Presumptive increase in protein‐bound serum calcium in a dog with multiple myeloma

Abstract: An 11‐year‐old male castrated Australian Shepherd was presented with a history of lethargy, panting, and weight loss for 1 month. Physical examination revealed a moderately enlarged spleen. Laboratory abnormalities included thrombocytopenia and marked hypercalcemia, with hyperglobulinemia, hypoalbuminemia, and a monoclonal spike in the β‐globulin region on serum protein electrophoresis. Serum total calcium concentration was markedly increased (16.5 mg/dL, reference interval 8.9–11.4 mg/dL) but ionized calcium concentration (1.39 mmol/L) was within the reference interval (1.25–1.45 mmol/L). Isosthenuria was noted, but the dog was not polyuric or polydipsic. Serum parathyroid hormone concentration was within reference limits and parathyroid hormone‐related peptide concentration was 0 pmol/L. Radiographic findings were largely unremarkable. Results of cytologic evaluation of a fine‐needle aspirate specimen from the spleen indicated plasma cell neoplasia. Based on the results of the electrophoresis, splenic aspirates, radiographs, and hypercalcemia, a diagnosis of splenic multiple myeloma was made. The marked hypercalcemia, normal ionized calcium and parathyroid hormone concentrations, and lack of osteolytic lesions indicated a presumptive increase in protein‐bound serum calcium, likely due to binding to molecules of the paraprotein (M protein). Protein binding of calcium in dogs with multiple myeloma should be considered as a potential mechanism of elevated total serum calcium concentration.     

Effect of conditioned medium of human multiple myeloma RPMI 8226 cells on osteoclastogenesis of RAW264.7 cells

AIM: To investigate the osteoclastogenic effect of conditioned medium of human multiple myeloma RPMI 8226 cells on preosteoclast RAW264.7 cells. METHODS: The protein expression of soluble receptor activator of NF-κB ligand (sRANKL) was detected by Western blotting. The morphological changes of RAW264.7 cells were observed after tartrate-resistant acid phosphatase (TRAP) staining. The mRNA expression of TRAP and cathepsin K was evaluated by RT-PCR. RESULTS: The result of Western blotting showed that conditioned medium of RPMI 8226 cells contained sRANKL. RPMI 8226 cell conditioned medium induced RAW264.7 cells to differentiate into TRAP-positive multinuclear osteoclasts. Human neutralized RANKL monoclonal antibody suppressed the differentiation of preosteoclasts in a dose-dependent manner, which was induced by 30% RPMI 8226 cell conditioned medium. RPMI 8226 cell conditioned medium increased the mRNA expression of TRAP and cathepsin K in RAW264.7 cells. CONCLUSION: The sRANKL in conditioned medium of human multiple myeloma RPMI 8226 cells has the bioactivity to induce preosteoclast RAW264.7 cells to differentiate into TRAP-positive multinuclear osteoclasts. Human neutralized RANKL monoclonal antibody suppresses the differentiation of preosteoclasts induced by sRANKL in a dose-dependent manner.     

冬虫夏草菌丝水提液对SP2／0细胞凋亡的影响

在冬虫夏草菌丝水提液诱导下，采用Hoechst33342荧光染色、MTT、琼脂糖凝胶电泳和流式细胞仪等方法，观察和分析了SP2／0细胞的凋亡情况。结果显示，冬虫夏草菌丝水提液可抑制SP2／0细胞增殖，在荧光显微镜下，凋亡细胞呈亮绿色，DNA琼脂糖电泳可见典型的“阶梯状”条带，Annexin—V—FITC和碘化丙啶（PI）双染后流式细胞仪分析显示，细胞凋亡数量明显增多。表明，冬虫夏草菌丝水提液对SP2／0细胞增殖的抑制作用可能与诱导其凋亡有关。     

ATYPICAL LYTIC PROLIFERATIVE SKELETAL LESIONS ASSOCIATED WITH PLASMA CELL MYELOMA IN A DOG

A 10-year-old female Labrador Retriever with intermittent lameness, lethargy, pancytopenia, hyper-calcemia, and severe hyperproteinemia was diagnosed as having plasma cell myeloma. Diagnosis was confirmed by finding a monoclonal gammopathy, extensive plasmacytic bone marrow infiltration, and radiographic evidence of skeletal disease. Survey radiography revealed atypical lytic proliferative osseous lesions involving the spinous process of the third thoracic vertebrae, left third rib, and both left and right fifth ribs. Nutritional support and specific chemotherapy with Melphalan have resulted in long-term clinical remission. This report describes the radiographic and clinicopathologic features of plasma cell myeloma in the dog. A discussion of the previously reported radiographic features of this neoplasm is also included.     

Mass spectrometric-based assessment of the serum kappa to lambda immunoglobulin light chain ratio (κ:λ) in dogs with immunoglobulin secretory diseases

The ratio of κ light chains to λ light chains (κ:λ) in serum is used as a biomarker of immunoglobulin secreting neoplasia in humans but has not been evaluated in dogs. A mass-spectrometry based method for determining the canine serum κ:λ was developed and used to evaluate samples from control dogs, dogs with an infectious aetiology, dogs with secretory plasma cell tumours (sPCT) and dogs with non-secretory B cell neoplasia. A human-targeted immunoturbidometric κ:λ assay and immunofixation using antisera targeting human κ light chain or λ light chain was also performed on all samples. Using whole serum samples, the MS-based κ:λ method identified 5 sPCT as κ-predominant (mean κ:λ = 3.307) and 5 sPCT as λ-predominant (mean κ:λ = 0.023) and documented differences between these groups and all other groups (p < 0.05 for all). The infectious aetiology group had a lower mean κ:λ ratio (mean κ:λ = 0.069) than control samples (mean κ:λ = 0.103, p = 0.035). Similar results were obtained when samples were enriched for proteins between 10 and 50 kDa using size exclusion chromatography, except for the statistical difference between the control and infectious aetiology group. All λ-predominant cases had only anti-human λ light chain labelling by immunofixation. Three κ-predominant cases had only anti-human κ-light chain labelling and the remaining two cases did not label with either antisera by immunofixation. The immunoturbidometric method had high analytical CV% (λ light chain CV = 13%, κ light chain CV = 50%), was unable to measure light chains in 20.5% of samples and did not distinguish groups. The data suggests that the human-targeted immunoturbidometric method would not be diagnostically useful and that the MS-derived serum κ:λ may be a useful biomarker of canine immunoglobulin secretory neoplasia which may have the ability to distinguish neoplasia from infectious causes of immunoglobulin secretion.     

A retrospective study of canine oral extramedullary plasmacytoma over a 15-year period (July 2004–July 2019): Treatment,histologic parameters and clinical outcomes

A total of 45 cases of canine oral extramedullary plasmacytomas (EMPs) presented to a tertiary referral institution over a 15-year period were examined. Histologic sections of 33 of these cases were examined for histopathologic prognostic indicators. Patients underwent variable treatment including surgical intervention, chemotherapy and/or radiation therapy. Long term survival was observed in the majority of dogs with a median survival time of 973 days (2–4315 days). However, almost 1/3 of dogs had progression of plasma cell disease, including two cases with myeloma-like progression. Histologic characterization of these tumours did not reveal criteria to predict tumour malignancy. However, cases without tumour progression did not exceed 28 mitotic figures in ten 400× fields (2.37 mm²). All cases with tumour related death showed at least moderate nuclear atypia. Oral EMPs may represent a local manifestation of systemic plasma cell disease or singular focal neoplasia.     

Cyclical 10-day dosing of melphalan for canine multiple myeloma

Canine multiple myeloma (MM) is typically treated with melphalan chemotherapy. A protocol with repeated 10-day cyclical dosing of melphalan has been used at our institution but has not been described in the literature. Our objectives were to describe the outcome and adverse events of this protocol in a retrospective case series. We hypothesised the cyclical 10-day protocol would have similar outcomes compared to other reported chemotherapy protocols. Dogs diagnosed with MM that received melphalan treatment at Cornell University Hospital for Animals were identified through a database search. Records were retrospectively reviewed. Seventeen dogs met inclusion criteria. Lethargy was the most common presenting complaint. The median duration of clinical signs was 53 days (range, 2–150 days). Seventeen dogs had hyperglobulinemia with 16/17 having monoclonal gammopathies. Sixteen dogs had bone marrow aspiration and cytology performed at initial diagnosis and plasmacytosis was diagnosed in all. Based on serum globulin concentrations, 10 of 17 dogs (59%) achieved complete response (CR), and 3 dogs (18%) achieved partial response (PR), for an overall response rate of 76%. The median overall survival time was 512 days (range, 39–1065). Retinal detachment (n = 3) and maximum response of CR/PR (n = 13) were associated with overall survival on multivariate analysis (p = .045 and .046, respectively). Adverse events were minimal with diarrhoea being the most reported (n = 6). This cyclical 10-day protocol was better-tolerated with fewer adverse events than with other reported chemotherapy protocols, but response rate was also lower, likely due to a lower dosing intensity.     

Effects of fucoidan on angiogenesis of human multiple myeloma RPMI 8226 cells

AIM: To investigate the effects of fucoidan on the angiogenesis of multiple myeloma cells in vitro, and its related mechanisms. METHODS: The human multiple myeloma RPMI 8226 cells and human endothelial cells were cultured in vitro. The growth inhibition rate of RPMI 8226 cells was examined by MTT assay. The cell cycle and apoptosis rate were measured by flow cytometry. RPMI 8226 cells were treated with fucoidan for 72 h, and the cell culture supernatant was collected. The VEGF concentration was examined by ELISA, and the tube formation assay was applied to assess the angiogenic activity. After treatment with fucoidan for 72 h at different concentrations, the protein levels of HIF-1α, VEGF, p-AKT and p-ERK1/2 were detected by Western blot. RESULTS: Fucoidan inhibited the growth of RPMI 8226 cells in a dose- and time-dependent manner. After treatment with fucoidan for 72 h, the cell cycle was arrested at G₁ phase, and the apoptotic rate of RPMI 8226 cells was increased with the increasing concentration of fucoidan, which was much higher than that in control group (P<0.05). The VEGF concentration was significantly decreased with the increa-sing concentration of fucoidan. The numbers and areas of the capillary-like structures decreased while the concentration of fucoidan increased, and those at 100 mg/L were less than those in the control (P<0.05). The protein levels of HIF-1α, VEGF, p-AKT and p-ERK1/2 in fucoidan group were significantly lower than those in control group (P<0.05). CONCLUSION: Fucoidan inhibits the secretion of VEGF in multiple myeloma cells, and reduces angiogenesis induced by multiple myeloma cells. It inhibits the protein expression of HIF-1α and VEGF, which may be related to inhibiting the phosphorylation of AKT and ERK1/2.