Ablation of VLA4 in multiple myeloma cells redirects tumor spread and prolongs survival.

Multiple myeloma (MM) is a cancer of bone marrow (BM) plasma cells, which is increasingly treatable but still incurable. In 90% of MM patients, severe osteolysis results from pathological interactions between MM cells and the bone microenvironment. Delineating specific molecules and pathways for their role in cancer supportive interactions in the BM is vital for developing new therapies. Very Late Antigen 4 (VLA4, integrin α4ß1) is a key player in cell-cell adhesion and signaling between MM and BM cells. We evaluated a VLA4 selective near infrared fluorescent probe, LLP2A-Cy5, for in vitro and in vivo optical imaging of VLA4. Furthermore, two VLA4-null murine 5TGM1 MM cell (KO) clones were generated by CRISPR/Cas9 knockout of the Itga4 (α4) subunit, which induced significant alterations in the transcriptome. In contrast to the VLA4+ 5TGM1 parental cells, C57Bl/KaLwRij immunocompetent syngeneic mice inoculated with the VLA4-null clones showed prolonged survival, reduced medullary disease, and increased extramedullary disease burden. The KO tumor foci showed significantly reduced uptake of LLP2A-Cy5, confirming in vivo specificity of this imaging agent. This work provides new insights into the pathogenic role of VLA4 in MM, and evaluates an optical tool to measure its expression in preclinical models.

A kinetic coupling between protein unfolding and aggregation controls time-dependent solubility of the human myeloma antibody light chain.

Protein aggregation is one of the most critical processes affecting protein solubility in various contexts-from protein therapeutics formulation to protein diseases. In general, time-dependent changes in protein solubility are complex kinetically driven processes that often involve a triggering event that consists of a protein unfolding/misfolding followed by the assembling of aggregation-competent protein species. In this study, we have examined the relation between stability and time-dependent solubility of the recombinant human antibody light chain, hLC, which was found to form renal tubular casts in the multiple myeloma patient. To analyze the aggregation quantitatively, the hLC stability and protein solubility assays were performed in vitro at elevated temperatures. A differential acceleration of the processes at high temperatures enabled us to dissect observed kinetics of irreversible hLC unfolding and aggregation. We find that for hLC these processes have different molecularity and activation energy barriers. While the irreversible unfolding of hLC is a unimolecular step with a substantial activation energy barrier of 260 kJ/mol, the aggregation is rate-limited by the bimolecular reaction, which is characterized by a lower activation energy barrier of 40 kJ/mol. By the combination of experimental assays at different temperatures, different protein concentrations and kinetic modeling using ordinary differential equations, we were able to extrapolate time-dependent protein solubility to temperatures where both unfolding and aggregation processes are strongly kinetically coupled. Our study enables mechanism-based evaluation and interpretation of different physico-chemical factors contributing to the hLC unfolding and aggregation and their effect on the formation of extracellular protein deposits.

Serum lipidomics for exploring biomarkers of bortezomib therapy in patients with multiple myeloma.

Although the proteasome inhibitor bortezomib (BTZ) shows excellent efficacy in multiple myeloma (MM), a fraction of patients has a suboptimal or no response to this agent. In addition, BTZ-induced peripheral neuropathy (BiPN), a frequent side-effect of this therapy, limits its use in some patients. This study aimed to explore serum lipid biomarker candidates to predict the response to BTZ and the severity of BiPN. Fifty-nine serum samples were collected from patients with MM prior to receiving BTZ plus low-dose dexamethasone therapy. Serum levels of phospholipids, sphingolipids, neutral lipids, and polyunsaturated fatty acids and their oxidation products were measured by a comprehensive lipidomic study. Overall, 385 lipid metabolites were identified in patients' sera; lower levels of several glycerophospholipids, sphingolipids, and cholesteryl esters were associated with a poor treatment response. Metabolites related to platelet-activating factor biosynthesis and cholesterol metabolism appeared particularly relevant. Furthermore, several lysophosphatidylcholines, phosphatidylcholines, ceramides, neutral lipids, and oxidative fatty acids were significantly increased or decreased in patients with BiPN grades ranging from G0 to G3. Among these compounds, mediators reportedly inducing myelin breakdown and stimulating inflammatory responses were prominent. Although further study is necessary to validate these biomarker candidates, our results contribute to the development of predictive biomarkers for response to BTZ treatment, or ensuing severe BiPN, in patients with MM.

An Algorithm for Building Multi-State Classifiers Based on Collision-Induced Unfolding Data.

Collision-induced unfolding (CIU) has emerged as a valuable method for distinguishing iso-cross-sectional protein ions through their distinct gas-phase unfolding trajectories. CIU shows promise as a high-throughput, structure-sensitive screening technique with potential applications in drug discovery and biotherapeutic characterization. We recently developed a CIU classification workflow to support screening applications that utilized CIU data acquired from a single protein charge state to distinguish immunoglobulin (IgG) subtypes and membrane protein lipid binding. However, distinguishing highly similar protein structures, such as those associated with biotherapeutics, can be challenging. Here, we present an expansion of this classification method that includes CIU data from multiple charge states, or indeed any perturbation to protein structure that differentially affects CIU, into a combined classifier. Using this improved method, we are able to improve the accuracy of existing, single-state classifiers for IgG subtypes and develop an activation-state-sensitive classifier for selected Src kinase inhibitors when data from a single charge state was insufficient to do so. Finally, we employ the combination of multiple charge states and stress conditions to distinguish a highly similar innovator/biosimilar biotherapeutic pair, demonstrating the potential of CIU as a rapid screening tool for drug discovery and biotherapeutic analysis.

Expression analysis of two SLAM family receptors, SLAMF2 and SLAMF7, in patients with multiple myeloma.

Monoclonal antibodies against surface antigens on MM cells, such as anti-SLAMF7 and anti-CD38 antibodies, represent an attractive therapeutic modality for the eradication of multiple myeloma (MM) cells. However, further exploration of target molecules is urgently needed for the development of more effective therapies. In the present study, we studied the expression of CD48 in a total of 74 primary MM samples derived from patients to evaluate SLAMF2 (CD48) as a candidate in mAb therapy for MM. Of 74 samples, 39 were subjected to SLAMF7 analysis. Most of the MM cells, defined as CD38 and CD138 double-positive cells, showed strong expression of CD48 or SLAMF7 independent of disease stage or treatment history. In these 39 samples, most MM cells showed expression of both SLAMF7 and CD48; however, several samples showed expression of either only CD48 or only SLAMF7, including seven cases that were only highly positive for SLAMF7, and five that were only highly positive for CD48. Our study demonstrates that the immune receptor CD48 is overexpressed on MM cells together with SLAMF7, and that CD48 may be considered as an alternative target for treatment of MM in cases showing weak expression of SLAMF7.

Myeloma-derived macrophage inhibitory factor regulates bone marrow stromal cell-derived IL-6 via c-MYC.

Multiple myeloma (MM) remains an incurable malignancy despite the recent advancements in its treatment. The protective effects of the niche in which it develops has been well documented; however, little has been done to investigate the MM cell's ability to 're-program' cells within its environment to benefit disease progression. Here, we show that MM-derived macrophage migratory inhibitory factor (MIF) stimulates bone marrow stromal cells to produce the disease critical cytokines IL-6 and IL-8, prior to any cell-cell contact. Furthermore, we provide evidence that this IL-6/8 production is mediated by the transcription factor cMYC. Pharmacological inhibition of cMYC in vivo using JQ1 led to significantly decreased levels of serum IL-6-a highly positive prognostic marker in MM patients. CONCLUSIONS: Our presented findings show that MM-derived MIF causes BMSC secretion of IL-6 and IL-8 via BMSC cMYC. Furthermore, we show that the cMYC inhibitor JQ1 can reduce BMSC secreted IL-6 in vivo, irrespective of tumor burden. These data provide evidence for the clinical evaluation of both MIF and cMYC inhibitors in the treatment of MM.

Human myeloma IgG4 reveals relatively rigid asymmetric Y-like structure with different conformational stability of CH2 domains.

Human IgG4 (hIgG4) has weak pro-inflammatory activity. The structural basis for this is still unclear. Here a 3D model of myeloma hIgG4 was created at ∼3nm resolution using electron microscopy (EM) with negative staining and single-particle 3D reconstruction. The hIgG4 model reveals relatively rigid asymmetric Y-like structure. The model shows that one Fab subunit is closer to the upper portion of the Fc subunit (CH2 domain) than the other Fab. This is in agreement with X-ray crystallography and X-ray/neutron scattering, recently published by others. The same hIgG4 sample was studied with differential scanning calorimetry (DSC) and fluorescence. The thermodynamics and fluorescence observations indicate that one CH2 domain displays less conformational stability than the other. This finding is consistent with the flipping of one CH2 domain, observed in pembrolizumab (recombinant hIgG4) by X-ray crystallography. The specific feature of hIgG4 CH2 domains together with relatively rigid asymmetric Y-like structure, in which one Fab subunit is closer to the upper portion of the Fc subunit (CH2 domain) than the other Fab, can explain the unique biological properties of hIgG4, such as its weak pro-inflammatory activity.

Raman spectroscopy differentiates between sensitive and resistant multiple myeloma cell lines.

Current methods for identifying neoplastic cells and discerning them from their normal counterparts are often nonspecific and biologically perturbing. Here, we show that single-cell micro-Raman spectroscopy can be used to discriminate between resistant and sensitive multiple myeloma cell lines based on their highly reproducible biomolecular spectral signatures. In order to demonstrate robustness of the proposed approach, we used two different cell lines of multiple myeloma, namely MM.1S and U266B1, and their counterparts MM.1R and U266/BTZ-R subtypes, resistant to dexamethasone and bortezomib, respectively. Then, micro-Raman spectroscopy provides an easily accurate and noninvasive method for cancer detection for both research and clinical environments. Characteristic peaks, mostly due to different DNA/RNA ratio, nucleic acids, lipids and protein concentrations, allow for discerning the sensitive and resistant subtypes. We also explored principal component analysis (PCA) for resistant cell identification and classification. Sensitive and resistant cells form distinct clusters that can be defined using just two principal components. The identification of drug-resistant cells by confocal micro-Raman spectroscopy is thus proposed as a clinical tool to assess the development of resistance to glucocorticoids and proteasome inhibitors in myeloma cells.

CEA-producing multiple myeloma with meningeal invasion during relapse.

Here we describe the case of a 62-year-old woman diagnosed with multiple myeloma (IgA-κ type) who had a high serum CEA level of 27.7 ng/ml. Upper and lower gastrointestinal endoscopy and PET/CT scan showed no abnormality. After two courses of VAD therapy, the serum CEA level decreased to 5.7 ng/ml, with a decrease in the IgA level, suggesting the diagnosis of CEA-producing myeloma. After 4 years and 1 month, she had a relapse with an increase in the LDH level and myeloma cells in the blood, followed by cognitive loss and convulsion. She died 1 month after the onset of neurological symptoms. Several myeloma cells were detected in the cerebral spinal fluid, which suggested the diagnosis of myelomatous meningitis. Myelomatous meningitis is a rare disease and accounts for 1% of all myelomas. This is the fourth reported case of CEA-producing myeloma.

Myeloma-Derived Light Chain Paired with a Diagnostic Monoclonal Antibody Hinders Immunoassay Performance.

Monoclonal antibodies are widely used as the capture and detection reagents in diagnostic immunoassays. In the past, myeloma fusion partners expressing endogenous heavy and/or light chains were often used to generate hybridoma cell lines. As a result, mixed populations of antibodies were produced that can cause inaccurate test results, poor antibody stability, and significant lot-to-lot variability. We describe one such scenario where the P3U1 (P3X63Ag8U.1) myeloma fusion partner was used in the generation of a hybridoma producing protein induced vitamin K absence/antagonist-II (PIVKA II) antibody. The hybridoma produces three subpopulations of immunoglobulin as determined by ion exchange (IEx) chromatography that exhibit varying degrees of immunoreactivity (0%, 50%, or 100%) to the target antigen as determined by Surface Plasmon Resonance. To produce an antibody with the highest possible sensitivity and specificity, the antigen-specific heavy and light chain variable domains (VH and VL) were cloned from the hybridoma and tethered to murine IgG1 and kappa scaffolds. The resulting recombinant antibody was expressed in Chinese hamster ovary cells and is compatible for use in a diagnostic immunoassay.

[Clinicopathologic characteristics of plasma cell myeloma with marrow fibrosis].

Objective: To study the clinicopathologic features of plasma cell myeloma(PCM) with bone marrow fibrosis (MF). Methods: The clinicopathologic data of 175 cases of newly diagnosed PCM patients were retrospectively analyzed. Based on reticular fiber staining, these cases were divided into PCM-MF and non-PCM-MF groups. Results: Sixty-three cases were PCM-MF(36%), 112 were non-PCM-MF (64%). No statistical difference in gender, age, hemoglobin level, platelet counts, the classification of immunoglobulin, ISS staging, immunohistochemical phenotypes and genetic features was found between PCM-MF and non-PCM-MF groups (P>0.05). Compared to non-PCM-MF group, lactate dehydrogenase (LDH)level and renal impairmentrate were higher in PCM-MF group (P<0.05). The degree of bone marrow hyperplasia, the percentage of myeloma cells and cells with plasmablastic morphology were significantly higher in PCM-MF group(P<0.05). Conclusion: The higher LDH level, renal impairment rate, and more significant bone marrow hyperplasia, proliferation of plasma cells and plasmablastic myeloma cells infiltration indicate poor prognosis of PCM-MF patients.

Plasmablastic lymphoma versus plasmablastic myeloma: an ongoing diagnostic dilemma.

AIMS: To determine the utility of clinical, morphological and phenotypical features in the differential diagnosis of plasmablastic lymphoma and myeloma with plasmablastic features. METHODS: All plasmablastic neoplasms identified from a 15-year retrospective search were reviewed and classified into 'lymphoma', 'myeloma' or 'indeterminate'. The classification was then compared with the previously established clinical diagnosis. Lessons learned from this review were used to design a diagnostic algorithm for pathologists to use in the absence of known clinical history. RESULTS: The classification was possible in 10 of 11 cases, 8 lymphomas and 2 myelomas (n=2). No distinctive morphological or phenotypical features were identified. The most useful histopathological parameter was a positive Epstein-Barr virus in situ hybridisation. Presence of associated lymphadenopathy and/or oral mass in the absence of complete myeloma-defining signs was used to favour a diagnosis of lymphoma in 4 of 8 cases. CONCLUSIONS: The distinction between plasmablastic lymphoma from plasmablastic myeloma warrants detailed knowledge of clinical, radiological and laboratorial findings. New studies identifying distinctive phenotypical or genetic features are needed to improve the histopathological differentiation of plasmablastic neoplasms.

[Expression of microRNA-221/222 in patients with monoclonal gammopathy of undetermined significance and multiple myeloma].

Objective: To detect the expression of miR-221/222 in serum and plasma cells in patients with monoclonal gammopathy of undetermined significance(MGUS) and multiple myeloma(MM), and to explore the possibility of miR-221/222 as biomarkers in the diagnosis and prognosis predicting of MGUS and MM. Methods: Bone marrow and serum samples from 14 patients with newly diagnosed MGUS, 81 patients with newly diagnosed or relapsed MM and 10 controls were collected from Sir Run Run Shaw Hospital of Zhejiang University and Tongde Hospital of Zhejiang Province during January 2013 and December 2015. The expressions of miR-221/222 in serum and in sorted CD138 positive plasma cells were detected by qRT-PCR, and the relative expression of miR-221/222 (Δct) was compared between the groups. Serum levels of miR-221 before and after treatment were compared in both remission group (n=22) and refractory group (n=13) in MM patients, and its correlation with serum level of ß2-MG was assessed using Pearson's correlation analysis. Results: Serum levels of miR-221/222 in MGUS and MM groups were significantly higher than those in control group (all P<0.01), while miR-221/222 levels in plasma cells were significantly lower in MGUS and MM groups than those in the control group (P<0.05 or<0.01). No significant difference in miR-221/222 levels in serum and plasma cells was observed between MGUS group and MM group (all P>0.05). There was no correlation between miR-221/222 levels in serum and plasma cells (r=0.024 and -0.127, all P>0.05), but miR-221 levels were correlated with miR-222 levels in both serum and plasma cells (r=0.534 and 0.552, all P<0.01). Receiver operating characteristic (ROC) curves showed that the areas under the curve (AUCs) of serum miR-221/222, plasma cell miR-221/222 in diagnosis of MGUS/MM were 0.968, 0.976, 0.801 and 0.727, respectively. There was no significant difference in serum level of miR-221 among MM patients with different paraprotein isotypes (P>0.05), but serum level of miR-221 in patients with relapsed MM was higher than that in patients with newly diagnosed MM (P<0.01). Compared with the patients with MGUS or MM stageⅠ and Ⅱ, patients with MM stage Ⅲ were of higher serum levels of miR-221 (P<0.01). Serum level of miR-221 decreased after chemotherapy in the remission group (U=51.5, P<0.01), but such decrease was not observed in the refractory group (U=67.5, P>0.05). Serum level of ß2-MG was positively correlated with serum level of miR-221 (r=0.524, P<0.01). Conclusion: miR-221/222 in serum and plasma cells may be biomarkers for early diagnosis of MGUS, and are helpful for diagnosis and efficacy evaluation of MM.

More frequent IgD and reduced CD200 expression in Chinese patients younger than 50 years old with multiple myeloma: a multicenter analysis.

We retrospectively analyzed the presenting features and survival of 194 newly diagnosed patients with multiple myeloma in the People's Republic of China. Compared with older patients, younger patients had a higher percentage of IgD isotype, lower percentage of International Staging System Stage 3 disease, higher albumin level, and lower frequency of high ß2-microglobulin and CD200 expression. There was no difference in sex, Durie-Salmon stage, bone lesion degree, creatinine, lactate dehydrogenase, fluorescence in situ hybridization, and expression of other antigens. Among all 940 newly diagnosed patients with multiple myeloma, those younger than 50 years had better overall survival and progression-free survival than older patients. Of these patients, 457 were treated with a bortezomib-containing regimen, and 450 received conventional therapy. Younger patients treated with bortezomib had better overall survival and progression-free survival than older patients. However, younger patients treated with conventional therapy had the same survival as older patients.

Bone Marrow Urokinase Plasminogen Activator Receptor Levels are Associated with the Progress of Multiple Myeloma.

Objective To determine the mRNA and protein levels of urokinase plasminogen activator receptors (uPAR) in bone marrow fluid and bone marrow tissue from multiple myeloma (MM) patients and assess association of uPAR level with prognosis of MM. Methods uPAR levels in bone marrow fluid of 22 MM patients at the stable and progressive stages and 18 iron deficiency anemia patients with normal bone marrow (control) were examined by ELISA. Furthermore, uPAR expression in bone marrow tissue was investigated by RT-PCR and Western blot, respectively. The distribution of uPAR in MM cells was examined using immunofluorescence staining. The pathological changes in different stages of MM patients were studied by HE staining. Results uPAR level in bone marrow fluid of MM patients (1.52±0.32 µg/ml) was found to be higher than that in the control group (0.98±0.15 µg/ml). Interestingly, uPAR protein (0.686±0.075 vs. 0.372±0.043, P<0.05) and mRNA (2.51±0.46 vs. 4.46±1.15, P<0.05) expression levels of MM patients at the progressive stage were significantly higher than those at the stable stage. The expression of uPAR in MM bone marrow was confirmed by immunofluorescence staining. Moreover, HE staining revealed a great increased number of nucleated cells and severe impairment of hematopoietic function in the bone marrow of patients with progressive-stage myeloma. Conclusion Our study reveals that uPAR expression is positively correlated with the development and progress of MM.

[Transition to aggressive phase in a multiple myeloma patient with IgH/CCND1 translocation and diffuse osteosclerotic lesions].

CASE: A 71-year-old woman had been diagnosed as having osteosclerotic myeloma (BJP-λ type) three years prior to the current presentation, based on tumor biopsy from the forehead showing plasmacytoma with systemic osteosclerotic lesions. At 71 years of age, she underwent transverse colectomy for a tumor in the hepatic flexure of the large intestine, and it was diagnosed as IgH/CCND-1-positive plasmacytoma of the large intestine. Although serum vascular endothelial cell growth factor (VEGF) was not elevated, the plasmacytoma was largely positive for VEGF staining. She subsequently experienced transformation to aggressive myeloma over a short period of time. Osteosclerotic myeloma is a rare disease that accounts for less than 3% of all myelomas, and requires differentiation from POEMS syndrome. In this case, peripheral nerve symptoms, which are necessary for the diagnosis of POEMS syndrome, were not confirmed. Thus, this case was diagnosed as having osteosclerotic myeloma. By contrast, abnormal IgH/CCND-1 is confirmed in 15% of patients with myeloma, and 25% of those with POEMS syndrome. While it is unclear whether this genetic abnormality is involved in the development of an osteosclerotic lesion, it is expected that data from patients with osteosclerotic myeloma and POEMS syndrome will be accumulated in the future, allowing clarification of the relationship between the genetic abnormality and osteosclerosis.

[A case of renal Fanconi syndrome due to Bence Jones' protein K-type multiple myeloma].

A 60-year-old man, who had been treated for chronic kidney disease and chronic hepatitis B infection, was referred to our hospital following presentation with thoracic bone pain and exacerbation of proteinuria and hematu- ria. On admission, laboratory test results showed evidence of hypophosphatemia, glucosuria and elevated levels of both urinary NAG and 62MG.The patient was diagnosed with Fanconi syndrome based on findings indicating the presence of pan-aminoaciduria, elevated urinary excretion of uric acid and an increased phosphorus reabsorption rate. Furthermore, bone scintigraphy showed increased multiple symmetric uptake of radiotracer in both sides of the ribs, leading to the diagnosis"of hypoposphatemia-related osteomalacia with renal Fanconi syndrome. Urinary immunoelectrophoresis indicated the presence of K Bence Jones' protein (BJP). A bone marrow biopsy examina- tion showed that the plasma-to-cell ratio was less than 10%. However, the patient had over lg/day of proteinuria and suppression of serum IgM (18mg/dL) and was, therefore, diagnosed with multiple myeloma based on SWOG criteria. Light microscopic examination showed evidence of glomerulosclerosis, intimal thickness of interlobular arteries and acidophilic granular deposits in the cytoplasm of the proximal epithelial tubular cells. Immunofluores- cence indicated positive anti-K staining in these regions. Electron microscopic examination of the proximal tubular epithelial cells revealed the presence of numerous diamond-shaped and oval crystals, thought to be the K light chain of BJP. In general, cast nephropathy, light chain deposition disease (LCDD) and AL amyloidosis are recog- nized renal injuries caused by myeloma. However, there have been few clinical reports of Fanconi syndrome with multiple myeloma, such as the case study we have described here. In addition, histological examination of a biopsy sample provided further evidence of K BJP in the proximal epithelial tubular cells.

Mieloma múltiple en neuroradiólogo vascular intervencionista: ¿Enfermedad profesional? / Multiple myeloma in a vascular and interventional neuroradiologist: Occupational disease?

Varón de 59 años, neuroradiólogo vascular intervencionista con una antigüedad laboral de 35 años. Sin antecedentes patológicos de interés. Asintomático hasta enero de 2015 que inicia dorsalgia súbita tras sobresfuerzo. Acude a consulta de salud laboral con el diagnóstico de mieloma múltiple. Solicita informe para la determinación de la contingencia como enfermedad profesional. Investigamos el caso revisando los efectos biológicos de las radiaciones ionizantes (deterministas y estocásticos), el cuadro de enfermedades profesionales provocadas por radiaciones ionizantes (grupo 2 y 6) y haciendo una profunda búsqueda bibliográfica acerca de los trastornos hematopoyéticos y la exposición a radiaciones ionizantes. Finalmente apoyamos la determinación de enfermedad profesional (AU)

A 59 years-old 4male, vascular and interventional neuroradiologist with 35 years of seniority.Without pathological backgrounds of interest. Asymptomatic until January 2015 when he starts with sudden dorsalgia after muscular effort. Attends to consultation of occupational health service with the diagnosis of Multiple Myeloma. He seeks for determination of occupational contingency. We researched the case checking for the biological effects of the ionizing radiations (deterministic and stochastic effects), the occupational disease chart caused by ionozing radiations (group 2 and 6) and making an exhaustive bibliographical search of the hematopoietic disorders and the exposition to ionizing radiations. Finally we support the determination of occupational contingency (AU)