Comprehensive histopathological diagnostics of aggressive B-cell lymphomas based on the updated criteria of the World Health Organisation’s 2017 classification

Abstract

Revision of the fourth edition of the World Health Organisation (WHO) Classification of Haematopoietic and Lymphatic Tissues, which was published in 2017, introduced important changes updating the biology, pathology, genetics, and clinical presentation of aggressive B-cell lymphomas. High grade B-cell lymphomas (HGBLs) replaced B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, the new provisional entity Burkitt-like lymphoma with 11q aberration was identified, and some categories were upgraded, e.g. EBV-positive diffuse large B-cell lymphoma, not otherwise specified. Still the histopathological diagnostics is based on morphology and immunoprofile, but to define the HGBLs evaluation of MYC, BCL2, and BCL6 gene statuses is required. According to the presented WHO criteria, in the comprehensive histopathological diagnostics of aggressive B-cell lymphomas a highly specialised diagnostic team including a pathologist, a molecular biologist, a geneticist, a haematologist, and immunophenotyping technicians is needed.

Introduction

Aggressive lymphomas from mature B cells are a heterogeneous group of diseases that differ in biological, pathological, and clinical features. The World Health Organisation (WHO) classification updated at the end of 2017 distinguishes 18 entities (Table I) with distinct clinical, pathological, and genetic features [1]. Some types of aggressive B-cell lymphomas are relatively frequent, i.e. diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS) which represents 25-35% non-Hodgkin’s lymphomas depending on the population; others occur rarely and refer to specific groups of patients. Prognosis and response to therapy are different in each clinical-pathological unit. Although currently used treatment regimens are effective in the majority of patients, about 30% of patients develop progression to incurable disease [1, 2]. The results of genetic and molecular research conducted in recent years allow a better understanding of the mechanisms underlying the clinical and biological diversity of these tumours and provide valuable information about new therapeutic options, and at the same time allow improvement of diagnostic criteria. The aim of this study is to present practical diagnostic recommendations necessary for the correct diagnosis of aggressive B-cell lymphomas. The novelty concerning the three aggressive B-cell lymphomas has been discussed in detail: DLBCL, NOS, high-grade B-cell lymphomas (HGBLs), and Burkitt lymphoma/Burkitt-like lymphoma with 11q aberration (BL/BLL,11q); for the remaining units, the most important features regarding clinical and pathological presentation as well as genetic changes are highlighted [1, 2]. Moreover, general rules for preservation of biological material and tips on supporting diagnostic techniques (immunohistochemistry, flow cytometry, genetics) are given.

Preservation of biological material

Various materials are evaluated in the diagnosis of lymphoid tumours. Samples most often originate from the main structures of the lymphatic system, which include lymph nodes, spleen and thymus, Waldeyer’s ring (lymphatic tissue band surrounding the oral throat with palatal tonsils, and lingual and pharyngeal tonsils), appendix, and Peyer’s patches in the ileum. Other areas containing lymphatic tissue concern: bone marrow, mediastinum, liver, skin, pleura and gonads. Patients admitted with suspicion of lymphoma for pathological diagnosis require biological material protection also for molecular, and/or cytogenetic tests. Therefore, it is necessary to provide a standard operating procedure for material processing, including the following major steps:

– tissue obtained for testing should be fresh and processed according to type of material; entirely excised lymph nodes should be cut into at least 5-mm thick slices along the long axis to ensure optimal penetration of a fixative;

– description of fresh material should include the size, colour, texture, and the presence or absence of macroscopically visible haemorrhages, necrosis fields, or nodular architecture;

– touch imprint cytological slides are made from freshly cut material surface and are fixed in alcohol or dried in the air;

– for cytogenetic tests (karyotype) a fresh tissue sample or viable cells from fine-needle aspiration biopsy (FNAB) should be collected into a sterile container with cell culture medium and antibiotics, always considering the requirements of the laboratory performing the test;

– immunophenotyping by flow cytometry (FCM) requires protection of a fresh tissue sample or viable cells from FNAB in an appropriate transport medium (e.g. RPMI-1640), always considering the requirements of the laboratory performing the test;

– always indicate the type of fixative used and if possible determine the estimated time from tissue collection to fixation, because this is important for the recovery of RNA and phosphorylated proteins;

– for histopathological examination it is recommended to use a fixative based on 10% buffered formaldehyde (so-called formalin); – most suitable for additional studies such as immunohistochemistry and fluorescence in situ hybridisation (FISH); – to avoid dilution and formalin buffering problems the use of commercially available ready-to-use (RTU) solutions is strongly advised;

– to ensure optimal antibody reactivity preservation (for immunohistochemistry) extension of fixation time (over 24 hours for small tissue sections fixated in formalin) should be avoided;

– if the size/volume of biological material and infrastructure of the diagnostic centre allow, some samples should be biobanked through deep freezing (short-term storage in –80°C freezers, long-term storage in liquid nitrogen at –170°C). The freezing technique should be complementary to further material processing methodology; the most popular techniques are tumour snap frozen sections or tumour viable cell freezing.

General remarks on pathological diagnostics of aggressive B-cell lymphoma:

– histopathological examination sustains a gold standard in diagnostics; however, in most cases precise determination of lymphoma type requires at least one additional method including immunophenotyping, and molecular and/or cytogenetic tests [4, 5, 6, 7, 8, 9, 10]; in cases where material is inadequate or insufficient for diagnostic purposes, the clinician should receive feedback and justify the above condition;

– in certain circumstances, when a lymph node is not easily accessible for surgical biopsy and in patients requiring immediate treatment, FNAB biopsy in conjunction with FCM, karyotype, FISH for major translocation, PCR for IGH, and TCR gene rearrangement may be sufficient for diagnosis;

– haematopatholgy consultation should include review of all slides with at least one paraffin block representative of the tumour. Re-biopsy is recommended if material is non-diagnostic;

– immunophenotyping can be performed by immunohistochemistry or flow cytometry [8]; each method has its own advantages and disadvantages. Flow cytometry is fast (hours) and quantitative method with evaluation of multiple antigens simultaneously. Antigen detection, however, does not allow to correlate with tumour architecture and its cytological features. Immunohistochemistry requires hours, sometimes days, and the quantitative assessment is subjective, but its most important feature is the possibility of correlation of reaction with architecture and tumour cytology. Moreover, not all antibodies are available for immunohistochemical assessment, especially for fixed tissues, but the advantage of this method is the possibility of using it in the archival materials embedded in paraffin. Both techniques can be used in the lymphomas diagnostics and are a source of clinically relevant information (e.g. identification of molecules necessary for the use of targeted therapies such as CD20);

– the importance of molecular research in lymphoid malignancies is constantly growing and allows the determination of clonality and the origin of neoplastic cells. In specific entities, these tests are necessary to make a definitive diagnosis i.e., Burkitt lymphoma, Burkitt-like lymphoma, or high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements;

– for an interpretation of immunohistochemical reactions a descriptive semi-quantitative scale is adopted: (+) positive reaction, (+/–) partially positive reaction, (–/+) positive reaction in a few, (–) negative reaction in the majority of cells; the percentage of positive cells for each staining may be presented in square brackets;

– panB panel includes antibodies: CD19, CD20, CD22, CD79a, PAX5;

– typical DLBCL immunophenotype includes: CD45(+), CD20(+), CD3(–) Ki-67 > 40% positive cell nuclei;

– panel of IHC antibodies to establish DLBCL diagnosis and GCB vs. non-GCB origin: CD20, CD3, CD5, CD10, BCL2, BCL6, Ki-67, IRF4/MUM1, MYC with or without cell surface analysis by flow cytometry: k/l, CD45, CD3, CD5, CD19, CD10, CD20, CD71;

– additional IHC studies to establish lymphoma subtype: Cyclin D1, PAX5, CD30, CD15, CD138, CD38, Epstein Barr virus (EBV) in situ hybridisation (ISH), EBV/LMP1, ALK, HHV8, SOX11, CD23, BOB1, OCT2, CD56, k/l, EMA;

– the following cutoff points are recommended according to WHO classification: BCL2 ≥ 50% strong positive cells, MYC ≥ 40% strong positive cell nuclei, for CD10, BCL6, IRF4/MUM1 ≥ 30% positive cells/nuclei. The importance of choosing an appropriate antibody clone, especially among those that show reaction liability (i.e. BCL2, CD10, MYC), is highlighted. For routine pathological diagnostics only certified antibodies (in vitro diagnostics, IVD) simultaneously with positive and negative controls are advised;

– presence of EBV virus should be confirmed by intra-tissue hybridisation (EBER-ISH); it is a superior technique to immunohistochemistry (LMP1) (Fig. 1);

– karyotype and/or FISH for MYC, BCL2, BCL6 rearrangements are recommended especially in cases with double expression of MYC and BCL2 and/or having a GCB phenotype and high-grade morphology;

– despite advanced diagnostic methods, still there is a group of cases whose image does not meet all criteria and “escapes” from diagnostic guidelines; the pathologist, taking into account the clinical presentation and course, should maximally narrow the differential diagnosis. It is worth remembering that the most powerful predictive factor determining the therapeutic approach remains the pathologically confirmed type of lymphoma;

– diagnosis of lymphomas requires close cooperation between haematopathologists and specialists in flow cytometry and genetics as well as clinicians (oncologists, haematologists).

Diffuse large B-cell lymphoma, not otherwise specified

Diffuse large B-cell lymphoma, not otherwise specified (DLBCL, NOS) is morphologically, clinically and biologically heterogeneous. In developing countries and in selected Eastern European countries, such as Poland it represents 25-35% of non-Hodgkin’s lymphomas and is a constant leader among all aggressive mature B-cell lymphomas [3, 4]. The identification of two molecular subtypes of this lymphoma based on gene expression profiling (GEP) was one of the most important achievements in understanding its diversity. Subtyping DLBCL, NOS is based on different cell origins and includes lymphomas derived from germinal centre cells (GCB) or from activated B-cells (ABC) [5]. Additionally, these DLBCL subgroups vary in the activated molecular pathways, chromosomal changes, and the occurrence of somatic mutations. In GCB type, the activation of the PI3K/AKT signalling pathway and over-expression of BCL6 protein are observed, whereas in ABC type there is a constitutive activation of the NF-kB pathway in the course of various mechanisms. Unique genetic changes, especially mutations such as GNA13 and EZH2 in GCB and MYD88, CARD11, and CD79B in ABC are found, respectively [6, 7]. Chromosomal translocations in DLBCL involving regions with BCL6 (3q27), BCL2 [t(14;18)(q32;q21.3)], and MYC (single hit) comprise, respectively, approximately 30% (with predominance in ABC subtype), 20-30% (more commonly in GCB subtype), and 8-14% (similar distribution among ABC and GCB subtype) [8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18]. It is estimated that approximately 50% of DLBCL with MYC translocation demonstrate BCL2 and/or BCL6 rearrangement and that cases should be transferred to the high-grade B-cell lymphoma category [1, 19, 20]. DLBCL with typical morphology and isolated MYC translocation usually present higher mitotic index, but still such cases meet the criteria of diagnosis of DLBCL, NOS [8, 21]. The pathologists are also discouraged from preselecting aggressive B-cell lymphomas to FISH testing upon Ki-67 result, while its range can be variable [21]. Biological differences translate into a distinct clinical course; so far in the majority of studies, a worse prognosis was observed among patients with DLBCL, ABC in comparison with GCB subtype. Moreover, the latest results of clinical trials concerning combining R-CHOP regimen with bortezomib, lenalidomide, and ibrutinib in the ABC subtype significantly stress the potential benefits of such treatment [22, 23]. The optimal method for the molecular classification of DLBCL is the study of the gene expression profile, but it requires fresh tissue and is not widely used in routine diagnostics. Due to its clinical impact, pathologists are still obligated to precisely identify the molecular subtype. An up-to-date Hans algorithm based on a panel of antibodies (CD10, BCL6, and IRF4/MUM1) determined by immunohistochemistry is available in most pathology departments and should be continuously applied [24, 25, 26, 27]. The compliance of IHC with GEP reaches about 80-90%, but a small group of about 10% of DLBCL cases “slip out” of molecular classification and the ABC/GCB match is not possible [28]. One of the limitations of IHC is also standardisation between haematopatholgy centres, including quality of staining and evaluation reproducibility among pathologists [28, 29]. Recently developed GEP tests based on RNA extracted from formalin-fixed and paraffin-embedded tissues (e.g. the Lymph2Cx assay from NanoString Technologies, Seattle, WA, USA) can identify DLBCL molecular subtypes [30]. Moreover, those methods are characterised by high compliance level with GEP microarrays and satisfactory reproducibility between laboratories [30, 31, 32]. Possibly, tests based on GEP will be a more precise diagnostic method than currently approved by the WHO IHC methods in future [1, 20]. The summary of DLBCL, NOS characteristic is presented in Table II.

High-grade B-cell lymphomas

High-grade B-cell lymphoma (HGBL), with gene expression profile intermediate between molecular signatures of Burkitt lymphoma (BL) and non-BL (mostly DLBCL), is a heterogeneous group of aggressive, mature B-cell lymphomas, which should not be classified separately due to biological and clinical reasons [1, 2]. High-grade B-cell lymphoma is a newly introduced category in the updated 2017 WHO classification, which primarily replaces B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma (BCLU, DLBCL/BL) and at the moment comprises two entities: HGBL with MYC and BCL2 and/or BCL6 chromosomal rearrangements (HGBL, R) and high grade B-cell lymphomas, not otherwise specified (HGBL, NOS). HGBL, R is also called double/triple-hit lymphoma (DH/THL). Most patients develop de novo HGBL, R, while a minority have a history of FL that progress to DH/THL secondarily, presumably by acquisition of a MYC translocation [1]. Double/triple-hit lymphomas are characterised by various morphologies including DLBCL, BL and intermediate features between DLBCL and BL (DLBCL/BL). HGBL, which lack co-occurring MYC and BCL2 and/or BCL6 rearrangements, falling in the category HGBL, NOS; such cases appear as blastoid or DLBCL/BL morphology (eventually resembling more closely BL than DLBCL), and in up to half of DLBCL/BL cases MYC rearrangements as one separate hit are found [1, 2, 33, 34] (Fig. 2). Former criteria for BCLU, DLBCL/BL were vague, and the diagnosis was not used uniformly, limiting its utility as a diagnostic category [1, 2]. The morphological appearance should always be specified in a comment in the pathological report referring to HGBL, R and HGBL, NOS because the DLBCL morphology may predict a better outcome compared to DLBCL/BL [35]. Neither morphology nor proliferation index assessed by Ki-67 have sufficient sensitivity and specificity to identify DH/THLs. MYC and BCL2 protein expression, although independently valuable prognostic factors, are similarly not ideal predictors of DH/THLs, and hence FISH (whenever the karyotype assessment is not available) is the recommended modality to identify DH/THLs (Fig. 3) [1, 2]. Despite the fact that a clear consensus has not yet been reached to provide molecular testing guidelines, the 2017 WHO update strongly advises that all DLBCL cases should undergo genetic studies for the detection of MYC, BCL2, and BCL6 rearrangements. Nevertheless, some pathologists and clinicians suggest that only cases with a GCB phenotype and/or high-grade morphology or with > 40% MYC immunohistochemically positive cells are worthy of deeper molecular testing [1] (Fig. 2). Nearly 100% of DHLs with MYC and BCL2 rearrangements harbour the t(14;18) translocation, which results in BCL2 overexpression by IHC and flow cytometry. BCL2 protein is expressed (in 30% of DHLs with MYC and BCL6 rearrangements) or overexpressed (occurrence of BCL2 extra copy/amplification) in a higher proportion of DLBCL and HGBL, NOS. That phenomenon is often associated with a concomitant expression of MYC, but still IHC evaluation cannot be used as a surrogate marker for DHL. Most DLBCL and HGBL do not carry both rearrangements (MYC and BCL2) and are referred to as “double-expressor lymphomas” (DELs) [1, 2, 33, 36]. DH/THL and DEL patients usually progress more rapidly, are resistant to R-CHOP chemo-immunotherapy [33, 35], and have very poor prognosis. Moreover, such cases may harbour TP53 mutations or deletion, frequently observed in MYC/BCL2 DHLs and blastoid morphology [37, 38, 39]. Worse prognosis in HGBL, R and HGBL, NOS compared with the DLBCL groups were published lately [38, 40]. DH/THLs show a common GCB immunophenotype (CD10+/CD81+higher/ BCL6+/CD44– or CD44+/–dim) by FCM, often presenting decreased expression of CD20 or CD19, over expression of CD38 and BCL2 (Fig. 4) [41]. An aggressive biologic behaviour and poor clinical outcome of DH/THL may shortly influence to therapy selection including still controversial more intensive regimens initiation and CNS-directed prophylaxis consideration. Under consideration is cytogenetic testing by metaphase or cytogenetic analysis by karyotyping if biological material (fresh cells from FNAB or tissue surgical section) is available. As far as possible, a comprehensive histopathological diagnostics of HGBL should include IHC, FISH, FCM and cytogenetic studies (Table III) [42, 43, 44].

Burkitt Lymphoma and Burkitt-like lymphoma with 11q aberration

Burkitt lymphoma (BL) is defined by the WHO classification as a highly aggressive lymphoid neoplasm, often presenting with extra nodal site involvement or as an acute leukaemia composed of monomorphic, medium-sized B-cells with basophilic cytoplasm and a high mitotic index. Translocation involving the MYC oncogene (8q24) and immunoglobulin IG genes is the constant features in 90% of cases [1]. Case analysis without MYC rearrangement have shown at least several mechanisms for the alternative activation of MYC i.e. microRNA, amplification, transcriptional increase of MYC activity. Results of next-generation sequencing revealed the BL profile of somatic mutations; in about 70% of classic BL mutations of transcription factor TCF3 (E2A) and its negative regulator ID3 were found and its role in PI3K pathway signalling was observed. Furthermore CCND3, RHOA, TP53, ARID1A and SMARCA4 mutations were identified in 30% of patients with BL.

Recently, a subset of MYC translocation-negative aggressive B-cell lymphomas resembling BL, characterized by proximal gains and distal losses of the long arm of chromosome 11 was described [45, 46]. In the 2017 WHO classification, these MYC-negative lymphomas were recognized as a new provisional entity, “Burkitt-like lymphoma with 11q aberration” (BLL,11q) [1] (Fig. 5). MYC-negative BLL,11q shows a number of clinico-pathological similarities to MYC-positive BL, but also harbour some significant differences in immunoprofile [1, 45, 46, 47, 48, 49, 50]. BLL,11q usually express CD43/LMO2/CD56 in IHC and CD16/CD56/CD38/CD45/CD8/CD43 in FCM. That characteristics may contribute to the differential diagnosis of BLL, 11q and BL [49]. The 11q aberrations in BLL, 11q (11q-gain/loss) were described as an inverted duplication of a part of the long arm of chromosome 11 with mono- or biallelic telomeric deletion of 11q (Figure 6) [45, 46, 47, 49]. Coincidence of duplication and deletion of 11q (11q23 and 11q24-qter, respectively) suggests a possibility of simultaneous up-regulation of oncogenes and down-regulation of tumour suppressor genes. The candidate oncogene is commonly up-regulated PAFAH1B2. FLI1 and ETS1, located in the region of deletion, are often down-regulated and/or mutated, and are postulated to be candidate tumour suppressor genes affected by this aberration [46, 47]. The diagnostic algorithm for diagnosing BL and BLL, 11q, besides standard histopathological and immunohistochemical examination, incorporates flow cytometry with a broad panel of monoclonal antibodies as well as genetic analysis (conventional cytogenetic analysis with fluorescence in situ hybridisation and molecular techniques) [1, 48]. Some cases with 11q aberration also have MYC rearrangement and are diagnosed as BL or high-grade B-cell lymphoma, not otherwise specified (HGBL, NOS). From the other point of view, the 11q-gain/loss are not exclusively specific for BLL, 11q [47]. Therefore, the conventional cytogenetic analysis by metaphase karyotyping is needed to provide correct BLL, 11q diagnosis (Table IV) [49].

Other aggressive B-cell lymphomas

Changes in the WHO 2017 classification and diagnostic criteria of other aggressive lymphomas from mature B cells are less significant.

In the group of large B-cell lymphomas associated with infection with EBV the most common lymphoma is EBV-positive diffuse large B-cell lymphoma, not otherwise specified [DLBCL EBV(+), NOS]. In the previous WHO 2008 classification, this unit was called EBV-positive DLBCL of the elderly, due to significantly more frequent occurrence in patients over the age of 50 years with a peak of incidence in the eighth decade of life [51]. Recent studies have shown that these lymphomas may also occur in younger patients (mainly in the third decade of life and almost three times more often in men) [1, 20]. DLBCL EBV(+), NOS is characterised by a different clinical picture and pathological features comparing with DLBCL, NOS. Mainly it occurs in patients with immunological disorders that impair the control of viral infections. Histopathology reveals large atypical B cells that may resemble Hodgkin’s and Reed-Sternberg cells, and variable inflammatory infiltrates involving cytotoxic T lymphocytes [CD8(+)], plasmocytes, and histiocytes. In addition, tumour necrosis is present in most cases. Histopathological features may suggest a diagnosis, but it should be confirmed by testing for the presence of EBV virus by in situ hybridisation, which is a superior method to IHC staining. EBV is found in most lymphoma cells and is usually in the second and third phase of latency [52]. Pictures of EBER-ISH and EBV-LMP1(IHC) are shown in Fig. 1.

In the group of large B-cell lymphomas classified on the basis of topography, the changes concern mainly primary mediastinal (thymic) large B-cell lymphoma (PMBL) and primary DLBCL of central nervous system (DLBCL, CNS). PMBL cells frequently express PDL1/PDL2 markers (rearrangement in about 20% of cases), CD30 and CD23, and usually are negative for IG and HLA class I and II antigens [53]. Primary mediastinal lymphoma has a specific gene expression profile that can be useful in differential diagnosis of PMBL and DLBCL, NOS involving mediastinum or other outside thorax locations. Differences between PMBL and DLBCL at the molecular level relate to CIITA rearrangements (38% vs. rarely respectively), which leads to the activation of NFB and JAK/STAT signalling pathways and the reduction of the antigen expression of the major histocompatibility complex of class II [5, 54, 55].

The primary DLBCL, CNS constitutes less than 1% of non-Hodgkin’s lymphomas and approximately 2.4-3% of all brain tumours. It has separate biological features related to the immunologically privileged location in which it develops and the lack of expression of HLA class I and II proteins, which allow tumour cells to avoid immune control. It should be differentiated with other large cell lymphomas occurring in the CNS, particularly with those associated with immunosuppression [56]; they are characterised with deletions and loss of gene expression within the HLA system and MYD88 L265P [> 50%], CD79B [20%], and CARD11 [16%] mutations are often observed, which may have potential therapeutic significance [57, 58].

B-cell lymphomas with terminal B-cell differentiation include a heterogeneous group of aggressive lymphomas characterised by immunoblastic or plasmablastic cell morphology, a plasma cell phenotype with no or reduced expression of B-cell markers (CD20 and PAX5), and strong expression of plasma cell antigens (CD38, CD138, IRF4/MUM1, and PRDM1/BLIMP1) [1]. Highly aggressive plasmablastic lymphoma (PBL) develops in immunocompromised patients, mainly caused by HIV infection, as well as during iatrogenic immunosuppression (after transplantations, autoimmune diseases). It is usually located extranodally within the head and neck, including the oral cavity, also in the gastrointestinal tract. In majority of cases the generalised disease stage is determined at the time of diagnosis (over 75% of patients with HIV infection) [59, 60, 61]. In some cases, PBL involves bones and with its morphological and immunophenotype features overlaps with plasmablastic plasma cell myeloma. The differential diagnosis should take into account the clinical picture of PBL and immunodeficiency and EBV infection. The EBV virus in the first type of latency is present in about 70% of cases. MYC translocation occurs in about 50% of patients, usually with the IG partner [62].

In Table V pathological and immunohistochemical presentation of other aggressive B cell lymphomas were presented.

Summary

In the diagnosis of aggressive B-cell lymphomas, parallel genetic tests should be done to determine the status of the MYC gene; these changes are complex and, although mostly involving classic translocations with one of the IG partners, also translocations with non-IG partners, mutations, transcriptional up-regulations, amplifications, and microRNA-dependent mechanisms are possible. This has a direct impact on the amount of protein product and its expression. BCL2 and BCL6 rearrangement and translocation mechanisms are well established. Two basic methods include the fluorescence in situ technique in break-apart probe or dual-fusion; these are convenient and stable methods and can be used on formalin-fixed paraffin-embedded material. It makes these methods more available and allows the FFPE to be sent to a standardised FISH laboratory. FISH analysis can detect critical gene rearrangements or abnormalities in aggressive B-cell lymphomas. If fresh biopsy or tissue material is available karyotype analysis can reveal additional chromosome aberrations giving a more accurate picture of genetic changes clarifying the diagnosis.

In the comprehensive histopathological report, the following components should be determined:

- cell morphology [blastoid, DLBCL, BL, DLBCL/BL];

- immunophenotype with reference to origin according to Hans’s algorithm [GCB vs. non-GCB subtype];

- MYC and BCL2 immunohistochemistry [double expressor vs. non-double expressor];

- rearrangement of MYC, BCL2, BCL6 genes [presence or absence of MYC and BCL2 and/or BCL6 rearrangements].

A summary of all diagnostic procedures in aggressive B-cell lymphomas is shown in Fig. 7.

This work has been implemented using the Project infrastructure POIG.02.03.00-14-111/13 funded by Operational Programme Innovative Economy 2007-2013, Priority II. R&D Infrastructure, Measure 2.3. Investments connected with development of IT infrastructure of Science.

The authors declare no conflict of interest.

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