Morphology of 9p21 homozygous deletion‐positive pleural mesothelioma cells analyzed using fluorescence in situ hybridization and virtual microscope system in effusion cytology

Abstract

BACKGROUND:In malignant pleural mesothelioma (MPM), most patients first present with pleural effusion; thus, cytologic analysis is the primary diagnostic approach. However, the cytologic distinction between MPM and reactive mesothelial cells (RMCs) in effusions can be extremely difficult due to the lack of both well-established immunocytochemical markers and definite cytological criteria for MPM. Moreover, the existence of both MPM cells and RMCs in effusions from the same patient makes the differentiation even more challenging. Homozygous deletion of the 9p21 locus, the site of the cyclin-dependent kinase inhibitor 2A/p16 (CDKN2A/p16) gene, frequently occurs in MPM but has never been reported in RMCs. The aim of this study was to define the cytomorphological characteristics of MPM cells, identified by the presence of 9p21 homozygous deletion by fluorescence in situ hybridization (FISH). METHODS: For this purpose, cells on smear preparations were recorded using a virtual microscope system and were subjected to FISH analysis. Thereafter, 9p21 homozygous deletion-positive cells were identified in the recorded virtual slides, followed by analysis of their morphological characteristics. RESULTS: Mesothelioma cells positive for the 9p21 homozygous deletion exhibited significantly more frequent cellin-cell engulfment, multinucleation (more than 2 nuclei), and larger multicellular clusters composed of more than 10 cells than did 9p21 deletion-negative RMCs. Possible cutoff values are also proposed for these morphological markers to differentiate MPM cells from RMCs. CONCLUSIONS: These morphological differences and cutoff values are useful for cytological differentiation of mesothelioma cells from RMCs. In addition, the novel technique of a combination of virtual microscopy and FISH is introduced for tumor morphological analysis. Cancer (Cancer Cytopathol) 2013;121:415-22. V C 2013 American Cancer Society.KEY WORDS: pleural mesothelioma; cytology; fluorescence in situ hybridization; 9p21 homozygous deletion; p16.

Pre-analytical issues in effusion cytology

“…The same solution was applied in analysis of CDKN2A, the gene coding for p16 at chromosome 9p21, which is deleted in different cancers, including malignant mesothelioma [47,48]. De-staining of Diff-Quik-stained slides was applied in another study in which changes in chromosomes 7 and 9 were assessed [49].…”

Guidelines for the Cytopathologic Diagnosis of Epithelioid and Mixed-Type Malignant Mesothelioma

“…The main cytologic findings of MM in effusions have been well described in the literature [8,17], and the criteria have recently been reevaluated [18]. The experienced cytopathologist will in many cases recognize the MM effusion, but the definite diagnosis should be supported by ancillary techniques.…”

p16/CDKN2A FISH in Differentiation of Diffuse Malignant Peritoneal Mesothelioma From Mesothelial Hyperplasia and Epithelial Ovarian Cancer

“…7,[16][17][18][19][20][21][22][23][24][25] Correct diagnosis of mesothelioma requires the detection of invasion of stroma and/or adipose tissue, but this is difficult in small biopsy specimens and/or effusion cytology. 28 Moreover, no reliable immunohistochemical markers have been established to differentiate diffuse malignant mesothelioma from benign mesothelial proliferations.…”

“…25 Briefly, sections were deparaffinized and rehydrated with descending alcohol dilutions. This was followed by treatment with 2× saline-sodium citrate (2× SSC) containing 0.3% Tween 20 (Sigma, St Louis, MO), washed with 2× SSC, and then treated with pretreatment solution (20× dilution) at 95°C for 10 minutes and digested with pepsin solution at 37°C for 5 minutes.…”

“…[12][13][14] Detection of the homozygous deletion of p16 by fluorescence in situ hybridization (FISH) was shown to be feasible and helpful in confirming a diagnosis of mesothelioma in cytologic and surgical specimens, especially in the differentiation of malignant pleural mesothelioma from RMH. 7,[16][17][18][19][20][21][22][23][24][25] Fewer reports are available for p16 FISH in DMPM. However, some studies have reported that p16 homozygous deletion, detected by FISH, was found in about 25% to 51% of DMPM cases.…”