Adaptation of a commercial fluorescent in situ hybridization test to the diagnosis of malignant cells in effusions
Introduction
An effusion is often the first material available for the diagnosis of malignant involvement of serous cavities. Cytological examination enables a definite diagnosis of malignancy in many although not all cases. In fact, reported sensitivities for detecting a malignant condition by effusion cytology are often around 60% [1].
The limitations with effusion cytology mainly relate to the representativity of the material and cellular preservation. Regressive changes may hamper an accurate diagnosis in some cases. Apart from possibly malignant cells, effusions normally contain macrophages and exfoliated mesothelial cells. If there is a growth stimulus, as is the case in inflammatory conditions, mesothelial cells may react by increasing their proliferation rate [2], [3]. A third diagnostic problem comes from such reactive changes in benign mesothelial cells that may imitate malignancy, malignant mesothelioma (MM) being the most difficult differential diagnosis.
Once malignancy has been established, a further challenge is to distinguish metastatic tumors from the primary MM. The experienced cytologist can in many cases reach this diagnosis without false positive errors, providing that today's ancillary techniques are adequately used. At the Karolinska University Hospital the diagnosis of a MM is routinely based on electron microscopy, analysis of hyaluronan contents and/or extensive immunocytochemistry.
This diagnostic accuracy of effusion cytology requires a frequent use of adjuvant techniques. Immunocytochemistry is well established to determine tumor type. In some cases, however, the difficulties in sorting out reactive changes remain. Fluorescence in situ hybridization (FISH) of interphase cells has more recently been available for use in diagnostic routine. This analysis is an accurate method to monitor the genetic status of cells, detecting aneuploid signals and gene deletions. Of particular interest for effusion cytology is the possibility to detect deletions of the 9p21 locus, containing the p16INK4A gene. Homozygous deletions of the tumor suppressor gene p16INK4A can be found in most MMs [4], suggesting that the test also might be used to identify a MM.
The UroVysion FISH-analysis kit was originally set to detect genetic changes in bladder cells from patients with urothelial cell malignancy. The kit contains four directly labelled probes that hybridize to the centromere region on chromosomes 3, 7 and 17 and to the p16 INK4A gene locus at 9p21.
The present study was performed (i) to adapt the UroVysion™ Bladder Cancer kit for the analysis of pleural effusions, (ii) to study its diagnostic utility in discerning malignant cells and (iii) to see to what extent the test can be used to distinguish a MM from metastatic cancers.
Section snippets
Samples
A total of 70 pleural effusions were recruited from the Department of Clinical Pathology and Cytology, Karolinska University Hospital in Huddinge, Sweden. Two cases were excluded because of too poor preservation of the cellular content. The remaining 68 cases, collected between January 2006 and January 2009, included 29 samples primarily diagnosed as malignant, 24 samples with inconclusive cytology and 15 samples with only benign cells detected.
In 29 cases immunocytochemistry or subsequent
Results
Since cell groups hamper the FISH analysis, trypsin digestion was tested to dissolve such groups (Fig. 1). In some cases the procedure resulted in a poorer nuclear morphology and DNA-splitter, although a sufficient number of preserved cells were present in most cases. Effusions with a high content of degenerated cells showed dispersed and less distinct hybridization signals (Fig. 2), and only 2 out of 70 samples had to be excluded from the study for this reason. To avoid DNA deterioration the
Discussion
The commercially available FISH-analysis UroVysion™-test was primed for the diagnosis of urothelial cells but can be adapted to the analysis of cells in effusions. We were successful when the material was washed and fixed in the methanol-based fixatives. These fluids permeabilize the cell membrane, increasing accessibility to the DNA. Trypsin digestion proved to be crucial in the preparation of samples with cell groups, in which interpretation otherwise would be difficult. The digestion,
Conflicts of interest
None declared.
Acknowledgement
This study was supported by the Swedish Heart- and Lung Foundation and The AFA Funds.
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