Immunotherapy is an important treatment option in multiple myeloma (MM), with allotransplantation demonstrating an inducible graft-versus-myeloma effect.1  This could be potentiated by vaccination, first requiring knowledge of tumor-associated antigens.

The cancer testis antigens (CTAs) are exemplary, as normal expression is restricted to testis, an immune-privileged site. CTAs are being identified in a number of malignancies. In lymphoma, serologic analysis of recombinant cDNA expression (SEREX) analysis has revealed PASD1, a gene encoding the CT antigen OX-TES-1, spliced alternatively to PASD1_v1 and PASD1_v2.2 PASD1 appears to map to chromosome band Xq28 (Unigene Hs.160594), also flanked by the cluster of MAGE gene families.3  With MAGE antigens of importance as immunotherapeutic targets in MM, additional CTA genes at this locus may be significant. Here, we report on PASD1 expression in MM as one such antigen.

We observed PASD1 expression in 5 of 11 MM cell lines by reverse-transcription–polymerase chain reaction (RT-PCR), and in 8 of 11 by increasing sensitivity (Figure 1A). In primary MM, PASD1 expression was found in 14 of 16 samples using quantitative PCR (Q-PCR), both in presentation (MM1-9) and pretreated cases (MM10-16) (Figure 1B). Protein expression was tracked immunohistochemically, using a novel monoclonal antibody 2ALCC128, recognizing a unique C-terminal epitope in PASD1_v2.4  The MM cell lines THIEL and RPMI8226 expressed both mRNA and protein for PASD1, whereas JJN3 lacked both (Figure 1A,C). Protein expression was also confirmed in 2 of 4 primary MM tumor samples examined (Figure 1C).5 

Figure 1.

Analysis of CTA expression by RT-PCR, Q-PCR, and antibody staining in bulk populations and in single MM cells. Primer design for all CTAs examined spanned intronic DNA (available on request). Expression of PASD1 was examined by RT-PCR for 30 cycles and compared with known CTAs in MM using identical amounts of cDNA input from RNA (2 μg), reverse transcribed with oligo-dT (A). PASD1 expression was further examined after 40 cycles. Several normal lymphoid tissues were also analyzed, including specific lymphocyte populations not probed previously. In purified B cells, in vitro–generated plasmablasts, plasma cells, T cells, and spleen and bone marrow mononuclear cells (MNCs), we failed to detect PASD1 expression either by high amplification cycle numbers or by Q-PCR (data not shown). Two Burkitt lymphoma cell lines (Ramos, Daudi) were used as negative controls (A). Q-PCR used Taqman primers (PASD1: Hs00542865_m1; β-actin: Hs99999903_m1; Applied Biosystems, Warrington, United Kingdom). Relative expression was calculated against reference sample (Ramos) as 2–[DCt(Sample) – DCt(Reference)], where DCt indicates Ct(PASD1) – Ct(β-actin). Presentation diagnostic samples were MM-(P1-9), pretreated MM-(T10-16); ND indicates not detected (B). Immunoperoxidase labeling showing PASD1 protein expression in testis and in the nuclei of THIEL cells, with JJN3 cells negative (inset), and in the nuclei (arrowed) of tumor cells in 1 of the 2 cases of MM found to be positive (C; RPMI8226 and second MM case staining not shown). Single cells obtained by flow cytometry deposition of 2 MM cell lines expressing high (Jim1) or low (RPMI 8226) levels were analyzed by nested RT-PCR for PASD1 expression, and for each line, upper and lower panels correspond to cell numbers 1 to 24 and 25 to 48, respectively (D). Both splice variants were detected in the 2 lines (± exon 8), yielding 298- and 215-bp amplification products. Each amplification product from bulk populations or single tumor cells was eluted and identity confirmed by DNA sequence analysis. Images were obtained using a Zeiss Axioskop microscope (Zeiss, Welwyn Garden City, United Kingdom) with nonimmersion 10×/0.25 NA (testis, JNN, and MM) and 40×/0.65 (THIEL and MM) Acroplan objectives. Image acquisition was performed using a Micropublisher 5 camera (Q-Imaging, Wokingham, United Kingdom) and Adobe Photoshop 8.0 software (Adobe Systems, San Jose, CA). All subsequent processing was performed using Adobe Photoshop 8.0.

Figure 1.

Analysis of CTA expression by RT-PCR, Q-PCR, and antibody staining in bulk populations and in single MM cells. Primer design for all CTAs examined spanned intronic DNA (available on request). Expression of PASD1 was examined by RT-PCR for 30 cycles and compared with known CTAs in MM using identical amounts of cDNA input from RNA (2 μg), reverse transcribed with oligo-dT (A). PASD1 expression was further examined after 40 cycles. Several normal lymphoid tissues were also analyzed, including specific lymphocyte populations not probed previously. In purified B cells, in vitro–generated plasmablasts, plasma cells, T cells, and spleen and bone marrow mononuclear cells (MNCs), we failed to detect PASD1 expression either by high amplification cycle numbers or by Q-PCR (data not shown). Two Burkitt lymphoma cell lines (Ramos, Daudi) were used as negative controls (A). Q-PCR used Taqman primers (PASD1: Hs00542865_m1; β-actin: Hs99999903_m1; Applied Biosystems, Warrington, United Kingdom). Relative expression was calculated against reference sample (Ramos) as 2–[DCt(Sample) – DCt(Reference)], where DCt indicates Ct(PASD1) – Ct(β-actin). Presentation diagnostic samples were MM-(P1-9), pretreated MM-(T10-16); ND indicates not detected (B). Immunoperoxidase labeling showing PASD1 protein expression in testis and in the nuclei of THIEL cells, with JJN3 cells negative (inset), and in the nuclei (arrowed) of tumor cells in 1 of the 2 cases of MM found to be positive (C; RPMI8226 and second MM case staining not shown). Single cells obtained by flow cytometry deposition of 2 MM cell lines expressing high (Jim1) or low (RPMI 8226) levels were analyzed by nested RT-PCR for PASD1 expression, and for each line, upper and lower panels correspond to cell numbers 1 to 24 and 25 to 48, respectively (D). Both splice variants were detected in the 2 lines (± exon 8), yielding 298- and 215-bp amplification products. Each amplification product from bulk populations or single tumor cells was eluted and identity confirmed by DNA sequence analysis. Images were obtained using a Zeiss Axioskop microscope (Zeiss, Welwyn Garden City, United Kingdom) with nonimmersion 10×/0.25 NA (testis, JNN, and MM) and 40×/0.65 (THIEL and MM) Acroplan objectives. Image acquisition was performed using a Micropublisher 5 camera (Q-Imaging, Wokingham, United Kingdom) and Adobe Photoshop 8.0 software (Adobe Systems, San Jose, CA). All subsequent processing was performed using Adobe Photoshop 8.0.

To examine intraclonal PASD1 expression, single cells were examined in 2 cell lines. PASD1 transcripts were found in 27% to 40% of cells, a significant fraction, but not all cells (Figure 1D). Parallel findings were confirmed in 2 primary MM samples analyzed at the single-cell level (data not shown). Antibody staining in THIEL, however, indicated a more prevalent expression (Figure 1C), and together with staining in MM cases (Figure 1C) revealed variability between tumors.

PASD1 expression was compared with known MM-associated CTAs. MAGE-A6 (8/11) was the most frequently expressed CTA in cell lines (Figure 1A). PASD1 expression (5/11) was comparable in frequency with MAGE-A2 (4/10) and NY-ESO-1 (4/10) among cell lines, using 30 cycles. The levels of PASD1 detected by RT-PCR in the 5 cell lines (Jim1, U266B1, L363, KMS-12-BM, THIEL; Figure 1A) appeared to parallel known CTA expression in MM, at this level of amplification. In 4 (25%) of 16 primary tumors (3/4 at presentation, 1/4 partial progression), PASD1 levels by Q-PCR were comparable with the highest levels seen in THIEL, U266B1, and Jim1 (Figure 1A-B), suggesting that PASD1 levels in many primary tumors will be therapeutically relevant. However, actual levels of specific CTAs overall vary and are differentially expressed within individual tumors. This suggests a specific induction of genes that may be relevant to tumor survival. The observed intraclonal variation in PASD1 expression paralleled known features of MAGE-A3/6 and NY-ESO-1 expression in MM.6,7  NY-ESO-1 positivity was reported in less than 25% of tumor cells,7  lower than the fraction observed here for PASD1 transcripts. These data indicate that PASD1 is an MM-associated gene, which could be exploited as an antigen for targeted therapy.

The authors declare no competing financial interests.

The assistance of Giovanna Roncador from the Centro Nacional de Investigaciones Oncológicas (CNIO) in obtaining samples is gratefully acknowledged. This work was supported by the Leukaemia Research Fund UK, ISIS University Innovation Fund, and in part by the Research Fund for Waldenstrom USA.

S.S.S., K.P., and A.H.B. designed research, analyzed data, and wrote the paper; C.M.G., C.D.O.C., A.P.L., and K.A.-T. carried out research and analyzed data; N.Z. performed clinical evaluation of patients, carried out research, and analyzed data; F.K.S. analyzed data and wrote the paper. A.H.B. and K.P. contributed equally to this work.

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