Establishment and characterization of pleomorphic adenoma cell systems: an in-vitro demonstration of carcinomas arising secondarily from adenomas in the salivary gland

Abstract

Background: Among the salivary gland carcinomas, carcinoma in pleomorphic adenoma has been regarded as a representative carcinoma type which arises secondarily in the background of a pre-existent benign pleomorphic adenoma. It is still poorly understood how and which benign pleomorphic adenoma cells transform into its malignant form, carcinoma ex pleomorphic adenoma.

Methods: We have established five cell systems from a benign pleomorphic adenoma of the parotid gland of a 61-year-old woman. They were characterized by immunofluorescence, classical cytogenetics, p53 gene mutational analysis, fluorescence in-situ hybridization, and histopathological and immunohistochemical examinations of their xenografts, to demonstrate their potency of secondary transformation.

Results: We established and characterized five cell systems (designated as SM-AP1 to SM-AP5) from a benign pleomorphic adenoma of the parotid gland. SM-AP1 to SM-AP3 showed polygonal cell shapes while SM-AP4 and SM-AP5 were spindle-shaped. SM-AP1-3 cells were immunopositive for keratin only, indicating their duct-epithelial or squamous cell differentiation, while SM-AP4/5 cells were positive for both keratin and S-100 protein, indicating their myoepithelial cell differentiation. Chromosome analyses showed numeral abnormalities such as 5n ploidies and various kinds of structural abnormalities, such as deletions, translocations, derivatives and isodicentric chromosomes. Among them, der(9)t(9;13)(p13.3;q12.3) was shared by all five of the cell systems. In addition, they all had a common deletion of the last base G of codon 249 (AGG to AG_) of the p53 gene, which resulted in generation of its nonsense gene product. Transplanted cells in nude mice formed subcutaneous tumors, which had histological features of squamous cell carcinoma with apparent keratinizing tendencies. In addition, they had ductal arrangements or plasmacytoid appearances of tumor cells and myxoid or hyaline stromata, indicating some characteristics of pleomorphic adenoma.

Conclusion: This study demonstrates in vitro that certain cell types from pleomorphic adenoma are able to clone and survive over a long term and develop subcutaneous tumors in nude mice. The histological features of squamous cell carcinoma from the transplanted cell systems in nude mice might suggest a secondary onset of malignancy from a pre-existing benign adenoma.

Figure 1

Histopathology of pleomorphic adenoma from which cells were isolated (A, HE stain, × 40; B, HE stain, × 200) and phase-contrast microscopy of pleomorphic adenoma cells (C, primary culture, ×100; D, colony with bizarre cells, ×135; E, established cell system SM-AP3, × 150; F, SM-AP5, × 150). The surgical specimen showed typical features of benign pleomorphic adenoma (A), containing only a small number of atypical tumor cells (B). Cells in the primary culture were mainly spindle in shape mixed with fewer amounts of polygonal cells (C). In the fourth passage, aggregates of polygonal and bizarre epithelioid cells appeared in the background of spindle-shaped cells (D). From the following passage, five clones were successfully grown and isolated. They were classified into two groups according to their cell shapes. One was a polygonal shape represented by SM-AP3 (E), and the other was a short spindle shape one as shown by SM-AP5 (F).

Figure 2

Immunohistochemistry for keratin, duct epithelial marker (A-B) and S-100 protein, myoepithelial marker (C-D) in pleomorphic adenoma cell systems: SM-AP1 (A, C), SM-AP4 (B, D) at day 6 after plating, indirect immunofluorescence, × 200. All of the cell systems, SM-AP1 to SM-AP5, were equally immunopositive for keratin (A-B). SM-AP1, SM-AP2, and SM-AP3 cells were not definitely positive for S-100 protein (C), while SM-AP4 and SM-AP5 cells were strongly positive for S-100 protein (D).

Figure 3

Cytogenetic analysis of pleomorphic adenoma cell systems. Panel A, chromosome numbers of pleomorphic adenoma cell systems: SM-AP1 (a), SM-AP2 (b), SM-AP3 (c), SM-AP4 (d), SM-AP5 (e), and those of cells in the primary culture (f) in histograms. Panel B, G-banded karyotyping of SM-AP5. Chromosome numbers of the five cell systems varied between 64 and 123, which were within tetraploid or pentaploid ranges, with a modal chromosome number of 108. Those of primary culture varied between 107 and 122 with a modal of 113.

Figure 4

Fluorescence in-situ hybridization (FISH) for screening of break points for the translocation, t(9;13)(p13;q12) in SM-AP5, by using BAC clones. Panel A, mapping of BAC clones of chromosome 9 (a) and FISH for 9p13.2 by BAC 1537E12 (b) and for 9p13.1 by BAC 1405F1 (c). Panel B, mapping of BAC clones of chromosome 13 (a) and FISH for 13q12.2 by BAC 1325C2 (b) and for 13q13.1 by BAC 1213F4 (c). × 800. Signals of BAC clones, 1537E12 and1405F1 for chromosome 9p13.2 (A-b) and 9p13.1(A-c), 1325C2 for chromosome 13q12.2 (B-c) were detected as clear single and/or paired fluorescence dots on the translocation t(9;13)(p13;q12) chromosome, while no signal for BAC clone 1213F4 for chromosome 13q13.1 was seen (B-b).

Figure 5

Mutational analysis of the p53 gene in genomic DNA from pleomorphic adenoma cells in the primary culture as well as from the five SM-AP cell systems. PCR-amplicons for exons 5, 6 and 7 of the p53 gene were directly sequenced, and the deletion of the last base G of codon 249 (AGG to AG-) in exon 7 was shared by all of the cell systems and the primary culture.

Figure 6

Transplanted tumors of SM-AP cell systems in nude mice. Macroscopic view of a tumor mass by SM-AP5 in lateral back in a nude mouse (A); cut surface view of a subcutaneous tumor by SM-AP1 (B); histopathology of transplanted tumors by SM-AP4 (C), SM-AP1 (D, E), SM-AP5 (F, G), and SM-AP3 (H). HE stain, C, × 100; D-G, × 320; H, × 240; immunoperoxidase stains of SM-AP3 transplants for perlecan (I) and fibronectin (J), × 200, hematoxylin counterstain. SM-AP cells formed subcutaneous tumors measuring about 10 mm in diameter in nude mice within one to four months (A). The tumors were rather limited to the dermis expanding into the superficial part of the muscle layer but had no capsular structure (B). Histopathologically, the tumors were basically squamous cell carcinomas with definite tendencies towards keratinization with invasive natures, although there was no basal cell alignment along the periphery of the tumor cell nests (C). Around the tumor cell nests, myxoid stroma was induced. SM-AP1 to SM-AP3 cells formed mimics of ductal structures (D), and at the same time, SM-AP1 and SM-AP2 showed plasmacytoid appearances (E). SM-AP4 and SM-AP5 cells formed less differentiated carcinomas composed of tumor cells with ground-glass-like cytoplasm (F). Irrespective of tumors, mitotic figures were frequently observed (G), and the stromata were wide, hyaline, and poor in vascularity and lymphocytic infiltration (H). The hyaline stroma was immunopositive for perlecan (I) and fibronectin (J).