bluuebunny/crossref_metadata_2025 huggingface dataset:
Subclonal IKZF1 Deletions Indicate a Multiclonal Evolution In BCR-ABL1-Positive B-Cell Precursor ALL
Authors: Boehm Verena , Lebenatus Annett , Bartels Marius , Nagel Inga , Trautmann Heiko , Gökbuget Nicola , Hoelzer Dieter , Pfeifer Heike , Siebert Reiner , Kneba Michael , Ottmann Oliver , Brüggemann Monika
Published: October 2019

The transcription factor Ikaros, encoded by the IKZF1 gene, has multiple functions in different steps of hematopoiesis. Loss of function mutations in IKZF1 are frequently associated with BCR-ABL1-positive ALL. IKZF1 deletions comprise whole gene deletions as well as smaller intragenic deletions. All IKZF1 aberrations are associated with a poor prognosis in terms of overall survival and frequency of relapse.

IKZF1 deletions were evaluated in a cohort of 180 adult BCR-ABL1-positive B-cell precursor ALL cases. MLPA analysis was used to assess both whole gene and intragenic deletions, whereas breakpoint-specific consensus multiplex PCR [Caye et al., Haematologica, 2013] and real-time quantitative (RQ)-PCR were used to specifically detect the recurrent intragenic deletions Δ2-7, Δ2-8, Δ4-7 and Δ4-8. Intragenic IKZF1 deletions were detected in 129 patients (71.7%). More than one intragenic deletion was detected in a subset of 44 patients. Unexpectedly, in the majority of these patients (41/180 patients; 22.8%), significant differences in relative frequencies of single intragenic deletions were detected by RQ-PCR analysis. Together with the finding of more than two distinct intragenic deletions in 19/44 patients this clearly indicates the presence of subclonal or oligoclonal intragenic IKZF1 deletions. In the remaining 3/44 patients, number and frequencies of detected intragenic deletions rather suggested the presence of bi-allelic deletions within the whole leukemic bulk. Specificity of individual (sub-) clonal deletions was validated by Sanger sequencing. These data indicate that IKZF1 deletions arise independently in different subclones more frequently than it was estimated in earlier reports.

The quantitative breakpoint-specific PCR approach was additionally used to correlate the detection of intragenic IKZF1 mutations with established Ig/TCR-based monitoring of minimal residual disease (MRD) during/after treatment in a subset of 17 patients. Comparative IKZF1- and Ig/TCR-based MRD monitoring was performed on a total of 207 samples of initial diagnosis and follow-up. Whereas good correlation of Ig/TCR-MRD results and detection of IKZF1 deletions was seen in 9 out of 17 patients, different kinetics of Ig/TCR-MRD results and IKZF1-based leukemia monitoring in the remaining 8 patients were additionally indicative for the existence of IKZF1 deletions in leukemic subclones besides the major leukemic clone with their own kinetics of clearance and persistence.

Illegitimate RAG-mediated recombination has been proposed as responsible mechanism for recurrent intragenic deletions in IKZF1. Our data suggest that these lesions occur repeatedly during leukemogenesis and support a model of multiclonal evolution in BCR-ABL1-positive B-cell precursor ALL.

Disclosures:

No relevant conflicts of interest to declare.
Activation Of EphrinB2/EphB4 Influences Myeloid Leukemia Cell Migration and Invasion
Authors: Zhou Xuan , Xiaoli Liu , Xu Na , Xiao Yajuan , Zhang Jinfang , Huang Bintao , Du Qingfeng
Published: October 2019

Eph receptors and ephrin ligands are cell-surface molecules capable of bidirectional signaling that control cell-cell interactions, migration and invasion. However, their role and regulation in myeloid leukemia cells remain to be defined. To address the hypothesis that Ephrin/EphB is an important regulator of myeloid leukemia cell migration and invasion, we first screened the mRNA levels of 23 eph and ligand ephrin RTK family members in myeloid leukemia cells (K562, HL-60, THP-1) and mononuclear cells from healthy donors, then found that EphB4, EphA5, EfnA1 highly expressed in most myeloid leukemia cells compared to healthy donors(P<0.05). Both the mRNA and protein levels of EphB4 and EphA5 were detected in 13 primary myeloid leukemia cells (5 from patients with extramedullary leukemia among 13 cases) and 10 mononuclear cells from healthy donors by real-time RT-PCR and Immunoblot analysis. The results showed that both the mRNA and protein levels of EphB4 and EphA5 were higher in 13 primary myeloid leukemia cells relative to the 10 healthy donors (P=0.046). Moreover, the EphB4 were highly expressed in 5 patients with extramedullary leukemia compared with 8 patients without extramedullary leukemia. These findings indicated that EphB4 and EphA5 expression were correlated with the development of myeloid leukemia cells, moreover, EphB4 may be closely related with myeloid leukemia cell migration or invasion. To further clarified the question, migration were determined in leukemia cell lines (K562 cells) which were treated with clustered ephrinA1–Fc proteins, ephrinB2–Fc proteins and Fc proteins by transwell migration assay. Invasion were also determined by matrigel invasion assay. The results showed that, after ephrinB2–Fc stimulation, the numbers of K562 cells migrating through transwell chamber were signiﬁcantly enhanced compared to Fc proteins stimulation (1.8 to 2.5-fold, P<0.05), meanwhile, the numbers of K562 cells invading the matrigel also enhanced (1.2 to 1.8-fold, P<0.05). However, after ephrinA1–Fc stimulation, the numbers of K562 cells migrating through transwell chamber didn’t signiﬁcantly increase compared to Fc proteins stimulation (P>0.05), and the numbers of K562 cells invading the matrigel also didn’t enhanced (P>0.05). These findings indicated that ephrinB2–Fc could activate EphB4, leading to the change of myeloid leukemia cell migration and invasion. Further study may help to assess a promising potential of this protein to be used as a prognostic marker or as a target for a molecular therapy.

Disclosures:

No relevant conflicts of interest to declare.
Copy Number Variations and IKZF1 Mutations In Pediatric CML
Authors: Krumbholz Manuela , Tauer Josephine , Kronnie Geertruy te , Ekici Arif , Suttorp Meinolf , Metzler Markus
Published: October 2019

Chronic myeloid leukemia (CML) occurring in children and adolescents is associated with a number of clinical and molecular features that differ from the correspondent disease in patients in older age. Detailed molecular analyses of the genomic BCR-ABL1 breakpoints in a cohort of 60 pediatric individuals from the German CML-paed I and CML-paed II trial revealed a significantly different breakpoints distribution compared to adult CML. Especially the observed bimodal breakpoint distribution pattern in the BCR gene and a higher proportion of breakpoints within Alu repeat regions vary between pediatric CML and adult CML and resembles the pattern observed in adult BCR-ABL1-postitive acute lymphoblastic leukemia.

To identify additional genetic variations, in addition to the BCR-ABL1 fusion gene, high resolution whole-genome microarray analyses using Affymetrix Cytogenetics Whole-Genome 2.7M or CytoScan® HD arrays were performed in a selected sub-cohort of pediatric CML patients from the German CML-paed II trial; twenty individuals diagnosed in chronic phase (CP) and two individuals diagnosed in blast crisis (BC) were screened. In contrast to adult CML patients in whom about 25% of individuals in CP exhibit detectable CNVs, nearly 60% of pediatric patients have CNVs (1.9 CNVs per case) in this initial stage of CML. All identified CNVs were private and no recurrent genetic aberration was associated with early disease manifestation in children. Two patients in BC showed an increased number of CNVs (6.5 CNVs per case) which is in accordance with data from adult patients, indicating that additional secondary genetic events are associated with disease progression. One of the patients in lymphoid CML-BC harbored a deletion involving the IKZF1 gene, an aberration frequently observed in advanced disease stages in adult CML and ALL.

To further evaluate the incidence of IKZF1 mutations and their impact on the disease progression in pediatric CML patients, deep sequencing of IKZF1 was performed in 55 patients; 52 individuals in CP and 3 individuals in BC. Previously performed whole genome array analyses showed a deletion of exon 4 to 7 in the IKZF1 gene in one of the three analyzed patients in BC. Deep sequencing identified no additional mutation within IKZF1 in both remaining individuals. Interestingly, two patients in CP showed a single nucleotide insertion resulting in a premature stop codon, E35 and Y348 respectively. One patient developed imatinib resistantance 1.5 years after treatment onset. The second patient underwent stem cell transplantation 1 year after initial diagnosis. To date, with a follow-up of 7 and 6 years respectively, no patient progressed to advanced disease. These findings are in line with findings in adult CML, where IKZF1 aberrations are preferentially observed in advanced disease stages or imatinib resistant patients.

Disclosures:

No relevant conflicts of interest to declare.
Impact Of Myelofibrosis (MF) In MDS Treated With Azacitidine (AZA). A Single Center Study
Authors: Legoupil Clemence , Sebert Marie , Braun Thorsten , Gardin Claude , Martin Antoine , Brechignac Sabine , Harel Stephanie , Fenaux Pierre , Ades Lionel
Published: October 2019

Background

Bone marrow fibrosis is observed in 10-20% of MD, and is a poor prognostic factor, both in lower and higher risk MDS (Della Porta, JCO 2009). AZA, the current reference treatment for higher risk MDS, approved in EU for patients with up to 30% bone marrow blasts not candidates to intensive chemotherapy (IC) or allogeneic SCT, gives 50-60% responses and improves OS in higher-risk MDS but its role in MDS with myelofibrosis remains unknown.

Methods

Between 2004 and 2012, we treated at our center 172 consecutive MDS patients (pts) including FAB RAEB-T / WHO AML with 20-30% blasts, with AZA (75 mg/m2/d x7 d every 4 weeks, for a median of 6 cycles). We assessed in those patients the impact of myelofibrosis (MF), evaluated on bone marrow trephine biopsy and graded according to the European consensus on grading bone marrow fibrosis.

Results

Median age of the 172 pts was 73 years and 67% were males. According to WHO classification, 1 had del(5q) syndrome, 4 RARS/RCMD-RS, 24 RCMD, 37 RAEB-1, 54 RAEB-2, 29 AML (20-30% blasts), 17 CMML and 6 MDS unclassified. Median Hb level, WBC, platelet count and marrow blast count were 9.4 g/dl (range 3.5-15), 1.6 G/L (0-58), 75G/l (8-1080) and 12%(1-29), respectively. IPSS was low, Int-1, Int-2, High and a failure in 1(>1%), 29(17%), 65 (38%), 59 (34%) and 18 (11%) patients, respectively. Twenty-three pts (13%) had grade 2-3 myelofibrosis (MF). Patients with MF were younger (median 68 vs. 74 years, p= 0.04), but had similar hematological characteristics: hemoglobin (median 9 vs. 9.45g/dl, p= 0.69); WBC (2.2 vs. 1.6 G/l, p= 0.48) Platelet (47 vs 77 G/l,p= 0.43) and bone marrow blast (10 vs. 12%, p=0.67). IPSS was int 1, int 2, high and a failure in 4, 7, 6 and 6 patients respectively without difference compared to patients without fibrosis. Cytogenetics was complex in 8, del(20q) +/- 1 additional abn in 5 patients, normal in 4 patients, failed in 6 patients (but with trisomy 8 and monosomy 7, resp, detected in 2 patients by FISH analysis). Overall, 73 (42%) patients achieved a response according to IWG2006 criteria, including 31 (18%) CR. The response rate and CR rate were 52% and 17% respectively in pts with MF, compared to 44% and 20% in pts without MF (p= 0.507 and p=1.00, respectively). With a median follow-up of 6.5 yr, median OS was 12 months in pts with MF, compared to 16 months in pts without MF (p=0.45).

Conclusion

In this MDS series, presence of MF was not associated with specific features (but blasts were often counted on marrow aspirates, with a possible underestimate in case of MF). Response to AZA and survival after AZA onset did not significantly differ based on the presence of MF.

Disclosures:

No relevant conflicts of interest to declare.
Aberrant Overexpression Of CD14 In Granulocytes Sensitizes Innate Immune Response In mDia1 Heterozygous Myelodysplastic Syndromes
Authors: Keerthivasan Ganesan , Zhao Baobing , Harris Chad E , Zhang Ling , Gao Juehua , McElherne James , Yang Jing , Basiorka Ashley A , Schipma Matthew , Boultwood Jacqueline , Verma Amit , List Alan , Williams David A. , Ji Peng
Published: October 2019

The myelodysplastic syndromes (MDS) are a group of pre-leukemic diseases characterized by increased risk of acute myeloid leukemia (AML). Heterozygous loss of chromosome 5q (5q-) is the most common cytogenetic abnormality in MDS. DIAPH1 is localized to 5q31 and encodes one of the formin family proteins, mDia1, involved in the regulation of linear actin polymerization. Mice with mDia1 deficiency develop hematologic features mimicking human myeloproliferative neoplasm, but its role in the pathogenesis of MDS is unclear. Here we report that mDia1 is largely dispensable for normal hematopoiesis. However, the committed or mature granulocytes in mDia1 heterozygous and knockout mice were activated and showed increased actin polarization. Strikingly, CD14 was aberrantly overexpressed in the bone marrow and peripheral granulocytes of mDia1 heterozygous and knockout mice in a cell-autonomous manner, leading to a hypersensitivity of the innate immune response to lipopolysaccharide (LPS) stimuli through CD14-Toll like receptor 4 (TLR4) signaling. Chronic stimulation with LPS accelerated the occurrence of MDS in mDia1 heterozygous and knockout mice. Similar findings of CD14 overexpression were observed in the bone marrow granulocytes of 5q- MDS patients, but not normal patients or MDS patients without 5q deletion. These patients exhibited relatively frequent infections with long duration of disease. Mechanistically, mDia1 was required for the endocytosis of CD14 upon LPS stimulation. Heterozygosity and loss of mDia1 led to diminished interferon expression that is dependent on CD14 endocytosis. Thus, our study revealed an essential role of the innate immune signaling in the pathogenesis of 5q- MDS. Specifically, heterozygosity or loss of mDia1 leads to a cell autonomous hypersensitivity of innate immune system in the granulocytes that causes myeloid dysplasia. Together with previous reported evidence of dyserythropoiesis due to loss of RPS14, and dysmegakaryopoiesis due to loss of mir-145/146a, these data fully recapitulate the pathogenesis of tri-lineage dysplasia in 5q- MDS. In addition, our study highlighted the significance of the activated CD14/TLR signaling pathway in the pathogenesis of MDS, which could serve as a novel target for therapeutic management.

Disclosures:

No relevant conflicts of interest to declare.
Germline JAK2 Mutations In The Kinase Domain Are Responsible For Hereditary Thrombocytosis and Are Resistant To JAK2 and HSP90 Inhibitors
Authors: Marty Caroline , Saint-martin Cecile , Pecquet Christian , Grosjean Sarah , Saliba Joseph , Mouton Celine , Emilie Leroy , Harutyunyan Ashot S. , Abgrall Jean-François , Favier Remy , Solary Eric , Kralovics Robert , Constantinescu Stefan N. , Najman Albert , Vainchenker William , Plo Isabelle , Bellanné-Chantelot Christine
Published: October 2019

The main molecular basis of essential thrombocytemia and hereditary thrombocytosis are acquired and germline activating mutations affecting the thrombopoietin signaling axis, respectively. We have identified two families with hereditary thrombocytosis presenting novel heterozygous germline mutations of JAK2. One family carries the JAK2 R867Q mutation located in the kinase domain, while the other presents two JAK2 mutations, S755R/R938Q, located in cis in both the pseudokinase and kinase domains. Expression of JAK2 R867Q and S755R/R938Q induced spontaneous growth of Ba/F3-MPL but not of Ba/F3-EPOR cells. Interestingly, both Ba/F3-MPL cells expressing the mutants and platelets from patients displayed a TPO-independent phosphorylation of STAT1. JAK2 R867Q and S755R/R938Q, compared to JAK2 V617F, had significantly longer half-lives correlating with an increased MPL cell surface expression. Moreover, these mutations conferred a resistance to JAK2 and HSP90 inhibitors compared to JAK2 V617F. Our results suggest that the mutations in the kinase domain of JAK2 may confer a weak activation of signaling specifically dependent on MPL, while inducing resistance to clinically available JAK2 inhibitors.

Disclosures:

No relevant conflicts of interest to declare.
Stereotyped Subset #4 In Chronic Lymphocytic Leukemia Is Associated With Distinct Gene and Microrna Transcriptional Profile
Authors: Maura Francesco , Mosca Laura , Cutrona Giovanna , Matis Serena , Lionetti Marta , Fabris Sonia , Carlotta Massucco , Reverberi Daniele , Gentile Massimo , Recchia Anna Grazia , Monica Colombo , Bossio Sabrina , Negrini Massimo , Tassone Pierfrancesco , Rossi Davide , Gaidano Gianluca , Di Raimondo Francesco , Morabito Fortunato , Ferrarini Manlio , Neri Antonino
Published: October 2019

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disorder characterized by the monoclonal accumulation of B lymphocytes and a variable clinical course. Specific B-Cell Receptor (BCR) utilized by leukemic cells may influence disease progression and outcome. Highly homologous BCR, “stereotyped BCR”, are expressed in a recurrent fraction of patients with CLL and in some cases they were associated with distinct biological and clinical features. Stereotyped subset #4 have been reported to exhibit a favorable clinical course and to be the most frequent stereotyped BCR among the IGHV mutated (IGHV-M) cases. In this study we performed a comprehensive clinical, biological and molecular characterization of leukemic cells from 16 patients utilizing stereotyped subset #4 BCR (IGHV4-34) among a representative prospective cohort of 462 Binet stage A CLL patients enrolled in O-CLL1 protocol (clinicaltrial.gov identifier NCT00917540). In all cases, biological and molecular analyses were performed in peripheral CD19+ B-cells. All subset #4 patients were characterized by lower CD38 expression, unique IGHV-M configuration and absence of NOTCH1 and SF3B1 mutations. None subset #4 patients showed unfavorable cytogenetic deletions (i.e del11q23 and del17p13). Gene expression profiling (GEP) analysis was performed on 217 patients, including 9 subset #4 cases for whom RNA material was available. Supervised analysis comparing subset #4 vs all other patients (208) revealed 14 differentially expressed genes. Furthermore subset #4 patients were characterized by a significant downregulation of WDFY4, MEF2A and upregulation of PDGFA, FGFR1 and TFEC genes when compared with the remaining IGHV-M patients. miRNA profiles were analyzed in 229 patients including 10 subset #4 patients for whom RNA material was available. A specific miRNA expression pattern involving the upregulation of miR-497 and miR-29c was found in subset# 4 cases. Furthermore, we demonstrated that transfection of the miR-497 mimic in primary leukemic CLL cells induces, after 48 and/or 72 h, a downregulation of BCL2, known to be a validated target in different solid cancers. Our data provide a contribution to the biological definition of CLL patients with specific stereotyped IGHV4-34 BCR and identify for the first time distinct gene and miRNA expression profiles associated with this subset, providing further evidence of the putative leading role of HCDR3 conformation in CLL.

Disclosures:

No relevant conflicts of interest to declare.
YM155, a Survivin Suppressant, Induces Cell Death Via Suppression Of c-Myc Expression In Multiple Myeloma Cells
Authors: Ito Shigeki , Asahi Maki , Sasaki Ryousei , Oyake Tatsuo , Tamura Hideto , Ishida Yoji
Published: October 2019

Survivin is a member of the inhibitor of apoptosis protein (IAP) family with its dual roles in mitosis and apoptosis, and emerges as an attractive target for cancer therapy. Recent reports have demonstrated that survivin overexpression is associated with drug resistance and poor outcome in hematological malignancy including multiple myeloma (MM). YM155, a novel molecular targeted agent, suppresses survivin through the inhibition of transcription. However, the effect of this agent on MM cells has not been elucidated. In this study, we investigated the effect of YM155 on proliferation and survival of five human MM cell lines. YM155 inhibited the proliferation of these cells in a dose- and time-dependent manner (IC50 = 10 nM in 3 and 100 nM in 2 cell lines, respectively). Annexin V assay showed that YM155 induced apoptosis in these cells. To better understand these effects of YM155 on MM cells, we evaluated the intracellular signaling and apoptosis-associated protein status. Immunoblot analyses showed that YM155 reduced not only survivin but also myeloid cell leukemia sequence 1 (Mcl-1) and X-linked inhibitor of apoptosis protein (XIAP) expression. We also observed the activation of caspase-3 and poly(ADP-ribose) polymerase in YM155-treated cells, indicating that YM155 induces caspase-dependent apoptosis. YM155 did not affect phosphorylation status of Erk1/2 and STAT3. Interestingly, we found that YM155 suppressed c-Myc and interferon regulatory factor 4 (IRF4) expression, both of which are recognized as an important oncogene in the pathogenesis of MM. In addition, c-Myc and IRF4 protein levels were reduced at 6 and 12 hours after treatment with YM155, respectively. As IRF4 and c-Myc form a positive feedback loop in myeloma cells, this observation indicates that c-Myc inhibition by YM155 treatment might lead to subsequent inhibition of IRF4 expression, and thus raises the possibility of YM155 target for c-Myc rather than IRF4. We next examined the mechanism of downregulation of c-Myc in RPMI8226 cells. Real-time quantitative RT-PCR assay showed that YM155 treatment reduced c-Myc mRNA level. On the other hand, proteasome inhibitor did not prevent the suppression of c-Myc expression by YM155 treatment. These Results suggest that YM155 transcriptionally at least in part represses c-Myc in RPMI8226 cells. In conclusion, YM155 suppresses cell proliferation and survival in MM cells in part via not only inhibiting anti-apoptotic proteins such as survivin, Mcl-1 and XIAP but also repressing c-Myc oncogene. Further study is needed to clarify the molecular mechanism of downregulation of c-Myc induced by YM155. Our Results may provide a platform for clinical trials of YM155 in MM.

Disclosures:

No relevant conflicts of interest to declare.
Survival In Adult Acute Lymphoblastic Leukemia Survival By Age, Gender and Ethnicity
Authors: Ghimire Krishna B , Shah Binay K.
Published: October 2019

Background

Studies have shown improvement in survival in adult acute lymphoblastic leukemia (ALL) with the use of risk-directed therapy pediatric-inspired regimens. We analyzed US Surveillance, Epidemiology and End Results (SEER) cancer registry database to evaluate whether survival of adult ALL patients has improved in general population.

Methods

We analyzed US Surveillance, Epidemiology and End Results (SEER) 18 registries using survival session to calculate relative survival rates (RS) during 1973-1989, 1990-1999 and 2000-2009 among adult (≥20 years) ALL patients. We used Z-test in the SEER*Stat software to compare the relative survival rates in several cohorts categorized by race, gender, and age groups (20-39, ≥ 40 years).

Results

There were 7,435 adult ALL patients reported in SEER 18 registries during 1973-2009. The majority of patients were Caucasian (85%) and Male (57%).

The relative survival rates for adult ALL patients between 1973-2009 were 55.8±0.6% and 25.0±0.5% at 1 year and 5 years respectively. The relative survival rates improved significantly for each successive time periods, with an improvement from 45.4%, to 51.6%, to 60.6% at 1 year and, from 15.3%, to 23.7%, to 29.0% at 5 years for time periods 1973-1989, 1990-1999 and 2000-2009 respectively. For younger patients (age 20-39 years), 1- and 5-year RS rates improved from 1973-1989 to 1990-1999 but not from 1990-1999 to 2000-2009. For older patients (age ≥40 years) RS improved for each successive time periods.

The 1- and 5- year relative survival rates improved significantly for both male and female patients during successive time periods. Interestingly, although there was significant improvement in survival rates at 1- and 5- years for Caucasians, improvement was not seen for AA adult ALL patients during subsequent time periods examined.

Conclusions

Although adult ALL survival rates have improved for most cohorts examined, the survival of African American patients has not improved. Similarly, survival rates of younger patients (20-39 years) has not improved from 1990-1999 to 2000-2009.

Disclosures:

No relevant conflicts of interest to declare.
Access To Therapeutic Surgical Procedures For Pathologic Fractures In Multiple Myeloma Patients: Analysis of Nationwide Inpatient Sample (NIS)
Authors: Shah Nishi N , Nooka Ajay K. , Lonial Sagar
Published: October 2019

Introduction

Myeloma patients that experience a pathological fracture have 44% increased risk of mortality compared to their counterparts without pathological fractures. Given the negative impact of pathological fractures on survival, we have explored if access to obtain a therapeutic procedure influences the survival. We analyzed the nationwide inpatient cohort to evaluate for access to procedures and other hospitalization variables for myeloma patients admitted to the hospital for the diagnosis of pathologic fractures.

Methodology

Myeloma patients admitted in the hospital for the primary diagnosis of pathologic fractures from 2001-2010 were analyzed from the Nationwide Inpatient Sample (NIS). Procedures and diagnoses were identified using ICD-9-CM and NIS CCS codes. Variables of therapeutic procedures, length of stay (LOS), in-hospital mortality (IHM) and hospital charges were explored using multivariate logistic regression. Costs are derived from total hospital charges using cost-to-charge ratios based on hospital accounting reports from the Centers for Medicare and Medicaid Services and reflect the actual costs to produce hospital services.

Results

Of the 5154 myeloma patient admitted for pathological fractures, 2422 patients (47%) underwent a therapeutic procedure. The mean LOS for the primary diagnosis was 7.23 days (SE 0.14); cost for the hospitalization was $19344 and the IHM is 2.32%. The probability of getting a therapeutic surgical procedure and the cost of hospitalization is lower in patients >65, but had similar LOS and IHM compared to patients <65. Similar probability of not getting a surgical procedure was seen in medicare patients; but longer LOS may have contributed to the similar cost. However, in the medicaid patients, the odds of getting a surgical procedure was 66% low; the LOS and the cost are significantly high compared to the private insurance. IHM did not differ significantly across all groups.

Conclusions

This analysis addresses the question that older myeloma patients (age >65) and those that have the payer status of medicare and medicaid are significantly associated with the probability of not getting a therapeutic surgical procedure when they are admitted with pathological fracture. However, this did not result in increased in-hospital mortality suggesting that probably pathological fractures may reflect the biology of the underlying disease with negative impact on survival or the morbidity associated with a pathological fracture may impede the long term survival in myeloma patients.

Disclosures:

Lonial: Millennium: Consultancy; Celgene: Consultancy; Novartis: Consultancy; BMS: Consultancy; Sanofi: Consultancy; Onyx: Consultancy.
Long-Term Sequelae and Late Effects In Childhood Acute Lymphoblastic Leukemia (ALL) Survivors: A Single AIEOP Center Experience
Authors: Parasole Rosanna , Petruzziello Fara , De Matteo Antonia , Mangione Argia , Sepe Gaia , Iuliano Annunziata , Pinto Annamaria , Ferrante Maria Rosa , Vitale Virginia , Poggi Vincenzo
Published: October 2019

The advent of more intensive chemotherapy and the improvement of supportive cares have dramatically changed the natural history of childhood acute lymphoblastic leukemia (ALL), with current estimated 5-year overall survival of about 80%. The increased survival rate and the establishment of follow-up survey for long term survivors (LTS) have allowed the identification of late chemo-radiotherapy adverse effects on psychological and general health. We retrospectively evaluate the incidence and type of sequelae and / or late effects in a cohort of 301 childhood ALL LTS, followed in a single pediatric AIEOP center.

From June 1986 to June 2013, 301 LTS (154 male and 147 female), aged <18 years at ALL diagnosis, were followed-up by a multidisciplinary team. The surveillance protocol is summarized in Figure 1. The timing of follow-up (FU) was modified, case by case, in relation to the appearance of adverse events or organ diseases. Survivors' results were compared with chronic medical and psychological conditions of siblings (n=89).

Mean FU time (time from the stop therapy to the last control) was 6 years (range 1.8-26.8 years). The majority of LTS were teenager or young adults : 35% ranged between 15 and 20 years; 19.7% was more than 21 years old and the 45.3% was less than 14 years old. During FU, 16 late recurrences (5.3%) were identified and 3 secondary malignancies (0.99%) such as one mesenteric paraganglioma and two Acute Myeloid Leukemia in second complete remission ALL LTS. 40 patients (13.3%) received cranial radiotherapy during treatment. 39 LTS (13%) reported at least one sequelae. The most frequent sequelae were neurological and orthopedic (6% and 3% respectively) as summarized in Figure 2 151 LTS (50.17%) presented at least one late effect as showed in Figure 3.

In our experience at least one late effect occurred in 50.17% of LTS; these late complications affect negatively the quality of life of survivors. Endocrine-metabolic events are the most frequent late effect (34,5%). 13% of LTS have at least one sequelae mainly neurological and orthopedic. Prevention and/or early identification of complications during follow-up survey of LTS are crucial in order to decrease the long-term health risks associated with curative treatment for childhood ALL.

Disclosures:

No relevant conflicts of interest to declare.
Radiation Therapy In Peripheral T-Cell Lymphoma Of The Head and Neck
Authors: Fernandes Annemarie T. , Svoboda Jakub , Nasta Sunita D. , Wilson Lynn D. , Plastaras John P.
Published: October 2019

Introduction

The role of radiation therapy in peripheral T-cell lymphoma (PTCL) is not well established. While the NCCN guidelines recommend combined modality treatment with consolidative radiation therapy after chemotherapy for localized (stage I and II) disease, the data supporting this recommendation is lacking and is extrapolated from B-cell lymphoma.

Methods

This is a retrospective analysis of outcome in patients with non-cutaneous peripheral T-cell lymphoma of the head and neck from the Surveillance, Epidemiology and End Results (SEER) database diagnosed between 1981-2010. Survival was estimated by Kaplan-Meier estimates using the log-rank test. Univariate and multivariable analyses were performed using Cox regression analysis.

Results

Of the 307 patients analyzed, 130 (43%) underwent radiation therapy. The median age was 59 years old, 60.3% were male, 73.9% were Caucasian and the median year of diagnosis was 2004. The majority of patients had PTCL, Not Otherwise Specified (74.3%), followed by anaplastic large cell histology (22.2%). Patients were grouped as stage I (47.2%), stage II (29.9%) and stage III/IV (22.8%). Head and neck sites included the nasal cavity (28.3%), oropharynx (25.7%), oral cavity (21.2%), salivary gland (11.7%), nasopharynx (7.8%) and larynx/hypopharynx (5.2%).

The median follow-up was 54 months for survivors. Radiation therapy was associated with a higher 5-year overall survival in all patients (56.7% vs. 38.4%, p=0.001), patients with stage I disease (63.4% vs. 53.4%, p=0.036) and patients with stage II disease (60.8% vs. 36.3%, p=0.034), see Figure 1. Radiation therapy was not associated with a difference in overall survival in patients with stage III/IV disease (p=0.91).

Univariate analysis demonstrated that age, stage, radiation therapy and marital status were predictive of overall survival, but sex, and race were not. The year (p=0.93) or decade of treatment (p=0.67) did not impact overall survival. On multivariable analysis, increasing age (HR: 1.02, p<0.001), increasing stage (HR: 1.42, p=0.004), radiation therapy (HR: 0.60, p=0.011) and marital status of not married (HR: 1.49, p=0.035) remained statistically significant predictors of overall survival.

An additional analysis was performed excluding patients who survived less than 3 months to account for mortality related to disease progression or chemotherapy toxicity. After excluding these 45 patients, radiation therapy was still associated with increased overall survival in all patients (p<0.001) and in patient with limited stage disease (p=0.021).

Conclusions

The integration of local radiation therapy to the treatment of PTCL of the head and neck may improve overall survival in patients with limited stage disease.

Disclosures:

No relevant conflicts of interest to declare.
C5a Complement Depletion Suppresses In Vivo B Lymphoma Progression and Enhances Development Of Cellular Anti-Tumor Immune Response
Authors: Veeramani Suresh , Weiner George J.
Published: October 2019

Background

Proteins within the complement system have complex effects on cellular immune responses. In previous studies, we found that active complement components, especially C5a, can dampen the development of antigen-specific immune responses following vaccination with a model antigen, in part by promoting generation of APC-induced T regulatory (Treg) cells. These studies also demonstrated that B lymphoma cell lines exposed to complement can induce Treg generation in vitro. The current study was designed to address whether depletion of C5a could enhance development of a cellular anti-lymphoma immune response in vivo.

Methods

Immunocompetent Balb/C mice were inoculated subcutaneously with syngeneic A20 B lymphoma cells mixed with either 10 μg of rat anti-mouse C5a monoclonal antibody (mAb) or 10 μg of isotype-matched Rat IgG2a control mAb. Tumor growth was followed. In select experiments, mice were sacrificed and analyzed for the percentage and activity of tumor-infiltrating T cells and A20-specific splenic T cell responses.

Results

1. Tumor progression. Lymphoma grew more slowly in mice treated with anti-C5a mAb compared to mice treated with control mAb (p<0.05) {Fig. 1).

2. Intratumoral T cells. Tumors from mice treated with anti-C5a mAb had higher CD8+ T cell infiltration compared to mice treated with control mAb (p=0.002) (Fig. 2). Tumor-infiltrating CD8+ T cells showed a trend towards higher intracellular IFNg production in mice treated with anti-C5a mAb compared to control mAb (p=0.051).

3. Splenic T cells. Splenic T cells from mice treated with anti-C5a mAb produced IFNg to a greater degree than did splenic T cells from control mice when splenocytes were cultured with irradiated A20 cells in vitro (p=0.041) (Fig. 3). There was a trend towards decreased numbers of splenic CD4+CD25highFoxp3+ Tregs in C5a-depleted mice compared to control mice.

Conclusions

Depletion of C5a at the site of tumor inoculation slows tumor growth and increases the number of tumor infiltrating CD8 T cells in a syngenic immunocompetent model of lymphoma. A trend towards enhanced production of IFNg in the tumor infiltrating T cells, increased numbers of tumor-specific splenic T cells, and reduced numbers of splenic Tregs, suggests intratumoral C5a depletion can enhance tumor-specific immune responses both within the tumor and systemically. Ongoing studies are exploring the molecular mechanisms involved in C5a-promoted tumor progression and the use of C5a depletion as a novel strategy to improve anti-tumor immunity.

Disclosures:

No relevant conflicts of interest to declare.
A Comparison Of Hemostatic and Inflammatory Markers In Overt and Non-Overt DIC
Authors: Hoppensteadt Debra , Tsuruta Kazuhisa , Hirman Joe , Kaul Inder , Osawa Yutaka , Pharm M , Fareed Jawed
Published: October 2019

Disseminated intravascular coagulation (DIC) is a complex pathophysiologic syndrome with marked alterations in the hemostatic system, which may lead to microvascular thrombosis and multi-organ failure. The International Society of Thrombosis and Hemostasis (ISTH) classifies DIC into two distinct categories; overt and non-overt DIC (NO). Overt DIC is characterized by a decompensated hemostatic system whereas non-overt is characterized as a stressed, compensated system. DIC is a frequently observed in sepsis patients. An association between the hemostatic aberrations and inflammation has been previously reported. Therefore we studied the level of hemostatic activation and inflammation in patients with sepsis and suspected DIC enrolled in the ART-123, Phase 2b study. The level of inflammatory mediators such as anaphylatoxin C5a (C5a), procalcitonin (PCT), interleukin 6 (IL-6), interleukin 10 (IL-10), myeloperoxidase (MPO), and circulating levels of hemostatic markers including protein C inhibitor (PCI), plasminogen activator inhibitor 1 (PAI-1), and protein C (Pr C) were evaluated in 617 subjects enrolled in a randomized, double-blind, placebo-controlled, Phase-2b study evaluating the safety and efficacy of recombinant thrombomodulin (ART-123) in subjects with sepsis and suspected DIC. When the subjects with overt DIC and with NO DIC were compared, the PrC level was lower and PCT, PAI-1, IL-6 and IL-10 levels were higher, in the overt DIC subjects (Table). The upregulation of the inflammatory cytokines observed in this study may contribute to the decreased levels of Pr C. Inflammatory cytokines may dysregulate the endothelial thrombomodulin resulting impaired hemostatic regulation. Pr C levels are also low due to consumption and impaired synthesis, especially in the overt DIC patients. These results suggest that profiling these mediators may be useful in the diagnostic and prognostic management of sepsis and differentiation between overt and non-overt DIC.BiomarkerGroupNMeanSEMMedianP ValuePr C (%)Normal4094.52.491.80.0043Overt9824.71.828.30.0043NO15746.52.637.00.0043PCTNormal240.260.020.270.0001(ng/ml)Overt9836.87.011.220.0001NO51918.031.45.330.0001PAI-1Normal3120.62.118.7<0.0001(ng/ml)Overt98232.925.0137.5<0.0001NO519147.18.676.0<0.0001Il-6Normal306.80.536.10.0003(pg/ml)Overt981017.6121.7374.40.0003NO519576.739.6174.70.0003IL-10Normal3210.42.43.50.0049(pg/ml)Overt98187.339.146.00.0049NO518108.114.831.00.0049

Disclosures:

Hoppensteadt: Asahi Kasei Pharma: Research Funding. Tsuruta:Asahi Kasei Pharma: Employment. Hirman:Asahi Kasei Pharma: Consultancy. Kaul:Asahi Kasei Pharma: Employment. Osawa:Asahi Kasei Pharma: Employment. Fareed:Asahi Kasei Pharma: Research Funding.
Characterization Of a New Recombinant Human Factor VIIa (LR769)
Authors: Jerry Grandoni , Perret Gerald , Forier Cynthia
Published: October 2019

Congenital Hemophilia A and B treatment may be complicated by the development of inhibitors to the coagulation factors used in the replacement therapies. In such cases factor replacement becomes ineffective, and use of a bypassing agent is needed to treat or prevent bleedings. A recombinant form of activated Factor VII has been available for this purpose. In this study, a new recombinant human Factor VIIa (rhFVIIa, LR769) produced by LFB/rEVO Biologics was tested. The goal is to provide patients with Hemophilia A and B who develop inhibitors a cost-effective alternative treatment option. Recombinant Human Factor VII was activated during the purification process to yield a highly homogenous rhFVIIa product (LR769). Kinetic enzyme assays and binding studies were used to characterize LR769. Active site titration demonstrated approximately 1 mole of active site per mole of protein. The Km and kcat for activation of FX and FIX were determined using an assay containing recombinant human tissue factor and phospholipid. The Kd for binding to soluble tissue factor was 22.3 ± 1.7 nM as measured using a FX activation assay. The apparent second order rate constant for inactivation by human plasma antithrombin was 5.9 ± 0.4 x103 M-1 sec-1. In all kinetic assays, LR769 behaved as expected for rhFVIIa. Binding studies with isolated human platelets were performed by FACS and indicated that binding was dependent on Ca+2. Activation of the platelets with thrombin and convulxin increased the binding of LR769. Binding of LR769 to Endothelial Protein C Receptor (EPCR) on the surface of cultured HEK 293 cells was determined by FACS analysis and confirmed that LR769 binds to EPCR. This was further demonstrated with a surface plasmon resonance assay using purified soluble EPCR. Binding to EPCR was dependent on Ca+2, and could be stimulated by Mg+2. Specificity of the binding was confirmed by the fact that it was inhibited by excess Protein C.

Disclosures:

No relevant conflicts of interest to declare.
Acquired Von Willebrand Factor Deficiency Caused By Left Ventricular Assist Devices Is Adamts-13 Dependent
Authors: Jilma-Stohlawetz Petra , Heinrich Schima , Stoiber Martin , Quehenberger Peter , Schranz Sabine , Jilma Bernd
Published: October 2019

Background

The high shear rates are induced by artificial heart valves or left ventricular assist devices cause acquired von Willebrand factor syndrome (aVWS). We hypothesised that an ex vivo model could be established to study whether mechanical shear alone causes aVWS or whether this process depends also on the VWF cleavage protein ADAMTS-13.

Methods

Healthy volunteers and two patients with congenital ADAMTS-13 deficiency donated blood. In vitro extracorporeal circuits were established using medically approved left ventricular assist devices (Heartware®) and silicone tubings with blood inlets and outlets. The pump speed and circulating blood volume were adjusted to correspond to in vivo use in humans. Anticoagulated blood circulated for 1-4 hours in the extracorporeal circuits. VWF multimers were quantified by sodium dodecyl sulphate-agarose discontinuous gel (1.2%) electrophoresis followed by Western-Blotting and consequent quantification with a luminescence image analyzer and a commercially available software, VWF antigen was measured by an STA assay and ristocetin co-factor activity (VWF:RCo) was quantified by turbidometry using a commercial kit (BC von Willebrand reagent; Dade Behring/Siemens, Marburg, Germany). Platelet function was measured by multiple electrode aggregometry in whole blood.

Results

The high shear stress in the extracorporeal circulation rapidly decreased VWF:RCo and thereby the VWF:RCo/VWF:Ag ratio by 47% (p<0.01) to pathologically low values. Concomitantly, high molecular weight multimers (HMWM) decreased, resembling the pathophysiological events that occur when an LVAD is implanted into humans. Up to 14-15 mers were visible on the gels at baseline, which were reduced by a maximum of 6-7 mers, corresponding to an average 68% lower densitometry signal of HMWM (p<0.01). This was accompanied by a 3-fold reduction in ristocetin induced aggregation (p<0.01). In contrast, the two patients with congenital thrombocytopenic purpura and virtually complete deficiency of ADAMTS-13 activity had only a minimal or no decrease in multimers (p<0.05 vs. healthy controls).

Conclusion

A model for LVAD associated aVWS was established, which demonstrated that ADAMTS-13 activity is essential for the depletion of HMWM of VWF that occurs in VAD-associated aVWS.

Disclosures:

No relevant conflicts of interest to declare.
RAP-011 Efficiently Rescues Erythropoiesis In Zebrafish Models Of Diamond Blackfan Anemia
Authors: Ear Jason , Huang Haigen , Tehrani Zahra , Sung Victoria , Daniel Thomas , Chopra Rajesh , Lin Shuo
Published: October 2019

Diamond Blackfan Anemia (DBA) is a bone marrow failure disorder characterized by low red blood cell count but normal levels of platelets and white blood cells. Ribosomal mutations in RPS19, RPS26, RPL5, and RPL11 have been identified in approximately 50% of all DBA cases. Corticosteriod therapy and bone marrow transplantion are the most common treatment options for DBA patients. However, corticosteroids have severe side effects and bone marrow transplantation is risky; thus, novel therapeutics for DBA are needed. Sotatercept (ACE-011), an activin receptor IIA ligand trap which rapidly increased hemoglobin and hematocrit in both pharmacologic models and in healthy volunteers, is currently being evaluated in diseases of ineffective erythropoiesis such as ß-thalassemia and MDS. Non-clinical studies in mice have demonstrated that RAP-011, a murine ortholog of sotatercept, stimulates RBC parameters in mice through stimulating expansion of late-stage erythroblasts through a mechanism distinct from EPO. Here, we evaluated the effect of RAP-011 in zebrafish models of ribosome insufficiency in RPS19 and RPL11 that recapitulate the anemic phenotype seen in DBA patients. Treatment with RAP-011 treatment dramatically restored hemoglobin levels compromised by ribosome stress. Furthermore, the beneficial effect of RAP-011 is synergistic with corticosteriod treatment. In zebrafish embryos, RAP-011 likely stimulates erythropoietic activity by altering the microenvironment of erythroid cells, reducing p21 levels through a p53-independent manner. These findings uncover a novel signaling pathway in the pathogenesis of DBA and support the potential use of Sotatercept for the treatment of DBA patients with ribosomal disorders. Our studies also demonstrate, for the first time, that protein drugs can be effectively evaluated in zebrafish human disease models, which offer a unique opportunity to identify the targets and study their mechanisms of action.

Disclosures:

Sung: Celgene Corp.: Employment. Daniel:Celgene: Employment. Chopra:Celgene: Employment, Equity Ownership. Lin:Celgene: Research Funding.