Tobacco, environmental and genetic factors and several lung diseases contribute to lung cancer carcinogenesis. Lung cancer is one of the most common cancer diagnosed, and has also the highest mortality rate due to the advanced stages at time of diagnosis, and when the options for treatment have to be understood as personalized therapy.1–8

Bronchial-pulmonary carcinomas consist of small cell lung cancer (SCLC), squamous cell carcinoma (SCC), adenocarcinoma, large cell carcinoma and sarcomatoid/pleomorphic carcinomas.8 Adenocarcinoma is the most common type in non-smokers and its incidence has increased in recent years, developing after terminal respiratory unit (TRU) but also recognized in respiratory epithelium.8–10 Adenocarcinoma pure patterns are recognized [acinar, papillary, micropapillary, BA (bronchioloalveolar/lepidic/AIS-adenocarcinoma in situ), solid with mucin production] but mixed type predominates. Differences between the subtype/patterns of adenocarcinoma should be taken into account to improve diagnosis, prognosis and as an enhancement in therapy selection.8,10

Cytokeratin 7 (CK7), high weight molecular cytokeratins (HWMC) like CK5/6, CK20, chromogranin A and TTF1 are currently used in lung cancer diagnosis.11,12 Ki67 is a cell proliferation marker with prognostic value.13–15 In the apoptosis pathway, p53 induces Bax transcription and reduces bcl2. Bcl2/Bax ratio is negative in lung carcinomas.16 Inactivation of RB pathway is considered a requirement for lung carcinogenesis.16 Adenomatous polyposis coli (APC) gene is involved in the canonical Wnt/β-catenin signal transduction. APC is mutated in liver, colorectal adenomas and lung tumors.17 Cyclins D (D1, D2, and D3) are activators of cyclin-dependent kinases cdk4 and cdk6, to translate growth signals into cell cycle progression, leading to proliferation.18,19 Cyclin D1 overexpression and pRB inactivation indicates a poor prognosis.20

Bcl2 as an anti-apoptotic protein has a more important role in neuroendocrine (NE) lung cancer than in other bronchial-pulmonary carcinomas.21 Excision repair cross-complementing 1 (ERCC1) genetic polymorphisms may affect patient's response to platinum-based chemotherapy.22,23 However, another study concluded that patients with a bronchial-pulmonary carcinoma with high ERCC1 expression have a better prognosis than those with low expression.24 Multidrug Resistance Protein 1 (MRP-1) and lung resistance-related protein (LRP) may confer resistance to cytotoxic and antiviral drugs.25–31

EGFR and HER2 play a central role in tumorigenesis.32,33 Targeting those receptors provides a unique approach for treating EGFR/HER2 expressing cancers.34,35 The most common alteration of HER2 is overexpression/amplification; mutations are less frequent.33,35,36 The activated receptors trigger Ras-Raf-MEK (mitogen-activated and extracellular signal regulated kinase), ERK1 and ERK2 (extracellular-signal regulated kinase 1 and 2) pathways leading to cell growth, mTOR (mammalian target of rapamycin) pathway leading to protein synthesis, and PI3K-AKT (phosphatidylnositol-2 kinase Akt) pathway sustaining cell survival.36

The objectives of this work were to validate a small differentiation IHC panel, evaluate protein expression of different genes according to different adenocarcinoma patterns, and correlate this expression with cell pathways of progression and drug resistance, in order to understand bronchial-pulmonary adenocarcinoma carcinogenesis. Another objective was to verify possible differences of gene expression in adenocarcinoma taking into account variables such as gender, age, smoking and different tumor stages. The global intention of this project is to acquire knowledge which can be applied in the diagnosis and prognosis evaluation of bronchial-pulmonary adenocarcinoma and its subtypes in order to define individual treatment.

Predominantly mixed-type adenocarcinomas,40 three acinar adenocarcinomas, one solid mucin producing adenocarcinoma and one bronchioloalveolar (BA)/lepidic mucinous adenocarcinoma/mucinous adenocarcinoma in situ were studied to validate the different patterns. Forty-two cases (93.3%) with acinar pattern (being the most prevalent), 26 (57.8%) with BA/lepidic pattern, 15 (33.3%) with solid pattern, 5 (11.1%) with papillary and 15 (33.3%) with micropapillary pattern were registered.

Thirteen cases (28.9%) showed lymph node metastasis as follows: acinar pattern was present in 13 cases (100%); solid and micropapillary patterns were present in 3 cases each (23%); and papillary pattern in 2 cases (15.4%). Two metastasis with solid pattern belonged to primary adenocarcinomas which harbored, respectively, acinar and BA/lepidic and acinar, BA/lepidic and micropapillary patterns.

Seventeen cases (37.8%) were diagnosed in men and twenty-eight cases (62.2%) in women. Age at diagnosis ranged from 44 to 85 years with a median of 67 years. Fourteen cases (31.1%) were diagnosed in smokers (8 males and 6 females), four (8.9%) in ex-smokers (3 males and 1 female) and twenty-seven (60%) in never smokers (6 males and 21 females). Female patients were more frequently non-smokers than men (p=0.0251).

Seventeen cases (37.8%) were diagnosed as stage IA (5 males/12 females), twelve (26.7%) as IB (2 males/10 females), ten (22.2%) as IIA (8 males/2 females) and six (13.3%) as stage IIIA (2 males/4 females). Females were diagnosed more frequently in lower stages when compared to men (p=0.029). There were no differences between stages according to smoking habits.

All adenocarcinomas (n=45) expressed CK7. No expression was observed for CK20, HWMC or chromogranin A. TTF1 was expressed in the great majority of lung adenocarcinomas, with statistical significant higher expression in acinar pattern (p=0.0094), micropapillary pattern (p=0.012) and BA/lepidic patterns (p=0.00046). The expression was significantly higher in BA/lepidic pattern when compared to solid pattern (p=0.021).

Nuclear expression of P53 was statistically higher in all patterns when compared to normal tissue (p<0.05), without differences between patterns.

RB expression was higher in micropapillary pattern when compared to normal tissue (p=0.011). The other patterns showed no differences from basal expression in normal tissue.

There were no significant differences between primary adenocarcinomas and their metastasis, except for cyclin D1 whose expression was higher in the metastasis when compared to the primary neoplasia (p=0.0409).

APC expression was higher in all patterns when compared to normal tissues (p<0.05), with no differences between patterns.

BCL2 expression was higher in all patterns studied than normal adjacent tissue (p<0.05), without differences between patterns.

Cyclin D1 expression differed between normal tissue and solid (p=0.0002) and BA/lepidic (p=0.002) patterns, being higher in these patterns than in the normal tissue.

EGFR protein expression was higher in acinar (p=0.033) and solid (p=0.012) patterns when compared to normal tissues, concerning membrane expression only. If we take into account cytoplasmic expression the overall expression was significantly higher in all patterns without differences between them.

For HER2 we observed significantly higher expression in all patterns compared to normal tissue (p<0.05). Expression is also higher in acinar, papillary, micropapillary and BA/lepidic patterns compared to solid pattern (p<0.05).

ERCC1 expression was lower in acinar (p=0.0016) and papillary (p=0.019) patterns compared to normal tissue. Expression in the other patterns revealed no differences to basal expression in normal tissue. Papillary pattern has significant less expression than solid (p<0.05) and BA/lepidic patterns (p<0.05).

Ki67 expression was higher in all adenocarcinoma patterns when compared to normal lung tissue (p<0.05). There were no differences between the patterns.

LRP expression was different (higher) between normal and the different patterns of the adenocarcinomas (p<0.05).

MRP-1 expression was higher in all patterns compared to normal lung tissue, without differences between patterns (p<0.05).

Some immunohistochemical results are illustrated in Fig. 1.

Figure 1.

(A) Expression (++) of APC in BA pattern, 200×; (B) Expression (+++) of BCL2 in papillary pattern, 200×; (C) BA pattern with Cyclin D1 expression, 400×; (D) Cyclin D1 expression in solid pattern, 200×; (E) EGFR expression (+++) in acinar pattern, 200×; (F) EGFR expression (+++) in solid pattern, 200×; (G) ERCC1 expression (++) in BA pattern, 200×; (H) ERCC1 expression (++) in solid pattern, 200×; (I) HER2 expression (++) in acinar pattern, 400×; (J) Ki67 expression (+++) in lymph node metastasis, 200×; (L) Ki67 expression (+++) in acinar pattern, 400×; (M) LRP expression (+++) in acinar pattern, 400×; (N) MRP-1 expression (+++) in solid pattern, 200x; (O) RB expression (++) in papillary pattern, 200×; and (P) P53 expression (+++) in micropapillary pattern, 200×.

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The analysis of the dendrogram clearly showed that adenocarcinoma is genetically different from normal tissue (Fig. 2). Concerning the patterns, BA/lepidic and acinar are the more similar and papillary the most distinct or genetically separated pattern. By PCA analysis (Fig. 3) we were able to identify three clusters: the first composed of papillary pattern adenocarcinomas; the second corresponding to solid; and the third to the group of acinar, BA/lepidic and micropapillary patterns.

Figure 2.

Dendrogram for the hierarchical clustering analysis of transcripts according to their expression values, using Single linkage algorithm, Euclidean distances, targeting the various patterns and normal tissue.

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Figure 3.

The correlation of the patterns with the advanced PCA factor analysis.

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Looking at gender, we have identified higher expression of ERCC1 in female patients when compared to males (p=0.0008). We did not find any differences according to age. HER2 expression was higher in adenocarcinomas of non-smokers or ex-smokers (p=0.0384). Ki67 expression was higher in adenocarcinomas of smokers and ex-smoker patients (p=0.0034 and p=0.0064).

TTF1, Ki67, APC, RB, P53, BCL2, ERCC1 and EGFR showed significantly different expressions according to the stage of the tumor. TTF1 expression showed differences between stage IA and IIIA, being higher in stage IA (p=0.02). Ki67 was higher in IIIA compared to IA and IIA (p=0.02 and p=0.02). APC expression was higher in IIA and IIIA compared to IA (p=0.065 and p=0.025). RB expression was higher in stage IB tumors compared to stage IA (p=0.04). P53 expression was higher in IIA, IB and IIIA compared to IA (p=0.01; p=0.03; p=0.01). BCL2 expression was higher for IB compared to IA (p=0.039). ERCC1 was higher in stage IA compared to IIA (p=0.02) and in IIIA compared to IIA (p=0.01). EGFR expression was lower in stage IB compared to IA (p=0.032).

To sum up, almost all patterns showed differences of expression in relation to normal tissues with some exceptions for cyclin D1 (papillary and micropapillary patterns), for EGFR membrane staining (papillary and micropapillary patterns), for ERCC1 (in micropapillary, solid and BA/lepidic patterns), for RB (in acinar, papillary and solid patterns) and for TTF1 (in papillary and solid patterns).

Solid pattern demonstrated lower HER2 expression compared to acinar, papillary, micropapillary and BA/lepidic patterns. Papillary pattern had lower ERCC1 expression than solid and BA/lepidic patterns. Solid pattern showed lower TTF1 expression than BA/lepidic pattern.

Non-BA/lepidic adenocarcinomas are most often peripheral tumors with different growth patterns considered mixed type according to WHO classification, with at least two individual patterns; solid or acinar patterns are the most frequent in the central tumoral bulk and BA/lepidic at the periphery. Acinar has been described as the most common pure subtype and the most common in the mixed type. Papillary pattern was present in 10% of the adenocarcinomas, often difficult to differentiate from BA/lepidic, which has important therapeutic strategies’ implications.9 Micropapillary pattern is characterized by small papillary tufts growing from alveolar septa or floating within alveolar spaces lacking fibrovascular cores and is usually observed with papillary and mixed patterns; they are more aggressive due to their increased capacity for metastasize, indicating unfavorable prognosis.9 Solid pattern with mucin production has been reported in 15% of mixed-type adenocarcinomas.9,10 BA/lepidic pattern shows tumor cell growth along alveolar septa and aerogeneous spread through the lung. It is frequently diagnosed in former and non-smoking women. BA/lepidic is classified as non-mucinous, mucinous or mixed.9,10,38

Female adenocarcinoma patients were more frequently non-smokers and diagnosed in earlier stages, with higher ERCC1 expression involved in DNA repair.

Advanced stages (IIA and IIIA) of adenocarcinomas showed higher Ki67, APC, ERCC1 expressions and lower TTF1 expression reflecting a more aggressive, mitotically active and less differentiated adenocarcinoma and eventually a non-TRU adenocarcinoma.

There was generally higher expression of the products of genes studied in the adenocarcinomas compared to normal adjacent cells reinforcing their importance in lung adenocarcinoma carcinogenesis.

There were two specific gene expressions with differences between patterns, HER2 and TTF1 that interfere with gene transcription. Papillary and solid patterns revealed less TTF1 expression, identical to normal tissue exhibiting a non-TRU/bronchial phenotype. At the other end acinar, BA/lepidic and micropapillary showed higher TTF1 expression. Solid pattern revealed also lower HER2 and higher EGFR and ERCC1 (this compared to papillary) expression. Papillary showed higher HER2 and lower ERCC1 expressions. Adenocarcinomas showed higher TTF1 expression (type TRU) in acinar, BA/lepidic and micropapillary patterns, micropapillary with higher RB expression, acinar with lower ERCC1 and higher EGFR. Ciclin D1 seems to have more importance in acinar and BA/lepidic patterns than in micropapillary.

ERCC1 protein expression in micropapillary, solid and BA/lepidic patterns indicates DNA repair and in acinar and papillary patterns there was lower expression. BCL2 was overexpressed in all patterns, suggesting that there is inhibition of apoptosis. MRP-1and LRP were overexpressed in all patterns and it is important to further analyze these proteins for a better understanding of the response to therapy.

By hierarchical clustering test we highlight papillary pattern as the most different pattern followed by solid pattern; acinar and BA/lepidic patterns are most alike. Using PCA analysis we realized that acinar, micropapillary and BA/lepidic patterns are the most alike, so we made three clusters of patterns: papillary, solid and micropapillary/acinar/lepidic (BA).

Furthers studies are needed in order to interpret these results regarding therapeutic response in advanced staged bronchial-pulmonary carcinomas.

Funded by a grant from CIMAGO, Faculty of Medicine, University of Coimbra, Portugal.

The authors declare that they have no conflicts of interest.