ctDNA Variants Research Articles

Abstract PS2-08: Identification of incidental putative germline variants in circulating tumor DNA

Abstract Background: Circulating tumor DNA (ctDNA) has emerged as a potential tool for detecting disease recurrence, monitoring response to therapy, and identifying resistance mutations in the peripheral blood. With increased frequency of testing, there is an unmet need to recognize putative germline variants in ctDNA, and the probability that these variants are associated with inherited conditions. Here, we evaluated a large cohort of breast cancer patients who underwent ctDNA evaluation to determine the type and frequency of ctDNA mutations identified with confirmed germline testing. Methods: We reviewed ctDNA testing from a single institution (Northwestern University). All breast cancer patients who had next-generation sequencing (NGS) performed by Guardant Health (Redwood City, CA) from 2015-2020 were included in this retrospective study. An allele frequency cutoff of 30% was pre-established as a threshold to review patient charts to determine whether genetic counseling and germline testing were performed, along with the timeframe of this testing (e.g. before or after ctDNA evaluation). Clinical information including demographics, pathology, tissue NGS testing, and germline testing were collected. Descriptive analyses and statistical associations were performed using STATA. Results: The initial cohort consisted of 520 patients with breast cancer who underwent ctDNA testing. From this, we identified 84 patients (16.2%) who had at least one variant with allele frequency ≥30%. The most common variants identified were the following: TP53 (34%), PIK3CA (27%), BRCA1 (9%), BRCA2 (8%), and AKT1 (4%). Guardant360 classified 99% of these variants as pathogenic and 1% as a variant of unknown significance. Germline positivity using a separate CLIA-approved test for this indication was confirmed at the following frequencies: BRCA1 (2 of 8 positive, 25%), BRCA2 (2 of 5 positive, 40%), PIK3CA (0 of 5 positive), and TP53 (0 of 26 positive). In total, 14% of patients with ctDNA allele frequency ≥30% had a confirmed germline mutation. Lower age at breast cancer diagnosis was significantly associated with the probability of germline testing prior to ctDNA evaluation (P=0.0001). For patients who had a variant with allele frequency ≥30%, 24.3% never received genetic counseling or germline testing. Conclusion: High allele frequency ctDNA variants (≥30%) were present in 16% of patients who underwent ctDNA evaluation with 14% of these variants confirmed as true germline variants. Consenting patients for ctDNA testing should include the possibility of identifying putative germline variants, and criteria should be established to refer patients for subsequent genetic counseling and germline testing, given the potential implications for patients and their family members. Citation Format: Andrew A Davis, Michael C Burns, Lorenzo Gerratana, Paolo D'Amico, Saya Jacob, Ami Shah, Neelima Katam, Firas Wehbe, Qiang Zhang, Elena Vagia, Lisa Flaum, Kalliopi P. Siziopikou, Leonidas C Platanias, Amir Behdad, William J Gradishar, Massimo Cristofanilli. Identification of incidental putative germline variants in circulating tumor DNA [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-08.

Phase II study of apatinib in combination with oral vinorelbine in heavily pretreated HER2-negative metastatic breast cancer and clinical implications of monitoring ctDNA.

Objective:Apatinib is an oral TKI targeting VEGFR-2. Single-agent apatinib treatment has been shown to produce an objective response in patients with pretreated mBC. Oral vinorelbine also holds promise as a treatment of choice in patients with mBC. This study aimed to investigate the efficacy and safety of the oral vinorelbine-apatinib combination in patients with pretreated mBC. In addition, we detected gene variants in ctDNA to explore the therapeutic implications.Methods:This study enrolled patients with HER2-negative mBC who were pretreated with anthracycline/taxanes. Patients were treated with apatinib at 500 mg/425 mg daily plus oral vinorelbine 60 mg/m2 on days 1, 8, and 15 of every cycle (3 weeks). The primary endpoint was PFS. The secondary endpoints were ORR, CBR, OS, and safety. Patients eligible for ctDNA detection were evaluated before and during treatment.Results:Forty patients were enrolled. The median PFS was 5.2 months (95% CI, 3.4–7.0 months), and the median OS was 17.4 months (95% CI, 8.0–27.0 months). The ORR was 17.1% (6/35), and the CBR was 45.7% (16/35). The most common AEs included gastrointestinal reaction, myelosuppression, and hypertension. In 20 patients, ctDNA was detected at baseline and during treatment. A significant difference was found in PFS for undetected vs. detected baseline ctDNA (13.9 months vs. 3.6 months, P = 0.018).Conclusions:All-oral therapy with apatinib plus vinorelbine displayed objective efficacy in patients with heavily pretreated HER2-negative mBC, with acceptable and manageable toxicity profiles. Patients with no gene variant detected and lower variant allele frequencies in ctDNA at baseline showed longer PFS.

Anlotinib has good efficacy and low toxicity: a phase II study of anlotinib in pre-treated HER-2 negative metastatic breast cancer.

Objective:Anlotinib is a novel tyrosine kinase inhibitor blocking angiogenesis. This study was performed to assess the efficacy and safety of anlotinib in patients with metastatic breast cancer.Methods:Patients with HER2-negative breast cancer, who were pre-treated with anthracycline or taxanes in a neoadjuvant, adjuvant, or metastatic setting, and had treatment failure after at least one prior chemotherapy regimen in the metastatic setting were enrolled. Anlotinib was administered at 12 mg daily for 14 days in a 21-day cycle until disease progression or unacceptable toxicity occurred. Simultaneously, 5–10 mL of venous blood was collected to perform circulating tumor DNA (ctDNA) testing every 2 treatment cycles. The primary endpoint was the objective response rate (ORR). Secondary endpoints included the disease control rate (DCR), progression-free survival (PFS), overall survival, safety, and biomarkers.Results:Twenty-six eligible patients were enrolled, with a median age of 56 (30–75) years. The median follow-up time was 10.5 months. The ORR was 15.4%, the DCR was 80.8%, and the median PFS was 5.22 months (95% confidence interval 2.86–6.24). Fourteen (53.8%) patients survived for more than 10 months. The changes in the detectable ctDNA variant allele frequency were consistent with the tumor response. The most common treatment-related adverse events were hypertension (57.7%), thyroid-stimulating hormone elevation (34.6%), and hand-foot syndrome (23.1%).Conclusion:Anlotinib showed objective efficacy with tolerable toxicity in heavily pre-treated, metastatic HER2-negative breast cancer. The dynamic changes in the ctDNA variant allele fraction may be predictive of the tumor response.

Genotype-Specific Differences in Circulating Tumor DNA Levels in Advanced NSCLC

IntroductionPlasma-based circulating tumor DNA (ctDNA) is an established biomarker for molecular profiling with emerging applications in disease monitoring in multiple tumor types, including, NSCLC. However, determinants of ctDNA shedding and correlation with tumor burden are incompletely understood, particularly in advanced-stage disease. MethodsWe retrospectively analyzed ctDNA-based and tissue-based genomic data and imaging from 144 patients with NSCLC. Tumor burden was quantified with computed tomography (CT) and brain magnetic resonance imaging for the overall cohort and 18F-fludeoxyglucose positron emission tomography-CT in a subset of patients. ResultsThere was a moderate but statistically significant correlation between ctDNA variant allele frequency and multiple imaging measures of tumor burden such as CT volume (rho = 0.34, p ≤ 0.0001) and metabolic tumor volume (rho = 0.36, p = 0.003). This correlation was strongest in KRAS-mutant tumors (rho = 0.56, p ≤ 0.001), followed by TP53 mutants (rho = 0.43, p ≤ 0.0001), and weakest in EGFR-mutated (EGFR+) tumors (rho = 0.24, p = 0.077). EGFR+ tumors with EGFR copy number gain had significantly higher variant allele frequency than EGFR+ without copy number gain (p ≤ 0.00001). In multivariable analysis, TP53 and EGFR mutations, visceral metastasis, and tumor burden were independent predictors of increased ctDNA shedding. ConclusionsLevels of detectable ctDNA were affected not only by tumor burden but also by tumor genotype. The genotype-specific differences observed may be due to variations in DNA shedding and cellular turnover. These findings have implications for the emerging use of ctDNA in NSCLC disease monitoring and early detection.

Comparison of Circulating Tumour DNA and Extracellular Vesicle DNA by Low-Pass Whole-Genome Sequencing Reveals Molecular Drivers of Disease in a Breast Cancer Patient.

There is increasing recognition of circulating tumour DNA (ctDNA) as a non-invasive alternative to tumour tissue for the molecular characterisation and monitoring of disease. Recent evidence suggests that cancer-associated changes can also be detected in the DNA contained within extracellular vesicles (EVs). As yet, there has been limited investigation into the relationship between EV DNA and ctDNA, and no studies have examined the EV DNA of breast cancer patients. The aim of this study was to use low-pass whole-genome sequencing to identify copy number variants (CNVs) in serial samples of both ctDNA and EV DNA from a patient with breast cancer. Of the 52 CNVs identified in tumour DNA, 36 (69%) were detected in at least one ctDNA sample and 13 (25%) in at least one EV DNA sample. The number of detectable variants in ctDNA and EV DNA increased over the natural history of the patient’s disease, which was associated with progression to cerebral metastases. This case study demonstrates that, while CNVs are detectable in patient EV DNA, ctDNA has greater sensitivity than EV DNA for serial monitoring of breast cancer.

Mutational and Copy Number Profiling of Circulating Tumor DNA in Acute Myeloid Leukemia Using Targeted Next Generation Sequencing

The assessment of circulating tumor DNA (ctDNA), released by tumor cells undergoing apoptosis or necrosis, has established utility in solid tumors due to the advantage of a non-invasive "liquid biopsy" replacing multiple site-specific biopsies. However, its role in acute myeloid leukemia (AML) is uncertain, where a significant proportion of variants detected in the bone marrow (BM) may not be detected in ctDNA (Short, Blood Adv 2020). We have previously demonstrated the possibility of comprehensive genomic characterization of lymphoid malignancy from ctDNA using a single targeted next generation sequencing (NGS) hybridization-based panel (Blombery, BJH 2017). We aimed to assess the performance of this same genomic approach in ctDNA and to compare it against BM in AML. In addition, we aimed to assess the integration of a sensitive variant caller (Mutect2; Benjamin, bioRxiv 2019) to the bioinformatics suite in an attempt to improve low-level variant detection. Nineteen patients were identified from sequential patients with AML treated at our institutions where paired ctDNA and BM aspirate DNA were available. ctDNA was analyzed using a hybridization-based NGS panel targeting genes recurrently mutated in hematological malignancy followed by a suite of bioinformatics tools including HaplotypeCaller (GATK)/Mutect2 (GATK) for variant calling, CNSpector/CNSpectorX (Markham, Sci Reports 2019) for copy number variation (CNV) assessment and GRIDSS (Cameron, Genome Res 2017) for structural variant detection. The cohort clinical details are summarized in Table 1; none had documented extramedullary disease at time of collection. A total of 66 unique variants in 27 genes were detected, summarized in Figure 1. Median number of variants detected was 3 per patient sample, including NPM1 (n=6), IDH1/2 (n=4) and FLT3 point mutation (n=2). Three patient samples had FLT3-ITD detected by fragment length analysis; none were detected by the NGS panel in either ctDNA or BM. Variant allele frequency (VAF) from both compartments were highly correlated (R2 0.87). Higher VAFs in ctDNA were more commonly observed for kinase activating mutations (12/17 variants) and TP53 (5/6). Using HaplotypeCaller alone, 58 and 61 variants were detected in the BM and ctDNA, respectively. Of the 2 variants "specific" to the BM, both (IDH1 and NRAS) were called by Mutect2 in ctDNA and confirmed by visual inspection of sequence read alignments. Of the 5 variants "specific" to the ctDNA, 4 were detected in the BM at low VAF: CBL (n=2), KRAS and TET2. One discrepant case was patient #15 with prior breast cancer: KRAS G13D 21% in ctDNA but absent in the BM. Analysis by Mutect2 additionally detected 3 variants not called by HaplotypeCaller: NRAS and KIT in both ctDNA and BM, and TP53 P278R (VAF 6%) specific to the ctDNA in patient #9 with normal karyotype AML without history of prior malignancy. Overall, 3/5 variants in ctDNA and 6/6 in BM with low VAF were kinase activating mutations. We then performed genome-wide alignment of off-target reads to generate a low-resolution digital karyotype and CNV from ctDNA (Figure 2) which was compared with CNV and conventional karyotyping from BM. CNVs were detected in ctDNA in 10/11 patients with abnormal karyotype (Figure 1). Of these, 6/7 with non-complex abnormal karyotypes had consistent ctDNA CNVs including (i) 3 patients (#1, #10 and #15) with either rearranged/amplified KMT2A by FISH were all found to have gains at 11q23.3-qter, and (ii) 1 patient (#4) had 2 marker chromosomes of unknown origin on karyotyping which were resolved as additional copies of 4p using ctDNA sequencing. Although no CNVs were detected in all 8 patients with normal karyotype, analysis of B-allele frequency from ctDNA revealed one patient (#5) with copy neutral loss of heterozygosity (CN-LOH) in 7q (with a concurrent EZH2 mutation >95% VAF). In summary, we have demonstrated the ability to detect sequence variants, perform low-resolution digital karyotyping, CNV detection and CN-LOH detection from ctDNA in AML using a single hybridization based NGS assay. When using this approach, we show a high degree of concordance for both sequence variant and CNV detection, supporting the use of ctDNA as an alternative to BM (e.g. in cases of dry tap, hypocellular AML or failed karyotyping). Finally, using a sensitive variant caller, additional mutations were able to be detected in the ctDNA, the significance of which require evaluation in future studies. Disclosures Tiong: Amgen: Consultancy, Honoraria; Pfizer: Consultancy; Servier: Consultancy. Wilson:Illumina: Other: Illumina Diagnostic Genomics Scholarship. Bajel:Novartis: Honoraria; Astellas: Honoraria; Abbvie: Honoraria; Amgen: Honoraria, Speakers Bureau; Pfizer: Honoraria. Blombery:Invivoscribe: Honoraria; Novartis: Consultancy; Janssen: Honoraria; Amgen: Consultancy.

Ultradeep targeted sequencing of circulating tumor DNA in plasma of early and advanced breast cancer.

We present a study to evaluate the feasibility and clinical utility of amplicon‐based Oncomine Pan‐Cancer cell‐free assay to detect circulating tumor DNA (ctDNA) in patients with early or advanced breast cancer. In this study, 109 early and metastatic breast cancer patients were recruited before the initiation of treatment. ctDNA mutation profiles were assessed through unique molecular tagging (UMT) and ultradeep next generation sequencing (NGS). For patients with mutations, DNA from corresponding white blood cells (WBC) was sequenced to exclude variants of clonal‐hematopoietic (CH) origin. UMT targeted sequencing from plasma of 109 patients achieved a median total coverage of 55 498X and a median molecular coverage of 4187X. Among 53 ctDNA positive samples, 38% were mutation positive by WBC sequencing, indicating potentially false‐positive results contributed by CH origin. Prevalence of CH‐related mutations was associated with age (P = 7.51 × 10−4). After exclusion of CH mutations, ctDNA detection rates were 37% for local or locally advanced breast cancer (stage I‐III) and 81% for metastatic or recurrent breast cancer. The ctDNA detection rate correlated with disease stage (P = 2.60 × 10−4), nodal spread (P = 6.49 × 10−3) and the status of distant metastases (P = 5.00 × 10−4). ctDNA variants were detected mostly in TP53, PIK3CA and AKT1 genes, with variants showing therapeutic relevance. This pilot study endorses the use of targeted NGS for non‐invasive molecular profiling of breast cancer. Paired sequencing of plasma ctDNA and WBC should be implemented to improve accurate interpretation of liquid biopsy.

Detection of ctDNA in plasma of patients with clinically localised prostate cancer is associated with rapid disease progression

BackgroundDNA originating from degenerate tumour cells can be detected in the circulation in many tumour types, where it can be used as a marker of disease burden as well as to monitor treatment response. Although circulating tumour DNA (ctDNA) measurement has prognostic/predictive value in metastatic prostate cancer, its utility in localised disease is unknown.MethodsWe performed whole-genome sequencing of tumour-normal pairs in eight patients with clinically localised disease undergoing prostatectomy, identifying high confidence genomic aberrations. A bespoke DNA capture and amplification panel against the highest prevalence, highest confidence aberrations for each individual was designed and used to interrogate ctDNA isolated from plasma prospectively obtained pre- and post- (24 h and 6 weeks) surgery. In a separate cohort (n = 189), we identified the presence of ctDNA TP53 mutations in preoperative plasma in a retrospective cohort and determined its association with biochemical- and metastasis-free survival.ResultsTumour variants in ctDNA were positively identified pre-treatment in two of eight patients, which in both cases remained detectable postoperatively. Patients with tumour variants in ctDNA had extremely rapid disease recurrence and progression compared to those where variants could not be detected. In terms of aberrations targeted, single nucleotide and structural variants outperformed indels and copy number aberrations. Detection of ctDNA TP53 mutations was associated with a significantly shorter metastasis-free survival (6.2 vs. 9.5 years (HR 2.4; 95% CIs 1.2–4.8, p = 0.014).ConclusionsCtDNA is uncommonly detected in localised prostate cancer, but its presence portends more rapidly progressive disease.

Efficient mutation screening for cervical cancers from circulating tumor DNA in blood

BackgroundEarly diagnosis and continuous monitoring are necessary for an efficient management of cervical cancers (CC). Liquid biopsy, such as detecting circulating tumor DNA (ctDNA) from blood, is a simple, non-invasive method for testing and monitoring cancer markers. However, tumor-specific alterations in ctDNA have not been extensively investigated or compared to other circulating biomarkers in the diagnosis and monitoring of the CC. Therfore, Next-generation sequencing (NGS) analysis with blood samples can be a new approach for highly accurate diagnosis and monitoring of the CC.MethodUsing a bioinformatics approach, we designed a panel of 24 genes associated with CC to detect and characterize patterns of somatic single-nucleotide variations, indels, and copy number variations. Our NGS CC panel covers most of the genes in The Cancer Genome Atlas (TCGA) as well as additional cancer driver and tumor suppressor genes. We profiled the variants in ctDNA from 24 CC patients who were being treated with systemic chemotherapy and local radiotherapy at the Jeonbuk National University Hospital, Korea.ResultEighteen out of 24 genes in our NGS CC panel had mutations across the 24 CC patients, including somatic alterations of mutated genes (ZFHX3–83%, KMT2C-79%, KMT2D-79%, NSD1–67%, ATM-38% and RNF213–27%). We demonstrated that the RNF213 mutation could be used potentially used as a monitoring marker for response to chemo- and radiotherapy.ConclusionWe developed our NGS CC panel and demostrated that our NGS panel can be useful for the diagnosis and monitoring of the CC, since the panel detected the common somatic variations in CC patients and we observed how these genetic variations change according to the treatment pattern of the patient.

Molecular analysis of circulating tumor DNA from breast cancer patients before and after surgery and following adjuvant chemotherapy.

The primary aim of the present study is to provide a complex molecular profile of tumors using liquid biopsy and to monitor profile changes over time in association with surgery and administered adjuvant therapy. Our secondary aim was to compare the liquid biopsy profile with the tissue biopsy and assess concordance. A total of 27 samples of circulating tumor DNA (ctDNA) collected from 9 breast cancer patients at three different time points and their matched formalin-fixed and paraffin-embedded (FFPE) samples of primary tumor were analyzed with targeted next-generation sequencing. Somatic pathogenic variants were detected before surgery in samples from 5 patients (55.6%). The most frequently mutated genes were phosphatase and tensin homolog (4/9, 44.4%) and tumor protein 53 (4/9, 44.4%). Serial sampling of ctDNA enabled the detection of more variants compared with single-time tissue primary tumor biopsy. There were 17 ctDNA variants across all samples, but only 6 FFPE variants across all patients. In addition, the concordance between ctDNA and FFPE DNA was determined in only 1 patient, and this was connected with higher variant allele frequency. The findings of the present study suggest that liquid biopsy and tissue biopsy may be used as complementary analyses to adequately capture all tumor variants.

Abstract B20: Prolonged time to clearance of circulating-tumor DNA from patients with limited-stage small-cell lung cancer is associated with inferior progression-free and overall survival

Abstract Introduction: Despite recent incremental advances, patients with small-cell lung cancer (SCLC) continue to have a poor prognosis, with a median overall survival (OS) of 12-20 months in limited-stage disease (LS-SCLC) and approximately 12 months in extensive-stage disease (ES-SCLC). In an attempt to improve the detection and monitoring of SCLC, assays of circulating tumor DNA (ctDNA) via blood-based next-generation sequencing (NGS) have been validated. In this study, we used a blood-based 14 gene SCLC ctDNA NGS panel to evaluate the prognostic significance of the diagnostic maximum ctDNA variant allele frequency (VAF), diagnostic mean ctDNA VAF, and time to ctDNA clearance while on first-line therapy. Design: In our previous work, we developed a ctDNA assay to sequence 14 genes (TP53, RB1, BRAF, KIT, NOTCH1-4, PIK3CA, PTEN, FGFR1, MYC, MYCL1, and MYCN) that are commonly mutated in SCLC. A total of 104 plasma samples were analyzed from a cohort of 14 patients with LS-SCLC who completed definitive chemoradiation (n=13) or surgical resection (n=1) and had an end-of-treatment blood collection within nine weeks (mean 10.5 days, range 0-63 days) of completion of definitive initial therapy. We used a Cox Proportional Hazards model to estimate the hazard ratio (HR) for progression-free survival (PFS) or death based on the diagnostic maximum ctDNA VAF, diagnostic mean ctDNA VAF, and time to ctDNA clearance on first-line therapy. Results: In our 14-patient cohort, we did not observe any association between progression or death and maximum diagnostic ctDNA VAF (PFS HR: 1.01, CI 0.97-1.05; OS HR: 1, CI 0.95-1.04) or mean diagnostic ctDNA VAF (PFS HR: 1.01, CI 0.94-1.08; OS HR: 0.99, CI 0.91-1.07). Of the specific mutations representing the maximum diagnostic ctDNA VAF, TP53 represented 14.2% (n=2), while the remaining included RB1 associated mutations (n=5), PIK3CA (n=1), MYCL1 amplification (n=1), or no ctDNA at diagnosis (n=5). Among patients with clearance of ctDNA during first-line therapy (n=9), delayed time to ctDNA clearance was associated with inferior PFS (HR 1.1, CI 1.01-1.19) and OS (HR 1.07, CI 1.01-1.15), with median time to clearance of 63 days (range 29-92 days). Notably, 3 patients cleared ctDNA on their first on-treatment draw at approximately 30 days (29, days, 29 days, and 32 days). These patients have had no evidence of relapse and all remain alive since the start of first-line treatment (1467, 965, and 383 days of follow-up). Of the patients who had disease recurrence, the median time to clearance on first-line therapy was 65 days and median time to progression was 249 days, with all but 1 patient succumbing to their disease (median OS 437 days). Conclusion: In patients with LS-SCLC, prolonged time to ctDNA clearance during first-line therapy is associated with inferior PFS and OS. Larger patient cohorts are needed to validate this finding. Citation Format: Christopher Cann, Prasad Kopparapu, Yingjun Yan, Anel Muterspaugh, Heidi Chen, Zhiguo Zhao, Sally York, Leora Horn, Kristen Ancell, Kenneth Wyman, Caterina Bertucci, Tristan Shaffer, Lauren Hodson, Kavita Garg, Seyed Ali Hosseini, Lee Lim, Christine M. Lovly, Wade Iams. Prolonged time to clearance of circulating-tumor DNA from patients with limited-stage small-cell lung cancer is associated with inferior progression-free and overall survival [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B20.

Comprehensive modeling of longitudinal circulating tumor DNA dynamics to predict clinical response to first-line immunotherapy and chemoimmunotherapy in advanced non-small cell lung cancer.

9525 Background: First-line immunotherapy (IO) and chemo-immunotherapy (chemo-IO) are approved for PD-L1-expressing advanced non-small cell lung cancer (NSCLC), but PD-L1 expression does not reliably predict therapeutic response. Therefore, accurate on-therapy response assessment is needed to guide clinical decision-making. Conventional radiographic imaging is the gold-standard, but may not capture the nature and timing of an immune-mediated response. We investigated whether comprehensive longitudinal circulating tumor DNA (ctDNA) dynamics could enhance prediction of clinical response. Methods: We conducted targeted error correction sequencing (TEC-Seq) of longitudinal plasma specimens and matched white blood cells (WBCs) in patients with NSCLC treated with first-line IO or chemo-IO. Plasma ctDNA variants were identified by filtering out clonal hematopoiesis (CH) and germline (GL) variants found in matched WBCs. Clinical and pathological data, including PD-L1 tumor proportion score (TPS), and imaging response by RECIST1.1 were assessed. ctDNA dynamics were modeled to predict durable clinical benefit (at 6 months), progression-free survival (PFS), and overall survival (OS). Results: A total of 143 longitudinal plasma and 24 white blood cell samples underwent TEC-Seq for 31 patients with NSCLC treated with IO (n = 17) or chemo-IO (n = 14). ctDNA variants were found in 77% (n = 24) of patients after filtering out CH and GL variants, which comprised 53% (n = 196 of 373) of all variants. Molecular response, signified by elimination of ctDNA variants, was detected in 32% (n = 10) of patients and associated with improved PFS (p = 0.0004, log rank) and OS (p = 0.017, log rank). Time to molecular response was shorter than time to best RECIST response (median 3 vs. 7.71 weeks, p = 0.048, Mann-Whitney U test). A logistic regression model incorporating molecular response, recrudescence, or emergence of new variants predicted durable clinical benefit (sensitivity 84%, specificity 76%, AUC 0.88) better than PD-L1 TPS (AUC 0.67, p = 0.046, bootstrap method). Conclusions: Comprehensive modeling of ctDNA variant dynamics predicts clinical outcome independent of PD-L1 status in patients with advanced NSCLC treated with first-line IO or chemo-IO. Verification of bona fide ctDNA variants by matched WBC sequencing is essential. Molecular response can be identified earlier than imaging response and could enable on-therapy decision-making to alter clinical outcomes.

Phase II study assessing tolerability, efficacy, and biomarkers for durvalumab (D) ± tremelimumab (T) and gemcitabine/cisplatin (GemCis) in chemo-naïve advanced biliary tract cancer (aBTC).

4520 Background: In aBTC, GemCis is the standard of care as 1st-line treatment. Immunotherapies have shown early promising efficacy in some BTC patients (pts). We assessed D (anti-PD-L1) ± T (anti-CTLA-4) and GemCis in 1L BTC pts, including an extensive biomarker analysis (NCT03046862). Methods: Pts were first enrolled in the biomarker cohort (BMC) to receive 1 cycle of Gem 1000 mg/m2 + Cis 25 mg/m2 on D1 & D8, followed by GemCis + D 1120 mg and T 75 mg, Q3W until disease progression (PD). Subsequent pts were allocated to GemCis + D (3 combo cohort [3C]) or GemCis + D+T (4 combo cohort [4C]) until PD. In all cohorts, tumor biopsies were obtained pre-treatment, after 1 cycle, and at PD. Blood samples for ctDNA were obtained every cycle. Results: 121 pts were enrolled. Median follow-up durations were 28.5 months (m; 95% CI, 26.5-30.5), 11.3 m (95% CI, 9.1-13.5), and 11.9m (95% CI, 8.4-15.4) in the BMC, 3C, and 4C arms, respectively. Efficacy data are shown (Table). The most common adverse events (AEs, any grade) were neutropenia (54.5%), nausea (59.5%), and pruritus (55.44%). The most common grade 3/4 AEs were neutropenia (50.4%), anemia (35.5%), and thrombocytopenia (16.5%). In the BMC cohort, frequent mutations were found in DNA damage repair, cell cycle regulation, and genome instability genes (eg, ATM, BRCA2, POLE, MSH2, CDKN2A). Distinct somatic variants were detected in responders vs non-responders. Baseline tissue TMB did not correlate with PFS or OS. Reductions in ctDNA variant allele frequency (VAF) were more prominent among responders during early D+T cycles. ctDNA VAF on C3, D1 was significantly correlated with ORR ( P< 0.015). Pretreatment PD-L1 expression was not associated with efficacy, but PD-L1 expression after 1st GemCis cycle trended with improved PFS. Conclusions: These are the first clinical data of D±T plus chemotherapy in chemo-naïve aBTC pts. The addition of immunotherapy to chemotherapy was tolerable and showed very promising efficacy. Biomarker analyses show early signs of markers associated with response. The combination of GemCis + D is being investigated in the Phase 3 TOPAZ-1 trial (NCT03875235). Clinical trial information: NCT03046862 . [Table: see text]

Circulating tumor DNA (ctDNA) landscape in metastatic castrate-resistant prostate cancer patients with germline alterations.

200 Background: Circulating tumor-DNA (ctDNA) in mCRPC patients (pts) provides a viable approach for examining the genetic landscape of prostate cancer. In this follow-up, we report ctDNA variants in germline tested mCRPC pts. Methods: ctDNA alterations in 73 genes were detected using Guardant360 (G360) assays. Alteration types assessed were missense, frameshift, insertions, splice variants, truncations, amplifications (amp), deletions, and other. Pts included in the analysis received germline genetic testing (Invitae Corporation, San Francisco, CA) and ctDNA assays at various treatment timepoints. Statistical analyses were performed using chi-square and fisher exact test with p-value <0.05 for significance. Results: Germline and ctDNA testing was completed in 270 mCRPC pts. 13% (35/270) of pts had pathogenic germline alterations. Germline alterations detected were BRCA2 (43%, n=15), ATM (8.5%, n=3), CHEK2 (8.5%, n=3), and BRCA1 (6%, n=2). Of the 673 alterations detected in G360 assays, TP53 (25%, n=167) and AR (17%, n=117) were most commonly observed. ctDNA alteration breakdown for germline negative/positive pts is summarized in Tables A/B. Germline negative pts had more AR alterations compared to germline positive (p = 0.023). Also, germline negative pts presented with more amps (p < 0.001) and germline positive pts with more frameshift alterations (p = 0.005). The association of ctDNA alteration to clinical outcomes in germline positive/negative pts was also assessed and is ongoing. Conclusions: Pts with germline positive alterations had few somatic AR alterations and higher frequency of deleterious mutation in comparison to their germline negative counterparts.[Table: see text][Table: see text]

Abstract P5-01-19: Circulating tumor DNA (ctDNA) analysis for investigating resistance to chemotherapy with DNA-damage agents in patients with hereditary or sporadic triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC), a very aggressive breast cancer subtype, exhibits high variability at both molecular and clinical aspects. Loss of function (LOF) germline mutation in BRCA1 has a very close association with this subtype of breast cancer. BRCA1 impairment is associated with homologous recombination deficiency (HRD) and tumor with HRD may benefit from treatment with drugs that induces DNA damage and also with PARP1 inhibitors. We previously detected that a significant fraction of TNBC diagnosed in young Brazilian women exhibits BRCA1 impairment by mechanisms including germline pathogenic mutation and promoter hypermethylation and that this group of tumors presented better overall survival. In the current study our aim is to comprehensively characterize the resistance to chemotherapy (DNA-damage agents) in patients with Hereditary or Sporadic TNBC by investigating somatic mutations in circulating tumor DNA (ctDNA). Methods: A series of 34 TNBC patients were subjected to germline genetic testing using a 26- or 96-gene panel, including homologous recombination-related genes, for classifying the TNBC as Hereditary or Sporadic. Tumor mutation burden (TMB) analysis was assessed in 20 cases using a panel containing 409 cancer-associated genes. Tumor somatic mutations were detected by evaluating DNA from tumor biopsies (Formalin-fixed, paraffin-embedded tissue) and matched leucocyte and were screened in cell-free DNA from 6 serial plasma samples during neoadjuvant treatment and for 6 months after surgery. Results: Pathogenic germline mutations were identified in BRCA1 (17.6% - 6/34), BRCA2 (2.9% -1/34) and TP53 (2.9% - 1/34) genes. Additionally, variants of uncertain significance (VUS) were identified in 21 patients (61.7% - 21/34), being ATM, BUB1B and PMS2 the most affected genes by VUS. Based on the 34 patients that underwent pre-operative chemotherapy and surgery, 47% exhibited pathological complete response (pCR), 50% in hereditary and 48% in sporadic. In terms of somatic variants, tumor mutation burden (TMB) analysis showed that 20% had high and 80% low TMB, with no association with hereditary or sporadic status. Also, we found, on average, three somatic variants per tumor (range 0-7) and used them as tumor marks in the screening of ctDNA in plasma. Somatic mutations in TP53 were identified in 80% (16 of 21) tumor biopsy samples investigated. In DNA from plasma before treatment, confident detection of at least one tumor mutation (ctDNA) was observed in 60% of patients (10 of 16). Persistent ctDNA detection in plasma during treatment was observed in 6 out 10 (60%) patients with residual disease after surgery and only in 1 out of 6 (16%) in patients with pCR. For 3 patients persistent ctDNA was detected after surgery at increasing levels showing clear anticipation of disease progression or metastasis. Serial plasma ctDNA samples showed great association with the clinical response data suggesting that the chemotherapy-resistance mechanisms can be investigated by ctDNA in TNBC. Conclusions: Germline and somatic evaluation of TNBC can provide valuable information for improving the clinical management of patients. For somatic evaluation, development of gene panels optimized for FFPE samples and low DNA input is enabling a comprehensive detection of potentially clinically-actionable somatic variants with better success rate in cancer treatment. Although still challenging, investigation of somatic variants in ctDNA from plasma samples is feasible and presents huge potential in monitoring patients during chemotherapy, especially regarding both the presence of residual disease and monitoring of disease progression. Citation Format: Dirce M Carraro, Rafael C Brianese, Giovana T Torrezan, Marina de Brot, Claudia AA de Paula, Maria NC Formiga, Solange M Sanches, Vladmir CC de Lima, Fabiana BA Makdissi. Circulating tumor DNA (ctDNA) analysis for investigating resistance to chemotherapy with DNA-damage agents in patients with hereditary or sporadic triple-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-01-19.

Circulating DNA for Molecular Response Prediction, Characterization of Resistance Mechanisms and Quantification of CAR T-Cells during Axicabtagene Ciloleucel Therapy

Circulating DNA for Molecular Response Prediction, Characterization of Resistance Mechanisms and Quantification of CAR T-Cells during Axicabtagene Ciloleucel Therapy

A single institute analysis of cell-free DNA concentrations in plasma of patients with various cancer types

Abstract Liquid biopsy using blood is the next-generation technology in the field of cancer diagnosis. Since the proportion of circulating tumor DNA (ctDNA) in the total cell-free DNA (cfDNA) can be extremely low, it is important to use as much cfDNA input as possible to detect variant alleles by next-generation sequencing (NGS). NGS will lose their detection sensitivity when the input amounts are short due to low concentrations of cfDNA in blood. In this study, we asked whether concentrations of cfDNA in plasma were sufficient in patients across various cancer types including rare cancers such as soft tissue sarcoma for NGS-based liquid biopsies. Using a magnetic bead-based method, we extracted cfDNA in 22 plasma samples from 14 patients with advanced lung, gastrointestinal, head and neck, thymic, unknown primary cancer, and soft tissue sarcoma. Concentrations of cfDNA in plasma were then analyzed. Four samples from four patients with metastatic lung cancer showed cfDNA concentrations at a median of 10.3 ng/mL (1.7-15.6 ng/mL). Their concentrations were comparable to those in literatures. Five samples from three patients with advanced soft tissue sarcoma showed a median of 6.2 ng/mL (3.9-62.8 ng/mL). Concentrations in plasma of the other patients were at a median of 4.8 ng/mL (0-29.9 ng/mL, n = 13). Subsequently, we performed the IonPGM NGS to analyze single-nucleotide variants in ctDNA. This system required the input of 20ng and 5 ng of cfDNA to lower the detection limit at 0.1% and 0.25%, respectively. With the input of 20ng of cfDNA, we were able to detect a single-nucleotide variant in TP53 at an allelic frequency of 0.36% in a patient with metastatic soft tissue sarcoma. Our data showed that 11 out of 14 (79%) cancer patients had sufficient concentrations of cfDNA for the NGS-based liquid biopsy with the detection limit at 0.25%. Thus, liquid biopsy using blood may be a versatile tool for patients across various types of cancer.

173TiP - Cambridge liquid biopsy “CALIBRATION” study: Can changes in circulating tumour DNA (ctDNA) predict durable tumour responses in patients with advanced oesophageal cancer receiving MEDI4736?

BackgroundOesophageal cancer is the 6th most common cause of cancer death with incidence of adenocarcinomas (EAC) increasing in western countries. Long term survival is poor (<5%) (ESMO guidelines 2016) and mutational signatures of EAC suggest patients with "mutagenic" or "DDR" signatures can benefit from immune-checkpoint blockers (ICB) (Secriert et al. Nat gen 2016). However, there is uncertainty around the population to benefit from ICBs. Circulating tumour DNA (ctDNA) can be used to predict responses to immuno-oncology (IO) agents (Howell J, Trans res 2017). TP53 mutations is frequent in EAC 6 and ctDNA-TP53 mutations can be measured/followed in plasma to track tumour behaviour (response, clonal changes) during treatment (Zill O. Clin Can Res 2018, Fisher O. Gut 2017). Next-generation sequencing (NGS) provides high sensitivity, coverage and the possibility to interrogate tumour treatment-response and heterogeneity. Trial designCALIBRATION is a single-centre, open-label, pilot trial of durvalumab (1500mg/4w) for patients with EAC progressing to standard chemotherapy. Pts with measurable disease undergo biopsies at screening/C3/progression alongside weekly blood sampling. Primary objective: asses if early changes in TP53 ctDNA variant allele fraction (VAF) levels, by weeks 4 and/ or 7 can predict durable (6 month) RECIST V 1.1 responses (Complete or Partial Response, Stable Disease). We plan to recruit 19 pts with a 5 % significance (one-sided) and 80 % power to detect if ctDNA changes correctly predict radiological response in≥70 % pts. The trial opened to recruitment in October 2018, 13 pts have been pre-screened and 4 pts included. An interim analysis is planned for September 2019. Secondary endpoints include characterization of paired blood/biopsy samples from pts pre/post durvalumab of: • genomic heterogeneity (evolution of mutational signatures under IO) and changes in the tumour microenvironment (dynamics of T-cell populations). • changes in ctDNA and PBMC markers to identify biomarkers of resistance/response. • predictive value of the mutagenic and DDR signatures to predict response to IO compounds. Clinical trial identificationNCT03653052. Legal entity responsible for the studyCambridge University Hospitals NHS Foundation Trust and the University of Cambridge. FundingAstraZeneca. DisclosureS. Dovedi: Leadership role: AstraZeneca.

Circulating tumor DNA quantity is related to tumor volume and both predict survival in metastatic pancreatic ductal adenocarcinoma.

Circulating tumor DNA (ctDNA) is assumed to reflect tumor burden and has been suggested as a tool for prognostication and follow‐up in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC). However, the prognostic value of ctDNA and its relation with tumor burden has yet to be substantiated, especially in mPDAC. In this retrospective analysis of prospectively collected samples, cell‐free DNA from plasma samples of 58 treatment‐naive mPDAC patients was isolated and sequenced using a custom‐made pancreatobiliary NGS panel. Pathogenic mutations were detected in 26/58 (44.8%) samples. Cross‐check with droplet digital PCR showed good agreement in Bland–Altman analysis (p = 0.217, nonsignificance indicating good agreement). In patients with liver metastases, ctDNA was more frequently detected (24/37, p < 0.001). Tumor volume (3D reconstructions from imaging) and ctDNA variant allele frequency (VAF) were correlated (Spearman's ρ = 0.544, p < 0.001). Median overall survival (OS) was 3.2 (95% confidence interval [CI] 1.6–4.9) versus 8.4 (95% CI 1.6–15.1) months in patients with detectable versus undetectable ctDNA (p = 0.005). Both ctDNA VAF and tumor volume independently predicted OS after adjustment for carbohydrate antigen 19.9 and treatment regimen (hazard ratio [HR] 1.05, 95% CI 1.01–1.09, p = 0.005; HR 1.00, 95% CI 1.01–1.05, p = 0.003). In conclusion, our study showed that ctDNA detection rates are higher in patients with larger tumor volume and liver metastases. Nevertheless, measurements may diverge and, thus, can provide complementary information. Both ctDNA VAF and tumor volume were strong predictors of OS.

Abstract 413: Development of a Highly Customizable Platform for Variant Detection in Circulating Tumor DNA

Abstract Introduction: Variant detection in circulating tumor DNA (ctDNA) provides an unprecedented opportunity to study the mutational landscape of cancers with minimal patient invasiveness. Meanwhile, new variants providing information about drug response and disease prognosis are being rapidly discovered. Therefore, to fully harness the power of ctDNA variant detection it is critical to be able to rapidly modify the set of variants targeted in an assay. Here we present a platform for next generation sequencing-based ctDNA assays that allows researchers to readily customize their panel targets. Methods: Anchored Multiplex PCR (AMP™) is a molecular biology technique that allows for near infinite modularity of assay targets while maintaining variant detection sensitivity. In order to ensure assay specificity, AMP utilizes molecular barcoded adapters (MBC) to uniquely label input molecules prior to amplification, enabling post-sequencing error correction. This increases analytical sensitivity by reducing background noise. We also developed bioinformatics methods, which allow the characterization of position-specific error levels. By combining molecular barcode-based error correction and error profile characterization, we can reliably detect low frequency variants with high specificity. Results: We tested the modularity of the AMP ctDNA platform by creating ctDNA panels targeting either 6 kb of cancer variant hotspots or 34 kb of tumor suppressors plus the same 6kb of cancer hotspots (40 kb total target sequence). We then prepared and sequenced libraries using ctDNA-like inputs containing important cancer variants at low allele fractions (AF). We used these data to characterize the noise inherent in AMP library preparation and Illumina sequencing. We then performed a power analysis to measure the lowest AF at which we could detect variants at each target position given the noise level and sequencing coverage. Our results show that we can confidently detect variants with an AF of less than 0.18% in 50% of bases and 0.47% in 95% of bases covered in the 6kb panel. Variant detection sensitivity remained consistent over the 6kb of hotspot targets in the context of the 40kb panel (50% of bases powered to detect 0.17% AF variants and 95% powered to detect 0.45% variants). Across the full 40kb of the larger panel we had statistical power to call 0.23% AF variants at 50% of target bases and to call 0.56% variants at 95% of target bases. These data demonstrate that ctDNA panels can maintain assay performance upon panel modifications. Conclusions: The modular nature of AMP ctDNA panels allows for endless combinations of targeted genes to maximize detection sensitivity in relevant regions while also maintaining assay performance. Our experience with AMP assays indicates that the range in the number of targets that can be included in a panel is quite large, allowing broad implementation of the assays developed on the AMP platform. Citation Format: Matt Egleston, Paula Roberts, Ian Hoskins, Aaron Garnett, Laura Griffin, Abel Licon, Ryan Walters. Development of a Highly Customizable Platform for Variant Detection in Circulating Tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 413.