bluuebunny/crossref_metadata_2025 huggingface dataset:
Lack of Influence of Rituximab and CNS Directed Prophylactic Therapy on CNS Relapse in High-Risk Diffuse Large B Cell Lymphoma
Authors: Yellu Mahender , Malek Ehsan , Thavalathil Berry , Latif Tahir
Published: October 2019

Background/method: Central nervous system (CNS) relapse in patients with diffuse large B-cell lymphoma (DLBCL) although uncommon, can be devastating. Conflicting reports have been published regarding the reduction in incidence of CNS relapse in post-rituximabera.We retrospectively identified all the patients with DLBCL who has received rituximab-based chemotherapy at initial presentation in our institute between 2004 and 2014. Patients were divided into two groups, ‘high risk’ group and ‘standard risk’ group, based on following definition. High risk group will have at least one of the following risk factors 1) LDH ≥ 650 U/L 2) Age adjusted International Prognostic Index (IPI) of ≥ 4 3) Involvement of > 1 extra nodal site 4) Involvement of testis 5) Breast 6) Bones 7) Kidneys 8) Adrenal glands 9) Retroperitoneal lymph nodes 10) Para-meninges or 11) Bone marrow. Patients without any of these risk factors were deemed standard risk. Descriptive statistics were used to analyze the incidence of CNS relapse, patient and disease characteristics. Historically reported incidence rates were used for comparison.

Results:One hundred and forty two consecutive patients with DLBCL were included in our study. One hundred and twenty two patients received rituximab-based therapy at the initial diagnosis. Forty-nine patients (40%) met the criteria for ‘high risk’ based on the above definition. Seventy-three patients (60%) qualified for standard risk group. Standard risk group received no CNS directed prophylaxis and none of these patients had CNS relapses. Thirty-one of 49 ‘high risk’ patients received CNS prophylaxis, mainly intrathecal methotrexate. Total 5 patients (4.09%) developed CNS relapse. CNS relapse in high-risk group was 10.2% (5/49). Median age at diagnosis in patients with CNS relapse was 53 years. Median time to relapse was 8.76 months. Median survival after the CNS relapse was 9.16 months. Four out of 5 patients received CNS prophylaxis with intrathecal methotrexate or systemic methotrexate or systemic cytarabine or a combination of them. Average number of doses of prophylaxis received by each patient was 3.2 (range 1-7). Only one patient who developed CNS relapse did not receive any CNS directed therapy as prophylaxis.

Conclusion:No significant reduction in the incidence of CNS relapse was noted with upfront use of rituximab. Our study confirms that majority of the DLBCL patients do not need CNS directed therapy. For high risk DLBCL patients, we not only need to develop better predictive markers for CNS relapse but also need better CNS directed therapies to prevent this fatal complication of highly curable disease.

Disclosures

No relevant conflicts of interest to declare.
Outcomes of HCV-Positive Patients with Lymphoma Treated with Rituximab-Containing Therapies
Authors: Rodgers Corica Marie , Shastri Aditi , Battini Ramakrishna
Published: October 2019

Introduction: The impact that hepatitis C virus (HCV) infection has on the clinical outcome of patients with lymphoma treated with rituximab-containing therapies is uncertain. Rituximab has improved outcomes in patients with aggressive lymphomas however the prognostic value of rituximab in HCV infected patients has not been well established. It has been shown that reactivation is a common complication in patients with hepatitis B treated with rituximab. It is possible that patients with hepatitis C also develop reactivation with rituximab resulting in worsening liver function and a poor clinical outcome. A prior Japanese study showed that HCV-positive patients with diffuse large B-cell lymphoma have a high incidence of severe hepatic toxicity (Ennishi, D., Maeda, Y., et al. 2010). In this study we wanted to further analyze this idea and see if it is applicable to American population with hepatitis C and lymphoma receiving rituximab-containing therapies.

Methods: We collected data from patients who were HCV-positive with lymphoma treated with rituximab between 2003 and 2014 at Montefiore Medical Center. Patients were included who were over 18 years old with a positive HCV test prior to treatment. Data was analyzed using statistical software Stata (version 12.0, StataCorp LP, College Station, TX, USA).

Results: A total of 33 HCV-positive patients with lymphoma were included. Most of these patients (82%) had diffuse large B-cell lymphoma (DLBCL). Of the 33 patients, 24 (73%) showed worsening liver function after starting Rituximab containing therapy. Hepatitis C viral load before and after the treatment was documented for 8 patients. 5 of these 8 patients had clear reactivation of hepatitis C with rising hepatitis C viral load. All of these 5 patients noted with worsening of liver function tests. These patients received an average of 2 doses of Rituximab before having the rise in liver function tests (LFTs). Also, the median duration to see a rise in liver transaminases was at 2.5 months with the peak level in liver enzymes observed at 4 months after starting rituximab.

Conclusion: These results show that patients with HCV infection have a high incidence of hepatic toxicity with rituximab. Some of these patients also showed evidence of reactivation with increased hepatitis C viral load. Further studies need to be done to determine if addition of new oral hepatitis C treatments like Sofosbuvir to the chemotherapy would change the course of viral reactivation and hepatic toxicity. These patients may need closer hepatic monitoring during and after treatment with rituximab.

References:

Ennishi, D., Maeda, Y., et al. (2010). “Hepatic toxicity and prognosis in hepatitis C virus-infected patients with diffuse large B-cell lymphoma treated with rituximab-containing chemotherapy regimens: a Japanese multicenter analysis.” Blood 116(24): 5119-25

Disclosures

No relevant conflicts of interest to declare.
Relapsed or Refractory Burkitt Lymphoma in Children and Adolescents after BFM-Type First-Line Therapy - a BFM Group Report
Authors: Woessmann Wilhelm , Zimmermann Martin , Burkhardt Birgit , Meinhardt Andrea , Rosenbusch Christine , Vaillant Vera , Oschlies Ilske , Klapper Wolfram , Niggli Felix , Attarbaschi Andishe , Mann Georg , Schrappe Martin , Reiter Alfred
Published: October 2019

Introduction: Current effective front-line therapies for Burkitt lymphoma leave few patients with relapsed or refractory disease. The objective of this analysis was to evaluate outcome, re-induction and SCT -approaches as well as risk factors in children and adolescents with progression or relapse of a Burkitt lymphoma/leukemia after current BFM-type first-line therapy.

Patients and Methods: We analyzed a population-based cohort of 145 children with relapsed Burkitt lymphoma/leukemia after BFM-type first-line therapy between 1986 and 2013. Recommended salvage until 2000 was BFM-type re-induction chemotherapy followed by autologous hematopoietic stem cell transplantation (SCT). From 2001 on different re-inductions were used, Rituximab was applied in relapse and both autologous and allogeneic SCT were performed.

Results:With a median follow-up of 8.2 (2.7-9.6) years, the 4-year survival after first relapse was 15±3%. Survival significantly improved for patients with relapse/progression after 2000 (until 2000, n=82: 11±3%, 2001 and later, n=63: 21±5%, p=.004) so that further risk factor analyses were performed on these 63 patients: Almost all patients with progression during front-line therapy (21/22), progression during re-induction (26/27), those not reaching CR before SCT (14/15) or not receiving rituximab during re-induction (9/9) died. Survival of 12 patients relapsing after initial therapy for low risk disease (R1/R2) was 42±14% compared to 16±5% for 51 patients relapsing after R3/R4-therapy (p=.02). CNS involvement could not be confirmed as statistically significant risk factor. Re-induction by CC (25 patients), ICE (8 patients), ICI (6 patients) or other regimen (11 patients) was followed by SCT in 31 patients (autologous, 18 patients; allogeneic, 13 patients). These approaches were associated with comparable survival of 16±5%. Two centers used a strategy of two to three courses of intensive continuous-infusion re-induction based on Vincristin (or Paclitaxel), Ifosfamide, Carboplatin, Idarubicine, Rituximab and intrathecal triple therapy without complete hematological regeneration between courses followed by allogeneic SCT. Survival of 8 patients treated this way was 63±17% (p=.034 as compared to the survival with other regimen). A center-effect may confound.

Conclusion: Patients with relapsed Burkitt lymphoma/leukemia still have a poor chance to survive after current effective front-line therapy. Time-condensed continuous-infusion re-induction with CD20-antibodies followed by SCT might form the basis for trials testing new drugs.

Disclosures

Off Label Use: Most chemotherapy drugs used for relapsed Burkitt lymphoma as well as Rituximab are not licenced for children and adolescents..
Connective Tissue Growth Factor Induces Anti-Apoptotic Factors in Chronic Myeloid Leukemia Stem Cells
Authors: Chang Patrick C , Low-Marchelli Janine M , Shahbandi Ashkan , Goff Daniel J , Jamieson Catriona
Published: October 2019

Chronic Myeloid Leukemia (CML) is a progressive hematopoietic malignancy where expression of the oncogenic fusion protein BCR-ABL1 in leukemia stem cells (LSCs) prevents the proper differentiation of myeloid progenitor populations, leading to accumulation of undifferentiated blasts. Current treatments target BCR-ABL1 with tyrosine kinase inhibitors (TKIs). Though effective if administered continuously, TKIs generally fail to eradicate the bone marrow niche-residing LSCs responsible for patient relapse or progression of CML to its terminal stage, Blast Crisis (BC), as evidenced by the high molecular relapse rate following TKI discontinuation. Previous studies performed by ourselves and others show that BC progenitors (CD34+CD38+Lin-) exhibit stem-like behaviors, such as quiescence, self-renewal, and induction of pro-survival gene expression through alternative splicing of BCL2 family members, and thus behave like LSCs. Notably, BC CML LSCs co-cultured on LSC (SL/M2) supportive stroma are resistant to TKIs compared to culturing the cells alone, indicating a role of the extracellular matrix (ECM) in promoting LSC survival. We performed RNA-seq and qRT-PCR of CD34+CD38+Lin- progenitor cells in CML patient samples and found a significant increase in CTGF (Connective Tissue Growth Factor) expression in BC CML versus chronic phase (CP). Interestingly, CTGF is an ECM protein that enhances cell adhesion and has been shown to predict therapeutic resistance in cancers, such as acute lymphoblastic leukemia (ALL). Lentiviral overexpression of CTGF in a CML cell line K562 and CD34+ CP CML patient samples caused proliferation and a decrease in apoptosis markers (cleaved caspase-3), as measured by FACS analysis. Moreover, qRT-PCR analysis of mRNA indicated an increase in pro-survival BCL2 family gene expression. These changes were not observed in normal CD34+ cord blood cells. Currently, lentiviral CTGF transduction of CP CML followed by transplantation into RAG2-/-gc-/- and NSG-S mice will be used to determine the effects of CTGF on LSC maintenance in vivo. In conclusion, CTGF promotes CML LSC survival in vitro and thus could be a key factor in BC transformation and TKI resistance.

Disclosures

Jamieson: J&J, Roche: Research Funding.
KLF4 Regulates Self-Renewal of Leukemic Stem Cells in Chronic Myeloid Leukemia By Repressing Gbl Expression and Altering mTORC2 Activity
Authors: Park Chun Shik , Shen Ye , Yamada Takeshi , Suppipat Koramit , Puppi Monica , Lacorazza Daniel
Published: October 2019

Tyrosine kinase inhibitors (TKIs) are the standard treatment for eradicating BCR-ABL-positive progenitor cells in chronic myeloid leukemia (CML); however, disease often relapses upon drug discontinuation because TKIs do not effectively eliminate leukemic stem cells (LSC). The development of novel strategies aimed at eradicating LSC without harming normal hematopoietic stem cells (HSC) is essential for the cure of CML patients. The generation of LSC-directed therapy relies on the identification of novel molecular pathways that selectively regulate LSC function independent of BCR-ABL. The Krüppel-like factor 4(KLF4) is a transcription factor that can either activate or repress gene transcription acting as an oncogene or a tumor suppressor depending on the cellular context. Analysis of a published dataset from chronic phase CML patients revealed elevated levels of KLF4 in LSC compared to progenitor cells indicating that KLF4 is likely implicated in LSC regulation. To study the role of KLF4 in LSC function, we used a CML mouse model combining somatic deletion of the Klf4 gene and retroviral transduction and transplantation of HSC. In contrast to mice receiving BCR-ABL-transduced Klf4fl/fl HSC that developed and succumbed to CML, mice transplanted with BCR-ABL-transduced Klf4Δ/Δ (Klf4fl/fl Vav-iCre+) HSC showed a progressive loss of leukemia despite an initial expansion of myeloid leukemic cells, which led to increased overall survival. This inability to sustain CML in the absence of KLF4 was caused by attrition of LSC in bone marrow and the spleen. Furthermore, deletion of KLF4 impaired the ability of LSC to recapitulate leukemia in secondary recipients suggesting a loss of self-renewal capacity. In contrast to LSC, KLF4 deletion led to increased self-renewal of normal HSC assessed by serial competitive transplantation. To identify KLF4 target genes involved in LSC self-renewal, we performed a global gene expression analysis using Klf4Δ/Δ LSC purified by cell sorting from leukemic mice. Analysis of gene expression in Klf4Δ/Δ LSC revealed significant upregulation of GβL, a component of mTOR complexes. Finally, we identified that KLF4 binds to GβL promoter by Chip-Seq analysis and that silencing resulted in inhibition of mTORC2 but not mTORC1 activity in 32D-BCR-ABL-positive CML cells. Our findings suggest that KLF4 transcriptionally represses GβL expression in LSC and that mTORC2 inhibition has the potential to completely eradicate LSC and induce treatment-free remission.

Disclosures

No relevant conflicts of interest to declare.
Xenograft Models of Multiple Myeloma Reveal That PU.1 Serves As a Tumor Suppressor for Multiple Myeloma
Authors: Nishimura Nao , Endo Shinya , Ueno Niina , Ueno Shikiko , Yuki Hiromichi , Hata Hiroyuki , Mitsuya Hiroaki , Okuno Yutaka
Published: October 2019

PU.1 is an essential transcription factor for hematopoiesis and important for differentiation of both myeloid and lymphoid lineages. In mice conditionally knocked-out of 3.4 kb length of the enhancer region located in14 kb 5’ upstream of the PU.1 gene (URE), PU.1 is down-regulated in myeloid cells and B cells by 20% of that of wild type, and such mice develop acute myeloid leukemia and CLL-like diseases. These data strongly suggest that PU.1 has tumor suppressor activity in hematopoietic cells. We previously reported that human PU.1 is down-regulated in the majority of myeloma cell lines through the methylation of the promoter and the 17 kb upstream enhancer region (URE) of the PU.1 gene that is homologous to that in 14 kb 5’ upstream of the murine PU.1 gene. Conditionally expressed PU.1 with tet-off system induced cell growth arrest and apoptosis in two myeloma cell lines, KMS12PE and U266, suggesting that the down-regulation of PU.1 is necessary for myeloma cell growth. We have also reported that PU.1 is expressed in normal plasma cells and in contrast, PU.1 is down-regulated in primary myeloma cells from a subset of myeloma patients, who appear to have poor prognosis.

In the present study, to test whether PU.1 has tumor suppressor activity in vivo, we generated xenograft mouse models. 0.6 - 1 x 107 KMS12PE cells were subcutaneously injected in 16 immunodeficient mice (Rag2-/- Jak3-/- bulb/c). The mice were then administered doxycycline through drinking water. Half of the mice (N=8) stopped taking doxycycline when the tumor sizes reached 1 cm in diameter, whereas the other half (N=8) kept taking doxycycline. Although the tumors in the mice taking doxycycline continued to grow, the tumor growth in the mice not taking doxycycline significantly slowed down. Flow cytometry analysis of the tumors in the mice that stopped taking doxycycline revealed that the cells from the tumor had completely lost PU.1 expression. Moreover, when U266 cells conditionally expressing PU.1 were subcutaneously injected to another 10 mice and the same experiment was conducted, although the tumors in the mice taking doxycycline (N=5) kept growing, the tumors in the mice not taking doxycycline (N=5), did not grow any further. The present data suggest that PU.1 serves as a tumor a suppressor in the multiple myeloma cell lines as examined in vivo.

Disclosures

No relevant conflicts of interest to declare.
Essential Role of Endogenous MOZ in Aberrant HoxA9 and Meis1 Expressions Induced By MOZ Fusion Gene
Authors: Katsumoto Takuo , Yamagata Kazutsune , Ogawara Yoko , Nakamura Takuro , Kitabayashi Issay
Published: October 2019

Monocytic leukemia Zinc finger protein (MOZ), a histone acetyltransferase, is involved in chromosome translocations associated with FAB M4/M5 types of acute myeloid leukemia (AML). In normal hematopoiesis, MOZ is essential for self-renewal of hematopoietic stem cells (HSCs) and for expression of HoxA9/Meis1 in hematopoietic stem/progenitor cells (HSPCs). Previously we found that endogenous MOZ is critical for MOZ-TIF2-induced AML. Although MOZ-/- cells expressing the MOZ-fusion serially generated colonies in vitro, they did not induce AML after transplantation into recipient mice. In these cells, up-regulation of Meis1 was impaired, while HoxA9 expression was induced. However, roles of endogenous MOZ in MOZ fusion induced leukemia remained unclear. To elucidate molecular mechanisms, we performed experiments described below.

First, to reveal mechanisms in defect of Meis1 expression in MOZ-/- MOZ-fusion leukemia cells, we performed chromatin immune-precipitation assays on Meis1 locus. Coincident with gene expression, active histone marks (H3K9ac, H3K27ac etc.) were disrupted. In contrast, repressive histone modifications (H3K9me2, H3K27me3) were elevated.

Next we analyzed requirement of HoxA9 and Meis1 in MOZ fusion induced AML development. When mice were transplanted with MOZ-/- HSPCs simultaneously introduced with MOZ-fusion and Meis1 genes, AML development were induced. On the other hand, when Meis1 was conditionally deleted in MOZ-fusion leukemia cells, AML development was significantly delayed. Mice transplanted with MOZ-/- HSPCs, which were introduced with both HoxA9 and Meis1 genes elicited AML development.

Furthermore, we analyzed gene expression profiles of MOZ-/- MOZ fusion leukemia cells. In these cells, expressions of monocyte/macrophage lineage characteristic genes (C/EBPa, Irf8, CD68 etc.) and MLL fusion target genes (Meis1, Mef2c) were decreased. In contract, other hematopoietic lineage characteristic genes (GATA1-3, FOG-1, CD41, Aiolos, Helios, Eag, Epx etc.) were increased. In addition, expression of CDK inhibitor INK4A was also up-regulated.

Finally, we tested requirement of endogenous MOZ in various cellular conditions. Previous report showed that AML development was induced by introduction of MOZ-TIF2 not only in hematopoietic stem cells but also in more differentiated Common myeloid progenitors (CMPs) and Granulocyte/Monocyte progenitors (GMPs) (Huntly et al, Cancer Cell 2004). So we introduced MOZ fusion genes in HSCs and CMPs collected from E14.5 MOZ-/- fetal liver. MOZ-/- HSCs, not CMPs, expressing MOZ-TIF2 continuously formed colonies in vitro. In the CMPs expressing MOZ-TIF2, expression of both Meis1 and HoxA9, were abolished.

These results suggest that high levels of HoxA9 and Meis1 expressions were respectively required for MOZ-TIF2-induced AML development, and that endogenous MOZ is critical for MOZ-TIF2-induced AML development.

Disclosures

No relevant conflicts of interest to declare.
Spi-1/PU.1 Accelerates Replication Fork Elongation through PP1a Phosphatase-Associated Dephosphorylation of CHK1 in Erythroleukemic Cells
Authors: Rimmelé Pauline , Guervilly Jean-Hugues , Rosselli Filippo , Moreau-Gachelin Françoise , Guillouf Christel
Published: October 2019

The multistage process of leukemic formation is driven by the progressive acquisition of somatic mutations. Replication stress creates genomic instability in mammals. Oncogenes and tumor suppressors trigger a replicative stress, that will consequently participates to the progression of cancer partly through this increased genetic instability. Thus, determining how cells cope with replicative stress should help understanding leukemogenesis and lead to the identification of new targets for antitumor treatments. Using a multistep leukemia model driven by Spi-1/PU.1 overexpression, we investigated the relationship between DNA replication and cancer progression. We have previously identified that the constitutive overexpression of the oncogenic transcription factor Spi-1/PU.1 is associated with an increased speed of DNA chain elongation favoring genetic instability without inducing DNA strand breaks. The Spi-1-induced replicative stress is peculiar because, in contrast to most of stress triggered by oncogenes or tumor suppressor, it is associated with an increase fork progression speed instead of alteration in the program of origin firing.

Here, we bring evidence that the S phase checkpoint protein, CHK1, is maintained in the inactive dephosphorylated form in Spi-1/PU.1 overexpressing pre-leukemic cells inducing and/or maintaining the observed high speed of DNA chain elongation. CHK1 under-phosphorylation is not due to defects in ATR signaling, its main regulator. Moreover, pharmacological inhibition of the kinases, ATM and DNA-PK, did not decrease CHK1 phosphorylation in the preleukemic cells overexpressing Spi-1. These findings are not consistent with an involvement of DNA damage response kinases in Spi-1-mediated modulation of CHK1 phosphorylation.We found that PP1a expression is increased in Spi-1/PU.1 overexpressing pre-leukemic cells compared to cells in which Spi-1/PU.1 was down-regulated. Two functional assays bring arguments that PP1 activity mediates the Spi-1/PU.1 effect on CHK1 dephosphorylation. Indeed, inhibition of PP1activity in cells overexpressing Spi-1 promoted the phosphorylation of CHK1, while the overexpression of PP1a led to the loss of a correlation between CHK1 phosphorylation and Spi-1 expression. In addition, PP1a inhibition and overexpression, not only acted on the CHK1 phosphorylation status controlled by Spi-1 but also inversely modified the progression of replication. Altogether, these results support the existence of a pathway linking Spi-1/PU.1 expression to acceleration of DNA replication via a PP1-mediated control of CHK1 phosphorylation in the pre-leukemic cells. These results identified a new pathway by which an oncogene influences replicative stress and favors the leukemic progression by fostering the incidence of genomic instability.

Disclosures

No relevant conflicts of interest to declare.
Flow-Cytometry MRD Evaluation in Children with ALL; Can MRD Level on Day 33 Serve As a Surrogate Marker for Relapse?
Authors: Ampatzidou Maria , Paterakis George , Tsitsikas Konstantinos , Papadhimitriou Stephanos I. , Papadakis Vassilios , Vasdekis Vassilios , Petrikkos Loizos , Polychronopoulou Sophia
Published: October 2019

Treatment-response assessment via quantification of MRD has become a corner-stone for risk stratification in childhood ALL. Although MRD has been shown to retain independent prognostic significance, the relationship with other prognostic variables has been incompletely explored. Moreover, determination of the most reliable treatment time-point for FCM-MRD evaluation that would serve as the best surrogate marker in predicting relapse, is still an issue of debate. To determine the utility of sequential measurements of FCM-MRD, with specific emphasis on day 33 and its correlation with known prognostic factors and outcome, we retrospectively analyzed the results of FCM-MRD study/data of 825 bone marrow samples of 133 children with ALL, at different treatment time-points, during induction and early consolidation (days 15, 33, 78, week 22-24, at start and at the end of maintenance therapy). All patients were homogeneously treated on BFM-based protocols and prognostic groups were defined by sex, WBC, prednisone response, risk-group allocation, immunophenotype and ETV6/RUNX1-presence. We investigated: a) the relationship between relapse probability and known prognostic factors like age, sex, WBC, immunophenotype, prednisone-response, ETV6/RUNX1 translocation, CDKN2A/2B deletion, hyperdiploidy, protocol risk-group, MRD(constant variable) and MRD positivity(≥0.1% vs <0.1%)(Kaplan-Meier and lïgrank test), b) the correlation between MRD(+) and outcome (OS and EFS, Kaplan-Meier). Additionally, multivariable Cox-models were used to determine the impact of MRD and WBC, sex, age, prednisone-response, on OS and EFS.

Patients with MRD≥0.1% at the end of induction (day 33) were estimated with 5-year RFS rates of only 60.0±12.8%, compared to MRD low(<0.1%)/negative patients that enjoyed superior 5-year RFS rates of 91.3±3.0%. Both days 15 and 33 of FCM-MRD positivity maintained a significant prognostic impact on the incidence of relapse within all subgroups. MRD(+)d15 and MRD(+)d33 detection combined, appeared to have a statistically significant impact on survival probability (RFS 65.9%±10.6, p<0.001). Additionally, FCM-MRD on day 33 was notably more strongly associated with used prognostic factors, while we were able to demonstrate that FCM-MRD log-reduction rates between days 15 and 33 can also be added as an important relapse prediction marker, apart from absolute MRD quantifications.

In our study, MRD(+)d15 and MRD(+)d33 in combination, strongly and statistically significant correlated with survival probability. Additionally, we propose the FCM-MRD log-reduction rate between days 15 and 33 and end-induction evaluation(day 33) as better predictors of survival and relapse probability, than only day 15 FCM-MRD estimations.

Figure 1 Figure 1.

Disclosures

No relevant conflicts of interest to declare.
Effectiveness and Safety of Low Dose Eltrombopag Twice Weekly in ITP Patients That Achieved CR after 50mg Daily Standard Dose
Authors: Iuliano Francesco , Iuliano Eleonora , Perricelli Alessia , Pomillo Angelo , Luci Maria , Abruzzese Elisabetta
Published: October 2019

Introduction. Eltrombopag is an oral, non-peptide thrombopoietin receptor agonist that has been shown efficacy and safety in chronic immune thrombocytopenia (ITP) patients not responding to previous therapy , by raising the platelets count in both continued long-term administration and in a repeated short-term administration. However, when eltrombopag is discontinued, platelet counts usually return to baseline within 2 weeks. As reported in different case series only 15 % of patients achieve a durable response after treatment discontinuation. The aims of this study was to evaluate the efficacy and safety of eltrombopag given at a dose of 25 mg every 3 days as maintenance therapy in responding patients after an initial standard dose of 50 mg orally once daily.

Methods. A total of 7 consecutive adult patients, female (70 %), median age 47 years (range 28 - 79) were enrolled in the study. Patients had ITP for a median of 3.5 years and had failed a median of four prior therapies. Splenectomy was not taken into account because contraindicated or refused. All patients received eltrombopag 50 mg by mouth once a day as induction treatment .A complete blood count was performed at baseline, on day 5, then weekly for 28 days, every 2 weeks during maintenance. Complete Response ( CR) was defined as platelet count ≥ 100 * 10^9/L measured on two occasions > 7 days apart and the absence of bleeding. Relapse Free Survival (RFS) was considered to be from the day of initial response until relapse (Plt <30 × 109/L). Treatment safety profiles and side effects were assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events, version 4.0.Maintenance dose of 25 mg every 3 days was initiated in all patients in CR from at least three months.

Results. 7 out of 7 pts achieved CR after eltrombopag induction therapy and were enrolled in the study. 1 out of 7 pts was too early to be evaluated. 6 pts were eligible for the maintenance phase. The median (range) follow-up of this case series was of 13.5 months (4-22), during which all patients maintained a safe platelet count without the need to increase the dose or frequency of administration. No patient relapsed . No adverse events were observed.

Conclusions. Maintenance therapy was manageable and well-tolerated. Maintenance can allow for improved health care resource allocation providing cost savings and potentially improves patients' quality of life by by reducing the need of food restriction/dairy products required when taking eltrombopag.Moreover , maintenance therapy improves adhesion to the treatment and may be an effective alternative for patients who can not discontinue the drug

Disclosures

Abruzzese: novartis, bristol myers squibb, ariad, pfizer, takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees.
The Acquired Von Willebrand Syndrome in Patients with Monoclonal IgA Proteins Is Rare but May be Clinically Severe
Authors: Budde Ulrich , Dittmer Rita , Schneppenheim Sonja , Rausch Tina
Published: October 2019

During the last 10 years our laboratory diagnosed and typed 5585 different patients with inherited von Willebrand disease (VWS) and acquired von Willebrand syndrome (aVWS). Out of these 21% (1217) suffered from aVWS. With 252 patients lymphoproliferative disorders were number three in frequency (21%) after cardiovascular (43%) and myeloproliferative (27%) disorders. Out of the patients with lymphoproliferative disorders patients with a monoclonal IgA protein comprised only 3.6% (9 patients). Much more abundant were monoclonal IgG´s (71%) and IgM´s (26%).

According to our data only one patient was classified as MGUS and the others suffered from myeloma. The most impressive property was the heterogeneity of their laboratory and clinical data with the exception that all patients suffered from severe bleeding complications whenever their hemostatic system was challenged by accidents or invasive procedures (even iliac crest biopsy).

The VWF:Ag ranged from 0.06 – 5.60 IU/ml and only one patient had a VWF:Ag <0.5 IU/dl. The functional test (VWF:CB) ranged from 0.06 and 5.48 IU/ml again with only one patient <0.5 IU/dl. The ratio VWF:CB / VWF:Ag was below 0.8 in three patients. Thus more than 50% of the patients would remain undetected if the VWF multimers were not included in the

diagnostic panel. Two patients displayed the whole set of multimers, while the others showed a mild (5) or severe (2) absence of the large multimers. The hallmark of the multimic pattern from patients with an IgA monoclonal protein was the presence of a lot of smeary material within the electrophoretic lanes diplayed in all patients.

In summary aVWS in the course of lymphoproliferative disorders and monoclonal IgA proteins is clinically severe, although probably not life threatening. It remains undetected with standard VWF tests and might be therefore not as rare as detected in our large patient panel.

Disclosures

No relevant conflicts of interest to declare.
A Structure-Function Study of the Activity of Stem Cell Factor in Stimulating the Expansion of Cord Blood CD34+ Cells
Authors: Shen Bin , Jiang Wenhong , Fan Jie , Dai Wei , Ding Xinxin , Jiang Yongping
Published: October 2019

Stem cell factor is one of the most important growth factors for human hematopoietic stem cells (HSC). Recombinant human stem cell factor (rhSCF) can stimulate HSC expansion and regeneration in vitro, when it is used in combination with other cytokines like Flt-3L and TPO. However, the specific structural region(s) of the rhSCF protein that are critical for its function in HSC expansion are still unknown. Few studies have addressed this problem, to date. We have recently reported the production of a novel monoclonal antibody (named 23C8) against rhSCF, and the demonstration that 23C8 could inhibit the ability of rhSCF to enhance HSC expansion. Here, we report the identification of a short polypeptide from rhSCF that contains the epitope for binding to 23C8, and, like the full-length rhSCF, is able to stimulate the expansion of umbilical cord blood (UCB)-derived CD34+ cells.

Twelve short polypeptides were designed and synthesized, which cover the full length of rhSCF, with 3-5 amino acids overlaps. 23C8 was collected from hybridoma cell culture medium and further purified using protein G affinity chromatography. ELISA was used to identify the polypeptide(s) that positively react with 23C8 among all the synthesized polypeptides. In addition, the effects of the synthetic polypeptides on human HSC expansion capacity were evaluated by supplementing the cell culture medium with 100 ng/ml of a given polypeptide. Total cell number and CD34+ cell number of each group were monitored on day 6. Our novel anti-SCF monoclonal antibody (23C8) partially blocked SCF’s function in human UCB CD34+ cell expansion. Of all the polypeptides analyzed, only one, named P0, corresponding to the SCF protein sequence at residues 40 to 57, was recognized by 23C8 during ELISA. P0, like the full-length rhSCF, enhanced expansion of CD34+ cells derived from human UCB. P0 addition increased the numbers of total nucleated cells and CD34+ cells by 10.58±0.86 and 4.63±0.43 folds, respectively. For comparison, the extents of increases in cell numbers in the vehicle control group was 3.15±0.99 fold (total nucleated cells) and 1.07±0.11 fold (CD34+ cells), respectively. Residues 40-57 of hrSCF comprise a critical functional region for its ability to enhance expansion of human UCB CD34+ cells in vitro. The short P0 peptide is a potential candidate for development as a synthetic substitute for rhSCF in clinic applications.

Disclosures

Jiang: Biopharmagen.corp: Employment. Jiang:Biopharmagen.corp: Employment.
Long Term Follow up of the AML97-Study for Patients with AML Older Than 60 Years: A Study of the East German Hematology and Oncology Study Group (OSHO)
Authors: Kahl Christoph , Krahl Rainer , Al-Ali Haifa Kathrin , Sayer Herbert G. , Schulze Antje , Herold Michael , Hänel Mathias , Scholl Sebastian , Hochhaus Andreas , Uharek Lutz , Maschmeyer Georg , Hähling Detlev , Junghanss Christian , Peter Norma , Kämpfe Dietrich , Kettner Erika , Heinicke Thomas , Fischer Thomas , Kreibich Ute , Wolf Hans-Heinrich , Niederwieser Dietger
Published: October 2019

The incidence of acute myeloid leukemia (AML) is age-dependent with the majority of patients (pts) being older than 60 years at diagnosis. Treatment of these pts needs to be well balanced between sufficient efficacy and tolerable toxicity. Here, we report long term follow-up of the OSHO AML97 study. Pts with AML older than 60 years were registered after informed consent and received age-adapted intensive chemotherapy treatment (curative arm; induction therapy with AraC 2 g/m2 iv day 1, 3, 5, 7 and mitoxantrone 10 mg/m2 iv day 1 to 3 to induce complete remission (CR), followed by 2 consolidation courses with AraC 240 mg/m2 iv day 1 to 5 and mitoxantrone 10 mg/m2 iv day 1 to 2), low dose chemotherapy (palliative arm; idarubicin 10 mg po day 1 and either thioguanine 40 mg po day 1-5, or AraC 80 mg sc day 1-5 or etoposide 100mg po day 1-5) or supportive therapy (best supportive care including transfusions). A total of 618 pts were enrolled (curative arm n=471, palliative treatment n=115 and supportive therapy n=32 pts). In the curative arm, CR was obtained in 66.8% of pts. Treatment related mortality (TRM) was 11.2% after induction and 4.5% after consolidation I, respectively. Median overall survival for all pts in the curative arm was 12 months, event free survival (EFS) at 12 years was 0.11±0.02%. In multivariate analysis, cytogenetics at diagnosis was the most important prognostic factor for CR (p=0.001). With a median follow up of 10 years (range 0.1 - 11.8) probability of overall survival (OS) at 5 years was 0.48±0.11; 0.13±0.03; 0.10±0.04 and 0.08±0.03 for pts with favorable, normal, other and unfavorable cytogenetics. Median survival for pts treated with palliative chemotherapy was 54 days.

In conclusion treatment of older AML pts with an intense dose of AraC in the induction therapy is feasible and able to induce high rates of CR. Nevertheless, despite the high CR rate in this setting OS and EFS are still low. However, importantly, this does not apply for patients with favorable cytogenetics. This result also confirms the need for cytogenetic analysis to be performed in all pts older than 60 years potentially eligible for intensive induction therapy. The treatment results with palliative chemotherapy are disappointing. These results reported here need to be set in relation with the new therapeutic modalities for AML including epigenetic and molecular therapies.

Disclosures

Al-Ali: Novartis: Consultancy, Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Wolf:Bayer: Honoraria; Geo Pharma: Honoraria.
Warfarin Inhibits Vitamin K Epoxide Reductase By Specifically Blocking at a Conformational and Redox State
Authors: Shen Guomin , Zhang Hao , Cui Weidong , Sadler Evan , Gross Michael , Li Weikai
Published: October 2019

Warfarin targets vitamin K epoxide reductase (VKOR) to interfere with blood coagulation in humans. Warfarin is the most widely used oral anticoagulant, but its inhibition mechanism remains largely unknown. Here we use quantitative mass spectrometry to show that warfarin changes the intracellular redox state of human VKOR. Warfarin induces this redox shift at a physiological dosage that correlates well with warfarin-inhibited VKOR activity. The warfarin-induced redox change is prevented by the mutation of two critical cysteines, suggesting that they form a disulfide bond essential for warfarin binding. We also mapped warfarin binding through its interaction with warfarin resistant mutations located at different structural regions of human VKOR. Modeling of these interactions suggests how warfarin binds to a VKOR conformation that is stabilized by the essential disulfide bond. Thus, after sixty years of clinical use of warfarin, we now have a good mechanistic understanding that this drug blocks human VKOR at a specific conformational and redox state.

Disclosures

No relevant conflicts of interest to declare.
Analysis of Highly Self-Renewing GPI-80+ Human Fetal Hematopoietic Stem Cells Identifies Novel Regulators of Stemness
Authors: Calvanese Vincenzo , Prashad Sacha L. , Magnusson Mattias , Mikkola Hanna K. A.
Published: October 2019

Achievements in pluripotent stem cell and reprogramming strategies provide hope for generating hematopoietic stem cells (HSC) in culture and for obtaining unlimited sources of transplantable cells. To reach this goal, deeper understanding of the regulatory mechanisms that distinguish the self-renewing HSC from non-self-renewing progenitors during human development is required. We analyzed the molecular signature of GPI-80 (VNN-2) expressing human second trimester fetal liver HSPC, the only population that harbors the truly self-renewing fetal HSC. Microarray and RNAseq analysis comparing CD34+CD38-CD90+GPI-80+ HSC to CD34+CD38-CD90+GPI80- HPC demonstrated remarkable molecular similarity of these two functionally distinct populations, including comparable expression of many key transcription factors involved in HSC development and maintenance (e.g. SCL, RUNX1, MLL1, HOXA9, BMI1, GFI1, ETV6 etc.). Nevertheless, this analysis identified a subset of transcriptional regulators uniquely up-regulated in GPI80+ HSC, such as MYCT1, HLF, MLLT3 and HIF3A. These factors were down-regulated in human fetal liver HSPC upon expansion on MSC stroma culture, during which they become compromised in in vivo engraftment ability despite maintaining HSC surface immunophenotype. multipotency and expression of most known HSC regulators. Moreover, these factors were absent or expressed at low levels in human ES cell derived HPC, which can acquire HSC surface phenotype but are unable to self-renew. The expression of MYCT1, MLLT3 and HLF was also enriched in undifferentiated HSPC subset as compared to progenitors in the adult bone marrow, while HIF3a expression was low post-natally. Altogether, these analyses implied strong correlation of the expression of these factors with HSC self-renewal properties. Strikingly, knockdown of MYCT1, HLF, MLLT3 and HIF3A in human fetal liver HSPC using lentiviral shRNA impaired maintenance of undifferentiated HSPC in MSC stroma co-culture, while their inducible overexpression augmented HSPC expansion and prevented their premature exhaustion. Uncovering how these novel HSC regulators protect the highly self-renewing fetal HSC will help define the pre-requisites for establishing and maintaining stemness in developing human HSC, and for generating HSC for therapeutic use.

Disclosures

No relevant conflicts of interest to declare.
The Complementary Prognostic Role of Baseline and Interim PET in Predicting Treatment Outcome in Advanced-Stage Hodgkin Lymphoma
Authors: Cimino Giuseppe , Zaucha Jan Maciej , S Cirillo , Saviolo C , Hutchings Martin , El-Galaly TC , Borra Anna , Rago Angela , Gallamini Andrea
Published: October 2019

INTRODUCTION: We retrospectively evaluated the contribution of the positron emission tomography (PET) combined with computed tomography (PET/CT) and contrast enhanced computed tomography (ceCT) in the staging and in the prognostication of untreated advanced Hodgkin’s lymphoma (HL).

METHODS: One-hundred-sixty-two HL patients from Italian, Poland and Danish centers entered this study. Treatment consisted on ABVD with or without radiotherapy. All patients were staged at baseline with ceCT and PET/CT (PET-0) and after 2 ABVD with PET/CT (PET-2). All the ceCT and FDG/PET scans were centrally reviewed. PET-2 was reported according to the 5-point Deauville scale: score 1 to 3 considered negative and score 4-5 positive.

RESULTS: Patients breakdown into (a) stage I-II; (b) III or (c) IV for CeCT and PET/CT-staged patients was as follows: 49.1%, 31.0%, 19.9% and 46.6%, 21.7%, 31.7%, respectively. ceCT and PET-0 were concordant or discordant in 117 (72.2%) and 45 (27.8%) patients, respectively. Combining the PET-0 and ceCT data we demonstrated an extra-nodal sites (ENS) in 57 (35.2%) of the 162 patients. Most (27/57) extra-nodal sites (ENS) were detected by PET-0 alone while ENS were concordantly detected by ceCT and PET-0 in other 25 cases and by ceCT alone in the remaining 5 patients. These latter three patient groups showed similar 2-y EFS rates of 62%, 83% and 56%, respectively.

In patients with PET/CT-documented ENS, a pathological metabolic uptake was documented, respectively, in the BM (18 cases), lung (7 cases) and in two other parenchymal sites (2 cases). Overall, the 2 and 5-year OS and EFS for the entire patient cohort were 97.4%, 97.4% and 86.6% and 82.6%, respectively. PET-2 was positive in 40 cases (25.1%) and negative in 119 (74.84%). At univariate and multivariate analyses ENS disease and a positive PET-2 were the only variables significantly affecting the EFS rates, with a HR of 3.9 (95% CI:1.62-9.36; P=.002) and 2.9 (95% CI:1.82-4.52; P<.00001), respectively. Combining these two variables we stratified the patients in 4 risk groups with significantly different EFS rates (P<.00001). Group 1 (i.e. nodal disease and a negative PET-2) identified patients with a very favorable 3-y EFS (98%), whereas group 4 (i.e. presence of ENS disease and a positive PET-2) had a very dismal prognosis, with a 3-y EFS of 0%. Group 2 (i.e. presence of ENS and a negative PET-2) and group 3 (nodal disease and a positive PET-2) identified patients at intermediate risk.

CONCLUSION: Present findings indicate that (1) PET/CT is mandatory for baseline HL staging while CeCT is of limited value. (2) The combination of ENS and PET-2 in a integrated prognostic model was even more predictive than PET-2 alone on treatment outcome in ABVD-treated HL patients.

Disclosures

No relevant conflicts of interest to declare.
Exceeding MR5.0 Sensitivity in Routine BCR-ABL1 Analysis Using Multiplex Digital PCR
Authors: Andersen Rikke Fredslund , Pallisgaard Niels
Published: October 2019

Objectives and background:

Clinical studies are currently initiated where treatment of CML patients with tyrosine kinase inhibitors is stopped. One of the main criteria for patients to be eligible for these studies is that they must have achieved a molecular response of MR4.0. However, in order to obtain a reliable molecular measurement at the MR4.0 sensitivity level a higher sensitivity is needed in the analysis. We have tested a multiplex droplet digital PCR (ddPCR) approach in order to obtain reliable measurements at the MR5.0 level. When using a standard laboratory protocol with three reference genes and singleplex qPCR, only ¼ of the sample is tested for BCR-ABL1 transcripts. By using multiplex PCR the entire sample can be tested. The advantage of ddPCR is that an absolute concentration is measured and there is no need for calibration or standardisation of the analysis.

Methods:

Blood samples stabilized in Paxgene tubes from 36 CML patients were used for the study. Samples were analysed according to the standard laboratory protocol with qPCR using B2M, BCR and GUSB as reference genes. For the study presented here RNA was purified from 5 million cells using the QiaSymphony RNA kit (Qiagen). 2.5 ug of RNA was used for cDNA synthesis using SuperScript VILO cDNA synthesis kit (LifeTechnologies). Samples were analysed with multiplex dd PCR on the QX100 system (Biorad). The WHO reference genes BCR and GUSB were used as reference. Eight wells were analysed for BCR-ABL1 (FAM labelled probe). Four of the wells were also analysed for BCR and four were analysed for GUSB (HEX labelled probes).

Results:

The median number of GUSB transcripts in the samples was 605,000 copies (range 195,520-934,400). According to the EUTOS “Working definitions of Molecular Response in CML” (ref), MR5.0 sensitivity is obtained when analysing 240,000 GUSB transcripts. All samples except one were above this level. MR5.5 is obtained when analysing 759,000 GUSB transcripts and 20% of the samples (7/36) reached this level. The ddPCR results were compared to the qPCR results and a high concordance was seen in the positive samples (r2=0.94). 21 samples were positive by qPCR, and an additional 4 samples were positive by ddPCR.

Conclusions:

Using the multiplex ddPCR protocol developed 97% of the analysed samples had a sensitivity of MR5.0 or better. Compared to the labs routine qPCR protocol 20% more samples were found positive with ddPCR. Due to the multiplex set-up of the ddPCR the entire cDNA sample can be tested for BCR-ABL1 transcripts. Contrary to qPCR there is no need for calibration of the ddPCR analysis and the method has potential for routine use when a high sensitivity is needed.

References:

“Working definitions of Molecular Response in CML”, EUTOS Molecular Monitoring Steering Group, Nick Cross, Martin Müller, Fabrizio Pane, Andreas Hochhaus, November 2013.

Disclosures

No relevant conflicts of interest to declare.