**2.2 ALDH family proteins**

The cancer stem cell model proposes that tumor progression, drug resistance, metastasis, and relapse after therapy may be driven by a subset of cells within the tumor: the cancer stem cells (CSCs) [17–20]. Recent evidences suggest that like other tumors, human lung cancers may also harbor CSC populations. Human alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are the principal enzymes responsible for ethanol metabolism and have heterogeneous tissue distribution. Isoenzymes of ADH participate in bioamine, prostaglandin, and retinoid acid metabolism [21]. The second enzyme ALDH belongs to a large family of intracellular enzymes that participate in cellular detoxification, differentiation, and drug resistance through the oxidation of endogenous and exogenous aldehydes to carboxylic acids [22]. The ALDH superfamily currently consists of 19 known putatively functional genes in 11 families and 4 subfamilies with distinct chromosomal locations [23–25]. Several studies have explored the biological significance of ALDH in cancers such as head and neck cancer, colon cancer, breast cancer, papillary thyroid carcinoma, and specifically lung cancer, where they have provided supportive evidence for the association between ALDH activity and lung cancer

**109**

**Table 1.**

particularly in lung cancer [36–38].

**3. Lung cancer screening**

*Molecular Biology of Lung Cancer and Future Perspectives for Screening*

**Altered genes Histology Mutation frequency (%) References** *KRAS* Ade 30 [5] *BRAF* NSCLC 3 [6] *EGFR* ADC 19 [5] *HER2* Ade 10 11 *MET* NSCLC 8–10 [6] *FGFR1* SCC 22 [14, 15] *CD74-NRG1* Ade 27 [8] *NTRK1* Ade 3.3 [13] *KIF5B-* and *CCDC6-RET* Ade 0.9 [13] *EML4/ALK* NSCLC 6.7 [10] *DDR2* SCC 4 [16] *c-KIT* SCLC, NSCLC 30–40 (SCLC), 40 (NSCLC) [7] *PIK3CA* SCC 70 [7] *PTEN* SCLC 70 [7] *IGFR1* NSCLC 29.2 [80]

stem cells [26–32]. ALDH1A1 seems to be co-expressed with other NSCLC stem cell markers such as leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) in NSCLC tissues, and their expression is significantly associated with stage disease and poor prognosis [33]. It was reported that ALDH1A1-negative expression in lung cancer patients corresponds to shorter survival compared to those with ALDH1A1-positive expression and that ALDH1A1 overexpression was associated with a favorable outcome. Moreover, high expression of ALDH1A1 mRNA was found to be correlated to a better overall survival (OS) in all NSCLC patients followed for 20 years. In addition, high expression of ALDH1A1 mRNA was also found to be correlated to better OS in Ade patients but not in SCC patients. These results strongly support that ALDH1A1 mRNA in NSCLC is associated with better prognosis. However, there are other contradictory results indicating that ALDH1 cytoplasmic expression was associated with poor prognosis in several tumors, such as NSCLC [34]. Jiang et al. also showed that ALDH1A1 expression was positively correlated with the stage and grade of lung tumors and related to a poor prognosis [34]. A recent meta-analysis showed that increased ALDH1A1 expression is associated with poor OS and disease-free survival in lung cancer patients [35]. Previous studies showed that also several other ALDH isoforms are involved in lung cancer as ALDH3A1, highly expressed in two types of NSCLC, Ade and SCC, and ALDH3B1 expression was also found to be upregulated in a high percentage of human tumors,

Cancer screening is promising for malignancies with a stage-dependent prognosis, and it aims to reduce morbidity and mortality through detection of cancer at an early stage. In general, the screening programs have to be subjected to a rigorous risk-benefit assessment taking into account the endpoints as cancer-related

*DOI: http://dx.doi.org/10.5772/intechopen.85334*

*Recurrent somatic genetic alterations detected in lung cancer.*


*Molecular Biology of Lung Cancer and Future Perspectives for Screening DOI: http://dx.doi.org/10.5772/intechopen.85334*

#### **Table 1.**

*Mass Spectrometry - Future Perceptions and Applications*

biopsy to improve the early detection of this disease.

**2. The molecular landscape of lung cancer**

**2.1 Gene mutations**

**2.2 ALDH family proteins**

biology of lung cancer and conventional diagnostic methods currently used, with a particular attention on the development of new screening approaches such as liquid

Recent advances in next-generation sequencing (NGS) and other highthroughput genomic profiling platforms have allowed the examination of the breadth of genetic mutations within lung cancer. The most common mutation is in the Kirsten rat sarcoma (KRAS) oncogene, occurring in approximately 30% of adenocarcinomas (Ade) predominantly in patients with a history of smoking [5]. BRAF is mutated in approximately 3% of patients (with half of cases being the V660E mutation) [6]. Along with KRAS and BRAF, epidermal growth factor receptor (EGFR) mutations were discovered in patients with Ade and small cell lung cancer (SCLC) [5]. Moreover, mutations and amplifications in many oncogenes have been identified, including HER2, MET, as well as fusion oncogenes involving anaplastic lymphoma kinase (ALK), neuregulin 1 (NRG1), neurotrophic tyrosine kinase receptor type 1 (NTRK1), and RET [7–13]. Microtubule-associated protein-like 4 (EML4) and ALK fusion gene is another important driver gene in lung cancer, which was discovered by Soda et al. in 2007 [10]. In NSCLC, EML4/ ALK is an aberrant fusion gene that encodes a cytoplasmic chimeric protein with constitutive kinase activity. The incidence of EML4/ALK fusion in cohorts of patients with NSLCL ranges from 1.6% to as high as 19.3%. Genes such as discoidin domain-containing receptor 2 (DDR2); fibroblast growth factor receptor 1, 2, and 3 (FGFR1, FGFR2, FGFR3); and genes in the phosphatidylinositol 3 kinase (PI3K) pathway seem instead to be more commonly mutated in squamous cell carcinoma (SCC). Many of these mutations have been validated by preclinical studies as driver mutations [14–16]. Aberration in stem cell factor receptor tyrosine kinase (c-KIT), PI3K catalytic subunit alpha (PIK3CA), PI3K/AKT/mTOR, phosphatase and tensin homolog (PTEN), insulin-like growth factor receptor (IGFR1), and hedgehog (Shh) signaling pathways have been identified in lung cancer [7] (**Table 1**).

The cancer stem cell model proposes that tumor progression, drug resistance, metastasis, and relapse after therapy may be driven by a subset of cells within the tumor: the cancer stem cells (CSCs) [17–20]. Recent evidences suggest that like other tumors, human lung cancers may also harbor CSC populations. Human alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are the principal enzymes responsible for ethanol metabolism and have heterogeneous tissue distribution. Isoenzymes of ADH participate in bioamine, prostaglandin, and retinoid acid metabolism [21]. The second enzyme ALDH belongs to a large family of intracellular enzymes that participate in cellular detoxification, differentiation, and drug resistance through the oxidation of endogenous and exogenous aldehydes to carboxylic acids [22]. The ALDH superfamily currently consists of 19 known putatively functional genes in 11 families and 4 subfamilies with distinct chromosomal locations [23–25]. Several studies have explored the biological significance of ALDH in cancers such as head and neck cancer, colon cancer, breast cancer, papillary thyroid carcinoma, and specifically lung cancer, where they have provided supportive evidence for the association between ALDH activity and lung cancer

**108**

*Recurrent somatic genetic alterations detected in lung cancer.*

stem cells [26–32]. ALDH1A1 seems to be co-expressed with other NSCLC stem cell markers such as leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) in NSCLC tissues, and their expression is significantly associated with stage disease and poor prognosis [33]. It was reported that ALDH1A1-negative expression in lung cancer patients corresponds to shorter survival compared to those with ALDH1A1-positive expression and that ALDH1A1 overexpression was associated with a favorable outcome. Moreover, high expression of ALDH1A1 mRNA was found to be correlated to a better overall survival (OS) in all NSCLC patients followed for 20 years. In addition, high expression of ALDH1A1 mRNA was also found to be correlated to better OS in Ade patients but not in SCC patients. These results strongly support that ALDH1A1 mRNA in NSCLC is associated with better prognosis. However, there are other contradictory results indicating that ALDH1 cytoplasmic expression was associated with poor prognosis in several tumors, such as NSCLC [34]. Jiang et al. also showed that ALDH1A1 expression was positively correlated with the stage and grade of lung tumors and related to a poor prognosis [34]. A recent meta-analysis showed that increased ALDH1A1 expression is associated with poor OS and disease-free survival in lung cancer patients [35]. Previous studies showed that also several other ALDH isoforms are involved in lung cancer as ALDH3A1, highly expressed in two types of NSCLC, Ade and SCC, and ALDH3B1 expression was also found to be upregulated in a high percentage of human tumors, particularly in lung cancer [36–38].

### **3. Lung cancer screening**

Cancer screening is promising for malignancies with a stage-dependent prognosis, and it aims to reduce morbidity and mortality through detection of cancer at an early stage. In general, the screening programs have to be subjected to a rigorous risk-benefit assessment taking into account the endpoints as cancer-related

mortality, overall mortality, morbidity, patient-reported outcome, and costs. All the screening programs need a transparent system of quality assurance.

## **3.1 Low-dose computed tomography**

Several studies on lung cancer screening were conducted mainly by using chest X-rays (CXR) for imaging alongside sputum cytology. The National Lung Screening Trial (NLST) enrolled 53,000 individuals aged 55–74 years with a 30-pack-year smoking history, and participants were randomly assigned to radiography or low-dose CT. The low-dose CT group had a 20% reduction in lung cancer mortality and a 6–7% reduction in all-cause mortality [39]. The International Early Lung Cancer Action Program (I-ELCAP) analyzed retrospectively the outcomes of more than 21,000 patients after the completion of the NLST. Different size threshold for nodule diameters resulted in different cancer diagnosis rates. Increasing the threshold from 5 to 0 mm to 6–0, 7–0, 8–0, or 9–0 mm also changed the frequencies of positive results [40]. With respect to North American, European studies performed on a smaller number of individuals at risk of lung cancer showed somewhat inconsistent and less significant results [41–43]. Although these studies showed an improved stage distribution in favor of earlier stages, better resectability of the tumors, and also improved survival, an effect on overall mortality could not be demonstrated [39, 44, 45]. Aside from the morbidity and mortality that is not justified within this context, the expenses turn out to be substantial, as thoracic imagery can be repeated, leading also to debated benefit risks. Despite the progress made in imagery, which allowed the detection of nodules less than 3–4 mm and even the definition of the malignant or benign features, currently cancerous lesions less than 1 mm cannot be detected by imagery [46]. A major drawback of low-dose CT is the large number of false-positive tests and the diagnosis of indolent tumors which in turn lead to an increased morbidity from unnecessary surgical treatment [47–50]. Thus, even if the imagery can allow early stage asymptomatic and operable lung cancer detection, these approaches are not satisfactory because of high cost, high risk of radiation exposure, and poor sensitivity and specificity.

#### **3.2 Biomarkers for lung cancer detection**

The discovery of cancer biomarkers, specific molecules that help to distinguish between normal and cancerous conditions, may potentially be used to develop a more effective diagnostic tool for cancer. Body fluids (blood, pleural effusion, etc.) that are in contact with tumors are enriched with proteins shed from cancer cells. Proteins secreted from cancer cells could enter the blood circulation and have the potential to be monitored in plasma/serum. Carcinoembryonic antigen (CEA) is an oncofetal protein not typically expressed in adult tissues. In lung cancer the CEA levels in blood are elevated and are inversely correlated with the response to cancer therapy. Therefore, this marker is used for the detection of cancer recurrence and the prediction of a poor survival rate. CYFRA-21-1 is a fragment of cytokeratin 19 that is typically associated with epithelial cell cancers including NSCLC. This marker is correlated with disease response and the prognosis of cancer but cannot be used to identify cancer patients from patients with respiratory diseases. The sensitivity of CYFRA 21-1 for NSCLC ranges between 23 and 70% [51, 52]. Neuronspecific enolase (NSE) is a glycolysis enzyme produced in neuronal cells and cells with neuroendocrine differentiation. SCLC is of neuroendocrine origin, and therefore NSE is found to be elevated in patients' blood [53]. Tumor M2-pyruvate kinase (PKM2) is a dimeric form of the pyruvate kinase isoenzyme type M2 that is increased in various cancers [52, 54]. C-reactive protein (CRP) is an acute-phase

**111**

*Molecular Biology of Lung Cancer and Future Perspectives for Screening*

protein, the levels of which rise in response to inflammatory conditions such as lung cancer. However, recent studies suggested that CRP could be used as a prognostic biomarker of lung cancer and angiogenesis [55]. Serological markers such as CEA, NSE, and CYFRA 21-1 are used for the monitoring of treatment effects in lung cancer, but their diagnostic value as screening biomarkers is still being debated [56, 57]. To date, no useful marker has been identified for the screening of asymptomatic patients. Ideally, a biomarker should have a sensitivity and specificity of 100%, a goal that is almost never achieved. One strategy potentially increasing both parameters is to combine several biomarkers into a screening marker panel. Several studies with smaller panels encompassing few markers provided first evidence that simultaneous analysis of several antigens have a higher potential for separating patients with lung cancer from controls [56]. Combined with other noninvasive methods, this may allow for further refinement of lung cancer screening [58].

Proteomics studies showed new lung cancer biomarkers that can be tested in the blood (**Table 2**). Plasma kallikrein (KLKB1) enzyme cleaves Lys-Arg and Arg-Ser bonds in kininogen to release bradykinin and has functions related to blood coagulation. Studies evidenced how serum levels of its fragmentation form were increased in lung cancer samples compared with normal control sera [59, 60]. Serum amyloid A (SAA) proteins are a family of apolipoproteins associated with the high-density lipoprotein (HDL) complex that are secreted during the acute phase of inflammation. In particular, isoforms SAA1/2 were detected in Ade patients' sera but not in healthy donors' sera using liquid chromatography/mass spectrometry (LC-MS/ MS). This protein was also detected in tissue [59, 61]. Haptoglobin (Hp) is a free hemoglobin-binding glycoprotein that inhibits the oxidative stress of hemoglobin and assists in hemoglobin uptake. It is a tetramer constituted by two α and two β chains. High levels of Hp have been reported in various cancer types including lung cancer. Proteomics analysis showed Hp β chain peptide levels to be threefold higher in lung cancer patients' sera with respect to control subjects [59, 62]. Complement component 9 (C9) protein, a terminal constituent of the membrane attack complex, plays a role in the immune response by forming plasma membrane pores. This protein was identified in sera of patients with SCC by glycoproteomics approaches. Its protein levels were significantly higher in SCC patients than those in healthy donors and in patients with other cancer types [59, 63]. Insulin-like growth factorbinding protein-2 (IGFBP-2), member of the insulin-like growth factor-binding protein family, inhibits IGF-mediated growth and development rates. Increased levels of IGFBP-2 have been found in solid tumors and in blood from patients with glioma and colorectal, prostate, and breast cancers above all at advanced stage disease. Recently circulating anti-IGFBP-2 autoantibodies and IGFBP-2 combined markers showed increased diagnostic sensitivity and specificity for lung cancer with respect to IGFBP-2 alone [64]. Peroxiredoxin 1 (PRX1) and peroxiredoxin 2 belong to a family of ubiquitous multifunctional antioxidant proteins. The main function of PRX1 is to eliminate peroxides generated during metabolism. PRX1 is also involved in the inhibition of oncogenes, and its protein levels were found to be higher in human cancer cells and tissues. Recently, PRX1 was also identified in lung cancer patients' plasma by mass spectrometry-based screening technology. Plasma PRX1 levels were increased in patients with lung cancer and also in subjects exposed to asbestos [65]. Endoglin (CD105) is a major cell membrane glycoprotein

*DOI: http://dx.doi.org/10.5772/intechopen.85334*

**4. Future perspectives**

**4.1 New potential lung cancer biomarkers**

*Molecular Biology of Lung Cancer and Future Perspectives for Screening DOI: http://dx.doi.org/10.5772/intechopen.85334*

protein, the levels of which rise in response to inflammatory conditions such as lung cancer. However, recent studies suggested that CRP could be used as a prognostic biomarker of lung cancer and angiogenesis [55]. Serological markers such as CEA, NSE, and CYFRA 21-1 are used for the monitoring of treatment effects in lung cancer, but their diagnostic value as screening biomarkers is still being debated [56, 57]. To date, no useful marker has been identified for the screening of asymptomatic patients. Ideally, a biomarker should have a sensitivity and specificity of 100%, a goal that is almost never achieved. One strategy potentially increasing both parameters is to combine several biomarkers into a screening marker panel. Several studies with smaller panels encompassing few markers provided first evidence that simultaneous analysis of several antigens have a higher potential for separating patients with lung cancer from controls [56]. Combined with other noninvasive methods, this may allow for further refinement of lung cancer screening [58].
