Circulating tumor DNA is detectable in canine histiocytic sarcoma, oral malignant melanoma, and multicentric lymphoma.

Circulating tumor DNA (ctDNA) has become an attractive biomarker in human oncology, and its use may be informative in canine cancer. Thus, we used droplet digital PCR or PCR for antigen receptor rearrangement, to explore tumor-specific point mutations, copy number alterations, and chromosomal rearrangements in the plasma of cancer-affected dogs. We detected ctDNA in 21/23 (91.3%) of histiocytic sarcoma (HS), 2/8 (25%) of oral melanoma, and 12/13 (92.3%) of lymphoma cases. The utility of ctDNA in diagnosing HS was explored in 133 dogs, including 49 with HS, and the screening of recurrent PTPN11 mutations in plasma had a specificity of 98.8% and a sensitivity between 42.8 and 77% according to the clinical presentation of HS. Sensitivity was greater in visceral forms and especially related to pulmonary location. Follow-up of four dogs by targeting lymphoma-specific antigen receptor rearrangement in plasma showed that minimal residual disease detection was concordant with clinical evaluation and treatment response. Thus, our study shows that ctDNA is detectable in the plasma of cancer-affected dogs and is a promising biomarker for diagnosis and clinical follow-up. ctDNA detection appears to be useful in comparative oncology research due to growing interest in the study of natural canine tumors and exploration of new therapies.

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.

Immunocytochemical detection of the class A macrophage scavenger receptor CD204 using air-dried cytologic smears of canine histiocytic sarcoma.

BACKGROUND: Cytologic diagnosis of canine histiocytic sarcoma (CHS) can be challenging because neoplastic histiocytes commonly show marked nuclear and cellular atypia and may resemble other pleomorphic malignant round cell tumors. Therefore, even on histopathologic examination, immunostaining is often necessary for a definitive diagnosis. OBJECTIVES: The objective of this study was to validate an anti-human CD204 antibody for immunocytochemical staining of air-dried smears for a rapid definitive diagnosis of CHS. METHODS: Cytologic specimens were obtained from 10 dogs with CHS and 45 dogs with other tumors. After the cytologic evaluation of modified Giemsa-stained smears, acetone-fixed specimens were immunostained using mouse anti-human CD204 antibodies. All immunocytochemical specimens were assessed blinded and at high-power magnification (× 40 objective) in 10 randomly selected fields per sample. Parameters evaluated were the subjective staining intensity and location, and the proportion of positive cells. RESULTS: All 10 CHS samples showed intense positive staining for CD204 in ≥ 50% of the cells, whereas the 45 other tumors were negative for CD204 staining. CONCLUSIONS: Immunocytochemistry of air-dried cytologic smears of CHS for CD204 is useful for a rapid confirmation of a cytologic diagnosis of CHS.

Monocyte chemotactic protein-1 and other inflammatory parameters in Bernese Mountain dogs with disseminated histiocytic sarcoma.

The interaction between cancer and the immune system, and the production of cytokines by the tumour itself have been associated with altered levels of cytokines in human cancer patients. Bernese Mountain dogs with disseminated histiocytic sarcoma (DHS) show vague and non-specific clinical signs. Although histiocytes can secrete cytokines in response to inflammatory stimuli, serum cytokine concentrations in dogs with DHS have not previously been investigated. The aim of this study was to evaluate the immunological state of untreated Bernese Mountain dogs with DHS by assessing multiple serum cytokines and to correlate these with other inflammatory markers. As a prospective case control study, 17 Bernese Mountain dogs with DHS were included along with 18 healthy controls (12 Bernese Mountain dogs and 6 dogs of various breeds). Blood samples were examined for fibrinogen, C-reactive protein (CRP), white blood cell count, monocyte count and the following cytokines: interleukin (IL)-6, IL-10, IL-12, IL-15, IL-18, tumour necrosis factor and monocyte chemotactic protein (MCP)-1. Significant differences were observed in Bernese Mountain dogs with DHS compared to healthy control dogs for fibrinogen (P=0.002), CRP (P=0.02) and MCP-1 (P=0.004). Other important pro-inflammatory cytokines were not significantly increased in dogs with DHS and none of the measured cytokines were correlated to either WBC, monocyte count, CRP or fibrinogen concentration. The implications of this increased MCP-1 blood levels in Bernese Mountain dogs with DHS warrant further investigations.

Investigation of a screening programme and the possible identification of biomarkers for early disseminated histiocytic sarcoma in Bernese Mountain dogs.

The aim of the study was to construct a screening programme for disseminated histiocytic sarcoma (DHS) in Bernese Mountain dogs using diagnostic imaging and blood analysis and evaluate blood borne biomarkers as early disease detection biomarkers. Healthy Bernese Mountain dogs were screened on four occasions in an attempt to detect early disease. Eleven blood borne biomarkers were examined for their worth as early tumour biomarkers. During 2.5 years, five dogs with early DHS were identified; four of these by diagnostic imaging. No dogs developed symptomatic DHS without being detected within 6 months of the screening programme. Only serum ferritin showed potential as a blood borne marker of the disease. Median survival times for the dogs with early DHS were 226 days. Screening programmes every 6 months for Bernese Mountain dogs over 4 years of age including diagnostic imaging and ferritin measurements may identify early DHS.

Disseminated histiocytic sarcoma with peripheral blood involvement in a Bernese Mountain dog.

A 6-year-old Bernese Mountain dog was presented with a history of lethargy and weight loss of 2 weeks duration. On physical examination the dog had pale mucous membranes and tachypnea. Ultrasound examination revealed hepatomegaly, splenomegaly, and mesenteric lymphadenomegaly. Results of a CBC included marked normocytic normochromic nonregenerative anemia, marked thrombocytopenia, and moderate leukocytosis with mild neutrophilia and a large population of unclassified round cells (6.2 x 10(3)/microL). The unclassified cells occasionally were bi- or multinucleated and had variably abundant pale basophilic cytoplasm that contained multiple irregular clear vacuoles and occasionally erythrocytes. Fine needle aspirate specimens of the mesenteric lymph nodes and spleen were composed of a population of round pleomorphic cells with the same features as the circulating cells. On flow cytometric analysis of peripheral blood, the unclassified cells expressed CD18, CD45, CD11c, CD1c, and CD14; immunocytochemical analysis of blood smears also indicated the cells were positive for CD1c, CD1a, and CD11c. The dog died a few hours after referral. The histologic interpretation of samples collected from spleen, liver, and lymph nodes was malignant neoplasia of histiocytic origin. Immunohistochemical staining yielded negative results for CD11d, a marker of red-pulp macrophages, ruling out hemophagocytic histiocytic sarcoma. Based on clinical and pathologic findings, the final diagnosis was disseminated histiocytic sarcoma (DHS) with peripheral blood involvement. To our knowledge, DHS in a dog with evidence and immunophenotyping of neoplastic cells in peripheral blood has been reported only rarely.

[Allogeneic bone marrow transplantation for a patient with malignant histiocytosis].

OBJECTIVE: To observe the efficacy and side effects of allogeneic bone marrow transplantation (allo-BMT) in the treatment of malignant histiocytosis (MH). METHODS: After conditioning with chemotherapy and total body irradiation, HLA-A, B, DR, DQ compatible allo-BMT was performed in a patient with MH. RESULTS: DNA fingerprinting showed engraftment on day 22, bone marrow aspiration showed hypercellularity on day 50, and no abnormal cell was found. The patient developed acute graft-versus-host disease ( II degrees) on day 40 and herpes zoster 6 months post-transplantation, and the patient has survived disease-freely for 24 months after allo-BMT. CONCLUSION: This is the first report of treatment of MH with allo-BMT in China, showing that allo-BMT may significantly prolong the survival of MH.

Higher in vivo protein binding of etoposide in children compared with adult cancer patients.

Etoposide is bound to plasma albumin (94%). Previous studies have revealed altered protein binding of etoposide in cancer patients. This has clinical implications since only the free fraction is considered pharmacologically active. We have studied the etoposide protein binding in 11 children (eight acute lymphocytic leukemia, two malignant histiocytosis, and one oligodendroglioma; age 1-17 years) and 46 adult patients (28 acute myelocytic leukemia, eight lymphoma, one multiple myeloma, and nine small cell lung cancer; age 38-81 years). All patients were treated with etoposide 50-200 mg/m2 i.v. or orally. Plasma from ten healthy volunteers, 26-50 years of age, was spiked with etoposide, 10 micrograms/ml, and the protein binding was compared with that in patient samples. The free etoposide concentration was determined by high performance liquid chromatography (HPLC) after ultrafiltration at room temperature. The free etoposide fraction was lower, 2.5 +/- 0.6% (mean +/- SD), in the children compared with 5.0 +/- 3.6% in adult cancer patients. In plasma from healthy adults it was 3.2 +/- 0.3%. It is concluded that children have significantly lower levels of free etoposide compared with adult patients (P = 0.03) as well as with healthy subjects (P = 0.001), which is likely to affect metabolism and renal clearance as well as cellular uptake of the drug.

Hyperferritinemia associated with malignant histiocytosis in a dog.

A 3.5-year-old male Golden Retriever with lethargy, generalized lymphadenopathy, and hepatosplenomegaly was determined to have malignant histiocytosis, based on the morphologic appearance of neoplastic cells by light and electron microscopic examination, evidence of erythrophagocytosis, the presence of diffuse nonspecific esterase activity, and immunohistochemical demonstration of vimentin intermediate filaments. Because of the appearance of abundant iron stores in the bone marrow, serum was obtained for determination of iron, total iron binding capacity, and ferritin values. Serum ferritin concentrations were markedly increased. Measurement of serum ferritin concentrations may be useful in supporting a diagnosis of malignant histiocytosis, and the monitoring of serum ferritin concentrations may be useful for assessing treatment and regression of the disease.

Malignant histiocytosis in the leukaemic stage: a new entity (M5c-AML) in the FAB classification?

Malignant histiocytosis (MH) is a rare, rapidly fatal disorder, characterized by systemic proliferation of abnormal histiocytes. Most patients present with pancytopenia. We report the case of a patient with MH in the leukaemic stage, who presented extremely pronounced general symptoms and multisystemic involvement. Determination of serum cytokines showed high levels of tumor necrosis factor (TNF), interleukin 6 (IL-6) and interleukin 1 receptor antagonist (IL-1ra). Cytogenetic studies proved the monoclonality of the histiocytic proliferation. These findings strongly suggest that we are dealing with a proliferation of activated macrophages (= histiocytes). By analogy with the monoblastic (M5a) and monocytic (M5b) acute leukaemia of the French-American-British (FAB) classification, we propose a new entity, 'M5c', designating acute histiocytic leukaemia.

[The morphofunctional characteristics of the mononuclear phagocytes in the concentrated venous blood leukocytes of patients with histiocytosis]. / Morfofunktsional'naia kharakteristika mononuklearnykh fagotsitov leikokontsentrata venoznoi krovi bol'nykh gistiotsitozami.

Morphological and cytochemical examinations of mononuclear phagocytes (MP) from venous blood leukoconcentrate were carried out in 25 patients with chronic monocytic leukemia, 7 patients with malignant histiocytosis, 3 patients with Langerhans' cell histiocytosis and 26 patients with reactive proliferations of the cells belonging to MP system associated with autoaggressive, infectious diseases or tumors. Morphofunctional features of MP from the patients with tumor and reactive histiocytosis may serve additional criteria in differential diagnosis of the diseases in which pathological process runs with participation of MP system cells.

The diagnostic significance of serum ferritin indices in patients with malignant and reactive histiocytosis.

Iron metabolism was studied in 10 patients with malignant histiocytosis (MH), in 16 patients with histiocytosis-X (Langerhans cell histiocytosis) and in 34 patients with reactive proliferation of the mononuclear phagocytes (MPS). Eight MH patients had a considerably increased level of serum ferritin (SF). The average level of SF was 6070 +/- 957 mg/l for MH patients, which is significantly greater than the SF level for HX and RH patients. The study of the serum ferritin profiles suggests that the main pathogenetic mechanism of hyperferritinaemia in MH is the synthesis and secretion of ferritin by neoplastic mononuclear phagocytes. From this, hyperferritinaemia in MH may be considered as pathognomic of this disease and used as an additional diagnostic criterion. In addition, SF is the most informative parameter for the clinical practice among the indices of the functional state of MPS.

Malignant histiocytosis as a fatal complication of systemic lupus erythematosus.

We present the case of a 26-year-old Caucasian woman with systemic lupus erythematosus (SLE) of 9 years duration, mainly involving the kidney. Her clinical course was complicated in the last year by several infectious and noninfectious events, and after pancytopenia, fever, and extremely high levels of lactate dehydrogenase developed, the woman died. Light and electron microscopy of bone marrow, together with immunophenotyping, disclosed malignant histiocytosis, a complication of SLE that has not been reported.

Phenytoin-induced hemocytophagic histiocytosis indistinguishable from malignant histiocytosis.

We have reported a case of phenytoin-induced hemocytophagic histiocytosis indistinguishable on clinical and histopathologic grounds from malignant histiocytosis. We emphasize the need to investigate for microbiologic causes and drug ingestion, even if the diagnosis of malignant histiocytosis is plausible. We think that reactive and malignant histiocytosis are not really two distinct entities with different etiologies, but a continuum of host responses to several insults with different degrees of aggressiveness depending on the host immune status.

Prognosis of children with virus-associated hemophagocytic syndrome and malignant histiocytosis: correlation with levels of serum interleukin-1 and tumor necrosis factor.

To clarify the correlation of cytokine level with the severity and prognosis of children with the hemophagocytic syndrome, we analyzed serum interleukin-1 (IL-1) and tumor necrosis factor (TNF) levels in 26 children with either the virus-associated hemophagocytic syndrome (VAHS, n = 12) or malignant histiocytosis (MH, n = 14). When compared to healthy controls, 13 children had an elevated IL-1 (greater than or equal to 20 pg/ml) and 21 children had an elevated TNF (greater than or equal to 10 pg/ml) level at diagnosis. There was however, no significant difference in the frequency of these high levels between the patients with VAHS and MH. Neither IL-1 nor TNF levels correlated with other clinical or laboratory findings in either VAHS or MH. Two of the 12 patients with VAHS died of an intracranial hemorrhage and 7 of the 14 patients with MH died despite chemotherapy. The MH patients who had a high TNF level (greater than or equal to 50 pg/ml) had a poorer prognosis than those with a low TNF level (less than 50 pg/ml; p less than 0.01). In MH patients, other parameters, such as coagulopathy and lactic dehydrogenase, ferritin and IL-1 levels, did not correlate with prognosis. In 3 patients (2 with VAHS and 1 with MH) analyzed periodically, the change in TNF level was closely associated with the clinical progression or regression of the diseases. Serum cytokine levels may thus be monitored not only for predicting the severity and prognosis of VAHS or MH but also for determining the indications for or timing of chemotherapy. Moreover, TNF may play an important role in the progression of VAHS and MH.

[Mechanism of hyperferritinemia in a case of malignant histiocytosis].

We report a case of malignant histiocytosis diagnosed by liver-spleen biopsy under laparoscopy. A 49-year-old woman was admitted to our hospital with thrombocytopenia, moderate anemia and hypoproteinemia. Her bone marrow findings revealed erythroid and megakaryocyte hyperplasia, and the serum ferritin concentration was 2,250 ng/ml though she had not received any blood transfusions. Ferrokinetics analysis showed the pattern of ineffective erythropoiesis, and the half-lives of erythrocytes and platelets were both shortened. Her hepatosplenomegaly gradually increased accompanied by increasing serum ferritin level to 10,000 ng/ml. Liver-spleen biopsy was carried out under laparoscopy and revealed infiltration of atypical histiocytes with erythrophagocytosis, which were positive for S-100 and ferritin but negative for lysozyme. The rate of glycosylation in whole serum ferritin, analyzed by using concanavalin-A binding method, showed that her glycosylated ferritin content was only 8.3%, whereas in sera after iron overloading, it was about 70%. Serum isoferritin profiles by isoelectric focussing were studied, and isoferritin pattern from malignant histiocytosis was the same as that in iron overloading after neuraminidase treatment. These findings suggest that serum ferritin is synthesized in proliferating histiocytes and released in the plasma as a nonsecretory type (nonglycosylated ferritin) in this case.

Spontaneous NBT reduction by monocytes as a marker of disease activity in children with histiocytosis.

In an attempt to define a biological marker of monocyte hyperactivation in the course of infantile histiocytosis, the spontaneous nitroblue tetrazolium (NBT) reduction assay was applied to monocytes from 13 children with Langerhans cell histiocytosis (LCH), familial haemophagocytic lymphohistiocytosis (FHL), juvenile xanthogranuloma or malignant histiocytosis. Significant increase in NBT reduction was observed in the patients with both active LCH and FHL in comparison with control subjects, who were either healthy or affected by different conditions. A close relationship between spontaneous reduction rate and clinical condition of the patients was evident in patients tested at diagnosis, during remission and during the course of disease reactivation. Interleukin-1 (IL-1) production by monocytes was also evaluated: the patients with LCH and FHL displayed a significant increase in in vitro IL-1 production by lipopolysaccharide-stimulated monocytes. In our experience the spontaneous NBT reduction assay was a sensitive, quite specific, low-cost and reproducible test for the evaluation of children with histiocytosis. Useful information may be obtained at diagnosis but also during the clinical course of disease by using this marker of monocyte spontaneous activation.

Peripheral T-cell lymphoma presenting as hemophagocytic syndrome.

Three patients with peripheral T-cell lymphoma presenting with pyrexia, wasting, hepatosplenomegaly and pancytopenia in the absence of myelophthisic lymphomatous involvement are reported. Early in the course of the disease when there was no significant lymphadenopathy, these cases created enormous diagnostic confusion. Although the clinical features were suggestive of malignant histiocytosis (MH), marrow findings showed phagocytic histiocytes which did not appear atypical, and the criteria for diagnosis of MH could not be satisfied. Lymph node enlargement was detected only after 14, 5, and 8 weeks from the onset of symptoms, and the diagnosis of T-lymphoma was then made on lymph node biopsies. Treatment with multiple agent chemotherapy was attempted. Two patients died 3 days and 11 weeks after treatment was started and the third was lost to follow-up. In contrast with most of the cases reported in the literature, our cases show that a reactive hemophagocytic syndrome can be an early and prominent manifestation of an underlying T-cell lymphoma. Differentiation from other causes of hemophagocytic syndrome can be difficult and lack of histological proof of malignancy in the initial stage often delays definitive diagnosis and treatment.