Phase II study in children and adults under 40 years with newly diagnosed Langerhans cell histiocytosis: protocol for an LCH-19-MSMFB clinical trial in Japan.

INTRODUCTION: Although the prognosis of Langerhans cell histiocytosis (LCH) is excellent, the high recurrence rate and permanent consequences, such as central diabetes insipidus and LCH-associated neurodegenerative diseases, remain to be resolved. Based on previous reports that patients with high-risk multisystem LCH show elevated levels of inflammatory molecules, we hypothesised that dexamethasone would more effectively suppress LCH-associated inflammation, especially in the central nervous system (CNS). We further hypothesised that intrathecal chemotherapy would effectively reduce CNS complications. We administer zoledronate to patients with multifocal bone LCH based on an efficacy report from a small case series. METHODS AND ANALYSIS: This phase II study (labelled the LCH-19-MSMFB study) is designed to evaluate the significance of introducing dexamethasone and intrathecal chemotherapy for multisystem disease and zoledronate for multifocal bone disease in previously untreated, newly diagnosed children, adolescents (under 20 years) and adults under 40 years. The primary endpoint is the 3-year event-free survival rate by risk group of under 20 years and the 3-year event-free survival rate of 20 years and over. ETHICS AND DISSEMINATION: This study was approved by the Central Review Board of the National Hospital Organisation Nagoya Medical Centre (Nagoya, Japan) on 21 January 2022 and was registered in the Japan Registry of Clinical Trials (https://jrct.niph.go.jp/en-latest-detail/jRCTs041210027). Written informed consent will be obtained from all patients and/or their guardians. TRIAL REGISTRATION NUMBER: jRCTs041210027.

Genome-wide analysis of DNA methylation in pseudomyxoma peritonei originated from appendiceal neoplasms.

INTRODUCTION: Pseudomyxoma peritonei (PMP) is a disease characterized by progressive accumulation of intraperitoneal mucinous ascites produced by neoplasms in the abdominal cavity. Since the prognosis of patients with PMP remain unsatisfactory, the development of effective therapeutic drug(s) is a matter of pressing concern. Genetic analyses of PMP have clarified the frequent activation of GNAS and/or KRAS. However, the involvement of global epigenetic alterations in PMPs has not been reported. METHODS: To clarify the genetic background of the 15 PMP tumors, we performed genetic analysis using AmpliSeq Cancer HotSpot Panel v2. We further investigated global DNA methylation in the 15 tumors and eight non-cancerous colonic epithelial cells using Methylation EPIC array BeadChip (Infinium 850k) containing a total of 865,918 probes. RESULTS: This is the first report of comprehensive DNA methylation profiles of PMPs in the world. We clarified that the 15 PMPs could be classified into at least two epigenotypes, unique methylation epigenotype (UME) and normal-like methylation epigenotype (NLME), and that genes associated with neuronal development and synaptic signaling may be involved in the development of PMPs. In addition, we identified a set of hypermethylation marker genes such as HOXD1 and TSPYL5 in the 15 PMPs. CONCLUSIONS: These findings may help the understanding of the molecular mechanism(s) of PMP and contribute to the development of therapeutic strategies for this life-threatening disease.

Hyperactive Natural Killer cells in Rag2 knockout mice inhibit the development of acute myeloid leukemia.

Immunotherapy has attracted considerable attention as a therapeutic strategy for cancers including acute myeloid leukemia (AML). In this study, we found that the development of several aggressive subtypes of AML is slower in Rag2-/- mice despite the lack of B and T lymphocytes, even compared to the immunologically normal C57BL/6 mice. Furthermore, an orally active p53-activating drug shows stronger antileukemia effect on AML in Rag2-/- mice than C57BL/6 mice. Intriguingly, Natural Killer (NK) cells in Rag2-/- mice are increased in number, highly express activation markers, and show increased cytotoxicity to leukemia cells in a coculture assay. B2m depletion that triggers missing-self recognition of NK cells impairs the growth of AML cells in vivo. In contrast, NK cell depletion accelerates AML progression in Rag2-/- mice. Interestingly, immunogenicity of AML keeps changing during tumor evolution, showing a trend that the aggressive AMLs generate through serial transplantations are susceptible to NK cell-mediated tumor suppression in Rag2-/- mice. Thus, we show the critical role of NK cells in suppressing the development of certain subtypes of AML using Rag2-/- mice, which lack functional lymphocytes but have hyperactive NK cells.

Reduced DEFA5 Expression and STAT3 Activation Underlie the Submucosal Invasion of Early Gastric Cancers.

INTRODUCTION: Submucosal invasion is a core hallmark of early gastric cancer (EGC) with poor prognosis. However, the molecular mechanism of the progression from intramucosal gastric cancer (IMGC) to early submucosal-invasive gastric cancer (SMGC) is not fully understood. The objective of this study was to identify genes and pathways involved in the submucosal invasion in EGC using comprehensive gene expression analysis. METHODS: Gene expression profiling was performed for eight cases of IMGC and eight cases of early SMGC with submucosal invasion ≥500 µm. To validate the findings of gene expression analysis and to examine the gene expression pattern in tissues, immunohistochemical (IHC) staining was performed for 50 cases of IMGC and SMGC each. RESULTS: Gene expression analysis demonstrated that the expression levels of small intestine-specific genes were significantly decreased in SMGC. Among them, defensin alpha 5 (DEFA5) was the most downregulated gene in SMGC, which was further validated in SMGC tissues by IHC staining. Gene set enrichment analysis showed a strong association between SMGC, the JAK-STAT signaling pathway, and the upregulation of STAT3-activating cytokines. The expression of phosphorylated STAT3 was significant in the nucleus of tumor cells in SMGC tissues but not in areas expressing DEFA5. CONCLUSION: The results of this study strongly suggest that the downregulation of DEFA5 and the activation of STAT3 play a significant role in the submucosal invasion of EGC.

Case report: Irinotecan-induced interstitial lung disease in an advanced colorectal cancer patient resurfacing decades after allogeneic bone marrow transplantation for aplastic anemia; a case report and narrative review of literature.

Two mechanisms of drug-induced interstitial lung disease (DILD) have been reported: 1) direct injury of lung epithelial cells and/or endothelial cells in lung capillaries by the drug and/or its metabolites and 2) hypersensitivity reactions. In both mechanisms, immune reactions such as cytokine and T cell activation are involved in DILD. While past and present lung diseases and accumulative lung damage due to smoking and radiation are risk factors for DILD, the association between the immune status of the host and DILD is not well known. Herein, we report a case of advanced colorectal cancer with a history of allogeneic bone marrow transplantation for aplastic anemia more than 30 years prior, in which DILD occurred early after irinotecan-containing chemotherapy. Bone marrow transplantation might be a potential risk factor for DILD.

Clinical and prognostic features of Langerhans cell histiocytosis in adults.

Langerhans cell histiocytosis (LCH) is a rare disease characterized by clonal expansion of CD1a+ CD207+ myeloid dendritic cells. The features of LCH are mainly described in children and remain poorly defined in adults; therefore, we conducted a nationwide survey to collect clinical data from 148 adult patients with LCH. The median age at diagnosis was 46.5 (range: 20-87) years with male predominance (60.8%). Among the 86 patients with detailed treatment information, 40 (46.5%) had single system LCH, whereas 46 (53.5%) had multisystem LCH. Moreover, 19 patients (22.1%) had an additional malignancy. BRAF V600E in plasma cell-free DNA was associated with a low overall survival (OS) rate and the risk of the pituitary gland and central nervous system involvement. At a median follow-up of 55 months from diagnosis, six patients (7.0%) had died, and the four patients with LCH-related death did not respond to initial chemotherapy. The OS probability at 5 years post-diagnosis was 90.6% (95% confidence interval: 79.8-95.8). Multivariate analysis showed that patients aged ≥60 years at diagnosis had a relatively poor prognosis. The probability of event-free survival at 5 years was 52.1% (95% confidence interval: 36.6-65.5), with 57 patients requiring chemotherapy. In this study, we first revealed the high rate of relapse after chemotherapy and mortality of poor responders in adults as well as children. Therefore, prospective therapeutic studies of adults with LCH using targeted therapies are needed to improve outcomes in adults with LCH.

Controlling genetic heterogeneity in gene-edited hematopoietic stem cells by single-cell expansion.

Gene editing using engineered nucleases frequently produces unintended genetic lesions in hematopoietic stem cells (HSCs). Gene-edited HSC cultures thus contain heterogeneous populations, the majority of which either do not carry the desired edit or harbor unwanted mutations. In consequence, transplanting edited HSCs carries the risks of suboptimal efficiency and of unwanted mutations in the graft. Here, we present an approach for expanding gene-edited HSCs at clonal density, allowing for genetic profiling of individual clones before transplantation. We achieved this by developing a defined, polymer-based expansion system and identifying long-term expanding clones within the CD201+CD150+CD48-c-Kit+Sca-1+Lin- population of precultured HSCs. Using the Prkdcscid immunodeficiency model, we demonstrate that we can expand and profile edited HSC clones to check for desired and unintended modifications, including large deletions. Transplantation of Prkdc-corrected HSCs rescued the immunodeficient phenotype. Our ex vivo manipulation platform establishes a paradigm to control genetic heterogeneity in HSC gene editing and therapy.

Ultrasonographic Evaluation of Intravenous Lobular Capillary Hemangioma in the Cephalic Vein.

A 60-year-old man presented with focal swelling on the dorsal surface of the left wrist. The sonographic exam revealed the presence of a smoothly rounded hypoechoic mass, with internal blood flow in the lumen of the vein. The histopathological findings led to the diagnosis of intravenous lobular capillary hemangioma (LCH). Here, we reported an intravenous LCH arising from the cephalic vein on the dorsal surface of the left wrist hand and discussed the related ultrasonographic findings.

Diffuse Large B-cell Lymphoma with Adrenal Involvement Presenting as Other Iatrogenic Immunodeficiency-associated Lymphoproliferative Disorders: Sustained Remission with Methotrexate Termination Alone in Two Cases.

Methotrexate-associated lymphoproliferative disorders (MTX-LPDs) with diffuse large B-cell lymphoma (DLBCL) pathology present with high rates of spontaneous regression after methotrexate (MTX) termination, especially in Epstein-Barr virus-encoded RNA (EBER)-positive cases. DLBCL with adrenal involvement is known for an extremely dismal prognosis. However, the prognosis of adrenal DLBCL in the context of MTX-LPD is unknown. We herein report two EBER-positive adrenal DLBCL MTX-LPD patients who achieved long-term remissions of 22 and 40 months with MTX termination alone. Both patients are doing well with no relapse at the time of reporting. Unlike adrenal DLBCL in general, adrenal involvement may not be a poor prognostic factor when restricted to DLBCL MTX-LPDs.

Identification of odontogenic ameloblast associated as a novel target gene of the Wnt/ß-catenin signaling pathway.

The Wnt/ß-catenin signaling pathway plays a key role in development and carcinogenesis. Although some target genes of this signaling have been identified in various tissues and neoplasms, the comprehensive understanding of the target genes and their roles in the development of human cancer, including hepatoma and colorectal cancer remain to be fully elucidated. In this study, we searched for genes regulated by the Wnt signaling in liver cancer using HuH-7 hepatoma cells. A comparison of the expression profiles between cells expressing an active form of mutant ß-catenin and cells expressing enhanced green fluorescent protein (EGFP) identified seven genes upregulated by the mutant ß-catenin gene (CTNNB1). Among the seven genes, we focused in this study on ODAM, odontogenic, ameloblast associated, as a novel target gene. Interestingly, its expression was frequently upregulated in hepatocellular carcinoma, colorectal adenocarcinoma, and hepatoblastoma. We additionally identified a distant enhancer region that was associated with the ß-catenin/TCF7L2 complex. Further analyses revealed that ODAM plays an important role in the regulation of the cell cycle, DNA synthesis, and cell proliferation. These data may be useful for clarification of the main molecular mechanism(s) underlying these cancers.

Case report: Common clonal origin of concurrent langerhans cell histiocytosis and acute myeloid leukemia.

Langerhans cell histiocytosis (LCH) and acute myeloid leukemia (AML) are distinct entities of blood neoplasms, and the exact developmental origin of both neoplasms are considered be heterogenous among patients. However, reports of concurrent LCH and AML are rare. Herein we report a novel case of concurrent LCH and AML which shared same the driver mutations, strongly suggesting a common clonal origin.An 84-year-old female presented with cervical lymphadenopathy and pruritic skin rash on the face and scalp. Laboratory tests revealed pancytopenia with 13% of blasts, elevated LDH and liver enzymes, in addition to generalised lymphadenopathy and splenomegaly by computed tomography. Bone marrow specimens showed massive infiltration of MPO-positive myeloblasts, whereas S-100 and CD1a positive atypical dendritic cell-like cells accounted for 10% of the atypical cells on bone marrow pathology, suggesting a mixture of LCH and AML. A biopsy specimen from a cervical lymph node and the skin demonstrated the accumulation of atypical cells which were positive for S-100 and CD1a. LCH was found in lymph nodes, skin and bone marrow; AML was found in peripheral blood and bone marrow (AML was predominant compared with LCH in the bone marrow). Next generation sequencing revealed four somatic driver mutations (NRAS-G13D, IDH2-R140Q, and DNMT3A-F640fs/-I715fs), equally shared by both the lymph node and bone marrow, suggesting a common clonal origin for the concurrent LCH and AML. Prednisolone and vinblastine were initially given with partial response in LCH; peripheral blood blasts also disappeared for 3 months. Salvage chemotherapy with low dose cytarabine and aclarubicin were given for relapse, with partial response in both LCH and AML. She died from pneumonia and septicemia on day 384. Our case demonstrates a common cell of origin for LCH and AML with a common genetic mutation, providing evidence to support the proposal to classify histiocytosis, including LCH, as a myeloid/myeloproliferative malignancy.

Intratumoral oncolytic herpes virus G47∆ for residual or recurrent glioblastoma: a phase 2 trial.

This investigator-initiated, phase 2, single-arm trial primarily assessed the efficacy of G47∆, a triple-mutated, third-generation oncolytic herpes simplex virus type 1, in 19 adult patients with residual or recurrent, supratentorial glioblastoma after radiation therapy and temozolomide (UMIN-CTR Clinical Trial Registry UMIN000015995). G47Δ was administered intratumorally and repeatedly for up to six doses. The primary endpoint of 1-yr survival rate after G47∆ initiation was 84.2% (95% confidence interval, 60.4-96.6; 16 of 19). The prespecified endpoint was met and the trial was terminated early. Regarding secondary endpoints, the median overall survival was 20.2 (16.8-23.6) months after G47∆ initiation and 28.8 (20.1-37.5) months from the initial surgery. The most common G47∆-related adverse event was fever (17 of 19) followed by vomiting, nausea, lymphocytopenia and leukopenia. On magnetic resonance imaging, enlargement of and contrast-enhancement clearing within the target lesion repeatedly occurred after each G47∆ administration, which was characteristic to this therapy. Thus, the best overall response in 2 yr was partial response in one patient and stable disease in 18 patients. Biopsies revealed increasing numbers of tumor-infiltrating CD4+/CD8+ lymphocytes and persistent low numbers of Foxp3+ cells. This study showed a survival benefit and good safety profile, which led to the approval of G47∆ as the first oncolytic virus product in Japan.

Altered mucosal immunity in HIV-positive colon adenoma: decreased CD4+ T cell infiltration is correlated with nadir but not current CD4+ T cell blood counts.

BACKGROUND: People living with HIV (PLWH) face greater risks of developing non-AIDS-defining cancers (NADCs) than the general population; however, the underlying mechanisms remain elusive. The tumor microenvironment plays a significant role in the carcinogenesis of colorectal cancer (CRC), an NADC. We studied this carcinogenesis in PLWH by determining inflammatory phenotypes and assessing PD-1/PD-L1 expression in premalignant CRC stages of colon adenomas in HIV-positive and HIV-negative patients. METHODS: We obtained polyp specimens from 22 HIV-positive and 61 HIV-negative participants treated with colonoscopy and polyp excision. We analyzed adenomas from 33 HIV-positive and 99 HIV-negative patients by immunohistochemistry using anti-CD4, anti-CD8, anti-FoxP3, and anti-CD163 antibodies. Additionally, we analyzed the expression levels of immune checkpoint proteins. We also evaluated the correlation between cell infiltration and blood cell counts. RESULTS: HIV-positive participants had fewer infiltrating CD4+ T cells than HIV-negative participants (p = 0.0016). However, no statistical differences were observed in infiltrating CD8+ and FoxP3+ T cells and CD163+ macrophages. Moreover, epithelial cells did not express PD-1 or PD-L1. Notably, CD4+ T cell infiltration correlated with nadir blood CD4+ T cell counts (p <  0.05) but not with current blood CD4+ T cell counts. CONCLUSION: Immune surveillance dysfunction owing to decreased CD4+ T cell infiltration in colon adenomas might be involved in colon carcinogenesis in HIV-positive individuals. Collectively, since the nadir blood CD4+ T cell count is strongly correlated with CD4+ T cell infiltration, it could facilitate efficient follow-up and enable treatment strategies for HIV-positive patients with colon adenomas.

MDS cells impair osteolineage differentiation of MSCs via extracellular vesicles to suppress normal hematopoiesis.

Myelodysplastic syndrome (MDS) is a clonal disorder of hematopoietic stem cells (HSCs), characterized by ineffective hematopoiesis and frequent progression to leukemia. It has long remained unresolved how MDS cells, which are less proliferative, inhibit normal hematopoiesis and eventually dominate the bone marrow space. Despite several studies implicating mesenchymal stromal or stem cells (MSCs), a principal component of the HSC niche, in the inhibition of normal hematopoiesis, the molecular mechanisms underlying this process remain unclear. Here, we demonstrate that both human and mouse MDS cells perturb bone metabolism by suppressing the osteolineage differentiation of MSCs, which impairs the ability of MSCs to support normal HSCs. Enforced MSC differentiation rescues the suppressed normal hematopoiesis in both in vivo and in vitro MDS models. Intriguingly, the suppression effect is reversible and mediated by extracellular vesicles (EVs) derived from MDS cells. These findings shed light on the novel MDS EV-MSC axis in ineffective hematopoiesis.

Hodgkin Lymphoma on Hemodialysis Successfully Treated with Extended Courses of Brentuximab Vedotin.

Chemotherapy for hemodialysis (HD) patients is a challenging situation because HD patients are generally frail, and the pharmacokinetics and pharmacodynamics of most chemotherapeutics in HD patients are unknown. We report a classical Hodgkin lymphoma (cHL) patient successfully treated with 34 courses of brentuximab vedotin (BV) monotherapy, of which 30 courses were carried out during HD. Although grade 2 peripheral sensory neuropathy and one occasion of febrile neutropenia were observed, treatment was well-tolerated overall and effective. This is the first report of successful BV administration in a cHL patient on HD, and also the first to report efficacy and safety of extended courses of BV in an HD patient. Treatment options for cHL in the HD patient are limited, and extended courses of BV monotherapy may be an optimal treatment approach for some patients.

CHIP-associated mutant ASXL1 in blood cells promotes solid tumor progression.

Clonal hematopoiesis of indeterminate potential (CHIP) is an age-associated phenomenon characterized by clonal expansion of blood cells harboring somatic mutations in hematopoietic genes, including DNMT3A, TET2, and ASXL1. Clinical evidence suggests that CHIP is highly prevalent and associated with poor prognosis in solid-tumor patients. However, whether blood cells with CHIP mutations play a causal role in promoting the development of solid tumors remained unclear. Using conditional knock-in mice that express CHIP-associated mutant Asxl1 (Asxl1-MT), we showed that expression of Asxl1-MT in T cells, but not in myeloid cells, promoted solid-tumor progression in syngeneic transplantation models. We also demonstrated that Asxl1-MT-expressing blood cells accelerated the development of spontaneous mammary tumors induced by MMTV-PyMT. Intratumor analysis of the mammary tumors revealed the reduced T-cell infiltration at tumor sites and programmed death receptor-1 (PD-1) upregulation in CD8+ T cells in MMTV-PyMT/Asxl1-MT mice. In addition, we found that Asxl1-MT induced T-cell dysregulation, including aberrant intrathymic T-cell development, decreased CD4/CD8 ratio, and naïve-memory imbalance in peripheral T cells. These results indicate that Asxl1-MT perturbs T-cell development and function, which contributes to creating a protumor microenvironment for solid tumors. Thus, our findings raise the possibility that ASXL1-mutated blood cells exacerbate solid-tumor progression in ASXL1-CHIP carriers.

Dual-antigen targeted iPSC-derived chimeric antigen receptor-T cell therapy for refractory lymphoma.

We generated dual-antigen receptor (DR) T cells from induced pluripotent stem cells (iPSCs) to mitigate tumor antigen escape. These cells were engineered to express a chimeric antigen receptor (CAR) for the antigen cell surface latent membrane protein 1 (LMP1; LMP1-CAR) and a T cell receptor directed to cell surface latent membrane protein 2 (LMP2), in association with human leucocyte antigen A24, to treat therapy-refractory Epstein-Barr virus-associated lymphomas. We introduced LMP1-CAR into iPSCs derived from LMP2-specific cytotoxic T lymphocytes (CTLs) to generate rejuvenated CTLs (rejTs) active against LMP1 and LMP2, or DRrejTs. All DRrejT-treated mice survived >100 days. Furthermore, DRrejTs rejected follow-up inocula of lymphoma cells, demonstrating that DRrejTs persisted long-term. We also demonstrated that DRrejTs targeting CD19 and LMP2 antigens exhibited a robust tumor suppressive effect and conferred a clear survival advantage. Co-operative antitumor effect and in vivo persistence, with unlimited availability of DRrejT therapy, will provide powerful and sustainable T cell immunotherapy.

Retrospective analyses of other iatrogenic immunodeficiency-associated lymphoproliferative disorders in patients with rheumatic diseases.

Other iatrogenic immunodeficiency-associated lymphoproliferative disorders (OIIA-LPDs) occur in patients receiving immunosuppressive drugs for autoimmune diseases; however, their clinicopathological and genetic features remain unknown. In the present study, we analysed 67 patients with OIIA-LPDs, including 36 with diffuse large B-cell lymphoma (DLBCL)-type and 19 with Hodgkin lymphoma (HL)-type. After discontinuation of immunosuppressive drugs, regression without relapse was achieved in 22 of 58 patients. Spontaneous regression was associated with Epstein-Barr virus positivity in DLBCL-type (P = 0·013). The 2-year overall survival and progression-free survival (PFS) at a median follow-up of 32·4 months were 92·7% and 72·1% respectively. Furthermore, a significant difference in the 2-year PFS was seen between patients with DLBCL-type and HL-type OIIA-LPDs (81·0% vs. 40·9% respectively, P = 0·021). In targeted sequencing of 47 genes in tumour-derived DNA from 20 DLBCL-type OIIA-LPD samples, histone-lysine N-methyltransferase 2D (KMT2D; eight, 40%) and tumour necrosis factor receptor superfamily member 14 (TNFRSF14; six, 30%) were the most frequently mutated genes. TNF alpha-induced protein 3 (TNFAIP3) mutations were present in four patients (20%) with DLBCL-type OIIA-LPD. Cases with DLBCL-type OIIA-LPD harbouring TNFAIP3 mutations had shorter PFS and required early initiation of first chemotherapy. There were no significant factors for spontaneous regression or response rates according to the presence of mutations. Overall, OIIA-LPDs, especially DLBCL-types, showed favourable prognoses.

Systematic Review of Spinal Lymphomatoid Granulomatosis Cases.

Lymphomatoid granulomatosis (LYG) is a rare Epstein-Barr virus-associated systemic angiocentric and angiodestructive lymphoproliferative disorder. It commonly involves the lungs and can also affect the skin, liver, kidney, and central nervous system. It can rarely occur in the spine, however, the details are unclear. We performed a systematic review of published cases (including our 1 case) of spinal LYG. We performed a systematic search of studies in English on spinal LYG, focusing on its clinical features, imaging, and treatments, according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines on the PubMed database. We identified 14 patients from the literature. We also found 1 case of isolated cervical LYG (grade 3) who was treated with steroid and radiation therapy for the spinal lesion after pathologic diagnosis. We performed a pooled analysis of these 15 cases. The mean age was 43.4 years, and 13 of the 15 patients were male. Brain lesions were present in 11 of 12 intramedullary spinal lesions, and only 1 was an isolated spinal LYG case. Regarding the diagnostic methods, 1 case was not described. Of the 14 cases described, 12 patients underwent biopsies (7 brain, 4 lung, and 1 spinal cord lesion) and 2 underwent surgical removal for an extramedullary lesion. In the overall prognosis from a mean follow-up period of 21.6 months, 4 patients died despite several treatments. Spinal LYG, particularly isolated spinal LYG, is rare. Thus further accumulation of cases may be necessary to better understand its characteristics.