Plasma Circulating Tumor DNA Sequencing Predicts Minimal Residual Disease in Resectable Esophageal Squamous Cell Carcinoma

Tao Liu, Qianqian Yao, Hai Jin, Tao Liu, Qianqian Yao, Hai Jin

Abstract

Esophageal squamous cell carcinoma (ESCC) is lethal as tumors are rarely detected at an early stage and have a high recurrence rate. There are no particularly useful biomarkers for the prognostic prediction of ESCC. Circulating tumor DNA (ctDNA) is becoming an important biomarker for non-invasive diagnosis and monitoring tumor prognosis. Here, we aimed to analyze variations in plasma cell-free DNA (cfDNA) amount to search for minimal residual disease (MRD). Plasma and white blood cells (WBCs) of 60 patients were collected before tumor resection and a week after surgery. Tumor specimens were also collected as formalin-fixed paraffin-embedded (FFPE) samples. All samples were extracted to analyze the genetic alterations of 61 genes using capture-based next-generation sequencing (NGS). Tumor variants were detected in 38 patients with ESCC, and the two driver genes with the highest mutation frequency were TP53 and PIK3CA. Of the pre-surgical plasma cfDNA samples, 73.7% of identified variants matched the tissue. In patients who did not receive adjuvant therapy after surgery, postoperative cfDNA-positive patients had shorter overall survival (hazard ratios (HR), 25.8; 95% CI, 2.7-242.6; P = 0.004) and were more likely to relapse than postoperative cfDNA-negative patients (HR, 184.6; 95% CI, 3.6-9576.9; P = 0.01). Detection of ctDNA after surgical tumor excision is associated with tumor relapse and disease-specific survival, and can be used as a prognostic biomarker for MRD detection in ESCC.

Keywords: circulating tumor DNA; esophageal squamous cell carcinoma; minimal residual disease; next-generation sequencing; prognostic marker.

Conflict of interest statement

QY was employed by Shanghai AccuraGen Biotechnology Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2021 Liu, Yao and Jin.

Figures

Figure 1
Figure 1
Overview of patient enrollment and sample collection.
Figure 2
Figure 2
Somatic mutations in FFPE tissues of 53 patients with ESCC. (A) Gender, age, tumor location, tumor stage, and p53 IHC status of 53 patients. (B) Mutation landscape of FFPE samples. FFPE, Formalin-Fixed Paraffin-Embedded; ESCC, esophageal squamous cell carcinoma; IHC, immunohistochemistry.
Figure 3
Figure 3
Postoperative ctDNA mutation status and prognosis of patients with ESCC without adjuvant therapy. (A) Kaplan-Meier survival curves for DFS analysis between postoperative ctDNA+ and ctDNA- patients. (B) Kaplan-Meier estimates of OS according to postoperative ctDNA status in patients with ESCC without adjuvant chemotherapy. ESCC, esophageal squamous cell carcinoma; ctDNA, circulating tumor DNA; DFS, disease-free survival; OS, overall survival, NR, not reach.

References

    1. Siegel RL, Miller KD, Jemal A. Cancer Statistics, 2017. CA Cancer J Clin (2017) 67(1):7–30. 10.3322/caac.21387
    1. Hiyama T, Yoshihara M, Tanaka S, Chayama K. Genetic Polymorphisms and Esophageal Cancer Risk. Int J Cancer (2007) 121(8):1643–58. 10.1002/ijc.23044
    1. Yang H, Liu H, Chen Y, Zhu C, Fang W, Yu Z, et al. . Neoadjuvant Chemoradiotherapy Followed by Surgery Versus Surgery Alone for Locally Advanced Squamous Cell Carcinoma of the Esophagus (Neocrtec5010): A Phase III Multicenter, Randomized, Open-Label Clinical Trial. J Clin Oncol (2018) 36(27):2796–803. 10.1200/JCO.2018.79.1483
    1. Su X-D, Zhang D-K, Zhang X, Lin P, Long H, Rong T-H. Prognostic Factors in Patients With Recurrence After Complete Resection of Esophageal Squamous Cell Carcinoma. J Thorac Dis (2014) 6(7):949–57. 10.3978/j.issn.2072-1439.2014.07.14
    1. Xu Y, Chen Q, Yu X, Zhou X, Zheng X, Mao W. Factors Influencing the Risk of Recurrence in Patients With Esophageal Carcinoma Treated With Surgery: A Single Institution Analysis Consisting of 1002 Cases. Oncol Lett (2013) 5(1):185–90. 10.3892/ol.2012.1007
    1. Scheuemann P Hosch SB, Izbicki JR. Prognostic Value of Minimal Residual Disease in Esophageal Cancer. In: Pantel K, editor. Micrometastasis. Cancer Metastasis - Biology and Treatment, vol. 5. Dordrecht: Springer; (2003). p. 127–38. 10.1007/978-1-4020-4460-1_7
    1. Lagergren J. Adenocarcinoma of Oesophagus: What Exactly is the Size of the Problem and Who is At Risk? Gut (2005) 54 Suppl 1:i1–5. 10.1136/gut.2004.041517
    1. Komatsu S, Ichikawa D, Hirajima S, Takeshita H, Shiozaki A, Fujiwara H, et al. . Clinical Impact of Predicting CCND1 Amplification Using Plasma DNA in Superficial Esophageal Squamous Cell Carcinoma. Dig Dis Sci (2014) 59(6):1152–9. 10.1007/s10620-013-3005-2
    1. Ueda M, Iguchi T, Masuda T, Nakahara Y, Hirata H, Uchi R, et al. . Somatic Mutations in Plasma Cell-Free DNA are Diagnostic Markers for Esophageal Squamous Cell Carcinoma Recurrence. Oncotarget (2016) 7(38):62280–91. 10.18632/oncotarget.11409
    1. Koppert LB, Wijnhoven BP, van Dekken H, Tilanus HW, Dinjens WN. The Molecular Biology of Esophageal Adenocarcinoma. J Surg Oncol (2005) 92(3):169–90. 10.1002/jso.20359
    1. Vallbohmer D, Lenz HJ. Predictive and Prognostic Molecular Markers in Outcome of Esophageal Cancer. Dis Esophagus (2006) 19(6):425–32. 10.1111/j.1442-2050.2006.00622.x
    1. Diaz LA, Jr, Bardelli A. Liquid Biopsies: Genotyping Circulating Tumor DNA. J Clin oncology: Off J Am Soc Clin Oncol (2014) 32(6):579–86. 10.1200/JCO.2012.45.2011
    1. Diehl F, Schmidt K, Choti MA, Romans K, Goodman S, Li M, et al. . Circulating Mutant DNA to Assess Tumor Dynamics. Nat Med (2008) 14(9):985–90. 10.1038/nm.1789
    1. Meng P, Wei J, Geng Y, Chen S, Terpstra MM, Huang Q, et al. . Targeted Sequencing of Circulating Cell-Free DNA in Stage II-III Resectable Oesophageal Squamous Cell Carcinoma Patients. BMC Cancer (2019) 19(1):818. 10.1186/s12885-019-6025-2
    1. Chaudhuri AA, Chabon JJ, Lovejoy AF, Newman AM, Stehr H, Azad TD, et al. . Early Detection of Molecular Residual Disease in Localized Lung Cancer by Circulating Tumor DNA Profiling. Cancer Discovery (2017) 7(12):1394–403. 10.1158/-17-0716
    1. Garcia-Murillas I, Schiavon G, Weigelt B, Ng C, Hrebien S, Cutts RJ, et al. . Mutation Tracking in Circulating Tumor DNA Predicts Relapse in Early Breast Cancer. Sci Transl Med (2015) 7(302):302ra133. 10.1126/scitranslmed.aab0021
    1. Reinert T, Henriksen TV, Christensen E, Sharma S, Salari R, Sethi H, et al. . Analysis of Plasma Cell-Free DNA by Ultradeep Sequencing in Patients With Stages I to III Colorectal Cancer. JAMA Oncol (2019) 5(8):1124–31. 10.1001/jamaoncol.2019.0528
    1. Gao XH, Li J, Gong HF, Yu GY, Liu P, Hao LQ, et al. . Comparison of Fresh Frozen Tissue With Formalin-Fixed Paraffin-Embedded Tissue for Mutation Analysis Using a Multi-Gene Panel in Patients With Colorectal Cancer. Front Oncol (2020) 10:310–0. 10.3389/fonc.2020.00310
    1. Xu T, Kang X, You X, Dai L, Tian D, Yan W, et al. . Cross-Platform Comparison of Four Leading Technologies for Detecting Egfr Mutations in Circulating Tumor DNA From Non-Small Cell Lung Carcinoma Patient Plasma. Theranostics (2017) 7(6):1437–46. 10.7150/thno.16558
    1. Li H, Durbin R. Fast and Accurate Long-Read Alignment With Burrows–Wheeler Transform. Bioinformatics (2010) 26(5):589–95. 10.1093/bioinformatics/btp698
    1. Lai Z, Markovets A, Ahdesmaki M, Chapman B, Hofmann O, McEwen R, et al. . VarDict: A Novel and Versatile Variant Caller for Next-Generation Sequencing in Cancer Research. Nucleic Acids Res (2016) 44(11):e108. 10.1093/nar/gkw227
    1. Paila U, Chapman BA, Kirchner R, Quinlan AR. GEMINI: Integrative Exploration of Genetic Variation and Genome Annotations. PloS Comput Biol (2013) 9(7):e1003153. 10.1371/journal.pcbi.1003153
    1. Huang M, Jin J, Zhang F, Wu Y, Xu C, Ying L, et al. . Non-Disruptive Mutation in TP53 DNA-Binding Domain is a Beneficial Factor of Esophageal Squamous Cell Carcinoma. Ann Transl Med (2020) 8(6):316. 10.21037/atm.2020.02.142
    1. Gao YB, Chen ZL, Li JG, Hu XD, Shi XJ, Sun ZM, et al. . Genetic Landscape of Esophageal Squamous Cell Carcinoma. Nat Genet (2014) 46(10):1097–102. 10.1038/ng.3076
    1. Soussi T, Legros Y, Lubin R, Ory K, Schlichtholz B. Multifactorial Analysis of p53 Alteration in Human Cancer: A Review. Int J Cancer (1994) 57(1):1–9. 10.1002/ijc.2910570102
    1. Li XC, Wang MY, Yang M, Dai HJ, Zhang BF, Wang W, et al. . A Mutational Signature Associated With Alcohol Consumption and Prognostically Significantly Mutated Driver Genes in Esophageal Squamous Cell Carcinoma. Ann Oncol (2018) 29(4):938–44. 10.1093/annonc/mdy011
    1. Hu N, Huang J, Emmert-Buck MR, Tang ZZ, Roth MJ, Wang C, et al. . Frequent Inactivation of the TP53 Gene in Esophageal Squamous Cell Carcinoma From a High-Risk Population in China. Clin Cancer Res (2001) 7(4):883–91.
    1. Hainaut P, Hollstein M. p53 and Human Cancer: The First Ten Thousand Mutations. Adv Cancer Res (2000) 77:81–137. 10.1016/S0065-230X(08)60785-X
    1. Zheng H, Wang Y, Tang C, Jones L, Ye H, Zhang G, et al. . Tp53, PIK3CA, FBXW7 and KRAS Mutations in Esophageal Cancer Identified by Targeted Sequencing. Cancer Genomics Proteomics (2016) 13(3):231–8.
    1. Hao JJ, Lin DC, Dinh HQ, Mayakonda A, Jiang YY, Chang C, et al. . Spatial Intratumoral Heterogeneity and Temporal Clonal Evolution in Esophageal Squamous Cell Carcinoma. Nat Genet (2016) 48(12):1500–7. 10.1038/ng.3683
    1. Li Q, Luo D, Zhu J, Yang L, Liu Q, Ma Y, et al. . AcrnaCT Trial Protocol: Efficacy of Adjuvant Chemotherapy in Patients With Clinical T3b/T4, N+ Rectal Cancer Undergoing Neoadjuvant Chemoradiotherapy: A Pathology-Oriented, Prospective, Multicenter, Randomized, Open-Label, Parallel Group Clinical Trial. BMC Cancer (2019) 19(1):1117. 10.1186/s12885-019-6289-6
    1. Matsumoto M, Natsugoe S, Nakashima S, Sakamoto F, Okumura H, Sakita H, et al. . Clinical Significance of Lymph Node Micrometastasis of pN0 Esophageal Squamous Cell Carcinoma. Cancer Lett (2000) 153(1-2):189–97. 10.1016/S0304-3835(00)00374-8
    1. Hosch S, Kraus J, Scheunemann P, Izbicki JR, Schneider C, Schumacher U, et al. . Malignant Potential and Cytogenetic Characteristics of Occult Disseminated Tumor Cells in Esophageal Cancer. Cancer Res (2000) 60(24):6836–40.
    1. Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, et al. . Detection of Circulating Tumor DNA in Early- and Late-Stage Human Malignancies. Sci Transl Med (2014) 6(224):224ra24. 10.1158/1538-7445.AM2014-5606
    1. Sausen M, Phallen J, Adleff V, Jones S, Leary RJ, Barrett MT, et al. . Clinical Implications of Genomic Alterations in the Tumour and Circulation of Pancreatic Cancer Patients. Nat Commun (2015) 6:7686. 10.1158/1538-7445.AM2015-619
    1. Majure M, Logan AC. What the Blood Knows: Interrogating Circulating Tumor DNA to Predict Progression of Minimal Residual Disease in Early Breast Cancer. Ann Transl Med (2016) 4(24):543. 10.21037/atm.2016.11.77
    1. Qing T, Zhu S, Suo C, Zhang L, Zheng Y, Shi L. Somatic Mutations in ZFHX4 Gene are Associated With Poor Overall Survival of Chinese Esophageal Squamous Cell Carcinoma Patients. Sci Rep (2017) 7(1):4951. 10.1038/s41598-017-04221-7
    1. Cho MS, Park CH, Lee S, Park HS. Clinicopathological Parameters for Circulating Tumor DNA Shedding in Surgically Resected non-Small Cell Lung Cancer With EGFR or KRAS Mutation. PloS One (2020) 15(3):e0230622. 10.1371/journal.pone.0230622
    1. Hsieh CC, Hsu HS, Chang SC, Chen YJ. Circulating Cell-Free Dna Levels Could Predict Oncological Outcomes of Patients Undergoing Esophagectomy for Esophageal Squamous Cell Carcinoma. Int J Mol Sci (2016) 17(12):2131. 10.3390/ijms17122131
    1. Azad TD, Chaudhuri AA, Fang P, Qiao Y, Esfahani MS, Chabon JJ, et al. . Circulating Tumor Dna Analysis for Detection of Minimal Residual Disease After Chemoradiotherapy for Localized Esophageal Cancer. Gastroenterology (2020) 158(3):494–505.e6. 10.1053/j.gastro.2019.10.039
    1. Reinert T, Henriksen TV, Rasmussen MH, Sethi H, Salari R, Shchegrova S, et al. . Serial Circulating Tumor DNA Analysis for Detection of Residual Disease, Assessment of Adjuvant Therapy Efficacy and for Early Recurrence Detection in Colorectal Cancer. Ann Oncol (2018) 29:viii151. 10.1093/annonc/mdy281.004
    1. Zhang R, Hu Y, Zhou T, Han W, Liu Y, Qian J, et al. . The Mutation Profiles of Cell-Free DNA in Patients With Oesophageal Squamous Cell Carcinoma Who Were Responsive and non-Responsive to Neoadjuvant Chemotherapy. J Thorac Dis (2020) 12(8):4274–83. 10.21037/jtd-20-230
    1. Li S, Lai H, Liu J, Liu Y, Jin L, Li Y, et al. . Circulating Tumor Dna Predicts the Response and Prognosis in Patients With Early Breast Cancer Receiving Neoadjuvant Chemotherapy. JCO Precis Oncol (2020) 4:244–57. 10.1200/PO.19.00292
    1. Raja R, Kuziora M, Brohawn PZ, Higgs BW, Gupta A, Dennis PA, et al. . Early Reduction in Ctdna Predicts Survival in Patients With Lung and Bladder Cancer Treated With Durvalumab. Clin Cancer Res (2018) 24(24):6212–22. 10.1158/1078-0432.CCR-18-0386
    1. Zhang Q, Luo J, Wu S, Si H, Gao C, Xu W, et al. . Prognostic and Predictive Impact of Circulating Tumor DNA in Patients With Advanced Cancers Treated With Immune Checkpoint Blockade. Cancer Discovery (2020) 10(12):1842. 10.1158/-20-0047
    1. Christensen E, Birkenkamp-Demtröder K, Sethi H, Shchegrova S, Salari R, Nordentoft I, et al. . Early Detection of Metastatic Relapse and Monitoring of Therapeutic Efficacy by Ultra-Deep Sequencing of Plasma Cell-Free DNA in Patients With Urothelial Bladder Carcinoma. J Clin Oncol (2019) 37(18):1547–57. 10.1158/1538-7445.AM2019-913
    1. Cristiano S, Leal A, Phallen J, Fiksel J, Adleff V, Bruhm DC, et al. . Genome-Wide Cell-Free DNA Fragmentation in Patients With Cancer. Nature (2019) 570(7761):385–9. 10.1038/s41586-019-1272-6
    1. Wang Y, Li L, Douville C, Cohen JD, Yen T-T, Kinde I, et al. . Evaluation of Liquid From the Papanicolaou Test and Other Liquid Biopsies for the Detection of Endometrial and Ovarian Cancers. Sci Trans Med (2018) 10(433):eaap8793. 10.1126/scitranslmed.aap8793
    1. Qu C, Wang Y, Wang P, Chen K, Wang M, Zeng H, et al. . Detection of Early-Stage Hepatocellular Carcinoma in Asymptomatic HBsAg-seropositive Individuals by Liquid Biopsy. Proc Natl Acad Sci USA (2019) 116(13):6308–12. 10.1073/pnas.1819799116
    1. Cohen JD, Li L, Wang Y, Thoburn C, Afsari B, Danilova L, et al. . Detection and Localization of Surgically Resectable Cancers With a Multi-Analyte Blood Test. Science (2018) 359(6378):926–30. 10.1126/science.aar3247
    1. Coombes RC, Page K, Salari R, Hastings RK, Armstrong A, Ahmed S, et al. . Personalized Detection of Circulating Tumor DNA Antedates Breast Cancer Metastatic Recurrence. Clin Cancer Res (2019) 25(14):4255–63. 10.1158/1078-0432.CCR-18-3663
    1. Oliveira KCS, Ramos IB, Silva JMC, Barra WF, Riggins GJ, Palande V, et al. . Current Perspectives on Circulating Tumor DNA, Precision Medicine, and Personalized Clinical Management of Cancer. Mol Cancer Res (2020) 18(4):517–28. 10.1158/1541-7786.MCR-19-0768
    1. Ptashkin RN, Mandelker DL, Coombs CC, Bolton K, Yelskaya Z, Hyman DM, et al. . Prevalence of Clonal Hematopoiesis Mutations in Tumor-Only Clinical Genomic Profiling of Solid Tumors. JAMA Oncol (2018) 4(11):1589–93. 10.1001/jamaoncol.2018.2297
    1. Fisher OM, Lord SJ, Falkenback D, Clemons NJ, Eslick GD, Lord RV. The Prognostic Value of TP53 Mutations in Oesophageal Adenocarcinoma: A Systematic Review and Meta-Analysis. Gut (2017) 66(3):399–410. 10.1136/gutjnl-2015-310888
    1. Zhang D, Zhang W, Liu W, Mao Y, Fu Z, Liu J, et al. . Human Papillomavirus Infection Increases the Chemoradiation Response of Esophageal Squamous Cell Carcinoma Based on P53 Mutation. Radiotherapy Oncol (2017) 124(1):155–60. 10.1016/j.radonc.2017.06.008
    1. Andolfo I, Petrosino G, Vecchione L, De Antonellis P, Capasso M, Montanaro D, et al. . Detection of erbB2 Copy Number Variations in Plasma of Patients With Esophageal Carcinoma. BMC Cancer (2011) 11:126. 10.1186/1471-2407-11-126
    1. Henriksen TV, Reinert T, Christensen E, Sethi H, Birkenkamp-Demtröder K, Gögenur M, et al. . The Effect of Surgical Trauma on Circulating Free DNA Levels in Cancer Patients-Implications for Studies of Circulating Tumor DNA. Mol Oncol (2020) 14(8):1670–9. 10.1002/1878-0261.12729
    1. Ignatiadis M, Lee M, Jeffrey SS. Circulating Tumor Cells and Circulating Tumor DNA: Challenges and Opportunities on the Path to Clinical Utility. Clin Cancer Res (2015) 21(21):4786–800. 10.1158/1078-0432.CCR-14-1190
    1. Sawada G, Niida A, Uchi R, Hirata H, Shimamura T, Suzuki Y, et al. . Genomic Landscape of Esophageal Squamous Cell Carcinoma In a Japanese Population. Gastroenterology (2016) 150(5):1171–82. 10.1053/j.gastro.2016.01.035

Source: PubMed

Sponsors and Collaborators

Medical Conditions

Drug Interventions

3
Subscribe