Liquid Biopsy Analysis Research Articles

Liquid and tissue profiling of targetable co-mutations with KRAS in non-small cell lung cancer.

e21201 Background: The primary objective of this study was to understand the co-occurrence of KRAS with other actionable drivers in Indian lung cancers patients, and to assess the utility of liquid biopsy in this cohort. Methods: We evaluated single nucleotide alterations, copy number alterations, and fusions in 507 tumor tissue and 1271 blood specimens with targeted NGS panels in NSCLC patients. Results: In tissue-based analysis (n = 507), KRAS mutations were detected in 68 (13.4%) samples. Of these, 63 (92.6%) samples did not show the presence of any targetable co-mutations. Targetable co-mutations were detected in 5 (7.4%), with concomitant EGFR mutations in all 5 samples(100%) and BRAF mutations in 2 samples (33.3%) In the liquid biopsy (n = 1271) analysis, KRAS mutations were observed in 94 (7.4%) samples. The liquid biopsy analysis showed higher presence of targetable co-mutations in 11.7% (n = 11). EGFR was the most common co-occurring targetable gene observed in 9 samples (81.8%). Mutations in BRAF were detected in 3 (27.3%) samples. ERBB2 Exon 20 insertion was detected in 1 sample (9.1%). No ALK, RET or ROS1 fusions were seen in our KRAS positive cohort. In the tissue cohort, one newly diagnosed adenocarcinoma case showed primary co-mutations in EGFR (Exon 19del VAF 76.8%) and KRAS (G13D VAF 10.5%), impacting utility of EGFR TKI as the first line therapy. Another EGFR Exon 19 del patient developed KRAS G12D mutation as an acquired resistance mutation post Osimertinib therapy. In the liquid biopsy cohort (n = 11), the KRAS positive cohort had four newly diagnosed patients with 3 having EGFR and 1 with BRAF co-mutation. Among the patients with prior therapy, one patient at presentations had EGFR Exon 19 deletion and developed EGFR T790M at progression on Afatinib. The baseline tissue did not have coexisting KRAS mutation. Fresh tissue biopsy was performed at the progression on Osimertinib. The noteworthy finding was the KRAS G12C (VAF 0.36%) presence in liquid biopsy but absences in the simultaneous fresh tissue sample. The EGFR Exon 19 was present at lesser frequency than KRAS in the liquid (VAF 0.23%). In the patients from liquid biopsy cohort with Osimertinib therapy (n = 4), all presented with KRAS as acquired resistance mutation. One among these also showed concomitant EGFR C797S mutation. Conclusions: KRAS mutation virtually makes it unlikely for other targetable mutations to coexist. It makes it thus compelling to further study the role of liquid biopsy with KRAS first and decide if chemotherapy with IO can be started considering the standard of care is to wait for the entire targeted panel before initiating IO. Equally its important to include KRAS in the lung panel and although small the concomitant KRAS may compromise the efficacy to TKI. Liquid biopsy is a promising alternative for detection of actionable alterations, including KRAS and can be harnessed for longitudinal profiling, monitoring tumor evolution and treatment outcome.

Assessing the utility of liquid biopsy for detecting actionable genetic mutations among indigenous Ghanaian women with metastatic breast cancer.

e13017 Background: Breast cancer is the leading cancer diagnosis in women worldwide, and the leading cause of cancer-related mortality in Ghanaian women. Up to 70% of breast cancer diagnoses in Ghanaian women are advanced-stage, leading to fewer opportunities for early intervention. Late presentation of breast cancer may be attributed to lack of comprehensive screening programs, among other factors. Next-Generation Sequencing (NGS)-based liquid biopsy of circulating free DNA (cfDNA) is non-invasive and been demonstrated to support diagnosis, monitoring and treatment selection in cancer. Unfortunately, people of African ancestry are under-represented in evaluations of this technology and little is known about the feasibility of such approaches in low-resource settings. Methods: The AMBER-01 study recruited 20 newly diagnosed, histologically confirmed, treatment-naive women with metastatic breast cancer at the Cape Coast Teaching Hospital. Testing was conducted at CAP and CLIA accredited laboratories (Lucence, Palo Alto; Singapore). Tissue NGS and cfDNA liquid biopsy analysis was ordered on a total of 20 patients following tissue quality analysis outcomes. All 20/20 (100%) liquid biopsy samples were acceptable for analysis, whereas only 6/20 (30%) passed QC for tissue NGS testing. cfDNA liquid biopsy and tissue NGS analyses were performed using Lucence LiquidHALLMARK and Tissue500 technology, respectively. Results: Liquid biopsy detected 42 cfDNA mutations in 17/20 patients. Of the 17 patients found to have mutations on liquid biopsy, 3 (17.6%) patients had mutations associated with African ancestry, including BRCA K719E, ARAF S262I and GATA3 G125dup. Eight actionable mutations specific to breast cancer were found in 7/17 (41.2%) patients while actionable mutations non-specific to breast cancer were detected in 12/17 (70.6%) patients. Tissue biopsy analysis detected mutations in all 6 patients tested, with 3/6 (50%) patients presenting actionable mutations specific to breast cancer. Of the 20 patients recruited, liquid biopsy was able to identify actionable treatments for 7 patients, while tissue was only able to identify actionable treatment for 3 patients. Of the 6 patients with matched liquid and tissue results, liquid biopsy detected additional mutations in 3 (50%) patients that were not detected by tissue analysis including: BRCA2 Q1379E, GNAQ Q176*, and a low Microsatellite Instability (MSI) reported as stable in tissue. Conclusions: Liquid biopsy detected multiple actionable variants in Ghanaian women with breast cancer that a tissue-only workflow missed. cfDNA analysis featured less variations in sample preparation which is a key consideration in resource-limited settings. Further work will optimize NGS panels to account for novel ancestry specific variants. Liquid biopsy presents a great opportunity to improve cancer care in Africa.

The role of ct-DNA in breast cancer surveillance assessing treatment response and recurrence: A community center experience.

e13021 Background: Breast cancer is a heterogeneous disease and primarily classified on immunohistochemistry. Currently treatment is based on biopsy but studies have showed changes in subtype and acquired resistance in the original subtype due to cancer therapies. While biopsy is gold standard, recently liquid biopsy from serum containing circulating tumor DNA (ct-DNA) has emerged as a noninvasive technique of tumor surveillance. Methods: This is a retrospective study approved by the IRB at Monmouth Medical Center. Objectives were to identify use of ct-DNA level and highlight the trends over course of treatment. Subjects were identified by reviewing patient’s charts and included on criteria of age > 18 years and in whom ct-DNA level was followed up. 27 patients were identified and results were obtained by categorizing the patients and calculating the proportions. Results: A total of 27 patients were included. The mean age at diagnosis of cancer was 56.29 years (95% CI 51.19-61.39). Among 27 patients, 11 had left sided and 16 had right sided tumor. The pathology identified was Invasive ductal in 18, adenocarcinoma in 2, lobular in 4, poorly differentiated in 2 and mesenchymal in 1 of the patients. Hormonal subtypes identified were triple negative in 11, ER/PR+ve/Her 2-ve in 8, triple positive in 2, ER/PR-ve/Her 2+ve in 1 and ER+ve in 5 of the patients. Among 27, 8 of the patients had stage I, 15 had stage II, 3 had stage III and 1 had stage IV disease. During the course of treatment, 7 of patients had left, 10 had right and 7 had bilateral mastectomy due to BRCA mutation identified on genetic testing. 3 of the patients did not undergo surgery. ct-DNA levels were elevated 8.05 and 1.31 MTM/ml in 2 patients and subsequently became undetectable and decrease in tumor size was noticed from 6.1 cm to 1.1 cm and 1.8 cm to 9mm. ct-DNA levels remained undetectable the rest of 25 patients and patient’s remained gross tumor free on interval MRI imaging of the breasts. Conclusions: The ct-DNA levels have a great utility in establishing the correlation of treatment effect on tumor volume. All this information is vital for clinical decision making and appropriate management. Therefore, the application of liquid biopsy and ct-DNA analysis in breast cancer opens a window of opportunity that encompasses all possible disease situations: from early diagnosis, through the detection of minimal residual disease, the early detection of relapse, and the monitoring and treatment planning for advanced disease. A limitation of study was low sample size. [Table: see text]

Circulating tumour DNA (ctDNA) as a predictor of clinical outcome in non-small cell lung cancer undergoing targeted therapies: A systematic review and meta-analysis.

e21124 Background: Liquid biopsy (LB) analysis using circulating-tumour DNA (ctDNA)/cell-free DNA (cfDNA) is an emerging alternative to tissue profiling in non-small cell lung cancer (NSCLC). LB is used to guide treatment decisions, detect resistance mechanisms, and predicts responses, and therefore outcomes. This systematic review and meta-analysis evaluated the impact of LB quantification on clinical outcomes in molecularly altered advanced NSCLC undergoing targeted therapies. Methods: We searched Embase, MEDLINE, PubMed and Cochrane Database, between 01/01/2000 and 01/08/2022. The primary outcome was progression-free survival (PFS) and overall survival (OS). Secondary outcomes included objective response rate (ORR), sensitivity and specificity of the test. Age stratification was performed based on the mean age of the individual study population. The quality of studies was assessed using the Newcastle-Ottawa Scale (NOS). Results: Twenty-seven studies reporting 2424 patients were included. Eleven studies (n = 1359) reported an association of baseline ctDNA levels, while 16 studies (n = 1649) reported an association of dynamic ctDNA changes to treatment, with clinical outcomes of PFS and/or OS. Baseline ctDNA-negative patients had higher PFS (pooled hazard ratio [pHR] = 2.97; (95%CI: 1.92-4.85; I2= 97.4%), and OS (pHR = 3.49; (95%CI: 1.73-6.95; I2= 84.2%), than ctDNA-positive patients. Egger’s test suggested publication bias (p < 0.001). Early reduction/clearance of ctDNA levels after treatment improved PFS (pHR = 3.78; 95%CI: 1.91-7.38; I2= 98.4%) and OS (pHR = 2.20; 95%CI: 1.49-3.28; I2= 90.6%), compared to those with no reduction/persistence of ctDNA levels. The PFS was comparable for patients stratified by age (age ≤60 years [pHR = 2.92; 95%CI: 1.43-5.93; I2= 98.3 %] vs. age > 60 years [pHR = 3.94; 95%CI: 1.54-10.07; I2= 83.6 %]). The sensitivity analysis based on study quality (NOS) demonstrated improved PFS only for good quality studies (pHR = 3.67; 95%CI: 1.79-7.54; I2= 91.6 %), but not for poor or fair quality studies. Conclusions: This large systematic review, despite heterogeneity, found that baseline negative ctDNA levels, and early reduction in ctDNA following treatment are strong prognostic markers for PFS and OS in patients undergoing targeted therapies for advanced NSCLC. Future randomised clinical trials should incorporate serial ctDNA monitoring to further establish the clinical utility in advanced NSCLC management. Systematic review registration: PROSPERO registration No. CRD42022347791 Keywords: non-small cell lung cancer; NSCLC; liquid biopsy; circulating tumour DNA; ctDNA; progression-free survival; targeted therapies.

The Use of ctDNA in the Diagnosis and Monitoring of Hepatocellular Carcinoma-Literature Review.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and is one of the leading causes of cancer-related deaths worldwide. Despite advances in medicine, it is still a cancer with a very poor prognosis. Both imaging and liver biopsy still have important limitations, especially in very small nodules and those which show atypical imaging features. In recent years, liquid biopsy and molecular analysis of tumor breakdown products have become an attractive source of new biomarkers. Patients with liver and biliary malignancies, including hepatocellular carcinoma (HCC), may greatly benefit from ctDNA testing. These patients are often diagnosed at an advanced stage of the disease, and relapses are common. Molecular analysis may indicate the best cancer treatment tailored to particular patients with specific tumor DNA mutations. Liquid biopsy is a minimally invasive technique that facilitates the early detection of cancer. This review summarizes the knowledge of ctDNA in liquid biopsy as an indicator for early diagnosis and monitoring of hepatocellular cancer.

Liquid biopsies and minimal residual disease in lymphoid malignancies.

Minimal residual disease (MRD) assessment using peripheral blood instead of bone marrow aspirate/biopsy specimen or the biopsy of the cancerous infiltrated by lymphoid malignancies is an emerging technique with enormous interest of research and technological innovation at the current time. In some lymphoid malignancies (particularly ALL), Studies have shown that MRD monitoring of the peripheral blood may be an adequate alternative to frequent BM aspirations. However, additional studies investigating the biology of liquid biopsies in ALL and its potential as an MRD marker in larger patient cohorts in treatment protocols are warranted. Despite the promising data, there are still limitations in liquid biopsies in lymphoid malignancies, such as standardization of the sample collection and processing, determination of timing and duration for liquid biopsy analysis, and definition of the biological characteristics and specificity of the techniques evaluated such as flow cytometry, molecular techniques, and next generation sequencies. The use of liquid biopsy for detection of minimal residual disease in T-cell lymphoma is still experimental but it has made significant progress in multiple myeloma for example. Recent attempt to use artificial intelligence may help simplify the algorithm for testing and may help avoid inter-observer variation and operator dependency in these highly technically demanding testing process.

Circulating Tumour DNA (ctDNA) as a Predictor of Clinical Outcome in Non-Small Cell Lung Cancer Undergoing Targeted Therapies: A Systematic Review and Meta-Analysis.

Liquid biopsy (LB) analysis using (ctDNA)/cell-free DNA (cfDNA) is an emerging alternative to tissue profiling in (NSCLC). LB is used to guide treatment decisions, detect resistance mechanisms, and predicts responses, and, therefore, outcomes. This systematic review and meta-analysis evaluated the impact of LB quantification on clinical outcomes in molecularly altered advanced NSCLC undergoing targeted therapies. We searched Embase, MEDLINE, PubMed, and Cochrane Database, between 1 January 2020 and 31 August 2022. The primary outcome was progression-free survival (PFS). Secondary outcomes included overall survival (OS), objective response rate (ORR), sensitivity, and specificity. Age stratification was performed based on the mean age of the individual study population. The quality of studies was assessed using the Newcastle-Ottawa Scale (NOS). A total of 27 studies (3419 patients) were included in the analysis. Association of baseline ctDNA with PFS was reported in 11 studies (1359 patients), while that of dynamic changes with PFS was reported in 16 studies (1659 patients). Baseline ctDNA-negative patients had a trend towards improved PFS (pooled hazard ratio [pHR] = 1.35; 95%CI: 0.83-1.87; p < 0.001; I2 = 96%) than ctDNA-positive patients. Early reduction/clearance of ctDNA levels after treatment was related to improved PFS (pHR = 2.71; 95%CI: 1.85-3.65; I2 = 89.4%) compared to those with no reduction/persistence in ctDNA levels. The sensitivity analysis based on study quality (NOS) demonstrated improved PFS only for good [pHR = 1.95; 95%CI: 1.52-2.38] and fair [pHR = 1.99; 95%CI: 1.09-2.89] quality studies, but not for poor quality studies. There was, however, a high level of heterogeneity (I2 = 89.4%) along with significant publication bias in our analysis. This large systematic review, despite heterogeneity, found that baseline negative ctDNA levels and early reduction in ctDNA following treatment could be strong prognostic markers for PFS and OS in patients undergoing targeted therapies for advanced NSCLC. Future randomised clinical trials should incorporate serial ctDNA monitoring to further establish the clinical utility in advanced NSCLC management.

Abstract CT212: A ctDNA-directed, multi-center phase II study of molecular response adaptive immuno-chemotherapy in patients with non-small cell lung cancer: Analysis of Stage 1 of CCTG BR.36

Abstract Introduction: Analyses of circulating tumor DNA (ctDNA) have shown promise in capturing tumor burden dynamics during immune checkpoint blockade (ICB), with the potential to allow patients with primary resistance to be rapidly identified and redirected to alternative therapies. Methods: BR.36 is an international multicenter, open label, biomarker-directed, phase II trial of molecular response-adaptive immuno-chemotherapy for patients with treatment-naïve non-small cell lung cancer-NSCLC (NCT04093167). The trial consists of two stages; stage 1 was a single-arm study to validate the ctDNA approach, with stage 2 focusing on ctDNA guided treatment selection. Key eligibility criteria included EGFR and ALK mutation negative, ICB and chemotherapy-naïve metastatic NSCLC with PD-L1 expression ≥1%. Primary objectives were to ascertain ctDNA molecular response, determine its timing and concordance with radiologic RECIST/iRECIST response. Secondary objectives included the evaluation of time to ctDNA molecular response and its correlation with progression-free (PFS) and overall survival (OS). Liquid biopsy analyses utilized a validated tumor-agnostic white blood cell (WBC) DNA-informed 33-gene panel approach. Ultra-sensitive error-correction ctDNA next-generation sequencing (NGS) was performed at baseline/cycle 1 (C1D1), cycle 2 (C2D1) and cycle 3 (C3D1) of pembrolizumab (200mg IV q3 weeks). The maximum variant allele fraction (maxVAF) of tumor-derived variants was tracked from the pre-treatment to on-therapy timepoints. Results: Activated October 17, 2019, stage 1 completed accrual of 50 patients on April 5, 2022; data lock date was September 20, 2022. Most patients were ever-smokers (98%), had no prior systemic therapy (92%), stage IV NSCLC (98%), adenocarcinoma (76%) with PD-L1 tumor proportion score of ≥ 50% (96%); cohort consisted of 82% white, 52% female, 56% age&amp;gt;65 and 76% ECOG PS 1 participants. Median follow-up was 13.5 months (range 2.5-23.0). Best overall response rate (ORR) by RECIST was 32% with a median duration of 10.1 months. Matched WBC DNA analyses allowed for effective removal of germline and clonal hematopoiesis variants, followed by evaluation of ctDNA response. MaxVAF clearance signified molecular response (mR), while maxVAF persistence indicated molecular disease progression (mPD). Among 35 evaluable patients (77.8%), 15 were classified in the mR category, with an evaluable mR rate of 43% (90% CI: 0.29-0.58). The median time to molecular response was 2.1 months (90% CI: 1.5-2.6). The sensitivity of ctDNA molecular response for RECIST and iRECIST response was 82% (90% CI: 52%-97%) and 83% (90% CI: 56%-97%), with a specificity of 75% (90% CI: 56.5%-88.5%) and 78% (90% CI: 60%-91%) respectively. Patients with mR attained longer PFS (5.03 vs 2.6 months,) and OS (not reached vs 7.23 months); ctDNA response also provided prognostic information for patients with RECIST stable disease. Conclusions: ctDNA molecular responses were concordant with radiologic response assessments, but importantly better predicted PFS and OS for patients with stable disease or better. These findings are now incorporated into the interventional second stage of the trial. Citation Format: Valsamo K. Anagnostou, Cheryl Ho, Garth Nicholas, Rosalyn Anne Juergens, Adrian Sacher, Andrea Fung, Paul Wheatley-Price, Scott Laurie, Benjamin Levy, Julie Brahmer, Archana Balan, Noushin Niknafs, Egor Avrutin, Liting Zhu, Mark Sausen, Penelope Bradbury, Jill O'Donnell-Tormey, Pierre-Olivier Gaudreau, Keyue Ding, Janet Dancey. A ctDNA-directed, multi-center phase II study of molecular response adaptive immuno-chemotherapy in patients with non-small cell lung cancer: Analysis of Stage 1 of CCTG BR.36 [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 CT212.

Electrochemical Cellulase-Linked ELASA for Rapid Liquid Biopsy Testing of Serum HER-2/neu.

Non-invasive liquid biopsy assays for blood-circulating biomarkers of cancer allow both its early diagnosis and treatment monitoring. Here, we assessed serum levels of protein HER-2/neu, overexpressed in a number of aggressive cancers, by the cellulase-linked sandwich bioassay on magnetic beads. Instead of traditional antibodies we used inexpensive reporter and capture aptamer sequences, transforming the enzyme-linked immuno-sorbent assay (ELISA) into an enzyme-linked aptamer-sorbent assay (ELASA). The reporter aptamer was conjugated to cellulase, whose digestion of nitrocellulose film electrodes resulted in the electrochemical signal change. ELASA, optimized relative aptamer lengths (dimer vs monomer and trimer), and assay steps allowed 0.1 fM detection of HER-2/neu in the 10% human serum in 1.3 h. Urokinase plasminogen activator and thrombin as well as human serum albumin did not interfere, and liquid biopsy analysis of serum HER-2/neu was similarly robust but 4 times faster and 300 times cheaper than both electrochemical and optical ELISA. Simplicity and low cost of cellulase-linked ELASA makes it a perspective diagnostic tool for fast and accurate liquid biopsy detection of HER-2/neu and of other proteins for which aptamers are available.

Multiplex Digital Methylation-Specific PCR for Noninvasive Screening of Lung Cancer.

There remains tremendous interest in developing liquid biopsy assays for detection of cancer-specific alterations, such as mutations and DNA methylation, in cell-free DNA (cfDNA) obtained through noninvasive blood draws. However, liquid biopsy analysis is often challenging due to exceedingly low fractions of circulating tumor DNA (ctDNA), necessitating the use of extended tumor biomarker panels. While multiplexed PCR strategies provide advantages such as higher throughput, their implementation is often hindered by challenges such as primer-dimers and PCR competition. Alternatively, digital PCR (dPCR) approaches generally offer superior performance, but with constrained multiplexing capability. This paper describes development and validation of the first multiplex digital methylation-specific PCR (mdMSP) platform for simultaneous analysis of four methylation biomarkers for liquid-biopsy-based detection of non-small cell lung cancer (NSCLC). mdMSP employs a microfluidic device containing four independent, but identical modules, housing a total of 40 160 nanowells. Analytical validation of the mdMSP platform demonstrates multiplex detection at analytical specificities as low as 0.0005%. The clinical utility of mdMSP is also demonstrated in a cohort of 72 clinical samples of low-volume liquid biopsy specimens from patients with computed tomography(CT)-scan indeterminant pulmonary nodules, exhibiting superior clinical performance when compared to traditional MSP assays for noninvasive detection of early-stage NSCLC.

DEcancer: Machine learning framework tailored to liquid biopsy based cancer detection and biomarker signature selection

Cancer is a leading cause of mortality worldwide. Over 50% of cancers are diagnosed late, rendering many treatments ineffective. Existing liquid biopsy studies demonstrate a minimally invasive and inexpensive approach for disease detection but lack parsimonious biomarker selection, exhibit poor cancer detection performance and lack appropriate validation and testing. We established a tailored machine learning pipeline, DEcancer, for liquid biopsy analysis that addresses these limitations and improved performance. In a test set from a published cohort of 1,005 patients including 8 cancer types and 812 cancer-free individuals, DEcancer increased stage 1 cancer detection sensitivity across cancer types from 48 to 90%. In addition, with a test set cohort of patients from a high dimensional proteomics dataset of 61 lung cancer patients and 80 cancer-free individuals, DEcancer's performance using a 14-43 protein panel was comparable to 1,000 original proteins. DEcancer is a promising tool which may facilitate improved cancer detection and management.

Abstract 6604: Analytical development and validation of a personalized hybrid capture based assay for high sensitivity monitoring minimal residual disease (MRD)

Abstract Introduction: 5-30% of patients with early-stage cancer relapse, even though most patients had undergone radical surgery. There is increasing evidence that circulating tumor DNA (ctDNA) minimal residual disease (MRD) predicts relapse following treatment for solid tumors. However, liquid biopsy analyses are challenged by the very low concentrations of ctDNA in blood samples. Existing ctDNA methods lack the sensitivity needed for detecting minimal residual disease (MRD) following therapy. Here, we describe a tumor-informed personalized hybrid capture based method using next-generation sequencing (NGS) to highly sensitive and specific detect MRD and predict recurrence in plasma cell-free DNA. To validate the performance of the assay, we present a technical validation study on DNA from cancer cell line material, including lung cancer cell line, reference material, Onco gDNA reference standard and wild type gDNA reference standard. Method: The performance of the assay was assessed using samples from Onco gDNA reference standard and lung cancer cell line. To estimate number of monitoring single nucleotide variants (SNVs) at input volume of 20-80ng, Onco gDNA reference standard was diluted into 8 concentrations (10, 20, 30, 40, 50, 60, 150 and 750 ppm with a variant allele frequency [VAF] range of 0.001% to 0.075%).To estimate limit of detection (LoD) of the assay at input volume of 20ng and 40ng, lung cancer cell line was diluted into 5 concentrations (10, 20, 40, 80 and 160 ppm with a variant allele frequency [VAF] range of 0.001% to 0.016%).10 samples were performed for each concentration, as well as 10 negative reference samples. Personalized panel was performed using ultradeep (100,000X) NGS with customized 55 or 60 SNVs. Result: Validation results of Onco gDNA reference standard: using 40 variants, sensitivity of input volume of 20ng and 40ng are 95% and 98% at 40 ppm, with a specificity of 100%, respectively. Using 40 variants, sensitivity of input volume of 60ng are 95% at 20 ppm, with a specificity of 100%. Using 50 variants, sensitivity of input volume of 60ng are 70% at 10 ppm, with a specificity of 100%. Validation results of lung cancer cell line: using 40 variants, sensitivity of input volume of 20ng and 40ng are 98% and 100% at 50 ppm, with a specificity of 100%, respectively. Conclusion: The assay was validated by sequentially diluting of lung cancer cell line and Onco gDNA reference standard. With input volume of 20ng, sensitivity of the assay is 95% at 40ppm when monitoring 40 variants, while with input volume of 60ng the LoD reached as low as 10 ppm with sensitivity of 70% when monitoring 50 variants. The assay demonstrates high sensitivity, while maintaining specificity above 99%. Citation Format: Xiaoqiang Wang, Lei Ye, Chao Zhang, Wanglong Deng, Yongjie Wei, Jing Chen, Xuan Tang, Xiaoxuan Wang, Chao Song, Qing Xu, Yong Ren. Analytical development and validation of a personalized hybrid capture based assay for high sensitivity monitoring minimal residual disease (MRD) [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 6604.

Abstract 5593: Clonal mutation burden and evolutionary dynamics analysis in metastatic gastro-esophageal adenocarcinoma (GEA) by error corrected whole-exome circulating tumor DNA sequencing

Abstract Introduction: The evolution of metastatic cancers over time can be assessed in circulating tumor DNA (ctDNA) but sequencing and bioinformatics tools for ultra-deep ctDNA whole-exome sequencing (WES) analysis are lacking. Methods: We developed ctDNA WES that only requires 15ng DNA to achieve sequencing depths of 1000-2000x. Error correction with molecular barcodes and duplex DNA detection allowed calling of mutations ≥0.5% variant frequency (VF). This pilot study applied ctDNA WES to plasma from 20 EGA patients (pts) and standard WES to matched biopsies in order to assess ctDNA WES performance and whether clonal mutation burden (cMB), a critical immunotherapy biomarker and important for neoantigen vaccine designs, differed between ctDNA and biopsies. We furthermore established a mutation and copy number data analysis pipeline using Bayesian clustering to define subclones and track their evolution during therapy in 3pts. Results: The median age of pts was 71y, 95% had distant metastases and 5% locally advanced EGAs. At a median sequencing depth of ????x after de-duplication, VFs of mutations in pre-treatment ctDNA was low (&amp;lt;2% VF) in 8 pts, and intermediate (2-10%) to high (&amp;gt;10%) in 12. Whether cMB differed in biopsies vs ctDNA was assessed in 7 pts with high VFs in ctDNA and good cancer purity in matching biopsy WES. The median cMB was 82 in biopsies and 111 in ctDNA. The increase was driven by 3 cases with 30%, 35% and 59% higher cMB in ctDNA vs biopsies. In two pts, most mutations that only appeared clonal in ctDNA were subclonal rather than absent in the primary tumor, indicating the subclonal presence of the metastasis progenitor clone. Subclonal intermixing in primary tumors hence limits the accurate identification of mutations that are clonal in metastatic disease. Evolutionary dynamics analyses in 3 pts who had good responses to chemotherapy before progression showed a major clonal sweep in one, supporting monoclonal resistance, and evolution of small subclones in two, indicating polyclonal resistance. A MEK1 K57T mutation evolved at resistance in a HER2 amplified EGA treated with trastuzumab+chemotherapy, demonstrating the utility to identify mechanisms of acquired resistance. Conclusions: ctDNA WES can assess the genetics of entire metastatic cancer cell populations over time and deconvolute their evolutionary trajectories. 43% pts had higher cMB in ctDNA compared to biopsies. Liquid biopsy analyses by WES may be superior to tissue-based WES for mutation burden analysis and neoantigen vaccine designs. ctDNA WES identified distinct evolutionary modes of resistance. A larger cohort is being analyzed to define how these differ clinically and whether they can be predicted from pre-treatment ctDNA WES. Efforts to increase the ctDNA WES sensitivity are ongoing as 8 pts had low ctDNA VFs which increased false negative rates. Citation Format: Neil McCafferty, Caroline Fong, Louise J. Barber, Andrew Woolston, Dimitrios Kleftogiannis, Taqia Rana, Susan Cromarty, Shannon Kidd, Ruwaida Begum, Ian Chau, Naureen Starling, David Cunningham, Marco Gerlinger. Clonal mutation burden and evolutionary dynamics analysis in metastatic gastro-esophageal adenocarcinoma (GEA) by error corrected whole-exome circulating tumor DNA sequencing. [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 5593.

Abstract 1040: Evaluation of blood collection tube’s performance for analysis of liquid biopsies by flow and mass cytometry

Abstract The detection of liquid biopsies, including circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) have demonstrated promise as non-invasive assays for monitoring response to therapy for patients with cancer. While ctDNA cannot predict transcriptomic and proteomic changes in response to therapy, we can measure direct cell surface antigen and gene expression changes in CTCs. In pediatric settings, blood samples are often collected from multi-institutional trials. Therefore, to translate liquid biopsies into routine practice, it is important to explore variables most likely to impact the functionality of CTCs including time to process the sample, prospective cryopreservation, blood collection tube and fixative reagents. Cell stabilizer tubes such as Streck Cell-Free DNA, CellSave, CellRescue, or PAXgene ccfDNA have been designed to prevent cell lysis and release of genomic DNA into the plasma, often observed in standard EDTA tubes. In pediatrics, we are often limited by the volume of blood we can draw for research. To this end, we have established an innovative pipeline to collect ctDNA, CTCs, and matched germline DNA from a single blood sample. Blood samples were processed for plasma extraction and CTC isolation after 4h, 24h, and 48h. We isolated rare CTCs from whole blood by combining peripheral blood cell depletion with antigen-based CTC identification. We then evaluated CTC recovery rate and cell membrane integrity (cell-membrane permeabilization) across all blood collection tubes by flow or mass cytometry. Additionally, we integrated whole transcriptome analysis of RNA transcripts in individually sorted CTCs. In this study, we detected similar levels of cell-free DNA, germline DNA and ctDNA in healthy donors and patient samples collected in the DNA stabilizer tubes up to 48h after blood collection. Surprisingly, frequent clotting was observed in the cell layer of samples collected in PAXgene tubes only 24h after blood collection, preventing further analysis of CTC quality in these tubes. At 4h, EDTA tubes and stabilizer tubes seem to perform similarly in preserving CTC integrity. However, at later timepoints, CTC recovery rate, RNA quality and cell membrane integrity were significantly higher in CellRescue tubes. Finally, we showed that CTC count, and RNA quality were slightly lower in patient samples enumerated after cryopreservation compared to the paired-samples processed the same day in both EDTA and cell-stabilizing tubes. In conclusion, we validated the CellRescue tubes as the best candidate to reduce cell lysis and protect against cell membrane permeabilization and mRNA degradation for up to 48 hours, allowing sample collection from multi-institutional studies. To the best of our knowledge, this study is the first to compare the key steps in handling blood samples and how this affects transcriptomic-based assay performance. Citation Format: Anne-Florence Blandin, Sarah Finstuen-Magro, Samuel Abbou, Kelly Klega, Alejandra E. Aguilar, Mohammad Tanhaemami, Maria Ana Isabel C. De Los Santos, Joadly Duplan, Brian Crompton. Evaluation of blood collection tube’s performance for analysis of liquid biopsies by flow and mass cytometry [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 1040.

Abstract 3377: Liquid biopsy analysis reveals orthologs of actionable human cancer variants in dogs

Abstract Genotype-matched therapeutics have become widely adopted in the management of human cancers and can be guided by testing tumor tissue or cell-free DNA from plasma. OncoKB (Oncology Knowledge Base) is the first FDA-recognized database that contains information about these targetable somatic variants and corresponding therapeutic agents, including levels of evidence. Many of the human OncoKB variants have orthologs in the canine genome. This study was conducted to determine the feasibility of detecting these orthologs in dogs using liquid biopsy. This study involved two cohorts: The first was a cohort of 619 client-owned dogs with a variety of cancer diagnoses across all disease stages enrolled in a clinical validation study for a liquid biopsy test (herein the “research cohort”). All dogs had pre-treatment plasma samples collected at enrollment, and a subset of dogs also had matched tumor tissue collected. The second cohort comprised 457 client-owned dogs that had testing as part of the clinical deployment of the above-mentioned liquid biopsy test (herein the “clinical cohort”); 31% of these dogs had samples submitted as an aid in diagnosis (for dogs in which cancer was suspected clinically), and 69% had samples submitted for screening (in dogs with no a priori suspicion of cancer). Blood samples (and tumor samples, when available) were subjected to DNA extraction, proprietary library preparation, and next-generation sequencing. Sequencing data were analyzed using an internally developed bioinformatics pipeline to detect genomic alterations associated with the presence of cancer. Only canine orthologs of variants in the OncoKB database were evaluated in this study. Variants were called if they were observed in plasma at an allele frequency of at least 0.5%, with at least 6 unique supporting reads. Variants identified from plasma were subsequently genotyped in tissue without additional variant-level filtering. Analysis of plasma identified canine orthologs of OncoKB variants in ~8% of dogs in the research cohort, and ~4% of dogs in the clinical cohort. The higher percentage observed in the research cohort is likely due to the higher percentage of patients with confirmed cancer diagnosis. In the patients with an OncoKB ortholog in the research cohort, the dog had a cancer type that matched the human indication for the detected variant in 15% of cases. Of dogs in which an ortholog was identified in plasma, 44% had matched tumor tissue; and the variant observed in plasma was confirmed in tissue in &amp;gt;50% of these subjects. This study demonstrates the feasibility of detecting canine orthologs of targetable human cancer somatic variants in the blood of dogs using next-generation sequencing. Although it is currently unclear how human databases can be used to inform targeted treatment decisions in dogs, these findings may have relevance for comparative oncology studies and future precision therapeutics development for both species. Citation Format: Daniel S. Grosu, Ilya Chorny, Kristina M. Kruglyak, John A. Tynan, Susan C. Hicks, Todd A. Cohen, Allison L. O'Kell, Jill M. Rafalko, Jason Chibuk, Angela L. McCleary-Wheeler, Andi Flory, Dana W. Tsui. Liquid biopsy analysis reveals orthologs of actionable human cancer variants in dogs [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 3377.

Abstract PD11-03: PD11-03 Comparison of a mono Atezolizumab window followed by Atezolizumab and chemotherapy with Atezolizumab and chemotherapy in triple negative breast cancer – an interim analysis of the adaptive randomized neoadjuvant trial NeoMono

Abstract Introduction: Improvement of systemic treatment of TNBC represents an unmet medical need. Targeted therapy of regulatory immune pathways has become an important option in the treatment of many malignant diseases including breast cancer. Neodjuvant trials combining chemotherapy and checkpoint inhibitors (KEYNOTE-522 and IMpassion031) have demonstrated a meaningful benefit regarding pathological complete remission (pCR) for the addition of PD-1- or PD-L1-inhibitors to chemotherapy in patients with TNBC. In the KEYNOTE-522 trial, the addition of an immune checkpoint inhibitor (ICI) to neoadjuvant chemotherapy also had a beneficial impact on event-free survival even in patients who did not achieve a pCR. Of note, in the neoadjuvant GeparNuevo trial only those patients with TNBC who received a 2-week checkpoint inhibitor monotherapy window before the start of neoadjuvant chemotherapy in combination with checkpoint inhibition, achieved a significant pCR benefit from the addition of the PD-1 inhibitor Durvalumab to neoadjuvant chemotherapy alone. Methods: NeoMono is a phase 2 randomized multicenter trial recruiting male and female patients with primary TNBC (defined as ER/PR &amp;lt; 10% and HER2 negative). Neoadjuvant treatment in Arm A and B consists of Atezolizumab 1200 mg every 3 weeks in addition to neoadjuvant chemotherapy (i.e., 12 x Carboplatin and Paclitaxel q1w followed by Epirubicin and Cyclophosphamide q3w). Combination therapy in arm A is preceded by an Atezolizumab monotherapy window (i.e., 840 mg Atezolizumab once two weeks prior to initiation of combination therapy). Study goals are to compare the efficacy of neoadjuvant chemotherapy with Atezolizumab with and without an Atezolizumab two-week monotherapy window (primary endpoint: pCR) and the identification of biomarkers predicting (early) response to or resistance against Atezolizumab. The extensive translational program of the neoMono trial aims at identifying these biomarkers on tumor and patient level through analysis of sequential tissue and liquid biopsies. The NeoMono statistical design adapts the idea of a proof-of-concept trial and uses Bayesian posterior and predictive probabilities for inference about the primary hypothesis. Up to four planned efficacy interim analyses provide decision points for early stopping for success or futility. The expected maximum number of patients to be recruited is 458. Results: The predefined number of 50 patients in each arm being evaluable for the primary endpoint pCR has been reached and the results of the first planned interim analysis will be presented at the meeting. Conclusion: The addition of an ICI to state of the art neoadjuvant chemotherapy has recently been established as a new standard of care in TNBC. NeoMono has the potential to answer the question if the beneficial effect of the ICI can be increased by a chemotherapy free ICI monotherapy window prior to a combination with neoadjuvant chemotherapy. Citation Format: Hans-Christian Kolberg, Johannes Schumacher, Ramona Erber, Michael Braun, Bernhard Heinrich, Oliver Hoffmann, Peter A. Fasching, Georg Kunz, Michael P. Lux, Christian Schem, Eva-Maria Grischke, Mustafa Deryal, Kristina Lübbe, Arndt Hartmann, Sabine Kasimir-Bauer, Cornelia Kolberg-Liedtke. PD11-03 Comparison of a mono Atezolizumab window followed by Atezolizumab and chemotherapy with Atezolizumab and chemotherapy in triple negative breast cancer – an interim analysis of the adaptive randomized neoadjuvant trial NeoMono [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD11-03.

A platform for high-resolution immune liquid biopsy analysis to predict response in patients with renal cell carcinoma treated with nivolumab or cabozantinib: Preliminary data from I-RENE trial (Meet-URO 8 study).

712 Background: Nivolumab, an immune checkpoint inhibitor of PD-1, demonstrated a significant OS benefit in patients with metastatic renal cell carcinoma (mRCC), in progression after a previous line of therapy with anti-VEGFR agents. However, the features of effective immune response and predictive biomarkers of clinical benefit to PD-1 blockade have not yet been recognized. Methods: I-RENE is a prospective translational multicenter Italian study of a real-life mRCC patients treated with nivolumab or cabozantinib after failure of therapy with anti-VEGFR agents. 82 patients were enrolled from December 2018 to August 2022 (nivo 60, cabo 22), with blood samples obtained at baseline and at different time points in both treatment groups. An extended concept of "immune liquid biopsy" is being applied to the study, consisting in the phenotypic and transcriptional profile of lymphoid and myeloid subsets, immune-related miRNA quantification, cyto/chemo-kinome and RNAseq of extracellular vesicle. Results: Multiparametric flow cytometry, performed to monitor the blood frequency and different myeloid and lymphoid cells, show that monocyte subsets (classical, intermediate and non-classical CD14+ cells), monocytic myeloid derived suppressor cells (MDSC, such as CD14+HLA-DRneg and CD14+PD-L1+) and polymorphonucleate (PMN)-MDSC, remain either stable or increase during treatment; concomitantly, CD8+PD-1+ T cells (detected by anti-nivolumab IgG4) increment frequency, acquire the effector CD45RA-CCR7+ phenotype and express the proliferating marker Ki67. Patients receiving cabozantinib display instead a remarkable decrease of all myeloid cell subsets, paired by the boost of cytotoxic CD3-CD16+CD56dim NK cells and more marginally of CD8+PD1+ cells, Preliminary correlations indicate that clinical benefit of nivolumab seems to cluster with the lack of CD14+ cells and M-MDSC increase and blood frequency of total, and the boost in CD8+CD45RA-CCR7+Ki67+ effector T cells. In contrast, the cabozantinib-induced immune modulation occurring in patients treated with does not associate with clinical response Conclusions: This first set of data indicate blood as promising source of dynamic biomarkers for the development of algorithms predicting response to PD-1 blockade. Furthermore, the results so far collected suggest that the potent immunomodulation induced by cabozantinib on the immunosuppressive myeloid compartment may not lead to any tumor control in the absence of specific immune stimulation by checkpoint inhibitors. This study was supported by the Italian Ministry of health (RF-2016_02363001). Clinical trials.gov: NCT04891055 Clinical trial information: NCT04891055 .

Efficacy and safety of a biomarker-driven cetuximab-based treatment regimen over 3 treatment lines in mCRC patients with RAS/BRAF wild type tumors at start of first line: The CAPRI 2 GOIM trial.

Monoclonal antibodies targeting EGFR such as cetuximab or panitumumab represent a major step forward in the treatment of RAS wild type (WT) metastatic colorectal cancer (mCRC). Unfortunately, primary and acquired resistance mechanisms occur, with a huge percentage of patients succumbing to the disease. In the last years, RAS mutation has been identified as the main molecular driver that determine resistance to anti-EGFR monoclonal antibodies. Liquid biopsy analysis allows to a dynamic and longitudinal assessment of mutational status during mCRC disease and has provided important information on the use of anti-EGFR drugs beyond progression or as rechallenge strategy in patients with RAS WT tumors. The phase II CAPRI 2 GOIM trial investigates the efficacy and safety of a bio-marker-driven cetuximab-based treatment regimen over 3 treatment lines in mCRC patients with RAS/BRAF WT tumors at start of first line. The aim of the study is to identify patients with RAS/BRAF WT tumors defined as "addicted" to an-anti EGFR based treatment along three lines of therapy. Moreover, the trial will evaluate the activity of cetuximab re-introduction in combination with irinotecan as 3rd line therapy as rechallenge for patients that will be treated in second line with FOLFOX plus bevacizumab, having a RAS/BRAF mutant disease at progression after FOLFIRI plus cetuximab first line. A novel characteristic of this program is that the therapeutic algorithm will be defined at each treatment decision (first line, second line and third line) in a prospective fashion in each patient by a liquid biopsy assessment of RAS/BRAF status by a comprehensive 324 genes Foundation One Liquid assay (Foundation/Roche). EudraCT Number: 2020-003008-15, ClinicalTrials.gov identifier: NCT05312398.

Mutation Spectrum in Liquid Versus Solid Biopsies From Patients With Advanced Gastroenteropancreatic Neuroendocrine Carcinoma.

Gastroenteropancreatic neuroendocrine carcinomas (GEP-NEC) are rare and have a poor prognosis. Most GEP-NEC are diagnosed with metastatic disease, with only minor biopsies available for molecular diagnostics. We assessed the applicability of liquid biopsies for molecular profiling of GEP-NEC. We performed massive parallel sequencing of 76 cancer-related genes in circulating tumor DNA from 50 patients with advanced GEP-NEC and compared findings to previous analyses of solid tumor biopsies from the same patients. Plasma samples were collected before therapy, and the median time span between blood and tissue sampling was 25 days. We detected 178 somatic mutations in the liquid biopsies, 127 (71%) were also detected in the solid biopsies, whereas 51 (29%) were unique to the liquid biopsies. In the same 76 genes, we previously detected 199 somatic mutations (single nucleotide variants) in solid biopsies, of which 127 (64%) were also now detected in liquid biopsies. In exploratory subgroup assessments, concordance was higher in patients with liver metastases (P = 1.5 × 10-5) and increasing with level of liver involvement (P = 1.2 × 10-4). The concordance was similar between GEP-NEC with different primary sites, except being lower in esophageal cases (P = .001). Concordance was not associated with tumor mutation burden. Tumor tissue mutations also detected in liquid biopsies was lower for MSI (40%) versus MSS tumors (70%; P = 7.8 × 10-4). We identified potentially targetable mutations in plasma of 26 (52%) of patients with GEP-NEC; nine patients (18%) had potentially targetable mutation detected only in liquid biopsies. Liquid biopsy analyses may be an applicable alternative to solid biopsies in GEP-NEC. Liquid biopsies may add additional mutations compared with tumor biopsies alone and could be useful for biomarker assessment in clinical trials for these patients.

Patterns of Somatic Variants in Colorectal Adenoma and Carcinoma Tissue and Matched Plasma Samples from the Hungarian Oncogenome Program.

Analysis of circulating cell-free DNA (cfDNA) of colorectal adenoma (AD) and cancer (CRC) patients provides a minimally invasive approach that is able to explore genetic alterations. It is unknown whether there are specific genetic variants that could explain the high prevalence of CRC in Hungary. Whole-exome sequencing (WES) was performed on colon tissues (27 AD, 51 CRC) and matched cfDNAs (17 AD, 33 CRC); furthermore, targeted panel sequencing was performed on a subset of cfDNA samples. The most frequently mutated genes were APC, KRAS, and FBN3 in AD, while APC, TP53, TTN, and KRAS were the most frequently mutated in CRC tissue. Variants in KRAS codons 12 (AD: 8/27, CRC: 11/51 (0.216)) and 13 (CRC: 3/51 (0.06)) were the most frequent in our sample set, with G12V (5/27) dominance in ADs and G12D (5/51 (0.098)) in CRCs. In terms of the cfDNA WES results, tumor somatic variants were found in 6/33 of CRC cases. Panel sequencing revealed somatic variants in 8 out of the 12 enrolled patients, identifying 12/20 tumor somatic variants falling on its targeted regions, while WES recovered only 20% in the respective regions in cfDNA of the same patients. In liquid biopsy analyses, WES is less efficient compared to the targeted panel sequencing with a higher coverage depth that can hold a relevant clinical potential to be applied in everyday practice in the future.