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Molecular & Cellular Proteomics 5:1072-1081, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

From Gene Expression Analysis to Tissue Microarrays

A Rational Approach to Identify Therapeutic and Diagnostic Targets in Lymphoid Malignancies^*

Sara Ek^,, Ulrika Andréasson, Sophia Hober^¶, Caroline Kampf^¶, Fredrik Pontén^¶, Mathias Uhlén^¶, Hartmut Merz^|| and Carl A. K. Borrebaeck

From the Department of Immunotechnology, Lund University, SE-220 07 Lund, Sweden, ^¶ Department of Biotechnology, Royal Institute of Technology, AlbaNova University Center, SE-106 91 Stockholm, Sweden, and ^|| Institut für Pathologie, Medizinische Universität Schleswig Holstein, Campus Lübeck, 235 38 Lübeck, Germany

	ABSTRACT

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Mantle cell lymphoma (MCL) is an aggressive lymphoid malignancy for which better treatment strategies are needed. To identify potential diagnostic and therapeutic targets, a signature consisting of MCL-associated genes was selected based on a comprehensive gene expression analysis of malignant and normal B cells. The corresponding protein epitope signature tags were identified and used to raise monospecific, polyclonal antibodies, which were subsequently analyzed on paraffin-embedded sections of malignant and normal tissue. In this study, we demonstrate that the initial selection strategy of MCL-associated genes successfully allows identification of protein antigens either uniquely expressed or overexpressed in MCL compared with normal lymphoid tissues. We propose that genome-based, affinity proteomics, using protein epitope signature tag-induced antibodies, is an efficient way to rapidly identify a number of disease-associated protein candidates of both previously known and unknown identities.

To allow analysis of the numerous deregulated gene products identified by gene expression profiling, a parallel approach is also necessary for downstream investigations. To study individual tumor-associated proteins and their expression pattern, highly specific affinity probes are required. We addressed the challenge of generating such antibodies in a high throughput manner using an approach recently described by Agaton et al. (22) who showed that protein epitope signature tags (PrESTs), having 100% homology with the target, can be used to raise monospecific antibodies.

Analysis of the MCL-derived proteome has been performed previously using commercially available antibody microarrays containing 512 different antibodies, and deregulated proteins were found as compared with normal CD19⁺ B cells (23). Similarly whole-proteome analysis, based on 2D gels and MALDI-TOF MS, was used to analyze biopsies from two MCL tumors, and nine proteins were found to be up-regulated in MCL compared with reactive lymph nodes (24). These approaches to the analysis of differences in protein content in malignant and non-malignant samples are based on a low number of analytes in comparison with gene expression profiling where >45,000 transcripts can be screened using e.g. an Affymetrix U133 Plus 2.0 array.

Tissue microarrays or staining of full tissue sections is commonly used to confirm the expression of proteins or gene products shown to be deregulated in different types of malignancies. However, due to limited availability of antibodies targeting many of the poorly characterized gene products that can be identified using whole genome analysis, new strategies to confirm expression of the corresponding gene products need to be established. Consequently in a rational genomic and proteomic approach we narrowed down the number of potential targets in MCL by confirming gene expression data in a high throughput manner on the protein level using novel PrEST-specific antibodies against MCL-associated antigens. This enabled us to analyze the expression and localization of both known and unknown gene products in different tissues and cells, independently of the availability of commercially available antibodies, and to initiate the direct process to identify potential diagnostic and therapeutic lymphoma targets.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Selection of Candidate Genes—
The MCL-associated genes were derived using gene expression profiling (U95v2, Affymetrix, Santa Clara, CA) of tumor samples from 19 patients diagnosed with MCL. As reference, 11 samples from five different B cell populations, derived from normal pediatric tonsils (Malmoe University Hospital), were used as described previously (9, 12). The different B cell populations used were: naïve B cells (CD19⁺, IgD⁺, CD38^–), preactivated B cells (CD19⁺, CD23⁺, CD38^–), centroblasts (CD19⁺, CD38⁺, CD77⁺), centrocytes (CD19⁺, CD38⁺, CD77^–), and memory B cells (CD19⁺, CD38^–, IgD^–). When filtering the data for differentially regulated genes, all samples were scaled to a fixed median value as recommended by Affymetrix. When represented in the heat map (Fig. 1), all samples were normalized to the naïve B cell population (two replicates) using "Normalize to specific samples" in Gene Spring 7.0 (Agilent Technologies Inc., Palo Alto, CA).

View larger version (69K): [in this window] [in a new window]   FIG. 1. Heat map representing the differential gene expression of the MCL-associated genes (n = 96) that were chosen for PrEST design. All MCL and normal B cell populations are normalized to the naïve B cell population. Red color indicates increased expression, black color indicates no change in expression, and green color indicates decreased expression, all compared with the naïve B cell population. The color bar shows the -fold change in gene expression compared with the naïve B cell population. Each row represents one gene.

1. Genes that were quantitatively overexpressed (>2-fold) in the majority (>80%) of MCL patient samples compared with normal B cell populations.
   
2. Genes that were qualitatively (i.e. present versus absent) overexpressed in the majority (>80%) of MCL patient samples compared with normal B cell populations. These genes were not expressed in any of the normal B cell populations analyzed.
   
3. Genes that were qualitatively overexpressed in a subgroup (>30%) of MCL patients compared with normal B cell populations. These genes were not expressed in any of the normal B cell populations analyzed.
   

Generation and Analysis of the Affinity Reagents—
Suitable PrESTs, representing unique regions for each target protein were designed using bioinformatic tools (25) and with the human genome sequence as template (26). In the design of the PrESTs, transmembrane regions and signal peptides were avoided, and an amino acid sequence, with a size between 100 and 150 amino acids, with low homology to other human proteins was selected to decrease the risk of cross-reactivity of antibodies to other human proteins. The cloning and protein expression were performed as described previously (22).

The monospecific antibodies were subsequently obtained by affinity purification of rabbit polyclonal antisera (27). The antibodies were quality-controlled using a protein array and Western blots procedure as described by Uhlen et al. (28).

Case Selection—
A total of 25 cases of non-Hodgkin lymphoma were collected from the files of the Department of Pathology, The University of Schleswig-Holstein, Campus Lübeck, between 2004 and 2005. All cases were diagnosed and classified using morphologic and immunophenotypic criteria specified in the World Health Organization classification of lymphoid neoplasms (29). The group of B cell non-Hodgkin lymphoma cases assessed included the following: eight mantle cell lymphoma (six small cell or classical and two blastoid), four follicular lymphoma (all grade 1 or 2), one nodal marginal zone B cell lymphoma, two lymphoplasmacytic lymphoma, and three small lymphocytic lymphoma/chronic lymphocytic leukemia. Non-neoplastic lymphoid tissues were included, represented by tonsils with follicular hyperplasia and tonsils with Epstein-Barr virus infection. All cases were analyzed using either tissue microarrays (n = 25) or full tissue sections (n = 2). The tissue microarrays included duplicate cores from all cases and were constructed using a manual tissue arrayer (AlphaMetrix Biotech GmbH, Rödermark, Germany). All neoplasms assessed were fixed in 10% buffered formalin, routinely processed, and embedded in paraffin.

Immunohistochemical Methods—
The immunohistochemical methods used in this study have been described previously (30). Briefly heat-induced antigen retrieval was performed prior to immunohistochemical staining. Sections were incubated with a polyclonal antibody (0.1–1 µg/slide) at room temperature for 30 min. Detection of signal was achieved using the ChemMate^TM EnVision^TM detection kit peroxidase/DAB (3,3'-diaminobenzidine), rabbit/mouse LSAB+ kit (DAKO, Hamburg, Germany) that contains secondary biotinylated goat anti-rabbit/mouse antibody and streptavidin/horseradish peroxidase complex according to the manufacturer’s recommendations. 3,3'-Diaminobenzidine/H₂O₂ (DAKO) was used as chromogen, and slides were counterstained with hematoxylin.

Scoring of Positive Cells in IHC—
Defined staining patterns were noticed for the various antibodies, and a cutoff of 2% of stained cells was used to categorize an antibody as positive. Cytoplasmic or nuclear staining was also considered, and expression was graded visually as negative, weak, or moderate/strong. A lymphoma case was considered weakly positive if cytoplasmic staining was similar to or less than that of normal cells and moderate to strongly positive if cytoplasmic staining was similar to or greater than that of normal cells.

	RESULTS

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Filtering for MCL-associated Genes and Subsequent PrEST Selection—
After comprehensive analysis of differentially expressed genes in which 19 MCL samples and five normal tonsil-derived B cell populations were compared we chose 96 genes (see "Experimental Procedures") for subsequent PrEST identification. Because the most important aim of the study was to identify gene products potentially useful for prognosis and therapy, all the selected genes were quantitatively or qualitatively overexpressed in MCL as compared with normal B cell populations (Fig. 1 and Table I). Furthermore the selection of genes was focused mainly on poorly or presently non-characterized gene products.

Immunohistochemistry Analysis—
The quality-controlled, monospecific antibodies were then evaluated using IHC on paraffin-embedded tissue sections from MCL, follicular lymphoma (FL), chronic lymphocytic lymphoma (CLL), marginal zone (MGZ) lymphoma, and tonsils. A 2% cutoff of positive cells was set to determine the antibody reactivity, although in most positive cases the percentage of positive cells was over 50% of tumor cells or defined cell populations, respectively. Most of the tested antibodies (84%, 70 of 83) showed reactivity to one or more of the tissues as shown in Fig. 2. In accordance with the preselection strategy of gene products, the majority (80%, 56 of 70) of the reactive antibodies showed staining of MCL tumor cells (Fig. 2). Among these 56 MCL-reactive antibodies, 45% (25 of 56) showed a tumor specificity and did not react with either normal B cell-containing regions, such as the mantle zone cells and the germinal center (GC), or with plasma cells (Fig. 3a). Staining of non-malignant cells was mostly seen in professional antigen-presenting cells, such as follicular dendritic cells (FDCs) and macrophages, whereas T cells were negative. Furthermore of the 25 MCL-, non-B cell-reacting antibodies, 14 (HPRK250003, -6, -7, -26, -27, -33, -34, -35, -51, -53, -54, -57, -60, and -78) strictly recognized lymphoma tissues and none of the tested normal tissue(s). Four antibodies (HPRK250033, -34, -53, and -60) showed an even further tumor selectivity in that they only recognized MCL and no other tested tissue. The reactivity pattern of these antibodies are of interest and are specifically marked (*) in Fig. 3a. One example of immunohistochemical staining of an MCL-reactive, non-B cell-reactive antibody (HPRK250026) on MCL and tonsil tissue sections is shown in Fig. 3b; it also illustrates the distinct reactivity pattern often seen with the polyclonal, anti-PrEST antibodies. Furthermore when we analyzed the reactivity pattern of the MCL-associated antibodies we found that nine antibodies (HPRK250015, -33, -34, -36, -38, -41, -53, -60, and -93) were able to separate MCL from the other lymphoma types that were tested, such as FL and CLL. One example of such an antibody (HPRK250027) is shown in Fig. 4 where it is shown to be able to discriminate between a mantle cell and a follicular lymphoma.

View larger version (28K): [in this window] [in a new window]   FIG. 2. Heat map representing differential protein expression in IHC for the generated PrEST-specific antibodies (n = 83). The different malignant and normal cells and morphological compartments that are represented are MCL cells, FL cells, CLL cells, MGZ lymphoma cells, mantle zone cells (tonsil), GC cells (tonsil), plasma cells (tonsil), T cell zone (tonsil), FDCs (tonsil), and macrophages (tonsil).

View larger version (47K): [in this window] [in a new window]   FIG. 3. a, heat map representing differential protein expression in IHC for the MCL-reactive antibodies (n = 25) that did not react with normal lymphoid tissue enriched in B cells (represented by either mantle zone cells, GC cells, or plasma cells). The antibodies that are specific for MCL and thus not reactive with any other tissue are marked (*) in the figure. b, examples of immunohistochemical staining of an antibody (HPRK250026) that show distinct reactivity differences in normal (tonsil, 20x magnification) and malignant tissues (MCL tissue, 20x magnification).

View larger version (86K): [in this window] [in a new window]   FIG. 4. Example of an antibody (HPRK250027) that distinguishes MCL from FL tissue.

View larger version (53K): [in this window] [in a new window]   FIG. 5. a, heat map representing the IHC results for the antibodies (n = 26) that recognize at least three of four tested lymphoma entities (MCL, FL, CLL, and MGZ lymphoma). The subgroup of lymphoma-associated antibodies (n = 6) that did not recognize normal B cells are marked (**). b, example of an lymphoma-reactive antibody (HPRK250057).

View larger version (17K): [in this window] [in a new window]   FIG. 6. Schematic overview of the different groups of antibody reactivity.

	DISCUSSION

TOP ABSTRACT EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In this study we have designed a rational approach based on PrESTs (22) to produce 83 antibodies specific for 57 gene products associated with MCL as deciphered by gene expression profiling. The affinity-purified antibodies were then used to analyze the corresponding protein expression in both malignant and normal tissues to validate our process. The MCL-associated genes were initially selected using extensive gene expression analysis of normal and malignant B cells (see "Experimental Procedures"). Genes coding for uncharacterized or poorly characterized gene products were considered particular interesting for this type of analysis. Consequently the aim of the study was to (i) demonstrate a rational process, based on genomic information, for the generation of antibodies against differentially expressed proteins; (ii) investigate the protein expression patterns and localization in different normal and malignant tissues; and (iii) identify lead protein targets potentially useful in diagnostic and/or therapeutic applications.

To validate the differences seen in gene expression at the protein level, patient tissue material needs to be evaluated. Most tissue banks that are available consist of paraffin-embedded material as it takes less time to prepare, stores well, and retains cellular details better than frozen material (39). Because routine, diagnostic immunohistochemical analyses are performed using this material, it is of importance that the generated antibodies are not only able to recognize native but also fixed antigen. Analysis of the different antibodies using IHC (Figs. 2–5) showed that 84% of the tested antibodies reacted toward one or more paraffin-treated and formalin-fixed tissue. Even if there are no, to our knowledge, previous report regarding the success rate of generating antibodies reactive toward paraffin-treated antigens, the achieved success rate was considered very satisfactory. The high success rate seems to be a positive feature of the PrEST-derived antibodies because reactivity against paraffin-treated and fixed tissue often is poor for most monoclonal antibodies (40, 41). The ability of the different antibodies to recognize native antigens expressed by different lymphoma cell lines was also successfully assessed using flow cytometry, although this needs to be confirmed on primary tumor cells (data not shown).

When analyzing the IHC data, several interesting subgroups of antibody specificity were identified as summarized in Fig. 6. The gene selection strategy was clearly reflected in the reactivity pattern of the antibodies as the majority of functional antibodies (80%) reacted with MCL tissue (Fig. 2). It was not surprising to find that not all of the functional antibodies reacted with MCL tissue as it is generally known that the correlation between mRNA and protein level is not absolute. Recently a 40% correlation between transcript and protein level was reported (42) that emphasizes the importance of integrated genomic and proteomic studies. Among the MCL-reactive antibodies, 45% showed no reactivity to B cells or cells found in either the mantle zone or the germinal center (Fig. 3). These antibodies are of interest as diagnostic and/or therapeutic candidates, and they are presently being further validated. Staining of normal cells was mostly seen in antigen-presenting cells, such as FDCs and tissue macrophages, whereas T cells showed no staining at all. One explanation for this observation might be the fact that antigen-presenting cells take up, process, and display tumor-associated antigens on their surface as part of their normal immune surveillance. Thus, the majority of the antibodies in Fig. 3a may potentially be specific for malignant compared with normal B cells. We also identified a number of antibodies that specifically recognize MCL as compared with the other lymphomas and that may represent antibodies of a diagnostic value. One example of such an antibody (HPRK250027) is shown in Fig. 4 where a very distinct staining pattern is displayed.

Interestingly a number of antibodies (n = 25) not only recognized MCL but also other lymphomas, such as FL, CLL, and MGZ lymphomas (Fig. 5). Six of these antibodies with lymphoma-associated reactivity did not recognize any of the tested normal B cells but seemed to identify tumor-associated antigens.

In therapeutic settings, antibodies targeting pan-B cell antigens have shown a clinical effect in the treatment of B cell lymphomas and are surprisingly well tolerated by the patients with no or only mild side effects (43). This demonstrates that antibodies recognizing both MCL and normal B cells have a therapeutic value, although our focus has been to identify MCL-associated antigens not found in normal B cells. The reason for this is that these antigens might have an even greater clinical benefit to the patient as therapeutic targets. In contrast, depletion of the T cell compartment is a potential life-threatening condition (44) that must be prevented, and it is therefore of interest to note that only seven of 83 antibodies analyzed seemed to recognize cells in the T cell zone (Fig. 2). Detailed analysis of clinically well documented patient material is presently being performed before selecting antigens for further clinical studies and for generation of human recombinant antibodies, which are more suitable for clinical development and therapy (31).

In summary, we have used gene expression analysis as a preselection tool to identify genes that are overexpressed in MCL, identifying potentially interesting targets for diagnosis and/or therapy. These genes formed the basis for a successful strategy using the PrEST concept to derive specific, polyclonal antibodies to identify lymphoma-associated protein signatures. Furthermore the derived antibodies displayed an ability to discriminate between tumor and normal tissue as well as to specifically recognize either mantle cell lymphomas alone or a group of several different lymphomas, which served as a validation of the strategy.

	ACKNOWLEDGMENTS

 
We thank A.-C. Olsson and I. Schliephake for expert technical assistance and Swegene Microarray Resource Center for experimental support.

	FOOTNOTES

 
Received, March 6, 2006

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Published, MCP Papers in Press, March 8, 2006, DOI 10.1074/mcp.M600077-MCP200

¹ The abbreviations used are: MCL, mantle cell lymphoma; CLL, chronic lymphocytic lymphoma/leukemia; FDC, follicular dendritic cell; FL, follicular lymphoma; GC, germinal center; IHC, immunohistochemistry; MGZ, marginal zone; PrEST, protein epitope signature tag; 2D, two-dimensional.

^* The Human Proteome Resource (www.hpr.se) program is funded by the Knut and Alice Wallenberg Foundation. This work was supported in part by the Faculty of Technology at Lund University, BioInvent International AB, and Leukemia and Lymphoma Society Grant 6085-06.

To whom correspondence should be addressed: Dept. of Immunotechnology, Lund University, P O. Box 7031, SE-220 07 Lund, Sweden. Tel.: 46462223824; Fax: 46462224200; E-mail: sara.ek{at}immun.lth.se

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