Lung cancer (LC) remains the worldwide leading cause of death from cancer. Unfortunately, approximately 75% of patients are diagnosed at an advanced stage of the disease (III, IV) [1]. Despite significant investment and advancement in LC research, only 16% of LCs are detected at the early stages [2]. Thus, even with recent advancements in treatment, survival remains poor. Developing early detection diagnostic methods, especially non-invasive methods, is a critical component in raising the overall survival rate and prognosis for lung cancer [3].

Current diagnostic methods (e.g., Computed Tomography—CT, Positron Emission Tomography—PET, Low-dose CT- LDCT, radiography) have high sensitivity but low specificity. False positive rates of 96.4% for LDCT and 94% for radiography [4,5,6,7] lead to a large number of unnecessary follow-up procedures. These procedures are expensive, invasive and can have significant complication risks. These can be pronounced in the elderly where para-physiological changes occur in the lungs which can lead to inappropriate interpretation of radiological findings that put patients at risk of over or under treatment as Baratella et al. report [8]. Recent work demonstrates that core-needle biopsy performed under CT leads to accurate histological diagnosis of LC with high sensitivity and specificity [9]. While it is less invasive than other procedures used to obtain tissue from the lung nodule, it is not without complication risks [9]. Invasive follow-up procedures are expensive and can have significant complication risks. Nuñez et al. reported [10] high frequency of complication rates, and factors associated with complications in a national sample of veterans screened for lung cancer by invasive procedures such as bronchoscopy, transthoracic needle lung biopsy and thoracic surgery. Shin et al. [11] demonstrated that after lung cancer surgery, pulmonary function and patient-reported outcomes noticeably decreased in the immediate postoperative period and improved thereafter, except for dyspnea and lack of energy. Hence, in recent years, several alternative liquid biopsy approaches such as metabolomic, transcriptomic, genomic, and proteomic [1, 12,13,14,15,16] for the identification of cancerous biomarkers have been explored for the early detection of LC. These approaches use different pathological, molecular, and biochemical analyses. Unlike invasive lung tissue biopsy to detect LC biomarkers, a liquid biopsy such as blood sample or other body fluid is non-invasive. For example, biomarkers as circulating cell-free tumor DNA (cf DNA), cell-free RNA (cf RNA), exosomes, tumor-educated platelets (TEP), and circulating tumor cells (CTCs) can be detected in blood to detect LC [17, 18]. Common to all of these diverse methods is that the detection of LC in its early stage has low sensitivity and/or specificity. Klein et al. validated a targeted methylation-based test to detect cancer and reported sensitivities of 16.8% to detect stage I and 40.4% to detect stage II [19, 20]. Xue et al. stated in their review of molecular technologies in liquid biopsy that early detection still needs to be improved [21].

Studies show that activation of immune cells requires changes in the way metabolic energy (ATP molecules) is generated. Immune system cells alter their energy generation in order to obtain an effector function. Usually, the shift is from the oxidative phosphorylation cycle into an aerobic glycolysis cycle. This shift provides immediate energy that gives the immune system the ability to attack the foreign antigen [22,23,24]. Hence, it appears that the activation state of the immune system, in response to tumor development, differs from the non-cancerous state [25,26,27,28,29,30]. These important discoveries corroborate our hypothesis that changes caused by cancer are reflected in different metabolic activity profiles of immune cells such as Peripheral Blood Mononuclear Cells (PBMCs) in response to various antigenic stimulants. In general, an effective in vitro response of the immune cells to re-stimulation with a LC tumor-associated antigen (TAA) stimulant indicates that the immune cells were previously exposed to the specific stimulant. Importantly, it indicates that the cells are able to produce an immunological response to it.

This article describes an improved immunometabolism blood test that measures the function of the immune cells in response to antigenic stimuli based on changes in the metabolic pathways of cells. There are several classical methods to test lymphocytes’ function. Mixed leukocyte culture (MLC) determines histocompatibility by co-culturing PBMCs of a potential donor with those of an allograft recipient. MLC takes 3–8 days to get results and involves the use of H3 thymidine radiolabeling [31]. Limiting dilution assay (LDA) also assesses histocompatibility between two parties. It determines the precursor frequencies of cytotoxic and helper T lymphocytes. The duration of this test is generally longer than MLC and takes 7–18 days [32]. Lymphocyte transformation test (LTT), in contrast to MLC and LDA, measures lymphocyte responses toward nonspecific stimuli (mitogens/drugs) or specific stimuli (antigen). A proliferative response shows that antigens of the respective microorganism are presented by antigen-presenting cells, and are recognized by pre-existing, antigen-specific T lymphocytes. The duration of this test is 8–10 days [33]. A more recent method to test the function of lymphocytes is the enzyme-linked immunospot (ELISpot) assay. It is a sensitive and quantitative method to detect cytokine production level in cell culture supernatant after growing cells with stimulant antigen. The duration of this ELISpot test, including cell culturing, is 2–12 days [34, 35]. Various flow cytometry assays that measure lymphocyte functionality include tests that are based on the detection of cell divisions by fluorescent CFSE staining, use of multimer staining of human leukocyte antigen (HLA) restricted peptides with their T cell receptor, use of other staining of cell’s receptors, or measurement of proteins that correlate with cell activation [36]. Like ELISpot, these types of tests need cell culturing for 2–12 days. ImmuKnow test measures the response of CD4+ T-helper lymphocytes to the mitogen phytohaemagglutinin-L (PHA), a general stimulator. It measures the amount of ATP produced by the cells following nonspecific stimulation. The duration of this test is 2 days [37]. While the methods described are non-invasive or devoid of the radiation risk of imaging, they all require days of execution, are cumbersome to perform, and there are no uniform standards (positive and negative controls, measurement units and working protocols) in performing these methods by different users. Therefore, the need for an assay that monitors in vitro cellular immune responses (primarily T and B cells) to antigenic stimuli with TAA, within a few hours, to determine immune activation levels is important.

In a previous publication, we presented a novel, non-invasive, cancer detection platform [38]. Our platform, named Liquid ImmunoBiopsy™, is based on measurements of metabolic activity profiles of immune cells. In our previous study we showed that by using machine learning methods to get a multivariate prediction model and training on the metabolic profiles, we were able to differentiate between blood samples of LC patients (n = 100, all stages) and control subjects (n = 100) with 91% sensitivity and 80% specificity in a cross-validation statistical evaluation. Since the clinical benefits for early detection of LC are demonstrated, we continued to develop the metabolic activity (MA) test protocol. The objective of this presented research is to investigate the accuracy of the metabolic activity test for lung cancer (MA-LC) in its improved protocol version versus the previous version by comparing MA-LC results from two additional clinical trials. The first clinical trial (n = 328) is referred to here as the “earlier” clinical trial, and the second additional clinical trial (n = 245) is referred to here as the “later” clinical trial. The earlier MA-LC protocol was used in the earlier clinical trial (n = 328), and an improved protocol was used in the later clinical trial (n = 245). We tested whether the improved protocol does, in fact, increase the sensitivity and specificity of the MA-LC to detect stage I and stage II LC.