Cancer remains a leading cause of death worldwide. More than 19 million new cancer cases were reported in 2020, and cancer caused 10 million deaths in 2020 globally (1). In general, most cancers are curable through appropriate early screening. Immune-related biomarkers are commonly applied in cancer diagnosis, prognosis, and treatment monitoring. For instance, a high neutrophil-to-lymphocyte ratio has been reported to be associated with poor survival outcomes in a variety of cancers (2–5). However, these measurements often require multiple laboratory procedures including centrifugation to extract certain cells from whole blood or plating cells on glass slides for cell activation. Unfortunately, those procedures may disrupt the natural presentation of immune cells and trigger false cell activation or damage natural cell–cell interaction in the circulation (6). Other inflammatory markers for cancer diagnosis are erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), procalcitonin (PCT), and D-dimer. Nevertheless, the poor sensitivity of these inflammatory markers hinders their ability to be an independent predictor of cancer (7). Therefore, affordable, simple, and easily accessed immune-related tests are needed for the diagnosis and prognosis of cancer.

An innovative technique termed Leukocyte ImmunoTest™ (LIT™, Seroxo Ltd., UK), also known as the leukocyte coping capacity test, has been developed to quantify the reactivity of leukocytes (primarily neutrophils) in response to phorbol 12-myristate 13-acetate (PMA) stimulation (8). The amount of reactive oxygen species (ROS) produced by circulating leukocytes during the PMA challenge is measured through a real-time chemiluminescent measurement (9, 10). This technique has been previously used to assess subjective mental workload (9) and physiological stress levels (10) of healthy volunteers when given various stress challenges. A recent study has demonstrated the LIT assay to be a novel biomarker of neutrophil function (11). However, its other applications including the predictive value of cancer diagnosis or prognosis have not been established. The objective of this study was to investigate the pre-treatment immune status of cancer patients with LIT and to correlate LIT with other cancer diagnostic approaches to evaluate its cancer predicting potential.

Our study found that a significantly high level of the LIT was associated with malignant tumors; in particular, those patients who were in the advanced T4 tumor stage had high readings of LIT. The LIT level also showed a positive correlation with ANC in cancer patients. Further data analysis showed that LIT may be a predicting factor for tumor being malignant or benign with the cutoff value of LIT of 267.3.

LIT test was initially developed by McLaren et al., measuring the activity change of leukocytes (mainly, but not limited to neutrophils) as a bio-indicator for mental stress load (8). This technique is based on detecting PMA-induced burst production of ROS from circulating leukocytes, primarily neutrophils. LIT luminometer measured the emission of photons released during the interaction between ROS and luminol. In our study, the ability of leukocytes to react to PMA stimulation and release ROS was likely affected by the tumor environment (such as tumor-derived cytokines) per se. It is likely that patients with advanced tumors tend to have more tumor-activated leukocytes, favoring the production of higher levels of PMA-induced ROS during the LIT test ( Figures 1D and 3 ).

It is well known that immune cells work as the first-line defense against external molecules including infectious pathogens and early-stage tumors in the host. Nevertheless, immune cells tend to have a multifaceted function in tumor development due to tumor-induced phenotype alteration. Despite that, the role of immune cells in tumorigenesis remains controversial (12, 13), with a large variety of pro-tumoral leukocytes being recruited to tumor sites via tumor-released cytokines and chemokines. For instance, an increased level of infiltrated tumor-associated macrophages is found around tumors, promoting tumor progression by enhancing vascular endothelial growth factor A-mediated angiogenesis (14) and suppressing cytotoxic T cell-regulated anti-tumor response (15). Another good example is tumor-associated neutrophils (TANs), which are largely recruited and activated by Ras-regulated CXC chemokines in the tumor microenvironment (16). Previous studies also demonstrated increased TANs in patients with lung cancer, melanoma, head and neck cancer, pancreas cancer, prostate cancer, and breast cancer (17–23). In line with these studies, our data ( Table 1 ) also suggested significantly higher levels of ANC in the cancer group compared to the control group. Moreover, a positive correlation between ANC and LIT was only found in the cancer group but not in the control group. This indicates that neutrophils in cancer patients were more susceptible to PMA stimulation than those in non-cancer patients. Despite the anti-tumoral roles of TANs, accumulated TANs are often linked with poor overall survival, recurrence-free survival, and disease-specific survival outcomes (24). Patients at the T4 stage had highly activated leukocytes (more capable of producing ROS with external stimulation). These cells facilitated cancer cells outgrowth and invasion via extracellular matrix remodeling and immunosuppression (25, 26). As the TNM stage is clinically used to assess cancer prognosis, the strong correlation between LIT and the TNM stage may suggest the potential prognostic value of LIT.

In our univariate analysis, patients’ age, smoking status, WBC count, CEA level, AFP level, tumor being malignant or benign, tumor stage T4, lymph node stage N1, and specific tumor types showed a strong association with LIT. However, multivariate analysis assisted to exclude any significance caused by interaction between variables, identifying only tumors being malignant or benign, WBC count, smoking status, and tumor stage affected LIT in all patients in our study. Particularly, ANC data showed a positive correlation with LIT in cancer patients but not in controls. It is worth pointing out that we found large variations of ANC and NLR in the cancer group ( Table 1 ). This was due to one single patient whose ANC is 5,096 × 10⁹/L, leading to a large sample variation. This patient was with T3 esophageal cancer and was also a smoker. Esophageal cancer presents very high mortality that contributes to 5.3% of all cancer-related deaths (27). Patients with advantage esophageal cancer usually show poor outcomes with 5-year survival between 15% and 20% (28). It is known that immune cells show a sensitive response to tumor growth and act as potential prognostic markers (29). The high neutrophil level has been reported to be an independent factor for the poor prognosis of esophageal cancer (30). Several studies suggested the association of high NLR with worse overall survival in esophageal cancer (31–33). Furthermore, previous studies demonstrated that the number of peripheral blood neutrophils significantly increased after smoking (34, 35). Although this patient’s ANC was extremely high, all patients’ data were included for further analysis to reflect the nature of the proof-of-concept study. However, follow-up of this patient and further study may assist us to understand the cancer biology of patients with extremely high neutrophil levels.

Apart from identifying the predictive value of LIT in cancer diagnosis, we also compared the sensitivity and specificity between LIT and other diagnostic markers. Our data indicate relatively high sensitivity (87.01%) and moderate specificity (46.91%) of LIT in the studied population. The moderate specificity of LIT might be due to the choice of the control group, who were patients with benign tumors rather than healthy volunteers. Benign tumors, especially sizable benign tumors, usually exist with chronic inflammation and can also trigger complex immune responses (36). Thus, the immune status of the control group and cancer group might share a certain level of similarity and lead to decreased specificity for cancer detection in this study. We noticed that inflammatory marker PCT presented an extremely high level of sensitivity (100%) and an extremely low level of specificity (29.17%) in our study. PCT is one of the most sensitive parameters in detecting bacteria-associated infections. For cancer patients with infections, the PCT concentration may therefore be increased regardless of the existence of a malignant tumor, leading to poor sensitivity for cancer (37). Compared to inflammatory markers, tumor markers including CEA, AFP, HCG, and PSA generally showed better sensitivity for cancers. However, there are concerns about the accuracy of these tumor markers as well. For example, the positive serum concentration of CEA varies dramatically among colorectal cancer, pancreatic cancer, and breast cancer (38). A high level of CEA was reported in various non-cancer conditions such as hepatitis, alcoholic cirrhosis, and ulcerative colitis (38).

There were limitations to this study. For instance, LIT is highly associated with leukocyte activity, which is known to be susceptible to various factors. In our study, patients with immune function disorders and infections that may influence LIT reading were excluded to minimize confounding factors, but other factors that may affect our data remain unknown. For example, smoking is a factor found in this study that was unexpected, although it is well known that cigarette smoking triggers inflammatory responses including increased WBC count and inflammatory mediators (39). A relatively small sample size may not represent patients’ generality. Lastly, despite patient recruitment being random, more female patients were involved in this study, leading to gender imbalance. All these warrant further studies to verify the value of LIT for clinical use and to justify its several technical advantages: i) no centrifugation or plating of blood cells is required, preventing procedure-induced leukocyte activation; ii) a relatively small amount of blood is needed, and the device is portable, making this test easily accessible; iii) chemiluminometer reading data can be given within 10 min, reducing cost and waiting time.

In conclusion, this proof-of-concept study proposes the potential value of LIT in rapidly identifying whether a tumor is malignant or benign, as a predictor to be considered by clinicians and pathologists. Compared to biopsy or routine imaging approaches, LIT is a non-invasive, low-cost, and rapid measurement for cancer diagnosis. Nevertheless, larger sample sizes and cancer type-specific clinical studies are required to further evaluate the diagnostic value of LIT. A long-term follow-up study is also needed to assess the prognostic potential of LIT.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Ethics Committee of Henan Provincial People’s Hospital, Zhengzhou University, Zhengzhou, China. The patients/participants provided their written informed consent to participate in this study.

XL coordinated the study and contributed to the experimental design, interpretation of results, and writing of the paper. XM conducted a study on-site, patient recruitment, and data collection. YL contributed to the interpretation of the results. EC contributed to patient recruitment. JC provided study support and writing of the paper. JZ, DM and XL conceived the study design, supervised the project, and drafted and corrected the manuscript. All authors contributed to the article and approved the submitted version.

The study was supported by Seroxo Ltd. The company also supplied the regents and monitor free of charge. The funder had no role in study design, data collection and analyses, and manuscript preparation for publication. JZ’s current research was supported by the Natural Science Foundation (No. 82071217, 81771149), Beijing, China, and the Department of Science and Technology (No. GZS2021005, 202102310124), Henan Province, China. Prof. Ma’s current research was supported by a grant from the National Institute of Health Research and British Journal of Anaesthesia, London, UK, and, the European Society of Anaesthesiology and Intensive Care (ESAIC), Brussels, Belgium.

We thank Seroxo Ltd. for its generosity in supporting this research.

The 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.

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