No abstract

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.

Background: We present a case of gamma-delta T-cell lymphoma that does not fit the current World Health Organization classifications. Case presentation: A 74-year-old Caribbean-American woman presented with lymphocytosis, pruritus, and nondrenching night sweats. Bone marrow and peripheral blood analyses both confirmed the diagnosis of gamma-delta T-cell lymphoma. An axillary lymph node biopsy was negative for lymphoma. Clinically absent hepatosplenomegaly and skin lesions with biopsy-proven gamma-delta T-cell lymphoma suggest that she is unclassifiable within the current classification system. Conclusions: We believe this is a case of not otherwise specified gamma-delta T-cell lymphoma. Accumulation of these rare not otherwise specified cases will be important for future classification which further defines the biology of this disease.

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. We conclude that 1) PD-1, PD L-1 expression is not altered in MPN patients. However, MPN patients have significantly increased MDSC's. 2) Pomalidomide, in vitro, significantly reduces the expression of PD-1 in CD4+, CD8+ cells and decreases MDSC. This data may be added to be one of the important mechanisms of immunomodulatory drugs in the treatment of Myelofibrosis. Disclosures: No relevant conflicts of interest to declare.

Endogenous erythroid colony (EEC) formation is one of the minor criteria for diagnosing polycythemia vera (PV) according to 2008 WHO diagnostic criteria. But EEC requires bone marrow aspiration and sophisticated laboratory procedures; therefore, practically it is rarely used to diagnose PV. Insulin-like growth factor 1 receptor (IGF-1R) was found to be constitutively phosphorylated and was responsible for the EEC formation in PV; therefore, we measured IGF-1R levels in the peripheral blood of 26 PV patients and compared them with those of 33 patients with secondary polycythemia and 29 normal controls. Among the PV patients, 16 were treated with only phlebotomy, 9 received hydroxyurea, and 1 was treated with ruxolinitinib. We found that PV patients treated with only phlebotomy had significantly higher IGF-1R levels than did those PV patients treated with hydroxyurea or ruxolinitinib. None of the secondary PV patients or normal controls had elevated IGR-1R levels, while 14 of 16 (87%) PV patients had significantly elevated IGF-1R levels. The new 2016 WHO has eliminated EEC as a minor criterion for diagnosing PV, but there are still some cases that cannot be definitively diagnosed by the current criteria. Therefore, we suggest that quantifying the IGF-1R level in peripheral blood by flow cytometry to replace EEC as the minor criterion for diagnosing PV.