Fixation effect of SurePath preservative fluids using epidermal growth factor receptor mutation-specific antibodies for immunocytochemistry

Authors


Abstract

BACKGROUND

Cytological diagnosis of respiratory disease has become important, not only for histological typing using immunocytochemistry (ICC) but also for molecular DNA analysis of cytological material. The aim of this study was to investigate the fixation effect of SurePath preservative fluids.

METHODS

Human lung cancer PC9 and 11-18 cell lines, and lung adenocarcinoma cells in pleural effusion, were fixed in CytoRich Blue, CytoRich Red, 15% neutral-buffered formalin, and 95% ethanol, respectively. PC9 and 11-18 cell lines were examined by ICC with epidermal growth factor receptor (EGFR) mutation-specific antibodies, the EGFR mutation DNA assay, and fluorescence in situ hybridization. The effect of antigenic storage time was investigated in lung adenocarcinoma cells in pleural effusion by ICC using the lung cancer detection markers.

RESULTS

PC9 and 11-18 cell lines in formalin-based fixatives showed strong staining of EGFR mutation-specific antibodies and lung cancer detection markers by ICC as compared with ethanol-based fixatives. DNA preservation with CytoRich Blue and CytoRich Red was superior to that achieved with 95% ethanol and 15% neutral-buffered formalin fixatives, whereas EGFR mutations by DNA assay and EGFR gene amplification by fluorescence in situ hybridization were successfully identified in all fixative samples. Although cytoplasmic antigens maintained high expression levels, expression levels in nuclear antigens fell as storage time increased.

CONCLUSIONS

These results indicate that CytoRich Red is not only suitable for ICC with EGFR mutation-specific antibodies, but also for DNA analysis of cytological material, and is useful in molecular testing of lung cancer, for which various types of analyses will be needed in future. Cancer (Cancer Cytopathol) 2014;122:145–52. © 2013 American Cancer Society

INTRODUCTION

In the field of lung cancer, the detection of epidermal growth factor receptor (EGFR) mutation in non–small-cell lung cancer tissue can be an important factor in predicting the response to EGFR tyrosine kinase inhibitor (TKI) drugs such as gefitinib and/or erlotinib.[1] There are 3 main types of EGFR antibodies: immunohistochemistry (IHC) for total EGFR, IHC for phosphorylated EGFR, and IHC for mutated forms of EGFR.[2] IHC for total EGFR is not recommended for selection of EGFR TKI therapy.[2-4] Through IHC for mutated forms of EGFR, we have previously demonstrated the use of EGFR mutation-specific antibodies, such as delE746-A750 and L858R, in IHC and immunocytochemistry (ICC).[5-8] Also, we have suggested that EGFR mutation-specific antibodies are suitable for screening of patients with EGFR mutations and are a useful indicator with regard to patient management.[8]

For cytologic analysis, liquid-based cytology (LBC) is a method of producing diagnostic specimens from cell samples that have been liquefied. LBC for gynecological cytology was introduced approximately 2 decades ago. In recent years, LBC preparations have increasingly come into use in nongynecological cytology and there are already several reports in the literature dealing with nongynecological cytology.[9, 10] In a previous study, we recommended the use of LBC in ICC, because 95% ethanol fixative cytological samples produced many equivocal results on ICC, compared with resection and biopsy samples fixed in 15% neutral-buffered (NB) formalin on IHC.[8] Loss of antigenicity is a potential cause of staining variability. Although antigen loss may be due to excessive time after fixation or to technical problems with immunostaining, the major cause is related to fixation. SurePath (BD Diagnostics, Burlington, NC) is a thin-layer, LBC technique that is based on a sedimentation process using CytoRich Red and CytoRich Blue in nongynecologic cytology. We have previously reported the expression levels of biomarkers in bronchial brushing samples fixed in CytoRich Red, and both nuclear and cytoplasmic expressions allowed for good visualization of cancer cells.[11] However, it is unclear whether SurePath preservative fluids are suitable for molecular testing of lung cancer, where several types of molecular analyses may be required.

The aim of this study was to investigate the fixation effect of SurePath preservative fluids in ICC, EGFR mutation analysis, and fluorescence in situ hybridization (FISH).

MATERIALS AND METHODS

Cell Culture and Clinical Sample

Human lung cancer cell lines PC9 [EGFRexon19 del] and 11-18 [EGFRexon21 L858R] were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum in an atmosphere of 5% CO2 as described in our previous studies.[5] Clinical samples used were lung adenocarcinoma cells in pleural effusion.

Fixation and Cell Block Preparation

PC9 cells, 11-18 cells, and lung adenocarcinoma cells from pleural effusion were separately fixed (unmixed) in a disposable tube of 4 different fixatives: CytoRich Blue, CytoRich Red (Becton Dickinson, Franklin Lakes, NJ), 95% ethanol, and 15% NB formalin. CytoRich Blue is an alcohol-based fixative including ethanol and methanol. CytoRich Red is a formalin-based fixative including isopropanol, ethylene glycol, methanol, and formalin. After 5 or 40 days fixation at room temperature, the disposable tubes were centrifuged at 800 g for 10 minutes, and the fixation fluid was decanted. After that, cell blocks were prepared from residues after LBC preparation.

ICC of Cell Block Section and LBC Smear

Each cell block was cut into 4-μm slices and examined on a coated glass slide, and labeled with the following antibodies using the BenchMark XT (Ventana Automated Systems, Tucson, Ariz) and Bond-Max autostainer (Leica Microsystems, Newcastle, UK); Ki67 (×200; DakoCytomation, Glostrup, Denmark): Napsin A (×200; American Research Products, Belmont, Mass), thyroid transcription factor-1 (TTF-1) (×200; DakoCytomation, Glostrup, Denmark), EGFR mutation-specific antibodies recognizing the E746-A750 del–specific (6B6) in exon 19 and the L858R mutant-specific (43B2) in exon 21 (×200; Cell Signaling Technology, Danvers, Mass), and cytokeratin 7 (CK7) (×400; DakoCytomation, Glostrup, Denmark). For Ki67, Napsin A, TTF-1, and CK7, the BenchMark XT was used. Briefly, each slide was heat-treated using Ventana's CC1 retrieval solution for 30 minutes, and incubated with each antibody for 30 minutes. This automated system used the streptavidin–biotin complex method (Ventana iVIEW DAB detection kit). ICC of cell block sections and LBC smears with EGFR mutation-specific antibodies were performed on the same fully automated Bond-Max system using onboard heat-induced antigen retrieval with ER2 for 30 minutes and a Refine polymer detection system (Leica Microsystems, Newcastle, UK). Another method of ICC was also used as described in our previous studies on EGFR mutation-specific antibodies.[6] Briefly, the specimens were boiled in a microwave for 30 minutes in target retrieval solution (DAKO, Glostrup, Denmark), and were incubated with primary antibody at room temperature for 30 minutes. All smears were incubated with labeled polymer–horseradish peroxidase secondary antibody (ChemMate ENVISION Kit; DAKO, Glostrup, Denmark) for 30 minutes at room temperature.

ICC Scoring Assessments

The cytoplasmic expression levels were assessed using the following score: 0, none; 1, little demonstration of antigen; 2, very weak demonstration of antigen; 3, weak demonstration of antigen; 4, good demonstration of antigen in most of the cells expected to be positive; and 5, excellent demonstration of antigen in cancer cells.[12] For nuclear antigens, the fractions of Ki-67–positive and TTF-1–positive tumor cells (labeling index) were calculated. All procedures were done by 2 independent assessors—1 cytotechnologist (T.T.) and 1 pathologist (M.K.)—both of whom were blind to the fixative used. Individual specimens with discordant results among the investigators were reevaluated.

DNA Extraction and EGFR Mutation Analysis

Mutation of the EGFR gene was examined in exons 19 (delE746-A750) and 21 (L858R) by both peptide nucleic acid (PNA)–locked nucleic acid (LNA) polymerase chain reaction (PCR) clamp and TaqMan assay.[5-8] In brief, genomic DNA was purified from the fixed PC9 and 11-18 cell lines using a QIAamp DNA Micro kit (Qiagen, Valencia, Calif). In PNA–LNA PCR clamp assay, the PCR primers employed were synthesized by Invitrogen (Carlsbad, Calif). PNA clamp primers and LNA mutant probes were purchased from FASMEC (Kanagawa, Japan) and IDT (Coralville, Iowa), respectively. TaqMan detection assay using a combination of mutant allele assay and gene reference assay was used to detect the EGFR mutations.[13] PNA–LNA PCR clamp and TaqMan assay were performed using the SDS-7500 System (Applied Biosystems, Foster City, Calif). Exon sequences for EGFR kinase domain were amplified with a specific primer by nested PCR.

FISH Analysis

FISH was performed on unstained 4-μm cell block sections with the use of an EGFR Spectrum Orange/CEP 7 Spectrum Green Probe (Vysis LSI; Abbott Molecular, Abbott Park, Ill) according to the manufacturer's instructions. Nuclei were counterstained with DAPI (Vektor Laboratories, Ab-Cys, Paris, France).

RESULTS

Molecular Analysis in PC9 and 11-18 Cell Lines

Expression levels of ICC for EGFR mutation-specific antibodies

On Papanicolaou staining, chromatin patterns of PC9 cells showed clearly in CytoRich Blue, CytoRich Red, and 95% ethanol fixative; however, those of PC9 cells in 15% NB formalin fixative were condensed. LBC smears and cell block sections were successfully stained by EGFR mutation-specific antibodies. In LBC smears, PC9 cells were strongly stained for E746-A750del–specific antibody in all fixative samples. In contrast, 11-18 cells were weakly stained for L858R mutant-specific antibody in samples of CytoRich Blue and 95% ethanol fixative in both DAKO and BOND detection systems (Fig. 1). Cell block sections of PC9 cells were weakly stained for E746-A750del–specific antibody in samples of 95% ethanol fixative in both DAKO and BOND detection systems (Fig. 2). The expression levels of 11-18 cells in cell block sections were similar to these of LBC smears. The expression levels of EGFR mutation-specific antibodies presented a stable immunostaining profile in formalin-based fixatives.

Figure 1.

Representative immunocytochemistry (ICC) of liquid-based cytology smears in PC9 and 11-18 cell lines. PC9 cells are clearly stained for ICC with E746-A750 del Specific antibody in all fixative samples, whereas 11-18 cells are weakly stained for ICC with L858R mutant Specific antibody in CytoRich Blue and 95% ethanol fixative samples. (Immunocytochemistry; ×400).

Figure 2.

Representative immunocytochemistry (ICC) of cell block sections in PC9 and 11-18 cell lines. PC9 cells are weakly stained for ICC with E746-A750 del Specific antibody in only 95% ethanol fixative sample. 11-18 cells are strongly stained for ICC with L858R mutant Specific antibody in CytoRich Red and 15% neutral-buffer formalin fixative samples. (Immunocytochemistry; ×400).

Detection of EGFR mutations

To examine the quality of the DNA in PC9 and 11-18 cell lines in each fixative, we first performed DNA ladder analysis[14] by electrophoresis (Fig. 3). Clear bands appeared around approximately 20 kilobase-pair (kbp) marker in both CytoRich Blue and CytoRich Red. However, bands of PC9 and 11-18 cell lines in 95% ethanol and 15% NB formalin fixatives were unclear, suggesting that CytoRich preservative fluids had superior DNA preservation. We next examined whether EGFR mutations were detected by both PNA–LNA PCR clamp and TaqMan assay. EGFR mutations were successfully identified in all fixative samples of PC9 and 11-18 cells (data not shown).

Figure 3.

DNA ladder analysis by Electrophoresis. Clear bands appear around approximately 20 kbp in both PC9 and 11-18 fixed in CytoRich Blue and Red.

Detection of EGFR gene by FISH

PC9 cell block sections were subjected to FISH. EGFR Spectrum Orange/CEP 7 Spectrum Green probe clearly showed the amplification signals of EGFR gene in all fixative samples.

Effect of Antigenic Storage Time Using Lung Adenocarcinoma Cells in Pleural Effusion

We also investigated changes in expression levels of lung cancer detection markers by storage time using cell block sections. In cytoplasmic antigen, all fixative samples were strongly stained for CK7 and Napsin A. However, the frequency of immunoreactivity with Napsin A was lower on CytoRich Blue and 95% ethanol fixatives than on CytoRich Red and 15% NB formalin fixatives. On the other hand, all fixative samples showed high Ki67 labeling index in nuclear antigen, whereas loss of TTF-1 immunoreactivity was seen in 95% ethanol and CytoRich Blue fixative samples (Fig. 4).

Figure 4.

Immunocytochemistry of lung adenocarcinoma cells in plural effusion after 5 days storage time using cell block sections. CytoRich Red and 15% neutral-buffer formalin fixative samples were strongly stained for lung cancer detection markers, whereas CytoRich Blue and 95% ethanol fixative samples were not stained for thyroid transcription factor-1 antibodies. (Immunocytochemistry; ×400).

Change in expression levels for different periods of storage time is shown in Table 1. Although CK7 and Napsin A maintained high expression scores of 4 and 5 as storage time increased, a decrease of expression levels in nuclear antigens were seen in all fixative samples.

Table 1. Alternation of Expression Levels Over Time by Immunocytochemistry Using Lung Adenocarcinoma Cell Block
AntibodyStorage TimeCytoRich BlueCytoRich Red95% Ethanol15% Neutral-Buffered Formalin
Cytokeratin 75 daysScore 5Score 5Score 5Score 5
 40 daysScore 5Score 5Score 5Score 5
 AlterationNoneNoneNoneNone
Napsin A5 daysScore 4Score 5Score 4Score 5
 40 daysScore 4Score 5Score 4Score 5
 AlterationNoneNoneNoneNone
Ki675 days44.3%34.9%48.5%62.0%
 40 days34.2%24.1%32.3%27.1%
 Alteration−10.1%−10.8%−16.2%−34.9%
TTF-15 days0%67.5%0%73.6%
 40 days0%56.3%0%57.8%
 AlterationNone−11.2%None−15.8%

DISCUSSION

It is well known that IHC/ICC can cause instability of protein expression depending on fixation method. We investigated the fixation effect of SurePath preservative fluids, focusing on the expression of EGFR mutation-specific antibodies. We observed that formalin-based fixatives such as CytoRich Red and 15% NB formalin were superior in ICC for EGFR mutation-specific antibodies and lung cancer detection markers, compared with ethanol-based fixatives. Furthermore, we confirmed that CytoRich Blue and CytoRich Red both achieved excellent DNA preservation, as compared with 95% ethanol and 15% NB formalin fixatives. These results indicate that CytoRich Red is not only suitable for ICC, but also for DNA analysis.

Cytologic analysis plays an important role in the diagnosis of lung cancer, particularly in histological typing, and cytologic samples are also suitable for molecular testing. ICC is performed using various methodologies of fixation, such as 95% ethanol, buffered formalin, and methanol-based fixatives.[15] ICC and IHC are both known to produce good results.[12] We have previously demonstrated the usefulness of EGFR mutation-specific antibodies in ICC, and recommended a specific algorithm for rapid response EGFR-TKI therapy.[5, 6, 8] Our strategy was to use the DNA-based assay to clarify EGFR status only in patients with equivocal or borderline immunostaining results. In fact, we had previously pointed out the instability of protein expression in cytological samples in 95% ethanol fixative.[8] Gong et al demonstrated significantly lower detection rates of Ki67, PCNA, and p53 with the ethanol-based fixative ThinPrep as compared with formalin-fixed cell-block slides in malignant cases.[16] In addition, Hudock et al[17] evaluated various fixatives on estrogen receptor and progesterone receptor detection, and demonstrated that cell-block materials fixed in formalin yielded a higher frequency of positivity for both markers. However, ethanol-fixed materials, either smears or cell blocks, resulted in a significantly lower positivity. The fixative has a significant effect on the immunoreactivity of numerous antigens, and immunoreactivity is also affected by differences in the clone of the antibody. Therefore, each laboratory should carefully evaluate their fixation methods when carrying out ICC for EGFR mutation-specific antibodies, or other antibodies.

It is known that long-term cell storage induces degeneration of both DNA and antigenicity of protein.[18] Fixation method is more important than storage time, especially for nuclear antigens, but the expression levels of stored tumor cells decreases compared with tumor cells fixed shortly after collection, suggesting that ICC should be carried out at the time of diagnosis.

Human papillomavirus DNA testing using gynecological smear is routinely performed worldwide, and a comparison study of the 2 LBC systems based on SurePath and ThinPrep have reported the accuracy, rates of unsatisfactory cytology, and sufficiency of residual LBC specimens for human papillomavirus DNA testing.[19] In respiratory disease, determination of EGFR status is most important for EGFR-TKI therapy in patients with lung cancer, and Echinoderm microtubule-associated protein-like 4 and anaplastic large cell kinase (EML4-ALK) have been newly identified as transforming fusion oncogenes causing non–small-cell lung cancer.[21] Personalized medicine requires molecular analysis of tumor cells, and we have reported EGFR mutations assay and ALK FISH analysis using cytological material.[6, 8, 21] Surgery is usually not performed in patients with advanced lung cancer, and cytological material may be the only material available for EGFR mutations and ALK analysis when it is most needed. Therefore, cytological samples such as formalin-fixed paraffin-embedded samples are important materials for personalized medicine in patients with lung cancer. LBC is well established in gynecological cytology and has several potential advantages when the material is being used for molecular techniques. When comparing CytoRich Blue, CytoRich Red, 95% ethanol, and 15% NB formalin fixatives, we found that the quality of extracted DNA was superior in CytoRich Blue and CytoRich Red as compared with 95% ethanol and 15% NB formalin fixatives. In the quality analysis of the extracted DNA, Dejmek et al[22] found that spray fixation was superior for preserving up to the tested limit of 760 bp, and air-dried specimens gave the shortest fragment lengths. Furthermore, they reported that DNA preservation with CytoRich Red was nearly as good as that of spray fixation. This means that clinical samples fixed in CytoRich Red can be used for DNA analysis, although CytoRich Red contains a small amount (0.4%) of formalin.

In summary, CytoRich Red can be used for several different analyses including morphological diagnosis, EGFR mutation, FISH, and especially ICC for EGFR mutation antibodies, and it is a fixative fluid that will be useful in molecular testing of patients with lung cancer.

FUNDING SOURCES

Dr. Kawahara was supported by a Japan Society for the Promotion of Science KAKENHI Grant-in-Aid for Young Scientists.

CONFLICT OF INTEREST DISCLOSURE

The authors made no disclosures.

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