A novel approach for the non-invasive diagnosis of pulmonary nodules using low-depth whole-genome sequencing of cell-free DNA
Original Article

A novel approach for the non-invasive diagnosis of pulmonary nodules using low-depth whole-genome sequencing of cell-free DNA

Bin Zhang1#, Han Liang2,3#, Weiran Liu4#, Xinlan Zhou2,3#, Sitan Qiao2,3#, Fuqiang Li2,3, Pengfei Tian1, Chenguang Li1, Yuchen Ma1, Hua Zhang1, Zhenfa Zhang1, Shigeki Nanjo5, Alessandro Russo6, Joan Anton Puig-Butillé7,8, Kui Wu2,3, Changli Wang1, Xin Zhao2,9, Dongsheng Yue1

1Department of Lung Cancer, Tianjin Lung Cancer Center, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China; 2The Institute of Precision Health, BGI-Shenzhen, Shenzhen, China; 3Guangdong Provincial Key Laboratory of Human Disease Genomics, Shenzhen Key Laboratory of Genomics, BGI-Shenzhen, Shenzhen, China; 4Department of Anesthesiology, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin, China; 5Division of Medical Oncology, Kanazawa University Cancer Research Institute, Kanazawa, Japan; 6Medical Oncology Unit, Papardo Hospital, Messina, Italy; 7Thoracic Oncology Unit, Hospital Clínic, Barcelona, Spain; 8Molecular Biology CORE, Hospital Clínic, Barcelona, Spain; 9Department of Biology, University of Copenhagen, Copenhagen, Denmark

Contributions: (I) Conception and design: D Yue, X Zhao, C Wang, H Liang, B Zhang; (II) Administrative support: C Wang, Z Zhang; (III) Provision of study materials or patients: W Liu, P Tian, Y Ma, H Zhang, C Li, Z Zhang; (IV) Collection and assembly of data: D Yue, X Zhao, C Wang, H Liang, B Zhang, S Qiao, W Liu, P Tian, Y Ma, H Zhang, C Li, Z Zhang; (V) Data analysis and interpretation: B Zhang, H Liang, D Yue, X Zhao, S Qiao; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors.

#These authors contributed equally to this work.

Correspondence to: Dongsheng Yue. Department of Lung Cancer, Tianjin Lung Cancer Center, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China. Email: yuedongsheng@tmu.edu.cn; Xin Zhao. The Institute of Precision Health, BGI-Shenzhen, Shenzhen 518083, China. Email: zhaoxin@genomics.cn; Changli Wang. Department of Lung Cancer, Tianjin Lung Cancer Center, National Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin’s Clinical Research Center for Cancer, Tianjin Medical University Cancer Institute and Hospital, Tianjin 300060, China. Email: wangchangli@tjmuch.com; Kui Wu. The Institute of Precision Health, BGI-Shenzhen, Shenzhen 518083, China. Email: wukui@genomics.cn.

Background: Differentiating between benign and malignant pulmonary nodules is a diagnostic challenge, and inaccurate detection can result in unnecessary invasive procedures. Cell-free DNA (cfDNA) has been successfully utilized to detect various solid tumors. In this study, we developed a genome-wide approach to explore the characteristics of cfDNA sequencing reads obtained by low-depth whole-genome sequencing (LD-WGS) to diagnose pulmonary nodules.

Methods: LD-WGS was performed on cfDNA extracted from 420 plasma samples from individuals with pulmonary nodules that were no more than 30 mm in diameter, as determined by computed tomography (CT). The sequencing read distribution patterns of cfDNA were analyzed and used to establish a model for distinguishing benign from malignant pulmonary nodules.

Results: We proposed the concept of weighted reads distribution difference (WRDD) based on the copy number alterations (CNAs) of cfDNA to construct a benign and malignant diagnostic (BEMAD) algorithm model. In a training cohort of 360 plasma samples, the model achieved an average area under the receiver operating characteristic (ROC) curve (AUC) value of 0.84 in 10-fold cross-validation. The model was validated in an independent cohort of 60 plasma samples, obtaining an AUC value of 0.87. The BEMAD model could distinguish benign from malignant nodules at a sensitivity of 74% and a specificity of 86%. Furthermore, analysis of the critical features of the cfDNA using the BEMAD model identified repeat regions that were associated with microsatellite instability, which is an important indicator of tumorigenesis.

Conclusions: This study provides a novel non-invasive diagnostic approach to discriminate between benign and malignant pulmonary nodules to avoid unnecessary invasive procedures.

Keywords: Cell-free DNA (cfDNA); diagnostic algorithm; non-small cell lung cancer (NSCLC); whole-genome sequencing (WGS); copy number alterations (CNAs)

Submitted Jun 29, 2022. Accepted for publication Oct 10, 2022.

doi: 10.21037/tlcr-22-647


The introduction of low-dose computed tomography (CT) for lung cancer screening, as well as CT imaging for other indications, has significantly increased the detection rate of pulmonary nodules in recent years, with approximately 1.5 million cases estimated per year in the United States (1,2). Despite reducing lung cancer mortality through the detection of pulmonary nodules, low-dose CT has an extremely high false-positive rate (96.4%), which leads to unnecessary workups of benign nodules (3-5). The detective methods to make a difference between benign and malignant pulmonary nodules mainly include CT/positron emission tomography (PET)-CT, long-term follow-up CT scans or even invasive procedures like bronchoscopy, transthoracic fine-needle aspiration biopsy, and surgery. Size and growth rate of pulmonary nodules are the main indicators to assess probability of nodule malignancy. Usually, lung nodules measured more than 30 mm in diameter on CT imaging are more likely to be cancerous than smaller nodules. It is still challenging to evaluate the malignancy risk for lung nodules with diameters of less than 30 mm only depending on CT scan. Long-term follow-up CT scans and invasive procedures result in a significant burden as most small lung nodules are benign (6-9). Thus, non-invasive methods are urgently required to aid in the differentiation of benign from malignant pulmonary nodules.

Plasma cell-free DNA (cfDNA) are short DNA fragments of double-stranded DNA ranging around a modal size of ~166 bp circulating in the blood that are released by apoptotic or necrotic cells but also by active secretion of non-damaged cells. The half-life of cfDNA is relatively short with 16 min to 2.5 h in the blood. The level of cfDNA is much lower in plasma from healthy individuals than cancer patients. Plasma cfDNA have been successfully utilized in the detection and diagnosis of various cancer types, including hematological malignancies, breast cancer, osteosarcoma, and ovarian cancer (10,11). Circulating tumor DNA (ctDNA) accounts for a variable proportion of the total cfDNA (12-14). CtDNA is believed to contain the same genetic aberrations as the corresponding tumor. The potential utility of ctDNA for early diagnosis, detection of minimal residual disease and molecular genotyping has been explored in lung cancer (15,16). However, it is difficult to reliably detect mutated ctDNA in early-stage non-small cell lung cancer (NSCLC) due to the low abundance of ctDNA in cfDNA, which is estimated to be less than 0.01% in 50% of patients with stage I NSCLC (17,18). Mutation-based cfDNA tests, which included the whole-exome sequencing (WES) and targeted multiple gene sequencing (Panel-seq) in early-stage NSCLC require ultra-deep coverage that are costly and technically challenging due to sequencing artifacts, vast mutational heterogeneity between patients, and the frequent occurrence of non-malignant somatic mutations in cfDNA, e.g., those that drive clonal hematopoiesis (12,19-22). Therefore, new non-invasive approaches other than mutation-based cfDNA tests should be explored.

Whole-genome sequencing (WGS) of cfDNA can identify chromosomal abnormalities such as copy number alterations (CNAs) in cancer (23,24). CNAs usually range from 1 Mb to 100+ Mb, spanning large genomic regions. Some studies have reported that, compared with point mutations, the detection of cancer-derived CNA events in cfDNA is likely to be a superior approach for ctDNA-based early cancer detection because ctDNA CNAs contribute a much larger number of ctDNA fragments to the total cfDNA per CNA event (24,25). CNAs have been previously identified to be specific biomarkers of lung malignancy (26,27). CfDNA fragmentomics, other than CNA events in cfDNA, also provides a way for non-invasive detection of lung cancer. It was reported that genome-wide cfDNA fragmentation analysis such as DNA evaluation of fragments for early interception (DELFI) approach can discriminate lung cancer patients from non-cancer individuals (28-30). Non-invasive prenatal testing (NIPT) utilizes low genomic depth (<1×) to detect large chromosomal aberrations. With the widespread use of NIPT, some women have been found to have CNAs in their plasma originating from undiagnosed maternal cancer (31,32). These findings highlight the potential of detecting cfDNA CNAs in early-stage cancer.

The feasibility of discriminating between benign and malignant pulmonary nodules according to the WGS-derived characteristics of cfDNA has not been adequately investigated (33). Compared to WES of plasma cfDNA, low-depth WGS (LD-WGS) was more reliable, more efficient, less expensive for the detection of CNAs. In this study, we developed a novel genome-wide approach to distinguish benign from malignant pulmonary nodules using LD-WGS of cfDNA. This method has the potential to provide a cost-effective, non-invasive diagnostic approach for the accurate diagnosis of pulmonary nodules. We present the following article in accordance with the TRIPOD reporting checklist (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-647/rc).


Study population

Patients undergoing treatment for suspected lung cancer at Tianjin Medical University Cancer Institute and Hospital between June 2015 and August 2017 were considered for this study. All of the following inclusion criteria had to be fulfilled: (I) lung nodules were no more than 30 mm in diameter as determined by CT scan; (II) lung nodules were evaluated as malignant by radiologists and physicians, were undiagnosed, or were evaluated as benign but the patients preferred surgery; and (III) patients underwent surgical excision, bronchoscopic biopsy, or transthoracic biopsy. The exclusion criteria were as follows: (I) patients with other malignant tumors; (II) those with no pathological diagnosis; and (III) subtypes of lung malignancy were small cell lung cancer or pulmonary large cell neuroendocrine carcinoma. A total of 420 cases, including 131 patients with benign pulmonary nodules and 289 patients with NSCLC, were analyzed.

The initial diagnoses were obtained by CT. Plasma samples were collected 1–5 days before treatment and within 1 month after nodule detection. Pathological diagnosis was made on the basis of biopsy or surgical samples. The staging was according to the 8th Edition of the International Staging of Thoracic Malignancies and the histological subtype was according to the 2015 World Health Organization (WHO) classification of lung tumors (34,35). We collected the following clinicopathological data: age, sex, smoking status, histological subtype, stage, and CT results reported by the radiologist. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). This study was approved by the ethical committee of Tianjin Medical University Cancer Institute and Hospital (approval Nos. bc2016014, bc2018009, and bc2019091), and all participants provided written informed consent.

External independent dataset from The Chinese University of Hong Kong (CUHK) including 38 healthy controls and 10 lung cancer patients was used for validation (36).

Sample preparation, library construction, and sequencing

A 5-mL sample of whole blood was collected from each patient in an ethylenediaminetetraacetic acid (EDTA) tube and processed immediately. Plasma and cellular components were separated by centrifugation at 1,600 ×g for 10 min at 4 ℃. Plasma was further centrifuged for 10 min at 16,000 ×g at 4 ℃ to remove any remaining cellular debris and then stored at −80 ℃. CfDNA was extracted using a MagPure Circulating DNA KingFisher (KF) Kit (Magen, Guangzhou, China). The concentration of the extracted cfDNA was quantified by a Qubit 3.0 fluorometer (Life Technologies, Paisley, UK), and the size distribution was detected using an Agilent DNA High Sensitivity Kit on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The total cfDNA of each plasma sample was input for library preparation using the MGIEasy cfDNA Library Prep Set (MGI-Tech, Shenzhen, China).

CfDNA isolation and WGS library construction were both performed using a MGISP-960 High-Throughput Automated Sample Preparation System (MGI-Tech) according to the manufacturer’s protocol. Briefly, purified cfDNA was subjected to end repair, A-tailing, ligation modules, polymerase chain reaction (PCR) amplification, and single-strand circularization. All single-strand circular DNA libraries were sequenced on the MGISEQ-2000 platform (MGI-Tech) with paired-end reads to generate approximately 1.5–3 Gb of whole-genome data for each sample with a coverage of 37% at 1× depth (MGI-Tech). The quantity of cfDNA is shown in (available online: https://cdn.amegroups.cn/static/public/tlcr-22-647-1.xlsx).

Calculation of weighted reads distribution difference (WRDD)

Following WGS, the sequencing reads were aligned to the hg38 reference genome. All autosomes were joined together and divided into a series of fixed-length windows (30 kb/window) along the DNA. We then counted the read number of each sample within each window of DNA. Differences in the read counts along a series of regions comprised the read distribution pattern, which reflected the bias of the read distribution related to the sample type.

For each sample, we combined the varying number of read windows to create a region and observed the features of this region. To identify the critical distribution characteristics of the reads across the genome, we defined the WRDD, which emphasizes CNAs in regions of the whole genome and provides additional genomic information. If a region consisted of n windows, the read number matrix x of a sample set with m samples within the region could be described as:


where xi,j is the read number of sample i in window j. The weight value w of these n windows could be calculated as:


where the function variance calculates the variance value of the read numbers of m samples in each window. The average read number of each window was used as the benchmark. The read numbers of benchmark b of these n windows could be calculated as:


where the function mean calculates the mean value of the read number of m samples in each window. The weighted sum value Si of sample i could be calculated as:


where the function scale calculates the scaled values of a given number list. Read numbers were scaled by sample. If the sum of the weighted differences in the read number of benign values in a given region had a higher variance than that of the malignant samples, that region was referred to as a benign variable (BV) region; otherwise, it was referred as a malignant variable (MV) region. The WRDD value was calculated differently for BV regions than for MV regions, as follows:


Calculation of t-values

For a given region, a t-value was calculated to measure the ability of the region to differentiate sample types. For a specific region, the t-value was calculated according to the WRDD values of the two types of samples using the following formula:


where x1¯ and x2¯ are the means of the two WRDD value lists that originate from the two sample sets, while n1 and n2 are the element numbers of the two sample sets, and S1 and S2 are their standard deviation (SD) values, respectively. A higher t-value was indicative of a better ability of a particular region to differentiate between the sample types.

Design of the genetic algorithm

To explore regions with high t-values, we developed a modified genetic algorithm. First, a simple strategy was adopted to generate a series of original regions. The genetic algorithm was then used to perform continuous combination and separation operations on these regions to obtain new regions with higher t-values.

  • Generation of the original regions. A continuous series of n windows at location i was combined with another series of n windows located 2jn windows downstream of the location to obtain an original region consisting of 2n windows. The original region that began at the window i was defined as:


    • where n is the number of a series of continuous windows (n=5) and N is the total number of windows (N=95,833). A certain distance between the two series of windows was specified to ensure that the regions possessed discriminatory abilities across long distances.
  • Combination and separation of regions. The genetic algorithm involved the combination of two parental regions for information exchange and the generation of offspring regions. All original regions were put in a regional pool for the random selection of parental regions to generate offspring regions. The probability that region i was selected as one of the parental regions was as follows:


    • where N is the total number of all existing regions and ti is the t-value of the i-th region. Given that region x had been selected as the first parental region, the probability that another region i was selected as the second parental region was as follows:


    • where N is the total number of all existing regions, mi is the mean of the positions of all windows contained by the region i, and ti is the t-value of the i-th region. The inclusion of the term |mx−mi| was set for the preferential selection of regions with a shorter inter-regional distance as the parental regions. Following the selection of both parental regions, 20% of the unified windows included in the two parental regions were randomly selected by sampling with the replacement and deleting it to form the offspring region. The obtained offspring region was then added to the regional pool for the next round of selection. The parental regions were not deleted during the entire process. The offspring region F generated by two parental regions P1 and P2 and probability p were defined as:


    • where S(p,s) is the subset of p% elements sampled randomly from set s with replacement (in this work, p=20%). The offspring generation was analyzed 300,000 times to generate the same number of regions, which balanced the costs and potential effects.

Benign and malignant diagnostic (BEMAD) model construction and prediction

BV and MV regions with the highest t-values were selected to construct the BEMAD model. When using the model with N regions to determine a sample’s type, the sum of the WRDDs of the sample in the N responding regions was calculated and used as the score of the sample for prediction.

To eliminate the influence of the imbalance in t-values between regions of different types, we separately calculated the sample scores for the MV and BV regions. The sums of the scores in BV and MV regions were called the BV and MV region scores, respectively. We then scaled the BV and MV region scores of a sample and combined them to obtain its final score:


where scorebenign is the BV region score of a sample, scoremalignant is the MV region score, scorebengintrain¯ is the mean BV score of the corresponding training cohort, Sbengintrain is its SD value, scoremalignanttrain¯ is the mean of the MV region score of the corresponding training cohort, and Smalignanttrain is its SD value. We determined whether a sample was benign or malignant based on its score.

The process of developing the BEMAD model according to the WRDD was as follows (Figure 1):

  • A region was generated by combining windows in the genome (Figure 1A). The whole genome was divided into a series of 30-kilo-base pair fixed-length and non-overlapping windows, which resulted in 95,833 windows including trans-chromosomal windows. The average read number in a series of benign and malignant samples was calculated as the benchmark for each window. Regions were generated by combining windows that were selected with a modified genetic algorithm.
  • The WRDD value of a sample in a region was calculated by converting the sum of the weighted differences of read numbers between the sample and the benchmark of the windows contained within a region (Figure 1B). To eliminate differences in the read number of the sample and its benchmark in that region, we scaled the read numbers of the sample in all windows within a region to ensure that the total read numbers were the same at the regional level. The read numbers of the benchmarks were also scaled in the same way. Given that the sum values of benign samples were further away from 0 (above or under) in the BV regions, while those of malignant samples were closer to 0, we converted the sum values to their absolute values to clearly separate them, which resulted in higher absolute values for the benign samples. The sum values in MV regions were converted using the same method, resulting in higher absolute values for the malignant samples. To ensure that the absolute values of the malignant samples were higher than those of the benign samples in both the BV and MV regions, we further converted the absolute values in the BV regions to their inverse values. To summarize, the WRDD of BV regions = −|sum|, while the WRDD for MV regions = |sum|. A t-value was used to evaluate the discrimination capacity of the regions in the benign and malignant groups. The two WRDD value sets obtained from the two sample groups were then used to calculate the t-value of a region; a higher t-value was indicative of a greater ability of a region to distinguish the respective sample group.
  • Regions with the top x of t-values were selected for the model construction (Figure 1C). The sum of the WRDDs within the regions generated the score of a sample, which was used to predict whether the sample was benign or malignant.
Figure 1 Flowchart of the BEMAD algorithm model development. (A) Generation of region. (B) Calculation of WRDD. (C) Model-construction & prediction. WRDD, weighted reads distribution difference; MV, malignant variable; BV, benign variable; BEMAD, benign and malignant diagnostic.

Statistical analyses

All statistical analyses were performed using R version 3.6.3. The area under the receiver operating characteristic (ROC) curve (AUC) values, 95% confidence intervals (CIs), cut-off value related to the maximum AUC, and the corresponding specificity and sensitivity from the model output were obtained with the pROC (v.1.16.2) R package (37). The Mann-Whitney U test was performed to determine the differences in score distributions between two groups. The Student’s t-test was used to measure the differences in clinicopathological factors with continuous variables (e.g., age) between patients with benign and malignant pulmonary nodules, and Fisher’s exact test was performed to measure the differences in clinicopathological features with discrete variables (e.g., sex) using the built-in functions, “t.test” and “fisher.test”, respectively, of the current version of R. The counting of read numbers was performed using “readcounter”, a tool in HMMcopy (Bioconductor R package version 1.32.0.). The investigators were not blinded to the groups during experiments and outcome assessments. A P value of <0.05 indicated statistical significance.


Patients’ features

We retrospectively analyzed plasma samples from 420 individuals with lung nodules of ≤30 mm diameter according to CT images (Table 1). Of these, 289 (69%) were NSCLC patients [adenocarcinoma, n=240 (83%); and squamous cell carcinoma, n=49 (17%)], which were referred to as malignant samples. The remaining 131 (31%) patients had benign pulmonary nodules [cyst, n=4 (3%); fibrosis, n=7 (5%); granuloma, n=24 (18%); hamartoma, n=34 (26%); inflammation, n=35 (27%); tuberculosis, n=17 (13%); other cases, n=10 (8%)], which were referred to as benign samples. Of the 289 NSCLC patients, 247 (85%) were stage I (229 of whom were stage IA and 18 were stage IB).

Table 1

The clinicopathological characteristics of the included patients

Characteristics Total Training cohort Validation cohort
Benign Malignant P value Benign Malignant P value Benign Malignant P value
Count, n [%] 131 [31] 289 [69] 101 [28] 259 [72] 30 [50] 30 [50]
Age, mean ± SD 53±10 59±8.4 7.70E-07 54±10 59±8.3 2.40E-05 52±12 57±9.3 0.037
Sex, n [%] 0.074 0.24 0.29
   Female 55 [42] 149 [52] 45 [45] 134 [52] 10 [33] 15 [50]
   Male 76 [58] 140 [48] 56 [55] 125 [48] 20 [67] 15 [50]
Smoking, n [%] 0.83 0.81 1
   No 67 [51] 153 [53] 51 [50] 135 [52] 16 [53] 17 [57]
   Yes 64 [49] 136 [47] 50 [50] 124 [48] 14 [47] 13 [43]
Smoking-years (for smokers only), mean ± SD 29±12 32±13 0.088 29±12 32±13 0.14 29±13 34±13 0.35
Pack-years (for smokers only), mean ± SD 26±17 22±14 0.18 25±16 22±14 0.26 28±23 24±13 0.59
Nodule size (mm), mean ± SD 19±8.1 21±7.4 0.042 19±8.2 21±7.5 0.029 21±8 21±7.1 0.72
Pathological stage, n [%]
   I 247 [85] 224 [86] 23 [77]
   II 15 [5] 12 [5] 3 [10]
   III 27 [9] 23 [9] 4 [13]
Subtype, n [%]
   AD 240 [83] 216 [83] 24 [80]
   SC 49 [17] 43 [17] 6 [20]
   Cyst 4 [3] 3 [3] 1 [3]
   Fibrosis 7 [5] 4 [4] 3 [10]
   Granuloma 24 [18] 20 [20] 4 [13]
   Hamartoma 34 [26] 29 [29] 5 [17]
   Inflammation 35 [27] 29 [29] 6 [20]
   Tuberculosis 17 [13] 11 [11] 6 [20]
   Others 10 [8] 5 [5] 5 [17]
Procedure type, n [%]
   Surgical excision 119 [91] 284 [98]
   Bronchoscopic biopsy 0 [0] 3 [1]
   transthoracic biopsy 12 [9] 2 [1]

When comparing age/smoking-years/pack-years/nodule size, Student’s t-test was used to calculate the P value; when comparing sex/smoking, Fisher’s exact test was used to calculate the P value. SD, standard deviation; AD, adenocarcinoma; SC, squamous cell carcinoma.

The malignant and benign samples were matched for sex, smoking status, and pack-years of smoking (P>0.05). NSCLC patients were older compared to individuals with benign pulmonary nodules (P=7.70e–7; Student’s t-test). The mean nodule size of the malignant samples was larger than that of the benign samples (P=0.042; Student’s t-test). The samples were randomly divided into a training cohort (n=360, including 101 benign samples and 259 malignant samples) and an independent validation cohort (n=60, including 30 benign samples and 30 malignant samples) at a ratio of 6:1. For the independent validation cohort, we limited the ratio of benign to malignant samples to 1:1. A workflow of the study is shown in Figure S1.

Development of the BEMAD model

We designed a procedure with 10-fold cross-validation in the training cohort (Figure 2). Higher t-values were identified in the BV regions compared with the MV regions. Approximately 99% of regions with the top 1,000 t-values were BV regions. Owing to the large differences in t-values between the BV and MV regions, the two types of regions were analyzed separately. BV regions with the 10 highest t-values were chosen first for the model construction (Figure 2A, available online: https://cdn.amegroups.cn/static/public/tlcr-22-647-2.xlsx). The mean AUC in 10 test sets of the BV region-based model was 0.8 (95% CI: 0.73–0.86). To explore the distribution of sample scores in different histological subtypes, we compared them in 10 test sets. We first normalized the scores from the same dataset to eliminate the differences in scores among the different test sets. We found that the average score of the malignant samples was higher than that of the benign samples (P=2.6e–20; Mann-Whitney U test; Figure 2A). There was no difference between the average scores of adenocarcinomas and squamous cell carcinomas.

Figure 2 BEMAD model construction and score distribution. The testing procedure was designed on the basis of 10-fold cross-validation. (A) Ten BV regions with the highest t-values were used to construct the BEMAD model. Left: 10 AUCs generated from 10 test sets in the 10-fold cross-validation and the mean AUC are shown. Right: Distribution of normalized scores of BV regions in samples of different histological subtypes. Differences between the scores were compared. (B) Ten MV regions with the highest t-values were used to construct the BEMAD model. (C) The regions in (A,B) were combined (20 regions in total) for the BEMAD model construction. BV, benign variable; MV, malignant variable; AD, adenocarcinoma; SC, squamous cell carcinoma; BEMAD, benign and malignant diagnostic; AUC, area under the receiver operating characteristic curve.

Subsequently, MV regions with the 10 highest t-values were selected to run the same procedure as the construction of the BV-based model (Figure 2B, available online: https://cdn.amegroups.cn/static/public/tlcr-22-647-2.xlsx). The MV region-based model obtained a mean AUC value of 0.72 (95% CI: 0.65–0.78). The score distributions also revealed that malignant samples had a higher average score (P=4.0e–11; Mann-Whitney U test; Figure 2B), and there was no difference between the average scores of adenocarcinomas and squamous cell carcinomas.

Considering the significant differences between the performances of the two models constructed using the top 10 BV regions and the top 10 MV regions, we combined these 20 regions for the construction of the BEMAD model (Figure 2C). Since the t-values of the top 10 BV regions were higher than those of the top 10 MV regions, the direct combination of the regions would have weakened the contribution of the MV regions toward the prediction results. To resolve this issue, we separately normalized the scores of the MV and BV regions of each sample and summed the two normalized scores to obtain the final score. The average AUC increased to 0.84 (95% CI: 0.80–0.89), with a sensitivity of 80% and a specificity of 83%.

We compared our method to other cfDNA genome-wide cancer detection approaches, including ichorCNA and DELFI using the LUCAS cohort (29,38). The DELFI approach achieved an AUC of 0.94 (95% CI: 0.91–0.98). Our BEMAD model provided a similar performance, with an average AUC of 0.93 (95% CI: 0.91–0.95). Meanwhile, the ichorCNA approach obtained an AUC of 0.80 (95% CI: 0.73–0.87) (Figure S2). These results suggested that our method was comparable to the DELFI approach and superior to the ichorCNA approach.

BEMAD algorithm model validation

The BEMAD algorithm model was validated in an independent validation cohort containing 30 benign samples and 30 malignant samples. The model obtained an AUC value of 0.87 (95% CI: 0.82–0.97) (Figure 3A), and the sensitivity and specificity were 74% and 86%, respectively. The distributions of the benign and malignant sample scores were similar to those observed in the training cohort, with a higher average score obtained in the malignant samples (Figure 3B). Additionally, we used another external independent dataset from The CUHK including 38 healthy control and 10 lung cancer patients for validation (36). Our model’s performance achieved 0.84 (95% CI: 0.70–0.97) of AUC, with a sensitivity of 0.8 and a specificity of 0.82 (Figure S3).

Figure 3 BEMAD algorithm model validation in the independent validation cohort. (A) AUC values of the independent validation cohort, which consisted of 30 benign and 30 malignant samples. (B) Distribution of the normalized scores of samples with different histological subtypes. AUC, area under the receiver operating characteristic curve; AD, adenocarcinoma; SC, squamous cell carcinoma; BEMAD, benign and malignant diagnostic.

A retrospective review of the patients’ CT reports revealed that there were 47 undiagnosed lung nodules in the training cohort and nine undiagnosed lung nodules in the validation cohort. According to the pathological diagnoses, the BEMAD model correctly identified 25 of 28 (89%) benign lung nodules and 15 of 19 (79%) malignant lung nodules in the training cohort, and 7 (100%) benign and 2 (100%) malignant lung nodules in the validation cohort. This indicated that our cfDNA-based method could further stratify undiagnosed lung nodules, aiding in CT to improve the differential diagnosis of lung nodules and reducing unnecessary invasive procedures.

Influence of clinicopathological parameters on the performance of the BEMAD algorithm model

To determine whether clinicopathological factors affected the performance of the BEMAD model, we analyzed the effects of sex, age, smoking status, and nodule size on the score distribution of the samples. The differences in the score distributions of benign and malignant samples were compared according to sex (female vs. male), smoking status (smoking vs. non-smoking), age (<60 vs. ≥60 years), and nodule size (≤10 vs. >10 mm) (Figure 4A-4D). All differences in score distributions in the benign and malignant samples were non-significant (P>0.05; Mann-Whitney U test), indicating that the BEMAD model was robust and unlikely to be influenced by clinicopathological factors.

Figure 4 Comparisons of the score distributions between benign and malignant samples with clinicopathological factors in the training cohort. Comparisons of the score distributions between benign and malignant samples in terms of sex (female vs. male) (A), smoking status (smoking vs. non-smoking) (B), age (<60 vs. 60 years) (C), and nodule size (10 and >10 mm) (D).

Characteristics of critical windows in the BEMAD model

To better understand the characteristics of selected genomic regions in the BEMAD model and their potential correlation with tumorigenesis, we analyzed the features of windows within each genomic region that was used for the construction of the BEMAD model. We found that some windows were critical in distinguishing malignant from benign samples, as demonstrated by the highest t-value among the windows in that region. We also observed that the critical window for 50% of the MV regions was window #57113 (chr10:38503000–38532999 in the hg38 reference genome), while the critical window for the remaining MV regions was window #92938 (chr21:10657000–10686999), and that for all BV regions was #92863 (chr21:8407000–8436999).

Interestingly, we found that many repeated motifs within these three windows could be classified into one of three types: “AATGG” for #57113, “TCCAT” for #92938, and “TCTC” for #92863. The first two repeated motifs were associated with microsatellite repeats, influencing microsatellite instability, which is a critical marker of tumorigenesis (39,40). The third repeated motif was novel and should be studied in the future.

Indeed, we found that in the region with numerous “AATGG” and “TCCAT” motifs, the differences in read numbers between benign and malignant samples were more significant compared with their upstream regions (Figure 5A,5B). However, this was not observed for the “TCTC” motif. These findings indicated that microsatellite instability is a feature of critical windows in the BEMAD model.

Figure 5 Relationship between the frequency of repeated motifs and differences in read number. (A) Analysis of the “AATGG” motif, aligned by the AATGG nucleotide base. Each window covered a 30-kilo-base pair region in the reference and all 30 windows covered the range of chr10:38443000–38532999. A total of 131 benign samples and 289 malignant samples were analyzed. Top: differences in the read numbers between benign and malignant samples. Middle: transformed values of −log(P); P values were calculated using the Student’s t-test. P values of 0.05 are highlighted in maroon. Bottom: frequency of repeated motif. (B) Analysis of the “TCCAT” motif, aligned by the TCCAT nucleotide base. All 30 windows covered the range of chr21:10597000–10686999.


The precise diagnosis of lung nodules is challenging. In clinical practice, 10–30% of resected lung nodules are found to be benign, which is a result of the limitations of radiological detection and the inability of CT to accurately distinguish benign from malignant nodules (41). For these patients, surgery represents overtreatment. Therefore, the development of economical non-invasive approaches that can discriminate benign from malignant pulmonary nodules is extremely valuable to aid diagnosis and reduce the number of unnecessary surgeries performed in patients with benign tumors. In this study, we developed an algorithm that utilized cfDNA LD-WGS data to differentiate between benign and malignant pulmonary nodules.

Cancer-associated CNAs have been detected in the cfDNA of patients with cancer, underscoring their potential clinical applications for the screening, early detection, and monitoring of human cancer (23,24,42-44). However, CNAs only reflect copy number variation relative to normal controls in some specific locations in the genome. Our approach introduces a new bioinformatics analysis to reflect genomic changes, namely WRDD. WRDD identifies differences in read numbers at multiple locations within a genomic region and amplifies local differences through weighting. In addition, it offers more information than CNAs, which is critical for capturing the extremely weak signals of cfDNA in early-stage NSCLC. Furthermore, WRDD allows for discontinuity within a region, which enables the elimination of unimportant windows when a region spans a large area. Although both CNAs and WRDD reflect abnormal gains or losses in the genome, WRDD serves as an extension of CNAs that can provide additional signal details and can also reflect the early characteristics of genomic instability in NSCLC.

To confirm the suitable performance of our method, we compared it to the other approaches including ichorCNA and DELFI using their cohort (29,30). Our BEMAD model achieved an average AUC of 0.93, which was almost identical to that obtained by DELFI (AUC of 0.94) and superior to that of ichorCNA (AUC of 0.80). Notably, the percentage of stage I patients in their cohort (training cohort 15/129, validation cohort 28/46) in which detection is more challenging than the later stages was far smaller than ours (training cohort 224/259, validation cohort 23/30). Therefore, the superior approach still needs to be validated with cohorts that include a large number of early-stage patients.

In recent years, cfDNA methylation patterns have demonstrated their potential utility in the early detection of several cancer types including lung cancer. However, various methods have been applied in different studies, including cfDNA methylation and bioinformatics analyses. Moreover, most of these studies focused on distinguishing cancer patients from healthy individuals, and few studies have explored the differentiation of benign from malignant lesions (33,45-49). Liang et al. attempted to discriminate between benign and malignant pulmonary nodule samples using cfDNA whole-genome bisulfite sequencing data; their model produced an AUC value of 0.839 in a training cohort containing 40 malignant and 26 benign samples, and an AUC of 0.816 in an independent validation cohort containing 39 malignant and 27 benign samples. In comparison, this study obtained AUC values of 0.84 and 0.87, respectively (33). Therefore, the performance of our WRDD-based cfDNA detection method for the discrimination of benign and malignant pulmonary nodules is comparable to that of the methylation signal-based cfDNA diagnostic method. Notably, inflammation-related benign diseases (inflammation and inflammatory granuloma), which are often erroneously classified as malignant in clinical practice, accounted for a smaller proportion of the benign samples in the study by Liang et al. (11/53=20.7%, as compared with 59/131=45.0% in the present study) (33). In addition, our LD-WGS approach is easier to implement in molecular testing laboratories. DNA methylation analysis requires a higher cfDNA amount, at 10 ng, which is 4–5 times greater than that required by our model. DNA methylation analysis also requires the genome coverage to be greater than 30×, which limits the use of this technique in clinical practice.

There were reports about the potential application of cfDNA from other liquid biopsy sources like bronchoalveolar lavage fluid (BALF) for identifying lung cancer (50,51). However, the performance of BALF cfDNA was not superior to that of plasma cfDNA to differentiate malignant from benign pulmonary nodules by analyzing the methylation and mutation profiling of cfDNA (50). It will be interesting to compare CNAs signatures from BALF cfDNA to our approach from plasma cfDNA for identifying lung cancer. Considering that obtaining BALF is invasive compared with collecting plasma, plasma cfDNA could be more suitable for early detection of lung cancer.

The differential diagnosis of pulmonary nodules of 6–20 mm is challenging for radiologists and thoracic surgeons, and usually requires long-term CT surveillance and even invasive procedures (8). In our cohort, the AUC value of pulmonary nodules of 10–20 mm was 0.83, while that of nodules ≤10 mm was 0.86 (Figure S4). The performance of the BEMAD model in differentiating benign from malignant lung nodules was not influenced by nodule size. For the undiagnosed lung nodules in our training cohort, the BEMAD model correctly identified 89% of benign nodules and 79% of malignant lung nodules. The real-world impact of this model is that approximately 50% of patients with benign lung nodules would avoid longitudinal radiographical follow-up, as recommended by the Fleischner Society guidelines (8), or invasive procedures. These results suggest that our model has the potential to be used as an adjuvant with CT in most patients with benign nodules to avoid unnecessary surgery.

To explore the underlying biological explanations of differentiation between benign and malignant samples based on the critical window for BV and MV regions used for the construction of the BEMAD model, we annotated the regions. These regions are mostly intergenic regions, which could act to control the expression of nearby genes. We noticed that windows #57113 and #92863 included a significant number of non-coding RNAs, while window #92938 nearby included immune-related genes (available online: https://cdn.amegroups.cn/static/public/tlcr-22-647-3.xlsx). It is known that non-coding RNAs and immune regulation play important roles in carcinogenesis (52,53), which could be involved in the differentiation of benign and malignant samples.

The present study has some limitations that should be noted. Firstly, the study lacks completely independent validation cohorts from another center. Thus, it would be useful to validate the model at other centers to assess the scalability of our results in other populations. Secondly, radiomic nodule characteristics such as nodule quality (solid, part-solid, or non-solid) and spiculation were not available in our cohort; therefore, it may be useful to assess how radiomic characteristics affect the performance of our model, which would in turn enable a direct comparison with the Veterans Affairs SNAP Cooperative Study Group and Mayo Clinic models (54,55). Thirdly, all samples included in this study were retrospective; thus, a prospective study to assess the performance of the BEMAD model is required for clinical application. It is worth noting that NIPT has been widely used in reproductive medicine for robust clinical evaluation due to the relatively low amount of cfDNA required, the use of genomic sequencing data, and the convenient experimental process with high sensitivity and specificity. Our experimental process could be easily plugged into the current NIPT clinical workflow for whole-genome library construction and data generation using cfDNA. When the BEMAD model was deployed in the diagnostic computational system, it could efficiently provide an accurate evaluation of benign and malignant pulmonary nodules. Hence, our approach could guide subsequent clinical decisions to improve patient care.

The advantages of our approach are that LD-WGS is inexpensive, the method referring to the workflow of NIPT is feasible, and there are no training issues that arise when introducing this approach to clinical practice. So it is worth wide application in diagnosis of pulmonary nodules in clinical. As an auxiliary screening technology for lung cancer, the input data (LD-WGS cfDNA) can be generated by existing conventional non-invasive prenatal screening and diagnostic technologies (such as NIPT), with lower sampling requirements and easier experimental accessibility than methylation sequencing technology. Furthermore, the cost of our approach is one-tenth that of conventional WGS (3 Gb base pair/sample vs. 30 Gb base pair/sample), and much lower than mutation-based methods, such as WES and Panel-seq (a minimum raw data of 100G base pair/sample and 20G base pair/sample). It will be important to explore combining the optimized BEMAD algorithm model with multi-omics biomarkers like the critical features from the miRNA expression and certain methylation variable positions of cfDNA used for early pan-cancer screening to further improve the sensitivity and specificity of benign and malignant diagnoses of pulmonary nodules before its clinical application (56).


In conclusion, we developed a novel non-invasive approach for discriminating between benign and malignant pulmonary nodules. This approach does not necessitate of prior knowledge of the tumor mutation profile, only requires a small amount of plasma and less than 3 Gb genomic data for analysis. In addition, the BEMAD algorithm model is highly robust and is not affected by the most common clinicopathological risk factors. CfDNA LD-WGS could serve as a parallel technique alongside CT imaging to further stratify undiagnosed lung nodules, reducing unnecessary invasive procedures in patients with benign lung nodules.


The authors appreciate the academic support from the AME Lung Cancer Collaborative Group. We thank Giuseppe Giaccone and Olivier Elemento from Weill-Cornell Medicine for helpful comments. We also thank Li Zhang from Sun Yat-sen University Cancer Center for helpful discussions. Computational and sequencing resources were provided by China National GeneBank (CNGB). This study made use of data generated by The Chinese University of Hong Kong (CUHK) Circulating Nucleic Acids Research Group, as reported by Jiang et al. in Cancer Discov and by the Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA, as reported by Mathios et al. in Nat Commun. We thank Hu Xi from The CUHK (Data for EGAS00001003409) and Jillian Phallen from Johns Hopkins University School of Medicine (Data for EGAS00001005340) for providing help with data access (https://ega-archive.org/studies/EGAS00001003409, Accession No. EGAS00001003409; and https://ega-archive.org/studies/EGAS00001005340, Accession No. EGAS00001005340). Finally, we thank H. Nikki March, PhD, from Liwen Bianji (Edanz) (https://www.liwenbianji.cn/) for editing the English text of a draft of this manuscript.

Funding: This work was supported by the National Key Research and Development Program of China Grant (No. 2016YFC0905501 to CW), the National Natural Science Foundation of China (No. 82173038 to DY), and the Guangdong Enterprise Key Laboratory of Human Disease Genomics (No. 2020B1212070028 to KW).


Reporting Checklist: The authors have completed the TRIPOD reporting checklist. Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-647/rc

Data Sharing Statement: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-647/dss

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-22-647/coif). AR reports consultancy/advisory board role for Astra Zeneca, MSD, Pfizer, and Novartis. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). This study was approved by the ethical committee of Tianjin Medical University Cancer Institute and Hospital (approval Nos. bc2016014, bc2018009, and bc2019091), and all participants provided written informed consent.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.


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(English Language Editor: A. Kassem)

Cite this article as: Zhang B, Liang H, Liu W, Zhou X, Qiao S, Li F, Tian P, Li C, Ma Y, Zhang H, Zhang Z, Nanjo S, Russo A, Puig-Butillé JA, Wu K, Wang C, Zhao X, Yue D. A novel approach for the non-invasive diagnosis of pulmonary nodules using low-depth whole-genome sequencing of cell-free DNA. Transl Lung Cancer Res 2022;11(10):2094-2110. doi: 10.21037/tlcr-22-647

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