Christian S. R. Hatton

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.

MicroRNA expression distinguishes between germinal center B cell-like and activated B cell-like subtypes of diffuse large B cell lymphoma

Diffuse large B cell lymphoma (DLBCL) is an aggressive malignancy that accounts for nearly 40% of all lymphoid tumors. This heterogeneous disease can be divided into germinal center B cell‐like (GCB) and activated B cell‐like (ABC) subtypes by gene expression and immunohistochemical profiling. Using microarray analysis on prototypic cell lines, we identified microRNAs (miR‐155, miR‐21 and miR‐221) that were more highly expressed in ABC‐type than GCB‐type cell lines. These microRNAs were over‐expressed in de novo DLBCL (n = 35), transformed DLBCL (n = 14) and follicular center lymphoma cases (n = 27) compared to normal B cells. Consistent with the cell line model, expression levels were higher in DLBCL cases with an ABC‐type immunophenotype than those that were GCB‐type (p < 0.05). Moreover, using multivariate analysis we found that expression of miR‐21 was an independent prognostic indicator in de novo DLBCL (p < 0.05). Interestingly, expression levels of both miR‐155 and miR‐21 were also higher in nonmalignant ABC than in GCB cells. As we also demonstrate that expression of microRNAs can be measured reliably from routine paraffin‐embedded biopsies of more than 8‐years‐old (p < 0.001), we suggest that microRNAs could be clinically useful molecular markers for DLBCL as well as other cancers.

Expression of microRNAs in diffuse large B cell lymphoma is associated with immunophenotype, survival and transformation from follicular lymphoma

MicroRNAs are naturally occurring small RNA species that regulate gene expression and are frequently abnormally expressed in cancers. However, the role of microRNAs in lymphoma is poorly understood. Therefore, we undertook a comprehensive study of microRNA expression in two of the most common lymphomas: diffuse large B‐cell lymphoma (DLBCL) (n= 80) and follicular lymphoma (FCL) (n= 18) using microarrays containing probes for 464 human microRNAs. Unsupervised cluster analysis revealed distinct expression patterns between these two lymphomas and specific microRNA signatures (including members of the miR‐17–92 cluster) were derived that correctly predicted lymphoma type in >95% of cases. Furthermore, we identified microRNAs in de novo DLBCL (n= 64) associated with germinal centre‐like and non‐germinal centre‐like immunophenotypes, international prognostic index status and event‐free survival in CHOP and rituximab (R)‐CHOP treated patients. Despite the indolent nature of FCL a significant proportion of cases undergo high‐grade transformation to more aggressive DLBCL. In order to see if transformation is associated with changes in microRNA expression we compared transformed DLBCL cases (n= 16) with de novo DLBCL, as well as FCL cases that underwent subsequent transformation (n= 7) with FCL cases that had not transformed at a median follow‐up of 60 months (n= 11). Differential expression of 12 microRNAs correctly predicted >85% of transformed versus de novo DLBCL cases; six microRNAs (miR‐223, 217, 222, 221 and let‐7i and 7b) were found which could similarly predict or transformation in FCL (P< 0.05). These data suggest that microRNAs have potential as diagnostic and prognostic markers in these lymphomas and may be used to identify FCL patients at risk of high‐grade transformation.

MicroRNA expression in Sezary syndrome: identification, function, and diagnostic potential.

MicroRNAs are commonly aberrantly expressed in many cancers. Very little is known of their role in T-cell lymphoma, however. We therefore elucidated the complete miRNome of purified T cells from 21 patients diagnosed with Sézary Syndrome (SzS), a rare aggressive primary cutaneous T-cell (CD4(+)) lymphoma. Unsupervised cluster analysis of microarray data revealed that the microRNA expression profile was distinct from CD4(+) T-cell controls and B-cell lymphomas. The majority (104 of 114) of SzS-associated microRNAs (P < .05) were down-regulated and their expression pattern was largely consistent with previously reported genomic copy number abnormalities and were found to be highly enriched (P < .001) for aberrantly expressed target genes. Levels of miR-223 distinguished SzS samples (n = 32) from healthy controls (n = 19) and patients with mycosis fungoides (n = 11) in more than 90% of samples. Furthermore, we demonstrate that the down-regulation of intronically encoded miR-342 plays a role in the pathogenesis of SzS by inhibiting apoptosis, and describe a novel mechanism of regulation for this microRNA via binding of miR-199a* to its host gene. We also provide the first in vivo evidence for down-regulation of the miR-17-92 cluster in malignancy and demonstrate that ectopic miR-17-5p expression increases apoptosis and decreases cell proliferation in SzS cells.

MicroRNA expression in multiple myeloma is associated with genetic subtype, isotype and survival

BackgroundMicroRNAs are small RNA species that regulate gene expression post-transcriptionally and are aberrantly expressed in many cancers including hematological malignancies. However, the role of microRNAs in the pathogenesis of multiple myeloma (MM) is only poorly understood. We therefore used microarray analysis to elucidate the complete miRNome (miRBase version 13.0) of purified tumor (CD138+) cells from 33 patients with MM, 5 patients with monoclonal gammopathy of undetermined significance (MGUS) and 9 controls.ResultsUnsupervised cluster analysis revealed that MM and MGUS samples have a distinct microRNA expression profile from control CD138+ cells. The majority of microRNAs aberrantly expressed in MM (109/129) were up-regulated. A comparison of these microRNAs with those aberrantly expressed in other B-cell and T-cell malignancies revealed a surprising degree of similarity (~40%) suggesting the existence of a common lymphoma microRNA signature. We identified 39 microRNAs associated with the pre-malignant condition MGUS. Twenty-three (59%) of these were also aberrantly expressed in MM suggesting common microRNA expression events in MM progression. MM is characterized by multiple chromosomal abnormalities of varying prognostic significance. We identified specific microRNA signatures associated with the most common IgH translocations (t(4;14) and t(11;14)) and del(13q). Expression levels of these microRNAs were distinct between the genetic subtypes (by cluster analysis) and correctly predicted these abnormalities in > 85% of cases using the support vector machine algorithm. Additionally, we identified microRNAs associated with light chain only myeloma, as well as IgG and IgA-type MM. Finally, we identified 32 microRNAs associated with event-free survival (EFS) in MM, ten of which were significant by univariate (logrank) survival analysis.ConclusionsIn summary, this work has identified aberrantly expressed microRNAs associated with the diagnosis, pathogenesis and prognosis of MM, data which will prove an invaluable resource for understanding the role of microRNAs in this devastating disease.ReviewersThis article was reviewed by Prof. Neil Smalheiser, Prof. Yuriy Gusev, and an unknown reviewer.

B and CTL responses to the ALK protein in patients with ALK‐positive ALCL

Anaplastic lymphoma kinase (ALK)‐positive anaplastic large cell lymphoma (ALCL) has a good prognosis compared to ALK‐negative ALCL, possibly as a result of the immune recognition of the ALK proteins. The aim of our study was to investigate the presence of both a B and cytotoxic T cell (CTL) response to ALK in ALK‐positive ALCL. We confirmed the presence of an antibody response to ALK in all 9 ALK‐positive ALCL patients investigated. An ELISpot assay was used to detect a γ‐interferon (IFN) T cell response after short term culture of mononuclear blood cells with 2 ALK‐derived HLA‐A*0201 restricted peptides: ALKa and ALKb. A significant γ‐IFN response was identified in all 7 HLA‐A*0201‐positive ALK‐positive ALCL patients but not in ALK‐negative ALCL patients (n = 2) or normal subjects (n = 6). CTL lines (>95% CD8‐positive) raised from 2 ALK‐positive ALCL patients lysed ALK‐positive ALCL derived cell lines in a MHC‐Class I restricted manner. This is the first report of both a B cell and CTL response to ALK in patients with ALK‐positive ALCL. This response persisted during long‐term remission. The use of modified vaccinia virus Ankara (MVA) to express ALK is also described. Our findings are of potential prognostic value and open up therapeutic options for those ALK‐positive patients who do not respond to conventional treatment.

Cancer-associated carbohydrate identification in Hodgkin's lymphoma by carbohydrate array profiling

Tumor‐associated carbohydrates have potential not only as diagnostic tools but also as specific therapeutic targets. Their identification, however, has been hampered by the lack of suitable technologies. We used carbohydrate array technology to compare serum antibody (IgG and IgM) levels against 37 different carbohydrates between classical Hodgkins lymphoma (cHL) patients and age/sex‐matched healthy controls. Serum IgM levels measured by ELISA against 2 of the 5 carbohydrates identified using this technique, L‐α‐arabinose (L‐Araf) and α‐N‐acetylgalactosamine (GalNAc(α)), were higher (F values of 11.30 and 18.27, respectively) in a cohort of cHL patients (n = 16) than either diffuse large B‐cell lymphoma patients (n = 18) or control sera (n = 12). Higher anti‐L‐Araf IgM levels in cHL patients were associated with cytosine arabinoside treatment (p < 0.05). The GalNAc(α) glycotope, Tn, was found to be heterogeneously expressed in the Reed‐Sternberg cells of 9/20 (45%) cHL cases, but not in malignant cells of 25 cases of lymphocyte‐predominant HL or another 21 hematological disorders (291 cases) examined immunohistochemically. Tn was expressed in 41/238 (17%) classical HL cases present on a tissue microarray. Expression was associated with CD79a and LMP1 expression and negatively with p27KIP1 expression (p < 0.05). Kaplan‐Meier survival analysis revealed a trend towards improved relapse‐free survival with Tn expression although this was not statistically significant (p = 0.271). We suggest that this technique could provide a powerful tool for identifying novel carbohydrates in other cancers.

Detection of metastatic tumour cells in routine bone marrow smears by immuno‐alkaline phosphatase labelling with monoclonal antibodies

Summary. The present study describes 11 cases (10 carcinomas, one rhabdo‐myosarcoma) in which immuno‐alkaline phosphatase labelling with monoclonal antibodies was used to demonstrate metastatic cells in routine smears of aspirated bone marrow. Carcinoma cells were detected using antibodies against epithelial cytokeratins, milk fat globule membrane antigen and carcinoem‐bryonic antigen, and rhabdomyosarcoma cells with monoclonal anti‐desmin. In four of the carcinoma cases it had not been possible to identify malignant cells in routinely stained marrow smears, whilst the case of disseminated rhabdomyosarcoma had initially been diagnosed (and treated) as a case of acute lymphoblastic leukaemia. The anti‐cytokeratin antibody was found to be the most valuable of the anti‐epithelial reagents used, since it labelled malignant cells in all of the 10 cases of carcinoma and gave the strongest reactions. These results suggest that immunocytochemical labelling should be used in cases of suspected carcinoma whenever conventional examination of marrow smears yields negative results, and furthermore (as illustrated by the case of rhabdomyosarcoma) that the technique is of value for identifying the true nature of poorly differentiated neoplasms in bone marrow.

Inter- and intra-observational variability in immunohistochemistry: a multicentre analysis of diffuse large B-cell lymphoma staining.

Lawrie C H, Ballabio E, Soilleux E, Sington J, Hatton C S R, Dirnhofer S & Tzankov A 
(2012) Histopathology 61, 18–25

Aberrant expression of microRNA biosynthetic pathway components is a common feature of haematological malignancy.

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