ctDNA Next-generation Sequencing Research Articles

KIT resistance mutations identified by circulating tumor DNA and treatment outcomes in advanced gastrointestinal stromal tumor.

11514 Background: Tyrosine kinase inhibitors (TKIs) are the cornerstone treatment for advanced GIST via pharmacologic targeting of driver oncogenes such as KIT. Detection of KIT alterations through tissue-based next-generation sequencing (NGS) is common, but circulating tumor DNA (ctDNA)-based NGS is a less invasive alternative to identify driver and resistance mutations in advanced GIST. Patients (pts) with KIT-mutant GIST benefit from first-line (1L) imatinib; however, KIT resistance mutations may confer imatinib-resistance and differential sensitivity to subsequent TKIs. We sought to analyze ctDNA from GIST pts to determine whether certain resistance mutations were associated with superior outcomes with particular TKIs in the second-line and beyond (2L+). Methods: Under an approved institutional review board protocol, a retrospective analysis was performed with available ctDNA NGS results (Guardant360; Redwood City, CA) from pts (N = 104) who progressed on 1L imatinib between 2017-21. Using R statistical programming, we identified pts with primary KIT alterations (N = 64) and known resistance mutations in KIT exons 13 (N = 25) and 17 (N = 35). We studied the median time to treatment failure (mTTF), defined as the time from treatment start to treatment end (months) due to progressive disease or toxicity, for each 2L+ drug. Using Kaplan-Meier methods, we calculated Cox proportional-hazard ratios (HR) with confidence intervals (CI) and p-values for statistical significance. Results: 49% were male (median age 66; range, 31-94). Driver oncogenes were detected in 80% (N = 83), including KIT, NF1, PDGFRA and BRAF. Of those with a KIT alteration, 12 (19%) had KIT exon 9 mutations and 52 (81%) had KIT exon 11 mutations. KIT resistance mutations were observed in KIT exons 13 (N = 25; V654), 14 (N = 2; T670), and 17 (N = 45; D816, D820, N822, Y823). Pts with KIT resistance mutations received 2L+ therapy with avapritinib, dose-escalated imatinib, nilotinib, pazopanib, ponatinib, regorafenib, ripretinib, or sunitinib. mTTF for KIT exon 13 V654 pts treated with 2L+ sunitinib, imatinib 800mg, or other was 10.8, 7.5, and 3.7 months, respectively. TTF for sunitinib vs other 2L+ drugs showed a HR of 0.51 (CI 0.33-0.8), p = 0.003. mTTF for KIT exon 17 (non-V654) pts treated with 2L+ regorafenib, imatinib 800mg, or other was 4.6, 1.2, and 6.3 months, respectively. Comparison of mTTF for regorafenib vs other 2L+ drugs was not statistically significant. Conclusions: ctDNA is a noninvasive tool for detecting driver and resistance mutations in pts with advanced GIST. GIST pts with KIT exon 13 V654 resistance mutations had superior outcomes in the 2L+ setting with sunitinib. Regorafenib was not superior to other 2L+ TKIs in pts with KIT exon 17 resistance mutations, possibly due to their own activity against exon 17 resistance alterations. ctDNA-guided therapy warrants evaluation in a prospective clinical trial.

Decoding circulating tumor DNA to identify durable benefit from immunotherapy in lung cancer

ObjectivesPredicting the outcome of immunotherapy-treated non-small cell lung cancer (NSCLC) patients is challenging. Measuring circulating tumor DNA (ctDNA) in plasma is promising, but its application for outcome delineation needs further refinement. Since most information from the next-generation sequencing (NGS) panel is typically left unused, we aim to integrate more information. Materials and MethodsPatient and ctDNA data were compiled from five published studies involving advanced NSCLC. Plasma samples collected prior (t0) and early during (t1) immunotherapy were selected, tracking the changes of the highest t0 variant per gene. Durable benefit (DB, defined as progression free survival ≥ ½ year) was predicted. Performance was quantified using the integrated receiver operating characteristic curve (ROC AUC) and compared with the traditional molecular response (MR). ResultsA total of 365 patients were pooled. Seven recurrently mutated genes were selected which optimally predicted DB (ROC AUC: 0.77-0.11+0.10), outperforming the MR predictor (with a ROC AUC: 0.64-0.11+0.11). Inclusion of patient characteristics led to a slight further improvement (ROC AUC: 0.80-0.10+0.09). The model performed satisfactory across all ctDNA platforms despite differences in panel size and content. ConclusionRelative to a non-informative classifier (ROC AUC: 0.5), a twofold improvement in predictive value was achieved compared to MR by an integration of changes across seven selected genes in immunotherapy-treated NSCLC patients, whilst being broadly applicable across ctDNA NGS panels.

Blood-Based Next-Generation Sequencing in Adrenocortical Carcinoma.

BackgroundAdrenocortical carcinoma (ACC) is a rare and heterogeneous malignancy with poor prognosis. We aimed to evaluate the feasibility of next-generation sequencing (NGS) testing of circulating cell-free tumor DNA (ctDNA) in patients with ACC, to characterize the genomic landscape of alterations, and to identify potential clinically actionable mutations.MethodsRetrospective analysis of genomic data from 120 patients with ACC who had ctDNA testing between 12/2016 and 10/2021 using Guardant360 (Guardant Health, CA) was performed. ctDNA NGS analysis interrogated single nucleotide variants, fusions, indels, and copy number amplifications of up to 83 genes. The frequency of genomic alterations, landscape of co-occurring mutations, and pathogenic/likely pathogenic alterations with potential targeted therapies was identified. The prevalence of alterations identified in ctDNA was compared to those detected in tissue using a publicly available database (cBioPortal).ResultsThe median age of this cohort was 53 years (range 21-81), and 56% of patients were female. Ninety-six patients (80%) had ≥1 somatic alteration detected. TP53 (52%), EGFR (23%), CTNNB1 (18%), MET (18%), and ATM (14%) were found to be the most frequently altered genes in ACC samples. Pathogenic and/or likely pathogenic mutations in therapeutically relevant genes were observed in 56 patients (47%) and included EGFR, BRAF, MET, CDKN2A, CDK4/6, and ATM. The most frequent co-occurring mutations were EGFR + MET (9%), MET + CDK4 (7%), EGFR + CDK4 (7%), and BRAF + MET (7%). The frequencies of mutations detected in ctDNA were similar to those detected in tissue.ConclusionsUtilizing blood-based NGS to characterize genomic alterations in advanced ACC is feasible in over 80% of patients. Almost half of the patients had actionable mutations with approved therapies in other cancers. This approach might inform the development of personalized treatment options or identify clinical trials available for this aggressive malignancy.

Blood-based tumor mutational burden from circulating tumor DNA and immune checkpoint inhibitors in advanced prostate cancer.

165 Background: A prior study of patients with advanced cancers treated with immune checkpoint inhibitors (ICI) associated improved overall survival with tissue tumor mutational burden above the 80th percentile in each histology (tTMB-H). TMB can also reliably be calculated via liquid biopsy (bTMB), and trends higher than tTMB in analogous tumors. Here, we generate a plasma-informed benchmark for bTMB-H in advanced prostate cancer and report preliminary data from an ongoing multi-institutional case series of patients treated with ICI. Methods: Circulating tumor DNA (ctDNA) next generation sequencing (NGS) results from 3,504 patients with advanced prostate cancer who received a Guardant360 liquid biopsy within a 12-month period were analyzed. The 80th percentile of TMB was defined as the benchmark for bTMB-H in this cohort, and prevalence of microsatellite instability (MSI-H) was determined. Subsequently, clinical data were collected from patients across five institutions who received pembrolizumab based on bTMB scores or other tumor characteristics (i.e. CDK12 mutation). Results: Overall, mean and median bTMB were calculated as 12.34 mut/Mb and 8.61 mut/Mb, respectively (range: 0 – 1164.75 mut/Mb). The 80th percentile of bTMB was 13.4 mut/Mb and the 90th percentile was 16.6 mut/Mb. 2.6% of patients were both MSI-H and bTMB-H. No patients with bTMB < 13.4 mut/Mb (TMB-L) were found to be MSI-H. Ten patients from five institutions received pembrolizumab (8 monotherapy) after a median of 4.5 lines of prior therapy (Table). Four patients were MSI-H. The 3 patients in the cohort with PSA decreases > 50% had both MSI-H and bTMB ≥ 13.4 mut/Mb. Per RECIST criteria, disease was controlled (CR, PR, SD) in 6 of 7 (86%) patients with bTMB scores ≥ 13.4 mut/Mb. Conclusions: These data continue to affirm that ctDNA NGS is a practical and cost-effective means of assessing potential predictors of ICI activity in patients with advanced prostate cancer. Preliminary data from this growing cohort of patients who have received ICI guided by bTMB scores and other molecular parameters identified via ctDNA NGS, including MSI-H, demonstrate a clinical benefit from therapy. Further studies are warranted for contextualizing individual tumor bTMB scores with established, pathology-specific benchmarks.[Table: see text]

Abstract P2-01-15: Developing highly sensitive high NGS data efficient ctDNA detection assays for breast cancer surveillance

Abstract Introduction: Growing data established the importance of monitoring dynamic changes in circulating tumor DNA (ctDNA) to identify early signs of therapeutic responses, allowing for timely management of treatment to achieve more effective personalized therapy. Higher assay accuracy and consistency, and lower assay cost will support more clinical validation trials and benefit more cancer patients with non-invasive ctDNA NGS tests that can simultaneously map multiple genomic alterations at an affordable price. Method: The NVIGEN X - Precision Cancer Profiling test is a next generation sequencing (NGS) based circulating tumor DNA detection assay using the hybridization capture approach with customized gene panels. Our ctDNA NGS assay was developed with the use of high performance magnetic nanobeads, which enhances assay workflow at key steps including cfDNA extraction, NGS library preparation, and target enrichment. Experiments with individual plasma samples and DNA mutant fragments spiked in plasma samples were carried out to establish the assay performance such as sensitivity, specificity, consistency and data efficiency. NGS data QC metrics of the NVIGEN assay were compared with other assays in peer reviewed publications. Results: We developed a focus 32 gene panel that covers 144 kb of gene regions of clinical significance for breast cancer treatment monitoring and guidance, such as AKT1, ERBB2, PIK3CA, EGFR, ESR1, BRCA1/2, and CD274. Our results demonstrated the capability of NVIGEN X ctDNA NGS assay to detect rare copies (8 cp) of gene mutation at 0.07% MAF from DNA mutant fragments spiked into plasma samples. The NVIGEN X ctDNA NGS assays consistently presented 2-5% duplication rate, >80% on-target rate, <10% CV for key NGS data metrics, and on average required 1.36X paired reads per 1X unique coverage. Compared with the Roche Avenio assays (targeted, expanded and surveillance panels) as published in 2020 which on average required 9.36X paired reads per 1X unique coverage, the NVIGEN X -precision cancer profiling assays demonstrated 85% reduction in NGS data need to generate each unique coverage. Compared with the original Capp-seq data as published in the 2014 Nature Medicine paper which required in average 13.78 or 27.56 paired reads per unique coverage, the NVIGEN X assay demonstrated >90% reduction in NGS data need per unique coverage. Conclusion: The NVIGEN X - Precision Cancer Profiling assay provided high NGS assay performance with high sensitivity, specificity, and consistency, and significantly improved NGS data efficiency. This allows for dramatically reduced assay cost and will help support routine applications of ctDNA NGS tests to improve cancer patient treatment. Experiments of applying NVIGEN X assays for clinical research with patient samples are ongoing and will be presented. Citation Format: Aihua Fu, Wenwu Cui, Minh V. Ton, Kevan Wang, Weiwei Gu, Tianhong Li, Heather A. Parsons, Minetta C. Liu, George W. Sledge. Developing highly sensitive high NGS data efficient ctDNA detection assays for breast cancer surveillance [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-15.

Abstract PD6-08: Exploring the interplay among ESR1/PIK3CA codon variants, oncogenic pathway alterations and clinical phenotype of metastatic breast cancer (MBC) through circulating tumor DNA (ctDNA) next-generation sequencing (NGS)

Abstract Background: High throughput genomic technologies such as NGS are enhancing the ability to dynamically characterize MBC but their role in describing biological evolution of multiple mutations together remains unclear. ESR1 and PIK3CA are central mutations related to the biology and druggability of hormone-receptor positive, HER2 negative (luminal-like) MBC. The aim of this study was to explore the interplay between oncogenic pathway alterations and ESR1 and PIK3CA codon variants on the impact and clinical phenotype of luminal-like MBC. Methods: The study retrospectively analyzed a multi-institutional cohort comprising 1047 MBC patients (pts) characterized for ctDNA through NGS before treatment start at Northwestern University (Chicago, IL), Massachusetts General Hospital (Boston, MA) and Washington University in St. Louis between 2015-2020. The analysis was then focused on luminal-like MBC. Pathway classification was defined based on previous work (Sanchez-Vega F et al, Cell. 2018) (i.e., RTK, RAS, RAF, MEK, NRF2, ER, WNT, MYC, P53, cell cycle, notch, PI3K). Single nucleotide variations (SNVs) were annotated for their oncogenicity through OncoKB and ClinVar. Only pathogenic variants were included in the models. Associations among, pathway classification, and ESR1/PIK3CA codon variants were explored through stepwise logistic regression. Overall survival (OS) was tested through Cox regression. Results: The luminal-like cohort comprised 702 pts. ESR1 mutations were detected in 166 pts (24%) and PIK3CA in 214 pts (31%). The most common ESR1 gene mutations were found in codons 537 (31%), 538 (21%), 536 (8%) and 380 (7%), while alterations in codons 1047 (38%), 545 (25%), and 542 (20%) were the most common for PIK3CA. Other pathogenic SNVs were observed in 33% and 17% of pts for ESR1 and PIK3CA, respectively with the former being polyclonal. SNVs alterations were mainly observed in the PI3K (35%), P53 (32%), ER (28%), RAS (8%), RTK (8%) and cell cycle (5%) pathways, while copy number variations (CNVs) were detected in the RTK (15%), cell cycle (11%), MYC (7%) PI3K (6%) and RAF (5%) pathways. ESR1 537 variants were associated with alterations in the ER and WNT pathways, 538 with cell cycle, 380 with P53 and ER, 536 with RTK. PIK3CA 1047 variants were associated with alterations in the RTK and P53 pathways, 542 with RTK, RAS and RAF, E545 with PI3K, RAS, cell cycle and P53. 1047 and 542 were also associated with CNVs in the PI3K pathway. Independent prognostic factors in terms of OS were ESR1 537/380 codon variants (HR 1.94 P = 0.001 and HR 2.29 P = 0.047), SNVs in the RAS, cell cycle, and P53 pathways (HR 1.74 P = 0.003 HR 1.84 P = 0.009 and HR 1.56 P < 0.001) and CNVs in the cell cycle pathway (HR 1.96 P < 0.001). Conclusions: This study demonstrated how ESR1 and PIK3CA codon variants, together with alterations in specific oncogenic pathways, can differentially impact the biology and clinical phenotype of luminal-like MBC. As novel endocrine therapy agents such as selective estrogen receptor degraders (SERDs) and PI3K inhibitors are being developed, these results highlight the pivotal role of ctDNA NGS to describe tumor evolution and optimize clinical decision making. Odds Ratio95% Confidence IntervalPESR1 Y537ER SNVs3.341.487.530.004WNT SNVs6.251.4127.740.016ESR1 D538cell cycle SNVs5.221.7915.230.003ESR1 E380P53 SNVs4.801.4116.310.012ER SNVs5.331.3321.400.018ESR1 L536RTK CNVs4.511.1517.690.031PIK3CA H1047RTK SNVs3.751.708.290.001P53 SNVs2.611.584.34< 0.001PI3K CNVs6.082.4515.08< 0.001PIK3CA E542RTK SNVs5.001.9412.880.001RAS SNVs3.651.369.770.01RAF SNVs6.011.0733.870.042PI3K CNVs6.302.2917.36< 0.001PIK3CA E545PI3K SNVs2.881.276.530.011RAS SNVs2.871.186.980.02cell cycle SNVs3.071.088.740.035NRF2 SNVs21.431.29356.520.033P53 SNVs3.752.046.89< 0.001 Citation Format: Lorenzo Gerratana, Andrew A Davis, Marko Velimirovic, Katherine Clifton, Whitney L Hensing, Ami N Shah, Charles S Dai, Carolina Reduzzi, Paolo D’Amico, Qiang Zhang, Firas Wehbe, Seth Wander, William J Gradishar, Amir Behdad, Fabio Puglisi, Cynthia X Ma, Aditya Bardia, Massimo Cristofanilli. Exploring the interplay among ESR1/PIK3CA codon variants, oncogenic pathway alterations and clinical phenotype of metastatic breast cancer (MBC) through circulating tumor DNA (ctDNA) next-generation sequencing (NGS) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD6-08.

Abstract P2-01-13: Longitudinal circulating tumor DNA (ctDNA) monitoring by digital droplet PCR (ddPCR) in metastatic breast cancer

Abstract Background: A common challenge in treating patients with metastatic breast cancer (MBC) is ensuring adequate therapeutic efficacy, maintenance of quality of life, and avoidance of drug toxicity by preventing exposure to ineffective treatment. Critical to balancing these goals is the ability to quickly identify progression in order to minimize exposure to ineffective therapies. Imaging is the current standard-of-care for disease response evaluation, but high cost, relative low sensitivity, and tumor flare phenomenon remain significant issues. Circulating tumor DNA (ctDNA) testing based on next generation sequencing (NGS) is emerging as an alternative to imaging-based response assessment, but remains an expensive proposition. Here we evaluate a hybrid monitoring strategy using an initial baseline NGS-based ctDNA analysis to identify mutant alleles, followed by response monitoring with a low-cost, personalized digital droplet PCR (ddPCR) assay. Methods: Blood for ddPCR ctDNA analysis was collected at the same timepoint as a sample for commercial ctDNA analysis by NGS. PCR primer and probe sets were designed against clinically relevant mutations identified from each patient’s commercial ctDNA NGS report. ddPCR was then performed. Variant allele fractions (VAFs) detected by ddPCR were compared to allelic fractions reported by commercial NGS using Bland-Altman analysis and significance was determined by paired t-test. To demonstrate the potential for longitudinal ddPCR-based ctDNA monitoring, serial samples from the same patient were analyzed. Results: In our pilot validation set, 10 paired patient samples were analyzed by NGS and ddPCR. NGS-reported VAFs ranged from 0.30% to 16.20%. High comparative performance was observed between NGS and ddPCR platforms over expected mutant fractional abundance ranges. There was no significant quantitative difference between the two approaches (p=0.45). Longitudinal monitoring of ctDNA by ddPCR was performed on a metastatic hormone-positive breast cancer patient and revealed a significant rise in the clinically-relevant PIK3CA H1047L mutation that preceded imaging-based documentation of progression by 3 months. A final timepoint was taken several months before the patient’s death with a further significant increase in ctDNA burden. Conclusions: Here we demonstrate that highly-sensitive ddPCR-based ctDNA assays provide comparable results to next generation sequencing, but at considerably lower cost. These results indicate that bespoke tracking of select alleles by ddPCR offers an inexpensive and rapid approach to serial ctDNA monitoring in metastatic breast cancer patients. A larger prospective study has been designed and is underway. Citation Format: Nicole Higashiyama, LaTerrica Williams, Bryant McCue, Alphi Kuriakose, Tiffaney Tran, Stephanie Gonzalez, Mayra Licerio, Carol Chenault, Heidi Dowst, Susan Hilsenbeck, Matthew Ellis, Bora Lim, George Miles. Longitudinal circulating tumor DNA (ctDNA) monitoring by digital droplet PCR (ddPCR) in metastatic breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-01-13.

Abstract PD2-05: Genomic profiling of PAM50-based intrinsic subtypes in HR+/HER2- advanced breast cancer (ABC) across the MONALEESA (ML) studies

Abstract Background: In the ML program, PAM50-based intrinsic subtypes (ie, luminal A [LumA], luminal B [LumB], HER2 enriched [HER2E], and basal-like [Basal]) were found to be prognostic and predictive of ribociclib benefit in ABC (Prat et al. J Clin Oncol. 2021). While ribocliclib demonstrated benefit in all subtypes (except Basal, with a limited sample size), LumB and HER2E derived the largest benefit. However, DNA features of the intrinsic subtypes in the advanced setting remain unknown. Here, we report the results of genomic profiling of baseline circulating tumor DNA (ctDNA) by PAM50-based intrinsic subtypes across ML studies. Methods: A total of 883 of 2066 patients recruited in the ML-2, -3, and -7 phase 3 trials had both tumor intrinsic subtype and plasma ctDNA next-generation sequencing (NGS)-based data obtained at baseline (ie, before starting treatment). The NGS-based panel targeted exonic regions in approximately 550 genes sequenced on an Illumina HiSeq instrument. A total of 130 patients had the normal-like subtype and were excluded from further analyses. For genes altered in > 5% of patients, we assessed the differences in frequency across intrinsic subtypes. Genetic alterations included mutations, indels, and copy number alterations. For each gene, a Fisher exact test was used to test for differences in frequency across the subtypes. A false discovery rate (FDR) correction was used to adjust for multiple testing. For genes with FDR < 0.10, a logistic regression model was used to quantify the relationship between subtypes and alteration status. Also, we evaluated differences across subtypes for tumor mutational burden (TMB) using analysis of variance and for ctDNA fraction using a Kruskal-Wallis test. Results: Overall, gene amplifications were more frequent in the LumB, HER2E, and Basal subtypes. CCND1 (and genes FGF3/4/19 found in the same amplicon) was more frequently altered in HER2E (14.6%) and LumB (14.3%) than in the LumA (4.8%) subtype. Similarly, FGFR1 and MYC were more frequently altered in HER2E (13% and 9.8%), Basal (12.5% and 12.5%), and LumB (8.6% and 10%) than in the LumA (3.3% and 2.3%) subtype. PIK3CA alterations, including hotspot somatic mutations, were less frequent in Basal (12.5%) than in the LumB (27.6%), LumA (33.8%), and HER2E (37.4%) subtypes. In contrast, TP53 alterations were more frequent in Basal (66.7%) and HER2E (29.3%) than in the LumB (16.2%) and LumA (12.4%) subtypes. ERBB2 alterations (n = 25) were found in the LumA, LumB, and HER2E subtypes at similar frequencies (3%-4%). ESR1 did not show any significant difference across subtypes. TMB did not differ by subtype (P = .20), even when a TMB cutoff ≥ 10 was used (P = .23). Finally, ctDNA fraction differed across subtypes (P < .001), being significantly higher in the LumB (P < .001) and HER2E (P < .001) than in the LumA subtype. Conclusions: This is the first combined report of ctDNA NGS profiling and intrinsic molecular subtype in ABC. Differences in tumor DNA profiles were observed across PAM50 subtypes, with a trend for higher copy number alterations in HER2E and LumB than in the LumA subtype. LumA and Basal subtypes were found to have the most distinct genomic features. The Basal subtype is known to be similar to triple-negative BC from a clinical and biological perspective, which may explain the limited activity of ribociclib in this subgroup, as shown previously (Prat et al. J Clin Oncol. 2021). The pronounced activity of ribociclib in HER2E and LumB subtypes, which are enriched with somatic alterations associated with endocrine therapy resistance and tend to have a worse prognosis, warrants further investigation. Citation Format: Aleix Prat, Nadia Solovieff, Faye Su, Aditya Bardia, Patrick Neven, Gabriel N. Hortobagyi, Debu Tripathy, Stephen Chia, Dennis Slamon, Yen-Shen Lu, Tetiana Taran, Agnes Lteif, Carlos L. Arteaga, Fabrice André. Genomic profiling of PAM50-based intrinsic subtypes in HR+/HER2- advanced breast cancer (ABC) across the MONALEESA (ML) studies [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD2-05.

Circulating Cell-Free Tumor DNA in Advanced Pancreatic Adenocarcinoma Identifies Patients With Worse Overall Survival.

BackgroundPlasma-based circulating cell-free tumor DNA (ctDNA) genomic profiling by next-generation sequencing (NGS)is an emerging diagnostic tool for pancreatic cancer (PC). The impact of detected genomic alterations and variant allele fraction (VAF) in tumor response to systemic treatments and outcomes is under investigation.MethodsPatients with advanced PC who had ctDNA profiled at time of initial diagnosis were retrospectively evaluated. We considered the somatic alteration with the highest VAF as the dominant clone allele frequency (DCAF). ctDNA NGS results were related to clinical demographics, progression-free survival (PFS) and overall survival (OS).ResultsA total of 104 patients were evaluated. Somatic alterations were detected in 84.6% of the patients. Patients with ≥ 2 detectable genomic alterations had worse median PFS (p < 0.001) and worse median OS (p = 0.001). KRAS was associated with disease progression to systemic treatments (80.4% vs 19.6%, p = 0.006), worse median PFS (p < 0.001) and worse median OS (p = 0.002). TP53 was associated with worse median PFS (p = 0.02) and worse median OS (p = 0.001). The median DCAF was 0.45% (range 0-55%). DCAF >0.45% was associated with worse median PFS (p<0.0001) and median OS (p=0.0003). Patients that achieved clearance of KRAS had better PFS (p=0.047), while patients that achieved clearance of TP53 had better PFS (p=0.0056) and OS (p=0.037).ConclusionsInitial detection of ctDNA in advanced PC can identify somatic alterations that may help predict clinical outcomes. The dynamics of ctDNA are prognostic of outcomes and should be evaluated in prospective studies.

EGFR-dependent mechanisms of resistance to osimertinib determined by ctDNA NGS analysis identify patients with better outcome.

BackgroundOsimertinib is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) that is highly selective for EGFRT790M subclones in patients with EGFRsensitizing non-small cell lung cancer (NSCLC). Unfortunately, all patients develop resistance through EGFR-dependent or EGFR-independent pathways. Recently, circulating tumoral DNA (ctDNA) analysis has highlighted the usefulness of plasma genotyping for exploring patient survival outcomes after disease progression under osimertinib.MethodsPlasma samples from patients treated with osimertinib as a second-line therapy were collected and the presence of molecular alterations of acquired resistance was evaluated after relapse under osimertinib using ctDNA molecular profiling by next-generation sequencing (NGS) assays. The clinical implications of these genomic alterations for the efficiency of the third-generation TKI were further assessed.ResultsOur ctDNA molecular profiling of plasma samples highlighted large number of actionable genomic alterations. According to ctDNA NGS results, patients were classified as having developed an EGFR-dependent or EGFR-independent mechanism of resistance. Thus, patients who developed an EGFR-dependent mechanism of resistance responded longer to osimertinib (13.8 vs. 4.6 months; P<10−4) and have a better post-osimertinib clinical outcome than EGFR-independent resistant patients. Moreover, the development of an EGFR-dependent mechanism of osimertinib resistance was identified as the best fit to determine patients’ clinical outcome compared with EGFRT790M status alone (P=0.003).ConclusionsOur study highlights the potential of ctDNA NGS to rapidly select the appropriate drug after osimertinib failure and to determine clinical outcomes of patients. We suggest that ctDNA NGS should be more intensively used in clinical practice to follow patients under third-generation TKIs.

BIOM-23. A PILOT STUDY OF WHOLE GENOME SEQUENCING (WGS) OF PLASMA CELL-FREE DNA (cfDNA) FOR ULTRASENSITIVE DETECTION OF TUMOR DNA IN PATIENTS WITH GLIOBLASTOMA (GBM)

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

1267P Clinical utility of ctDNA for detection of EGFR, ALK, BRAFV600E alterations and resistance mutations in patients with NSCLC at failure to targeted therapy

The comprehensive genomic profile by next generation sequencing (NGS) of circulating tumour DNA (ctDNA) can identify a wide spectrum of genomic alterations (GAs) in non-small cell lung cancer (NSCLC). We aimed to assess the clinical utility of amplicon-based ctDNA NGS for detecting GAs and resistance mutations at time of progression (PD).

Clinical Outcomes in Non-Small-Cell Lung Cancer Patients Treated With EGFR-Tyrosine Kinase Inhibitors and Other Targeted Therapies Based on Tumor Versus Plasma Genomic Profiling.

PURPOSETo compare clinical outcomes in a cohort of patients with advanced non–small-cell lung cancer (NSCLC) with targetable genomic alterations detected using plasma-based circulating tumor DNA (ctDNA) or tumor-based next-generation sequencing (NGS) assays treated with US Food and Drug Administration–approved therapies at a large academic research cancer center.METHODSA retrospective review from our MD Anderson GEMINI database identified 2,224 blood samples sent for ctDNA NGS testing from 1971 consecutive patients with a diagnosis of advanced NSCLC. Clinical, treatment, and outcome information were collected, reviewed, and analyzed.RESULTSOverall, 27% of the ctDNA tests identified at least one targetable mutation and 73% of targetable mutations were EGFR-sensitizing mutations. Among patients treated with first-line epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) therapies, there were no significant differences in progression-free survival of 379 days and 352 days (P value = .41) with treatment based on tissue (n = 40) or ctDNA (n = 40), respectively. Additionally, there were no differences in progression-free survival or objective response rate among those with low (n = 8, 0.01%-0.99%) versus high (n = 16, ≥ 1%) levels of ctDNA of the targetable mutation as measured by variant allele frequency (VAF). Overall, there was excellent testing concordance (n = 217 tests) of > 97%, sensitivity of 91.7%, and specificity of 99.7% between blood-based ctDNA NGS and tissue-based NGS assays.CONCLUSIONThere were no significant differences in clinical outcomes among patients treated with approved EGFR-TKIs whose mutations were identified using either tumor- or plasma-based comprehensive profiling and those with very low VAF as compared with high VAF, supporting the use of plasma-based profiling to guide initial TKI use in patients with metastatic EGFR-mutant NSCLC.

A case report of response to crizotinib in chemotherapy-refractory metastatic gallbladder cancer with met amplification and acquired resistance resulting from the loss of MET amplification.

Gallbladder cancer (GBC) is a highly invasive disease and the most prevalent malignancy of the biliary system. Patients with GBC are commonly diagnosed at a late stage and have an unfavorable prognosis. Palliative chemotherapy has been the standard care for recurrent or metastatic disease in the past decades. Recently, several targeted therapies have been investigated in advanced biliary tract cancer (BTC) including inhibitors of genes or pathways such as FGFR2 fusions or rearrangements, IDH1 mutations, and NTRK gene fusions. Also, several clinical studies involving molecular stratification have been performed in defined patient groups, for example, BRAF V600E and HER2. Mesenchymal epithelial transition(MET)encodes a tyrosine kinase receptor and its ligand hepatocyte growth factor is a proto-oncogene. Targeting the MET signaling pathway is an effective strategy in numerous cancer types. However, the poor efficacy of MET inhibitors has been demonstrated in several phase II studies, but currently no reports have explained the potential mechanisms of resistance to MET inhibitors in BTC. In this article, we report a case of metastatic GBC with MET amplification that exhibited a rapid response to crizotinib after the failure of two lines of chemotherapy. After the patient had progressed and discontinued crizotinib, cabozantinib was introduced. Analysis of circulating tumor DNA (ctDNA) by next-generation sequencing (NGS) indicated a loss of MET amplification status. To our knowledge, this is the first case study demonstrating the use of NGS in ctDNA to monitor the development of acquired resistance during anti-MET treatment in GBC.

Clinical utility of next-generation sequencing-based ctDNA testing for common and novel ALK fusions

ObjectivesLiquid biopsy for plasma circulating tumor DNA (ctDNA) next-generation sequencing (NGS) can detect ALK fusions, though data on clinical utility of this technology in the real world is limited. Materials and Methods: Patients with lung cancer without known oncogenic drivers or who had acquired resistance to therapy (n = 736) underwent prospective plasma ctDNA NGS. A subset of this cohort (n = 497) also had tissue NGS. We evaluated ALK fusion detection, turnaround time (TAT), plasma and tissue concordance, matching to therapy, and treatment response. Results: ctDNA identified an ALK fusion in 21 patients (3%) with a variety of breakpoints and fusion partners, including EML4, CLTC, and PON1, a novel ALK fusion partner. TAT for ctDNA NGS was shorter than tissue NGS (10 vs. 20 days; p < 0.001). Among ALK fusions identified by ctDNA, 93% (13/14, 95% CI 66%–99%) were concordant with tissue evaluation. Among ALK fusions detected by tissue NGS, 54% (13/24, 95% CI 33%–74%) were concordant with plasma ctDNA. ctDNA matched patients to ALK-directed therapy with subsequent clinical response, including four patients matched on the basis of ctDNA results alone due to inadequate or delayed tissue testing. Serial ctDNA analysis detected MET amplification (n = 2) and ALK G1202R mutation (n = 2) as mechanisms of acquired resistance to ALK-directed therapy. Conclusion: Our findings support a complementary role for ctDNA in detection of ALK fusions and other alterations at diagnosis and therapeutic resistance settings.

Reliable Detection of Somatic Mutations for Pancreatic Cancer in Endoscopic Ultrasonography-Guided Fine Needle Aspirates with Next-Generation Sequencing: Implications from a Prospective Cohort Study

Background or PurposePancreatic ductal adenocarcinoma (PDAC) is commonly diagnosed by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA). However, the diagnostic adequacy of EUS-FNA is often limited by low cellularity leading to inconclusive results. We aimed to investigate the feasibility and added utility of targeted next-generation sequencing (NGS) on PDAC EUS-FNAs. MethodsEUS-FNAs were prospectively performed on 59 patients with suspected PDAC (2014-2017) at a high-volume center. FNAs were analyzed for the presence of somatic mutations using NGS to supplement cytopathologic evaluations and were compared to surgical specimens and circulating tumor DNA (ctDNA). ResultsFifty-nine patients with suspected PDAC were evaluated, and 52 were diagnosed with PDAC on EUS-FNA. Four of the remaining seven patients had inconclusive EUS-FNAs and were ultimately diagnosed with PDAC after surgical resection. Of these 56 cases of PDAC, 48 (85.7%) and 18 (32.1%) harbored a KRAS and/or TP53 mutation on FNA NGS, respectively. Particularly, in the four inconclusive FNA PDAC diagnoses (false negatives), half harbored KRAS mutations on FNA. No KRAS/TP53 mutation was found in remaining three non-PDAC cases. All EUS-FNA detected KRAS mutations were detected in 16 patients that underwent primary tumor NGS (100% concordance), while 75% KRAS concordance was found between FNA and ctDNA NGS. ConclusionTargeted NGS can reliably detect KRAS mutations from EUS-FNA samples and exhibits high KRAS mutational concordance with primary tumor and ctDNA. This suggests targeted NGS of EUS-FNA samples may enable preoperative ctDNA prognostication using digital droplet PCR and supplement diagnoses in patients with inconclusive EUS-FNA.

Abstract 2064: Homologous recombination repair somatic/germline mutation detection in plasma by next-generation sequencing in Chinese solid tumor patients

Abstract Purpose: PARP inhibitors (PARPis) have been approved for germline and/or somatic BRCA-mutated ovarian cancer, breast cancer, pancreatic cancer and homologous recombination repair gene (HRR)-mutated prostate cancer. Emerging evidence has shown that antitumor activity of PARPis could expand to other HRR-mutated solid tumors. Therefore, efficient and timely methods to detect both somatic and germline HRR mutations using tissue or blood are urgently needed. Here, we first specially reported the prevalence of HRR mutation in plasma samples using Next-generation sequencing (NGS). Methods: A total of 1601 paired plasma-white blood cell samples and 3404 paired tumor-white blood from Chinese solid tumor patients were collected between October 2019 and October 2020. The samples were assessed with targeted NGS of circulating tumor DNA (ctDNA) or tumor tissue DNA (ttDNA) to detect deleterious somatic and germline mutations in 16 core HR genes in a CLIA-certified laboratory. Results: Overall, 165/1601 (10.3%) patients had at least one somatic or germline HRR gene mutations by ctDNA-based NGS analysis. The most commonly mutated tumor types were endometrium (42.9%, n=7), ovary (38.5%, n=13), prostate (27.8%, n=108), neuroendocrine (20%, n=5), breast (19%, n=42), melanoma (16.7%, n=6), stomach (15.8%, n=101), biliary tract (15.6%, n=64), cervix (12.5%, n=8), pancreas (11.4%, n=70), colorectum (9.8%, n=174) and bladder urinary tract (9.5%, n=21). Except for the four tumor types (endometrium, neuroendocrine, melanoma and cervix) with less than ten samples, the prevalence of HRR mutation was comparable with the ttDNA-based NGS data. Among HRR mutations, BRCA2 was the most commonly mutated gene (3.2%), followed by ATM (2.4%), BRCA1 (1.4%), CHEK2 (1.2%), BARD1 (0.7%), PALB2 (0.7%), CDK12 (0.7%) and FANCA (0.5%). Most of these data was similar to the results of ttDNA-based NGS. Moreover, slightly higher prevalence was found in BRCA2 (3.2% vs 2.5%) and CHEK2 (1.2% vs 0.7%), and slightly lower prevalence was found in ATM (2.4% vs 3.2%) and CDK12 (0.7% vs 1.4%). Conclusion: We first described the prevalence of somatic and/or germline HRR mutations detected by ctDNA-based NGS in a Chinese solid tumor cohort. Further prospective study is needed to compare the impact of the HRR status identified by ctDNA and ttDNA NGS on PARPis efficacy. Citation Format: Wei Zhu, Hui Chen, Bei Zhang, Xiaochen Zhao, Yuezong Bai. Homologous recombination repair somatic/germline mutation detection in plasma by next-generation sequencing in Chinese solid tumor patients [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 2064.

Identification and monitoring of mutations in circulating cell-free tumor DNA in hepatocellular carcinoma treated with lenvatinib

BackgroundThere has been a recent surge in interest in predicting biological effects associated with genomic alterations in order to implement personalized cancer treatment strategies. However, no reports have yet evaluated the utility of profiling blood-based circulating tumor DNA (ctDNA) in hepatocellular carcinoma (HCC) patients treated with lenvatinib (LEN).MethodWe retrospectively performed ctDNA next-generation sequencing (NGS) analysis in 24 patients with advanced HCC at baseline and 4 weeks after initiation of LEN. Association of the changes in variant allele frequencies (VAFs) during treatment and clinical outcome were evaluated.ResultsIn total, 131 single nucleotide variants, 17 indels, and 23 copy number variations were detected as somatic alterations in 28, 6, and 12 genes, respectively in 23 of 24 patients. The most frequently altered genes were TP53 (54%), CTNNB1 (42%), TERT (42%), ATM (25%), and ARID1A (13%). The reduction in the mean frequency of variants (VAFmean) following 4 weeks of LEN treatment was associated with longer progression-free survival. The specificity and sensitivity of the reduction of VAFmean for predicting partial response were 0.67 and 1.0, respectively, which were higher than those of serum α-fetoprotein level (0.10 and 0.93, respectively). No association between the mutation status at baseline and the effectiveness of LEN was observed.ConclusionOur study demonstrated that somatic alterations could be detected in the majority of advanced HCC patients by ctDNA profiling and that ctDNA-kinetics during LEN treatment was a useful marker of disease progression. These results suggest that ctDNA profiling is a promising method that provides valuable information in clinical practice.

Molecular characterization of ctDNA from Chinese patients with advanced gastric adenocarcinoma reveals actionable alterations for targeted and immune therapy.

Circulating tumor DNA (ctDNA) is considered an ideal sample type for genotyping patients with advanced unresectable cancer to inform treatment decision. It may better capture tumor heterogeneity, especially in gastric adenocarcinoma (GAC). However, there exists little evidence regarding genomic profiling of Chinese advanced GAC patients from ctDNA. Blood samples were obtained from 200 advanced GAC patients. Next-generation sequencing (NGS) was performed on ctDNA using a validated 150-gene panel. Blood tumor mutation burden (bTMB) was calculated according to the NGS results. Blood microsatellite instability (bMSI) status was determined by targeted sequencing of 100 microsatellite loci. One hundred sixty-nine (84.5%) patients carried at least one genomic alteration and 138 (69%) patients had at least one deleterious or likely deleterious alteration (del-alteration). The clonal fraction of del-alterations was higher than that of non-del-alterations (80.1% vs 54.5%, P < 0.0001). The most frequently altered genes were TP53 (38%), LRP1B (20%), MYC (13.5%), ERBB2 (12.5%), and KRAS (11.5%). The alterations were most enriched in the TP53/cell cycle (52%) and the RTK-Ras-MAPK pathway (51.5%). The median bTMB was two (range 0 to 42). Eight patients were identified to be high bMSI, with higher median bTMB than the blood microsatellite stable (bMSS) patients (15 vs 2, P = 0.0062). Patients harboring del-alterations of the DDR pathway had significantly higher percentages of high bTMB and bMSI-H patients than the wild-type subgroup (61.1% vs 6.5%, P < 0.0001; 33.3% vs 1.7%, P = 0.0002). A total of 45.5% cases harbored at least one potentially actionable alteration and one patient achieved complete response after receiving matched targeted therapy. Our study uncovered the molecular characterization of Chinese patients with advanced GAC from ctDNA, including genomic alteration, bTMB, and bMSI status. The findings suggested that targeted NGS-based ctDNA analysis may help inform the clinical decision in advanced GAC. KEY MESSAGES: We report the molecular profiling of the largest Chinese advance stage GACs cohort using a CLIA-certified ctDNA assay. Potentially actionable genomic alterations were identified in 45.5% of patients, suggesting clinical utility for ctDNA NGS in advance stage GACs. There was evidence of clinical benefit in one GAC patient with MET amplification treated with MET inhibitor.

Assessment of HER2 (ERBB2) amplification (HER2amp) using blood-based circulating tumor DNA (ctDNA) next generation sequencing (NGS) and correlation with tissue-based testing in metastatic colorectal cancer (mCRC).

3589 Background: HER2 amplified mCRC has emerged as a unique clinical subset, characterized by resistance to anti-EGFR therapy and response to anti-HER2 strategies. Accurate identification and quantification of HER2amp has predictive value for efficacy of anti-HER2 therapies and appropriate patient selection. Despite availability and use of various tumor tissue-based and blood-based assays for detecting HER2amp, data on cross-performance of these platforms are lacking. Methods: Leveraging a multicenter international consortium (Italy, Japan and USA), we generated a large cohort (N = 353) of mCRC patients (pts), tested for HER2amp using both tissue and blood. Tissue testing was done using immunohistochemistry (IHC), in-situ hybridization (ISH) and (NGS). ctDNA NGS was performed using CLIA-certified Guardant360 ctDNA assay, capable of detecting HER2 copy number (CN) variations. The primary endpoint was to correlate HER2 gene CNs in tissue (tCN) and plasma (pCN). Descriptive statistics, spearman correlation (r) and Fisher’s exact test were used. Results: Baseline tumors characteristics included right-sided primary in 234 (23%), proficient mismatch repair in 264 (98%) and RAS/BRAF wild type (WT) genotype in 194 (67%) pts. Tissue testing was done by IHC, ISH and NGS in 76%, 64% and 74% pts, respectively. A total of 177 pts had HER2amp detected by at least one test: 116 (66%), 157 (89%) and 96 (54%) of which had tissue +, ctDNA +, and both tissue and ctDNA + disease, respectively. Discordant cases consisted of 20 (6%) with positivity in tumor only and 61 (17%) in ctDNA only. Sensitivity, specificity, positive and negative predictive values of ctDNA assay (vis-à-vis tissue) were 83%, 74%, 61% and 90% respectively. Among HER2amp pts, median (range) HER2/CEP17 (ISH) ratio, tCN and pCN were 5.2 (2–12), 11.6 (2–700) and 3.5 (2–122), respectively. The pCN showed strong correlation with ISH ratio (r = 0.69) and tCN (r = 0.68) (P &lt; 0.001). Median pCN differed significantly between pts with HER2 IHC 3+ (12.0), 2+ (2.2) and 0/1+ (2.0) tumors (P &lt; 0.001). High HER2amp (pCN &gt; 4.0) appeared to be enriched with tissue + cases (69% vs 8% [OR 24.6, P &lt; 0.001]), tumor tissue HER2 + status (IHC3+ [75%] vs IHC2+ISH+ [50%] vs IHC2+/ISH- or IHC0/1+ [12%], P &lt; 0.001), HER2 tCN &gt; 6 (79% vs 31% [OR 8.7, P &lt; 0.001]) and RAS/BRAF WT tumors (41% vs 17% [OR 3.5, P = 0.064) but not left sidedness (41% vs 38%; OR 1.1; P = 0.82). Conclusions: In this large diverse cohort of mCRC, we demonstrated correlation of HER2 tCN and pCN obtained by tissue-based and blood-based ctDNA assay. Further prospective efforts are needed to standardize this cross-platform quantification of HER2amp to facilitate robust clinical application of HER2 therapies. This effort shows the value of strategic international partnership in furthering research for rare cancer subsets.