bluuebunny/crossref_metadata_2025 huggingface dataset:
A Somatic Variant in MYD88 (L265P) Revealed by Whole Genome Sequencing Differentiates Lymphoplasmacytic Lymphoma From Marginal Zone Lymphomas
Authors: Xu Lian , Sohani Aliyah R. , Arcaini Luca , Hunter Zachary , Yang Guang , Zhou Yangsheng , Liu Xia , Cao Yang , Manning Robert , Patterson Christopher J , Ioakimidis Leukothea , Tripsas Christina , Pinkus Geraldine S. , Harris Nancy Lee , Rodig Scott J. , Treon Steven
Published: October 2019

Abstract 261

Lymphoplasmacytic (LPL) and marginal zone lymphoma (MZL) are distinct clinicopathological entities under the WHO classification system for B-cell lymphomas. Differentiation of LPL from MZL has been difficult due to overlapping clinical, morphological, histopathological, immunophenotypic, and cytogenetic features. We therefore sought to identify a molecular marker by which LPL could be differentiated from MZL. Using paired normal/tumor tissues from 10 LPL patients, whole genome sequencing was utilized to identify somatic variants. These studies identified a somatic variant at position 38182641 in chromosome 3p22.2 with a single nucleotide change from T→C in the myeloid differentiation primary response (MYD88) gene, and a predicted non-synonymous change at amino acid position 265 from leucine to proline (L265P) in 10 of 10 LPL patients. MYD88 L265P is an oncogenically active mutation in DLBCL ABC cell lines via activation of IRAK1/4/TRAF-6/NF-κβ signaling, and is present in tumors from 29% of patients with ABC subtype of DLBCL, and 6% of patients with MALT lymphomas (Ngo et al, Nature 2011, 470:115–119). Further to these efforts, we performed Sanger sequencing of MYD88 in malignant cells obtained from 51 patients with LPL, 49 of whom had an IgM monoclonal protein and were therefore classified as Waldenstrom's Macroglobulinemia (WM), and 2 with an IgG monoclonal protein, along with 46 patients with MZL, which included 21 Splenic (SMZL), 20 Extranodal (EMZL), and 5 Nodal (NMZL) Subtypes, as well as B-cells from 15 healthy donors. Among LPL patients, the MYD88 L265P variant was found in malignant cells from 46/51 (90.1%) cases, which included 44 patients with WM, and 2 patients with IgG LPL. Expression of the MYD88 L265P variant was heterozygous in 42, and homozygous in 4 LPL patients. By comparison, only 3/46 (6.5%) patients with MZL (1 SMZL; 1 EMZL; 1 NMZL) exhibited the MYD88 L265P variant which was heterozygous (p<0.0001), and included 2 patients (1 SMZL, 1 NMZL) with extensive bone marrow involvement, a monoclonal IgM protein, and whose clinicopathological characteristics overlapped with LPL. By comparison, the MYD88 L265P variant was absent in CD19+ cells from all 15 healthy donors. The results of this study demonstrate that the MYD88 L265P mutation is widely expressed in patients with LPL, and can be used to differentiate LPL from MZL.

Disclosures:

Treon: Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.
KPT-SINE, a Potent, Small Molecule Inhibitor of CRM1-Dependent Nuclear-Cytoplasmic Shuttling, with Potent Activity Against T-ALL and AML
Authors: Etchin Julia , Kentsis Alex , Sanda Takaomi , Kung Andrew L. , Stone Richard M. , McCauley Dilara , Kauffman Michael , Shacham Sharon , Look Thomas
Published: October 2019

Abstract 2622

Background:

CRM1 (XPO1) is the major nuclear exporter that mediates transport of a variety of cargos, including proteins involved in tumor suppressor and cellular proliferation pathways (e.g. p53, PI3K/AKT, and NF-kB). CRM1 is upregulated in a range of solid and hematologic malignancies and its overexpression is correlated with poor prognosis, and with resistance to chemotherapy. Recently, the crystal structure of CRM1, in complex with its export cargo, Snurportin 1, has been resolved. The structure elucidates the key contact residues required for the formation of the CRM1-cargo export complex. The molecular understanding of the CRM1-cargo binding interface has led to the development of novel small molecule inhibitors of CRM1-cargo interaction, termed KPT-Selective Inhibitors of Nuclear Export (SINE). KPT-SINE are potent, drug-like CRM1 inhibitors that irreversibly inactivate the CRM1-directed protein export by covalent modification of the essential CRM1-cargo binding residue Cys528. The inhibition of the CRM1 nuclear export has been shown to lead to selective apoptosis in cancer cells when compared to normal cells. Here, we assess the efficacy of the KPT-SINE in human T-ALL, AML, and normal hematopoietic cells. Methods: The viability of a panel of human T-ALL and AML cell lines and normal CD34+ progenitor cells upon treatment by the KPT-SINE was assessed. Dose-response measurements were done using serial dilutions of KPT-SINE from 1 μM to 0.3 nM and luminescent cell viability assay. Apoptosis was measured using Annexin V staining and TUNEL assays. Results: KPT-SINE induces rapid apoptosis in 12 of 15 T-ALL and 10 of 14 AML cell lines with 50% inhibitory concentrations (IC50s) of 15–100 nM. In the KPT-SINE-sensitive cell lines, BCL2 overexpression suppresses KPT-SINE-induced apoptosis, indicating its intrinsic pathway mediation. Importantly, KPT-SINE exhibits selective cytotoxicity against tumor cells when compared to peripheral blood mononuclear cells (PBMCs) and CD34+ progenitor cells in vitro. KPT-SINE at 57 and 150 mg/kg induced 100% inhibition of tumor growth in vivo in a MOLT-4 T-ALL xenograft study. Other xenograft studies to address the potency of the KPT-SINE on selected AML lines in vivo are ongoing. These studies emphasize the clinical promise of the KPT-SINE as a novel and selective drug candidate for the treatment of T-ALL and AML.

Disclosures:

McCauley: Karyopharm Therapeutics: Employment. Kauffman:Karyopharm: Equity Ownership. Shacham:Karyopharm: Equity Ownership.
Improved Survival in Chronic Myeloid Leukemia (CML) Since the Introductin of Imatinib Therapy - A Single Institution Historical Experience
Authors: Lee Hun , Cortes Jorge E. , O'Brien Susan , Jabbour Elias , Garcia-Manero Guillermo , Quintas-Cardama Alfonso , Shan Jenny , Rios Mary Beth , Ravandi Farhad , Faderl Stefan , Kadia Tapan , Borthakur Gautam , Huang Xuelin , Kantarjian Hagop M.
Published: October 2019

Abstract 2750

Background:

Outcome of CML since introduction of imatinib therapy has improved. Aims: analyze improvement of CML outcome in different phases. Study Group: A total of 1,569 patients with CML referred since 1965, within 1 month from diagnosis, were reviewed and used to identify phase-specific prognostic factors: 1,148 chronic, 175 accelerated, 246 blastic. Results: The median survival was 8.9 years in chronic, 4.8 years in accelerated, and 6 months in blastic phase. In chronic phase, the 8-year survival was ≤ 15% before 1983, 42–65% from 1983 to 2000, and 87% since 2001 (Figure 1). Survival was worse in older patients (p=0.004), but less significant since 2001 (p=0.07). Survival by Sokal risk was significantly different before 2001 (p<0.001), but not since 2001 (p=0.4). In accelerated phase, survival improved over time (p<0.001); the 8-year survival in patients treated since 2001 was 75% (Figure 2). Survival by age was not different in years < 2001 (p=0.09), but was better since 2001 in patients ≤ 70 years (p=0.004). Multivariate analysis derived adverse factors since 2001: older age (p=0.049), increased marrow blasts (p=0.03). In blastic phase, the median survival improved over time (p<0.001), although it is only 7 months since 2001. Conclusions: Survival in CML significantly improved significantly since 2001, particularly so in chronic and accelerated phases. Imatinib therapy minimized the impact of known prognostic factors and Sokal risk in chronic phase, and accentuated the impact of age in accelerated and blastic phases.

Disclosures:

Cortes: Novartis: Consultancy; Novartis: Research Funding; BMS: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Kantarjian:Novartis: Consultancy; Novartis: Research Funding; Pfizer: Research Funding; BMS: Research Funding.
Resveratrol Enhances NKG2D-Mediated Cytotoxicity Against Leukemia Cells by Upregulating Both NKG2D Receptor on NK Cells As Well As NKG2D Ligands on Target Cells,
Authors: Espinoza Luis J. , Takami Akiyoshi , Nakata Katsuya , Trung Ly Quoc , Yamada Kayoko , Touda Saki , Nakao Shinji
Published: October 2019

Abstract 3236

NKG2D is a powerful activating receptor expressed by natural killer (NK) cells that promotes cytotoxic lysis of cancer cells expressing NKG2D ligands (NKG2D-Ls). Pharmacological induction of NKG2D-Ls in malignant cells has been an attractive therapeutic approach but has gained poor clinical utility because currently available NKG2D-Ls inducers have been hampered either by their limited efficacy or by their associated toxicity. Resveratrol (RVT), a compound derived from several natural sources, has proved in vivo and in vitro potent anti-tumor effects against various cancers. Extensive research in the last decade has shown that such effects are mediated by targeting various molecules involved in the regulation of proliferation and cell survival and those include, NFκB, STAT3, ATM/ATR and ERK1/2. To date, it is unknown whether RVT has any effect on NKG2D-Ls expression. We report here NKG2D-Ls induction by RVT in a broad range of leukemia cells. RVT upregulated the NKG2D-Ls MICA/B, ULBP1, ULBP2 and ULBP3 in the myeloid leukemia cells OUN-1, NB4, THP-1 and KG1 and upregulated MICA/B, ULBP-1 and ULBP3 ligands in the lymphoid leukemia cells Jurkat and Molt-4. The upregulation of NKG2D-Ls by RVT was associated with increased transcription of each NKG2D-L gene. Ligand upregulation induced by RVT was prevented by cell pretreatment with caffeine, and inhibitor of ATM/ATR, which is the main signal regulator of NKG2D-Ls expression. Leukemia cells treated with RVT were more susceptible to killing by NK cells than untreated cells and the enhanced cytotoxicity of NK cells was blocked by the treatment of NK cells with anti-NKG2D monoclonal antibodies. Interestingly, the same concentration of RVT that effectively induced NKG2D-Ls in tumor cells, consistently upregulated NKG2D receptor expression in primary NK cells from healthy individuals and in the NK cell lines NKL and NK-92 and this effect was also associated with enhanced NKG2D-mediated NK cells cytotoxicity. RVT-induced NKG2D receptor enhancement in NK cells associated with the activation of the MAP kinase ERK1/2 and was prevented by the ERK1/2 specific inhibitor PD98059. Thus, RVT represents the first identified agent capable of activating both arms of the NKG2D axis. Since several clinical trials on RVT are ongoing, these previously unrecognized properties of this non toxic compound have an attractive immunotherapeutic potential.

Disclosures:

No relevant conflicts of interest to declare.
A Disease and Stage Risk Grouping System for Patients Undergoing Allogeneic Stem Cell Transplantation
Authors: Armand Philippe , Gibson Christopher J , Cutler Corey , Ho Vincent T , Koreth John , Alyea Edwin P. , Antin Joseph H. , Soiffer Robert J. , Kim Haesook T
Published: October 2019

Abstract 327

Background:

The outcome of patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT) varies significantly based on the patients' disease and stage at the time of HSCT. When analyzing outcomes of HSCT in heterogeneous patient populations, whether in retrospective or prospective studies, it is therefore necessary to stratify patients based on their disease/stage risk. At present, there is no robust method for doing so; the most commonly used breakdowns are based on older data that may not be applicable today, and do not incorporate cytogenetics for myeloid diseases, which are an important prognostic factor.

Methods:

We analyzed a cohort of 1539 patients transplanted between 2000 and 2009 at Dana-Farber/Brigham and Women's Hospital, and reviewed their disease type (including cytogenetics) and stage at HSCT. Based on proportional hazards modeling for overall survival (with a median follow-up of 35 months), we defined disease and stage risk groups, with independent analyses performed in the 812 patients who underwent myeloablative conditioning (MAC) and the 727 who underwent reduced intensity conditioning (RIC). We used the results to define overall disease/stage risk groups for both MAC and RIC HSCT.

Results:

Interestingly, the disease risk groups turned out to be identical for MAC and RIC; the stage risk groups were very similar, except for the assignment of CR>1 to low risk in MAC but high risk in RIC. The groups were as follows:

Low-risk disease:

AML with favorable cytogenetics, CLL, CML, Hodgkin lymphoma, and non-Hodgkin lymphoma (excluding extranodal T-cell lymphomas)

Intermediate-risk disease:

ALL, AML or MDS with intermediate cytogenetics, myeloproliferative neoplasms, and multiple myeloma

High-risk disease:

AML or MDS with adverse cytogenetics, extranodal T-cell lymphomas

Low-risk stage:

CR1, CR>1 (for MAC), PR1, untreated disease, CP CML

High-risk stage:

CR>1 (for RIC), PR>1, induction failure or active relapse, accelerated or blast phase CML

Those groups could be combined to form 4 overall groups with highly significantly different OS and PFS (Table and Figure).

Conclusion:

We propose a disease/stage risk grouping scheme for patients undergoing HSCT, applicable to both myeloablative and reduced intensity conditioning transplantation, which separates patients into 4 groups with significantly different OS and PFS. This scheme could be used for prognostic purposes, and to stratify patients in retrospective studies or in clinical trials. In the future, it may be further validated and refined through registry studies.

Disclosures:

No relevant conflicts of interest to declare.
Cyclin E Regulation by the Fbw7 Ubiquitin Ligase Pathway Is Required for Normal Mitophagy and Restraining Reactive Oxidative Species During Erythroid Cell Maturation In Vivo,
Authors: Xu Yanfei , Siu Ka Tat , Verma Amit , Minella Alexander C.
Published: October 2019

Abstract 3393

We previously described a knock-in mouse model that permits study of the physiologic consequences of cyclin E deregulation, by selective ablation of its regulation via the SCFFbw7 ubiquitin ligase. We found that erythroid progenitor cells in our cyclin ET74A T393A knock-in mice exhibit abnormally increased proliferation, increased apoptosis, impaired maturation, and dysplastic morphologies. Most prominent among the gene expression alterations we have identified in the cyclin E knock-in erythroid cells is induction of multiple p53 target genes, consistent with p53 pathway activation. In contrast to several recently described models of ribosomal protein gene mutations, in which p53 activation appears to induce dyserythropoiesis, we determined that p53 function actually maintains partially compensated erythroid cell maturation in vivo, in the context of impaired Fbw7-mediated cyclin E degradation. We next found that dysregulated cyclin E-CDK2 activity in cyclin ET74A T393A erythroid cells is associated with increased reactive oxidative species and increased mitochondrial mass and activity. These results coincide with findings of abnormal mitochondria retention in late-stage erythroid cells and significantly down-regulated expression of BNIP3L (NIX). BNIP3L encodes a critical regulator of erythroid cell mitophagy, and the transcriptional controls maintaining its expression in maturing erythroid cells likely account for why this lineage is acutely sensitive to deregulated cyclin E activity. Finally, we show evidence that reduced expression of BNIP3L may play a role in some cases of early-stage myelodysplastic syndromes.

Disclosures:

No relevant conflicts of interest to declare.
Circulating Mesenchymal Stromal Cells As a New Prospective Cancer Marker,
Authors: Kolonin Mikhail , Zhang Yan , Simmons Paul J. , Bellows Charles
Published: October 2019

Abstract 3404

Mobilization of progenitor cells is implicated in pathology and can be indicative of disease progression. Recently, we reported the influence of body mass index (BMI) on level of circulating progenitor cells (Bellows et al., Obesity 2011). Comparative analysis of peripheral blood mononuclear cells (PBMC) from 12 non-obese (BMI < 30) and 14 obese (BMI > 30) disease-free donors by flow cytometry revealed that obesity is associated with a 10-fold increased frequency of circulating mesenchymal stromal progenitor cells (MSC), which circulate at a very low level in healthy lean individuals. We showed that obesity is also associated with a 5-fold increased frequency of circulating progenitor cells (CPC), a population consisting of hematopoietic and endothelial precursors, while the frequencies of mature endothelial cells (EC) and CD34-bright leukocytes (CD34b LC) are unaffected by BMI. Here, we followed up on the assessment of circulating MSC as a potential surrogate pathology marker by analyzing the frequency of circulating CD34-positive progenitor and endothelial cells in a cohort of colorectal cancer patients. PBMC were collected from 45 obese and lean cancer patients and compared to control cancer-free donors. Flow cytometric enumeration of cells was performed based on established immunophenotypes: CD34brightCD31dimCD45dim (CPC), CD34dimCD31brightCD45- (EC), CD34brightCD31-CD45- (MSC) and CD34brightCD45bright CD34b (LC). Groups were compared using multivariate regression analysis. After adjusting for co-founders such as age and BMI, the mean frequencies of MSC and CD34bLC, but not of CPC and EC, were found to be significantly higher in the circulation of CRC patients compared to cancer-free donors. Interestingly, the frequency of circulating MSC, but not of the other cell populations, was also found to be significantly higher in the circulation of obese CRC patients compared to lean CRC patients and obese cancer-free controls. We conclude that markedly increased frequency of MSC in the peripheral blood may represent a new diagnostic CRC marker. BMI-dependent changes in circulating MSC, potentially mobilized from adipose tissue may reveal their trafficking to tumors, which could be one of the mechanistic links between obesity and cancer progression. Validation of MSC as a new surrogate marker of cancer could provide a tool for determining prognosis, predicting response to therapy, and detecting relapse following treatment.

Disclosures:

No relevant conflicts of interest to declare.
High Frequency of Large Gene Deletions Detected by Multiplex Ligation-Dependent Probe Amplification in Diamond Blackfan Anemia,
Authors: Quarello Paola , Garelli Emanuela , Carando Adriana , Pappi Patrizia , Brusco Alfredo , Dianzani Irma , Ramenghi Ugo
Published: October 2019

Abstract 3428

Background:

Diamond-Blackfan anemia (DBA,#MIM105650) is a rare congenital pure red cell aplasia characterized by normochromic macrocytic anemia, reticulocytopenia, and normocellular bone marrow with a selective deficiency of erythroid precursors.

Although mutations of eleven ribosomal protein (RP) genes have been detected in more than 50% of DBA patients the remaining patients appear to have intact ribosomal protein genes using stadard sequencing methods (Boria et al. Hum Mut 2010).

We previously described the detection of three large RPS19 deletions using the MLPA (Multiplex Ligation-dependent Probe Amplification) technique (Quarello et al. Haematologica 2008). As MLPA is an efficient and rapid technique that detects gene dosage alterations, we thus decided to apply this approach also to other RP genes.

Aim:

To search for unidentified RP large deletions we applied the MLPA technique in Italian DBA patients who have been found mutation-negative by sequencing.

Methods:

Italian DBA patients without RP genes mutations (73/156, 47%) were included in this study. The analysis was performed using a homemade MLPA kit following the recommendations provided by MRC Holland (Amsterdam, The Netherlands, www.mlpa.com). The probes were designed to detect deletions of six RP genes (RPS17, RPS19, RPS26, RPL5, RPL11, RPL35A). Deletions of probe recognition sequences were apparent by a 35–50% reduced relative peak area of the amplification product of that probe.

Results:

The results of the MLPA assay revealed that 13 out of the 73 probands (18%) had a multi-exonic deletion in one of the six DBA genes analyzed. We identified four deletions of the RPS17 gene, three of the RPS26 gene, three of the RPL35A, two of the RPL11 gene and one of the RPL5 gene. No additional RPS19 deletions were found. DBA patients with deletions showed a severe phenotype with a very high percentage of transfusion dependence (85%). Somatic malformations were observed only in two patients.

Conclusion:

We detected a high percentage of deletions of known DBA genes in a cohort of patients in whom no mutations were found by RP sequencing.

Mutation screening of the RP genes with a combination of sequencing and MLPA reached an overall detection rate of 61.5% (96/156). In our cohort, large genomic deletions represent up to 18% of all mutations detected.

In conclusion, we stress the high percentage of identified RP genes deletions in DBA patients. We also highlight that a gene-dosage technique, such as MLPA, should complement sequencing in a clinical environment since only a combined approach of this kind permits the comprehensive detection of all mutations in the DBA RP genes.

Disclosures:

No relevant conflicts of interest to declare.
Successive Development of T-Cell Acute Lymphoblastic Leukemia, Non-Langerhans-Cell Histiocytose and Histiocytic Sarcoma with a Common Origin: Evidence for Common Oncogenetic Transformation Before Full Lineage Commitment,
Authors: te Loo D. Maroeska W.M. , Waanders Esmé , Groenen Patricia JTA , Simons Annet , van Dongen Jacques J.M. , Hoogerbrugge Peter M. , Hebeda Konnie M
Published: October 2019

Abstract 3470

Hematopoiesis is traditionally seen as the unidirectional maturation of stem cells into lineage committed cells. Recent data are suggestive for some degree of lineage flexibility in both normal as malignant cells. We here present a boy that presented with T-cell acute lymphoblastic leukemia (T-ALL). During T-ALL treatment, the patient developed in short time a non-Langerhans-cell histiocytose in the ileum, and subsequently a disseminated form of histiocytic sarcoma (a.o. also in the liver). Interestingly, the same clonal T cell receptor (TCR) gene rearrangements were found in all three malignancies (TCRB-VJ, TCRG-VJ and TCRB-DJ) indicating they were related. There were no immunoglobulin rearrangements. To understand the evolution from T-ALL to non-Langerhans-cell histiocytose and to histiocytic sarcoma, an extensive genetic analysis was performed. Using a SNP6.0 array platform we analyzed DNA isolated from T-ALL cells, a bone marrow sample obtained during complete remission from theT-ALL, a biopsy from the ileum tumor, the liver tumor, and DNA isolated from skin fibroblasts.

The three tumor samples were not fully concordant in the SNP analysis, with lesions acquired but also absent from subsequent samples. All three tumor samples showed identical TCR gene rearrangements as well as a loss of the CDKN2A/B region, the only gene-containing copy number aberration (CNA) present in the T-ALL. The T-ALL sample showed a homozygous loss of the CDKN2A/B region, whereas both the ileum and liver samples showed a heterozygous loss. In addition, the three tumor samples shared two 1Mb regions of loss of heterozygosity (LOH) on chromosomes 6p and 11p, containing DDB2, MADD and RUNX2, amongst others. A gain on chromosome 19q containing 12 genes was shared between the ileum and liver sample only. In addition, the ileum tumor showed 14 non-shared CNAs of which 11 losses, 2 homozygous losses, and 1 gain, affecting 11 genes including ZFAT, PTPRK and beta-catenin (CTNNB1). The liver tumor acquired gains of a large part of chromosome 5, the p-arm of chromosome 11 and the q-arm of chromosome 22 reminiscent of a chromothrypsis event. The PAX5 gene indicated in the progression of an ALL to histiocytic sarcoma was not affected in either of the samples. These results indicate that the three tumors are related but did not evolve from each other in a linear manner. Apparently, the common oncogenetic transformation occurred at an early differentiation stage with lineage plasticity, i.e. with some lineage specification but without full lineage commitment.

Disclosures:

No relevant conflicts of interest to declare.
Long Term Followup of Bystander Vaccine Therapy in Mantle Cell Lymphoma (MCL),
Authors: Shah Bijal D. , Tao Jianguo , Sokol Lubomir , Chervenick Paul A. , Tomblyn Marcie , Pinilla-Ibarz Javier , Moscinski Lynn , Antonia Scott , Sotomayor Eduardo M. , Dessureault Sophie
Published: October 2019

Abstract 3720

Background:

A Phase I study revealed that vaccination of cancer patients with irradiated autologous tumor cells and GM.CD40L bystander cells (engineered to secrete GM-CSF and express CD40L) is safe, recruits/activates dendritic cells, and elicits tumor-specific T cell responses. We report the long term followup using this vaccine strategy in patients with MCL, an aggressive and incurable B-cell malignancy.

Methods:

After lymph node resection for autologous tumor harvest, 4–6 cycles of chemotherapy, and restaging (CT, endoscopy, bone marrow biopsy), patients with usable vaccine who achieved a PR or CR lasting 1 month were vaccinated x4 at 28-day intervals with IL-2 (0.5 × 106 U SC BID × 14 d/cycle). Patients were monitored for toxicity, tumor response, tumor-specific immune responses, and EFS/OS.

Results:

43 were enrolled, including 21 with relapsed MCL, 20 with int/high MIPI, and 6 with blastoid MCL. Twenty never received vaccine: 2 withdrew consent, 1 progressed rapidly prior to lymph node harvest, 7 had insufficient or contaminated specimens, and 10 progressed/died during chemotherapy. The unvaccinated were older (68.4y vs 62.8y; p=.026) but otherwise did not differ significantly by stage, LDH, MIPI, de novo status, or number of prior treatments.

Among 23 treated, 10 had relapsed disease, 10 had an int/high MIPI, and 2 had blastoid MCL. Pre-vaccination response following chemotherapy included 7 CR, 15 PR, and one SD. At 6 months after vaccination, 2 pts in PR had resolution of MRD within the bone marrow, 10 progressed (including 3 who progressed after only 1–2 vaccines), and 11 had no change. Two are still in CR at 49 & 50 months. Median EFS and OS are calculated from receipt of first vaccine. Median EFS is 9 mo, however, this high risk cohort continues to show a prolonged median OS, which has not yet been reached (median follow-up 43 mo, range 4–79 mo). An increase in interferon gamma secretion as measured by ELISPOT assays was observed in 15 of the 23 patients (65%). Additionally, this was accompanied by an increase in EFS among those with a positive response.

Conclusions:

These phase 2 data are analogous to what we have seen among 4 patients with relapsed MCL treated on our phase 1 trial (EFS 6.5 mo, OS 82 mo), and support the need for further studies using GM.CD40L bystander vaccination. Similar discrepancies in EFS and OS have also been observed with other approved immunotherapeutics, ipilumumab & sipleucel-T (Hodi et al. 2010, Kantoff et al. 2010).

Disclosures:

No relevant conflicts of interest to declare.
Angiogenic Factors eNOS and VEGF Have Increased Expression in Granulocytes of JAK2V617F Homozygous Forms of Polycythemia Vera,
Authors: Cokic Vladan P , Markovic Dragana , Mitrovic Olivera , Vignjevic Sanja , Djikic Dragoslava , Beleslin-Cokic Bojana B , Perunicic Maja , Ilic Bojana , Jovcic Gordana , Gotic Mirjana , Noguchi Constance Tom , Schechter Alan N
Published: October 2019

Abstract 3847

The microvessel density of bone marrow is increased in myeloproliferative neoplasms (MPN) parallel with vascular endothelial growth factor (VEGF). VEGF-mediated angiogenesis requires nitric oxide (NO) production from activated endothelial NO synthase (eNOS). NO as well as hypoxia stimulate the VEGF gene expression and angiogenesis by enhancing hypoxia inducible factor (HIF)-1 activity. We studied 126 newly diagnosed patients with BCR-ABL− MPN: 64 polycythemia vera (PV), 36 essential thrombocythemia (ET), 26 primary myelofibrosis (PMF) and 12 healthy individuals. We performed a combined analysis of hematopoietic CD34+ progenitor cells and granulocytes in peripheral blood of these individuals. The eNOS protein level is more than three-fold elevated in granulocytes of JAK2V617F homozygous PV patients. The essential inducer of angiogenesis VEGF-A has also about three-fold elevation at the protein level in granulocytes of PV patients, with major increases in JAK2V617F homozygous forms. Immunohistochemical analysis reveal that the percentage of VEGF-A-positive cells is increased in bone marrow of PV (5.58±0.7%) compared to normal controls (2.78±0.7%) and VEGF-A mRNA levels are increased in hematopoietic progenitor cells of PV origin. Transcription factor HIF-1α gene expression is decreased in hematopoietic progenitor cells and increased in granulocytes of PV patients. Negative regulator of HIF-1α activity, a transcription factor HIF-3α, has decreased expression in hematopoietic progenitor cells and not changed in granulocytes. In contrast to PV patients, PMF and ET disorders with a minor JAK2 mutation burden demonstrate reduced eNOS and VEGF protein levels and decreased HIF-1a gene expression in peripheral blood granulocytes, although the increase in percentage of VEGF-A-positive cells in bone marrow observed in PV patients is also evident. The present results expand the significance of JAK2V617F mutation in induction of angiogenic factors eNOS and VEGF in granulocytes of PV patients with enhanced HIF-1α presence. Moreover, the stromal and hematopoietic cells also show increased VEGF protein expression in bone marrow of PV patients. Therefore, we find that variations in angiogenic factors expression among MPN patients appear to be related to JAK2V617F mutation allele burden.

Disclosures:

No relevant conflicts of interest to declare.
CD38 Regulates Homing and Engraftment of CLL Cells,
Authors: Vaisitti Tiziana , Serra Sara , Audrito Valentina , Pepper Chris , Rossi Davide , D'Arena Giovanni , Laurenti Luca , Zucchetto Antonella , Gattei Valter , Gaidano Gianluca , Malavasi Fabio , Deaglio Silvia
Published: October 2019

Abstract 3873

Chronic lymphocytic leukemia (CLL) is considered the result of a dynamic balance between proliferating cells in lymphoid organs and circulating cells resisting apoptosis. Re-circulation of leukemic cells from blood to growth-permissive niches represents an essential step in the maintenance and progression of the disease. This equilibrium is finely tuned by a set of surface molecules expressed by CLL cells and modulated in response to environmental conditions.

We previously reported that CD38, an enzyme and a receptor, functionally cooperates with the CXCL12/CXCR4 axis, enhancing the ability of CLL cells to home to bone marrow and lymph nodes. In addition, the use of anti-CD38 mAbs can enhance or impair the chemotactic behavior of the neoplastic cells. New evidence also indicates that CD38 synergizes with the CD49d integrin, increasing adhesion of CLL cells to VCAM-1 or the CS-1 fibronectin fragment, two known ligands of CD49d. To complete the picture, CD38 expression denotes a CLL subset with increased activity of the matrix metalloproteinases MMP-9. Ligation of CD38 with specific antibodies increases MMP-9 secretion and the invasive properties of CLL cells, using in vitro assays. The effects on chemotaxis, adhesion and invasion are obtained through modulation of a ERK1/2-dependent pathway.

To further confirm the involvement of CD38 in CLL homing to specific niches, in vivo experiments have been set using NOD/SCID/γ chain−/− (NSG) mice. The CLL-like cell line Mec-1, constitutively CD38−/CD49d+, was adopted as a model and compared to transfectants stably expressing wild-type (wt) CD38, as well a mutant lacking enzyme activities. Results after i.v. injections of tumor cells indicate that de novo expression of CD38 by Mec-1 cells increases growth kinetics in vivo with a higher proliferation rate and metastatic potential, as compared to the Mec-1 mock-trasfected cells. Both these features are lost when the animals are injected with the enzyme-deficient variant of CD38, suggesting that the enzymatic activity is critical for in vivo growth and re-circulation of Mec-1 cells. Microarray data confirm that the genetic signature of the CD38-enzyme mutant overlaps with the wild-type cell line, clearly distinct from cells transfected with CD38. The latter cell line shows up-modulation of several genes involved in chemotaxis and adhesion.

All together, these results support the notion that CD38 is part of a complex network of molecules and signals, that regulate homing of CLL cells to growth-permissive niches, suggesting a relationship between the expression of CD38, the ability to migrate and invade and the poor clinical outcome of the CD38+ subset of patients.

Disclosures:

No relevant conflicts of interest to declare.
Prognostic Significance of Telomere Length in Chronic Lymphocytic Leukemia Patients in Early Stage Disease,
Authors: Fabris Sonia , Hoxha Mirjam , Agnelli Luca , Dioni Laura , Bollati Valentina , Ciceri Gabriella , Mosca Laura , Barbieri Marzia , Cutrona Giovanna , Matis Serena , Gentile Massimo , Deliliers Giorgio Lambertenghi , Cortelezzi Agostino , Morabito Fortunato , Ferrarini Manlio , Baccarelli Andrea , Neri Antonino , Bertazzi Pier Alberto
Published: October 2019

Abstract 3890

Chronic lymphocytic leukemia (CLL) is a genetically heterogeneous disease with a variable outcome. The identification of factors that could predict the clinical course of early-stage CLL represents a crucial objective. Although previous studies indicated that telomere length may be a useful independent prognostic factor in the risk stratification of CLL, limited information has been reported in asymptomatic early stage patients (Binet stage A). We investigate the association of telomere length with the major biological and cytogenetic markers known to predict clinical outcome in CLL. The global DNA methylation levels of Alu and LINE sequences, was also investigated. Correlation with disease progression, measured as the time elapsed from diagnosis to first treatment, was evaluated. We measured relative telomere length (RTL) by real-time PCR in a panel of highly purified (>90%) peripheral mononuclear CD19+ cells from 7 healthy donors and 77 untreated CLLs. All the cases were characterized by FISH for the most frequent chromosomal aberrations (Fabris et al. GCC, 2008). Molecular markers including mutation status of the heavy chain variable regions of immunoglobulin genes (IGVH), the expression of the 70-kd zeta-chain T-cell receptor-associated protein kinase (ZAP-70) and CD38 cell surface antigen protocols were previously reported (Cutrona et al., Haematologica, 2008). A quantitative bisulfite-PCR Pyrosequencing method was used to evaluate methylation of Alu and LINE-1. We found a significantly lower RTL values in CLLs (median RTL=0.4 IQR 0.3–0.6) as compared with controls (median RTL=1.0 IQR 0.9–1.3) (P <0.001). A progressive and significant RTL decrease in low (13q- and normal karyotype), intermediate (+12) and high (11q- and 17p-) cytogenetic risk categories (P for trend =0.008) was observed. Patients with IGVH mutated genes had longer telomeres than patients with unmutated genes (P <0.001). No significant association between telomere length and either CD38 or ZAP-70 expression was found. Telomere shortening was significantly correlated with hypomethylation of Alu (P =0.048) and LINE-1 (P =0.001), indicating a contribution to chromosome instability. Finally, follow-up analysis showed a significantly higher risk of starting treatment for patients with shorter telomeres (P =0.0037). Our results extended previous evidence that telomere length could be used as marker for the identification of CLLs with a different prognostic risk.

Disclosures:

No relevant conflicts of interest to declare.
An International Study of High Cut-off Hemodialysis for the Management of Myeloma Kidney,
Authors: Hutchison Colin A , Bevins Anne , Mead Graham , Cook Mark
Published: October 2019

Abstract 3974

Background:

An early reduction in serum free light chains (FLC) improves outcomes for patients with myeloma kidney. The purpose of this study was to assess how high cut-off (HCO) dialysers are being used internationally to achieve this target.

Methods:

Data was collected for 54 patients, from 18 centers in 10 countries. Clinical presentation and treatment parameters, including details of removal of FLCs with high cut off hemodialysis and chemotherapy treatment, were captured using a web-based collection database.

Demographics:

All patients were Caucasian, median age of 65 years (range 43–81). Median biochemistry at presentation was: GFR of 8mls/min/1.73m2 (1–27). creatinine 633.5μmol (168–2263); calcium 2.3mmol/L (0.91–3.83); albumin 34g/L (14–46) and β2M 9.45mg/L (0–55.7). Baseline monoclonal κ and λ FLCs levels were: 5070mg/L (range 2250–20200) and 4200mg/L (range 300–13300), respectively. Of the patients who received a renal biopsy, 81% had myeloma kidney as their primary diagnosis. For initial chemotherapy prescriptions: 78% received bortezomib and 34% received thalidomide. 68.75% of the patients were treated with the Theralite HCO dialyzer; the other 31.25% received treatment with the HCO1100 dialyzer. There was a total of 626 HCO dialysis sessions recorded, with each patient receiving on average 13 session each (median, range 3–35) treatments per patient. Of the 54 patients, only 3 were treated with HDF.

Results:

73.2% of patients demonstrated a decreased serum FLC level over the course of treatment, with 86.7% of these patients demonstrated decreased levels by day 12. The median FLC reduction achieved was 72.96% (15.09–99.62%) by day 12 and 93.03% (40.23–99.96%) by the last dialysis treatment. There was no difference in the percentage FLC reduction achieved between bortezomib (median: total 94.09%, day 12 79.14%) and thalidomide (median: total 77.65%, day 12 66.51%) treatment groups (total p=0.179, day 12 p=0.300). FLC removal was significantly increased in patients receiving Theralite treatment over those receiving HCO1100 at both day 12 (p=0.030) and by the end of treatment (p=0.031) Dialysis independence occurred in 68.2% of patients, median time 32 days (10–249). Patients who became independent of dialysis had significantly greater reductions in serum FLC by day 12 (p=0.032). No significant adverse events related to the study device were reported.

Conclusion:

Reducing FLC in patients with myeloma kidney is associated with increased levels of dialysis independence. This study adds further evidence that a combination of chemotherapy and FLC removal by HCO hemodialysis improves patient outcomes following acute kidney injury secondary to multiple myeloma.

Disclosures:

Hutchison: The Binding Site: Honoraria. Bevins:The Binding Site Group Ltd: Employment. Mead:The Binding Site Group Ltd: Employment.
Lentiviral Transduction of Patient Derived Leukaemic Blasts Allowing In Vivo Bioluminescent Monitoring in An NSG Model of Leukaemia Stem Cell Maintenance,
Authors: Bomken Simon , Buechler Lars , Rehe Klaus , Ponthan Frida , Blair Helen , Heidenreich Olaf , Vormoor Josef
Published: October 2019

Abstract 4000

Serial transplantation of patient derived acute lymphoblastic leukaemia (ALL) blasts continues to contribute to our understanding of the biology of leukaemia stem cells (LSC). Refinement of techniques, and in particular intrafemoral injection and development of the highly immunocompromised NOD/scid IL2Rγ null (NSG) mouse have demonstrated B precursor ALL propagating cells to be both common and present in diverse immunophenotypes. These studies must now be complemented by interrogation of the biological pathways underpinning leukaemia stem cell behaviour and clonal propagation in ALL. We have developed a lentiviral based approach to such studies, using the transfer vector pSLIEW, encoding both enhanced green fluorescent protein (EGFP) and firefly luciferase (luc).

We have recently replaced a bone marrow stromal feeder based transduction protocol with a feeder free protocol, removing the risk of co-transduction of feeder cells. Using the feeder free protocol, we have achieved transduction of primary (n=4) and primograft (n=3) material with between 13.0 and 51.4% eGFP positive cells.

Transplantation of transduced cells by intrafemoral injection into NSG mice resulted in engraftment and disease dissemination. This process was monitored using an IVIS Spectrum bioluminescence imaging system. This technique demonstrated progression of disease to the contralateral femur, spleen, CNS and vertebrae. Disease progression was also monitored by serial bone marrow punctures and 5-colour flow cytometry, which demonstrated no immunophenotypic bias amongst the transduced cells. Flow cytometry of harvested bone marrow and spleen showed between 5.5% and 10.2% eGFP positive cells representing only a moderate decrease from 26.3% eGFP positivity at initial transplantation. This confirms the relative resistance of the SFFV promoter to silencing, making this approach suitable for serial transplantation.

Harvested bone marrow and splenic cells were re-transplanted at 5.5 × 103 – 1.0 × 104 SLIEW+ cells per mouse (total 1 × 105 cells transplanted). Bioluminescent imaging has shown engraftment and dissemination of leukaemia within five weeks, confirming transduction of the leukaemia stem cell compartment.

Further development of the pSLIEW vector to include shRNA sequences now offers the potential for functional studies using patient derived material, transduced with a single lentivector construct and serially engrafted in the NSG assay for leukaemic stem cell maintenance. We believe that this approach will allow us to investigate the genetic programmes underpinning leukaemia stem cell self-renewal.

Disclosures:

No relevant conflicts of interest to declare.
Mitochondrial DNA Minisatellites Showed Higher Informativeness and Sensitivity Than the Nuclear DNA Markers for the Quantitative Determination of Chimerism After Allogeneic Stem Cell Transplantation,
Authors: Kim Hye Ran , Won Eun-Jeong , Choi Hyun-Jung , Kim Hwan-Young , Moon James , Shin Jong-Hee , Suh Soon-Pal , Ryang Dong-Wook , Shin Myung-Geun
Published: October 2019

Abstract 4004

Background:

Mitochondrial DNA (mtDNA) is widely used in forensic identification and anthropologic studies on account of its abundance resulting in preferential amplification, sequencing and inherent variability. We developed mtDNA markers to monitor donor cell engraftment after allogeneic stem cell transplantation(SCT), then compared with nuclear short tandem repeat (STR) markers. Patients and methods: The mtDNA control regions and six mtDNA minisatellites (mtMS) (303 poly C, 16184 poly C, 514 (CA) repeat, 3566 poly C, 12385 poly C and 12418 poly A) from the total DNA samples of 215 cases (donor, recipient and after allogeneic SCT) were amplified using the designated specific primers and PCR. The results were compared with those from the six short tandem repeat (STR) markers (D12S391, D18S51, F13A1, HUM RENA-4, HUM FABP2 and Amelogenin). Results: Polymorphisms in the mtDNA control region identify an informative marker in 88% (189 cases) of all cases. Among the six mtMS markers, the informativeness of 303 poly C and 16184 poly C mtMS was 63% and 67% respectively. A combination of direct sequencing through the mtDNA control region, 303poly C and 16184 poly C mtMS could completely distinguish the donor cells from the recipient cells. The results from a typical mixing experiment to determine the sensitivity revealed a detection limit (DL) of the gene scan analysis in a mtDNA mixture to be visible at 1% heteroplasmy in 303 poly C mtMS marker. However, the DL from D12S391 in the same mixing experiment was 5–10% heteroplasmy. Conclusions: mtMS markers, especially 303 poly C and 16184 poly C markers, can provide a sensitive, accurate and quantitative determination of mixed chimerism after a SCT.

Disclosures:

No relevant conflicts of interest to declare.
Selective Targeting of B Cells with Mesenchymal Stromal Cells Treatment Is An Efficacious Strategy for Cutaneous Cgvhd,
Authors: Lai Peilong , Weng Jianyu , Luo Chenwei , Guo Rong , Lu Zesheng , Geng Suxia , Du Xin , Wu Suijing
Published: October 2019

Abstract 4086

Background:

Cutaneous chronic graft versus host disease (cGVHD) is an increasingly common complication in long term survivors of hematopoietic stem cell transplantation, but currently available therapies have demonstrated limited efficacy. Furthermore, Mesenchymal Stromal Cells (MSCs) have been reported to be effective in various immune-mediated disease models, but their therapeutic potentials versus cutaneous cGVHD have not been determined.

Methods:

We enrolled 23 patients suffering from cutaneous cGVHD who had failed to respond to conventional immunosuppressive therapy or relapsed after reduction of prednisone. They received intravenous in vitro expanded bone marrow (BM)-derived MSCs. The median follow-up period was 90 days (range 60–146 days).The dermal manifestations of cGVHD were monitored, and a score was given to the cutaneous response by the physician. B lymphocyte subsets and ratio of CD4/CD8 were detected by flow cytometry before the first and after the last MSC infusion. The plasmatic levels of TNF-α, sICAM-1 and Th1 or Th2 factors, including IL-2 and IL-4, were measured by enzyme-linked immunosorbent assay (ELISA).

Results:

Nineteen patients (82.61%) had skin symptom abatement after MSCs treatment with the skin scores improved according to the NIH criteria. Most patients had healing of skin ulceration, regression on skin findings and increased flexibility of involved joint. Steroid sparing or discontinuation of immunosuppressive medications was possible in all of the patients. Clinical improvement was accompanied by increasing levels of CD19+CD5+ B cells and elevated ratio of CD4/CD8. In addition, after the last MSCs treatment, the patients had significantly higher levels of IL-2 compared to before the first MSC infusion, while they had lower levels of TNF-α, sICAM-1 and IL-4.

Conclusion:

MSCs infusion represents an effective treatment option for cutaneous chronic GVHD. This is of high interest in patients suffering from cutanous cGVHD resistant to conventional immunosuppressive therapy. MSCs alter allo-reactivity by affecting CD19+CD5+ B cells, possibly normalize the ratio of CD4/CD8 and Th1/Th2 and decreased the level of proinflammatory cytokine TNF-α and proadhesive cytokine sICAM-1. Our study demonstrates CD19+CD5+ B cells could be a possible target for therapeutic intervention in cutaneous cGVHD.

Disclosures:

No relevant conflicts of interest to declare.