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With the explosion in knowledge about the molecular landscape of lymphoid malignancies and the increasing availability of high throughput techniques, molecular diagnostics in hematopathology has moved from isolated marker studies to a more comprehensive approach, integrating results of multiple genes analyzed with a variety of techniques on the DNA and RNA level. Although diagnosis of lymphoma still relies on the careful integration of clinical, morphological, phenotypic, and, if necessary molecular features, and only few entities are defined strictly by genetic features, genetic profiling has contributed profoundly to our current understanding of lymphomas and shaped the two current lymphoma classifications, the International Consensus Classification and the fifth edition of the WHO classification of lymphoid malignancies. In this review, the current state of the art of molecular diagnostics in lymphoproliferations is summarized, including clonality analysis, mutational studies, and gene expression profiling, with a focus on practical applications for diagnosis and prognostication. With consideration for differences in accessibility of high throughput techniques and cost limitations, we tried to distinguish between diagnostically relevant and in part disease-defining molecular features and optional, more extensive genetic profiling, which is usually restricted to clinical studies, patients with relapsed or refractory disease or specific therapeutic decisions. Although molecular diagnostics in lymphomas currently is primarily done for diagnosis and subclassification, prognostic stratification and predictive markers will gain importance in the near future., (© 2023. The Author(s).)

Despite high cure rates in classic Hodgkin lymphoma (cHL), relapses are observed. Whether relapsed cHL represents second primary lymphoma or an underlying T-cell lymphoma (TCL) mimicking cHL is underinvestigated. To analyze the nature of cHL recurrences, in-depth clonality testing of immunoglobulin (Ig) and T-cell receptor (TCR) rearrangements was performed in paired cHL diagnoses and recurrences among 60 patients, supported by targeted mutation analysis of lymphoma-associated genes. Clonal Ig rearrangements were detected by next-generation sequencing (NGS) in 69 of 120 (58%) diagnoses and recurrence samples. The clonal relationship could be established in 34 cases, identifying clonally related relapsed cHL in 24 of 34 patients (71%). Clonally unrelated cHL was observed in 10 of 34 patients (29%) as determined by IG-NGS clonality assessment and confirmed by the identification of predominantly mutually exclusive gene mutations in the paired cHL samples. In recurrences of >2 years, ∼60% of patients with cHL for whom the clonal relationship could be established showed a second primary cHL. Clonal TCR gene rearrangements were identified in 14 of 125 samples (11%), and TCL-associated gene mutations were detected in 7 of 14 samples. Retrospective pathology review with integration of the molecular findings were consistent with an underlying TCL in 5 patients aged >50 years. This study shows that cHL recurrences, especially after 2 years, sometimes represent a new primary cHL or TCL mimicking cHL, as uncovered by NGS-based Ig/TCR clonality testing and gene mutation analysis. Given the significant therapeutic consequences, molecular testing of a presumed relapse in cHL is crucial for subsequent appropriate treatment strategies adapted to the specific lymphoma presentation., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Two new classifications were recently released: the 5th edition of the WHO classification of hematolymphoid tumors and the International Consensus Classification (ICC) drafted by the Clinical Advisory Committee. In the preparation of both classifications, the previously existing lymphoma categories were reevaluated according to recently obtained data on clinical, morphological, and molecular findings. In this review we summarize the current placements of classic and nodular lymphocyte predominant Hodgkin lymphoma and their relevant differential diagnoses., (© 2023. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

With the introduction of large-scale molecular profiling methods and high-throughput sequencing technologies, the genomic features of most lymphoid neoplasms have been characterized at an unprecedented scale. Although the principles for the classification and diagnosis of these disorders, founded on a multidimensional definition of disease entities, have been consolidated over the past 25 years, novel genomic data have markedly enhanced our understanding of lymphomagenesis and enriched the description of disease entities at the molecular level. Yet, the current diagnosis of lymphoid tumors is largely based on morphological assessment and immunophenotyping, with only few entities being defined by genomic criteria. This paper, which accompanies the International Consensus Classification of mature lymphoid neoplasms, will address how established assays and newly developed technologies for molecular testing already complement clinical diagnoses and provide a novel lens on disease classification. More specifically, their contributions to diagnosis refinement, risk stratification, and therapy prediction will be considered for the main categories of lymphoid neoplasms. The potential of whole-genome sequencing, circulating tumor DNA analyses, single-cell analyses, and epigenetic profiling will be discussed because these will likely become important future tools for implementing precision medicine approaches in clinical decision making for patients with lymphoid malignancies., (© 2022 by The American Society of Hematology.)

Since the publication of the Revised European-American Classification of Lymphoid Neoplasms in 1994, subsequent updates of the classification of lymphoid neoplasms have been generated through iterative international efforts to achieve broad consensus among hematopathologists, geneticists, molecular scientists, and clinicians. Significant progress has recently been made in the characterization of malignancies of the immune system, with many new insights provided by genomic studies. They have led to this proposal. We have followed the same process that was successfully used for the third and fourth editions of the World Health Organization Classification of Hematologic Neoplasms. The definition, recommended studies, and criteria for the diagnosis of many entities have been extensively refined. Some categories considered provisional have now been upgraded to definite entities. Terminology for some diseases has been revised to adapt nomenclature to the current knowledge of their biology, but these modifications have been restricted to well-justified situations. Major findings from recent genomic studies have impacted the conceptual framework and diagnostic criteria for many disease entities. These changes will have an impact on optimal clinical management. The conclusions of this work are summarized in this report as the proposed International Consensus Classification of mature lymphoid, histiocytic, and dendritic cell tumors.

Background: Malignant lymphomas of the eye and its adnexal structures account for approximately 5-15% of extranodal lymphomas. According to anatomic and biological criteria, two large groups of lymphomas in and around the eye need to be distinguished: (1) primary lymphomas of intraocular structures and (2) primary lymphomas of ocular adnexa. Furthermore, there is a large spectrum of secondary manifestations of malignant lymphomas in ocular and periocular structures., Objective: This article gives a summary of the classification and molecular pathology of various intraocular and periocular lymphomas as well as oncological systemic treatment with a focus on primary vitreoretinal lymphomas., Methods: A selective literature search was carried out in PubMed on the topic of intraocular and periocular lymphomas and own experiences are presented., Results: The treatment of primary vitreoretinal lymphomas (PVRL) is an interdisciplinary challenge and despite the apparently localized disease, systemic treatment concepts are necessary to reduce the high risk of secondary involvement of the central nervous system (CNS). Therefore, it is crucial that the substances used can penetrate the CNS, and protocols should be chosen in accordance with the treatment concepts for primary CNS lymphomas. The knowledge on the genetics and biology of ocular lymphomas generated by modern high throughput methods enable not only improved diagnostics using molecular methods but also provide rationales for targeted therapeutic approaches., Conclusion: A deep understanding of the biological and molecular principles of intraocular and periocular lymphomas forms a basic prerequisite for precise diagnostics and the use of targeted systemic treatment., (© 2022. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Background: The classification of intraocular lymphomas is based on their anatomical location. They are divided into uveal lymphomas with involvement of the choroid, ciliary body or iris and vitreoretinal lymphomas with isolated or combined involvement of the vitreous body and/or retina. Over the last decades it has become increasingly possible to work out the clinical and pathobiological features of the various subtypes, thereby reducing the diagnostic hurdles and creating improved treatment options., Objective: A summary of the various types of intraocular lymphoma in terms of clinical features, diagnostics, treatment and prognosis is given as well as recommendations for follow-up care., Methods: A selective literature search was carried out on the subject of intraocular lymphomas using PubMed and Google Scholar., Results: Intraocular lymphomas affect different structures, so that the symptoms can also be very different. The diagnostic spectrum ranges from typical ocular examination methods to sample biopsies with subsequent cytological, histological and molecular pathological processing. The treatment pillars available are percutaneous irradiation and intravitreal drug administration as local treatment and systemic treatment or a combination of systemic and local treatment. The prognosis depends mainly on the subtype of the lymphoma and the extent of the infestation when the diagnosis is confirmed. Even though some effective treatment options are now available, it has not yet been possible to significantly reduce the mortality rate., Conclusion: Many different options are available for the diagnostics and treatment of intraocular lymphomas, which require close interdisciplinary cooperation. The further developments in the field of molecular pathology allow a faster and more accurate diagnosis and could open up new treatment options in the future., (© 2022. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Epstein-Barr-virus-associated lymphoproliferations (EBV-LPD) constitute a wide spectrum from benign, self-limiting lymphoproliferations to malignant lymphoma. Because of the clinical and morphological heterogeneity of EBV-LPD and the high prevalence of EBV infections, knowledge of the diagnostic criteria is of great importance for the practice of diagnostic pathology. In this review, clinical and pathological characteristics of the non-malignant EBV-related LPD in patients with and without immunosuppression are presented and their current classification is discussed.EBV-LPD can be of B‑cell or T/NK-cell origin. Identification of EBV latency type and information about possible (iatrogenic) immunodeficiency of the patient are critical for diagnostic evaluation and separation from malignant lymphoma. The clinical context and the detection of EBV in T‑ and NK-cells are both essential for the diagnosis of EBV+ T/NK-cell LPD, which are rare in European countries., (© 2022. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Malignant lymphomas are derived from a common progenitor cell with a unique rearrangement of immunoglobulin or T‑cell receptor genes. Polymerase chain reaction (PCR)-based analyses allow detection of the clone and are an important adjunct for the diagnosis of difficult lymphoproliferations, e.g. for the discrimination of reactive versus malignant lesions. Further applications are detection of disease dissemination and evaluation of the clonal relationship of two lymphomas. However, clonality analysis is not a stand-alone test and must always be considered in context with clinical, histological and immunophenotypic data. For the correct use of clonality analysis, comprehensive knowledge of the biological basis, technical requirements and interpretation are needed in order to avoid incorrect conclusions.

After 8 years, the WHO has now published the updated version of the 4th edition of the classification of hematopoietic and lymphoid tumors. This update provides a conceptual rewrite of existing entities as well as some new provisional entities and categories, particularly among the aggressive B‑cell lymphomas. Important new diagnostic categories include the high-grade B‑cell lymphomas, the large B‑cell lymphoma with IRF4 rearrangement, and the Burkitt-like lymphoma with 11q aberrations. Of particular importance, new concepts concerning the taxonomy and classification of early lymphoid lesions or precursor lesions are included, such as the in situ follicular neoplasia or the in situ mantle cell neoplasia. In addition, the concept of indolent lymphoproliferations, such as breast-implant-associated anaplastic large cell lymphoma and the indolent T‑cell lymphoproliferative disorder of the gastrointestinal tract, has been strengthened. Finally, diagnostic criteria for existing lymphoma entities have been refined.

The facts that cancer represents tissues consisting of heterogeneous neoplastic, as well as reactive, cell populations and that cancers of the same histotype may show profound differences in clinical behavior have long been recognized. With the advent of new technologies and the demands of precision medicine, the investigation of tumor heterogeneity has gained much interest. An understanding of intertumoral heterogeneity in patients with the same disease entity is necessary to optimally guide personalized treatment. In addition, increasing evidence indicates that different tumor areas or primary tumors and metastases in an individual patient can show significant intratumoral heterogeneity on different levels. This phenomenon can be driven by genomic instability, epigenetic events, the tumor microenvironment, and stochastic variations in cellular function and antitumoral therapies. These mechanisms may lead to branched subclonal evolution from a common progenitor clone, resulting in spatial variation between different tumor sites, disease progression, and treatment resistance. This review addresses tumor heterogeneity in lymphomas from a pathologist's viewpoint. The relationship between morphologic, immunophenotypic, and genetic heterogeneity is exemplified in different lymphoma entities and reviewed in the context of high-grade transformation and transdifferentiation. In addition, factors driving heterogeneity, as well as clinical and therapeutic implications of lymphoma heterogeneity, will be discussed., (© 2017 S. Karger AG, Basel.)

The contribution of Epstein Barr virus (EBV) and Kaposi sarcoma herpes virus (KSHV) to the development of specific types of malignant lymphomas occurring in the human immunodeficiency virus (HIV) setting has been extensively studied since the beginning of the HIV epidemic 35 years ago. The introduction of highly active antiretroviral therapies (HAART) in 1996 has changed dramatically the incidence of HIV-related malignancies. Nevertheless, malignant lymphomas continue to be the major group of malignances observed in HIV infected individuals, and the most common cause of cancer related-deaths. Common features of the predominant B-cell lymphomas in the HIV + setting are the frequent plasmacytoid morphology of the neoplastic cells, advanced stage, aggressive disease and frequent extranodal involvement. In this article, we review the evolving concepts and definitions of the various EBV-associated lymphomas in HIV + patients, including diffuse large B-cell lymphoma, Burkitt lymphoma, classical Hodgkin lymphoma, plasmablastic lymphoma and primary effusion lymphoma. The current knowledge regarding the pathogenesis of these malignancies, the interplay between HIV and EBV co-infection in the development of certain HIV related lymphomas, and the emerging paradigm that suggests that HIV may play a direct role in lymphomagenesis are explored as well., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The rapid technological development in diagnostic pathology, especially of immunohistochemical and molecular techniques, also has a significant impact on diagnostic procedures for the evaluation of bone marrow trephine biopsies. The necessity for optimal morphology, combined with preservation of tissue antigens and nucleic acids on one hand and the wish for short turnaround times on the other hand require careful planning of the workflow for fixation, decalcification and embedding of trephines. Although any kind of bone marrow processing has its advantages and disadvantages, formalin fixation followed by EDTA decalcification can be considered a good compromise, which does not restrict the use of molecular techniques. Although the majority of molecular studies in haematological neoplasms are routinely performed on bone marrow aspirates or peripheral blood cells, there are certain indications, in which molecular studies such as clonality determination or detection of specific mutations need to be performed on the trephine biopsy. Especially, the determination of B- or T-cell clonality for the diagnosis of lymphoid malignancies requires stringent quality controls and knowledge of technical pitfalls. In this review, we discuss technical aspects of bone marrow biopsy processing and the application of diagnostic molecular techniques.

The trephine bone marrow (BM) biopsy is an important diagnostic tool in patients with malignant lymphoma. BM examination can serve to establish or confirm a primary diagnosis of lymphoma or to determine the extent of disease dissemination for staging purposes. BM histology renders information which cannot be gained equally from aspirate material, such as spacial distribution and extent of infiltrates, BM cellularity and fibrosis. Furthermore, cytology including flow cytometric immunophenotyping can give false-negative results in BM involvement by lymphoma due to intralesional fibrosis. In addition to morphological examination, the availability of a broad panel of antibodies suitable for paraffin-embedded tissues, in conjunction with less damaging decalcification procedures, nowadays enables us to perform complete immunophenotyping on BM trephines and allows for classification of lymphoma infiltrates according to established algorithms. Molecular determination of clonality and interphase fluorescent in situ hybridization can be employed selectively to resolve difficult cases. This review describes important diagnostic features of malignant lymphoma in the BM, relevant differential diagnoses, and the proper use of ancillary techniques.

The diagnosis of gastric lymphoma in endoscopic biopsy specimens remains difficult despite the emergence of accepted criteria for the histologic diagnosis of lymphomas originating from mucosa-associated lymphoid tissue (MALT). The sensitivity and validity of immunoglobulin (Ig) gene rearrangement analysis of mucosal biopsies for the diagnosis of malignant B-cell lymphoma were investigated in comparison with conventional histology and immunohistology. Biopsy specimens from 34 different endoscopies of 20 patients with a previous history, or tentative diagnosis of gastric lymphoma, and 12 control samples were analyzed for the presence of clonal Ig gene rearrangements. A clonal B-cell population was detected by Southern blot analysis in all patients with a definitive histologic diagnosis of lymphoma. In addition, in two patients the detection of clonal rearrangements in biopsy specimens preceded by several months the histologic diagnosis of lymphoma, and clonality was confirmed in three further patients where histology remained inconclusive. In some cases of low-grade MALT-lymphoma, discrete spreading of malignant cells within chronically inflamed mucosa was suggested by the presence of identical clonal rearrangements in all simultaneously obtained biopsies, with or without histologically detectable involvement by lymphoma. Our results show that immunoglobulin gene rearrangement studies of endoscopic biopsy samples are an additional powerful tool for the diagnosis of gastric lymphoma, especially for detecting early recurrence, and improve the preoperative assessment of the extent of mucosal involvement.