THE BRONCHIAL PRENEOPLASTIC LESIONS. DIAGNOSIS AND CHEMOPREVENTION POSSIBILITIES

Dr. IULIAN POPESCU

The Clinical Department of Radiobiology of the Fundeni Clinical Institute

Abstract:

The issue of early detection is critical in the fight against Lung Cancer. The pre-invasive bronchial lesions represent an antechamber in the evolution to invasive cancer. The histological forms of these lesions as well as the early,
intermediate and tardive molecular changes will be depicted. Also, will be mentioned the possibilities of early detection:

1. Bronchoscopy with laser-induced self-fluorescence, guided by Optical Coherence Tomography, which can view cellular and extra cellular structures on the cell surface or beneath it.
2. PET-CT which may detect lesions of up to 6 mm diameter.
3. the bio-markers: they have a sensitivity of 60-70%. When they are researched jointly, their prediction value increases. The p53 and p16-INK4A genes seem to have the highest predictive value.

Highlighting these modifications leads to increased possibilities for chemo-prevention

(I) INTRODUCTION

THE LUNG CANCER (LC) is a huge challenge.

As for the histological and biological point of view, this cancer is very complex, probably having multiple preneoplastic ways [1, 2]. LC results from multiple changes in the genome of lung cells caused by the exposure to carcinogens. LC is thus the result of some sequential accumulation of specific genetic and morphological changes in the epithelial cells [3].

A) THE WAYS to escape of the cellular control mechanism [3] are as follows:

- activation of oncogenes	self-suficiency in the cell growth
- DNA methylation	irreversibility for anti-growth signals
- evasion of apoptosis	potency of limitless multiplication
- sustained angiogenesis	invasion and metastasis

B) JUSTIFICATION OF THE EARLY DIAGNOSIS

LC represents the main cause of the death from cancer. LC has more deaths than breast, colon and prostate cancer taken together.

The 5 years survival is below 15%. Depending on the stage, it is 60% at 5 years for the 1st stage. At the other stages [2, 3, 4], it decreases from 41% to 5% [4, 5, 6].

Most cases are discovered too late. At the moment of detection, 2/3 of the cases have ganglionic metastases. Thus, the fight against LC is compromised from the very beginning.

Hence the need for early detection through very sensitive tests of the pre-neoplastic lesions in order to establish screening programs.

C) THE METHODS FOR EARLY DETECTION are as follows:

1) radiological examination (radiography, computed tomography)

2) bronchoscopy examination

3) cytological examination of sputum, bronchial aspiration, bronchoalveolar lavage, bronchial biopsy following bronchoscopy

4) Bronchoscopy with laser induced self-fluorescence for detecting pre-neoplastic lesions [1, 7] guided by O.C.T[Optical Coherence Tomography = an optical imaging method , which can view cellular and extra cellular structures on the cell surface or beneath it] can view technically the preneoplastic lesions [48]

5) PET-CT which can highlight a malignant lesion of 6 mm diameter.

6) Bio-markers. Their highlighting makes possible a high probability diagnosis and further the chemo-prevention evolution towards invasive cancer

(II) THE HISTOLOGIC PRENEOPLASTIC CHANGES

THE squamos cancer

Like other epithelial cancers, it is believed they form after a series of progressive pathologic modifications (pre-neoplastic lesions) [8]. They have been best described in squamous cancer.

There are 6 stages: 

- Squamous hyperplasia and metaplasia
- Minimum dysplasia	With a low degree of malignant transformation
- Moderate  dysplasia
- Severe dysplasia	With a high degree of malignant transformation
- Plus the angiogenic form
- „In situ” carcinoma [8,11]

The first 2 changes do not fall within our problems, being considered reactive, inflammatory and disappear after stopping the smoking.

The natural history of the preneoplastic lesions and the risk of developing LC is not clear and the views are differentiated.

Saccomanno [9] has observed at the miners from uranium mines in the sputum cytology, atypia or in situ carcinoma [ISC] 4-10 years before the onset of LC.

The moderate sputum ayipia leads to cancer in 10% of cases, in a 9 years span, while those with severe atypia develop cancer in 40% of cases in 9 years.

Auerbach [10], in his studies of premalignant epithelium brings the proof of the steps progression in squamous cancer. The severe atypical cytology shows a risk to develop LC in 40-50% of cases, over the next 2 years. He also states that ISC occurs in most of the smokers. But because only 10% of the smokers get invasive cancer of squamous type, he concludes that most preneoplastic lesions do not lead to invasive cancer. Still the opinion closest to the truth is that patients with severe preneoplastic lesions have an increased risk to develop LC.

Philip Jeremy George et al. [11,12] kept trace of the preneoplastic lesions by self-fluorescence bronchoscope at a distance between 4-12 months and a computed tomography scan at 1 year. The tracking lasted between 1 year and 7 years. Out of 22 patients, 9 developed invasive cancer. Among these, 5 cancers have developed in distant loci in patients with severe dysplasia. All cases were NoMo and 8 cases were treated. The risk of getting invasive cancer in patients with severe dysplasia with a high degree of malignant transformation. Thus, 33% of them developed cancer after 1 year and 54% had cancer in the next two years. Among the lesions with a low degree of malignant transformation, none led to invasive cancer.

Still Breuer at all [13] has not seen a evolution in steps of the preneoplastic lesions. The regression rate of the preneoplastic lesions is 54%. The rate of progression to ISC and invasive cancer fluctuates between 26% for the low degree malignant transformation dysplasia and 39% for dysplasia with an increased degree of malignant transformation. The overall evolution towards malignant histological forms of preneoplastic lesions is 13.4%. The preneoplastic lesions with p53 mutations have a higher rate of progressing to invasion than the p53 negative lesions. The over expression of Bcl2 and Bax sub regulation are observed in the preneoplastic lesions and persist during invasion and contribute to the expansion.

The authors found no differences as concerns the progression rate and time. They did not notice a stepped progression to malignancy. The progression may also start from mild dysplasia in the absence of severe dysplasia.

This behavior suggests the presence of malignant clones in the bronchial mucosa at risk. Each lesion should be seen as likely to contain clonal cell with malignant potential.

On the whole it results that the patients with severe dysplasia lesions and ISC have an increased risk of getting invasive cancer. The risk is due to multi-focal lesions distribution in more remote places.

Thus appeared the concept of "cancerization field" where the entire bronchial epithelium is exposed to carcinogen. Giving up smoking only influences the evolution to malignancy and the dysplastic lesions do not disappear completely [14].

AdenocarcinomA

This is accompanied by hyperplastic or dysplastic changes [15] in peripheral bronchi and includes the ATYPICAL adenomatous hyperplasia [15] It is described by SHIMOSATO as precursor to the bronchiolo-alveolar cancer. This is known as an adenocarcinoma in which the neoplastic cell growth is done on the pre-existing alveolar structures [lepidic growth] without invading the pleura, vessels, stroma [16 ]

There are 3 forms of bronchioloalveolar carcinoma:

- mucinous form	25% of the cases
- non-mucinous form	most of the cases
- mixed form	infrequent

The non-mucinous, solitary, peripheral form - has a favorable prognosis.

Compared to the invasive adenocarcinoma can be cured by pulmonary resection. Responds to the treatment with EGFR inhibitors. The mucinous form does not respond to the treatment with EGFR inhibitors [16]

SMALL CELL CANCER

It is not observed any sequence of pre-neoplastic changes. These tumors may appear directly from the normal or hyperplastic epithelium, without going through intermediate levels. This led to the theory of cancer developing in “parallel”.

The Idiopathic pulmonary hyperplasia with NEURO ENDOCRINE CELL is proven to be a precursor of carcinoid. It has not been noticed a connection with the small cell cancer [8]

HISTOLOGICAL IMAGES K. M .KERR şi H.H PoPPER[46]

(with the approval of the author  and editorial office of the "European respiratory journal" ReVIEW)

Minimum dysplasia with nuclear irregularities in the basal third

Severe dysplasia with atypical cytology and mitoses in the medium third of epithelium

In situ carcinoma pointing a squamous, atypical differentiation extending up to the surface

Severe dysplasia / „in situ” carcinoma in a bronchial biopsy

Squamous angiogenic dysplasia. Clumps of connective tissue containing many capillaries are covered by a thin layer of squamous epithelium with moderate atypia

Irregular islands of infiltrated invasive carcinoma around some uninvolved glands

Atypical adenomatous hyperplasia lying outside the lung structures

Diffuse idiopathic pulmonary hyperplasia with neuroendocrine cells

a) mural nodule with neuroendocrine lung cells entering the lumen

Diffuse idiopathic pulmonary hyperplasia with neuroendocrine cells

b) Bronchiolus obliterated by a neuroendocrine cell nodule [46]

(III) THE CHANGES OF MOLECULAR BIOLOGY in the Bronchitis pre-neoplastic lesions

THE SQUAMOUS CANCER

The molecular analysis of bronchial epithelium appeared in 1990 and is relevant to squamous cancer [17]

The LC early diagnosis contributes to a better understanding of its biology. Special efforts are made for the early detection of LC [18]

The genetic abnormalities begin in the normal histological epithelium and grow with the increasing of the histological changes severity [13].

The molecular changes of bronchial epithelium are extensive and multi-focal in the bronchial tree in smokers indicating a ”field cancerization”. Multiple areas of the bronchial epithelium have been mutagenised after the exposure to carcinogen. The multiple clonal and sub-clonal areas may be detected in bronchial epithelium in patients with lung cancer [19]

In the preneoplastic lesions we found changes similar to the primary tumor. The genetic instability is observed at two levels [4]:

a) at the chromosomal level including a large scale of losses and gains

b) at the nucleotide level including changes of single or multiple bases [20]

Three categories of cellular genes are involved:

a) proto-oncogenes activated through:

• point mutations
• gene amplification
• chromosomal rearrangements

b) tumor suppressor genea: they are inactivated through:

• allela losses combined with point mutations
• methilation [4]

c) DNA repair genes = have not been found to be involved in carcinogenesis [4]

The molecular biology studies have been better studied - so far – in the squamous cell cancer. In active smokers and ex-smokers it is noticed a large increase of allele losses (LOH = loss of heterozygosity) and micro-satellite alterations (MA) both in normal and in the dysplastic bronchial epithelium. These allele losses are observed at multiple chromosomal sites [8]. The analysis of preneoplastic lesions by micro-dissection shows a sequential loss of heterozygosity in the chromosomal areas : 3p21, 9p, 8p21-22, 17p, 5q [8, 12]. Out of these genetic changes, the heterozygous loss at the level of chromosomes 3p and 9p are the most common and the earliest in the LC carcinogenesis [12]. The allele losses are more frequent in severe dysplasia and ISC [8]. The allele losses in the area of the 8p21-22 chromosome is seen somehow late in the ISC and the squamous invasive cancer.

We also find the damage of FHIT gene located at the 3p14, 2 chromosome, and the 5q chromosome [21].

The Kras and p53 mutations are also late phenomena of the preneoplastic lesions in invasive cancer.

Other changes in pre-neoplastic lesions include markers of cell cycle regulation or cell proliferation - c myc, K ras, cyclin D1, cyclin E, the product of Rb gene Rb[retinoblastoma][22].

The p53 gene mutations appear lately and are more common in severe dysplasia, ISC and invasive cancer [23]. Histo-chemical studies have shown the over expression of p53 in preneoplastic lesions [24]. The preneoplastic lesions with over expression of p53 have a higher probability to get lung cancer compared to the preneoplastic lesions with low expression of p53 [23, 24]

Following the study of JAY BOLE [21] it is shown that the chromosomal losses are most commonly found in severe dysplasia, ISC and invasive cancer (consistent).

Thus we have the following percent of heterozygosity loss:

- normal bronchial epithelium	27%
- severe dysplasia	18-45%
- squamous cancer	18-73%

But there are also inconsistent situations: some areas of the normal bronchial epithelium may have genetic changes more similar to invasive cancer than dysplasia.

Thus: in biopsies from the normal epithelium of smokers are shown losses of heterozygosity more than chromosomal sites, a phenomenon that is commonly seen in ISC and invasive cancer [25]

Normal bronchial epithelium specimens in smokers have indicated heterozygosity losses in 50% of cases. But only in ISC the heterozygosity frequency corresponds to the severity of histological modifications.

In normal histological epithelium of the active smokers and former smokers are found clonal areas with genetic alterations, which also persist after the smoking interruption [25]

These changes match with the theory of some converging or diverging clonal selection ways in the squamous cancer carcinogenesis.

It is stated that each marker taken alone has a sensitivity of 60-70%. When taken together, their sensitivity increases [22] Recent data show that the p53 and p16-INK4A genes seem to have a more promising predictive power than the KRAS genes, RASSF1A [49] Until today, these particularly deep studies show that we can diagnose with high probability but not certainly yet. We are not yet able to predict who is going to develop invasive cancer, but we can choose on objective bases the patients with high-risk. Identification of the disordered molecular signaling pathways in preneoplastic lesions is becoming a rational basis for early detection and chemoprevention.

ADENOCARCINOMA

Genetic studies has shown that atypical adenomatous hyperplasia has frequent losses of heterozygosity, aneuploidy, Kras mutations in smokers and fewer p53 mutations (10%). In non-smokers the presence of EGFR gene indicates the suspicion of adenocarcinoma [27, 31].

SMALL CELL CANCER

It is not known any precursor of small cell cancer. The pulmonary idiopathic hyperplasia with neuro-endocrine cell is valid for carcinoid [17]. The small cell cancer in the normal bronchial epithelium has an incidence and an extension of allele losses higher than AdC and squamous.

Thus we have: 90% in small cell cancer compared to 54% in the squamous and 10% in adeno-carcinoma. It also shows deletions in the areas 5q21-22 and 17p13 (TP53 gene) in the normal epithelium accompanying the invasive cancer. The allele losses in the 4 chromosome are more common than in other forms. During the pathogenesis of small cell and squamous cancer are noticed genetic changes in the central bronchi unlike adenocarcinoma that occurs at the periphery.

Table 1: Molecular alterations in different stages of bronchial carcinogenesis [European R. J., Hirsch at all, 2002;19:1151-1158]

Crt. No.	MOLECULAR ALTERATION	FREQUENCY
	Early changes = normal epithelium, hyperplasia and metaplasia
1	3pLOH/ small telomeres deletions	80%
2	micro-satellite alterations	50%
3	9p21 deletions	70%
4	Telomerase disorder	80%
5	myc over expression	60%
	Intermediate modifications = in bronchial dysplasia
6	8p21-23 LOH	80%
7	Neoangiogenesis	40%
8	Loss of immuno-coloration of the FHIT gene	70%
9	P53 LOH	70%
10	aneuploidy	80%
11	methylation	100%
	Late changes = in ISC and invasive cancer
12	TP53mutations	70%
13	5Q21-APL-MCC LOH	30%
14	Kras mutations	20%

Very recently it has been discovered that the 15q25chromosomal region is associated with the lung cancer. The region contains several genes that include 3 subunits codifying the nicotinic acetylcholine receptor.

These subunits have the names: CHRNA 5, CHRNA 3 şi CHRNB 4.

They are found in neurons, but also in the alveolar epithelial cells.

The 15q25 locus predisposes to lung cancer and increases the interest that the nicotinic acetylcholine receptors become future targets for chemoprevention [47].

All these successes have made it possible to switch to targeted therapy. It has entered into the daily practice, either as single treatment course or in combination with classical treatments [28].

The growth factors and their receptors have become a target of the tyrosine-kinases inhibitors. They are under erlotinib and gefitinib.

Cetuximab (Erbitux) - monoclonal antibody - in combination with the palliative chemotherapy are in the 3rd phase of clinical experiment and other monoclonal antibodies directed to EGFR are in the first stages of experiment.

The inhibition of angiogenesis through the VEGF / VEGFR blocking is done with the help of a Bevacizumab (Avastin) monoclonal antibody. There are also inhibitors of the matrix metallo-proteinase, the farnesyl transferase, but which have not improved their patients’ fate.

The molecular pathology is necessary for establishing the targets of attack [46].

CONCLUSIONS

Lung Cancer [LC] is a complex cancer, resulting from multiple changes in the genome of lung cells caused by the exposure to carcinogen and it appears due to some sequential accumulation of specific genetic and morphological alterations in the epithelial cells.

The necessity for early detection is due the fact that at the moment of current detection, 2/3 of cases have metastases in the ganglia. This makes the survival in 5 years to be up to 15%.

LC arises after a number of progressive histopathological changes - preneoplastic lesions. They have been best studied in the suqamous cancer.

For early detection, the most important are severe dysplasia (including angiomatous form) and ISC.

They have an increased risk of malignant transformation, but this risk is not absolute. The dysplasia forms with minimal or moderate lesions may cause direct invasive cancer, without going through all the steps, due to some multifocal clones. Any clone may contain a malignant potential.

The risk of malignant transformation for all forms of dysplasia is 13%, but for severe dysplasia and ISC, the risk is around 39%. Time is variable, but is shorter for severe dysplasia and ISC.

Adenocarcinoma is accompanied by hyperplastic and dysplastic changes in the peripheral bronchii and includes atypical adenomatous hyperplasia. It is the precursor of bronchiolo-alveolar cancer.

Small cell cancer has no intermediate structure. The pulmonary idiopathic hyperplasia with neuro-endocrine cells is proven as a precursor of the carcinoid.

The genetic abnormalities begin in the normal histological epithelium and increase with the growth of the histological changes severity. These molecular changes are extensive and multi focal in smokers, indicating an "cancerization field" effect. We distinguish several markers that appear early, intermediately and lately. The earliest changes are the heterozygosity loss and micro-satellite anomalies to 3p and 9p chromosome and the late modifications are represented by the TP53 gene mutations (for small cell and squamous cancer) and the Kras mutations for adenocarcinoma in smokers and EGFR for adenocarcinoma in non-smokers.

When taken together, their sensitivity increases. These histological and genetic proofs are elements of very high probability diagnosis, but it is not certain yet. They are rational data, for the continuous monitoring of high risk patients. And the genetic modifications are an objective target for chemoprevention - which is ongoing.

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GLOSSARY:

Allele: is a member of a pair or a series of various forms of a gene. Usually alleles are sequences which encode, but sometimes are sequences which do not encode.

Homozygote: an organism in which the two copies of the gene are identical, meaning that it has the same alleles for this gene.

Heterozygote: an organism having 2 different alleles of the gene.

Markers micro-satellites: are repeatable DNA sequences that are found in introns [non-codifiable area of the gene]. The expansion and deletion of these sequences are termed micro-satellite alterations.

Aneuploidy: abnormal number of chromosomes [more or less]

FHIT: fragile hystidin triad tumor suppressor gene

Kras proto-oncogene

c.Myc: proto-oncogene [prevails in small cell lung cancer]

cyclin D1: proto-oncogene

p53; tumor-suppressor gene

p16-ink-4ª: tumor-suppressor gene located in the chromosome 9 region 9p21

RASSF1A: tumor-suppressor gene

EGFR: receptor of the epithelial growth factor

CHRNA : gene [acetylcholine nicotinic receptor]