MD PhD, pathologist Philip Kluin
email: p.m.kluin@path.umcg.nl

PhD, molecular-biologist Ed Schuuring

WHERE DO WE STAND IN 2010?

Keywords: lymphoma, review, genetic, translocation, cytogenetics molecular genetics of lymphoma lymphoma, cytogenetics, review

For the past 20 years most malignant lymphomas have been classified as clinico-pathologic entities, each with its own combination of clinical, morphologic, immunophenotypic and molecular genetic characteristics. Molecular and cytogenetic abnormalities can be detected by a wide range of techniques, ranging from conventional karyotyping to single nucleotide polymorphism analysis. In this review, we consider the common genetic abnormalities found in lymphoma and discuss the advantages and disadvantage of individual techniques used in their detection. Finally, the authors briefly discuss possible novel developments in the field of lymphoma diagnostics.

Introduction

During the past two decades molecular diagnostics have become an integral part of the laboratory repertoire in haematopathology. Conventional and molecular cytogenetic techniques are used to detect recurrent chromosomal translocations or breakpoints, as well as specific deletions, gains and point mutations. These abnormalities may be used in the primary classification of a tumour or as prognostic factors. This review aims to give an overview of the currently available molecular and cytogenetic methods, the lymphoma specific targets that can be analyzed by these methods and to provide guidelines for their use in everyday practice.

The important molecular abnormalities found in the common types mature B and T cell lymphomas as known in 2010 1 are shown in table 2 Molecular cytogenetic methods.

1. Karyotype analysis / classical cytogenetics.

Only three years after the discovery of the Philadelphia chromosome in 1960, [START_REF] Nowell | Chromosome studies on normal and leukemic human leukocytes[END_REF] the first recurrent cytogenetic abnormalities were described in Burkitt lymphoma. [START_REF] Jacobs | Cytogenetic studies in Burkitt's lymphoma[END_REF] This was later found to be the classical translocation t(8;14)(q24;q32) in which the MYC gene and immunoglobulin heavy chain gene (IGH) complex are involved. This was followed by the discovery of many more recurrent chromosomal translocations. Some of these chromosomal translocations are considered as the diagnostic hallmark of specific disease entities, whereas others may be secondary and involved in tumour progression. [START_REF] Siebert | Mature B-and T-cell Neoplasms and Hodgkin Lymphoma[END_REF] Recurrent translocations can be detected in > 90% of the cases of Burkitt lymphoma, mantle cell lymphoma and follicular lymphoma grade 1 and 2, and are considered as the cancer initiating events in these diseases.

In other lymphomas, and in particular in diffuse large B cell lymphoma, multiple breakpoints and translocations with a plethora of numerical abnormalities (gains and losses) are seen. In these cases it is often impossible to decide which events are involved in the primary initiation and subsequent progression of the tumour.

A good example of this complexity is the involvement of MYC and the 8q24 breakpoint in lymphomagenesis. MYC rearrangements are widely regarded as the primary event in Burkitt lymphoma (the t (8;14) or variant t(2;8)(p12;q24) and t(8;22)(q24;q11) translocations), but MYC is also frequently involved in tumour progression, either by chromosomal translocation, gain or amplification in other types of lymphoma.. Rearrangements of MYC can be found in In more than half of these cases additional abnormalities, most commonly the t (14;18) ,can be identified. These "double-hit" or "dualhit" lymphomas [START_REF] Barrans | Rearrangement of MYC is associated with poor prognosis in patients with diffuse large B-cell lymphoma treated in the era of rituximab[END_REF][START_REF] Carbone | B-cell lymphomas with features intermediate between distinct pathologic entities. From pathogenesis to pathology[END_REF][START_REF] Johnson | Lymphomas with concurrent BCL2 and MYC translocations: the critical factors associated with survival[END_REF][START_REF] Hasserjian | Gray zone" lymphomas overlapping with Burkitt lymphoma or classical Hodgkin lymphoma[END_REF][START_REF] Tomita | Clinicopathological features of lymphoma/leukemia patients carrying both BCL2 and MYC translocations[END_REF][START_REF] Gouill | The clinical presentation and prognosis of diffuse large B-cell lymphoma with t(14;18) and 8q24/c-MYC rearrangement[END_REF] often have complex cytogenetic alterations including occasional breakpoints at 3q27/BCL6 ("triple hit" lymphomas). This complexity can be best detected by conventional cytogenetics, since it represents a genome wide method by which many structural and numerical abnormalities can be simultaneously detected, albeit at a low resolution.

The genome wide scope of conventional cytogenetic analysis means that it is often considered to be the "gold standard" for lymphoma diagnostics raising the question as to why is it not part of routine practice. Several logistic and technical problems hamper the routine use of this method. These include the need to obtain fresh tumour tissue which must be processed within few hours after removal from the patient, the need for a relatively large volume of tissue, and the high level of skill required to culture the lymphoma cells, to prepare high quality metaphase cells, and to analyse the often complex karyograms. This makes the method labour intensive, time-consuming and expensive. Moreover, the failure rate is relatively high. In particular in indolent lymphomas and myelomas the tumour cells may fail to grow in culture only normal karyotypes may be seen.

Given these problems the possibility that fluorescent in situ hybridization (FISH) or other methods could completely replace conventional cytogenetics needs to be considered. FISH is a rapid, robust, cheap and relatively easily applicable technique that can be applied to formalin fixed and paraffin embedded (FFPE) tissues and does not need vital, growing cells or specific culture systems (Table 1). [START_REF] Ventura | FISH analysis for the detection of lymphoma-associated chromosomal abnormalities in routine paraffin-embedded tissue[END_REF][START_REF] Haralambieva | Detection of three common translocation breakpoints in non-Hodgkin's lymphomas by fluorescence in situ hybridization on routine paraffin-embedded tissue sections[END_REF] A further advantage is a short turnaround time which is usually in the order of 2 or 3 workings days. The major limitation of FISH is that is targeted to the detection of specific abnormalities and the genome wide perspective offered by conventional cytogenetics is lost. This may be clinically important as additional aberrations (or their absence) can be diagnostic or predictive for the course of disease (see Table 2). For example, the diagnosis of Burkitt lymphoma depends not only on the presence of a MYC or 8q24 breakpoint in Burkitt lymphoma, but on the absence of complex genomic complexity abnormalities. 30-40% of all Burkitt lymphoma cases harbour only the t (8;14) or its variants, the rest exhibiting only one to two additional abnormalities (mostly alterations at 1q, trisomy7, trisomy12 or alterations at 13q involving the microRNA 17-92 cluster) are found. [START_REF] Siebert | Mature B-and T-cell Neoplasms and Hodgkin Lymphoma[END_REF][START_REF] Boerma | Translocations involving 8q24 in Burkitt lymphoma and other malignant lymphomas: a historical review of cytogenetics in the light of todays knowledge[END_REF][START_REF] Toujani | High resolution genome-wide analysis of chromosomal alterations in Burkitt's lymphoma[END_REF] In contrast, the genomic complexity in DLBCL with an 8q24 breakpoint is usually much F o r P e e r R e v i e w 5 higher. [START_REF] Boerma | Translocations involving 8q24 in Burkitt lymphoma and other malignant lymphomas: a historical review of cytogenetics in the light of todays knowledge[END_REF][START_REF] Hummel | A biologic definition of Burkitt's lymphoma from transcriptional and genomic profiling[END_REF][START_REF] Seegmiller | Simple karyotype and bcl-6 expression predict a diagnosis of Burkitt lymphoma and better survival in IG-MYC rearranged high-grade B-cell lymphomas[END_REF][START_REF] Salaverria | Chromosomal alterations detected by comparative genomic hybridization in subgroups of gene expression-defined Burkitt's lymphoma[END_REF] . In a recent study of aggressive B cell lymphomas with a MYC-IG breakpoint, 22 Burkitt lymphomas had a mean of 1,7 additional conventional cytogenetic abnormalities, whereas 12 other (non Burkitt) lymphomas with a MYC-IG breakpoint had a mean of 8.8 abnormalities with three DLBCL showing over 21,cytogenetic abnormalities. [START_REF] Seegmiller | Simple karyotype and bcl-6 expression predict a diagnosis of Burkitt lymphoma and better survival in IG-MYC rearranged high-grade B-cell lymphomas[END_REF] In another study using FISH and array CGH on a large series of lymphomas, an almost similar genomic complexity of 2 additional abnormalities in the 39 molecular Burkitt lymphomas versus 9 in the 35 other aggressive B cell lymphomas with a MYC breakpoint was observed. [START_REF] Hummel | A biologic definition of Burkitt's lymphoma from transcriptional and genomic profiling[END_REF] Based on this data in Burkitt lymphoma and DLBCL, it now seems justifiable to regard the presence of a complex karyotype with a t(8;14) as a diagnostic criteria for distinguishing Burkitt Lymphoma from Diffuse Large B-cell Lymphoma. The importance of this distinction argues for the retention of techniques that provide a genome wide survey of genetic abnormalities lymphoma.

An intrinsic problem with conventional cytogenetic analysis is that the resolution offered by banding techniques is low at approximately 4-5 Mb (Table 1). This means that structural alterations like translocation breakpoints may look similar at the light microscopic level but are different at the molecular level. For instance approximately 15% of all myelomas contain a t(11;14)(q13;q32) which appears identical to the characteristic translocation seen in mantle cell lymphoma. However, at the molecular level the breakpoint in myeloma affects the IGH locus at switch regions instead of the VDJ complex in mantle cell lymphoma and thus imply a different cellular origin. [START_REF] Chesi | Dysregulation of cyclin D1 by translocation into IgH gamma switch region in two multiple myeloma cell lines[END_REF][START_REF] Janssen | Concurrent activation of a novel putative transforming gene, MYEOV, and cyclin D1 i a subset of multiple myeloma cell lines with t(11;14)(q13;q32)[END_REF] In spite of these limitations conventional cytogenetics remains a useful method to detect both specific primary and secondary abnormalities and an overall assessment of genetic complexity in lymphomas. It remains a valuable method to find new recurrent abnormalitie and the systematic collection of data in the Mitelman database , which in July 2010 contained karyotypic data of 7583 mature B cell and 1265 mature T cell lymphoma cases, is a highly valuable source for data-mining (http://cgap.nci.nih.gov/Chromosomes/Mitelman).

Multicolour fluorescent in situ hybridization (FISH) on chromosomes.

A ability of conventional metaphase cytogenetics to detect genome-wide genetic changes has been expanded by the introduction of multicolour painting of chromosomal preparations, by multiplex FISH, spectral karyotyping or COBRA FISH (combined binary ratio labelling [START_REF] Speicher | Karyotyping human chromosomes by combinatorial multi-fluor FISH[END_REF][START_REF] Tanke | New strategy for multi-colour fluorescence in situ hybridisation: COBRA: COmbined Binary RAtio labelling[END_REF][START_REF] Gisselsson | Refined characterisation of chromosome aberrations in tumours by multicolour banding and electronic mapping resources[END_REF][START_REF] Veldman | Hidden chromosome abnormalities in haematological malignancies detected by multicolour spectral karyotyping[END_REF][START_REF] Kearney | Multiplex-FISH (M-FISH): technique, developments and applications[END_REF] In these methods multiple labels (up to > 50) are generated by using mixtures of different fluorochromes. These labels are used to generate probes to multiple genomic regions including individual chromosomes or long and short chromosomal arms.are labelled with such mixtures. These labelled genomic sequences are subsequently hybridized to a metaphase chromosome preparations. Complex structural abnormalities can be visualized readily and the chromosomal origin of abnormal structures like marker chromosomes can be identified much more easily than by conventional cytogenetics. However, these methods require specific interpretative skills and more complex equipment than conventional cytogenetics, and therefore are only accessible to limited number of investigators.

Conventional comparative genomic hybridization (CGH) and array comparative genomic hybridization (aCGH)

Genome wide methods that do not need dividing tumour cells are comparative genetic hybridization (CGH) [START_REF] Lichter | Comparative genomic hybridization: uses and limitations[END_REF][START_REF] Bentz | Identification of genetic imbalances in malignant lymphoma using comparative genomic hybridization[END_REF][START_REF] Salaverria | Genomic profiling reveals different genetic aberrations in systemic ALK-positive and ALK-negative anaplastic large cell lymphomas[END_REF] and its successor array CGH (aCGH). The principles of both methods are shown in Figure 1. A major drawback of both techniques is that balanced chromosomal alterations like translocations cannot be detected. Conventional CGH is based on a competitive hybridization between normal (for instance Cy3-labeled) DNA and tumour cell derived (for instance Cy5-labeled) DNA, where chromosomes obtained from normal individuals are used as a template for hybridisation. Thus the availability and quality of normal chromosome preparations may be a limiting factor. Moreover, the resolution is not much better than of conventional cytogenetics (Table 1).

Conventional CGH has been replaced by array CGH or "matrix CGH" in which the DNA fragments used for competitive hybridization are spotted on a glass slide and not on normal chromosomes. Depending on the number of (overlapping) probes used to create the platform, aCGH has a very high resolution at less than 50-100 kb. DNA isolated from paraffin tissue blocks can be successfully used for hybridization. Up to now approximately 65 publications have appeared on the application of array CGH in mature B cell and T cell lymphomas. [START_REF] Seto | Diversity of genome profiles in malignant lymphoma[END_REF][START_REF] Takeuchi | The potential of copy number gains and losses, detected by array-based comparative genomic hybridization, for computational differential diagnosis of B-cell lymphomas and genetic regions involved in lymphomagenesis[END_REF][START_REF] Booman | Genomic alterations and gene expression in primary diffuse large B-cell lymphomas of immune-privileged sites: the importance of apoptosis and immunomodulatory pathways[END_REF][START_REF] Schraders | Integrated genomic and expression profiling in mantle cell lymphoma: identification of gene-dosage regulated candidate genes[END_REF][START_REF] Wessendorf | Further delineation of chromosomal consensus regions in primary mediastinal B-cell lymphomas: an analysis of 37 tumor samples using high-resolution genomic profiling (array-CGH)[END_REF][START_REF] Thorns | Chromosomal aberrations in angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma unspecified: A matrix-based CGH approach[END_REF][START_REF] De Leeuw | Comprehensive whole genome array CGH profiling of mantle cell lymphoma model genomes[END_REF] and these show distinct patterns of gains and losses characteristic of particular lymphoma subtypes. Within the group of DLBCL, activated B cell (ABC) type DLBCL have a different pattern from germinal centre B cell (GCB) type DLBCL, with recurrent trisomy 3, gain of 18q and 19q, as well as loss of 6q in ABC type and gain/amplification of 2p and loss of 1p and 13q in GCB type of DLBCL as well as frequent gain/amplification of 9p in primary 2). [START_REF] Lenz | Molecular subtypes of diffuse large B-cell lymphoma arise by distinct genetic pathways[END_REF] Similarly, distinct features were found between different types of T cell lymphomas.

Single nucleotide polymorphism (SNP) analysis.

SNP analysis is the most recently introduced whole genome method and has the potential advantage of an even higher resolution. [START_REF] Heinrichs | SNP array analysis in hematologic malignancies: avoiding false discoveries[END_REF][START_REF] Wang | Genome-wide SNP analysis in cancer: leukemia shows the way[END_REF] Figure 2 illustrates the analysis of a series of paediatric BL using either aCGH (n=15; figure 2A) or SNP analysis (n=18; figure 2B) (Kluin et al, unpublished data). The higher resolution with SNP analysis is also shown in Figure 2C, in which we encountered a homozygous deletion at chromosome 9p21 affecting CDKN4 and CDKN6, two important tumour suppressor genes, that was missed by both conventional and array based CGH analysis. A unique advantage of SNP analysis over all other methods is that not only changes in copy number but also regions of loss of heterozygosity without loss or gain of DNA can be detected, indicating that both alleles are homozygous and derived from one parent. This loss of heterozygosity without changes in copy numbers is called uniparental disomy (UPD). [START_REF] Bea | Uniparental disomies, homozygous deletions, amplifications, and target genes in mantle cell lymphoma revealed by integrative highresolution whole-genome profiling[END_REF][START_REF] Schwindt | Chromosomal imbalances and partial uniparental disomies in primary central nervous system lymphoma[END_REF][START_REF] O'shea | Regions of acquired uniparental disomy at diagnosis of follicular lymphoma are associated with both overall survival and risk of transformation[END_REF][START_REF] Fitzgibbon | Genome-wide detection of recurring sites of uniparental disomy in follicular and transformed follicular lymphoma[END_REF][START_REF] Nielaender | Partial uniparental disomy: a recurrent genetic mechanism alternative to chromosomal deletion in malignant lymphoma[END_REF] UPD may be important to detect since regions of UPD may harbour one or more genes with inactivating mutations (classical tumour suppressor genes). Since in a region with UPD both alleles will have the mutation, the Knudson two hit rule for tumour suppressor genes is fulfilled. Alternatively, regions with UPD may contain bi-allelic hypermethylated and thus silenced (candidate tumour suppressor) genes. SNP analysis is a promising approach to genome wide analysis in lymphoma and is now supported by efficient commercially available platforms which are widely available.

The major problem of SNP analysis is the difficulty in distinguishing acquired abnormalities from polymorphisms. This can only be conclusively resolved by analysing paired germ line (for instance derived from a buccal swab) and tumour material from the same patient. [START_REF] Heinrichs | SNP array analysis in hematologic malignancies: avoiding false discoveries[END_REF] Comparing the data from the individual cancer with large SNP databases of germ line material to correct for all potential polymorphisms is still not reliable and this analysis often results in the exclusion of many regions smaller than 250kb. Polymorphic regions, can even span several megabases, indicating that such a threshold may be even insufficient. Therefore, at the present time, except for the detection of UPD, SNP analysis on archival materials where germline DNA is not available is not superior to aCGH for the genome wide assessment of structural abnormalities. 1). Interestingly, due to the fact that many structural abnormalities like chromosomal breakpoints in lymphomas are associated with mutations and also small deletions or duplications of several nucleotides, such abnormalities may in fact be detectable by SNP analysis. However, this potential of SNP analysis has not been systematically addressed so far.

Interphase (and metaphase) fluorescent in situ hybridization

Interphase fluorescent in situ hybridization (interphase FISH) on cytospin preparations, imprinted fresh cells or paraffin tissue sections or metaphase cells of the tumour (metaphase FISH) has become a routine technique in many laboratories. [START_REF] Ventura | FISH analysis for the detection of lymphoma-associated chromosomal abnormalities in routine paraffin-embedded tissue[END_REF][START_REF] Haralambieva | Detection of three common translocation breakpoints in non-Hodgkin's lymphomas by fluorescence in situ hybridization on routine paraffin-embedded tissue sections[END_REF][START_REF] Haralambieva | Interphase fluorescence in situ hybridization for detection of 8q24/MYC breakpoints on routine histologic sections: Validation in Burkitt lymphomas from three geographic regions[END_REF][START_REF] Martin-Subero | Interphase cytogenetics of hematological neoplasms under the perspective of the novel WHO classification[END_REF][START_REF] Wolff | Guidance for fluorescence in situ hybridization testing in hematologic disorders[END_REF][START_REF] Barrans | The detection of t(14;18) in archival lymph nodes: development of a fluorescence in situ hybridization (FISH)-based method and evaluation by comparison with polymerase chain reaction[END_REF][START_REF] Vaandrager | Interphase FISH detection of BCL2 rearrangement in follicular lymphoma using breakpointflanking probes[END_REF] The technique is mainly used to detect well characterised structural chromosomal abnormalities such as recurrent chromosomal breakpoints or juxtaposition of two different loci involved in chromosomal translocations. The method can also be used to detect amplification or low level gains and losses of loci. However, real amplifications as seen in solid tumours are relatively rare in lymphomas. Moreover, the detection of low copy gains and losses in tissue sections is much more problematic than detection of breakpoints for which sets of probes are available that allow easy "pattern recognition" (see below). [START_REF] Haralambieva | Detection by the fluorescence in situ hybridization technique of MYC translocations in paraffin-embedded lymphoma biopsy samples[END_REF] Identification and counting of signals per cell on tissue sections is impeded by many factors like cutting artefacts and nuclear overlap related to the thickness and homogeneity of the tissue sections, the size of the nuclei, as well as the fixation. Thus for a reliable assessment of subtle copy number variations (low levels of gains and losses) FISH on isolated nuclei either prepared from intact cell suspension or nuclear suspensions from paraffin blocks is preferable. [START_REF] Thompson | Cytogenetic profiling using fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH)[END_REF][START_REF] Qian | Comparison of fluorescence in situ hybridization analysis of isolated nuclei and routine histological sections from paraffin-embedded prostatic adenocarcinoma specimens[END_REF][START_REF] Jordanova | Flow cytometric sorting of paraffinembedded tumor tissues considerably improves molecular genetic analysis[END_REF][START_REF] Jordanova | Hemizygous deletions in the HLA region account for loss of heterozygosity in the majority of diffuse large B-cell lymphomas of the testis and the central nervous system[END_REF] For detection of the most common recurrent translocations including those affecting 8q24/MYC, 18q21/BCL2, 3q27/BCL6, 18q21 / MALT1 efficient probe sets and protocols for hybridization procedures are now commercially available. These are based on two colour probe sets which generate patterns that allow normal cells to be distinguished from those with a mono-allelic or bi-allelic breakpoint. [START_REF] Haralambieva | Detection of three common translocation breakpoints in non-Hodgkin's lymphomas by fluorescence in situ hybridization on routine paraffin-embedded tissue sections[END_REF][START_REF] Haralambieva | Detection by the fluorescence in situ hybridization technique of MYC translocations in paraffin-embedded lymphoma biopsy samples[END_REF] A good example of the usefulness of interphase FISH is detection of 8q24/MYC breakpoints and exclusion of other breakpoints in Burkitt lymphoma. In >90% of all Burkitt lymphomas a t(8;14)(q24;q32), t(2;8) or t(8;22) will be found whereas t(14;18)(q32;q21) affecting BCL2 and 3q27 breakpoints affecting BCL6 should be absent. In contrast in 5-20% of all diffuse large B-cell lymphoma MYC breakpoints can also be detected, more than half of these cases sharing a t(14;18)(q32;q21) and / or a 3q27 breakpoint, thus representing a so called "double hit lymphoma". [START_REF]WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues[END_REF] The latter lymphomas were previously diagnosed as diffuse large B cell lymphoma, Burkitt-like lymphoma, atypical Burkitt lymphoma or aggressive B cell lymphoma NOS and represent very aggressive lymphomas that behave differently from classical Burkitt lymphoma and are difficult to treat with regimens like R-CHOP or high intensity -short duration polychemotherapy. [START_REF] Barrans | Rearrangement of MYC is associated with poor prognosis in patients with diffuse large B-cell lymphoma treated in the era of rituximab[END_REF][START_REF] Mead | A prospective clinicopathologic study of dosemodified CODOX-M/IVAC in patients with sporadic Burkitt lymphoma defined using cytogenetic and immunophenotypic criteria (MRC/NCRI LY10 trial)[END_REF] MYC breakpoints can be detected by FISH in different ways. The most widely used approach is a segregation or split assay using two differently coloured probes that flank the MYC locus at 8q24. In case of a green and red label, the two probes are seen as a yellow or red/green signal in normal interphase nuclei. In case of a mono-allelic break, the nucleus will show one normal signal derived from the normal 8q24 allele and one split signal with separate green and red signals. It is estimated that more than 90% of the breakpoints in Burkitt lymphoma are detected by the currently available probe sets. The few failures are partly due to the presence of cryptic rearrangements that can be missed by some assays. [START_REF] Haralambieva | Interphase fluorescence in situ hybridization for detection of 8q24/MYC breakpoints on routine histologic sections: Validation in Burkitt lymphomas from three geographic regions[END_REF][START_REF] May | Detection of cryptic and variant IGH-MYC rearrangements in high-grade non-Hodgkin's lymphoma by fluorescence in situ hybridization: implications for cytogenetic testing[END_REF] In this MYC segregation assay, only a MYC breakpoint is detected and no information is given on the partner that is juxtaposed to MYC. In Burkitt lymphoma the partners are exclusively immunoglobulin loci (85% IGH, >10% lambda and <5% kappa), so for the diagnosis of Burkitt lymphoma the segregation assay could be replaced by more specific co-localization assays for MYC with IGH, or MYC with IGL and MYC with IGK. However, in other lymphomas like diffuse large B-cell lymphoma with a MYC breakpoint, IGH/IGK/IGL and MYC are juxtaposed in only two thirds of the cases. In the other cases loci like a locus at 9p13 or BCL6 at 3q27 are the partners of MYC. Thus, to detect all 8q24/MYC breakpoints in Burkitt lymphoma and diffuse large B-cell lymphoma (and other lymphomas), all cases should always first be assessed by a simple MYC segregation assay. After this screening, a FISH test for juxtaposition of the MYC locus to one of the immunoglobulin loci should be applied, since absence of any type of co-localization with the immunoglobulin genes is strong evidence against a diagnosis of Burkitt lymphoma. [START_REF] Boerma | Translocations involving 8q24 in Burkitt lymphoma and other malignant lymphomas: a historical review of cytogenetics in the light of todays knowledge[END_REF] Figure 3 shows an example of a MYC-IGH colocalisation FISH pattern in a case of Burkitt lymphoma with a MYC-break.

Because of the diagnostic and therapeutic consequences it is recommended that 8q24/MYC FISH analysis is carried out on all B cell lymphomas that share some features with Burkitt One major drawback of FISH tests is the need to have immunofluorescence microscopy equipment and expertise to analyse the slides in a dark room. It sometimes is difficult to find the optimal regions with most tumour cells in such slides. Several companies are currently preparing bright field in situ hybridization methods (BRISH) making use of chromogenic substances and/or silver that could replace dual colour FISH tests, allowing pathologists to read slides at the normal bright-field microscope and to better correlate the signal pattern with the microscopic anatomy of the tumour. [START_REF] Nitta | Automated brightfield break-apart in situ hybridization (ba-ISH) application: ALK and MALT1 genes as models[END_REF][START_REF] Stasik | Increased MYC gene copy number correlates with increased mRNA levels in diffuse large B-cell lymphoma[END_REF][START_REF] Van Rijk | Double-staining chromogenic in situ hybridization as a useful alternative to split-signal fluorescence in situ hybridization in lymphoma diagnostics[END_REF] An additional advantage of BRISH over FISH is that the specimen can be archived and that inter-laboratory quality control programs are much easier to perform. However, the major problem of BRISH is the visualization of exactly colocalizing signals.

Polymerase chain reaction (PCR), real time polymerase chain reaction PCR (RT-PCR).

In the diagnosis of lymphoma PCR tests at the DNA level are used mostlyfor clonality analysis of the immunoglobulin genes or T cell receptor genes. Highly sensitive and specific primer combinations and protocols have been developed most notably by the BIOMED consortium (http://www.euroclonality.org/). These tests are currently used worldwide and enable to detect clonality in fresh tissues as well as in archival, FFPE materials. [START_REF] Van Krieken | Improved reliability of lymphoma diagnostics via PCR-based clonality testing: report of the BIOMED-2 Concerted Action BHM4-CT98-3936[END_REF][START_REF] Langerak | Polymerase chain reaction-based clonality testing in tissue samples with reactive lymphoproliferations: usefulness and pitfalls. A report of the BIOMED-2 Concerted Action BMH4-CT98-3936[END_REF][START_REF] Sandberg | BIOMED-2 multiplex immunoglobulin/T-cell receptor polymerase chain reaction protocols can reliably replace Southern blot analysis in routine clonality diagnostics[END_REF][START_REF] Van Dongen | Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936[END_REF] Some translocations, such as the t(14;18) involving BCL2, can be detected at the genomic level by PCR. However in view of the diversity of breakpoints multiple tube tests with many sets of different primers are necessary to detect most t(14;18) breakpoints. [START_REF] Van Dongen | Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936[END_REF] This problem in detecting chromosomal breakpoints is even more evident for the t(11;14)(q13;q32) in mantle cell lymphoma. Extensive mapping studies have shown that only 30-40% of all t (11;14) breakpoints cluster in a small region (MTC) in which the breakpoint can be detected by PCR. 62 63 Since FISH probes that cover all breakpoints can be easily designed, this technique is superior. Another disadvantage of PCR is that this method in general makes use of primers that flank the breakpoint, and therefore the primers have to be derived from both partners. In consequence both partners should be consistently involved, otherwise breakpoints will be missed. On the other hand, if a breakpoint is amplifiable by PCR, it is a far superior method for the detection and quantification of small numbers of cells, and thus in the detection of minimal residual disease (MRD). PCR has been used to detect MRD after intensive chemotherapy and stem cell transplant in patients with mantle cell lymphoma with an amplifiable t (11;14) breakpoint. [START_REF] Andersen | Pre-emptive treatment with rituximab of molecular relapse after autologous stem cell transplantation in mantle cell lymphoma[END_REF] Quantitative RT-PCR is very useful for the detection of chromosomal translocations that lead to the production of novel fusion mRNA transcripts. Such events form the minority of translocations in lymphomas (but the majority in myeloid neoplasms), since most translocations are derived from RAG1/2 or AICDA mediated juxtaposition of oncogenes to the enhancers of immunoglobulin gene or T cell receptor genes, leaving the protein-encoding domains intact. Examples of translocation that generate fusion transcripts and proteins are those involving ALK1 in ALK+ anaplastic large cell lymphomas (ALK+ ALCL) and ALK+ large B cell lymphoma (ALK+ DLBCL). In the great majority ALK and NPM are fused giving rise to a fusion protein with nuclear and cytoplasmic localization. In addition in 5-30% of the gastric and in 30-50% of pulmonary marginal zone B cell (MALT) lymphomas, a t(11;18)(q21;21) with fusion of API2 and MALT1 is present. In gastric lymphomas the presence of this translocation is associated with resistance to Helicobacter pylori eradication therapy. Because of the therapeutic consequences it is recommended to either FISH or RT-PCR to detect this translocation routinely in marginal zone lymphomas. [START_REF] Liu | T(11;18) is a marker for all stage gastric MALT lymphomas that will not respond to H. pylori eradication 3[END_REF] The better performance of RT-PCR on cDNA than PCR on DNA in the detection of translocations is because the number of exons involved in the breakpoints is limited and RNA splicing eliminates long stretches of (intronic) nucleotides that can not be efficiently amplified by PCR on DNA. In consequence, most fusion breakpoints can be detected by RT-PCR with a very limited number of primer combinations. Also because of these characteristics, RT-PCR can be used on FFPE material. Formalin fixation has the advantage that endonucleases that degrade RNA are rapidly inactivated. On the other hand, apart from cross-linking and fragmentation of RNA, formalin also induces the addition of monoethylol groups to adenosine and consequently inhibits primer annealing for reverse transcription. Specific protocols are now for optimal RNA extraction and efficient cDNA synthesis from FFPE material. 12 Despite all optimized procedures, the efficiency of RT-PCR using RNA extracted from FFPE material remains approximately 30-50-fold lower than RNA extracted from matched frozen materials. This topic has been extensively reviewed by Farragher et al and the reader should consult these sources for more detailed information on the protocols that are now available 66- 69 ..

Other molecular methods.

Several other molecular methods can be used for quantification of DNA targets and thus for the detection of copy number variations. They include quantitative fluorescent PCR of short tandem repeats (QF-PCR of STR), multiplex PCR of short exonic sequences (QMPSF) and multiplex ligation probe amplification (MLPA). [START_REF] Gouas | Gene dosage methods as diagnostic tools for the identification of chromosome abnormalities[END_REF] In principle all techniques enable to assay multiple loci for gains or losses, and therefore they could be helpful in the assessment of the genomic complexity, for instance as a tool to differentiate between Burkitt lymphoma and diffuse large B cell lymphoma. As far as known, none of these techniques have been introduced yet into a diagnostic haematopathology setting. Moreover, like for CGH methods and SNP analysis, these methods do not allow detecting reciprocal translocations.

Immunohistochemisty and molecular cytogenetics.

This review would not be complete without a comment on the use immunohistochemistry for the detection of genetic abnormlaties. In fact immunohistochemistry may in some instances replace molecular cytogenetics. [START_REF] Falini | Proteins encoded by genes involved in chromosomal alterations in lymphoma and leukemia: clinical value of their detection by immunocytochemistry[END_REF] Chromosomal abnormalities cause deregulation of genes that are not expressed in normal B cells or T cells, or are not expressed in a specific stage of maturation of normal B or T cells. An example is the t(11;14)(q13;q32) present in >95% of all mantle cell lymphomas. This translocation gives rise to activation of CCND1 by juxtaposing to an immunoglobulin gene enhancer, and in consequence CCND1 expression is induced while it is not expressed in normal B and T cells. Previous work has shown that there is an excellent correlation between the presence of a translocation and expression of CCND1 at the RNA and protein level (western blot and immunohistochemistry). [START_REF] Boer | BCL1/cyclin D1 in malignant lymphoma[END_REF][START_REF] Boer | Cyclin D1 protein analysis in the diagnosis of mantle cell lymphoma[END_REF] Excellent monoclonal antibodies against CCND1 have been developed and in consequence immunohistochemistry for CCND1 has almost completely replaced the molecular methods to detect this translocation. A second example is the detection of ALK translocations in ALK+ anaplastic large cell lymphoma (ALCL) and ALK+ diffuse large B cell lymphoma. These lymphomas and also a subset of inflammatory myofibroblastic tumours contain a fusion of the ALK gene at 2p23 to a large variety of other partners (NPM being the most frequent partner). Since ALK is not expressed in normal adult tissues, ALK expression in these tumours as observed by immunohistochemistry shows that a fusion protein containing ALK is present (table 2). In all lymphomas and inflammatory myofibroblastic tumours with an ALK breakpoint this expression is high and can be easily detected by immunohistochemistry. In consequence, immunohistochemistry has completely replaced molecular methods. Notably, the pattern of its expression within the cells as seen in tissue slides informs us also on the ALK fusion partner used in the translocation. [START_REF] Stein | CD30(+) anaplastic large cell lymphoma: a review of its histopathologic, genetic, and clinical features 140[END_REF] Recent research has shown that ALK inversions involving the EML4 gene occur in approximately 4-9% of all adenocarcinomas of the lung. However, in these cases a very low level of ALK protein expression is present that only can be detected using specific antibodies and very sensitive assays. [START_REF] Mino-Kenudson | A novel, highly sensitive antibody allows for the routine detection of ALK-rearranged lung adenocarcinomas by standard immunohistochemistry[END_REF] A third example is the use of antibodies against SOX11 in the diagnosis of mantle cell lymphoma. SOX11 encodes a member of the SOX (SRY-related HMG-box) family of transcription factors involved in the regulation of embryonic development. Based on gene expression studies this gene appeared to be highly expressed in almost all mantle cell lymphomas in addition to some acute lymphoblastic lymphomas / leukaemias, Burkitt lymphomas and hairy cell leukaemias. Interestingly, t(11;14) and CCND1 protein negative mantle cell lymphomas are SOX11 positive. [START_REF] Ek | Nuclear expression of the non B-cell lineage Sox11 transcription factor identifies mantle cell lymphoma[END_REF][START_REF] Mozos | SOX11 expression is highly specific for mantle cell lymphoma and identifies the cyclin D1-negative subtype[END_REF][START_REF] Chen | Nuclear expression of sox11 is highly associated with mantle cell lymphoma but is independent of t(11;14)(q13;q32) in nonmantle cell B-cell neoplasms[END_REF] Other publications suggest that very indolent but also rare very aggressive mantle cell lymphomas may be negative for SOX11. [START_REF] Wang | The subcellular Sox11 distribution pattern identifies subsets of mantle cell lymphoma: correlation to overall survival[END_REF][START_REF] Fernandez | Genomic and gene expression profiling defines indolent forms of mantle cell lymphoma[END_REF]

Molecular cytogenetic aspects of individual lymphomas

In Table 2, a brief summary is given of the most common molecular abnormalities in specific lymphomas, [START_REF]WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues[END_REF] the methods to detect these abnormalities, as well as their diagnostic and clinical relevance. Excellent web resources include (http://atlasgeneticsoncology.org/ , http://www.progenetix.net/ and http://www.ncbi.nlm.nih.gov/sites/entrez?db=cancerchromosomes) and elsewhere. [START_REF] Siebert | Mature B-and T-cell Neoplasms and Hodgkin Lymphoma[END_REF][START_REF] Ventura | FISH analysis for the detection of lymphoma-associated chromosomal abnormalities in routine paraffin-embedded tissue[END_REF] Conclusions and future perspectives The introduction of reliable BRISH assays will allow many more pathologists to perform and interpret these tests.

High throughput methods such array CGH, SNP analysis and whole genome expression analysis have not been introduced into routine diagnostic laboratories. This may be facilitated in the near future by providing tailor made platforms with a limited number of targets to be detected. There is clearly a place for such assays, for instance for the differential diagnosis between Burkitt lymphoma and diffuse large B cell lymphoma. Similar approaches could likely be followed to distinguish indolent from conventional mantle cell lymphomas, since there are indications that indolent mantle cell lymphomas have a relatively simple karyotype. [START_REF] Fernandez | Genomic and gene expression profiling defines indolent forms of mantle cell lymphoma[END_REF] At present, these assays require fresh or freshly frozen material and the question whether these methods will become part of our routine diagnostics is very much dependent on the question whether these techniques will be applicable in routinely FFPE materials.

Finally, it is obvious that the development of new drugs that target specific intracellular or intercellular pathways in specific types of lymphoma, will drive the development of new molecular tests in malignant lymphoma. One example will be new drugs that interfere with specific signalling pathways used in ABC type diffuse large B-cell lymphoma but not GC type. [START_REF] Hailfinger | Essential role of MALT1 protease activity in activated B cell-like diffuse large B-cell lymphoma[END_REF] This will require better methods to distinguish these types of diffuse large B-cell lymphoma, and the detection of the specific molecular abnormalities in the pathway targeted by the drug. Developments of this type are already taking place in other types of tumour. Normal DNA and tumor DNA are differentially labeled, often with Cy3 and Cy5. After blocking of (ubiquitously present and thus hindering) repetitive DNA, these labeled DNAs are competitively hybridized to either normal chromosome preparations or a high density array of (preferably) overlapping probes on a glass slide. In case of conventional CGH, the preparations can be directly analyzed on a fluorescence microscope, in cases of an array, dedicated hardware (a microarray scanner) and software is necessary. Gains are indicated by green bars richt of the chromosomes, losses by red bars (left of the chromosome). Note the higher resolution but also the higher complexity, with in particular a larger number of smaller events, in particular around the centromeres of chromosomes. A large number of these alterations reflect copy number variations between individuals and are not tumour specific.

Panel C shows a case analyzed by SNP's with a small homozygous deletion on 9p21, containing only the CDKNBA and CDKN2B genes. In a second case (not shown) there was a large hemizygous deletion and a similar small deletion on the other allele. These small deletions were not detected by either conventional CGH or array CGH (data not shown). Note the higher resolution but also the higher complexity, with in particular a larger number of smaller events, in particular around the centromeres of chromosomes. A large number of these alterations reflect copy number variations between individuals and are not tumour specific.

F o r P e e r R e v i e w

Panel C shows a case analyzed by SNP's with a small homozygous deletion on 9p21, containing only the CDKNBA and CDKN2B genes. In a second case (not shown) there was a large hemizygous deletion and a similar small deletion on the other allele. These small deletions were not detected by either conventional CGH or array CGH (data not shown). Note the higher resolution but also the higher complexity, with in particular a larger number of smaller events, in particular around the centromeres of chromosomes. A large number of these alterations reflect copy number variations between individuals and are not tumour specific.

Panel C shows a case analyzed by SNP's with a small homozygous deletion on 9p21, containing only the CDKNBA and CDKN2B genes. In a second case (not shown) there was a large hemizygous deletion and a similar small deletion on the other allele. These small deletions were not detected by either conventional CGH or array CGH (data not shown).

  of diffuse large B cell lymphoma.

  and SNP analysis all have the intrinsic disadvantage that balanced genetic abnormalities (abnormalities without any gain or loss of DNA), and thus most translocations and inversions are missed (Table

  in adult patients. The analysis should include a segregation assay for 8q24/ MYC, and preferably also a co-localization assay for MYC and the IG loci, as well as a segregation assay for both BCL2 and BCL6 to rule out a double hit lymphoma.

75

 75

  now become an integral part of routine lymphoma diagnostics. In many laboratories, PCR based clonality tests and FISH investigations for specific chromosomal abnormalities are performed on a routine basis. One challenge for the future will be to develop multicolour BRISH instead of fluorescent in situ hybridization (FISH) assays for detection of all breakpoints and relevant numerical abnormalities in lymphomas.

Figure 1 .

 1 Figure 1. Principles of conventional CGH and array CGH.

Figure 2 .

 2 Figure 2. Array CGH analysis and SNP analysis of pediatric Burkitt lymphoma.Panel A shows conventional CGH analysis of a series of 15 paediatric Burkitt lymphomas is show. Each black bar to the right of a chromosome represents gain in an individual case; each grey bar to the left side of a chromosome represents loss. This analysis shows a limited number of recurrent aberrations, such as copy number increase of chromosome 13q31-32 and loss of chromosome 17p.

Figure 3 .

 3 Figure 3. Colocalization of MYC and IGH in Burkitt lymphoma. A case of paediatric Burkitt lymphoma with presentation in the tonsil with a MYC-break (detected with the Vysis LSI MYC Dual Color, Break Apart Rearrangement Probe set from Abbott molecular; data not shown), was hybridized with the Vysis LSI IGH/MYC, CEP 8 Tricolor, Dual Fusion Translocation Probe. Many cells show fusion of the MYC (green) and IGH (red) signals (arrows). Note that the percentage of spurious co-localizing signals can be high in normal cells, up to 20% of all cells.

Figure 1 .Figure 2 .

 12 Figure 1. Principles of conventional CGH and array CGH. Normal DNA and tumor DNA are differentially labeled, often with Cy3 and Cy5. After blocking of (ubiquitously present and thus hindering) repetitive DNA, these labeled DNAs are competitively hybridized to either normal chromosome preparations or a high density array of (preferably) overlapping probes on a glass slide. In case of conventional CGH, the preparations can be directly analyzed on a fluorescence microscope, in cases of an array, dedicated hardware (a microarray scanner) and software is necessary. 254x190mm (96 x 96 DPI)

Figure 2 .

 2 Figure 2. Array CGH analysis and SNP analysis of pediatric Burkitt lymphoma. Panel A shows conventional CGH analysis of a series of 15 paediatric Burkitt lymphomas is show. Each black bar to the right of a chromosome represents gain in an individual case; each grey bar to the left side of a chromosome represents loss. This analysis shows a limited number of recurrent aberrations, such as copy number increase of chromosome 13q31-32 and loss of chromosome 17p. Panel B shows a SNP analysis of 18 paediatric Burkitt lymphomas is shown. The human CNV370-DUO BeadChips (Illumina Inc, San Diego, CA, USA) array was used. SNP array data were imported into BeadStudio (Illumina Inc, San Diego, CA, USA) and analyzed using the Chromozone bookmark plug-in and a modified version of SOMATICs. Directly to the right of each chromosome, the black bar indicates the region that is covered by the probes. Gains are indicated by green bars richt of the chromosomes, losses by red bars (left of the chromosome).Note the higher resolution but also the higher complexity, with in particular a larger number of smaller events, in particular around the centromeres of chromosomes. A large number of these alterations reflect copy number variations between individuals and are not tumour specific.

Figure 3 .

 3 Figure 3. Colocalization of MYC and IGH in Burkitt lymphoma. A case of paediatric Burkitt lymphoma with presentation in the tonsil with a MYC-break (detected with the Vysis LSI MYC Dual Color, Break Apart Rearrangement Probe set from Abbott molecular; data not shown), was hybridized with the Vysis LSI IGH/MYC, CEP 8 Tri-color, Dual Fusion Translocation Probe. Many cells show fusion of the MYC (green) and IGH (red) signals (arrows). Note that the percentage of spurious co-localizing signals can be high in normal cells, up to 20% of all cells. 254x190mm (96 x 96 DPI)

TABLE 1 .

 1 INTRINSIC FEATURES OF MOLECULAR GENETIC METHODS

	Method	Resolution Possible on	Levels of	Individual locus			Ability to
		formalin	required	unique probes /			detect:
		fixed		skills &	primers required
		materials	equipment	for the assay
						genome-wide	genome-wide	Specific	Specific	Low copy	UPD
						structural	numerical	translocations	amplifications	number
						abnormalities	abnormalities			changes
	karyotyping	>4-5 mb	-	++	-	++	+	+	±	±	-
	Spectral karyotyping	>1-5 mb	-	+++	-	+++	+	+	±	±	-
	CGH	>5mb	+	++	-	-	++	-	±	+	-
	Array CGH	>50kb	+	+++	-	-	+++	-	++	++	-
	SNP array analysis	>50kb a	+	+++	-	-	+++	-	++	++	++
	Interphase
	FISH	>50kb	+	+	+	-	-	++	++	+	-
	(sections)
	PCR	<400bp b	+	+	+	-	-	± c	++	-	-
	RT-PCR	<150bp d	+	+	+	-	-	±	-	-	-
	UPD: uniparental disomy; CGH: comparative genomic hybridization; SNP: single nucleotide polymorphism; FISH: fluorescence in situ hybridization; PCR: polymerase chain
	reaction; RT-PCR: reverse transcription PCR. a : Theoretically very high resolution, practically limited by polymorphisms. b : <400 bp in formalin fixed materials. c : very much
	dependent on the individual translocations, see text. d : in formalin fixed materials; only for fusion genes, see text

Published on behalf of the British Division of the International Academy of Pathology Histopathology


Rebecca L Auer

The coming of age of microRNA for B cell lymphomas

Keywords: B cell lymphoma, microRNAs, diagnosis, prognosis

The coming of age of microRNA for B cell lymphomas

F o r P e e r R e v i e w

sequence specific base pairing. The result is a reduction in translation of the corresponding protein [START_REF] Miska | How microRNAs control cell division, differentiation and death[END_REF] . miRNAs by their nature are involved in virtually all biological processes, including cell differentiation, apoptosis and proliferation, in which they exert their effects through alteration of expression of a range of target genes [START_REF] Bartel | MicroRNAs: genomics, biogenesis, mechanism, and function[END_REF][START_REF] Chen | The regulation of genes and genomes by small RNAs[END_REF] . miRNAs are relatively fewer and less complex in numbers compared to the human complement of genes (in excess of 20,000), but, are individually considerably more influential in the expression of large groups of genes, through their shared sequence specificity, and thus cell growth, development and death. A single miRNA can target many hundred genes and the 3'UTR of a single gene can also bind multiple miRNAs cooperatively [START_REF] Lewis | Prediction of mammalian microRNA targets[END_REF] . Currently approximately 10-30% of all human genes are thought to be a target for miRNA regulation [START_REF] Lewis | Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets[END_REF] .

Despite their fundamental role in biology, these critical molecules were only discovered in 1993 following identification of a post-transcriptional regulator sequence, lin-4, in a developmental gene of C.elegans [START_REF] Wightman | Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans[END_REF][START_REF] Lee | The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14[END_REF] . It was here that the key and unique characteristics of the miRNA molecule were first described; small transcript size (~22 nt), non-coding and derivation from a hairpin structured RNA. However, it was another 7 years later until the term "miRNA" was first coined when Ruvkun's group identified another C.elegans miRNA, let-7 8 9 . Following on from this discovery, conserved regulatory RNAs were identified throughout the genome of prokaryotic and eukaryotic organisms with an explosion in their characterization [START_REF] Lee | An extensive class of small RNAs in Caenorhabditis elegans[END_REF][START_REF] Griffiths-Jones | miRBase: microRNA sequences, targets and gene nomenclature[END_REF] . miRNA are common to all living organisms with more than 14000 miRNA sequences discovered to date in 133 species. However within a single organism the numbers are limited so that there are only approximately 800 human sequences (www.mirbase.org, Release 15, April 2010) influencing some 20,000 genes.

The mature miRNA (often designated miR) arises from a characteristic stem-loop sequence (called a pre-mir) and it is this structure that gives it the ability to "silence" gene expression. The loop is excised from a longer primary transcript (or pri-mir), through a series of processing reactions which begin in the nucleus. Only a handful of primary transcripts have been fully described, but evidence suggests that miRNAs are RQ-PCR assays) with a number of studies defining the signature pattern of normal cells [START_REF] Zhang | Patterns of microRNA expression characterize stages of human B-cell differentiation[END_REF][START_REF] Basso | Identification of the human mature B cell miRNome[END_REF][START_REF] Malumbres | Differentiation stage-specific expression of microRNAs in B lymphocytes and diffuse large B-cell lymphomas[END_REF][START_REF] Tan | miRNA profiling of B-cell subsets: specific miRNA profile for germinal center B cells with variation between centroblasts and centrocytes[END_REF] . A highly unique miRNA profile has led to an understanding of specific miRNAs critical to normal B cell differentiation.

Global profiles

There have been 4 major studies [START_REF] Zhang | Patterns of microRNA expression characterize stages of human B-cell differentiation[END_REF][START_REF] Basso | Identification of the human mature B cell miRNome[END_REF][START_REF] Malumbres | Differentiation stage-specific expression of microRNAs in B lymphocytes and diffuse large B-cell lymphomas[END_REF][START_REF] Tan | miRNA profiling of B-cell subsets: specific miRNA profile for germinal center B cells with variation between centroblasts and centrocytes[END_REF] , that have attempted to define the miRNA profile of normal B cells during mature B cell differentiation as a basis for understanding the role of miRNAs in B-cell function and lymphomagenesis. These studies have employed various techniques analysing purified tonsillar subsets. An earlier study [START_REF] Landgraf | A mammalian microRNA expression atlas based on small RNA library sequencing[END_REF] cloned and sequenced small RNA libraries from a large panel of different cell types and tissue and suggested that most miRNAs are ubiquitously expressed with little tissue specificity.

These discordant results are rather out of line with more recent data and go against the more recent understanding that there is considerable variation in miRNA expression during cell development.

Further confirmation of this was supported by a combination of cloning and computational analysis by Dalla-Favera's group who reported on the identification of 178 miRNAs that represent the mature B cell miRNome [START_REF] Basso | Identification of the human mature B cell miRNome[END_REF] . From their analysis naïve and memory B cells appeared similar, sharing a large fraction of the most abundant miRNAs whilst germinal centre (GC) B cells appeared to be quite distinct in terms of their miRNA profile.

Similarly, Malumbres et al [START_REF] Malumbres | Differentiation stage-specific expression of microRNAs in B lymphocytes and diffuse large B-cell lymphomas[END_REF] showed that the pattern of miRNA expression in centroblasts was clearly distinguishable from the other cell subpopulations with specific increased expression of 51 miRNAs. Although naive and memory B cells could be distinguished based on their miRNA expression pattern, the observed differences were less striking compared with the GC lymphocytes. They constructed a "cell of origin classifier" consisting of 39 miRNAs that could consistently differentiate between the distinct B cell subpopulations. Twenty-one of the 39 miRNAs in this "cell of origin These studies have employed genome wide expression profiling of tonsillar subsets and demonstrate that mature B cell subsets have distinct patterns of miRNA expression with a regulatory role for miRNAs at every stage of the B cell differentiation process. It has, however, been difficult to collate the published information from these studies to produce a specific miRNA signature for each mature B cell subset and this in part may be due to the different technological approaches taken by different groups. However, from these studies some consensus arises (summarised in Figure 1) and it can be anticipated that the mature B cell miRNome may predict the signature of their malignant counterparts.

Although an important component of the biology of the malignant lymphoma cell is inherited from its non-transformed cellular progenitor, it is clear that aberrant miRNA expression is acquired upon cell transformation (see below) and may in turn play an important role in initiating or promoting malignant lymphoma. Early B cell development (summarized in Figure 2)

Specific miRNAs control B cell development

miR-150
miR-150 is selectively expressed in mature resting B and T cells but not their progenitors [START_REF] Monticelli | MicroRNA profiling of the murine hematopoietic system[END_REF][START_REF] Zhou | miR-150, a microRNA expressed in mature B and T cells, blocks early B cell development when expressed prematurely[END_REF] and appears to function by influencing the transcription factor c-Myb which is critically important for the multiple steps of lymphocyte development required for the generation of B1 cells [START_REF] Xiao | MiR-150 controls B cell differentiation by targeting the transcription factor c-Myb[END_REF] . Its expression is highly up-regulated during the development of mature B and T cells. Ectopic expression of miR-150 in haemopoietic stem cells reduces mature B cell levels in the circulation, spleen and lymph nodes but does not effect T cell or myeloid cell numbers [START_REF] Zhou | miR-150, a microRNA expressed in mature B and T cells, blocks early B cell development when expressed prematurely[END_REF] . Detailed analysis of miR-150 in B cell maturation showed that its premature expression blocked the differentiation of pro-B cells to pre-B cells [START_REF] Zhou | miR-150, a microRNA expressed in mature B and T cells, blocks early B cell development when expressed prematurely[END_REF] .

By combining loss-and gain-of-function approaches for miR-150 with conditional and partial ablation of c-Myb, Xiao et al showed in vivo that miR-150 controls c-Myb expression in a dose-dependent manner over a narrow range of miRNA and c-Myb concentrations. This dramatically affects lymphocyte development and response [START_REF] Xiao | MiR-150 controls B cell differentiation by targeting the transcription factor c-Myb[END_REF] . It can be seen how miR-150 disruption could lead to maturation arrest of B cells and contribute to abnormal lymphogenesis.

miR-17~92

The miRNA cluster miR-17~92 (comprising miR-17, miR-18a, miR-19a, miR-20a, miR-

miR-34

The most recently identified miRNA with a significant role in B cell development is miR-34a, a transcriptional target of p53 [START_REF] He | A microRNA component of the p53 tumour suppressor network[END_REF][START_REF] Chang | Transactivation of miR-34a by p53 broadly influences gene expression and promotes apoptosis[END_REF] . Overexpression of miR-34a causes a partial block in B cell development at the pro-B cell to pre-B-cell transition, leading to a reduction in mature B cells. This has been shown to be mediated primarily by inhibited expression of the transcription factor Foxp1, a direct target of miR-34a [START_REF] Rao | MicroRNA-34a perturbs B lymphocyte development by repressing the forkhead box transcription factor Foxp1[END_REF] .

Dicer

The [START_REF] Eis | Accumulation of miR-155 and BIC RNA in human B cell lymphomas[END_REF]33 . It was the first oncogeneic miRNA to be identified and plays a significant role in carcinogenesis being up-regulated in various haematological and solid malignancies. However, it also plays a pivotal role in normal haematopoietic cell differentiation [START_REF] Georgantas | CD34+ hematopoietic stemprogenitor cell microRNA expression and function: a circuit diagram of differentiation control[END_REF] being expressed in both myeloid and lymphoid lineages. Higher levels of miR-155 expression are observed in activated B, T cells and macrophages [START_REF] Eis | Accumulation of miR-155 and BIC RNA in human B cell lymphomas[END_REF][START_REF] Van Den Berg | High expression of B-cell receptor inducible gene BIC in all subtypes of Hodgkin lymphoma[END_REF] at critical points for normal lymphocyte development and immune function. BIC/miR-155 deficient mice display defective B and T cell immunity and abnormal function of antigenpresenting cells. [START_REF] Rodriguez | Requirement of bic/microRNA-155 for normal immune function[END_REF] In a comparable mouse model, Thai et al [START_REF] Thai | Regulation of the germinal center response by microRNA-155[END_REF] showed that miR-155 mutant mice displayed reduced numbers of GCs and impaired T cell dependent antibody response due to impaired cytokine production. TNF production was reduced in the B cells whilst T cells showed significantly increased IL-10 production. Furthermore, miR-155 has been shown to play a key role in antigen-driven B cell maturation and is required for the normal production of isotype-switched, high-affinity antibodies and a memory response [START_REF] Vigorito | microRNA-155 regulates the generation of immunoglobulin class-switched plasma cells[END_REF] . A potential mechanism for miR-155 functionality has been identified through direct regulation of the transcription factor Pu.1. miR-155 is required for the downregulation of Pu.1, and, in the absence of miR-155, excessive Pu.1 results in reduced numbers of IgG1 switched cells [START_REF] Vigorito | microRNA-155 regulates the generation of immunoglobulin class-switched plasma cells[END_REF] . The activation induced cytidine deaminase (AID) enzyme responsible for deaminating cytosine to produce U:G mismatches in DNA and thus initiate Ig gene diversity in activated B cells is a direct target of miR-155. In this way miR-155 acts as a negative regulator of AID downmodulating expression [START_REF] Dorsett | MicroRNA-155 suppresses activation-induced cytidine deaminase-mediated Myc-Igh translocation[END_REF][START_REF] Teng | MicroRNA-155 is a negative regulator of activation-induced cytidine deaminase[END_REF] .

Consequently miR-155 can act as a tumour suppressor by reducing potentially oncogenic translocations generated by AID [START_REF] Dorsett | MicroRNA-155 suppresses activation-induced cytidine deaminase-mediated Myc-Igh translocation[END_REF] .

microRNAS in B cell lymphomas (summarised in Table 1)

Following identification of disease specific signatures, studies are now being undertaken to develop specific miRNAs for classification and to determine whether these can replace standard immunohistochemical techniques. Whilst many studies have looked for global changes in miRNA expression, the evidence from mouse models for a causal role of specific miRNAs in lymphomagenesis is more limited. The miR-15a/16-1 cluster at chromosome 13q14, within the most commonly deleted region in CLL, was found to be downregulated in 68% of cases with the deletion 14 .

Chronic Lymphocytic

Subsequently these miRNAs have been shown to target Bcl-2 and remove the suppressor influence on antiapoptotic Bcl-2 [START_REF] Cimmino | miR-15 and miR-16 induce apoptosis by targeting BCL2[END_REF] which is consistently overexpressed in CLL. These observations have not, however, been confirmed by subsequent studies [START_REF] Fulci | Quantitative technologies establish a novel microRNA profile of chronic lymphocytic leukemia[END_REF][START_REF] Ouillette | Integrated genomic profiling of chronic lymphocytic leukemia identifies subtypes of deletion 13q14[END_REF] . Following this original report, a number of groups [START_REF] Fulci | Quantitative technologies establish a novel microRNA profile of chronic lymphocytic leukemia[END_REF][START_REF] Calin | MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias[END_REF] have undertaken genome wide expression profiling studies to identify a miRNA signature for CLL compared to normal B lymphocytes as well as to characterise a signature associated with the different prognostic sub-groups of CLL (V H mutation status, ZAP70, 13q14 deletion, 11q22-23 deletion and 17p13/TP53 deletion). There is, however, significant variation in terms of the miRNAs identified as being differentially expressed between the studies. These discrepancies can be attributed to the differences in methodology between the studies (northern blot in early studies, miRNA cloning and RQ-PCR / array analysis in more recent) as well as the use of different controls used (CD5+ cells from tonsils 14 and CD5+ cells from cord blood [START_REF] Fulci | Quantitative technologies establish a novel microRNA profile of chronic lymphocytic leukemia[END_REF] ).

In addition the miRdatabase has expanded rapidly since the earlier studies with ongoing identification of novel miRNA sequences.

Unique miRNA signatures have been generated dependent upon V H gene mutation and ZAP70 status [START_REF] Fulci | Quantitative technologies establish a novel microRNA profile of chronic lymphocytic leukemia[END_REF][START_REF] Calin | MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias[END_REF][START_REF] Calin | A microRNA signature associated with prognosis and progression in chronic lymphocytic leukemia[END_REF] . The miR-29 family and miR-223 have been consistently found to be down-regulated in association with unmutated V H genes [START_REF] Fulci | Quantitative technologies establish a novel microRNA profile of chronic lymphocytic leukemia[END_REF][START_REF] Calin | MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias[END_REF][START_REF] Calin | A microRNA signature associated with prognosis and progression in chronic lymphocytic leukemia[END_REF][START_REF] Stamatopoulos | microRNA-29c and microRNA-223 down-regulation has in vivo significance in chronic lymphocytic leukemia and improves disease risk stratification[END_REF] and are dramatically decreased in poor prognostic patients independently of the prognostic marker classification used [START_REF] Stamatopoulos | microRNA-29c and microRNA-223 down-regulation has in vivo significance in chronic lymphocytic leukemia and improves disease risk stratification[END_REF] .

Similarly, miRNA expression data have been analysed to distinguish the subtypes of CLL with the most common genomic aberrations. Although Calin et al identified low levels of miR-15-a/16-1 in patients with the 13q deletion 14 , further work suggests that approximately 15% of CLL cases display marked reductions in miR15-a/miR16-1 expression often but not invariably associated with bi-allelic 13q14 loss [START_REF] Ouillette | Integrated genomic profiling of chronic lymphocytic leukemia identifies subtypes of deletion 13q14[END_REF] . Although there has been discordance over the significance of miR-15a/16-1 loss in CLL [START_REF] Ouillette | Integrated genomic profiling of chronic lymphocytic leukemia identifies subtypes of deletion 13q14[END_REF] , mice with a conditional knockout of miR-15a/16-1 develop a lymphoproliferative phenotype analogous to CLL confirming a tumour suppressor role for miR-15a/16-1 in CLL [START_REF] Klein | The DLEU2/miR-15a/16-1 cluster controls B cell proliferation and its deletion leads to chronic lymphocytic leukemia[END_REF] . Possibly of as much importance in CLL associated with the very poor prognostic genotype, 17p13/TP53 deletion, is the consistently associated downregulation of miR-34a [START_REF] Zenz | miR-34a as part of the resistance network in chronic lymphocytic leukemia[END_REF][START_REF] Mraz | miR-34a, miR-29c and miR-17-5p are downregulated in CLL patients with TP53 abnormalities[END_REF][START_REF] Zenz | Detailed analysis of p53 pathway defects in fludarabine-refractory chronic lymphocytic leukemia (CLL): dissecting the contribution of 17p deletion, TP53 mutation, p53-p21 dysfunction, and miR34a in a prospective clinical trial[END_REF][START_REF] Dijkstra | 17p13/TP53 deletion in B-CLL patients is associated with microRNA-34a downregulation[END_REF][START_REF] Visone | Karyotype-specific microRNA signature in chronic lymphocytic leukemia[END_REF][START_REF] Asslaber | microRNA-34a expression correlates with MDM2 SNP309 polymorphism and treatment-free survival in chronic lymphocytic leukemia[END_REF][START_REF] Rossi | microRNA fingerprinting of CLL patients with chromosome 17p deletion identify a miR-21 score that stratifies early survival[END_REF][START_REF] Merkel | Interdependent regulation of p53 and miR-34a in chronic lymphocytic leukemia[END_REF] . Members of the miR-34 family have been discovered to be direct p53 targets and mediate some of the p53-dependent effects. Whilst miR-34 b/c are not expressed in CLL cells, low miR-34a levels are a sensitive indicator of the activity of the p53 axis in B-CLL [START_REF] Rossi | microRNA fingerprinting of CLL patients with chromosome 17p deletion identify a miR-21 score that stratifies early survival[END_REF][START_REF] Moussay | Determination of genes and microRNAs involved in the resistance to fludarabine in vivo in chronic lymphocytic leukemia[END_REF] and are associated with p53 inactivation and chemotherapy-refractory disease irrespective of 17p deletion/TP53 mutation status [START_REF] Zenz | Detailed analysis of p53 pathway defects in fludarabine-refractory chronic lymphocytic leukemia (CLL): dissecting the contribution of 17p deletion, TP53 mutation, p53-p21 dysfunction, and miR34a in a prospective clinical trial[END_REF] . Amongst patients harboring a 17p deletion, low expression of miR-223, miR-29c, miR-29b, and miR-181 family were strongly associated with disease progression [START_REF] Rossi | microRNA fingerprinting of CLL patients with chromosome 17p deletion identify a miR-21 score that stratifies early survival[END_REF] confirming the adverse effect of low levels of miR-29 and miR-223 in CLL progression [START_REF] Stamatopoulos | microRNA-29c and microRNA-223 down-regulation has in vivo significance in chronic lymphocytic leukemia and improves disease risk stratification[END_REF] . In a separate study the 17p deleted miRNA signature comprised high levels of miR-15a, miR-21,and miR-155 and low levels of miR-34a and miR-181b [START_REF] Rossi | microRNA fingerprinting of CLL patients with chromosome 17p deletion identify a miR-21 score that stratifies early survival[END_REF] . In addition, miR-21 expression stratified survival in patients with 17p deleted CLL and amongst cohorts of patients with CLL with a variety of chromosomal aberrations [START_REF] Rossi | microRNA fingerprinting of CLL patients with chromosome 17p deletion identify a miR-21 score that stratifies early survival[END_REF] . Amongst both the good-and poor-prognosis subgroups, miR-181b levels identified patients who required early therapy and who thus need a closer follow-up [START_REF] Rossi | microRNA fingerprinting of CLL patients with chromosome 17p deletion identify a miR-21 score that stratifies early survival[END_REF] . Further to this, studies have identified miRNAs that may predict clinical resistance of CLL to fludarabine and a similar expression profile emerges with miR-29a, miR-181a and miR-221 amongst those being differentially expressed [START_REF] Moussay | Determination of genes and microRNAs involved in the resistance to fludarabine in vivo in chronic lymphocytic leukemia[END_REF] .

Despite the variation between studies, a number of miRNAs have been have been identified as being consistently and differentially expressed (Table 1) in CLL cells. The miR-15a/16-1 cluster lies at 13q14, targets Bcl-2 and is down regulated in a proportion of patients. It is clear from in vivo studies that loss of miR-15a/16-1 accelerates the proliferation of both human and mouse B cells by modulating the expression of genes controlling cell-cycle progression [START_REF] Klein | The DLEU2/miR-15a/16-1 cluster controls B cell proliferation and its deletion leads to chronic lymphocytic leukemia[END_REF] although it is not necessarily CLL specific and is deleted in other low grade lymphomas [START_REF] Ferreira | Comparative genome profiling across subtypes of low-grade B-cell lymphoma identifies type-specific and common aberrations that target genes with a role in B-cell neoplasia[END_REF] . miR-34a is a surrogate for p53 inactivation and predicts for a shorter time to treatment [START_REF] Zenz | Detailed analysis of p53 pathway defects in fludarabine-refractory chronic lymphocytic leukemia (CLL): dissecting the contribution of 17p deletion, TP53 mutation, p53-p21 dysfunction, and miR34a in a prospective clinical trial[END_REF] . Downregulation of miRs-29c and -223 are associated with unmutated VH genes providing a surrogate marker of mutational status. It is not possible from the published data as it stands to define a consensus miRNA signature for all CLL that can be useful for diagnosis and prognosis but some clarity is appearing that may be tested prospectively to fully understand their potential utility. miR-155 was the first oncogenic miRNA to be described following the observation that this miRNA is increased in several types of B cell lymphomas [START_REF] Eis | Accumulation of miR-155 and BIC RNA in human B cell lymphomas[END_REF]33 . Studies from transgenic mice confirm the oncogenic role of this miRNA in lymphoma. Mice overexpressing miR-155 in B cells consistently developed polyclonal pre-B cell proliferation followed by B cell malignancy [START_REF] Costinean | Pre-B cell proliferation and lymphoblastic leukemia/high-grade lymphoma in E(mu)-miR155 transgenic mice[END_REF] . On the other hand, sustained expression in haemopoietic stem cells causes a myeloproliferative disorder with a phenotype consistent with AML [START_REF] O'connell | Sustained expression of microRNA-155 in hematopoietic stem cells causes a myeloproliferative disorder[END_REF] . These observations suggest miR-155 is a significant regulator of haematopoetic cell development from an early stage.

Whilst its elevated expression is not specific to DLBCL [START_REF] Eis | Accumulation of miR-155 and BIC RNA in human B cell lymphomas[END_REF][START_REF] Roehle | MicroRNA signatures characterize diffuse large B-cell lymphomas and follicular lymphomas[END_REF] , significantly higher levels are present in DLBCLs with an activated B cell-like (ABC) phenotype in comparison with the germinal center B cell-like (GCB) phenotype [START_REF] Eis | Accumulation of miR-155 and BIC RNA in human B cell lymphomas[END_REF][START_REF] Pedersen | Onco-miR-155 targets SHIP1 to promote TNFalpha-dependent growth of B cell lymphomas[END_REF][START_REF] Lawrie | MicroRNA expression distinguishes between germinal center B cell-like and activated B cell-like subtypes of diffuse large B cell lymphoma[END_REF] . SHIP1 has been identified as a target of miR-155 and is downregulated by miR-155 in DLBCL as a consequence of autocrine stimulation by TNFα [START_REF] Pedersen | Onco-miR-155 targets SHIP1 to promote TNFalpha-dependent growth of B cell lymphomas[END_REF] . Another target of miR-155 is SMAD which is suppressed by high levels of miR-155 and protects DLBCL cells from the growthinhibitory effects of TGF beta [START_REF] Rai | Targeting of SMAD5 links microRNA-155 to the TGF-beta pathway and lymphomagenesis[END_REF] .

High resolution profiling integrating genome-wide copy number analysis with geneexpression by the Staudt group identified amplification of the miR-17~92 cluster in GCB DLBCL but not in ABC type [START_REF] Lenz | Molecular subtypes of diffuse large B-cell lymphoma arise by distinct genetic pathways[END_REF] having been previously identified as over-expressed in DLBCL [START_REF] Ota | Identification and characterization of a novel gene, C13orf25, as a target for 13q31-q32 amplification in malignant lymphoma[END_REF] . Data from mice confirm the transforming potential of this miRNA in lymphoma whereby microRNAs from the miR-17~92 cluster cooperate with MYC to transform mouse B cells, and lymphomas that develop with miR-17~92 overexpression have less apoptosis than those expressing MYC alone [START_REF] O'donnell | c-Myc-regulated microRNAs modulate E2F1 expression[END_REF] . Further work investigating the targets of miR-17~92 in lymphomagenesis demonstrated that CDKN1A/p21 is an essential target but that miR-17~92 can regulate different subtypes of B-cell lymphomas through down-regulation of different target genes (eg, Bim or/and p21) [START_REF] Inomata | MicroRNA-17-92 down-regulates expression of distinct targets in different B-cell lymphoma subtypes[END_REF] .

Profiling studies have continued to look for ways of differentiating GCB and ABC DLBCL based on miRNA expression. Lawrie et al identified three miRNAs, miR-155, miR-21 and miR-221 that were more highly expressed in ABC-type than GCB-type cell lines [START_REF] Lawrie | MicroRNA expression distinguishes between germinal center B cell-like and activated B cell-like subtypes of diffuse large B cell lymphoma[END_REF] . In a much larger series a 5-miRNA (miR-182, miR-150, miR- [START_REF] Li | Copy number abnormalities, MYC activity, and the genetic fingerprint of normal B cells mechanistically define the microRNA profile of diffuse large B-cell lymphoma[END_REF] where an alternative classifier has been put forward which robustly segregated DLBCL into three groups based on transcriptional activity of MYC. Of those miRNAs previously identified as dysregulated in DLBCL were the miR-17~92 cluster but this was unable to differentiate between GCB and ABC DLBCL in this series. Some of these differences may be explained by the derivation of the miRNA signature from cell lines 20 rather than primary patient material [START_REF] Li | Copy number abnormalities, MYC activity, and the genetic fingerprint of normal B cells mechanistically define the microRNA profile of diffuse large B-cell lymphoma[END_REF] . Further work and analysis of larger sample series is clearly needed before a reproducible diagnostic miRNA score exists that can be put forward for routine practice.

In terms of a correlate of outcome, miR-222 has been shown to predict survival in DLBCL treated with R-CHOP chemotherapy where high levels of expression were associated with inferior outcome [START_REF] Malumbres | Differentiation stage-specific expression of microRNAs in B lymphocytes and diffuse large B-cell lymphomas[END_REF] . The prognostic role of other miRNAs is not as yet defined. There remains considerable potential for miRNA biomarkers to be examined in clinical trials as a better marker of disease stratification.

Follicular Lymphoma (FL)

The published literature defining a miRNA signature for FL is limited. miR-155, miR-221 and miR-21 are similarly overexpressed in FL as they are in DLBCL with no significant differences between expression levels in these different lymphomas [START_REF] Lawrie | MicroRNA expression distinguishes between germinal center B cell-like and activated B cell-like subtypes of diffuse large B cell lymphoma[END_REF] .

A signature of four miRNAs (miR-330, -17-5p, -106A, -210) has been proposed as a diagnostic classifier to discriminate FL, DLBCL and non-neoplastic lymph node 60 but this has not been confirmed by other series.

Mantle Cell Lymphoma (MCL)

The first description of miRNA involvement in MCL came from the observation that miR-16-1 regulates the expression of cyclin D1 (CCND1) [START_REF] Chen | Truncation in CCND1 mRNA alters miR-16-1 regulation in mantle cell lymphoma[END_REF] . This control is lost in cases with truncated CCND1 mRNA and associated with a poor prognosis [START_REF] Wiestner | Point mutations and genomic deletions in CCND1 create stable truncated cyclin D1 mRNAs that are associated with increased proliferation rate and shorter survival[END_REF] was found to be a direct target of miR-29.

miRNAs as new diagnostic and prognostic markers in lymphoma

The majority of early reports detailing miRNA expression changes were performed by Southern blot analysis with probes specific for the miRNA. Array platforms subsequently developed using oligonucleotides optimized for detection have progressed with the expansion of the database. As the field has exploded, so the methods of detection have provides the opportunity for the first time of a lymphoma biomarker to monitor the progression and response to therapy that can be measured from a peripheral blood sample. However, it is important to bear in mind that the expression profile of circulating miRNA may change depending on the condition of the patient and that large studies will be needed to provide the evidence. Thus miRNA has the potential to change the diagnostic methodology available and opens up the door to rapid diagnosis and prognostic analysis from a blood sample in the near future.

F

2 Introduction

 2 Fig 1

  evolutionarily conserved non-protein-coding sequences of between 19-23 nucleotides that control gene expression at the post-transcriptional level by binding to the 3' untranslated region (UTR) of target messenger RNA (mRNA) through

  nucleus by RNA polymerase II, and that the transcripts are capped and polyadenylated[START_REF] Kim | MicroRNA biogenesis: coordinated cropping and dicing[END_REF] . Following cleavage by the microprocessor complex, Drosha, to form the hairpin structure pre-mir of 60-70 nucleotides, this is exported by the cytoplasmic exporter Exportin5 and cleavage occurs by Dicer and accessory proteins to produce the mature miRNA. The mature miRNA is loaded onto the miRNA-induced silencing complex (RISC) and together recognize complementary sequences in the 3'UTR of the target gene resulting in either degradation of the mRNA or inhibition of translation (reviewed in Kim

  specifically up-regulated in centroblasts and almost two-thirds belonged to several clusters whose miRNA members demonstrated similar temporal changes during B-cell differentiation. Moreover, paralog groups of miRNA clusters also exhibited similar profiles of expression in distinct cell subpopulations, suggesting the presence of common regulatory networks controlling distinct paralogs.A more quantitative RT-PCR (RQ-PCR) approach to miRNA analysis for B cells has produced two interesting studies. Dave's group[START_REF] Zhang | Patterns of microRNA expression characterize stages of human B-cell differentiation[END_REF] used a 384-well multiplexed RQ-PCR assay containing 365 miRNAs (based on miRBase 9.2) and detected a total of 113 unique miRNAs in the B cell populations. Similarly, Tan et al[START_REF] Tan | miRNA profiling of B-cell subsets: specific miRNA profile for germinal center B cells with variation between centroblasts and centrocytes[END_REF] determined a miRNA profile of naïve, GC and memory B cells sorted from normal tonsils and verified their RQ-PCR results of differentially expressed miRNAs using in situ hybridisation in tonsil tissue sections. A total of 86 miRNAs were found to be expressed amongst the 3 normal B cell subsets and 16 miRNAs showed at least 4.5-fold difference between GC and non-GC B cells, suggesting these are important throughout the GC response. More than 4.5-fold changes in the expression levels of miR-17-5p, miR-106a, miR-146a, miR-150 and miR-181b were found in GC B cells as compared with naïve and memory B cells.

6 A

 6 number of specific miRNAs have been identified in controlling early B cell and peripheral B cell development as well as defined by studies of the signature pattern for normal cells[START_REF] Zhang | Patterns of microRNA expression characterize stages of human B-cell differentiation[END_REF][START_REF] Basso | Identification of the human mature B cell miRNome[END_REF][START_REF] Malumbres | Differentiation stage-specific expression of microRNAs in B lymphocytes and diffuse large B-cell lymphomas[END_REF] demonstrating a highly unique miRNA profile. Certain critical miRNAs have been identified during normal B cell differentiation and warrant greater consideration as outlined below.

19b- 1 ,

 1 miR-92a-1) promotes survival of early B cell progenitors. Absence of miR-17~92 leads to inhibition of B cell development at the pro-B cell to pre-B cell transition.Ventura et al[START_REF] Ventura | Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters[END_REF] demonstrated that expression of miR-17~92 is tightly regulated during B cell development, reaching a maximum at the pre-B cell stage and then rapidly returning to basal levels at later stages. It acts by repressing the expression of pro-apoptotic genes including Bim, whose levels increase in miR-17~92 deficient murine cells[START_REF] Ventura | Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters[END_REF] . Conversely, overexpression leads to a lymphoproliferative phenotype probably as a consequence of tumour suppressor gene PTEN and Bim[START_REF] Xiao | Lymphoproliferative disease and autoimmunity in mice with increased miR-17-92 expression in lymphocytes[END_REF] . It is of note that Bim inhibition is notable in CLL and could indicate aberration of the miR-17~92 pathway.

  Dicer molecule plays a pivotal part in miRNA processing to its active form by means of cleavage and processing of the miRNA molecule. The effects of miRNAs in B cell development have been assessed globally as well as in terms of specific miRNAs with analysis of aberrant expression/activity of the components of the miRNA biosynthetic machinery. The deletion of Dicer at the earliest stages of B cell development leads to a block in development at the pro-B cell to pre-B cell transition 31 . Koralov and others explored the effects of deleting the Dicer gene (dcr-1) conditionally at the earliest stage of B cell development. This led to a dramatic block in this process at the pro-B cell stage but could be partially overcome by genetically promoting B cell survival either through the enforced expression of the prosurvival gene BCL2 or ablation of BIM[START_REF] Koralov | Dicer ablation affects antibody diversity and cell survival in the B lymphocyte lineage[END_REF] . Deregulated control of cell survival is at least partly responsible for the developmental block imposed by Dicer deficiency, and Bim is a major player in this context consistent with its key role in the control of lymphocyte survival. Upregulated genes in Dicer deleted cells shared a signature consistent with gene-expression control by three miRNA families related to the miR-17 92 cluster which includes Bim. Their results also established that the basic mechanism of V(D)J recombination is independent of Dicer, although Dicer has an impact on the antibody repertoire expressed following V(D)J recombination[START_REF] Koralov | Dicer ablation affects antibody diversity and cell survival in the B lymphocyte lineage[END_REF] .The germinal centre response miR-155miR-155 is processed from an exon of a non-coding RNA transcribed from the B-cell Integration Cluster (BIC), originally identified through its aberrant expression in various

  advanced to enable large scale detection in small sample sizes, with more reliance on RQ-PCR based techniques. Whilst the early means of detection required large amounts of total RNA, more recently developed techniques now allow smaller archival material to be used. Although extraction techniques still require solvent based methodology, it is possible to obtain reproducible and reliable miRNA profiles from FFPE tissue, comparable to fresh paired samples. The PCR based assays are widely available and only require picogram amounts of RNA making them suitable for diagnostic assays on very small amounts of tumour material. For lymphoma diagnostics this raises the possibility of detecting malignant cells from the peripheral blood if a unique miRNA signature can be , detection of lymphoma miRNA profiles from serum samples[START_REF] Lawrie | Detection of elevated levels of tumourassociated microRNAs in serum of patients with diffuse large B-cell lymphoma[END_REF]

20 Figure 1 21 Figure 2

 201212 Figure 1 Distinct miRNA profiles of mature B cells. Consensus data are shown from global profiling studies. Only miRNAS whose expression is confirmed in two or more studies are included. Blue = upregulation, Red = downregulation.

  [START_REF] Kim | MicroRNA biogenesis: coordinated cropping and dicing[END_REF]

  ). miR genes are either present as single units scattered throughout the genome located within introns and intergenic regions or organized as gene clusters. A typical miR gene cluster is miR-17~92 which comprises 6 miRNAs (miR-17, miR-18a,

	study of murine bone marrow, miR-181 was one of three miRNAs specifically expressed
	in hematopoietic cells and found to regulate the differentiation of the multipotent
	progenitor in the lymphoid lineage 17 . Involvement of miRNAs in B cell development
	have been assessed globally using high throughput based technologies (microarrays and
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	lymphocytic leukaemia when in 2002 Croce's group demonstrated two microRNAs, e e r miR-15a and miR-16-1 from within the commonly deleted region 13q14 to be down-regulated in the majority of cases 14 . Following this discovery, further evidence of a link between miRNAs and cancer pathogenesis stemmed from the identification of a genome-wide correlation between the genomic location of miRNAs and regions including areas frequently involved in deletion, amplification, chromosomal breakpoint and loss of heterozygosity in malignancy 15 . miRNAs appear to play a direct role in oncogenesis and can function as both oncogenes (eg. miR-155 and members of the miR-17~miR-92 R e v i e w
	cluster) and tumour suppressors (eg. miR-15a and miR-16-1) in all cancer types. The
	definition of a cellular miRNA signature ('miRNome') has been shown to permit
	diagnostic differentiation of normal from malignant tissue and definition of subsets
	within a tumour type 16 with improved accuracy over traditional gene expression analysis.
	microRNAs in normal B cell development
	miRNAs play a critical role in lymphocyte development by exerting control of lineage-
	specific transcription factors crucial to haemopoietic differentiation 17 . In the first such
	4

miR-19a, miR-20a, miR-19b-1, miR-92a-1)

[START_REF] Olive | miR-19 is a key oncogenic component of mir-17-92[END_REF]

. miRNA clusters are typically transcribed together in polycistronic transcripts and usually control mRNAs with related functions.

It is clear that miRNAs control several pathways including developmental timing, haematopoiesis, organogenesis, apoptosis, cell proliferation as well as oncogenesis. A role in cancer by aberrant or failure of expression was first postulated for chronic

  It was in CLL that a role for miRNAs in cancer was first reported byCalin et al in 2002.

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	Leukaemia (CLL)
	9

  Two subsequent studies have performed global profiling in an attempt to identify the miRNA signature of MCL. In the first study 86 mature miRNAs were analysed, chosen by their location at the most frequently altered chromosomal regions in MCL[START_REF] Navarro | MicroRNA expression, chromosomal alterations, and immunoglobulin variable heavy chain hypermutations in Mantle cell lymphomas[END_REF] . Based on the miRNA expression profile they identified two distinct sub-sets of

	MCL is altered. MCL showing significant differences in the IgV H mutational status, proliferation
	signature, and number of chromosomal alterations. miR-17-5p/miR-20a from the miR-
	17~92 cluster were expressed at high levels concomittantly with MYC and associated
	with poor prognosis disease 71 . Subsequently Zhao et al have performed miRNA using a
	platform of 515 miRNAs 72 . They found that eighteen miRNAs were down-regulated and
	21 were up-regulated in MCL compared with normal B lymphocytes with the most F o frequent alterations being decrease of miR-29a/b/c, miR-142-3p/5p, and miR-150 and
	r increase of miR-124a and miR-155. Expression levels of miR-29 family were associated
	with prognosis and patients with significantly down-regulated miR-29 had a shorter P survival compared with those who expressed relatively high levels of miR-29. Down-e r e regulation of the miR-29 family was associated with CDK6 overexpression and CDK6
	R e v i e w
	13

whereby miR-16-1 binding sites within the CCND1 mRNA 3' UTR are deleted and miRNA regulation of

Table 1 microRNAs consistently altered in B cell lymphomas

 1 miRNAs written in blue are known to be commonly altered in lymphoid malignancies and are not disease specific.

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Published on behalf of the British Division of the International Academy of Pathology Histopathology