A novel non-invasive exhaled breath biopsy for the diagnosis and screening of breast cancer
Journal of Hematology & Oncology volume 16, Article number: 63 (2023)
Early detection is critical for improving the survival of breast cancer (BC) patients. Exhaled breath testing as a non-invasive technique might help to improve BC detection. However, the breath test accuracy for BC diagnosis is unclear.
This multi-center cohort study consecutively recruited 5047 women from four areas of China who underwent BC screening. Breath samples were collected through standardized breath collection procedures. Volatile organic compound (VOC) markers were identified from a high-throughput breathomics analysis by the high-pressure photon ionization–time-of-flight mass spectrometry (HPPI-TOFMS). Diagnostic models were constructed using the random forest algorithm in the discovery cohort and tested in three external validation cohorts.
A total of 465 (9.21%) participants were identified with BC. Ten optimal VOC markers were identified to distinguish the breath samples of BC patients from those of non-cancer women. A diagnostic model (BreathBC) consisting of 10 optimal VOC markers showed an area under the curve (AUC) of 0.87 in external validation cohorts. BreathBC-Plus, which combined 10 VOC markers with risk factors, achieved better performance (AUC = 0.94 in the external validation cohorts), superior to that of mammography and ultrasound. Overall, the BreathBC-Plus detection rates were 96.97% for ductal carcinoma in situ, 85.06%, 90.00%, 88.24%, and 100% for stages I, II, III, and IV BC, respectively, with a specificity of 87.70% in the external validation cohorts.
This is the largest study on breath tests to date. Considering the easy-to-perform procedure and high accuracy, these findings exemplify the potential applicability of breath tests in BC screening.
To the editor
Breast cancer (BC) is one of the most common cancers and a leading cause of death worldwide . Early BC detection improves survival . However, imaging-based BC screening methods are prone to being expensive and overdiagnosed.  By detecting volatile organic compounds (VOCs) during exhalation , breath biopsy is a promising non-invasive strategy for early cancer detection . However, the accuracy of the breath test for BC diagnosis has not been verified by multi-center clinical trials with sufficient sample sizes .
Herein, we enrolled 5047 women who underwent BC screening from six hospitals in four areas of China (Fig. 1 and Additional file 1: Figure S1). The discovery set included 216 BC patients and 2959 non-cancer women from three hospitals in Beijing, and the external validation set included 249 BC patients and 1545 non-cancer women from another three hospitals in Yantai, Wenzhou, and Guiyang, respectively (Additional file 1: Tables S1, S2). Most BC patients were diagnosed at early stages (Additional file 1: Table S3).
Breath samples of 1.2L for each participant were collected according to established procedures and analyzed by high-pressure photon ionization time-of-flight mass spectrometry (HPPI-TOFMS) (Additional file 1: Supplementary methods) . HPPI-TOFMS has a higher throughput than earlier technologies and does not require the pretreatment of exhaled breath . Each VOC ion’s peak area was then computed. Spectrum peak patterns and VOC correlation modules of the BC patients and controls differed (Additional file 1: Figures S2 and S3). Ten optimal VOC features were selected to differentiate the BC patients and non-cancer controls in the discovery cohort (Fig. 2A). Eight VOCs showed significantly higher peak areas in BC patients than controls, and two VOCs were substantially lower (Fig. 2B and Additional file 1: Table S4). Significant fold changes and diagnostic performances were identified in these 10 VOC ions (Additional file 1: Figure S4). The m/z values of 28.0 and 40.0, which may contain ethylene and propyne or fragment ions, showed the highest AUCs (Fig. 2C and Additional file 1: Table S4).
The random forest algorithm  was employed as the classifier. The discovery dataset was randomly split 5:2:3 into training, internal validation, and test datasets for model construction. We constructed two BC detection models, BreathBC and BreathBC-Plus, using only the 10 VOC markers and both VOC markers and risk factors, respectively (Fig. 2A).
BreathBC scores were higher in BC patients than controls (0.66 ± 0.31 vs. 0.11 ± 0.15, p = 1.29 × 10−153), regardless of tumor size, lymph node status, and molecular subtypes (all p < 0.01, Additional file 1: Figure S5), and collinear with tumor size (r = 0.41, p = 0.05; Additional file 1: Figure S6). The diagnostic AUC of the BreathBC model was 0.96 (95%CI, 0.94–0.97) in the internal validation cohort and 0.95 (95%CI, 0.93–0.90) in the test cohort (Fig. 2D, E, Additional file 1: Table S5). The performances are higher than all the results of previous studies using the gas chromatography-mass spectrometry (GC–MS) (AUC = 0.67–0.93) [9,10,11] but lower than the electronic nose (AUC = 0.99; Additional file 1: Table S6) . However, no external validation was conducted for the previous methods, and their sample sizes were relatively small. In external validation cohorts, the BreathBC model achieved an AUC of 0.87, a sensitivity of 92.37% (230/249), and a specificity of 60.45% (934/1545; Additional file 1: Table S7).
Furthermore, the BreathBC-Plus diagnostic model was developed in the discovery cohort, combining BreathBC scores with traditional risk factors (Additional file 1: Supplementary methods). The combined model outperformed the BreathBC model in the internal validation cohort (AUC = 0.98), the test cohort (AUC = 0.97), and external validation cohorts (AUC = 0.94) (Fig. 2F, G, Additional file 1: Table S5). In external validation cohorts, BreathBC-Plus produced sensitivity and specificity of 89.16% (222/249) and 87.70% (1355/1545; Additional file 1: Table S7). Collectively, the total detection rates were 96.97% (32/33) in ductal carcinoma in situ (DCIS), 85.06% (74/87), 90.00% (99/110), 88.24% (15/17), and 100% (2/2) for stages I, II, III, and IV BC in external validation cohorts, respectively (Additional file 1: Table S8). Intriguingly, breathBC-Plus outperformed mammography and ultrasound in diagnosis (Additional file 1: Figure S7, Table S9).
There are some limitations of this study. First, although the HPPI-TOFMS provided a high-throughput methodology for VOC analysis, it is still being determined which chemical compound is associated with each MS peak. Second, as most previous studies on VOCs were only focusing on one cancer type, we also aimed to identify the BC-specific VOC markers in this study.
To our knowledge, this is the largest breathomics analysis study to date. Collectively, breath-based methods may provide supplemental or alternative screening strategies to detect early-stage BC and DCIS at comparable performance to imaging-based technologies.
Availability of data and materials
The supplementary data supporting this study's findings are openly available in the supplemental materials. Deidentified participant data and analytic code are available upon reasonable request to Dr. Jiaqi Liu (email@example.com).
Area under the curve
Ductal carcinoma in situ
Gas chromatography–mass spectrometry
High-pressure photon ionization-time-of-flight mass spectrometry
Receiver operating characteristic
Volatile organic compound
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We thank all the individuals, families, and physicians involved in the study for their participation.
This research was funded in part by the National Natural Science Foundation of China (82272938 to Jiaqi Liu), Beijing Nova Program (20220484059 to Jiaqi Liu), the CAMS Innovation Fund for Medical Sciences (2021-I2M-1-014 to Jiaqi Liu and 2021-I2M-C&T-A-015 to Xiang Wang), and the Beijing Hope Run Special Fund (LC2020B05 to Jiaqi Liu).
Ethics approval and consent to participate
This study was reviewed and approved by the ethics committees at each participating hospital (Ethics number: 22/290-3492). Written informed consent was obtained from each participant.
The authors have no conflict of interest to declare.
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Liu, J., Chen, H., Li, Y. et al. A novel non-invasive exhaled breath biopsy for the diagnosis and screening of breast cancer. J Hematol Oncol 16, 63 (2023). https://doi.org/10.1186/s13045-023-01459-9
- Breast cancer
- Breath test
- Volatile organic compound
- Early diagnosis
- Cancer screening