Molecular analysis of the pleural fluid of patients using fluorescence in situ hybridisation to discriminate those with benign and malignant pleural effusions has not been well studied. We hypothesized that in patients in whom malignant cells may not be detectable in the pleural fluid by conventional cytology, tumour-associated aneuploidy would be present in sufficient quantities to be detected, and that the sensitivity of this approach would improve the overall diagnostic sensitivity of conventional cytology. Our data show that even for multiple tumor types, and with a limited cohort size, we were able to improve the diagnostic performance of cytologic analysis by measuring tumour-associated aneuploidy in the pleural fluid. We employed a molecular technique and observed that conventional cytology and FISH analysis alone have similar sensitivities (63% and 67%, respectively), but that the sensitivity improves to 88% when the results of the FISH and cytology techniques are combined. It is noteworthy that cytology analysis cost $60 for each case vs $625 of cytology analysis combined with FISH. It has been proposed that the use of tissue microarray for FISH might provide satisfactory results with a markedly cost reduction.
Tumor cell detection in effusions can be significantly improved by FISH and PCR techniques applying appropriate molecular markers. PCR method targeted at tumor-specific genetic abnormalities could detect a small number of cancer cells mixed within a large number of normal cells. This method offered considerable promise for early diagnosis of malignant pleural effusions and detection of tumor progression before clinically evident metastasis. However, the clinical application has been limited so far. The primary limitation had been the lack of suitable target genes common to most tumors. The other limitation of RT-PCR is related to laboratory method per se.
FISH analysis for the diagnosis of malignancy is based on the fact that tumor cells are regularly chromosomally aberrant, mostly harboring complex polysomies for one or multiple chromosomes. The methodical approach used here is based on the previous observation that the identification and quantification of aneuploidy by FISH can be used as a sensitive and highly specific marker of malignancy in metastatic cells [11, 14–17]. We selected chromosomes 7, 11, and 17 for this study, which were found to be prognostic markers in malignant tumour [18–21]. We chose to use a combination of probes to these 3 chromosomes as the basis for our FISH test to detect genetically abnormal pleural effusions. Although we had tumors from 11 different organ sites, we designed a broad panel of genes to detect primarily breast and lung tumors since the most common malignancies that metastasize to the pleural fluid are, in order of frequency, lung and breast. Our chromosome panel was successful in detecting breast and lung neoplasms with sensitivities of 100% and 75%, respectively. Moreover, 15 of the 24 tumors that FISH alone failed to detect were not covered by our panel, namely, 1 patient with acute myelocytic leukemia, 1 with renal cell carcinoma, 1 with cervical, 1 with chrondrosarcoma, 2 gastric,2 with ovarian, 2 with skin, 2 with colon and 3 with NSCLC. Indeed, it will be a challenge to be borne out of subsequent studies to determine how large of a panel of chromosomes will be required to identify the variety of neoplasms that metastasize to the pleural cavity.
The survival rate correlated predominant aneusomy for chromosomes 11 has been previously reported in breast cancer and NSCLC. For breast cancer, patients with no exhibiting aneuploidy for chromosome 11 in cancerous effusions had a significantly shorter overall survival rate than did have patients who had aneuploidy . Other studies also found that poor prognosis was associated with aneusomy of chromosome 17 in breast cancer [22, 23]. In the present work, our results were consistent with the above-mentioned studies showing that aneuploidy for chromosome 17 was associated with poor prognosis in patients with malignant pleural effusions. But survival rate was not found to correlate with the predominant aneusomy for chromosomes 7 and 11.
We also observed in the present study that pleural fluids positive by the combined FISH and cytology analysis correlated with pleural invasion. These data suggest that there is a higher yield of malignant cells in the pleural fluid after the pleura is invaded by tumor. This is in agreement with recent studies performed using pleural lavages during pulmonary resections which have shown increased detection of positive pleural lavage cytology when there is parietal pleura invasion by malignancy . Combined cytology and FISH analysis were also more accurate in discerning exudative samples from patients with a primary malignancy. In general, exudative malignant pleural effusions have higher cell counts, lower glucose and pH levels, and are cytologically positive. But these criteria are far from absolute , and in our dataset, cytologic analysis only detected 57% of exudative pleural fluids from patients with a known primary malignancy compared with 64% for FISH alone and 79% for the chromosome and cytology assays combined.
Limitations of the present study include the small cohort size that precludes detailed analysis of specific tumor subtypes. Only descriptive, observational conclusions about the FISH analyses in patients with NSCLC and breast cancer can be made. Second, only FISH technology with 3 probes were used in this study. The patients mainly came from Chinese north areas. Therefore, it is need more study in a large patient group and varieties areas in China in order to figure out specific chromosome anomalies to specific tumors in large population. Finally, the patients with benign disease in this study only had 6 months of longitudinal follow-up to determine if they remained cancer free. That is usually insufficient time to allow preneoplastic events to progress to clinically detectable cancer. Despite our ability to detect tumour-associated aneuploidy in pleural fluid, the sensitivity of this assay would most likely be enhanced by designing separate chromosome panels for tumors originating from specific organs. Chromosome profiles of tumors from different organs do seem to be distinct [26, 27], but more validity studies are necessary before these profiles can be optimized for routine clinical diagnostic use. Improving the diagnostic performance of any test has the potential to alter medical practice, even if, as in this case, it simply means eliminating the need for repeated thoracentesis or other invasive procedures to secure the diagnosis of metastatic disease in a patient with a know primary tumor. Although a more sensitive test to diagnose patients earlier may have limited clinical value in extending survival in patients with metastatic disease, this assay may have important diagnostic implications particularly in patients who have unilateral pleural effusions as their principal presenting clinical sign. It is conceivable that chromosome profiles of the pleural fluid could also be used to identify the site of origin of the metastatic malignant cells in the chest. But this technology requires much validation before it becomes clinically widespread.