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Early detection of primary liver cancer using plasma cell‐free DNA fragmentomics: Do all the pieces come together?

HCC, the most common primary liver malignancy, occurs predominantly in patients with liver cirrhosis or chronic HBV infection[1] and is the thirdleading cause of cancerrelated mortality worldwide.[2] Despite a clear delineation of an atrisk population, the low sensitivity of current screening modalities for detecting earlystage disease,[3] coupled with their woefully low use,[4] results in the majority of patients being diagnosed with advancedstage, incurable disease. As such, improving the early detection of HCC, which has been shown to increase the ability to offer curative therapy and improve prognosis,[5] is a critical unmet need. Liquid biopsy (LB) approaches, which involve analysis of circulating tumor cells, extracellular vesicles (EVs), and cellfree DNA (cfDNA) with a simple blood draw, have shown great promise as a potential effective surveillance strategy.[6] In the current issue, a study by Zhang et al.[7] describes a novel bloodbased assay using analysis of plasma cfDNA fragmentomics— specifically examining the fragment size ratios and fragment size distribution— for the early detection of HCC. The modeling approach was based on the landmark study by Cristiano et al.,[8] which showed that cfDNA fragmentation patterns in cancer patients differ dramatically depending on tissue of origin and accurately discriminate healthy patients from those with malignancy. In this singlecenter, phase 2 biomarker development study, the researchers developed a stacked ensemble machine learning model trained on 192 primary liver cancer (PLC) patients (including HCC, intrahepatic cholangiocarcinoma [ICC], and biphenotypic tumors [combined HCCICC]) and 170 noncancer controls (NCCs). The model was then assessed in an independent validation cohort of 189 PLC patients with 164 NCCs, resulting in an excellent performance for cancer detection with an AUC of 0.995 (96.8% sensitivity at 98.8% specificity). In further sensitivity analysis, the AUC remained excellent for detecting earlystage PLC, small tumors <3 cm, and both HCC and ICC. The researchers are to be congratulated on the impressive performance of their promising novel model. In fact, the minimal deterioration of the performance characteristics at lowerdepth wholegenomesequencing coverage has the potential for a lowcost assay in clinical practice. However, there are several areas that deserve more scrutiny, including concerns about the design of the clinical study cohort, potential logistical and technical limitations in the preanalytical blood processing, and, finally, concerns about the ease of reproducibility of the complex bioinformatic analyses, all of which raise concerns about the applicability and generalizability of this assay to other populations and centers. According to the International Liver Cancer Association White Paper on biomarker development for HCC,[9] phase 2 biomarker studies should ideally include only earlystage HCCs, the appropriate atrisk population, and compare the novel biomarker’s performance to the standard ultrasound ± alphafetoprotein [AFP] screening modality. The current work unfortunately falls short on all three fronts. First, the test subjects are comprised by an extremely heterogeneous liver cancer population, where ICCs and biphenotypic combined HCCICCs— fundamentally different tumors— are lumped together with HCC in both the testing and validation cohorts, comprising 17.1% (33 of 192 PLCs) and 16.9% (32 of 189 PLCs) of the “cases” in each population, respectively. HCC differs significantly from ICC, both radiographically and histologically, and on bloodbased biomarkers (AFP vs. carbohydrate antigen 199). Given the relatively lower discrimination of their assay in distinguishing HCC from ICC (AUC, 0.776), one is left to wonder whether the assay is detecting the presence of any cancer compared to healthy patients, and not specifically primary liver malignancies from the appropriate atrisk population. Inclusion of test patients with nonliver primary malignancies, as well as metastatic liver lesions from extrahepatic primary tumors, would be essential to establish the specificity of this assay for PLC. Furthermore, both the training and validation cohorts include significant numbers of moreadvancedstage patients, including 38% (72 of 192 PLCs) and 33.9% (64 of 189 PLCs) with tumor node Received: 1 February 2022 | Accepted: 1 February 2022