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The prognosis of patients affected by myelofibrosis (MF) is usually dismal and allogeneic hematopoietic stem cell transplantation (HSCT) remains the only cure. The number of HSCTs in MF patients has recently increased. However, a major obstacle is still represented by primary graft failure (PGF). Currently there are no definitive guidelines for the treatment of PGF and a second HSCT can be performed only when an allogeneic donor is rapidly available. Herein we report on a MF patient with PGF after an unrelated HSCT, who was rescued by a non-myeloablative, unmanipulated, haploidentical HSCT that resulted in persistent engraftment and bone-marrow fibrosis regression, but not in a long-term disease control. Based on this experience we briefly review the role of different conditioning regimens and hematopoietic stem cell sources in the setting of HSCT for MF patients with PGF. The role of haploidentical donors in MF patients lacking HLAmatched relatives is also discussed. [ABSTRACT FROM AUTHOR]

p63, a member of the p53 family, is involved in the survival and differentiation of reserve/stem cells in different epithelia. To unveil the possible role of p63 in thymic physiology and pathology, we investigated the expression of p63 isoforms in normal thymus, thymomas and other mediastinal tumours. All samples were analysed using immunohistochemistry with three different antibodies: 4A4 antibody recognising all p63 isoforms, p40 antibody reacting only with truncated dominant-negative isoforms (DeltaN-p63) and H-129 antibody recognising all alpha-isoforms. Reverse-transcription polymerase chain reaction (RT-PCR), and real-time PCR analyses were performed on RNA extracted from frozen samples of four thymomas and two primary-mediastinal large-B-cell lymphoma (PMLBCL). In normal thymus, DeltaN-p63alpha was expressed in all cortical and medullary epithelial cells, with decreasing intensity in Hassall's corpuscles. This phenotype was conserved in neoplastic transformation since all 54 investigated thymomas (World Health Organization types A, AB, B1, B2, B3, C) expressed DeltaN-p63alpha (virtually 100% cells). The predominance of DeltaN-p63alpha isoform mRNA was confirmed by real-time PCR. Among other mediastinal tumours, DeltaN-p63alpha was only expressed in those displaying either a stratified epithelial component (teratomas) or epidermoid differentiation (lung carcinoma). Among lymphomas, T-cell-precursor lymphomas did not express p63, whereas most PMLBCL expressed TA-p63alpha (7/8). [ABSTRACT FROM AUTHOR]

In this paper we present VDJSeq-Solver, a methodology and tool to identify clonal lymphocyte populations from paired-end RNA Sequencing reads derived from the sequencing of mRNA neoplastic cells. The tool detects the main clone that characterises the tissue of interest by recognizing the most abundant V(D)J rearrangement among the existing ones in the sample under study. The exact sequence of the clone identified is capable of accounting for the modifications introduced by the enzymatic processes. The proposed tool overcomes limitations of currently available lymphocyte rearrangements recognition methods, working on a single sequence at a time, that are not applicable to high-throughput sequencing data. In this work, VDJSeq-Solver has been applied to correctly detect the main clone and identify its sequence on five Mantle Cell Lymphoma samples; then the tool has been tested on twelve Diffuse Large B-Cell Lymphoma samples. In order to comply with the privacy, ethics and intellectual property policies of the University Hospital and the University of Verona, data is available upon request to supporto.utenti@ateneo.univr.it after signing a mandatory Materials Transfer Agreement. VDJSeq-Solver JAVA/Perl/Bash software implementation is free and available at http://eda.polito.it/VDJSeq-Solver/.

Calreticulin (CALR) mutations are detected in the majority of JAK2 wild type patients with essential thrombocythemia (ET). Unlike JAK2V617F and MPL point mutations, CALR mutations are highly heterogeneous, with several types of indels being reported so far. CAL2 is a monoclonal antibody specifically recognizing the C-neoterminal peptide derived from all the frameshift mutations of CALR. We retrospectively analysed 172 ET patients diagnosed at our Institution from 1980 to 2015. In JAK2V617F- and MPLW515K/L-wild type patients CALR mutations were searched on peripheral blood and CAL2 immunostaining was performed on bone marrow. In addition, bone marrow biopsies were histologically reviewed for megakaryocytic features. Thirty-one patients (18%) were CALR-mutated. Concordance between molecular and immunohistological detection of CALR mutations was near complete, albeit a single patient was found to be positive by molecular tests only. Two patterns were defined in CAL2-positive bone marrow samples, characterized by staining of almost only megakaryocytes (pattern A: 41%) or staining of megakaryocytes and ≥ 2% small non megakaryocytic elements (pattern B: 59%), at least partially being myeloid precursors. Pattern B biopsies had higher cellularity and number of megakaryocytes compared to pattern A samples. In this series, CAL2 allowed rapid and cost-efficient identification of CALR-mutated ET patients. The biological significance of different staining pattern should be confirmed in wider and independent series. [ABSTRACT FROM AUTHOR]

Lipoblastoma is a rare benign mesenchymal neoplasm that typically occurs in infancy but may also occur in older age groups and various locations. Thus, there are often numerous clinical differential diagnoses. Moreover, lipoblastomas can show a broad histologic spectrum, which can hamper the correct diagnosis, particularly in small biopsies. At the genomic level, lipoblastomas are characterized by chromosomal fusions involving the PLAG1 gene. We investigated 11 lipoblastoma samples from 10 pediatric patients (age range five months to 12 years), including one patient with local recurrence, in view of their histopathological features, and performed targeted RNA sequencing. We found a broad histological spectrum with some tumors with prominent myxoid changes, but also tumors composed mainly of mature adipocytic cells, and classified the cases according to the literature as classic (mixed), maturing, or myxoid subtype. By targeted RNA sequencing analysis, we identified characteristic PLAG1 rearrangements in 70% of the investigated cases. Moreover, these analyses revealed three novel gene fusions, two affecting the PLAG1 gene and one involving HMGA2. Besides, we performed PLAG1 immunohistochemistry and identified positive cells, typically immature adipocytic cells and spindle cells, at various numbers in all cases. However, in the maturing areas, only very sparsely positive cells were found, limiting the value of the PLAG1 immunohistochemistry as an adjunct in the diagnosis of lipoblastoma, particularly for the maturing subtype and small biopsies. The presented case series confirms the broad morphological spectrum of lipoblastoma described in the literature and underlines the value of modern molecular diagnostic approaches as a supportive diagnostic tool in challenging cases and for gaining further insights into the molecular basis of this rare mesenchymal tumor. [ABSTRACT FROM AUTHOR]

Session 3 of the lymphoma workshop of the XXI joint meeting of the European Association for Haematopathology and the Society for Hematopathology took place in Florence, Italy, on September 22, 2022. The topics of this session were splenic and nodal marginal zone lymphomas, transformation in marginal zone lymphomas, and pediatric nodal marginal zone lymphomas and their differential diagnosis as well as related entities. Forty-two cases in these categories were submitted to the workshop, including splenic lymphomas (marginal zone and diffuse red pulp lymphomas), transformed marginal zone lymphomas (splenic and nodal), nodal marginal zone lymphomas with increased TFH-cells, and pediatric nodal marginal zone lymphomas. The case review highlighted some of the principal problems in the diagnosis of marginal zone lymphomas, including the difficulties in the distinction between splenic marginal zone lymphoma, splenic diffuse red pulp lymphoma, and hairy cell leukemia variant/splenic B-cell lymphoma with prominent nucleoli which requires integration of clinical features, immunophenotype, and morphology in blood, bone marrow, and spleen; cases of marginal zone lymphoma with markedly increased TFH-cells, simulating a T-cell lymphoma, where molecular studies (clonality and mutation detection) can help to establish the final diagnosis; the criteria for transformation of marginal zone lymphomas, which are still unclear and might require the integration of morphological and molecular data; the concept of an overlapping spectrum between pediatric nodal marginal zone lymphoma and pediatric-type follicular lymphoma; and the distinction between pediatric nodal marginal zone lymphoma and "atypical" marginal zone hyperplasia, where molecular studies are mandatory to correctly classify cases., (© 2023. The Author(s).)

The 2022 European Association for Haematopathology/Society for Hematopathology lymphoma workshop session on cavity-based lymphomas included sixty-eight cases in seven sections. The disease entities discussed include primary effusion lymphomas (PEL), extracavitary primary effusion lymphomas and confounding entities (ECPEL), HHV8-negative B-lineage lymphomas-effusion based (EBV-negative, EBV-positive, and plasmablastic types), diffuse large B-cell lymphoma associated with chronic inflammation, fibrin-associated diffuse large B-cell lymphoma (FA-DLBCL), breast implant-associated anaplastic large cell lymphoma (BIA-ALCL), and other lymphomas presenting as an effusion. All entities above are discussed; however, three are delved into greater detail given the challenges with classification: ECPEL, HHV8-negative effusion-based lymphomas, and FA-DLBCL. Cases exemplifying the diagnostic difficulty in differentiating ECPEL from HHV8-positive diffuse large B-cell lymphoma and germinotropic lymphoproliferative disorder were discussed. The more recently recognized effusion-based HHV8-negative large B-cell lymphoma is explored, with several cases submitted raising the question if this subset should be carved out as a specific entity, and if so, what should be the refining diagnostic criteria. Case submissions to the FA-DLBCL section yielded one of the largest case series to date, including classic cases, cases furthering the discussion on disease sites and prognosis, as well as novel concepts to be considered in this entity. The 2022 EA4HP/SH workshop cases allowed for further confirmation of the characteristics of some of the more historically accepted cavity-based lymphomas, as well as further inquiry and debate on relatively new or evolving entities., (© 2023. The Author(s).)

The 5th edition of the WHO classification (WHO-HAEM5) and the International Consensus Classification (ICC) have considerable overlap but also some distinct differences in categorizing indolent B‑cell lymphomas. Most differences with the expected impact on the daily diagnostic routine relate to follicular lymphoma (FL). Grading of FL remains mandatory only in the ICC; a diffuse growth pattern in an FL with > 15 blasts per high-power field (FL grade 3A) is not automatically classified as DLBCL according to WHO-HAEM5, and an FL subtype with unusual morphology (blastoid or large centrocyte) and biology is recognized as an entity only in the WHO-HAEM5. With the exception of B‑prolymphocytic leukemia, which is no longer acknowledged in WHO-HAEM5, there are only minor differences between both classifications and include updated names of entities, improved diagnostic criteria, and upgrades from provisional to definite entities., (© 2023. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

The subclassification of diffuse large B-cell lymphoma (DLBCL) into germinal center B-cell-like (GCB) and activated B-cell-like (ABC) subtypes has become mandatory in the 2017 update of the WHO classification of lymphoid neoplasms and will continue to be used in the WHO 5 th edition. The RNA-based Lymph2Cx assay has been validated as a reliable surrogate of high-throughput gene expression profiling assays for distinguishing between GCB and ABC DLBCL and provides reliable results from formalin-fixed, paraffin-embedded (FFPE) material. This test has been previously used in clinical trials, but experience from real-world routine application is rare. We routinely applied the Lymph2Cx assay to day-to-day diagnostics on a series of 147 aggressive B-cell lymphoma cases and correlated our results with the immunohistochemical subclassification using the Hans algorithm and fluorescence in situ hybridization findings using break-apart probes for MYC, BCL2, and BCL6. The routine use of the Lymph2Cx assay had a high technical success rate (94.6%) with a low rate of failure due to poor material and/or RNA quality. The Lymph2Cx assay was discordant with the Hans algorithm in 18% (23 of 128 cases). Discordant cases were mainly classified as GCB by the Hans algorithm and as ABC by Lymph2Cx (n = 11, 8.6%). Only 5 cases (3.9%) were classified as non-GCB by the Hans algorithm and as GCB by Lymph2Cx. Additionally, 5.5% of cases (n = 7) were left unclassified by Lymph2Cx, whereas they were defined as GCB (n = 4) or non-GCB (n = 3) by the Hans algorithm. Our data support the routine applicability of the Lymph2Cx assay., (© 2022. The Author(s).)

We previously reported that t(14;18)-negative follicular lymphomas (FL) show a clear reduction of newly acquired N-glycosylation sites (NANGS) in immunoglobulin genes. We therefore aimed to investigate in-depth the occurrence of NANGS in a larger cohort of t(14;18)-positive and t(14;18)-negative FL, including early (I/II) and advanced (III/IV) stage treatment-naive and relapsed tumors. The clonotype was determined by using a next-generation sequencing approach in a series of 68 FL with fresh frozen material [36 t(14;18) positive and 32 t(14;18) negative]. The frequency of NANGS differed considerably between t(14;18)-positive and t(14;18)-negative FL stage III/IV, but no difference was observed among t(14;18)-positive and t(14;18)-negative FL stage I/II. The introduction of NANGS in all t(14;18)-negative clinical subgroups occurred significantly more often in the FR3 region. Moreover, t(14;18)-negative treatment-naive FL, specifically those with NANGS, showed a strong bias for IGHV4-34 usage compared with t(14;18)-positive treatment-naive cases with NANGS; IGHV4-34 usage was never recorded in relapsed FL. In conclusion, subgroups of t(14;18)-negative FL might use different mechanisms of B-cell receptor stimulation compared with the lectin-mediated binding described in t(14;18)-positive FL, including responsiveness to autoantigens as indicated by biased IGHV4-34 usage and strong NANGS enrichment in FR3., (© 2021 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.)

In recent years, the outcome of mantle cell lymphoma (MCL) has improved, especially in younger patients, receiving cytarabine-containing chemoimmunotherapy and autologous stem cell transplantation. Nevertheless, a proportion of MCL patients still experience early failure. To identify biomarkers anticipating failure of intensive chemotherapy in MCL, we performed target resequencing and DNA profiling of purified tumor samples collected from patients enrolled in the prospective FIL-MCL0208 phase 3 trial (high-dose chemoimmunotherapy followed by autologous transplantation and randomized lenalidomide maintenance). Mutations of KMT2D and disruption of TP53 by deletion or mutation associated with an increased risk of progression and death, both in univariate and multivariate analysis. By adding KMT2D mutations and TP53 disruption to the MIPI-c backbone, we derived a new prognostic index, the "MIPI-genetic" ("MIPI- g"). The "MIPI-g" improved the model discrimination ability compared to the MIPI-c alone, defining three risk groups: i) low-risk patients (4-year progression free survival and overall survival of 72.0% and 94.5%); ii) inter-mediate-risk patients (4-year progression free survival and overall survival of 42.2% and 65.8%) and iii) high-risk patients (4-year progression free survival and overall survival of 11.5% and 44.9%). Our results: i) confirm that TP53 disruption identifies a high-risk population characterized by poor sensitivity to conventional or intensified chemotherapy; ii) provide the pivotal evidence that patients harboring KMT2D mutations share the same poor outcome as patients harboring TP53 disruption; and iii) allow to develop a tool for the identification of high-risk MCL patients for whom novel therapeutic strategies need to be investigated. ( Trial registered at clinicaltrials.gov identifier: NCT02354313 )., (Copyright© 2020 Ferrata Storti Foundation.)

Terminal tissue differentiation and function of slan + monocytes in cancer is largely unexplored. Our recent studies demonstrated that slan + monocytes differentiate into a distinct subset of dendritic cells (DC) in human tonsils and that slan + cells colonize metastatic carcinoma-draining lymph nodes. Herein, we report by retrospective analysis of multi-institutional cohorts that slan + cells infiltrate various types of non-Hodgkin lymphomas (NHL), particularly the diffuse large B-cell lymphoma (DLBCL) group, including the most aggressive, nodal and extranodal, forms. Nodal slan + cells displayed features of either immature DC or macrophages, in the latter case ingesting tumor cells and apoptotic bodies. We also found in patients with DLBCL that peripheral blood slan + monocytes, but not CD14 + monocytes, increased in number and displayed highly efficient rituximab-mediated antibody-dependent cellular cytotoxicity, almost equivalent to that exerted by NK cells. Notably, slan + monocytes cultured in conditioned medium from nodal DLBCL (DCM) acquired a macrophage-like phenotype, retained CD16 expression, and became very efficient in rituximab-mediated antibody-dependent cellular phagocytosis (ADCP). Macrophages derived from DCM-treated CD14 + monocytes performed very efficient rituximab-mediated ADCP, however, using different FcγRs from those used by slan + macrophages. Our observations shed new light on the complexity of the immune microenvironment of DLBCL and demonstrate plasticity of slan + monocytes homing to cancer tissues. Altogether, data identify slan + monocytes and macrophages as prominent effectors of antibody-mediated tumor cell targeting in patients with DLBCL. Significance: slan + monocytes differentiate into macrophages that function as prominent effectors of antibody-mediated tumor cell targeting in lymphoma. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/13/3544/F1.large.jpg Cancer Res; 78(13); 3544-59. ©2018 AACR ., (©2018 American Association for Cancer Research.)

First line drug treatment of follicular lymphoma (FL) patients is followed by a highly variable disease-free time before relapse in about one third of patients. No molecular marker is able to predict efficiently the risk of relapse. We investigated the expression profile of microRNAs (miRNAs) by microarrays and of the tumor microenvironment by immunohistochemistry in 26 FLs and 12 reactive lymph nodes (rLN) as reference. Twenty-nine miRNAs were differentially expressed in FLs compared to rLNs and some of them discriminated grade 1 from 3a FLs. Both FLs and rLNs displayed molecular heterogeneity. FLs grouped into two clusters mostly driven by the tumor T-cell content. Among 21 drug-treated FL patients with an average follow-up of 13.5 years, eight cases relapsed. Twenty-six miRNAs discriminated between relapsed and non-relapsed FLs. Ten miRNAs also correlated with Foxp3 + cells number. Notably, Foxp3 + cells were significantly less in relapsed patients and lower Foxp3 + cell number associated with shorter time-to-relapse. Foxp3 + cells did not co-expressed follicular helper T-cell markers and were therefore classified as regulatory T cells rather than follicular regulatory T-cells. These findings introduce new knowledge about the relationship between miRNA alterations and infiltrating immune cells and show that Foxp3 + cells might be predictive of disease relapse., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Background and Objectives: Mantle cell lymphoma (MCL) cell lines are difficult to generate; only nine lines have been described so far and few of them have been thoroughly characterized., Design and Methods: We established MAVER-1, a new MCL cell line, obtained from a leukemic MCL harboring both a t(11;14) translocation and a MYC rearrangement, and used immunohistochemistry, flow cytometry, molecular biology and cytogenetic techniques in order to characterize the cell line precisely., Results: By immunohistochemistry and flow cytometry MAVER-1 displayed a classical MCL phenotype (IgM+, l+, CD5+, CD10-, CD19+, CD20+, CD23-, CD79a+, cyclin D1+) and genetic analysis showed a typical V/D/J rearrangement with naïve mutational status. According to both classic cytogenetic analysis and spectral karyotyping, MAVER-1 harbored the t(11;14) translocation associated with a complex karyotype. Molecular analysis by polymerase chain reactions showed that the t(11;14) breakpoint is within the major translocation cluster. Other important abnormalities of MAVER-1 include TP53 gene inactivation by a combined mutation of exon 8 and chromosome 17p13 deletion, ATM deletion, 8q24 (MYC) rearrangement and 8p22 deletion., Interpretation and Conclusions: The new cell line will be useful for in vitro studies regarding MCL pathogenesis and drug sensitivity, as well as a diagnostic control material.

Background and Aim of the Work: Idiopathic Pulmonary Fibrosis/Usual Interstitial Pneumonia (IPF/UIP) is a diffuse and progressive lung disease whose pathogenesis is incompletely understood. Recently, the presence of Herpesvirus-specific DNA was detected in the large majority of cases of a series of IPF/UIP and other lung interstitial diseases. We have therefore tested our own IPF/UIP series for the presence of HHV-8 and EBV proteins., Methods: We used a variety of sensitive technologies including immunohistochemistry with NCL-HHV8-LNA and EBV-LMP1 antibodies, as well as of EBV RNA (EBER) by in-situ hybridisation; the presence of HHV-8 and EBV DNA was also investigated by means of PCR and subsequent analysis using a microfluidic apparatus., Results: Despite the good sample quality, immunohistochemical, in-situ hybridisation and PCR results were negative for both EBV and HHV-8., Conclusions: We conclude that EBV and HHV-8 are not involved in the pathogenesis of IPF/UIP.