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We present a series of 9 follicular lymphomas that progressed/transformed into classical Hodgkin lymphoma (CHL). Three cases of CHL showed a syncytial pattern (SCHL) making the differential diagnosis to Gray zone lymphoma (GZL) challenging. None of these three cases presented in the mediastinum. Based in all molecular data analyzed (BCL2/BCL6 FISH studies, IgH PCR and TNGS with a customized gene panel) we did find clonal relationship between the BCL2-positive FL cases and their CHL components in all cases. The three SCHL/GZL cases showed an activated phenotype according to Hans algorithm, presented the t(14; 18)(q32; q21), two out of three showed B cell markers and all expressed CD30 and p53. Interestingly, we identified three BCL2-negative FL cases with a further diagnosis of CHL expanding the spectrum of these association. In one of these three cases a different mutational profile was found in both the FL and the CHL components. All this data together suggests that CHL associated to BCL2-positive FL could be originated in a common progenitor cell (CPC) that give rise to both FL and CHL, acquiring this last component further genetic events in a linear fashion. On the other hand, no clonal relationship between CHL and BCL2-negative FL could be found, suggesting a fortuity association. Nevertheless, ample series of cases studied with more sensitive techniques are needed to confirm our hypothesis., Competing Interests: Declaration of competing interest None of the authors have any relevant conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Lymphomatoid papulosis (LyP) has several histopathologic presentations. LyP featuring gamma-delta (γδ) T-cell receptor expression may masquerade as and may be misdiagnosed as aggressive cutaneous T-cell lymphoma, particularly primary cutaneous γδ T-cell lymphoma (PCGDTL) or γδ mycosis fungoides. We performed a clinicopathologic analysis of the largest series of LyP featuring γδ T-cell expression. We identified 26 patients with a diagnosis of LyP with γδ T cells from our institutions, as well as through a comprehensive review of the literature, and characterized these cases. Most cases were treated with topical steroids or not treated at all. The majority of cases showed a CD4 - CD8 + phenotype and featured at least one cytotoxic marker. Histopathologic features included an intraepidermal or dermal infiltrate with large cells and frequent angiotropism. One case was initially misdiagnosed as PCGDTL, requiring further therapy. Our case series, the largest international cohort of γδ T cell predominant LyP cases, confirms marked clinicopathologic heterogeneity that may contribute to misdiagnosis, reasserting the need to identify classic clinical features, CD30 + T-cell components, and markers of cytotoxicity when dealing with this differential diagnosis. A limitation of this study includes somewhat limited follow-up, histologic, and immunophenotypic information for some cases., Competing Interests: Conflicts of Interest and Source of Funding: A.L.F. receives research funding from Seattle Genetics, is an inventor of technology for which Mayo Clinic holds an unlicensed patent, and has intellectual property licensed to Zeno Pharmaceuticals. A.A.G. is an investigator for StemLine Therapeutics, Innate Pharma, and Dren Bio. He is also consultant for Kyowa Kirin and BluePrints Medicine. For the remaining authors none were declared., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

EBV+ diffuse large B cell lymphoma (DLBCL) not otherwise specified (NOS) is a new entity confirmed by the World Health Organization (WHO) in 2017. In this new entity, the virus may contribute to a tolerogenic microenvironment. Traces of the virus have been described in DLBCL with more sensitive methods, in cases that were originally diagnosed as negative. The aim of this study was to analyze the expression of immune response genes in the tumor microenvironment to disclose the role of the virus and its traces in DLBCL. In 48 DLBCL cases, the expression of immune response genes and the presence of molecules that induce tolerance, such as TIM3, LAG3 and PDL1 by immunohistochemistry (IHC), were studied. To broaden the study of the microenvironment, tumor-associated macrophages (TMAs) were also explored. No significant differences were observed in the expression of immune response genes in the EBV+ DLBCL and those cases that were EBV- DLBCL but that exhibited viral traces, assessed by ViewRNA assay. Only the EBV+ DLBCL cases displayed a significantly higher increase in the expression of CD8 and cytotoxic T cells detected by gene expression analysis, and of PDL1 in tumor cells and in the expression of CD68 in the tumor microenvironment detected by IHC, not observed in those cases with viral traces. The increase in CD8 and cytotoxic T cells, PDL1 and CD68 markers only in EBV+ DLBCL may indicate that traces of viral infection might not have influence in immune response markers., (© 2024. The Author(s).)

Aggressive B-cell non-Hodgkin lymphomas are a heterogeneous group of diseases and our concepts are evolving as we learn more about their clinical, pathologic, molecular genetic features. Session IV of the 2020 EAHP Workshop covered aggressive, predominantly high-grade B-cell lymphomas, many that were difficult to classify. In this manuscript, we summarize the features of the submitted cases and highlight differential diagnostic difficulties. We specifically review issues related to high-grade B-cell lymphomas (HGBCLs) with MYC and BCL2 and/or BCL6 rearrangements including TdT expression in these cases, HGBCL, not otherwise specified, large B-cell lymphomas with IRF4 rearrangement, high-grade/large B-cell lymphomas with 11q aberration, Burkitt lymphoma, and pleomorphic mantle cell lymphoma. Since the workshop, the 5th edition of the WHO Classification for Haematolymphoid Tumours (WHO-HAEM5) and International Consensus Classification (ICC) 2022 were published. We endeavor to use the updated terminology., (© 2023. The Author(s).)

Session 3 of the 2021 European Association for Haematopathology/Society for Hematopathology Workshop focused on mediastinal large B cell lymphomas and surrounding gray areas. One half of the session was dedicated to primary mediastinal large B cell lymphoma (PMBL) and included cases with classic clinicopathologic features, as well as cases with either morphologic or immunophenotypic variation, and PMBL-like cases with primary extramediastinal disease. The role of additional immunophenotyping and/or molecular testing to aid in the diagnosis of PMBL was discussed. The second half of the session focused on mediastinal and non-mediastinal gray zone lymphomas (GZL) with features intermediate between diffuse large B cell lymphoma (DLBCL) and classic Hodgkin lymphoma (CHL). Several cases illustrating the current challenges in separating this entity from PMBL/DLBCL and CHL were presented. There was discussion regarding the clinical and genetic differences between mediastinal and non-mediastinal GZLs. Rare cases of PMBL and GZL associated with EBV or follicular lymphoma were reviewed. Finally, several cases included in the session highlighted composite or sequential CHL and PMBL/DLBCL and/or GZL, highlighting challenges in separating such cases from GZL., (© 2023. The Author(s).)

Lymphomas with plasmablastic features are a heterogeneous group of aggressive and mostly uncommon neoplasms of varied aetiologies, presenting in immunocompetent individuals as well as in immunodeficiency, associated with EBV and Kaposi sarcoma virus infections, and some as progression from indolent B-cell lymphomas. They show overlapping diagnostic features and pose a differential diagnosis with other aggressive B-cell lymphomas that can downregulate the B-cell expression programme. The spectrum of rare reactive proliferations and all lymphomas defined by plasmablastic features, together with an expanding range of poorly characterised, uncommon conditions at the interface between reactive lymphoid proliferations and neoplasia submitted to the session V of the 20 th European Association for Haematopathology/Society for Hematopathology lymphoma workshop are summarised and discussed in this paper., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Competing Interests: Conflicts of interest M.A.P. declares having received lecture fees and advisory board fees from Celgene, Gilead, Jansen, Kyowa Kirin, Millenium/Takeda and NanoString. P.L.O.-R. declares having received advisory fees from 4SC, Helsinn, Innate Pharma, Kyowa Kirin, Mallinckrodt, Recordati Rare Diseases and Takeda. The authors declare that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article.

Competing Interests: The authors report no conflict of interest.

We investigated the clinicopathological features and prognostic factors of patients with peripheral T-cell lymphoma (PTCL) in 13 sites across Spain. Relevant clinical antecedents, CD30 expression and staining pattern, prognostic indices using the International Prognostic Index and the Intergruppo Italiano Linfomi system, treatments, and clinical outcomes were examined. A sizeable proportion of 175 patients had a history of immune-related disorders (autoimmune 16%, viral infections 17%, chemo/radiotherapy-treated carcinomas 19%). The median progression-free survival (PFS) and overall survival (OS) were 7·9 and 15·8 months, respectively. Prognostic indices influenced PFS and OS, with a higher number of adverse factors resulting in shorter survival (P < 0·001). Complete response (CR) to treatment was associated with better PFS (62·6 vs. 4 months; P < 0·001) and longer OS (67·0 vs. 7·3 months; P < 0·001) compared to no CR. CD30 was expressed across all subtypes; >15% of cells were positive in anaplastic lymphoma kinase-positive and -negative anaplastic large-cell lymphoma and extranodal natural killer PTCL groups. We observed PTCL distribution across subtypes based on haematopathological re-evaluation. Poor prognosis, effect of specific prognostic indices, relevance of histopathological sub-classification, and response level to first-line treatment on outcomes were confirmed. Immune disorders amongst patients require further examination involving genetic studies and identification of associated immunosuppressive factors., (© 2020 The Authors. British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

Cutaneous eruption of lymphocyte recovery (ELR) during bone marrow (BM) aplasia recovery after intensive chemotherapy has been reported in very few patients. The presence of skin rashes in patients with acute leukemia who are undergoing intensive chemotherapy and BM transplantation is a diagnostic challenge because of the clinical similarity between drug eruptions, infiltrates related to the relapse of the underlying disease, cutaneous graft-versus-host disease, and ELR. IDH1 mutations have been identified as a recurrent genetic anomaly in acute myeloid leukemia and myelodysplastic syndromes. However, until now, this IDH1 mutation has not been reported as being shared by myeloid cells and non-neoplastic inflammatory cells in this clinical setting. Here, we present the rare case of a woman diagnosed with myelodysplastic syndrome that evolved into an acute myelogenous leukemia with leukemic cutaneous infiltrate. The patient developed ELR after the intensive chemotherapy administered before BM transplantation. The IDH1 mutation was identified in BM cells and in myeloid and inflammatory cells in skin biopsies before allogeneic BM transplantation. We discuss the main aspects of the differential diagnosis of these cutaneous reactions in leukemic patients and the biological significance of the IDH1 mutation.

Background: The malignant mechanisms that control the development of cutaneous T-cell lymphoma (CTCL) are beginning to be identified. Recent evidence suggests that disturbances in specific intracellular signalling pathways, such as RAS-mitogen-activated protein kinase, T-cell receptor (TCR)-phospholipase C gamma 1 (PLCG1)-nuclear factor of activated T cells (NFAT) and Janus kinase (JAK)-signal transducer and activator of transcription (STAT), may play an essential role in the pathogenesis of CTCL., Objectives: To investigate the mechanisms controlling disease development and progression in mycosis fungoides (MF), the most common form of CTCL., Methods: We collected 100 samples that were submitted for diagnosis of, or a second opinion regarding, MF between 2001 and 2018, 80% of which were in the early clinical stages of the disease. Formalin-fixed paraffin-embedded tissues were used for histological review and to measure the expression by immunohistochemistry of surrogate markers of activation of the TCR-PLCG1-NFAT, JAK-STAT and NF-κB pathways. Folliculotropism and large-cell transformation were also examined., Results: NFAT and nuclear factor kappa B (NF-κB) markers showed a comparable activation status in early and advanced stages, while STAT3 activation was more frequent in advanced stages and was associated with large-cell transformation. Consistently with this observation, STAT3 activation occurred in parallel with MF progression in two initially MF-negative cases. A significant association of NFAT with NF-κB markers was also found, reflecting a common mechanism of activation in the two pathways. Genomic studies identified nine mutations in seven genes known to play a potential role in tumorigenesis in T-cell leukaemia/lymphoma, including PLCG1, JAK3 and STAT3, which underlies the activation of these key cell-survival pathways. A higher mutational allele frequency was detected in advanced stages., Conclusions: Our results show that STAT3 is activated in advanced cases and is associated with large-cell transformation, while the activation of NFAT and NF-κB is maintained throughout the disease. These findings could have important diagnostic and therapeutic implications. What's already known about this topic? Mycosis fungoides is characterized by a clonal expansion of T cells in the skin. The mechanisms controlling disease development and progression are not fully understood. What does this study add? An association of the nuclear factor of activated T cells and nuclear factor kappa B pathways was found, which could reflect a common mechanism of activation. These pathways were activated in early and advanced stages at the same level. Signal transducer and activator of transcription 3 activation was associated with large-cell transformation and was more frequent in advanced stages. A genomic analysis of cutaneous T-cell lymphoma-associated genes was performed. Nine mutations were detected. What is the translational message? These results could have important implications for the treatment of MF in the near future., (© 2019 British Association of Dermatologists.)

Follicular helper T (T FH ) cells are the providers of T-cell help to B-cells in the development of germinal centers and for the generation of most class-switched antibodies. The markers most commonly associated with T FH activity are IL21, IL4, CD40L, BCL6, SAP, CXCR5/CXCL13, and ICOS. T-cell lymphoma genomic studies have shown that different T-cell lymphoma types express signatures typical for T FH cells, this including angioimmunoblastic T-cell lymphoma (AITL), a related condition termed peripheral T-cell lymphoma with T FH phenotype and primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder. Angioimmunoblastic T-cell lymphoma is a well-established entity, a clinically aggressive disease with a survival of 30% OS after 5 years. Molecular and clinical studies have confirmed this as a well-established clinicopathological entity with relatively specific gene mutations, including mutations found in hematopoietic precursor cells and others. Peripheral T-cell lymphoma with T FH phenotype is an associated disorder with histology of PTCL but a T FH phenotype, as defined by the expression of 2-3 immunohistochemical markers. Molecular studies on this entity are showing a partial overlap with AITL. Primary cutaneous CD4+ small/medium lymphoproliferative disorder is an entirely different process that takes place in the skin, showing frank cytologic atypia, monoclonal TCR rearrangement and T FH phenotype in the context of a clinically benign lesion. Here we review the main clinical, molecular and diagnostic features of these three lymphoproliferative processes., Competing Interests: Declaration of Competing Interest This work was supported by grants from the Instituto de Salud Carlos III (ISCIII) of the Spanish Ministry of Economy and Competence (MINECO, RTICC ISCIII and CIBERONC) (SAF2013-47416-R, RD06/0020/0107-RD012/0036/0060 and Plan Nacional I+D+I: PI17/2172, PI16/01294 and PIE15/0081), AECC, and the Madrid Autonomous Community., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The presence of CD30 cells in cutaneous lymphomas has come to prominence in recent years as a potential diagnostic and therapeutic marker. In primary cutaneous marginal zone B-cell lymphomas, the presence of large CD30 cells with Hodgkin-like features and their significance have not yet been studied. Here we describe the main clinical, histologic, immunophenotypic, and molecular characteristics of 13 cases of primary cutaneous marginal zone lymphomas featuring >10% of CD30 large cells, and analyze their relationship with histologic and clinical progression of the disease and with other morphologic and immunophenotypic features. We report 10 male and 3 female patients, 4 with early-local disease and 8 with locoregional advanced disease without extracutaneous involvement but with a high relapse rate of 69%. We describe an association between a high level of CD30 expression and disease progression, with increased clinical recurrence in cases with >15% of CD30 cells. We also discuss the differential diagnosis with other cutaneous and systemic lymphomas, especially Hodgkin lymphoma.

Primary cutaneous CD30-positive T-cell lymphoproliferative disorders are the second most common subgroup of cutaneous T-cell lymphomas. They include two clinically different entities with some overlapping features and borderline cases: lymphomatoid papulosis and primary cutaneous anaplastic large cell lymphoma. Molecular studies of primary cutaneous anaplastic large cell lymphoma reveal an increasing level of heterogeneity that is associated with histological and immunophenotypic features of the cases and their response to specific therapies. Here, we review the most significant genetic, epigenetic and molecular alterations described to date in primary cutaneous CD30-positive T-cell lymphoproliferative disorders, and their potential as therapeutic targets., (Copyright © 2019 Ferrata Storti Foundation.)

Background: Hereditary breast cancer comprises 5-10% of all breast cancers. Mutations in two high-risk susceptibility genes, BRCA1 and BRCA2, along with rare intermediate-risk genes and common low-penetrance alleles identified, altogether explain no more than 45% of the high-risk breast cancer families, although the majority of cases are unaccounted for and are designated as BRCAX tumours. Micro RNAs have called great attention for classification of different cancer types and have been implicated in a range of important biological processes and are deregulated in cancer pathogenesis., Methods: Here we have performed an exploratory hypothesis-generating study of miRNA expression profiles in a large series of 66 primary hereditary breast tumours by microarray analysis., Results: Unsupervised clustering analysis of miRNA molecular profiles revealed distinct subgroups of BRCAX tumours, 'normal-like' BRCAX-A, 'proliferative' BRCAX-B, 'BRCA1/2-like' BRCAX-C and 'undefined' BRCAX-D subgroup. These findings introduce a new insight in the biology of hereditary breast cancer, defining specific BRCAX subgroups, which could help in the search for novel susceptibility pathways in hereditary breast cancer., Conclusion: Our data demonstrate that BRCAX hereditary breast tumours can be sub-classified into four previously unknown homogenous groups characterised by specific miRNA expression signatures and histopathological features.

SPIB is an Ets transcription factor that is expressed exclusively in mature B cells, T-cell progenitors, and plasmacytoid dendritic cells. In the present study, we developed a novel mAb against the SPIB protein and characterized its expression in major hematolymphoid neoplasms, including a series of 45 cases of blastic plasmacytoid dendritic cell (BPDC) neoplasms and their potential cutaneous mimics. We found that SPIB is expressed heterogeneously among B- and T-cell lymphoma types. Interestingly, SPIB is expressed in a large proportion of nongerminal center type DLBCLs. In cutaneous neoplasms, SPIB is overexpressed in all BPDC neoplasms, but none of its cutaneous mimics. SPIB remains overexpressed in all cases that lack 1 or 2 of the markers used for BPDC neoplasms (ie, CD4, CD56, TCL1, and CD123). We conclude that SPIB expression can be used as a tool for diagnosing BPDC neoplasms, but it needs to be tested in conjunction with the growing arsenal of markers for human plasmacytoid dendritic cells.

Angioimmunoblastic T-cell lymphoma (AITL) is the second most common peripheral T-cell lymphoma with unusual clinical and pathologic features and a poor prognosis despite intensive chemotherapy. Recent studies have suggested AITL derives from follicular helper T (T(FH)) cells, but the causative molecular pathways remain largely unknown. Here we show that approximately 50% of mice heterozygous for the "san" allele of Roquin develop tumors accompanied by hypergammaglobulinemia by 6 months of age. Affected lymph nodes displayed the histologic features diagnostic of AITL, except for the presence of expanded FDC networks. Accumulation of T(FH) cells preceded tumor development, and clonal rearrangements in the TCR-β genes were present in most tumors. Furthermore, T(FH) cells exhibited increased clonality compared with non-T(FH) cells from the same lymph nodes, even in the absence of tumors. Genetic manipulations that prevent T(FH) development, such as deletion of ICOS, CD28, and SAP, partially or completely abrogated tumor development, confirming a T(FH)-derived origin. Roquin(san/+) mice emerge as a useful model to investigate the molecular pathogenesis of AITL and for preclinical testing of therapies aimed at targeting dysregulated T(FH) cells or their consequences.

Background: Plasmablastic lymphoma has recently come to be considered a distinct entity among mature B cell neoplasms, although the limits with diffuse large B-cell lymphoma (DLBCL) need to be more accurately defined., Design and Methods: Here we show the results of an immunohistochemical study of 35 cases of plasmablastic lymphoma compared with a set of 111 conventional DLBCLs., Results: Our results demonstrate that the use of a limited combination of immunohistochemical markers (PAX5&CD20, PRDM1/BLIMP1 and XBP1s) enables the identification of a plasmablastic immunophenotype highly characteristic of plasmablastic lymphoma cases and associated with an aggressive clinical behavior. Additionally, the study shows that the acquisition of a partial plasmablastic phenotype (PRDM1/BLIMP1 expression) in DLBCL is associated with shorter survival in R-CHOP-treated patients., Conclusions: The use of a restricted combination of immunohistochemical markers (PAX5&CD20, PRDM1/BLIMP1 and XBP1s) enables a more accurate definition of terminal differentiation for large B-cell lymphoma.

Background: Splenic diffuse red pulp small B-cell lymphoma is an uncommon B-cell lymphoma, now recognized as a provisional entity in the 2008 update of the WHO Classification. Additional work is required to review this entity and establish its diagnostic features., Design and Methods: We have retrospectively analyzed the disease features in a highly selected series of 17 patients diagnosed as splenic diffuse red pulp small B-cell lymphoma., Results: The median age was 65.5 years (range 40-79 years) and there was a predominance of males (male/female ratio: 2.4). Clinical manifestations were mainly derived from splenomegaly. Splenectomy was the front-line treatment in 11 symptomatic patients; the remaining 6 received chemotherapy initially followed by splenectomy. After a mean follow-up of 72 months, the five-year overall survival was 93%. All cases showed a purely diffuse pattern of splenic infiltration by monomorphous small cells with small round nuclei and pale cytoplasm. All bone marrow biopsies showed tumoral infiltration, with intrasinusoidal infiltration. Peripheral blood cells were small to medium-sized, with clumped chromatin and round nuclear outline and villous cytoplasm. Neoplastic cells had a CD20(+), CD23(-), bcl6(-), Annexin A1- phenotype, with frequent expression of DBA44+ (15/17) and IgG (10/15). FCM data had a B-cell phenotype (CD19(+), CD20(+), CD22(+)) with FMC7 (10/11) and CD11c (5/8) expression. Clonal IgH rearrangement studies in 4 cases showed IgVH mutations in all cases, without VH1.2 usage., Conclusions: Our data suggest that splenic diffuse red pulp small B-cell lymphoma is a distinct entity with morphological and immunophenotypical features that differ from those of other splenic lymphomas.

Breast cancer is a heterogeneous disease caused by the accumulation of genetic changes in neoplastic cells. We hypothesised that molecular subtypes of breast cancer may be driven by specific constellations of genes whose expression is regulated by gene copy number aberrations. To address this question, we analysed a series of 48 microdissected grade III ductal carcinomas using high resolution microarray comparative genomic hybridisation and mRNA expression arrays. There were 5,931 genes whose expression significantly correlates with copy number identified; out of these, 1,897 genes were significantly differentially expressed between basal-like, HER2 and luminal tumours. Ingenuity Pathway Analysis (IPA) revealed that 'G1/S cell cycle regulation' and 'BRCA1 in DNA damage control' pathways were significantly enriched for genes whose expression correlates with copy number and are differentially expressed between the molecular subtypes of breast cancer. IPA of genes whose expression significantly correlates with copy number in each molecular subtype individually revealed that canonical pathways involved in oestrogen receptor (ER) signalling and DNA repair are enriched for these genes. We also identified 32, 157 and 265 genes significantly overexpressed when amplified in basal-like, HER2 and luminal cancers, respectively. These lists include known and novel potential therapeutic targets (e.g. HER2 and PPM1D in HER2 cancers). Our results provide strong circumstantial evidence that different patterns of genetic aberrations in distinct molecular subtypes of breast cancer contribute to their specific transcriptomic profiles and that biological phenomena characteristic of each subtype (e.g. proliferation, HER2 and ER signalling) may be driven by specific patterns of copy number aberrations.

Background: WT1, first recognized as a tumor suppressor gene involved in the development of Wilms' tumor, may have apparently contradictory findings and functions. As WT1 has been identified as a molecular target for cancer immunotherapy, immunodetection of WT1 in tumor cells has become an essential step in cancer studies., Methods: We compare the expression of this protein among different types of melanocytic nevi and among stages in primary melanoma progression. Tissue microarrays containing normal tissues and 271 primary melanocytic lesion samples (163 primary melanomas and 108 nevi) were studied by immunohistochemistry using monoclonal antibody against WT1., Results and Discussion: The present study shows these: 1. WT1 protein is predominantly expressed in the cytoplasm of the neoplastic cells. 2. A higher rate of WT1 staining in melanocytic nevi against melanomas has been observed. 3. WT1 expression is increased in advanced stages of melanoma progression: a significant (p < 0.05) increase of expression of WT1 was detected in vertical cases 46.5% vs. radial cases 16.0%, in high levels of Clark (IV, V) 57.4% vs. low levels (I, II, III) 33.0% and when comparing depth of invasion within thickness subgroups. 4. Finally, this study establishes an association of WT1 protein expression with shorter overall survival in melanoma.

Background: T follicular helper (T(FH)) cells reside in the light zone of germinal centers and are considered the cell of origin of angioimmunoblastic T-cell lymphoma. Recently, CXCL13, PD-1 and SAP were described as useful markers for T(FH) cells and angioimmunoblastic T-cell lymphoma but also reported in some peripheral T-cell lymphomas, not otherwise specified., Design and Methods: In the present study the expression pattern of ICOS protein was investigated by immunohistochemistry-based techniques in routine sections of normal lymphoid tissues and 633 human lymphomas., Results: Cells strongly positive for ICOS were restricted to the light zone of germinal centers and co-expressed T(FH)-associated molecules. In addition, weak to moderate ICOS expression was observed in a small proportion of FOXP3-positive cells. In lymphomas, ICOS expression was confined to angioimmunoblastic T-cell lymphoma (85/86), peripheral T-cell lymphomas of follicular variant (18/18) and a proportion of peripheral T-cell lymphomas, not otherwise specified (24/56) that also expressed other T(FH)-associated molecules., Conclusions: ICOS is a useful molecule for identifying T(FH) cells and its restricted expression to angioimmunoblastic T-cell lymphoma and a proportion of peripheral T-cell lymphomas, not otherwise specified (showing a T(FH)-like profile) suggests its inclusion in the antibody panel for diagnosing T(FH)-derived lymphomas. Our findings provide further evidence that the histological spectrum of T(FH)-derived lymphomas is broader than previously assumed.

Loss of the long arm of chromosome 16 (16q) is observed in the vast majority of low grade/grade I (GI) invasive ductal carcinomas of no special type (IDC-NSTs), whereas this event is uncommonly seen in high grade/grade III (GIII) IDC-NSTs. Together with data on the pathology and genetics of breast cancer recurrences, this has led to the proposal that GI and GIII breast cancers evolve through distinct genetic pathways and that progression from GI to GIII is an unlikely biological phenomenon. We compared the genomic profiles of GIII-IDC-NSTs with 16q whole arm loss (16qWL) according to estrogen receptor (ER) status. 16qWL was found in 36.5% of cases and was significantly associated with ER expression and luminal phenotype. ER+ GIII-IDC-NSTs with 16qWL displayed significantly higher levels of genomic instability than ER+ IDC-NSTs without 16qWL. Furthermore, ER+ and ER- IDC-NSTs stratified according to the presence of 16qWL harbored distinct patterns of genetic aberrations. Interestingly, ER+/16qWL tumors displayed genetic features usually found in tumors with homologous DNA repair defects and significantly more frequently harbored heterozygous loss of BRCA2 than the remaining ER+ cancers. Our results demonstrate that approximately one third of GIII tumors harbor 16qWL, confirming that progression from low to high grade breast cancer is not found in the majority of breast cancers. 16qWL was significantly more prevalent in ER+/luminal GIII-IDC-NSTs. Given that GI breast cancers harbor a luminal phenotype, our results suggest that if progression from GI to GIII breast cancer does happen, it may preferentially occur in breast cancers of luminal phenotype.

The assembly of a collection of gene-expression signatures of the major types of B-cell non-Hodgkin's lymphoma has identified increased T-cell leukemia/lymphoma 1A (TCL1) expression in multiple lymphoma types and cases, and has enabled the investigation of the functional and clinical importance of TCL1 expression. Specifically, Burkitt's lymphoma cases show a homogeneously strong expression of TCL1, whereas diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia, nodal marginal zone lymphoma, and splenic marginal zone lymphoma display a striking variability in the intensity of TCL1 staining. This was validated in two independent series. A Gene-Set Enrichment Analysis of the genes correlated with TCL1A expression found that variation in the level of expression of TCL1A was significantly associated with some of the most important gene signatures recognizing B-cell lymphoma pathogenesis and heterogeneity, such as germinal center, B-cell receptor, NF-kappaB (and its target genes), death, MAP kinases, TNFR1, TOLL, and IL1R. Additionally, TCL1 expression was correlated with shorter time to treatment in chronic lymphocytic leukemia cases and shorter lymphoma-specific survival in mantle cell lymphoma series, thus indicating the clinical and biological significance of TCL1 expression, and suggesting TCL1A as a potential therapeutic target.

Expression profiling studies have suggested that HER2-amplified breast cancers constitute a heterogeneous group that may be subdivided according to their ER status: HER2-amplified ER-positive breast carcinomas that fall into the luminal B cluster; and HER2-amplified ER-negative cancers which form a distinct molecular subgroup, known as the erbB2 or HER2 subgroup. ER-negative breast cancer differs significantly from ER-positive disease in the pattern, type, and complexity of genetic aberrations. Here we have compared the genomic profiles of ER-positive and ER-negative HER2-amplified cancers using tiling path microarray-based comparative genomic hybridization (aCGH). Validation of the differentially amplified regions was performed in an independent series of 70 HER2-amplified breast cancers. Although HER2-amplified cancers had remarkably complex patterns of molecular genetic aberrations, ER-positive and ER-negative HER2-amplified breast carcinomas shared most molecular genetic features as defined by aCGH. Genome-wide Fisher's exact test analysis revealed that less than 1.5% of the genome was significantly differentially gained or lost in ER-positive versus ER-negative HER2-amplified cancers. However, two regions of amplification were significantly associated with ER-positive carcinomas, one of which mapped to 17q21.2 and encompassed GJC1, IGFBP4, TNS4, and TOP2A. Chromogenic in situ hybridization analysis of an independent validation series confirmed the association between ER status and TOP2A amplification. In conclusion, although hormone receptor status does not determine the overall genetic profile of HER2-amplified breast cancers, specific genetic aberrations may be characteristic of subgroups of HER2 breast cancers.

HER2 and TOP2A are targets for the therapeutic agents trastuzumab and anthracyclines and are frequently amplified in breast cancers. The aims of this study were to provide a detailed molecular genetic analysis of the 17q12-q21 amplicon in breast cancers harbouring HER2/TOP2A co-amplification and to investigate additional recurrent co-amplifications in HER2/TOP2A-co-amplified cancers. In total, 15 breast cancers with HER2 amplification, 10 of which also harboured TOP2A amplification, as defined by chromogenic in situ hybridisation, and 6 breast cancer cell lines known to be amplified for HER2 were subjected to high-resolution microarray-based comparative genomic hybridisation analysis. This revealed that the genomes of 12 cases were characterised by at least one localised region of clustered, relatively narrow peaks of amplification, with each cluster confined to a single chromosome arm (ie 'firestorm' pattern) and 3 cases displayed many narrow segments of duplication and deletion affecting the vast majority of chromosomes (ie 'sawtooth' pattern). The smallest region of amplification (SRA) on 17q12 in the whole series extended from 34.73 to 35.48 Mb, and encompassed HER2 but not TOP2A. In HER2/TOP2A-co-amplified samples, the SRA extended from 34.73 to 36.54 Mb, spanning a region of approximately 1.8 Mb. Apart from HER2 and TOP2A, this region encompassed four additional genes whose expression levels as defined by quantitative real-time PCR are significantly higher in HER2/TOP2A-co-amplified vs HER2-amplified breast cancers: CASC3, CDC6, RARA and SMARCE1. Of the cell lines studied, SKBR3 and UACC812 showed HER2/TOP2A co-amplification. In conclusion, this is the first detailed genome-wide characterisation of HER2/TOP2A-amplified breast cancers; cell lines were identified that can be used to model these cancers in vitro. The 17q12 amplicon is complex and harbours multiple genes that may be associated with breast cancer development and progression, and potentially exploitable as therapeutic targets.

Epithelial-mesenchymal transition (EMT) is defined by the loss of epithelial characteristics and the acquisition of a mesenchymal phenotype. In carcinoma cells, EMT can be associated with increased aggressiveness, and invasive and metastatic potential. To assess the occurrence of EMT in human breast tumors, we conducted a tissue microarray-based immunohistochemical study in 479 invasive breast carcinomas and 12 carcinosarcomas using 28 different markers. Unsupervised hierarchical clustering of the tumors and statistical analysis showed that up-regulation of EMT markers (vimentin, smooth-muscle-actin, N-cadherin, and cadherin-11) and overexpression of proteins involved in extracellular matrix remodeling and invasion (SPARC, laminin, and fascin), together with reduction of characteristic epithelial markers (E-cadherin and cytokeratins), preferentially occur in breast tumors with the "basal-like phenotype." Moreover, most breast carcinosarcomas also had a basal-like phenotype and showed expression of mesenchymal markers in their sarcomatous and epithelial components. To assess whether basal-like cells have intrinsic phenotypic plasticity for mesenchymal transition, we performed in vitro studies with the MCF10A cell line. In response to low cell density, MCF10A cells suffer spontaneous morphologic and phenotypic EMT-like changes, including cytoskeleton reorganization, vimentin and Slug up-regulation, cadherin switching, and diffuse cytosolic relocalization of the catenins. Moreover, these phenotypic changes are associated with modifications in the global genetic differentiation program characteristic of the EMT process. In summary, our data indicate that in breast tumors, EMT likely occurs within a specific genetic context, the basal phenotype, and suggests that this proclivity to mesenchymal transition may be related to the high aggressiveness and the characteristic metastatic spread of these tumors.

Background: TOP2A gene encodes topoisomerase II alpha, the direct molecular target of anthracyclines. This gene is frequently coamplified with HER2. The aims of this study were to analyse the pattern of TOP2A amplification and protein expression in relation to the molecular subgroups of breast cancers; and to define the impact of TOP2A amplification on the outcome of a series of patients homogeneously treated with adjuvant anthracyclines., Methods: A cohort of 245 patients with early breast cancer homogeneously treated with anthracyclines in the adjuvant setting was selected. A tissue microarray containing these cancers was used to determine HER2 and TOP2A gene copy number by means of chromogenic in situ hybridization. Immunohistochemical staining of topoisomerase II alpha was also performed using a monoclonal antibody (Ki-S1). TOP2A amplification and protein expression were correlated with classical prognostic parameters, expression of immunohistochemical markers and with a gene expression profiling classification using surrogate immunohistochemical markers. Kaplan-Meier method was used to construct survival curves and results were compared with log-rank test., Results: TOP2A amplification was restricted to tumours with HER2 amplification and was significantly associated with ER positivity. In the subgroup of patients with HER2 amplified tumours, TOP2A amplification predicted a better overall survival and disease free survival (P = 0.028 and 0.026, respectively). On multivariate analysis, TOP2A amplification maintained its predictive value for DFS., Conclusion: TOP2A amplification is likely to be a useful marker to predict the subset of patients who will benefit from anthracyclines.

Aims: To analyse the correlation between MYC amplification and various clinicopathological features and outcome in a cohort of 245 patients with invasive breast carcinoma treated with surgery followed by anthracycline-based chemotherapy. Given the high prevalence of MYC amplification in tumours of BRCA1 mutation carriers and the similarities between these and sporadic "basal-like" carcinomas, the prevalence of MYC amplification in "basal-like" breast carcinomas was investigated., Methods: MYC gene copy number was assessed on tissue microarrays containing duplicate cores of 245 invasive breast carcinomas by means of chromogenic in situ hybridisation using SpotLight C-MYC amplification probe and chromosome 8 centromeric probe (CEP8). Signals were evaluated at 400x magnification; 30 morphologically unequivocal neoplastic cells in each core were counted for the presence of the gene and CEP8 probes., Results: Amplification was defined as a MYC:CEP8 ratio >2. Signals for both MYC and CEP8 were assessable in 196/245 (80%) tumours. MYC amplification was found in 19/196 cases (9.7%) and was not associated with tumour size, histological grade, positivity for oestrogen receptor, progesterone receptor, HER2, epidermal growth factor, cytokeratins 14, 5/6 and 17, MIB1 or p53. Only 4% of basal-like carcinomas showed MYC amplification, compared to 8.75% and 10.7% of luminal and HER2 tumours respectively. On univariate analysis, MYC amplification displayed a significant association with shorter metastasis-free and overall survival and proved to be an independent prognostic factor on multivariate survival analysis., Conclusion: MYC amplification is not associated with "basal-like" phenotype and proved to be an independent prognostic factor for breast cancer patients treated with anthracycline-based chemotherapy.

Tumours arising in BRCA1 mutation carriers and sporadic basal-like breast carcinomas have similar phenotypic, immunohistochemical and clinical characteristics. SOX2 is an embryonic transcription factor located at chromosome 3q, a region frequently gained in sporadic basal-like and BRCA1 germline mutated tumours. The aim of the study was to establish whether sox2 expression was related to basal-like sporadic breast tumours. Two hundred and twenty-six sporadic node-negative invasive breast carcinomas were immunohistochemically analysed for oestrogen receptor (ER), progesterone receptor (PR), CK5/6, EGFR, vimentin, HER2, ki67, p53 and sox2 using tissue microarrays. Tumours were considered to have basal-like phenotype if they were ER/HER2-negative and CK5/6 and/or EGFR-positive. Thirty cases of this series (13.7%) displayed a basal-like phenotype. Sox2 expression was observed in 16.7% of cases and was significantly more frequently expressed in basal-like breast carcinomas (43.3% in basal-like, 10.6% in luminal and 13.3% in HER2+ tumours, P<0.001). Moreover, Sox2 showed a statistically significant inverse association with ER and PR (P=0.001 and 0.017, respectively) and direct association with CK5/6, EGFR and vimentin (P=0.022, 0.005 and <0.001, respectively). Sox2 is preferentially expressed in tumours with basal-like phenotype and may play a role in defining their less differentiated/'stem cell' phenotypic characteristics.

Recently, it has been shown that mice deficient in the proapoptotic protein prostate apoptosis response 4 (Par-4) are specifically prone to develop endometrial carcinomas. Based on this, we have examined here the possible role of Par-4 as a tumor suppressor gene in human endometrial cancer. Using cDNA arrays, quantitative reverse transcription-PCR, and immunohistochemistry, we detected Par-4 down-regulation in approximately 40% of endometrial carcinomas. This alteration was not associated with phosphatase and tensin homologue (PTEN), K-RAS, or beta-catenin mutations, but was more frequent among tumors showing microsatellite instability (MSI) or among tumors that were estrogen receptor positive. Mutational analysis of the complete coding sequence of Par-4 in endometrial cancer cell lines (n = 6) and carcinomas (n = 69) detected a mutation in a single carcinoma, which was localized in exon 3 [Arg (CGA) 189 (TGA) Stop]. Interestingly, Par-4 promoter hypermethylation was detected in 32% of the tumors in association with low levels of Par-4 protein and was more common in MSI-positive carcinomas. Par-4 promoter hypermethylation and silencing was also detected in endometrial cancer cell lines SKUT1B and AN3CA, and reexpression was achieved by treatment with the demethylating agent 5'-aza-2'-deoxycytidine. Together, these data show that Par-4 is a relevant tumor suppressor gene in human endometrial carcinogenesis.