bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinical Research: Treatment with Warfarin Followed By Hydroxyurea for Thrombosis Caused By JAK2V617F-Positive Essential Thrombocythemia Associated with Mutation in the Thrombomodulin Gene and Deficiency of Protein S
Authors: Sun Xingfu , Fu Jianxin , Li Jianyong , Lu Hua
Published: November 2020

JAK2V617F-positive essential thrombocythemia is rarely associated with mutation in the thrombomodulin gene and deficiency of protein S without being reported in the literature. A case of a previously healthy 50-year-old Chinese female presenting with superior mesenteric vein thrombosis and portal thrombosis showed by enhanced computed tomography(CT) scan is reported. Blood platelet count showed 644×109/L. Molecular testing for genetic mutation found a JAK2V617F mutation by 11089 copies per 100ng DNA revealed by real-time polymerase chain reaction (RT-PCR), with the mutation rate being over ten percent. Level of free protein S decreased to 30.4%(reference:70%-140%). Heterozygous missense mutation(c.404C>G, i.e.nucleotide 404 of encoding region was mutated from cytosine to guanine) in exon 1 in the thrombomodulin gene was detected in peripheral blood, which caused p.Pro135Arg(i.e.amino acid No.135 was changed from proline to arginine). The chromosomal position of thrombomodulin gene was at chr20:23029738. Family investigation showed that a similar mutation in the thrombomodulin gene was detected in peripheral blood in her daughter. Low molecular weight heparin was given at the beginning of treatment, and bleeding was monitored. In the meantime, warfarin was administered orally and the dosage of which was adjusted to maintain the international normalized ratio of prothrombin time at the range of 2 to 3. Adjust the dosage of hydroxyurea according to blood cell count. A month later, no new thrombosis was found in enhanced CT scan. It was an amazing feat that the patient was in a state of remission without new thrombosis being found for a year. JAK2V617F-positive essential thrombocythemia with mutation in the thrombomodulin gene and deficiency of protein S tends to be incurable. Warfarin alone with hydroxyurea may serve as an effective option to those cases.

Disclosures

No relevant conflicts of interest to declare.
Late Splenic Sequestration: A Consequence of Early Hydroxyurea Use?
Authors: Raghunandan Sharmila , Goubeaux Derrick L , Hollenkamp Christine R , Blaylark Rae M , Nelson Stephen C
Published: November 2020

Background: Acute splenic sequestration is a potentially life-threatening complication in patients with sickle cell disease. It is the second leading cause of death in young children with hemoglobin SS/Sβ0 thalassemia (SCD). The incidence approaches 35% during the first two years of life in SCD. Although splenic sequestration can occur in patients with any sickle phenotype, patients with SCD tend to be significantly younger when compared to patients with hemoglobin SC disease. Splenic function may be preserved in children treated with hydroxyurea. We recommend hydroxyurea for all SCD patients diagnosed via newborn screening, with a goal of starting therapy before 12 months of age. With this aggressive approach and potential for maintaining some splenic function, this study evaluates acute splenic sequestration in this young cohort and compares this to institutional and historical controls

Methods: We performed a retrospective search of electronic medical records at Children's Minnesota from 1/1/1999 through 12/31/2019 to identify patients with SCD and splenic sequestration. We used a broad array of ICD-9 an ICD-10 codes in order to capture all appropriate patients. Following this initial search, in-depth chart review was performed to identify patients with SCD and confirmed splenic sequestration. Data collected includes age, sex, hydroxyurea use, splenectomy, and hematologic results including hemoglobin F percentage, if available. Student t-tests for independent samples were performed to compare cohorts. This study was granted exemption by our Institutional Review Board.

Results: Over this 21 year period, the initial search revealed 151 patients. Of those, 74 had SCD. In-depth chart review found 38 (24M, 14F) of these children were confirmed to have splenic sequestration. Twelve patients started hydroxyurea before 2 years of age at a mean age of 12.6 months, median 11 months (cohort A). Of these, six started hydroxyurea during infancy at a mean and median age of 6.5 months (cohort B). The remaining 26 patients were not on hydroxyurea at the time of splenic sequestration (cohort C). Children in cohorts A and B were significantly older at the time of splenic sequestration compared to patients in cohort C (mean 58 and 45.8 months vs. 14.9 months, p< 0.0001).

Conclusions: Children with SCD who start hydroxyurea at a young age may develop splenic sequestration much later in life than previously expected. More study is needed to confirm this finding. It will be important to educate families and healthcare providers about this potential unintended consequence of early hydroxyurea use.

Disclosures

No relevant conflicts of interest to declare.

OffLabel Disclosure:

Hydroxyurea use in children with sickle cell disease < 2 years of age
Developing a Simple Algorithm Based on Multiparameter Flow Cytometry for Fast Screening of Acute Promyelocytic Leukemia
Authors: Ceni Vitória Ceni , Pagnano Katia B , Duarte Gislaine B O , Pellegrini Marina DB , Duarte Bruno Kosa , Metze Konradin , Lorand-Metze Irene
Published: November 2020

Introduction: Acute promyelocytic leukemia (APL) is a genetically and molecularly well-defined type of acute leukemia that is curable but has a frequent early mortality due to bleeding. So, there is a need for a fast diagnostic screening in order to start appropriate therapy. Multiparameter flow cytometry (MFC) is usually performed in all types of acute myeloid leukemias (AMLs) but only few features have been described as characteristic of APL.

Aim: to develop a diagnostic algorithm based on the intensity of expression of several antigens examined by MFC in AML that could reliably discriminate between APL and the other types of AML.

Material and Methods: Consecutive newly diagnosed AMLs treated in our Institution during the last 2 years entered the study. Immunophenotyping was included in the diagnostic workup. An 8-color platform based on the Euroflow recommendations was used. The mean fluorescence intensity (MFI) of each antigen tested was assessed and those best discriminating between APL and all other types of AML were obtained by a discriminant analysis. Phenotypic characteristics of normal myeloblasts taken from examinations of bone marrow (BM) MFC performed for the diagnosis of cytopenias were used as controls.

Results: 24 cases of APL and 56 cases of other primary AML entered the study. Median age: 39 (23-56) and 62(26-81) years respectively. Concerning ELN risk groups of non-APL cases, 13 were favorable risk, 26 were intermediate and 09 were adverse risk. In 8 cases risk assessment was not possible due to the absence of cytogenetics. Moreover, among APL patients, 7 cases had a FLT3-ITD mutation. Among non-APL AMLs, 4 had FLT3-ITD mutation, 4 had NPM1 and 10 had FLT3-ITD and NPM1mutation.

Concerning antigen expression, CD34 was expressed in only 1/24 APL samples, and in 18/56 samples from non-APL AMLs. The following flow features were differentially expressed in both groups: SSC (p <0.0001), CD45 (p=0.02), CD13 (p=0.001), CD64 (p=0.004), HLA-DR (p<0.0001) and CD33 (p<0.0001) (Table 1). In the discriminant analysis, MFI CD34 and MFI HLA-DR were able to accurately classify APL and non-APL AML in only 62.5%. However, after the addition of the ratio of SSC between blasts and lymphocytes, these 3 parameters were able to differentiate APL from non-APL AML in 91.2% of the cases.

Conclusion: MFC was adequate for a fast screening of APL in most cases. Expression of CD34 was not very useful, as many AMLs do not express this antigen, similar to APL, but SSC, together with HLA-DR could discriminate both types of leukemia in most cases.

Disclosures

Pagnano: Astellas: Other: Advisory Board and lecture; Novartis: Other: Advisory Board; Pintpharma: Other: Lecture; EMS: Other: Lecture. Duarte:Janssen: Other: Lecture; Astellas: Other: Lecture.
Use of Ppmx-T003 As a Potent Inhibitor of Erythrocytosis in Polycythemia Vera
Authors: Masubuchi Nami , Araki Marito , Shioiri Kaho , Kitazawa Sota , Kihara Yoshihiko , Ohira Yuta , Nomura Fumiko , Matsuura Tadashi , Komatsu Norio
Published: November 2020

Iron incorporation through transferrin (Tf) and its receptor, TfR1, plays a critical role in erythropoiesis. We hypothesized that blockade of TfR1 may be a promising strategy to suppress erythrocytosis observed in polycythemia vera (PV). To examine this possibility, we developed PPMX-T003, a monoclonal antibody against human TfR1, through phage display screening. PPMX-T003 was observed to bind to TfR1 with a high affinity(Kd= 3.56×10-10)and presented strong ability to suppress the growth of PV model cell lines such as HEL (EC50= 0.07 nM). PPMX-T003 induced TfR1 internalization but blocked the recycling of the receptor presumably because it was strongly bound, even in the endosome, which switched the fate of TfR1 from recycling to degradation. Consequently, the PPMX-T003 treatment greatly reduced the intracellular iron concentration, along withcell cycle arrest in the G2/M phase in HEL cells. In contrast, PPMX-T003 failed to block cell proliferation in human umbilical vein endothelial cells that expressed a subtle level of TfR1. These findings suggest that PPMX-T003 is a potent inhibitor for cells whose growth is dependent on iron incorporation by TfR1. This led us to examine whether PPMX-T003 blocked the endogenous erythroid colony (EEC) formation in erythroid progenitor cells in PV. We observed that PPMX-T003, at a concentration of 40 ng/mL, exhibited complete blockade of EEC formation inJAK2 V617F-positive PV patients' cells. Although hydroxycarbamide (HU) and phlebotomy are well-established treatment strategies for the management of hematocrit in PV patients, there is a risk of secondary leukemia associated with HU treatment and side effects such as fatigue and restless leg syndrome due to phlebotomy-induced iron deficiency. Therefore, we suggest PPMX-T003, which may preferentially suppress erythrocytosis without changing the iron level in peripheral blood, as an alternative treatment for PV patients.

Disclosures

Ohira: PPMX: Current Employment, Current equity holder in private company. Nomura:PPMX: Current Employment. Matsuura:PPMX: Current Employment. Komatsu:Takeda Pharmaceutical Co., Ltd, Novartis Pharma KK, Shire Japan KK: Speakers Bureau; AbbVie: Other: member of safety assessment committee in M13-834 clinical trial.; PPMX: Consultancy, Research Funding; Meiji Seika Pharma Co., Ltd.: Patents & Royalties: PCT/JP2020/008434, Research Funding; Otsuka Pharmaceutical Co., Ltd., PharmaEssentia Japan KK, AbbVie GK, Celgene KK, Novartis Pharma KK, Shire Japan KK, Japan Tobacco Inc: Consultancy; Otsuka Pharmaceutical Co., Ltd., Shire Japan KK, Novartis Pharma KK, PharmaEssentia Japan KK, Fuso Pharmaceutical Industries, Ltd., Fujifilm Wako Pure Chemical Corporation, Chugai Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Takeda Pharmaceutica: Research Funding.
A Novel t(9;22;11) Translocation Variant Involving 11q23 in a Patient with Chronic Myeloid Leukemia
Authors: Yuehui Lin , Zheng Qinglong , Wu Tong , LIU DAN
Published: November 2020

Background:The progression of Philadelphia chromosome positive chronic myeloid leukemia (CML) is frequently accompanied by cytogenetic evolution, commonly unbalanced chromosomal changes. but balanced chromosomal translocations are very rare in CML, especially translocations involving the 11q23. The few reported cases with blast phase (BP) of CML carrying a 11q23 rearrangement results in insufficient responses to tyrosine kinase inhibitors (TKIs) and possess a poor prognosis.

Methods:Cytogenetic analysis , fluorescence in situ hybridization (FISH), RNA sequencing (RNA-seq), targeted genomic sequencing and multi-parametric flow cytometry analysis were performed to identify the chromosome translocations and pathogenic gene alterations in a 36-year-old female with myeloid BP of CML.

Results: In BP, the bone marrow (BM) aspiration showed 61% myeloid blasts; Multi-parametric flow cytometry analysis revealed the abnormal myeloid blasts expression of the following antigens: CD117, CD13, CD33, CD38, partially expressed CD15, CD64. Chromosome analysis revealed a t(11;22)(q23;q11) translocation in addition to the t(9;22)(q34;q11). Fluorescence in situ hybridization (FISH) test confirmed that the t(11;22)(q23;q11) involved the mixed lineage leukemia(MLL) gene on 11q23 and RNA sequence revealed MLL-SEPT5 and BCR/ABL1(p210) fusion transcripts positive. Mutations on 339 commonly mutated genes in hematologic malignancies were analyzed by targeted next-generation sequencing showed ASXL1 p.G949Vfs*2 mutation.

The patient failed to respond to both imatinib and dasatinib despite the absence of resistance-associated mutations in the BCR/ABL1 gene and she had a myeloid blast crisis at 19 months after initiation of first- and second-generation TKI treatment. After BP, she received ponatinb, a third-generation TKI with chemotherapy. Regretly she didn't achieve a complete remission(CR) and was in the process of salvage transplantation at present.

Conclusions:The presence of 11q23 rearrangements in BC of CML is rare and most likely accounts for the adverse clinical outcome. We first report a patient who diagnosed CML with t(11;22)(q23;q11) and MLL-SEPT5 fusion gene positive in BP of CML. The clinical course was aggressive, and therapy was poorly tolerated.

Disclosures

No relevant conflicts of interest to declare.
Activating NOTCH1 Mutations Alter T-ALL Chemosensitivity By PREX2-AKT Pathway
Authors: Yu Hui , DU Yuxin , Xu Ji , Sun Zhenchang , Fu Xiaorui , Young Ken H. , Zhang Mingzhi
Published: November 2020

Mutations on NOTCH1 gene are the most commonly found mutations in T-cell acute lymphoblastic leukemia (T-ALL) and they are reported to be favorable indicators for T-ALL patients' prognosis. However, the effects of activating NOTCH1 mutations on T-ALL's chemosensitivity have not been studied. We reported that NOTCH1 inhibition by γ-secretase inhibitors or shRNA knockdown in MOLT-3 cells could reduce the T-ALL cells' sensitivity to chemotherapeutic drugs. However, this effect was absent in Jurkat and CUTLL cells. We further demonstrated that NOTCH1 inhibition could activate the PI3K-AKT pathway in a cell specific pattern similar as their effects on chemosensitivity. RNA-seq revealed that Phosphatidylinositol-3,4,5-Trisphosphate Dependent Rac Exchange Factor 2 (PREX2) is a target gene of NOTCH1 and may mediate the effects of activating NOTCH1 mutations on chemosensitivity. Consistently, we proved that overexpression of PREX2 could mimic the effects of NOTCH1 inhibition on chemosensitivity. Our study has highlighted the effects of activating NOTCH1 mutations on T-ALL's chemosensitivity by altering PREX2-AKT pathway, which may explain the favorable effects of NOTCH1 mutations on T-ALL patients' prognosis, as well as provided potential targets to alter T-ALL cells' chemosensitivity.

Disclosures

No relevant conflicts of interest to declare.
Anti CD19/22 Cocktail CAR T-Cell Therapy Can Improve the Outcomes of Patients with TP53-Mutated Relapsed/Refractory B-Cell Lymphoma
Authors: Wei JIA , Xiao Min , Mao Zekai , Wang Na , Li Chunrui , Cao Yang , Sun Weimin , Xiao Yi , Gu Chaojiang , Zhang Shangkun , Zhang Yicheng , Zhang Tongcun , Zhou Jianfeng , Huang Liang
Published: November 2020

Mutations of the Tp53 gene occur in a subset of patients with refractory/relapse B-cell lymphoma and confer an exceedingly adverse prognosis. Chimeric antigen receptor-modified (CAR) T-cell immunotherapy represents a novel promising treatment and has achieved impressive anti-tumor responses in patients with refractory or relapsed B cell malignancies. Whether CAR T cell therapy can improve the outcomes of refractory/relapse B-cell lymphoma with Tp53 mutations or high-risk functional classification of Tp53 mutations has not been fully investigated yet.

In phase 2 trial, we evaluated the anti-CAR 19/22 cocktail therapy in patients with relapsed or refractory B-cell lymphoma with Tp53 mutations. The primary endpoint was the percentage of patients with an objective response (complete and partial response). The high-risk Tp53 mutations were defined as missense mutations, 'disruptive' mutations and the 'high-risk group of evolutionary action (EAp53) score. The outcome impact of CAR T cell therapy on high-risk Tp53 mutations were also assessed.

Between September 2016 to January 2020, a total of 30 patients [median age, 44.0 years (range: 28.0 to 61.0)] were enrolled. The primary efficacy analysis showed that 76.7 % (95%CI: 61% to 93%) patients have best objective response(Figure 1A), while 60.0 % (95%CI: 41% to 79%) patients at month three maintained the objective response(Figure 1B). At a median follow-up of 7.95 months (range: 1.13 to 42.76), the 12-months progression-free survival and overall survival rate were 43.33% and 45.33% respectively(Figure 1C-D). The therapy led to similar serious and life-threatening toxic effects with those reported with other CAR T-cell therapies. For all 30 enrolled patients, total 32 Tp53 Mutations were detected in this cohort including 23 missense, 4 splice site mutations, 4 insertions, 2 deletions and 1 nonsense mutation. Two Tp53 mutations were found in 2 patients. 21 patients were assessed with Tp53 FISH and 14 patients (66.66%) had 17p-. There is no difference on ORR,OS and PFS in patients based on three different Tp53-specific scoring systems. The ORR at three months predicts the long-term survival of this group of patients (Figure 1E-F).

CAR19/22 T-cell cocktail therapy can improve the outcome of r/r B-cell lymphoma with high-risk of functional classification of Tp53 mutation. The patients who achieved overall response at month three can greatly benefit from CAR19/22 T-cell cocktail therapy with long-term favorable outcome.

Trial registration: ChiCTR-OPN-16008526.

Key words: B cell lymphoma; CAR T-cell therapy; Tp53 mutation; outcome

Figure 1

Disclosures

No relevant conflicts of interest to declare.
The Cytogenetic Aberrations of Lymphoplasmacytic Lymphoma/ Waldenström's Macroglobulinemia in China
Authors: Wenjie Xiong , Yi Shuhua , Liu Huimin , Wang Tingyu , Liu Wei , Zou Dehui , Qiu Lugui
Published: November 2020

Object:Lymphoplasmacytic lymphoma/ Waldenström's macroglobulinemia (LPL/WM) is a rare B-cell proliferative malignancy characterized by immunoglobulin M monoclonal gammopathy and /or bone marrow infiltration by lymphoplasmacytic cells. Recently, there are no large-scale studies to detect the clinical features, genetic background in patients with LPL/WM in China even Asia. The aim of this study is to explore the clinical and genetic differences, observe the impact of cytogenetic abnormalities on survival and prognosis in Chinese patients.

Methods:305 patients with LPL/WM admitted in our hospital from June 1998 to December 2018 were retrospectively analyzed. Baseline characteristics, laboratory detection and therapy were compared using Chi-square test and Kaplan-Meier methodology was undertaken for survival analysis.

Results:The median age of 305 patients in the study was 62 years (range 32-87) and male to female ratio was 2.45:1(216/89). The most common symptoms at diagnosis were fatigue (64.9%) and bleeding (14.1%).The patients in low, intermediate and high risk group of the international prognostic scoring system for WM (ISSWM) was 17.6%, 36.5% and 45.9%,respectively.The median hemoglobin was 8.5g/dl(range 2.4-18.7). About 41.3% patients had IgM higher than 4000mg/dl. The cytogenetic abnormalities were uncommon in LPL/WM patients. The chromosomal karyotypes were successful in 194 cases, of which 20 patients showed clonal abnormalities. 12 patients had two or more chromosome abnormalities. The most common chromosome abnormalities were deletion 6/6q(25%) and Y chromosome loss (20%). The median progression-free survival (PFS) and overall survival (OS) in patients with normal and abnormal chromosome karyotypes were similar. 194 patients had FISH detection, of which 33 patients (17%) had FISH abnormalities, 9 (4.6%) patients had two kind of abnormalities. The patients with more than one cytogenetic abnormality had lower survival than just had one abnormality or normal patients. The most common aberrations were 6q deletion and trisomy 12. 17p deletion was detected in 11 patients (11/184,6.0%) and it was a poor prognostic marker of PFS and OS. Single 13q14 deletion had not impact on PFS or OS, but when companied with other cytogenetic aberration, it could worsen the PFS and OS . Besides related to survival, cytogenetic abnormal was also related to the clinical features.

Conclusions:Despite the low incidence and abnormal cytogenetic detection rate of LPL/WM, the clinical and genetic features are unique in Chinese patients. The cytogenetic aberrations play an important role in predicting prognosis.

Disclosures

No relevant conflicts of interest to declare.
Consecutive Hypoalbuminemia Predicts Inferior Outcome in Patients with Diffuse Large B-Cell Lymphoma
Authors: Wei Xiaolei , Zheng Jingxia , Zhang Zewen , Liu Qiongzhi , Zhan Minglang , Huang Weimin , Liu Jianbo , Wei Qi , Wei Yongqiang , Feng Ru
Published: November 2020

Albumin at diagnosis can be used to predict survival in diffuse large B-cell lymphoma (DLBCL). But whether consecutive albuminemia, which seen in DLBCL patients from time to time, could be more accuracy to predict outcome remains unknown.

We retrospectively analyzed 574 de novo DLBCL patients treated with R-CHOP from our and other 2 centers. All the patients were divided into a training cohort (n=278) and a validation cohort (n=296) depending on the source of the patients. Overall survival (OS) and progression-free survival (PFS) were analyzed by the method of Kaplan-Meier and the differences between groups were compared by the log-rank test. Multivariable analysis was conducted by Cox proportional hazard regression model.

The cutoff value of albumin for survival analysis was 39.2g/L with an area under the curve value of 0.589±0.034 (p=0.009) by receiver operating characteristic curve. In the training cohort, 163(58.6%) patients had low serum albumin at diagnosis and 80 of them were present with consecutive hypoalbuminemia. Patients with consecutive hypoalbuminemia showed inferior OS and PFS (p=0.010 andp=0.079, respectively). Similar survival differences were also observed in the independent validation cohort (p=0.006 andp=0.030, respectively). Multivariate analysis revealed that consecutive hypoalbuminemia was an independent prognostic factor OS (relative ratio [RR], 0.499; 95% confidence interval [CI], 0.317-0.786,p=0.003) and PFS (RR, 0.576; 95% CI, 0.413-0.804,p=0.001) in all entire DLBCL patients independent of IPI.

In conclusion, our study suggests that consecutive hypoalbuminemia is a simple and effective prognostic factor in DLBCL patients, allowing the identification of an inferior outcome subgroup in DLBCL patients, which may need to closely follow up.

Disclosures

No relevant conflicts of interest to declare.
Rapid Turnaround ADAMTS13 Testing Is Likely to Improve Diagnosis of Thrombotic Thrombocytopenic Purpura and Avoid Unnecessary Plasma Exchange
Authors: McGinnis Eric , Martin Spencer D. , Smith Tyler W.
Published: November 2020

Introduction: Thrombotic thrombocytopenic purpura (TTP) is a life-threatening thrombotic microangiopathy (TMA) resulting from severe ADAMTS13 deficiency, which is generally treated with therapeutic plasma exchange (PLEX). Although ADAMTS13 activity is often assayed to differentiate TTP from TMAs not requiring PLEX, technical and logistical constraints often limit rapid turnaround of results, with PLEX initiated based on clinical suspicion of TTP while awaiting ADAMTS13 activity results. We estimated the potential reduction in plasma product use if rapid turnaround ADAMTS13 activity testing were available in our centre.

Methods: We reviewed medical records for all Vancouver General Hospital patients with ADAMTS13 activity testing since assay implementation. Patients receiving PLEX but ultimately diagnosed with a disease not requiring PLEX were identified as "potentially avoidable PLEX" (paPLEX), and their plasma product exposures and related blood product costs were estimated. Laboratory results, ADAMTS13 activity, and PLASMIC scores (a validated clinical tool for TTP diagnosis) of this group were compared to those of newly diagnosed TTP patients (N=35).

Results: We identified 16 paPLEX patients, including TMAs secondary to malignant hypertension, infection, hemolytic uremic syndrome, illicit drug use, autoimmune renal disease, and malignancy (Table 1). These patients underwent 104 total PLEX cycles (3-12 per patient, median 6), involving 1,428 plasma units (28-199 per patient, median 71.5) and estimated product-associated costs of $187,759 CAD ($140,889 USD). Median platelet counts were significantly lower in TTP than the paPLEX group (7x109/L versus 38x109/L), as was serum creatinine (98µmol/L versus 224µmol/L). PLASMIC scores indicating low or intermediate likelihood of TTP were observed in 63% of patients receiving paPLEX and 17% of patients with TTP. All patients with TTP had ADAMTS13 activity < 10%, while all patients receiving paPLEX had ADAMTS13 activity ≥ 30%.

Conclusions: Unnecessary PLEX carries significant patient blood product exposure risks and system costs that may be circumvented if TTP can be reliably distinguished from other TMAs at the time of initial presentation. In our cohort, ADAMTS13 activity results provided clear separation of these groups and improved upon TTP diagnosis by clinical judgement and PLASMIC scores. Rapid turnaround of ADAMTS13 activity testing results has the potential to reduce the unnecessary costs and blood product exposures resulting from PLEX administration to patients with non-TTP TMAs.

Figure 1

Disclosures

Smith: Alexion: Other: Participated in an advisory board without receiving financial compensation.
CD19/CD22 CAR-T Cell Cocktail Therapy Following Autologous Transplantation in Patients with Relapsed/Refractory B-Cell Lymphomas
Authors: Cao Yang , Xiao Yi , Wang Na , Wang Gaoxiang , Zhou Xiaoxi , Huang Liang , Chen Liting , Xiao Min , Zhang Tongcun , Zhang Yicheng , Zhou Jianfeng
Published: November 2020

High-dose chemotherapy followed by autologous stem cell transplantation (HDT-ASCT) is the standard of care for relapsed or refractory(R/R) B-cell lymphoma. Eligibility for HDT-ASCT is based upon the presence of chemo-sensitive disease to salvage therapy. Patients with a positive PET scan prior to ASCT have a substantially inferior suvival. Furthermore, the outcomes of patients who have progressive disease after salvage treatment is extremely dismal. We hypothesized that, following HSCT, the immunosuppressive microenvironment would be diminished as a result of myeloablative conditioning and that administering CAR-T cell therapy during the hematopoietic reconstitution could eradicate posttransplant residual disease, leading to lower relapse rates after HSCT. Based on our previous work that a dual-targeting CAR T cell cocktail of sequential cell infusions targeting CD19 and CD22 may help overcome antigen escape relapse in B-cell malignancies, we therefore conducted an open-label, single-center, and single-arm pilot study of a treatment comprising the sequential infusion of CD22 and CD19 CAR-T cells following autologous hematopoietic stem cell transplantation (HSCT). We aimed to evaluate the safety and efficacy of the treatment in R/R B cell lymphoma patients with residual or progression disease after salvage treatment. Between November 14, 2016 and August 15, 2019, 42 patients underwent BEAM conditioned HDT-ASCT and followed by CAR 19 /22 T cells infusion. The overall response rate was 90.5% (95% CI, 77.4-97.3). At a median follow-up of 13.8 months, the median progression-free survival (PFS) and overall survival (OS) were not reached. The 12-month PFS rate was 85.7 % (95% CI, 70.9 to 93.3). Grade 3 cytokine release syndrome (CRS) occurred in only 2 (5%) patients. 21% patients experienced any grade of neurotoxicity, 5% was severe Grade 3. All CRS and neurotoxicity were reversible. Beyond its extremely high complete response rates, the durability of remission and low toxicity of the autologous HSCT plus CD19/CD22 CAR-T cell cocktail therapy examined in this study emphasize strong potential of such myeloablative conditioning and dual-targeting approaches for R/R B-cell lymphomas.

Disclosures

No relevant conflicts of interest to declare.
Iron Trafficking through Macrophages Regulates Signaling Pathways in Myeloma
Authors: Tan Xuelian , Ashby Timothy Cody , Zhu Yuqi , Li Can , Shen Xuxing , Brockman Qierra , Gai Dongzheng , Johnson Sarah K , Yong Donghoon , Schinke Carolina D. , Zangari Maurizio , van Rhee Frits , Shaughnessy John , Huang Dingming , Frech Ivana , Tricot Guido J. , Zhan Fenghuang
Published: November 2020

Background

Iron is an essential element for cell growth, including cancer cells, and is present in the microenvironment. We have shown that multiple myeloma (MM) cells have abnormal iron metabolism and harbor increased intracellular iron. However, the mechanism by which MM cells retain iron has remained largely elusive.

Methods

Expression and clinical relevance of the transferrin receptor in MM samples were analyzed in publicly available microarray and RNA-sequencing databases. Macrophages were isolated from C57BL/6J mice and were induced to specific subtypes by cytokines or culturing with MM cells. The 5TGM1-KaLwRij MM mice were used to confirm whether MM cells induce macrophage polarization in vivo. Specific subtypes of macrophage and transferrin receptor expression in MM cells were assessed by flow cytometry. Expression of ferroportin (FPN1) and ferritin in MM cells and/or macrophages were analyzed by Western blots. Single-cell RNA-sequencing (scRNA-seq), RNA-seq, and gene expression profiles (GEPs) were employed to identify ferroportin-signaling pathways in both tumor cells and macrophages of primary human MM samples.

Results

MM cells induced polarization with a significant increase of CD38+CD206- M1 macrophages both in vitro and in vivo. We also confirmed that the tumor associated macrophages (TAMs) were increased in the 5TGM1-KaLwRij MM mice. MM cells upregulated ferroportin expression in macrophages to provide iron to MM cells in co-culture studies and in vivo models. The transferrin receptor antibody treatment prevented MM cells from taking up iron from macrophages. scRNA-seq identified a subset of FPN1+ TAMs in human bone marrow aspirates, which are assumed to provide iron to MM cells. Using RNA-seq and GEPs analyses in primary human samples, multiple signaling pathways were differentially modulated in FPN1+ versus FPN1- TAMs, including those related to inflammation and apoptosis

Conclusions

Increased expression of the transferrin receptor in MM cells strongly suggests that tumor cells take up iron from its environment. MM cells promote intracellular iron mobilization in macrophages, which provide iron to MM cells in a transferrin-dependent manner. Blockade of iron trafficking between MM cells and macrophages might be a promising approach to MM therapy.

Disclosures

van Rhee: EUSA: Consultancy; CDCN: Consultancy; Karyopharm: Consultancy; Adaptive Biotech: Consultancy; Takeda: Consultancy.
The Role of Alpha 2 Macroglobulin in Chronic Activation of the Complement System in Patients with Chronic Lymphocytic Leukemia
Authors: Naseraldeen Naseba , Michelis Regina , Barhoum Masad , Chezar Judith , Tadmor Tamar , Aviv Ariel , Shvidel Lev , Litmanovich Adi , Yuklea Mona , Shehadeh Mona , Shaoul Ety , Braester Andrei
Published: November 2020

The immunotherapy treatments offered for Chronic lymphocytic leukemia (CLL) activate various cellular and biochemical mechanisms, including the complement system. Recently it was shown that the classical complement pathway (CP) in CLL patients is persistently activated at a low level through (Michelis et al. PlosOne, 2019), and that the mechanism of chronic CP activation involves the formation of IgG-hexamers (Michelis et al. PlosOne, 2020). According to recent studies, formation of ordered IgG-hexamers occurs on cell surfaces via specific interactions between Fc regions of the IgG monomers, which occur only after antigen binding. The study investigated the formation of IgG-hexamers in CLL patients and normal controls (NC), their ability to activate complement, their incidence as cell-free and cell-bound forms and the identity of the antigen causing their formation.

Sera from 30 patients and NC were used for separation of IgG-hexamers. The obtained IgG-hexamers were measure and used for assessment of CP activity. For evaluation of the presence of IgG-hexamers on blood cells, whole blood samples were stained and assessed by flow cytometry.

Serum levels of IgG-hexamers were higher in patients compared to NC. Also, they activated the complement system to a higher extent in patients than in NC. Alpha 2 macroglobulin (A2M) was recognized as the antigen causing the hexamerization of IgG, and was found to be part of the hexamer structure by mass spectrometry, Western blot and flow cytometry analysis. The presence of A2M-IgG-hexamers on B-cells suggests that it may be formed on B cells surface and then be detached to become cell-free. Alternatively, it may form in the plasma and then attach to the surface of the cells. The exact course of A2M-IgG-hexamers formation in CLL should be further studied. The results in this study may be useful for improvement of current immunotherapy regimens.

Disclosures

Tadmor: Neopharm: Consultancy, Speakers Bureau; AbbVie: Consultancy, Speakers Bureau; Janssen: Consultancy, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau; Medison: Consultancy, Speakers Bureau; 6. Novartis Israel Ltd., a company wholly owned by Novartis Pharma AG: Consultancy, Speakers Bureau. Aviv:Abbvie: Honoraria.
IDH Mutations Identify a Subgroup of NPM1 Patients with a More Favorable Prognosis. a Retrospective Multicenter Study of the Filo Group
Authors: Garciaz Sylvain , Hospital Marie Anne , Saillard Colombe , Hicheri Yosr , D'Incan Evelyne , Rey Jerome , Lacombe Catherine , Pigneux Arnaud , Béné Marie-Christine , Recher Christian , Mozziconacci Marie-Joelle , Vey Norbert
Published: November 2020

IDH mutations are strongly enriched in cytogenetically normal AML harboring NPM1 mutation (CN-NPM1mut-AML). The impact of these mutations on response to treatment is still a matter of debate. In the ELN 2017 classification, NPM1mut/FLT3-ITD allelic ratio >0.5 (FLT3-high) are considered intermediate-risk AML, whereas NPM1mut/ FLT3-ITD neg or <0.5 (FLT3-low) are low-risk. We aimed to evaluate the impact of IDH mutation in CN-NPM1mut-AML patients (pts) treated intensively. For this purpose, we retrospectively analyzed 177 CN-NPM1-AML pts from the Paoli-Calmettes Institute and from the French Innovative leukemia organization (FILO) databases who had received conventional intensive chemotherapy according to the FILO protocols (anthracycline-cytarabine based regimen for induction and High-intermediate dose cytarabine (HIDAC) for consolidation. Forty-seven (26%) AML pts had an IDH mutation -18 IDH1-R132 (10%), 27 IDH2-R140 (15%) and 2 IDH2-R172 (1%) - while 130 AML pts were IDHwt. Pts characteristics are presented in the Table.The complete response rate after one or two courses of chemotherapy (CR1) was 100% and 90% (p-value=.03) in the IDHmut and IDHwt groups, respectively. For pts in CR1, NPM1 molecular residual disease after the first consolidation (MRD2) was negative (>4 Log reduction) in 86% vs 53% of pts (p-value=.04). Nine (19%) and 24 (18%) pts received an allogeneic transplantation in CR1. The median time between CR1 and relapse was 11 months and 8 months, in IDHmut and IDHwt pts, respectively (p-value=.008). Day-100 non-relapse mortality was 8% and 12% respectively (p-value=ns). Median follow-up is 45 months (range, 2.4-115). Median EFS and OS are 21 months vs 12 months (p-value=.01) and 112 vs 23 months (p-value=.02), in the IDHmut vs IDHwt groups respectively (Figure). No survival differences were observed between IDH1mut and IDH2mut AML patients. Multivariate analyses with age>65, FLT3-high and IDHmut as covariates showed that IDHmut was independently associated with a higher EFS (HR=1.7, ranges 1.1-2.6) and OS (HR=1.7, ranges 1.1-2.7). Our results suggest that IDHmut is associated with a better response and a good disease control with high-dose chemotherapy. Nevertheless, some relapses still occur justifying the use of an IDH inhibitor combined with first-line chemotherapy or in a post-remission maintenance setting.

Figure

Disclosures

No relevant conflicts of interest to declare.
Adenylate Kinase 2 Is a Selective Dependency in NSD2-High Multiple Myeloma
Authors: Sobh Amin , Kaestner Charlotte , Li Jianping , Riva Alberto , Bennett Richard Lynn , Licht Jonathan D.
Published: November 2020

Background: Multiple myeloma (MM)-associated t (4;14) chromosomal translocation leads to overexpression of NSD2, the histone H3 lysine 36 specific methyltransferase. t(4;14) MM patients have a high risk of relapse and NSD2 overexpression drives an oncogenic epigenetic and transcriptional program promoting clonogenicity, proliferation, altered adhesion and chemoresistance in MM cells. The lack of a specific and potent NSD2 inhibitors mandates finding alternative strategies for treating NSD2-high MM.

Aim: This study aims to test the hypothesis that NSD2 overexpression in MM cells generates cellular vulnerabilities that can be therapeutically exploited for treatment of t (4;14) MM.

Methods: We conducted a genome wide CRISPR-based loss-of-function genetic screen using the human Brunello library in isogenic NSD2-high (NTKO) and NSD2-low (TKO) KMS-11 derived MM cells to define genes whose loss is selectively detrimental to cells with NSD2 overexpression. The cellular dependency of each identified candidate was then investigated across hundreds of human cell lines using the Cancer Dependency Map portal (www.Depmap.org). Candidate genes were validated using CRISPR-Cas9 gene knockout and shRNA knockdown of individual target genes followed by in vitro competitive growth assays and cell viability assays.

Results: Our study revealed multiple candidate genes with increased dependency in NSD2-high cells including the adenine nucleotide regulator Adenylate Kinase 2 (AK2). AK2 catalyzes the reversible conversion of ADP to AMP and ATP and can thus modulates energy balance within the cell. Dependency map analysis showed that AK2 is not a commonly essential gene. The top enriched lineages with AK2 dependency included MM with notable representation of t(4;14)-positive MM cell lines. The increased dependency of NTKO and other t (4;14) MM cells on AK2 was confirmed by in vitro competition assays. Disruption of AK2 in TKO cells had a minimal effect on cellular fitness but the dependency on AK2 was restored upon engineered overexpression of NSD2 in these cells. In addition, NSD2-high cells displayed higher sensitivity to the proteasome inhibitor bortezomib than NSD2-low cells suggesting elevated levels of endoplasmic reticulum (ER) stress in cells overexpressing NSD2. Elevated ER stress necessitates increased levels of ATP to refold proteins and could underlie the increased dependency of NSD2-high cells on AK2. Notably, suppression of AK2 increased bortezomib sensitivity in t (4;14) MM cell lines.

Conclusions: Our findings indicate that NSD2 high t(4;14) MM may have a vulnerability due to increased proteostatic stress. Accordingly, AK2 inhibition could be used in combination with proteasome inhibitors to treat MM patients with t (4;14) translocations by inducing the accumulation of lethal levels of unfolded proteins.

Disclosures

No relevant conflicts of interest to declare.
Incidence and Outcomes of Heparin-Induced Thrombocytopenia Associated with a Heparin Shortage at a Large Academic Medical Center
Authors: Seto Stephanie L. , Barra Megan E. , Roberts Russel J. , Rosovsky Rachel P.
Published: November 2020

Heparin-induced thrombocytopenia (HIT) is a life-threatening complication associated with significant morbidity and mortality in hospitalized patients. The 2019 African swine fever outbreak in China resulted in a critical national shortage of porcine-derived heparin products in the United States. As a result, our institution implemented a multitude of mitigation strategies to reduce heparin utilization by >80% and optimize safe and effective alternative therapies. The aim of this study was to determine whether this change in clinical practice impacted the incidence of HIT and associated outcomes.

A single-center, retrospective cohort study was performed on patients admitted to Massachusetts General Hospital who were ≥ 18 years of age and had a positive platelet factor 4 (PF4) drawn between February 2019 and January 2020. Patient demographics, comorbidities, baseline labs, serotonin release assay results, timing and magnitude of platelet count fall, and characteristics of heparinoid and non-heparinoid anticoagulant use were collected from the medical record. Thrombotic and hemorrhagic outcomes were characterized.

Seventy-five patients were included in the final analysis, of which 56 (75%) were critically-ill. Baseline characteristics were similar between groups including median age 66.8 years, 49% male, 80% Caucasian. Forty-four (59%) patients underwent surgery, 23 (31%) required continuous renal replacement therapy, and 13 (17%) underwent extracorporeal membrane oxygenation. Incidence of HIT with any exposure to heparinoid product was 0.3% and 0.15% (p=0.002) in the pre-shortage and shortage periods, respectively. In those who received therapeutic dose unfractionated heparin, incidence of HIT was 1.26% and 1.05% (p=0.63) in the pre-shortage and shortage groups, respectively. Thrombotic complications were observed in 19 (41%) patients in the pre-shortage group and 11 (38%) in the post-shortage group (p=0.77). Bleeding events were observed in 8 (17%) and 5 (17%) (p=0.99).

We observed a lower incidence of HIT resulting from our institution's efforts to conserve unfractionated heparin supply and utilize alternative anticoagulants during a critical national drug shortage. There were no significant differences in associated thrombotic and bleeding events.

Disclosures

Rosovsky: Bristol-Myers Squibb, Dova, Janssen, Portola: Consultancy; Bristol-Myers Squibb, Janssen: Research Funding.
Recombinant Human Thrombopoietin in the Treatment of Chemotherapy-Induced Thrombocytopenia in Acute Myeloid Leukemia
Authors: Can Can , Ding Lu , Tan Yuxin , Wu Balu , Zhang Dongdong , Chen Yanling , Luo Ziyi , Liu Xiaoyan , Liu Li , Shen Hui , Zhou Fuling
Published: November 2020

Objective: To investigate the efficacy and safety of recombinant human thrombopoietin(rhTPO) in the treatment of thrombocytopenia after chemotherapy for acute myeloid leukemia in vivo and in vitro.

Methods: A retrospective collection of 95 patients with acute myeloid leukemia who suffered thrombocytopenia after chemotherapy in Zhongnan hospital from July 2014 to April 2019 were collected. The patients were divided into two groups according to different medications, one group was treated with reorganized human thrombopoietin, the other one with recombinant IL-11, the platelet counts between two groups was analyzed and the platelet recovery under different ages, medical conditions and other factors were analyzed; mice patient derived xenograft model of acute myeloid leukemia with thrombocytopenia after chemotherapy was established to verify the efficacy and the mechanism of rhTPO.

Results: Since the platelet counts began to recover, the platelet increase in rhTPO group was significantly greater than that in IL-11 group, the platelet count is significantly higher than IL-11 group at Day42 (P<0.05). Age and whether it is chemotherapy-naive patient has no significant effect on the function of rhTPO in restoring platelet counts. It is verified that rhTPO can increase platelet counts, but has no significant effect on leukocytes, erythrocytes. The injection of rhTPO leads to more synthesis of TPO in liver by activating the JAK2-STAT3 pathway.

Conclusion: Our work has paved the way for further study on the clinical application of rhTPO in managing thrombocytopenia for AML after chemotherapy.

Disclosures

No relevant conflicts of interest to declare.
Binding of FANCD2 to SRSF1 Splicing Factor Prevents Genomic Instability through R Loop Regulation
Authors: Olazabal-Herrero Anne , Green Allison M. , Chen Xiaoyong , Sung Patrick , Pillai Manoj M , Kupfer Gary M.
Published: November 2020

BACKGROUND. Fanconi Anemia (FA) is a rare disease characterized by congenital abnormalities, bone marrow failure and cancer susceptibility. The pivotal role of the FA pathway is DNA interstrand crosslink (ICL) repair, where FANCD2-FANCI complex monoubiquitination is the key regulatory step. Recent studies have linked defects in the FA pathway to R-loop metabolism and implicated R-loops as an endogenous source of genomic instability that contributes to the FA phenotype, although the mechanism remains elusive. Splicing factors, such as SRSF1 (ASF/SF2), have also been linked to R loops, cancer, and myelodysplastic syndrome (MDS), so we hypothesized a link between SRSF1 and FA.

METHODS. Cells: FA-D2 mutant (PD20) and corrected (D2), HeLa. siRNA transfections were used to reduce SRSF1 and FANCD2 protein levels. Survival assays were performed to determine cell sensitivity to DNA damaging agents. In vitro immunoprecipitation was performed using SRSF1 purified from bacteria and FANCD2-FANCI purified from insect cells. R-loops were studied using the Damage At RNA Transcription (DART) assay and S9.6 immunofluorescence.

RESULTS. In order to study the involvement of splicing factors in the FA pathway, we knocked down SRSF1 by using siRNA. SRSF1 knockdown lead to impaired FANCD2 monoubiquitination after DNA damage as well as diminished cell survival, thus creating a FA-like phenotype (A). In addition, immunoprecipitation in cells (B) and in vitro showed direct interaction between FANCD2 and SRSF1. We found that double FANCD2+SRSF1 knockdown led to increased R-loops compared to FANCD2/SRSF1 single knockdown visualized by S9.6 immunofluorescence. In addition, DART assay revealed colocalization of SRSF1 and FANCD2 with R-loops in highly transcribed genomic sites, known to be R-loop hotspots (C). Finally, we found that SRSF1 interacts with the mRNA export factor NFX1 in a FANCD2-dependent fashion (D) through RNA, suggesting that they could cooperate in the prevention of R-loop formation by exporting accumulated RNA in the nucleus.

CONCLUSION. Our findings suggest a novel non-canonical mechanism of FANCD2 via regulation of SRSF1 in R-loop prevention via facilitation of mRNA export, thus preventing genomic instability (E).

CLINICAL APPLICABILITY. These results will reveal a novel mechanism for a non-canonical role of the FA pathway in prevention of genomic instability and will shed light upon the most basic cellular processes of the FA pathway, which is germane for delineating more generalized mechanisms of oncogenesis and genome maintenance. As accumulation of R-loops is associated with FA, we propose that R-loops and R-loop processing proteins (such as SRSF1) offer new avenues as relevant therapeutic targets not only for FA but also for multiple cancers.

Disclosures

No relevant conflicts of interest to declare.