An improved flow cytometric assay for detection and discrimination

Cytometry Part B (Clinical Cytometry) 80B:324–334 (2011)

Original Article

An Improved Flow Cytometric Assay for Detection and Discrimination Between Malignant Cells and Atypical Mesothelial Cells, in Serous Cavity Effusions Nektaria A. Kentrou,1 Nikolaos J. Tsagarakis,1* Konstantina Tzanetou,2 Maria Damala,2 Konstantinos A. Papadimitriou,1 Dimitra Skoumi,1 Aimilia Stratigaki,3 Nikolaos I. Anagnostopoulos,4 Eleni Malamou-Lada,2 Pauline Athanassiadou,5 and George Paterakis1 1

Department of Immunology, Flow Cytometry Laboratory, Athens Regional General Hospital ‘‘G. Gennimatas,’’ 11527 Athens, Greece 2 Department of Microbiology, Athens Regional General Hospital ‘‘G. Gennimatas,’’ 11527 Athens, Greece 3 Cytology Laboratory, Athens Regional General Hospital ‘‘G. Gennimatas,’’ 11527 Athens, Greece 4 Department of Clinical Hematology, Athens Regional General Hospital ‘‘G. Gennimatas,’’ 11527 Athens, Greece 5 Cytology Department, Pathology Laboratory, University of Athens Medical School, Athens, Greece

Background: The aim of this study was to evaluate a flow cytometric assay for the detection of malignant effusions. Methods: During the last 4-year period, 125 effusions suspicious for malignancy were prospectively analyzed by flow cytometry and conventional cytology. A three-step flow cytometric assay was performed, beginning with an initial informative panel of two protocols, containing SYTO-16, 7-AAD, CD71PE, CD45-ECD, and CD66abce-FITC, CD64-PE, CD45-ECD, CD16-PECy5, CD14-PECy7, respectively. This was followed by a basic immunophenotypic panel of seven three-color combinations, containing in the first position, EMA, Ber-EP4, CD66abce, CD56, and intracellular desmin-33, combined with CD71-PE and CD45-PeCy5 in each tube. Finally, a cytokeratin-FITC/propidium iodide DNA panel was conducted, for the detection of aneuploidy in cytokeratin positive cells. Results: The sensitivity and specificity of flow cytometry were 85.1 and 97.8%, and of cytology 93.2 and 95.6%, respectively. A significant association was observed between the results of the two techniques (P < 0.001). Among eight atypical cases detected by cytology, five had been precisely characterized as malignant by flow cytometry. EMA and Ber-EP4 proved the most sensitive markers for malignancy diagnosis, while the detection of desmin-33 negative/cytokeratin positive cells had the simultaneous highest positive and negative predictive values. CD66abce was very specific, although nonsensitive, while DNA ploidy analysis was nonspecific, as hyperploidy was observed in reactive mesothelial cells. Conclusions: A flow cytometric assay of high sensitivity and specificity is proposed for the routine identification of carcinoma cells in effusions and their distinction from atypical mesothelial cells, as an ancillary to conventional cytology. VC 2011 International Clinical Cytometry Society Key terms: flow cytometry; malignant effusions; atypical mesothelial cells

How to cite this article: Kentrou NA, Tsagarakis NJ, Tzanetou K, Damala M, Papadimitriou KA, Skoumi D, Stratigaki A, Anagnostopoulos NI, Malamou-Lada E, Athanassiadou P, Paterakis G. An improved flow cytometric assay for detection and discrimination between malignant cells and atypical mesothelial cells, in serous cavity effusions. Cytometry Part B 2011; 80B: 324–334. *Correspondence to: Nikolaos J. Tsagarakis, Department of Immunology, Flow Cytometry Laboratory, Athens Regional General Hospital ‘‘G. Gennimatas’’, 154 Messogion Avenue, Athens, 11527, Greece. E-mail: [email protected].

C 2011 International Clinical Cytometry Society V

Received 19 February 2011; Revision 13 May 2011; Accepted 19 May 2011 Published online 25 May 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/cyto.b.20608

FLOW CYTOMETRIC DETECTION OF MALIGNANT CELLS IN EFFUSIONS

Although the main current application of flow cytometry (FCM) is in the diagnosis and prognosis of hematolymphoid neoplasia, it has also been extensively used for cell cycle and DNA ploidy investigation of malignant effusions. DNA aneuploidy and the increased percentage of cells in the S phase of the cell cycle (proliferation phase) were the most frequent observations in studies of malignant tumors (1–4). A wide variety of reports published on the determination of the sensitivity and specificity of FCM technology in the detection of malignancy, based on DNA ploidy investigation (5–7), document sensitivity of 55–100% and specificity 86–100%. The combination of detection of aneuploidy with immunophenotyping appears to be needed for definitive diagnosis (8,9). The positive and negative predictive values of conventional cytology (CC) for the detection of malignancy have been calculated to be 89.3 and 64.9%, respectively (10). However, a ‘‘gray zone’’ sometimes occurs, where CC cannot distinguish between reactive, atypical cells, and malignant cells (11). The purpose of this study was to evaluate the efficacy of FCM for the rapid identification of malignant effusions in routine clinical practice. The FCM assay comprised a three-step procedure: an initial informative antibody panel, a second panel for extended immunophenotyping of the suspicious cell population, and a DNA ploidy analysis to define the aneuploidy of malignant cells. PATIENTS, MATERIALS, AND METHODS Patients The study was conducted on effusions derived from 125 patients hospitalized in the Departments of Internal Medicine, Cardiology and Surgery, of whom 60 were males and 65 females, with a median age of 65 years (range, 15–91 years). Of the 125 patients, 74 had been previously, or were later, diagnosed with malignancy, based on histological diagnosis using hematoxylin and eosin (H and E) staining or on clinical follow-up, and specifically, 18 tumors of the gastrointestinal tract, 8 cases of breast cancer, 9 cases of lung cancer, 8 pancreatic tumors, 8 ovarian tumors, 6 cases of hepatocellular carcinoma, 8 cases of metastatic adenocarcinoma of unknown origin, 2 tumors of the uterus, 2 cases of melanoma, 2 of sarcoma, and 3 cases of disseminated tumor disease. Materials Pleural, peritoneal, and pericardial effusions were included in the study. Every effusion, which was morphologically considered suspicious for malignancy, or that was derived from a patient with known malignancy, was further processed by FCM and CC, and in total, 59 pleural, 58 peritoneal, and 8 pericardial samples of fluid were analyzed. Effusions derived from hematopoietic neoplasias were retrospectively excluded. The ‘‘gold standard’’ used to define a sample, as being infiltrated by malignant cells was a positive finding on at least two of the following examinations, in any combination: 1) CC, 2) Biopsy, and 3) imaging (computed tomography, ultrasound).

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Methods Giemsa morphology. Giemsa stained centrifuged slide preparations were studied. The morphological features taken into account for the characterization of an effusion as ‘‘suspicious for malignancy’’ were: cells in aggregation or clusters, large discrete isolated malignant cells, large cytoplasmic vacuoles, signet-ring cells, basophilic or eosinophilic cytoplasm, presence of mitoses, central or eccentric large nucleus, prominent nucleolus, and presence of nucleoli (1). CC. Centrifuged slide preparation was performed on each specimen, followed by Papanicolaou staining. Effusions were classified as: 1) malignant, 2) benign/reactive, and 3) atypical/suspicious for malignancy, according to previously described criteria (1). FCM. An initial informative panel, a basic immunophenotype panel and a DNA-ploidy analysis were performed for every sample. The informative panel consisted of two protocols with 4- and 5-color combinations, respectively. In the first protocol, CD71-PE, CD45ECD, and two fluorescent nucleic acid stains, SYTO-16 and 7-AAD, were used. SYTO-16 (Invitrogen, Eugene, OR) was provided as stock solution of high density, stored at 20 C. A solution of 1 mM concentration was prepared, after the dilution of 5 lL stock solution in 1000 lL PBS. For further analysis, 5 lL of the latter solution (SYTO-16, 1 mM) was used at the first week of storage, while 10 lL/sample were used for later analysis. The second protocol used CD66abce-FITC, CD64-PE, CD45-ECD, CD16-PE-cy5, and CD14-PE-Cy7. The basic immunophenotype panel consisted of two parts, one surface direct staining and the other intracellular indirect staining, combined with surface direct staining. Specifically, in the first part, 3-color FCM surface direct staining was performed, by the following FITC antibody conjugates: IgG1(isotype control), EMA, Ber-EP4, CD66abce, and CD56, which were combined in every tube with a common backbone of the two conjugates, CD71-PE and CD45-PE-Cy5. Indirect fluorescence was performed intracellularly (see below) using FITC-polyclonal rabbit anti-mouse immunoglobulin with two other unconjugated antibodies, IgG1 (pure isotype control), and desmin-33, combined in each tube with the common backbone conjugates CD71-PE and CD45PE-Cy5 (Table 1). For the detection of surface antigens, after 15-minute incubation at room temperature of fresh cell suspensions (100 lL) with 10 lL labeled antibodies, the cells were lyzed (BD FACS Lysing Solution, BD Biosciences, CA) for 10 minutes and centrifuged (1,800 rpm, for 5 minutes). The supernatant fluid was discarded and the cells were resuspended in 0.5 mL of PBS for analysis. For the detection of desmin-33 intracellular expression, the following procedure was conducted. After the first incubation period with conjugated CD71-PE and CD45PeCy5, 100 lL Fix & Perm reagent ‘‘A’’ (Fix & Perm reagent kit, Caltag Laboratories, San Francisco, CA) was added for a second 15-minute incubation. After being

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Table 1 Antibodies and Stains Used for Flow Cytometric Analysis Antibody

Description

Source

Clone

Fluorochrome

EMA

Monoclonal Mouse Anti-Human Epithelial Membrane Antigen Monoclonal Mouse Anti-Human Epithelial Membrane Antigen Monoclonal Mouse Anti-Human Myeloid Cell CD66abce Monoclonal Mouse Anti-Human neural cell adhesion molecule Monoclonal Mouse Anti-Human Desmin Negative Control Mouse Rabbit F(ab0 )2

Dako, Denmark

E29

FITC

Dako, Denmark

Ber-EP4

FITC

Dako, Denmark

kat4c

FITC

BD, NJ, USA

NCAM16.2

FITC

Dako, Denmark

D33

Indirect staining with secondary antibody

Ber-EP4 CD66 CD56 (N-CAM) Desmin IgG1(pure) Polyclonal Rabbit Anti-Mouse Immunoglobulins IgG Cytokeratin IO Test Human CD71 CD45-ECD IO Test CD45-PC5 IO Test SYTO-16 7-aminoactinomycin D (7-AAD)

Negative Control Mouse IgG1 Monoclonal Mouse Anti-Human IgG1 Monoclonal Mouse Anti-Human Transferrin Receptor Antigen Monoclonal Mouse Anti-Human Leucocyte Common Antigen Monoclonal Mouse Anti-Human Leucocyte Common Antigen Green fluorescent Nucleic acid stain Viability Dye

washed once, the cells were permeabilized by the addition of 100 lL Fix & Perm reagent ‘‘B’’ and the unconjugated antibodies were added; 5 lL of IgG1 (pure isotype control) and 5 lL desmin-33, respectively, followed by a 15-minute incubation period, one wash-step and a 10-minute incubation period with 10 lL polyclonal rabbit IgG1-FITC (diluted, 1/10 with PBS) for indirect immunofluorescence. After one further wash the cells were resuspended in PBS for analysis. DNA index (DI) was evaluated with Coulter DNA Prep Reagent kit, with propidium iodide and RNAse. Two tubes of the sample were prepared according to instructions, while 10 lL cytokeratin-FITC and IgG1-FITC (control tube) were added, respectively (30 minutes incubation). FCM was performed on a 5-color flow cytometer, FC-500 (Beckman Coulter), where at least 60,000 events were counted for each sample. Analysis was made with CXP software (Beckman Coulter). FLOW CYTOMETRIC GATING STRATEGY Initial Panel The aim of the initial informative panel was the investigation of CD45 negative/CD71 positive cells, suspected

Dako, Denmark Dako, Denmark

Secondary antibody FITC FITC

Dako, Denmark Immunotech, Beckman Coulter, Marseille, France Invitrogen, Camarillo, USA

J1B3

FITC

T56/14

R-PE

Immunotech, Beckman Coulter, Marseille, France Immunotech, Beckman Coulter, Marseille, France Invitrogen, Eugene, Oregon, USA Immunotech, Beckman Coulter, Marseille, France

J.33

ECD

J.33

PE-Cy5

to be malignant, or mesothelial cells. In addition, an extended differential leukocyte count of the effusion cells was conducted. Nuclear dyes SYTO-16 (Molecular Probes) and 7-AAD were combined with CD45-ECD and CD71-PE to identify CD45, SYTO-16þ, and 7-AAD cells, excluding nonspecific interference from unlyzed erythrocytes, platelet aggregates, debris and dead, or apoptotic cells (SYTO-16, 7-AADþ). After the exclusion of the above elements, suspicious cells were gated, related to the scatter characteristics (usually high forward and side-scatter signals). This strategy ensured avoidance of false positive CD45 events that could have been considered malignant, or mesothelial cells (Fig. 1). CD71 was used as a marker to localize a suspicious CD45 negative cell population, which could be of malignant or other origin. Although it is not a common tumor marker, it is upregulated in proliferating cells (such as tumor cells) (12– 14). It is also expressed by normal mesothelial cells (15), so it could operate as a linkage marker for their differential diagnosis. The detection of CD71þ cluster (cluster positivity and not intensity positivity) of high side- and forward scatter-characteristics was used as evidence of need for further analysis with the basic panel.


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FIG. 1. Flow cytometry on first sample case: SYTO-16þ/7-AAD negative cells were gated (Gate A and C) in order to exclude apoptotic and necrotic elements (upper panels). The remaining viable cells were examined in SS/CD45 diagram for the detection of CD45-cluster. This cluster was easily differentiated from white blood cells, while debris (cell fractions and membrane fragments) was excluded (gating the high side-scatter cluster). The remaining viable cells were examined in CD71/CD45 diagram for the detection of CD71þ/CD45-cluster (even with dim CD71 positivity). This atypical cluster was finally backgated in FS/CD45 and SS/CD45 diagram (to confirm large dimensions, due to cell clustering or isolated large malignant cells), not shown. The detection of CD71þ cluster of high side- and forward scatter-characteristics was used as evidence for further analysis with the basic panel. (Upper and middle panels are from the same sample/case, while lower panels are from a different sample/case, FS: forward scatter, SS: side scatter). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

The use of CD66abce, CD64, CD16, and CD14 in the second tube was mainly used to distinguish neutrophils (CD66abceþ and CD16þ), eosinophils (CD66abceþ and CD16), lymphocytes (CD45brightþ and side scatter


low), and macrophages/monocytes (CD14þ and CD64þ); macrophages are CD14þ, CD64þ, and CD16þ (16,17). The side-scatter characteristics and CD45 expression of the cells were also helpful. It is significant

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FIG. 2. Flow cytometry on second sample case: CD45 negative cells of relevant high FS were examined for CD71 expression. The isotype control IgG1-FITC was always used to define the cut-off intensity, so to further evaluate the expression of EMA, BerEp4, and CD66 expression on malignant or mesothelial cells (CD45/CD71þ/high FS). The positivity threshold was defined at 20% expression. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

that a high CD64 expression on neutrophils was supportive of an infectious etiology (18), while an increased number of eosinophils was indicative of a possible underlying malignancy (19). If a hematological malignancy was suspected in the initial panel (usually CD45 positive), we did not proceed to the basic panel, but to T-cell and B-cell immunophenotype and clonality assessment, with different panels. Basic Immunophenotypic Panel The aim of the basic panel was the immunophenotyping of the suspicious population of CD45/CD71þ cells, for the expression of EMA, Ber-EP4, CD66abce, CD56, and desmin-33. The expression of EMA, Ber-EP4 and CD66abce was considered positive when >20% (ensuring bright expression and avoiding false positive results), compared with the isotype control (adjusted to be 20%) 59 1 0 60/73 (82.2%) Ber-EP4 þ (>20%) 58 0 0 58/72 (80.6%) CD66abce þ (>20%) 38 0 1 39/68 (57.3%) CD56 þ (>20%) 1 2 2 5/74 (6.7%) Desmin-33 65 1 1 67/74 (9.5%) DNA index 1.4 45 0 0 45/68 (66.2%) Informative immunophenotypes for adenocarcinomas Desmin-33/cytokeratinþ 62/73 (84.9%) EMAþ/Ber-EP4þ 55/71 (77.5%) Desmin-33/EMAþ/BerEP4þ 54/71 (76.1%) EMAþ/Ber-EP4þ/desmin-33/cytokeratinþ 54/70 (77.1%) Informative immunophenotype for NS and M malignancies CD56þ/cytokeratin 4/4 Informative immunophenotypes for mesothelial cells in reactive effusions Desmin-33þ/cytokeratinþ 6/73 (8.2%) EMA/Ber-EP411/71 (15.5%) EMA/Ber-EP4/desmin-33þ/cytokeratinþ 5/70 (7.1%)

6/45 4/43 1/45 0/45 4/45 20/39 1/44 3/43 1/43 1/42

P*