Amanda P. Liggins

Detection of elevated levels of tumour-associated microRNAs in serum of patients with diffuse large B-cell lymphoma.

Circulating nucleic acids have been shown to have potential as non‐invasive diagnostic markers in cancer. We therefore investigated whether microRNAs also have diagnostic utility by comparing levels of tumour‐associated MIRN155 (miR‐155), MIRN210 (miR‐210) and MIRN21 (miR‐21) in serum from diffuse large B‐cell lymphoma (DLBCL) patients (n = 60) with healthy controls (n = 43). Levels were higher in patient than control sera (P = 0·009, 0·02 and 0·04 respectively). Moreover, high MIRN21 expression was associated with relapse‐free survival (P = 0·05). This is the first description of circulating microRNAs and suggests that microRNAs have potential as non‐invasive diagnostic markers for DLBCL and possibly other cancers.

Cytolytic T-cell response to the PASD1 cancer testis antigen in patients with diffuse large B-cell lymphoma

The identification of immunogenic cancer testis antigens (CTAs) as immunotherapeutic targets represents one approach to improve treatment options for diffuse large B‐cell lymphoma (DLBCL). We previously identified PASD1 [PAS (Per ARNT Sim) domain containing 1 (PASD1)], a DLBCL‐associated CTA that was expressed in a range of hematopoietic malignancies. The aim of the present study was to investigate the presence of a cytotoxic T‐cell (CTL) response to PASD1 in DLBCL patients. A significant γ‐interferon (IFN) release was detected in 21/29 HLA‐A*0201‐positive DLBCL patients (18 de novo DLBCL, two transformed DLBCL and one T‐cell rich B‐cell lymphoma) following short‐term culture of their peripheral blood mononuclear cells stimulated with five HLA‐A*0201‐restricted PASD1 peptides. No significant responses were detected in 21 HLA‐A*0201‐negative DLBCL patients (12 de novo DLBCL, seven transformed DLBCL, two T‐cell rich B‐cell lymphoma) or six normal subjects. CTL cell lines were able to lyse PASD1‐positive tumour cells in a major histocompatibility complex‐Class I dependent manner. The presence of a γ‐IFN response correlated with PASD1 protein expression in the tumour cells in 12/15 cases studied. This is the first report of a CTL response to a CTA in DLBCL. Our results provide additional valuable evidence supporting PASD1 as a potential immunotherapeutic target for the treatment of DLBCL and other malignancies.

Serologic detection of diffuse large B-cell lymphoma-associated antigens.

Diffuse large B‐cell lymphoma (DLBCL) accounts for 30–40% of all adult non‐Hodgkins lymphomas, yet the understanding of its underlying genetic abnormalities remains poor. Our present study used the serological analysis of recombinant cDNA expression libraries (SEREX) technique to identify DLBCL‐associated antigens. SEREX screening of testis libraries has previously identified cancer‐testis antigens (CTAs) that may act as disease‐specific targets for immunotherapy. Screening a testis cDNA expression library with serum from a DLBCL patient identified a total of 94 positive clones, representing 28 distinct antigens. Two of these antigens were novel, 8 were previously uncharacterised, and the remainder were proteins of known function. Screening of the antigens with sera from DLBCL (n = 10), acute myeloid leukaemia (AML, n = 10) and chronic myeloid leukaemia (CML, n = 10) patients, alongside normal healthy donor controls (n = 20), revealed that 7 of the antigens were recognised by DLBCL sera but not normal donor sera, whilst 2 of these antigens were also recognised by leukaemic sera. Some of the genes identified here were already known to be transcribed in DLBCL. The mRNA expression of the majority of the remaining antigens was confirmed in DLBCL cell lines using reverse‐transcriptase PCR (RT‐PCR). Our study identified a number of DLBCL associated antigens that may be suitable as prognostic/diagnostic markers and/or for the immunotherapy of haematologic malignancies.

MORC4, a novel member of the MORC family, is highly expressed in a subset of diffuse large B-cell lymphomas.

The OX‐TES‐4 antigen originally elicited an antibody response in 50% of diffuse large B‐cell lymphoma (DLBCL) patients but not in control subjects. OX‐TES‐4 is encoded by a novel gene, MORC4, located at chromosome Xq22.3. The MORC4 protein contains a HATPase‐c domain, CW zinc finger motif, nuclear localisation signals and a nuclear matrix‐binding domain, together with a coiled‐coil region. MORC4 mRNA is widely expressed at low levels in normal tissues, showing highest expression levels in placenta and testis. mRNA levels were increased in non‐germinal centre‐derived DLBCL and Hodgkin lymphoma cell lines, compared with germinal centre‐derived DLBCL cell lines and normal B cells. Nineteen DLBCL patients (66%) expressed significantly higher levels of MORC4 mRNA than normal B cells (P = 0·0031). The differential expression of MORC4 identifies this molecule as a potential lymphoma biomarker, whose overexpression may contribute to the immunological recognition of MORC4 by a subgroup of DLBCL patients.

CD4-positive T-helper cell responses to the PASD1 protein in patients with diffuse large B-cell lymphoma.

Background Vaccine development targeting the novel immunogenic Per ARNT Sim Domain containing 1 (PASD1) cancer testis antigen represents an attractive therapeutic approach for the significant number of patients with diffuse large B-cell lymphoma who are refractory to conventional treatment. Since CD4-positive T helper cells have crucial roles in promoting and maintaining immune responses to tumor antigens, the presence of a CD4-positive T-helper immune response to the PASD1 antigen in patients with diffuse large B-cell lymphoma was investigated in the current study. Design and Methods Thirty-one patients with diffuse large B-cell lymphoma (25 with de novo, five with transformed and one with T-cell-rich B-cell lymphoma) were studied. Five immunogenic PASD1 peptides predicted to bind to several major histocompatibiliy complex, class II DR beta 1 alleles were identified using web-based algorithms. Peripheral blood mononuclear cells from patients were used to investigate the immunogenicity of these DR beta 1-restricted peptides in vitro using both gamma-interferon release enzyme-linked immunospot and cytolytic assays. Results Two of the five PASD1 peptides, PASD1(6) and PASD1(7), were shown to be immunogenic in 14 out of 32 patients studied in a gamma-interferon release assay. CD4-positive T-helper cell lines from two patients raised against PASD1 peptides were able to lyse cell lines derived from hematologic malignancies expressing endogenous PASD1 protein. Conclusions This is the first report of a CD4-positive T-helper response to the PASD1 protein in patients with lymphoma. The immunogenic peptides described here represent valuable additional candidates for inclusion in a vaccine to treat patients with PASD1-positive diffuse large B-cell lymphoma whose disease is refractory to conventional therapies.