bluuebunny/crossref_metadata_2025 huggingface dataset:
Acquired Secondary Erythrocytosis in Long-Term Survivors of Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Kibirova Albina , Najeeb Sidra , Craig Michael
Published: October 2019

Background: Erythrocytosis is a condition of increased red blood cell mass, as reflected by laboratory findings of elevated hemoglobin (HGB) and hematocrit (HCT). Erythrocytosis is classified into primary (polycythemia) and secondary, depending on its etiology. Secondary polycythemia most commonly develops in the setting of chronic hypoxemia which triggers increased production of erythropoietin by the kidneys. We have observed several patients in our hematology clinic who were long-term survivors after allogeneic hematopoietic stem cell transplant (allo-HSCT), and subsequently developederythrocytosisduring their follow up without obvious secondary causes. On literature review we found two single-institutional studies with estimated incidence of this unexpected complication reported as 0.45 and 1.1% respectively. Majority of described patients in the literature were young males with aplastic anemia (AA).

Methods: To establish the incidence of post-transplant erythrocytosis in long ter m survivors of allo-HSCT, we conducted a retrospective single site chart review study using the WVU database . All patients above age of 18 at the time of transplantation regardless the initial diagnosis that lead to transplantation and type of allo-HSCT were included. Patients with disease relapse and follow up less than 12 months were excluded. We also excluded patients with underlying conditions that are commonly associated with secondary erythrocytosis. Following definition of polycythemia or erythrocytosis was used: a) HCT >48% in women or >49% in men and/or HGB >16.0 g/dL in women or >16.5 g/dL in men (as extrapolated from WVO 2016 PV criteria); b) HGB/HCT elevation consistent on at least two measurements 3 months apart.

Results: We identified 133 eligible patients for final analysis. Among them, 12 (9.0% ) developed erythrocytosis in a median time from initial allo-HSCT 44.5 months (11.8-60.1). Peripheral blood Jak2V617F mutation was tested in 8/12 patients - negative in all. Serum erythropoietin (EPO) level was tested for 9/12 patients and found to be not suppressed in all cases to the level of HGB/HCT, median 11.1 mU/ml (4.9-18.6). All patients were non-smokers and 11/12 patients had different degree of GVHD requiring immunosuppressive therapy. The highest levels of HGB/HCT were observed in two patients with aplastic anemia and one of them required periodic therapeutic phlebotomies. In rest of the patients erythrocytosis was relatively mild but persistent. Details on our findings are presented in a table below.

Conclusions: Acquired secondary erythrocytosis is not uncommon in long-term survivors of allo-HSCT, estimated at 9.0% in our patient cohort. The exact mechanisms of erythrocytosis in this setting are unknown but probably related to dysregulated EPO production and response.

Disclosures

Craig: Celgene: Research Funding; Novartis: Research Funding; Actinium Pharmaceuticals: Research Funding.
Longitudinal Flow Cytometry Identified "Minimal Residual Disease" (MRD) Evolution Patterns for Predicting the Prognosis of Patients with Transplant-Eligible Multiple Myeloma
Authors: Gu Jingli , Liu Junru , Chen Meilan , Huang Beihui , Li Juan
Published: October 2019

Background: Many questions about "minimal residual disease" (MRD) still need to be answered for multiple myeloma (MM). Methods: Flow MRD were monitored in 104 consecutive patients with MM after induction and at the 3rd, 6th, 9th, 12th, 18th, and 24th months post-transplant. Results: Four MRD evolution patterns were revealed: pattern 1 patients had persistent MRD-negative after post-induction with no progression; pattern 2 patients had MRD-positive status post-induction but became MRD negative within 24 months post-transplant; pattern 3 patients had MRD-negative post-induction but became MRD-positive within 24 months post-transplant; and pattern 4 patients had persistent MRD-positive after post-induction. Patients with MRD evolution pattern 1 had a better time to progression (TTP) than did the patients with the other evolution patterns (not reached vs. not reached, vs. 15.4 ± 2.4 months, vs. 16.9 ± 3.0 months; log-rank test, p=0.003, 0.000, 0.000). Patients with MRD pattern 1 had a significantly longer OS than did patients with pattern 3 (not reached vs. 35.2 ± 18.6 months; log-rank test, p=0.000) and pattern 4 (not reached vs. 23.8 ± 15.0 months, log-rank test, p=0.000), but had a similar OS as did pattern 2 patients (not reached vs. not reached; log-rank test, p=0.229). For progressing patients with MRD evolution pattern 2 or 3, the median interval of a sustained MRD-negative status was only 17 months and the median time from MRD reappearance to disease progression was only 4.6 months. Conclusions: (1) A more complete MRD evolution pattern was developed to predict the outcomes for patients with MM. (2) The optimal time of MRD assessment should include post-induction, 3rd month and 24th month post-transplant. (3) Regular MRD assessments will help detect relapse early. (4) A sustained negative MRD status should last for at least 24 months.

Disclosures

No relevant conflicts of interest to declare.
Mutational Phasing: Clinical Relevance in Tyrosine Kinase Domain Mutations Using Next Generation Sequencing in Chronic Myeloid Leukemia
Authors: Bhatwadekar Seema S. , Shah Parth
Published: October 2019

Background: Tyrosine kinase mutation analysis in BCR/ABL1 gene is important for management of patients with chronic myeloid leukemia. Sanger Sequencing has been the mainstay for testing with Next Generation Sequencing (NGS) now becoming the primary technology. In this study we show a comparison between NGS versus Sanger Seqencing based ABL kinase domain mutation analysis with a likely trend of clinical relevance based on a compound versus polyclonal state of mutational distribution which may also need to be considered for patient management and therapy.

Methodology:

A total of 213 Imatinib-resistant patients with CML for BCR-ABL1 mutation analysis were processed on both technologies.Initial blood counts were assessed and RNA was extractedfollowed by cDNA conversion. NGS libraries were prepared with 400bp multiplexed amplicons to allow optimal phasing.

Results:

179 samples were negative by both technologies. A total of only 20 samples were positive and concordant by both technologies(58.2%). Mutations in 14 other samples however were only detected in NGS(41.17%). In these 14 samples (41.17%), NGS was able to detect 23 mutations with mutation frequencies of 3-28%, which were missed by Sanger.

Conclusions:

Moreover 11/34 patients had 2 or >2 mutations. An inhouse script delineated mutations as compound or polyclonal from NGS data. 2/11 cases demonstrated compound mutations (Mutations in the same clone) while 7/11 cases were polyclonal per NGS. Sanger sequencing cannot differentiate between polyclonal and compound mutations. 2/11 cases appeared to have polyclonal and compound mutations. 4/11 patients presented in a blast crisis or accelerated phase CML. Interestingly, most of these patients hadat leasttwo mutations and were polyclonal(3/4).

Significantly previously archived samples patients with polyclonal mutations showed polyclonality at extremely low frequency percentages in initial samples.

None of the single mutation patients had presented in a blast crisis or an accelerated phase.

Disclosures

No relevant conflicts of interest to declare.
Oncogenic KRASG12D in the Hematopoietic System Causes NLRP3 Inflammasome Activation Leading to Myeloproliferative Syndrome
Authors: Hamarsheh Shaima'A , Erlacher Miriam , Osswald Lena , Gorka Oliver , Duyster Justus , Groß Olaf , Lengerke Claudia , Börries Melanie , Brummer Tilman , Zeiser Robert
Published: October 2019

Oncogenic Ras mutations occur frequently in myelodysplastic (MDS) and myeloproliferative syndromes (MPN). It was unclear if besides the direct transforming effect via constant RAS/MEK/ERK signaling, an inflammation related effect of KRAS contributes to the development of the disease.

To address this question we performed a microarray based analysis of bone marrow derived from Rosa26CreERT2; LSL-KrasG12D mice after induction of KrasG12D with tamoxifen. We found that the NLRP3 inflammasome and related genes were upregulated. In agreement with increased NLRP3 sensitivity, KrasG12D bone marrow derived dendritic cells (BMDCs) showed increased inflammasome activation upon stimulation with LPS and ATP as quantified by intracellular levels of IL-1β and caspase-1 p20 subunit.

To validate the functional role of the NLRP3 inflammasome for the MDS phenotype in vivo we generated Rosa26CreERT2; LSL-KrasG12D; NLRP3-/- mice. We transferred BM (CD45.2) from WT mice, Rosa26CreERT2; LSL-KrasG12D or Rosa26CreERT2; LSL-KrasG12D; NLRP3-/- mice into C57BL/6 WT recipients (CD45.1) after myeloablative conditioning and induced KrasG12D by tamoxifen treatment.

Rosa26CreERT2; LSL-KrasG12D BM recipient mice developed MPN stigmata including anemia, leukopenia and thrombocytopenia. The mice also presented weight loss, splenomegaly, and an increase in myeloid cells in peripheral blood, spleen and BM, as well as increased CD11b+ckit+ immature myeloid progenitors. In contrast, Rosa26CreERT2; LSL-KrasG12D; NLRP3-/- recipient mice developed no disease phenotype.

BMDCs isolated from Rosa26CreERT2; LSL-KrasG12D recipient mice exhibited strong NLRP3 inflammasome activation and high IL-1β levels which was not found in BMDCs of Rosa26CreERT2; LSL-KrasG12D; NLRP3-/- mice.

CD11b+ cells of a Juvenile myelomonocytic leukemia (JMML) patient with an activating KRAS mutation showed increased inflammasome activation as compared to a non-mutant patient, which is in agreement with our observations in mouse models.

Our findings support the concept that oncogenic KrasG12D does not only act via its oncogenic driver function, but also enhances activation of the NLRP3/IL-1β axis. This could lead to novel therapeutic approaches combining inhibition of oncogenic signaling and immune modulation via IL-1β blockade.

Disclosures

No relevant conflicts of interest to declare.
Temporal Trends in Early Mortality in Acute Promyelocytic Leukemia in the United States
Authors: Xavier Ana C. , Kutny Matthew A. , Jamy Omer , Costa Luciano J
Published: October 2019

Background: Survival of patients with acute promyelocytic leukemia (APL) has dramatically improved with the use of all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). Despite this, due to the complexity of initial management and the high risk of fatal thrombotic and hemorrhagic complications at presentation, early mortality (EM) remains the major contributor for treatment failure. It is less known whether advances in treatment, improvements in supportive measures, urgent access to specialized care and broad availability of ATRA and ATO have reduced EM in the last two decades.

Methods: We used data from the National Cancer Institute's Surveillance Epidemiology and End Results program (SEER-13) to determine the rates of EM (death within the first 30 days from diagnosis) and overall survival (OS) in patients with APL. Inclusion criteria was the diagnosis of APL as first malignant neoplasm among patients of all ages between 1992 and 2015. Follow up was updated to the end of 2015. Cases were grouped and analyzed according to age, children, adolescent and young adults (< 40 years) and older adults (≥ 40 years), race/ethnicity, gender, county-level income and educational achievement, and residency in rural or urban county. Trends in EM and OS were analyzed across consecutive 4-year eras.

Results: A total of 2,224 APL cases (895 <40 and 1,329 ≥ 40 years) were included in the analysis with median follow up of 41 months (range 0-287 months). Median age of patients was 45, and 1,090 (49%) were male. Most patients were White (1,228; 55.2%), 199 (9%) Black, 785 (35.3%) other ethnicity, and 12 (0.5%) unknown. Three-year OS for APL patients diagnosed in 1992-1995 was 49.2% ± 3.5% vs. 76.4 ± 2.1% for patients diagnosed in 2012-2015 (p < 0.001), Figure 1. Early mortality improved for patients < 40 years (27.4% in 1992-1995 vs. 5.4% in 2012-2015, p < 0.001), but not at the same extent for patients ≥ 40 years (35.2% in 1992-1995 vs. 22.2% in 2012-2015, p = 0.02), Figure 2. Improvements in EM are displayed in Table 1. Importantly, improvements in EM were not seen among patients of residents of rural counties.

Conclusion: These findings confirm consistent improvements in EM and OS for APL patients in the US and point to the challenge of further extending these improvements in EM rates to older patients and for those living in rural areas.

Disclosures

Costa: Celgene: Honoraria, Research Funding; Sanofi: Honoraria; Karyopharm: Research Funding; Janssen: Research Funding; BMS: Research Funding; Abbvie: Research Funding; Amgen: Honoraria, Research Funding.
Validation Study of Logistic Regression in Classifying Primary Vitreoretinal Lymphoma Vs. Uveitis By IL-6 and IL-10 Levels
Authors: Kuo David , Wei Maggie , Knickelbein Jared , Armbrust Karen , Yeung Ian , Chan Chi-Chao , Sen Nida
Published: October 2019

Background/Aim:

The aim of this study was to assess the utility of intraocular IL-10 and IL-6 analysis by logistic regression in classifying primary vitreoretinal lymphoma (PVRL) vs. uveitis using a logistic regression model trained on a single-center retrospective cohort as well as the previously published ISOLD score compared against the IL-10/IL-6 ratio.

Methods:

Patient diagnoses of PVRL vs. uveitis and associated aqueous and/or vitreous IL-6 and IL-10 levels were retrospectively collected. From this data, cytokine levels were compared between diagnoses with the Mann-Whitney U test and a logistic regression model was developed to classify PVRL vs. uveitis from aqueous and vitreous IL-6 and IL-10 by nested cross-validation. ROC curves were plotted and AUCs were calculated for the IL-10/IL-6 ratio, ISOLD score, and our logistic regression model. Optimal cut-offs for each classifier were determined by the maximal Youden index; and sensitivity, specificity, PPV, and NPV were determined for each cut-off.

Results:

79 lymphoma (10 aqueous, 69 vitreous) and 84 uveitis patients (19 aqueous, 65 vitreous) between 10/5/1999 and 9/16/2015 were included in the study. IL-6 was higher in uveitis vs. lymphoma patients while IL-10 was higher in lymphoma vs. uveitis patients (p <0.01 for all comparisons). For vitreous samples, our logistic regression model achieved an AUC of 98.3%, while ISOLD achieved an AUC of 97.8%, and the IL-10/IL-6 ratio achieved an AUC of 96.3%. The optimal cut-offs for our logistic regression model, ISOLD, and the IL-10/IL-6 ratio achieved sensitivity/specificity of 92.7%/100%, 94.2%/96.9%, 94.2%/95.3% respectively, corresponding to PPV/NPV of 100%/92.9%, 97%/94%, and 95.6%/93.9% respectively. For aqueous samples, all three classifiers achieved 100% AUC with 100% sensitivity/specificity. Odds ratios of PVRL vs. uveitis were 0.981 (aqueous) and 0.992 (vitreous) for IL-6 and 1.030 (aqueous) and 1.060 (vitreous) for IL-10 according to our logistic regression model.

Conclusion:

In this study, logistic regression, as demonstrated by our model and the ISOLD score, showed strong classification performance and generalizability with high sensitivity and specificity. These results, in addition to logistic regression's ability to further improve with more training data suggest a promising step forward in intraocular cytokine analysis for the early diagnosis of primary vitreoretinal lymphoma. Additional validation studies, especially with cohorts that have proven challenging for the IL-10/IL-6 ratio, would further elucidate the strengths and weakness of this approach.

Disclosures

No relevant conflicts of interest to declare.
F5 Sequence Variant Identified in 7 Patients Evaluated for Hypercoagulable State with a Factor V (FV) HR2 Assay
Authors: Leech Margaret M. , Bellissimo Daniel B. , Uhrmacher John A. , Gunawardena Sriya W. , Meyer Michael J. , Bontempo Franklin A.
Published: October 2019

A 15-year-old African-American male with a history of thrombosis was examined for the Factor V HR2 sequence variation (c.3980A>G, p.H1327R) using PCR followed by RsaI digestion. An anomalous restriction pattern was observed: one strand was uncut (wild type) and the other was cleaved once. However, the fragment sizes were 516bp and 312bp instead of the 447bp and 381bp expected from HR2. Sanger sequencing of the PCR product detected a sequence variant (c.3845A>G) in this patient that creates a RsaI site explaining the novel band pattern. The c.3845A>G variant is found within one of four 27bp repeats found in exon 13 of the F5 gene. The HR2 variant is also present in one of the repeats. In this patient's case, the cleavage occurred in an abnormal location (c.3845A>G instead of c.3980A>G), creating the novel banding pattern observed.

Six additional patients being tested for hypercoagulable states were evaluated with the FV HR2 assay and were found to have the same abnormal restriction pattern. The ethnicity of these other patients was not available to our laboratory. Sanger sequencing confirmed the presence of c.3845A>G sequence variant in each of these patients. Unlike the HR2 and FV Leiden variants, the c.3845A>G, p.H1282R variant (rs143333036) is most common in African populations (Minor Allele Frequency 0.012) but the clinical consequences of this variant are unclear. The variant lies within a 27bp repeat similar to the HR2 site and might cause it to be associated with a thrombotic risk similar to the HR2 variant. Further investigation will be required to determine whether the c.3845A>G variant is a population variant with no clinical consequences or whether it represents a genetic prothrombotic risk factor in certain patient populations. Nevertheless, in the laboratory the c.3845A>G, p.H1282R sequence variant may confound results when testing patients for the HR2 (rs1800595) variation.

Disclosures

No relevant conflicts of interest to declare.
Intensity and Duration of Anticoagulation in Non-Cirrhotic, Non-Malignant Portal Vein Thrombosis
Authors: Sukumar Senthil , Shen Yu-Min
Published: October 2019

Introduction:

Portal vein thrombosis (PVT) can result in significant morbidity and mortality. There are currently no clinical studies reviewing either the intensity or duration of anticoagulant therapy. There is a paucity of data to guide the overall management of this entity and current practice is extrapolated from studies on management of deep vein thrombosis of the lower extremities and pulmonary embolism.

Methods:

All available electronic medical records with a diagnosis of PVT were reviewed from 2005-2016 at hospitals affiliated with the University of Texas Southwestern Medical Center. Patients with cirrhosis, active malignancy, or procedure within the preceding 3 months were excluded. Eighty-four patients met inclusion criteria. The charts were screened for patient demographics, etiology of PVT, type of anticoagulation therapy, and duration and response to therapy.

Results:

Demographic, treatment and outcome data is summarized in table 1. Patient ages ranged 18-84 years; 49 male. Etiology identified include Infectious (18%), Locally Inflammatory (34%), Systemic Hypercoagulable State (24%), or Idiopathic (24%). The majority of patients were treated with Vitamin K Antagonist (61%) followed by no treatment (17%). Average duration of therapy was categorized by months: <3 (20%), 3-6 (15%), 6-12 (16%), or >12 (49%). Outcome of treatment included resolved (28%), chronic (34%), or not reported (39%). When outcomes were stratified by etiology, intensity (in those patients treated with VKA for whom INR data was available), or duration there was no apparent difference in rate of clot resolution compared with progression to chronic cavernous transformation (tables 2-4).

Conclusions:

This first retrospective review studying the efficacy of treatment in PVT reveals that a similar number of patients develop cavernous transformation (indicating chronic PVT) regardless of the etiology, intensity, or duration of treatment. This data suggests that treatment with anticoagulation may not be impactful in this select group of patients. However, it is still recommended to pursue treatment in those patients presenting with a symptomatic acute thrombus given risk of mortality associated with clot progression. Furthermore, no single modality of anticoagulation has shown to be superior in the presented outcome data. Larger prospective studies are needed to further clarify if anticoagulation is effective in this clinical entity.

Disclosures

No relevant conflicts of interest to declare.
Cumulative Incidence of Blood Stream Infections during Acute Myeloid Leukemia Treatment in the Netherlands: A 15-Year Overview
Authors: Schwartz Emily Risa , Klein Kim , de Haas Valerie , Lissenberg-Witte Birgit , Ball Lynn , Dors Natasja , Van den Heuvel-Eibrink Marry M , Knops Rutger , Tissing Wim , Versluys Birgitta A. , Zwaan Christian M. , van Litsenburg Raphaele R.L. , Kaspers Gertjan J.L.
Published: October 2019

Background/Objectives

Despite the use of antibiotic prophylaxis, bacterial infections remain the leading cause of treatment-related morbidity in children with acute myeloid leukemia (AML). The objectives of this study were to: 1) evaluate the cumulative incidence of blood stream infection (BSI) during treatment according to the last three consecutive protocols of the Dutch Childhood Oncology Group (DCOG); 2) determine the most common bacterial pathogens; and 3) determine risk factors.

Design/Methods

A retrospective chart review was performed on 246 patients (DCOG treatment protocols ANLL-97/AML-12 n=119, AML-15 n=60, DB-AML01 n=67) diagnosed with de novo AML (APL and Down syndrome excluded) between 1998-2013. All patients received prophylaxis with co-trimoxazole. From AML-15 onwards, patients also received prophylactic fluoroquinolones. BSIs were defined as a positive blood culture confirmed by the hospital's microbiologist. The cumulative incidence of BSIs was determined using competing risk analysis with death, switch to another protocol, or stem cell transplantation as competing events. Age, sex, BMI, white blood cell count (WBC) at diagnosis, cytogenetics, and treatment protocol were evaluated as risk factors.

Results

Median age was 6.4 years [range 17.4], 58% were male. The cumulative incidence of BSIs was 78.2%. The most common pathogens were Staphylococcus epidermidis (15.6%), Streptococcus mitis (14.9%) and other Staphylococci (11.6%). ANLL-97/AML-12 (78%) and AML-15 (72%) showed a lower BSI incidence than DB-AML01 (84%; hazard ratio(HR)s 0.69 [0.50-0.97], p=0.033 and 0.59 [0.40-0.89], p=0.012, respectively). WBC >20x109/l was associated with a lower risk of infections (HR 0.72 [0.54-0.96], p=0.026). The other risk factors were not statistically significant.

Conclusion

The most recent DCOG protocol was associated with the highest incidence of BSIs, probably as a result of the high intensity of this protocol. Furthermore, the use of prophylactic antibiotics with gram-negative coverage may have resulted in a relatively higher incidence in gram-positive BSI. The high incidence stresses the urge to improve anti-infective supportive care measures.

Disclosures

No relevant conflicts of interest to declare.
Donor-Derived Second Generation of CD19 CAR-T Cell Therapy for Relapsed B-Cell Acute Lymphoblastic Leukemia after Allogenic Stem Cell Transplantation
Authors: Zhang Rongli , He Yi , Yang Donglin , Wang Ying , Feng Sizhou , Wang Jianxiang , Han Ming-Zhe
Published: October 2019

Background: Patients with B cell acute lymphoblastic leukemia (B-ALL) relapsed after allogenic hematopoietic stem cell transplantation have poor prognosis. Donor lymphocyte infusion (DLI) have shown limited success in the setting of relapse by a mere increase in median survival by 6 months and a significant risk of acute and chronic graft-versus-host disease (GVHD) and additional risk of marrow aplasia. The second generation chimeric antigen receptor-T cell (CAR-T) for CD19 is a promising treatment for relapsed and refractory B-ALL, but the effectiveness and safety of donor-derived second generation of CD19 CAR-T cell infusion for relapsed B-ALL after allogenic stem cell transplantation have not been determined.

Methods: Between July 2017 and June 2018, 6 adult patients with B-ALL relapsed (2 patients were hematologic relapsed; 4 patients were minimal residual disease(MRD)-positive) after allogenic stem cell transplantation were enrolled, including 5 sibling-matched stem cell transplantation and 1 haploidentical transplantation . Donor's T cells were infected with lentivirus carrying CD19 CAR plasmid which containing CD19 scfv (HI-19 clone) and 4-1BB-CD3ζ signaling domains to generate CAR-T cells. Patients received FAC (fludarabine:25-30mg/m2/d*3, cyclophosphamide:350mg/m2/d*2, cytosine arabinoside:100mg/m2/d*4) pretreatment and then total (1.25-3.5)*106/kg donor-derived 4-1BB CAR-T cell were infused in consecutive 2 or 3 days .

Results: All the patients achieved MRD negative remission and complete donor chimerism. Three patients experienced grade 2 cytokine release syndrome (CRS) and received 6-8mg/kg interleukin-6 receptor blocker (tocilizumab) treatment ; the other 3 just experienced grade 1 CRS. None of these patients needed glucocorticoid treatment. No patients developed acute or chronic graft-versus-host disease (GVHD). Now all the 6 patients are alive and show complete donor chimerism with MRD negative remission. The median follow-up time are 243.5days.

Conclusion: Donor-derived second generation of CD19 CAR-T cell treatment for relapsed B-ALL after allogenic stem cell transplantation were effective and safe, which may be confirmed with more clinical studies.

Key words: donor-derived CD19 CAR-T cell therapy, allogenic stem cell transplantation, B cell acute lymphoblastic leukemia, relapse

Disclosures

No relevant conflicts of interest to declare.
Donor-Specific Isoagglutinin Clearance in ABO Mismatched Stem Cell Transplant Recipients: How Long Should It Normally Take?
Authors: Aneke John Chinawaeze , Sondi Nayana , Thyagu Santhosh , Cserti-Gazdewich Christine M. , Pendergrast Jacob
Published: October 2019

Introduction: In patients undergoing ABO-incompatible stem cell transplants, recipient isoagglutinins with specificity for donor antigens are expected to slowly become undetectable during the post-transplant period, presumably secondary to the loss of recipient plasma cells via either conditioning chemotherapy or graft-versus-host-disease. Conversely, the persistence or return of these anti-donor isoagglutinins may induce hemolytic anemia or pure red cell aplasia, and may even signify disease relapse. In order to define the expected time to clearance of these donor isoagglutinins at our institution, a retrospective study was performed

Methods: The transfusion records of all major-side or bidirectionally ABO-incompatible stem cell transplants performed at an academic cancer hospital between May 2009 and July 2018 were reviewed. Patients without detectable anti-donor isoagglutinins at time of transplant were excluded. Isoagglutinin clearance was defined as non-reactivity of patient plasma against donor-type reverse grouping cells occurring during the post-transplant period; relapse was defined as a 2+ or stronger reaction after one or more non-reactive results

Results: A total of 280 patients were included in analysis, of which 44 were native group A, 42 group B and 194 group O. Average age was 52 (±13) years and 130 (46%) were female. Compatibility samples were sent to the UHN blood bank during the post-transplant period for a median of 156 days (IQR 74-370 days). During this time period, donor-targeting/host-derived isoagglutinin clearance was documented in 192 (69%) patients at a median of 48 days (IQR 23-116.5) post-transplant. Of these, 52 (30%) experienced a subsequent return of reactivity at a median 98 days post-transplant (IQR 54-216). Amongst the 128 patients with at least 28 days of post-transplant monitoring and no return of previously cleared isoagglutinins, the median time to clearance was 60 days (IQR 26.5 - 127.5 days). There was no statistically significant difference in time to isoagglutinin clearance between those with and without a subsequent return of reactivity, and time to durable clearance did not correlate with patient age, sex or native ABO group.

Conclusions: Amongst patients undergoing major-side or bidirectionally ABO incompatible stem cell transplants, the majority will have cleared their donor-specific isoagglutinins within 120 days of stem cell infusion, although this does not guarantee against later return of reactivity. Thus, the persistence of donor-specific isoagglutinins beyond 120 days post-transplant, or the subsequent return of previously cleared isoagglutinins, may be considered abnormal results which warrant notification of the clinical team by the hospital transfusion service.

Disclosures

No relevant conflicts of interest to declare.
The Nek2 Genetic Knockout Mouse Model Provides Novel Treatment Strategies in Myeloma and Lymphoma
Authors: Can Li , Nadiminti Kalyan , Zhu Yuqi , Jethava Yogesh , Frech Ivana , Tricot Guido J. , Zhan Fenghuang
Published: October 2019

Background; Major progress in the treatment of B cell tumors has been made in the past decades. Nevertheless, relapses and refractoriness to currently available chemotherapy and even to high dose therapy with stem cell transplantation still cause significant mortality. NEK2, NEver in Mitosis Gene A (NIMA)-Related Kinase 2, is a serine/threonine kinase. High expression of NEK2 increases cell survival and drug resistance, resulting in poor clinical outcome in multiple cancers including multiple myeloma and lymphoma. In this study, we used genetic mouse models to evaluate whether NEK2 is a druggable target in the treatment of B cell tumors including myeloma and diffuse large B-cell lymphoma (DLBCL).

Materials and Methods: We have generated Nek2 knockout mice and crossed these with Eµ-Myc mice. RNA-sequencing was performed to determine signaling pathways related to Nek2 inhibition. Both NEK2 and USP7 (a protein interacting with NEK2) inhibitors were applied to treat myeloma and DLBCL in vitro and in vivo.

Results: Mouse studies showed that Nek2 played a critical role in B cell tumor development and progression. Specifically, in genetic Eμ-MYC transgenic mice, which spontaneously develop DLBCL and Burkitt lymphoma, knockout of Nek2 prevented B cell tumor development and significantly extended mouse survival. Further, immunohistochemistry analyses showed that Nek2 was highly detected in biopsies from aggressive Burkitt lymphoma patients. Our data also indicate that both NEK2 and USP7 inhibitors significantly inhibited myeloma cell and lymphoma cell growth in vitro and in vivo models and without apparent toxicity to normal tissues. Intriguingly, the combination of USP7 inhibitor P5091 with doxorubicin blocked B cell lymphoma development and extended lymphoma mouse survival.

Conclusions: Our studies demonstrate the importance of Nek2 function in tumorigenesis and progression in B cell lineage malignancies. Both NEK2 and USP7 inhibitors showed excellent efficacy in the treatment of myeloma and B-cell lymphoma.

Disclosures

No relevant conflicts of interest to declare.
Suppression of FVIII Antibody Production By FVIII BAR T-Cell in Vitro in the Presence of Inhibitors
Authors: Parvathaneni Kalpana , Zhang Ai-Hong , Scott David W.
Published: October 2019

To modulate B-cell responsiveness to FVIII, we previously generated cytotoxic cells expressing FVIII C2 or A2 immunodominant domains as chimeric receptors. We termed these antigen-expressing engineered T cells, "BARs", for B-cell Antibody Receptor. These CD8 T cells directly interact and kill FVIII-specific B cells and anti-FVIII hybridomas in prophylactic experiments in vitro and in vivo. It was not known whether these BAR CD8s could function or would be blocked in the presence of circulating antibodies to the expressed BAR domains. To test this, we cultured FVIII C2 or A2 BAR CD8 T cell with a mixture of monoclonal antibodies specific for these domains (up to 10 BU), and then added them to spleen cells from FVIII-immunized mice. These spleen cells were then re-stimulated with FVIII and the antibody response was determined after 5 days. Our results showed that these BAR CD8 T cells were not blocked in their ability to suppress the antibody response to FVIII under these conditions. Coupled with the observation that BAR-T cells can be stimulated to proliferate by anti-FVIII monoclonals, these results suggest that BAR cytotoxic activity may still be effective in the presences of inhibitors. (Supported by NIH grant R01 HL126727)

Disclosures

No relevant conflicts of interest to declare.
Clinical Outcomes Using Mycophenolate and Tacrolimus for Graft Versus Host Disease Prophylaxis in Patients Undergoing Allogenic Stem Cell Transplant: A Single Institution Experience
Authors: Hashmi Hamza , Bhandari Shruti , Wu Xiaoyong , Rai Shesh Nath , Tse William , Emmons Robert V.B.
Published: October 2019

Introduction: Graft versus host disease [GVHD] remains the major factor underlying transplant related morbidity and mortality among recipients of allogeneic stem cell transplant [allo-SCT]. The choice of GVHD prophylaxis is largely determined by HLA disparity between the donor and the recipient and the intensity of the conditioning regimen. Mycophenolate mofetil [MMF] plus calcineurin inhibitor [CNI] combination is mostly used in reduced intensity [RI] and non-myeloablative conditioning [NMAC] whereas methotrexate and CNI combination is preferred in myeloablative conditioning [MAC]. We present single institution outcomes in patients undergoing allo-SCT with MMF and CNI as the only combination for GVHD prophylaxis.

Methods: Data from all adult patients who underwent allo-SCT at our institution from 2007 to 2016 was collected from our Center for Blood and Marrow Transplant Research [CIBMTR] portal. Only patients who received both MMF and CNI as GVHD prophylaxis were included in the analysis.

Results: Total 141 patients [table 1] were included with the median age of 50 years. Most patients were female [52%] and white [85%]. Disease characteristics included AML 56.7%, ALL 14.2%, CML 7.7%, MDS/MPD 7.7%, HL/NHL 6.4% and others 7%. MAC was used in 75.2% whereas NMAC/RI conditioning was used in 24.8% of the patients. Peripheral blood was the predominant source of stem cell graft [81%]. Donor status included 30.5% HLA matched related, 63.8% HLA mismatched/matched unrelated and 4.9% HLA mismatch related. Incidence of acute GVHD at day 100 was 49.6% [grade I 16.5%; grade II-IV 33.1%], acute GVHD at day 365 was 59.6%, and chronic GVHD at day 365 was 48.1% [limited 13.5%; extensive 34.6%]. One year overall survival [OS] was 51% [95% CI 42-59%]. OS was significantly associated with race (p = 0.033) and conditioning regimen (p = 0.036), favoring better survival with white race and NMAC/RIC regimen.

Discussion: Based on historical cohort, combination of methotrexate and tacrolimus is associated with acute graft versus host disease in 89% [grade I 11%; grade II-IV 78%], chronic graft versus host disease in 94% [limited 32%; extensive 65%] and one year overall survival of 47%. Hence our single institutional analysis shows that the combination of mycophenolate and tacrolimus is superior to methotrexate and tacrolimus combination for prevention of acute and chronic graft versus host disease as well as 1-year overall survival. We suggest that well-designed randomized controlled clinical trials be conducted to verify these findings.

Disclosures

No relevant conflicts of interest to declare.
The Efficacy and Safety of a Catheter Removal Only Strategy for the Treatment of PICC Line Thrombosis Vs Standard of Care Anticoagulation: A Retrospective Review
Authors: Shatzel Joseph J. , Mart Dylan , Bien Jeffrey Y. , Maniar Ashray , Olson Sven R , Liem Timothy , Deloughery Thomas G.
Published: October 2019

Background: Peripherally-inserted central catheters (PICCs) are commonly used during hospitalization for ease of access with blood draws and fluid and medication administration. PICC use is frequently complicated by catheter-related thrombosis (CRT). Current guidelines recommend 3-6 months of anticoagulation for patients with CRT after catheter removal. This recommendation is based on extrapolation of data on lower extremity thrombosis, as data is lacking regarding the efficacy and safety of more specific management strategies in upper extremity cases. Retrospective data exists to suggest catheter removal without anticoagulation is a sufficient and reasonable treatment option, particularly for patients at risk for bleeding; no prospective randomized data have been reported in this population. We aimed to examine the incidence of progressive thrombosis and bleeding in a retrospective cohort of patients with PICCs.

Methods: We performed a retrospective analysis of hospitalized adult patients diagnosed with CRT at our center from 2012 to 2018. We determined rates of progressive thrombosis and bleeding in cohorts of patients who underwent catheter removal vs. those who had catheters removed and received anticoagulation.

Results: Among 83 total patients, 62 were treated with PICC removal alone, while 21 underwent PICC removal and received therapeutic anticoagulation. Patients treated with PICC removal alone were more likely to have hematologic malignancy, receive chemotherapy, develop thrombocytopenia, and have brachial vein thrombosis. No patients in the PICC removal plus anticoagulation arm developed progressive thrombosis, while 6.4% of patients treated with catheter removal alone developed a secondary VTE event, including one PE, three DVTs; this difference did not reach statistical significance. Five patients (8%) treated with catheter removal alone also developed progressive symptoms leading to initiation of anticoagulation. Major bleeding was significantly more common in the PICC removal + anticoagulation arm (28.5% vs 4.8% p=0.007).

Conclusions: Catheter-removal alone results in significantly reduced major bleeding compared with catheter-removal plus anticoagulation. In select patients, catheter removal alone may be considered as an option for CRT, though larger, prospective trials are needed to clarify whether this practice is effective at reducing rates of progressive thrombosis.

Disclosures

No relevant conflicts of interest to declare.
Heterogeneous Responses Among Ph+-B-ALLs to Imatinib with NRAS Mutant Being One Mechanism of Complete Resistance
Authors: An Xiaoyu , Liu Jinping , Wang Na , Wang Di , Huang Liang , Cai Jie , Zhou Demin , Li Henry
Published: October 2019

Background. Philadelphia Chromosome (Ph+) characterized by BCR-BAL fusion genes are one of the major leukemogenic drivers for a number of leukemia, including acute lymphoid leukemia (ALL) and chronic myeloid leukemia (CML). Experimental models are important to study disease mechanisms and assess pharmaceuticals, including drug resistances/mechanisms. Patient derived xenografts (PDXs) are stem cell driven diseases and considered to be the most closely resembling the original patients including leukemia [1], in terms of histo-/molecular pathology and pharmacology [2]. As far as we know, there has yet to be reported of PDXs of Ph+ leukemia, and thus such models could be particular useful for drug evaluation and study drug resistance.

Methods. Different B-lineage acute lymphoid leukemia (B-ALL) adult and pediatric patient bone marrow (BM) cells were engrafted into NOD/SCID mice or NSG-like mice, via IV injection (also intra-BM). The disease development is performed by monitoring peripheral blood for human CD45+ cells and cage-side clinical observations. At the terminal leukemia stage, the mice were gross-/histo-pathologically analyzed and immune-phenotyped for different affected tissues. The diseases are passed in mice by serially transplantations, and are by transcriptome sequenced-/pharmacologic-tested, enabling biomarker discovery.

Results. We achieved aggressive and serially transferrable full-blown ALL in mice for a number of patient samples that cause 100% mortality. The diseases of the established B-ALL-PDXs (patient-derived xenografts) start at BM followed by the quick expansion to peripherals, e.g. spleen and blood. The leukemic cells in mice have similar morphology, phenotypes and genotypes, including five ALLs with different forms of BCR-ABL fusion as seen in the original patients. The treatments of these five Ph+-ALLs with imatinib, a TKI targeting BCR-ABL fusion, caused heterogeneous remissions in peripheral blood, spleen and bone among the five PDXs, along with the correspondingly relief of the symptoms and extension of life. This is largely as anticipated and confirms that BCR-ABL fusion are likely key leukemogenic drivers to the majority of these PDXs. One of the five Ph+-ALLs, AL7267, displayed complete resistance to imatinib, which could potentially be explained by the identification of NRAS-G12S mutation that is absent in other 4 BCR-ABL bearing PDXs and likely responsible for AL7267's non-response to imatinib.

Conclusions. Our observations in the mouse trial of the five different Ph+ B-ALL-PDXs would suggest that individual patients of the same diseases could be greatly different in their response to TKIs targeting BCR-ABL, and NRAS mutation could be one of the important resistant mechanisms. These models could be useful tools to investigate drug resistance and the combination therapy to overcome the resistance.

Disclosures

No relevant conflicts of interest to declare.
DNA Damage in Haemopoeitic Stem Cells Impacts on Neutrophil and Platelet Engraftment Following Autologous Transplantation
Authors: Bai Lijun , Best Oliver Giles , Xia Wei , Peters Lyndsay , Wong Kelly , Ward Christopher , Greenwood Matthew
Published: October 2019

Background and Aim: Processing and cryopreservation is known to reduce the viability and absolute number of CD34+ haematopoetic stem cells (HSCs). DNA damage and subsequent apoptosis of stem cells may occur as a result of prior treatment regimens or from the accumulation of reactive oxygen species (ROS) during the freeze/thawing process. In our recent study we demonstrated that two distinct populations of HSCs are present in all cryopreserved patient samples, based on their ROS expression; these we defined as ROShigh and ROSlow. The significant correlation between the percentage of ROShigh HSCs in the graft and neutrophil recovery following transplant shown in our study suggests that some degree of ROS accumulation may play an important role in promoting neutrophil engraftment following autologous HSC transplantation (AHSCT) (Bai et al 2018). The current study examined the association between ROS levels and DNA damage in cryopreserved HSCs and whether these correlated with time to neutrophil and platelet engraftment following AHSCT.

Methods: Cryopreserved HSC samples from 51 patients who underwent AHSCT were studied. HSC's were defined as CD45+/CD34+. DNA damage and intracellular ROS levels were assessed using an antibody against the phosphorylated form of histone H2Ax (γH2Ax) and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA), respectively. Data acquisition and analysis were performed by flow cytometry.

Results: We observed a proportion of HSC in each sample that expressed elevated levels of gH2Ax. The median percentage of γH2Ax expressing CD34+ cells was 54.6% (range 3.4 - 98.4%). We observed a strong correlation between ROShigh and γH2Ax levels in terms of the dose of cells/kg infused (p < 0.001, r = 0.72). The ratio of ROShigh/γH2Ax-expressing HSCs was inversely associated with delayed neutrophil engraftment (p = 0.03, r = -0.31).

The cohort was divided into group 1 and group 2 based on ROShigh/γH2Ax ratios of >1 (n = 32) and ≤1 (n = 18) respectively. Median days to neutrophil engraftment were 12 (range 9-18), in group 1 and 15 (range 9-28) in group 2, (p = 0.007). Median days to platelet engraftment were 17 (range 10-28) in group 1 and 21.5 (range 14-55) in group 2, (p = 0.001). Median days to platelet count of >50 (plt50) was 18.5 (range 12-28) in group 1 and 22 (range 16-75) in group 2, (p = 0.001). In group 2, significant correlations were noted between the ROShigh/γH2Ax ratio and time to neutrophil engraftment (r = -0.54, p=0.025), plt20 (r = -0.64, p=0.004) and plt50 (r = -0.7, p = 0.002).

Conclusion: Our data suggest that DNA damage in CD34+ and accumulation of ROS may impact upon the time to engraftment of HSCs. Calculating a ratio of ROShigh/γH2Ax expression may be useful in predicting engraftment time following autologous transplantation.

Disclosures

No relevant conflicts of interest to declare.
Palliative Care Physicians' Views Toward Hematology and the Care of Patients with Hematologic Diseases: Results of a Nationwide Survey
Authors: Santivasi Wil L. , Wu Kelly M. , Litzow Mark R. , LeBlanc Thomas W. , Strand Jacob J.
Published: October 2019

Background: Palliative care specialists provide supportive care for patients with hematologic diseases. However, the degree of engagement by palliative care physicians may be limited by the views of both palliative care physicians and hematologists. Prior studies have surveyed hematologists to identify barriers to delivery of palliative care, however palliative care physician views are unclear. This study aimed to examine views of palliative care physicians toward hematology.

Methods: A survey was mailed to a random sample of the AAHPM physician contact list in 2017. Items focused on perceptions of their understanding of hematologic diseases, comfort providing care, opinions regarding palliative care and hospice involvement, and beliefs about hematologists. Anonymized responses were assessed on a Likert scale. Statistical testing was based on logistic regression models with generalized estimating equations to account for correlated data within respondents.

Results: 538 of 1000 surveys were completed. 51.9% of respondents were male. Community (37.2%), academic (36.7%) and hospice (26.2%) physicians were represented. Respondents were likelier to believe they understand the trajectories of lymphoma and myeloma than leukemia or patients undergoing hematopoietic stem cell transplantation (HSCT) (p < 0.001). They were more comfortable discussing prognosis (p =< 0.001) and managing symptoms (p < 0.001) in lymphoma and myeloma than leukemia and HSCT. They were likelier to believe that hematologists' perceptions of palliative care physicians limit collaboration rather than their own views of hematologists (p < 0.001). 80.2% agreed that hospice referrals are not made early enough.

Conclusions: Palliative care physicians' understanding of trajectories and comfort caring for patients varies by hematologic disease. They perceive that hematologists' perceptions are a larger barrier than their own and hospice referrals are delayed. These results provide insights into opportunities for better collaboration with hematologists.

Disclosures

No relevant conflicts of interest to declare.