The relative frequency of adenocarcinoma as compared to squamous cell carcinoma of lung has increased 8-fold in North America since 1970, and is now the most common histological type of lung cancer [1]. Therefore, it is imperative to determine clinical and pathological factors related to prognosis. Published data on the subject in South East Asian population is limited, however, as the area’s population is rapidly expanding, understanding unique patient and disease characteristics of this population is important. This study examines several possible prognostic factors from a small medical center in Pakistan, including: certain patient demographics, tumor stage, mucin production, measures of angiogenesis, and mast cell density.

Mucins are high molecular weight glycoproteins synthesized, stored and secreted by the epithelial mucosal cells of various tumors, including adenocarcinoma of lung. While previously studied by immunohistohemisty, effective histochemical stains have recently gained favor as an easy, cost-effective alternative [2]. It is widely accepted that mucinous adenocarcinomas of lung, colon, ovary and other body organs have worse prognosis as compared to their nonmucinous site-specific counterparts [3,4].

Tumor angiogenesis is a multifaceted phenomenon regulated by the delicate balance between a number of pro- and anti-angiogenic molecules released by tumor cells and host cells, including endothelial cells, macrophages, mast cells and stromal components reviewed by a number of studies [5-8]. Measured in terms of microvascular density, angiogenesis is indispensable in order to supply adequate oxygenation and nutrition to tissues, and, it is widely accepted that increased angiogenesis is necessary for tumor growth and metastasis. Angiogenesis is believed to depend on an ‘angiogenic switch’, a cascade of events starting with the discharge of proangiogenic factors including fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF), ultimately leading to the activation of endothelial cells, proteolytic enzymes such as matrix metalloproteinases (MMPs), digestion of the basement membrane, followed by endothelial cell migration, proliferation, and capillary tube formation [9]. The neocapillaries formed in tumors are deficient in the similar supporting architecture as their parent vessels: they are thin walled and leaky, providing access to the circulation for tumor cells to metastasize [10]. This forms the basis for use of antiangiogenic chemotherapeutic agents for the treatment of a variety of human cancers including non-small cell lung cancers (NSCLC) such as adenocarcinoma. Angiogenesis has been associated with poor survival in patients with pulmonary adenocarcinoma. Incorporating anti-angiogenic agents (like bevacizumab) for the treatment regimens of these patients have significantly improved patient survival [11-13].

Mast cells are widely known for their pivotal role in allergic, inflammatory and immune reactions. In addition, they are implicated in pain, tissue damage, and healing. In recent past, mast cells have been shown to have a strong link with cancer progression and mediate their effect through a torrent of cytokines, chemokines, pro-angiogenic factors and heparin release [14]. High mast cell density may be simply a consequence of tumor invasiveness, or, mast cells themselves may play a role in tumor progression [15]. Tumorigenic effects of mast cells may be mediated through modulation of immunosupression, angiogenesis, degradation of extracellular matrix and mitogenesis [16]. In last two decades, many investigators working on NSCLC have failed to show a direct relationship between mast cell density and patient survival, likely due to the extremely complicated role of mast cells in tumor biology [17].

This is an observational, descriptive study in which 56 consecutive patients presenting to Gulab Devi Chest Hospital in Lahore, Pakistan between the dates of September 2010 and May 2013 with biopsy confirmed primary adenocarcinoma of the lung were reviewed. Biopsy slides were reviewed by two independent pathologists (AHN, MA), and were diagnosed according to WHO classification 2004 for adenocarcinoma. Of note, since 2011, a new multidisciplinary classification of adenocarcinoma has been proposed by the European Respiratory Society, the American Thoracic Society and the International Association for the Study of Lung Cancer [2,18]. TTF 1 was used for confirmation of adenocarcinoma of the lung. ALK, KRAS, and EGFR mutations were not assessed in this study. No patient had received chemotherapy or radiation therapy prior to the initial diagnostic biopsy. However, patients were offered adjuvant chemotherapy, and surgical excision was offered for resectable tumors. Paraffin blocks containing sufficient formalin-fixed tumor samples were obtained from a total of 56 patients. Figure 1 depicts representative stained sections of tumor in hematoxylin and eosin, mucicarmine, PAS, and Anti-CD34 staining.

Figure 1: Selected microphotographs: A. Adenocarcinoma of lung (Haematoline and Eosin x10); B. Mucin in adenocarcinoma of lung (Mucicarmine x20); C. Mucin in adenocarcinoma of lung (PAS, x40); D. Microvessels (low microvascular density in a case of mucinous adenocarcinoma of lung (CD34, x20).

Mucin was observed in Hematoxylin and Eosin, Periodic Acid Schiff, Mucicarmine and Alcian Blue stained sections of adenocarcinoma, details on histochemical methods is given by Ali et al. [19].

Microvascular density (MVD) or angiogenesis per section was measured using immunostaining with a CD34 monoclonal antibody (Clone QBEnd10; BioSB, USA), which required phosphate buffer saline based antigen retrieval and 30 minutes incubation of the primary antibody (prediluted). Vessel count was assessed in areas of the biopsy containing the highest numbers of capillaries and small venules, based on the criteria of Weidner et al. [20]. Vessels in five high-power fields (200× magnifications) were counted by two independent experienced pathologists without knowledge of the patient outcome. An average value of the two scores was used in the current study. MVD was defined as the mean number of CD34+ vessels per section, and was then classified as either high or low compared to the mean observed in this study, which were 13 microvessels per high power field.

Mast cell counts per high power field (HPF) were calculated in the same manner as MVD as described above, as counted in toulodine blue stained sections. The mean mast cell density (MCD) was 3.27 mast cells per HPF.

Data was collected and stored on Microsoft excel, and subsequently analyzed using SPSS version 17 (IBM Inc. USA). Mean ± S.E. were given for quantitative variables, whereas frequencies and percentages were given for qualitative variables. Means were compared using Independent Samples t-test and ANOVA. Correlation between continuous variables was determined with Pearson’s correlation. Spearman’s rho was used to measure the correlation co-efficient when one of the variables was rank-scale. Kaplan-Meier survival curves were plotted and Log Rank tests were applied observe the effect of various factors i.e. age, gender, stage, nodal status, mucin production, angiogenesis and mast cell density on survival. P value <0.05 was considered to be statistically significant.

Average age at time of diagnosis was 55.96 ± 1.67 years. Male (n = 34, 60.7%) to female (n = 22, 39.3%) ratio was nearly 1.5:1. About half of the patients (n = 29, 51.8%) were current smokers. A significant proportion (n = 26, 46.4%) of the patients had significant unintentional weight loss (≥10%) before diagnosis was established. Thirty-seven (66.1%) patients had locally advanced (defined as any tumor 5 cm (T3) or greater, or positive lymph nodes) or distant metastatic disease. 36 (64.3%) had biopsy-proven involvement of ipsilateral supraclavicular lymph nodes. Regarding histological grading, 15 (26.8%) were well-differentiated, 25 (44.6%) were moderately differentiated and remaining 16 (28.6%) were poorly differentiated adenocarcinomas. Out of total 56, forty-one (73.2%) tumors did not show mucinous material within the glandular structures whereas almost 1/4th (n = 15, 26.8%) tumors showed mucin by one or more histochemical methods. High mast cell density was

While a third (n = 19, 33.1%) of patients presented with early stage (Stage I or II) disease, only 7 (12.5%) underwent surgical resection i.e. lobectomy (2 cases), pneumonectomy (5 cases). Other patients could not undergo resection due to various reasons (such as significant medical comorbidities) and were offered chemotherapy. The patients who underwent surgical resection were given chemo-radiotherapy with curative/therapeutic intent. The average disease progression free survival (PFS) was 6.08 ± 0.32 months (Range: 1-15 months). Table 1 shows the effect of various parameters on the patient survival in adenocarcinoma of lung. It is evident from the p values that significant weight loss, advanced stage (III or IV), positive supraclavicular lymph node(s), positive mucin production and high micro vessel density are associated with worse prognosis. However, gender, age, smoking status, mode of therapy, and mast cell density did not show statistically significant effect on patient survival in this study population. The decreased survival with relation to stage, micro vascular density, and mucin production along with the lack of difference in survival with respect to mast cell density is well visualized in the Kaplan-Meyer curves in Figure 2A-2D.

Table 1: Predictors of patient surviva lina denocarc inomaoflung.

Figure 2: Selected Kaplan-Meier survival curves for: A. Stage of cancer; B. Mucin production; C. Microvascular density; D. Mast cell density.

Lung cancer is the second most common malignancy, and leading cause of cancer death worldwide [21]. Adenocarcinoma has now surpassed other histologic types as the most common [1]. Adenocarcinoma of lung is a disease commonly afflicting younger patients, females, and non-smokers – in drastic distinction to squamous cell carcinoma of lung which frequently occur in older, male, smokers [22-25].

Similarly to this small observational study, A recent study by Ma et al. (2010) involving 100 patients of all histological types of non-small cell lung cancer (NSCLC) including 36 patients of adenocarcinoma of lung showed that age, gender and histological grade has insignificant effect on the patient survival [26].

The observed result in this study may be confounded due to the limited number of patients studied, as well as the low percentage of patients proceeding with curative surgical intervention despite tumor resectability at the time of diagnosis. Lack of advanced radiodiagnostic measures such as positron emission tomography (PET) scans in small centers, such as this one, may also play a role. Furthermore, there is dearth of skilled thoracic oncologic surgeons not only in the city, but, in Pakistan as a whole. While this is an important limiting factor of this study, it is an important epidemiologic reminder that a very large number of individuals world-wide with various cancers are diagnosed and managed by centers with limited resources compared to those so often taken for granted in larger institutions in developed countries.

Average age of lung cancer patients has increased in developed countries leading to changes in the management of disease [27]. Asmis et al. working with National Cancer Institute of Canada Clinical Trials Group concluded that age more than 65 years was associated with poor survival [28]. However, in the current study, the mean age of patients was only 55 years. Effect of patient age on survival was conducted by separating the patients into two groups: below 55 vs. 55 and older. None of the patients in current study were older than 57. The relative lack of variability in age likely contributed to age not having a significant effect of overall survival in our study. Moreover, previous work by Båtevik et al. demonstrated that female gender has a positive prognostic implications following surgical resection of primary NSCLC [29]. Due to very small number presenting for surgical resection in our study, this was not observed here.

As discussed previously, mucinous adenocarcinomas are generally expected to have a poorer prognosis than their non-mucinous counterparts based on previous studies. Our study lends further support to these findings, as we demonstrated decreased survival, 4.13 ± 0.42 months vs. 6.79 ± 0.36 months (p = 0.0001) among mucinous and non-mucinous pulmonary adenocarcinomas. Similarly, weight loss, stage of tumor and lymph node status are well-known predictors of survival in patients of adenocarcinoma of lung and the same has been demonstrated here.

We conclude that weight loss, tumor stage and nodal status, mucinous characteristics, and increased angiogenesis (as determined by vessel density) are predictors of poor survival in this group of patients with pulmonary adenocarcinoma. In this small cohort, age, gender, smoking status, histologic grade, and mast cell density did not reach statistical significance with respect to survival.

Authors are grateful to the laboratory staff of Department of Morbid Anatomy and Histopathology, University of Health Sciences and Gulab Devi Chest Hospital Lahore for their technical assistance. Authors also acknowledge the University of Healthy Sciences Lahore for the financial grant of this project.

University of Health Sciences, Lahore for financial grant for this project.