatively high levels in the plasma of patients with CRLM. Liquid biopsy ctDNA allows for longitudinal follow-up and gives the possibility to track intratumor heterogeneity caused by different sub-clones without a repeated tumor biopsy. Here, we aim to assess the use of molecular profiling using serial liquid biopsies as a biomarker for treatment response evaluation. Methods: Tumor tissue obtained prior to treatment as well as longitudinal liquid biopsies were collected from patients with CRLM who participated in a prospective clinical trial with CRC and were treated with panitumumab and doublet chemotherapy. Liquid biopsy ctDNA was isolated and analyzed by targeted sequencing using a panel of 33-genes, allowing to map dynamic molecular changes during treatment. ctDNA sequencing results were corrected for germline variants and clonal hematopoiesis variants by using targeted sequencing data of patient-matched tumor tissue DNA and white blood cell (WBC) genomic DNA, respectively. ‘Molecular response' was defined as the elimination of more than 95% of ctDNA after treatment compared to the measurement before treatment initiation. Detection of disease progression by ctDNA was compared to CT imaging. Results: At present, 110 longitudinal plasma samples as well as WBC genomic DNA and tumor tissue DNA are analyzed from a cohort of 33 patients. Currently, we are expanding the number of patients and samples. Molecular responders to treatment showed a significantly longer overall survival than non-responders (median 51 vs 25 months; p=0.033; HR=3.7). In addition, significantly earlier detection of disease progression was observed using ctDNA compared to radiological imaging (median difference of 4.8 months; p=0.006). Conclusion: Serial plasma ctDNA analyses in patients with mCRC provide a minimally-invasive tool for longitudinal treatment response evaluation of dynamic genomic alterations and creates an opportunity for patient subset selection for possible adaptation of the treatment regimen. Citation Format: Iris van 't Erve, Jamie E. Medina, Alessandro Leal, Eniko Papp, Karen Bolhuis, John K. Simmons, Samuel Angiuoli, Cornelis J. Punt, Gerrit A. Meijer, Victor E. Velculescu, Remond J. Fijneman. Molecular response evaluation of patients with metastatic colorectal cancer using circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 540.

Abstract 549: Genomic characterization and monitoring molecular response to treatment in African American (AA) advanced prostate cancer (PC) patients (pts) via next-generation sequencing (NGS): Real-world experience in a safety net hospital Oncology clinic

Abstract Background: AA men have higher PC incidence and mortality than white men. Healthcare access and other socioeconomic factors contribute to these disparities. The high cost of NGS can become an additional factor for disparity. Indeed, minority and uninsured pts were underrepresented in prior NGS studies and NGS-based Precision Oncology clinical trials. Methods: Ben Taub Hospital (BTH) is a safety net hospital serving a racially/ethnically diverse pt population (91% minorities). We retrospectively analyzed NGS data obtained via Tempus|xT tissue assay (DNA sequencing of 648 genes in tumor tissue at 500x depth) and/or Tempus|xF liquid biopsy assay (ctDNA sequencing of 105 genes at 5,000x depth) for germline and/or somatic mutations detected in 117 BTH pts [61 self-identified AA and 56 white (including white Hispanic)] receiving treatment for locally advanced, biochemically recurrent or metastatic PC. We also analyzed de-identified NGS data from a nationwide cohort of 2090 metastatic PC pts (225 AA) previously sequenced with xT and/or xF by Tempus Labs (Chicago, IL). Results: AA BTH PC pts exhibited high frequencies of BRCA2 (14.8%), SPOP (19.7%), AR (16.4%) and TP53 (49.1%) mutations (respective frequencies in white BTH pts were 9%, 7%, 3.6% and 12.5%). TMPRSS2 fusions were less frequent in AA than white pts. Upon treatment with active therapy (hormonal or taxane), the allele frequency of PC-derived ctDNA mutations in the liquid biopsy declined significantly and concordantly to the biochemical (PSA) response. In addition, two out of 61 (3.3%) AA BTH PCs had microsatellite instability [a frequency comparable to that seen in white BTH PC pts (3.6%) and in the nationwide Tempus Labs cohort (3.6% of AA PC pts)], and experienced prompt and durable clinical, biochemical and molecular responses to pembrolizumab, including disappearance of PC-derived mutant ctDNA. On the other hand, four AA PC pts who had progressed on abiraterone or enzalutamide with ctDNA findings of AR mutations previously reported as sensitive in vitro to the newest FDA-approved AR antagonist darolutamide, did not achieve clinical or biochemical response on darolutamide treatment and that was paralleled by lack of molecular (ctDNA fraction) response. Conclusions: The high mutation frequency in key PC drivers in AA pts at our safety net hospital can be attributed to underlying disease biology or the more advanced disease at presentation in AA pts with socioeconomic factors delaying access to healthcare. Wide use of NGS testing is necessary to improve early access of underserved minority populations to novel targeted therapies and to biomarker-based Precision Oncology clinical trials. Liquid biopsy is a minimally invasive tool that allows longitudinal monitoring of response (or lack thereof) to treatment. Citation Format: Tamer Khashab, Salma Kaochar, Alexander D. Le, Samantha Cohen, Attiya B. Noor, Heidi Dowst, Neda Zarrin-Khameh, Michael A. Brooks, Guilherme Godoy, Maria A. Berezina, Anna E. Schwarzbach, Michael E. Scheurer, Martha P. Mims, Nicholas Mitsiades. Genomic characterization and monitoring molecular response to treatment in African American (AA) advanced prostate cancer (PC) patients (pts) via next-generation sequencing (NGS): Real-world experience in a safety net hospital Oncology clinic [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 549.

Monitoring of Circulating Tumor DNA Improves Early Relapse Detection After Axicabtagene Ciloleucel Infusion in Large B-Cell Lymphoma: Results of a Prospective Multi-Institutional Trial.

Although the majority of patients with relapsed or refractory large B-cell lymphoma respond to axicabtagene ciloleucel (axi-cel), only a minority of patients have durable remissions. This prospective multicenter study explored the prognostic value of circulating tumor DNA (ctDNA) before and after standard-of-care axi-cel for predicting patient outcomes. Lymphoma-specific variable, diversity, and joining gene segments (VDJ) clonotype ctDNA sequences were frequently monitored via next-generation sequencing from the time of starting lymphodepleting chemotherapy until progression or 1 year after axi-cel infusion. We assessed the prognostic value of ctDNA to predict outcomes and axi-cel-related toxicity. A tumor clonotype was successfully detected in 69 of 72 (96%) enrolled patients. Higher pretreatment ctDNA concentrations were associated with progression after axi-cel infusion and developing cytokine release syndrome and/or immune effector cell-associated neurotoxicity syndrome. Twenty-three of 33 (70%) durably responding patients versus 4 of 31 (13%) progressing patients demonstrated nondetectable ctDNA 1 week after axi-cel infusion (P < .0001). At day 28, patients with detectable ctDNA compared with those with undetectable ctDNA had a median progression-free survival and OS of 3 months versus not reached (P < .0001) and 19 months versus not reached (P = .0080), respectively. In patients with a radiographic partial response or stable disease on day 28, 1 of 10 patients with concurrently undetectable ctDNA relapsed; by contrast, 15 of 17 patients with concurrently detectable ctDNA relapsed (P = .0001). ctDNA was detected at or before radiographic relapse in 29 of 30 (94%) patients. All durably responding patients had undetectable ctDNA at or before 3 months after axi-cel infusion. Noninvasive ctDNA assessments can risk stratify and predict outcomes of patients undergoing axi-cel for the treatment of large B-cell lymphoma. These results provide a rationale for designing ctDNA-based risk-adaptive chimeric antigen receptor T-cell clinical trials.

Elucidating Prostate Cancer Behaviour During Treatment via Low-pass Whole-genome Sequencing of Circulating Tumour DNA

BackgroundBetter blood tests to elucidate the behaviour of metastatic castration-resistant prostate cancer (mCRPC) are urgently needed to drive therapeutic decisions. Plasma cell-free DNA (cfDNA) comprises normal and circulating tumour DNA (ctDNA). Low-pass whole-genome sequencing (lpWGS) of ctDNA can provide information on mCRPC behaviour. ObjectiveTo validate and clinically qualify plasma lpWGS for mCRPC. Design, setting, and participantsPlasma lpWGS data were obtained for mCRPC patients consenting to optional substudies of two prospective phase 3 trials (FIRSTANA and PROSELICA). In FIRSTANA, chemotherapy-naïve patients were randomised to treatment with docetaxel (75 mg/m2) or cabazitaxel (20 or 25 mg/m2). In PROSELICA, patients previously treated with docetaxel were randomised to 20 or 25 mg/m2 cabazitaxel. lpWGS data were generated from 540 samples from 188 mCRPC patients acquired at four different time points (screening, cycle 1, cycle 4, and end of study). Outcome measurements and statistical analysislpWGS data for ctDNA were evaluated for prognostic, response, and tumour genomic measures. Associations with response and survival data were determined for tumour fraction. Genomic biomarkers including large-scale transition (LST) scores were explored in the context of prior treatments. Results and limitationsPlasma tumour fraction was prognostic for overall survival in univariable and stratified multivariable analyses (hazard ratio 1.75, 95% confidence interval 1.08–2.85; p = 0.024) and offered added value compared to existing biomarkers (C index 0.722 vs 0.709; p = 0.021). Longitudinal changes were associated with drug response. PROSELICA samples were enriched for LSTs (p = 0.029) indicating genomic instability, and this enrichment was associated with prior abiraterone and enzalutamide treatment but not taxane or radiation therapy. Higher LSTs were correlated with losses of RB1/RNASEH2B, independent of BRCA2 loss. ConclusionsPlasma lpWGS of ctDNA describes CRPC behaviour, providing prognostic and response data of clinical relevance. The added prognostic value of the ctDNA fraction over established biomarkers should be studied further. Patient summaryWe studied tumour DNA in blood samples from patients with prostate cancer. We found that levels of tumour DNA in blood were indicative of disease prognosis, and that changes after treatment could be detected. We also observed a “genetic scar” in the results that was associated with certain previous treatments. This test allows an assessment of tumour activity that can complement existing tests, offer insights into drug response, and detect clinically relevant genetic changes.

Circulating Tumor DNA Testing for Homology Recombination Repair Genes in Prostate Cancer: From the Lab to the Clinic.

Approximately 23% of metastatic castration-resistant prostate cancers (mCRPC) harbor deleterious aberrations in DNA repair genes. Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) therapy has shown improvements in overall survival in patients with mCRPC who harbor somatic and/or germline alterations of homology recombination repair (HRR) genes. Peripheral blood samples are typically used for the germline mutation analysis test using the DNA extracted from peripheral blood leucocytes. Somatic alterations can be assessed by extracting DNA from a tumor tissue sample or using circulating tumor DNA (ctDNA) extracted from a plasma sample. Each of these genetic tests has its own benefits and limitations. The main advantages compared to the tissue test are that liquid biopsy is a non-invasive and easily repeatable test with the value of better representing tumor heterogeneity than primary biopsy and of capturing changes and/or resistance mutations in the genetic tumor profile during disease progression. Furthermore, ctDNA can inform about mutation status and guide treatment options in patients with mCRPC. Clinical validation and test implementation into routine clinical practice are currently very limited. In this review, we discuss the state of the art of the ctDNA test in prostate cancer compared to blood and tissue testing. We also illustrate the ctDNA testing workflow, the available techniques for ctDNA extraction, sequencing, and analysis, describing advantages and limits of each techniques.

Costs, throughput time, and proportion of valid test results, a model-based analysis of liquid biopsies for stage IV non-small cell lung cancer.

e18844 Background: Tissue diagnostics for metastasized non-small cell lung cancer (NSCLC) is hampered by the difficulty of obtaining tissue biopsies, and sequential testing. This leads to patient burden, and a time-consuming diagnostic trajectory, sometimes without valid test results. We analyzed the diagnostic process of liquid biopsies (ctDNA) as an addition to tissue diagnostics, for patients with stage IV NSCLC. Methods: Data was retrieved from stage IV patients (N=209) included in the observational, Lung cancer Early Molecular Assessment trial (LEMA; NCT02894853). Within this study, plasma and tissue was collected upfront. Sequencing of ctDNA was performed retrospectively, and compared to results from tissue diagnostics, as performed in the including hospital, including test results, success-rates, and identified targets per patient. Throughput time for tissue, and costs for diagnostics were calculated within a single specialized institute. Because of the retrospective nature of the ctDNA assessment, diagnostics for implementation of ctDNA was modelled (1 run per week), based on estimations for the trajectory and throughput time. A discrete-event simulation model was developed with three scenarios: 1) diagnostics with tissue biopsy alone (comparator); 2) diagnostics with ctDNA first, and tissue biopsy if required; 3) ctDNA if biopsy failed. The process scope ranged from tissue/blood retrieval to outcome in terms of targets. Simulation analysis was performed, calculating mean price per patient (pp), median throughput time pp, and targets found per scenario. Results: In scenario 1, 85% of the patients had a clinically relevant test result, compared to 93% and 92% when ctDNA was added in scenario 2 and 3, respectively. Scenario 2 was the fastest, but also the costliest scenario: median throughput time 15 days pp (IQR 8-30) and mean costs of €2.716 pp (standard deviation [SD] €789) (Table). In this scenario, 9.9% of the biopsies could be cancelled, and in 39.6% a simple biopsy with immunohistochemistry was sufficient. Conclusions: The addition of ctDNA may lead to better informed treatment decisions in patients with stage IV NSCLC. Discrete-event simulation analysis showed that the addition of ctDNA in the diagnostic process increased proportion of patients with a clinically relevant test-result in both scenarios. Throughput time is faster, and additional costs per patient are limited, especially when ctDNA is added after biopsy failure. Clinical trial information: NCT02894853. [Table: see text]

Genomic profiling by cell-free circulating tumor DNA in patients with recurrent adenoid cystic carcinoma (ACC) and the potential activity of selective fibroblast growth factor receptor (FGFR) inhibitors.

e15018 Background: Few effective systemic therapies are available for adenoid cystic carcinoma (ACC). Multitargeted Tyrosine Kinase inhibitors (TKIs) as Lenvatinib and Rivoceranib can provide disease control and limited radiographic response in unselected ACC patients. Next-generation sequencing in tissue (tNGS) has identified MYB gene fusions in [patients with ACC but currently, only Notch1 mutations have an available targeted agent. The fibroblast growth factor receptor (FGFR) pathway is a downstream target of MYB. Methods: We retrospectively reviewed adult patients with a diagnosis of recurrent ACC seen at the University of Miami Sylvester Comprehensive Cancer Center between January 1, 2019 and October 31, 2020. Commercially available cell free DNA panel of 73 genes was used for analyzing circulating tumor DNA (ctDNA). Patients included had radiographically measurable disease and both tNGS and ctDNA data available. Results: Twelve patients were included. Median age at diagnosis was 54 years (28 - 68), and 69% were males. 83% of the patients had genomic abnormalities reported by tNGS, with the same number by ctDNA sequencing. Seven patients (58%) had genomic abnormalities in ctDNA of potentially therapeutic significance which were not found on tNGS, including FGFR1 amplification as well as FGFR2, BRAF, KRAS and JAK2 mutations. Three patients with FGFR genomic abnormalities only seen by ctDNA had been previously treated with Lenvatinib. One patient with primary lacrimal gland ACC metastatic to the liver and tNGS positive for NOTCH 1 had disease progression despite treatment with Lenvatinib and a NOTCH γ-secretase inhibitor. The patient’s ctDNA reported a FGFR2 mutation. The patient was started on Erdafitinib, an oral selective FGFR inhibitor, achieving a partial response at the primary site and metastatic liver lesions and remained on therapy for 7 months. Follow-up ctDNA after 3 months of therapy noted resolution of both FGFR and NOTCH abnormalities. Conclusions: Patients treated with lenvantinib or other TKIs may develop acquired resistance mutations and other genomic abnormalities. ctDNA can reveal additional therapeutic targets not present on tNGS and can be repeated serially. FGFR genomic abnormalities can be a driver mutation in patients with refractory advanced ACC and the activity of Erdafinib should be evaluated in this subset of patients with few therapeutic options.

Rarity of acquired mutations (MTs) after first-line therapy with anti-EGFR therapy (EGFRi).

3514 Background: Colorectal cancers (CRC) lacking RAS MTs treated with EGFRi are thought to evolve by a repetitive process of genetic diversification and clonal evolution. Acquired MTs in KRAS, NRAS, BRAF, MAP2K1, and EGFR are known mechanisms of acquired resistance in the EGFRi refractory population. However, the prevalence of MTs in the first line (1L) setting is not well established as most experience with EGFRi has been beyond the 1L setting. Methods: We analyzed paired plasma samples from RAS/BRAF/EGFRWT mCRC patients (pts) enrolled in 3 large randomized phase 3 trials who had been treated with EGFRi and in whom paired baseline (BL) and time of progression (PRO) plasma samples had been collected for sequencing of ctDNA on a platform optimized for very low allele frequencies (Plasma Select-R™ and Resolution Bio™). Prevalence of MTs at BL and PRO from a 1L study (‘203; FOLFOX ± panitumumab) were compared with 2 studies in the third line setting (3L; ‘007; panitumumab + best supportive care [BSC] vs BSC; and 3L; ‘763; panitumumab vs. cetuximab), to assess the frequency of acquired resistance mutations via ctDNA analysis. Results: For pts with available paired plasma samples (n = 112 for ‘203; n = 89 for ‘007; n = 274 for ‘763), acquisition of at least one KRAS, NRAS, BRAF, MAP2K1, or EGFR MT was significantly less common in post-progression samples in the EGFR containing arms of the 1L ‘203 study compared to the 3L ‘763 and ‘007 studies (6.8% vs 50.4% vs 39.6%, respectively; p &lt; 0.001). In the non EGFR containing arms of the ‘203 and ‘007 study, the rate of acquired MTs was 7.5% and 0%, respectively (p = 1). While this difference in the rate of acquired MTs between the EGFR and non EGFR containing arms was statistically significant for the 3L study (p &lt; 0.001) it was not significant for the 1L study. Further, pts on both 3L studies treated with EGFRi who experienced CR, PR or SD acquired more MTs than those who had PD as best response (53.6% vs 33.3%, respectively; p &lt; 0.001). This relationship was not significant in the 1L setting (7.7% vs 0%; p = 1). Subclonal MTs (rMAF &lt; 25%) in KRAS, NRAS, EGFR, BRAF and MAP2K1 were present at BL in 129 pts (27%). Based on the hypothesis that EGFRi is selecting for rare existing mutated cells in the tumor, we would expect expansion of any preexisting subclones in the BL samples. However, in contrast to expectations, these subclones rarely expanded to become clonal at the time of progression (12.4%). Conclusions: In contrast to expectations, acquired KRAS, NRAS, BRAF, EGFR, or MAP2K1 MTs rarely develop after 1L therapy. While selective pressure appears to increase the frequency of acquired MTs in the 3L setting, preexisting subclonal MTs do not appear to be the dominant source of acquired MTs at progression, implying that there may also be a transient mutational process driving resistance rather than expansion of preexisting clones. These findings have significant implications for ongoing and planned EGFRi rechallenge studies.

Targeted ctDNA sequencing analysis reveals concurrent genomic alterations and its impact on TKI and immune checkpoint inhibitor therapy in advanced NSCLC from Asian population.

3042 Background: For advanced non-small cell lung cancer (NSCLC), liquid biopsy followed by targeted DNA sequencing is used in the clinical setting for driver-gene identification in circulating tumor DNA (ctDNA), tumor evolution and treatment monitoring. To understand the complexity and diversity of genetic alterations in advanced NSCLC, we examined the genetic landscape of late-stage NSCLC from an Asian population and its association with tyrosine kinase inhibitor (TKI) or immune checkpoint inhibitor (ICI) treatment. Methods: Plasma cell-free DNA (cfDNA) from 142 Asian patients with advanced NSCLC underwent real-world testing in a CAP-accredited and CLIA-certified central laboratory. We analyzed genetic alterations in cfDNA using an amplicon-based next-generation sequencing assay that covers 61 NSCLC related genes. 88 patients (62%) had received one or more lines of treatment. A similar cohort from Caucasian NSCLC was used for comparison (Blakely et al. Nat Genet 2017). Systematic co-mutation analysis, functional annotation and pathway enrichment analysis were performed. Results: The top 10 mutated genes in the Asian cohort were EGFR (66.2%), TP53 (48.6%), KRAS (11.3%), BRCA2 (7%), CTNNB1 (6.3%), PIK3CA (6.3%), SMAD4 (5.6%), BRCA1 (4.9%), MET (3.5%) and RB1 (3.5%). EGFR mutations occurred at hotspots in exons 18 – 21, with exon19 deletion, L858R, T790M, C797S, and G719A being the top-ranking alleles, similar to the Caucasian NSCLC cohort. Concurrent KRAS and EGFR mutations were higher in Asian cohort (8.5%) than in Caucasian cohort (0.56%). A rare PD-L1 structural variant (1.1%), reported to be related to immune evasion in other cancers (Kataoka et al. Nature 2016), was detected in the EGFR-mutated subgroup. Within the first line TKI-treated group, responders (CR+PR) harbored more alterations than non-responders (p &lt; 0.017). Among ICI-treated NSCLC patients (anti-PD-1/PD-L1 mono- or polytherapy), responders from 1st line or 2nd line ICI treatment harbored fewer mutations than non-responders, but the distribution of mutational allele frequency (MAF) in responders was shifted to the right (more clonal than subclonal mutations). Functional annotation suggests that concurrent mutated genes and copy number alterations in advanced EGFR-mutant NSCLC were enriched in cell cycle, DNA repair, PI3K-AKT signaling pathways. Conclusions: We characterized the ctDNA-derived genetic landscape of advanced NSCLC from an Asian population and dissected mutated genes with the outcome of TKI or ICI treatment. We also report a rare PD-L1 structural variant in the EGFR mutated subgroup, which could be associated with immune evasion. Our analyses support the occurrence of clonal and subclonal driver co-alterations in EGFR-driven NSCLC, underlining the clinical utility of ctDNA detection for NSCLC diagnosis and treatment selection.

Circulating tumor DNA as markers of dynamic recurrence risk and adjuvant chemotherapy benefit in resected non-small cell lung cancer.

3028 Background: A significant proportion of non-small cell lung cancer (NSCLC) patients relapse after surgical resection with or without adjuvant therapy. The detection of molecular residual disease (MRD) has great potentials to stratify postoperative risk and facilitate early recurrence diagnosis. Here, we aim to evaluate the clinical utility of serial plasma circulating tumor DNA (ctDNA) in MRD detection, adjuvant therapy guidance, and recurrence risk prediction in resected NSCLC patients. Methods: This prospective cohort study recruited 116 patients with NSCLC following surgery and/or adjuvant therapy. Thirteen patients discontinued, leaving 103 patients for analysis. Tumor samples were obtained at surgery. Plasma samples were collected at baseline, after surgery, after adjuvant therapy and every 3 months thereafter and were analyzed by ultradeep (30000×) next-generation sequencing with molecular barcode and in-silico error correction. Results: Pretreatment ctDNA was detected in 69.8% of patients. ctDNA positivity after surgery and after completion of adjuvant chemotherapy (ACT) were significantly associated with worse recurrence-free survival (RFS) (HR: 4.0; 95% CI: 2.0-8.0; P&lt;.001 and HR:3.2; 95% CI: 1.3-8.2; P &lt;.05, respectively). In stage II-III patients who were positive for ctDNA after surgery, ACT-treated patients had a better RFS than those without ACT ( P&lt;.05), whereas ACT did not confer significant clinical benefits in patients with negative postsurgical ctDNA. During surveillance after definitive therapy, ctDNA positivity was associated with worse RFS (HR: 8.5, 95% CI: 3.7-20, P &lt;.001) and preceded radiological recurrence by a median of 88 days. Using joint modeling of serial ctDNA and time-to-recurrence, we accurately predicted patients’ 12-month and 15-month recurrence status, with areas under receiver-operating characteristics curves (AUROC) of 0.88 and 0.84, respectively. Conclusions: These results indicate that ultradeep ctDNA sequencing could sensitively detect MRD, thus identifying patients with high recurrence risk and guiding the adjuvant therapy decision in resected NSCLC. We also demonstrate that joint modeling of serial ctDNA levels and time to recurrent can provide an accurate dynamic risk prediction for NSCLC patients during surveillance. Clinical trial information: ChiCTR1900024656.

Distribution of BRCA1/2 germline and somatic alterations across cancer type.

10590 Background: PARP inhibitors (e.g. Olaparib or niraparib) have been approved by FDA as a targeted therapy for many tumors harboring germline or somatic BRCA1/2 (g or sBRCA1/2), including ovarian cancer, prostate cancer, breast cancer and pancreases cancer. It is imperative to study the distribution of BRCA1/2 across cancer type. In this study, we aim to assess the landscape of BRCA1/2 alterations in solid tumors and evaluate the feasibility of circulating tumor DNA (ctDNA) tested by next-generation sequence (NGS) as a tool to detect BRCA1/2 alterations. Methods: For tissue specimen, genomic DNA from formalin fixed paraffin-embedded (FFPE) tumor specimens or fresh tumor tissues was used for sequence analysis. Genomic DNA (gDNA) from white blood cells was extracted using the QIAamp DNA Mini Kit (Qiagen). For ctDNA, cell-free DNA libraries were prepared using the KAPA Hyper Prep Kit following the manufacturer’s protocol. The captured libraries were loaded onto a NovaSeq 6000 platform (Illumina) for 100bp paired-end sequencing. The testing was performed in the College of American Pathologists (CAP) and Clinical Laboratory Improvement Amendments (CLIA) -certified 3D Medicines Library. Results: A total of 27, 835 patients were tested using tumor tissue during Jan. 1th 2017 to June 1th 2020, including 43% (N = 12089) of non-small cell lung cancer, 19% (N = 5357) of colorectal cancer, 8% (N = 2181) of liver cancer, 6% (N = 1621) of gastric cancer, 5% (N = 1479) of biliary tract cancer, 4% (N = 1084) of kidney cancer, 4% (N = 1045) of pancreas cancer, 3% (N = 689) of breast cancer and 2% (N = 599) of ovarian cancer. Across all tumor types, the known or likely deleterious BRCA1/2 alterations were identified in 2147 (7.7%) patients. Ovarian cancer had the highest frequency of BRCA1/2 alteration (23.4%), followed by endometrial cancer (12.7%) and breast cancer (10.6%). BRCA1/2 alteration was found in 8.8% prostate cancer and 4.2% pancreas cancer respectively. Across all tumor types, the known or likely deleterious gBRCA1/2 alterations were identified in 369 (1.3%) patients. Notably, ovarian cancer had the highest frequency of gBRCA1/2 alteration (13.9%), followed by breast cancer (7%), prostate cancer (4.4%) and endometrial cancer (4.1%). No clear hotspot mutations and mutated codons were spread throughout g or sBRCA1/2 mutations. Additionally, among 15699 patients who suffered ctDNA sequencing, any known or likely deleterious sBRCA1/2 alterations were identified in 358 (2%) patients. Similar to the results of tissue sequencing, ovarian cancer had the highest frequency of sBRCA1/2 alteration (16.67%), followed by endometrial cancer (9.68%), prostate cancer (7.18%) and breast cancer (5.58%) in the blood cohort. Conclusions: BRCA1/2 alterations existed across tumor types and the landscape of g or sBRCA1/2 alterations varied according to cancer type. Furthermore, ctDNA can be used as a potential tool to detect BRCA1/2 alterations.

Residual of circulating tumor DNA (ctDNA) indicated therapeutic efficacy of sintilimab plus docetaxel in patients with previously treated advanced non-small cell lung cancer (NSCLC).

e21207 Background: The synergetic effect of ICIs plus chemotherapy has been demonstrated in first line setting for patients with advanced NSCLC. As previously reported, sintilimab plus docetaxel in advanced Chinese NSCLC pts who had failed first-line chemotherapy showed encouraging efficacy and tolerable safety profile. This exploratory study aims to investigate putative biomarker(s) predicting therapeutic response and long-term outcome for eligible patients. Methods: Advanced NSCLC pts who had failed standard platinum doublet without receiving any ICIs before would receive docetaxel (75mg/m2, day 1) plus sintilimab (200mg, day 3) every 3 weeks for 4-6 cycles followed by sintilimab maintenance until disease progression, unacceptable toxicity, or up to 2 years. Thirty-nine eligible patients received comprehensive genomic profiling of circulating tumor DNA (ctDNA) via a 448-gene panel before treatment. ctDNA from twenty-three patients were dynamically assessed after two courses (at 6th week). Eventually, 22 patients were enrolled into analysis, one patient was lost. White blood cells were used to filter germline variants from ctDNA sequencing data. Results: Of 22 patients with paired ctDNA profiling results at 6th week, 11 patients (50%) were defined as ctDNA residual due to presence of ≥2 somatic variants; Another 11 patients (50%) who had ≤1 somatic variant were defined as non-ctDNA residual. Significant difference of best objective response rate (ORR) (63.64% vs 0%, P=0.0039, two-sided Fisher’s Exact Testing for non-ctDNA residual vs ctDNA residual patients) was observed between these two populations. And numerically higher disease control rate (DCR) was seen in non-ctDNA residual patients (100% vs 63.64%, non-ctDNA residual vs ctDNA residual). Further, patients with ctDNA residual after 2 courses of sintilimab plus docetaxel (at 6th week) displayed higher risk of disease progression [Hazard Ratio (95%CI), 9.91(2.09-46.97), P=0.0038] and inferior prognosis (median PFS, ctDNA residual vs non-ctDNA residual, 3.0 months vs NR, P=0.0007). In addition, mutations of EGFR and LRP1B were enriched in ctDNA residual group. Especially, LRP1B gene mutations associated with shorter PFS period, which should be further investigated. Conclusions: Residual of ctDNA at 6th week was able to indicate inferior response to sintilimab plus docetaxel in patients with previously treated advanced NSCLC. Further validation of ctDNA residual as a robust predictive biomarker is warranted. [Table: see text]

CtDNA analysis in patients with cancer of unknown primary and its value in guiding targeted treatment when combined with predicted cancer types by 90-gene reverse-transcription polymerase chain reaction assay.

e18730 Background: No targeted agents except for drugs against NTRK fusion, dMMR/MSI-H or TMB-H are recommended for the treatment of cancer of unknown primary (CUP), despite of the occurrence of multiple actionable mutations identified by NGS. We aimed to explore the characteristics of circulating tumor DNA (ctDNA) and its value in guiding targeted treatment in combination with predicted cancer types by 90-gene reverse-transcription polymerase chain reaction assay for tissue origin. Methods: 172 treatment-naïve CUP patients with ctDNA testing were retrospectively included between April 2017 and Oct 2020. Of them, 98 patients had primary site predicted. Genetic alterations were reclassified based on their predicted primary site and the oncoKB scale. 153 patients treated with the first-line therapy and available survival data were used to explore prognostic value of detected genetic alterations in ctDNA. Results: We identified 82.6% (142/172) of patients had alterations detected, with the most commonly seen mutations of TP53 (51%), ARID1A (11%), KRAS (10%), RB1 (9%), APC (8%), PI3KCA (8%) and NFE2L2 (7%). The most commonly observed actionable mutations were PIK3CA (n=14, 9.8%), ERBB2 (n=7, 4.9%), BRAF (n=6, 4.2%), MET (n=6, 4.2%) and EGFR (n=5, 3.5%). After introducing predicted primary site in the 98 patients, 6.1% (n=6) of patients upgraded to a Level 1 alteration, 1.0% (n=1) to a Level 2 alteration (Table). In these 7 patients, only one patient with predicted lung cancer and EGFR 19 del received gefitinib with partial response for 5+ months. In multivariate analysis, NFE2L2 (hazard ratio [HR] = 2.96, 95%CI=1.32-6.61, P = .008) and CDKN2A mutation (HR = 2.50, 95%CI=1.26-4.96, P = .009) were independently associated with shorter PFS. Furthermore, NFE2L2 (HR = 4.96, 95%CI=1.98-12.43, P ＜ .001) and CDKN2A mutation (HR = 4.84, 95%CI=1.63-14.40, P = .005) were correlated with worse OS. Conclusions: This is the first attempt to integrate ctDNA sequencing and gene expression profiling for tissue of origin in OncoKB classification schema, resulting in 7.1% (7/98) of the genetic alterations reclassified to level 1 or 2, which might identify patients benefiting from corresponding targeted treatment. NFE2L2 and CDKN2A mutations in ctDNA were associated poor prognosis.[Table: see text]

The role of comprehensive analysis with circulating tumor DNA in advanced non-small cell lung cancer patients considered for osimertinib treatment.

Background EGFR mutations are good predictive markers of efficacy of EGFR tyrosine kinase inhibitors (EGFR‐TKI), but whether comprehensive genomic analysis beyond EGFR itself with circulating tumor DNA (ctDNA) adds further predictive or prognostic value has not been clarified.MethodsPatients with NSCLC who progressed after treatment with EGFR‐TKI, and with EGFR T790 M detected by an approved companion diagnostic test (cobas®), were treated with osimertinib. Plasma samples were collected before and after treatment. Retrospective comprehensive next‐generation sequencing (NGS) of ctDNA was performed with Guardant360®. Correlation between relevant mutations in ctDNA prior to treatment and clinical outcomes, as well as mechanisms of acquired resistance, were analyzed.ResultsAmong 147 patients tested, 57 patients received osimertinib, with an overall response rate (ORR) of 58%. NGS was successful in 54 of 55 available banked plasma samples; EGFR driver mutations were detected in 43 (80%) and T790 M in 32 (59%). The ORR differed significantly depending on the ratio (T790 M allele fraction [AF])/(sum of variant AF) in ctDNA (p = 0.044). The total number of alterations detected in plasma by NGS was higher in early resistance patients (p = 0.025). T790 M was lost in 32% of patients (6 out of 19) after acquired resistance to osimertinib. One patient with RB1 deletion and copy number gains of EGFR, PIK3CA, and MYC in addition to T790 M, showed rapid progression due to suspected small cell transformation.ConclusionsNGS of ctDNA could be a promising method for predicting osimertinib efficacy in patients with advanced NSCLC harboring EGFR T790 M.

Integrating Cycled Enzymatic DNA Amplification and Surface-Enhanced Raman Scattering for Sensitive Detection of Circulating Tumor DNA.

Circulating tumor DNA (ctDNA) represents an emerging biomarker of liquid biopsies for the development of precision cancer diagnostics and therapeutics. However, sensitive detection of ctDNA remains challenging, due to their short half-life and low concentrations in blood samples. In this study, we report a new method to address this challenge by integrating cycled enzymatic DNA amplification technique and Au nanoparticle@silicon-assisted surface-enhanced Raman scattering (SERS) technique. We have demonstrated a reproducible identification of a single-base-mutated ctDNA sequence of diffuse intrinsic pontine gliomas (DIPGs), with the limit of detection (LOD) as low as 9.1 fM in the spiked blood samples. This approach can be used to analyze trace amounts of ctDNA in translational medicine for early diagnosis, therapeutic effect monitoring, and prognosis of patients with cancer.

Evaluating the analytical validity of circulating tumor DNA sequencing assays for precision oncology.

Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the accuracy, sensitivity, and reproducibility of ctDNA assays is poorly understood. Here we report the findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments, and proficiency testing on standardized cell line–derived reference samples. Above 0.5% variant allele frequency, ctDNA mutations were detected with high sensitivity, precision and reproducibility by all five assays, whereas below this limit detection became unreliable and varied widely between assays, especially when input material was limited. Missed mutations (false-negatives) were more common than erroneous candidates (false-positives), indicating that the reliable sampling of rare ctDNA fragments is the key challenge for ctDNA assays. This comprehensive evaluation of the analytical performance of ctDNA assays serves to inform best-practice guidelines and provides a resource for precision oncology.