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The analysis of circulating tumor DNA (ctDNA) in liquid biopsy specimens has an established role for the detection of predictive molecular alterations and acquired resistance mutations in several tumors. The low-invasiveness of this approach allows for repeated sampling and dynamic monitoring of disease evolution. Originating from the entire body tumor bulk, plasma-derived ctDNA reflects intra- and interlesional genetic heterogeneity. In the management of lymphoma patients, ctDNA quantification at various timepoints of the patient's clinical history is emerging as a complementary tool that may improve risk stratification, assessment of treatment response and early relapse detection during follow-up, most prominently in patients with diffuse large B-cell lymphoma or classic Hodgkin lymphoma. While liquid biopsies have not yet entered standard-of-care treatment protocols in these settings, several trials have provided evidence that at least a subset of lymphoma patients may benefit from the introduction of liquid biopsies into daily clinical care. In parallel, continuous technological developments have enabled highly sensitive ctDNA assessment methods, which span from locus-specific techniques identifying single hotspot mutations, to sequencing panels and genome-wide approaches that explore broader genetic and epigenetic alterations. Here, we provide an overview of current methods and ongoing technical developments for ctDNA evaluation. We also summarize the most important data from a selection of clinical studies that have explored the clinical use of ctDNA in several lymphoma entities., (Copyright © 2024. Published by Elsevier Inc.)

Somatic variant testing through next-generation sequencing (NGS) is well integrated into Swiss molecular pathology laboratories and has become a standard diagnostic method for numerous indications in cancer patient care. Currently, there is a wide variation in reporting practices within our country, and as patients move between different hospitals, it is increasingly necessary to standardize NGS reports to ease their reinterpretation. Additionally, as many different stakeholders-oncologists, hematologists, geneticists, pathologists, and patients-have access to the NGS report, it needs to contain comprehensive and detailed information in order to answer the questions of experts and avoid misinterpretation by non-experts. In 2017, the Swiss Institute of Bioinformatics conducted a survey to assess the differences in NGS reporting practices across ten pathology institutes in Switzerland. The survey examined 68 reporting items and identified 48 discrepancies. Based on these findings, the Swiss Society of Molecular Pathology initiated a Delphi method to reach a consensus on a set of recommendations for NGS reporting. Reports should include clinical information about the patient and the diagnosis, technical details about the sample and the test performed, and a list of all clinically relevant variants and variants of uncertain significance. In the absence of a consensus on an actionability scheme, the five-class pathogenicity scheme proposed by the ACMG/AMP guideline must be included in the reports. The Swiss Society of Molecular Pathology recognizes the importance of including clinical actionability in the report and calls on the European community of molecular pathologists and oncologists to reach a consensus on this issue., (© 2024. The Author(s).)

Background: Diagnosis of primary diffuse large B-cell lymphoma of the central nervous system (PCNSL) is challenging and often delayed. MRI imaging, CSF cytology and flow cytometry have a low sensitivity and even brain biopsies can be misleading. We report three cases of PCNSL with various clinical presentation and radiological findings where the diagnosis was suggested by novel CSF biomarkers and subsequently confirmed by brain biopsy or autopsy., Case Presentations: The first case is a 79-year-old man with severe neurocognitive dysfunction and static ataxia evolving over 5 months. Brain MRI revealed a nodular ventriculitis. An open brain biopsy was inconclusive. The second case is a 60-year-old woman with progressive sensory symptoms in all four limbs, evolving over 1 year. Brain and spinal MRI revealed asymmetric T2 hyperintensities of the corpus callosum, corona radiata and corticospinal tracts. The third case is a 72-year-old man recently diagnosed with primary vitreoretinal lymphoma of the right eye. A follow-up brain MRI performed 4 months after symptom onset revealed a T2 hyperintense fronto-sagittal lesion, with gadolinium uptake and perilesional edema. In all three cases, CSF flow cytometry and cytology were negative. Mutation analysis on the CSF (either by digital PCR or by next generation sequencing) identified the MYD88 L265P hotspot mutation in all three cases. A B-cell clonality study, performed in case 1 and 2, identified a monoclonal rearrangement of the immunoglobulin light chain lambda (IGL) and kappa (IGK) gene. CSF CXCL-13 and IL-10 levels were high in all three cases, and IL-10/IL-6 ratio was high in two. Diagnosis of PCNSL was later confirmed by autopsy in case 1, and by brain biopsy in case 2 and 3., Conclusions: Taken together, 5 CSF biomarkers (IL-10, IL-10/IL-6 ratio, CXCL13, MYD88 mutation and monoclonal IG gene rearrangements) were strongly indicative of a PCNSL. Using innovative CSF biomarkers can be sensitive and complementary to traditional CSF analysis and brain biopsy in the diagnosis of PCNSL, potentially allowing for earlier diagnosis and treatment., (© 2024. The Author(s).)

Mutations affecting codon 172 of the isocitrate dehydrogenase 2 ( IDH2 ) gene define a subgroup of sinonasal undifferentiated carcinomas (SNUCs) with a relatively favorable prognosis and a globally hypermethylated phenotype. They are also recurrent (along with IDH1 mutations) in gliomas, acute myeloid leukemia, and intrahepatic cholangiocarcinoma. Commonly reported mutations, all associated with aberrant IDH2 enzymatic activity, include R172K, R172S, R172T, R172G, and R172M. We present a case of SNUC with a never-before-described IDH2 mutation, R172A. Our report compares the methylation pattern of our sample to other cases from the Gene Expression Omnibus database. Hierarchical clustering suggests a strong association between our sample and other IDH-mutant SNUCs and a clear distinction between sinonasal normal tissues and tumors. Principal component analysis (PCA), using 100 principal components explaining 94.5% of the variance, showed the position of our sample to be within 1.02 standard deviation of the other IDH-mutant SNUCs. A molecular modeling analysis of the IDH2 R172A versus other R172 variants provides a structural explanation to how they affect the protein active site. Our findings thus suggest that the R172A mutation in IDH2 confers a gain of function similar to other R172 mutations in IDH2 , resulting in a similar hypermethylated profile.

Primary bone lymphoma (PBL) is rare and mostly represented by diffuse large B-cell lymphomas (DLBCL). Follicular lymphoma (FL), albeit commonly disseminating to the bone marrow, rarely presents primarily as bone lesions. Here, we studied 16 patients (12 men:4 women, median age 60 years) who presented with bone pain and/or skeletal radiologic abnormalities revealing bone FL. Lesions were multifocal in 11 patients (spine ± appendicular skeleton), and unifocal in 5 patients (femoral, tibial, or vertebral). An infiltrate of centrocytes and centroblasts (CD20+ CD5- CD10+ BCL2+ BCL6+) with abundant reactive T cells and an increased reticulin fibrosis massively replaced the marrow spaces between preserved bone trabeculae. The pattern was diffuse ± nodular, often with paratrabecular reinforcement and/or peripheral paratrabecular extension. Ki-67 was usually <15%. Two cases had necrosis. BCL2 rearrangement was demonstrated in 14 of 14 evaluable cases (with concomitant BCL6 rearrangement in one). High-throughput sequencing revealed BCL2, KMT2D, and TNFRSF14 to be the most frequently mutated genes. After staging, 5 qualified for PBL (3 limited stage) and 11 had stage IV systemic FL. All patients received rituximab ± polychemotherapy as firstline treatment, and 7 received local therapy (6 radiotherapy and 2 surgery). Three patients experienced transformation to DLBCL. At the last follow-up (15/16, median 48 months), 11 patients achieved complete remission, including all cases with PBL and most patients with limited extraosseous disease (3-year progression-free survival 71%). One patient died of unrelated cause (3-year overall survival 91%). FL may manifest as a localized or polyostotic bone disease. A minority represent PBL, whereas most reveal systemic disease., (Copyright © 2024 United States & Canadian Academy of Pathology. Published by Elsevier Inc. All rights reserved.)

Aims: Follicular helper T-cell (TFH) lymphoma of the angioimmunoblastic-type (AITL), one of the most prevalent T-cell lymphomas, typically encompasses proliferation of high endothelial venules and Epstein-Barr virus-positive immunoblasts, but neither infection with HHV8 nor association with Kaposi's sarcoma (KS) have been described. The aims of this study are to characterise the association between AITL and HHV8 infection or KS., Methods and Results: Three male patients aged 49-76 years, HIV-negative, with concurrent nodal involvement by AITL and KS, were identified from our files and carefully studied. Two patients originated from countries where endemic KS occurs, including one with cutaneous KS. The lymphomas featured abundant vessels, expanded follicular dendritic cells and neoplastic TFH cells [PD1+ (three of three), ICOS+ (three of three), CXCL13+ (three of three), CD10 + (two of three), BCL6 (two of three)] but lacked EBV+ immunoblasts. The foci of KS consisted of subcapsular proliferations of ERG+, CD31 + and/or CD34 + , HHV8+ spindle cells. High-throughput sequencing showed AITL-associated mutations in TET2 (three of three), RHOA (G17V) (three of three) and IDH2 (R172) (two of three), which were absent in the microdissected KS component in two cases. Relapses in two patients consisted of AITL, without evidence of KS. No evidence of HHV8 infection was found in a control group of 23 AITL cases., Conclusion: Concurrent nodal involvement by AITL and KS is rare and identification of both neoplastic components may pose diagnostic challenges. The question of whether the association between AITL and KS may be fortuitous or could reflect the underlying immune dysfunction in AITL remains open., (© 2023 The Authors. Histopathology published by John Wiley & Sons Ltd.)

Squamous cell carcinoma (SCC) comprises one of the major groups of non-small-cell carcinoma of the lung, and is subtyped into keratinising, non-keratinising and basaloid SCC. SCC can readily be diagnosed using histomorphology alone in keratinising SCC. Confirmatory immunohistochemical analyses should always be applied in non-keratinising and basaloid tumours to exclude differential diagnoses, most prominently adenocarcinoma and high-grade neuroendocrine carcinoma, which may have important therapeutic consequences. According to the World Health Organisation (WHO) classification 2015, the diagnosis of SCC can be rendered in resections of morphologically ambiguous tumours with squamous immunophenotype. In biopsies and cytology preparations in the same setting the current guidelines propose a diagnosis of 'non-small-cell carcinoma, favour SCC' in TTF1-negative and p40-positive tumours to acknowledge a possible sampling bias and restrict extended immunohistochemical evaluation in order to preserve tissue for molecular testing. Most SCC feature a molecular 'tobacco-smoke signature' with enrichment in GG > TT mutations, in line with the strong epidemiological association of SCC with smoking. Targetable mutations are extremely rare but they do occur, in particular in younger and non- or light-smoking patients, warranting molecular investigations. Lymphoepithelial carcinoma (LEC) is a poorly differentiated SCC with a syncytial growth pattern and a usually prominent lymphoplasmacytic infiltrate and frequent Epstein-Barr virus (EBV) association. In this review, we describe the morphological and molecular characteristics of SCC and LEC and discuss the most pertinent differential diagnoses., (© 2023 The Authors. Histopathology published by John Wiley & Sons Ltd.)

The treatment of BRAFV600E mutant melanoma has been revolutionized by BRAF inhibitors. Furthermore, the BRAF/MEK combination has shown further improvement in clinical outcomes in advanced and in adjuvant melanoma patients. In low-grade ovarian tumors, BRAF inhibitor use has been also proposed. Here we present a patient with an excellent, lasting response to BRAF therapy alone. At first progression, after more than two years on BRAF monotherapy, we could not identify any molecular mechanisms explaining resistance. After a switch to dual BRAF/MEK therapy, the patient responded. However, despite the initial response clinical the patient again progressed, this time with the appearance of a KRAS G12C mutation, which could not be overcome by BRAF/MEK therapy. We provide evidence that BRAF inhibitor alone can be highly beneficial in BRAF mutant low-grade ovarian tumors and the resistance mechanisms are similar to that of other BRAF mutant tumors, including in melanoma.

In this review focus article, we highlight the main modifications introduced in the latest 2022 International Consensus Classification and World Health Organization classification (ICC and WHO-HAEM5) of mature T (and NK) cell neoplasms (PTCLs) and consequent implications for diagnostic practice. The changes result from recent advances in the genomic and molecular characterization of PTCLs and enhanced understanding of their pathobiology. Specifically, consideration is given to the following groups of diseases: Epstein-Barr virus (EBV)-associated neoplasms; follicular helper T cell lymphoma; anaplastic large cell lymphomas; primary intestinal T and NK cell lymphomas and lymphoproliferative disorders; and PTCL, not otherwise specified., (© 2023. The Author(s).)

Predominantly nodal is the most common clinical presentation of peripheral T- (and NK-) cell lymphomas (PTCL), which comprise three main groups of diseases: (i) systemic anaplastic large cell lymphomas (ALCL), whether positive or negative for anaplastic lymphoma kinase (ALK); (ii) follicular helper T-cell lymphomas (TFHL); and (iii) PTCL, not otherwise specified (NOS). Recent advances in the genomic and molecular characterization of PTCL, with enhanced understanding of pathobiology, have translated into significant updates in the latest 2022 classifications of lymphomas. ALK-negative ALCL is now recognized to be genetically heterogeneous, with identification of DUSP22 rearrangements in approximately 20-30% of cases, correlated with distinctive pathological and biological features. The notion of cell-of-origin as an important determinant of the classification of nodal PTCL is best exemplified by TFHL, considered as one disease or a group of related entities, sharing oncogenic pathways with frequent recurrent epigenetic mutations as well as a relationship to clonal hematopoiesis. Data are emerging to support that a similar cell-of-origin concept might be relevant to characterize meaningful subgroups within PTCL, NOS, based on cytotoxic and/or Th1 versus Th2 signatures. The small group of primary nodal Epstein-Barr virus-positive lymphomas of T- or NK-cell derivation, formerly considered PTCL, NOS, is now classified separately, due to distinctive features, and notably an aggressive course. This review summarizes current knowledge of the pathology and biology of nodal-based PTCL entities, with an emphasis on recent findings and underlying oncogenic mechanisms.

ALK-negative anaplastic large cell lymphoma (ALCL) comprises subgroups harboring rearrangements of DUSP22 (DUSP22- R) or TP63 (TP63-R). Two studies reported 90% and 40% 5-year overall survival (OS) rates in 21 and 12 DUSP22-R/TP63- not rearranged (NR) patients, respectively, making the prognostic impact of DUSP22-R unclear. Here, 104 newly diagnosed ALK-negative ALCL patients (including 37 from first-line clinical trials) from the LYSA TENOMIC database were analyzed by break-apart fluorescence in situ hybridization assays for DUSP22-R and TP63-R. There were 47/104 (45%) DUSP22-R and 2/93 (2%) TP63-R cases, including one DUSP22-R/TP63-R case. DUSP22-R tumors more frequently showed CD3 expression (62% vs. 35%, P=0.01), and less commonly a cytotoxic phenotype (27% vs. 82%; P<0.001). At diagnosis, DUSP22- R ALCL patients more frequently had bone involvement (32% vs. 13%, P=0.03). The patient with DUSP22-R/TP63-R ALCL had a rapidly fatal outcome. After a median follow-up of 4.9 years, 5-year progression-free survival (PFS) and OS rates of 84 patients without TP63-R treated with curative-intent anthracycline-based chemotherapy were 41% and 53%, respectively. According to DUSP22 status, 5-year PFS was 57% for 39 DUSP22-R versus 26% for 45 triple-negative (DUSP22-NR/TP63-NR/ALK-negative) patients (P=0.001). The corresponding 5-year OS rates were 65% and 41%, respectively (P=0.07). In multivariate analysis, performance status and DUSP22 status significantly affected PFS, and distinguished four risk groups, with 4-year PFS and OS ranging from 17% to 73% and 21% to 77%, respectively. Performance status but not DUSP22 status influenced OS. The use of brentuximab vedotin in relapsed/refractory patients improved OS independently of DUSP22 status. Our findings support the biological and clinical distinctiveness of DUSP22- R ALK-negative ALCL. Its relevance to outcome in patients receiving frontline brentuximab vedotin remains to be determined.

Bone marrow (BM) cellularity assessment is a crucial step in the evaluation of BM trephine biopsies for hematologic and nonhematologic disorders. Clinical assessment is based on a semiquantitative visual estimation of the hematopoietic and adipocytic components by hematopathologists, which does not provide quantitative information on other stromal compartments. In this study, we developed and validated MarrowQuant 2.0, an efficient, user-friendly digital hematopathology workflow integrated within QuPath software, which serves as BM quantifier for 5 mutually exclusive compartments (bone, hematopoietic, adipocytic, and interstitial/microvasculature areas and other) and derives the cellularity of human BM trephine biopsies. Instance segmentation of individual adipocytes is realized through the adaptation of the machine-learning-based algorithm StarDist. We calculated BM compartments and adipocyte size distributions of hematoxylin and eosin images obtained from 250 bone specimens, from control subjects and patients with acute myeloid leukemia or myelodysplastic syndrome, at diagnosis and follow-up, and measured the agreement of cellularity estimates by MarrowQuant 2.0 against visual scores from 4 hematopathologists. The algorithm was capable of robust BM compartment segmentation with an average mask accuracy of 86%, maximal for bone (99%), hematopoietic (92%), and adipocyte (98%) areas. MarrowQuant 2.0 cellularity score and hematopathologist estimations were highly correlated (R 2  = 0.92-0.98, intraclass correlation coefficient [ICC] = 0.98; interobserver ICC = 0.96). BM compartment segmentation quantitatively confirmed the reciprocity of the hematopoietic and adipocytic compartments. MarrowQuant 2.0 performance was additionally tested for cellularity assessment of specimens prospectively collected from clinical routine diagnosis. After special consideration for the choice of the cellularity equation in specimens with expanded stroma, performance was similar in this setting (R 2  = 0.86, n = 42). Thus, we conclude that these validation experiments establish MarrowQuant 2.0 as a reliable tool for BM cellularity assessment. We expect this workflow will serve as a clinical research tool to explore novel biomarkers related to BM stromal components and may contribute to further validation of future digitalized diagnostic hematopathology workstreams., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Monomorphic epitheliotropic intestinal T-cell lymphoma (MEITL) is a rare aggressive T-cell lymphoma most reported in Asia. We performed a comprehensive clinical, pathological and genomic study of 71 European MEITL patients (36 males, 35 females, median age 67 years). The majority presented with gastrointestinal involvement and had emergency surgery, and 40% had stage IV disease. The tumors were morphologically classified into two groups: typical (58%) and atypical (i.e., non-monomorphic or with necrosis, angiotropism or starry-sky pattern) (42%), sharing a homogeneous immunophenotypic profile (CD3+ [98%] CD4- [94%] CD5- [97%] CD7+ [97%] CD8+ [90%] CD56+ [86%] CD103+ [80%] cytotoxic marker+ [98%]) with more frequent expression of TCRgd (50%) than TCRab (32%). MYC expression (30% of cases) partly reflecting MYC gene locus alterations, correlated with non-monomorphic cytology. Almost all cases (97%) harbored deleterious mutation(s) and/or deletion of the SETD2 gene and 90% had defective H3K36 trimethylation. Other frequently mutated genes were STAT5B (57%), JAK3 (50%), TP53 (35%), JAK1 (12.5%), BCOR and ATM (11%). Both TP53 mutations and MYC expression correlated with atypical morphology. The median overall survival (OS) of 63 patients (43/63 only received chemotherapy after initial surgery) was 7.8 months. Multivariate analysis found a strong negative impact on outcome of MYC expression, TP53 mutation, STAT5B mutation and poor performance status while aberrant B-cell marker expression (20% of cases) correlated with better survival. In conclusion, MEITL is an aggressive disease with resistance to conventional therapy, predominantly characterized by driver gene alterations deregulating histone methylation and JAK/STAT signaling and encompasses genetic and morphologic variants associated with very high clinical risk.

Dasatinib, a second-generation tyrosine kinase inhibitor (TKI), used as treatment for chronic myeloid leukemia, BCR::ABL1-positive (CML), is complicated by pleural or pericardial effusions in about one-third of patients. Besides, in exceptional instances, effusion-based neoplastic B-cell lymphoproliferations have been described. Here, we report an HHV8-negative, EBV-positive large B-cell lymphoma presenting as a pericardial effusion in a patient with CML treated with dasatinib for 23 months, without associated tumor mass or lymphadenopathies. Large tumor cells showed a B-cell phenotype (CD20+, CD79+), with evidence of EBV infection (EBER-ISH+), but HHV8 (LANA-1) negative. Monoclonal IG gene rearrangements were identified. BCL2, BCL6, and MYC genes were not rearranged. Despite the aggressive cytomorphology the patient was in complete remission after 4 cycles of R-CHOP after 8 months follow-up. Four other cases of large B-cell effusion-based lymphomas developed in the setting of dasatinib therapy for CML have been reported in the literature. The four cases were HHV8-negative and one case was EBV-positive. Three of the four patients experienced a benign clinical course, which is in contrast to HHV8-positive primary effusion lymphoma (PEL). The mechanisms of development of these effusion-based B-cell lymphoproliferations in patients receiving TKI are not completely elucidated. Acute or relapsing effusions during TKI treatment in the setting of CML should be cytologically examined to exclude clonal B-cell lymphoproliferations., (© 2022 Wiley Periodicals LLC.)

Nodal peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS) with cytotoxic phenotype is overall rare, with most reports coming from Asia. Given its elusive pathobiology, we undertook a clinicopathological and molecular study of 54 Western patients diagnosed with PTCL, NOS expressing cytotoxic molecules, within a lymph node. More commonly males (M/F-2,6/1) with median age of 60 years were affected. Besides lymphadenopathy, 87% of patients had ≥1 involved extranodal site. High-stage disease (III-IV), International Prognostic Index >2, B symptoms, LDH level, and cytopenia(s) were observed in 92, 63, 67, 78, and 66% of cases, respectively. Ten patients had a history of B-cell malignancies, one each of myeloid neoplasm, breast or prostate cancer, and 4 others had underlying immune disorders. Most patients (70%) died, mostly of disease, with a median overall survival of 12.7 months. Immunophenotypically, the neoplastic lymphocytes were T-cell receptor (TCR) αβ + (47%), TCR-silent (44%) or TCRγδ+ (10%), commonly CD8 + (45%) or CD4-CD8- (32%). All except one had an activated cytotoxic profile, and 95% were subclassified into PTCL-TBX21 subtype based on CXCR3, TBX21, and GATA3 expression pattern. Seven patients (13%) disclosed EBER + tumor cells. Targeted DNA deep-sequencing (33 cases) and multiplex ligation-dependent reverse transcription-polymerase chain reaction assay (43 cases) identified frequent mutations in epigenetic modifiers (73%), including TET2 (61%) and DNMT3A (39%), recurrent alterations affecting the TCR (36%) and JAK/STAT (24%) signaling pathways and TP53 mutations (18%). Fusion transcripts involving VAV1 were identified in 6/43 patients (14%). Patients with nodal cytotoxic PTCL, NOS have an aggressive behavior and frequently present in a background of impaired immunity, although the association with Epstein-Barr virus is rare. The recurrent alterations in genes involved in DNA methylation together with genes related to cytokine or TCR signaling, suggest that co-operation of epigenetic modulation with cell-signaling pathways plays a critical role in the pathogeny of these lymphomas., (© 2022. The Author(s), under exclusive licence to United States & Canadian Academy of Pathology.)

Intravascular large B-cell lymphoma is a rare and aggressive EBV-negative large B-cell lymphoma with a dismal outcome. Here, we describe the case of a 76-year-old HIV-positive patient with an acute presentation of systemic symptoms and rapidly fatal outcome. Autopsy revealed a disseminated large B-cell lymphoma with an intravascular distribution involving the liver, lymph nodes, spleen, and bone marrow and associated to fibrin thrombi in hepatic capillary haemangiomas. The neoplastic B cells (CD79a + / - , CD20 + / - , CD30 + , MUM1 + , PD-L1 +) showed a Hodgkin and Reed-Sternberg-like morphology and were EBV-positive with a latency type II (LMP1 + , EBNA2-). Haemophagocytosis was documented in the bone marrow and lymph nodes. This case illustrates the diagnostic challenges of large B-cell lymphoma with intravascular presentation. We found only five other cases of EBV-positive large B-cell lymphoma with an intravascular presentation in the literature, three of which had an underlying immunodeficiency adding to the broad spectrum of EBV-associated lymphoma in the setting of immunosuppression., (© 2021. The Author(s).)