Disciplines such as morphology, immunophenotyping and genetics widely contributed over decades to the understanding of the cellular mechanisms of cancer. To obtain a greater insight into the complex processes of tumorigenesis, scientists have joined their efforts to combine many of the available techniques. Here, we report on the development of a FICTION (Fluorescence Immunophenotyping and Interphase Cytogenetics as a tool for the Investigation of Neoplasms) technique that allows a simultaneous detection of immunophenotypic markers and genetic aberrations on routinely processed lymphoma samples. As the antigen retrieval method seems to play an important role in the final results, we tested the pressure-cooking method at different times (2, 4 and 8 min) using three different buffers (EDTA, Tris-EDTA and citrate), resulting in improved sensitivity for the detection of both immunophenotypic markers and genetic aberrations. We also applied this method to different types of lymphoma using double immunofluorescence assays (including CD30, CD20, CD8 monoclonal antibodies) and several fluorescence in situ hybridization probes to demonstrate that the FICTION technique could be easily applied on paraffin sections in different combinations for the diagnosis and research of cancer.
In 1992, Klaus Weber-Matthiesen and co-workers described a novel method called FICTION (Fluorescence Immunophenotyping and Interphase Cytogenetics as a tool for the Investigation of Neoplasms). This technique preserves cell morphology and combines immunofluorescence to detect cellular antigen and fluorescence in situ hybridization (FISH) to detect chromosomal abnormalities.1
So far, FICTION has been applied to bone marrow smears, peripheral blood, cytospin samples and cryostat sections,2,3,4 with good results, allowing the simultaneous detection of the genotypic and phenotypic aspects of tumour cells.
The use of directly labelled anti-light-chain antibody for recognition of bone marrow plasma cells and FISH centromeric probes directed against chromosomes 3, 7, 9 and 11 has demonstrated that monoclonal gammopathy of undetermined significance patients acquire slow but inevitable chromosome changes.5 The FICTION technique has permitted the detection and study of T-cell receptor-associated chromosomal aberrations in T-cell neoplasias.6 This technique has also been used for the detection of the Rel protein in the nucleus of Hodgkin and Reed–Sternberg (HRS) cells in cases of classical Hodgkin's lymphoma (HL) with REL gene rearrangements.7 Recently, an improved FICTION method, multicolour FICTION (M-FICTION), has been developed, permitting the simultaneous analysis of multiple chromosomal aberrations in a single experiment. This method has been applied to the study of B-cell non-Hodgkin's lymphoma (B-NHL) and anaplastic large-cell lymphoma (ALCL), allowing the detection of the most important chromosomal aberrations in the diagnosis of these tumour types.8
However, the successful application of this technique on formalin-fixed and paraffin-embedded tissues has been hindered by some technical problems, such as poor target signal intensities and nonspecific background. These problems have been attributed to the intra- and extracellular protein crosslinkages caused by the fixation process, which result in a reduction in the accessibility of the target DNA to the specific FISH probes. Recently, the pressure-cooking method in combination with Tris-EDTA or EDTA buffer as antigen retrieval has been applied in some FISH studies on paraffin tissue,9,10,11 resulting in good signal intensity. On the other hand, heat-retrieval and calcium-chelating agent such as EDTA have been shown to improve the quality of immunostaining when used on paraffin sections.12,13 Our aim was to identify the optimal conditions of antigen retrieval in order to obtain consistently good results with the FICTION method on routinely processed tissue samples. The duration of antigen retrieval could be critical in obtaining a good equilibrium between FISH and immunofluorescence. Long periods of pressure-cooking could increase FISH signal but could also damage the cell membrane, resulting in a decrease of fluorescence staining. In order to optimize the equilibrium between optimal FISH and immunofluorescence staining, we have compared different times of pressure-cooking (2, 4 and 8 min) and three different buffers: EDTA, Tris-EDTA and citrate. For this experiment, a case of ALCL was selected. CD20 and CD30 monoclonal antibodies have been used for the double immunofluorescence technique in combination with the break-apart ALK FISH probe that detects any chromosome translocation involving the ALK gene. The optimal FICTION conditions were identified and applied to a variety of FISH and immunostaining combinations, demonstrating its easy application in routinely fixed paraffin sections.
The combination of double fluorescence staining for different antigens with the FISH technique offers various advantages, in particular to define the cell type, carrying the specific genetic lesion. We have studied one case of ALCL, two cases of HL and some B-NHL cases to demonstrate that the FICTION technique can be applied to different types of lymphoma, using various combinations of FISH probe sets and immunological markers.
Materials and methods
Biopsy samples (HL, ALCL, mantle cell lymphoma (MCL), and follicular cell lymphoma (FCL)) were obtained from the Tumour Bank Network of the Centro Nacional de Investigaciones Oncologicas (CNIO). The tissue samples originated from different pathology departments in Spain and had been fixed in formalin and embedded in paraffin by conventional techniques. Tissue sections (2–4 μm) were cut onto Dako slides (DakoCytomation, Glostrup Denmark), incubated at 60°C overnight and deparaffinized twice using xylene (10 min, room temperature). The slides were hydrated in a series of ethanol solutions (100, 95 and 75% for 5 min each) and then washed with PBS.
For the study of the effect of formalin fixation on the already optimized FICTION method, we obtained samples of fresh tonsil and FCL from the Tumour Bank Network. The samples were cut into five pieces, four having been fixed in 10% neutral-buffered formalin (Panreac, Barcelona, Spain) for different times (12 h, 24 h, 48 h and 1 week, respectively). The last piece was fixed for 1 h in B5 (Panreac). All samples were then processed and embedded in paraffin.
The tested retrieval solutions were: 1 mM EDTA (pH 8), 0.01 M citrate buffer (pH 6.5) and 50mM Tris–2 M EDTA (pH9). Each retrieval solution (2 l) were placed in a stainless-steel 6-l pressure-cooker with an operating pressure of 0.90 bars at 117°C (WMF perfect plus, Spain) and brought to boil on an electric hot plate without sealing the lid. The slides were placed in a metal rack and lowered into the boiling buffer. The pressure-cooker was then sealed and brought to full pressure. In this study, heating times of 2, 4 and 8 min (after full pressure was reached) were employed. Thereafter, the pressure-cooker was depressurized using running tap water and the lid was then removed. The slides were placed in PBS and stored at 4°C if not used immediately.
After antigen retrieval, the slides were incubated for 1 h at room temperature in a humid chamber with a mixture of two primary antibodies diluted in PBS+10% FCS (Australian Foetal Calf Serum, Life Technologies Inc., USA). The exact dilution values are shown in Table 1. Slides were then washed in PBS 0.5% Tween 20 three times for 5 min each. The slides were incubated for 1 h with fluorochrome-conjugated antibodies against the different Ig isotypes, diluted in PBS (Molecular Probes, Leiden, Netherlands) (dilution values are shown in Table 1) in a humid chamber in the dark. Subsequently, slides were washed in PBS 0.5% Tween 20 three times for 5 min each. Before performing FISH analysis, the slides were covered with a coverslip and revised by fluorescence microscopy for the presence of good immunofluorescence staining. Slides were kept at 4°C if not used immediately.
Fluorescence in situ hybridization
Slides were dehydrated through a series of ethanol washes (70, 80 and 100%) for 2 min each and denatured in the presence of the specific probes at 75°C for 90 s and left overnight in a humid chamber at 37°C in the dark. A rapid wash was performed as follows: 0.4 × SSC/0.3% NP-40, 72°C (±2°C), 2 min; 2 × SSC/0.1% NP-40, room temperature, 5 min; 1 × PBS, room temperature, 5 min. Following washing, antifading (Qbiogene, Illkirch, France) was added. Locus-specific probes were used for the detection of amplification and rearrangement in genes associated with different lymphoid malignancies. The probes and the colour labelling schemes are shown in Table 2. Tissue images were captured using a CCD camera (Photometrics SenSys camera) connected to a PC running the Cytovision image analysis system (Applied Imaging Ltd., UK) with focus motor and Z stack software, which is very important for obtaining quality images from 2 to 4 μm slides.
Results and conclusion
The results of each assay with the tested times and retrieval solutions are shown in Table 3. Optimal results were obtained using pressure-cooking for 2 min with EDTA (Figure 1a) or Tris-EDTA. The nuclear morphology and tissue architecture were well preserved. Both the FISH and immunofluorescence signals showed excellent intensity. As expected, increasing the duration of pressure-cooking damaged the nuclear morphology, while a significant improvement of the FISH signals was not observed. However, in the case of EDTA with 8 min of pressure-cooking, the FISH signal was significantly decreased.
The worst FICTION results were obtained using citrate buffer (Figure 1b), at all durations of pressure-cooking, with a slight improvement of the immunofluorescence signals after 8 min of pressure-cooking. The citrate buffer still provided good FISH results. In the slides treated with EDTA buffer, 8 min, and in all cases using citrate buffer, immunofluorescence staining was weak and diffuse, creating signal background, and nuclear morphology and tissue architecture were damaged. The equilibrium between FISH and immunofluorescence signals is achieved after short antigen retrieval with the best results for both techniques after 2 min pressure-cooking in buffer containing a chelating agent (EDTA and Tris-EDTA).
Enzymatic digestion, using pepsin or protease K, is consistently present in most of the routinely used FISH protocols. Logically, it will interfere with a simultaneous immunodetection of the different epitopes by destroying the antigens or stripping the antibody complex. We illustrated that, with the introduction of the pressure-cooking method, enzymatic digestion, it is not always necessary. We have found that pepsin treatment is only needed in a small number of cases due to the apparent suboptimal fixation (data not shown). In optimally fixed tissues samples, the pepsin treatment does not increase the intensity of the FISH signals and damages the cell morphology and tissue architecture and interferes with the immunostaining. The antigen retrieval by pressure-cooking with chelating agent or citrate buffer is sufficient to overcome the complex protein crosslinkages caused by fixation and insures the accessibility of the target DNA to the probes.
Duration of the denaturation/hybridization step is another critical issue when performing FICTION. Three different denaturing times have been compared (60, 90 and 120 s). Denaturation (60 s) resulted in reduced FISH signal intensity. On the contrary, immunofluorescence staining was well preserved. Denaturation (2 min) resulted in a good FISH signal but the immunofluorescence staining was completely lost. We found that 90-s denaturation gave the best equilibrium between FISH and immunofluorescence signals.
We also tested the effect of formalin fixation on the quality of the FICTION results. We applied the procedure, using three different pressure-cooking times, EDTA buffer and 90 s denaturation, in one case of FCL and one case of normal tonsil that had been fixed with different fixatives and/or different durations of fixation (12 h, 24 h, 48 h or 1 week in 10% formalin or 1 h in B5 fixative). For this experiment, we used two FISH probes: IGH/BCL-2 for the FCL case and ALK for the normal tonsil. As shown in Table 4, the best results are achieved after 24 h formalin fixation, the standard method used in most laboratories.14 This is independent of the probes and tissue used. Fixation times appear to be critical, particularly for the intensity of the FISH signals. Overfixation drastically decreases the FISH signal culminating in complete loss of signal in the case of 1-week fixation. The B5-fixed samples show weak FISH signal and poor immunofluorescence staining. This indicates that B5 fixation is not suitable for performing FICTION analyses with the protocol presented herein. These results reconfirm previous findings that optimum results are achieved using tissue fixed for 24 h in analyses involving immunofluorescence, FISH and immunohistochemistry. Furthermore, increased pressure-cooking time did not improve the FISH and immunofluorescence intensities in the cases of suboptimal fixation (Table 4).
This FICTION technique was applied to a variety of lymphoma tissue samples (two cases of HL, one case of ALCL, one MCL, one FCL and one composite HL-MCL lymphoma). This allowed the identification of malignant and nonmalignant cell subsets and permits the analysis of the genetic lesions in their histological context. The most valuable application of the FICTION technique is in cases where the percentage of tumour cells is low. In the two HL cases (Figures 2a and b), we were able to identify CD30+ HRS cells and the normal CD20+ B-cell population. We combined the immunofluorescence labelling with the cyclin D1 probe (CCND1) (Figure 2a) and with the BCL6 probe (Figure 2b). As expected, the two populations identified showed no rearrangements of these genes. In the ALCL case (Figure 2c), two morphologically distinct populations can be distinguished, with the malignant CD30+ cells marked red and the normal CD20+ B-lymphocytes marked in green. Only the CD30+ tumour cells show the translocation breakpoint in the ALK gene, characteristic of this type of lymphoma, as shown by FISH analysis. Double immunofluorescence (CD20 and CD8) was used in the MCL and FCL cases. The FISH probes used were CCND1/IGH and BCL2/IGH, respectively (Figures 2d and e). The presence of the translocation (seen as two fusion signals) could only be demonstrated in the CD20+ cells.
The FICTION technique is extremely helpful in the analysis of composite tumors. We present a case where FICTION permitted the identification of two different tumour populations in the same section (Figures 2f and g). With the CD30 monoclonal antibody, it has been possible to identify a small population of HRS cells that showed the presence of several copies of the CCND1 gene, suggesting a hyperdiploid karyotype which is a common cytogenetic feature of HL.15,16 CD20− cells, that show normal expression of CCND1, surround the CD30+ cells. These cells probably represent the T-cell population that has been previously described associated with HRS cells.17 Supporting this observation, the FISH probe did not show rearrangements of CCND1 (Figure 2g). In the same case, it was also possible to identify a population of CD20+ cells that showed the t(11;14) translocation involving CCND1 and IGH genes (Figure 2f). This data strongly suggest the presence of HD and MCL in the same patient.
This technique is potentially very useful due to its easy applicability, rapidity and amenability to automation. Owing to its capacity to improve the efficacy of result interpretation, resulting from the combination of more than one technique on the same sample, FICTION could be extremely useful in tumour diagnosis, especially in the detection of low-level disease, minimal residual disease or diagnosis of composite tumors. Using our optimized FICTION method, the hybridization and the immunofluorescent signals on paraffin sections were strong and distinct, while nonspecific background staining was insignificant. The application of the FICTION technique on paraffin sections provides a powerful tool for all laboratories where molecular cytogenetic analysis is routinely used. In addition, the use of the FICTION technique on paraffin sections will permit retrospective studies on archived cases and large-scale studies using tissue microarrays.
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Angel Martinez-Ramírez is a fellow of the Instituto de la Salud Carlos III. Sandra Rodriguez is a fellow of the Fundacion Inocente Inocente. We thank the Immunohistochemistry Unit of the Centro Nacional de Investigaciones Oncologicas (CNIO) for providing monoclonal antibodies and technical support, and the Tumour Bank Network for providing the clinical samples. Finally, we thank Lorraine Tracey for her help.
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Martínez-Ramírez, A., Cigudosa, J., Maestre, L. et al. Simultaneous detection of the immunophenotypic markers and genetic aberrations on routinely processed paraffin sections of lymphoma samples by means of the FICTION technique. Leukemia 18, 348–353 (2004). https://doi.org/10.1038/sj.leu.2403230
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