bims-preonc Biomed News
on Precision oncology
Issue of 2024–10–27
ten papers selected by
Ankita Daiya, OneCell Diagnostics Inc.
  1. Discrepancy Between Liquid Biopsy and Tumor Next-Generation Sequencing Due to Low Tumor Fraction in a Patient With Lung Adenocarcinoma.
  2. Circulating tumor DNA (ctDNA)-the next generation biomarker in non-small cell lung cancer patients treated with immunotherapy?
  3. Utility of Circulating Tumor DNA Assay in Identifying Mutations and Guiding Matched Targeted Therapy in Lung Cancers.
  4. Concordance of ctDNA and tissue genomic profiling in advanced biliary tract cancer.
  5. Feasibility of ctDNA in detecting minimal residual disease and predicting recurrence for colorectal cancer liver metastases.
  6. Clinical impact of large genomic explorations at diagnosis in 198 pediatric solid tumors: a monocentric study aiming practical feasibility of precision oncology.
  7. Predicting non-small cell lung cancer lymph node metastasis: integrating ctDNA mutation/methylation profiling with positron emission tomography-computed tomography (PET-CT) scan: protocol for a prospective clinical trial (LUNon-invasive Study).
  8. Real-world performance of integrative clinical genomics in pediatric precision oncology.
  9. Evaluation of a multimodal ctDNA-based assay for detection of aggressive cancers lacking standard screening tests.
  10. Circulating DNA in Rectal Cancer to Unravel the Prognostic Potential for Radiation Oncologist: A Meta-analysis.
  1. Cureus. 2024 Sep;16(9): e70119
    Discrepancy Between Liquid Biopsy and Tumor Next-Generation Sequencing Due to Low Tumor Fraction in a Patient With Lung Adenocarcinoma.
    Madeline C Baker, Trevor A Rose, Bruna Pellini.
      Tumor next-generation sequencing (NGS) is the gold standard molecular testing for driver genomic alterations in patients with advanced non-small cell lung cancer (NSCLC). However, it requires a biopsy, which is an invasive procedure. In contrast, a liquid biopsy is a minimally invasive test that measures circulating tumor DNA (ctDNA) in the plasma and can be also used for molecular profiling. We report a case of a patient with stage IV metastatic lung adenocarcinoma with a negative liquid biopsy for tumor-derived genomic alterations but positive tissue NGS for mutations, including a driver KRASG12C mutation. The discrepancy between the two results can be attributed to low levels of ctDNA determined by tumor fraction below 1%, which prevents the liquid biopsy assay from detecting genomic alterations when the tumor shedding into the blood is below the detection threshold. It is well known in the literature that false negative liquid biopsies are possible, but a significant finding this case highlights is the clinical importance of tumor fraction in a liquid biopsy report. We conclude that patients with a liquid biopsy with low tumor fraction need further testing with tumor NGS to determine the presence of driver genomic alterations.
    Keywords:  circulating tumor dna (ctdna); liquid biopsy; metastatic adenocarcinoma of lung; next generation sequencing (ngs); non-small cell lung carcinoma (nsclc); tumor fraction
    DOI:  https://doi.org/10.7759/cureus.70119
  2. Transl Lung Cancer Res. 2024 Sep 30. 13(9): 2103-2105
    Circulating tumor DNA (ctDNA)-the next generation biomarker in non-small cell lung cancer patients treated with immunotherapy?
    Per Hydbring.
      
    Keywords:  Non-small cell lung cancer (NSCLC); circulating tumor DNA (ctDNA); immune checkpoint inhibitor (ICI); liquid biopsy; whole exome sequencing (WES)
    DOI:  https://doi.org/10.21037/tlcr-24-308
  3. Clin Med Insights Oncol. 2024 ;18 11795549241285238
    Utility of Circulating Tumor DNA Assay in Identifying Mutations and Guiding Matched Targeted Therapy in Lung Cancers.
    Kun Li, Nana Zhang, Bing Xu, Zichen Liu, Dan Zhao, Yujie Dong, Jing Mu, Haifeng Lin, Guangyu Shan, Sihang Gao, Bo Yu, Xiaoxi Pan, Yanrong Wang, Dongxing Zhang, Nanying Che, Xiaoyong Ji.
       Background: Tumor genomic profiling has a significant impact on the selection of targeted therapy. Circulating tumor DNA (ctDNA) has emerged as a noninvasive, and reproducible assay compared with tissue biopsy. We aimed to evaluate its utility in identifying mutations and guiding targeted therapy for lung cancer.
    Methods: A total of 173 lung cancer patients underwent next-generation sequencing (NGS) using a targeted enrichment panel covering 20 lung cancer-related genes. The performance of the ctDNA NGS assay in identifying genetic mutations or alterations was compared with tissue biopsy and droplet digital PCR (ddPCR). The treatment response to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) therapies based on the ctDNA assay results was also assessed.
    Results: The ctDNA was detected in 61.85% of patients. Tissue mutations were detected in paired ctDNA in 38.57% of cases, while ctDNA mutations were detected in paired tissues in 89.1% of cases. The ctDNA increased the number of advanced non-small cell lung cancer (NSCLC) patients who received NCCN-recommended genetic testing by 12%. The concordance between ddPCR and ctDNA was relatively high reaching 99.43%. EGFR T790M/C797S c.G2390C and EGFR T790M/C797S c.T2389A were detected in tissue and ctDNA, respectively, in patient 01015. Moreover, ctDNA assay identified the EGFR T790M mutation, which was missed by tissue biopsy in patient 01149, who developed drug resistance after 1 year of EGFR-TKI therapy. Of the 17 patients who received EGFR-TKI targeted therapies based on the ctDNA NGS results, 12 patients achieved a partial response and two patients had stable disease.
    Conclusions: The results demonstrated that the ctDNA assay could partially overcome tumor heterogeneity in detecting mutations and provide complementary information on tumor genomic profiles. Moreover, the presence of EGFR mutations in ctDNA could offer valuable guidance for selecting appropriate EGFR-TKI treatment for advanced lung cancer patients. However, it is important to note that the ctDNA NGS assay has certain limitations in fully identifying all genomic alterations present in the tumor.
    Keywords:  Circulating tumor DNA; liquid biopsy; lung cancer; tumor biopsy; tumor heterogeneity
    DOI:  https://doi.org/10.1177/11795549241285238
  4. J Hepatol. 2024 Oct 21. pii: S0168-8278(24)02641-2. [Epub ahead of print]
    Concordance of ctDNA and tissue genomic profiling in advanced biliary tract cancer.
    Sohyun Hwang, Seonjeong Woo, Beodeul Kang, Haeyoun Kang, Jung Sun Kim, Sung Hwan Lee, Chang Il Kwon, Dong Soo Kyung, Hwang-Phill Kim, Gwangil Kim, Chan Kim, Hong Jae Chon.
       BACKGROUND & AIMS: Recent advances in molecular profiling enable the identification of potential therapeutic targets for biliary tract cancer (BTC). However, in patients with BTC, molecular profiling is hindered by challenges in obtaining adequate tissue samples. Circulating tumor DNA (ctDNA) may offer an alternative to tissue-based analysis. Here we aimed to assess the concordance between ctDNA and tissue genomic profiling in a large cohort of Asian patients with advanced BTC, and to evaluate the feasibility of liquid biopsy in BTC treatment.
    METHODS: This study included patients with systemic treatment-naive advanced BTC, treated at CHA Bundang Medical Center between January 2019 and December 2022. We enrolled patients with available baseline tissue-based next-generation sequencing (NGS), and sufficient plasma samples for ctDNA analysis (AlphaLiquid®100 from IMBdx).
    RESULTS: Among 102 enrolled patients, 49.0% had intrahepatic cholangiocarcinoma, 26.5% extrahepatic cholangiocarcinoma, and 24.5% gallbladder cancer. The concordance between intra-patient ctDNA and tumor tissue mutations revealed a sensitivity of 84.8%, and positive predictive value of 79.4%. ctDNA revealed targetable alterations in 34.3% of patients-including FGFR2 fusion, IDH1 mutation, microsatellite instability (MSI)-high, ERBB2 amplification, PIK3CA mutations, BRCA1/2 mutations, and MET amplification. Notably, a novel FGFR2-TNS1 fusion was identified in ctDNA, which was not targeted in the tissue NGS panel. A high maximum somatic variant allele frequency in ctDNA was associated with poor prognosis after gemcitabine/cisplatin-based chemotherapy, in terms of both overall survival (p = 6.9 × 10-6) and progression-free survival (p = 3.8 × 10-7).
    CONCLUSIONS: Among patients with advanced BTC, ctDNA-based genotyping showed acceptable concordance with tissue genomic profiling. Liquid biopsy using ctDNA could be a valuable complement to tissue-based genomic analysis in BTC.
    IMPACT AND IMPLICATIONS: Our study is the first large-scale investigation of the clinical utility of liquid biopsy, focusing on ctDNA, as an alternative to conventional tumor tissue analysis, among Asian patients with advanced BTC. The results demonstrated acceptable concordance between analysis of ctDNA versus tissue for identifying therapeutic targets and potentially actionable genetic alterations. This indicates that ctDNA analysis can provide critical insights regarding advanced BTC treatment, particularly in cases where it is challenging to obtain or analyze tumor tissue.
    Keywords:  NGS; biliary tract cancer; circulating tumor DNA; liquid biopsy
    DOI:  https://doi.org/10.1016/j.jhep.2024.10.020
  5. Front Oncol. 2024 ;14 1418696
    Feasibility of ctDNA in detecting minimal residual disease and predicting recurrence for colorectal cancer liver metastases.
    Jennifer A Kalil, Lucyna Krzywon, Stephanie K Petrillo, Migmar Tsamchoe, Oran Zlotnik, Anthoula Lazaris, Peter Metrakos.
       Introduction: Approximately 50% of patients diagnosed with colorectal cancer develop colorectal cancer liver metastases (CRLM). Although curative intent liver resection provides 5-year survival of 40-50%, up to 70% of patients develop recurrence of CRLM. Detection of minimal residual disease (MRD) is essential for timely, optimized treatment. This study evaluated the feasibility and utility of using circulating tumor DNA (ctDNA) to identify MRD and predict disease recurrence.
    Methods: Patients with CRLM that underwent liver resection and had known KRAS or PIK3CA mutations were retrospectively identified. Serial blood samples were collected every 3 months following surgery for disease surveillance. ctDNA was isolated from the samples and analyzed with digital PCR (dPCR).
    Results: KRAS and PIK3CA mutations were identified by dPCR in 29 patients over 115 timepoints. In patients with detectable ctDNA at time of liver resection, 81% (13/16) developed disease recurrence, while 46% (6/13) of the patients with undetectable ctDNA recurred (p=0.064). Presence of ctDNA was detected in 27.6% (8/29) of the initial postoperative samples. Radiologic recurrence was later diagnosed in 100% (8/8) of these patients, while 52% (11/21) who had undetectable ctDNA postoperatively recurred (p=0.026). Detectable ctDNA postoperatively was associated with a shorter disease-free survival (DFS) of 9 months vs 13 months in patients who had undetectable ctDNA (HR 2.95, 95% CI 1.16-7.49; p=0.02).
    Conclusion: Liquid biopsy using dPCR can identify low levels of ctDNA, enabling early detection of disease recurrence. Additionally, the presence of ctDNA postoperatively was predictive of recurrence. This study corroborates current literature and provides rational for moving toward a clinical trial using ctDNA and dPCR to detect MRD after CRLM resection.
    Keywords:  colorectal cancer liver metastases (CRLM); ctDNA; digital PCR; disease recurrence; liquid biopsy; liver resection; minimal residual disease (MRD)
    DOI:  https://doi.org/10.3389/fonc.2024.1418696
  6. BMC Cancer. 2024 Oct 21. 24(1): 1296
    Clinical impact of large genomic explorations at diagnosis in 198 pediatric solid tumors: a monocentric study aiming practical feasibility of precision oncology.
    Juliette Simon, Damien Reita, Eric Guerin, Benoit Lhermitte, Noelle Weingertner, François Lefebvre, Marie Karanian, Julien Masliah-Planchon, Veronique Lindner, Alina Onea, Sarah Jannier, Alexandra Salmon, Guillaume Bergthold, Florence Vincent, Marlène Deschuyter, Marie-Odile Barbaza, Natacha Entz-Werlé.
       INTRODUCTION: Faced to the growing development of collecting systematic molecular analyses in relapsed pediatric cancers to transform their targeted matched therapies, this study aimed to assess the clinical and therapeutic indications of systematic diagnostic genomic explorations performed in pediatric solid cancers to determine which type of screening and if it afford at relapse time an accurate targeted strategy.
    METHODS: A total of 280 patients less than 22 years, referred at the University Hospitals of Strasbourg for a newly diagnosed solid tumor from January 2015 to December 2021, were prospectively genomically investigated since diagnosis. Using 7 different molecular tests going from single-gene methods (IHC, FISH, RT-PCR, Sanger sequencing, droplet digital PCR) to largescale analyses (Next-Generation sequencing, RNAsequencing and FoundationOne®CDx), we explored retrospectively the molecular findings in those pediatric solid tumors (except hematolymphoid cancers) to improve diagnosis, prognosis assessment and relapse therapeutics.
    RESULTS: One hundred and ninety-eight patients (71%) underwent molecular biology (MB) at diagnosis. Thirty-eight different histologies were grouped into cerebral tumors (30%), sarcomas (26%, bone and soft tissues), various blastomas (27%), and other entities (17%). Over a median 40-month follow-up, the overall survival rate of patients was 85% and the relapse rate 28%. Of the 326 analyses carried out, 245 abnormalities (single nucleotide variations: 50%, fusions: 25%, copy number alteration: 20%) concerning 70 oncogenes were highlighted. The overall clinical impact rate was 84%. Broad-spectrum analyses had a higher therapeutic impact (57%) than the targeted analyses (28%). 75% of broad-spectrum tests found an actionable variant conducting 23% of patients to receive rapidly a matched targeted therapy since first relapse.
    CONCLUSION: Our experience highlighted the clinical utility of molecular profiling of solid tumors as soon as at diagnosis in children to expect improving access to innovative agents at relapse.
    Keywords:  Next-generation sequencing; Pediatric brain tumors; Precision medicine; Solid malignancies; Targeted therapies
    DOI:  https://doi.org/10.1186/s12885-024-13034-7
  7. J Thorac Dis. 2024 Sep 30. 16(9): 6272-6285
    Predicting non-small cell lung cancer lymph node metastasis: integrating ctDNA mutation/methylation profiling with positron emission tomography-computed tomography (PET-CT) scan: protocol for a prospective clinical trial (LUNon-invasive Study).
    Haitang Yang, Xiaoran Gu, Zhexin Wang, Gang Liu, Yongliang Niu, Xufeng Pan, Feng Yao.
       Background: Non-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancer cases and remains a leading cause of cancer-related death. Lymph node metastasis (LNM) significantly affects recurrence, survival rates, and treatment options. While lymph node sampling is standard for surgically removing operable NSCLC, it can lead to complications. Positron emission tomography-computed tomography (PET-CT) helps assess preoperative LNM despite false positive or negative rates. Additionally, circulating tumor DNA (ctDNA) detects minimal residual disease with high sensitivity and specificity. Whether ctDNA can predict LNM in operable NSCLC remains uncertain. Our goal is to develop a precise model for predicting NSCLC LNM using non-invasive ctDNA/methylation profiling combined with PET-CT imaging.
    Methods: This is a prospective study conducted in three stages. We will enroll patients with clinical stage I-IIIB [8th tumor, node, metastasis (TNM) staging] NSCLC requiring lobectomy plus lymph node sampling/dissection. The distribution of clinical stages in the enrolled population is as follows: clinical stage cN0 (n=100) and cN1/cN2 (n=100). During Stage 1, we will establish LNMs-specific ctDNA methylation signatures and compare negative predictive value (NPV) rates of LNMs using preoperative blood ctDNA somatic mutation/methylation alone or combined with PET-CT across different groups. For Stage 2, we will compare detection rates between ctDNA somatic mutation/methylation profiles alone or combined with PET-CT and traditional mediastinoscopy/endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA). As for Stage 3, ctDNA-free interval (CFI) and disease-free survival between systematic lymph node presence and absence in patients will be compared with preoperative negative ctDNA profiling and/or PET-CT. In Stage 3, patients will be followed up for 5 years to collect recurrence and survival data. Post-surgery follow-up ctDNA tests will be conducted every 3 months for the first 2 years, every 6 months for years 3-4, and annually in year five. Demographics and baseline data will be summarized with mean, standard deviation, median, max, and min values. Tests will include t-tests, Welch/Behren-Fisher test, and Wilcoxon rank-sum test for continuous variables. Categorical data will be presented as counts/percentages and compared using χ2 test or Fisher's exact test.
    Discussion: By utilizing preoperative ctDNA/methylation profiling in conjunction with PET-CT, this study is expected to yield substantial evidence for accurately predicting LNM before surgery. This will help inform surgeons in selecting the appropriate intraoperative lymph node dissection strategy for operable NSCLC patients.
    Trial Registration: This study is registered on www.clinicaltrials.gov (NCT06358222).
    Keywords:  Non-small cell lung cancer (NSCLC); circulating tumor DNA (ctDNA); lymph node metastasis (LNM); methylation; positron emission tomography-computed tomography (PET-CT)
    DOI:  https://doi.org/10.21037/jtd-24-1033
  8. Lab Invest. 2024 Oct 21. pii: S0023-6837(24)01839-7. [Epub ahead of print] 102161
    Real-world performance of integrative clinical genomics in pediatric precision oncology.
    Petra Pokorna, Hana Palova, Sona Adamcova, Robin Jugas, Dagmar Al Tukmachi, Michal Kyr, Dana Knoflickova, Katerina Kozelkova, Vojtech Bystry, Sona Mejstrikova, Tomas Merta, Karolina Trachtova, Eliska Podlipna, Peter Mudry, Zdenek Pavelka, Viera Bajciova, Pavel Tinka, Marie Jarosova, Tina Catela Ivkovic, Sibylle Madlener, Karol Pal, Natalia Stepien, Lisa Mayr, Boris Tichy, Klara Drabova, Marta Jezova, Sarka Kozakova, Jitka Vanackova, Lenka Radova, Karin Steininger, Christine Haberler, Johannes Gojo, Jaroslav Sterba, Ondrej Slaby.
      Despite significant improvement in the survival of pediatric cancer patients, treatment outcomes for high-risk, relapsed, and refractory cancers remain unsatisfactory. Moreover, prolonged survival is frequently associated with long-term adverse effects due to intensive multimodal treatments. Accelerating the progress of pediatric oncology requires both therapeutic advances and strategies to mitigate the long-term cytotoxic side effects, potentially through targeting specific molecular drivers of pediatric malignancies. In this report, we present the results of integrative genomic and transcriptomic profiling of 230 patients with malignant solid tumors (the "primary cohort") and 18 patients with recurrent or otherwise difficult-to-treat nonmalignant conditions (the "secondary cohort"). The integrative workflow for the primary cohort enabled the identification of clinically significant single-nucleotide variants, small insertions/deletions, and fusion genes, which were found in 55% and 28% of patients, respectively. For 38% of patients, molecularly informed treatment recommendations were made. In the secondary cohort, known or potentially driving alteration was detected in 89% of cases, including a suspected novel causal gene for patients with inclusion body infantile digital fibromatosis. Furthermore, 47% of findings also brought therapeutic implications for subsequent management. Across both cohorts, changes or refinements to the original histopathological diagnoses were achieved in 4% of cases. Our study demonstrates the efficacy of integrating advanced genomic and transcriptomic analyses to identify therapeutic targets, refine diagnoses, and optimize treatment strategies for challenging pediatric and young adult malignancies and underscores the need for broad implementation of precision oncology in clinical settings.
    Keywords:  next-generation sequencing; pediatric oncology; precision medicine
    DOI:  https://doi.org/10.1016/j.labinv.2024.102161
  9. Future Oncol. 2024 Oct 21. 1-11
    Evaluation of a multimodal ctDNA-based assay for detection of aggressive cancers lacking standard screening tests.
    Chi Van Thien Nguyen, Thi Hue Hanh Nguyen, Dac Ho Vo, Thi Tuong Vi Van, Giang Thi Huong Nguyen, Trung Hieu Tran, Trong Hieu Nguyen, Le Anh Khoa Huynh, Thanh Dat Nguyen, Nhat-Huy Tran, Thi Minh Thi Ha, Phan Tuong Quynh Le, Xuan Long Truong, Hong-Dang Luu Nguyen, Uyen Vu Tran, Thanh Quang Hoang, Viet Binh Nguyen, Van Cuong Le, Xuan Chung Nguyen, Thi Minh Phuong Nguyen, Van Hung Nguyen, Nu Thien Nhat Tran, Thi Ngoc Quynh Dang, Manh Hoang Tran, Phuc Nguyen Nguyen, Thi Huyen Dao, Huu Tam Phuc Nguyen, Nhat-Thang Tran, Thi Van Phan, Duy Sinh Nguyen, Hung Sang Tang, Hoa Giang, Minh-Duy Phan, Hoai-Nghia Nguyen, Le Son Tran.
      Aim: Cancers lacking standard screening (LSS) options account for approximately 70% of cancer-related deaths due to late-stage diagnosis. Circulating tumor DNA (ctDNA) is a promising biomarker for multi-cancer early detection. We previously developed SPOT-MAS, a multimodal ctDNA-based assay analyzing methylation and fragmentomic profiles, effective in detecting common cancers (breast, colorectal, liver, lung and gastric). This study extends the analysis to five LSS cancers: endometrial, esophageal, head and neck, ovarian and pancreatic.Methods: SPOT-MAS was applied to profile cfDNA methylation and fragmentomic patterns in 739 healthy individuals and 135 LSS cancer patients.Results: We identified 347 differentially methylated regions and observed genome-wide hypomethylation across all five LSS cancers. Esophageal and head and neck cancers showed an enrichment of short cfDNA fragments (<150 bp). Eleven 4-mer end motifs were consistently altered in cfDNA fragments across all LSS cancers. Many significant signatures were consistent with previous observations in common cancers. Notably, SPOT-MAS achieved 96.2% specificity and 74.8% overall sensitivity, with a lower sensitivity of 60.7% in early-stage cancers.Conclusion: This proof-of-concept study demonstrates that SPOT-MAS a non-invasive test trained on five common cancer types, could detect a number of LSS cancer cases, potentially complementing existing screening programs.
    Keywords:  MCED; ctDNA; endometrial cancer; esophageal cancer; head and neck cancer; methylation and fragmentomic; ovarian cancer; pancreatic cancer
    DOI:  https://doi.org/10.1080/14796694.2024.2413266
  10. Am J Clin Oncol. 2024 Oct 23.
    Circulating DNA in Rectal Cancer to Unravel the Prognostic Potential for Radiation Oncologist: A Meta-analysis.
    Francesco Fiorica, Marta Mandarà, Jacopo Giuliani, Umberto Tebano, Antonella Franceschetto, Milena Gabbani, Elvira Rampello, Giorgia Condarelli, Giuseppe Napoli, Nicoletta Luca, Daniela Mangiola, Marco Muraro, Navdeep Singh, Andrea Remo, Carlotta Giorgi, Paolo Pinton.
       OBJECTIVES: Liquid biopsy, with its noninvasive nature and ability to detect tumor-specific genetic alterations, emerges as an ideal biomarker for monitoring recurrences for locally advanced rectal cancer (LARC). Completed studies have small sample sizes and different experimental methods. To consolidate and assess the collective evidence regarding the prognostic role of circulating DNA (ctDNA) detection in LARC patients undergoing neoadjuvant chemoradiotherapy (nCRT).
    METHODS: Computerized bibliographic searches of MEDLINE and CANCERLIT (2000 to 2023) were supplemented with hand searches of reference lists. Study selection: studies evaluating oncological outcomes of patients with LARC treated with a nCRT comparing patients with positive and negative liquid biopsy at baseline and after nCRT. Data extraction: data on population, intervention, and outcomes were extracted from each study, in accordance with the intention to treat method, by 2 independent observers, and combined using the DerSimonian method and Laird method.
    RESULTS: Nine studies follow inclusion criteria including 678 patients treated with nCRT. The pooled RD rate of ctDNA negative between measure at baseline and after nCRT is statistically significant 61% (95% CI: 53-70, P=0.0002). The hazard ratio (HR) of progression-free survival between ct-DNA negative and positive is significant 7.41 (95% CI: 4.87-11.289, P<0.00001).
    CONCLUSIONS: ctDNA can identify patients with different recurrence risks following nCRT and assess prognosis in patients with LARC. Further prospective study is necessary to determine the utility of ctDNA in personalised therapy for patients with LARC.
    DOI:  https://doi.org/10.1097/COC.0000000000001148
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