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Assessment of the presence of clonal lymphoproliferations via polymerase chain reaction (PCR)-based analysis of rearranged immunoglobulin (IG) or T-cell receptor (TR) genes is a valuable method in the diagnosis of suspect lymphoproliferative disorders. Additionally, this methodology can be used for evaluating dissemination of lymphoma cells and for studying the clonal relationship between multiple (different locations) and consecutive (over time) lymphomas. Here we describe an integrated approach to assess clonality via analysis of Ig heavy chain (IGH), Ig kappa (IGK), TCR beta (TRB), and TCR gamma (TRG) gene rearrangements, based on the standardized multiplex PCRs as originally developed by the European BIOMED-2 consortium (currently named EuroClonality). The described protocol covers the pre-analytical phase of DNA isolation (from formalin-fixed paraffin-embedded and fresh tissues, body fluids, peripheral blood, and bone marrow), the analytical phase of PCR GeneScan analysis, and the post-analytical interpretation of the obtained profiles, following established guidelines., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: Next generation sequencing (NGS) is an important tool used in clinical practice to obtain the required molecular information for accurate diagnostics of high-grade adult-type diffuse glioma (HGG). Since individual centers use either in-house produced or standardized panels, interlaboratory variation could play a role in the practice of HGG diagnosis and treatment. This study aimed to investigate the current practice in NGS application for both primary and recurrent HGG., Methods: This nationwide Dutch survey used the expertise of (neuro)pathologists and clinical scientists in molecular pathology (CSMPs) by sending online questionnaires on clinical and technical aspects. Primary outcome was an overview of panel composition in the different centers for diagnostic practice of HGG. Secondary outcomes included practice for recurrent HGG and future perspectives., Results: Out of twelve neuro-oncology centers, the survey was filled out by eleven (neuro)pathologists and seven CSMPs. The composition of the diagnostic NGS panels differed in each center with numbers of genes ranging from 12 to 523. Differences are more pronounced when tests are performed to find therapeutic targets in the case of recurrent disease: about half of the centers test for gene fusions (60%) and tumor mutational burden (40%)., Conclusion: Current notable interlaboratory variations as illustrated in this study should be reduced in order to refine diagnostics and improve precision oncology. In-house developed tests, standardized panels and routine application of broad gene panels all have their own advantages and disadvantages. Future research would be of interest to study the clinical impact of variation in diagnostic approaches., (© 2024. The Author(s).)

Patients with lymphoplasmacytic lymphoma/Waldenström macroglobulinemia (LPL/WM) occasionally develop diffuse large B-cell lymphoma (DLBCL). This mostly results from LPL/WM transformation, although clonally unrelated DLBCL can also arise. LPL/WM is characterized by activating MYD88 L265P (>95%) and CXCR4 mutations (~30%), but the genetic drivers of transformation remain to be identified. Here, in thirteen LPL/WM patients who developed DLBCL, the clonal relationship of LPL and DLBCL together with mutations contributing to transformation were investigated. In 2 LPL/WM patients (15%), high-throughput sequencing of immunoglobulin gene rearrangements showed evidence of >1 clonal B-cell population in LPL tissue biopsies. In the majority of LPL/WM patients, DLBCL presentations were clonally related to the dominant clone in LPL, providing evidence of transformation. However, in 3 patients (23%), DLBCL was clonally unrelated to the major malignant B-cell clone in LPL, of which 2 patients developed de novo DLBCL. In this study cohort, LPL displayed MYD88 L265P mutation in 8 out of eleven patients analyzed (73%), while CXCR4 mutations were observed in 6 cases (55%). MYD88 WT LPL biopsies present in 3 patients (27%) were characterized by CD79B and TNFAIP3 mutations. Upon transformation, DLBCL acquired novel mutations targeting BTG1, BTG2, CD79B, CARD11, TP53, and PIM1 . Together, we demonstrate variable clonal B-cell dynamics in LPL/WM patients developing DLBCL, and the occurrence of clonally unrelated DLBCL in about one-quarter of LPL/WM patients. Moreover, we identified commonly mutated genes upon DLBCL transformation, which together with preserved mutations already present in LPL characterize the mutational landscape of DLBCL occurrences in LPL/WM patients., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.)

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.)

Next-generation sequencing (NGS)-based clonality analysis allows in-depth assessment of the clonal composition of a sample with high sensitivity for detecting small clones. Within the EuroClonality-NGS Working Group, a protocol for NGS Ig clonality analysis was developed and validated previously. This NGS-based approach was designed to generate small amplicons, making it suitable for samples with suboptimal DNA quality, especially material derived from formalin-fixed, paraffin-embedded tissue. Using expert assessment of NGS Ig clonality results as a reference, a structured algorithmic approach to the assessment of NGS-amplicon-based B-cell clonality analysis was developed. A structured approach with the Detection of clonality through Evaluation of sample quality and assessment of Pattern, Abundance and RaTio (DEPART) algorithm was proposed, which consecutively evaluates sample quality, the pattern of the clonotypes present, the abundance of the most dominant clonotypes, and the ratio between the dominant clonotypes and the background to evaluate the different Ig gene targets. Specific issues with respect to evaluation of the various Ig targets as well as the integration of results of individual targets into a molecular clonality conclusion are discussed and illustrated with case examples. Finally, the importance of interpretation of NGS-based clonality results in clinical and histopathologic contexts is discussed. It is expected that these recommendations will have clinical utility to facilitate proper evaluation of clonality assessment., (Copyright © 2023 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Virtual pathology education has shown to enhance the students' learning experience. At the Radboud University, an E-learning platform-called the "PathoDiscovery"-was developed and first used in a course about neoplasm development amongst first year (bio)medical sciences students. The PathoDiscovery incorporates high-power microscopic images, histological annotations, interactive questions and pre-programmed feedback.The objective of our study was to develop and evaluate the PathoDiscovery within the "Neoplasm" course focusing on student perceptions of usability and utility. For this study the online feedback on the PathoDiscovery that was obtained anonymously from (bio)medical students over two consecutive academic years was analyzed. The responses of the first year were used to make improvements. After the second year, the feedback of the two academic years was compared. The rating of the E-learning increased from 6.8 (n = 285) to 7.4 (n = 247) after implementation of feedback obtained in the first year. The students judged the structure as logical (90%). The content was considered easy or just right (57%), matched the learning objectives (76%), and contributed to knowledge development (78%). We conclude that the first experiences with the PathoDiscovery are positive for both students and lecturers; it is an example of a dynamic online learning tool that is easily adaptable and is well suited for a blended learning approach., (© 2023 The Authors. APMIS published by John Wiley & Sons Ltd on behalf of Scandinavian Societies for Pathology, Medical Microbiology and Immunology.)

Clonality assessment by the detection of immunoglobulin (IG) gene rearrangements is an important method to determine whether two concurrent or subsequent lymphoid malignancies in one patient are clonally related. Here, we report the detailed clonality analysis in a patient with a diagnosis of B-cell acute lymphoblastic leukemia (B-ALL) followed by a histiocytic sarcoma (HS), in which we were able to study clonal evolution by applying next generation sequencing (NGS) to identify IG rearrangements and gene mutations. Using the sequence information of the NGS-based IG clonality analysis, multiple related subclones could be distinguished in the PAX5 P80R-mutated B-ALL. Notably, only one of these subclones evolved into HS after acquiring a RAF1 mutation. This case demonstrates that NGS-based IG clonality assessment and mutation analysis provide clear added value for clonal comparison and thereby improves clinicobiological understanding., (© 2022. The Author(s).)

Large B-cell lymphoma of immune-privileged sites (LBCL-IP) arise in immune sanctuaries including the testis and central nervous system (CNS). After initially reaching complete response, relapses occur in almost 50% of patients, typically at other immune-privileged sites. Resolution of the clonal relationships and evolutionary patterns of LBCL-IP is required to understand the unique clinical behavior. We collected a unique set of 33 primary-relapse LBCL-IP sample pairs and performed next-generation sequencing for copy number, mutation, translocation, and immunoglobulin clonality analysis. All LBCL-IP sample pairs were clonally related, and both tumors developed from a common progenitor cell (CPC) with MYD88 and TBL1XR1 mutations and/or BCL6 translocations in 30/33 cases, indicating that these are early genetic events. This was succeeded by intermediate genetic events including shared, as well as unique alterations in targets of aberrant somatic hypermutation (aSHM), CD79B mutations, and 9p21.3/CDKN2A loss. Genetic alterations in genes involved in immune escape (HLA, CD274/PDCD1LG2) were predominantly unique in primary and relapse samples and thus considered late genetic events. Together, this study indicates that primary and relapsed LBCL-IP follow an early parallel evolutionary pattern where the CPC contains genetic alterations that support prolonged survival/proliferation and retention in a memory B-cell state, followed by germinal center reentry, aSHM and immune escape., Significance: Genomic analyses reveal that primary and relapse LBCL-IP originate from a common progenitor cell with a small set of genetic alterations, followed by extensive parallel diversification, elucidating the clonal evolution of LBCL-IP., (©2023 American Association for Cancer Research.)

Aims: Large B cell lymphoma with IRF4 rearrangement (LBCL-IRF4) is a new entity in the 2017 revised World Health Organisation (WHO) classification that was initially mainly reported in children. After identification of a 79-year-old patient, we assessed how often IRF4 rearrangements can be detected in adult diffuse large B cell lymphomas (DLBCLs) which have to be reclassified to LBCL-IRF4 based on fluorescence in-situ hybridisation (FISH) for IRF4., Methods and Results: With FISH, we studied the presence of IRF4 rearrangements in 238 lymphomas that were diagnosed as DLBCL according to the previous WHO classification of 2008., Conclusions: In addition to the index patient, an IRF4 rearrangement was detected in another five of 237 patients (2%). The immunohistochemical profile of these five IRF4 rearranged lymphomas was consistent with previous reports of LBCL-IRF4. One case was recognised to represent transformation of follicular lymphoma rather than de-novo LBCL-IRF4. BCL6 rearrangements were found in two cases of LBCL-IRF4; BCL2 and MYC rearrangements were excluded. Patients presented with limited stage disease with involvement of the head and neck in three patients, and involvement of the lung and thyroid in two others. This study shows that, although rare, LBCL-IRF4 should also be considered in older patients and at localisations other than the head and neck region., (© 2023 The Authors. Histopathology published by John Wiley & Sons Ltd.)

Approximately one-third of patients with diffuse large B-cell lymphoma (DLBCL) relapse and often require salvage chemotherapy followed by autologous stem cell transplantation. In most cases, the clonal relationship between the first diagnosis and subsequent relapse is not assessed, thereby potentially missing the identification of second primary lymphoma. In this study, the clonal relationship of 59 paired DLBCL diagnoses and recurrences was established by next-generation sequencing-based detection of immunoglobulin gene rearrangements. Among 50 patients with interpretable results, 43 patients (86%) developed clonally related relapsed disease. This was observed in 100% of early recurrences (<2 years), 80% of the recurrences with an interval between 2 and 5 years, and 73% of late recurrences (≥5 years). On the other hand, 7 (14%) out of 50 patients displayed different dominant clonotypes in primary DLBCL and clinical recurrences, confirming the occurrence of second primary DLBCL; 37% of DLBCL recurrences that occurred ≥4 years after diagnosis were shown to be second primary lymphomas. The clonally unrelated cases were Epstein-Barr virus positive in 43% of the cases, whereas this was only 5% in the relapsed DLBCL cases. In conclusion, next-generation sequencing-based clonality testing in late recurrences should be considered in routine diagnostics to distinguish relapse from second primary lymphoma, as this latter group of patients with DLBCL may benefit from less-intensive treatment strategies., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Activated B cells play a key role in the pathogenesis of primary Sjögren's syndrome (pSS) through the production of autoantibodies and the development of ectopic germinal centers in the salivary glands and other affected sites. Around 5-10% of pSS patients develop B-cell lymphoma, usually extranodal marginal zone lymphomas (eMZL) of the mucosa-associated lymphoid tissue (MALT). The aim of the current study is to investigate if the eMZL clonotype is detectable in prediagnostic blood and tissue biopsies of pSS patients., Methods/results: We studied prediagnostic tissue biopsies of three pSS patients diagnosed with eMZL and four pSS controls through immunoglobulin (IG) gene repertoire sequencing. In all three cases, we observed the eMZL clonotype in prediagnostic tissue biopsies. Among controls, we observed transient elevation of clonotypes in two pSS patients. To evaluate if eMZL clonotypes may also be detected in the circulation, we sequenced a peripheral blood mononuclear cell (PBMC) sample drawn at eMZL diagnosis and two years prior to eMZL relapse in two pSS patients. The eMZL clonotype was detected in the peripheral blood prior to diagnosis in both cases. Next, we selected three pSS patients who developed eMZL lymphoma and five additional pSS patients who remained lymphoma-free. We sequenced the IG heavy chain (IGH) gene repertoire in PBMC samples taken a median of three years before eMZL diagnosis. In two out of three eMZL patients, the dominant clonotype in the prediagnostic PBMC samples matched the eMZL clonotype in the diagnostic biopsy. The eMZL clonotypes observed consisted of stereotypic IGHV gene combinations (IGHV1-69/IGHJ4 and IGHV4-59/IGHJ5) associated with rheumatoid factor activity, a previously reported feature of eMZL in pSS., Discussion: In conclusion, our results indicate that eMZL clonotypes in pSS patients are detectable prior to overt eMZL diagnosis in both tissue biopsies and peripheral blood through immunogenetic sequencing, paving the way for the development of improved methods of early detection of eMZL., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kolijn, Huijser, Wahadat, van Helden-Meeuwsen, van Daele, Brkic, Rijntjes, Hebeda, Groenen, Versnel, Thurlings and Langerak.)

Clonality assessment using the unique rearrangements of immunoglobulin (IG) and T-cell receptor (TR) genes in lymphocytes is a widely applied supplementary test for the diagnosis of B-cell and T-cell lymphoma. To enable a more sensitive detection and a more precise comparison of clones compared with conventional clonality analysis based on fragment analysis, the EuroClonality NGS Working Group developed and validated a next-generation sequencing (NGS)-based clonality assay for detection of the IG heavy and kappa light chain and TR gene rearrangements for formalin-fixed and paraffin-embedded tissues. We outline the features and advantages of NGS-based clonality detection and discuss potential applications for NGS-based clonality testing in pathology, including site specific lymphoproliferations, immunodeficiency and autoimmune disease and primary and relapsed lymphomas. Also, we briefly discuss the role of T-cell repertoire of reactive lymphocytic infiltrations in solid tumors and B-lymphoma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Groenen, van den Brand, Kroeze, Amir and Hebeda.)

The developments in targeted therapies and molecular pathology have changed the classification of tumors and precision oncology. Pathologists and clinical scientists in molecular pathology and oncologists have regular multidisciplinary meetings and are responsible for translating molecular results into an appropriate treatment plan. This requires expertise and skills to be effective team players. Interprofessional collaboration (IPC) is essential for professionals in medicine; however, learning opportunities in current resident training are limited. This narrative study explores the collaborative output and learning mechanisms of interprofessional learning (IPL) of residents of different disciplines in the Morphology & Molecular PLUS workshop and its preparation. Topics that were discussed in the workshop were technologies for the detection of mutations, copy number variations, tumor mutational burden, and circulating tumor DNA (ctDNA) analysis in the context of differential diagnosis and precision oncology. Data were collected by analyzing pre- and post-workshop questionnaires and interviews. An interprofessional team of three residents of each hospital had to be formed by one of the residents, which was challenging as not all residents from a hospital knew each other. Residents reported to have got to know each other and have learned about each other's roles and perspectives. They gained knowledge of molecular pathology and the added value of IPC, in particular, for residents early in their training. Enabling meetings for medical residents of different disciplines to get acquainted was perceived as the most facilitating factor for IPL. Time constraints as the biggest barrier in daily practice. We recommend offering IPL activities as early as possible in residency programs., Competing Interests: The authors report no conflicts of interest, no disclosures. The authors alone are responsible for the content and writing of this article., (© 2022 The Author(s).)

Classical Hodgkin lymphoma (cHL) represents a B-cell lymphoproliferative disease characterized by clonal immunoglobulin gene rearrangements and recurrent genomic aberrations in the Hodgkin Reed-Sternberg cells in a reactive inflammatory background. Several methods are available for the molecular analysis of cHL on both tissue and cell-free DNA isolated from blood, which can provide detailed information regarding the clonal composition and genetic alterations that drive lymphoma pathogenesis. Clonality testing involving the detection of immunoglobulin and T cell receptor gene rearrangements, together with mutation analysis, represent valuable tools for cHL diagnostics, especially for patients with an atypical histological or clinical presentation reminiscent of a reactive lesion or another lymphoma subtype. In addition, clonality assessment may establish the clonal relationship of composite or subsequent lymphoma presentations within one patient. During the last few decades, more insight has been obtained on the molecular mechanisms that drive cHL development, including recurrently affected signaling pathways (e.g., NF-κB and JAK/STAT) and immune evasion. We provide an overview of the different approaches to characterize the molecular composition of cHL, and the implementation of these next-generation sequencing-based techniques in research and diagnostic settings.

Clonality analysis in classic Hodgkin lymphoma (cHL) is of added value for correctly diagnosing patients with atypical presentation or histology reminiscent of T cell lymphoma, and for establishing the clonal relationship in patients with recurrent disease. However, such analysis has been hampered by the sparsity of malignant Hodgkin and Reed-Sternberg (HRS) cells in a background of reactive immune cells. Recently, the EuroClonality-NGS Working Group developed a novel next-generation sequencing (NGS)-based assay and bioinformatics platform (ARResT/Interrogate) to detect immunoglobulin (IG) gene rearrangements for clonality testing in B-cell lymphoproliferations. Here, we demonstrate the improved performance of IG-NGS compared to conventional BIOMED-2/EuroClonality analysis to detect clonal gene rearrangements in 16 well-characterized primary cHL cases within the IG heavy chain (IGH) and kappa light chain (IGK) loci. This was most obvious in formalin-fixed paraffin-embedded (FFPE) tissue specimens, where three times more clonal cases were detected with IG-NGS (9 cases) compared to BIOMED-2 (3 cases). In total, almost four times more clonal rearrangements were detected in FFPE with IG-NGS (N = 23) as compared to BIOMED-2/EuroClonality (N = 6) as judged on identical IGH and IGK targets. The same clonal rearrangements were also identified in paired fresh frozen cHL samples. To validate the neoplastic origin of the detected clonotypes, IG-NGS clonality analysis was performed on isolated HRS cells, demonstrating identical clonotypes as detected in cHL whole-tissue specimens. Interestingly, IG-NGS and HRS single-cell analysis after DEPArray™ digital sorting revealed rearrangement patterns and copy number variation profiles indicating clonal diversity and intratumoral heterogeneity in cHL. Our data demonstrate improved performance of NGS-based detection of IG gene rearrangements in cHL whole-tissue specimens, providing a sensitive molecular diagnostic assay for clonality assessment in Hodgkin lymphoma., (© 2021. The Author(s).)

Objective: To comparatively analyse the aberrant affinity maturation of the antinuclear and rheumatoid factor (RF) B cell repertoires in blood and tissues of patients with Sjögren's syndrome (SjS) using an integrated omics workflow., Methods: Peptide sequencing of anti-Ro60, anti-Ro52, anti-La and RF was combined with B cell repertoire analysis at the DNA, RNA and single cell level in blood B cell subsets, affected salivary gland and extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT) of patients with SjS., Results: Affected tissues contained anti-Ro60, anti-Ro52, anti-La and RF clones as a small part of a polyclonal infiltrate. Anti-Ro60, anti-La and anti-Ro52 clones outnumbered RF clones. MALT lymphoma tissues contained monoclonal RF expansions. Autoreactive clones were not selected from a restricted repertoire in a circulating B cell subset. The antinuclear antibody (ANA) repertoires displayed similar antigen-dependent and immunoglobulin (Ig) G1-directed affinity maturation. RF clones displayed antigen-dependent, IgM-directed and more B cell receptor integrity-dependent affinity maturation. This coincided with extensive intra-clonal diversification in RF-derived lymphomas. Regeneration of clinical disease manifestations after rituximab coincided with large RF clones, which not necessarily belonged to the lymphoma clone, that displayed continuous affinity maturation and intra-clonal diversification., Conclusion: The ANA and RF repertoires in patients with SjS display tissue-restricted, antigen-dependent and divergent affinity maturation. Affinity maturation of RF clones deviates further during RF clone derived lymphomagenesis and during regeneration of the autoreactive repertoire after temporary disruption by rituximab. These data give insight into the molecular mechanisms of autoreactive inflammation in SjS, assist MALT lymphoma diagnosis and allow tracking its response to rituximab., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Immunoglobulin (IG) clonality assessment is a widely used supplementary test for the diagnosis of suspected lymphoid malignancies. The specific rearrangements of the immunoglobulin (IG) heavy and light chain genes act as a unique hallmark of a B-cell lymphoma, a feature that is used in clonality assessment. The widely used BIOMED-2/EuroClonality IG clonality assay, visualized by GeneScanning or heteroduplex analysis, has an unprecedented high detection rate because of the complementarity of this approach. However, the BIOMED-2/EuroClonality clonality assays have been developed for the assessment of specimens with optimal DNA quality. Further improvements for the assessment of samples with suboptimal DNA quality, such as from formalin-fixed paraffin-embedded (FFPE) specimens or specimens with a limited tumor burden, are required. The EuroClonality-NGS Working Group recently developed a next-generation sequencing (NGS)-based clonality assay for the detection of the IG heavy and kappa light chain rearrangements, using the same complementary approach as in the conventional assay. By employing next-generation sequencing, both the sensitivity and specificity of the clonality assay have increased, which not only is very useful for diagnostic clonality testing but also allows robust comparison of clonality patterns in a patient with multiple lymphoma's that have suboptimal DNA quality. Here, we describe the protocols for IG-NGS clonality assessment that are compatible for Ion Torrent and Illumina sequencing platforms including pre-analytical DNA isolation, the analytical phase, and the post-analytical data analysis., (© 2022. The Author(s).)