bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression Proteomics As Surrogate Biomarkers For Early Treatment Response In Chronic Myeloid Leukemia (CML) Patients
Authors: Alaiya Ayodele , Chaudhri Naeem , Owaidah Tarek , Fox Jonathan , Shinwari Zakia , Barhoush Eman , Alsharif Fahad , Mohamed Said Y , Rasheed Walid , Alfraih Feras , Aljurf Mahmoud
Published: October 2019

Purpose

Despite advancements in diagnosis and treatment modalities for hematological malignancies, presently, there are no reliable blood tests for disease prognosis or prediction of patient’s response to particular treatments. Identification of such biomarkers for diagnostic or therapeutic response in clinical monitoring of hematologic diseases such as Chronic Myeloid Leukemia (CML) would be very useful.

Experimental Design

The ability to identify biomarkers in body fluids (i.e., plasma or serum) will not only involve minimally invasive procedures, but also more importantly, the potential for monitoring disease progression and effective response to therapy in relatively early stages. In this study, global proteome analysis of serum, plasma, and bone marrow samples from 28 patients with newly diagnosed chronic–phase CML were analyzed by using expression proteomics technology (label free quantitative liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The goal was to evaluate for early treatment response and discovery disease–specific /disease-associated proteins for prognostic monitoring and patient’s response to therapy at protein level.

Results

We identified a panel of 15 differentially expressed proteins (> 2- °- fold change, p< 0.001) to accurately discriminate between CML patients that achieved Major Molecular Response (MMR) vs. No-Major Molecular Response (NoMMR) at 6 months (Figure 1 & Table 1). Six (6) of the 15 identified proteins were filtered and mapped as potential biomarkers using Ingenuity Pathway Analysis. Among the identified proteins implicated in hematological diseases include Group-specific component (vitamin D binding protein), haptoglobin and vitronectin. Others were inter-alpha-trypsin inhibitor heavy chain 1, leucine-rich alpha-2-glycoprotein 1 and metallophosphoesterase 1. We also observed most of these proteins to be located in extracellular space, acts as transporters and have been implicated as markers of hematological disease and inflammatory response. Our data indicates that multivariate analysis of quantitative proteome data can potentially be useful as a means of unsupervised artificial intelligence algorithm for classification and stratification of clinical CML patients.

Conclusion

Our results highlight the power of proteomics in the discovery of biomarkers for more accurate prediction of prognosis and monitoring treatment response in CML patients. These proteins might be valuable, once validated, to complement the currently existing parameters for reliable and objective prediction of disease progression, monitoring treatment response and clinical outcome of CML patients.

Disclosures:

No relevant conflicts of interest to declare.
Pharmacokinetic (PK) and Pharmacodynamic (PD) Study Of Dasatinib In Chronic Phase Of Newly Diagnosed Chronic Myeloid Leukemia (CML-CP)
Authors: Ishida Yoji , Murai Kazunori , Yamaguchi Kohei , Miyagishima Takuo , Shindo Motohiro , Ogawa Kazuei , Nagashima Takahiro , Sato Shinji , Watanabe Reiko , Yamamoto Satoshi , Hirose Takaaki , Kato Yuichi , Yonezumi Masakatsu , Kondo Takeshi , Mochizuki Nobuo , Ohno Keiko , Kishino Satoshi , Ito Shigeki , Kubo Komei , Saito Sohichi
Published: October 2019

Purpose

Dasatinib is a novel kinase inhibitor of BCR-ABL and SRC family kinases. Dasatinib has shown promising anti-leukemic activity in chronic myeloid leukemi (CML). Tounderstand how to administer dasatinib with best efficacy and less adverse events, the pharmacokinetic (PK) properties of dasatinib and the relationship of PK and pharmacodynamic (PD) characteristics were investigated in newly diagnosed CML-chronic phase (CP) patients.

Methods and Materials

The PK analysis of dasatinib: The plasma concentrations of dasatinib (1, 2, 4 hr after taking dasatinib post 28 days) were determined by Francia's method using high performance liquid chromatography with mass spectrometry. PK analysis was performed using Phoenixâ NLMEâ1.2. The maximum plasma concentration (Cmax) was determined by visually inspecting the profiles of plasma drug levels.

PD analysis of dasatinib: Phospho-CrkL in CD 34 positive cells was analyzed by flow cytometry using anti-phospho-CrkL (Tyr207) antibody after incubation of bone marrow mononuclear cells in the presence of dasatinib for 2 hours.

PK/PD analysis: Area under the curve (AUC) and time above IC50 were calculated using Phonix WinNonlin 6.3.

Results

Twenty-eight newly diagnosed CML-CP patients were included. The correlation between the efficacy and PK/PD parameters were analyzed using Peason's correlation coefficient test (Table1). The efficacy(expression of bcr/abl at 1 month) was not correlated with AUC, Cmax, AUC/IC50 and Cmax/IC50 but significantly correlated with TAIC50 (r=0.4763, p=0.0104). The reduction rate at 1 month was correlated significantly with only TAIC50(r=0.5136, p=0.0052)(Figure 2). Eight cases reached MMR at 3 month among 15, whose TAIC50s were more than 12 hrs(53.3%), while only 2 cases reached MMR among 13, whose those were less than 12 hrs(15.4%).

Conclusion

Dasatinib has anti-leukemic activity in a time dependent manner. Exposure more than 12 hrs at TAIC50 will get benefits of better prognosis.

Disclosures:

No relevant conflicts of interest to declare.
The Polymorphism Of Base Excision Repair Gene, XRCC1 Arg399Gln Is Associated With The Therapy-Related Myelodysplastic Syndromes (MDS)
Authors: Norjmaa Batchimeg , Saitoh Takayuki , Iwasaki Atsushi , Nitta Yasuhiro , Masuda Yuta , Suda Itsumi , Kaneko Ayaka , Shimizu Hiroaki , Handa Hiroshi , Yokohama Akihiko , Tsukamoto Norifumi , Sakura Toru , Murakami Hirokazu
Published: October 2019

Background

Base excision repair (BER) is critical for genome maintenance, and is mainly responsible for the correction of small base changes of DNA damage. BER pathway involved many enzymes including OGG1, XRCC1, APE1 and MUTYH. Single nucleotide polymorphisms (SNPs) in DNA repair genes result reduced DNA repair capacity, have been reported to be associated with an increased risk of various cancers including hematologic malignancies. However, it is unclear that these polymorphisms alter the susceptibility and clinical outcome of myelodysplastic syndromes (MDS) patients. The aim of this study is to evaluate the association of polymorphisms in gene encoding four key proteins of DNA BER: OGG1 Ser326Cys, XRCC1 Arg399Gln, APE1 Asp148Glu, and MUTYHGln324His with the susceptibility and clinical features of MDS.

Methods

Our study included 113 MDS patients [median 68.3 years, range 17.1-86.5 years; male/female 76/37; RCUD (n=37), RARS (n=6), RCMD (n=21), MDS-u (n=11), RAEB-1(n=14), RAEB-2 (n=11), others (n=13)] and 192-health control group. Twenty four patients with MDS had the history of cancer. Genetic polymorphisms in BER pathway genes were examined using PCR and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Genotype and allele frequencies were compared between patients group and control group by using χ2-test. All patients and healthy controls received written information about the study. This study was approved by the International Research Board of Gunma University Hospital.

Results

There was no statistically significant difference in the allele and genotype frequencies of the OGG1 Ser326Cys, XRCC1 Arg399Gln, APE1 Asp148Glu, and MUTYH Gln324His polymorphisms between the MDS patients and the control group. In the analysis of clinical characteristics, XRCC1 non Arg/Arg genotype (low DNA repair type) was significantly associated with lower Hb level (8.64±2.29g/dL vs. 9.96±2.08 g/dL, p<0.005) and higher frequency of the complex karyotype (14.9% vs. 2.8%, p=0.05). Furthermore, XRCC1 non Arg/Arg genotype was associated with therapy- related MDS (OR 3.15, 95% CI 1.24-7.98, p=0.02) and especially the past history of radiotherapy (14.3% vs. 0%, p<0.005). In contrast, the polymorphisms in OGG1 Ser326Cys, APE1 Asp148Glu, and MUTYH Gln324His were not involved in the clinical features of MDS.

Conclusion

The low DNA repair polymorphism, XRCC1 Arg399Gln is associated with the clinical features of MDS, including therapy- related MDS. Further investigation of BER polymorphisms will provide the understanding of pathogenesis of therapy- related MDS in a larger sample size analysis.

Disclosures:

No relevant conflicts of interest to declare.
Fibrotic Tissues In Human Primary Myelofibrosis With JAK2 V617F Are Developed Clonally From Malignant Hematopoietic Stem Cells
Authors: Yamauchi Takuji , Shima Takahiro , Takenaka Katsuto , Miyamoto Toshihiro , Akashi Koichi
Published: October 2019

Primary myelofibrosis (PMF) is a clonal stem cell disorder, characterized by deregulated proliferation of myelofibroblasts. Pathogenesis of PMF has been intensively investigated by analyzing clonality of the disease mainly by X-linked gene inactivation assays. These studies suggested that hematopoiesis in PMF is clonal, whereas fibroblasts are polyclonal not belonging to the PMF clone but they secondarily proliferate in response to cytokines produced by malignant megakaryocytes or monocytes. However, we have previously reported that PMF-initiating cells exist within the circulating CD34+CD38- fraction expressing CD45, whose phenotype is analogous to normal hematopoietic stem cells (HSCs), and this population can reconstitute myelofibrosis after xenotransplantation into NOD/SCID/IL2rg-null mouse newborns (Saito et al. ASH Annual Meeting 2007). In this assay, human PMF HSCs reconstitute PMF-like disease in mouse after transplantation, and strikingly the vast majority of myelofibroblasts were of human origin on FISH analysis, suggesting that myelofibroblasts in PMF can be generated directly from circulating PMF stem cells. We here tried to test this hypothesis by directly analyzing human PMF samples. We performed clonal analyses of fibrotic tissues in PMF patients at the single cell level by utilizing JAK2 V617F mutation as a clonal marker. Nine patients of PMF with JAK2 V617F mutation were enrolled in this study. The fibrotic bone marrow tissues were stained with vimentin, CD34 and KP1 to identify fibroblast-like cells, endothelial cells and myeloid cells, respectively. Single cells of fibrotic tissues were sampled by a laser micro-dissection system, and CD34+CD38-CD45+ HSCs were purified from the blood by FACS. Genomic DNA from single sampled cells was tested for JAK2 V617F mutation. This extensive analysis showed that ∼20-40% of single HSCs and granulocyte/monocyte progenitors (GMPs) had JAK2 V617F. Similar percentages of single vimentin+ fibroblast-like cells, CD34+ endothelial cells and KP-1+ myeloid cells had JAK2 V617F in each patient. Furthermore, each population also constituted similar percentages of heterozygous and homozygous JAK2 V617F as HSCs and GMPs did in each case. Because JAK2 V617F exists in each cell component of myelofibrotic tissues, whose frequency was almost equal to that of PMF HSCs, this study strongly suggests that the majority of myelofibrotic tissue cells belong to the PMF clone, and are differentiated directly from PMF HSCs. These data are compatible with our previous xenotransplantation data. Finally, it is also suggested that JAK2 V617F signaling is not necessary for PMF development. The new understanding of PMF pathophysiology shown here by our experiments might be useful to develop new treatment strategies for human PMF in future studies.

Disclosures:

Miyamoto: Kyushu University Hospital: Employment.
Signal Transducer and Activator Of Transcription 3 Activates and Deregulates Micro RNA Expression In Chronic Lymphocytic Leukemia Cells
Authors: Uri Rozovski , Calin George , Setoyama Tetsoro , Lucilla Abundu , David Harris , Li Ping , Zhiming Lui , Srdana Grugurevic , Ferrajoli Alessandra , Faderl Stefan H. , Burger Jan A. , O'Brien Susan , Wierda William G. , Keating Michael J. , Estrov Zeev
Published: October 2019

MicroRNA (miR) deregulation is a hallmark of chronic lymphocytic leukemia (CLL). However, little is known about the mechanisms of miR gene transcription. In CLL, signal transducer and activator of transcription 3 (STAT3) is constitutively phosphorylated on serine 727 residues, and phosphoserine STAT3 activates protein-coding genes known to be induced by phosphotyrosine STAT3. Therefore, we sought to determine whether phosphoserine STAT3 also activates miR genes. Because an analysis of publically available data, revealed that STAT3 binds to the promoter of nearly 25% of all human miR genes, we transfected unstimulated CLL cells with STAT3 small hairpin (sh)-RNA. Using an RNA microarray, we found that STAT3-shRNA downregulated the levels of 63 miR genes and upregulated the levels of 9 miR genes. Using qRT-PCR, we confirmed the microarray results in 5 of the 6 differentially expressed miR genes. Together, these data suggested that STAT3 affects miR gene levels. An analysis of chromatin immunoprecipitation followed by sequencing (ChIP-seq) data revealed STAT3-miR promoter binding sites in only 60% of the STAT3-regulated miR genes. This suggested to us that mechanisms unrelated to miR promoter binding are involved in the STAT3-mediated regulation of miR expression. Indeed, computer simulation of complementary binding of STAT3-targeted microRNA genes to STAT3 mRNA provided indirect evidence that STAT3 mRNA acts as a “sponge”, soaking up miRs and altering their level and function. Several miRs that are downregulated by STAT3-shRNA, including miR-15, miR-16, miR-21, and miR-155, play crucial roles in CLL pathobiology. In particular, miR-155 is involved in tumorigenesis, and its overexpression induces lymphoma in mice; also, CLL has a high expression level of miR-155. Therefore, we sought to confirm that STAT3 directly regulates the transcription of miR-155 in CLL cells. Electrophoretic mobility shift assay analysis revealed that STAT3 bound to the miR-155 promoter in CLL cells, and ChIP analysis revealed that STAT3 bound the 700–709 bp but not the 615–624 bp in the miR-155 promoter. We then co-transfected MM1 cells with truncated miR-155 promoter constructs plus STAT3 small interfering (si)-RNA and found that the STAT3-siRNA reduced the luciferase activity of the construct harboring the 700–709 bp miR-155 promoter region. Taken together, our data suggest that STAT3 directly and indirectly deregulates miR gene levels in CLL.

Disclosures:

No relevant conflicts of interest to declare.
Dnmt3a Deletion Predisposes Hematopoietic Stem Cells To Malignant Transformation
Authors: Mayle Allison , Yang Liubin , Challen Grant A , Zhou Ting , Rebel Vivienne I. , Goodell Margaret A.
Published: October 2019

DNA methyltransferase 3A (DNMT3A), a de novo DNA methyltransferase, is mutated in various hematological malignancies affecting both myeloid (20%), mixed (50%), and lymphoid (18%) malignancies and is associated with poor prognosis. The most frequently reported DNMT3A mutation is R882 in acute myeloid leukemia (AML), which results in altered enzyme activity, but various missense and nonsense mutations have also been found throughout the gene, suggesting that loss-of-function mutations in DNMT3A may also contribute to leukemogenesis. Our group recently showed that transplantation of HSCs from Dnmt3a knock-out (KO) mice led to increased hematopoietic stem cell (HSC) self-renewal and inhibition of differentiation, but was insufficient to cause transformation. However, in these experiments, Dnmt3a-KO HSCs were transplanted alongside wild-type whole bone marrow to quantitate HSC function, potentially protecting against malignant transformation.

We hypothesized that if Dnmt3a-KO HSCs were transplanted alone, a predisposition to transformation would be uncovered. We established a large non-competitive transplantation cohort receiving 500 control or Dnmt3a-KO HSCs and monitored the mice closely for disease. Strikingly, mice with Dnmt3a-KO HSCs had significantly shorter survival (246d vs 467d, p<0.0001, Figure 1). As mice succumbed to disease, we analyzed histological changes in hematopoietic organs and performed CBCs and immunophenotyping to diagnose the diseases. We identified multiple disease classes within the Dnmt3a-KO recipients, including T-cell acute lymphoblastic leukemia, myeloproliferative disease (MPD), myelofibrosis (MF), and myelodysplastic syndromes (MDS). The relatively long disease latency suggests that acquisition of secondary hits promotes disease; identification of these secondary mutations is ongoing.Figure 1Mice transplanted with Dnmt3a-KO HSCs succumb to hematologic malignanciesFigure 1. Mice transplanted with Dnmt3a-KO HSCs succumb to hematologic malignancies

Here, we show that Dnmt3a deletion in noncompetitive transplanted HSCs leads to an array of hematologic disorders that models the spectrum of disorders seen in human malignancies. Since DNMT3A mutations are known early genetic lesions in leukemia development, mutations that cooperate with DNMT3A might influence the type of disease developed. This mouse model serves to validate an important role for Dnmt3a in the development of hematologic malignancies, and is also valuable for the study of future targeted therapies.

Mice transplanted with Dnmt3a-KO HSCs died significantly earlier than mice transplanted with control HSCs (median survival 246 days and 467 days, p<0.0001). Fifty and 20 female mice were transplanted with 500 Dnmt3a-KO or control HSCs respectively, all at 6-8 weeks of age. Censored points indicate mice that were euthanized for unrelated reasons.

Disclosures:

No relevant conflicts of interest to declare.
Eptifibatide Reduced Cell Adhesion and Recruitment Of The Myeloid Sarcoma-Inducing Leukemia Cells
Authors: Fu Jen-Fen , Shih Lee-Yung
Published: October 2019

AML patients with myeloid sarcoma (MS) usually had a poor outcome. Our clinical data revealed that AML patients harboring MLL/AF10 and RAS gene mutations were associated with MS formation. By using retroviral transduction/transplantation assay, we demonstrated that the mice transplanted with bone marrow (BM) cells carrying cooperating MLL/AF10(OM-LZ) and KRAS-G12C mutations induced MPD-like myeloid leukemia and MS. Gene expression analyses identified Gpr125, an adhesion G protein-coupled receptor, was up-regulated in the cells carrying cooperating mutations than the cells carrying MLL/AF10(OM-LZ) alone. Knockdown of Gpr125 by RNA interference reduced the number and the size of MS, suggesting that Gpr125 was involved in the MS formation. As Gpr125 contains a HormR domain with Lysine-Glycine-Aspartic acid (KGD) motif which is known to involve in the cell-extracellular matrix (ECM) and cell-cell adhesion, we investigated whether a cyclic RGD peptide drug, eptifibatide (Ep), could interfere MS formation. An in vitro cell-ECM binding assay showed that Gpr125 interacted with fibronectin. Ep reduced leukemia cell-fibronectin binding. Ep also reduced homotypic leukemia cell adhesion and leukemia cell-adipocyte adhesion. In vivo assay demonstrated that Ep reduced leukemia cells recruitment to the adipose tissues, spleen and bone marrow. Our results suggested that blocking Gpr125-mediated cell-ECM and cell-cell adhesion might be helpful to interfere MS formation and BM/spleen recruitment of leukemia cells.

Disclosures:

Off Label Use: Eptifibatide (Integrilin, Millennium Pharmaceuticals, also co-promoted by Schering-Plough/Essex), is an antiplatelet drug of the glycoprotein IIb/IIIa inhibitor class.
The Prognostic Impact Of Gene Rearrangements and Protein Expression Of MYC, BCL2 and BCL6 In Young High-Risk Patients With DLBCL
Authors: Horn Heike , Ziepert Marita , Barth Thomas F.E. , Bernd Heinz-Wolfram , Wartenberg Martin , Feller Alfred C. , Klapper Wolfram , Hummel Michael , Stein Harald , Lenze Dido , Hartmann Sylvia , Hansmann Martin-Leo , Möller Peter , Cogliatti Sergio , Pfreundschuh Michael , Trümper Lorenz , Löffler Markus , Ott German , Schmitz Norbert , Rosenwald Andreas
Published: October 2019

Purpose

A number of retrospective analyses have looked into the prognostic implication of MYC, BCL2 and BCL6 rearrangements and protein expression for MYC, BCL2 and BCL6 in patients with diffuse large B-cell lymphoma (DLBCL). Many of these studies suffer from small patient cohorts and differing or unknown treatment strategies (with or without Rituximab) administered to patients under study. Furthermore, the median age of these patients was relatively high. We for the first time report on the prognostic consequences of MYC, BCL2 and BCL6 alterations in younger (18-60 years) high-risk patients (aaIPI 2 or 3) with DLBCL.

Patients and Methods

The MegaCHOEP study (Schmitz et al. Lancet Oncology 2012: 13, 1250) randomized patients to 8xCHOEP-14 or sequential high-dose therapy supported by repeated infusions of autologous stem cells. Both treatment arms included 6 infusions of R (375 mg/ sqm). The median patient age was 48 years, and 27% of patients scored an aaIPI of 3. Paraffin-embedded tumor samples from 112 de novo DLBCL were investigated using immunohistochemistry for MYC, BCL2, and BCL6, and fluorescence in-situ hybridisation (FISH) to detect breaks of MYC, BCL2 and BCL6.

Results

Rearrangements of MYC, BCL2 and BCL6 were detected in 13.6%, 20.7% and 30.9% of DLBCL, respectively. A double or triple hit constellation occurred in 10.8%. Presence of BCL2 breaks (RR=4.7, 95% CI: 1.8-12.2) and MYC breaks (RR=2.4, 95% CI: 0.8-7.5), but not of BCL6 breaks were associated with inferior overall survival in univariate and multivariate analyses adjusted for aaIPI and treatment arm. Protein overexpression of MYC ≥30% (RR=2.4, 95% CI: 0.9-6.5), but not BCL2 (≥60%) or BCL6 (≥30%) indicated inferior overall survival. BCL2 overexpression was associated with inferior EFS (RR=2.2, 95% CI: 0.9-5.5) and PFS (RR=2.8, 95% CI: 1.0-8.2). If the same cutpoint for BCL2 used for a similar analysis in elderly patients treated on the RICOVER-60 trial (Horn et al. Blood 2013) was applied, no differences in EFS or PFS were observed.

Conclusion

Rearrangements of MYC and BCL2 seem to be relevant prognostic factors also in young high-risk patients. The frequencies of MYC and BCL2 rearrangements are not substantially higher than reported for other, mostly elderly patient groups carrying IPI scores from 0 - 5; it seems unlikely, therefore, that the rearrangements described here (completely) explain the poor prognosis of young, high-risk patients. The prognostic role of BCL2, BCL6, and MYC as well as other biological risk factors must be validated in independent data sets.

Disclosures:

No relevant conflicts of interest to declare.
An Essential Role For Transferrin Receptor 2 In Erythropoiesis During Iron Restriction
Authors: Wallace Daniel F , McDonald Cameron J , Secondes Eriza S , Ostini Lesa , Rishi Gautam , Hooper John D , Velasco Gloria , Ramsay Andrew J , López-Otín Carlos , Subramaniam V Nathan
Published: October 2019

Iron deficiency and iron overload are common clinical conditions that impact on the health and wellbeing of up to 30% of the world’s population. Understanding mechanisms regulating iron homeostasis will provide improved strategies for treating these disorders. The liver-expressed proteins matriptase-2 (encoded by TMPRSS6), HFE and transferrin receptor 2 (TFR2) play important and opposing roles in systemic iron homeostasis by regulating expression of the iron regulatory hormone hepcidin. Mutations in TMPRSS6 lead to iron refractory iron deficiency anemia, whereas mutations in HFE and TFR2 lead to the iron overload disorder hereditary hemochromatosis. To elucidate the competing roles of these hepcidin regulators, we created mice lacking matriptase-2, Hfe and Tfr2.

Tmprss6 -/-/Hfe-/-/Tfr2-/- mice had iron deficiency anemia resulting from hepatic hepcidin over-expression and activation of Smad1/5/8, indicating that matriptase-2 predominates over Hfe and Tfr2 in hepcidin regulation. Surprisingly, this anemia was more severe than in the Tmprss6-/- mice, demonstrated by more extensive alopecia, lower hematocrit and significant extramedullary erythropoiesis in the spleen. There was increased expression of erythroid-specific genes in the spleens of Tmprss6-/-/Hfe-/-/Tfr2-/- mice, consistent with the extramedullary erythropoiesis. Expression of Tfr2 but not Hfe in the spleen was increased in the Tmprss6-/- mice compared to wild type and correlated with the expression of erythroid genes, suggesting that Tfr2 is expressed in erythroid cells. Further analysis of gene expression in the bone marrow suggests that the loss of Tfr2 in the erythroid cells of Tmprss6-/-/Hfe-/-/Tfr2-/- mice causes a delay in the differentiation process leading to a more severe phenotype.

In conclusion, our results indicate that Hfe and Tfr2 act upstream of matriptase-2 in hepcidin regulation or in a way that is overridden when matriptase-2 is deleted. These results indicate that inhibition of matriptase-2 would be useful in the treatment of iron overload conditions such as hereditary hemochromatosis. We have also identified a novel role for Tfr2 in erythroid differentiation that is separate from its canonical role as a regulator of iron homeostasis in the liver. This important role of Tfr2 in erythropoiesis only becomes apparent during conditions of iron restriction. Our results provide novel insights into mechanisms regulating and linking iron homeostasis and erythropoiesis.

Disclosures:

No relevant conflicts of interest to declare.
Either Combined-Modality Or Radiotherapy Alone Provide Favorable Outcome In Stage I-II Mantle Cell Lymphoma: A Report Of 82 Patients From The International Lymphoma Radiation Oncology Group (ILROG)
Authors: Dabaja Bouthaina S. , Tsang Richard , Qi Shunan , Allen Pamela , Hodgson David C. , Ricardi Umberto , Hoppe Richard T. , Ng Andrea , Specht Lena , Li Ye-xiong , Terezakis Stephanie , Constine Louis , Yahalom Joachim
Published: October 2019

Patients with Mantle cell lymphoma (MCL) often present with advanced-stage and have a poor prognosis. Although MCL is highly radiosensitive, RT is rarely used other than for palliation. Stage I-II presentation is rare, and there is only limited data on treatment outcome with less aggressive combined modality approaches or RT alone. To assess the outcome of this group of patients, we retrospectively reviewed 82 patients referred to radiation oncology at 9 ILROG institutions between 1990 and 2012. Of the 82 patients, 65 (79%) were males, 44 (54%) were > 60 years; 47 (57%) were stage I, 61 (74%) presented in the head and neck areas, 51 (62%) had extranodal presentation, 14 (17%) were bulky (>5 cm). Treatment approaches consisted of combined modality (CM) with chemotherapy and consolidative radiation therapy (RT) in 66 (80%) and definitive RT alone in 16 (20%). The median radiation dose used was 35 Gy (range 12-45). Chemotherapy consisted of cyclophosphamide, adriamycin, vincristine, and prednisone (CHOP) or CHOP-like, regimens in 62 (57%), rituximab was added to chemotherapy in 51 (62%). The median follow-up was 60 months (range 5-245). At last follow up, 26 (32%) patients relapsed (70% of relapses were outside the radiation field) and 21 (26%) died (2 deaths unrelated to disease or treatment).

The 5-and 10-years overall survivals (OS) were 71% and 63%, respectively. The 5-and 10-years disease free survivals (DFS) were 75% and 39%, respectively.

On univariate analysis, the following characteristics were significantly associated with DFS and OS: age as a continuous variable (HR=1.05, p=0.005 for DFS; HR=1.05, p=0.006 for OS) and bulky disease (HR=2.78, p=0.03 for DFS; HR= 3.18, p=0.02 for OS). On multivariate analysis, both DFS and OS were significantly influenced by: age as a continuous variable (HR=2.09, p=0.03; HR=1.05, p=0.005), bulky disease (HR=2.23, p=0.05; HR=3.1, p=0.03), and female gender only affected DFS (HR=2.16, p=0.03).SurvivalAll (%)Combined modality (%)RT alone up-front(%)5-years OS71689210-years OS6362745-years DFS75738910-years DFS393657

Conclusion

Patients who presented with stage I-II MCL and were treated with programs that included consolidative local radiotherapy or even radiotherapy alone demonstrated favorable disease control and relatively long survival. It appears that radiotherapy as a sole modality can offer in selected patients a comparable DFS and OS to a combined modality approach.

Disclosures:

No relevant conflicts of interest to declare.
Using Forced Chromatin Looping To Overcome Developmental Silencing Of Embryonic and Fetal β-Type Globin Genes In Adult Erythroid Cells
Authors: Rupon Jeremy W , Deng Wulan , Wang Hongxin , Gregory Philip D , Reik Andreas , Dean Ann , Blobel Gerd A.
Published: October 2019

The β-genes undergo developmental activation and silencing in part by competing for their upstream enhancer, the locus control region (LCR). In adult erythroid cells, the LCR contacts the β-globin gene promoter by forming a loop that precludes interaction with the embryonic and fetal β-type globin genes. Reversing this developmental gene expression switch in favor of embryonic/fetal genes has therapeutic implications for patients with hemoglobinopathies. Here we employed a forced chromatin looping approach to activate the silenced murine embryonic βh1-globin gene and the human fetal γ-globin gene in adult erythroid cells. We have previously shown that forced recruitment via artificial zinc finger proteins of Ldb1, a protein necessary for long-range chromatin interactions at the β-globin locus, can trigger chromatin loop formation and transcription initiation. Here, we designed a zinc finger protein targeting the βh1 promoter, fused it to the self-association domain of Ldb1 (βh1-Ldb1), and introduced it into an adult murine erythroid cell line that normally produces nearly 100% adult β-globin. βh1-Ldb1 expression activated βh1-globin transcription up to 3000-fold accounting for ∼20% of total β-globin expression. βh1-Ldb1 similarly increased expression of βh1-globin in fetal liver derived primary erythroid cells. These results are striking given the degree to which murine embryonic globin genes are normally repressed. To test whether the activity βh1-Ldb1 was due to a looped interaction of the βh1 promoter with the LCR, we introduced βh1-Ldb1 into fetal liver derived erythroblasts from mice in which the LCR had been deleted. βh1-Ldb1 was virtually inactive in the absence of the LCR, demonstrating the dependence on the LCR and, by inference, long range looping of βh1-Ldb1 function. We next extended this approach to the human β-globin locus in an effort to activate expression of the fetal γ-globin gene in adult erythroid cells. Ldb1 was fused to a previously described γ-globin promoter binding zinc finger protein, GG1, to generate GG1-Ldb1. Introduction of GG1-Ldb1 into adult primary human erythroid cells strongly activated γ-globin expression with a concomitant reduction in β-globin transcription. Strikingly, γ-globin accounted for nearly 90% of total β-type globin transcription. Furthermore, fetal hemoglobin expression was nearly pan-cellular as determined by flow cytometry. These results demonstrate the power of forced chromatin looping to reprogram developmental regulation of gene expression, and provide a novel proof of concept for activating the γ-globin gene for the benefit of patients with hemoglobinopathies.

Disclosures:

Gregory: Sangamo BioSciences: Employment. Reik:Sangamo BioSciences: Employment.
Primary Cold Agglutinin Associated Lymphoproliferative Disease: A B-Cell Lymphoma Of The Bone Marrow Distinct From Lymphoplasmacytic Lymphoma
Authors: Randen Ulla , Trøen Gunhild , Tierens Anne Maria , Steen Chloe , Abdirashid Warsame , Klaus Beiske , Geir Tjønnfjord , Sigbjørn Berentsen , Delabie Jan
Published: October 2019

Primary chronic cold agglutinin disease (CAD) is a rare hemolytic disease caused by the production of cold agglutinin antibodies that bind to erythrocyte I/i blood group antigens at low temperatures. The binding is mediated by monoclonal IGHV4-34-encoded cold agglutinins. CAD is a disease of older individuals with a median age of 76 years and accounts for about 15 % of all cases of autoimmune hemolytic anemia. The estimated incidence of CAD is 1-4/106 worldwide. The aim of this study is to better characterize the underlying lymphoproliferative disorder in CAD patients by review of bone marrow histology, immunophenotypic and genetic analysis. The bone marrow trephine biopsies of 54 patients with CAD showed circumscribed intraparenchymatous nodules with small monotonous monoclonal B-cells (median infiltration: 10% of marrow cells) with a CD20+, IgMs+, IgDs+, CD27+, CD5-/+, CD11c-/+, CD23-, CD38- immunophenotype. Neither plasmacytoid cytological features nor expression of plasma cell differentiation-associated transcription factors IRF4, BLIMP1 and XBP1 were expressed. However, a limited number of mature monoclonal IgM+, IgD- plasma cells were present outside the lymphoid nodules and were diffusely scattered throughout the marrow. Thus, the bone marrow in CAD shows a characteristic lymphoid infiltration pattern that is different from lymphoplasmacytic lymphoma that typically shows an admixture of lymphoid cells, plasmacytoid cells and plasmacells and that, not infrequently, shows a peritrabecular localisation. Of interest, the MYD88 L265P mutation, typical of lymphoplasmacytic lymphoma, was not detected (17/17 cases, sensitivity 3 %). Somatically mutated monoclonal IGHV4-34 gene rearrangement was demonstrated in eight patients with frozen samples (mean sequence homology 95.4%). Unexpectedly, no mutations of BCL6 intron 1 were demonstrated, except in one patient. BCL6 intron 1 mutations usually accompany immunoglobulin gene mutations in B lymphocytes that have matured in the germinal center. In conclusion, the histological, immunophenotypic and genetic properties of CAD are proof that CAD is a lymphoproliferative disease that is distinct from lymphoplasmacytic lymphoma with which it has previously been confounded. The disease likely originates from B lymphocytes that have not matured in the germinal center.

Disclosures:

No relevant conflicts of interest to declare.
Lenalidomide, Bendamustine, and Rituximab As First-Line Therapy For Patients >65 Years With Mantle Cell Lymphoma: Preliminary Results From The Nordic Lymphoma Group MCL4 (LENA-BERIT) Phase I-II Trial
Authors: Jerkeman Mats , Albertsson-Lindblad Alexandra , Kolstad Arne , Laurell Anna , Räty Riikka , Grønbæk Kirsten , Sundberg Jan , Pedersen Lone Bredo , Geisler Christian
Published: October 2019

Background

Mantle cell lymphoma is a disease of the elderly, with a median age of 70 years. Younger patients may be treated with potentially curative treatment including high dose chemotherapy. For elderly patients, however, no standard therapy has been defined. In the current trial, we investigate if the addition of lenalidomide (LEN) to rituximab (R)+bendamustine (B) (B 90 mg/m2 D1-2 and R 375 mg/m2 D1) followed by maintenance with LEN for 7 months may enhance efficacy, with manageable toxicity, for the older population of MCL patients.

Methods

Eligibility criteria were age > 65 years, or ≤ 65 years, unable to tolerate high dose chemotherapy, with untreated mantle cell lymphoma, stage II-IV. BR was given for 6 cycles q4w. In the phase I part, the MTD of LEN was established as 10 mg days 1-14 during the induction phase, cycles 2-6. Prednisolone 20 mg days 1-14 was given during cycle 2. When LEN was initially given from cycle 1, we encountered unexpected grade III-IV toxicity in the form of cutaneous and allergic reactions. In the maintenance phase, LEN single therapy was given as follows: cycles 7-8 - 10 mg days 1-21, cycles 9-13 - 15 mg days 1-21.

Results

The trial was concluded June 1, 2013, after inclusion of 51 patients, of whom 24 were in the phase I part. The median age is 72 years. According to MIPI, 55% were high risk. Presently, 29 patients are evaluable for response after 6 cycles LBR. ORR is 28/29 (97%), CR+CRu 23 (79%). 17 out of 28 evaluable patients (61%) were MRD-negative after 6 cycles. After a median follow-up of 18 months, the median PFS has not been reached, and the estimated PFS at 2 years is 74%. Eight patients have died, 3 due disease progression, 3 due to treatment related toxicity, 1 of lung cancer in a heavy smoker, 1 of CMML. Overall survival at 2 years is 87%.

Conclusions

When omitted in cycle 1, lenalidomide in combination with R-bendamustine is feasible as first-line therapy in older patients with MCL, and is associated with a high response rate, also as assessed by MRD. The long term efficacy of this regimen remains to be established by longer follow-up.

Disclosures:

No relevant conflicts of interest to declare.
Altered Expression and Mutation Analysis Of Heterogeneous Nuclear Ribonucleoprotein k In Bone Marrow Of Primary Myelofibrosis Patients
Authors: Bozinovic Ksenija , Manshouri Taghi , Post Sean M. , Zhang Ying , Gallardo Miguel , Zhang Xiaorui , Verstovsek Srdan
Published: October 2019

The heterogeneous nuclear ribonucleoproteins (hnRNPs) are RNA/DNA-binding proteins that consist of several family members: A1, A2, B1, C2 and K. hnRNPs have been implicated in numerous cellular processes and when dysregulated, have been suggested to impact tumorigenesis. hnRNP A2/B1 is overexpressed in some lung cancers and has been suggested to be a useful early detection marker for lung carcinoma. hnRNP K has been reported to associate with colon cancer and directly interacts with DNA, RNA, and proteins to regulate gene expression in numerous cellular processes involved in mitogenic responses and tumorigenesis. Loss of hnRNP K expression results in defects in differentiation and apoptosis, and increased hnRNP K expression has been associated with loss of apoptosis and an increase in cellular proliferation.

To explore the possibility of altered hnRNP K expression or mutations in primary myelofibrosis, we isolated mRNA from primary blood or bone marrow mononuclear cells from patients with myelofibrosis (n=62) and healthy controls (n=19). We examined hnRNP K levels and determined that hnRNP K is significantly overexpressed in myelofibrosis (p= <0.0001). Sequencing of the hnRNP K locus revealed a single nucleotide alteration (A-to-G) one allele of intron 5 in 43% of myelofibrosis patients (n= 21) In contrast, only one out of 11 control samples harbored this alteration ((x2 p = 0.05). At present, we are investigating whether G allele is a mutation or a SNP. Furthermore, western blotting indicates an increase in phosphorylation of serine-248 in myelofibrosis patients. Together, these data suggest altered hnRNP K expression and mutations in myelofibrosis that might play a significant role in myeloproliferation and hematopoietic differentiation in myelofibrosis patients. Given the significant impact that hematopoietic differentiation has on leukemogenesis, it is imperative to decipher

Disclosures:

No relevant conflicts of interest to declare.
The PTPN22 R620W Polymorphism Is Not Associated With Clinical and Molecular Correlates Of Chronic Lymphocytic Leukemia (CLL) In Patients Originating From a Region With Low Prevalence Of The Autoimmunity Risk Variant
Authors: Panovska-Stavridis Irina , Trajkova Sanja , Eftimov Aleksandar , Ivanovski Martin , Dukovski Dusko , Marija Popova-Simjanovska , Marija Staninova , Cevreska Lidija , Dimovski Aleksandar
Published: October 2019

PTPN22 is a protein tyrosine phosphatase that modulates antigen-receptor signaling in T and B cells. A PTPN22 R620W polymorphic allele (C1858T, rs2476601) has been associated with increased risk for the development of multiple autoimmune diseases in patients originating from Northwestern Europe, whereas this association is less clear in patients originating from Southern Europe where the 620W (1858T) allele is less frequent. A higher frequency of the 620W risk allele has recently been reported in Chronic lymphocytic leukemia (CLL) patients originating from Northwest Europe. These data adds to the recent findings that PTPN22 is markedly overexpressed in the majority of CLL patients and possibly contributes to the pathogenesis of the disease by affecting apoptosis/survival of antigen stimulated CLL B-lymphocytes.

In order to extend further those observations we conducted a retrospective study on the frequency of the PTPN22 R620W variant in 87 CLL patients and 108 age/sex matched normal controls without a history of malignant disease originating from the Republic of Macedonia (Southeastern Europe) and compared the association of the variant with various clinical/molecular correlates.

Similar allele frequency of the minor T allele (0.06 and 0.063) and genotype distribution (0.88, 0.115 and 0.005 of CC, CT and TT genotypes) was found in CLL patients and normal controls, respectively. Both study groups were in Hardy-Weinberg equilibrium. Also, the PTPN22 R620W variant was not associated with any of the studied clinical and biological parameters, such as blood counts, CD38 expression, IGHV mutation status, incidence of autoimmune hemolytic anemia and autoimmune thrombocytopenia, immunoglobulin levels, time-to-treatment, progression free survival and overall survival.

We conclude that the PTPN22 R620W variant is not a risk factor for the development or progression of CLL in patients originating from the Southeastern European region. The differences of our data from the reported findings for CLL patients originating from Northwest Europe mirrors the differences observed in patients with various autoimmune diseases, suggesting a possible gene-environmental factor that affects the influence of the R620W variant on lymphocyte homeostasis.

Disclosures:

No relevant conflicts of interest to declare.
CD1d Expression Is Higher In Chronic Lymphocytic Leukemia Patients With Unfavorable Prognosis
Authors: Bojarska-Junak Agnieszka , Hus Iwona , Dmoszynska Anna , Rolinski Jacek
Published: October 2019

Chronic lymphocytic leukemia (CLL) is characterized by a very heterogeneous clinical course, which is slow and indolent in most of the patients, however some patient experience rapid disease progression and anticancer therapy is required shortly after the diagnosis. Many issues in CLL development and progression are still unclear. The functional consequences of CD1d expression on tumour cells are not well understood. However, increasing evidence suggests that they may affect iNKT cells.The role of CD1d expression in CLL immunopathogenesis remains undefined. In this study, we investigated the potential role of CD1d in CLL by analyzing the level of CD1d expression on leukemic B cells in peripheral blood of120 patients and assessed its correlation with prognostic markers such as ZAP-70 and CD38 expression, Rai stages and unfavourable cytogenetic changes.Measuring CD1d expression by flow cytometry and qRT-PCR, we showed lower CD1d molecule and CD1d mRNA expression in B cells of CLL patients than of healthy controls. The frequency of CD1d+/CD19+ cells, CD1d staining intensity and CD1d transcript levels increased with the disease stage. CD1d expression was positively associated with ZAP-70 and CD38 expressions as well as with unfavourable cytogenetic changes (17p deletion, 11q deletio),. We established the relationship between high CD1d expression and shorter time to treatment and overall survival. The percentage of CD1d+/CD19+cells inversely correlated with the percentage of iNKT cells. iNKT cells ζ-chain expression was downregulated in the high-CD1d group.These results suggest that high CD1d expression is associated with poor prognosis of CLL and might be involved in disease progression.

Disclosures:

No relevant conflicts of interest to declare.
Extramedullary Multiple Myeloma Associated With Reduced Overall Survival: A Retrospective Single-Center Study
Authors: Barkay Ido , Maziarz Richard T. , Chen Andy I. , Dibb William , Chen Yiyi , Scott Emma C.
Published: October 2019

Introduction

Multiple Myeloma (MM) is an incurable neoplasm, however recent advancements in therapies have led to median overall survival of 7-10 years in standard risk patients. High risk patients still succumb to the disease within 3 to 5 years. Traditionally used clinical prognostic markers such as the International Staging System (ISS), the Durie-Salmon (DS) staging system, and cytogenetics do not adequately predict response to novel agents or overall prognosis. We previously presented data identifying high risk sub-groups that succumb to shorter progression free survival (PFS) and overall survival (OS) after hematopoietic stem cell transplant (HSCT). Extramedullary disease (EMD) was a highly significant risk factor for poor survival.

Methods

A retrospective single institution cohort study was performed of 251 patients who underwent HSCT for MM between 1/1/2001 and 12/31/2011. Of these 251 patients, 18 were identified as having EMD, which was defined by the presence of ≥ 2 plasmacytomas in organs other than bone marrow or bone at any stage of disease. Data points collected included patient and disease characteristics, ISS and DS stage, cytogenetics and FISH, induction therapy, type and number of HSCT, treatment type and cycles, as well as maintenance therapy. Outcome measures included overall response rate (ORR), PFS and OS. Descriptive statistical analysis was conducted for all primary and secondary endpoints. Univariate and multivariate analysis were conducted using the Cox proportional hazards regression model.

Results

Patients with EMD represented 6.6% of our total population. Patient and disease characteristics are found in table 1. Seven of 18 patients had died by the time of data collection. With a median follow up time of 29 months, the median PFS and OS for the entire group (n= 251) compared to the EMD group (n= 18) were PFS: 22.3 months (95% CI 19.75 – 30.32) vs. 12.9 and OS: 57.3 months (95% CI: 46.52 - 77.77) vs. 17.2 (Figs 1 and 2). Results of univariate and multivariate analysis are in table 2.

Conclusion

Extramedullary multiple myeloma represents a small but highly aggressive subgroup of multiple myeloma. It prevails as a poor prognostic indicator despite the use of novel agents, yet it is not included in traditionally used staging systems. Further confirmatory studies are needed, and likely, new therapeutic approaches will be required for MM patients with EMD.

Disclosures:

Chen: Genzyme: supported database used for this study Other. Scott:Millenium Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees, Research Funding.