Aplasia In Patients Research Articles

Relapsing Parvovirus B19 Infection after Intravenous Immunoglobulin in Renal Transplant Recipient: A Case Report

Parvovirus B19 has special affinity to erythroid progenitor cells leading to destruction and inhibition of erythropoiesis. One of its presentations is pure red cell aplasia in patients receiving immunosuppressive medications after organ transplantation. Intravenous immunoglobulin (IVIG) and reduction of immunosuppressive medication has been used for treatment but the effects of IVIG may be temporary. Here we present a case of Relapsing infection due to Parvovirus B19 after IVIG in a renal transplant recipient in Nepal. This case shows that in renal transplant patients presenting with anemia and low reticulocyte count, Parvovirus B19 infection should be suspected. Treatment with IVIG has good results but there may be relapse of infection.

Uncovering the Enemy: Single-Cell Transcriptional Profiling of Measurable Residual Disease (MRD) Cells

Albeit novel treatment concepts have entered the therapeutic landscape of AML, relapse rates remain high. This is attributed to treatment-refractory leukemic cells, which can be quantified by flow cytometry and molecular techniques (Measurable Residual Disease, MRD). MRD positivity is a significant prognostic biomarker for relapse and low overall survival rates. Post-remission therapy for the elimination of MRD remains a major challenge. We previously confirmed the prognostic value of early flow MRD assessment during aplasia in patients who achieve complete remission after intensive induction therapy (Koehnke et al., 2019). Here, we hypothesized that single-cell transcriptional profiling of refractory leukemic cells using flow cytometry (flow MRD) during aplasia will help to understand resistance mechanisms to therapy and to identify suitable targets for cellular immunotherapy. Flow MRD detection is based on the definition and characterization of leukemia-associated aberrant immunophenotypes (LAIPs) at the time of initial diagnosis, as described previously (Koehnke et al., 2015). A similar approach was applied to define LAIP+ cells at the time of aplasia (d16-d21 after initiation of induction therapy). For single-cell RNA-seq, LAIP+ cells from paired initial diagnosis and aplasia were sorted and subjected to transcriptional profiling using the mcSCRB-seq (molecular crowding single-cell RNA barcoding and sequencing) protocol (Bagnoli et al., 2018). We included 7 AML patients of different ELN genetic risk group (favorable, n=1; intermediate, n=4; adverse, n=2). In total, 854 LAIP+ cells from paired initial diagnosis-aplasia samples were projected using uniform manifold approximation and projection (UMAP) visualizations. We observed a distinct clustering of LAIP+ cells per patient and time point, revealing high inter-patient heterogeneity (Figure 1A). To identify common transcriptional changes across patients, we performed differential gene expression analysis of LAIP+ cells, comparing aplasia to initial diagnosis. We identified 93 genes significantly upregulated and 41 genes significantly downregulated (p < 0.01, FC >1 or <-1; Figure 1B). Among markedly upregulated genes across patients at the time of aplasia, we found 3 genes related to leukemic cell maintenance. Interestingly, SMARCA4, an ATPase subunit of the SWI/SNF chromatin remodeling complex, known to be essential for the proliferation and survival of AML cells (Buscarlet et al., 2014; Shi et al., 2013), showed a 1.99-fold increase in expression (p < 0.00). In addition, TRAP1, a candidate target in cancers with antiapoptotic function, was upregulated 1.5-fold (p < 0.00). And lastly, LIN28B, the cancer stem cell and leukemia driver gene, was also found to be upregulated by 0.7-fold in aplasia LAIP+ cells (p < 0.00). In addition, several immune-related genes including DC marker gene, LRP1 (CD91; p < 0.00, FC = 1.3) and the neutrophil/GMP- specific gene, cathepsin G (CTSG; p < 0.01, FC = 0.5) showed a significant up-regulation in aplasia LAIP+ cells. The latter was previously identified as a leukemia-associated antigen and a potential target for immunotherapy (Alatrash et al., 2017). Strikingly, LAIP+ cells at the time of aplasia harbored a LSC gene expression pattern in a patient-specific manner. Lastly, cell type prediction using the annotated dataset (van Galen et al., 2019) assigned the phenotype of LAIP+ cells mainly to GMP-like and cDC-like cells in variable abundance across patients. In summary, our data showed high inter-patient heterogeneity and phenotypic differences, making general statements on chemo-resistance challenging. However, despite this heterogeneity, LAIP+ cells at the time of aplasia harbor remarkable proliferation and survival advantage (stem cell-like phenotype), specifically, due to the upregulation of SMARCA4, TRAP1, and LIN28B genes. This phenotype is accompanied by patient-specific enrichment of LSC signature and upregulation of immune-related genes. Our data open up the path to better understand chemo-resistance and to identify targets that need to be addressed to eradicate MRD and improve relapse-free survival. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Outcomes after Reinfusion of CD19-Specific Chimeric Antigen Receptor (CAR)-Modified T Cells in Children and Young Adults with Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia

Background: CAR-modified T cells targeting CD19 have produced remarkable responses in relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL); however, relapse continues to be a substantial challenge. CD19+ relapses, which account for 33-78% of relapses, are associated with loss of CAR T-cell surveillance due to short persistence. Thus, strategies to improve functional persistence to prevent and treat CD19+ relapsed disease are crucial. Here, we report our experience administering reinfusions of murine or humanized 4-1BB CD19 CAR T cells in an effort to prolong persistence in patients with demonstrated short persistence to mitigate relapse risk, treat CD19+ relapsed disease, and produce responses after nonresponse to initial CAR infusion.Methods: This analysis included patients aged <30 years treated with a murine CD19 CAR construct, either investigational CTL019 (NCT01626495, NCT02906371) or commercial tisagenlecleucel, or a humanized CD19 CAR construct, huCART19 (NCT02374333), who received ≥1 reinfusion of the same CAR product due to: 1) clinical signs of poor persistence within 6 months (mos) of initial infusion, including peripheral B-cell recovery (BCR) or CD19+ hematogones in the bone marrow, 2) new CD19+ minimal residual disease (MRD) or relapse, or 3) nonresponse to initial infusion. The huCART19 trial included patients who had previously received a CAR T cell product (CAR-exposed), whereas all patients reinfused with CTL019/tisagenlecleucel were CAR-naïve at initial infusion. The primary outcome was complete response (CR) at day 28 after reinfusion, defined as complete remission with establishment or maintenance of B-cell aplasia. Secondary outcomes included CRS incidence, cumulative incidence of relapse (CIR) and overall survival (OS).Results: Among 229 CAR-naïve and 33 CAR-exposed patients treated with CD19 CAR between 2012-2020, 81 received ≥1 reinfusion (investigational CTL019, n=44; commercial tisagenlecleucel, n=11; huCART19, n=13 CAR-naïve and n=13 CAR-exposed). In addition, 18 patients received PD-1 blockade after their first (n=11) or subsequent (n=7) reinfusions. Indications for first reinfusion were peripheral BCR (CAR-naïve, n=32; CAR-exposed, n=6), hematogones (CAR-naïve, n=21; CAR-exposed, n=4), CD19+ MRD/relapse (CAR-naïve, n=10, CAR-exposed, n=0), and nonresponse to initial infusion (CAR-naïve, n=5, CAR-exposed, n=3). CRS grade ≥2 (Penn scale) occurred in 19 patients (grade 2, n=13; grade 3, n=4; grade 4, n=2). Grade 3-4 events only occurred in patients with active disease at time of reinfusion. Twenty-two patients had an inpatient admission within 30 days of first reinfusion, of which 7 required intensive care unit admissionAmong the 63 patients reinfused for relapse prevention, 33 (52%) had a CR at day 28. With a median duration of follow-up of 38 mos, 13 experienced a subsequent relapse (7 CD19+, 4 CD19-, 2 CD19-subset negative), 4 received alternative therapy or allogeneic hematopoietic stem cell transplantation (HSCT) in remission, and 16 remain in remission without further therapy at a median of 39 mos after first reinfusion. The median duration of B-cell aplasia was 8 mos (IQR 2-35) after reinfusion. Of the 30 with no response (NR), 10 had a subsequent CD19+ relapse, 15 received alternative therapy or HSCT, and 5 remain in remission without further therapy at a median of 43 mos after reinfusion. CIR and OS were not statistically significantly different between patients with CR or NR (CIR, p=0.26; OS, p=0.25) (Figure A-B). However, at 24 mos after reinfusion, CIR was 29% (95% CI, 11-44%) for CR compared to 61% (95% CI, 24-80%) for NR; OS was 90% (95% CI, 80-100%) for both groups.Of the 10 patients reinfused for relapse, 5 (50%) had a CR; 2 subsequently experienced a CD19+ relapse, 2 received an HSCT in remission, and 1 remains in remission without further therapy at 18 mos after reinfusion. Of the 8 patients reinfused for nonresponse to initial infusion, 7 were evaluable; none had a CR, and all died at a median of 2.5 mos after reinfusion.Conclusions: Reinfusion of CTL019/tisagenlecleucel or huCART19 is safe, may prolong B-cell aplasia in patients with short CAR persistence and reduce relapse risk, and can induce remissions in patients with CD19+ relapsed disease. Thus, reinfusion may provide an alternative to HSCT for short persistence. However, reinfusion is not effective for patients with nonresponse to initial CAR infusion. [Display omitted] DisclosuresCallahan: Novartis: Speakers Bureau. Rheingold: Optinose: Other: Spouse's current employment; Pfizer: Research Funding. June: Tmunity, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Current equity holder in publicly-traded company; Novartis: Patents & Royalties; AC Immune, DeCART, BluesphereBio, Carisma, Cellares, Celldex, Cabaletta, Poseida, Verismo, Ziopharm: Consultancy. Grupp: Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy; Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards; Novartis, Kite, Vertex, and Servier: Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding. Maude: Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Wugen: Consultancy.

FLT4 Mutations Are Associated with Segmental Lymphatic Dysfunction and Initial Lymphatic Aplasia in Patients with Milroy Disease.

This study explored mutations in the Fms-related tyrosine kinase 4/vascular endothelial growth factor receptor 3 gene (FLT4) and lymphatic defects in patients with Milroy disease (MD). Twenty-nine patients with lower limb lymphedema were enrolled. Sixteen patients had a familial history of MD, while 13 patients exhibited sporadic MD. Clinical signs, FLT4 mutations, indocyanine green (ICG) lymphography findings, and skin tissue immunohistochemical staining results were evaluated. Twenty-eight variants in FLT4 were identified. Twelve of these have previously been reported, while 16 are novel. Of the 28 variants, 26 are missense mutations, and the remaining two comprise a splicing mutation and a non-frame shift mutation. Twenty-five variants are located in the intracellular protein tyrosine kinase domain; three are located in the extracellular immunoglobulin domain. Substantially delayed contrast-enhanced tortuous lymphatic vessels were visualized to the ankle or knee level in 15 of 23 patients who underwent ICG lymphography. No initial lymphatic vessels were visualized in skin specimens from four patients who did not exhibit lymphatic vessels during imaging analyses. No specific variant was identified in relation to the unique clinical phenotype. Segmental dysfunction of lymphatic vessels and initial lymphatic aplasia are present in MD patients with FLT4 mutations.

ALL-026: Evaluating Efficacy and Safety of Tisagenlecleucel Reinfusion Following Loss of B-Cell Aplasia in Pediatric and Young Adult Patients with Acute Lymphoblastic Leukemia: HESTER Phase II Study

Context: In the ELIANA trial, tisagenlecleucel, a chimeric antigen receptor (CAR)-T cell therapy, demonstrated efficacy (81% overall remission rate [ORR]; 60% complete remission [CR]) and safety in pediatric and young adult patients with relapsed/refractory (r/r) B-cell acute lymphoblastic leukemia (B-ALL) (Maude et al. N Engl J Med. 2018). Sustained remissions were associated with B-cell aplasia. In patients with short CAR-T cell persistence, reinfusion with tisagenlecleucel may restore B-cell aplasia in some patients (Maude et al. J Clin Oncol. 2016). Objective: To determine the efficacy and safety of tisagenlecleucel reinfusion in pediatric and young adult patients with B-ALL experiencing loss of B-cell aplasia. Design: HESTER (NCT04225676) is an ongoing phase II, open-label, multicenter trial being conducted in the United States. Clinical and laboratory assessments will be done at months 1, 3, 6, and 12 post-reinfusion. Patients: Eligible patients must be ≤25 years old, with CD19(+) B-ALL and a previous infusion of commercial tisagenlecleucel with ≥1 additional tisagenlecleucel dose available. Patients must exhibit loss of B-cell aplasia, defined as peripheral blood (PB) absolute B lymphocyte count ≥50/µL or PB B lymphocytes ≥10% of the total, but are not required to be minimal residual disease negative (MRD-). Patients with active central nervous system metastasis or treated with prior gene or other T-cell therapies are excluded. Estimated enrollment is approximately 54 patients. Interventions: Reinfusion with tisagenlecleucel will be given once during the study, with the dose range of 0.2-5.0×106 CAR-positive viable T-cells/kg for patients ≤50 kg body weight or 0.1-2.5×108 CAR-positive viable T-cells for patients >50 kg body weight. Reinfusion must occur within 9 months from manufacture. Main Outcomes Measures: The primary efficacy endpoint is the proportion of patients who reestablish B-cell aplasia (circulating B lymphocytes

Serotonin Targeting Using Common Antidepressants Induces Rapid Recovery of Cytopenia

Background. Hematopoiesis is a highly regulated system where multiple, yet undiscovered, factors orchestrate the self-renewal of bone marrow stem cells and their differentiation into blood cells. Following acute stresses like infections, inflammation, chemotherapy or radiation, the hematopoietic system quickly adapts by a process termed "emergency" or "stress" hematopoiesis. For instance, switches from steady state to emergency granulopoiesis or emergency erythropoiesis have been described in response to infection or bleeding. We recently identified, both in human and murine erythroid progenitors, a functional cell-autonomous serotonergic network with pro-survival and proliferative functions. Furthermore, pharmacologic restoration of serotonin levels using selective serotonin reuptake inhibitors (SSRIs) such as fluoxetine, a common antidepressant, was demonstrated to rescue the anemic phenotype in mice models. Here, we hypothesized that serotonin also has a role on other hematopoietic lineages, and that the serotonergic system could be a valuable therapeutic target in radiation or chemotherapy-induced cytopenia. Moreover, as our previous work suggested a cross-talk between serotonin and erythropoietin, we postulate that serotonin could act in cooperation with known hematopoietic growth factors. Material and method. For mice models, we submitted C57BL/6 wild-type 8-10 weeks old mice to sub-lethal irradiation and monitored hematopoietic recovery through complete blood counts. We compared mice treated with fluoxetine administrated orally (~ 20 mg/kg/day in drinking water) to a control group, with or without hematopoietic growth factors. For the retrospective human cohort, we used a computerized database to identify patients who underwent autologous hematopoietic stem cell transplantation (ASCT) in the adult hematology department of Necker hospital between 2008 and 2018. We compared 22 patients treated with an SSRI to 66 controls, matched according to number of injected CD34+ cells/kg, age, sex, conditioning chemotherapy, pathology, depth of response before transplantation, previous lines of chemotherapy, and year of transplantation. The study was conducted according to the Declaration of Helsinki. Results. First, we confirmed that, following sub-lethal irradiation, pharmacologic restoration of 5-HT levels in mice using fluoxetine improved normalization of the erythroid lineage (at day 17 post-irradiation, mean hemoglobin was 8.1 versus 2.8 g/dL in the fluoxetine versus control group (p=0.0002)). Second, the data revealed that the use of SSRI lead to a more rapid restoration of the leukocytes and platelets levels (at day 17 post-irradiation, mean leukocyte level was 1740 versus 314/mm3 (p&lt;0.0001) and mean platelet level was 216 versus 43/mm3 (p=0.002) in the fluoxetine versus control group, respectively). Third, we observed an additive effect between fluoxetine and Granulocyte-Colony Stimulating Factor (G-CSF) on the recovery of the three myeloid lineages (at day 17 post-irradiation, mean hemoglobin was 10.5 versus 4.0 g/dL (p&lt;0.0001), mean leukocyte level was 2900 versus 900/mm3 (p&lt;0.0001) and mean platelet level was 308 versus 84/mm3 (p=0.0009) in the fluoxetine + G-CSF versus control group, respectively). Finally, analysis of the in vivo murine model under steady state condition showed that without any hematopoietic stress, fluoxetine did not impact hematopoiesis. In human, analysis of the retrospective ASCT cohort demonstrated that patients treated with SSRI had a more rapid neutrophil recovery than matched control patients (mean duration of neutropenia &lt;500/mm3 was 12.2 days in the SSRI group and 14.26 days in the control group, p=0.0216). SSRI therapy was not associated with a higher rate or quicker relapses of underlying malignant hemopathy: mean progression free survival was 36 months in both groups. Conclusion. In this work, we report a previously unknown role of SSRI in the hematopoietic recovery of cytopenia, both after sub-lethal irradiation in mice and after autologous hematopoietic stem cell transplantation in human. We also observed a significant cooperation between SSRI and G-CSF on the three myeloid lineages. We propose that the serotonergic system could be a valuable therapeutic target in stress hematopoiesis such as in therapy-induced aplasia in patients. Disclosures Hermine: AB Science: Consultancy, Equity Ownership, Honoraria, Research Funding; Celgene: Research Funding; Novartis: Research Funding. OffLabel Disclosure: Fluoxetine is a selective serotonin reuptake inhibitor for oral administration. It is an antidepressant approved for major depressive disorder, obsessive compulsive disorder, bulimia nervosa and panic disorder. We believe its role on serotonin can improve hematopoiesis.

Switchable control over in vivo CAR T expansion, B cell depletion, and induction of memory.

Chimeric antigen receptor (CAR) T cells with a long-lived memory phenotype are correlated with durable, complete remissions in patients with leukemia. However, not all CAR T cell products form robust memory populations, and those that do can induce chronic B cell aplasia in patients. To address these challenges, we previously developed a switchable CAR (sCAR) T cell system that allows fully tunable, on/off control over engineered cellular activity. To further evaluate the platform, we generated and assessed different murine sCAR constructs to determine the factors that afford efficacy, persistence, and expansion of sCAR T cells in a competent immune system. We find that sCAR T cells undergo significant in vivo expansion, which is correlated with potent antitumor efficacy. Most importantly, we show that the switch dosing regimen not only allows control over B cell populations through iterative depletion and repopulation, but that the "rest" period between dosing cycles is the key for induction of memory and expansion of sCAR T cells. These findings introduce rest as a paradigm in enhancing memory and improving the efficacy and persistence of engineered T cell products.

Uric acid as a novel biomarker for bone-marrow function and incipient hematopoietic reconstitution after aplasia in patients with hematologic malignancies.

Prolonged aplasia and graft failure (GF) represent life-threatening complications after hematopoietic cell transplantation (HCT) requiring suitable biomarkers for early detection and differentiation between GF and poor graft function (PGF). Uric acid (UA) is a strong immunological danger signal. Laboratory results were analyzed from patients undergoing either allogeneic or autologous HCT or induction chemotherapy for acute leukemia (n = 50 per group, n = 150 total). During therapy, UA levels declined from normal values to hypouricemic values (all p < 0.001). Alongside hematopoietic recovery, UA serum levels returned to baseline values. During aplasia, UA levels remained low and started steadily increasing (defined as >two consecutive days, median one 2-day increase) at a median of 1 day before rising leukocytes in allogeneic HCT (p = 0.01) and together with leukocytes in autologous HCT (median one 2-day increase). During induction chemotherapy, a UA increase was also observed alongside rising leukocytes/neutrophils but also several times during aplasia (median 3 increases). Most HCT patients had no detectable leukocytes during aplasia, while some leukocytes remained detectable after induction therapy. No increase in UA levels was observed without concomitant or subsequent rise of leukocytes. Changes in UA serum levels can indicate incipient or remaining immunological activity after HCT or induction therapy. They may, therefore, help to differentiate between PGF and GF.

Efficacy and Safety of Humanized Chimeric Antigen Receptor (CAR)-Modified T Cells Targeting CD19 in Children with Relapsed/Refractory ALL

Background Targeted immunotherapy with T cells expressing a chimeric antigen receptor (CAR) can produce dramatic anti-tumor responses. We previously demonstrated complete remissions (CRs), prolonged persistence, and sustained responses in children and adults with relapsed/refractory (r/r) acute lymphoblastic leukemia (ALL) treated with CD19-specific CAR-modified T cells (CTL019). However, a subset (~25%) of patients has limited persistence of CTL019, which can increase the risk of relapse. We hypothesized that repeat infusion may prolong persistence in some patients but would be ineffective in patients with immune-mediated rejection. As most CAR single chain variable fragment (scFv) domains, including that of CTL019, are of murine origin, anti-mouse reactivity is one potential cause of immunogenicity that may be overcome by fully human or humanized CAR design. We now report on retreatment with murine or humanized CD19-directed CAR T cells. Aims We sought to 1) prolong persistence by reinfusion of murine CTL019 in children with r/r CD19+ ALL and evidence of poor persistence and 2) determine the safety and efficacy of CD19-directed CAR T cells with a humanized scFv domain in children with B cell recovery or CD19+ relapse after prior CAR T cell therapy. Method T cells collected from patients were transduced with a lentiviral vector encoding a CAR composed of murine or humanized anti-CD19 scFv, CD3z, and 4-1BB domains, activated/expanded ex vivo with anti-CD3/anti-CD28 beads, cryopreserved, and then infused. The humanized scFv was developed by grafting the complementary determining regions of both the heavy and light chains onto human germline acceptor frameworks. Results Of 50 patients in CR at 1 month after CTL019 infusion (50/53 CR), 14 patients received a repeat infusion of murine CTL019 at 3 and/or 6 months after initial infusion and 11 are evaluable for response. Indications were B cell recovery (n=4), CD19+ minimal residual disease (MRD, n=2), or undetectable CTL019 in peripheral blood by flow cytometry (≤0.1% CTL019+ of CD3+, n=5). Minimal toxicities were observed and included fever and fatigue. CTL019 reinfusion induced B cell aplasia for a second time in 1 of 4 children treated for B cell recovery. Two of these patients later relapsed, 1 with CD19+ and 1 with CD19- disease. Of 2 patients with CD19+ MRD, 1 had no response and subsequently experienced a CD19+ relapse and the second became MRD-negative but had B cell recovery. All 5 children reinfused for poor CTL019 persistence demonstrated continued B cell aplasia 3-15 months after repeat infusion with detectable CTL019 in peripheral blood by flow cytometry (0.2-2.8% CTL019+ of CD3+) in 4 patients. Of this group, 4 patients remain in remission 12-21 months after initial infusion, and 1 experienced a CD19- relapse. To overcome the potential for anti-murine immune-mediated rejection, patients previously treated with CAR-modified T cells were eligible for a phase 1 study of humanized CD19-directed CAR T cells (CTL119). Five children were infused for B cell recovery (n=3) or CD19+ relapse (n=2). Cytokine release syndrome (CRS), seen in 2 patients, was limited to fever, headache, and nausea and did not require vasopressor or respiratory support. Three patients showed no response and progressed (n=1) or relapsed with CD19+ (n=1) or CD19- (n=1) ALL. Responses were demonstrated in 2 patients: 1) MRD-negative CR in a patient treated for CD19+ relapse and 2) reduction in MRD and return of B cell aplasia in a patient treated for CD19+ MRD and B cell recovery, with subsequent progression to CD19+ relapse. Both responding patients were previously resistant to murine CTL019 reinfusion, suggesting a possible immune-mediated rejection mechanism. Ongoing studies are investigating anti-CAR responses. Conclusion Repeat infusion of murine CTL019 may prolong B cell aplasia in patients with early evidence of poor persistence and in a fraction of patients with B cell recovery. In the first demonstration of humanized anti-CD19 CAR efficacy, retreatment with CTL119 expressing a humanized scFv induced remission of ALL refractory to prior CD19-directed murine CAR T cell therapy, suggesting that immune-mediated rejection may be an important mechanism of resistance in the subset of patients with rapid CAR cell loss and loss of B cell aplasia. Further investigation into anti-CAR responses will be vital to improve durable remission rates in this highly refractory population. Disclosures Maude: Novartis: Consultancy, Research Funding. Off Label Use: CTL019 and CTL119 for relapsed/refractory ALL. Rheingold:Endo: Other: Husband's employer, has equity interest; Novartis: Consultancy. Aplenc:Sigma Tau: Consultancy. Teachey:Novartis: Research Funding. Shaw:Novartis: Research Funding. Brogdon:Novartis: Employment. Loew:Novartis: Employment. Zheng:Novartis: Patents &amp; Royalties. Levine:Novartis: Patents &amp; Royalties, Research Funding. Porter:Novartis: Patents &amp; Royalties, Research Funding. Lacey:Novartis: Research Funding. Melenhorst:Novartis: Research Funding. June:Novartis: Research Funding; University of Pennsylvania: Patents &amp; Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight. Grupp:Novartis: Consultancy, Research Funding.

Pulsating Enophthalmos in Neurofibromatosis Type 1

Neurofibromatosis type 1 (NF-1) is a neurocutaneous syndrome with multisystem involvement, especially the central nervous system, eyes, and skin. In this article, we describe a patient with NF-1 with radiologically-imaged sphenoid wing aplasia that led to pulsatile enophthalmos. Although sphenoid wing aplasia in patients with NF-1 frequently occurs as painless pulsatile exophthalmos in clinical practice, as in our case it can rarely present as pulsatile enophthalmos. Globe pulsation occurs as a reflection of intracranial pulsation from direct contact of meningeal structures and globe, secondary to the defect in the posterior orbital wall. The diagnosis is made by the presence of characteristic bone defects on radiological imaging modalities and by the absence of vascular malformations on doppler ultrasonography or computed tomography angiography.

Acquired Pure Red Cell Aplasia in patients with plasma cell neoplasm and long term remission with bortezomib therapy

We report two cases of Pure Red Cell Aplasia (PRCA) associated with plasma cell dyscrasia. The first case is of an elderly woman with PRCA and light chain disease (LCD)/multiple myeloma (MM). She received multiple blood transfusions and was refractory to conventional therapies. Subsequent bortezomib therapy resulted in symptomatic improvement and normalization of her bone marrow and transfusion independence. This unique case of LCD/MM and PRCA treated with bortezomib induced long lasting remission of myeloma and resulted in reversal of PRCA and hence transfusion independence. This novel therapy is useful in elderly patients even with a high tumour burden of multiple myeloma. PRCA can also be associated in patients with low tumour bulk plasma cell disorders such as Monoclonal Gammopathy of Uncertain Significance (MGUS) as evidenced by the second case. Combination of prednisolone and oral cyclo - phosphamide therapies controlled her anaemia. Hence, a tailored therapy approach is recommended. Furthermore, patients with PRCA would benefit from thorough investigation to exclude plasma cell disorders.

Risk of Pure Red Cell Aplasia in Patients With Hepatitis C Receiving Antiviral Therapy and an Erythropoiesis-Stimulating Agent

Antibody-mediated pure red cell aplasia (PRCA) has been primarily observed in patients with chronic kidney disease treated with an erythropoiesis-stimulating agent (ESA); only a few anecdotal cases have been reported in other patient populations. We searched the Amgen Global Safety Adverse Event Database and identified 14 patients with hepatitis C who developed severe anemia, anti-erythropoietin antibodies, and bone marrow biopsy-proven PRCA, while receiving interferon therapy (with or without ribavirin) and an ESA. During the follow-up period and after ESA treatment stopped, 11 patients no longer required transfusions and 3 did. Analysis of antibody isotypes showed that, contrary to reports of patients with chronic kidney disease, immunoglobulin G1 was the predominant isotype rather than immunoglobulin G4 (immunoglobulin G4 was detected in only 1 of 6 patients). Epitope mapping showed the anti-erythropoietin antibodies bound domains required for receptor binding. Therefore, the potential benefits of ESA therapy must be weighed against the risk for PRCA in patients with hepatitis C who are receiving treatment with interferon and ribavirin.

Early intervention with allogeneic hematopoietic cell transplantation during chemotherapy-induced aplasia in patients with high-risk acute myeloid leukemia

Early intervention with allogeneic hematopoietic cell transplantation during chemotherapy-induced aplasia in patients with high-risk acute myeloid leukemia

CD19-Targeted Donor T Cells Exert Potent Graft Versus Lymphoma Activity and Attenuated Gvhd

AbstractAbstract 451Chimeric antigen receptors (CAR) represent a potent strategy to target T cells against selected tumor antigens. Ongoing clinical trials indicate that autologous T cells expressing CARs targeting CD19, a B cell-associated antigen, can induce complete remission and B cell aplasia in patients with B cell malignancies. Donor CD19-CAR+ T cells could potentially be used to treat recipients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT), but the risk of alloreactivity mediated by endogenous T cell receptors (TCR) triggering an acute GVHD is not known. This is partly due to the absence of in vivo models to study the relative effects of CAR and endogenous TCR signaling. For the first time, we have evaluated the relative effects of CD19-targeted donor T cells on the elimination of CD19+ B cells and endogenous TCR-mediated alloreactivity in mouse models of allo-HSCT.We generated a panel of retroviral vectors encoding mouse CD19-specific CARs: as a control, CD19-delta, a tail-less CAR lacking the CD3ζ signaling domain; CD19z1, which signals through its CD3ζ endodomain; and CD19-28z, which signals through CD28 and CD3ζ (Figure 1A). CD19z1+ and CD19-28z+ T cells mediated specific lysis of CD19-expressing tumors in vitro, while CD19-delta+ T cells did not. In order to assess the anti-tumor capacity of CD19-CAR+ T cells in vivo, we transferred the transduced B6 donor T cells into lethally irradiated BALB/c recipients that were administered T cell-depleted allografts and CD19+ lymphoma A20-TGL (B6–> BALB/c+A20-TGL). CD19-CAR+ T cells (CD19z1 and CD19-28z) mediated clearance of A20 tumor cells visualized by in vivo imaging of luciferase-expressing tumor cells (Figure 1B and data not shown) and significantly improved tumor free survival. CD19-CAR+ B6 T cells could sustain prolonged B cell hypoplasia when adoptively transferred into lethally irradiated haploidentical CBF1 recipients of T cell-depleted allografts (B6–> CBF1, Figure 1C). These data indicate that under alloreactive conditions, donor CD19-CAR+ T cell signaled through the CAR leading to specific elimination of CD19+ tumors and B lineage cells.In order to determine the risk of GVHD, we transferred the donor CD19-CAR+ T cells into haploidentical HSCT recipients. Interestingly, CD19-CAR+ T cells mediated significantly less acute GVHD, resulting in improved survival and lower GVHD scores (Figure 1D). Donor CD19-delta+ T cells however mediated lethal GVHD, indicating that the endogenous TCR mediated strong alloreactivity in the absence of CAR signaling. Similar results were obtained from experiments using MHC-mismatched (B6–> BALB/c) models. It is known that signaling through endogenous TCR is accompanied by down-regulation of surface TCR expression. We found significant decreases in surface CD3ε, TCRβ and CD90 expressions in donor CD19-delta+ T cells under alloreactive conditions. In contrast, donor CD1928z+ T cells failed to down-regulate surface TCR expression under similar conditions, suggesting that endogenous TCR function was altered in CAR-activated T cells. In the context of allo-HSCT, preferential CAR signaling at the expense of alloreactive endogenous TCR signaling may thus lead to reduced alloreactivity and attenuation of GVHD. These results provide the first pre-clinical evidence suggesting that CAR-modified, unselected donor T cells may be safely applied in an allogeneic context. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Parvovirus B19 seroconversion in a cohort of human immunodeficiency virus-infected patients

Erythrovirus B19 (B19V) infection may cause red cell aplasia in patients infected with human immunodeficiency virus (HIV). The introduction of highly active antiretroviral therapy (HAART) has improved the immune function of these patients by modifying the course of B19V infection. The purpose of this study was to estimate the frequency of B19 seroconversion in a cohort of HIV-infected patients and evaluate the occurrence of B19V-related anaemia during the seroconversion period. Adult HIV-infected patients were studied at a public hospital in Niterói, state of Rio de Janeiro, Brazil. IgG and IgM antibodies against B19V were detected by an enzyme-linked immunosorbent assay and B19 viraemia was assayed by polymerase chain reaction. Medical records were reviewed for any clinical evaluation of anaemia. Seroconversion was detected in 31.8% of the 88 individuals who began the study as anti-B19V IgG-negative. No clinical manifestations of B19V infection were detected during the period of seroconversion. Patients who seroconverted were 5.40 times more likely to have anaemia than those who did not [odds ratio 5.40 (95% confidence interval: 1.33-22.93)]. Anaemia was detected in eight patients. All patients recovered from anaemia by either beginning or continuing HAART, without requiring blood transfusions. In the HAART era, B19V infection may only be associated with a course of disease characterised by less severe chronic anaemia. This milder course of B19V-associated disease is likely due to the increased immune function of HAART-treated patients.

Parvovirus-B19 and hematologic disorders.

Parvovirus-B19 (PV-B19) is a member of Parvoviridae, which is one of the smallest DNA viruses. PV-B19-associated diseases usually serve as a good representation of the balance of virus, host response and the immune system. The diseases manifested with PV-B19 are erythema infectiosum, which is common in children, hydrops fetalis, transient pure red cell aplasia in patients with chronic hemolytic anemia, arthralgia - mostly observed in women, and chronic pure red cell aplasia in immunocompromised individuals. Cytopenia (bicytopenia, monocytopenia or pancytopenia) may also accompany the diseases mentioned above. On the other hand, there are many diseases, including neurologic, vasculitic, hepatic, rheumatoid, nephritic, autoimmune, myocardial, and others in which the mechanisms of the diseases are not clear, which may be associated with PV-B19. The virus may manifest with unexpected and unexplained clinical pictures and lead to misdiagnosis. Therefore, hematologic disorders in any unestablished clinical diagnosis should be investigated for PV-B19 infection. However, serologic examination for PV-B19 diagnosis is not sufficient in immunocompromised status. The virus can be determined with polymerase chain reaction (PCR) in the serum or tissue samples. Supportive therapy, blood transfusion and immunoglobulin are the conventional therapeutic interventions for PV-B19 today. Vaccination studies are under examination.

Pretreatment with Ascorbic Acid Prevents Lethal Gastrointestinal Syndrome in Mice Receiving a Massive Amount of Radiation

While bone marrow or stem cell transplantation can rescue bone marrow aplasia in patients accidentally exposed to a lethal radiation dose, radiation-induced irreversible gastrointestinal damage (GI syndrome) is fatal. We investigated the effects of ascorbic acid on radiation-induced GI syndrome in mice. Ascorbic acid (150 mg/kg/day) was orally administered to mice for 3 days, and then the mice underwent whole body irradiation (WBI). Bone marrow transplantation (BMT) 24 h after irradiation rescued mice receiving a WBI dose of less than 12 Gy. No mice receiving 14 Gy-WBI survived, because of radiation-induced GI syndrome, even if they received BMT. However, pretreatment with ascorbic acid significantly suppressed radiation-induced DNA damage in the crypt cells and prevented denudation of intestinal mucosa; therefore, ascorbic acid in combination with BMT rescued mice after 14 Gy-WBI. DNA microarray analysis demonstrated that irradiation up-regulated expressions of apoptosis-related genes in the small intestine, including those related to the caspase-9-mediated intrinsic pathway as well as the caspase-8-mediated extrinsic pathway, and down-regulated expressions of these genes in ascorbic acid-pretreated mice. Thus, pretreatment with ascorbic acid may effectively prevent radiation-induced GI syndrome.

Preliminary MRI study in patients with congenital complex strabismus

Objective Although the ocular motility examination has been traditionally used in the diagnosis of complex strabismus resulting from cranial nerve (CN) and extraocular muscles (EOM) abnormalities, magnetic resonance imaging (MRI) now permits the direct imaging of lesions in CNs and EOMs. Methods Twenty-six patients with complex strabismus underwent MRI examination on 1.5 T MR unit (Twinspeed, GE). Nerves to EOMs were imaged with T1 weighted in orbits in all patients using phase array surface colls. Results Patients with Duane syndrome ( 15 cases, 19 eyes) all exhibited absence or hypoplasia of abducens nerve (CN6), always with mild hypoplasia and apparent misdirection of oculomotor nerve ( CN3 ) to the lateral rectus muscle in the orbit, and there were no hypoplasia of EOMs. Patients with congenital fibrosis of EOMs (9 cases, 16 eyes) exhibited severe hypoplasia of CN3 and CN6, and EOMs appeared hypoplasia to a different degree, particularly severe for the superior rectus and levated palpted muscles. Multiple nerves displayed aplasia in patients with Mobius syndrome (1 case, 2 eyes) and there was abnormal branch from CN3 to lateral rectus. The tendons of bilateral superior oblique muscles were thin in the patients with Brown syndrome ( 1 case, 2 eyes). Conclusion MRI can directly demonstrate absence or hypoplasia of CNs and corresponding EOM abnormalities in congenital complex strabismus, which suggests that the mechanism of congenital complex strabismus is perhaps abnormal innervation or displasia of the ocular motor nerves. Key words: Strabismus; Oculomotor muscles; Ophthalmic nerve; Magnetic resonance imaging

Pure Red Cell Aplasia Induced by Erythropoiesis-Stimulating Agents

Pure red cell aplasia in patients who are treated for anemia of chronic kidney disease with erythropoiesis-stimulating agents such as epoetin was first reported in 1998. Although the incidence of pure red cell aplasia peaked in 2002, it remains important for nephrologists to know how to investigate a suspected case of pure red cell aplasia and how to identify other causes of hyporesponsiveness to erythropoiesis-stimulating agents, which account for the vast majority of such cases. The authors reviewed the current status of information in the literature and drew on their personal experiences with patients regarding the diagnosis and management of epoetin-induced pure red cell aplasia. The mechanism for development of epoetin-induced pure red cell aplasia remains unconfirmed. It generally occurs after the production of neutralizing anti-erythropoietin antibodies. Elucidation of a suspected pure red cell aplasia case requires a systematic approach, beginning with simple measurements such as blood cell counts, because most cases of erythropoiesis-stimulating agent hyporesponsiveness are attributable to other causes. If these criteria indicate that the patient's response to erythropoiesis-stimulating agent therapy is very poor, then bone marrow examination and measurement of anti-erythropoietin antibodies is justified. If pure red cell aplasia is confirmed, then cessation of erythropoiesis-stimulating agent therapy and initiation of immunosuppressive therapy are recommended. Continued study of epoetin-induced pure red cell aplasia is needed to help nephrologists prevent or manage future cases and will have implications for the use of other protein-based therapeutic agents.

Non‐Clinical Safety Studies on Biosimilar Recombinant Human Erythropoietin

Recombinant human erythropoietin (rhEPO) is widely used for the treatment of patients with anaemia and its loss of patent protection has stimulated the development of cheaper biosimilar products. However, the quality and comparability of rhEPO products recently marketed in several developing countries is questionable. Paying attention to quality in its isolation, purification and analytical characterization, it has been possible to produce a biosimilar rhEPO that is comparable with the originator product. Non-clinical safety testing was initially carried out in the absence of a regulatory framework and contributed to the receipt of marketing approval for biosimilar rhEPO in Eastern Europe. Subsequently, this non-clinical testing was extended to take into account the recent guidelines for similar biological medicinal products published by the European regulatory authorities, which were markedly influenced by the intervening occurrence of pure red cell aplasia in patients taking what proved to be an impure rhEPO product. This Mini Review discusses the challenges faced, approaches taken and lessons learned in developing a biosimilar rhEPO product, both before and after the publication of the regulatory guidelines.