Expression of Human Tissue Kallikreins (KLKs) in Polymorphous Low Grade Adenocarcinoma (PLGA)

Polymorphous low grade adenocarcinoma (PLGA) is the second most common malignant salivary gland tumor of the minor salivary glands [1]. It is characterized by asymptomatic, indolent growth and low propensity for metastatic disease; however, delayed recurrence is not uncommon, warranting careful clinical follow up. Furthermore, recent literature reports PLGA may not be as indolent as it was previously thought, describing histologic transformation from low to high grade malignancies [2].  

Human tissue kallikreins (KLKs) are a family of highly conserved serine proteases encoded by 15 genes. They are expressed by various tissue and organ systems throughout the body and are involved in the regulation of both physiologic and pathologic processes. Specifically, KLKs have been linked to different stages of cancer development and progression, and as a result, have become powerful tumor markers for the diagnosis, prognosis and management of the cancer patient [3]. Most notable is prostate specific antigen (KLK3). The literature demonstrates a link between KLKs and salivary gland neoplasms.

The aim of this project is to determine levels of KLK mRNA in tissue samples of formalin fixed paraffin embedded polymorphous low grade adenocarcinoma. Secondly, we wish to determine if KLK expression is limited to tumor cells alone. It is our hypothesis that the expression pattern of KLKs for PLGA will be markedly different compared to normal salivary gland tissue. The consequences of altered expression will be explored with hopes of predicting tumor behaviour and its implications on diagnosis and treatment. 

 Cases of polymorphous low grade adenocarcinoma were reviewed at the Department of Pathology, Schulich School of Medicine and Dentistry, Western University from 1987-2013. A diagnosis of PLGA was confirmed, demographic data was collected, and formalin fixed paraffin-embedded PLGA and normal salivary gland tissue samples were obtained. RNA isolation from these tissue samples was achieved, followed by conversion to complementary DNA via reverse transcription. Synthesized DNA primers were added to target kallikrein DNA and through polymerase chain reaction, the quantitative level of expression of KLKs 1-15 was recorded. Samples exhibiting high and low KLK expression were selected for immunohistochemistry staining, using a standard protocol.

 In total, 19 samples of polymorphous low grade adenocarcinoma were obtained from the archives at the Department of Pathology, Western University. The patient population ranged in age from 17 to 78, with a mean age of 68, and 64% of the patients were female. Most tumors were located on the palate (64%), with others sources being the buccal mucosa (21%) and the lips (14%). RNA isolation yielded 0.32ng of RNA from 16/19 PLGA tissue samples. Preliminary PCR data reveals an increase in the mean KLK (1-15) mRNA expression in all of the PLGA tissue samples, as compared with normal salivary gland tissue. KLK1, KLK4, KLK10, KLK12, and KLK15 showed statistically significant upregulation of mRNA expression in PLGA samples as compared to normal salivary gland tissue (Mann Whitney U test, p<0.05). Immunohistochemistry results using KLK7 primary antibody demonstrate specific staining of PLGA tumor cells. Immunohistochemistry staining for other KLKs is underway.

In conclusion, KLK mRNA demonstrates an increase expression in tissue samples of polymorphous low grade adenocarcinoma. Furthermore, the tumor cells stain positively, and specifically, for kallikreins. Further interpretation of this preliminary data, along with clinical correlation, is needed to elucidate the role of kallikreins as cancer biomarkers for patients with polymorphous low grade adenocarcinoma. 

 References:

1.  El-Naaj, I.A. et al. Polymorphous Low Grade Adenocarcinoma: Case Series and Review of Surgical Management.

2.  Olusanya, A.A. et al. Polymorphous Low Grade Adenocarcinoma: Literature Review and Report of Lower Lip Lesion with Suspected Lung Metastasis.

3. Pampalakis G, Sotiropoulou G. Tissue kallikrein proteolytic cascade pathways in normal physiology and cancer.