MOLECULAR PATHOLOGY OF LUNG ADENOCARCINOMA

POPESCU IULIAN PhD, MD, Clinical Department of Radio-Biology at the Fundeni Clinical Institute in Bucharest

e-mail: popdociul@yahoo.com

              ALINA HALPERN PhD, SF.ŞTEFAN Hospital  Bucharest

INTRODUCTION.

Lung Cancer (C:P.) develops through the accumulation of multiple alterations molecular, genetic and epigenetic causing the aberrant gene functioning. Aneusomya (the presence of an abnormal number of chromosomes) is associated with lung cancer. It has not been defined yet whether aneumosomya also exists in pre-neoplastic lesions (3)). Smoking is an important etiologic factor. The tobacco components facilitates tumorigenesis through genotoxic efects and by modeling the signaling pathways.
            Lung cancer still remains a serious illness, the number of deaths exceeding deaths following the cancer of prostate, breast and colon taken together. The five-year survival remained 15 %, instead in the other locations increased up to 64 % of colon cancer , 88 % breast cancer and 99 % prostate cancer  (4.1)

Today, the lung cancer classification is made by the morphological appearance of the cells and the surrounding tissue.

 The limits are due to the degree of knowledge and experience of the pathologist (therefore is partially subjective). The classification gives a limited or minimal information on the chance of successful treatment. Also tumors with identical pathology may have different origins and different responses to treatment. (6).

Perfection of the human genome sequence using gene expression analysis has the potential to revolutionize the cancer diagnosis and its treatment.

The classification of cancerous tissue based on molecular profile breaks these limits. Today, with the new techniques, we can obtain information from multiple genes simultaneously.

The molecular profile informs us on the potential of disease prognostic, helping us to choose the therapy method /(5). Also, the expression profile helps us to distinguish primary tumor from metastases of extra pulmonary origin (6).

Adenocarcinoma forms a part of the non-small cell lung cancer. It is treated similarly, regardless of its biological and histological heterogeneity and the low response rates to treatment would be due to a homogeneous treatment method to a heterogeneous disease.

The molecular biology and genetic data are helping us today to prove the heterogeneity of lung adenocarcinoma (ADC) (7)

LUNG ADENOCARCINOMA 

Adenocarcinoma is one of the three types of small cell lung cancer. In the 1940-1950 adenocarcinoma was rare, in LC prevailing micro-cell and squamous (epidermoid) forms.

An anatomical-pathological study performed between 1980-1986 (1pg. 251) indicated that the ADC was a rarity, namely 12% compared to 35% in squamous cancer and 25% in microcell cancer.

At the same institution, between 1990-1996, ADC hit the first place with 37%, reaching 42% in 2001 (1 pg251). As location only 20% of ADC arises in the central bronchial area, the rest arises in terminal respiratory units.

The simplest explanation is the following (in the absence of other arguments): in 1950 it was established that there is a connection between LC and smoking. Gradually, the filter cigarettes have been introduced. They led to a decrease in the nicotine content from 2.7 mg to 1mg and the tar lowered from 38 mg to 13.5 mg between 1950-1993.

Due to this, the volatile part began to prevail. In this way, the smoke was inhaled deeper and more intense, spreading throughout the lung field. Thus the peripheral lung is exposed to high levels of carcinogenic and may explain the increase of adenocarcinoma frequency. Thus the vaporized toxins and carcinogen go towards the periphery. In cigarette smoke, the nitrate content increases from 0.5% to 1.2-1.5%. Nitrogen oxides and nitrosamines raise 2-3 times. Among these nitrosamines NNK is a strong carcinogenic having the formula 4-(methyilnitrosamino)-1- (3-pyridyl)-1-butanone. NNK may induce ADC in lungs (1, 8). NNK is formed by nicotine nitrosation. From the molecular pathology point of view, NNK induces a functional cooperation between Bcl-2 and c-Myc, facilitating the survival and proliferation (1-9). Witschi et al showed that the 1,3-butadiene contributes to lung carcinogenesis (1,10).

ROLE OF  STEM CELLS   IN  ADENOCARCINOMA

The most widely accepted theory for ADC development is the mutation caused by carcinogen that acts on the genome integrity of the local stem cells in the lung. Stem cells are multi-potent cells, able to differentiate into one or more histological types {11}. Stem cells respond immediately to aggression by activating certain renewal programs. This hypothesis would show that the transition from stem cells to cancer cell is shorter than the multi-step model (1.12). The ontogeny of lung tumor cell is determined by gene expression, which repeats the major events from the embryonic lung developments (11).

In experimental models it is implied that lung contains distinct populations of stem-cell both anatomically and functionally. They are found in the bronchoalveolar duct junction and are resistant to alterations of alveoli and bronchioles and proliferate during epithelium recovery. These data lead to the hypothesis that bronchioloalveolar stem cells are a population of stem-cells, which maintain the CLARA bronchiolar cells and alveolar cells in the distal lung area and therefore could lead through their transformation to adenocarcinoma development (1-13)}. In 20% of cases ADC arises in the central bronchial area, that is bronchial basal cells. The peripheral ADC may arise from the CLARA bronchioloalveolar stem cells (expressing CC10) and the type II pneumocyte (expressing surfactants and their transcription factor, TTF1). In the central area, stem cells might lead to squamous cancer or small cell cancer (2 pg1486).

There is another theory, according to which lung cancer is a disease derived from the bone marrow stem cells, but it is necessary to deepen this theory.

It is not known yet the origin of tumor stromal cells (1pg.254).

PRE-NEOPLASTIC LESIONS IN ADENOCARCINOMA

AAH (atypical adenomatous hyperplasia) is regarded as a precursor of bronchioloalveoolar adenocarcinoma (1.14, .15, .16.). It consists of the atypical proliferation of pneumocyte II and cells similar to CLARA cells, covering the alveolar septa in a lepidic manner.

AAH is the classic version, but are not known yet the precursors for mucinous forms, tall-columnar and sclerosing bronchoalveolar cancer. Recently has been described the bronchiolar columnar cell dysplasia (BCCD) as a precursor for the ADC coming from the bronchioles (I-17) and bronchial epithelial dysplasia for the ADC derived from the large bronchi (I-18)

The proof that BCCD is a precursor is the fact that chromosomal aberrations increase from 2.6 in BCCD to 14.7 in ADC simultaneously (I, 16). Some aberrations were the losses in chromosomes 3p, 9p, 13p, 14p, and gains in 1q, 17, 19 q 20q and (I-17)}. In the lung ADC precursors it was seen a overexpression of hTERT{human telomerase reverse transcriptase } in adenomatous atypical hyperplasia (77%) and 97% in non-mucinous bronchioloalveolar carcinoma (II,87)

Other data (1,19) have confirmed the  pre-neoplastic nature of globet cell proliferation that can lead to mucinous ADC (1.19). There have been found gains in chromosomes 2 and 4 of both forms, matching the prevalence in the ADC gains from smokers.

In AAH loss of heterozygosity (LOH) is rare compared to the ADC simultaneously where LOH is markedly high. A partial loss of heterozygosity  in TSC1 and TSC2 genes LOH was observed in AAH associated with ADC (1-20). These data show that AAH is a pre-neoplastic lesion in ADC.

Aoyagi et al (1,21) revealed the progression from AAH to invasive bronchiolo-alveolar adenocarcinoma. According to authors, progression follows from AAH to non invasive bronchoalveolar atelectasis (type B) cancer and then invasive brochoalveolar adenocarcinoma (type C). From AAH to type C- allelic losses significantly increase in the areas 3p, 17p, 18q, 22q(1-21).

Also, in smoker cancer, chromosomal losses or gains in the areas 3p, 4q, 9p, 17p, and 19p are more common in ADC from smokers compared to ADC with non-smokers. Allelic imbalance and chromosomal aberrations are rarer in non-smokers ADC. Thus raises the idea that ADC in non-smokers arise by genetic alterations distinct from events of tumors occurring in smokers (1-22)

GENETIC  ASPECTS IN LUNG ADENOCARCINOMA

Lung adenocarcinoma is a subtype of non-small cell lung cancer which is characterized by multiple somatic alterations of ADN(7)

Chromosomal aberrations in ADC are less balanced than in other forms of non-small cell lung cancer. These are relevant for prognosis, metastasis, survival.

Losses of heterozygosity in the 9p area have been found at an early stage of  LC(1-91) and in the areas 3p and 17p(1-92).  In 9q area we have chromosomal losses. Here it is also located the TSC1{Tuberous sclerosis  complex} gene, while the TSC2 gene is in the area 16p(I-93).

Loss of heterozygosity in the 9q34 area (the TSC1 gene position) is frequent in ADC. TSC1 and TSC2 genes appear to be involved in ADC development (1-20).

Loss of heterozygosity is rare in AAH and increases as frequency in ADC, proving that AAH is a precursor lesion in ADC (1 to 92,93). Between ADC and squamous subtype there are common and different chromosomal changes. There are also differences between ADC in smokers and non-smokers. Thus, the chromosomal gains and losses are more frequent in smokers ADC (in the areas: 3p, 6q, 9p, 16p, 17p, 19p) than in non-smokers with ADC (at chromosomes 1-5) (1-22).  AOYAGI has gradually shown the tumor progression. Thus we have a significant increase of allelic losses from AAH in bronchoalveolar cancer, in areas 3p, 17p, 18q and 22q (1-21). These data support the opinion that in non-smokers, LC arises through genetic alterations distinct from tumors of smokers (1-22).

The 3q chromosome amplification in lung cancer is the main signature neoplastic transformation. It is seen in bronchial dysplasia up to the metastatic stage.(97)

GENE  IN ADC

Mutations of genes in ADC. They meet early in the ADC development. Li Ding et al (23), studying 188 adenocarcinomas, have found 26 genes with significant mutations that are likely to be involved in carcinogenesis. Among the most common mutated - in decreasing order - are the genes: TP53, Kras, STK11, NF1, EPHA3, ERBB4, ELDK4, FGFR4, INHBA.

Weir et al(24) found that the areas of  amplification and deletions include 14q.13.3,12q15,8q29-21, 7p11.2 and 8q21.23.

TTF1 is frequently amplified in ADC. It is a transcription factor located on the 14q13.3 chromosome.

The amplification events are important in the initiation, differentiation and especially progression of adenocarcinoma (24).

Both ADC and its precursors have more than over-regulations than the squamous form and inhibitors are effective to ADC, but not to the squamous form. It is not known why a cancer over-regulates the expression of a gene and another lesion subregulates the same gene product.

It is possible that COX2 inhibition to prevent ADC and simultaneously to speed the development of a neuroendocrine tumor (25).

Some of the most studied genes in ADC are EGFR and Kras. The over-expression of the EGFR gene is frequent in 80% of cases in ADC. The number of gene copies correlates with the protein expression. However, they do not influence the prognosis (1,26).

The EGFR mutations are related with sensitivity to inhibitors (27). In ADC there are mutations in two ERBB genes: EGFR(ERBB1) and HER2-neu(ERBB2) (27).

 The Kras gene is associated with ADC. It is found in 40% in codon 12 (1.28). Kras mutations in codon 12 have been detected in 39% of the atypical adenomatous hyperplasia and 42% in ADC (1.29)

Among patients with AAH and ADC synchronously, one-third have Kras mutations in ADC but not in AAH; one third have mutations in AAH but not in ADC. The rest either do not have mutations, or have in both histological forms. In ADC precursors is also observed an overexpression of hTERT mainly in hyperplastic adenomatous hyperplasia (77%) and in non-mucinous bronchiolo-alveolar carcinoma in 97% (2.94). All these suggest the neoplastic nature of AAH. They also suggest that the glandular neoplasm arises on the background of cancerization field (1.30).

The Kras mutations are related to smoking. They represent 15% in non-smokers,  increase 22% in ex-smokers and 25% in smokers (30). Thus ADC becomes a heterogenity of KRAS mutations (30).

It has been attempted a subclassification of ADC correlated with the degree of tumor differentiation and survival (1.31). Also based on histological classification has also been added the manner of gene expression. ShiNgal et al have identified 40 genes differently expressed in lung ADC (1.32). In another study ShiNgal et al (1,33) have used 308 apoptotic genes, 24 of them showed differently expressed in ADC, among them being Akt, Bcl-cl PTEN and Fas.

The VEGF gene expression is important in angiogenesis. SU et al (1.34) showed that the level of COX-2 in ADC is linked with VEGF-C which over-regulates it and with the lymphatic vessel density (1.34). VEGF has been associated with angio-lymphatic invasion in the conventional adenocarcinoma (1.64). The number of vessels in the tumor correlates with the expression of IL-8mARN. The Interleukin-8mARN expression is associated not only with angiogenesis, but also with and tumor progression, survival, and time until relapse (1-36).

Another protein regulates VEGF, namely the RECK gene. The RECK gene is a favorable factor. It suppresses tumor invasion, metastasis and angiogenesis in ADC probably suppressing angiogenesis induced by VEGF (1.37).

In ADC is frequent the over-expression C-MET plus Hepatocit Growth Factor Receptor(HGFR).

Its over-expression has been detected  in peumocyte tipII. The C-MET over-expression is correlated to smoke and tumor stage(1,38).

The RUNX3 GENE plays an important role in ADC pathogeny. RUNX3 methylation is more frequent in non-smokers with histological ADC (1.39). The RUNX3 genes are transcription factors within the TGF-beta signaling pathway and are involved in cell cycle regulation, differentiation apoptosis and malignant transformation (1-40).

The TTF-1 GENE. The TTF-1 gene amplification was seen in ADC. The transition from epithelium to mesenchyme is a crucial event for the cancer cell in order to acquire invasive and metastatic phenotype. And this can be gained by using TGF-beta gene.TGF mediates the transition from epithelium to mesenchyme. TTF1 inhibits the transition from epithelium to mesenchyme mediated by TGF-beta and restores the epithelial phenotype in ADC. TTF-1 diminishes the production of TGF-beta and, vice versa, increases the expression of TGF-beta-1 leads to the decrease of TTF-1. Modulating the expression of TTF-1 may become a treatment strategy of ADC.(41,42, 100,101).

OTHER GENETIC CHANGES IN LUNG ADENOCARCINOMA

The p16 gene. In ADC is frequently inactivated by homozygous deletions. Homozygous deletions have been found in 29% of primary tumors, 25% in bronchiolo-alveolar carcinoma and 26% in brain metastases. Tobacco does not induce homozygous deletions in ADC progression.(43)

The BRAF gene. Is an oncogene belonging the RAF family of serin-threonine protein kinaze. Plays a role in regulation of signalling pathways. Its mutations are only seen in ADC(95,96).

The FHIT gene is a  tardy element in the progression of carcinogenesis in ADC(44)

RRS-82.(relapse-related molecular signature  reprezented by 82 probe) is useful in identifying the patients with high risk of relapse, even if they are in stage I (45).

MEK-1(mitogen-activated protein kinase 1). It has been found mutated in a subset of ADC. In this subset have been not found mutations of some genes encoding the EGFR signaling pathway components (EGFR,Her2,Kras,PI3KCA and BRAF are missing)(46)

MCM.2.(minicrosomial  maintenance protein 2). It has been noticed a high expression of MCM2 and Ki67 in impure bronchiolo-alveolar carcinoma and with an unfavorable prognostic compared to the pure bronchiolo-alveolar carcinoma. It is a factor independent of gender or disease stage (47).

Three transcription factors have been identified which are over-regulated in adenocarcinoma:

TTF1,DAT1 and TF2. Also  metalo-proteinaza-2 and urokinase plasminogen activator-alpha have been related to metastasis, thus predicting the global survival (1,48).

The histological types my be differentiated by basing us on the ADN profile methylation. Thus, in adenocarcinoma have been methylated the  genes TEMF2, MGMT, and CDKNIC. Instead, in squamous cancer have been methylated the genes ARH1,MGMT,GP1beta, RAR beta and TMEF2(1,49).

The STK11 (serine-threonine kinase 11) or (LKB1) gene lung ADC is frequently moved from smokers and non-Asian populations and of course are associated with KRAS mutations. It is located on chromosome 9(2,88,89).

The Cyclin D1(CCND1) and  Cyclin E(CCNE2) genes are found in the main areas of amplification in lung ADC (2,90).

IRAK-1(IL-1R associated kinase) is a protein kinase which has a role in activation of  NFkB and MAPK (mitogen-activated protein kinase). In non-small cell cancer is seen a significant increase in the cytoplasm and a nuclear decrease in the expression of IRAK-1 versus normal epithelium. The high level of expression in the cytoplasm has been correlated with a decrease of disease-free time in women compared to men.

In ADC the low level of IRAK-1 and NFkB expression correlates with the decrease of survival time and disease-free stage in patients of stage 1(103).

The risk of lung cancer is associated with the locus on the chromosome15q25. On this locus is found the CHRNA5 gene (choline-nicotinic receptor A5). This is 30 times over-regulated in comparison with the normal case. Within the CHRNA5 gene the D398N polymorphism - was associated with the risk of ADC (102).

The EML 4-ALK is observed in around 5% from ADC and defines  a group which may respond to treatment with inhibitors. The low ALK protein expression is a characteristic of ADC showing the gene EML4-ALK(104)

The genes: TAL2(T-cell acute lymphocytic Leukemia 2) and ILF3(Inter-leukin enhancer-binding factor 3) have been confirmed in lung ADC. Their proteins over-expression was correlated with the development and progression of lung cancer.

In some adenocarcinomas from women, non-smokers, East Asians is seen the presence of the E6 oncoprotein of Human Papillomavirus. In these cases, it is observed that the E6 oncoprotein leads to the expression increases of hTERT (human telomerase reverse transcriptase), which in turn increases the oncogenic potential of E6 positive neoplastic cells.

Overregulation of the E6 oncogene leads to the inactivation of p53 gene (106,107).

SIGNALLING PATHWAYS

From the above data it can be deduced that one single gene may be responsible for the development and progression of lung ADC. It is responsible for the disorder of one or more signaling systems. In addition, the cascade disorder of the growth signaling pathways occurs simultaneously to the disordered apoptosis (1,50).

Several signaling pathways have their functions altered in lung cancer. This disorder is important because it becomes therapeutic target. Most signaling pathways are driven by oncogenes. They lead the cells to malignant phenotype, proliferation and apoptosis escape (2,51).

The signaling pathways of growth stimulation:

1) EGFR is the prototype of a family of 4 receptor-kinases, namely: EGFR (ERBB1,HER1), ERBB2(HER2,neu), ERBB3(HER3) şi ERBB4(HER4)

The members of the ERBB family of receptor tyrozin-kinases are tightly linked to the malignant cell proliferation (2, pg.1487).

The EGFR over expression is one of the most early and frequent abnormalities in the bronchial epithelium from heavy smokers. It has been found in basal cell hyperplasia, squamous metaplasia, dysplasia and in situ carcinoma (1.52)
  EGFR and HER2 are frequent in pre-neoplastic lesions. In invasive tumors the EGFR is expressed in ADC, squamous cancer and large cell cancer. HER2 is over expressed in ADC. The genetic mechanisms responsible for EGFR and HER2 genes overexpression consists in over representation or amplification, translational and post-translational mechanisms. Instead, EGFR and HER2 amplification is rare in carcinomas (1;53)

The EGFR mutations are more common in well moderate ADC, regardless of age, disease stage or survival. EGFR mutations are not found in tumors with KRAS mutations. EGFR mutations are also independent of TP53 mutations. EGFR mutations define a distinct subset in lung ADC without KRAS mutations in non-smokers (1,54)

At EGFR mutations it has been observed an increased activity of tyrozin-kinases, which shows a sensitivity to the action of inhibitors (1.55). Mutations are an early phenomenon in the multi-stage pathogenesis, while the frequency of copies is a late phenomenon and is associated with tumor phenotype with metastasis (2,56)

2)Other genes in the EGFR signalling pathway

a)      The Ras pathway disorder was observed in most ADC and would play a role in the lung ADC development. (98) The KRAS gene is one of the most well-known oncogenes. It is detected in 20% of non-small cell cancer, and in particular in the ADC and smokers. KRAS and EGFR mutations are mutually exclusive (2.57). The fact that the EGFR and Her2 gene mutations are seen in non-smokers while Kras mutations are observed in smokers is a proof that there are different pathogenic pathways (1 pg1488).

b)      PI3K  It is a lipid kinase heterodimer. Has somatic changes in lung cancer. A sub-unit of it, PI3KCA (alpha catalyst), is one of the most common mutated gene, along with the Kras gene in LC (2.58). Number of copies of PI3KCA is higher in squamous form than in ADC. It is located in the area 3q26 (2,59).

c)      ALK(anaplastic lymphoma kinase proteins). Observed in non-smokers. It plays a role in the pathogenesis of LC. Plays a role in the ras activation. Does not associate in the presence of Kras and EGFR mutations and facilitates ADC in non-smokers.(2,60)

d)     THYROID TRANSCRIPTION FACTOR 1(NKX2-1) It is a transcription factor essential to the development of peripheral airways (2.61). It is over-expressed and amplified occasionally peripheral ADC. (2,24,61). TTF-1 expression is crucial for the diagnosis of lung peripheral ADC.

e)      PDGF(The system of platelet-derived growth factor). It and its receptors are known in normal peripheral cell epithelium. It is noted in all cases of small cell cancer (100%) in squamous cancer in 40% of cases and 55% in lung adenocarcinoma. The PDGF receptor has also been found in the tumor stroma. It is correlated to a low prognosis with in LC regardless of age, sex, stage of disease and degree of differentiation. PDGF-beta mRNA was detected in the ADC at a rate of 85% and in squamus forms in 100% of cases.(1,63).

f)       INSULIN-LIKE  GROWTH  FACTOR  SYSTEM(IGF) In experiments on mice the transgenic over-expression of IGF-2 has been found in 69% of lung tumors induced from the lung epithelium. These tumors have the characteristics of human lung adenocarcinoma. The IGF-II receptor induces the proliferation of cell lines from human lung cancer and CREB (cAMP- Regulatory element binding protein) -transcription factor-induced phosphorylation. These data suggest that IGFII and CREB contribute to the growth of lung tumors (1,64).

2) THE VASCULAR ENDOTHELIAL GROWTH FACTOR AND ANGIOGENESIS SYSTEM

VEGF is a protein frequently expressed in non-small cell cancer. It plays a major role to the development of blood and lymph vessels. VEGF plays an important role in the progression of LC. VEGF and VEGF receptors are connected to other signaling pathways.

In non-small cell cancer and mainly in ADC are observed frequent positive reactions for VEGF-C. Instead the VEGF-3 expression correlates with squamous cancer, age and sex.

Both  VEGF-c and VEGF-3 are low prognostic signs. VEGF -3 expression is independent of the other factors with respect to the reserved prognostic (1.65). The increased VEGF expression is observed in 73 % of ADC and 75 % in squamous cancer. The rich vascularization has been related to an increased expression of VEGF. The VEGF expression and small vessel density correlates with a low differentiation (I, 66). The expression of VEGF - C gene was found high in cells that had an overexpression of COX -2 . The COX-2 level correlates very well with VEGF.C and lymphatic vascular density (1,34,35). In differentiated ADC has been observed an over expression of VEGF and protein-3 related to IGF (IGF - binding protein -3 ) mRNA.

Lung adenocarcinoma is also characterized by the high correlation between VEGF and Bradikinin B2 receptor. (1.67).

Reversely – in a favorable manner -another protein - CTGF (connective tissue growth factor) inhibits the metastatic action of lung cancer cells. Tumors of patients with the same stage, but with a high expression of CTGF have a low vascular density (1,68)

3) HEPATIC GROWTH FACTOR (C-MET)

The over expression of this pathway was observed in 35% of ADC. In lung cancer C-MET has a role in cancer invasion and differentiation (1,69).

EPIGENETIC CHANGES

Epigenetic changes are a number of molecular mechanisms that regulate gene expression, without causing changes in the DNA sequence. Methylation is a physiological function. These changes include:

a)      alterations in the DNA methylation status within the CpG islands leading to hyper methylation of tumor-suppressor gene and their inactivation (silencing),

b)      histone changes and

c)      genes regulation through  micro –ARN(mi-RNA)(2  pg 1481)

Epigenetic  changes of histones

Histones play a crucial role in the regulation of chromatin packaging, in nuclear architecture, gene expression and genomic stability (2,70,71,72). Cooperation between acetylases: NF-kB, STAT, CREB and RNA polymerase-2 ADN prepared by acetylation of histones and make chromatin easily accessible for transcription.

Reversly, HDAC(histone deacetylase) together with methyl CpG binding protein2 and other factors methylate DNA, deacetylase histones and make DNA inaccessible to transcription.

Cancerous tissues can be divided into 3 groups depending on the level of HDAC gene expression. The group with reduced expression of HDAC has a poor prognosis. HDAC low expression facilitates the lung cancer progression (1,73).
It was further observed in non-small cell cancer as well as in dysplastic bronchial lesions an excessive acetylation of H4K5/H4K8 and loss of H4K20 trimethylation. Loss of H4K20 trimethylation was observed in a subpopulation of stage 1 of ADC which has a low survival ( 2,74 ). In ADC subregulation of H4K20me3 was 28 % . H4K20me3 is a candidate as a bio-marker for early diagnosis and targeted therapy ( 2,74 ). Changes in histones acetylation and trimethylation of H3 and H2 were seen both in small cell cancer, and in the non-small cell one. In this way could have been detected subpopulations with differential prognosis suggesting that epigenetic modifications of the histone code have important role in LC tumorigenesis. The global changes of histones may predict the evolution of a non-small cell lung cancer after resection (2.75 ) The alteration way of histones and is connected to DNA methylation and is a cause of lung cancer (2,76). 

miRNA  IN LUNG  CANCER

miRNAs are a class of non - protein small molecules ( about 22 nucleotides ) encoding RNA that regulates the gene expression, modulating the activity of the specific targets of messenger RNA ( 2,77,78,79 ). The miARN expression is usually disordered in several cancers including lung cancer (2,80,81,82). They are an important class of biomarkers that are released into the bloodstream by the tumor cells, becoming blood markers. .

miRNAs can function both as oncogenes and as tumor - suppressor genes being  over or sub-regulated in different cancers. They can play an important role in the pathogenesis of cancer (2,83). miRNAs regulate the mRNA expression, as well as translation within the proteins (1,84). miARN expression profiles are diagnostic and prognostic markers for lung cancer (2.81 ). In ADC it was observed an increased expression of hsa - mir- 99b and hsa-mir -102 (99). miARN expression profile was correlated with survival in lung ADC, including those patients with stage I ( 2.81 high profile of expression)

 miARN hsa -mir -155 as well as the low expression of miARN hsa - let- 7a -2 correlates with low survival ( 1,2,80,84 ). The LET family 7miARN inhibits the expression of the Ras protein, which is mutated and super-active  in ADC from smokers (2.85). Since RAS mutations are rare in non-smokers, the expression of LET-7 family shows us the difference between tumors from smokers and those of non- smokers.

 

The above data show the complex structure of lung ADC. They demonstrate the heterogeneity of this family member of non-small cell lung cancer. They highlight several sub-sets of lung adenocarcinoma, which can be treated more precisely, targeted and efficient. Molecular pathology data help us to estimate the subsequent evolution of the disease.

 Many of these molecular changes become diagnostic factors and future targets for treatment. Future treatment of ADC will be much more differentiated and precise and might be able to overcome this sinister survival barrier of only 15% over 5 years.

                                             CONCLUSIONS

Adenocarcinoma, a member of non-small cell lung cancer is a frequent entity (40%-42%). Nitrosamine, with role in the development of ADC is NNK.

ADC arises through the modification of genomic structure of local pluri-potent stem cells.

Precursor lesions have neoplastic character based on molecular data. In addition to the histological form was added as the expression mode of the genes. Genetic changes in the various forms of lung cancer may be common and /or specific. In ADC was found a large number of genes differently expressed. Gene mutations in ADC are an early phenomenon.

Within the growth signaling pathway has been shown that mutations in EGFR and HER2-neu genes are early, frequent and specific for ADC from women and non-smokers. Kras mutation genes occur in smokers in 40% of ADC. EGFR and HER2-neu genes are exclusive compared to Kras genes.

The TTF-1 gene is amplified in ADC and is crucial for the diagnosis of peripheral ADC. Along with the TGF-beta gene- being opposites – modulates the epithelial to mesenchyme transformation, a phenomenon that precedes metastasis.

The VEGF gene is over-regulated in the ADC and correlated with vascular development and VEGF-C expression correlates with increase of lymph vessels and over-expression of COX-2.

Among epigenetic changes, we distinguish:

a)      alteration of DNA methylation status inside the CpG islands.

b)      changes in histones. In non-small cell lung cancer we notice an excessive acetylation of H4K5/H4K8 and loss of H4K20 trimethyilation. The latter was observed in a case of stage I ADC with reticent prognosis. In ADC subregulation of H4K20me3 is 28%, as a biomarker and treatment target.

c)      profiles of the expression of micro-RNA (RNA I) are markers for diagnosis and prognosis in lung cancer. High expression of hsa-miR-155 and low expression of hsa-let-7a-2 is correlated with low survival. LET family expression specifies the difference between smokers and non-smokers.

ADC is a heterogeneous entity, with many sub-sets, with its specific, that must be treated differently. Some data of molecular pathology become biomarkers for early diagnosis and future therapeutic targets.                                                                                  

GLOSSARY

TTF-1.             Thyroid transcription factor-1 is a protein encoded by the gene NKX2-1. Regulates the gene transcription in lung and thyroid. The positive cells are found in the lung in type II CLARA cells pneumocyte. It is a marker for peripheral ADC

VEGF-C         It is a chemical signal produced by cells that stimulates the growth of new  vessels

CpG islands   are regions of ADN wherein a nucleotide cytozine occurs next to a guanine nucleotide in a linear sequence of bases. Their methylation leads to silencing the tumor-supressor genes. Instead their hypomethylation is associated with over-expression of oncogenes.

HISTONELE  are strongly alkaline proteins that are found in the nucleus and which packs and coordinates DNA in a structural unit called nucleosome. They are the main chromatin proteins. The assembly of histone and DNA is called chromatin.

Histone 4 lysine 20(K20) is monomethylated by  histone methyl transferase (PR-SET7).

H4K20            is essential for cell proliferation.

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