sj-docx-1-tah-10.1177_20406207221127532 – Supplemental material for Avatrombopag for the treatment of thrombocytopenia post hematopoietic stem-cell transplantation

Supplemental material, sj-docx-1-tah-10.1177_20406207221127532 for Avatrombopag for the treatment of thrombocytopenia post hematopoietic stem-cell transplantation by Meng Zhou, Jiaqian Qi, Chengyuan Gu, Hong Wang, Ziyan Zhang, Depei Wu and Yue Han in Therapeutic Advances in Hematology</p

Societal implications of medical insurance coverage for imatinib as first-line treatment of chronic myeloid leukemia in China: a cost-effectiveness analysis

<p><b>Objectives:</b> Imatinib (Glivec) and nilotinib (Tasigna) have been covered by critical disease insurance in Jiangsu province of China since 2013, which changed local treatment patterns and outcomes of patients with chronic myeloid leukemia (CML). This study evaluated the long-term cost-effectiveness of insurance coverage with imatinib as the first-line treatment for patients with CML in China from a societal perspective.</p> <p><b>Methods:</b> A decision-analytic model based on previously published and real-world evidence was applied to simulate and evaluate the lifetime clinical and economic outcomes associated with CML treatments before and after imatinib was covered by medical insurance. Incremental cost-effectiveness ratio (ICER) was calculated with both costs and quality-adjusted life years (QALYs) discounted at 3% annually. Different assumptions of treatment benefits and costs were taken to address uncertainties and were tested with sensitivity analyses.</p> <p><b>Results:</b> In base case analysis, both cost and effectiveness of CML treatments increased after imatinib was covered by the medical insurance; on average, the incremental QALY and cost were 5.5 and ¥277,030 per patient in lifetime, respectively. The ICER of insurance coverage with imatinib was ¥50,641, which is less than the GDP per capita of China. Monte Carlo simulation resulted in the estimate of 100% probability that the insurance coverage of imatinib is cost-effective. Total cost was substantially saved at 5 years after patients initiated imatinib treatment with insurance coverage compared to no insurance coverage, the saved cost at 5 years was ¥99,565, which included the cost savings from both direct (e.g. cost of bone marrow or stem cell transplant) and indirect costs (e.g. productivity loss of patients and care-givers).</p> <p><b>Conclusions:</b> The insurance coverage of imatinib is very cost-effective in China, according to the local cost and clinical data in Jiangsu province. More importantly, the insurance coverage of imatinib and nilotinib have changed the treatment patterns of CML patients, thus dramatically increasing life expectancy and quality-of-life (QoL) saving on productivity losses for both CML patients and their caregivers.</p

GAS2 is up-regulated in chronic myeloid leukemia.

<p>(A) The cells from chronic myeloid leukemia (CML) patients and healthy donors were collected and processed to yield nucleated cells, and then CD34⁺ cells were enriched with immunomagnetic method. The gene expression of <i>GAS2</i> was assessed with nucleated cells and CD34⁺ cells, respectively. For the nucleated cells, 7 healthy donors and 25 CML patients were recruited; for the CD34⁺ cells, 3 healthy donors and 8 CML patients were recruited. (B) Immunofluorescence assay was used to detect the expression of GAS2 (green) in CD34⁺ cells of normal bone marrow (NBM) and CML patient, together with K562 and MEG-01 cells. Hoechst 33342 was used to visualize the nuclei (blue). The representative graphs of individual section with confocal microscopy analyses were shown. Original magnification: ×150. The data were shown as mean ± S.E.M.; **mean <i>p</i><0.01, which was estimated with student <i>t</i>-test in a two-tailed fashion.</p

The expression and activity of beta-catenin are not affected by GAS2 targeting.

<p>(A) Immunofluorescence assay was used to detect the expression of beta-catenin (red) in Venus and GAS2DN transduced cells with Hoechst 33342 to visualize the nuclei (blue). The representative graphs of individual section with confocal microscopy analyses were shown. (B) The expression of beta-catenin was assessed quantitatively with flow cytometry in various virally transduced K562 cells. (C) The transcription activity of beta-catenin was measured in SW620 (human colorectal adenocarcinoma cell, as a positive control of beta-catenin activity), K562 and MEG-01 cells (upper panel), Venus and GAS2DN transduced K562 and MEG-01 cells (middle panel), and shNC, shGAS2#1 and shGAS2#2 transduced K562 cells (lower panel). The beta-catenin activated reporter vector (TOPflash) or its mutant vector (FOPflash) plus renilla reporter vector were used for transfection. The ratio of normalized TOPflash versus FOPflash was used to represent the activity of beta-catenin. (D) The cytosol and nucleus protein from various virally transduced cells were purified, and then subjected to immunoblot with antibodies against Tublin, beta-catenin and histone H3, respectively. The representative graph of 3 independent experiments was displayed.</p

EZH2 Mutations Are Related to Low Blast Percentage in Bone Marrow and -7/del(7q) in De Novo Acute Myeloid Leukemia

<div><p>The purpose of the present work was to determine the incidence and clinical implications of somatic EZH2 mutations in 714 patients with de novo acute myelogenous leukemia by sequencing the entire coding region. EZH2 mutations were identified in 13/714 (1.8%) of AML patients were found to be more common in males (<i>P</i> = 0.033). The presence of EZH2 mutations was significantly associated with lower blast percentage (21–30%) in bone marrow (<i>P</i><0.0001) and -7/del(7q) (<i>P</i> = 0.025). There were no differences in the incidence of mutation in 13 genes, ASXL1, CBL, c-KIT, DNMT3A, FLT3, IDH1, IDH2, MLL, NPM1, NRAS, RUNX1, TET2, and WT1, between patients with and without EZH2 mutations. No difference in complete remission, event-free survival, or overall survival was observed between patients with and without EZH2 mutation (<i>P</i>>0.05). Overall, these results showed EZH2 mutation in de novo acute myeloid leukemia as a recurrent genetic abnormality to be associated with lower blast percentage in BM and -7/del(7q).</p></div

Silence of CALPAIN2 partically rescues the suppressed cell proliferation upon GAS2DN expression.

<p>In order to delineate the role of CALPAIN2 in the inhibitory effect of GAS2DN, a lentiviral vector to silence CALPAIN2 was used. (A) The mRNA expression of <i>CALPAIN2</i>, (B) activity of calpain, (C) cell proliferation in liquid culture and, (D) the CFC production of various transduced K562 cells were assessed. The data were shown as mean ± S.E.M. from more than 3 independent experiments; *mean <i>p</i><0.05, which was estimated with student <i>t</i>-test.</p

Targeting GAS2 suppresses the <i>in vitro</i> and <i>in vivo</i> growth of MEG-01 cells.

<p>(A) The expression of <i>GAS2</i> in shNC (control), shGAS2#1 and shGAS2#2 transduced MEG-01 cells was measured with Q-RT-PCR. (B) The expression of GAS2 protein in shNC, shGAS2#1 and shGAS2#2 transduced cells were analyzed with FACS. (C) The calpain activities of various virally transduced cells were measured. (D) & (E) The proliferation and CFC production of various virally transduced cells were assessed. (F) Tumour growth of the Venus and GAS2DN expressed cells in nude mice were observed, and the fluorescence was monitored with IVIS II imaging system as well. (G) Representative pictures of the H & E staining of the tumours were presented. (H) The average sizes of the tumours formed by the Venus and GAS2DN expressed cells were compared. The tumour size was estimated with the equation: volume = 1/2 × length × width². Original magnification: ×200. *mean <i>p</i><0.05 and **mean <i>p</i><0.01, which were estimated with student <i>t</i>-test in a two-tailed fashion.</p

The transcriptome analysis of GAS2DN transduced cells.

<p>(A) Van-diagram of meta-analysis of the gene expression data. The differentially expressed genes identified in MEG-01 cells (blue) upon GAS2DN were compared with 2 public accessible datasets, namely the comparison between CML and NBM CD34⁺ cells (brown) and the comparison between CD34⁺ cells in CP and those in BC (purple). The 388 up-regulated genes in MEG-01 cells upon GAS2DN was compared with the 1462 genes down-regulated genes in CD34⁺ cells along disease progress and 119 genes down-regulated genes comparing CD34⁺ cells in CML versus NBM (left panel). Conversely, The 343 down-regulated genes in MEG-01 cells upon GAS2DN was compared with the 1653 genes up-regulated genes in CD34⁺ cells along disease progress and 1402 genes up-regulated genes comparing CD34⁺ cells in CML versus NBM (right panel). The numbers of genes for comparisons were shown in the middle of each pie, and the numbers of overrepresented genes were indicated in italic. (B) The relative gene expression of <i>HNRPDL, PTK7</i> and <i>UCHL5</i> in GAS2DN transduced K562, MEG-01 and primary CML cells (n = 3) was compared to that of control cells. (C) The gene expression of <i>HNRPDL, PTK7</i> and <i>UCHL5</i> in CML and normal bone marrow cells was assessed. For the nucleated cells, the samples of 7 individual healthy donor and 23 CML patients were used; for CD34⁺ cells, the samples of 4 individual healthy donors and 8 CML patients were used. (D) The protein expression of HNRPDL was analyzed with western blot. A representative graph was shown (left panel) and, the quantitative relative expression of both isoforms of HNRPDL was summarized from 3 independent replicates (right panel). RE, relative expression. *mean <i>p</i><0.05, which was estimated with student <i>t</i>-test in a two-tailed fashion.</p

Targeting GAS2 with both RNAi and GAS2DN inhibits K562 cells.

<p>(A) The expression of <i>GAS2</i> was measured in shNC (control), shGAS2#1 and shGAS2#2 transduced cells with Q-RT-PCR. (B) The expression of GAS2 protein in shNC, shGAS2#1 and shGAS2#2 transduced cells were analyzed with FACS. (C) Calpain activities of shNC, shGAS2#1 and shGAS2#2 transduced cells were measured. (D) & (E) The proliferation and colony-forming cell (CFC) capacities of various virally transduced K562 cells were measured. (F) The schematic structure of lentiviral vector to express GAS2DN. LTR, long terminal repeat; pSFFV, spleen focus forming virus promoter; IRES, internal ribosome entry site; Venus, the enhanced yellow fluorescent protein; GAS2DN, the dominant negative form of GAS2. (G) The western blot was used to detect the expression of GAS2DN with an antibody recognizing N-terminus of GAS2 (N-GAS2). (H) Calpain activities of Venus and GAS2DN transduced cells were measured. (I) K562 cells were transduced with various lentiviral vectors. The FACS purified cells were plated in methylcellulose media with Imatinib mesylate (IM, final concentration as 1 µM and 2 µM), and then the colonies were numerated. The survival rates of CFC (IM treated versus IM untreated) of various transduced cells were calculated and compared. The data were shown as mean ± S.E.M. from at least 3 independent experiments; *mean <i>p</i><0.05, which was estimated with student <i>t</i>-test in a two-tailed fashion.</p

Growth Arrest Specific 2 Is Up-Regulated in Chronic Myeloid Leukemia Cells and Required for Their Growth

<div><p>Although the generation of BCR-ABL is the molecular hallmark of chronic myeloid leukemia (CML), the comprehensive molecular mechanisms of the disease remain unclear yet. Growth arrest specific 2 (GAS2) regulates multiple cellular functions including cell cycle, apoptosis and calpain activities. In the present study, we found GAS2 was up-regulated in CML cells including CD34⁺ progenitor cells compared to their normal counterparts. We utilized RNAi and the expression of dominant negative form of GAS2 (GAS2DN) to target GAS2, which resulted in calpain activity enhancement and growth inhibition of both K562 and MEG-01 cells. Targeting GAS2 also sensitized K562 cells to Imatinib mesylate (IM). GAS2DN suppressed the tumorigenic ability of MEG-01 cells and impaired the tumour growth as well. Moreover, the CD34⁺ cells from CML patients and healthy donors were transduced with control and GAS2DN lentiviral vectors, and the CD34⁺ transduced (YFP⁺) progeny cells (CD34⁺YFP⁺) were plated for colony-forming cell (CFC) assay. The results showed that GAS2DN inhibited the CFC production of CML cells by 57±3% (n = 3), while affected those of normal hematopoietic cells by 31±1% (n = 2). Next, we found the inhibition of CML cells by GAS2DN was dependent on calpain activity but not the degradation of beta-catenin. Lastly, we generated microarray data to identify the differentially expressed genes upon GAS2DN and validated that the expression of <i>HNRPDL</i>, <i>PTK7</i> and <i>UCHL5</i> was suppressed by GAS2DN. These 3 genes were up-regulated in CML cells compared to normal control cells and the growth of K562 cells was inhibited upon HNRPDL silence. Taken together, we have demonstrated that GAS2 is up-regulated in CML cells and the inhibition of GAS2 impairs the growth of CML cells, which indicates GAS2 is a novel regulator of CML cells and a potential therapeutic target of this disease.</p></div