"Next top" mouse models advancing CTCL research.

This review systematically describes the application of in vivo mouse models in studying cutaneous T-cell lymphoma (CTCL), a complex hematological neoplasm. It highlights the diverse research approaches essential for understanding CTCL's intricate pathogenesis and evaluating potential treatments. The review categorizes various mouse models, including xenograft, syngeneic transplantation, and genetically engineered mouse models (GEMMs), emphasizing their contributions to understanding tumor-host interactions, gene functions, and studies on drug efficacy in CTCL. It acknowledges the limitations of these models, particularly in fully replicating human immune responses and early stages of CTCL. The review also highlights novel developments focusing on the potential of skin-targeted GEMMs in studying natural skin lymphoma progression and interactions with the immune system from onset. In conclusion, a balanced understanding of these models' strengths and weaknesses are essential for accelerating the deciphering of CTCL pathogenesis and developing treatment methods. The GEMMs engineered to target specifically skin-homing CD4+ T cells can be the next top mouse models that pave the way for exploring the effects of CTCL-related genes.

Socs1-knockout in skin-resident CD4+ T cells in a protracted contact-allergic reaction results in an autonomous skin inflammation with features of early-stage mycosis fungoides.

Recent detailed genomic analysis of mycosis fungoides (MF) identified suppressor of cytokine signaling 1 (SOCS1), an inhibitor of JAK/STAT signaling, as one of the frequently deleted tumor suppressors in MF, and one-copy deletion of SOCS1 was confirmed in early-stage MF lesions. To better understand the functional role of SOCS1 in the genesis of MF, we used a genetically engineered mouse model emulating heterozygous SOCS1 loss in skin resident CD4+ T cells. In these mice an experimentally induced contact-allergic reaction was maintained for 20 weeks. Ten weeks after discontinuing contact-allergic challenges, only the skin with locally one-copy deletion of Socs1 in CD4+ T cells still showed high numbers of CD3+/CD4+ Socs1 k.o. cells in the dermis (p ＜ 0.0001) with prevalent Stat3 activation (p ＜0.001). And in one out of 9 mice, this had progressed to far more dramatic increases, including the thickened epidermis, and with an explosive growth of Socs1 k.o. T cells in circulation; indicative of cutaneous lymphoma. Hence, we show that Socs1 mono-allelic loss in CD4+ T cells locally in protractedly inflamed skin results in autonomous skin inflammation with features of early-stage MF.

In vivo modelling of cutaneous T-cell lymphoma: The role of SOCS1.

Introduction: Mycosis fungoides (MF), the most common type of Cutaneous T cell Lymphoma (CTCL), is characterized by an inflamed skin intermixed with proliferating malignant mature skin-homing CD4+ T cells. Detailed genomic analyses of MF skin biopsies revealed several candidate genes possibly involved in genesis of these tumors and/or potential targets for therapy. These studies showed, in addition to common loss of cell cycle regulator CDKN2A, activation of several oncogenic pathways, most prominently and consistently involving JAK/STAT signaling. SOCS1, an endogenous inhibitor of the JAK/STAT signaling pathway, was identified as a recurrently deleted gene in MF, already occurring in the earliest stages of the disease. Methods: To explore the mechanisms of MF, we create in vivo mouse models of autochthonous CTCLs and these genetically engineered mouse models (GEMMS) can also serve as valid experimental models for targeted therapy. We describe the impact of allelic deletion of Socs1 in CD4 T cells of the skin. To achieve this, we crossed inducible Cre-transgenic mice in the CD4 lineage with transgenic mice carrying floxed genes of Socs1. We first determined optimal conditions for Socs1 ablation with limited effects on circulating CD4 T-cells in blood. Next, we started time-course experiments mimicking sustained inflammation, typical in CTCL. FACS analysis of the blood was done every week. Skin biopsies were analyzed by immunocytochemical staining at the end of the experiment. Results: We found that the Socs1 knockout transgenic group had thicker epidermis of treated skin compared with the control group and had more CD3 and CD4 in the skin of the transgenic group compared to the control group. We also noted more activation of Stat3 by staining for P-Stat3 in Socs1 knockout compared to wt CD4+T cells in the skin. The results also indicated that single copy loss of Socs1 in combination with sustained inflammation is insufficient to start a phenotype resembling early stage mycosis fungoides within eight weeks in these mice. Conclusion: In sum, we developed and optimized an autochthonous murine model permitting selective knockout of Socs1 in skin infiltrating CD4 T-cells. This paves the way for more elaborate experiments to gain insight in the oncogenesis of CTCL.

Genetic Stability of Driver Alterations in Primary Cutaneous Diffuse Large B-Cell Lymphoma, Leg Type and Their Relapses: A Rationale for the Use of Molecular-Based Methods for More Effective Disease Monitoring.

Primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL-LT) is a rare, aggressive cutaneous lymphoma with a 5-year disease-specific survival of only ~55%. Despite high response rates to initial immune-polychemotherapy, most patients experience a disease relapse. The genetic evolution of primary and relapsed/refractory disease has only scarcely been studied in PCDLBCL-LT patients. Therefore, in this retrospective cohort study, 73 primary/pre-treatment and relapsed/refractory biopsies of 57 patients with PCDLBCL-LT were molecularly characterized with triple FISH and targeted next-generation sequencing for 52 B-cell-lymphoma-relevant genes, including paired analysis in 16 patients. In this cohort, 95% of patients harboured at least one of the three main driver alterations (mutations in MYD88/CD79B and/or CDKN2A-loss). In relapsed/refractory PCDLBCL-LT, these oncogenic aberrations were persistently present, demonstrating genetic stability over time. Novel alterations in relapsed disease affected mostly CDKN2A, MYC, and PIM1. Regarding survival, only MYC rearrangements and HIST1H1E mutations were statistically significantly associated with an inferior outcome. The stable presence of one or more of the three main driver alterations (mutated MYD88/CD79B and/or CDKN2A-loss) is promising for targeted therapies addressing these alterations and serves as a rationale for molecular-based disease monitoring, improving response evaluation and early identification and intervention of disease relapses in these poor-prognostic PCDLBCL-LT patients.

Cell-of-origin classification using the Hans and Lymph2Cx algorithms in primary cutaneous large B-cell lymphomas.

Primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL-LT) and primary cutaneous follicle center lymphoma with a diffuse population of large cells (PCFCL-LC) are both primary cutaneous B-cell lymphomas with large-cell morphology (CLBCL) but with different clinical characteristics and behavior. In systemic diffuse large B-cell lymphoma, not otherwise specified (DLBCL-NOS), gene-expression profiling (GEP) revealed two molecular subgroups based on their cell-of-origin (COO) with prognostic significance: the germinal center B-cell-like (GCB) subtype and the activated B-cell-like (ABC) subtype. This study investigated whether COO classification is a useful tool for classification of CLBCL. For this retrospective study, 51 patients with PCDLBCL-LT and 15 patients with PCFCL-LC were analyzed for their COO according to the immunohistochemistry-based Hans algorithm and the NanoString GEP-based Lymph2Cx algorithm. In PCFCL-LC, all cases (100%) classified as GCB by both Hans and Lymph2Cx. In contrast, COO classification in PCDLBCL-LT was heterogeneous. Using Hans, 75% of the PCDLBCL-LT patients classified as non-GCB and 25% as GCB, while Lymph2Cx classified only 18% as ABC, 43% as unclassified/intermediate, and 39% as GCB. These COO subgroups did not differ in the expression of BCL2 and IgM, mutations in MYD88 and/or CD79B, loss of CDKN2A, or survival. In conclusion, PCFCL-LC uniformly classified as GCB, while PCDLBCL-LT classified along the COO spectrum of DLBCL-NOS using the Hans and Lymph2Cx algorithms. In contrast to DLBCL-NOS, the clinical relevance of COO classification in CLBCL using these algorithms has limitations and cannot be used as an alternative for the current multiparameter approach in differentiation of PCDLBCL-LT and PCFCL-LC.

Genetic and epigenetic insights into cutaneous T-cell lymphoma.

Primary cutaneous T-cell lymphomas (CTCLs) constitute a heterogeneous group of non-Hodgkin T-cell lymphomas that present in the skin. In recent years, significant progress has been made in the understanding of the pathogenesis of CTCLs. Progress in CTCL classifications combined with technical advances, in particular next-generation sequencing, enabled a more detailed analysis of the genetic and epigenetic landscape and transcriptional changes in clearly defined diagnostic entities. These studies not only demonstrated extensive heterogeneity between different CTCL subtypes but also identified recurrent alterations that are highly characteristic for diagnostic subgroups of CTCLs. The identified alterations, in particular, involve epigenetic remodeling, cell cycle regulation, and the constitutive activation of targetable oncogenic pathways. In this respect, aberrant JAK-STAT signaling is a recurrent theme; however, it is not universal for all CTCLs and has seemingly different underlaying causes in different entities. A number of the mutated genes identified are potentially actionable targets for the development of novel therapeutic strategies. Moreover, these studies have produced an enormous amount of information that will be critically important for the further development of improved diagnostic and prognostic biomarkers that can assist in the clinical management of patients with CTCL. In the present review, the main findings of these studies in relation to their functional impact on the malignant transformation process are discussed for different subtypes of CTCLs.

Whole-genome profiling of primary cutaneous anaplastic large cell lymphoma.

Primary cutaneous anaplastic large cell lymphoma (pcALCL), a hematological neoplasm caused by skin-homing CD30+ malignant T cells, is part of the spectrum of primary cutaneous CD30+ lymphoproliferative disorders. To date, only a small number of molecular alterations have been described in pcALCL and, so far, no clear unifying theme that could explain the pathogenetic origin of the disease has emerged among patients. In order to clarify the pathogenetic basis of pcALCL, we performed high-resolution genetic profiling (genome/transcriptome) of this lymphoma (n=12) by using whole-genome sequencing, whole-exome sequencing and RNA sequencing. Our study, which uncovered novel genomic rearrangements, copy number alterations and small-scale mutations underlying this malignancy, revealed that the cell cycle, T-cell physiology regulation, transcription and signaling via the PI-3-K, MAPK and G-protein pathways are cellular processes commonly impacted by molecular alterations in patients with pcALCL. Recurrent events affecting cancer-associated genes included deletion of PRDM1 and TNFRSF14, gain of EZH2 and TNFRSF8, small-scale mutations in LRP1B, PDPK1 and PIK3R1 and rearrangements involving GPS2, LINC-PINT and TNK1. Consistent with the genomic data, transcriptome analysis uncovered upregulation of signal transduction routes associated with the PI-3-K, MAPK and G-protein pathways (e.g., ERK, phospholipase C, AKT). Our molecular findings suggest that inhibition of proliferation-promoting pathways altered in pcALCL (particularly PI-3-K/AKT signaling) should be explored as potential alternative therapy for patients with this lymphoma, especially, for cases that do not respond to first-line skin-directed therapies or with extracutaneous disease.

Deregulation of JAK2 signaling underlies primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma.

Primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma (pcAECyTCL) is a rare variant of cutaneous T-cell lymphoma with an aggressive clinical course and a very poor prognosis. Until now, neither a systematic characterization of genetic alterations driving pcAECyTCL has been performed, nor effective therapeutic regimes for patients have been defined. Here, we present the first highresolution genetic characterization of pcAECyTCL by using wholegenome and RNA sequencing. Our study provides a comprehensive description of genetic alterations (i.e., genomic rearrangements, copy number alterations and small-scale mutations) with pathogenic relevance in this lymphoma, including events that recurrently impact genes with important roles in the cell cycle, chromatin regulation and the JAKSTAT pathway. In particular, we show that mutually exclusive structural alterations involving JAK2 and SH2B3 predominantly underlie pcAECyTCL. In line with the genomic data, transcriptome analysis uncovered upregulation of the cell cycle, JAK2 signaling, NF-κB signaling and a high inflammatory response in this cancer. Functional studies confirmed oncogenicity of JAK2 fusions identified in pcAECyTCL and their sensitivity to JAK inhibitor treatment. Our findings strongly suggest that overactive JAK2 signaling is a central driver of pcAECyTCL, and consequently, patients with this neoplasm would likely benefit from therapy with JAK2 inhibitors such as Food and Drug Adminstration-approved ruxolitinib.

Cutaneous T cell lymphoma.

Primary cutaneous T cell lymphomas (CTCLs) are a heterogeneous group of lymphomas that present in the skin with no evidence of extracutaneous disease at the time of diagnosis. CTCL subtypes demonstrate a variety of clinical, histological, and molecular features, and can follow an indolent or a very aggressive course. The underlying pathogenetic mechanisms are not yet entirely understood. The pathophysiology of CTCL is complex and a single initiating factor has not yet been identified. Diagnosis is based on clinicopathological correlation and requires an interdisciplinary team. Treatment decision is made based on short-term and long-term goals. Therapy options comprise skin-directed therapies, such as topical steroids or phototherapy, and systemic therapies, such as monoclonal antibodies or chemotherapy. So far, the only curative treatment approach is allogeneic haematopoietic stem cell transplantation. Novel therapies, such as chimeric antigen receptor T cells, monoclonal antibodies or small molecules, are being investigated in clinical trials. Patients with CTCL have reduced quality of life and a lack of effective treatment options. Further research is needed to better identify the underlying mechanisms of CTCL development and course as well as to better tailor treatment strategies to individual patients.

Improved Sézary cell detection and novel insights into immunophenotypic and molecular heterogeneity in Sézary syndrome.

Sézary syndrome (SS) is an aggressive leukemic form of cutaneous T-cell lymphoma with neoplastic CD4+ T cells present in skin, lymph nodes, and blood. Despite advances in therapy, prognosis remains poor, with a 5-year overall survival of 30%. The immunophenotype of Sézary cells is diverse, which hampers efficient diagnosis, sensitive disease monitoring, and accurate assessment of treatment response. Comprehensive immunophenotypic profiling of Sézary cells with an in-depth analysis of maturation and functional subsets has not been performed thus far. We immunophenotypically profiled 24 patients with SS using standardized and sensitive EuroFlow-based multiparameter flow cytometry. We accurately identified and quantified Sézary cells in blood and performed an in-depth assessment of their phenotypic characteristics in comparison with their normal counterparts in the blood CD4+ T-cell compartment. We observed inter- and intrapatient heterogeneity and phenotypic changes over time. Sézary cells exhibited phenotypes corresponding with classical and nonclassical T helper subsets with different maturation phenotypes. We combined multiparameter flow cytometry analyses with fluorescence-activated cell sorting and performed RNA sequencing studies on purified subsets of malignant Sézary cells and normal CD4+ T cells of the same patients. We confirmed pure monoclonality in Sézary subsets, compared transcriptomes of phenotypically distinct Sézary subsets, and identified novel downregulated genes, most remarkably THEMIS and LAIR1, which discriminate Sézary cells from normal residual CD4+ T cells. Together, these findings further unravel the heterogeneity of Sézary cell subpopulations within and between patients. These new data will support improved blood staging and more accurate disease monitoring.

Cucurbitacin E and I target the JAK/STAT pathway and induce apoptosis in Sézary cells.

Cutaneous T-cell lymphomas and leukemias (CTCLs) are a heterogeneous group of extranodal non-Hodgkin's lymphomas. These are characterized by an accumulation of malignant CD4+ T-lymphocytes in the skin, lymph nodes, and peripheral blood. Novel treatment options are needed for patients who progress to advanced stage disease. Cucurbitacin I has previously shown promising results in Sézary syndrome (Sz). A plethora of cucurbitacins, however, have not yet been tested in CTCL. Herein, we investigated the effect of cucurbitacin E and I in two CTCL cell lines. We show that both cucurbitacins decrease viability and cause apoptosis in these cell lines, although HuT-78 was more affected than SeAx (IC50 of 17.38 versus 22.01 µM for cucurbitacin E and 13.36 versus 24.47 µM for cucurbitacin I). Moreover, both cucurbitacins decrease viability of primary cells of a Sz patient (56.46% for cucurbitacin E and 59.07% for cucurbitacin I). Furthermore, while JAK2 inhibition leads to decreased viability in SeAx cells (IC50 of 9.98 and 29.15 µM for AZD1480 and ruxolitinib respectively), both JAK1 and JAK3 do not. This suggests that JAK2 has a preferential role in promoting survival. Western blotting in SeAx cells revealed that both cucurbitacins inhibit STAT3 activation (P < 0.0001), while only cucurbitacin I inhibits STAT5 activation (P = 0.05). This suggests that STAT3 plays a preferential role in the mechanism of action of these cucurbitacins. Nevertheless, a role of STAT5 and JAK2 cannot be excluded and should be explored further. This knowledge could contribute to the development of effective therapies for CTCL and other malignancies involving dysfunction of the JAK/STAT pathway.

Whole-genome analysis uncovers recurrent IKZF1 inactivation and aberrant cell adhesion in blastic plasmacytoid dendritic cell neoplasm.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive hematological malignancy with a poorly understood pathobiology and no effective therapeutic options. Despite a few recurrent genetic defects (eg, single nucleotide changes, indels, large chromosomal aberrations) have been identified in BPDCN, none are disease-specific, and more importantly, none explain its genesis or clinical behavior. In this study, we performed the first high resolution whole-genome analysis of BPDCN with a special focus on structural genomic alterations by using whole-genome sequencing and RNA sequencing. Our study, the first to characterize the landscape of genomic rearrangements and copy number alterations of BPDCN at nucleotide-level resolution, revealed that IKZF1, a gene encoding a transcription factor required for the differentiation of plasmacytoid dendritic cell precursors, is focally inactivated through recurrent structural alterations in this neoplasm. In concordance with the genomic data, transcriptome analysis revealed that conserved IKZF1 target genes display a loss-of-IKZF1 expression pattern. Furthermore, up-regulation of cellular processes responsible for cell-cell and cell-ECM interactions, which is a hallmark of IKZF1 deficiency, was prominent in BPDCN. Our findings suggest that IKZF1 inactivation plays a central role in the pathobiology of the disease, and consequently, therapeutic approaches directed at reestablishing the function of this gene might be beneficial for patients.

Acquired N-Linked Glycosylation Motifs in B-Cell Receptors of Primary Cutaneous B-Cell Lymphoma and the Normal B-Cell Repertoire.

Primary cutaneous follicle center lymphoma (PCFCL) is a rare mature B-cell lymphoma with an unknown etiology. PCFCL resembles follicular lymphoma (FL) by cytomorphologic and microarchitectural criteria. FL B cells are selected for N-linked glycosylation motifs in their B-cell receptors (BCRs) that are acquired during continuous somatic hypermutation. The stimulation of mannosylated BCR by lectins on the tumor microenvironment is therefore a candidate driver in FL pathogenesis. We investigated whether the same mechanism could play a role in PCFCL pathogenesis. Full-length functional variable, diversity, and joining gene sequences of 18 PCFCL and 8 primary cutaneous diffuse large B-cell lymphoma, leg-type were identified by unbiased Anchoring Reverse Transcription of Immunoglobulin Sequences and Amplification by Nested PCR and BCR reconstruction from RNA sequencing data. Low BCR variation demonstrated negligible ongoing somatic hypermutation in PCFCL and primary cutaneous diffuse large B-cell lymphoma, leg-type, and indicated that the PCFCL microarchitecture does not act as a functional germinal center. Similar to FL but in contrast to primary cutaneous diffuse large B-cell lymphoma, leg-type, BCR genes of 15 PCFCLs (83%) had acquired N-linked glycosylation motifs. These motifs were located at the BCR positions converted to N-linked glycosylation motifs in normal B-cell repertoires with low prevalence but mostly at different positions than those found in FL. The cutaneous localization of PCFCL might suggest a role for lectins from commensal skin bacteria in PCFCL lymphomagenesis.

Array-based CGH of primary cutaneous CD8+ aggressive EPIDERMO-tropic cytotoxic T-cell lymphoma.

Primary cutaneous CD8+ aggressive epidermotropic cytotoxic T-cell lymphoma (pcAECyTCL) is a rare provisionally categorized cutaneous lymphoma characterized by an aggressive course. Its pathogenesis and molecular mechanisms are still unknown, and only two individual cases have so far been molecularly characterized. The aim of this study was to define the pattern of numerical chromosomal alterations in tumor samples taken from 20 patients with pcAECyTCL at the time of diagnosis by means of array-comparative genomic hybridization (a-CGH). a-CGH detected numerous genomic aberrations in all the patients and, putting these together as a whole, they affected all the chromosomes. However, no specific profile of recurrent copy number alterations (CNAs) was found. Most of the gains involved regions previously described in other aggressive cutaneous lymphomas such as 7q, 8q24.3, and 17q, whereas the most significant CNA was the loss of 9p21.3 (CDKN2A-CDKN2B), which has already been found in a variety of malignant tumors and is associated with aggressive cutaneous T-cell lymphomas. In brief, CGH analysis revealed a large number of CNAs with only few recurring regions that probably do not represent driving events. The genomic instability found in this aggressive variant of cutaneous lymphoma may therefore be a secondary event but, at the time of the diagnosis of pcAECyTCL, the genomic integrity of tumor cells is already compromised.

Genomic analysis reveals recurrent deletion of JAK-STAT signaling inhibitors HNRNPK and SOCS1 in mycosis fungoides.

Mycosis fungoides (MF) is the most common cutaneous T-cell lymphoma (CTCL). Causative genetic alterations in MF are unknown. The low recurrence of pathogenic small-scale mutations (ie, nucleotide substitutions, indels) in the disease, calls for the study of additional aspects of MF genetics. Here, we investigated structural genomic alterations in tumor-stage MF by integrating whole-genome sequencing and RNA-sequencing. Multiple genes with roles in cell physiology (n = 113) and metabolism (n = 92) were found to be impacted by genomic rearrangements, including 47 genes currently implicated in cancer. Fusion transcripts involving genes of interest such as DOT1L, KDM6A, LIFR, TP53, and TP63 were also observed. Additionally, we identified recurrent deletions of genes involved in cell cycle control, chromatin regulation, the JAK-STAT pathway, and the PI-3-K pathway. Remarkably, many of these deletions result from genomic rearrangements. Deletion of tumor suppressors HNRNPK and SOCS1 were the most frequent genetic alterations in MF after deletion of CDKN2A. Notably, SOCS1 deletion could be detected in early-stage MF. In agreement with the observed genomic alterations, transcriptome analysis revealed up-regulation of the cell cycle, JAK-STAT, PI-3-K and developmental pathways. Our results position inactivation of HNRNPK and SOCS1 as potential driver events in MF development.

Pathogenesis of Skin Carcinomas and a Stem Cell as Focal Origin.

UV radiation in sunlight has long been recognized as the main exogenous cause of skin carcinomas. We present a brief historical perspective on the progress in understanding the pathogenesis of skin carcinomas, and recent advances. Sun-exposed skin carries numerous UV-related mutations, and skin carcinomas rank among the tumors with the highest mutational loads. In this multitude of mutations only a few are crucial in driving the tumor. Some are known from hereditary (skin) cancer syndromes and other recurrent ones have been validated in transgenic mice. Considering the continuous renewal of the epidermis, the question arises whether the lifelong residing stem cells are the main targets in skin carcinogenesis, a multistep process that would require ample time to evolve. Therefore, classic quiescent stem cells have been studied as potential tumor-initiating cells, as well as more recently discovered actively dividing stem cells (either Lgr5+ or Lgr6+). Interesting differences have emerged between experimental UV and two-stage chemical carcinogenesis, e.g., the latter appears to originate from follicular stem cells, in contrast to the former.

Molecular advances in cutaneous T-cell lymphoma.

Cutaneous T-cell lymphoma (CTCL) is a group of malignancies derived from skin-homing T cells. Mycosis fungoides (MF) and Sézary syndrome (SS) are the most common CTCL variants. In recent years, the genetic landscape of SS/MF has been characterized using genome-wide nextgeneration sequencing approaches. These studies have revealed that genes subjected to oncogenic mutations take part in cell cycle regulation, chromatin modification, Janus kinase (JAK)-signal transducer and activator of transcription protein (STAT) signaling, T-cell receptor (TCR)/ nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, and microtubule associated protein kinase (MAPK) signaling, which suggests that deregulation of these cellular processes underlies lymphomagenesis. These studies provide the groundwork for functional and clinical studies that will lead to better risk assessment and more effective therapeutic approach in CTCL patients.