Matthias S. Matter 1,2, Esther Schwarz 1, Teresa Marafioti 3, Peter Schraml 1, Holger Moch 1

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1 Immunohistochemical detection of CD3 in T- cell lymphomas: superior sensitivity of rabbit monoclonal 2GV6 antibody compared to mouse monoclonal F antibody Matthias S. Matter 1,2, Esther Schwarz 1, Teresa Marafioti 3, Peter Schraml 1, Holger Moch 1 1 Department of Pathology, Institute of Surgical Pathology, University Hospital of Zurich, Zurich, Switzerland, 2 Current address: Institute of Surgical Pathology, University Hospital, Basel, Switzerland 3 Leukaemia Research Fund Immunodiagnostics Unit, Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, Oxford, United Kingdom Immunohistochemical detection of cluster of differentiation 3 (CD3) expression is important for the diagnosis of peripheral T-cell and NK-cell lymphomas. We compared CD3 staining intensity and percentage of CD3-positive cells in 28 T-cell lymphomas was compared using mouse anti-human CD3 antibody (clone F7.2.38) and rabbit anti-human CD3 antibody (clone 2GV6). Analysis was performed on tissue microarray sections containing 12 peripheral T-cell lymphomas unspecified, 4 angioimmunoblastic T-cell lymphomas, 6 adult T-cell leukemia/lymphomas, and 6 anaplastic large T-cell lymphomas. Compared to mouse antibody, CD3 staining intensity and the fraction of CD3-positive cells increased significantly with rabbit antibody in the vast majority of the T-cell lymphomas analyzed. The mean staining intensity of positive cells (weak: z1; moderate: z2; strong: z3) of all 28 lymphomas rose from 2.1 with mouse antibody to 2.5 with rabbit antibody. Notably, the mean fraction of strongly positive cells (z3) was 17.7% with mouse antibody and increased considerably to 49.1% with rabbit antibody. In addition, there was an overall 8% increase in CD3-positive cells if rabbit antibody was used. The results show that detection of CD3 expression in T-cell lymphomas with rabbit antibody represents an improved standard by increasing sensitivity and staining intensity. Keywords: CD3, Immunohistochemistry, Mouse monoclonal antibody, Pathology, Rabbit monoclonal antibody, T-cell lymphoma Introduction In industrialized countries, mature T-cell and natural killer (NK)-cell neoplasms account for 12% of all non- Hodgkin lymphomas. 1 The current World Health Organization classification of tumors of hematopoietic and lymphoid tissue recognizes 18 distinct clinicopathologic T-cell and NK-cell non-hodgkin lymphomas. 2 The most common subtypes of mature T-cell and NK-cell lymphomas are peripheral T-cell lymphoma unspecified (PTCL/U), angioimmunoblastic T-cell lymphoma (AITL), NK T-cell lymphoma, adult T- cell leukemia/lymphoma (ATLL), and anaplastic large T-cell lymphoma (ALCL). Correspondence to: MS Matter, Institute of Surgical Pathology, University of Hospital, 4031 Basel, Switzerland. matterm@uhb3.ch Mature T-cell and NK-cell neoplasms generally express cluster of differentiation 3 (CD3), therefore a reliable detection of CD3 is important for their diagnosis. CD3isnon-covalently associated with the T-cell receptor and consists of the four distinct polypeptides CD3c, CD3d,CD3e, and the disulfide-linked homodimer of the f chain. 3 CD3 is expressed in both major classes of T cells, the ab Tcellsandthecd T cells. NK cells do not have a complete T-cell receptor complex, but generally express the e chain of CD3 in the cytoplasm. Compared to antisera generated in mice, rabbit antisera have a higher affinity and recognize a greater variety of epitopes. 4 As a consequence, monoclonal antibodies derived from rabbit hybridomas are preferentially used for the detection of human epitopes, such as CD3. Because of the lack of myeloma-like tumors in ß National Society for Histotechnology 2012 DOI / Y Journal of Histotechnology 2012 VOL. 35 NO

2 rabbits, the production of monoclonal antibodies by rabbit hybridomas was difficult for a long time. The advent of c-myc/v-abl transgenic rabbits a few years ago has led to the establishment of rabbit plasmacytoma cell lines which allow the production of stable monoclonal rabbit antibodies. 5 In this study, CD3 staining intensity was compared in four subtypes of mature T-cell lymphomas using two consecutive tissue microarray (TMA) sections that were stained with the monoclonal mouse antihuman CD3e clone F and the new monoclonal rabbit anti-human CD3e clone 2GV6. Materials and Methods Tissue specimen and TMA construction Twenty-eight formalin fixed, paraffin-embedded T- cell lymphomas were provided by the John Radcliffe Hospital, University of Oxford (Oxford, UK). There were 12 PTCL/U, 6 ATLL, 6 ALCL, and 4 AITL. All six cases of ALCL were ALK-negative. Hematoxylin and eosin sections from each block were reviewed by one pathologist and the areas containing T-cell lymphoma were marked on the glass slides for TMA construction. Two to three cores were punched from each tumor as described. 7 Antibodies Anti-human CD3 mouse monoclonal antibody F was purchased from Dako (Baar, Switzerland). Antihuman CD3 rabbit monoclonal antibody 2GV6 was purchased from Ventana-Roche (Basel, Switzerland). Both antibodies recognize the e chain of the CD3 complex. Immunohistochemistry TMA sections of 3 mm thickness were cut from paraffin blocks and transferred to glass slides, then immunohistochemistry (IHC) was conducted. IHC with mouse monoclonal antibody F was performed using the automated Bond Max platform (Leica Microsystems, Heerbrugg, Switzerland). Heatinduced epitope retrieval pre-treatment was performed using H2 buffer (Leica Microsystems) and by boiling for 10 minutes. Afterwards, slides were incubated with mouse monoclonal antibody F (concentration 12 mg/ml) at 37uC. Immunoreactivity was detected with a corresponding secondary antibody included in the Refine-DAB detection kit (Leica Microsystems). The optimal staining protocol for F was elaborated by a surgical pathologist and a lab technician. First, several different pre-treatments at different boiling temperatures were tested at an antibody concentration of 1 mg/ml. Secondly, the antibody was titrated at different antibody concentrations and the best staining was determined. At each step, the best staining was defined as the condition with the lowest background and the highest specific signal. IHC with rabbit monoclonal antibody 2GV6 was performed on the Ventana Benchmark platform (Ventana-Roche) as recommended by the manufacturer. Heat-induced epitope retrieval pre-treatment was performed using CC1m buffer and by boiling for 18 minutes. Afterwards, slides were incubated with rabbit monoclonal antibody 2GV6 (concentration 0.45 mg/ml) at 37uC. Immunoreactivity was detected with a corresponding secondary antibody included in the UltraView DAB detection kit (Ventana-Roche). TMA analysis Stained TMA slides were digitally scanned with the Nano-Zoomer (Hamamatsu, Shizuoka, Japan). Analysis of the TMA cores was conducted using Analysis D software (Olympus, Hamburg, Germany). The staining intensity of cells for T-cell lymphomas and cells for tonsils and lymph nodes was determined in a representative area within each TMA core. The intensity of cytoplasmic CD3 staining was classified as negative (0), weak (z1), moderate (z2), or strong (z3) by one slide reviewer for T-cell lymphomas and by a different slide reviewer for tonsils and lymph nodes. A blinded analysis of T-cell lymphomas was conducted. Statistical analysis Mean values, contingency table analysis, and Chisquare tests for evaluating correlations between staining intensities of tumor subtypes were calculated using the statistic program Statview (SAS, Cary, NC, USA). Results The fractions of cells with different CD3 staining intensities (negative: 0; weak: z1; moderate: z2; strong: z3) from all 28 T-cell lymphoma cases are listed in Table 1. Representative examples of lymphoma cases stained with mouse and rabbit monoclonal antibodies are shown in Fig. 1. In 27 of 28 cases, the percentage of cells with strong (z3) staining intensity markedly increased if the cells were incubated with rabbit monoclonal antibody (Table 1). In summary, the mean percentage of strong (z3) CD3- positive cells increased from 17.7 to 49.1% with rabbit monoclonal antibody and was significantly higher with rabbit monoclonal antibody in all four T-cell lymphoma subtypes analyzed (Table 1). In ATLL, the fraction of strong (z3) CD3-positive cells increased by 24.2%, in AITL by 24.7%, in ALCL by 41.4%, and in PTCL/U by 36.2% (Fig. 2). In contrast, the mean percentage of moderately (z2) and weakly (z1) CD3-positive cells decreased markedly with 176 Journal of Histotechnology 2012 VOL. 35 NO. 4

3 rabbit monoclonal antibody (Table 1; Fig. 2). As a result, the mean intensity of positive cells in all four T- cell lymphoma subtypes was higher with rabbit monoclonal antibody (Tables 1 and 2). Importantly, in only one case (PTCL/U 2), the percentage of negative cells increased by 0.8% with rabbit monoclonal antibody. In all other 27 cases analyzed, the percentage of negative cells decreased if staining was performed with rabbit monoclonal antibody, and was reduced between 0.4 and 26.3% (Table 1). Therefore, the mean percentage of negative cells was significantly lower in all four T-cell lymphoma subtypes analyzed with rabbit monoclonal antibody, resulting in an overall 8% increase in CD3-positive cells (Table 1; Fig. 2). To extend the study to non-tumoral tissue, tissue samples from tonsils and lymph nodes were stained with both antibodies. Similarly, the fraction of Table 1 CD3 staining intensities in four T-cell lymphoma subtypes Mouse mab (clone F7.2.38) strong (z3) CD3-positive cells increased by 22.1% in tonsils and 45.1% in lymph nodes with rabbit monoclonal antibody (Table 1; Fig. 3a and b). However, the difference was statistically significant only in lymph nodes. As a consequence, the mean intensity of rabbit monoclonal antibody was higher for all analyzed T-cell lymphomas, tonsils and lymph nodes (Table 2). To exclude the possibility that increased staining intensity and percentage of positive cells by rabbit monoclonal antibody were due to non-specific staining, cores with lymphatic tissue harboring only few CD3-positive T cells and cores with muscle tissue were stained with both antibodies. As shown in Fig. 3c h, staining with rabbit monoclonal antibody did not result in increased non-specific background staining. Rabbit mab (clone 2GV6) Lymphoma case 0(%) z1(%) z2(%) z3(%) 0(%) z1(%) z2(%) z3(%) ATLL ATLL ATLL ATLL ATLL ATLL ATLL (mean) 13.9* * 7.1* * P AITL AITL AITL AITL AITL (mean) 41.5* * 34.4* * P ALCL ALCL ALCL ALCL ALCL ALCL ALCL (mean) 29.5* * 19.4* * P, PTCL/U PTCL/U PTCL/U PTCL/U PTCL/U PTCL/U PTCL/U PTCL/U PTCL/U PTCL/U PTCL/U PTCL/U PTCL/U (mean) 28.5* * 20.9* * P Tonsil Tonsil Tonsil (mean) 3.9* * 5.0* * P5NS Lymph node Lymph node Lymph node (mean) 8.3* * 4.7* * P, Notes: ATLL, adult T-cell leukemia/lymphoma; AITL, angioimmunoblastic T-cell lymphoma; ALCL, anaplastic large T-cell lymphoma; PTCL/U, peripheral T-cell lymphoma unspecified. NS represents non-significant. *Only fractions of negatively (0) and strongly (z3) stained cells were used for statistical calculations. Journal of Histotechnology 2012 VOL. 35 NO

4 Figure 3 CD3 staining (mouse mab or rabbit mab) of a tumor-free lymph node (a,b) muscle tissue (c,d) and areas from T-cell lymphomas containing only few CD3-positive cells (e h). Figure 1 Examples of CD3-positive T-cell lymphomas stained immunohistochemically with mouse monoclonal antibody (mouse mab) or rabbit monoclonal antibody (rabbit mab). ATLL (a d), AITL (e h), ALCL (i l), and PTCL/ U (m p). Two representative examples of each T-cell lymphoma type are shown. ATLL, adult T-cell leukemia/ lymphoma; AITL, angioimmunoblastic T-cell lymphoma; ALCL, anaplastic large T-cell lymphoma; PTCL/U, peripheral T-cell lymphoma unspecified. Discussion Immunohistochemical detection of CD3 is routinely performed for the diagnosis of T-cell and NK-cell lymphomas. The results of this work suggest that staining of human CD3 by monoclonal rabbit antibody (clone 2GV6) is significantly more sensitive than Figure 2 Percentage of cells staining positive (weak, moderate, and strong) or negative for CD3 with mouse mab or rabbit mab in all four T-cell lymphoma subtypes. Bars indicate mean standard error of the mean. ATLL, adult T-cell leukemia/lymphoma; AITL, angioimmunoblastic T-cell lymphoma; ALCL, anaplastic large T-cell lymphoma; PTCL/ U, peripheral T-cell lymphoma unspecified. monoclonal mouse antibody (clone F7.2.38) in selected cases of T-cell lymphomas, with no apparent loss of specificity. In particular, the mean percentage of strong (z3) CD3-positive cells increased significantly with rabbit monoclonal antibody in comparison to mouse monoclonal antibody (49.1% versus 17.7%). Notably, the working dilution was approximately 20 times higher with rabbit monoclonal antibody than with mouse monoclonal antibody. Therefore, the results suggest that the use of rabbit monoclonal antibody facilitates the interpretation of CD3 positivity in T-cell lymphomas. Similar results comparing staining intensity between mouse and rabbit monoclonal antibodies have been obtained by analyzing estrogen and progesterone receptor expression and Ki-67 in human breast cancer Only one study compared CD3 staining between monoclonal rabbit anti-human CD3 antibody (clone SP7) and monoclonal mouse anti-human CD3 Table 2 Mean staining intensities* of CD3-positive cells in four T-cell lymphoma subtypes, and tonsils and lymph nodes F GV6 All cases (n528) Mean intensity ATLL (n56) Mean intensity AITL (n54) Mean intensity ALCL (n56) Mean intensity PTCL/U (n512) Mean intensity Tonsils (n52) Mean intensity Lymph nodes (n52) Mean intensity Notes: ATLL, adult T-cell leukemia/lymphoma; AITL, angioimmunoblastic T-cell lymphoma; ALCL, anaplastic large T-cell lymphoma; PTCL/U, peripheral T-cell lymphoma unspecified. *Mean staining intensity was calculated by: [(number of cells at 1z staining intensity61)z(number of cells at 2z staining intensity62)z(number of cells at 3z staining intensity63)]/total number of cells. 178 Journal of Histotechnology 2012 VOL. 35 NO. 4

5 antibody (clone F7.2.38). 10 In that particular study, staining was concordant; however, non-neoplastic CD3 cells in tonsils were analyzed and the mean staining intensity in the two groups was not reported. With the exception of ALCL, % of T-cell and NK-T cell lymphoma cases have been shown to be positive for CD3. 6,11 16 These earlier reports did not mention the exact percentage of CD3-expressing cells in each single lymphoma case. Yet, some studies report that a lymphoma case was regarded as positive for CD3 if more than 30 or 50% of the cells stained for CD3. 11,14 If a threshold for positivity is set at 30% in our data, all lymphoma cases are CD3 positive with the exception of one PTCL/U case, independent of whether monoclonal mouse or rabbit antibody was used. However, if a threshold is set, for example at 60% positivity, six lymphoma cases must be regarded as negative with mouse monoclonal antibody and only two lymphoma cases as negative with rabbit monoclonal antibody (Table 1). This reveals the consequences that antibody selection can have. In addition, the data demonstrate that with the monoclonal rabbit antibody, the percentage of total CD3-positive cells clearly increased in 27 of 28 cases analyzed. This result suggests that some cells were not recognized by the mouse monoclonal antibody. Therefore, one assumes that, depending on the antibody and the threshold used for analyzing CD3 positivity, some T-cell and NK-cell tumors might have been regarded as falsenegative for CD3 expression in earlier investigations. It would be important to re-evaluate CD3 positivity with rabbit monoclonal antibody in T-cell and NKcell lymphomas, which have been diagnosed as negative for CD3 with mouse monoclonal antibody. In accordance with our data, two separate studies have described that detection of estrogen receptor by rabbit monoclonal antibody increased the percentage of estrogen receptor-positive breast cancers in comparison to mouse monoclonal antibody. 9,10 In conclusion, rabbit monoclonal antibodies seem to provide higher sensitivity than monoclonal mouse antibodies with no apparent loss of specificity. They can be used at high working dilutions and may contribute to reliable reproducibility which consequently leads to better standardization. 17 Acknowledgements We would like to thank Silvia Behnke and Martina Storz for excellent technical assistance. IHC only was supported by a grant of the Ventana Medical Systems Inc. (Tucson, AZ, USA). References 1 A clinical evaluation of the International Lymphoma Study Group classification of non-hodgkin s lymphoma. The Non-Hodgkin s Lymphoma Classification Project. Blood. 1997;89: Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, et al. 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Ann Oncol. 2005;16: Benharroch D, Meguerian-Bedoyan Z, Lamant L, Amin C, Brugières L, Terrier-Lacombe MJ, et al. ALK-positive lymphoma: a single disease with a broad spectrum of morphology. Blood. 1998;91: Rhodes A, Jasani B, Balaton AJ, Barnes DM, Anderson E, Bobrow LG, et al. Study of interlaboratory reliability and reproducibility of estrogen and progesterone receptor assays in Europe. Documentation of poor reliability and identification of insufficient microwave antigen retrieval time as a major contributory element of unreliable assays. Am J Clin Pathol. 2001;115: Journal of Histotechnology 2012 VOL. 35 NO