Differential diagnostics of non-small-cell and small-cell lung cancer: modern approaches and promising technologies

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List of abbreviations

IHC — immunohistochemical examination

CT — computed tomography

VOC — volatile organic compounds

SCLC — small-cell lung cancer

NSCLC — non-small-cell lung cancer

PCR — polymerase chain reaction

PET-CT — positron-emission tomography, combined with computed tomography

LC — lung cancer

CTC — circulating tumor cells

FDA — USA Food and Drug Administration

NGS — next generation sequencing

INTRODUCTION

Lung cancer (LC) is a heterogeneous group of malignant neoplasms that differ by the histogenesis, the molecular-genetic profile, the clinical course and the treatment approaches. The source of tumor growth is the cells of the surface epithelium of bronchial mucosa, the mucosa of the bronchiolar glands and the pulmonary alveoli [1]. As for the mortality, LC takes the first place among men and second — among women both in Russia and worldwide; as for the incidence rates — also the first place among men, among women — the second and the fifth worldwide and in Russia, respectively [2, 3]. In total, LC remains the main cause of cancer-related death with the approximate statistics of 2.48 mln of new cases and 1.84 mln of fatal outcomes annually [3], despite the decrease in LC mortality in general within the last 10 years [4]. Though the incidence of LC among men is higher than among women, two tendencies were simultaneously observed — the decrease among men and the increase among women [5]. The decrease of the incidence among men is directly related to the decease in the rates of smoking, at the same time, the incidence in women gets increased to a greater extent among the non-smokers and at the age older than 60 years [6]. Women are more prone to the development of LC, not related to smoking, which the investigators associate with the intersexual differences of the rates of mutations in the epidermal growth factor receptor, in the anaplastic lymphoma kinase and in the Kirsten rat sarcoma homologue gene (Kirsten rat sarcoma, KRAS) [7, 8].

CLASSIFICATION OF LUNG CANCER

The classification of LC is based on the histological type of tumor and has the fundamental value for the diagnostics, the prognosis and the choice of treatment tactics. In the international clinical practice, the most widely used classification is the one from the World Health Organization along with dividing LC into two main groups: the non-small-cell lung cancer (NSCLC), which includes the most widespread adenocarcinoma, as well as the squamous and the large cell subtypes, as well as the small-cell lung cancer (SCLC), Fig. 1 [9].

 

Fig. 1. Classification of lung cancer [8].

 

SCLC and NSCLC significantly differ by the origin, by the interrelation with smoking, by the course, the prognosis and the effective treatment (table 1)^{1, 2}[1, 2, 10–22].

 

Table 1

Non-small-cell and small-cell lung cancer: key differences

Parameter	NSCLC	SCLC	Source
Incidence rate	86–89% — in males 90–93% — in females	11–14% — in males 7–10% — in females	[10, 11]
Origin	Adenocarcinoma — glandular cells producing the mucus. Squamous carcinoma — epithelial cells	Neuroendocrine cells of the basal epithelium in the bronchi	[1, 12, 13]
Location and features	More commonly seen in the peripheral zone (especially adenocarcinoma), less frequently — in the central one (more often — squamous carcinoma). The tumor cells have signs of malignant transformation of the epithelium	Central tumor, developing from the submucosal layers of the airways as the perihilar mass. The cells appear as the spindle-like or round cells with sparse cytoplasm, grained chromatin, necrosis is often found	[12–14]
Relation to smoking	~84% of the cases (adenocarcinoma can develop in non-smokers)	>96% of the cases	[15]
Subtypes	Adenocarcinoma, squamous, large cell	Pure SCLC, combined (with elements of NSCLC)	[1]
Typical mutations	EGFR, KRAS, ALK, ROS1	Loss of TP53 and RB1	[16–21]
Course	Slow growth, late metastatic activity	Aggressive growth, early metastatic activity (into the brain and liver)	[1]
Treatment	Surgery — at early stages. Chemotherapy — at stages II, III and in some cases — at the В stage. Radiation therapy. Target therapy (inhibitors of EGFR, ALK). Immunotherapy (PD-1/PD-L1)	Surgical treatment is rarely used due to the early metastatic spreading, only for stage I (IА and IВ) and in separate cases at the stage II with an obligatory adjuvant chemotherapy. Systemic chemotherapy (etoposide + platinum) + immunotherapy (Atezolizumab) + radiotherapy (optional). Rapid development of resistance to chemotherapy (often related to the loss of TP53/RB1)	[2, 13]
Prognosis	Better (five-years survival rate ~32% in total for all the stages)	Worse (five-years survival rate <9% in total for all the stages)	-
Paraneoplastic syndromes	Rare	Often (SIADH, Cushing syndrome)	[22]

Note. NSCLC/SCLC — non-small-cell/small-cell lung cancer. SIADH — syndrome of inappropriate antidiuretic hormone secretion.

 

SCLC is an aggressive and rapidly growing tumor, often metastasizing into the liver, the brain and the bones, also, despite the initially reported sensitivity to chemo- and radiotherapy, frequently and rapidly developing the resistance with further development of recurrences. Within this context, the timely differential diagnostics of NSCLC and SCLC is an important task for the modern Medicine.

DIFFERENTIAL DIAGNOSTICS OF NON-SMALL-CELL AND SMALL-CELL LUNG CANCER IN THE SETTINGS OF MODERN CLINICS

In the modern clinical practice, the differential diagnostics of SCLC and NSCLC is arranged in a combined manner using the methods of radiography, computed tomography (CT), positron-emission tomography combined with the computed one (PET-CT), bronchology examination with further histological and immunohistochemical diagnostics (the main method), as well as the method of molecular-genetic testing for NSCLC (Fig. 2). The clinical indications for conducting these examinations include such symptoms as coughing, difficulty of respiration, shortness of breath, chest pain, rales, blood spitting, weakness, fatigability, decreased appetite, frequent infections of the chest cavity organs, constant pain in the chest or shoulders, hoarseness or depression of voice and the unexplainable loss of body weight [2, 13].

 

Fig. 2. The methods of differential diagnostics of non-small-cell and small-cell lung cancer. NSCLC/SCLC — non-small-cell/small-cell lung cancer; CT — computed tomography; PET-CT — positron-emission tomography, combined with computed tomography.

 

Methods of diagnostic visualization

The methods of diagnostic visualization include the radiography, the CT and the PET-CT. The chest cavity radiography is not recommended as the population screening of LC, for the prospective randomized research works did not reveal a significant decrease in LC-related mortality when using this method as the screening one. The sensitivity of radiography for detecting the early stages of LC is less than 50%, which is why, when suspecting the presence of tumor, CT is obligatory, however, radiography is still the main method of detecting the primary evidences of LC with the present clinical indications. For the purpose of further evaluating the pathological changes revealed using the radiography, CT examination is used with intravenous bolus contrasting [2].

Though the exact subtype of LC is impossible to detect using CT, nevertheless, there are indirect signs characteristic for SCLC, which include the central location of tumor with massive mediastinal lymphadenоpathy, as well as the rapid growth and early metastatic activity. At the same time, NSCLC can be both the peripheral (adenocarcinoma) and the central (squamous), and, generally, it progresses at a slower rate.

The evaluation of the metabolic activity using the radiopharmaceutical (usually the radio-labeled glucose analogue, into the molecules of which, the radioactive fluoride-18 isotope is introduced (18F-FDG)) is based on the property of malignant tumors to actively absorb glucose, which helps differentiating them from benign changes (granulomas and scars). The sensitivity of PET-CT in detecting the malignant nodes is >90%, with the specificity of 70–85% (false-positive results are possible in cases of inflammation or infections). This method allows for staging the tumor process, classifying them using the TNM system (International classification of stages for malignant neoplasms: Tumor — tumor, Nodus — lymph nodes, Metastasis — metastases), detecting the metastases, with this, SCLC usually demonstrates a high SUVmax (maximal ¹⁸F-FDG uptake coefficient) comparing to the NSCLC. The additional benefit of precise defining the tumor margins using the PET-CT is the possibility to optimize the exposure dosage and, as a result, to minimize the damage of healthy tissues.

Bronchological examination

Bronchological examinations, such as bronchoscopy and endobronchial ultrasound examination, are assigned as the main and the obligatory methods of LC diagnostics, which allow for not only visually examining the throat, the trachea and all the bronchi, for directly observing the location of the tumor, for defining the margins of its spreading, for indirectly judging on the enlargement of the lymph nodes in the root of the lung and the mediastinum, but also for conducting the biopsy for histological examination (fine-needle and trephine biopsy), for obtaining the material (brush-bioptates, histological biopsy, impression smears, scrapings or washings from the bronchial tree) for further assessment. However, bronchoscopy has substantial limitations in the diagnostics of pre-cancerous lesions, for they are difficult to detect visually, as they consist of several layers of cells with a thickness of 0.2–1 mm and with a diameter of several millimeters [2, 12, 13].

Histological and immunohistochemical diagnostics

Histological and immunohistochemical (IHC) diagnostics is the main method of modern differential diagnostics for NSCLC and SCLC. The histological criteria for SCLC include the small cells (measuring ~2–3 lymphocyte diameters), the small-grained pattern of chromatin in the cell nucleus (“salt and pepper”), the nuclear molding, the specific IHC-markers (CD56, Synaptophysin, Chromogranin A — neuroendocrine differentiation), as well as the frequent loss of RB1 and TP53 genes (detected using the molecular-genetic methods).

The criteria for NSCLC include the following: large cells with clear cytoplasm and IHC-markers [adenocarcinoma: TTF-1, Napsin A; squamous: p40 (more specific), p63, CK5/6].

Molecular-genetic testing

Molecular-genetic testing is carried out for cases of NSCLC, while the adenocarcinoma cases include the detection of such mutations as EGFR, ALK, ROS1, BRAF as the obligatory minimal set along with the KRAS G12C (for which there are specific medications), MET, RET, HER2 as an addition.

The results of genetical testing define the treatment tactics, the possibility of effective application of target therapy. At the same time, for the SCLC, the molecular-genetic testing is not routinely conducted, for in Russia there are no registered target therapy medications. The DLL3 testing is not yet included into the routine practice in our country, but it is accessible within the frameworks of clinical research on target therapy (Ampuliximab).

Thus, the gold standard of differentiation between the SCLC and the NSCLC in the clinical settings is the IHC, but the development of new methods of diagnostics, especially the minimally invasive and non-invasive ones, is justified due to several key reasons:

1) in 15–20% of the patients, the biopsy is inaccessible due to the presence of concomitant diseases or due to the hardly accessible tumor location;

2) the invasiveness and the risks related to it (when conducting the bronchoscopy, there is a risk of pneumothorax and hemorrhages; transthoracic biopsy is especially dangerous in cases of central tumors or severe conditions);

3) high probability of false results due to the low quality of collecting the material and its fixation, as well as due to the heterogeneity of the tumor (biopsy can examine a limited area of the material and can miss the key mutations);

4) errors are possible that are caused by the complexity of interpretation (for example, large cell neuroendocrine cancer can mimic the SCLC);

5) the duration of testing procedures (sample preparation and IHC take up to 7–14 days, while in the regions with the deficit of histopathologists, the testing duration can be longer, which is critical for cases of aggressive SCLC);

6) IHC detects the subtype, but it does not replace the molecular test, as a consequence of which, the NSCLC requires further diagnostics using the method of polymerаse chain reaction (PCR) / next generation sequencing (NGS).

All the problems described above create a strong need for developing the alternative approaches to differential diagnostics. A special interest is arising in the methods of fluid biopsy, such as the analysis of circulating tumor cells and of the extracellular DNA (ctDNA), the microRNA (miRNA), as well as the use of artificial intelligence in processing the radiography images.

PROMISING NON-INVASIVE ALTERNATIVES FOR THE DIFFERENTIAL DIAGNOSTICS OF NON-SMALL-CELL AND OF SMALL-CELL LUNG CANCER

Circulating tumor cells

The differentiation between the SCLC and the NSCLC using the circulating tumor cells (CTC) is based on the fact that SCLC shows a significantly higher concentration of CTC comparing to NSCLC, which allows for differentiating the tumor type [23]. The number of CTC correlates with the metastatic potential of the tumor, with the level of angiogenesis and with the prognosis for the patient [23–25]. Multiple research works confirm that the number of CTC positively correlates with the poor prognosis [26, 27].

The isolation and the analysis of CTC for the primary diagnostic purpose opens the additional functional possibilities, such as creating the cell lines and testing the in vitro and in vivo treatment sensitivity, which can become an important step in personalized treatment. Special diagnostic interest arises from investigating the CTC clusters as an indicator of aggressiveness and of potential metastatic spreading. It is known that the presence of CTC clusters is indicative for high metastatic activity, especially in cases of SCLC [23].

The modern approaches for the detection of CTC include the following:

1) methods based on the EpCAM epithelial markers (epithelial cellular adhesion molecule) [28];

2) methods of negative selection (for example, CD45) [29];

3) microchip technologies (CTC-chip) [30];

4) size-dependent methods (ISET, ScreenCell, MCA) [31].

It is necessary to point out that the use of markers is characterized by low sensitivity, for it allows for detecting only the part of CTC population, missing the EpCAM-negative and the mesenchymal forms when using the EpCAM epithelial markers, as well as the CD45-negative cells when using the CD45-marker.

The key problems of CTC analysis for differential diagnostics are the insufficient standardization of methods and the biological heterogeneity of CTC. Significant complexity is added by the biological heterogeneity, when the differences between CTC-subpopulations (for example, by the degree of epitheliality /mesenchymality, by the presence of clusters) significantly complicate their precise identification, classification and developing the diagnostic and prognostic methods on their basis. Currently, the only system approved by the USA Food and Drug Administration (FDA) for the detection and quantitative determination of CTC in blood is still the CellSearch (CELLSEARCH®), while the majority of methods require further development and standardization [23]. The CellSearch system uses magnetic nanoparticles, covered in antibodies to the EpCAM epithelial marker for the detection of CTC, and the system is approved as the marker of predicting the survival rate in cases of metastatic breast cancer, prostate cancer and as a progression marker for metastatic colorectal cancer (CELLSEARCH®).

Thus, the use of CTC for the differentiation of SCLC and NSCLC represents a promising method, though still having a limited use in clinical practice. The most justified directions of development are deemed the combination of CTC analysis with testing the circulating extracellular tumor DNA and the implementation of the new uptake methods, based on the physical properties or negative selection, which could allow for taking into account the EpCAM-negative CTC subpopulations and for significantly expanding the diagnostic possibilities of this approach.

Circulating tumor DNA

DNA of the tumor cells, originating from the processes of apoptosis, necrosis and active secretion [24], enters the blood and other biological fluids, for example, the saliva, the sputum and others, potentially being the promising marker for non-invasive diagnostics of oncological diseases. Multiple research works have revealed the principal differences in the mutation profiles of SCLC and NSCLC. Thus, the SCLC is characteristic by the inactivation of the TP53 and RB1 suppressor genes [15, 21], as well as the mutually exclusive expression of MYCL, MYC or MYCN [15, 32–35]. These changes are considered the most important for the development of small-cell phenotype and the ones that are closely related to its neuroendocrine nature. NSCLC, unlike the SCLC, demonstrates a more variable spectrum of genetic changes, significantly differing between the histological subtypes. Lung adenocarcinomas — the most widespread NSCLC variants — often carry the activating mutations within the EGFR and KRAS genes, as well as the rearrangements in the ALK and ROS1 genes [17–20]. Squamous carcinoma, on the contrary, rarely has these changes, but often contains amplifications in the SOX2, PDGFRA, FGFR1/WHSC1L1 genes along with the deletions of CDKN2A [36, 37] and the mutations of PIK3CA [38]. Modern methods of molecular diagnostics, such as NGS and PCR, allow for highly precise detecting these differences. NGS-panels provide the possibility of combined evaluation of the mutation status, while PCR has an exclusive sensitivity for the detection of specific mutations, such as the EGFR and others. As an example of the NGS-panel, one can take MSK-IMPACT — the first FDA-approved combined genomic test for onco-diagnostics, developed at the Memorial Sloan Kettering Cancer Center in the USA, which analyses more than 400 cancer-associated genes and which is being used in clinical practice predominantly for the diagnostics and personalized treatment of solid tumors, including the NSCLC [39]. In Russia, there are already registered NGS-panels for the diagnostics of LC, for example, the ones manufactured by the companies “OnkoAtlas” and “Helikon”.

Not less important are the differences in the copy number variations (CNV) between SCLC and NSCLC. The small-cell cancer is characteristic by the amplification of the oncogenes MYC (20% of the cases [40]) and SOX2 (27% [35]), as well as frequent deletions in the 3p chromosome region, involving the FHIT gene [41]. These changes are associated with especially aggressive disease course. With this, the increased expression of SOX2 was also found in cases of SCLC, while in the NSCLC (adenocarcinoma and squamous LC), as it appears, the specific marker functions of SOX2 are still to be determined. Nevertheless, even now, a high potential was defined for using SOX2 as the target for therapeutic procedures [42]. The NSCLC, on the contrary, often has amplifications of EGFR and MET — 5–15% (in case of adenocarcinoma [43, 44]) or CCND1 (in squamous-cell cancer [45]), as well as the deletions of CDKN2A [46]. The CNV analysis allows for not only differentiating the subtypes of LC, but also to isolate the prognostically unfavorable variants, such as the SCLC with MYC amplification [40]. Modern approaches combining the analysis of the mutation profile and the copy number variations, significantly increase the precision of diagnostics (by 27%), while using the methods of fluid biopsy is topical for the diagnostics of SCLC [47]. These data, as well as the methods of their collection and analysis, are already used in the development of modern clinical recommendations in America and Europe, including the NCCN and ESMO guidelines.

The assessment of abberrant methylation of the circulating DNA

DNA methylation is an epigenetic modification, in which the cytosine in the CpG-dinucleotides gets a methyl group added. This modification influences the expression of genes by suppressing the transcription: the methylation of promotor regions blocks the binding of transcriptional factors and attracts the proteins, compacting the chromatin, which “switches off” the gene. In the pathogenesis of LC, methylation abnormalities manifest as the two opposite processes. On the one hand, there occurs the global hypomethylation, activating the mobile genetic elements and the oncogenes, which promotes to the genomic instability. On the other hand, hypermethylation of promotor regions of the suppressor genes of tumor growth, such as p16INK4a and BRCA1, lead to their functional inactivation and to the acceleration of tumor cell proliferation.

As of today, there is a large number of methods for analyzing the methylation status of specifically the extracellular DNA [48], while the modern research works clearly demonstrate their diagnostic value. It was found that various histological subtypes of LC are characterized by the unique methylation patterns of the circulating free DNA (cell-free DNA, cfDNA): for example, the analysis of the APC, HOXA9, RARβ2 and RASSF1A genes can define the types of LC and the stage of the disease [49]. Moreover, the classifiers based on the analysis of the cfDNA methylation, investigated in various research works, have allowed for isolating not only the types of LC between each other [50], but also the subtypes of SCLC [51, 52] and NSCLC [50, 53]. However, the high specificity of analyzing the methylation of separate markers (often >80%) in these research works is accompanied by low sensitivity (~50–65%). The decision for this problem could be the expansion of the panels for the simultaneous analysis of methylation among the several markers.

Beside the use for the diagnostic purposes, the analysis of methylation profile could also be informative for the evaluation of treatment tolerance [52]. A separate problem when developing various diagnostic methods is the risk of false-positive results due to the presence of background methylation, especially in smokers [54], which is critically important in case of LC and requires additional research.

The proof of the efficiency of using the methylated markers for fluid biopsy in LC is the presence in the European and Chinese markets of several diagnostic tests aimed at the diagnostics of LC (CE-IVD mark [55]) and approved by the Chinese National Medical Products Administration (NMPA) [56] and by the American FDA [57].

Thus, the analysis of cfDNA methylation represents a promising tool for the non-invasive and specific diagnostics of SCLC and NSCLC, nevertheless, further development is needed in the field of compiling the panels for the evaluation of multi-gene methylation and for the automated analytical platforms, which requires arranging additional validation studies.

Aberrant expression of microRNA

MicroRNA are considered the promising biomarkers for the differential onco-diagnostics due to their stability in biological fluids and tissues, as well as due to the ability to reflect the molecular specific features of the tumor. Multiple research works demonstrate that the microRNA expression profiles both in blood plasma and in blood exosomes significantly differ in cases of NSCLC and SCLC, which opens new possibilities for developing the non-invasive diagnostic tests [58, 59]. Indeed, microRNA can be found in blood both at the free state and as a part of membrane-coated micro-vesicules [60–64]. The significant part of blood micro-vesicules is represented by exosomes — the vesicules with a diameter of 30–150 nm, which get released by the normal and by the tumor cells and take part in the intercellular communication. Exosomes/micro-vesicules of tumor origin contain proteins, nucleic acids and lipids reflecting the molecular profile of the tumor. Blood micro-vesicules are evidently a more acceptable source of microRNA for diagnostic purposes than blood plasma: data from a number of research works provide strong evidence of the greater enrichment of this microRNA pool with tumor-specific microRNA [65].

The majority of studies is devoted to evaluating the expression of microRNA in NSCLC and its subtypes, while the SCLC, due to its lesser incidence has gained insufficient attention. A comprehensive research on the microRNA-31 expression using the method of quantitative polymerаse chain reaction with reverse transcription (RT-PCR) in lung tissue samples obtained after surgical resection in cell lines and in tumor xenotransplants from mice, along with the available data from the Cancer Genome Atlas (TCGA) has shown that microRNA-31 is variously expressed in the tumors of various histological types of LC. In particular, excessive microRNA-31 expression was found in the NSCLC samples (lung adenocarcinoma, squamous carcinoma, adeno-squamous carcinoma and large cell neuroendocrine carcinoma), however, the samples of small-cell carcinoma and atypical carcinoids did not show any increase of the expression [66], which allows for suggesting the high potential of using microRNA-31 as the molecular marker of NSCLC.

In another research, the evaluation of the microRNA expression profiles in the SCLC and NSCLC cell lines along with the normal immortalized human bronchial epithelium cells using the microchips test has revealed a number of differentially expressed microRNA. In total, 29 microRNA were statistically significantly differentially expressed in the SCLC and NSCLC cell lines, of which 19 (-15a,b, 16, 195, 135, 106a,b, 101, 338, 1, 98, 103, 107, 17-5р, 92, 93, 326, 328, 96) were hyper-expressed in the SCLC cell lines comparing to the NSCLC, and 10 (-21, 22, 23a,b, 24, 27a, 29a,b,c, 31) — had a decreased expression [67]. Significantly differing microRNA expression in SCLC comparing to NSCLC and to the normal immortalized human bronchial epithelium cells allows for suggesting that the microRNA expression profiles can be successfully used for the differential diagnostics of these LC variants.

Another research has developed and validated a panel of eight microRNA (106a, 125a-5p, 129-3p, 205, 21, 29b, 375, 7) using the pathological and the cytological LC samples. It was found that this panel, named as the “miRview lung”, can be used for the differentiation of SCLC and NSCLC (in particular, the squamous and the non-squamous lung carcinoma or carcinoid). The research work was arranged in three stages: detection stage, at which the potential biomarkers were identified; the test development stage, during which the marker microRNA were selected and the classifier was compiled; and the validation stage, at which the diagnostic protocol was tested using the blinded independent sample. The total precision of the analysis was 93.7% (95% CI 90.8–95.8) [68]. Despite the optimistic results, the research did not result in the appearance of the LC diagnostic system on the market, which seems to be related to the specific features of the analytical system or to the group of donors and patients involved into the research.

NSCLC is the most widespread type of LC, being responsible for up to 85% of all the cases. For this reason, multiple research works were arranged for the identification of microRNA, which can differentiate the histological subtypes of NSCLC, in particular, the lung adenocarcinoma and the squamous LC. For example, in the research based on the analysis of blood microRNA in 90 LC patients and in 85 healthy volunteers, microRNA-944 has shown the diagnostic efficiency for operative detection of squamous LC (area under the curve, AUC, 0.982), while the microRNA-3662 was effective for detecting the operable lung adenocarcinoma (AUC 0.926) [69]. In another research of microRNA expression in blood plasma, a panel was compiled for the diagnostics of lung adenocarcinoma, consisting of seven circulating microRNA (9-3p, 96-5p, 147b-3p, 196a-5p, 708-3p, 708-5p, 4652-5p), as well as the panel for the diagnostics of squamous LC, containing nine various microRNA (130b-3p, 269-3p, 301a-5p, 301b-5p, 744-3p, 760, 767-5p, 4652-5p, 6499-3p) [70].

Summarizing the abovementioned, modern research works demonstrate the significant potential of microRNA as the specific biomarkers for the differential diagnostics of LC subtypes. As shown by the data, the unique microRNA expression profiles in the tissues, blood plasma and exosomes allow for clearly differentiating the NSCLC and the SCLC with an accuracy of up to 93.7% [68]. Special attention deserve the microRNA panels effectively differentiating the SCLC from the NSCLC, and the developments based on the analysis of the circulating microRNA, opening the possibilities for the non-invasive diagnostics [69]. For the standardization of the panels and their implementation into the Clinical laboratories, further multi-center research works are necessary with the unified protocols of the isolation, the analysis of microRNA and the normalization of the obtained data. Nevertheless, already as of today, microRNA represent a powerful tool in personalized oncology, capable of improving the precision of diagnostics and the treatment tactics for the LC patients.

Protein markers

Modern research works differentiate several classes of candidate proteins, demonstrating the differential expression in cases of SCLC and NSCLC. These include the neuroendocrine differentiation proteins (ProGRP, NSE), the cytokeratins and their fragments (CYFRA 21-1), the adhesive molecules (EpCAM, CEACAM), the embryonic antigens (CEA), the carbohydrate antigens (CA 125, CA 19-9). High level of neuroendocrine differentiation proteins is observed in cases of SCLC, while the other protein markers are elevated in cases of NSCLC. Thus, the levels of ProGRP (pro-gastrin-releasing peptide) — one of the most SCLC-specific markers, related to the neuroendocrine origin of the tumor — in the blood serum clearly correlate with the histological type of LC: abnormal levels of ProGRP are detected in 60–70% of the patients with localized stage of SCLC and in 75–90% of the patients with the advanced stage of the disease, while the elevation of ProGRP level to 120 pg/ml was observed only in 4% of the NSCLC cases. As for the adjacent areas of application, according to the data from the multifactorial analysis, ProGRP has no independent prognostic value [71]. Another protein — NSE (neuron-specific enolase) — acts as the highly specific marker for the neurons and peripheral neuroendocrine cells. Taking into consideration the location of NSE in certain tissues at the normal state, the elevation of its level in biological fluids can indicate the presence of malignant proliferation and have a diagnostic value for the detection, the staging and the treatment of neuroendocrine tumors. NSE is more often increased in cases of SCLC, however, its specificity is lower comparing to the ProGRP, due to the possible elevation in a number of other diseases (for example, in cases of neuroblastoma, melanoma, seminoma etc.) [72].

The levels of serum biomarkers, such as the serum amyloid А (SAA) and CYFRA 21-1, are increased in cases of NSCLC, especially in squamous-cell cancer and adenocarcinoma, which allows for using them for the differentiation with SCLC [73]. Carcinoembryonic antigen CEA and the CEACAM carcinoembryonic antigen-related cell adhesion molecule are more often associated with adenocarcinoma and other forms of NSCLC, but can be also elevated in cases of SCLC, which decreases their specificity. In the detailed review of the available literature data, it was shown that serum CEA has an independent prognostic and predictive value in cases of NSCLC regardless of the type of treatment, however, its diagnostic value is insignificant [74]. SCC (squamous-cell carcinoma antigen) — the marker of squamous carcinoma, one of the subtypes of NSCLC, is useful for the differentiation of SCLC. The CA 125, CA 19-9 and CA 15-3 markers are being investigated in a context of NSCLC, especially the adenocarcinoma. It was shown that the levels of CEA, SCC, СА 125, СА 15-3 and TAG-72-3 were significantly higher in NSCLC cases as compared to the SCLC [75].

A number of research works have shown that various combinations of markers, for example, ProGRP and NSE, CYFRA21-1 and SCC-Ag, or CEA + CYFRA 21-1 + SCC/СА 15.3, allow for considerably increasing the sensitivity and specificity parameters comparing to the single marker protein [74–77]. Special diagnostic interest arises from the fact that many of these markers not only reflect the histogenetic features of the tumor, but also correlate with the activity of specific molecular pathways, which opens the potential possibilities for using them in personalized therapeutic strategies.

Thus, the special interest for the differential diagnostics is gained specifically by the proteins associated with the neuroendocrine differentiation (ProGRP, NSE) for the SCLC and with the epithelial tumors (CYFRA 21-1, CEA etc.) for NSCLC. Their detection in blood serum bears a significant diagnostic potential, allowing for not only differentiating the cancer subtypes, but also for evaluating the dynamic changes of the disease during the treatment course. However, none of the known markers shows the absolute specificity, which dictates the necessity of searching the optimal combinations and developing the standardized algorithms of interpretation.

Exosomal proteins

Low concentrations of tumor-specific proteins in blood plasma/serum oftentimes do not allow to reliably detect these proteins using the accessible methods, with this, the exosomes secreted by the tumor cells (and other micro-vesicules) can be concentrated and “washed off” from the ballast blood plasma proteins, or the proteins produced during the blood cell lysis, which will significantly simplify their further analysis. Indeed, it was shown that exosomal proteins can be used for the diagnostics of oncological diseases: for example, the exosomal markers CD151, CD171 and tetraspanin 8 have a significant potential for the diagnostics of oncological diseases in the lungs in general [78]. As for the differential diagnostics of LC types, encouraging results were obtained: for example, integrin αV is expressed in the exosomes of cancer cells of both LC types, while the epithelial-specific heterodimer of integrin α6β4 was selectively expressed in the exosomes of NSCLC [79]. At the same time, there is only a small number of comparative research works on the exosomes in NSCLC and SCLC patients. In particular, it was found that the expression of JUNB and CXCR4 is increased in the exosomes of SCLC patients comparing to the healthy donors, however, it remained unclear if there are differences in the expression levels of these proteins in the exosomes of NSCLC patients [80]. The most part of research works is devoted to studying the most widespread NSCLC. Thus, M. Bao et al. [81] have found the exosomal proteins having the potential for the diagnostics, the staging and the prognosis of this disease.

It is necessary to note that there is a number of problems limiting the use of exosomes for the diagnostics of oncological diseases, including the LC. First of all, the applied methods of extraction and analysis of the exosomal proteins are heterogenous and require unification. Secondly, the exosomes present in the biological fluids have various origin, including the non-tumor one. Such exosomes have a set of markers typical for healthy cells, which can complicate the identification of marker proteins. Thirdly, the majority of models were built using the small and limited samples, while it is necessary to arrange the large prospective research, solving the validation objective. Thus, the exosomal proteins demonstrate strong potential for the differential diagnostics, for the predicting and the monitoring of SCLC and NSCLC, however, the evaluation of their clinical value requires the development of standardized protocols, large-scale multi-center research and, probably, the inclusion of proteomic data into the multiomic diagnostic systems.

Radiomics

Radiomics — the extraction and analysis of data obtained from the medical images — is a rapidly developing modern field of Medicine. The development of the reliable systems for computed diagnostics using artificial intelligence is already acknowledged as important by the part of research works in medical visualization. The algorithms based on artificial intelligence are learning to process the visualization data with further setting the diagnosis. There is a whole number of research works demonstrating the possibilities of artificial intelligence in the diagnostics, the staging, in predicting and sub-typing the NSCLC [82–85]. In general, the majority of models demonstrate the diagnostic efficiency that is comparable or even exceeding the efficiency of the experts, while the general problems are the reproducibility and the adaptation for use in clinics [82]. At the same time, despite the active usage of the artificial intelligence approaches for sub-typing the NSCLC, very few research works are aimed at solving the tasks of differential diagnostics of SCLC and NSCLC using CT/PET data [86-89]. The specific features of the clinical course of SCLC (higher mortality) and the diagnostics routing often result in the lesser extent of visualization data (especially PET/CT), applicable for radiomic analysis, which, in turn, explains the significant sub-representativity of clinically and morphologically confirmed cases of SCLC in the public and institutional PET/CT visualization data sets. For example, the data set (the structurized collection of data) from the TCIA (Cancer Imaging Archive) for non-small-cell lung cancer (NSCLC) of the Radiogenomics dataset (cancerimagingarchive.net) (https://www.cancerimagingarchive.net/collection/nsclc-radiogenomics/), compiled in 2018, includes only the cases of non-small-cell cancer, and the widely used LIDC-IDRI, compiled by the Lung Image Database Consortium and by the Image Database Resource Initiative (cancerimagingarchive.net) (https://www.cancerimagingarchive.net/collection/lidc-idri/) contains no histological verification. Thus, there is an urgent need for creating the SCLC specialized data sets.

Nevertheless, several commercial platforms are already available for solving the similar tasks by means of radiomics: they include the OncoRadiomics (OncoRadiomics SA — the compilation of the prognostic model for NSCLC), the IBEX (IBM Watson Health — research on NSCLC), Mirada Medical (Canon Medical Systems — building the validated models for predicting the response to immunotherapy; HealthMyne — compiling the prognostic models for SCLC).

Thus, the statistically insignificant share of SCLC cases in the data sets and the insufficient standardization of visualization protocols significantly restrict the possibilities of developing and validating the artificial intelligence models, directed at the non-invasive differential diagnostics of SCLC and NSCLC, nevertheless, separate research works devoted to this problem [85–88] and the advance in the adjacent fields indicate that radiomics based on machine learning can be used for the differentiation of SCLC from the NSCLC along with other lung neoplasms, it also can be included into the multimodal diagnostic systems (for example, including the CT, the PET, the clinical data and the molecular markers).

Volatile organic compounds

Volatile organic compounds (VOC) represent the low molecular mass (<300 Da) metabolites, excreted by tumor cells as a result of abnormal metabolism. These compounds (alkanes, ketones, aldehydes, aromatic carbohydrates) enter the circulation and become excreted via the respiratory system, which makes them the promising non-invasive biomarkers [90]. The biological significance of VOC comes from their direct relation to the key oncological processes. This field is being actively developed, for it allows for detecting the molecular patterns reflecting the differences in the metabolism of various types of tumor cells. The method of gas chromatography–mass-spectrometry (combining the gas chromatography and mass spectrometry — GC-MS) is the gold standard in detecting the LOC, that allows to precisely identify the individual volatile compounds.

Non-invasive tests based on detecting the VOC are also being developed for the differential diagnostics of SCLC and NSCLC [91]. Specific metabolites were identified that differentiate the VOC-profiles of SCLC from the NSCLC. Some compounds, such as alkanes, show the presence of high correlation with LC, which indicates the high practicability of using the specific VOC for its diagnostics [92]. As for the differential analysis of SCLC and NSCLC, controversial results were obtained here. Thus, in the research involving the number of cell lines, the analysis of VOC and metabolites has allowed for significantly differentiating the LC and the normal cells, as well as the SCLC and the NSCLC, including various NSCLC subtypes. SCLC differed from NSCLC by m- and p-xylenes, ethyl-benzol, styrene, о-xylene, 1.3-bis(1.1-dimethylethyl)-benzol and 2.4-bis(1.1-dimethylethyl)-phenol, and each of these VOC had an AUC value above 0.95 [93]. Another research has analyzed VOC in patients with NSCLC and SCLC along with healthy donors, and, though it successfully helped differentiating the patients and the healthy donors, it was not successful in the differentiation of LC subtypes [94]. The analysis including the research on the differences of VOC profiles between the SCLC and the NSCLC, has revealed an elevation of hexanal levels (p <0.006) in cases of SCLC [95], however, the observed differences were presumably related to higher malignancy and increased tumor cell activity in the SCLC. Thus, the issue on how effectively it is possible to differentiate SCLC and NSCLC based on the VOC, remains unsolved and requires further research.

The promising approach to analyzing the VOC are the artificial sensory systems, or the “electronic nose” (eNose). eNose is the integrated system, mimicking the olfaction with the main task being the recognition and classification of complex VOC-mixtures; it includes a sensor module and the data processing system (algorithms of machine learning, the method of main component, the linear discriminant analysis etc.), which classifies the tested “odor”. The sensor module can use various technologies: gas chromatography, gas sensors based on semiconductors with metal oxide (metal-oxide-semiconductor, MOS), devices with combined sensors made of conducting polymers, quartz microbalance (QMB), the colorimetric sensors, the chemical resistors and the superficial acoustic waves [96]. “Electronic noses”, generally, do not identify specific compounds, but operate with a combined odor “fingerprint”. The artificial sensory systems quickly analyze the VOC-profiles of the expiratory air and use the machine learning algorithms for classifying the type of tumor. Currently there is a limited number of research works evaluating the efficiency of such an odor “fingerprint” for the diagnostics of SCLC and NSCLC patients, with this, encouraging results were obtained that show the sensitivity/specificity of the differential diagnostics of SCLC and NSCLC when using the eNose equaling 87% [97]. Despite the optimistic findings in this field, there is still a number of problems to solve, among which, for example, is the effect of unspecified factors on the precision of detecting the LOC, the contribution of individual specific features of the patients into the specificity of diagnostics (for example, VOC characteristic for LC, can be also excreted in cases of chronic bronchitis or chronic obstructive pulmonary disease) [98]. Besides, an important contribution to the concentration of VOC is made by the individual variations, for the metabolism of VOC depends on the age, the gender, the diet, the smoking and the microbiome of the oral cavity [99, 100]. VOC analysis based on the eNose is a promising tool for the non-invasive differentiation of SCLC and NSCLC, especially for developing the screening tests, for it allows for conducting a rapid and non-invasive testing, however, its sensitivity and specificity require large-scale and profound research.

CONCLUSION

The modern differential diagnostics of SCLC and NSCLC is experiencing a period of active transformation, transitioning from the traditional invasive methods to the combined non-invasive approaches. Despite the undisputed significance of the immunohistochemical examination as the golden standard, its limitations stimulate the development of principally new diagnostic strategies.

The promising directions including the analysis of circulating tumor cells, of the extracellular DNA, of the exosomal markers, the microRNA and the volatile organic compounds, demonstrate the significant diagnostic potential. Parallel development is observed in the methods of radiomics and artificial intelligence, opening new possibilities in processing the medical images and multiomic data. However, the transition of these technologies into clinical practice meets a number of methodological and practical difficulties. The key issues remaining are the standardization, the reproducibility and the validation of new methods. With this, the differential diagnostics of rare and aggressive forms of cancer, such as the SCLC, represents a special complexity.

The future of differential onco-diagnostics, undoubtedly, belongs to the multimodal methods, integrating the advances in the fluid biopsy, the radiomics and the artificial intelligence into the unified diagnostic algorithms. It is specifically the synergy of various diagnostic approaches that will allow for overcoming the limitations of separate methods and for achieving the new level of precision. And, in achieving it, the important role in the processing of multimeric data, will be played by the modern algorithms, including the method of the main components, the linear discriminant analysis, the random forest method, the reference vectors method and the neuron networks (including the deep learning). The combined approach to the diagnostics of lung cancer with taking into consideration the individual characteristics of the patient will also open new possibilities for the personalized treatment, which ultimately will significantly improve the quality and the duration of the patients’ life.

ADDITIONAL INFORMATION

Author contributions: M.Yu. Konoshenko, concept of the review, literature analyses, writing the manuscript; P.P. Laktionov, manuscript critical revision, editing; O.E. Bryzgunova, editing, preparation of tables, writing the “Molecular markers of lung cancer” chapter; E.V. Shutko, writing the “MiRNA markers of lung cancer” chapter; A.A. Ilyushchenko, Ya.M. Danilova, S.D. Gorbunkov, writing the “Clinical diagnosis of lung cancer” chapter; K.A. Zykov, methodological support, technical editing of the review. Thereby, all authors provided approval of the version to be published and agree to be 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.

Funding sources: The study was funded by the Russian state-funded project for the Federal State Budgetary Institution “Pulmonology Scientific Research Institute under Federal Medical and Biological Agency of Russian Federation” (grant number 388-03-2024-136) and supported by the Russian state-funded project for Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (grant number 125012900932-4).

Disclosure of interests: The authors declare no conflict of interests.

Statement of originality: The authors did not utilize previously published information (text, illustrations, data) in creating this article.

Data availability statement: The editorial policy regarding data sharing does not apply to this work.

Generative AI: Generative AI technologies were not used for this article creation.

 

¹ National Cancer Institute [Internet]. Cancer Stat Facts: lung and bronchus cancer. Available at: https://seer.cancer.gov/statfacts/html/lungb.html

² American Cancer Society [Internet]. Lung cancer survival rates. Available at: https://www.cancer.org/cancer/types/lung-cancer/detection-diagnosis-staging/survival-rates.html

About the authors

Maria Yu. Konoshenko

Institute of Chemical Biology and Fundamental Medicine; Pulmonology Scientific Research Institute under Federal Medical and Biological Agency of Russsian Federation

Email: lacyjewelrymk@gmail.com
ORCID iD: 0000-0003-2925-9350
SPIN-code: 9374-8489

PhD

Russian Federation, Novosibirsk; Moscow

Ekaterina V. Shutko

Institute of Chemical Biology and Fundamental Medicine; Pulmonology Scientific Research Institute under Federal Medical and Biological Agency of Russsian Federation

Author for correspondence.
Email: katshutko@gmail.com
ORCID iD: 0009-0004-3004-8969
SPIN-code: 3627-2494
Russian Federation, 8 Lavrentyeva ave, Novosibirsk, 630090; Moscow

Olga E. Bryzgunova

Institute of Chemical Biology and Fundamental Medicine; Pulmonology Scientific Research Institute under Federal Medical and Biological Agency of Russsian Federation

Email: olga.bryzgunova@niboch.nsc.ru
ORCID iD: 0000-0003-3433-7261
SPIN-code: 9752-3241

PhD

Russian Federation, Novosibirsk; Moscow

Antonina A. Ilyushchenko

Pulmonology Scientific Research Institute under Federal Medical and Biological Agency of Russsian Federation

Email: Kdlmedwans@gmail.com
ORCID iD: 0009-0003-9068-5401
Russian Federation, Moscow

Yaroslava M. Danilova

Pulmonology Scientific Research Institute under Federal Medical and Biological Agency of Russsian Federation

Email: yaroslava.danilova.82@mail.ru
ORCID iD: 0009-0003-6679-9185
Russian Federation, Moscow

Stanislav D. Gorbunkov

Pulmonology Scientific Research Institute under Federal Medical and Biological Agency of Russsian Federation

Email: sdgorbunkov@mail.ru
ORCID iD: 0000-0002-8899-4294
SPIN-code: 7473-0530

MD, PhD, Assistant Professor

Russian Federation, Moscow

Kirill A. Zykov

Pulmonology Scientific Research Institute under Federal Medical and Biological Agency of Russsian Federation

Email: kirillaz@inbox.ru
ORCID iD: 0000-0003-3385-2632
SPIN-code: 6269-7990

MD, PhD, corresponding member of the Russian Academy of Sciences, Professor of the Russian Academy of Sciences

Russian Federation, Moscow

Pavel P. Laktionov

Institute of Chemical Biology and Fundamental Medicine; Pulmonology Scientific Research Institute under Federal Medical and Biological Agency of Russsian Federation

Email: lakt@1bio.ru
ORCID iD: 0000-0002-0866-0252
SPIN-code: 4114-3170

PhD

Russian Federation, Novosibirsk; Moscow

References

Supplementary files