MOLECULAR PATHOLOGY OF THE SQUAMOUS FORM OF NON-SMALL CELL LUNG CANCER

 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

Study of the molecular pathology of lung cancer (LC) has contributed and is further contributing to a more deepened knowledge of pre-neoplastic lesions, of carcinogenesis process, of metastatic invasion and disseminations. By simultaneously highlighting multiple markers the early diagnosis becomes possible in the targeted treatment of LC. So far the LC treatment was a uniform treatment for a heterogeneous disease. These cumulated data give hope for the increase of survival rate, which currently is one of the lowest
(15% over 5 years) (1).

Among the subtypes of non-small cell lung cancer, the squamous form has prevailed as frequency percentage compared to the other three subtypes.

After 1950 the scientific world was convinced of the smoking role in the etiology of LC (85 % of cases are due to smoking) (2). At that time of cigarettes without filter the squamous form was predominant as frequency and location in the central bronchial. By introducing filter cigarettes the tar disappeared (or lowered) and increased the volatile part. Following this the frequency of adenocarcinoma (ADC) was higher and the location became predominantly peripheral (2,3).

Most LC subtypes derive from various lung components. Thus, in non-small cell cancer, in squamous form, and in 20% of the ADC cases they arise in the central compartment of the bronchial pathway from STEM cells, represented by bronchial basal cells. These candidate stem cells differentiate in ciliated or mucous cells ( the latter can give rise to central adenocarcinoma and, possibly, also the neuroendocrine cells of the terminal bronchia(2,3,4).

The terminal breathing units consist of the respiratory bronchioles and alveolar cells. The STEM cells capable to proliferate are CLARA cells (expressing CC10) and type II pneumocyte expressing surfactant and transcription factor TTF -1 ( thyroid- transcription factor  1 (2).

Under the action of environmental factors at the bronchial epithelium level occur histopathological changes. They may evolve, but may also regress and others may remain stationary for years (5,6).

Development is gradual, both temporally and spatially (multistep, multicenter).

Due to the predominance of tobacco’s volatile aspect appeared the notion of cancerization (SLAUGHTER) field (7). This means that it is possible to find more areas with genetically altered cells. These areas (clones) play a central role in cancer development (5, 8, 9). The large number of preneoplastic cell in the proliferation field increases the risk of cancer. This explains the high incidence of primary type cancers, but occurring secondarily (3%) (2).

Exposure to carcinogens results in the formation of a field of altered cells which may evolve by altering the DNA then further by genetic and epigenetic changes, which also adds to the genome alteration. These phenomena further lead to local invasion and then to metastatic disease (5)

                           MOLECULAR PATHOLOGY

                                            OF  THE

                            SQUAMOUS FORM OF LUNG CANCER

GENETICAL ALTERATIONS

At the DNA level we distinguish deletions, insertions, point mutations of some dominant genes such as the Ras gene, some tumorsuppressor genes such as p53, Rb gene, p16 gene.

At chromosome level we distinguish the loss of heterozygosity (LOH) of the p53, p16 genes, and DNA aneuploidy.

Epigenetic changes – hypermethylation of tumorsupressing genes, histone deacetylation (5.10 11). Epigenetic changes in LC contribute to cellular transformation by:

-          Modification of the chromatin structure

-          Modification of the specific gene expression, This includes DNA methylation, change of histone and chromatin proteins.

-          Changes in the micro-RNA (mi-RNA) responsible for silencing tumor suppressor genes and encoding oncogenes expression ( 2 )

In squamous cancer we have deletions in areas 3p  and 13q , leading to molecular alterations in normal tissue.

Loss of heterozygosity is a common occurrence in carcinogenesis. Loss of heterozygosity in 3p chromosome is an early event in cancer evolution and it is observed, in 90% of small cell and squamous cancer cases. Instead in ADC is seen in only 50% of cases (5,12 )

There are also other areas with  loss of heterozygosity such as -3p, 3p12, 3p14.2  3p21.3, 3p24, 3p25. These areas contain several tumor-supressor genes ( 5, ).

Loss of heterozygosity is also noticed in areas-2q, 9p.21, 8.21.22, 13q14 (Retinoblastom Rb gene) and 17p.13 ( p53 gene). Allelic losses in areas 2q,  3p and 22q play an important part in the development of squamous cancer (5,13 ).

P53 gene mutations

The p53 gene is located on the 17p.13 chromosome. It encodes a transcription factor, activating the transcription of many correlated genes p21, MDM2,GAAD5, Bax..

The p53 gene, binding to p 21 gene, induces its expression, which in turn has a inhibiting function of the cell cycle in the G/S area.

The p53 gene binds to MDM2 gene, a protein that affects cell cycle, apoptosis, tumorigenesis.

The Bax gene is forming a hetero-dimer with the Bcl-2 gene and plays a role in apoptosis.

The p53 gene has multiple roles in maintaining stability of the cell genom during cell stress due to DNA alteration and hypoxia, when proto-oncogenes are activated.

P53 dysfunction is the most frequent genetic alteration and important in lung carcinogenesis and the p53 gene alteration area is 17p.13.

The p53 gene mutations are more frequent in non-small cell lung cancer (90%) and 65% in non-small lung cancer. Within non-small cell lung cancer there is a higher frequency in squamous form and in large cell lung cancer, but less in ADC (14 ).

Mutations of the p53 gene lead to impossibility to activate the p21  gene and produce a supression of the function of tumorsupression gene, which facilitates cell proliferation ( 15 ).

A single point mutation in bronchial epithelium consisting of transversion G: C to T: A in l245 codon is morphologically highlighted by changes of squamous metaplasia type, moderate and minimum dysplasia (16).

The P53 gene mutations are hosted in the middle of gene, in codons 157, 245, 248, 273. These mutational areas correlate to the effect of tobacco carcinogens. Mutations in codon 157 seem to be unique in LC (17 ).

The p53 mutations are observed in one third of the minimum and moderate dysplasia and reach half of the cases of severe dysplasia and CIS. In invasive cancer they reach to over 75% of cases (Beneth 18). We thus have a continuous growth, which is parallel to the morphological development.

P53 mutations are found in 50% of smokers, are early both in squamous form, as well as in ADC (18.19).

P53 mutations frequent in lung cancer generate the p53 protein level, which is highlighted by histochemical methods. Thus the frequency of p53 mutations increases continuously from minimum dysplasia up to invasive cancer.

           we have - 29% in minimum dysplasia

                      26% in moderate dysplasia

                      59% in severe dysplasia

                       58,5% in CIS

                       67,5% in micro-invasive

                       79,5% in invasive cancer (Beneth18)

The gradual increase of p53 mutations is a marker for pre-neoplastic lesions.

Bcl-2 proto-oncogene

This gene encodes a protein that inhibits apoptosis. This protein is expressed in the basal cells of normal bronchial epithelium. Bcl -2 protects the cells against programmed death.

The positive expression of Bcl-2 was found in 25% of squamous cancer and only in 12% of ADC cases. Bcl-2 is a proto-oncogene involved in the translocation 14:18, which is a chromosomal abnormality. The survival is higher in Bcl-2 positive patients and patients over 60 years have a better prognosis.( 20,21,22 )

The p53-Sp1 disorder

DNMT (DNA-5.cytosine-methyl-transferaza). It is an enzyme that methylates the cytosine residues from CpG areas (islands containing multiple tumor-suppressor genes). Its over-expression was related to p53 mutations and high expression of Sp-1. Patients with DNMT over-expression and high expression of Sp-1-1 have a reticent prognosis. DNMT Disorder is associated with p53 mutations, and high expression of the Sp-1. The epigenetic alteration of tumor suppressor genes leads to DNMT over-expression, which further leads to tumor-genesis and a reticent prognosis (23)

The PTEN(MMAC-1) tumor suppressor gene

The PTEN(phosphatase and tensin homolog on chomosome 10) tumor suppressor gene, which is also named MMAC-1(mutated in multiple advanced cancers) is located in 10q.23( 5,24,25 ) area.

PTEN plays a role in apoptosis, cell migration and cell dissemination (5,26,27). In non-small cell lung cancer, are found deletions and mutations of PTEN only in 11% of the cases (5,28,29)

Aberrant methylation of the p16 (INK4) pathway

P16 (INK4 ) is an inhibitor of cyclin-dependent kinase 4 and inhibits Rb phosphorylation , making this as a tumor - suppressor gene. It is found in the 9p.21 area.

Disruption of the p16 function leads to Rb hyper-phosphorylation and inactivation. This Rb inactivation leads in turn to a Rb dissociation from the transcription factor E2F (a transcription factor that activates the S-phase genes ) leading to cell proliferation .

Along with Rb pathway inactivation is also observed losses of RB alleles in the 13q14 chromosome area, but only in 15 % of non-small cell lung cancer ( 5,30,31 ).

Abnormalities of p16 , cyclin D -1 and CDK -4 are seen in 70 % of cases of small cell lung cancer.

The P16INK4 gene is altered particularly in non-small cell lung cancer by aberrant methylation in 25 % of cases and through deletions or point mutations in 10 % -14 % of cases. The p16INK 4 gene inactivation by methylation is noticed 17 % in preneoplastic hyperplasia  , 24 % squamous metaplasia and in CIS reaches 50 % .

Homozygous mutations and deletions are seen especially in late-stage of non-small cell lung cancer  (32,33,34 ).

TELOMERASE

Telomerase is a reverse transcriptase that stabilizes telomere length by hexameric fragment addition - TTA : GGG at telomere ends and compensate losses ( 5 ).

The hTERT catalytic subunit (human telomerase reverse transcriptase) is expressed in embryonic stem cells and germ cells of the adult male. In adults is not seen but in the areas of cellular proliferation for tissue recovery (hematopoietic stem cells, activated lymphocytes , epidermis basal cells and in the proliferative endometrium and in the cells from intestinal crypts. (5)

Telomere shortening is an early genetic abnormality in bronchial carcinogenesis, predominantly in pre-invasive lesions that precede the expression of telomerase and p53 - Rb pathway inactivation (5.35)

Reactivation of telomerase expression is essential for cell proliferation, leading to immortality.

Telomerase positive activity was observed in 26% of normal bronchial epithelial cells and in 23 % of the epithelium of the small and periphery bronchioles.

Together with the histological changes, the increase in telomerase activity reaches 71 % in hyperplasia, 80% in metaplasia and 100% in dysplasia and CIS ( 5.36 ) . After this gradual increase, we have a decrease in invasive cancer.

Increased expression (ectopic) bronchial epithelia telomerase prior to transformation into cancer. Detection of telomerase protein in non-cancerous bronchial epithelium, becomes a marker to developments CP (37   ).

Disordering telomerase occurs as -

a)      the majority of lesions express hTERT, but with low enzymatic activity when compared to the terminal area. We have 4% in the tissue adjacent to tumor and 80% in the cancer area (5.38)

b)      cells express hTERT, but an intense over-regulation is observed in CIS from the adjacent invasive lesions, indicating an imminent invasion.

c)      In pre-neoplastic lesions in ADC there are no telomerase changes ( 39 )

The hTERT expression is correlated with p53, Ki67 (proliferation index) and with the ratio of Bax-Bcl-2. We thus have a coupling between telomerase reactivation- proliferation, and apoptosis resistance (35). The detection of hTERT protein in the non-cancer bronchial epithelium becomes a marker for patients with high risk (40). Recently it has been noticed that the hTERT gene also presents a functional polymorphism that contributes as prognostic factor in non-small cell lung cancer.

EGFR(epithelial growth factor receptor)

EGFR is a tyrosin-kinase receptor. It is released into the epithelial tissue. EGFR induces cancer through three major mechanisms for epithelial developments (5) –

a)      over expression of EGFR ligands

b)      EGFR amplification

c)      mutational activation of EGFR

a) EGFR over expression in non-small cell lung cancer is between 50% -80%, of which 70% in squamous cancer and 50% in the ADC. We find an over expression also in pre-neoplastic lesions. EGFR over expression and activation are important signs in non-small cell lung cancer (41). Over expression and amplification are commonly associated with squamous cancer. When EGFR is activated it induces Ras signaling pathway and thus is involved in cytoplasmic-skeletal rearrangement ( 42  )

b) EGFR amplification is seen in –

       non-small cell cancer 33%-45%

       squamous cancer        10%-27%

       adenocarcinoma         10%-33%

c) mutations are noticed in –

       non-small cell cancer  13%

       squamous cancer          0%

       adenocarcinoma          20%-55%

Therefore the squamous cancer is characterized by over expression of EGFR (70%), amplification (10% -27%) and lack of mutations.

Activation of EGFR leads to influencing MAPK (mitogen activated protein kinase), which is a signal of transduction and Keratin5. Keratin 5 is specifically expressed in the basal layer of epithelium. In squamous cancer there is an over expression of Keratin 5 (41).

Recently it has been described a new molecular circuit in non-small cell cancer comprising

-          ADAM17(a disintegrin and metallo-proteinase) which is a regulator of the EGFR expression through the activation of Notch-1. It is also called TACE(tumor necrosis factor-alfa converting enzime). Is an enzime being part of the ADAM protein family of metalloproteinases and disintegrines.

-          NOTCH-1 is a transduction signal a control pathway of ADAM17

There is a significant correlation between ADAM17 , NOTCH-1 signalling and the increased level of the EGFR expression. They have a role in tumor genesis and neoplastic cell survival. ADAM17 and NOTCH-1 constitute future therapeutic target(42)

                     

RECENT HIGHLIGHTED MECHANISMS  IN SQUAMOUS CANCER 

Nrf-2 and KEAP-1

Nrf-2 (nuclear factor erytroid-2-related factor -2) Is a key factor of redox-sensitive transcription involved in mitochondrial biogenesis. It regulates the enzymes expression of electrophilic and xenobiotic detoxification and protein efflux conferring a cytoprotection against the oxidative stress and apoptosis in normal cells.
Nuclear expression of NRF-2 is-

       in non-small cell LC 26%

       in the squamous form of LC is of 38%

       in Adenocarcinoma is of  18% ( 43 )

Nuclear expression of Nrf-2 was related to a low survival.

Nuclear expression of Nrf-2 was also related to the decrease of period –disease free- in squamois cancer after adjuvant chemotherapy

Nuclear expression of NRF-2 plays a role in the squamous resistance to the treatment with Cis-Platinum

KEAP-1.(Kelch-like Ech-associated protein-1)   It is a protein encoded by the gene of the same name. It interacts with the Nrf-2 in a redox-sensitive.

KEAP.1 is an inhibitor of the Nrf-2

Her expression is low or absent in non-small cell LC. It predominates in ADC (62%) compared to the squamous form (46%)

Increased expression of Nrf-2 and KEAP-1 expression decreases are abnormalities in non-small cell LC and are related with a poor prognosis.

The structural base of the KEAP-1 activity defects is caused in LC by point mutations. KEAP-1 mutations lead to activation of Nrf-2 in non-small cell LC (43,44 ). Further the activation of Nrf-2 facilitates tumorigenicity and contributes to chemotherapy resistance.

Conversely, reducing the expression of Nrf-2 (ARM-1-mediated) lung cancer induces generation of oxygen-reactive species, suppress cell growth and increases sensitivity to chemotherapy.

Therefore by targeting Nrf-2 activity in lung cancer-particularly in cancer with KEAP-1 mutations  we can have a strategy for inhibiting the tumor growth and can hamper the emergence of chemical resistance to chemotherapy ( 43,44  )

SOX2

It is a key transcription factor in mammalian for the development and maintenance of both embryonic stem cells and of stem cells from other tissues (45).

Its over expression was found in several tumors. SOX2 is an oncogene.

SOX2 is over-expressed in squamous cancer.

In squamous cancer out of 20 cases, 15 cases were stained for SOX2.

Instead, for adenocarcinoma were stained for SOX2 20cazuri only 4 cases of 20 ADC cases. Also they have stained positively for both, 14 of 20 cases. Instead no ADC cancer was not stained positively for both ( 45 )

FUS-1

FUS-1 gene is a gene located in the chromosome p16 area.112. Plays a role in maintaining the genomic integrity.

Protein of FUS-1 gene shows a very frequent decrease and a lowering in the expression of squamous cancer. Reducing the protein may play a role in the early pathogenesis of squamous cancer. Tumors show a low expression of FUS-1 protein, as compared to normal epithelium.

Reducing the FUS-1 expression was observed in 100% of cases of small cell LC in 80% of cases in non- small cell LC. In squamous cancer there was an expression decrease in 87% of cases and in adenocarcinoma in 79% of cases.

Loss of FUS-1 expression has prognostic-independent value, concerning the survival. ( 46  )

MARKERI  SPECIFIC TO SQUAMOUS FORM

Markers specific to the squamous form are :

-          Tumor protein 63(transformation related protein 63) is encoded by the p63 gene. It belongs to the family of  p53, p63 ,p73. It is a transcription factor. It is highlighted poorly differentiated squamous cancer, differentiating it from ADC or small cell cancer. (wikipedia)

-          Cito-Keratin 5/6. It is seem in the basal-like carcinomas. The immunoreactivity degree correlates with the degree of survival. Survival increases along with the increase of immunoreactivity. Immunoreactivity is higher in men than women. It is noticed in most non-small cell cancers. It distinguishes mesothelioma from pleural metastasis and from adenocarcinoma. It is very common in squamous cancer, namely squamous cancer with positive CK5 /6 and negative TTF-1 (thyroid trasforming factor1). (Wikipedia)

-          Desmoglein 3 is a protein encoded by the DSG3 gene. It is interacting with PKP 3 (plakophilin 3 - is a protein encoded by the PKP 3 gene). It acts on cellular adhesion-dependent on desmosomes and with signaling pathways.

The desmoglein 3 expression shows in squamous cancer a specificity of 90%, but only a sensitivity of just 88% (47,48 )

Also, micro-RNA(mi RN$A) are an alternative for completing the histological type of squamous cancer. Thus hsa-miR.-205 has a high sensitivity of 96% and a specificity of 90% in specifying the histological diagnosis of squamous form (47.49)

Finally, in antineoplastic treatment, Pemetrexed has won currently a leading role. He is a multi-targeted chemotherapeutic agent. It acts on the expression of thymydilat-synthase. (47,50) There have been obtained promising results in ADC compared to the squamous form. This is due to the fact that the squamous form shows a high expression of thymydilat-synthetase. Thus the combination of pemetrexed with Cisplatinum increases the survival which is higher in ADC than the squamous form. Instead Cisplatinum combination with gemcitabine is more effective in squamous cancer than in ADC (47, 51, 52).

The squamous form of non-small cell lung cancer is clinically and genetically heterogeneous (53)

Four subtypes have been highlighted  depending on the mARN expression. Tumor differentiation and sex of patients were associated with that subtype.

The MRNA expression profile of these 4 subtypes contains different biological processes. Thus:

-          First subtype called - primitive - is correlated with proliferation. It has a severe prognosis on survival.

-          Second subtype called – classic- shows a xenobiotic metabolism

-          Third subtype called –secretory – is related to the immune process.

-          Fourth subtype called – basal – is related to cell adhesion.

This stratification leads to a more accurate prognosis. They have a different survival prognosis and will be distinct targets in pharmacological treatment (53)

The squamous form represents about 30% of small cell lung cancer.(54) and

The squamous form is defined by the presence of cytoplasmic keratinization and (or) desmosomes (bridges between cells) (53). Squamous form predominates in men and smokers. Histologically we distinguish papillary, small cell, clear cell and basaloid form (53)

Classification by mRNA expression is a progress. The four subtypes are distinguished by survival duration, demographics aspects, physical characteristics, biological processes. They have correspondence with normal lung cell types.

.

I) PRIMITIVE type. At this subtype the main functional data is proliferation. It includes the following genes

-          MCM10 (mini chromosome maintenance 10) The overexpression of this gene characterises this subset and is a biomarker of it.

-          E2F3 (E2F transcription factor 3) E2F transcription factor modulator of proliferation in which the main members of E2F family are –E2F3 and E2F8(58)

-          TYMS(thymidilate sintethase)(57)

-          POLA1(polimerase alfa 1)

II) CLASSICAL type. At this subtype the distinctive functional aspect xenobiotic metabolism that detoxifies foreign particles. This form is seen in smokers compared to non-smokers. It includes the following genes

-          GPX2

-          ALDH3A1(59))

-          TP63 is a transcription factor essential for developing the stratified epithelium. It is found in 3q27-28area. It is the main bio-marker for this subtype (60)

In squamous form it is overexpressed and amplified (61)

III) At this SECRETORY subtype the main functional theme is immune. This subtype includes genes-

-          1)ARHGD6B – Rho GDP dissociation inhibitor Beta

-          2)TNFRSP14_tumor necrosis factor receptor14

-          3)NF-Kappa Beta with over expression

The main biomarker of this subtype is KRT7

                 4) over expression of the secretory lung cell markers such as

-          MUC-1(mucin)

-          SFTPC,SFTPB,SFTPD(62). These are lung surfactant proteins

In this SECRETORY subtype is also found an increased expression of TTF-1 as in adenocarcinoma (similarity by glandular cells)(63)

IV) BASAL SUBYPE. The functional aspect is related to cell adhesion function.
It includes genes such as:

a)      Laminines (LAMB3 and LAMC2)

b)      Colagene (COL1A1 and COL17A1)

c)      Integrine (ITCGB4 and ITCGB5)

d)     Claudin 1

In addition are added genes related to epidermal developments such as:

a)      Keratin5(KRT5)

b)      S100A

c)      GJB5(GAP jonction proteinA)

Under the basal type also presents over expression of several genes of the S100 family, such as -S100A2,S100A3,S100A7,S100A8,S100A9.S100A12,S100A14. But S100A8 and S100A9 sunt overexpressed in the basal layer in psoriatic epidermal tissue (64)

S100A2 is overexpressed in the basal layer of the lung epithelium and in squamous form (65)

KRT5 is a marker of the basal layer of the epithelial tissue (66)

Besides this basal subtype is enreached with genes whose product is located in cell membrane

COMPARATIVE DATA OF THE 4 SUBTYPES OF THE SQUAMOUS FORM

In morfological terms, grading is related to that subtype.

-          PRIMITIVE subtype has an overrepresentation of poorly diferentiated tumors.

-          BASAL subtype has an overrepresentation of well-diferentiated tumors.

-          CLASSIC and SECRETORY subtypes have a high proportion of 3rd stage tumors.

In terms of gender,

- Women are represented by the primitive type

- Men are overrepresented by the classic type.

In terms of smoking

-          the classic subtype has smokers with high doses of tobacco and have the lowest proportion of non-smokers.

In terms of time, global surviving (OS) and (or) the relapse-free survival are different and each subtype has its survival:

-          PRIMITIVE subtype has the worst figures concerning overall survival (OS) and relapse-free survival (RFS) in all stages of the disease, including Stage 1

-          Other subtypes have an equal survival

The percentage of subtypes within the squamous form of non-small cell lung cancer -

-          16% PRIMITIV subtype

-          36% CLASSIC subtype

-          26% SECRETORY subtype

-          21% BASAL subtype

These subtypes have similarities- depending on the expression- with various populations of lung normal cells. Thus-

-          The BASAL subtype has characteristics similar to normal basal cells, namely cell adhesion and main development functions of epidermis. Thus for example genes S100A2 and KRT5. They are markers of the BASAL subtype are similar to normal basal cells

-          The SECRETOR subtype has similarity to lung secretory cells and submucosal glands.

-          The primitive subtype is distinguished by functional cell proliferation, reduced survival, is predominant in women and non-smokers and has an over abundance of poorly differentiated tumors.

-          The CLASSIC subtype is the usual type of squamous form of non-small cell lung cancer. It has the highest prevalence (37%), predominant in male, smokers. The TP63 gene overexpression presents a possible TP63 gene amplification and locus amplification of in the 3q27-28 area

-           

These subtypes have cells similar to the ancestral cells. MRNA expression persists in the progeny of tumor cells.

Subtypes of the squamous form of non-small cell lung cancer are useful for the targeted therapy and for selecting groups of patients.

CONCLUSIONS

Lung cancer in squamous form and 20% of adenocarcinomas arise in the central compartment of the airways, from stem cells represented by basal bronchial cells, which differentiate in ciliated or mucus cells (for 20% of adenocarcinomas from the center area).
Its development is progressive and gradual (multistep, multicenter).
In squamous cancer we have deletions in areas 3p şi13q. Loss of heterozygosity (LOH) is seen at 3p chromosome, early event in 90% of cases.
The P53 gene dysfunction is the most frequent genetic alteration and important for carcinogenesis.The p53 mutations are early and frequent. They gradually increase from 26% in moderate dysplasia to 79.5% in invasive cancer.
The increased expression of the telomerase precedes to neoplastic transformation. Increased expression of telomerase in normal epithelium starts (26%) and increased to 80% in squamous metaplasia, and reaches 100% in severe dysplasia and CIS. Its expression correlates with p53, Ki67şi ratio Bax/Bcl 2.
EGFR amplification is shown in 10%.-27%
There is a significant correlation between high levels of EGFR and EGFR- ADAM17 expression regulator and NOTCH.1 signaling pathway, which is a signal transduction. They control tumorigenesis and neoplastic cell survival. They constitute future therapeutic targets.
KEAP-1 interacts with Nrf2 and is an inhibitor thereof.
Increasing the Nrf2 expression and decreasing the KEAP-1 expression through mutation is associated with poor prognosis, facilitates tumorigenesis and increases resistance to chemotherapy.
The reduced expression of FUS-1 has a value-independent – to other factors concerning the global surviving. Reduced expression is
All these new conquests in molecular pathology of non-small cell lung cancer will improve early diagnosis, prognostic and targeted treatment.

The p16(cyclinD1-cyclin-dependent kinase4) retinoblastoma pathway is found in 9p.21 area.
Inactivating the p16 pathway through aberrant methylation increases from 24% in squamous metaplasia to 50% in CIS.

Homozygous deletions and mutations are observed in the late stage.

EGFR gene. Its over expression is seen in non-small cell LC of 50% to 80%, of which 70% in squamous form and 50% in adenocarcinoma. Its over expression correlates with a severe prognosis.
The EGFR mutations is not seen in squamous cancer.


Nrf-2 and KEAP-1
The Nrf 2 nuclear expression (transcription factor regulating the expression of the enzymes in the detoxification process) is observed 26% in non-small cell LC, 38% in squamous form and in adenocarcinoma in 18% of cases. Its expression correlates with low survival and resistance to chemotherapy.

SOX2
Is a transcription factor. Its overexpression sa is oncogene. SOX2 is overexpressed in squamous cancer.

FUS-1
The FUS-1 protein expression is low in squamous cancer.
100% in the case of small cell LC
82% in the case of non-small cell LC
87% in cases of squamous cancer
The squamous form of non-small cell cancer shows depending on the mRNA expression four sub-types: primitive, classical, secretory and basal, with biologically distinct shapes and different prognosis.

.

GLOSSARY (wikipedia)

1)      CC10    Clara Cell protein10. It is an inhibitor of phospholipase 2. It is regulated by the transcription factors AP-1 and Hepatocyte nuclear factor 3 (HNF-3). It is expressed by non ciliated epithelial cells of the airway. CC10 contribute to the loss of non-small cell cancer carcinogenesis. Its overexpression reduces invasion in the absence of matrix-metalloproteinases MMP-2 and MMP-9.

2)      MDM2(murine double minute 2). Is an oncogene. Is a negative regulator of p53, that is subregulates p53 protein.

3)      SP-1(specific protein-1). Is a human transcription factor

4)      GADD45(growth arrest and DNA damage gene 45) Is a response gene for DNA alterations. ZBRK1 has a repressive action on GADD45.

5)      p16INK4A. Is a tumor supressor gene participating in stopping the cell in G phase. A member of the IN K4 protein family inhibits CDK4 and Rb phosphorylation mediated by CDK6. It is encoded by the gene Cyclin Dependent Kinase Inhibitor 2A.

6)      ADAM 17(A Disintegrin And  Metalloproteinase). It is an enzyme (glycoprotein) which is part of the ADAM family protein, a member of disintegrines and metalloproteinases and is involved in the processing of TNF-alpha (tumor nuclear factor-alpha) to cell-surface, TNF-alpha being a mediator of inflammation. Plays a role in the Notch signaling pathway.

7)      NOTCH. Is a signaling pathway. It has 4 Notch receptors, which are transmembrane -proteins receptors. Notch signaling pathway is important for cell-cell communication involving gene regulation mechanisms which control the cell differentiation process

8)      Disintegrines are a family of small proteins, which act as platelet aggregation inhibitors and integrin-dependent cell adhesion.

9)      Desmosomes. They are a specialized cell structure in inter-cellular adhesion.

10)  POLA-1(polymerase(DNA directed) alpha-1.It has a role in DNA replication.

11)  GPX2 (glutathione peroxidase2)  is a human enzyme encoded by the GPX2 gene

12)  ALDH3A1(aldehyde dehydrogenase3 family,member A1). ALDH plays a key role in the detoxification of alcohol-derived aceto-aldehydes. They are involved in the metabolism of corticosteroids, biogenic amines, neurotransmitters, and lipid peroxidation. Usually it oxidizes the aromatic-aldehyde substrate. It has a role in the oxidation of toxic aldehydes.

13)  TNFSP14(tumor necrosis factor ligand super family member 14). Also called CD258(LIGHT). The protein encoded by this gene is a member of the family. It stimulates lymphoid cells, T cells, influences apoptosis in various tumors. It prevents apoptosis mediated by TNF-alpha.

14)  NF-kappa Beta. It is a protein complex that controls DNA transcription. It is involved in cellular response to various stimuli - cytokines, free radicals.

15)  KERATYN 7 (keratyn type1 cytoskeletal 7(cytokeratin7) is a protein encoded by  KRT7 gene. Is located in the 12q12-q13 area. It has a role in epithelial cell differentiation.

16)  MUC-1(cell surface associated (MUC-1), or polymorphic epithelilal mucin (PEM) is a mucin encoded by the humana MUC-1 gene. It is found in the apical area of epithelial cells (lung, stomach, gut). Its overexpression is related with lung, stomach, colon, ovary, pancreas.

17)  SFTB Gene, which encodes the surfactant-proteine B related to lung. The lung surfactant is a lipoprotein complex consisting of 90% fat and 10% protein, which includes plasma-proteins and SPA, SPC, SPD apolipoproteins. The surfarctant is secreted by the alveolar cells and maintains stability of the lung tissue. SPA increases the rate of expansion and raises the stability of the surfactant monolayer.

18)  LAMININES. They are major protein BASAL lamina, one of the layers of basement membrane. Laminines are influencing cell differentiation, migration, adhesion as well as phenotype and survival.

19)  INEGRINES are a family of transmembrane proteins involved in the extracellular matrix. They are receptors that mediate attachment from cells and over-added tissue. Play a role in cell signaling, on cell conformation, motility and cell cycle.

20)  KERATIN5(Keratin type II cytoskeletal5) is a protein encoded by KRT5 gene. It is found in the 12q12-q23 area. It is expressed in the basal layer of the epidermis with family KRT14.

21)  S100. S100 proteins are located in the cytoplasm and (or) nucleus and are involved in many cellular processes. They are located in the 1q21 area. They are involved in motility, invasion, and tubulin polymerization. The chromosomal rearrangements and the altered expression of this gene are involved in tumor metastasis.

22)  GJB5(GAP JONCTION BETA-5). It is also known as CONNEXIN-31.1. It is a protein encoded in humnas by GJB5 gene.

GAP JONCTION are conduits that allow direct passage from one cell to another cell of the small cytoplasmic molecules (ions, intermediate products, secondary messengers). Gap junction channels have subunits of the connexin protein, which are encoded by multiple gene families.

BIBLIOGRAFIE

1)         H Popper Adenocarcinoma and its Precursors lesions. Helmut. in Molecular pathologyof lung disease. pg 251-269Edited by Dani S.Sander,Helmuth H Popper.Jaishree Jagirdar et.al .Springer Verlag 2008

2)         E. Brambilla, A Gazdar Pathogenesis of lung cancer signaling pathways:roads map for therapies Eur.Resp.journal 2009;33/6:1485-1497

3)         Sun S. Schiller J.H, Gazdar A.F.Lung cancer in never smokers-a different disease Nat.Rev.cancer 2007;7:778-790

4)         Yatabe Y,Kosaka T,Takahashi,T et alEGFR mutations is specific for terminal respiratory unit type adenocarcinomaAm J. Surg  pathol. 2005:22:633-639

5)         Soon Hee,Bihong G, Zhao Li Mao and Jae Y..RMolecular Pathology of squamos cell carcinoma and its precursors inMolecular Pathology of Lung Diseases. Springer Verlag pg.:276-278

6)         Rocha A.T,McCormick M, Montana G Schreober GAssociation between lower lobe location and upstanding for early stage non-small lung cancerCHEST 2004;25;1424-1430

7)         Braakhuis B.J,Tabor M.P,Kummer J.A A genetic explications of SLAUGHTER-s concept of Field cancerization evidence and clinical implications Cancer Research 2003;15;1727

8)         Miller Y.E Pathogenesis of lung cancer;100 years report Am. J. Resp.Cell. Mol Biol. 2005;:r:216-223

9)         Hirsch T.R,Franklin W.A Gazdar A.F,Bunn jr.P.A Early detection of lung cancer: clinical perspectives of recent advances in biology and radiology Clinical cancer research 2001;7:5-22

10)       Yokota j,Takashi K Molecular foot prints of human lung cancer progression Can.Sci.2004;95:197-204

11)       Wistuba I.I,Gazdar A.F Characteristic genetic alterations in lung cancer In:Dricolli B .Ed.Lung Cancer.molecular Pathology.Metods and reviews. Vol 1 Los Angeles Human Press 2003;3:-28

12)       Forgacs E ,Zochbauer-Mueller S,Olah E, Minna J.D Molecular genetic abnormalities in the pathogenesis of human lung cancer Pathol Oncol.Res 2001;6-13

13)       ThibervilleL,Payne p,Vielkinds J et al Evidence of cumulative losses of premalignant epitehelial lesions to carcinoma of the bronchus Cancer Research  1995;55:5133-5139

14)       Vincenzi B,Schiavon G, Silleta M et al Cell cycle alterations and lung cancer Histol.Histopathol 2006;21:423-435

15)       Greenblatt M.S,Benett W.P,Hollstein M,Harris C.C Mutations in the p53 tumor-supressor gene:clues to cancer etiology and molecular pathogenesis Camcer Reaerch 1994;54:4855-4878

16)       Franklin W.A,Gazdar A.F,Haney G et al Widely dispersed p53 mutations in respiratory epithelium.A novel mechanism for field carcinogenesis J.Cli.Invest 1997;100:2133-2137

17)       Ramet M,Casten K,Jarvinen K et al p53 protein expression is correlated with benzo(a)pyrene-Dna adducts in carcinoma cell lines Carcinogenesis1996;16:2117-2124

18)       Benett  W.p,Colby T.V,Travis W.D et al p53 protein accumulates frequently in early bronchial neoplasia Cancer Reaerch1993;53:4817-482

19)       Pfeifer G.P,Denissenko M.F,Olivier M et al Tobacco smoke carcinogens,DNA damage and p53 mutations in smoking associated cancers Oncogene2002;21:7435-7451

20)       Pazzella F,Turley H,Kuzu I et al Bcl-2 protein in  non small lung cancer N.Engl.J.Med1993;329:690-694

21)       Walker C,Robertson L, Miskow M,Dixon G Expression of Bcl-2 protein in normal and dysplastic bronchial epithelium and in lung carcinoma Br.J.Cancer 1995;72:164-169

22)       Uren A.G,Vaux D.L Molecular and clinical aspects of apoptosis Pharmacol. Therap1996 ;72:37-50

23)       Zuo Kai Lin,Chun Yi-Wei,Jer-Wei Chang Dysregulation of p53/Sp-1 control leads to DNA methyl transpherase-1 over expression in lung cancer Cancer Research 2010;70(14):5802-5819

24)       Li J Yen C,Lian D et al PTEN,a putative protein-kinase phosphate gene mutated in human brain ,breast and prostata cancers Science 1997;275;1943-1947

25)       Steck P.A, Pershouse M.A,  Jasser S.A et al Identification of candidate tumor suppressor gene,MMAC 1 at chromosome 10q-23.3,that is mutated in multiple advanced cancers Nat. Genet 1997;15:356-362

26)       Weng l, Brown J,Eng C PTEN induces apoptosis and cell-cycle arrest trough phospho-inositol-3-kinase/AKT dependent degradation and –independent pathway Hum.Mol.Genet 2000;10;237-242

27)       Tamura M, Gu J, Matsumoto K et al Inhibitiopn of cell migration ,spreading and focal adhesion by tumor suppressor PTEN Science1998;28:1614-1617

28)       Forgacs E,Biesterfeld E.J,Sekido Y et al Mutation analysis of PTEN/(MMAC) gene in lung cancer Oncogene 1998;17:1557-1565

29)       Petersen S ,Rudolf J ,Dockmuhl et al Distinct region of allelic imbalance on chromosome 10q.22-q26 in squamous cell carcinoma of the lung Oncogene 1998;17:449-454

30)       Gorgoulis V.G,Zacharatos P,Kostinas A et al Alterations of the p16-Rb pathway and the chromosome locus 9p21-22 in nonsmall cell lung carcinoma.Relationship with p53 and MDM2 protein expression AM J. Pathol.1998;153:1749-1765

31)       Bates S ,Phillips A.C,  Clark D.A  et al. p-14 ARF links  the tumor-supressor Rb and p53 Nature 1998;;395:124-125

32)       Zochbauer Mueller S, Lam S,  Toyoka S  et al Aberrant methylation of multiples  genes in the upper aero-digestive tract epithelium of heavy-smokers Int. J. Cancer 2993;;107:612-616

33)       Belinski R.A, Nikula k.J,Palmisano N.A et al Aberrant  methylation of p16INK4 is an early event in Lung Cancer and a potential biomarker for early diagnosis Proc. Natl. Acad. Sci USA 1998;95:11891-11896

34)       Brambilla E,Moro D, Gazzeri S,Brambilla C Alteration of expression of Rb,p16INK4 and Cyclin D1 in non small cell carcinoma and their clinical significance J.Pathol.1999;188:351-360

35)       Sylvie Lantuejoul,Jean Charles Soria,Luc Morat Telomere shortening and  Telomerase reverse transcriptase expression in pre-invasive bronchial lesions Clinical Cancer Research 2005;11:1074-2082

36)       Yashima K,Lityzky L.A,Kaiser L et al Telomerase expression in respiratory epithelium during multi-stage pathogenesis of Lung carcinomas Cancer Rsearch 1997;57:2373-2374

37)       Yuka Matsuoka,Truomi Miazawa, Keiko Hiyama Telomerase  expression in non cancerous bronchial epithelia is a possible marker of  early development of lung cancer Cancer Research 2005;65:9623-9627

38)       Hiyama K,Hiyama E,Ishioka S et al Telomerase activity in small cell and non small cell lung cancers J.Nat.cancer Inst 1995;87:895-902

39)       Kauzo Yashima,Leslie A, Litzki Laray Kaiser et al Telomerase expression in respiratory epithelium during multi stage pathogenesis of lung cancer Cancer research 1997;57:2373-

40)       Miyazu Y.M,Myazawa T, Hiyama  K et al Telomerase expression in noncancerous bronchial epithelia is a possible marker of early development of lung cancer Cancer  research 2005;65;9623-9627

41)       Borzuck A.C,Gorenstein L,Walter K   Non small lung cancer :molecular signatures recapitulate lung developmental pathway   Am J. Path.2003:;63;1949-1960    

42)       Anja Baumgarten,Stefan Seidl, Petras Vlachom et al ADAM17 reglates epidermal growt factor receptor expression trough the activation in non small cell lung cancer Cancer Research 2010;70:5368-5378

43)       Luisa M Solis, Carmen \Behrens, Wenli Dong Nrf2 and Keap-1  abnormalities in non small cell lung carcinoma and association with clinico-pathologic features Clinical Cancer Research 2010;11(14):3743-3753

44)       Anju Singh,Swetlana  Boldin-Adamski,Rajek a Thimmulappa RNAi mediated silencing of nuclear fator erythroid-2-related factor2  gene expression in non small lung cancer inhibits tumor growth and increase efficacy of chemo-therapy Cancer Rsearch 2008;68(19):7975-7984

45)       Yun Lu,  Christopher Futtner, Jason R.Rock Evidence that SOX2 over expression is oncogene in the lung PlusOne 2010;June 5/6:

46)       Ludmila Prudkin,Carmen Behrens,Diane D-Liu Loss and reduction of FUS-1 protein expression is a frequent phenomen in the pathogenesis of lung cancer Clinical Cancer Research 2008;14:41-47

47)       Zalcman G,E.Bergot,E.Lechapt Up-date on small cell lung cancer Eur.  Resp. Rev.2010;19?117:173-185

48)       Savci-Heijink  CD,Kosari F,Aubry MC et al The role of desmoglein-3 in the diagnosis of squamous  cell carcinoma of lung. Am J.Pathol. 2009;174:1629-1637

49)       Lebanomy D,Benjamin h,Gilad S et al  Diagnostic assay based on has-miR-205 expression distinguishes squamous from non-sqamous non smallcell lung carcinoma j.Clin .Oncol.2009;27;2030[2037

50)       Ceppi P, Volpnite M,Saviozzi  et al Squamous cell carcinoma of the lung compared with other histo-types show higher messenger RNA and protein level for thymidylate synthase Cancer 2006;107:1586-1589

51)       Scagliotti G.V Efficacy of pemetrexed according to NSCLC histology:a review of the phase III studies Oncologist 2009;14:253-263

52)       Scagliotti G.V, Parika P, von Pavel J et al Phase I I I study comparing cis-platinum plus gemcitabine with cis-platinum  plus pemetrexed in chemotherapy-naïve patients with advance-stage non-small cell lung  carcinoma J.Clin.Oncol.2008;21:3543-3547

53)       Mathew D. Wilkerson, Xiaoying Yin,Katherine A.Hoadle,D.Neil Hayes  et al Lung squamous cell lung carcinoma mRNA expression subtypes are reproductible clinically important and correspond to normal cell types Clinical cancer research 2010;16:4613-

54)       Koyi H,Hillerdal G,branden e et al A prospective study of a total material of lung cancer from a county in SWEDEN 1997-1999:gender, type, stage  and smoking habits Lung Cancer 2002;36:9-14

55)       Travis W.D;World Health Organization:histological typing of lung and pleural tumours Berlin Springer verlag

56        Golub T.R,Slonim D.K,Tamayo P et al  Molecular classification of cancer: class discovery and class prediction by gene expression monitoring Science 1999;286;531-537

57)       Whitfield M.L, George L.K, Grant G.D, Perou C.M Commun markers of proliferation Nat. Rev.Cancer 2006;6;99-106

58)       Polager S,Ginsberg D. E2F at the crossroads of life and death Trends cell boil 2008;18:528-535

59)       Spira A, Beane J, Saah V et al Effect of cigarette smoke on the human airway epithelial cell transcriptome Proc.Natl. Acad.Sci2004;101:10143-10148

60)       King  K.E, Weinberg W.C p63 defining roles in morphogenesis,homeostasis and neoplasia of the epidemis Mol.Carcinog 2007;46:716-724

61)       Massion P.P, .Taflan P.M,Jamshedur Rahman s.M. et al Signifiance of p63  amplification and overexpression in lung cancer development and prognosis Cancer Research 2003;63:7113-7121

62)       Gaillard D,Puchelle E Differentiation  and maturation of airway epithelial cells;role of extracelular matrix and growth factors in:Gauthier C,Bourbon J.R.Post M editors

1999; p 46-76

63)       Garber M.E,Troyanskaya O.G,Schluens K et al Diversity of gene expression in adenocarcinoma of the lung Proc. Natl acad Sci USA 2001;98;13784-13789

64)       Brome A.M. Ryan D, Eckert R.L. S100 protein subcellular localization during epidermal differentiation and psoriasis J.Histochem.Cytochem 2003;51:675-685

65)       Smith S.C.  Guger M, Huban P et al S 100A2 is strongly expressed in airway basal cell, preneoplastic bronchial lesions and primary non-small cell lung carcinoma Br. J.Cancer 2004;91:1515-1524

66)       CHU P.g, Weiss L.M Keratin expression in human tissues and neoplasms Histo-pathology 2002;40;403-439

.