Intratumoral Canine Distemper Virus Infection Inhibits Tumor Growth by Modulation of the Tumor Microenvironment in a Murine Xenograft Model of Canine Histiocytic Sarcoma.

Histiocytic sarcomas refer to highly aggressive tumors with a poor prognosis that respond poorly to conventional treatment approaches. Oncolytic viruses, which have gained significant traction as a cancer therapy in recent decades, represent a promising option for treating histiocytic sarcomas through their replication and/or by modulating the tumor microenvironment. The live attenuated canine distemper virus (CDV) vaccine strain Onderstepoort represents an attractive candidate for oncolytic viral therapy. In the present study, oncolytic virotherapy with CDV was used to investigate the impact of this virus infection on tumor cell growth through direct oncolytic effects or by virus-mediated modulation of the tumor microenvironment with special emphasis on angiogenesis, expression of selected MMPs and TIMP-1 and tumor-associated macrophages in a murine xenograft model of canine histiocytic sarcoma. Treatment of mice with xenotransplanted canine histiocytic sarcomas using CDV induced overt retardation in tumor progression accompanied by necrosis of neoplastic cells, increased numbers of intratumoral macrophages, reduced angiogenesis and modulation of the expression of MMPs and TIMP-1. The present data suggest that CDV inhibits tumor growth in a multifactorial way, including direct cell lysis and reduction of angiogenesis and modulation of MMPs and their inhibitor TIMP-1, providing further support for the concept of its role in oncolytic therapies.

Histiocytic and dendritic cell neoplasms: Reappraisal of a Japanese series based on t(14;18) and neoplastic PD-L1 expression.

Histiocytic and dendritic cell (H/DC) neoplasms are heterogeneous, originating from myeloid- or stromal-derived cells. Multiple reports describe the cross-lineage transdifferentiation of neoplastic B cells into H/DC neoplasms. Most such cases are from Western countries, and rarely from Japan or East Asia. Here we report 17 cases of H/DC neoplasms in Japanese patients, with analysis of t(14;18) by fluorescence in situ hybridization, and of neoplastic programmed death-ligand 1 (PD-L1) expression by immunostaining (clones SP142, E1J2J, and 28-8). These 17 cases were diagnosed according to the 2017 World Health Organization (WHO) classification, and included two histiocytic sarcomas (HS), two interdigitating cell (IDC) sarcomas, one Langerhans cell sarcoma, two dendritic cell sarcomas, and 10 follicular dendritic cell (FDC) sarcomas. No case had any past history of follicular lymphoma (FL). Two cases of HS and one IDC sarcoma, all of which were myeloid-driven, were found to exhibit t(14;18). In the latter case, at 30 months after IDC sarcoma diagnosis, FL development was detected. Three (30%) FDC sarcoma cases exhibited neoplastic PD-L1 expression with all the three PD-L1 antibody clones. This is the first report of t(14;18) and neoplastic PD-L1 expression on H/DC neoplasms among Japanese patients, each of which appeared to be associated with HS and FDC sarcoma, respectively.

Mesenchymal to epithelial transition driven by canine distemper virus infection of canine histiocytic sarcoma cells contributes to a reduced cell motility in vitro.

Sarcomas especially of histiocytic origin often possess a poor prognosis and response to conventional therapies. Interestingly, tumours undergoing mesenchymal to epithelial transition (MET) are often associated with a favourable clinical outcome. This process is characterized by an increased expression of epithelial markers leading to a decreased invasion and metastatic rate. Based on the failure of conventional therapies, viral oncolysis might represent a promising alternative with canine distemper virus (CDV) as a possible candidate. This study hypothesizes that a CDV infection of canine histiocytic sarcoma cells (DH82 cells) triggers the MET process leading to a decreased cellular motility. Immunofluorescence and immunoblotting were used to investigate the expression of epithelial and mesenchymal markers followed by scratch assay and an invasion assay as functional confirmation. Furthermore, microarray data were analysed for genes associated with the MET process, invasion and angiogenesis. CDV-infected cells exhibited an increased expression of epithelial markers such as E-cadherin and cytokeratin 8 compared to controls, indicating a MET process. This was accompanied by a reduced cell motility and invasiveness. Summarized, these results suggest that CDV infection of DH82 cells triggers the MET process by an increased expression of epithelial markers resulting in a decreased cell motility in vitro.

Cancer-testis antigens in canine histiocytic sarcoma and other malignancies.

Cancer-testis antigens (CTAs) are a category of self proteins aberrantly expressed in diverse malignancies, mostly solid tumours, due to epigenetic de-repression. Normally expressed only in fetal or gametogenic tissues, CTAs are tantalizing immunotherapy targets, since autoimmunity risks appear minimal. Few prevalent CTAs have been identified in human hematologic cancers, and just two in their veterinary counterparts. We sought to discover new CTAs in canine hematologic cancers such as histiocytic sarcoma (HS) and lymphoma to foster immunotherapy development. To accomplish this, the ligandome binding the dog leukocyte antigen (DLA)-88*508:01 class I allele overexpressed in an HS line was searched by mass spectrometry to identify possible CTA-derived peptides, which could serve as CD8+ T-cell epitopes. Twenty-two peptides mapped to 5 human CTAs and 12 additional proteins with CTA characteristics. Expression of five promising candidates was then evaluated in tumour and normal tissue by quantitative and end-point RT-PCR. The ortholog of an established CTA, IGF2BP3, had unexpectedly high expression in peripheral blood mononuclear cells (PBMCs). Four other testis-enhanced proteins were also assessed. AKR1E2, SPECC1 and TPX2 were expressed variably in HS and T-cell lymphoma biopsies, but also at high levels in critical tissues, including kidney, brain and marrow, diminishing their utility. A more tissue-restricted candidate, NT5C1B, was detected in T-cell lymphomas, but also at low levels in some normal dog tissues. These results illustrate the feasibility of discovering canine CTAs by a reverse approach, proceeding from identification of MHC class I-presented peptides to a comparative RNA expression survey of tumours and normal tissues.

KRAS mutation in secondary malignant histiocytosis arising from low grade follicular lymphoma.

BACKGROUND: Transformation of follicular lymphoma most typically occurs as diffuse large B-cell lymphoma, however other forms of transformation such as classic Hodgkin lymphoma and lymphoblastic transformation can occur. Secondary malignant histiocytosis also represents a rare form of transformation, which is thought to occur due to a process of transdifferentiation whereby the lymphoma cells exhibit lineage plasticity and lose all evidence of B-cell phenotype and instead acquire the phenotype of a histiocytic neoplasm. Little is known about the underlying genetic alterations that occur during this unusual process. Comparative genetic analysis of pre- and post-transformation/transdifferentiation would be one tool by which we could better understand how this phenomenon occurs. CASE PRESENTATION: Here we report the clinical, immunophenotypic and genetic features of a rare case of secondary malignant histiocytosis, Langerhans cell-type (Langerhans cell sarcoma) arising from a previous low grade follicular lymphoma. FISH analysis confirmed the presence of IgH/BCL2 rearrangement in both the low grade follicular lymphoma (FL) and transformed Langerhans cells sarcoma (LCS) samples, demonstrating a clonal relationship. Comparative whole exome sequencing was then performed, which identified a KRAS p.G13D mutation in the LCS that was not present in the FL. CONCLUSIONS: This report highlights genetic alterations, in particular an acquired somatic KRAS mutation, that may occur during transdifferentiation, with additional significance of KRAS mutation as a possible therapeutic target in cases which otherwise would have limited treatment options.

Primary histiocytic sarcoma of the central nervous system: a case report with platelet derived growth factor receptor mutation and PD-L1/PD-L2 expression and literature review.

BACKGROUND: Histiocytic sarcoma (HS) is an aggressive malignant neoplasm. HS in the central nervous system is exceptionally rare and associated with a poor prognosis. This report documents a case of primary HS of the central nervous system with treatment including surgery, radiotherapy, and chemotherapy. CASE PRESENTATION: Our patient was a 47 year old female presenting with progressive ataxia, headaches, imbalance, nausea, vomiting, and diplopia. MRI showed a heterogeneously enhancing lesion approximately 2.9 × 3.0 × 2.3 cm centered upon the cerebellar vermis with mild surrounding vasogenic edema and abnormal enhancement of multiple cranial nerves. The patient underwent surgical debulking, which revealed histiocytic sarcoma with grossly purulent drainage. Staging revealed diffuse leptomeningeal involvement, primarily involving the brain and lower thoracic and lumbar spine. She underwent adjuvant radiotherapy to the brain and lower spine and was started on high dose methotrexate. However, she experienced progressive disease in the cervical and thoracic spine as well as pulmonary involvement. Genomic sequencing of her tumor showed a mutation in the platelet-derived growth factor receptor A (p.V0681) which could be targeted with Dasatinib. However, she did not tolerate Dasatinib and she succumbed to progressive disseminated disease eight months from original diagnosis. Our pathologic evaluation also revealed expression of PD-L1 and PD-L2 by tumor cells raising the potential therapeutic role for immune checkpoint inhibition. CONCLUSIONS: This case provides an example of effective CNS control with resection and moderate doses of radiation therapy. A review of the literature confirms aggressive multidisciplinary treatment is the most effective treatment against this disease. In addition, genomic sequencing may play an important role in determining new therapeutic options. However, CNS histiocytic sarcoma remains an aggressive disease with a propensity for early widespread dissemination and few long term survivors.

Histiocytic Sarcoma with p16-Overexpression Presenting as Uterine Cervical Mass.

A 67-year-old woman presented with post-menopausal bleeding. Physical examination and imaging revealed a cervical mass with extensive pelvic involvement and supraclavicular lymphadenopathy. Clinical diagnosis was stage IVB cervical carcinoma. Cervical biopsy revealed high-grade squamous intraepithelial lesion and epithelioid tumor cells in the stroma. Lymph node biopsy revealed metastasis by similar epithelioid tumor cells. The epithelioid tumor cells were positive for p16, CD68, and CD163 on immunohistochemical study. The diagnosis of histiocytic sarcoma (HS) was rendered. This case demonstrated a rare presentation of HS mimicking cervical carcinoma. Awareness of this rare entity with appropriate ancillary tests is essential for accurate diagnosis. In contrast to the previous postulation of p16-inactivation as the pathogenesis of HS, p16-overexpression might suggest a different pathway for tumorigenesis.

Histiocytic sarcoma: New insights into FNA cytomorphology and molecular characteristics.

BACKGROUND: Histiocytic sarcoma (HS) is a rare malignant neoplasm showing morphologic and immunophenotypic features of histiocytes. Molecular characteristics of HS and fine-needle aspiration (FNA) criteria for its diagnosis have not been established. METHODS: A case series of HS in 8 FNA samples from 6 patients was reviewed along with histopathologic and clinical data. Immunohistochemistry was performed on cell blocks (3 cases), core biopsies (5 cases), and surgical specimens (4 cases). Targeted-exome next-generation sequencing (NGS) was performed on surgical resection specimens in 4 cases. RESULTS: Four patients had a known history of hematolymphoid malignancy. Cytomorphologic features included variably cellular smears composed of large epithelioid cells with reniform nuclei and abundant vacuolated cytoplasm, in an inflammatory background, with occasional cytophagocytosis and lymphoglandular bodies. Marked pleomorphism, multinucleated monster cells, and binucleated histiocytoid cells with partially overlapping, eccentrically placed nuclei resembling Pac-Man were common. Most cases expressed histiocytic markers CD68 (6 of 7 cases), CD163 (5 of 5 cases), and PU.1 (3 of 4 cases). In 3 cases, NGS analysis revealed alterations in lysine methyltransferase 2D (KMT2D)/mixed-lineage leukemia 2 (MLL2), a gene involved in chromatin regulation and previously implicated in the pathogenesis of follicular lymphoma. CONCLUSIONS: Although diagnosing HS with FNA alone is extremely challenging, the presence of pleomorphic and epithelioid large cells with binucleation and/or multinucleation in an inflammatory background should prompt the diagnosis of HS with judicious use of confirmatory histiocytic lineage markers. The detection of recurrent KMT2D/MLL2 alterations implicates epigenetic regulation in the pathogenesis of HS and supports the notion of transdifferentiation from a genetically similar but phenotypically distinct tumor of a different lineage. Cancer Cytopathol 2017;125:604-14. © 2017 American Cancer Society.

Syndrome of inappropriate antidiuretic hormone secretion in a dog with a histiocytic sarcoma.

A 7-year-old female neutered Bernese mountain dog was presented in a semi-comatose state. Based on serum hypo-osmolality with inappropriate urine hyper-osmolality and urine sodium excretion, the dog was diagnosed with a syndrome of inappropriate antidiuretic hormone secretion secondary to a histiocytic sarcoma. This report describes the first case of this syndrome in a dog with histiocytic sarcoma.

Syndrome de sécrétion inappropriée d'hormone antidiurétique chez un chien avec un sarcome histiocytaire. Une chienne stérilisée de 7 ans, Bouvier bernois, est présentée dans un état semi-comateux. Basé sur une hypo-osmolalité sérique associée à une hyper-osmolalité urinaire et une excrétion sodique urinaire toutes deux inappropriées, un diagnostic de syndrome de sécrétion inappropriée d'hormone antidiurétique secondaire à un sarcome histiocytaire est posé. Ce cas est le premier cas de ce syndrome chez un chien avec un sarcome histiocytaire.(Traduit par les auteurs).

Circulating let-7g is down-regulated in Bernese Mountain dogs with disseminated histiocytic sarcoma and carcinomas - a prospective study.

Cancer is a prevalent cause of mortality in Bernese mountain dogs (BMDs). Circulating microRNAs (miRNAs) are found in blood and have been identified as promising biomarkers in various neoplastic diseases in humans. In the current study, the expression profile of different types of miRNAs was investigated in healthy BMDs and BMDs with cancer. Seven healthy and six non-treated BMDs with cancer [four with disseminated histiocytic sarcomas (DHS)] were enrolled in this study. Clinical evaluations including physical examination, blood analysis, urinalysis and diagnostic imaging were performed on all dogs. Twenty-four different miRNAs were profiled from RNA isolated from whole blood preserved in PAXgene® tubes using quantitative real-time PCR (qPCR). The miRNA let-7g was significantly down-regulated in dogs with cancer (P = 0.002) and dogs with DHS (P = 0.011) compared with healthy controls. This miRNA is a known tumour suppressor and further analyses are warranted to assess its value as a non-invasive biomarker for early detection of different types of cancer in BMDs.

Persistent Morbillivirus Infection Leads to Altered Cortactin Distribution in Histiocytic Sarcoma Cells with Decreased Cellular Migration Capacity.

Histiocytic sarcomas represent rare but fatal neoplasms in humans. Based on the absence of a commercially available human histiocytic sarcoma cell line the frequently affected dog displays a suitable translational model. Canine distemper virus, closely related to measles virus, is a highly promising candidate for oncolytic virotherapy. Therapeutic failures in patients are mostly associated with tumour invasion and metastasis often induced by misdirected cytoskeletal protein activities. Thus, the impact of persistent canine distemper virus infection on the cytoskeletal protein cortactin, which is frequently overexpressed in human cancers with poor prognosis, was investigated in vitro in a canine histiocytic sarcoma cell line (DH82). Though phagocytic activity, proliferation and apoptotic rate were unaltered, a significantly reduced migration activity compared to controls (6 hours and 1 day after seeding) accompanied by a decreased number of cortactin mRNA transcripts (1 day) was detected. Furthermore, persistently canine distemper virus infected DH82 cells showed a predominant diffuse intracytoplasmic cortactin distribution at 6 hours and 1 day compared to controls with a prominent membranous expression pattern (p ≤ 0.05). Summarized, persistent canine distemper virus infection induces reduced tumour cell migration associated with an altered intracellular cortactin distribution, indicating cytoskeletal changes as one of the major pathways of virus-associated inhibition of tumour spread.

NLRX1 suppresses tumorigenesis and attenuates histiocytic sarcoma through the negative regulation of NF-κB signaling.

Histiocytic sarcoma is an uncommon malignancy in both humans and veterinary species. Research exploring the pathogenesis of this disease is scarce; thus, diagnostic and therapeutic options for patients are limited. Recent publications have suggested a role for the NLR, NLRX1, in acting as a tumor suppressor. Based on these prior findings, we hypothesized that NLRX1 would function to inhibit tumorigenesis and thus the development of histiocytic sarcoma. To test this, we utilized Nlrx1-/- mice and a model of urethane-induced tumorigenesis. Nlrx1-/- mice exposed to urethane developed splenic histiocytic sarcoma that was associated with significant up-regulation of the NF-κB signaling pathway. Additionally, development of these tumors was also significantly associated with the increased regulation of genes associated with AKT signaling, cell death and autophagy. Together, these data show that NLRX1 suppresses tumorigenesis and reveals new genetic pathways involved in the pathobiology of histiocytic sarcoma.

Osteocalcin and Osteonectin Expression in Canine Osteosarcoma.

Osteosarcoma (OSA) is a malignant heterogeneous primary bone tumor responsible for up to 90% of all primary bone tumors in dogs. In this study, osteocalcin (OC) and osteonectin (ON) immunoreactivity was evaluated in 23 canine OSAs, 4 chondrosarcomas, 4 fibrosarcomas, 2 hemangiosarcomas, and 4 histiocytic sarcomas. The effects of three different decalcification agents (ethylenediaminetetraetic acid [EDTA], formic acid and hydrochloric acid [HCl]) on the immunoreactivity for OC and ON was also assessed. Immunoreactivity to OC was present in 19/23 (83%) cases of OSA and all cases of chondrosarcoma. In three OSAs the extracellular matrix showed immunoreactivity to OC. None of the fibrosarcomas, histiocytic sarcomas or hemangiosarcomas showed immunoreactivity to OC. The sensitivity and specificity for OC in canine OSA in this study was 83% and 71% respectively. For ON, 100% of both OSAs (23/23) and non-OSAs (14/14) showed cytoplasmic immunoreactivity to this antibody, giving a sensitivity of 100% but a complete lack of specificity. There were no significant differences in immunoreactivity for OC and ON between the different decalcification agents used. In conclusion, OC showed high sensitivity for identifying OSA but it failed to distinguish between OSA and chondrosarcoma, and the osteoid produced by neoplastic cells in most cases did not show immunoreactivity to OC. These factors may limit the practical utility of OC in the diagnosis of OSA in dogs when chondrosarcoma is a differential diagnosis. ON showed no specificity in detecting OSA and has little practical application for the diagnosis of OSA in dogs.

Cooperation of BRAF(F595L) and mutant HRAS in histiocytic sarcoma provides new insights into oncogenic BRAF signaling.

Activating BRAF mutations, in particular V600E/K, drive many cancers and are considered mutually exclusive with mutant RAS, whereas inactivating BRAF mutations in the D(594)F(595)G(596) motif cooperate with RAS via paradoxical MEK/ERK activation. Due to the increasing use of comprehensive tumor genomic profiling, many non-V600 BRAF mutations are being detected whose functional consequences and therapeutic actionability are often unknown. We investigated an atypical BRAF mutation, F595L, which was identified along with mutant HRAS in histiocytic sarcoma and also occurs in epithelial cancers, melanoma and neuroblastoma, and determined its interaction with mutant RAS. Unlike other DFG motif mutants, BRAF(F595L) is a gain-of-function variant with intermediate activity that does not act paradoxically, but nevertheless cooperates with mutant RAS to promote oncogenic signaling, which is efficiently blocked by pan-RAF and MEK inhibitors. Mutation data from patients and cell lines show that BRAF(F595L), as well as other intermediate-activity BRAF mutations, frequently coincide with mutant RAS in various cancers. These data define a distinct class of activating BRAF mutations, extend the spectrum of patients with systemic histiocytoses and other malignancies who are candidates for therapeutic blockade of the RAF-MEK-ERK pathway and underscore the value of comprehensive genomic testing for uncovering the vulnerabilities of individual tumors.

Flow cytometry used to identify histiocytic sarcoma: A case report.

Histiocytic sarcoma (HS) is an extremely rare but aggressive malignancy of hematopoietic origin. The diagnosis of HS mainly relies on pathological morphology and immunohistochemical staining combined with clinical symptoms. However, whether histiocytic sarcoma can be detected using flow cytometry (FCM) is not clear. We report the case of a 61-year-old female patient with a group of abnormal cells identified primarily by FCM. Largeabnormal cells -CD68+ CD11c+ CD14+ HLA-DR+ CD123+ CD45+ presented in upper forward scatter and side scatter, indicating that a malignancy originally derived from monocyte/macrophage could be histocytic sarcoma. This patient with histiocytic sarcoma was finally confirmed with pathology and immunohistochemistry. In conclusion, HS can be detected by assessment of CD68+ CD11c+ CD14+ HLA-DR+ CD123+ CD45+ cells using FCM, suggesting that FCM can be an effective tool for early finding of HS. © 2015 International Clinical Cytometry Society.