Supplementary Figures 1-7 from Antimyeloma Activity of the Orally Bioavailable Dual Phosphatidylinositol 3-Kinase/Mammalian Target of Rapamycin Inhibitor NVP-BEZ235
Supplementary Figures 1-7 from Antimyeloma Activity of the Orally Bioavailable Dual Phosphatidylinositol 3-Kinase/Mammalian Target of Rapamycin Inhibitor NVP-BEZ235
e20013 Background: Multiple Myeloma (MM) is an incurable plasma cell (PC) malignancy and high risk (HR) MM remains an unmet clinical need. Translocation 4;14 occurs in 15% of MM and is associated with an adverse prognosis. A deeper understanding of the biology and immune micro-environment of t(4;14) MM is necessary for the development of effective targeted therapies. Single Cell multi-omics provides a new tool for phenotypic characterization of MM. Here we used Proteona’s ESCAPE™ single cell multi-omics platform to study a cohort of patients with t(4;14) MM. Methods: Diagnostic bone marrow (BM) samples from 14 patients with t(4;14) MM were analysed using the ESCAPE platform from Proteona which simultaneously measures gene and cell surface protein expression in single cells. Cryopreserved BM samples were stained with 65 DNA barcoded antibodies and subsequently sorted on CD138 expression. The CD138 positive and negative fractions were recombined at a known ratio for analysis using the 10x Genomics 3’ RNAseq kit. Resulting data were analyzed with Seurat and MapCell. Results: The patients had a median age of 63 years. All received novel agent based induction. Median progression free and overall survival (PFS and OS) were 22 and 34 months respectively. MMSET was overexpressed in all PCs while FGFR3 expression could be categorized into zero cells expressing FGFR3, low expression (< 10% of cells expressing FGFR3) or high expression (> 80% of cells expressing FGFR3). We also found heterogeneity in the expression of cancer testis antigens (CTA) such as FA133A and CTAG2 between PC clusters across samples. Variation in the immune microenvironment of the BM was seen across all patient samples with no correlation between cell types and PFS or OS. However, an analysis of BM samples at diagnosis and relapse in one patient showed a shift in the ratio of T cells to CD14 monocytes with a ratio of 5.7 at diagnosis compared to 0.6 at relapse. Further analysis of PCs in this patient found 8 PC populations, each containing variable numbers of cells from both the diagnostic and relapse samples. This suggests that all populations present at relapse were also present at diagnosis, although at variable proportions. Increased expression of RCAN3 (associated with cereblon depletion) was detected at relapse. Conclusions: We present the first application of single cell multi-omics immune profiling in high risk MM. The heterogeneity in expression of CTA has implications for the application of immunotherapies, while the upregulation of RCAN3 may explain failure of immunomodulatory therapy. Our small sample size may explain the lack of correlation between gene or protein expression with clinical outcomes. We propose that t(4;14) MM is a genomically and immunologically heterogeneous disease. Single cell analysis of larger cohorts is required to build on our findings.
Abstract The phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway mediates proliferation, survival, and drug resistance in multiple myeloma (MM) cells. Here, we tested the anti-MM activity of NVP-BEZ235 (BEZ235), which inhibits PI3K/Akt/mTOR signaling at the levels of PI3K and mTOR. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric survival assays showed that MM cell lines exhibited dose- and time-dependent decreased viability after exposure to BEZ235 (IC50, 25–800 nmol/L for 48 hours). MM cells highly sensitive (IC50, <25 nmol/L) to BEZ235 (e.g., MM.1S, MM.1R, Dox40, and KMS-12-PE) included both lines sensitive and resistant to conventional (dexamethasone, cytotoxic chemotherapeutics) agents. Pharmacologically relevant BEZ235 concentrations (25–400 nmol/L) induced rapid commitment to and induction of MM.1S and OPM-2 cell death. Furthermore, normal donor peripheral blood mononuclear cells were less sensitive (IC50, >800 nmol/L) than the majority of MM cell lines tested, suggesting a favorable therapeutic index. In addition, BEZ235 was able to target MM cells in the presence of exogenous interleukin-6, insulin-like growth factor-1, stromal cells, or osteoclasts, which are known to protect against various anti-MM agents. Molecular profiling revealed that BEZ235 treatment decreased the amplitude of transcriptional signatures previously associated with myc, ribosome, and proteasome function, as well as high-risk MM and undifferentiated human embryonic stem cells. In vivo xenograft studies revealed significant reduction in tumor burden (P = 0.011) and survival (P = 0.028) in BEZ235-treated human MM tumor-bearing mice. Combinations of BEZ235 with conventional (e.g., dexamethasone and doxorubicin) or novel (e.g., bortezomib) anti-MM agents showed lack of antagonism. These results indicate that BEZ235 merits clinical testing, alone and in combination with other agents, in MM. [Cancer Res 2009;69(14):5835–42]
Cutaneous plasmacytosis (CP) is a rare skin disorder characterized by multiple reddish brown nodules with polyclonal plasma cell proliferation. It has most often been reported to affect the trunk but is also known to affect the face and extremities in adults and is predominantly seen in Asians. The etiology is poorly understood, and there is no consensus on treatment methods.Five cases diagnosed to have CP were collated from our institution. Their clinicopathologic features and treatment outcomes were reviewed.Four of the 5 patients presented with lesions that affected multiple sites of the body including the trunk, axillae, face, and limbs. The remaining patient had lesions localized to his axillae. The lesions were generally asymptomatic. All patients had hypergammaglobulinaemia but only one had a faint monoclonal band detected on immunofixation. Common findings in the biopsy results for all patients were perivascular plasma cell infiltrates without light chain restriction on kappa/lambda staining, as well as mast cell infiltrates. Partial remission of cutaneous lesions was observed in 3 of the patients, with 2 of them responding well to psoralen and ultraviolet A radiation therapy.CP presents with distinctive clinical features and characteristic histological features including polyclonal perivascular plasma cell infiltrates. The axilla seems to be a frequent and characteristic site of involvement and may be a useful clinical clue to the condition. In the management of patients with CP, it is important to exclude secondary causes of plasmacytic infiltrates. While there are no clearly established treatment modalities for CP, psoralen and ultraviolet A radiation therapy may be a viable option in view of the clinical improvement observed in our patients who received it.
Introduction: This study aimed to evaluate the clinical utility of positron emission tomography/magnetic resonance imaging (PET/MRI), especially in comparison with PET/computed tomography (CT), which has been widely used in clinical practice in multiple myeloma. Method: F-18 fluorodeoxyglucose PET/MRI and PET/CT studies were done at baseline and when at least a partial response to treatment was achieved. These were done for newly-diagnosed myeloma patients who have not had more than 1 cycle of anti-myeloma treatment, or for relapsed and/or refractory myeloma patients before the start of next line of therapy. Results: PET/MRI correlated significantly with PET/CT, in terms of number of lesions detected, standardised uptake value (SUVmean and SUVmax, both at baseline and post-treatment. PET/MRI and PET/CT correlated with survival at baseline, but not post-treatment. Conclusion: In this study, PET/MRI was more sensitive in detecting early disease and disease resolution post-treatment, compared with PET/CT. However, PET/MRI was less sensitive in detecting lesions in the ribs, clavicle and skull.
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