The Gender Difference and Prognostic Factors in Lung Cancer

Lung cancer has become a global health problem, accounting for 12% of new cases of cancer worldwide, followed by cancers of the breast and colo-rectum. Lung cancer is the leading cause of death from cancer among men and women in many Western countries, including the U.S. and countries in Europe. It is relatively more important in developed rather than developing countries, as it accounts for 22% versus 14.6% of cancer deaths.
Lung cancer results from complex, genetic and epigenetic changes characterized by stepwise malignant progression of cancer cells in association with accumulation of genetic alteration. This process, referred to as multistep carcinogenesis, develops through the clonal evolution of initiated lung cells. Initiation consists in the acquisition of defined genetic alterations in a small number of genes that confer a proliferative advantage that facilitates progression towards invasive carcinoma. Many environmental factors, including smoking and air pollution, could act as an initiator of carcinogenesis in bronchial or bronchiolar-alveolar epithelial cells.
The male to female ratio of lung cancer patients throughout the world is 2.7:1. In men, the areas with the highest incidence and mortality are Europe (especially Eastern Europe), North America, Australia/New Zealand, and South America. The rates in China, Japan, and South East Asia are moderately high, while the lowest rates are found in southern Asia (India, Pakistan), and sub-Saharan Africa. In women, the geographic pattern reflects different historical patterns of tobacco smoking. The highest incidence rates are observed in North America and North Western Europe (U.K., Iceland, Denmark) with moderate incidence rates in Australia, New Zealand, and China.
In the United States, lung cancer is the most common cause of death from cancer in both males and females. For the past 50 years, lung cancer is the leading cause of death from cancer for males. Since 1987, lung cancer has become the most common cause of cancer death for females. There are substantial differences in incidence and mortality in the different regions and populations within Europe. In men, the incidence is highest in Eastern Europe. In women, the highest incidence of lung cancer is in Northern Europe, which is almost twice as high as Western Europe. There is a similar pattern in the distribution of mortality. Predictions for the year 2010 show that most European countries carry a decline in mortality for males, but an increase in females.
In Asia, the mortality rates of lung cancer in women are generally lower than those in western countries. However, there has been a significant increase in the mortality rate from lung cancer in Asian women over the past few decades. In Taiwan, the mortality rates of lung cancer are high in both genders. For males, lung cancer is the second most common cause of death from cancer, accounting for 43.03 deaths per 100, 000 people. For females, lung cancer has been the leading cause of death from cancer, accounting for 19.71 deaths per 100,000 people.
The research into prognostic factors helps clinicians to give personal advice, to choose treatment modalities, and to understand the pathogenesis of the disease. The prognostic factors might be different among the various histologic types of lung cancer. For small cell lung cancer, studies on prognostic factors are limited. It is well known that performance status and extent of disease are important prognostic factors. For laboratory parameters, serum lactate dehydrogenase is frequently reported as a prognostic factor. In contrast, the prognostic factors of non-small cell lung cancer have been widely studied. Those factors could be divided into two categories: tumor-related factors and host-related factors. Among these factors, molecular biologic factors increased rapidly with the progression of genomic and proteomic technologies. However, clinical and pathologic staging according to the TNM system is still the most powerful prognostic factor for non-small cell lung cancer.
Although more and more prognosis research data for non-small cell lung cancer have been published, there are still some important issues that should be addressed: most studies are retrospective; the number of significant prognostic factors identified is still small; and study results of most prognostic factors identified were controversial or contradictory.
In the previous literature, lung cancer patients with various ages and genders demonstrated different clinical characteristics. Epidemiologic studies of lung cancer have shown geographic and ethnic differences, especially for female lung cancer patients. Therefore, we reviewed and analyzed the clinical data of lung cancer patients at National Taiwan University Hospital in order to characterize lung cancer patients of different age groups and genders in regards to the distribution of histologic types, clinical stages, treatments, and survival. In addition, we tried to combine the molecular biological experiment and clinical data analysis to clarify the value of the known prognostic factor of lung cancer. Through the integration of bioinformatics method and conventional biotechnology, we also try to establish a new research model to find more novel prognostic factors.
Our study consists of three sections: gender differences in lung cancer, the research of known prognostic factors for lung cancer, and identification of novel prognostic factors for lung cancer.

Gender differences in lung cancer
The importance of gender and age differences in lung cancer needs to be elucidated because of its implication in the design of experimental protocols for targeted chemoprevention, early disease screening, molecular markers-based staging, and individualized treatment. This study was designed to characterize lung cancer patients of different age groups and genders in regard to the distribution of histologic types, clinical stages, treatments, and survival. A multivariate analysis was also performed to identify the clinical prognostic factors for lung cancer patients in Taiwan.
Patients with a diagnosis of lung cancer admitted to National Taiwan University Hospital between January 1991 and December 1999 were identified from a computer registration database through the ICD-9 coding system. The clinical records of the patients included were reviewed and analyzed for age at diagnosis, gender, family history of malignancy, history of smoking, stages of cancer, histology, treatment modalities, and survival.
This study demonstrated the trend evolution of the distribution in histologic cell types and stages of lung cancer patients in Taiwan with respect to different genders and age groups. The annual number of lung cancer patients had a trend of increase from 1991 to 1999. The male to female ratio was constantly 2:1 during this period. The trend of increase was demonstrated in all age groups except for those patients at ages < 40 years old. The trend of increase in lung cancer patients was mainly due to the annually increase of patients with adenocarcinoma.
Adenocarcinoma has been shown to be a predominant histologic type in both male and female lung cancer patients. It has become an emerging problem more than the other cell types of lung cancer. Females (73.5%) had a significantly higher percentage of adenocarcinoma than males (42.1%) (p<0.001). For the histological distribution among different age groups, a trend of decrease in adenocarcinoma and a trend of increase in squamous cell carcinoma were demonstrated, as the age increased.
A multivariate analysis using Cox forward regression model was conducted for gender, age group, histology, stage, and treatment modalities. The adenocarcinoma group had better survival than other cell type groups. Female lung cancer patients had a better prognosis than males, which occurred mainly at the age of 50-69 years. We considered that physiological change in middle ages, such as change of sex hormone status due to menopause, might influence the prognosis. Further population-based studies on gender and age differences in lung cancer patients will help to reinforce these conclusions.

Research of known prognostic factors for lung cancer
Determining the prognosis for an individual patient with non-small cell lung cancer is difficult, in part because of the marked clinical heterogeneity of patients. Most patients presenting with one or more potential constellations in progression are, in turn, due to multiple potential manifestations of the primary tumor of involved metastatic sites and of paraneoplastic syndromes.
Human telomerase is a ribonucleoprotein that adds repeated units of TTAGGG to the ends of telomeres. Telomerase activity was detected in almost all cases of small cell lung cancer and in 80% of non-small cell lung cancer cases. In contrast, in normal somatic cells, telomerase activity is usually undetectable. Telomerase activity is one of the most important prognostic factors in patients with non-small cell lung cancer.
The detection of telomerase might be of little use in predicting the prognosis of lung cancer in patients since most of the patients have positive telomerase activity. Little has been studied regarding the quantitative analysis of telomerase activity and subsequent outcomes. We hypothesized that the quantification of telomerase activity, rather than the detection, would predict the prognosis of lung cancer. Thus, we used a non-radioactive quantitative method to investigate the correlation between the level of telomerase activity and the clinical features in patients with non-small cell lung cancer.
Patients who underwent surgery for non-small-cell lung cancer at National Taiwan University Hospital were included as potential subjects. The level of telomerase activity for lung cancer tissue and adjacent non-cancerous tissue obtained at surgery was accessed according to the telomeric repeat amplification protocol (TRAP) method with modifications using a TeloTAGGG Telomerase PCR ELISAplus kit. Clinical and pathologic parameters, including age, gender, histology, pathological staging, tumor invasion, and lymph node metastasis, were evaluated with respect to the level of telomerase activity. The high relative telomerase activity (RTA) group was defined as a RTA level above the median value. Otherwise, patients were categorized in the low RTA group.
Prominent telomerase-mediated 6-bp ladders using modified TRAP assay were detected in 58 (85.3%) lung cancer tissues and 21 (30.9%) adjacent non-neoplastic tissues. There was no difference between patient groups of positive and negative telomerase activity with respect to age, gender, tumor invasion, lymph node metastasis, histological type, and pathological staging. The median of the RTA value in non-cancerous tissues was 3.95, whereas that of cancer specimens was 536.95. There was no difference between the groups with respect to age, gender, and histological type of malignancy. However, there was a trend of increasing RTA in relation to the advance of pathological staging, depth of tumor invasion and lymph node metastasis.
The difference was statistically significant for patients with stage I disease. The mean disease-free period was longer in the low RTA group than in patients with high RTA in patients with stage I disease. A high level of telomerase activity tended to correlate with a higher risk of recurrence. For patients with stage II, III and IV disease, no statistical significance of overall and disease-free survival difference between the low and high RTA groups could be demonstrated.
Using a Cox regression analysis, we found that every 100 units of increase in telomerase activity cause the hazard ratio of death to increase by 13%. The effect did not change even after controlling for other variables such as age, gender, and stage. For the subset of patients with stage I disease, an increase of every 100 units in telomerase activity causes an even higher increase of 33% in the hazard ratio of death. For every 100 units of increase in telomerase activity, there was a 13% increase in the hazard ratio of recurrence for patients with stage I disease. After controlling for age and gender, for every 100-unit increase in telomerase activity, there was a 16% increase in the hazard of disease recurrence.
Our findings support the conclusion that the quantification of telomerase activity, rather than its presence, predicts the prognosis of lung cancer patients. This finding is helpful for application of telomerase activity to predict patient outcomes. Our data demonstrated the same trend of increase in telomerase activity as previously reported with the advance of tumor invasion, node metastasis, and pathological staging. These results showed a more detailed increase when using a non-radioactive method. These results indicated that the level of telomerase activity can be a more useful marker for tumor aggressiveness and may distinguish between primary tumors with potential for lymphatic spread and limited local tumors. Further study with a larger case number is warranted to clarify the trend of increase along with the advance of tumor invasion, nodal metastasis and pathological staging.

Identification of novel prognostic factors for lung cancer
Genome-wide microarray technologies, which are widely used to monitor global gene expression in cancer, have identified numerous differentially expressed genes suggesting that microarray has promised to shed light on the identification of disease markers for clinical use. However, researchers often faced the dilemma of effective utilization of the vast information source gathered through these microarray studies. The unmet needs include how to efficiently translate existing raw microarray data into a workable model to aid in the greater understanding of target prioritization. The combination of in silico biology and empirical methods may circumvent the bottlenecks from microarray profiling to target identifications.
In this study, a methodology was first set up to extract useful information from a microarray dataset for identification of new prognostic factors of lung cancer. We hypothesized that those genes showing similar expression patterns in a microarray database, which might belong to the same categories of biological functions, may also have the same impact on clinical outcomes. Microarray profiling of lung adenocarcinoma was performed on Affymetrix Human Genome U133A microarray chips. Since survivin (BIRC5) is a poor prognostic factor for non-small cell lung cancer, we hypothesized those genes exhibit expression profiling similar to survivin might be of clinical prognostic importance for lung cancer. Trophinin (TRO) is one of the best-correlated genes. Considering its role in embryonic implantation with blastocyst invading the endometrium, trophinin may also mediate the invasion process of cancer cells. Immunohistochemical staining of trophinin in lung cancer specimens was also performed. The results were correlated with clinical characteristics and outcomes. Ectopic expression of trophinin and inhibition by small interfering RNA (siRNA), which were monitored for cell invasion abilities, were also attempted.
Paraffin sections of lung cancer tissue from 141 patients (41 female and 100 male) were subjected to immunohistological assessment. The proportion of positive trophinin expression was significantly higher for female patients (p=0.003) and patients with an age less than 65 years (p=0.027). No statistically significant correlations were found between trophinin expression and histological type, pathological staging, or lymph node metastasis.
For patients with lung adenocarcinoma, the proportion of positive trophinin expression was also significantly higher for female patients (p=0.028) and patients with T1-2 disease (p=0.021). For patients with squamous cell carcinoma, no statistically significant correlations were found between trophinin expression and age, gender, pathological staging, tumor invasion, or lymph node metastasis. We found that among patients with stage I disease, the survival period for individuals without trophinin expression was significantly longer (80.4 vs. 50.2 months, p=0.042). A multivariate analysis, using Cox forward regression model, was conducted for gender, age, histology, type of surgery, together with trophinin expression for patients with stage I disease. The patients with positive trophinin expression had a poor survival after adjusting for other prognostic variables.
As for the various histologic types of lung cancer, the difference of survival between patients with and without trophinin expression was statistically significant for adenocarcinoma. The difference for overall survival and disease-free survival period between patients with and without trophinin expression was even more significant in stage I lung adenocarcinoma (overall survival: 78.8 vs. 39.6 months; p=0.006, Figure 3, disease-free survival period: 69.8 vs. 34.5 months; p=0.03, Figure 4). For squamous cell carcinoma, there was no difference between patients with and without trophinin expression.
To provide a mechanistic basis for trophinin as a novel poor prognostic factor and to delineate the biological effects of trophinin in cellular invasion, we took two approaches. First, we used MDCK renal epithelial cells as our model system, which is suitable for the cell invasion assay, to establish various trophinin stable clones. These stable clones were used in the invasion assay. Overexpression of trophinin in two different MDCK cell clones resulted in a two-fold increase in invasion ability compared with those of vector control (vehicle) or parental MDCK cells.
Second, we employed three different chemical synthesized siRNA to knock down the expression of trophinin in a highly invasive lung adenocarcinoma cell line, CL1-5. Endogenous trophinin RNA was knocked down by 80-90% in all three trophinin siRNAs. Knock down of endogenous trophinin in CL1-5 led to a decrease in invasion ability as compared to the vehicle transfected parental CL1-5 cells. Together, these data are in agreement with the idea that trophinin may promote cell invasion.
Survivin and trophinin have similar expression patterns in lung adenocarcinoma microarray dataset, which suggests trophinin and survivin might have some similar characteristics of expression regulation in common. Survivin is an anti-apoptosis gene; however, whether trophinin has a role in anti-apoptosis remains to be determined. Interestingly, trophinin might be an enhancer of cancer invasion/metastasis, suggesting these two genes of different functions leading to the same prognosis of non-small cell lung cancer. As such, further investigation is warranted to clarify the regulation mechanism of trophinin during cancer metastasis as compared with the findings of studies concerning regulation of survivin expression.
Through clinical observation and epidemiological analysis, we found significant gender differences in carcinogenesis, treatment response, mortality rates and prognosis. Emerging reports in the literature have provided the biological basis of explanation for the gender differences. However, there are still limitations for the basis. The main reason is that most large-scale studies are retrospective. These studies have some case selection bias and discrepancy in methodology. Prospective studies with population-based screening are necessary for further investigation of gender differences. In Taiwan, more than 90% of female lung cancer patients are non-smokers. Thus, risk factors other than smoking are critical for female lung cancer patients in Taiwan. The important issue for lung cancer carcinogenesis is the following: Why are women more susceptible to lung cancer than men? Factors including cooking, passive smoking, gene polymorphism, hormone factors, and air pollution should be carefully investigated. Complete epidemiological data is helpful to provide insight toward the explanation for such a high proportion of female lung adenocarcinoma in Taiwan. The interaction between genetic risk factors and environmental ones should be investigated. A multiple-center research team would be able to integrate the variety of lung cancer patients, to perform large-scale screening by using biotechnologies, and to identify novel risk and prognostic factors.
The major research subjects for prognostic factors of lung cancer are patients with resectable non-small cell lung cancer. For most patients with unresectable non-small cell lung cancer and small cell lung cancer patients who will not undergo operation, much is left to be studied regarding prognostic factors, especially for molecular biological factors. It would be more useful to develop methods for clinicians to obtain prognostic information from small tissue specimen via biopsy or from scanty cells via aspiration. Analysis of the changes of tumor-specific gene products in body fluids, including blood, pleural effusion, or urine, would be highly practical for clinicians in predicting patient outcomes. Our results support the importance of quantitative assay for prognostic factors. Through the emergence of identified prognostic factors, it will be necessary to perform quantitation and correlate with clinical data to confirm prognostic values.
In the future, while searching for the novel prognostic factors for lung cancer, it will become a great task to screen candidate genes or gene products from the genomic or proteomic database. We established a research model, in which we used a known prognostic factor as a template, and then searched for genes or gene products with similar expression patterns. The expression of genes or gene products was examined in the tumor specimens. The factors with prognostic values are investigated by experimental methods to clarify the role in cancer invasion/metastasis. Through this methodology, we could identify the novel prognostic genes more efficiently. The prognostic factor identified might have a different function in cell biology from the template gene. We might be able to find the links between different cell signal transduction pathways by molecular biological approaches. The research model is helpful not only for identification of prognostic factors, but also for understanding the mechanism of cancer invasion/metastasis.