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Combination of Cytology, Fluorescence In Situ Hybridization for Aneuploidy, and Reverse-Transcriptase Polymerase Chain Reaction for Human Mammaglobin/Mammaglobin B Expression Improves Diagnosis of Malignant Effusions

From the Departments of Internal Medicine, Pathology, and Obstetrics and Gynecology, Divisions of Hematology and Oncology and General Internal Medicine, and Institute of Medical Biology and Human Genetics, University of Innsbruck, Innsbruck; Department of Pulmonology, Natters Hospital, Natters, Austria

Address reprint requests to Michael Fiegl, MD, Division of Hematology and Oncology, Innsbruck University Hospital, Anichstraße 35, A-6020 Innsbruck, Austria; e-mail: michael.fiegl{at}uibk.ac.at

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION Authors’ Disclosures of... REFERENCES

 
PURPOSE: The identification of malignant cells in effusions by conventional cytology is hampered by its limited sensitivity. The aim of this study was to improve tumor cell detection in effusions by molecular approaches.

MATERIALS AND METHODS: A total of 157 effusions from patients with tumors and 72 effusions from patients without a history or evidence of malignancy were included in this study. All effusion specimens were evaluated in parallel by cytology, fluorescence in situ hybridization (FISH) for aneuploidy, and reverse-transcriptase polymerase chain reaction (RT-PCR) for expression of human mammaglobin (hMAM) and mammaglobin B (hMAM-B).

RESULTS: In effusions from patients with tumors, the sensitivities of tumor cell detection by cytology, FISH, and hMAM and hMAM-B detection were 46.2%, 53.3%, 36.4%, and 57.7%, respectively. The corresponding specificities were 94.4%, 97.0%, 87.1%, and 88.6%. Notably, a high percentage of effusions containing malignant cells were in fact transudates, indicating the necessity for molecular diagnostic work-up of transudates collected from patients with tumors. Dependent on the tumor type, the use of appropriate marker combinations improved tumor cell detection in effusions significantly. By combining all four diagnostic tests, a positive test result indicating the presence of malignancy was achieved in 81.1%, with a fairly good specificity of 70.1%.

CONCLUSION: Molecular techniques are definitely useful to detect malignancy in cytologically negative effusions. Tumor cell detection in effusions can be significantly improved by FISH and PCR techniques applying appropriate molecular markers. This finding should help to improve tumor staging, prognostic assessment, and treatment monitoring.

	INTRODUCTION

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In the majority of patients with malignant disease, the occurrence of effusions in the pleural, pericardial, or peritoneal cavity indicates metastatic disease. However, in patients with tumors, causes other than metastasis may also elicit effusions, such as lymph drainage congestion, hypoproteinemia, portal hypertension owing to liver metastasis, or yet poorly defined paraneoplasia [1]. Furthermore, comorbidity such as pulmonary embolism, congestive heart failure, infection, or hepatopathy needs to be considered when effusions arise in patients with a history or evidence of cancer. Thus sensitive and reliable detection of tumor cells in effusions is desirable, as the documentation of their presence or absence often determines disease stage and prognosis.

The detection of malignancy in effusions by conventional cytology is hampered by the problem of differentiating malignant cells from reactive mesothelial cells [2]. Thus new methods complementing cytology for the diagnostic work-up of effusions need to be evaluated. We and others used interphase cytogenetics by fluorescence in situ hybridization (FISH) to detect aneuploidy in effusions [3-5]. FISH testing of tumor-associated aneusomies proved to be a sensitive and specific diagnostic approach to identify malignancy in effusions, liquor, urine, and aspirated cytologic material [6-8]. Furthermore, in our hands, reverse-transcriptase polymerase chain reaction (RT-PCR) for human mammaglobin (hMAM) gene expression turned out to be a highly sensitive assay to detect occult tumor cells in effusions from patients with gynecologic malignancies [9]. To date, only a few studies have addressed the issue of tumor cell detection in effusions using high-performance techniques like RT-PCR [10-16]. hMAM belongs to the uteroglobin family, and its function is still unknown. hMAM was thought to be solely expressed in normal and malignant mammary tissue [17]; recently, however, hMAM-specific mRNA was detected in a high percentage of normal and malignant epithelial tissues from the gynecologic tract [9]. Human mammaglobin B (hMAM-B), another protein of the uteroglobin family, was found to be normally expressed in benign and malignant breast and gastrointestinal epithelium and to indicate malignancy when expressed ectopically [18,19].

In this study, we aimed to compare the sensitivity and specificity of different diagnostic methods and their combined use to detect tumor cells in a large series of effusions from patients with malignant or nonmalignant disease. Indeed, we found that cytology, FISH analysis for aneuploidy, and RT-PCR for hMAM and hMAM-B expression can be efficiently combined to diagnose malignant effusions.

	MATERIALS AND METHODS

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Effusion sampling and investigational analyses had been approved by the institutional ethics committee. A total of 229 effusion samples were prospectively collected from 199 patients with various malignant (Table 1) and nonmalignant diseases. Seventy-two effusions taken from 67 patients with nonmalignant diseases served as controls (22 of ascitic and 50 of pleural origin). One hundred fifty-seven effusions (including 98 pleural and 52 ascitic, two cyst fluids, and five peritoneal lavage specimens) were derived from 132 patients with a former or current histologically confirmed malignant disease. Effusion samples taken by repeated punctures were reanalyzed when two subsequent punctures were performed at least 1 month apart. In this case, a significant alteration of the cellular composition could be expected. All specimens were subjected to routine cytology (hematoxylin/eosin or Papanicolau staining) and molecular analyses as outlined below. Routine cytologic examination was performed by an experienced cytopathologist and by one of the authors (J.K. or E.M.) independently. Effusion specimens were classified as being benign/reactive or malignant using generally accepted criteria. Malignancy as defined by cytology was also diagnosed when only a few suspicious single cells or cell clusters were present.

Analysis by Fluorescence Microscopy
All effusion cells in a field except for polynucleated granulocytes, which are easily distinguishable by nuclear shape, were evaluated (Fig 1). Stringent criteria of FISH signal assessment were applied to avoid overestimation of hyperdiploidy, which may result from cellular and technical factors [21]. FISH evaluation was performed by either one of two investigators (M.F., A.M.) in a blinded fashion. Signal counting on 100 to 500 cells was performed in the single-color FISH evaluation mode when the percentage of aneusomic effusion cells was greater than 5% for hyperdiploidy and greater than 15% for monosomy. The cut-offs used in this study for true aneuploidy are higher than the cut-offs for chromosomes 11 and 17 necessary to unequivocally distinguish normal/reactive cells from truly malignant cells [3,22]. In effusion specimens with an apparent aneuploidy rate of less than 5% for hyperdisomic cells, an alternative mode of quantification of aneuploid cells was used, as detailed in our previous study [22]. Briefly, a rapid visual screening of 10,000 to 20,000 cells was performed in the dual-color FISH evaluation mode, and only cells with hyperdisomy for one or both chromosomes observed were scored. Dual-color FISH allowed for detection of rare aneuploid cells indicating low-level content of malignant cells. To achieve a specificity of greater than 95%, the presence of rare aneuploid cells evidencing malignancy was diagnosed if at least 20 cells with hyperdisomy were detected and if at least 60% of these cells showed intranuclear complexity (ie, 4/2-signal; 2/3-signal, 5/3-signal cells, and so on). In summary, a FISH result was considered positive for malignancy if aneuploid cells were present above a cutoff level of 5% (hyperdisomy) or 15% (monosomy) in the single-color FISH evaluation mode or if rare aneuploid cells were detected in the dual-color FISH evaluation mode.

View larger version (51K): [in this window] [in a new window]   Fig 1. Cytologic and fluorescence in situ hybridization (FISH) evaulation in effusion cells from a patient with metastatic non-small-cell lung cancer. (A) Cytologically malignant cells showing multinucleated cells with irregular nuclei and a mitotic figure (arrow); (B) complex aneuploidy with pronounced heterogeneity of FISH signals, with a gain of chromosome 17 copy numbers (orange signals); chromosome 11 (green) was predominantly disomic in this case, with a few cells showing trisomy (arrow) and tetrasomy. Several granulocytes represent a reactive background without FISH abnormalities.

PCR and Gel Electrophoresis
Specific cDNA sequences were amplified in a reaction mix composed of 2 µL of cDNA, 5 µL of 10x PCR buffer (100 mmol/L of Tris HCl pH 8.3, 500 mmol/L of KCl, 15 mmol/L of MgCl₂, and 0.1% gelatin), 50 µmol/L of dNTPs, 400 nmol/L of each specific sense and antisense primer, and 1.5 units of AmpliTaq DNA polymerase (Perkin Elmer, Vienna, Austria) in a total volume of 50 µL. The second round of PCR using nested primers and 1 µL of outers product was performed identically, except that the dNTP concentration was increased to 100 µmol/L.

For paraffin-embedded tissues and effusion cells, other primer sets were used because of partial degradation of these RNAs. All primers were synthesized at the MWG-Biotech Laboratory (Ebersberg, Germany). The primer sequences and the specific annealing temperatures are listed in Table 2. The cycling conditions for fresh samples were 40 cycles (30 seconds at 95°C, 60 seconds annealing at specific temperature, and 60 seconds at 72°C) and for paraffin-embedded samples were 40 cycles (30 seconds at 95°C, 30 seconds annealing at specific temperature, and 30 seconds at 72°C). As a first step of the PCR, reaction samples were heated up to 95°C for 3 minutes. At the end of all PCR runs, a last extension was done at 72°C for 5 minutes. The RT-PCR products were analyzed in 2% agarose gels stained with ethidium bromide. A 100-bp DNA ladder (Life Technologies, Vienna, Austria) was used as a size marker. The presence of intact RNA was confirmed by a single-round RT-PCR using the house-keeping gene glyceraldehye-3-phosphate dehydrogenase (GAPDH; Fig 2).

View larger version (72K): [in this window] [in a new window]   Fig 2. Nested reverse-transcriptase polymerase chain reaction for human mammoglobin (hMAM), mammaglobin B (hMAM-B), and glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Ethidium bromide stained agarose gel: 100 bp molecular marker (lane M); effusions from lung cancer (lane 1); ovarian cancer (lanes 2, 3); breast cancer (lanes 4-6); MD-MBA 361 (lane 7); healthy blood donor (lane 8); effusion from breast cancer (lane 9).

	RESULTS

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Comparison of Cytology, FISH^aneusomy, RT-PCR^hMAM, and RT-PCR^hMAM-B in Effusions From Patients With Malignant and Nonmalignant Disease
The characteristics of effusions from cancer patients in terms of origin (pleural or ascitic) and underlying malignancy as well as the results obtained from the four diagnostic tests applied are summarized in Table 1. The diagnostic sensitivities indicating malignancy (ie, the rate of positive results for each single marker in the whole series of effusion fluids collected from cancer patients, including five peritoneal lavages and two cyst fluids) are listed in Table 4. In summary, cytologic evaluation was positive for malignant cells in 46.2% of the effusions from cancer patients. FISH analysis yielded a positive result for malignancy in 53.3% of effusions, whereas hMAM and hMAM-B expression was found in 36.4% and 57.7% of effusions, respectively. A significantly higher sensitivity than that of cytology was achieved by use of RT-PCR for molecular markers in certain diagnostic subgroups (Table 4).

Clinically useful and perhaps more cost-effective test combinations for the respective tumor entity are highlighted in Table 4. For effusions from patients with gynecologic carcinomas, the combination of cytology and RT-PCR^hMAM yielded a positive result in 85.7% (P < .001 compared with cytology). For effusions from gastrointestinal carcinomas, the addition of RT-PCR^hMAM-B to cytology enhanced diagnostic sensitivity from 25.8% to 51.7% (P < .016). For effusions from lung cancer, the combination of cytology and FISH or cytology and RT-PCR^hMAM-B enhanced the sensitivity from 59.5% to 81.1% and 83.3%, respectively (P < .008 for both comparisons with cytologic evaluation only). Of note, for effusions from hematologic malignancies, the use of the molecular markers in addition to cytology did not enhance diagnostic sensitivity.

Malignancy in Effusions Classified as Exudates and Transudates
Finally, we addressed the question of whether the various diagnostic tests (and combinations thereof) would be suitable to detect malignancy in transudates from cancer patients. The total protein content was determined in all test samples and a transudate was defined by a protein concentration of less than 25 g/L [26]. Overall, 29.6% (45 of 152) of the effusions in cancer patients were transudates. Remarkably, in a significant proportion of these transudates, positive results for malignancy were obtained by cytology, FISH, RT-PCR^hMAM, RT-PCR^hMAM-B, and test combinations (Table 7).

	DISCUSSION

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To date, cytology remains the diagnostic standard for examining effusion samples from patients with a former or current malignancy, irrespective of whether the effusion is a transudate or exudate. This rule is corroborated by our findings in transudates from patients with tumors in which malignancy was detected by cytology and molecular markers in a significantly higher proportion than previously reported [27,28]. In the literature it is widely accepted that cytologic evaluation of transudates is not usually necessary. Recently, however, this topic has been intensively debated [29,30]. Our data strengthen the view that tumor cells are frequently occurring in effusions from cancer patients, including transudates.

Does the analysis of molecular markers reliably contribute to the diagnosis of malignancy in effusions? In this study, the specificities for the evaluation of molecular markers were determined in a large series of control effusions from patients with nonmalignant disease and range between 70% and 80%. This is an acceptable range of specificity considering the rather low sensitivities at acceptable specificities of biochemical assays used in effusions (eg, in pleural effusions, for lactate dehydrogenase [31], sensitivity 81%, specificity 85%; for carcinoembryonic antigen [32], sensitivity 48%, specificity 78%). FISH analysis is a microscopic evaluation method directly complementing cytology by which tumor-associated aneusomies are visualized. Thus positive FISH testing for aneusomy provides direct evidence for the presence of tumor cells with a higher specificity, ie, 90.9% in combination with cytology (Table 4). We and others established a definite role of FISH analysis in the evaluation of effusion fluids and CSF where the diagnostic yield of cytology is often unsatisfying as a result of difficulties in distinguishing tumor cells from a reactive cellular background [3-6]. 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 [22,33,34]. In breast cancer, we could demonstrate by FISH that primary tumors as well as corresponding axillary and distant metastases exhibited nearly identical aberration patterns [3,22]. Similarly, it has been unequivocally proven by FISH that circulating tumor cells in blood are true neoplastic (sub)clones derived from the respective primary tumors [34]. Thus tumor cell detection with enhanced specificity can be achieved by directed use of appropriate FISH probes, selected according to the FISH karyotype as determined in the initial work-up of the primary tumor. However, because FISH analysis is rather laborious, it would be most applicable as a confirmatory assay when a PCR-based approach proves positive and relevant clinical consequences arise from the test result.

Because of the lack of PCR-suited markers specifically differentiating tumor cells from normal background cells, there are only a few studies addressing the role of PCR-based tumor cell detection in effusions. Identification of K-ras oncogene and p53 mutations (by restriction enzyme mismatch PCR) was used to complement cytologic diagnosis in effusions from pancreatic cancer [12,13]. RT-PCR detection of tumor-associated mRNAs coding for epithelial glycoprotein 2, CD44 variants, certain mucins, and matrix metalloproteinases was attempted in smaller series of potentially malignant effusions [10,11,14,16]. Other PCR-based approaches to complement cytologic diagnosis in effusions include the detection of telomerase activity [15] and of single nucleotide polymorphisms for several markers featuring malignant cells [35]. Recently, we have shown that RT-PCR-based identification of hMAM expression can be helpful in diagnosing malignancy in effusions from gynecologic cancers [9]. The gene encoding hMAM belongs to the uteroglobin gene family. The function of hMAM protein is as yet poorly defined. Initially, hMAM expression was considered to be tightly restricted to mammary tissue, but we could demonstrate its frequent expression in epithelial tissues of female genital tract origin. hMAM-B, a related protein with a presumably exocrine function, is known to be preferentially expressed in mammary, gastrointestinal, and salivary gland tissue [18]. By RT-PCR, mRNA for hMAM or hMAM-B was detectable in histologically negative regional lymph nodes of breast, esophageal, gastric, colon, and biliary carcinoma [19,36-39], indicating micrometastatic disease. Based on these findings, both mammaglobin markers were chosen for our study.

As a first step, the specificities of the four diagnostic markers (ie, cytology, FISH for aneusomy, and RT-PCR for hMAM and hMAM-B expression) were determined in control effusions from patients with nonmalignant diseases. In four of 72 control effusions, single cells or cell clusters were found by cytology to be highly suspicious for malignancy. In none of these patients, cancer was presently or had been previously diagnosed. The resulting specificity of 94.4% for cytology is quite comparable with data from other studies [40,41]. These four pleural effusions classified as false-positive came from patients with chronic disease (tuberculosis, pulmonary embolism, lung transplantation, or right heart insufficiency), and further follow-up did not confirm the diagnosis of cancer. This finding documents the difficulties in using current cytologic criteria [42] for the examination of effusions, a fact of which cytopathologists are well aware [40,43]. FISH analysis was highly specific (97%, Table 5) because of stringent cutoff thresholds chosen for evidencing aneusomy. RT-PCR-based detection of hMAM and hMAM-B expression was noted in 12.9% and 11.4% of control effusions, respectively, resulting in a specificity of 87.1% and 88.6%. These false-positive RT-PCR test results are most likely due to ectopic expression of hMAM and hMAM-B genes by nonmalignant cells, for example, when endometriosis or activated inflammatory cells are shed into effusions [44,45].

As shown, the combination of diagnostic methods can definitely enhance sensitivity, but at the expense of reduced specificity. Combining all four diagnostic markers increased the sensitivity by approximately 30% to 40% (Table 4), but specificity decreased from maximal 97% (FISH) to 70.1%. From our experience, a tailored application of a set of marker pairs can be recommended for achieving high sensitivity and reasonably good specificity. For example, to test effusions of unknown origin or effusions from patients with documented gastrointestinal or lung cancer, a highly efficient combination would be cytology/RT-PCR^hMAM-B. For testing effusions from patients with gynecologic cancers, the most sensitive combination would be cytology/RT-PCR^hMAM. A test set with enhanced specificity proved to be the combination cytology/FISH for aneusomy, with a specificity of 90.9%. This test combination can be recommended for effusions from patients with carcinomas of the lung and gastrointestinal or female genital tract.

The use of molecular markers can be crucial for the proper evaluation of effusions and the staging of malignancies. In the majority of patients with tumors, the development of effusions is indicative for locally advanced disease or distant metastasis. The detection of malignancy in effusion samples will frequently lead to a disease upstaging and affect prognosis and treatment planning. In lung cancer, for instance, positive cytology discriminates limited (eg, T1) from locally advanced stages (eg, T4), with profound therapeutic consequences. Furthermore, cytologic detection of tumor cells in intraoperative lavages before the resection of cancerous lung was shown to be of a definite prognostic significance [46]. However, no analytic test except cytology has yet been accepted for the classical International Union Against Cancer /American Joint Committee on Cancer tumor-node-metastasis system [47]. Studies like ours create a database to adapt currently accepted diagnostic rules over time. However, large prospective studies will be required to demonstrate whether molecular upstaging (eg, in cytologically negative effusions) correlates with dismal prognosis, and the therapeutic management should be adapted accordingly.

	Authors’ Disclosures of Potential Conflicts of Interest

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The authors indicated no potential conflicts of interest.

	NOTES

 
Supported in part by the "Tiroler Verein zur Förderung der Krebsforschung," by the "Verein zur Förderung von Forschung und Fortbildung in molekularer Genetik und Diagnostik internistischer Erkrankungen," and by the "Hans und Blanca Moser-Stiftung."

Authors’ disclosures of potential conflicts of interest are found at the end of this article.

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Submitted June 16, 2003; accepted November 24, 2003.

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