Serial ctDNA Research Articles

Abstract P3-01-07: Phase II trial of palbociclib with fulvestrant in individuals with hormone receptor-positive, HER2-negative metastatic breast cancer with disease progression after palbociclib with an aromatase inhibitor

Abstract Background: CDK4/6 inhibition (CDK4/6i) with endocrine therapy (ET) is a standard first line treatment for individuals with HR+, HER2- metastatic breast cancer (MBC). The anti-proliferative effect of CDK4/6i may reverse upon discontinuation, and the role of continuing CDK4/6i after progression on a CDK4/6i with aromatase inhibitor (AI) is not known. We examine the clinical activity of palbociclib and fulvestrant after disease progression on palbociclib with AI in a phase II trial in individuals with HR+, HER2- MBC (NCT02738866). Methods: We conducted a prospective, phase II, multicenter, open-label study of palbociclib with fulvestrant in patients with HR+ HER2- MBC with disease progression after at least 6 months of first line palbociclib and AI. Previous chemotherapy was allowed. Eligible participants received fulvestrant and continued on the same palbociclib dose. Premenopausal women received a concomitant GnRH agonist. We obtained a baseline tumor biopsy, when accessible, for correlative studies, and serial blood samples for ctDNA and circulating tumor cells (CTCs) analysis. Participants received disease assessment with imaging every 2 cycles (8 weeks) for the first year, followed by every 3 cycles (12 weeks) for participants who had been on study for >12 months. The primary endpoint was progression-free survival (PFS), estimated using the Kaplan Meier method. Other key endpoints included objective response rate (ORR) and clinical benefit rate (CBR) per RECIST v1.1. Results: As of June 1, 2022, a total of 58 of 60 participants were enrolled, with median age 57.9 (range 28.2-82.1 years). 17 patients (29%) were premenopausal. 41 (71%) of participants were white and 12 (21%) of participants were black/African American. Median PFS (mPFS) among the entire cohort was 3.7 months; among pre-menopausal patients, mPFS was 2.5 months and among post-menopausal patients, mPFS was 4.0 months). 22 (38%) of patients were on treatment for 6 or more months, with 11 (19%) of patients experiencing long term response beyond one year (Table 1). 13/58 (22%) of participants required further dose reductions of palbociclib after initiating fulvestrant despite previously being on a stable dose of palbociclib with an AI; no unexpected treatment-related toxicities were noted. Updated results for the entire 60 patient cohort will be presented at the meeting. Conclusions: Second line ET with palbociclib and fulvestrant following palbociclib and AI resulted in limited overall PFS. However, 38% of participants remained on therapy for greater than 6 months, suggesting clinical benefit in a subset of participants. Correlative studies, including of tissue, serial ctDNA and CTCs, are ongoing to further characterize this subset. Table 1: Preliminary efficacy of palbociclib and fulvestrant Citation Format: Jessica J. Tao, Amanda L. Blackford, Raquel Nunes, Cristina I. Truica, Justin Mahosky, Mary Kate Jones, Nick C. Leasure, Terrence Cescon, Antonios Christou, Jeanine L. Werner, Rima Couzi, Karen L. Smith, Cesar Augusto Santa-Maria, Antonio C. Wolff, Vered Stearns. Phase II trial of palbociclib with fulvestrant in individuals with hormone receptor-positive, HER2-negative metastatic breast cancer with disease progression after palbociclib with an aromatase inhibitor [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 P3-01-07.

Abstract PD17-03: Cell-free DNA monitoring in a phase II study of adjuvant endocrine therapy with CDK 4/6 inhibitor ribociclib for localized HR+/HER2- breast cancer (LEADER)

Abstract Background: While adjuvant endocrine therapy (ET) reduces recurrence risk in hormone receptor-positive (HR+) breast cancer, many patients still experience disease recurrence. Adjuvant therapeutic advances are needed to improve outcomes. Meanwhile, monitoring for circulating tumor DNA (ctDNA) in the adjuvant setting may detect molecular residual disease and/or emergences of molecular recurrence from tumor dormancy. Cyclin-dependent kinase 4 and 6 (CDK 4/6) inhibitors have shown efficacy in HR+/HER2- metastatic breast cancer, and abemaciclib is now approved for adjuvant use in high-risk HR+/HER2- breast cancer. Adjuvant clinical trials have evaluated upfront use of adjuvant CDK 4/6 inhibition; however, the optimal timing of adding a CDK 4/6 inhibitor for HR+/HER2- breast cancer remains unknown. We conducted a prospective phase II clinical trial to evaluate the addition of the CDK 4/6 inhibitor ribociclib in patients with at least one remaining year of adjuvant ET regardless of duration of ET prior to trial enrollment, and we prospectively collected plasma for ctDNA analysis. Methods: Eligible patients had Stage I-III HR+/HER2- breast cancer and had been on adjuvant ET (any number of years) with at least one year of treatment remaining. Patients were randomized to one of two ribociclib schedules: continuous (400 mg daily, 28-day cycle) or intermittent (600 mg daily days 1-21, 28-day cycle) for one year. Patients were concurrently treated with an aromatase inhibitor (plus GnRH agonist, if premenopausal). Time to recurrence was calculated using the Kaplan-Meier method. ctDNA monitoring was performed using the SignateraTM platform, a tumor-informed assay based on whole exome sequencing of the primary tumor for multiplex PCR-NGS ctDNA assay design with targeting of up to 16 single nucleotide variants. Plasma samples were collected at the start of ribociclib/ET and serially during follow-up visits. Results: Among 81 patients treated with adjuvant endocrine therapy and the CDK4/6 inhibitor ribociclib, 42 patients had samples suitable for ctDNA analysis: 3 (7%) had a single ctDNA test, 17 (40%) had 2 serial ctDNA tests, and 22 (52%) had 3 serial ctDNA tests. After a median follow-up of 20 months, 2 patients who received ribociclib (intermittent dosing) experienced disease recurrence with recurrence-free survival of 100% at 1 year from study entry and 97% (95% CI 88-99%) at 2 years. ctDNA was detected exclusively in the only 2 patients that experienced recurrence, with lead times of 7 months and 8 months prior to clinical recurrence. Both patients had no detectable ctDNA at the start of ribociclib/ET. One patient had detectable ctDNA [mean tumor molecules/mL (MTM/mL) = 0.1] while on ribociclib/ET for 5 months, after which she completed a full 12 months of treatment. One month after completing ribociclib/ET (8 months after ctDNA detection), she presented with metastases in the liver and bones. The second patient had 2 negative ctDNA tests at days 0 and 147 while receiving ribociclib/ET and became ctDNA positive (MTM/mL = 0.1) at day 350. She developed CNS-only metastatic disease 7 months after completing ribociclib/ET. Among the other 40 patients who did not have detectable ctDNA, none have experienced recurrence. Conclusions: Overall, only 2 patients had detectable ctDNA, and both patients developed recurrent metastatic disease after completion of ribociclib with ET. Notably, one of these patients developed CNS-only disease. While follow-up is early, the remaining patients did not have detectable ctDNA and have not developed recurrent disease. This study suggests monitoring for ctDNA may identify patients at increased risk for recurrence in the extended adjuvant period and potentially guide therapy escalation. Citation Format: Arielle J. Medford, Lauren Scarpetti, Andrzej Niemierko, Steven J. Isakoff, Beverly Moy, Seth A. Wander, Elizabeth Deluca, Elizabeth Abraham, Jennifer Shin, Lowell Schnipper, Amy E. Comander, Therese Mulvey, Erik Spickard, Ekaterina Kalashnikova, Angel Rodriguez, Leif Ellisen, Aditya Bardia, Laura M. Spring. Cell-free DNA monitoring in a phase II study of adjuvant endocrine therapy with CDK 4/6 inhibitor ribociclib for localized HR+/HER2- breast cancer (LEADER) [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 PD17-03.

Abstract P1-05-07: Detection of progression or regression of breast cancer by circulating tumor DNA (ctDNA)

Abstract Background: Circulating tumor DNA (ctDNA) are short DNA sequences shed by tumor cells into the systemic circulation. Studies have shown potential utility of the test to predict relapse or recurrence following treatment in solid tumors, but sensitivity and specificity have varied widely, ranging from 19-100% and 80-100% respectively, in breast cancer specifically. Moreover, literature describing the utility of monitoring dynamic changes in ctDNA trends is limited. We aim to evaluate the correlation between ctDNA test, both single test as well as dynamic trends in value over time, with imaging findings. Methods: We retrospectively analyzed the medical records of all adult patients diagnosed with breast cancer who underwent ctDNA testing at the hematology-oncology clinic at William Beaumont - Royal Oak and Troy Hospitals, Michigan, from August 2017 to June 2022. Patients who had ctDNA testing done but did not have imaging to correlate it with were excluded from the study. We calculated the sensitivity and specificity of a single positive ctDNA test to detect disease progression or residual disease on imaging. In patients with multiple ctDNA tests, we calculated the sensitivity and specificity of dynamic trends in ctDNA values to detect progression, regression, or absence of disease on imaging. Moreover, we calculated the lead time for positive ctDNA results to detect disease progression compared to imaging. Results: Nineteen patients were included in the study, with 34 total ctDNA test results, each utilized as a separate data point to compare with corresponding imaging findings (Table 1). Ten out of the 19 patients had multiple(>=2) ctDNA test results reported, with a total of 15 pairs of ctDNA values and each pair was analyzed separately as up trending (N=7), down trending (N=4), or persistent negative (N=4) to compare with a corresponding pair of imaging findings (Table 2). The median age at diagnosis was 55 years, and 94.7% were female. At diagnosis, majority of patients (68.4%) had either stage III or IV disease. Our primary endpoint, the correlation of single positive ctDNA result with imaging showing either progression or residual disease, showed a sensitivity and specificity of 100% and 93.3%, respectively. Secondarily, serial ctDNA trend analysis in ten patients revealed both sensitivity and specificity of 100% for up-trending ctDNA values to detect progression, down-trending to detect regression, and persistent negative results to detect absence of disease on imaging, respectively. The positive ctDNA results detected disease progression with a median lead time of 44.5 days compared to imaging. Conclusion: Given the high sensitivity and specificity to detect disease progression and regression in breast cancer patients by single ctDNA results and dynamic ctDNA trends in our study, we conclude that this may be a valid way to reliably monitor for changes in disease status before they become evident in imaging studies. Further clinical studies are required to prove the utility of ctDNA to detect changes in disease status and to guide therapeutic interventions in breast cancer. Table 1. Correlation of single ctDNA result with imaging findings. Table 2. Correlation of dynamic trends in ctDNA values with imaging findings. Citation Format: Ujjwal Karki, Bipin Ghimire, Emma Herrman, Siddhartha Yadav, Mohammad Muhsin Chisti. Detection of progression or regression of breast cancer by circulating tumor DNA (ctDNA) [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 P1-05-07.

Utility of circulating tumor DNA in monitoring treatment response to immune checkpoint inhibitors in patients with advanced genitourinary cancers.

721 Background: Current methodologies for monitoring treatment response are largely based on conventional scans and/or tumor biopsies, which may be limited in their ability to accurately assess disease burden at the molecular level. Circulating tumor DNA (ctDNA) detection in blood has emerged as a prognostic and predictive biomarker and has shown to better assess treatment response in patients receiving immune checkpoint inhibitors (ICI). We conducted a prospective, pilot study to investigate the concordance of serial ctDNA detection and dynamics with radiographic response in patients with advanced GU malignancies undergoing ICI-based treatment. Methods: Twenty patients with histologically confirmed advanced GU malignancies (renal, urothelial, and prostate) were enrolled in the prospective study. All eligible patients received ICI treatment for at least 12 weeks and were followed by serial collection of blood samples every 6-8 weeks until disease progression. Conventional scans were performed approximately every 12 weeks until disease progression. Overall response rate (ORR) by investigator was reported and associated with ctDNA detection. Results: ctDNA analysis was performed on 122 plasma samples obtained from 20 patients (N=15 renal cell carcinoma; N=4 urothelial carcinoma; N=1 prostate cancer). Prior therapies to ICI-based treatment included chemotherapy (10%), hormonal therapy (5%) and anti-VEGF (5%). After study enrollment, patients received anti-PD-1 (95%), anti-CTLA-4 (30%) or anti-PD-L1 (5%) with an ORR of 70% as best response. With a median follow-up of 19 months (range: 4-48), progressive disease was observed in 7 patients. Nineteen patients had longitudinal plasma samples available and ctDNA detection at any time point was 45% (9/20). The overall concordance between ctDNA dynamics and radiographic response at 12 weeks was observed in 89% (17/19) of patients. Of these 17 concordant patients, one patient showed transient ctDNA positivity followed by clearance at the last two timepoints on treatment. The two patients with discordant results included the ones with CNS-only metastasis (ctDNA negative). Of the 7 patients who progressed on ICI, ctDNA was detected in five (71%); the remaining two had CNS-only metastases. The last patient had a single time point available on treatment that showed ctDNA-positivity and passed away 7 weeks after molecular evidence of disease. Conclusions: In this study, serial collection of blood samples for ctDNA analysis to monitor response to ICI-based therapiesin patients with advanced GU tumors was feasible. There was a high concordance rate between radiological imaging and ctDNA data, especially in extra-CNS disease. Further studies are needed to validate ctDNA as a tool to aid disease monitoring in patients treated with ICI.

Performance characteristics of a tumor-informed circulating tumor DNA (ctDNA) minimal residual disease (MRD) assay in invasive bladder cancer in clinical practice.

542 Background: Detection of minimal residual disease (MRD) is emerging as a potential risk stratification and surveillance tool in patients with resected bladder cancer to potentially direct adjuvant therapy and early intervention. Recently, the sensitivity of MRD testing for early detection of recurrence has been questioned. We sought to evaluate the real world diagnostic performance characteristics of the Signatera ctDNA assay in patients with stage 1-4 bladder cancer who either had transurethral resection of bladder tumor (TURBT) in addition to either chemoradiation or radical cystectomy. Methods: Patients with bladder cancer who underwent ctDNA MRD testing after resection for stage 1-4 disease from 03/2021 to 09/2022 were evaluated retrospectively. Both patients with single and serial ctDNA assays were included. Individual chart review was performed to collect demographic and clinical variables such as diagnosis, stage, pathology, imaging results, and treatment course. Results: MRD results from 19 patients were available, 9 of which had serial assays. 13/19 patients were found to have ctDNA positive results at any time point. The positivity rate at initial testing was associated with stage: Stage 1: 3/5 patients, of which 2 had TURBT and 1 Radical cystectomy, stage 2: 2/5, stage 3: 6/8, and stage 4: 0/1. Notably, of the 9 patients with serial assays, one patient’s result converted from negative to highly positive during surveillance, with subsequent decrease to low positive after adjuvant therapy. One patient had a result of 2 consecutive low positives initially, which was converted to negative after systemic therapy and radiation. 7/19 patients completed testing 3 or more times over a span of approximately 4-13 months, 2 of whom had negative results throughout. Most patients who were found to have positive ctDNA did demonstrate radiographic recurrence within 2-3 months of ctDNA detection. Conclusions: Positive ctDNA MRD after resection is common in bladder cancer. Tumor-informed ctDNA MRD testing has very high sensitivity, but a one-time negative test result does not exclude the presence of metastatic disease. The above data shows an association between cancer stage and positivity results, particularly for advanced stages. Stage I cases with positive results can likely be explained by inadequate resection. Increases in ctDNA may represent early recurrence and can potentially be salvaged with early systemic therapy reverting to negative ctDNA. Prospective randomized and non-randomized studies are evaluating the clinical utility of ctDNA MRD testing in bladder cancer, and analysis of Signatera ctDNA assays will largely contribute to these efforts.

The role of serial circulating tumor DNA for predicting clinical outcomes of chemotherapy in patients with advanced gastric cancer.

433 Background: Serial circulating tumor DNA (ctDNA) analysis enables us to detect minimal residual disease, to estimate recurrence and to trace evolution of cancer. However, the role of ctDNA in patients with advanced gastric cancer (AGC) who received palliative chemotherapy is still unclear. We performed this study to evaluate if serial ctDNA can predict clinical outcomes of chemotherapy and propose evolutionary findings during chemotherapy. Methods: The patients with histopathologically confirmed AGC and who received palliative chemotherapy were enrolled. Serial blood sample for ctDNA sequencing were obtained at baseline (BL) (before first-line chemotherapy), after first cycle and every 2-3 months until disease progression. ctDNA sequencing was performed for all samples using the next-generation sequencing (NGS) platform with a targeted gene panel including 106 genes. Single nucleotide variants (SNVs), insertions and deletions (Indels), and copy number variants (CNVs) were identified by ctDNA sequencing while serial ctDNA were monitored by tracing changes in the variants. ctDNA clearance was defined as no detection of any kind of variants or variant allele frequency (VAF) less than 0.1%. Results: A total of 50 patients were registered and 248 samples were collected. Median age was 62. HER2-positive GC were 5 (10.0%) and 49 received fluoropyrimidine plus platinum (1 did not receive chemotherapy). Response rate and median progression-free survival (PFS) were 42.9% and 10.7 months (95% CI, 7.9-13.6). Among 40 who were available for BL ctDNA, patients who were detected with ctDNA variant (D group) were 21 (52.5%). TP53 mutation was the most frequently observed genetic alteration in BL ctDNA (76.2%) and median copy numbers of ERBB2 in HER2-positive GC were 28 (range 2-42). D group were more frequently observed in tubular or papillary adenocarcinoma than in poorly cohesive carcinoma (69.0% vs. 9.1%, p = 0.001). Patients who were not detected with any kind of ctDNA variant (ND group) showed a trend in longer PFS, compared to D group, however it was not significant (10.5 vs 7.9 months, p = 0.29). In serial analysis during first-line chemotherapy, ctDNA clearance was observed in 14 (70.0%) out of 20 in D group. Patients with ctDNA clearance showed significantly longer PFS, compared with patients without ctDNA clearance (9.8 vs. 3.6 months, p < 0.001). Among 32 patients who had disease progression, increased VAF or re-development of previous BL ctDNA variants were observed in 16 (50.0%). Increased VAF or re-development of BL ctDNA were detected in 2.1 months earlier than clinical or radiologic deterioration. Conclusions: Serial ctDNA could predict clinical outcomes of chemotherapy and detect disease progression earlier than clinical or radiological findings in AGC. Further study for serial ctDNA is needed to find out the resistant mechanism and evolutionary process in cancer.

Effect of dynamic circulating tumor DNA minimal residual disease detection on recurrence surveillance and second resection rate after curative intent resection of colorectal liver metastasis: A prospective cohort study.

242 Background: Recurrence rates after curative intent resection of colorectal cancer liver metastases (CRLM) are nearly 50%. Standard of care surveillance includes intensive computed tomographic (CT) imaging and CEA measurements. However, recurrence detection often happens too late to resume a second resection. Previous studies have shown that circulating tumor DNA (ctDNA) detection, as a biomarker of minimal residual disease (MRD), is a robust prognostic factor for patients with colorectal cancers. Here, we performed a prospective study to explore the potential use for improving postoperative CRLM surveillance by dynamic ctDNA detection and its impact on the second resection of recurrent patients. Methods: Enrolled patients received curative intent resection of CRLM and were confirmed at the status of no evidence of disease (NED) by CT about one month after surgery. All patients received stand-of-care postoperative surveillance and serial plasma samples were collected, including presurgical and postsurgical samples (4-6 weeks after surgery) and every 3rd month until relapse or up to 24 months after resection. A personalized tumor-informed assay (Signatera/HuaJianwei bespoke multiplex-PCR NGS) was used to quantify ctDNA in plasma samples. Results: A total of 318 plasma samples from 60 patients recruited from July 2020 to July 2021 were included in our analysis. Twenty-five (41.6%) patients received neoadjuvant chemotherapy, and 46 patients (76.7%) received adjuvant chemotherapy after surgery. The median follow-up time was 18.0 months (range, 7.5 to 25.3 months). ctDNA was detectable in 58/60 (96.7%) patients before surgery, in 35/60 (58.3%) patients after surgery, and in 23/58 (39.7%) patients at the end of treatment (EOT). Patients with ctDNA/MRD positive status post-surgery or EOT experienced a significant shorter recurrence-free survival (RFS) than patients with negative ctDNA/MRD [hazard ratio (HR) 4.2; P < 0.001 and HR 9.8; P < 0.001]. The recurrence rate in MRD-positive patients (aka. positive predictive value, PPV) was 91.4% (32/35) at post-surgery and 91.3% (21/23) at EOT, respectively. In 93.0% of postoperative MRD-positive patients, ctDNA detected early relapse with a lead time up to 9.0 months (median 3.4 months) compared with standard radiological imaging. Among 40 patients with recurrence, the total secondary resection rate was 52.5% (21/40) with 40% (21/40) cases reached secondary NED. While among 24 recurrent cases with only liver metastases, the secondary resection rate was 66.7% (16/24) with 58.3% (14/24) cases reached secondary NED. Conclusions: Postoperative ctDNA status is strongly associated with shorter RFS. Serial ctDNA monitoring assists early detection of relapse, and therefore, improves patient outcomes by secondary resection. Clinical trial information: ChiCTR2000035677 .

Bintrafusp alfa plus paclitaxel for advanced gastric cancer as a second-line treatment: An open label, single-arm, phase Ib/II study.

374 Background: Anti-PD-1 monotherapy did not improve overall survival compared with paclitaxel in second-line treatment for advanced gastric cancer (AGC) in KEYNOTE-061 trial. Recent studies suggest that inhibition of transforming growth factor beta (TGF-β) signaling reverses immunosuppressive tumor microenvironment into improved responses to cancer immunotherapy. Especially, genomically stable subtype of AGC showed the increased level of TGF-β pathway. Here, we report the results of phase Ib/II trial of bintrafusp alfa (M7824), a first-in-class bifunctional fusion protein composed of the extracellular domain of TGF-βRII fused to a human IgG1 mAb blocking PD-L1, in combination with paclitaxel as a 2nd line treatment for AGC. Methods: This investigator-initiated, single-arm, open-label, phase Ib/II study enrolled HER2-negative, histologically confirmed AGC patients with evaluable disease who failed prior palliative 1st line treatment. Prior immune checkpoint inhibitor was not allowed. Phase 1b was planned to enroll 3 to 12 patients in a 3+3 design (bintrafusp alfa 1200mg on days 1 and 15 plus paclitaxel 80 mg/m2 (level 1) or 70 mg/m2 (level -1) on days 1, 8 and 15) every 28 days). The primary endpoints were to determine recommended phase II dose (RP2D) for phase Ib, and progression-free survival (PFS) at 6 months for phase II. Secondary endpoints included PFS, OS, ORR, DCR, and safety. Exploratory endpoints included immune-related biomarker changes from serial liquid biopsies (NCT04835896). Results: In phase 1b, dose limiting toxicities were not observed, and the RP2D was determined as dose level 1. During phase II, enrollment was stopped after 30 patients were recruited, following negative readouts from bintrafusp alfa trials in other indications. With a median follow-up duration of 10.9 months (95% CI 9.5–12.6), 7 patients remained on treatment (treatment duration range: 1.0 to 15.1 months) as of September, 2022. Median PFS was 3.8 months (95% CI, 2.6-7.8) and PFS rate at 6 months was 36.7% (95% CI, 20.1-53.4). Median OS was 9.6 months (95% CI, 4.1-not reached). In patients with measurable lesions (n=25), ORR was 28% (PR, n=7) and DCR was 68%. No patient was EBV-related. Patients with MSI-H (n=3; mPFS, 7.8 months; mOS, not reached) or PD-L1-positive (CPS≥1) (n=9; mPFS, 7.8 months; mOS, not reached) showed durable efficacy. Treatment-related AEs (≥G3) occurred in 23 pts (76.7%), including anemia (G3, n=12, 40%) and neutropenia (G3, n=9, 30%). Immune-related AE included urticaria (G3, n=2, 6.7%; G2, n=4, 13.3%), rash (G3, n=1, 3.3%), and pneumonitis (G3, n=1, 3.3%). Conclusions: Combination of bintrafusp alfa and paclitaxel was feasible with modest efficacy as 2nd line treatment for AGC. There was subset of patients with durable responses. Analyses of targeted next-generation sequencing from serial ctDNA sampling are ongoing. Clinical trial information: NCT04835896 .

Effect of EGFR and ERBB2 amplifications and activating alterations on efficacy of lenvatinib in hepatocellular carcinoma.

600 Background: Hepatocellular carcinoma (HCC) is the most common primary liver cancer and is associated with a high mortality burden. Lenvatinib is an effective frontline tyrosine kinase inhibitor for HCC but is associated with primary and acquired resistance in most patients. Molecular mechanisms of resistance to lenvatinib remain unclear. Methods: We retrospectively analyzed 227 patients with HCC who underwent baseline ctDNA profiling as a component of routine clinical care, including 46 patients who had serial ctDNA analysis done at baseline and after progression to lenvatinib or ICI. Clinical data including demographics, treatment history, tumor responses, and survival outcomes were abstracted from the electronic medical record. Tumor response was categorized according to RECIST 1.1. A national registry of HCC that underwent ctDNA testing using Guardant360 was analyzed to determine the prevalence of EGFR/ERBB2 alterations in HCC. Results: Nearly a third of patients who progressed on lenvatinib demonstrated a gain in EGFR/ ERBB2 amplifications or activating mutations. No consistent trend in EGFR/ ERBB2 amplifications and alterations was found among ICI refractory HCC. Disease control rates (including complete response, partial response, and stable disease) were 20% in EGFR/ERBB2 altered HCC treated with lenvatinib, 62.5% in EGFR/ERBB2 wildtype HCC treated with lenvatinib (p = 0.05), and 58% in EGFR/ERBB2 altered HCC treated with ICIs (p = 0.09). PFS was also longer in EGFR/ERBB2 wildtype HCC treated with lenvatinib (5.9 months) and EGFR/ERBB2 altered HCC treated with ICIs (7.1 months) compared to EGFR/ERBB2 altered HCC treated with lenvatinib (2.2 months, p = 0.002). In a national cohort of 1739 patients with HCC, EGFR amplifications and activating mutations occurred in 6.5% and 1.5% of cases, respectively, and ERBB2 amplifications and activating mutations occurred in 1.9% and 0.7% of cases, respectively. Among EGFR/ERBB2 mutations, we identified recurrent gatekeeper mutations, including multiple A547T and T790M mutations, in 31% of patients. Conclusions: Our study substantiates EGFR/ERBB2 amplifications and mutations as putative drivers of lenvatinib resistance in patients with HCC and identifies the genetic mechanisms for EGFR pathway activation in refractory cancers. EGFR/ERBB2 alterations may thus represent predictive and pharmacodynamic markers of lenvatinib resistance. These findings lend support to the use of EGFR inhibitors combined with lenvatinib in previously untreated or lenvatinib refractory cancers.

APOLLO: A randomized phase II double-blind study of olaparib versus placebo following curative intent therapy in patients with resected pancreatic cancer and a pathogenic BRCA1, BRCA2 or PALB2 mutation—ECOG-ACRIN EA2192.

TPS763 Background: A meaningful subset of PDAC is characterized by a homologous recombination deficiency (HRD). The most well-defined patients within this group are those with pathogenic variants in BRCA1, BRCA2 and PALB2. In the metastatic setting, PARP inhibitor maintenance provides a progression-free survival benefit after a period of platinum based chemotherapy1,2, but the role of PARP inhibitors in the curative intent setting is undefined. The OlympiA study established one year of olaparib as the standard of care for patients with BRCA-related, early stage breast cancer who completed all other curative-intent treatment3. Therefore, we have designed a randomized, phase II double-blind study of one year of olaparib vs placebo in patients with pancreatic cancer and a germline or somatic variant in BRCA or PALB2 who have completed all curative intent therapy. Methods: We have enrolled and treated 23 of 152 planned patients on study NCT 04858334/EA2192. Eligibility criteria include: a pathogenic germline or somatic variant in BRCA1, BRCA2 or PALB2 as determined by local laboratory (central review required); completion of curative-intent resection and ≥ three months of multi-agent chemotherapy; no evidence of recurrent disease. At enrollment, patients must be within 12 weeks of their last anti-cancer intervention. Patients are randomized 2:1 to receive oral olaparib 300 mg twice daily or placebo for 12 28-day cycles. The primary endpoint is relapse-free survival. Overall survival is a secondary endpoint. Tumor tissue, fecal material (for microbiome analysis) and serial ctDNA samples are being collected. 1.Golan T, Locker GY, Kindler HL: N Engl J Med 381:1492-1493, 2019. 2. Reiss KA, Mick R, O'Hara MH, et al: J Clin Oncol 39:2497-2505, 2021. 3. Tutt ANJ, Garber JE, Geyer CE, Jr.: N Engl J Med 385:1440, 2021. Clinical trial information: NCT04858334 .

NRG GI008: Colon adjuvant chemotherapy based on evaluation of residual disease (CIRCULATE-US).

TPS260 Background: Currently, there are no biomarkers validated prospectively in randomized studies for resected colon cancer (CC) to determine need for adjuvant chemotherapy (AC). However, circulating tumor DNA (ctDNA) represents a highly specific and sensitive approach (especially with serial monitoring) for identifying minimal/molecular residual disease (MRD) post-surgery in CC patients (pts), and may outperform traditional clinical and pathological features in prognosticating risk for recurrence. CC pts who do not have detectable ctDNA (ctDNA-) are at a much lower risk of recurrence and may be spared the toxicities associated with AC. Furthermore, for CC pts with detectable ctDNA (ctDNA+) who are at a very high risk of recurrence, the optimal AC regimen has not been established. We hypothesize that for pts whose CC has been resected, ctDNA status may be used to risk-stratify for making decisions about AC. Methods: In this prospective phase II/III trial, up to 1,912 pts with resected stage III A, B (all pts) and stage II, IIIC (ctDNA+ only) CC will be enrolled. Based on the post-operative ctDNA status using personalized and tumor-informed assay (Signatera™, bespoke assay), those who are ctDNA- (Cohort A) will be randomized to immediate AC with fluoropyrimidine (FP) + oxaliplatin (Ox) for 3-6 mos per established guidelines vs . serial ctDNA monitoring. Patients who are ctDNA+ post-operatively or with serial monitoring (Cohort B) will be randomized to FP+Ox vs . more intensive AC with addition of irinotecan (I) for 6 mos. The primary endpoints for Cohort A are time to ctDNA+ status (phase II) and disease-free survival (DFS) (phase III) in the immediate vs . delayed AC arms. The primary endpoint for Cohort B is DFS in the FP+Ox vs FP+Ox+I arms for both phase II and phase III portions of the trial. Secondary endpoints include prevalence of detectable ctDNA post-operatively, time-to-event outcomes (overall survival and time to recurrence) by ctDNA status, and the assessment of compliance to adjuvant therapy. Biospecimens including archival tumor tissue, as well as post-operative plus serial matched/normal blood samples, will be collected for exploratory correlative research. Active enrollment across the NCTN started in June, 2022. Support: U10-CA-180868, -180822; UG1CA-189867; Natera, Inc. Clinical trial information: NCT05174169 .

Tumor-informed or tumor-agnostic circulating tumor DNA as a biomarker for risk of recurrence in resected colorectal cancer patients.

Circulating tumor DNA (ctDNA) has been increasingly recognized as a promising minimally-invasive biomarker that could identify patients with minimal residual disease and a high risk of recurrence after definitive treatment. In this study, we've compared the clinical utility and sensitivity of 2 different approaches to ctDNA analyses: tumor-informed and tumor-agnostic in the management of colorectal (CRC) patients. The clinical benefits of a single timepoint ctDNA analysis compared to serial ctDNA monitoring after definitive treatment were also evaluated to uncover the ideal surveillance protocol. Patient-paired resected tumor tissues, peripheral blood cells, and a total of 127 pre-operative and serial plasma cell-free DNA (cfDNA) samples after definitive treatment from 38 CRC patients that had undergone curative intent surgery were analyzed using a commercial NGS cfDNA panel. Up to 84% (32/38) of the recruited patients were detected with at least 1 genomic alteration from the tumor tissues that could be monitored using the tumor-informed ctDNA approach and none of the detected alterations were clonal hematopoiesis (CH) related. In contrast, 37% (14/38) of patients were detected with at least 1 monitoring alteration after exclusion of CH mutations using the tumor-agnostic approach. Serial plasma samples after definitive therapy were available for 31 patients. In the landmark ctDNA analysis, 24% (7/29) of patients had detectable ctDNA and were more likely to relapse than ctDNA-negative patients (p < 0.05). The landmark analysis sensitivity and specificity for recurrence were 67% and 87%, respectively. The incorporation of longitudinal ctDNA analysis at 6-months intervals improved the sensitivity to 100%. The median variant allele frequency (VAF) of the ctDNA mutations detected during surveillance was 0.028% (range: 0.018-0.783), where up to 80% (8/10) of the mutations were detected at VAF lower than the tumor-agnostic detection limit of 0.1%. Utilizing the tumor-agnostic approach reduced the recurrence detection sensitivity to 67% (4/6). Serial ctDNA analyses predicted disease recurrence at a median of 5 months ahead of radiological imaging. Longitudinal monitoring using tumor-informed ctDNA testing shows high analytical sensitivity, low probability of false-positive results due to CH mutations, and improved sensitivity in detecting recurrence which may modify the clinical management of CRC.

Truncal Dynamics May Trump: Serial ctDNA to Predict Early Therapeutic Response.

Promising utility of using serial ctDNA in metastatic colorectal cancer to both refine patient selection, reduce toxicity due to chemotherapy, and to evaluate emerging resistance mechanisms may lead the way to novel therapeutic strategies. However, important questions remain in validating its use as a predictive biomarker of treatment response. See related article by Vidal et al., p. 379.

S333 How Reliable Is Circulating Tumor DNA in Detecting Disease Progression or Regression of Non-Colorectal Gastrointestinal Cancers

Introduction: Circulating tumor DNA are short DNA sequences of tumor cells shed into the systemic circulation. Post-operative ctDNA positivity has been studied as a potential marker for disease recurrence, however, dynamic changes in its level and immediate correlation with imaging have not been well described. Methods: We conducted a retrospective study including adult patients with non-colorectal gastrointestinal (GI) cancer. We evaluated the correlation of ctDNA with imaging studies to detect disease progression or regression. Eighteen patients, with 33 ctDNA samples were included. Results: Out of the 18 patients, five had pancreatic, three each had hepatocellular and cholangiocarcinoma, two each had anal cancer and neuroendocrine tumor, and one each had gastric, small bowel, and GI malignancy of unknown primary. Among the patients, 50% were male, and the median age at diagnosis was 64 years. 72.2% of the patients had advanced disease (stage III/IV), and only 22.2% had a predisposing condition leading to malignancy. Our primary endpoint, the correlation of single positive ctDNA results with imaging showing either progression or residual disease, showed a sensitivity of 60% and specificity of 100%. Secondarily, serial ctDNA was analyzed in ten patients with at least two ctDNA test results. This revealed a sensitivity of 80% and specificity of 100% for up-trending ctDNA values to detect progression, down-trending to detect regression, and persistent negative results to detect the absence of disease. This calculated sensitivity was lower than our separate analysis of colorectal cancer, where the sensitivity of single and serial ctDNA was 84.8% and 92.9%, respectively. The specificity, however, was 100% in both cancer groups. The positive ctDNA results detected disease progression with a median lead-time of 44 days compared to imaging. Conclusion: Colorectal cancer is the most studied malignancy in regards to the use of circulating tumor DNA as a marker of tumor recurrence. Similar studies in non-colorectal GI cancers are lacking. However, limited studies have shown some promising results for the use of post-operative ctDNA. The test’s sensitivity in our study was inferior compared to colorectal cancer, but given high specificity and improvement in sensitivity with serial analysis, ctDNA can be a valid way to monitor disease progression or regression in non-colorectal GI cancers. Further clinical studies are required to prove its utility in the reliable detection of immediate changes in disease status.

Serial Profiling of Circulating Tumor DNA Identifies Dynamic Evolution of Clinically Actionable Genomic Alterations in High-Risk Neuroblastoma.

ctDNA is prevalent in children with neuroblastoma. Serial ctDNA profiling in patients with neuroblastoma improves the detection of potentially clinically actionable and functionally relevant variants in cancer driver genes and delineates dynamic tumor evolution and disease progression beyond that of standard tumor sequencing and clinical surveillance practices. See related commentary by Deubzer et al., p. 2727. This article is highlighted in the In This Issue feature, p. 2711.

Gemcitabine combined with apatinib and toripalimab in recurrent or metastatic nasopharyngeal carcinoma

The role of a triple combination of gemcitabine (chemotherapy) plus apatinib (anti-vascular endothelial growth factor [VEGFR]) and toripalimab (anti-PD-1) (GAT) in recurrent/metastatic nasopharyngeal carcinoma (RM-NPC) is unclear. Between August 2019 and April 2020, 41 patients with RM-NPC were enrolled and received GAT for up to 6 cycles followed by apatinib and toripalimab. The primary endpoint was the safety. The secondary endpoints included the objective response rate (ORR) and progression-free survival (PFS). Integrated genomic and transcriptional analyses were conducted to identify the patients who benefited in response to this novel combination therapy. As of April 1, 2022, treatment-related grade 3 or 4 adverse events (AEs) occurred in 23 of 41 patients (56.1%, 95% confidence interval [CI] 41%-70.1%). G3-4 nasopharyngeal necrosis was observed in 9 (9/41, 21.9%) patients. High-risk factors for necrosis included repeated radiotherapy and an interval of less than 12months from the last radiotherapy. The ORR was 90.2% (95% CI: 76.9%-97.2%). The median PFS was 25.8months (95% CI: not reached (NR)-NR), and the 24-month PFS rate was 50.7% (95% CI: 34.0%-67.4%). MAS-related GPR family member F (MRGPRF) high expression in tumors correlated with poor PFS from the GAT therapy, characterized by high epithelial mesenchymal transition signatures. Serial circulating tumor DNA (ctDNA) sequencing could predict PFS outcomes to combination therapy. GAT therapy exhibits a promising antitumor activity and manageable toxicities in patients with RM-NPC. Patients with repeated radiotherapy and an interval of less than 12months from the last radiotherapy should be carefully selected for antiangiogenic therapies. MRGPRF expression and serial ctDNA monitoring could identify patients that derive benefits from the combination therapy. ClinicalTrials.gov: NCT04073784. This research was funded by the National Natural Science Foundation of China (nos. 81772895 and 82002857), the Key-Area Research and Development of Guangdong Province (2020B1111190001), the Special Support Program for High-level Talents in Sun Yat-sen University Cancer Center, the Guangzhou Science and Technology Plan Project (202103010001), and the National "Ten Thousand TalentsProgram" Science and Technology Innovation Leading Talents (84000-41180005).

Serial Circulating Tumor DNA in Monitoring the Effect of Neoadjuvant and Adjuvant Immunotherapy in Patients With Colon Cancer: Case Series and Review of the Literature.

Although programmed death 1 blockade has significantly improved the survival of advanced colorectal cancer patients with DNA mismatch repair-deficient (dMMR)/microsatellite instability-high (MSI-H), clinical data in neoadjuvant and adjuvant setting are limited. The role of circulating tumor DNA (ctDNA) in precision oncology is promising, but its clinical significance in immunotherapy needs to be validated. We report a case series of 3 colon patients who received neoadjuvant and adjuvant immunotherapy and serial ctDNA analysis. This report summarizes clinical and molecular details for 3 patients with locally advanced or recurrent dMMR/MSI-H/polymerase epsilon ( POLE ) mutation-positive tumors treated with neoadjuvant/adjuvant immunotherapy. One stage IV recurrent colon cancer patient diagnosed with Lynch syndrome received adjuvant sintilimab monotherapy and had a progression-free survival (PFS) over 16 months, one stage Ⅲc colon cancer patient with MSI-H/high tumor mutation burden received neoadjuvant toripalimab monotherapy, was assessed as clinical complete response before surgery, continued with adjuvant sintilimab monotherapy and had a PFS over 17 months, one stage Ⅱ colon cancer patient with POLE P286R also received adjuvant sintilimab monotherapy and had a PFS over 17 months. All patients had detectable ctDNA after radical surgery and clearance of ctDNA during adjuvant immunotherapy. All 3 patients are free of tumor disease at the time of this report. Further studies are warranted to evaluate the long-term efficacy of neoadjuvant and adjuvant programmed death 1 blockade in locally advanced and metastasis in dMMR/MSI-H/ POLE mutated colorectal cancer and the role of ctDNA monitoring.

Investigation of PARP inhibitor resistance through the analysis of serially collected circulating tumor DNA (ctDNA) in ovarian cancer patients (027)

Objectives: An increasing number of ovarian cancer patients are becoming resistant to PARP inhibitor (PARPi). Aside from BRCA reversion, other patient-specific molecular alterations which underlie the development of acquired resistance are unknown. Moreover, the potential efficacy of serial circulating tumor DNA (ctDNA) in predicting response to PARP is yet to be explored. Thus, we have analyzed serially obtained ctDNA in samples from patients receiving PAPR inhibitors. Methods: Patients receiving PARPi were prospectively enrolled since January 2018. Whole blood samples were obtained prior to receiving PARPi and serially every three months until progression on PARPi. Every patient had tumor next-generation sequencing data. Cell-free DNA was extracted from plasma and germline DNA from buffy coats. Extracted DNA was target enriched with a panel targeting 531 cancer-related genes, and the resultant library was sequenced using the Illumina Novaseq 6000 sequencing system. The sequencing data was analyzed using our custom analysis pipeline. Results: A total of 72 patients have contributed at least one sample, and 29 patients have recurred so far. Preliminary analysis was performed for 17 patients with paired samples. When compared to tumor NGS data, analysis of ctDNA in patient-matched samples showed ctDNA specific variants. Analysis of patient-matched paired samples before and after progression on PARP inhibitor showed an increase in intratumoral heterogeneity in post-progression samples. Genes that were frequently modified after gaining PARPi resistance were BRCA1, TP53, BRCA2, and ATM. BRCA reversion was found in two patients. Moreover, new variants in genes that are linked to DNA repair pathways were found to be associated with increased tumor mutation burden. Conclusions: ctDNA, a less-invasive alternative to serial tumor biopsy, helps capture the changes in mutational status in patients receiving PARPi. In terms of prediction of PARPi response and investigation of resistance mechanism, serial ctDNA samples before and during PARPi may provide useful insights. Updated analysis with additional patients will be shared at the conference. Objectives: An increasing number of ovarian cancer patients are becoming resistant to PARP inhibitor (PARPi). Aside from BRCA reversion, other patient-specific molecular alterations which underlie the development of acquired resistance are unknown. Moreover, the potential efficacy of serial circulating tumor DNA (ctDNA) in predicting response to PARP is yet to be explored. Thus, we have analyzed serially obtained ctDNA in samples from patients receiving PAPR inhibitors. Methods: Patients receiving PARPi were prospectively enrolled since January 2018. Whole blood samples were obtained prior to receiving PARPi and serially every three months until progression on PARPi. Every patient had tumor next-generation sequencing data. Cell-free DNA was extracted from plasma and germline DNA from buffy coats. Extracted DNA was target enriched with a panel targeting 531 cancer-related genes, and the resultant library was sequenced using the Illumina Novaseq 6000 sequencing system. The sequencing data was analyzed using our custom analysis pipeline. Results: A total of 72 patients have contributed at least one sample, and 29 patients have recurred so far. Preliminary analysis was performed for 17 patients with paired samples. When compared to tumor NGS data, analysis of ctDNA in patient-matched samples showed ctDNA specific variants. Analysis of patient-matched paired samples before and after progression on PARP inhibitor showed an increase in intratumoral heterogeneity in post-progression samples. Genes that were frequently modified after gaining PARPi resistance were BRCA1, TP53, BRCA2, and ATM. BRCA reversion was found in two patients. Moreover, new variants in genes that are linked to DNA repair pathways were found to be associated with increased tumor mutation burden. Conclusions: ctDNA, a less-invasive alternative to serial tumor biopsy, helps capture the changes in mutational status in patients receiving PARPi. In terms of prediction of PARPi response and investigation of resistance mechanism, serial ctDNA samples before and during PARPi may provide useful insights. Updated analysis with additional patients will be shared at the conference.

Deep whole-genome ctDNA chronology of treatment-resistant prostate cancer.

Circulating tumour DNA (ctDNA) in blood plasma is an emerging tool for clinical cancer genotyping and longitudinal disease monitoring1. However, owing to past emphasis on targeted and low-resolution profiling approaches, our understanding of the distinct populations that comprise bulk ctDNA is incomplete2-12. Here we perform deep whole-genome sequencing of serial plasma and synchronous metastases in patients with aggressive prostate cancer. We comprehensively assess all classes of genomic alterations and show that ctDNA contains multiple dominant populations, the evolutionary histories of which frequently indicate whole-genome doubling and shifts in mutational processes. Although tissue and ctDNA showed concordant clonally expanded cancer driver alterations, most individual metastases contributed only a minor share of total ctDNA. By comparing serial ctDNA before and after clinical progression on potent inhibitors of the androgen receptor (AR) pathway, we reveal population restructuring converging solely on AR augmentation as the dominant genomic driver of acquired treatment resistance. Finally, we leverage nucleosome footprints in ctDNA to infer mRNA expression in synchronously biopsied metastases, including treatment-induced changes in AR transcription factor signalling activity. Our results provide insights into cancer biology and show that liquid biopsy can be used as a tool for comprehensive multi-omic discovery.

P749: PROGNOSTIC SIGNIFICANCE OF SERIAL CIRCULATING TUMOR DNA STATUS POST TREATMENT IN MYELODYSPLASTIC SYNDROMES AND ACUTE MYELOID LEUKEMIA

Background: A major advance in our understanding of Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML) biology has been the implementation of next generation sequencing (NGS) which influenced diagnostic, prognostic, and therapeutic decisions in myeloid malignancies. Circulating tumor DNA (ctDNA) sequencing, as as a novel and minimally invasive measure, was reported to exhibit excellent correlations with matched bone marrow NGS in MDS and AML. However, the clinical relevance of dynamic ctDNA monitor during active treatment is unknown. Aims: In this study, we assessed the role of ctDNA as a biomarker to monitor therapeutic response and clonal evolution in MDS and AML. Methods: Thirty-one MDS and AML patients who had both bone marrow NGS and ctDNA at baseline were included for concordance analysis. Twenty-seven patients who had at least two serial ctDNA assessment with at least one month interval were included for dynamic ctDNA analysis. Targeted deep sequencing was performed on bone marrow and ctDNA using a customized panel of 165 genes known to be recurrently mutated in MDS and acute myeloid leukemia. Results: Thirty out thirty-one patients were identified with ctDNA mutation at baseline. Diagnostic ctDNA and matched bone marrow DNA exhibited excellent correlations with variant allele frequencies (VAFs) (R2=0.721, p<0.001). For 27 patients who had ctDNA mutation in baseline and treated with HMA or chemotherapy, the average mutation VAFs at the end of treatment was lower in patients achieving a CR/CRi versus those with mCR, mLFS or SD (2.7% vs 24.1%, respectively, p<0.001). As expected, mutation VAFs from all patients with treatment failure did not decrease during treatment. (Figure 1). Similarly, the average decrease in mutation VAFs from pretreatment to end of treatment was greater in patients achieving a CR versus those who did not (87.7% vs 35.5%, respectively, p= 0.001). ctDNA negative post treatment was associated with longer PFS (median PFS not reached (NR) vs. 5.6 (95% CI 4.08-7.13) months; P=0.009)(Fig 2A) and OS (median OS NR vs. 12.0 (95% CI 9.08-15.0) months; P=0.023) (Fig 2B). Seven of ten MDS patients who transformed to AML were identified with lately acquired subclones harboring FLT3 or NF1, mutations involving in signaling pathway. Moreover, new subclones were detected 0.5 to 4 months prior to AML transformation in 4 cases, indicating that genetic progression can predate morphological progression. Image:Summary/Conclusion: ctDNA status post treatment was relevant to clinical response and was of prognostic significance for disease progress and relapse. Dynamic ctDNA changes revealed complex patterns of clonal structure of MDS and AML in response to treatment. ctDNA sequencing could be an attractive, prospective measure for disease monitoring.