Clinical Cancer Research Meeting Calendar Metabolism
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Heidebrecht, H.-J.
Right arrow Articles by Parwaresch, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Heidebrecht, H.-J.
Right arrow Articles by Parwaresch, R.
Clinical Cancer Research Vol. 12, 4804-4811, August 15, 2006
© 2006 American Association for Cancer Research

Human Cancer Biology

Characterization and Expression of CT45 in Hodgkin's Lymphoma

Hans-Jürgen Heidebrecht1, Alexander Claviez2, Marie Luise Kruse3, Marc Pollmann1, Friedrich Buck4, Sönke Harder4, Markus Tiemann1, Wolfgang Dörffel5 and Reza Parwaresch1,{dagger}

Authors' Affiliations: 1 Department of Hematopathology and Lymph Node Registry, 2 Department of Pediatrics, and 3 First Department of Medicine, University of Kiel, Kiel, Germany; 4 Institute for Clinical Chemistry, University of Hamburg, Hamburg, Germany; and 5 Second Department of Pediatrics, HELIOS-Klinikum Berlin-Buch, Berlin, Germany

Requests for reprints: Hans-Jürgen Heidebrecht, Department of Hematopathology, University of Kiel, Niemannsweg 11, D-24105 Kiel, Germany. Phone: 49-431-597-3393; Fax: 49-431-597-3428; E-mail: hheidebrecht{at}path.uni-kiel.de.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: The monoclonal antibody Ki-A10 (IgG1) generated after immunization of mice with Hodgkin's lymphoma cell line L428 detects a nuclear antigen in human tissues with a restricted distribution pattern similar to cancer/testis antigens. The aim of this study was to characterize the antigen and to determine the expression profile in Hodgkin's lymphoma.

Experimental Design: The half-life and phosphorylation of the antigen were determined by radiolabeling. The antigen was characterized by immunopurification and sequencing. Demethylation of genes is used to induce cancer/testis antigens. Ki-A10-negative cells were treated with 5-aza-2'-deoxycytidine. The Ki-A10 expression in paraffin-embedded tumors was determined immunohistochemically.

Results: Immunopurification of the 25/22-kDa antigen and sequencing revealed a peptide of 14 amino acids corresponding to the gene product of the newly described gene family MGC27005, located on chromosome Xq26.3, now termed CT45. CT45 is significantly phosphorylated and down-regulated during mitosis. Demethylation of CT45-negative HeLa cells and stimulated peripheral blood lymphocytes induced CT45 expression. Except testis, immunohistochemical stainings of normal tissues, reactive lymphoid lesions, and most malignant tumors were negative. In comparison, 54 of 99 (55%) samples from pediatric and adolescent Hodgkin's lymphoma patients enrolled in the multicenter trial HD-95 stained Ki-A10 positive. Ki-A10 expression correlated with histologic subtypes (nodular sclerosis Hodgkin's lymphoma 68% versus mixed cellularity Hodgkin's lymphoma 40% versus nodular lymphocyte predominant Hodgkin's lymphoma 9%; P < 0.001).

Conclusions: Ki-A10 is the first monoclonal antibody that detects CT45. As benign lymphoid lesions did not express CT45, the use of Ki-A10 antibody will facilitate the discrimination of Hodgkin's lymphoma from reactive lymphadenopathies.

The monoclonal antibody (mAb) Ki-A10, generated by immunizing mice with lysates of the Hodgkin's lymphoma–derived cell line L428, detects a nuclear antigen with a unique distribution pattern in normal human tissues. The antigen is exclusively expressed in premeiotic precursors of spermatogenesis but is totally absent in normal human somatic tissues (1). Most human tumors do not express this antigen. Only a proportion of germ cell–derived tumors and some malignant lymphomas are stained by the mAb Ki-A10 (1). This distribution pattern is reminiscent of that exhibited by the cancer/testis antigens. Cancer/testis antigens, a rapidly expanding tumor-associated protein family, are of broad interest because of their diagnostic and possible therapeutic implications (2–5). Only few studies thus far have investigated the cancer/testis gene expression in Hodgkin's lymphoma. Cancer/testis gene products, such as MAGE-A4 and SSX, were reported only in a subset of Hodgkin's lymphoma cases (6, 7).

The diagnosis of Hodgkin's lymphoma relies on morphologic criteria [i.e., the diagnostic Hodgkin and Reed-Sternberg cells in classic Hodgkin's lymphoma or lymphocytic and histiocytic cells in nodular lymphocyte predominant Hodgkin's lymphoma (NLPHL)]. In addition, the immunohistochemical expression pattern of CD30, CD15, CD20, and latent membrane protein-1 is of great help in the differential diagnosis of this lymphoma versus other lymphomas and reactive lymphoid entities (8). None of these markers, however, is specific to Hodgkin's lymphoma. All of them may be expressed in larger reactive cells, such as activated immunoblasts (CD20 and CD30) and macrophages (CD15; ref. 8). In addition, no Hodgkin-specific protein has been identified thus far. The diagnosis of Hodgkin's lymphoma may also be complicated by the paucicellular nature of the lymphoma, especially in cases with only partial infiltration. Along this line, distinguishing Hodgkin's cells from reactive lymphoid blasts in cases of EBV infection or other infectious or hyperimmune diseases may prove impossible. On such occasions, the detection of the Ki-A10 antigen in the dubious blasts should provide further support for their neoplastic nature.

The unique distribution pattern detected by the mAb Ki-A10 and its highly restrictive occurrence in neoplastic cells, such as Hodgkin and Reed-Sternberg cells, justifies a closer look into the biology of the antigen and the diagnostic potential of mAb Ki-A10.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Nonmalignant tissue samples. Nonmalignant tissue samples and reactive lymphoid lesions available from the Lymph Node Registry, German Society of Pathologists and the Department of Pathology, University of Kiel were screened. This included neural tissue (n = 7) and tissue from the cardiovascular system (n = 6); lungs and respiratory tract (n = 9); skin, connective, and fatty tissue (n = 10); bone, cartilage, ligaments, and synovia (n = 6); hepatobiliary system (n = 11); gastrointestinal tract (n = 19); endocrine and exocrine glands (n = 5); bone marrow, spleen, and thymus (n = 12); lymphatic tissue (n = 203); lymph nodes (n = 140); tonsils (n = 63); urovesical tract (n = 5); female genital tract (n = 5); and male genital tract (testis excluded; n = 5). In addition, seven testes were screened.

Malignant tumors. Various types of malignant tumors from the following organ systems were screened: respiratory tract (n = 27), gastrointestinal tract (n = 166), liver (n = 10), pancreas (n = 16), breast (n = 81), urogenital tract (n = 137), sarcomas (n = 23), brain tumors (n = 40), melanoma (n = 34), hematologic neoplasms (n = 263), and malignant lymphomas, excluding Hodgkin's lymphoma (n = 559).

Hodgkin's lymphomas. Diagnostic lymph node biopsy specimens from 99 consecutive patients (50 boys and 49 girls) diagnosed in 2000 and enrolled in the prospective pediatric multicenter trial HD-95 of the Society of Pediatric Oncology and Hematology were studied for the presence of the Ki-A10 antigen. The median age of the patients was 14.8 years (range, 4.1-17.4 years). All diagnoses were verified by a panel of experienced hematopathologists based on conventional paraffin sections and immunohistochemistry (8). The histologic subtypes were distributed as follows: 11 (11%) patients had NLPHL, 63 (64%) patients had nodular sclerosis Hodgkin's lymphoma (NSHL), and 25 (25%) patients had mixed cellularity Hodgkin's lymphoma. Of the patients with NSHL subtype, 54 had the Bennett 1 subtype and 9 patients had the blast-rich variant (Bennett 2; ref. 9).

Cell culture. All cell lines were grown in DMEM or RPMI 1640 without HEPES supplemented with 10% FCS, 10 mmol/L glutamine, and 50 µg/mL streptomycin and penicillin. Peripheral blood lymphocytes (PBL) from healthy donors were incubated with phytohemagglutinin (10 µg/mL; Sigma, Deisenhofen, Germany) and 100 units/mL cell culture tested human interleukin-2 (PromoCell, Heidelberg, Germany) with or without 1 µg/mL 5-aza-2'-deoxycytidine (DAC; Sigma; refs. 10, 11). HeLa cells were incubated on glass slides and cultured with 1 µg/mL DAC. From PBLs, conventional cytospin slides were prepared. They were fixed in acetone for 10 minutes at room temperature and kept until use.

Imunohistochemistry. Tissue samples fixed in 5% to 10% formaldehyde for various, mostly unknown, periods were processed routinely. Tissue sections (~4 µm thick) were fixed onto sialinized slides (12). Antigen retrieval was achieved by boiling the slides in 0.01 mol/L citric acid (pH 6.0) for 2 minutes in a pressure cooker (13). Cryostat sections and cytospin slides were fixed in acetone at room temperature for 10 minutes and incubated with the supernatant of the primary antibody for 60 minutes. The immunoreaction was visualized by the alkaline phosphatase-anti-alkaline phosphatase (APAAP) or streptavidin-biotin complex techniques (DAKO, Hamburg, Germany; refs. 12, 14). The slides were briefly counterstained with Meyer's hemalum (Fluka, Steinheim, Germany). As controls of the immunohistochemical stainings, sections of normal or reactive lymphoid tissues were prepared and treated in the same manner, except for the primary antibodies, which were replaced by an irrelevant isotype mAb. Latent EBV infection was documented by immunostaining against latent membrane protein-1 (DAKO) as described before (15).

Immunofluorescence. Cytospin preparations of L428 cells or adherently growing HeLa cells transfected with pRK5/MGC27005 were fixed with acetone and used for indirect immunofluorescence staining with mAb Ki-A10. Detection of the primary antibody was done with a goat anti-mouse IgG polyclonal antibody conjugated with Alexa 594 or 488 (Molecular Probes, Invitrogen, Karlsruhe, Germany). DNA staining was done with 4',6-diamidino-2-phenylindole (Molecular Probes, Invitrogen). Digital images were recorded using a Zeiss Axioplan 2 microscope (Zeiss, Jena, Germany).

Biolabeling and determination of the molecular mass of the Ki-A10 antigen. Cell lysates of L428 cells were subjected to SDS-PAGE on a gel gradient of 7.5% to 15% or 10% to 20%, transferred to nitrocellulose membranes overnight, and stained with mAb Ki-A10 (16).

To determine the turnover time of the Ki-A10 antigen, L428 cells were labeled for 1 hour with [35S]methionine (Amersham Biosciences, Freiburg, Germany). Subsequently, the cells were intensively washed and immunoprecipitation experiments were done at different time intervals after the end of labeling (17). To determine whether the Ki-A10 antigen is phosphorylated, radiolabeling experiments were done with [32P]orthophosphate (Amersham Biosciences; ref. 17).

For protein sequencing experiments, a lysate preparation of at least 2 x 108 L428 cells was used. The gel was stained with Coomassie blue; the relevant bands were cut out; the cysteine residues were modified with iodacetamide and the protein in-gel was digested with trypsin (18).

Expression of MGC27005 protein (Ki-A10 antigen) in HeLa cells. To analyze transient expression of MGC27005 in HeLa cells, the coding region of MGC27005 was PCR amplified using cDNA clone IMAGp958O222761Q (Deutsches Ressourcenzentrum für Genomforschung, Berlin, Germany) as template. Both primers used for amplification harbored BamHI restriction enzyme sites. After restriction digestion, the MGC27005 cDNA was ligated into the BamHI site of pRK5 vector for eukaryotic expression.

Transient transfection of pRK5/MGC27005 construct into HeLa cells grown on coverslips was done in 24-well cell culture plates. Plasmid DNA (200 ng) per 0.5 mL/well medium was transfected using FuGENE6 transfection reagent (Roche, Mannheim, Germany) according to the manufacturer's instructions. After 48 hours, the cells were fixed for 10 minutes with methanol or acetone at –20°C and incubated with Ki-A10 antibody for 1 hour. Detection was done as described above.

Recombinant expression of MGC27005 protein. To investigate recombinant expression of the MGC27005 protein, the TNT-coupled reticulocyte lysate system (Promega, Mannheim, Germany) was used. For this purpose, the coding region of MGC27005 was PCR amplified using cDNA clone IMAGp958O222761Q as template. Both primers used for amplification contained a BamHI restriction enzyme site. After restriction digestion, MGC27005 was ligated into the BamHI site of the pGEM-3Z expression vector (Promega). The correct orientation and authenticity of the insert's DNA sequence were confirmed via automated sequencing using an ABI PRISM 310 genetic analyzer. Nonradioactive expression of MGC27005 protein (amino acids 1-189) was achieved using 1 µg purified plasmid DNA in a total assay volume of 50 µL. All steps were done as suggested in the manufacturers' protocols. The human proliferation-associated protein repp86/hTPX2 (molecular mass, ~100 kDa) expressed in the same system served as negative control (17).

Statistical analysis. Categorical data were compared by the {chi}2 test. The tests were two-sided. P < 0.05 was considered statistically significant. Calculations were done with SPSS software for Windows version 11.5.1 (SPSS, Inc., Chicago, IL).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Ki-A10 antigen in cell lines
The mAb Ki-A10 was detected on cytospin preparations of the human Hodgkin's lymphoma–derived cell line L428, showing strong nuclear reactivity. Immunostainings of further human tumor cell lines with mAb Ki-A10 revealed that the majority of the cell lines of hematopoietic origin (e.g., U937, HL60, THP1, K562, and Raji) lacked any Ki-A10 expression. Only the plasmacytoma-derived cell line U266Bl and the two Hodgkin's lymphoma–derived cell lines L428 and L540 stained nearly all cell nuclei positively. In other Hodgkin's lymphoma–derived cell lines, such as HDLM-2 and KM-H2, <10% of the cells showed nuclear reactivity. HeLa cells were consistently negative.

Post-translational modification, sequencing, and induction of expression of the Ki-A10 antigen
Immunostaining of Western blots of nuclear extracts from L428 cells revealed a double band of 22- and 25-kDa apparent molecular mass (Fig. 1A ). Labeling of L428 cells with [32P]orthophosphate and subsequent immunoprecipitation with the mAb Ki-A10 showed significant phosphorylation of the Ki-A10 antigen (Fig. 1B). Pulse-chase experiments with [35S]methionine-labeled L428 cells followed by immunoprecipitation at various times with the mAb Ki-A10 revealed an obvious decrease in labeled Ki-A10 antigen 3 hours after the end of labeling (Fig. 1C). Densitometric evaluation of the immunoprecipitates showed a constant decrease in labeled Ki-A10 antigen. Three hours after the end of labeling, only 47% of the labeled antigen was detectable, indicating a half-life of ~3 hours (Fig. 1C).


Figure 1
View larger version (17K):
[in this window]
[in a new window]
  		Fig. 1. Characterization of Ki-A10 antigen (CT45). A, determination of the molecular weight of Ki-A10 antigen. A nuclear lysate of L428 cells stained with Ki-A10 (IgG1; b) shows two distinct protein bands corresponding to ~25 and 22 kDa; isotype control (a). B, immunoprecipitation experiments with mAb Ki-A10 on L428 cells after the cells were labeled with [32P]orthophosphate. c, isotype control; d, Ip Ki-A10. C, determination of the turnover time of CT45. L428 cells were labeled for 1 hour with [35S]methionine and then intensively washed. Immunoprecipitation experiments were done at the end of labeling (f; t0), 30 minutes (t30), 60 minutes (t60), and 180 minutes (t180) after the end of labeling. Densitometric analysis of the immunoprecipitated CT45. CT45 immunoprecipitated at t0 was designated as 100%. Isotype control experiments at each point in time (e). Left, molecular weight marker (A-C).

 
For immunopurification and sequencing of the Ki-A10 antigen, extracts of at least 2 x 108 cell nuclei of L428 cells were prepared. Immunoprecipitation was done with mAb Ki-A10 coupled to protein A-Sepharose. After intensive washing steps, the purified Ki-A10 antigen and, as a control, the Ki-A10 antibody without Ki-A10 antigen were separated on a 10% to 20% gradient gel. The Coomassie blue–stained bands of the Ki-A10 antigen were excised, pooled, and in-gel digested with 5 ng trypsin/µL. The light chain of mAb Ki-A10 was excised and digested for control as well. The resulting peptides were analyzed by nano-electrospray mass spectrometry. After exclusion of all peptides belonging to the light chain of mAb Ki-A10, one peptide of 14 amino acids (IFEMLGYQGPTAVR) was detected that corresponded to the gene product of a hitherto uncharacterized gene family of six members termed MGC27005 (AAH28711.1). This gene family, which differs by only 2 to 12 bp, has recently been designated CT45 (19). CT45 is a protein of 189 amino acids with a theoretical molecular mass of 21,159 Da containing two nuclear localization signals. It is located on chromosome Xq26.3. In addition, CT45 showed a significant homology with the COOH terminus of a human sarcoma antigen, which has already been characterized as a cancer/testis antigen (20).

Verification of the nature of the antigen
To confirm our results, the coding cDNA of MGC27005 (IMAGp958O222761Q) was cloned into the eukaryotic expression vector pRK5 and subsequently transfected into Ki-A10-negative HeLa cells. Forty-eight hours after transfection, some HeLa cells showed distinct nuclear expression of MGC27005 when immunostained with the mAb Ki-A10 (Fig. 2B ). Control HeLa cells transfected with pRK5 without insert stained with Ki-A10 displayed only weak cytoplasmic background staining (Fig. 2A). In addition, MGC27005 and the nuclear protein repp86 used as a control were expressed in the reticulocyte lysate system (17, 21). Western blots of these lysates showed that MGC27005 is detected by the mAb Ki-A10 (Fig. 2C, a), whereas the control repp86 expression was negative (Fig. 2C, b).


Figure 2
View larger version (21K):
[in this window]
[in a new window]
  		Fig. 2. Verification of the cancer/testis protein MGC27005 as Ki-A10 antigen. MGC27005 (CT45)–negative HeLa cells were transfected with pRK5/MGC27005 or pRK5 without insert. Cells were stained with mAb Ki-A10 48 hours after transfection. A, HeLa cells transfected with pRK5 without insert did not show any nuclear staining. B, in contrast, three pRK5/MGC27005-transfected cells showed distinct nuclear staining. DNA staining was done with 4',6-diamidino-2-phenylindole. C, expression of MGC27005 (CT45) and repp86, a different nuclear protein as negative control, in the reticulocyte lysate system. MGC27005 (CT45) and repp86 were transfected in the reticulocyte lysate system and blotted. Ki-A10 stained two protein bands of the expected size (a), whereas the control transfection with repp86, a protein of ~100 kDa, is completely negative for Ki-A10 (b).

 
Induction of CT45 expression by demethylation
DNA hypomethylation is a common mechanism for inducing cancer/testis antigen expression in human cell lines or PBLs that do not express cancer/testis antigens (10, 11, 22). The demethylating agent DAC was used to induce DNA demethylation in vitro (10, 23). CT45-negative HeLa cells and PBLs from various healthy donors stimulated with interleukin-2 (100 units/mL) and phytohemagglutinin (10 µg/mL) were incubated with 1 µg DAC/mL for at least 8 days. After 6 days, control HeLa cells (Fig. 3A ) did not express CT45, whereas ~50% of the DAC-treated HeLa cells strongly expressed CT45 in their cell nuclei (Fig. 3B). In addition, Western blotting was done with lysates of L428 cells serving as positive controls, with untreated HeLa cells, and with DAC-treated HeLa cells. Strong CT45 expression was detectable in lysates of L428 cells (Fig. 3E, a) and DAC-treated HeLa cells (Fig. 3E, c), whereas untreated cells remained negative (Fig. 3E, b). Stimulated PBLs showed weak but distinct nuclear staining with the mAb Ki-A10 after 3 days. After 6 days ~10% of PBLs strongly expressed CT45 in their cell nuclei (Fig. 3D). About 90% of these stimulated PBLs expressed CD3, and ~40% expressed CD30 (data not shown). Preliminary double immunofluorescence stainings showed Ki-A10 expression in CD3+ and CD20+ cells (data not shown). Mitogen-treated PBLs without DAC treatment remained negative (Fig. 3C). Unstimulated PBLs treated with DAC did not show any distinct nuclear staining within 2 weeks of cell culture. Strong nuclear CT45 expression was still detectable in mitogen- and DAC-treated PBLs for at least 2 weeks. One Ki-A10-positive cell showed an atypical nuclear morphology (Fig. 3D).


Figure 3
View larger version (77K):
[in this window]
[in a new window]
  		Fig. 3. Induction of CT45 by DAC in adherently growing HeLa cells (A and B) and mitogen-stimulated PBLs (C and D). HeLa cells were treated with 1 µg/mL DAC. A, after 6 days, no nuclear staining is detected in HeLa cells without DAC, whereas >50% of the DAC-treated cells show strong nuclear staining with mAb Ki-A10. B, PBLs from healthy donors were treated with phytohemagglutinin (10 µg/mL) and interleukin-2 (100 units/mL) without (C) and with (D) DAC. About 10% of DAC-treated PBLs revealed strong nuclear staining with mAb Ki-A10 after 6 days. One CT45-expressing cell showed a cell nucleus with an atypical morphology (APAAP staining). E, lysates of HeLa cells treated without (b) and with (c) DAC for 6 days were blotted and stained with Ki-A10; a, a lysate of L428 cells served as positive control. Only DAC-treated HeLa cells express the Ki-A10 antigen.

 
Tissue distribution of CT45 (Ki-A10 antigen)
CT45 expression in normal human tissues. No Ki-A10 antigen expression was detectable in sections of any of the 303 normal tissues, except testis. Lymph nodes and tonsils from patients with acute EBV infection showed the typical Hodgkin-like blasts and small necrotic areas. In no case were Ki-A10-positive cells detected; especially, immunoblasts and Hodgkin-like B blasts were negative. In the seven testes investigated, only spermatogonia and spermatocytes of first order showed nuclear positivity for Ki-A10 (Fig. 4A ).


Figure 4
View larger version (122K):
[in this window]
[in a new window]
  		Fig. 4. Immunohistochemistry of normal testis (A) and Hodgkin's lymphoma (B-F). A, ductus seminiferus with Ki-A10-positive spermatogonia and spermatocytes. Duct epithelium stains negative. Human testis (APAAP; magnification, x800). B, immunofluorescence staining of Hodgkin's lymphoma cell line L428 with Ki-A10 shows a distinct nuclear staining. During mitosis, CT45 expression is down-regulated (magnification, x1,200). C, selective nuclear Ki-A10 staining of neoplastic cells from a patient with NSHL. Human lymph node (APAAP; magnification, x120). D, same specimen as shown in (C) with higher magnification of a binucleated Hodgkin and Reed-Sternberg cell and two Hodgkin's cells. Human lymph node (APAAP; magnification, x800). E, blast-rich variant of a case of NSHL (subtype Bennett 2), showing sheets of Ki-A10-positive tumor cells. Human lymph node (APAAP; magnification, x120). F, Ki-A10-positive Hodgkin and Reed-Sternberg cells in a case of mixed cellularity Hodgkin's lymphoma. Human lymph node (APAAP; magnification, x240).

 
CT45 expression in malignant tumors and lymphomas, excluding Hodgkin's lymphoma. All tumors from the respiratory tract, hepatocellular carcinomas, renal cell carcinomas, adenocarcinomas of the uterus, prostate cancers, and brain tumors were consistently Ki-A10 negative. Tumors of the gastrointestinal tract were Ki-A10 negative, with the exception of one case of esophageal carcinoma and one case of breast cancer. Of 107 adenocarcinomas of the colon, 12 (11%) cases stained positive in the nuclei. In contrast, the majority of seminomas, dysgerminomas, and intratubular germ cell neoplasia [100 of 109 (92%)] stained positively for Ki-A10, a finding reported previously (1). Four of 34 melanomas, including metastases, were Ki-A10 positive, whereas the group of leukemias studied were Ki-A10 negative. Ki-A10 expression was observed only in a subset of malignant lymphomas, such as diffuse large B-cell lymphomas, anaplastic plasmacytomas, and large cell lymphomas of T phenotype (Table 1 ). Other tumor types showed only single positive cases.


View this table:
[in this window]
[in a new window]
  		Table 1. Immunohistochemical occurrence of CT45 antigen in various malignant tumors other than Hodgkin's lymphoma

 
CT45 expression in Hodgkin's lymphoma. Immunofluorescence staining of the Hodgkin's lymphoma cell line L428 showed a distinct nuclear positivity with Ki-A10 in interphase. Mitotic cells, however, were only weakly stained (Fig. 4B). Among 99 pediatric patients with Hodgkin's lymphoma, tumor cells from 54 (55%) cases were Ki-A10 positive. Ki-A10 positivity was confined to the nuclei of the Hodgkin and Reed-Sternberg and lymphocytic and histiocytic cells, whereas reactive cells were consistently negative (Fig. 4C-F). In sections of paraffin-embedded tissues, Ki-A10 showed distinct nuclear staining. In most cases, all neoplastic cells stained with the same strong intensity. Only in a few cases partial Ki-A10 expression was detectable (i.e., most cells were positive, but single cells were negative). This phenomenon was restricted to tumor cells in single nodules in the NSHL subtype.

All but one case of classic Hodgkin's lymphoma were CD30+ (98%) in contrast to 1 of 11 cases of NLPHL subtype (P < 0.001). Ki-A10-positive cases expressed CD30. A correlation was observed between Ki-A10 expression and histologic subtype. Of 88 cases with classic Hodgkin's lymphoma, 53 (60%) were positive in contrast to 1 of 11 (9%) of NLPHL (P = 0.001). In addition, significantly more patients with NSHL were Ki-A10 positive than those with mixed cellularity Hodgkin's lymphoma as shown in Table 2 . Ki-A10 positivity was significantly higher in the blast-rich NSHL subtype [Bennett 2; 9 of 9 (100%)] than in those cases with Bennett 1 [34 of 54 (63%), P = 0.03; ref. 9]. There was no significant difference between boys and girls with respect to Ki-A10 positivity (24 of 50 versus 22 of 49; P = 0.76).


View this table:
[in this window]
[in a new window]
  		Table 2. CT45 expression in pediatric Hodgkin's lymphoma patients with relation to histologic subtype

 
The Ki-A10 expression status was independent of latent EBV infection as determined by latent membrane protein-1 expression in Hodgkin's lymphoma tissue specimens. In 12 of 29 EBV-infected patients, Ki-A10 was positive, whereas 24 of 59 EBV-negative patients were Ki-A10 positive (P = 0.11).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In normal tissues, the mAb Ki-A10 identifies a nuclear antigen whose occurrence is strictly restricted to the human testis. Among all tissue types, only spermatogonia and first-order and second-order spermatocytes express the antigen, which is absent in spermatids. In a subset of germ cell–derived tumors, predominantly seminomas and dysgerminomas, a strong nuclear reactivity pattern was regularly detectable as shown previously (1). Screening of a wide range of other solid cancers with Ki-A10 revealed that only a few of them express this antigen. This unique expression pattern, which is shared by the normal testis and some malignancies of various histologic types, implies that the Ki-A10 antigen belongs to the growing family of cancer/testis antigens (2–4, 24, 25). To date, 44 cancer/testis gene families have been identified (3). In this article, we provide evidence that the mAb Ki-A10 detects the product of the MGC27005 gene family, which is localized on chromosome Xq26.3 (19). This gene family differs by only 2 to 12 bp. All members of this gene family comprise 189 amino acids and contain the sequenced peptide. The MGC27005 gene product is a new member of the chromosome X–associated cancer/testis antigens (CT-X antigens; ref. 26).

The MGC27005 gene family was identified by massively parallel signature sequencing as a gene strongly expressed in the testis and in some tumor cell lines. MGC27005 is designated CT45 in the cancer/testis nomenclature of Scanlan et al. (3, 19). The gene product of CT45 was identified after immunopurification with the mAb Ki-A10 and subsequent sequencing. CT45 is a protein of 189 amino acids with a theoretical molecular mass of 21,159 Da containing two nuclear localization sites, possible N-glycosylation sites and several possible phosphorylation sites. These theoretical data are compatible with the distinct nuclear localization of CT45 in immunostainings of human testis and tumor tissues and the significant phosphorylation in L428-arrested cells. The slightly different mass of the two proteins (22/25 kDa) could be caused by post-translational modifications of the protein. Preliminary characterizations of the CT45 protein showed significant phosphorylation and a short half-life of ~3 hours. Short turnover times of proteins offer cells a good possibility to regulate the protein expression. Transient transfection of CT45 into CT45-negative HeLa cells revealed distinct nuclear staining of transfected cells, evidence that the mAb Ki-A10 detects CT45.

Promoter methylation of many cancer/testis antigens is associated with gene silencing (26). Thus far, all CT-X genes studied have methylated CpG islands in normal somatic tissues (10, 27, 28). Demethylation of mitogen stimulated PBLs that did not express cancer/testis antigens with DAC-induced cancer/testis antigen expression (MAGE-A1, MAGE-A2, and MAGE-A3; refs. 10, 11, 22). Although agents, such as DAC, induce chromosomal damage and chromosomal rearrangements that could effect gene expression, our ability to induce CT45 expression in mitogen-stimulated PBLs from healthy donors or HeLa cells treated with the demethylating agent argues that the most likely possibility is demethylation of CpG islands in the promoter region of CT45 (29, 30). Direct proof of this hypothesis is impossible because the promoter and enhancer regions of CT45 have not yet been characterized. CT45 antigen is expressed in both CD3+ and CD20+ cells. DAC-treated PBLs without mitogenic stimulation did not express CT45, indicating that hypomethylation alone is not sufficient for CT45 expression. In human testis, spermatogonia and spermatocytes express CT45 antigen and harbor the Ki-67 antigen, a protein only expressed in proliferating cells from G1 until the end of mitosis (31). Immunostainings of Hodgkin's lymphoma with a mAb specific to the Ki-67 antigen showed that ~80% of the Hodgkin and Reed-Sternberg cells were at least in G1 cell cycle phase, showing that CT45 antigen expression is probably associated with cell proliferation in these cells (32). Nuclear expression of CT45 in a small fraction of resting cells of the Hodgkin's cell line L428 could be a consequence of the process of cell culturing. Promoter demethylation and additional mitogenic stimuli seem to be necessary for the expression of CT45. Thus far, the biological function of CT-X antigens in germ line and tumor cells is poorly understood. The central question is whether their expression contributes to tumorigenesis or not (26).

The expression of cancer/testis antigens in various human carcinomas and lymphomas is currently being studied by several groups with a view to therapy and diagnosis (7, 33–36). Because of the restricted expression pattern in normal tissues, various groups have studied cancer/testis antigens as possible targets for immunotherapy and gene therapy (5). Objective evidence for the presence of cancer/testis genes in Hodgkin's lymphoma has been presented (6, 7, 37). MAGE-A4 (CT1) was detected by reverse transcription-PCR in 5 of 18 (28%) cases and the protein was found by immunohistochemistry in 11 of 53 (21%) cases (6). The protein expression was confined to Hodgkin and Reed-Sternberg cells. SSX (CT15) expression was analyzed by reverse transcription-PCR and in situ hybridization (7). Reverse transcription-PCR revealed CT15 in the Hodgkin's lymphoma–derived cell lines L428, L540, KM-H2, and HD-MY-Z. In addition, by reverse transcription-PCR, CT15 was shown in tissue samples in 5 of 32 (16%) cases. In situ hybridization revealed CT15 to be positive in 6 of 11 cases. These cases were negative by reverse transcription-PCR (7). Because of the small number of cases investigated, no statistical analyses were done in these two studies.

In our series, 53 of 88 (60%) cases of various types of classic Hodgkin's lymphoma were Ki-A10 positive but only 1 of 11 cases of NLPHL. Only Hodgkin and Reed-Sternberg cells were Ki-A10 positive, whereas all cases of infectious mononucleosis and reactive lymphadenopathies were negative. This finding was in sharp contrast to the expression pattern of CD30 in virus-induced Hodgkin-like lymphadenopathies. mAb specific to cancer/testis antigens have been used to improve the diagnosis of various malignant tumors (1, 36). The diagnosis of Hodgkin's lymphoma may be difficult for a variety of reasons. These include the rarity of Hodgkin and Reed-Sternberg and lymphocytic and histiocytic cells, especially in cases in which the sampling was inadequate or where there is only partial tumor cell infiltration. Moreover, CD30 may be expressed in benign lymphadenopathies, such as infectious mononucleosis, whereas the Ki-A10 antibody is only found in malignant tumor cells. Consequently, analysis of CT45 antigen expression in addition to the use of established antibodies (CD30, CD15, and CD20) might be of great value for the discrimination between benign and malignant neoplasms, especially in a paucicellular tumor, such as Hodgkin's lymphoma.

It is noteworthy that there was a difference in CT45 expression between the histologic subtypes of Hodgkin's lymphoma (Table 2). Patients with classic Hodgkin's lymphoma more often expressed CT45 than those with NLPHL. Especially, in the aggressive variant of NSHL subtype, there was obvious Ki-A10 positivity, indicating that cancer/testis antigen expression may be associated with a more aggressive biological behavior of the disease. Notably, it has been recently reported that CT1 and CT7 expression in multiple myeloma correlated with elevated plasma cell proliferation and advanced disease stage (38). Therefore, a systematic study of CT45 in selected entities may help to define its role in diagnosis and prognosis.


   Acknowledgments
 
In memory of Prof. Reza Parwaresch, M.D., Ph.D., who died unexpectedly on November 1, 2005.

We thank the Deutsches Ressourcenzentrum für Genomforschung for providing the clone IMAGp958O222761Q, Dr. Bence Sipos for kindly providing tissue arrays, Monika Hauberg for excellent technical assistance, and Kay Dege for valuable help with the preparation of the manuscript.


   Footnotes
 
Grant support: Kinderkrebsinitiative Buchholz Holm-Seppensen, Germany.

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.

Deceased. Back

Received 1/26/06; revised 5/25/06; accepted 6/ 6/06.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Rudolph P, Kellner U, Schmidt D, et al. Ki-A10, a germ cell nuclear antigen retained in a subset of germ cell-derived tumors. Am J Pathol 1999;154:795–803.[Abstract/Free Full Text]
  2. Boon T, Old LJ. Cancer tumor antigens. Curr Opin Immunol 1997;9:681–3.[CrossRef][Medline]
  3. Scanlan MJ, Simpson AJ, Old LJ. The cancer/testis genes: review, standardization, and commentary. Cancer Immun 2004;4:1.[Medline]
  4. Sahin U, Tureci O, Pfreundschuh M. Serological identification of human tumor antigens. Curr Opin Immunol 1997;9:709–16.[CrossRef][Medline]
  5. Scanlan MJ, Gure AO, Jungbluth AA, Old LJ, Chen YT. Cancer/testis antigens: an expanding family of targets for cancer immunotherapy. Immunol Rev 2002;188:22–32.[CrossRef][Medline]
  6. Chambost H, Van Baren N, Brasseur F, et al. Expression of gene MAGE-A4 in Reed-Sternberg cells. Blood 2000;95:3530–3.[Abstract/Free Full Text]
  7. Colleoni GW, Capodieci P, Tickoo S, Cossman J, Filippa DA, Ladanyi M. Expression of SSX genes in the neoplastic cells of Hodgkin's lymphoma. Hum Pathol 2002;33:496–502.[CrossRef][Medline]
  8. Stein H, Delsol G, Pileri S, et al. Hodgkin lymphoma. In: Jaffe ES, Harris NL, Stein H, Vardiman JW, editors. WHO classification of tumours. Pathology and genetics of tumours of hematopoietic and lymphoid tissues. Lyon: IARC; 2001. p. 237–53.
  9. Bennett MH, MacLennan KA, Easterling MJ, Vaughan HB, Jelliffe AM, Vaughan HG. The prognostic significance of cellular subtypes in nodular sclerosing Hodgkin's disease: an analysis of 271 non-laparotomised cases (BNLI report no. 22). Clin Radiol 1983;34:497–501.[CrossRef][Medline]
  10. Weber J, Salgaller M, Samid D, et al. Expression of the MAGE-1 tumor antigen is up-regulated by the demethylating agent 5-aza-2'-deoxycytidine. Cancer Res 1994;54:1766–71.[Abstract/Free Full Text]
  11. Shichijo S, Yamada A, Sagawa K, et al. Induction of MAGE genes in lymphoid cells by the demethylating agent 5-aza-2'-deoxycytidine. Jpn J Cancer Res 1996;87:751–6.[CrossRef]
  12. Cordell JL, Falini B, Erber WN, et al. Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J Histochem Cytochem 1984;32:219–29.[Abstract]
  13. Taylor CR, Shi SR, Chaiwun B, Young L, Imam SA, Cote RJ. Strategies for improving the immunohistochemical staining of various intranuclear prognostic markers in formalin-paraffin sections: androgen receptor, estrogen receptor, progesterone receptor, p53 protein, proliferating cell nuclear antigen, and Ki-67 antigen revealed by antigen retrieval techniques. Hum Pathol 1994;25:263–70.[CrossRef][Medline]
  14. Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 1981;29:577–80.[Abstract]
  15. Claviez A, Tiemann M, Lüders H, et al. Impact of latent Epstein-Barr virus infection on outcome in children and adolescents with Hodgkin's lymphoma. J Clin Oncol 2005;23:4048–56.[Abstract/Free Full Text]
  16. Heidebrecht HJ, Buck F, Haas K, Wacker HH, Parwaresch R. Monoclonal antibodies Ki-S3 and Ki-S5 yield new data on the "Ki-67" proteins. Cell Prolif 1996;29:413–25.[Medline]
  17. Heidebrecht HJ, Buck F, Steinmann J, Sprenger R, Wacker HH, Parwaresch R. p100: a novel proliferation-associated nuclear protein specifically restricted to cell cycle phases S, G2, and M. Blood 1997;90:226–33.[Abstract/Free Full Text]
  18. Lellek H, Kirsten R, Diehl I, Apostel F, Buck F, Greeve J. Purification and molecular cloning of a novel essential component of the apolipoprotein B mRNA editing enzyme-complex. J Biol Chem 2000;275:19848–56.[Abstract/Free Full Text]
  19. Chen YT, Scanlan MJ, Venditti CA, et al. Identification of cancer/testis-antigen genes by massively parallel signature sequencing. Proc Natl Acad Sci U S A 2005;102:7940–5.[Abstract/Free Full Text]
  20. Martelange V, De Smet C, De Plaen E, Lurquin C, Boon T. Identification on a human sarcoma of two new genes with tumor-specific expression. Cancer Res 2000;60:3848–55.[Abstract/Free Full Text]
  21. Heidebrecht HJ, Adam-Klages S, Szczepanowski M, et al. repp86: a human protein associated in the progression of mitosis. Mol Cancer Res 2003;1:271–9.[Abstract/Free Full Text]
  22. Sigalotti L, Fratta E, Coral S, et al. Intratumor heterogeneity of cancer/testis antigens expression in human cutaneous melanoma is methylation-regulated and functionally reverted by 5-aza-2'-deoxycytidine. Cancer Res 2004;64:9167–71.[Abstract/Free Full Text]
  23. Guo ZS, Hong JA, Irvine KR, et al. De novo induction of a cancer/testis antigen by 5-aza-2'-deoxycytidine augments adoptive immunotherapy in a murine tumor model. Cancer Res 2006;66:1105–13.[Abstract/Free Full Text]
  24. Zendman AJ, Ruiter DJ, Van Muijen GN. Cancer/testis-associated genes: identification, expression profile, and putative function. J Cell Physiol 2003;194:272–88.[CrossRef][Medline]
  25. Kalejs M, Erenpreisa J. Cancer/testis antigens and gametogenesis: a review and "brain-storming" session. Cancer Cell Int 2005;5:4.[CrossRef][Medline]
  26. Simpson AJ, Caballero OL, Jungbluth A, Chen YT, Old LJ. Cancer/testis antigens, gametogenesis and cancer. Nat Rev Cancer 2005;5:615–25.[CrossRef][Medline]
  27. De Smet C, De Backer O, Faraoni I, Lurquin C, Brasseur F, Boon T. The activation of human gene MAGE-1 in tumor cells is correlated with genome-wide demethylation. Proc Natl Acad Sci U S A 1996;93:7149–53.[Abstract/Free Full Text]
  28. De Smet C, Lurquin C, Lethe B, Martelange V, Boon T. DNA methylation is the primary silencing mechanism for a set of germ line- and tumor-specific genes with a CpG-rich promoter. Mol Cell Biol 1999;19:7327–35.[Abstract/Free Full Text]
  29. Haaf T. The effects of 5-azacytidine and 5-azadeoxycytidine on chromosome structure and function: implications for methylation-associated cellular processes. Pharmacol Ther 1995;65:19–46.[CrossRef][Medline]
  30. Stresemann C, Brueckner B, Musch T, Stopper H, Lyko F. Functional diversity of DNA methyltransferase inhibitors in human cancer cell lines. Cancer Res 2006;66:2794–800.[Abstract/Free Full Text]
  31. Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 1984;133:1710–5.[Abstract]
  32. Tiemann M, Claviez A, Lüders H, et al. Proliferation characteristics in pediatric Hodgkin's lymphoma point to a cell cycle arrest in the G(1) phase. Mod Pathol 2005;18:1440–7.[CrossRef]
  33. Luo G, Huang S, Xie X, et al. Expression of cancer-testis genes in human hepatocellular carcinomas. Cancer Immun 2002;2:11.[Medline]
  34. dos Santos NR, Torensma R, de Vries TJ, et al. Heterogeneous expression of the SSX cancer/testis antigens in human melanoma lesions and cell lines. Cancer Res 2000;60:1654–62.[Abstract/Free Full Text]
  35. Xie X, Wacker HH, Huang S, et al. Differential expression of cancer testis genes in histological subtypes of non-Hodgkin's lymphomas. Clin Cancer Res 2003;9:167–73.[Abstract/Free Full Text]
  36. Luftl M, Schuler G, Jungbluth AA. Melanoma or not? Cancer testis antigens may help. Br J Dermatol 2004;151:1213–8.[CrossRef][Medline]
  37. Cossman J, Annunziata CM, Barash S, et al. Reed-Sternberg cell genome expression supports a B-cell lineage. Blood 1999;94:411–6.[Abstract/Free Full Text]
  38. Jungbluth AA, Ely S, Diliberto M, et al. The Cancer-Testis antigens CT7 (MAGE-C1) and MAGE-A3/6 are commonly expressed in multiple myeloma and correlate with plasma cell proliferation. Blood 2005;106:167–74.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 BloodHome page
C. Langer, M. D. Radmacher, A. S. Ruppert, S. P. Whitman, P. Paschka, K. Mrozek, C. D. Baldus, T. Vukosavljevic, C.-G. Liu, M. E. Ross, et al.
High BAALC expression associates with other molecular prognostic markers, poor outcome, and a distinct gene-expression signature in cytogenetically normal patients younger than 60 years with acute myeloid leukemia: a Cancer and Leukemia Group B (CALGB) study
Blood, June 1, 2008; 111(11): 5371 - 5379.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Heidebrecht, H.-J.
Right arrow Articles by Parwaresch, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Heidebrecht, H.-J.
Right arrow Articles by Parwaresch, R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online