Introduction . We had previously reported that TLR 2 levels were significantly elevated in patients with polycythemia vera and MPN ( PV , ET, MF grouped together ) and TLR2 levels were related to JAK2 mutation and leukocytosis ( Blood (2022) 140 (Supplement 1): 12185-12186.). Since MPN has increased incidence of thrombosis in patients with JAK2V617F mutation and leukocytosis , therefore , current studies were done to test whether TLR-2 is related to thrombosis in MPN . In vitro platelet activation ( an pre-thrombotic marker) and clinical episodes of thrombosis were then investigated. . Material and Methods. 119 patients ( 51ET, 37 PV, 31MF, 21 CTR) were studied . 1) platelet activation. : The diluted blood was incubated with platelet agonists Pam3CSK4 (TLR2 agonist) or lipopolysaccharide (LPS) (TLR4 agonist) for 15 minutes at room temperature , then platelet activation was measured by Flow cytometry assaying CD40L, P-selectin, PAC-1 and LPI ( leukocyte-platelet-interaction). We obtained more consistent results from the LPI methods . Therefore, LPI methods were used to measure the platelet activation. LPI method was measured by CD45 and CD41 antibodies (Miltenyi Biotec, Auburn, CA), and platelet activation were measured as the percentage of CD45 cells with aggregated CD41. To test whether TLR2 or TLR4 were responsible for the LPI response ,TLR2 inhibitor C29 (InvivoGen, San Diego, CA) or the TLR4 inhibitor C34 ((Sigma-Aldrich, St. Louis, MO) were added to the LPI assay. Effects of inhibitions were measured by fold-changes which were calculated by dividing the LPI values after activation by either Pam3CSK4 (TLR2 agonist) or LPS (TLR4 agonist), with or without added inhibitors, by the LPI values before activation or inhibition. Results . 1) As shown in Fig A, a) un-activated MPN (U-MPN) patients had more LPI than un-activated controls (u-CTR), with mean ± SE of 13.01 ± 1.12 and 5.01 ± 0.97 in U-MPN and U-CTR, respectively (P<0.01). This is consistent with previous reports that un-activated MPN patients have more intrinsically activated platelets than un-activated controls .b) Greater LPI response by activation by Pam3CSK4, (TLR2 agonist ) than by LPS ( lipopolysaccharide)) (TLR4 agonist) . MPN and controls ( mean ± SE ) were of 22.25 ± 0.99 and 10.46 ± 0.44 ( by Pam3CSK4) and 12.12 ± 2.16 and 4.34 ± 0.32 ( by LPS) (P<0.0005) respectively . 2). Fig B showed the TLR2 inhibitor C29 significantly suppressed LPI ( measured by fold-changes ) , in un-activated MPN (0.38 ± 0.05), activated controls (0.42 ± 0.11), and activated MPN (0.42 ± 0.05). Conversely, the TLR4 inhibitor C34 had no inhibitory effect on un-activated MPN (0.98 ± 0.03), activated MPN (1.02 ± 0.02), or activated controls (0.84 ± 0.06) . 3) As shown in Table 2 , TLR 2 levels elevated Patients ( TLR2-E) ( defined as more than mean plus 2 standard deviation of controls (> 310 MFI) , have more thrombosis episode (29%) than TLR2 -normal patients (11%) . Conclusion . TLR2 likely plays a significant role in thrombosis in MPN patients on the basis : 1) JAK2 mutated patients have more TLR2 levels 2) TLR2-E patients have more leukocytosis than TLR-N patients 3) LPI was significantly elevated with TLR2 agonist ( Pam3SK4) but not by TLR4 agonist ( LPS) , and LPI was significantly decreased by TLR2 inhibitor and not by TLR4 inhibitor. 4) clinical evidence of more thrombosis in TLR -E patients than TLR-N patients.

Abstract This study investigated TLRs on inflammatory pathways in Ph(-)MPNs. The results showed that TLR2 were increased in PV and MPN (PV + ET + MF), while TLR4 were only increased in MPN. TLR3, 7, and 9 were not elevated. We further cultured mdDC cells and demonstrating TLR2 elevated (TLR-E) patients secreted more cytokines than TLR2 normal (TLR-N), confirming the importance of TLR2. S100A9 and ROS were also measured, revealing S100A9 levels were increased in PV, MF, and MPN, while ROS levels increased only in MF and not in PV or ET. This data suggests that the pathogenesis of MPNs initially involves TLR2, minorly fromTLR4, along with S100A9 then induce ROS formation, JAK2 mutation,, leading ET and PV evolved into MF or leukemia. Further analysis of the data and studies, we found. 1) patients with Jak2 mutations or leukocytosis exhibited higher TLR2 expression, 2) cells from MPN patients displayed a stronger response to PM3CSK4 (TLR2 agonist) in leukocyte-platelet interaction compared to LPS ( TLR4 agonist). Adding TLR-2 inhibitor (not TLR-4 inhibitor) significantly attenuated this response, 3) TLR2-E patients experienced more thrombosis (29%) than TLR2-N (19%). These findings also suggest TLR2 plays a significant role in thrombosis in MPN.

Introduction There has been significant progress in immune checkpoint inhibitor (ICI) therapy in using anti-PD-1 and or anti-CTLA-4 in many solid tumor types. However, only a single failed study has been published in treating Ph(-) myeloproliferative neoplasm (MPN) (Mascarenhas et al 2020, Blood 2020, 135, supply, 1-60.). New inhibitory pathways are under investigation, and drugs blocking LAG-3, TIM-3, TIGIT, VISTA are being investigated and developed in treating other solid tumors. Therefore, to further possible advance ICI therapy in Ph(-) MPN , we measured these 2nd generation ICIs including LAG-3, TIM-3, TIGIT, VISTA in MPN cells. Material and Methods Flow cytometric analysis of 2nd ICI Expression 1) on MDSC : blood MNC cells after CD14 + microbeads (Miltenyi Biotec) isolation , then assayed by flow cytometry using monoclonal antibodies from BD Biosciences, with gating of HLA-DR - CD14 + CD33 + as M-MDSC (monocytic MDSC) , and HLA-DR - CD14 - CD33 + as G-MDSC (granulocytic ). 2) on Different population cells : Expression levels of the 2nd ICIon the CD3 +, CD4 +, CD8 +, CD14 +, CD34 +, CD41a +, and CD71b + were also assayed by flow cytometry . Human VSIG3 Fc treatment and T cell activation and proliferation: MNCs were cultured for three days with recombinant human VSIG3- IgG1 Fc chimera (R&D Systems). Recombinant human IgG1 Fc (R&D System)was used as control.The CD3 + cells were maintained in culture medium plus IL-2 (10ng/ml), and stained with 5uM CFSE according to the manufacturer's protocol (Thermo Fisher). The CD3 + cells were stimulated with T-Activator CD3/CD28, and combined VSIG3-IgG1 Fc or IgG Fc treated MNCs at 1:2 ratio for further culture of three days. CFSE cell counts were numerated in gated CD4 + and CD8 + cells. Data were processed using FlowJo VX. Results 1) 2nd Generation ICI (2nd ICI) expression on the MDSC a) Percentage of cells . Fig 1,showed no significant difference of the LAG3 , TIGIT , TIM3 on either G-MDSC or M-MDSC , however, VISTA expression was significantly higher in G-MDSC and M-MDSC from MPN patients than in controls. The mean + SE (% of positive cells)for G-MDSC in MPN was ( 23.97 + 6.86 ) and in controls was (0.00 + 0.00) ( P=0.003). For M-MDSC, the MPN was ( 31.56 + 6.62 ) and for controls was ( 1.47 + 1.47) ( p=0.003). b) MFI of the 2nd ICI . Fig 2, showed no significant difference of LAG3 , TIGIT , TIM3 (MFI) on MDSC between MPN patients and controls. However, VISTA ( MFI) was significantly higher in MPN patients than controls, in G-MDSC , with mean + SE in MPN (3085+ 783.6 ) and controls (179.7 + 64.66) ( P<0.0001) as well as in M-MDSC, with mean + SE in MPN ( 4241 +617.7 ) and controls ( 159.7 + 31.29) ( p<0.001). 2) 2nd ICI expression on different cell populations. a) Percentage of cells expressing 2nd ICI. Fig 3, LAG3 , TIGIT , TIM3 on the different population of cells in MPN were not significantly different from controls, but VISTA was significantly higher in MPN patients than controls on the CD3 +, CD14 +, CD34 +cells. CD3 + cells were with mean + SE in MPN (20.40 + 5.94 ) and controls (0.91 + 0.44) ( P<0.05), CD14 + cells were with MPN ( 36.86 +0.12 ) and controls ( 0.79 + 0.24) ( p<0.005) and CD34 + cells were with MPN (24.08 + 10.47) and controls (2.30 +1,28) (P<0.05) , while in CD41 + and CD71 + cells VISTA expression was not different in MPN and controls. b) MFI of the 2nd ICI in different population of cells. There was no significant difference in MFI of LAG3 , TIGIT , TIM3, but there was a significant difference in VISTA expression (MFI ) on the CD3 +, CD14 +, CD34 + cells in MPN patients and controls. MFI of CD3 + cells were with mean + SE in MPN (3257 + 673.4 ) and controls (457.0 +59.02) ( P<0.05) ; CD14 were in MPN ( 5399 + 994.3 ) and controls ( 879.3 + 325.2 ) ( p<0.005) and CD34 + Cells were in MPN (6447 + 3785) and controls (327 + 122.4) (P<0.05), while there were no significance between MPN patients and controls of MFI in CD41 +.and CD71 + cells.controls ( 879.3 + 325.2 ) ( p<0.005) and CD34 + cells. 3) Human VSIG3 Fc treatment. as shown in Fig 5, VSIG3 , a specific ligand for VISTA , decreased MPN T cells response in MPN patients Conclusion . We analyzed the 2nd G of ICI in Ph(-) MPN , We found there were a significant VISTA and not the others including LAG3 , TIGIT , TIM3 expression on the MDSC and different cell population. We also demonstrated by adding VISTA specific ligand , T cell response were blunted . Further studies employing the VISTA antibody and siRNA are in progress. This will form the basis of employing anti-VISTA therapy in the future clinical trials of ICI therapy in Ph(-) MPN.

Introduction. We previously reported that PD-1 and PD-L1 were increased in patients with Ph(-) myeloproliferative neoplasm (MPN) ( Wang et al., Leuk Res 2019). The PD-1 inhibitor therapy or immune checkpoint inhibitor therapy (ICI) trial in MPN by Hobbs et al. reported a negative result (Blood, 2020 (supplement )). Resistance to ICI therapy has been reported to be related to myeloid-derived suppressor cells (MDSCs) in melanoma and breast carcinoma. We also previously reported that MDSCs were increased in patients with Ph(-) MPN (Wang et al., Leu Res 2016). Regulation of immune suppression by MDSCs has been reported to be related to PD-1and PD-L1 expression on the MDSC. Therefore, the current study measured PD-1 and PD-L1 expression in MDSCs in patients with Ph(-) MPN.

Recent evidence of increased constitutional symptoms and inflammatory cytokines in Philadelphia chromosome negative (Ph (-)) MPN suggests that an inflammatory response is important in the pathogenesis of Ph (-) MPN. Toll-like receptors (TLR), Receptor for Advanced Glycation End products (RAGE) and High mobility group protein B1 (HMGB1) are the important pathways for the inflammatory response. All these three important pathway proteins were studied in MPN diseases in the current studies. Materials and Methods: TLR assay. TLR 2,3, 4, 7, 9 quantification was performed by immuno-staining of 1×106 mononuclear cells (peripheral blood) which were incubated with fluorescence-conjugated anti-TLR-2,3, 4, 7, 9 antibodies and assayed by flow cytometry. HMGB1assay:HMGB1 ELISA kit from Immuno-Biological Laboratories, Inc. (IBL-America) were used. The plasma samples were diluted four times with the provided sample dilution buffer, and assayed in duplicate according to the manufacturer's suggestion. RAGE (RT-PCR) Assay: Total RNA was extracted from normal control or patient mononuclear cells. Predesigned primers for RAGE, and internal control genes were ordered from Qiagen (Germantown, MD). Real-time PCR was performed using SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA) on Bio-Rad iQ5 Multicolor Real-Time PCR Detection System. At least three house-keeping genes (ribosomal protein L4, TATA box binding protein, and tubulin-α 1b) were used as normalization controls. The expression of RAGE were compared with each internal control. Average of three was used to calculate the ratio of final patient to normal Results: Total of 97 patients with MPN were studied 1) TLR: TLR 3,7,9 was not significantly different from controls. But TLR 2 was significantly increased in both PV, as well as in the MPN group when PV, ET and MF were grouped together as MPN (Fig A). TLR 4 was not significantly increased in PV, ET, MF individually but was found to be significantly increased than the controls, when they are grouped together as MPN (Fig B). 2) RAGE: No significant difference was found between ET, PV, MF individually or when they were grouped together as MPN than the controls (Fig C). 3) HMGB1: No significant difference was seen between ET, PV, MF or when they were grouped as MPN (Fig D). Conclusion: Current study suggests that TLR pathway especially TLR2, and to a lesser extent TLR4 are the important pathways for inflammatory response with increased inflammatory cytokines in MPN, while HMGB1 and RAGE pathways were not different from controls. Figure Disclosures No relevant conflicts of interest to declare.

We previously reported that by flow cytometry study of mononuclear cells (peripheral blood) of IGF-1 R (insulin growth factor -1 receptor) can help in differentiating polycythemia vera (PV) from secondary erythrocytosis (PLoS ONE 11(11): e016529), in patients negative for JAK2V617F mutation who requires frequent phlebotomy. EEC (Endogenous erythroid colony formation) formation which was shown to be related to IGF-1 signaling was employed as a minor criteria of PV prior to 2016 WHO criteria for the diagnosis of PV. IRS-2, an IGF-1 activation downstream signaling protein was found to be a docking protein, specific for erythropoietin-induced erythropoiesis. Current studies were designed to correlation between IGF-1R over-expression to EEC (Endogenous erythroid colony formation) formation and IRS-2 over expression in those patients who required frequent phlebotomy with elevated IGF-1R and JAK2 mutation negative patients. Materials and Methods: Patients: 8 patients withsecondary polycythemia (JAK2V617F mutation negative), and 12 MPN patients (6 ET, 3 PV and 3 MF) were studied. Of the 12 MPN patients, 11 were JAK2 mutation positive and 1 was CALR mutation positive. All 20 patients had elevated IGF-1 R. Methods: 1) IGF-1R expression: 106 cells blood mononuclear cells were incubated with 10 μL of PE-conjugated control or anti-IGF1R IgG (R&D Systems) for 30 minutes. Fluorescent intensity was detected by a flow cytometry. 2) EEC formation: Erythroid colony cultures using Methyl cellulose based medium without (MethoCult H4230) or with pre-inclusion of cytokines (MethoCult H4230, including erythropoitin) from StemCell Technologies Inc. were used. On day 10-12, colonies were scored for Burst forming unit-erythroid (BFU-e), Colony forming unit-erythroid (CFU-e), and Colony forming unit cells (CFU-c) according to the manufacturer's graphic guide. 3) IRS-2 Expression: RT-PCR Predesigned primers for IRS-2 and internal control genes were ordered from Qiagen (Germantown, MD). Real-time PCR blood mononuclear cells was performed using SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA) on Bio-Rad iQ5 Multicolor Real-Time PCR Detection System. At least three house-keeping genes (ribosomal protein L4, TATA box binding protein, and tubulin-α 1b) were used as normalization controls. The expression of IRS-2 was compared with each internal control and the final patient to normal control ratio was the average of the three. Results: 7 out of 8 patients with secondary PV, were found to have EEC formation. 12 MPN patients had increased expression of IRS-2 (Fig 1). 2 patients with secondary PV, so far studied also were found to have increased expression of IRS-2. Conclusion: 1) In patients with secondary PV (JAK2 mutation negative and increased IGF-1 and requiring frequent phlebotomy0, 7 out of 8 (87%) were found to have EEC formation. This suggests that IGF-1 by Flow cytometry can replace EEC as a supplement to the diagnosis of PV. 2) In JAK2 or CALR mutation positive patients , IGF-1R over-expression were found to have to have IRS-2 over-expression , 2 patients with secondary PV so far studied were also found to have elevated IRS-2 signaling. This suggests that the etiology of erythrocytosis in these secondary PV is likely through IRS-2 pathway. Further studies on this is still in progress. Disclosures No relevant conflicts of interest to declare.

Programmed Cell Death Receptor (PD-1) and its Ligand (PD-L1) pathway inhibitor therapy has been explored in the field of oncology treatment mainly for solid tumors. In hematologic malignancies, there is limited information except for Hodgkin's lymphoma, and there is even less information regarding myeloproliferative neoplasm (MPN). Therefore, we explored this by first measuring PD-1 and PD-L1 levels (percentage of positive cells) in 63 patients with Philadelphia chromosome-negative MPN (Ph(-) MPN), including 16 MF (12 PMF, 2 post-PV-MF, 2 post-ET-MF), 29 ET, and 18 PV. We found there was no significant difference in PD-1 or PD-L1 levels between the different MPN groups but that there was a significant difference when PV, ET and MF were grouped as MPN and compared with controls, of all immune cells including CD4(+), CD8(+), CD14(+) and CD34(+) progenitor cells. We further found a higher incidence of higher expression levels (more than 50% of cells with positive expression) of PD-1 and PD-L1 (20% and 26%, respectively) in the CD34(+) cells; in contrast, we found a low incidence (0.08-1.8%) in the immune cells in MPN patients. PD-1 and PD-L1 levels were also measured by MFI methods, and we obtained similar results except the measurements by percentage appeared to be more sensitive than the MFI methods. We found no correlation between PD-1 and PD-L1 expression levels and clinical features including WBC, platelet counts, hemoglobin levels, presence or absence of the JAK2, MPL, or CALR gene mutation, or splenomegaly. Since MPN represents stem cell disorders, the presence of elevated expression of PD-1 and PD-L1 in these cells suggests that the exploration of PD-1 and PD-L1 pathway inhibitor therapy may be worthwhile in Ph(-) MPN.

Introduction : PD-1 and PD-L1 pathway inhibitor therapy has been extensively explored in the field of oncology treatment recently, however there is no information so far about its usefulness in myeloid neoplasms (MPN). We have previously reported on PD-1 and PD-L1 expression in Philadelphia chromosome negative myeloid neoplasm (MPN), but now that we have accumulated more cases , we wish to report the final results.

Introduction. Evidences indicate that megakaryocyte/platelet lineage is crucial in BCR-ABL negative MPN, especially in myelofibrosis (MF) induction. Monocyte/Macrophage lineage were also reported to play a significant role . These Monocyte /Macrophage studies were on the blood monocytes such as have increased the intermediate forms of monocytes (CD14+ CD16+) and Substance P in MF , but in the bone marrow these cell lineages were not studied. Tumor-associated macrophages (TAM) have been classified into two subsets. as the M1 subset (CD86+ ) which produces Th1 cytokines such as interferon (IFN)-γ and pro-inflammatory cytokines. They are involved in the host defense against different pathogens and play a role in the anti-tumor immunity. The M2 subset, that secretes IL-10 and other iimmunosuppresive cytokines and are identified as CD206+ (in vitro induced by IL4) or CD163+(IL10). These M2 macrophages are associated with tumor progression and metastasis. Myeloid suppressor cells (MDSC ) also were found abundantly in the tumor matrix. We previously reported an increase in MDSC in the peripheral blood of BCR-ABL(-) MPN, therefore, these cells were also studied. Material and Methods: Patient and sample preparation: Peripheral blood, bone marrow (BM) aspirate and biopsy were obtained and used in isolation for mononuclear cells (MNCs). 11 MPN patients consisting of 5 ET, 1 PV , 2 PMF, 1 ET-MF, and 2 PV-MF were studied along with 5 controls ,including 4 questionable spike on protein electrophoresis with normal bone marrow exminations and one with leukemoid reaction. Flow cytometric quantification of monocytes, TAMs and MDSCs MNCs from peripheral blood or BM (aspirates and biopsy) were incubated (1X106/ml) with fluorophore-conjugated monoclonal antibodies against the following markers (CD14, HLA-DR, CD86, CD163, and CD206; BD Bioscience, Inc.) The raw flow data were analyzed using FlowJo (v 7.6.2).Thecell populations were gated and identified with the following immunophenotypes: CD14+HLA-DR-/low as the Mo-MDSCs , CD14+ CD86+as M1 macrophage, and CD14+ CD206+ or CD14+CD163+ as M2 TAMs . TAM or Mo-MDSC cells were presented in MFIs, as well as their percentages of total gated MNCs. Results: 1) Mo-MDSC were significantly elevated in MPN compared to that of controls both in the bone marrow aspirates and in the biopsy specimens (Fig1); 2) No difference was identified in M1 macrophages between MPN and controls in the percentage of total gated cells or by the MFI (data,not shown); 3) No difference was noted in M2 macrophages between MPN and controls in the percentage of total gated cells (Fig 2a) or by the MFI (Fig 2b). Conclusions: Mo-MDSC are increased in the bone marrow aspirates and biopsy specimens of patients with BCR-ABL(-) MPN. No difference was noted in the M1 and M2 TAM macrophages between the MPN and control.

Although BCR-ABL negative myeloproliferative neoplasms (MPN)--and especially myelofibrosis (MF)--are recognized to be associated with autoimmune phenomena, immune derangements in MPN have been much less studied. Myeloid-derived suppressor cells (MDSC) are one type of important immune modulator cell. Therefore, we studied MDSCs in MPN disease. MDSCs were studied in two cohorts: the first cohort was 55 patients including 16 primary myelofibrosis (PMF), 7 post-polycythemia vera (PV)-MF, 2 post-essential thrombocythemia (ET)-MF, 11 ET, 17 PV, 2 undefined MPN disorder, and 23 normal controls; the second cohort included 38 patients: 17 ET, 7 PMF, 3 ET-MF, 2 PV-MF, 9 PV patients, and 20 normal volunteers. The second cohort was studied using freshly collected specimens and a comparable age group as controls. CD11b(+), CD14(-), and CD33(+) cells were defined as MDSCs in both cohorts by flow cytometry. Since there are no differences in MDSC levels among different MPN categories, they were grouped as MPNs. The results showed that MDSCs were significantly elevated in MPNs compared with controls in both cohorts. We also performed RT-PCR and found that MPN patients have significantly elevated arginase-1 mRNA compared with controls, and sorted MDSCs were found to have suppressor T cell activity in MPNs, substantiating the hypothesis that levels of MDSCs are, in fact, deranged in MPNs. MDSC levels were not correlated with JAK2 status, white blood cells, Hb levels, platelet counts, splenomegaly, or the degree of bone marrow fibrosis (in MF). Further studies in immune therapy involving MDSC inhibitors or differentiation may be developed to treat MPN disease.

Myelofibrosis (MF), including primary myelofibrosis, post-essential thrombocythemia MF, and post-polycythemia vera MF, has been reported to be associated with autoimmune phenomena. IMiDs have been reported to be effective in some patients with MF, presumably for their immune-modulator effects. We therefore sought to elucidate the immune derangements in patients with MF. We found no differences in T regulatory cells (Treg) and T helper 17 (Th17) cells in MF patients and normal healthy controls. However, we found significantly elevated soluble interleukin 2 alpha (sIL2Rα) in MF patients compared to those with other myeloproliferative neoplasm diseases and normal healthy controls. Our studies with MF patients further revealed that Treg cells were the predominant cells producing sIL2Rα. sIL2Rα and IL2 complex induced the formation of Treg cells but not the formation of Th1 or Th17 cells. sIL2Rα induced CD8+ T cell proliferation in the presence of Treg cells. Monocytes or neutrophils had no effect on the production of sIL2Rα by Treg cells. Furthermore, we found plasma sIL2Rα levels were correlated to the auto-immune serology in MPN patients and ruxolitinib significantly inhibits the sIL2Rα production by the Treg cells in MF patients which may explain the effects of ruxolitinib on the relief of constitutional symptoms. All these findings suggest that sIL2Rα likely plays a significant role in autoimmune phenomena seen in patients with MF. Further studies of immune derangement may elucidate the mechanism of IMiD, and exploration of immune modulators may prove to be important for treating myelofibrosis.

Introduction. Ruxolitinib is the first JAK1/JAK2 inhibitor to be approved by the US Food and Drug Administration for the treatment of myelofibrosis (MF) and polycythemia vera (PV). Ruxolitinib is beneficial in relieving constitution symptoms and reducing splenomegaly. The main mechanism is the inhibition of T cell differentiation and function. Ruxulitinib inhibits Th1, TH17, Treg differentiation , and proliferation both in vivo and in vitro. Several clinical trials are under way to identify other Jak2 inhibitors to further improve efficacy and reduce adverse events. Notably momelotinib, a JAK1 and JAK2 inhibitor and pacritinib, a JAK2 and Fms-like tyrosine kinase 3 (FLT- 3) inhibitor that does not inhibit JAK1, have shown promise. Hence, we compared these three agents in their ability to interfere with T cell differentiation and function.

PD-1 and MDSC are two recently discovered important tumor immune escape mechanisms. These immune escape pathways have been studied and their levels were found to be elevated in tumors such as myeloma, pancreatic, renal, ovarian, and breast cancers. Therefore, we studied these two immune escape pathways in patients with myeloid neoplasms including polycythemia vera (PV), Essential thrombocythemia (ET), myelofibrosis (MF) including primary myelofibrosis (PMF) and Post-ET, Post-PV myelofibrosis. We also studied IMID drug in vitro to see if IMID drug mechanism is related to these two escape pathways. 51 patients with MPN and 15 normal volunteer controls were studied. For quantification of expression of PD-1 and PD L-1 in MNC subpopulations (CD4+, CD8+ or CD14+ cells). Ficoll-paque- isolated MNCs were stained with PD-1(CD279-APC) and PD L-1 (CD274-PE)BD Biosciences; CA) plus either CD4-FITC, CD8-FITC or CD14-FITC, along with 7AAD (6 ug/ml), and subjected to flow cytometric analysis. The flow data was analyzed using FlowJo (v 7.6.2). PD-1 or PD L-1 positive cells were expressed as percentage in the subpopulation of cells assayed. For MDSC's, after Ficoll-Paque density centrifugation of peripheral blood, 106 mononuclear cells were stained for flow cytometric analysis using CD11b-APC, CD33-PE, and CD14-FITC (BD Biosceinces; Carlsbad, CA), along with their matched isoptoye controls. 7AAD (6 ug/ml) was used to exclude dead cells to eliminate nonspecific antibody binding. 50,000 events per specimen were acquired and the resultant flow cytometric data was analyzed by FlowJo software. MDSC's were defined as CD11b+CD14-CD33+ cells and were calculated as percentage of viable gated MNC's. MDSC's were also tested for in vitro inhibitory effects on Tcells : MDSC cell sorting was performed by flow cytometry of CD33+ cells from CD11b+CD14- microbead selected cells. CD3 cells were then labeled with CFSE (Life Technologies; CA) and incubated with and without the sorted MDSC's in the presence of CD3+CD28+ microbeads for five days before assessing CFSE activity. For IMID drug in vitro experiments, Pomalidomide (Pm)( Celgene) (10ϰg/ml) was added to mononuclear cells in the presence of CD3+CD28+ microbeads in culture medium. After 5-7 days, cells were assayed for PD-1, PD-L1 expression and MDSC's. Since our data showed no significant difference between ET, PV and MF, we grouped ET, PV and MF together as MPN. The results showed that there is no significant difference between patients with MPN and controls of PD-1 and PD-L1 expression in CD4+, CD8+ and CD14+ cells. However, MDSC's were significantly elevated in patients with MPN compared to controls (Fig 1.). Pomalidomide significantly reduced the expression of PD-1 and MDSC's- expressed as fold change of Pm/DMSO (mean+ SE) in CD4 +,( 0.46 ± 0.120, p= 0.05) , CD8+ (0.66±0.07,p=0.02), MDSC ( 0.71 ± 0.09, P=0.01). Pomalidomide has no significant effect on PD-1 expression in CD14+ cells and PD1-L-1 expression in CD4+, CD8+ and CD14 + cells .Sorted MDSC's significantly suppressed proliferation of the CFSE labeled CD3+ T cells.

Abstract Patients with ET eventually transform into Post- ET MF in most cases after several years. The underlying mechanism of the transformation is still unknown. ET and Post-ET MF is characterized by abnormal megakaryocytic proliferation. Therefore we have performed microarray analysis of gene expression profiles to compare megakaryocytes derived from patients with Post-ET MF to ET. CD 34+ cells were obtained from peripheral blood, then cultured with Serum-Free Medium of stem cell factor (100 ng/mL) and thrombopoietin (TPO 100 ng/mL), and added murine bone marrow stromal cell line OP9 as feeder layer. Megakaryocytes were then obtained by MACS selection of CD 61+ cells after 10 days culture. Then oligonucleotide microarray analysis was performed by PhalanxBio Inc. (Palo, Alto, Ca). Gene expression profiling was achieved by using the comparative cycle threshold (CT) method of relative quantification (the calibration samples were MK pools from 10 ET patients with GAPDH used as internal controls). SLR (Signal log ratio) was then calculated for comparative analysis of 2 patients with post-ET MF to a pool of ET patients. The results of post-ET MF compared with ET were as follows: 1) with analysis of 4544 genes, SLR 1, 2) in apoptosis genes, over expressed genes were IGF-1R, CFLAR, NFKBA,(GLR between 1-1.5) down-regulated genes were BAX, LGALSI, PPPIR 15A, CASP3 (GLR between -0.5 to -0.1), 3) in cell cycle controlling genes, over expressed were IL8, INSIG1, IGF-1R, BCL3(GLR between 1 to 1.5), down-regulated were PPPIR15A (GLR = -0.7). 4) in megakaryocyte-important development genes, growth factor genes including PDGF A, C were up-regulated (GLR=1.8), and VEG B was down-regulated (GLR = -1. 4):; growth factor receptor genes including IL1R 1and 2, CXCR4, IL8, PF4 were up-regulated (GLR between 1-2), IL3RA, KIT were down-regulated (GLR between -1.6 to -3); cell cycle regulator genes including CCND1, 3, RASGRP3 were up-regulated (GLR between 1 to 2.5), CDK4 was down-regulated (GLR = -1); transcription regulators including CEBPE, CEBPG were up-regulated (GLR-1.5); all Signal transducers including all STAT 1-5, MAK14 were up-regulated (GLR between 1.5 to 2.4). There are 20 up-regulated genes and 20 down-regulated genes which need further analysis for their function. These preliminary analyses of MK genes comparison between Post ET-MF and ET suggest that many anti-apoptosis, cell cycle regulator genes, growth factor genes, growth factor receptor genes and all of the signal transducer genes for the MK development genes were up-regulated while many of the pro-apoptosis genes were down-regulated. Further work to dissect the genes using the microarray analysis for the mechanism for the transformation of ET into ET-MF will be important for understanding this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5010. doi:10.1158/1538-7445.AM2011-5010

Abstract Abstract 3845 Patients with ET eventually transform into Post- ET MF. The underlying mechanism of the transformation is still unknown. Megakaryocytes play an important role in the pathogenesis of bone marrow fibrosis in MF; ET and Post-ET MF also are characterized by abnormal megakaryocytic proliferation. Therefore we have performed microarray analysis of gene expression profiles to compare megakaryocytes derived from patients with Post-ET MF to ET. CD 34+ cells were obtained from peripheral blood, then cultured with Serum-Free Medium of stem cell factor (100 ng/mL) and thrombopoietin (TPO 100 ng/mL). Megakaryocytes (MK) were obtained by MACS selection of CD 61+ cells after 10 days culture. MK from two pooled ET-MF and a pooled from eight ET patients were extracted into RNA then converted to cDNA and amplified by PCR, followed by hybridization to an oligonucleotide microchip in PhalanxBio Inc. (Palo Alto, Ca). Gene expression levels in MK cells from ET-MF patients expressed in threshold cycle (CT) values were validated by statistical analysis of replicates and normalized to that from pooled ET patients to generate fold-change and statistical relevance. The resulting signal log ratios (SLRs) were used to classify the expressed genes into various groups according to its graded values. The results of post-ET MF compared with ET were as follows: 1) with analysis of 4544 genes, SLR <-1 were 6.58 %, and 10.12 % were >1, 2) in apoptosis genes, over expressed genes were IGF-1R, CFLAR, NFKBA,(GLR between 1–1.5) down-regulated genes were BAX, LGALSI, PPPIR 15A, CASP3 (GLR between -0.5 to -0.1), 3) in cell cycle controlling genes, over expressed were IL8, INSIG1, IGF-1R, BCL3(GLR between 1 to 1.5), down-regulated were PPPIR15A (GLR= -0.7). 4) in megakaryocyte-important development genes, growth factor genes including PDGF A, C were up-regulated (GLR=1.8), and VEG B was down-regulated (GLR= -1. 4):; growth factor receptor genes including IL1R 1and 2, CXCR4, IL8, PF4 were up-regulated (GLR between 1–2), IL3RA, KIT were down-regulated (GLR between -1.6 to -3); cell cycle regulator genes including CCND1, 3, RASGRP3 were up-regulated (GLR between 1 to 2.5), CDK4 was down-regulated (GLR = -1); transcription regulators including CEBPE, CEBPG were up-regulated (GLR-1.5); all Signal transducers including all STAT 1–5, MAK14 were up-regulated (GLR between 1.5 to 2.4). There are 20 up-regulated genes and 20 down-regulated genes which need further analysis for their function. These analyses of MK genes comparison between Post ET-MF and ET suggest that many anti-apoptosis, cell cycle regulator genes, growth factor genes, growth factor receptor genes and all of the signal transducer genes for the MK development genes were up-regulated while many of the pro-apoptosis genes were down-regulated. These gene changes found in the present study, when transformation of ET into post-ET MF occurs will be important for understanding this disease process and target therapy may be emerged. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 4798 Objectives: The role of steroids in mammalian erythropoiesis has not well defined. We have previously reported our observation on three human cases in which there was a synergism and accelerated response to the Erythropoietic Stimulating Agents (ESA) with simultaneous low and physiologic dose administration of glucocorticoids. In the current study, we investigated the additive effects of different dose schedules of steroids on hematopoietic effects of ESA in animal modules. Methods: A total of 74, four-weeks old male Sprague-Dawley rats were randomized to 6 groups; (A) control, (B) therapeutic doses of either erythropoietin, [Procrit Epoetin Alfa, 100 UI/kg], or (C) dexamethasome (300 mcg/kg), as well as combination of erythropoietin (Epoetin Alfa, 100 UI/kg) with (D) low, [25 mcg/kg], (E) physiologic, [300 mcg/kg], and (F) high, [2.5 mg/kg] doses of dexamethasone through abdominal hypodermal injection three times a week for a total of four weeks. At the conclusion of the study, peripheral blood sample, and Bone marrow mononuclear cells were collected through femur flushing. The samples were lysed and stored in RNA denaturation buffer at –80°C until use. Expressions of multiple hematopoietic major genes were assessed by real-time RT-PCR. Amplification data were processed using ΔΔCt method. Hemoglobin concentration and other CBC parameters were measured at the reference lab. Results: Mean hemoglobin concentrations were significantly higher in groups D (20.76 g/dl, 95% CI 20.08–21.45), E (20.45 g/dl, 95% CI, 19.97–20.94), and F (20.99 g/dl, 95% CI 20.55–21.42), compared to the controlled groups A, B, C (14.57, 15.68, 19.23 g/dl respectively) with two-tailed p-value of <.0001. (Figure-1) Real time RT-PCR based gene expression profiling of major hematopoietic regulators revealed robot increases of JAK2 gene expression in groups of animals treated with EPO only, or even higher increase with EPO plus either low or physiologic doses of dexamethasome. Similarly, GATA-1 levels are increased in groups treated with EPO only, or EPO with low or physiologic doses of dexamethasome. c-kit and NFkB1 expression levels are markedly higher in EPO plus dexamethasome groups. In contrast, the levels of EPOR are generally reduced in all groups receiving ESA. (Figure -2) Conclusion: The findings in this study is suggestive that simultaneous administration of ESAs with glucocorticoids is associated with significant additive elevation of the hemoglobin concentration; however, higher dose of dexamethasone is associated with more frequent adverse side effects such as significant weight loss. It is also suggested that the erythropoietic effect of steroid is concerted by up-regulation of the multiple erythropoietic gene expressions, such as JAK2, GATA-1, c-Kit, and NFkB1, while down regulations of EPOR is uniformly seen in the Epo-treated groups. This novel finding could be clinically utilized to accelerate the erythropoietic response of the ESA in selected cases. Disclosures: No relevant conflicts of interest to declare.

Abstract 5051 Primary myelofibrosis (PMF) and myelofibrosis post essential thrombocytosis (MF-ET) or myelofibrosis post polycythemia vera (MF-PV) have been reported to be associated with autoimmune phenomenon, such as Coomb positive anemia, lupus anticoagulant, positive ANA and the presence of circulating immune complex etc. Regulatory T cells (T reg ) also have known to play important roles in modulating immune responses. Therefore we studied T reg cells in 25 patients with MF including PMF (12), MF-ET (8), MF-PV (5) and compared with other MPD including ET (7), PV (8), and normal volunteer controls (17). Mononuclear cells (MNC) were separated from peripheral blood. 10 6 MNCs were stained for flow cytometry analysis using T reg Detection Kit from Miltenyi Biotec Inc (Auburn, CA). The numbers of T reg was calculated as the percentage of positive CD4 + CD25 + FoxP3 + T cells (T reg ) from the numbers of gated CD4 + cells. T reg function was evaluated by XTT cell proliferation assay (Invitrogen) with ratios of T reg vs. T-effector cells (CD4 + CD25 - ) at 1:1, 1:2, 1:4, and 0:1 in the presence of anti-CD3 CD28 microbeads (Invitrogen). The results (mean ± SE) showed numbers of T reg cells in MF were 0.73 + 0.08, other MPD were 1.37 ± 0.22, and normal controls were 0.96 ± 0.27 ( p =NS). T reg function was evaluated in 12 patients with MF and 6 normal volunteer controls. Four MF patients were found to have significant lower values than controls. We concluded that in MF, quantitatively T reg were not different from other MPD or normal controls but T reg dysfunction was observed in 30–40 % of MF patients. This could explain why some patients with MF are prone to develop autoimmune phenomenon. Further studies with more patients are in progress. Disclosures: No relevant conflicts of interest to declare.

Abstract Except allogenic bone marrow transplantation with either myeloablative or reduced intensity conditioning, there is no effective treatment for PMF. Age-related morbility and mortality factors limit this application. Anti-Jak2 treatment relieves splenomegaly but does not improve cytopenia. IMiDs on the other hand can improve anemia in up-to 20% of transfusion dependent anemic patients with PMF. In order to asses the underlying mechanism and to predict treatment responsiveness, in vitro suppression of Treg cells by lenalidomide correlated to clinical treatment response was performed. Seven patients with transfusion dependent and ESD (erythropoietin stimulating drugs) resistant PMF were studied. Mononuclear cells from blood in patients with PMF were cultured in the culture medium with FCS and Il-2 (100 IU/ml) in the presence of lenalidomide or DMSO for 6 days, the cells were then stained for Treg cells (CD4+, CD 25 +,and FoxP3+,) and analyzed by flow cytometry using Treg Detection Kit from Miltenyi Biotec Inc (Auburn, CA). Treg cells were enumerated as percentage of selected CD4 + cells. The results showed that in the 4 patients who responded to lenalidomide treatment, Treg cells were suppressed by the in vitro testing by lenalidomide, while in the three patients who were clinically refractory to lenalidomide treatment, their Treg cells were not suppressed by in vitro lenalidomide treatment. These results suggest that in transfusion dependent PMF, Treg cells may play a role in the pathogenesis of anemia and that effectiveness of lenalidomide treatment may be partly related to its suppression of Treg cells. Further studies to recruit more patients and to explore the role of Treg cells in modulating erythroipoiesis in PMF using the erythroid methylcellulose culture systems are currently in progress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5632.

Abstract Abstract 4622 Elevated histone deacetylase (HDAC) enzyme levels have been described in patients with carcinoma and leukemia. Using HDAC inhibitor (HDACi) to treat carcinoma has been promising and is currently undergoing intense research. In order to be better in using HDACi to treat chronic lymphocytic leukemia (CLL), HDAC isoenzyme levels were measured in 32 patients with CLL and compared with 17 normal volunteer controls. Peripheral blood CD20+ cells from patients with CLL and normal volunteer controls were isolated by using Progenitor Cell Isolation Kits (Miltenyi Biotec, Auburn, CA). Isolated CD20+ cells were confirmed by flow cytometry studies to have more than 92% purity. Patient and control CD20+ cells were then lysed in denaturation buffer. Total RNA was extracted, then cDNAs were prepared and quantitative RT-PCR was performed using premixed primer and FAM- and TAMRA-labeled probes obtained from ABI. The results showed: 1) HDAC1, -3, -6, -7, -9, and -10 and SIRT1, -2, and -6 were significantly over-expressed, suggesting that, in CLL, elevated HDAC activity are not restricted to one class. Therefore, HDAC inhibitor therapy may need to be directed to more than one specific class of HDAC; 2) Patients with ZAP70+ were associated with more advanced Rai clinical stage and higher HDAC activity than Zap70– patients and HDAC activity were correlated to the expression levels of CD44 and Pin1, suggesting that higher HDAC values may indicate a poor prognosis and more advanced disease stage. Disclosures: No relevant conflicts of interest to declare.