Abstract

Advances in gene expression profiling and immunolabeling techniques have provided evidence of clinically relevant subtypes within the heterogeneous disease entity diffuse large B-cell lymphoma (DLBCL). The development of improved treatment regimens still remains a priority as more than half of DLBCL patients are incurable using combination CHOP chemotherapy. Evidence that the immune system plays a critical role in cancer biology is rapidly accumulating with many tumor proteins being recognized by an anti-tumor immune response. Such tumor-associated antigens (TAAs) have the potential to provide new diagnostic, prognostic and therapeutic options for cancer patients. Of particular interest are the cancer testis antigens (CTAs), whose restricted normal tissue expression but widespread expression in tumors makes them attractive targets for therapy. SEREX was previously used to identify a DLBCL-associated TAA, PAS domain containing 1 (PASD1) that was immunologically recognized by multiple high-risk DLBCL patients. PASD1 maps to chromosome Xq28, in common with many other CTA genes, and encodes a novel putative transcription factor. Since PASD1 mRNA expression in normal tissues is restricted to testis with transcripts being expressed in both DLBCL cell lines and solid tumours, it therefore represents a new CTA and potential immunotherapeutic target. Two PASD1 transcripts were identified, v1 encoding a 639 aa N-terminus while v2 encodes a longer protein (773 aa) with a unique C-terminus. Both proteins were nuclear when expressed in transfected COS-1 cells. We have raised a panel of monoclonal antibodies against the PASD1 proteins, including two antibodies (2ALCC16 and 2ALCC136) recognizing both variants, and one antibody (antibody 2ALCC128) specific for the C-terminus of the longer protein, to study PASD1 protein distribution in both normal and neoplastic tissues. In normal tissues, PASD1 expression is restricted to the nuclei of cells in the basal layer of testis, a subset of primary spermatogonia (2ALCC16 also labeled the cytoplasm of spermatogonia) and a small subset of cells in the salivary gland. PASD1 protein was detected in a range of malignant cell lines with nuclear labeling being observed in OCI-Ly3 (DLBCL), KM-H2 (Hodgkin’s lymphoma), K562 (chronic myeloid leukaemia) and RPMI 8226 (myeloma) lines, whilst cytoplasmic labeling was detected in Karpas 299 (t(2;5) anaplastic large cell lymphoma), SUDHL6, SUDHL10 (both DLBCL) and Jurkat (T-acute lymphoblastic leukaemia) cell lines. Tumor tissue was available from two patients with seroreactivity to PASD1; PASD1-positive tumour cells were observed using all three antibodies in one patient, with scattered positive cells being observed with 2ALCC136 in the other. It is of note that, whereas strong cytoplasmic labeling was obtained with 2ALCC136, nuclear labeling was observed with 2ALCC128 recognizing the longer PASD1 variant. Variations in the staining patterns and western blotting data using different antibodies may reflect the presence of multiple PASD1 isoforms. Preliminary results demonstrate PASD1 expression in a subset of DLBCL, mantle cell lymphoma, follicular lymphoma, multiple myeloma and Hodgkin’s disease. Further studies of a larger series of hematological malignancies are in progress to determine the potential of this molecule, both in the identification of high-risk patients and as an immunotherapeutic target.

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