bluuebunny/crossref_metadata_2025 huggingface dataset:
Rituximab, Bendamustine, Mitoxantrone, Dexamethasone (R-BMD) in Patients with Follicular Lymphoma in Relapse or Refractory to First-Line Treatment with Immunochemotherapy. R-BMD Geltamo 08 Trial
Authors: Peñalver Francisco Javier , Márquez José Antonio , Durán Soledad , Giraldo Pilar , Montalban Carlos , Ramírez María José , Sancho Juan Manuel , Terol María José , Capote Francisco Javier , Gutiérrez Antonio , Sánchez Blanca , Canales Miguel , Caballero María Dolores
Published: October 2019

Abstract 1639

Objectives

To evaluate the efficacy and safety of rituximab-bendamustine-mitoxantrone-dexamethasone (R-BMD) in patients with relapsed or refractory follicular lymphoma, (R/R FL) to first-line therapy with R-chemotherapy (R-ChemoT), followed by maintenance with R.

Methods

Phase II trial including 61 patients with R/R LF, after a 1st R-ChemoT line. Induction treatment: Rituximab 375 mg/m2 iv, day 1; bendamustine 90 mg/m2 iv, days 1 and 2; mitoxantrone 6 mg/m2/day iv, day 1; oral dexamethasone 20 mg / day, days 1 to 5. Cycles of 28 days. Evaluation of response after 3rd cycle. If stable disease or progression: withdrawal from the study. If complete response (CR) or complete response unconfirmed (CRu): administration of a 4th cycle. If partial response (PR): administration up to 6 cycles. If CR, CRu or PR at the end of induction: patients receive maintenance with R 375 mg/m2/day every 12 weeks for 2 years. Primary objective: Complete responses (CR + CRu). Results are presented as valid % and median [range].

Results

Results from 46 patients who completed induction period. 52.2% women, age 63 [32–76] years. Ann Arbor stage III / IV 75.6% (31/41) and III / IV-B 22.6% (7/31). FLIPI: intermediate risk 28.9% (11/38); high-risk 23.7% (9/38). Number of administered cycles: 4 [1–6]. Overall response 93.5% (43/46); CR: see Table 1. Progression Free Survival –median (CI95%)-: 14.5 (11.6-NA) months. The most relevant grade 3/4 toxicity: neutropenia 52% (n = 24; 17 patients received G-CSF) and thrombocytopenia 4.3% (n = 2). Infections grade 3/4: 6.5% (n = 3). One patient died due to CMV reactivation. No skin reactions were reported. There are maintenance available data from 15 patients: 3 patients sustained CR at the end of this period, and 2 patients progressed.

Conclusions

R-BMD is a treatment schedule effective and a safe alternative for patients with R/R FL, after a 1st line with R-ChemoT. No skin reactions were reported, possibly due to the inclusion of dexamethasone in the treatment scheme. Additional follow up is required to achieve more conclusive findings.

Disclosures:

No relevant conflicts of interest to declare.
Progressive Peripheral Arterial Occlusive Disease and Other Vascular Events During Nilotinib Therapy in Chronic Myeloid Leukemia
Authors: Levato Luciano , Cantaffa Renato , Kropp Mariagrazia , Magro Domenico , Piro Eugenio , Molica Stefano
Published: October 2019

Abstract 1679

Recent data suggest that second-generation tyrosine-kinase inhibitor (TKI) dasatinib and nilotinib can be responsible for increased non-hematologic adverse events in comparison to imatinib. In particular, there are a few reports of severe peripheral arterial occlusive disease (PAOD) and other vascular occlusive events (infarction) in patients receiving nilotinib. With this in mind we retrospectively evaluated incidence of PAOD or other vascular occlusive events in our cohort including 82 consecutive chronic myeloid leukemia (CML) patients treated at our institution with imatinib alone (n=55) or nilotinib as first-line (n=17) or second-line treatment after imatinib failure (n=10). After a median time of exposition to nilotinib of 24 months (range, 7–34 months) 4 (14.8%) out of 27 patients developed an episode of severe and previously unrecognized PAOD or other vascular occlusive events (2 PAOD, 1 myocardial infarction, 1 ictus). All 4 patients were more than 60 years old and 3 out of 4 were male while obesity was never observed. A history of nicotine abuse could be found in 2 out of 4 patients. The same applied when we looking for the presence of arterial hypertension (2/4) or dyslipidemia (2/4). When 55 patients treated with imatinib were analyzed for PAOD or other vascular occlusive event incidence we detected only one patient who experienced myocardial infarction after 135 months of therapy.

We then evaluated the likelihood of developing PAOD in the subset of patients treated only with imatinib and in those who received nilotinib, respectively. The projected 10-year actuarial probability of remaining PAOD-free was 100% in the imatinib group and 67% in the nilotinib group (HR, 14.6; P=0.0008). Interestingly, the two patient cohorts were alike with respect to age (P=0.76), gender (P=0.80), number cardiovascular risk factors (P=0.62) and body mass index (P=0.59). The only difference we observed was a significantly longer exposition to drug among patients treated only with imatinib in comparison to those who received nilotinib (P<0.001).

In conclusion this study although hampered by the relatively small patient number suggests that in CML front-line and second-line treatment should be adapted to the individual situation in each patient. Parameters related to risk of developing TKI-associated adverse PAOD events should be considered before choosing a second-generation TKI.

Disclosures:

No relevant conflicts of interest to declare.
Genetic Predisposition to Molecular Response in Patients with Myeloproliferative Neoplasms Treated with Hydroxycarbamide
Authors: Angona Anna , Bellosillo Beatriz , Alvarez-Larrán Alberto , Martinez-Aviles Luz Maria , Camacho Laura , Pairet Silvia , Fernández Concepción , Besses Carles
Published: October 2019

Abstract 1738

Introduction:

Hydroxycarbamide (HC) is an antimetabolite used as first-line therapy in high risk essential thrombocythemia (ET) and polycythemia vera (PV). In treatment-naive patients, HC achieves complete hematological and partial molecular response in 80% and 50% of patients, respectively. Genetic factors involved in the pharmacokinetics of HC, as well as in the acquisition of the JAK2V617F mutation, could play a role in the variability among these patients in achieving a molecular response.

Objective:

To assess the influence of 46/1 JAK2 haplotype and urea transporters polymorphisms on the molecular response in JAK2V617F-positive PV and ET patients treated with HC.

Methods:

JAK2V617F allele burden was measured by quantitative PCR at diagnosis and every 6 months during follow-up in 53 PV patients and in 41 ET patients receiving HC therapy. Molecular response to HC treatment was defined according to European LeukemiaNet (ELN) criteria. SNPs rs12340895 and rs12343867 were used to determine 46/1 haplotype status and SNPs rs2298720 (SLC14A1), rs9960464 (SLC14A2), were used to genotype urea transporters. The rate of molecular response was compared according to 46/1 JAK2 haplotype status (negative, heterozygous, homozygous), and genotype of 2 urea transporters: SLC14A1 (GG, GA, AA) and SLC14A2 (GG, GA, AA). The study was approved by the local Ethics Committee and informed consent was obtained according to the Declaration of Helsinki.

Results:

Overall, molecular response (CR or PR) was achieved in 48 patients (51%), corresponding to 46% and 57% in PV and ET patients, respectively (p=0.3). In ET patients, there were no significant differences in the proportion of patients achieving a molecular response according to 46/1 JAK2 haplotype or to the different transporter genotypes. PV patients with the GG genotype in transporter SLC14A2 obtained more frequently a molecular response than those with AA or GA genotype (71% versus 36%, p=0.03). No significant differences in molecular response were observed in PV patients according to transporter SLC14A1 and 46/1 JAK2 haplotype.

Conclusion:

46/1 JAK2 haplotype status did not influence molecular response in PV or ET patients treated with HC. Polymorphisms in urea transporters showed a minimal effect on the rate of molecular response in these patients.

Disclosures:

No relevant conflicts of interest to declare.
PDE4 Inhibitors Block TLR7 and TLR9-Driven Signaling, Proliferation and Cytokine Secretion in CLL Cells As Well As Proliferation Driven by Exposure to Apoptotic Cells
Authors: Tan Ying , Watkins Amanda A. , Rifkin Ian R. , Lerner Adam
Published: October 2019

Abstract 1774

A growing body of literature supports the concept that at least a subset of chronic lymphocytic leukemia (CLL) B cell-receptors (BCRs) are reactive with antigens exposed on apoptotic cells. Work done in the autoimmunity field suggests that through their BCRs, autoreactive B cells can internalize RNA and DNA-containing autoantigens, thereby activating endosomal Toll-like receptor (TLR) 7 and TLR9, respectively, with subsequent TLR-mediated signaling, proliferation and cytokine secretion. cAMP signaling modulates inflammatory signaling in immune system cells and our prior studies have shown that PKA, EPAC and CREB-mediated cAMP signaling can be potently induced in CLL cells with inhibitors of the cyclic nucleotide phosphodiesterase (PDE) PDE4 family in the absence of additional exogenous stimulators of adenylate cyclase. We therefore examined the ability of PDE4 inhibitor-mediated cAMP signaling to regulate TLR7 and 9-mediated signaling in CLL cells. Treatment of CLL cells with the TLR7 and 9 agonists R848 and CpG-B oligonucleotide, respectively, induced IRAK1 degradation and activation of three signaling pathways known to be downstream of TLR7 and 9: the transcription factors NFkB and IRF5 as well as the MAP kinase JNK. The prototypic PDE4 inhibitor rolipram inhibited R848-induced TLR7 and CpG-B-induced TLR9 signaling in CLL cells as judged by Western analysis and immunofluorescent studies of NFkB and IRF5 nuclear translocation as well as JNK phosphorylation. Rolipram also blocked R848 and CpG-B-induced CLL proliferation and TNF secretion. In contrast, rolipram augmented R848 and CpG-B-induced CLL IL-10 synthesis, in keeping with prior studies documenting cAMP response elements in the IL-10 promoter. To test whether RNA and/or DNA antigens exposed in apoptotic cells might induce proliferation in CLL cells through BCR-mediated internalization and subsequent activation of TLR7 and TLR9, we irradiated CLL cells and added them in a graded fashion to live autologous CLL cells. Such exposure to irradiated cells undergoing apoptosis drove CLL proliferation in a manner that was sensitive to TLR7 and TLR9 antagonists, IRAK4 kinase inhibitors and the PDE4 inhibitor rolipram. Our work supports the concept that BCR and TLR-driven responses to apoptotic RNA and DNA-containing complexes may drive proliferation in CLL cells and that PDE4 inhibitors and other agents that target TLR signaling may prove to be effective therapeutic agents in this disease.

Disclosures:

No relevant conflicts of interest to declare.
B-Cell Receptor Configuration and Adverse Cytogenetics Are Associated with Autoimmune Hemolytic Anemia in Chronic Lymphocytic Leukemia
Authors: Maura Francesco , Visco Carlo , Falisi Erika , Gianluigi Reda , Fabris Sonia , Agnelli Luca , Tuana Giacomo , Lionetti Marta , Guercini Nicola , Novella Elisabetta , Nichele Ilaria , Montaldi Anna , Autore Francesco , Gregorini Anna Ines , Barcellini Wilma , Callea Vincenzo , Mauro Francesca Romana , Laurenti Luca , Foa Robin , Neri Antonino , Rodeghiero Francesco , Cortelezzi Agostino
Published: October 2019

Abstract 1780

Background:

Biological features related to the development of autoimmune hemolytic anemia (AHIA) in patients with chronic lymphocytic leukemia (CLL) are crucial insights in the understanding of the pathogenesis of autoimmune phenomena in the course of the disease.

Design and Methods:

We retrospectively analyzed 585 CLL patients with available immunoglobulin heavy-chain variable (IGHV) gene status and B-cell receptor (BCR) configuration (HCDR3). Of them, 73 developed AIHA. The clinical characteristics at CLL diagnosis and follow-up were available in all patients, while cytogenetic analysis at the time of diagnosis was available in 409 patients.

Results:

Occurrence of AIHA was significantly associated with an IGHV unmutated (UM) status (p<0.0001) and unfavorable cytogenetic lesions [del(17)(p13) and del(11)(q23)] (p<0.0001). Stereotyped HCDR3 sequences were identified in 173 of 585 patients (29.6%) and were similarly represented among patients developing AIHA (28,7%) or not (29.6%). Of the stereotyped subsets, subset #3 was associated with a significantly higher risk of AIHA occurrence than the other HCDR3 configurations (p=0.004). Restricting the analysis to UM patients, a strong association was found between AIHA and “truly” UM patients, defined as patients carrying a 100% identity with the germ line configuration. Multivariate analysis showed that “truly” UM IGHV, del(17)(p13) and del(11)(q23) were the strongest independent variables associated with risk of developing AIHA (p=0.02, p=0.0002 and p=0.01, respectively).

Based on the results of the multivariate analysis, we constructed a risk score of developing AIHA during time, according to the presence of none (low risk = favorable cytogenetics and mutated (M) IGHV), one (intermediated risk = unfavorable cytogenetic or UM), or two (high risk = unfavorable cytogenetic and UM) risk factors. This scoring system allowed a significant patient risk stratification (Figure 1).

Conclusions:

Taken together, our data indicate that an UM IGHV status and/or unfavorable cytogenetic lesions are associated with the risk of developing secondary AIHA in CLL patients and suggest a possible role of specific stereotyped BCR subsets in a proportion of cases.

Disclosures:

No relevant conflicts of interest to declare.
Stromal Tumor Microenvironment in CLL: Regulation of Leukemic Progression
Authors: Chaturvedi Nagendra K , Ahrens Adam K , Shukla Ashima , Gilling Christine E , Mittal Amit K. , Bierman Philip , Weisenburger Dennis D , Lu Runqing , Joshi Shantaram S
Published: October 2019

Abstract 1781

Chronic Lymphocytic Leukemia (CLL), the most prevalent adult B-cell malignancy in western countries, is a highly heterogeneous with a very variable clinical outcome. Emerging evidence indicates that the stromal tumor microenvironment (STME) play important roles in the pathogenesis of CLL. However, the precise mechanism and molecules of STME involved in this process remain unknown. In an attempt to explore the role of STME in this process, we examined the expression levels of stromal associated genes using gene expression profiling (GEP) of CLL cells from 53 patientsâ€™ lymph node (LN) (n=15), bone marrow (BM) (n =18), and peripheral blood (PB) (n=20). Using significant analyses of microarray (SAM), gene set enrichment analyses (GSEA), and ingenuity pathway analyses (IPA), among the major pathways associated with the differentially expressed genes, a cytoskeleton genes associated with stromal signatures are the focus of this report. Of these molecules, a significant number of molecules including: LUM, MMP9, MYLK, ITGA9, CAV1, CAV2, FBN1, PARVA, CALD1, ITGB5 and EHD2 were overexpressed and ITGB2, DLC1 and ITGA6 are under expressed in LN-CLL compared to BM-CLL and PB-CLL indicating a role of LN-mediated TME in CLL cell survival/progression. Among these genes, expression of myosin light chain kinase (MYLK), caveolin 1 (CAV1) and caveolin 2 (CAV2) correlated with clinical outcome (see adjacent Figure) as determined by time to treatment. We recently reported the role of a CAV1 in LN microenvironment-induced immune tolerance in CLL and possibility of their involvement in CLL cytoskeleton (Gilling et al, 2012). In the present study we report aberrant expression of other cytoskeleton genes such as MYLK and CAV2 are involved in the regulation of CLL cell survival in the stromal microenvironment affecting other members of the cytoskeletal signature via actin cytoskeleton signaling, integrin signaling and Pak signaling. In addition, MYLK and CAV2 are also involved in regulation of CLL proliferation. Together our studies show that members of the stromal signature particularly in the CLL cells from lymph nodes regulate the CLL cell survival and proliferation and thus leukemic progression.

Figure: Association of MYLK expression with time to first treatment Figure:. Association of MYLK expression with time to first treatment

Disclosures:

No relevant conflicts of interest to declare.
Characterization of Peripheral B Cells Expressing CLL-Associated Receptor Tyrosine Kinase ROR1 in Healthy Donors
Authors: Milleck Julia , Kreuzer Karl-Anton , Assenmacher Mario , Schmitz Jürgen , Jähn Peter S
Published: October 2019

Abstract 1787

Recent studies have described an extensive expression of the receptor tyrosine kinase-like orphan receptor 1 (ROR1) on the surface of malignant B cells in patients suffering from chronic lymphoid leukemia (CLL). ROR1 expression has also been detected in ovarian cancer, renal cancer, melanoma, and lung adenocarcinoma, suggesting a general role of ROR1 in cancer genesis and/or maintenance. However, low levels of ROR1 mRNA or protein expression have also been found on undifferentiated embryonic stem cells, adipose tissue and on early stage of B-cells in bone marrow, but not in major adult tissues and peripheral B cells.

In contrast, our results describe for the fist time expression of ROR1 on rare B cells in peripheral blood of healthy donors. Using magnetic cell sorting techniques we were able to enrich ROR1 positive B cells from PBMCs of healthy donors. The average frequency of ROR-1+ B cells in PBMCs ranged from 1×10−3 to 1×10−4. Interestingly, ROR1+ B cells revealed a heterogeneous phenotype and could further be classified into a CD19+/CD27+/CD38− and a CD19+/CD27−/CD38+ subpopulation as assessed by flow cytometry. Remarkably, in contrast to the CD27+ subpopulation the CD38+ subpopulation showed higher levels of CD5 and CD23 expression, which are both markers known to be upregulated on B cells of CLL patients. Both populations do not show elevated expression of transcription factor ZAP70 and Bcl-2 but expressed higher level of Bcl-6 compared to CD19 B cells. Bcl-6 is known to be required for pre-B cell self-renewal but has also been described to play an essential role in resistance of leukemic cells to therapeutic tyrosine kinase inhibitors. Moreover ROR1+ B cells revealed an immature and non-activated sate as evidenced by the expression of IgD and IgM, and the lack of IgG and CD69. Further functional examination and immunophenotyping of these rare cells are under investigation.

We have identified rare B cells expressing the tumor-associated receptor tyrosine kinase ROR1, which are heterogenic in CD27 and CD38 expression and show an immature phenotype. We are currently assessing a potential relationsphip ROR1+ peripheral B cells and ROR1 expressing B cells in malignancies like CLL, mantel cell (MCL) and marginal zone lymphoma (MZL).

Disclosures:

Milleck: Miltenyi Biotec GmbH: Employment. Assenmacher:Miltenyi Biotec GmbH: Employment. Schmitz:Miltenyi Biotec GmbH: Employment. Jähn:Miltenyi Biotec GmbH: Employment.
Minimal Residual Disease Stratification in Fludarabine Ccyclophosphamide Rituximab (FCR) Therapy in Chronic Lymphocytic Leukaemia (CLL
Authors: Hayat Amjad , O'Brien David , Quinn Fiona , Keane Fionnuala , Parker Imelda , Kelly Johanna , Wieczorkowska Marzena , Crotty Gerard , Leahy Maeve , Enright Helen , Hennessy Brian , Cahill Mary , Vandenberghe Elisabeth A.
Published: October 2019

Abstract 1791

Six courses of FCR is considered standard therapy for fit, non-(17p) deleted patients with Chronic Lymphocytic Leukaemia (CLL), however the optimal dose of Rituximab in combined chemotherapy for CLL or the number of FCR courses required has never been formally assessed. Minimal residual disease (MRD) eradication (assessed by 6 colour flow-cytometry(FC) is associated with an increased disease free interval and is a logical endpoint in determining length/efficacy of treatment. Serial MRD testing following therapy provides an objective test of disease re-emergence.

A multi-centre prospective phase II study in treatment-naïve CLL patients with modified FCR (Rituximab 375mg/m2) using MRD to determine treatment length/efficacy and identify disease emergence recruited from 2009–2012. Standard pre-treatment assessment plus C-T scan, FISH and Ig somatic hypermutation (SHM) analyses were performed. Patient in radiological and MRD-ve remission after 4 courses of FCR stopped therapy and the remainder received 6 courses. All were followed by 6 monthly MRD assessment.

Fifty-two patients were included (35M/17F), mean age 52 years (range 37–72), mean WCC 51 × 109/L (range 8–386), elevated LDH 23 of 45 (51%), lymphadenopathy 43 (83%) and hepatosplenomegaly in 37 (71%). Abnormal FISH results were, del(11q) in 15, +12 in 5, del(13q) in 18; SHM 15(29%) mutated and 37(71%) unmutated or with V3–21 gene usage. Post-treatment MRD is available in 43 patients; 36 (70%) were MRD –ve including 18 (42%) after 4 courses. Nine patients did not complete treatment (toxicity −7, progression-1, non-compliance-1). With a mean follow-up of 20.7 months (range 4.5–38), 6 patients have reverted to MRD +ve at a mean of 13 months (range 10–19) from therapy; only the patient with primary treatment failure has required further chemotherapy. Myelotoxoicty resulted in 23 NCI grade 3 episodes and 7 treatment delays of a mean duration of 25 days (range15–32). One further grade 3 toxicity of pneumonia was identified.

Modified FCR was effective in this patient cohort with high risk features (38% unfavourable FISH, 71% unfavourable SHM), with 70% patients achieving MRD-ve status on completion of therapy. 42% of patients became MRD-ve after 4 courses of FCR, suggesting that some patients may not require 6 courses of therapy. Myelotoxicity remains an issue with 7 patients not completing therapy. Longer follow-up will clarify whether shortened therapy will have an impact on MRD+ve reversion, time to re-treatment and survival.

Disclosures:

No relevant conflicts of interest to declare.
A 3-Dimensional Co-Culture Model to Investigate Adhesion-Mediated Drug Resistance in Multiple Myeloma
Authors: Aljitawi Omar S. , Xiao Yinghua , Ramachandran Karthik , Lipe Brea C. , Garimella Rama , Krishnan Amrita , Stehno-Bittel Lisa , Li Linheng
Published: October 2019

Abstract 1826

Background:

In multiple myeloma (MM), evidence suggests that the adhesion of malignant plasma cells to bone marrow microenvironment elements is central to MM growth and drug resistance, otherwise referred to as adhesion–mediated drug resistance. As other investigators have shown, the MM-stroma interaction is in part mediated by adhesion molecules like N-cadherin.

Goal:

To develop a 3-dimensional MM in vitro co-culture model where MM cells interact with stromal elements mimicking the bone marrow microenvironment and use the model to investigate N-cadherin mediated drug resistance.

Methods:

We co-cultured U266 cells, a MM cell line, with mesenchymal stromal cells (MSCs) isolated from Wharton's jelly (WJ) in a synthetic copolymer (polyglycolic acid/ poly L-lactic acid 90/10 copolymer) (3-dimensional culture conditions). For controls, we cultured U266 cells over a monolayer of WJMCS (2-dimensional conditions) or U266 cells in suspension. Cell proliferation was measured by Ki-67 immunohistochemistry (IHC) stain and lambda light chain expression was measure by enzyme-linked immunosorbent assay (ELISA) and IHC stains. Bortezomib in 1, 5, and 10 nM concentrations was used to treat U266 cells for 24 hours under the three culture conditions. Cytotoxicity was measured by modified Alamar Blue assay.

Results:

U266 cells in our 3-D model adhered to stromal cells and formed clusters close to matrix material. U266 cells proliferated as evidenced by positive Ki-67 stain, and expressed lambda light chain by IHC and ELISA. Lambda light expression measured by ELISA peaked at 1 week of co-culture in the 3-D model. Though N-cadherin was expressed in both stromal cells and U266 MM cells in 3-D, it was expressed only in stromal cells in 2-D culture conditions. Finally, pre-incubating U266 cells with N-cadherin blocking antibody resulted in less population of the 3-D model by U266 MM cells. Treatment of U266 with bortezomib resulted in less cell cytotoxicity in 3-D and 2-D co-culture conditions compared to U266 cells treated in suspension.

Conclusions:

Using a 3-D in vitro co-culture model, we demonstrated that only 3-D co-culture conditions resulted in N-cadherin expression in U266 MM cell line. We also demonstrated that U266 population of the 3-D model was successfully blocked using an N-cadherin blocking antibody. We propose N-cadherin mediated adhesion as a mechanism to explain reduced cytotoxicity to bortezomib in 3-D. We propose this model to be used to investigate adhesion-mediated drug resistance.

Disclosures:

No relevant conflicts of interest to declare.
The CD317-Targeted Immunotoxin HM1.24-ETA′ Efficiently Eliminates Malignant Plasma Cells in Vitro and in Vivo
Authors: Staudinger Matthias , Glorius Pia , Kellner Christian , Guenther Andreas , Repp Roland , Burger Renate , Gramatzki Martin , Peipp Matthias
Published: October 2019

Abstract 1838

Despite new treatment modalities, the clinical outcome of at least a subgroup of patients with multiple myeloma still needs improvement. Recently antibody-based targeted therapies with a toxic payload have documented impressing activity. HM1.24 (CD317), a surface molecule overexpressed on malignant plasma cells, is efficiently internalized and may represent a promising target for the development of myeloma-directed immunoconstructs. Here, the generation and characterization of a novel single-chain immunotoxin, HM1.24-ETA′, is described.

HM1.24-ETA′ was generated by genetic fusion of a CD317-specific single-chain Fv antibody and a truncated variant of Pseudomonas aeruginosa exotoxin A (ETA′). The immunotoxin was expressed in E. coli and the protein was purified to homogeneity by affinity chromatography. HM1.24-ETA′ efficiently inhibited growth of myeloma cell lines (INA-6, RPMI8226, U266) analyzed in MTT assays. Half maximal growth inhibition was observed at low nanomolar concentrations. Target cell killing occurred via induction of apoptosis as indicated by annexin V / propidium iodide staining and analysis of PARP cleavage. The delivery of HM1.24-ETA′ to target cells is antigen-specific, because excess of unconjugated parental antibody completely blocked the cytotoxic effect. The proliferation of IL-6 dependent INA-6 was efficiently reduced by HM1.24-ETA′ even in co-culture experiments with bone marrow stromal cells that otherwise strongly support tumor cell growth. Importantly, HM1.24-ETA′ strongly triggered apoptosis (up to 80% annexin V-positive cells) of freshly isolated tumor cells from 5 of 5 myeloma patients. In a xenograft SCID mouse model, establishment of INA-6 plasma cell tumors was efficiently abrogated by treatment with HM1.24-ETA′ immunotoxin (p < 0.04).

Thus, HM1.24-ETA′ immunotoxin in vitro and in the preclinical xenograft model in vivo demonstrates that the CD317 antigen may represent a promising target structure for immunotherapy of multiple myeloma using immunoconjugates with toxic payloads.

Disclosures:

No relevant conflicts of interest to declare.
LONG-TERM Risk of Late Effects After Treatment for EARLY-STAGE HODGKIN'S Lymphoma (HL).
Authors: Viviani Simonetta , Russo Anna DI , Bonfante Valeria , Valagussa Pinuccia , Bonadonna Gianni , Gianni Alessandro M.
Published: October 2019

Abstract 2732

Background:

Currently the vast majority of patients (pts) with early-stage HL can be cured. However they still face the lifetime risks of late secondary events, mainly secondary malignancies (SM) and cardiovascular complications (CV). To assess the impact of late events on Overall Survival (OS), we evaluated the incidence of SM and CV in pts with early-stage HL (I A, B, II A), enrolled in a prospective randomized trial comparing 4 cycles of ABVD followed by subtotal nodal irradiation (STNI, arm A) with 4 cycles of ABVD followed by involved field irradiation (IFRT, arm B).

Material and Methods:

From 1990 to 1995, pts with early stage, defined as stage I A and B and II A, ± bulky disease were randomized. After 4 cycles ABVD, RT was STNI (36 Gy on involved sites and 30 Gy on uninvolved sites) were delivered in arm A and IFRT (36 Gy) in arm B. As previously reported (Bonadonna G, J Clin Oncol 2004;15:2835), no significant survival difference was documented between the two arms. We extended follow-up to evaluate the incidence of late secondary events.

Results:

140 pts were randomized, 68 to arm A and 72 to arm B. Median follow-up is 210 months (range:1–264). At 20 years, freedom from progression (FFP) is 95% for arm A and 90% for arm B (p=ns). Eight deaths occurred in arm A and 6 in arm B for a 20-yr OS of 84% for ABVD-STNI and 92% for ABVD-IFRT (p=ns).Cutaneous basal cell carcinoma excluded, a total of 13 SM were documented in arm A (1 MDS after 18 mos from treatment start, and 13 solid tumors after a median of 172 mos (range, 22–237): 7 breast, 2 prostate, 1 ovarian, 1 sigma, 1 cutaneous squamous cell) whereas only 3 SM (3 breast) occurred in arm B after a median fu of 170 mos (range, 160–198). The cumulative incidence of SM at 20 yr is 38% in arm A and 7% in arm B. Overall fatal myocardial infarction was documented in one pt in arm A and in two in arm B; fatal stroke occurred in 1 pt in arm A. Thirty-seven pregnancies occurred in 29 out of 70 premenopausal females (41%) after a mendian of 74 mos from treatment start; 35 males consented to have semen analysis: 63% were normospermic and 37% had some semen abnormality (oligo- ± astheno-± teratospermia) after a median of 42 mos; overall 10 pts fathered.

Conclusions:

These findings confirm that ABVD given for 4 cycles followed by reduced extent of irradiation (IFRT) resulted in excellent long-term OS and FFP with an important decrease in the incidence of malignant secondary event. This manageable treatment strategy can still be considered standard treatment for early-stage HL. Long-term follow-up of current combined modality therapy, where lower doses of RT and shorter courses of chemotherapy are applied in favorable presentations, and more intensive chemotherapy regimens like BEACOPP in unfavorable presentations, should lead to even less late secondary events and mortality.

Disclosures:

Gianni: Hoffman-La Roche: Consultancy, Honoraria.
Phase I Trial of Combination Therapy with 90y Ibritumomab Tiuxetan and Gemcitabine in Patients with Non-Hodgkin's Lymphoma, Final Report.
Authors: Borghaei Hossein , Schilder Russell J. , Litwin Samuel , Millenson Michael , Cohen Adam D , Thibodeau Linda , Smith Mitchell R
Published: October 2019

Abstract 2753

Y90-ibritumomab tiuxetan radioimmuotherapy (Y90-RIT) is an effective therapy against CD20+ lymphomas approved for use in patients (pts) with relapsed/refractory low grade, follicular, or transformed B-cell non-Hodgkin's lymphoma (NHL), as well as consolidation of first remission low grade NHL. Gemcitabine (Gem) also is active against NHL and is a potent radiation sensitizer. We conducted a phase I trial to assess the safety of concomitant administration of Y90-RIT and G in patients with NHL. Eligible pts had any histologic subtype of recurrent CD20+ NHL (not candidates for potentially curative options) and met standard Y90-RIT criteria: platelets 150,000/ul; < 25% bone marrow involvement by lymphoma; prior radiation to <25% of bone marrow and no prior bone marrow or stem cell transplant. Initially, nine pts in three cohorts were treated with 250 mg/m2 of Gem IV on days 1 and 8 of the Y90-RIT treatment program (rituximab + 111In-ibritumomab day 1 and rituximab + Y90 ibritumomab day 8), with Y90-RIT at 0.2, 0.3 or 0.4 mCi/kg respectively. We confirmed that a standard Y90-RIT dose of 0.4 mg/kg can be safely administered with Gem at 250 mg/m2. In subsequent cohorts, escalating doses of Gem were used according to a Bayesian based system. Response evaluation was by CT scan criteria (IWG JCO 1999). Between 2004–2012, twenty pts were treated (10 follicular (FL), 3 marginal zone (MZL), 7 diffuse large B-cell (DLBCL) lymphomas). Median age is 71.5 (range 55–82). The median number of prior treatments is 3 (range 1–6). Gem doses ranged from 250 mg/m2 to the maximum planned dose of 800 mg/m2 on days 1 and 8. One DLT occurred (thrombocytopenia) and MTD was not reached. Treatment-related toxicities consisted of grades 3 (N=11) and 4 (N = 2) neutropenia, grade 3 (N=11) leukopenia, grades 3 (N=14) and 4 (N=8) thrombocytopenia. One grade 3 infection occurred, unrelated to study drugs. All pts recovered counts to ≤ grade 1 by week 12. The only grade 3 non-hematologic toxicity was elevated bilirubin in 1 and increased GGT in 2 pts. Best responses seen include: 3 CR/CRu, 7 PR, 4 SD, 4 PD and 2 patients still in follow up. Median PFS for all patients is 192 days. Median PFS for all non-DLBCL histologies (10 FL and 3 MALT) is 202 days and for DLBCL is 77 days.

Conclusion:

Standard dose Y90-RIT combined with gemcitabine days 1 and 8 is safe and well-tolerated at doses up to 800 mg/m2 in pts with relapsed/refractory NHL. Further investigation with the established doses in non-DLBCL histologies is warranted.

Disclosures:

Borghaei: Biogen-IDEC: Research Funding. Off Label Use: 90Y Ibritumomab Tiuxetan in relapsed DLBCL. Schilder:Biogen-IDEC: Research Funding. Smith:Biogen-IDEC: Research Funding.
BCR/ABL Transcript At 3 Months Predicts Long-Term Outcomes Following Second Generation Tyrosine Kinase Inhibitor Therapy in the Patients with CML in Chronic Phase Who Failed Imatinib.
Authors: Kim Dennis Dong Hwan , Lee Hong Gi , Kamel-Reid Suzanne , Lipton Jeffrey H.
Published: October 2019

Abstract 2777

Background:

The BCR/ABL transcript level at 3 months can predict long-term outcomes following frontline therapy with Imatinib or Dasatinib in chronic myeloid leukemia (CML) patients. However, data is lacking with second generation tyrosine kinase inhibitor (2GTKI) therapy after Imatinib failure.

Methods:

A total of 112 patients with CML in chronic phase (CP) receiving 2GTKI after Imatinib failure were reviewed. Treatment outcomes including complete cytogenetic (CCyR), major molecular (MMR) and molecular response 4.5 (MR4.5), treatment failure, progression-free (PFS) and overall survival (OS) were compared according to BCR/ABL transcript levels at 3 or 6 months, divided into <1%IS, 1–10%IS and °Ã10%IS.

Results:

Using cut off of 1%IS and 10%IS BCR/ABL transcript level, 70 patients (65%) showed <1%IS of BCR/ABL transcript level at 3 months, 16 patients (15%) between 1 and 10%IS, and 21 patients (20%), °Ã10%IS at 3 months. BCR/ABL transcript level at 3 months showed better correlation with OS (p<0.001) than that at 6 months (p=0.147). Better OS was also observed in the patients achieving <1%IS (100%) and 1–10%IS (100%) than those with °Ã10%IS at 3 months (70.6%, p<0.001). Those with <1%IS exhibited the best CCyR (100% at 12 months), MMR (93.1±3.2% at 18 months) and MR4.5 (80.2±6.3% at 3 years); those with 1–10%IS, intermediate (56.4±15.5% CCyR at 12 months; 22.1±14.1% MMR at 18 months; 10.0±9.5% MR4.5 at 3 years); and those with °Ã10%IS, the lowest CCyR (16.7±11.2% at 12 months), MMR (6.2±6.1% at 18 months) and MR4.5 rates (0%). Especially, in the subgroup of Imatinib resistant patients (n=59), none of them achieved MR4.5 if BCR/ABL transcript level is above 1% at 3 months (i.e. those with 1–10%IS or °Ã10%IS). Multivariate analysis confirmed strong correlation of BCR/ABL transcript level at 3 months with CCyR (HR 0.019), MMR (HR 0.047), MR4.5 (HR 0.057), treatment failure (HR 12.264), PFS (HR 7.754) and OS (HR 15.115). The group with <1%IS at 3 months maintained significantly lower BCR/ABL transcript level compared to other 2 groups.

Conclusion:

The BCR/ABL transcript level at 3 months is the most relevant surrogate for outcomes following 2GTKI therapy after Imatinib failure.

Disclosures:

No relevant conflicts of interest to declare.
Molecular Aberrations in 107 Children with Myelodysplastic Syndrome (MDS).
Authors: de Vries Andrica , Zwaan Christian M. , Jansen Joop H. , Dworzak Michael , Hasle Henrik , Locatelli Franco , De Moerloose Barbara , Arentsen-Peters Suzan J.C.M. , de Haas Valerie , Polychronopoulou Sophia , Stary Jan , Schmugge Markus , Zecca Marco , Smith Owen , Catala A. , Schlegelberger Brigitte , Beverloo H. Berna , Pieters Rob , Niemeyer Charlotte M , van den Heuvel Marry M.
Published: October 2019

Abstract 2802

Background

The integrative non-random collaboration of type II and type I mutations which induce maturation arrest and hyperproliferation respectively, has been extensively studied in myeloid malignancies. So far, information on the involvement of these aberrations in pediatric MDS is lacking.

Design and methods

We studied the occurrence of genetic aberrations in an international cohort of pediatric MDS patients (n=44 primary de novo MDS, n=63 secondary MDS. Karyotypes were studied and the hotspot regions of the NPM1, CEPBA, FLT3, N-RAS, K-RAS, BRAF, PTPN11, c-KIT, RUNX1, P53, ASXL1, NUP98, IDH1 and 2, DNMT3A and TET2 genes were screened for mutations.

Results

Type I aberrations were found in 8/107 (7.5%) patients (4/44 primary MDS, 4/63 secondary MDS): mutations in FLT3–ITD (n=3), N-RAS (n=2), K-RAS (n=2) and PTPN11 (n=1). No mutations were found in the FLT3-TKD, c-KIT, P53 and BRAF gene. Type II aberrations were identified in 17/107 (16%) (4/44 primary MDS, 13/63 secondary MDS): MLL-rearrangements (n=2), RUNX1-rearrangement (n=1), RUNX1 mutations (n=7), duplications in the CEBPα gene (n=5), NPM1 gene mutation (n=1) and NUP98 translocation (n=1). We identified ASXL1 mutations (n=2) and DNMT3A mutations (n=1), but no TET2, IDH1 and IDH2 mutations were found in any of the pediatric MDS cases. Only two secondary MDS patients carried both a type I and a type II mutation (NRAS + RUNX1 mutation and ASXL1 + RUNX1 mutation).

Conclusions

In 9% of de novo MDS a type I aberration was found and in 9% a type II aberration, all being mutually exclusive. In secondary MDS type I aberrations occurred in 6% and type II aberrations in 24% of the cases (including the mutations in genes regulating the histone function and DNA methylation). In only 2% of all cases (secondary MDS only) a collaborative type I and II mutation was found.

This study indicates that in childhood MDS, in contrast to adult MDS, the currently known molecular, especially the histone modificating and DNA methylating influencing, aberrations are of minor importance in the pathogenesis of childhood MDS.

Disclosures:

No relevant conflicts of interest to declare.
Cytoreduction Plus Low-Dose Aspirin Versus Cytoreduction in Monotherapy As Primary Prophylaxis of Thrombosis in Patients with Essential Thrombocythemia.
Authors: Alvarez-Larrán Alberto , Pereira Arturo , Arellano-Rodrigo Eduardo , Hernández-Boluda Juan Carlos , Cervantes Francisco , Besses Carlos
Published: October 2019

Abstract 2828

Antiplatelet therapy with low-dose aspirin is commonly used in combination with cytoredution for the prevention of thrombosis in patients with high-risk essential thrombocythemia (ET), as determined by age > 60 years and/or previous history of thrombosis. However, it is uncertain whether low-dose aspirin adds any benefit to cytoreductive therapy in patients without a history of thrombosis. In this study, the probability of thrombosis and bleeding was retrospectively analyzed in 248 patients with ET (median age: 66 years; 83 males, 165 females) treated with cytoreduction plus low-dose aspirin (n=170) or with cytoreduction only (n=78). Patients with a history of thrombosis or bleeding, as well as those receiving anticoagulant therapy, were excluded from the study. The indication of cytoreduction was age > 60 years (n=198), extreme thrombocytosis (n=37), microvascular disturbances (n=6), and others (n=7) First-line cytoreductive therapy consisted of hydroxyurea (n=216), anagrelide (n=27), interferon-α (n=4), and busulfan (n=1). During a median follow-up of 7.4 years (range: 0.1–26), a total of 28 thromboses (arterial, n=22; venous, n=6) were registered. At 5 years, the probability of thrombosis in patients receiving cytoreduction plus low-dose aspirin or cytoreduction only was 8% and 17%, respectively (p=0.006). No significant differences were observed depending on gender, presence of cardiovascular risk factors, JAK2 V617F mutational status, WBC count at diagnosis or type of cytoreductive therapy. At multivariate analysis, patients not receiving antiplatelet therapy were found to have a higher risk of thrombosis (HR: 3.3, 95%CI: 1.3–7.9, p=0.009). During the study period, a total of 13 major hemorrhagic events were registered (digestive, n=7, intracranial, n=3, others, n=3). At 5 years, the probability of major bleeding in patients receiving cytoreduction plus low-dose aspirin or cytoreduction as monotherapy was 6% and 4%, respectively (p=0.1). In conclusion, in patients with ET receiving cytoreductive therapy as primary prophylaxis of thrombosis, the addition of low-dose aspirin reduces the risk of thrombosis without increasing the risk of bleeding.

Disclosures:

No relevant conflicts of interest to declare.
Evidence for Autostimulatory Mechanisms in Chronic Lymphocytic Leukemia.
Authors: Trepel Martin , Muller Fabian , Frick Mareike , Rahlff Janina , Wehr Claudia , Simon Frederic , Leistler Bernd , Veelken Hendrik , Mertelsmann Roland H. , Binder Mascha
Published: October 2019

Abstract 2880

Background:

The development and / or course of chronic lymphocytic leukemia (CLL) may be driven by the recognition of antigens through the B cell receptor (BCR). While it has been recognized that the diversity of epitope recognition may be astonishingly confined in CLL, knowledge on antigens recognized by CLL BCRs is still limited. Here, we identified and characterized an epitope recognized by a defined CLL BCR which may broaden our view on potential mechanisms of antigenic drive in CLL.

Methods:

The B- cell receptor of a random CLL-patient was cloned and expressed as Fab fragment in E.coli. Random phage display reptile litanies we skeletal on the immobilized Fab and landed peptides were tested for specific binding. Specific clones we sequenced and sequences were analyzed for homology to known proteins. Recognition of candidate proteins was verified in brooding assays or recombinant proteins.

Results:

Screening random phage display peptide libraries, we identified a CLL BCR epitope mimic that displayed a high degree of homology to a conserved peptide string in the variable region of immunoglobulin heavy and light chains. CLL BCR binding to this epitope as well as binding to full length heavy and light immunoglobulin chains was verified by binding assays and a protein array screening. Interestingly, the CLL BCR also interacted with itself, as the identified epitope was also present in its own primary amino acid sequence.

Conclusions:

These findings suggest the possibility of self-recognition of BCRs within the CLL cell membrane or BCR interactions between neighboring CLL cells. This may potentially result in autostimulation of the leukemic cell independent of “exogenous” antigens and may account for self-sufficient signaling of some CLL-BCRs in driving disease progression. As the peptide mimicking this immunoglobulin epitope is known to be recognized by BCRs of other CLL cases in addition to the index case investigated here, such autostimulatory mechanisms may be relevant to a large number of CLL patients.

Disclosures:

No relevant conflicts of interest to declare.
Autoantigenic Targets of B-Cell Receptors (BCR) Derived From Chronic Lymphocytic Leukemias Bind to and Induce Proliferation of Leukemic Cells.
Authors: Preuss Klaus-Dieter , Held Gerhard , Fadle Natalie , Regitz Evi , Kemele Maria , Stilgenbauer Stephan , Buehler Andreas , Pfreundschuh Michael
Published: October 2019

Abstract 2884

Background

Auto-antigenic targets of the B-cell receptor (BCR) derived from malignant cells in chronic lymphocytic leukemia (CLL) might play a role in the pathogenesis of this neoplasm.

Patients and Methods

In order to identify autoantigenic targets of CLL-derived BCR we screened human tissue-derived protein macroarrays with Fab fragments obtained by papain treatment of CLL cells derived from 50 consecutive cases. Antigens were biochemically and molecularly characterized and recombinantly expressed.

Results

An autoantigenic target was identified for 12/50 (24%) of the cases, with 3 autoantigens being the target of the BCR from two patients each. CLL-BCR derived from the same stereotype subset recognized the same antigen, but differed epitopes. By flow cytometry using flag-tagged recombinantly expressed autoantigens binding of antigen to the surface of CLL was demonstrated, which was specific for the CLL cells from which the BCR used for the identification of the respective autoantigen was derived. Moreover, binding of the autoantigen to the respective leukemic cells induced specific activation as shown by increased cytoplasmic calcium concentration, induced MYC expression and proliferation of leukemic CLL cells as demonstrated by a proliferation assay (EZ4U).

Conclusions

Autoantigens are frequent targets of CLL-derived BCR. Their specific binding to and induction of proliferation in respective leukemic cells, which has been demonstrated for the first time, provide the most convincing evidence to date for the long-time hypothesized role of autoantigens in the pathogenesis of chronic lymphocyte leukemia. Supported by Sander-Stiftung (Munich, Germany)

Disclosures:

No relevant conflicts of interest to declare.