Antibodies Research Articles

Transbronchial cryobiopsy using an ultrathin cryoprobe with a guide sheath for the diagnosis of pulmonary mucosa-associated lymphoid tissue lymphoma.

Pulmonary mucosa-associated lymphoid tissue (MALT) lymphoma is difficult to diagnose and relatively rare. Tissue sampling through transbronchial biopsy is often inadequate, necessitating surgical lung biopsy. However, a recently developed technique, transbronchial lung cryobiopsy (TBLC), has shown promise for obtaining larger specimens. A 1.1 mm cryoprobe has recently become available, and its usefulness has been increasingly reported. Use of a conventional cryoprobe for TBLC in diagnosing pulmonary MALT lymphoma has been previously reported; however, there are no reports on the use of a 1.1 mm ultrathin cryoprobe and guide sheath (GS). We aimed to assess the effectiveness and safety of using a 1.1 mm ultrathin cryoprobe in combination with a GS for diagnosing pulmonary MALT lymphoma using a simpler and safer method. We retrospectively analyzed the findings for four patients showing characteristic computed tomography (CT) findings of MALT lymphoma, including peripheral pulmonary lesions, air bronchogram nodules, and bronchiectasis, at our hospital. Each patient underwent endobronchial ultrasound (EBUS) with a GS, followed by TBLC using a 1.1 mm cryoprobe. Morphological diagnosis, immunohistochemical examination, and molecular testing were performed on the biopsy specimens to establish the diagnosis. Complications during the procedure were also monitored. We obtained 8-16 biopsy specimens in all four cases using a cryoprobe. Histopathological analysis of two cases revealed the infiltration of small lymphocytes with numerous lymphoepithelial lesions, confirming MALT lymphoma. Immunohistochemical examination further demonstrated B-cell lymphocyte proliferation and light-chain restriction, confirming monoclonality and providing a definitive diagnosis. In the remaining two cases, histopathological evidence of pulmonary MALT lymphoma was lacking. However, molecular testing using polymerase chain reaction to analyze immunoglobulin gene rearrangements revealed B-cell clonality, which supported the diagnosis. Molecular testing proved particularly useful when histopathological diagnosis alone was inconclusive. No complications such as pneumothorax or hemorrhage occurred during the procedure. The combination of a GS and EBUS facilitated specimen collection at the same location as EBUS, with the GS providing compression hemostasis and eliminating the need for an additional hemostatic device. Therefore, TBLC with a GS is a useful and safe method for diagnosing pulmonary MALT lymphomas and reproducibly yielded sufficient quantities of good-quality biopsy specimens.

RSV Neutralizing Antibodies in Dried Blood.

The key correlate of protection of respiratory syncytial virus (RSV) vaccines and monoclonal antibodies (mAbs) is virus neutralization, measured via sera obtained through venipuncture. Dried blood obtained with a finger prick can simplify acquisition, processing, storage, and transport in trials and thereby reduce costs. In this study, we validate an assay to measure RSV neutralization in dried capillary blood. Functional antibodies were compared between matched serum and dried blood samples from a phase 1 trial with RSM01, an investigational anti-RSV prefusion F mAb. Hep-2 cells were infected with a serial dilution of sample-virus mixture by using RSV-A2-mKate to determine the half-maximal inhibitory concentration. Stability of dried blood was evaluated over time and during temperature stress. Functional antibodies in dried blood were highly correlated with serum (R2 = 0.98, P < .0001). The precision of the assay for dried blood was similar to serum. The function of mAb remained stable for 9 months at room temperature and frozen dried blood samples. We demonstrated the feasibility of measuring RSV neutralization using dried blood as a patient-centered solution that may replace serology testing in trials against RSV or other viruses, such as influenza and SARS-CoV-2. Clinical Trials Registration. NCT05118386 (ClinicalTrials.gov).

Neurodegenerative diseases associated antibody repertoire signatures in mimotope arrays based on cyclic versus linear peptides

The role of peptide probes’ conformational flexibility in extracting immunosignatures has not been sufficiently studied. Immunosignatures profile the antibody diversity and prove promising for early cancer detection and multi-disease diagnostics. A novel tool for modeling antibody repertoires, the concept of antibody reactivity graphs, proved instrumental in this respect. Serum samples from patients with Alzheimer’s disease (AD), frontotemporal dementia (FTD), dementia of unknown etiology (DUE), and healthy controls were probed using a set of 130 7-mer peptides relevant to neurodegenerative diseases. Results show that linear peptides probed with IgM yielded higher graph density compared to IgG, indicating different levels of polyspecificities. Additionally, the impact of peptide topology and antibody isotype on feature selection was studied using recursive feature elimination. Findings reveal that IgM assays on linear peptides offer superior diagnostic differentiation of neurodegenerative diseases and define the degree of agreement between IgG and IgM immunosignatures with linear or cyclic peptides.

Refining Immunogenicity through Intradermal Delivery of Outer Membrane Vesicles against Shigella flexneri in Mice

Shigellosis remains a global health concern, especially in low- and middle-income countries. Despite improvements in sanitation, the absence of a licensed vaccine for human use has prompted global health organizations to support the development of a safe and effective multivalent vaccine that is cost-effective and accessible for limited-resource regions. Outer Membrane Vesicles (OMVs) have emerged in recent years as an alternative to live attenuated or whole-inactivated vaccines due to their immunogenicity and self-adjuvating properties. Previous works have demonstrated the safety and protective capacity of OMVs against Shigella flexneri infection in mouse models when administered through mucosal or intradermal routes. However, some immunological properties, such as the cellular response or cross-protection among different Shigella strains, remained unexplored. In this study, we demonstrate that intradermal immunization of OMVs with needle-free devices recruits a high number of immune cells in the dermis, leading to a robust cellular response marked by antigen-specific cytokine release and activation of effector CD4 T cells. Additionally, functional antibodies are generated, neutralizing various Shigella serotypes, suggesting cross-protective capacity. These findings highlight the potential of OMVs as a promising vaccine platform against shigellosis and support intradermal administration as a simple and painless vaccination strategy to address this health challenge.

Tumor-Infiltrating Normal B Lymphocytes Have Remarkable Prognostic Effects and Are Crucial for Antitumor Immune Responses in Diffuse Large B-Cell Lymphoma

Introduction: In recent years, increasing evidence suggests an important role of tumor-infiltrating B lymphocytes (TIL-Bs) in cancer immunity. In various types of solid tumors, high density of TIL-Bs and presence of mature tertiary lymphoid structures consistently correlate with better prognosis and response to immune checkpoint blockade therapies. Diffuse large B-cell lymphoma (DLBCL) is a type of aggressive lymphoma arising from mature B cells. TIL-Bs have been identified in DLBCL based on single-cell gene-expression data in several previous studies, but their prognostic significance in DLBCL is unknown. In this study, we determined the clinical significance of TIL-B abundance and investigated the potential underlying mechanisms. Patients and Methods: This abstract includes a discovery study in which we distinguished TIL-Bs from malignant B-cells by immunoglobulin gene (IG) clonotype analysis and a validation study using flow cytometry. In the discovery study, a molecularly well-characterized DLBCL cohort assembled by the International DLBCL R-CHOP Consortium Program was analyzed. DNA samples from DLBCL patients were sequenced by high-throughput ultra-deep sequencing assays using multiplexed forward and reverse primers. Based on the IG heavy chain (IGH) sequencing results, malignant B-cell and TIL-B VDJ clonotypes were identified for 138 patients, and the frequencies and numbers of TIL-B clonotypes in individual sequence repertoires were calculated. In 79 DLBCL cases, a DJ-only sequence was predominant and no productive VDJ sequences met the criteria for diagnostic sequences. Total frequencies of productive VDJ sequences in the sequence repertoires were then used to access high and low TIL-B frequencies. TIL-B frequencies were also evaluated by IG light chain clonotypes for these 217 patients and additional 52 patients. The TIL-B frequencies were correlated with patient survival, gene-expression profiling (GEP) data GSE31312, and frequencies of DLBCL-infiltrating immune cells quantified by fluorescent multiplex immunohistochemistry (mIHC). To validate the prognostic significance of TIL-B frequencies, we assembled a validation cohort of 94 DLBCL patients treated at Duke University Medical Center. TIL-Bs in tissues were quantified by flow cytometry immunophenotyping. Ratios of TIL-Bs to abnormal B cells were assessed for prognostic effects. Results: First, we found thathigh frequencies of TIL-Bs and clonotypic diversity were associated with significantly favorable prognostic effects in 138 DLBCL patients with their malignant B-cell IGH-VDJ clonotypes identified (Figure A). The prognostic effects of TIL-Bs outweighed those of the germinal center B-cell-like/activated B-cell-like classification (Figure B). Second, patients with high frequencies of TIL-Bs compared with those without had significant upregulation of immune response genes by GEP (Figure C), higher CD4 T cell infiltration quantified by fluorescent mIHC (Figure D), and enrichment of EcoTyper-defined specific cell states, including significantly increased frequencies of B cell state S2 (pre-memory), CD4 T cell state S3 (naïve), follicular helper T cell state S3, and endothelial cell state S3, whereas lower frequencies of B cell state S5 (plasmablast) and follicular helper T cell state S1. Third, high TIL-B abundance in 79 ‘DJ-only’ clonotype cases and additional 52 cases assessed by light chain clonotype analysis showed similar favorable prognostic effects and associations with upregulated immune gene expression and CD4 T cell infiltration. Fourth, the identified GEP signatures, prominent with genes involved in T-dependent B cell activation and T cell activation and differentiation, showed significant prognostic effects in two GEO datasets. Fifth, TIL-B abundance showed significant prognostic effects in DLBCL cases infiltrated with T-cells in both the discovery study (Figure E) and validation study (Figure F, flow cytometry cohort). Conclusions: TIL-B frequencies and clonotypic diversity have remarkable prognostic and biological effects in DLBCL, which has significant implications for developing novel therapeutic strategies. Our study identified a new prognostic biomarker in DLBCL and an important player in anti-lymphoma immune responses, providing a new point of view to analyze the tumor biology in aggressive B-cell lymphoma and precisely identify prognostic determinants.

Transient CDK9 Inhibition with AZD4573 Effectively Induces Apoptosis in Burkitt Lymphoma As a Monotherapy in Pre-Clinical Models

Burkitt Lymphoma (BL) accounts for almost two thirds of all B-cell non-Hodgkin lymphoma cases in children and adolescents. It may affect the jaw, bones of the face, bowel, kidney, ovaries and in some cases spread to the central nervous system. Nearly all types of BL are characterized by c-MYC gene translocation of chromosome 8 with immunoglobulin genes at chromosome 2, 14 or 22 resulting in high levels of the transcription factor. Recently, MCL-1 was shown to be overexpressed in BL and is a critical survival factor in pre-clinical BL models using genetic and chemical approaches. CDK9, a serine/threonine kinase, regulates transcription elongation through phosphorylation of RNA polymerase II at serine 2 (pSer2-RNAPII). AZD4573 is a highly potent and selective CDK9 inhibitor that results in downregulation of short-lived transcripts and labile proteins such as c-MYC and key survival proteins including MCL-1 and BFL-1, resulting in apoptosis in several hematological malignancies (Cidado et al. 2020 &amp; Boiko et al. 2021) We selected several preclinical BL models to assess the rapid apoptogenic potential of AZD4573 in vitro and in vivo. To measure apoptosis, we evaluated cleaved caspase-3 (CC3) induction following acute treatment (6h) using Caspase-Glo 3/7. Using this assay, we found that 3 BL models were sensitive to CDK9 inhibition (EC50 &amp;lt; 100nM; max. CC3 &amp;gt; 50%) and 2 were resistant (EC50 &amp;gt; 100nM; max. CC3 &amp;lt; 50%). Treatment with AZD4573 in these sensitive BL cell line models decreased pSer2-RNAPII, and downregulation of MCL-1 and c-MYC within 6 hours. In AZD4573 resistant lines we observed relatively higher levels of anti-apoptotic proteins BCL2 and BCL-xL, compared to the sensitive cell lines. These increased levels potentially lead to the decreased sensitivity to AZD4573, which does not reduce levels of BCL-2 and BCL-xL due to their longer half-life. Using CRISPR genetic knockouts we demonstrate that BL cell lines are dependent on c-MYC and MCL-1 and that loss of these protein decreases caspase induction after AZD4573 treatment, highlighting the importance of these proteins in driving the efficacy of AZD4573. In vivo AZD4573 dosed on a once weekly schedule resulted in tumor growth inhibition in aggressive BL cell line xenografts Namalwa and Ramos (40-60%) and decreased pSer2-RNAPII, MCL-1 and c-MYC and increased CC3 8 hours after treatment. AZD4573 is currently in a phase 2 study (NCT05140382) to assess the efficacy, safety, and PK of AZD4573 in patients with relapsed or refractory Peripheral T-cell lymphomas (pTCL). Our findings demonstrate that targeting CDK9 with AZD4573 can effectively induce apoptosis in pre-clinical BL models and could be an effective therapy for BL patients.

Routine Whole Genome Sequencing Offers a More Comprehensive Assessment of IG/TCR Gene Rearrangements for MRD Targets in Acute Lymphoblastic Leukemia - Data from the First 62 Cases from the NHS England Genomic Medicine Service

Introduction Minimal residual disease (MRD) monitoring is fundamental for risk stratification and treatment in acute lymphoblastic leukemia (ALL). Sensitive molecular assay requires presentation samples to be screened for immunoglobulin and T-cell receptor (IG/TCR) gene rearrangements. Previously samples were screened by PCR, heteroduplex analysis and Sanger sequencing to identify clone defining IG/TCR gene rearrangements. Subsequently these sequences were used to develop qPCR assays for MRD follow-up monitoring. More recently, targeted IG/TCR high-throughput sequencing (HTS) has been used with similar panels to identify gene rearrangements. Both these methods have limitations, in that only rearrangements covered by the panels are detected, and the techniques are labour intensive and expensive. Whole genome sequencing (WGS) has potential to discover all IG/TCR rearrangements in a single assay giving greater potential to detect markers for follow up MRD monitoring. In England all patients with ALL are eligible for WGS at diagnosis via the National Health Service, Genomic Medicine Service (NHS GMS). Methods Sixty-two patients, presenting with ALL at either Great Ormond Street Hospital or Bristol Children's Hospital, had short-read WGS via the NHS GMS performed concurrently with standard of care (SOC) targeted HTS to identify IG/TCR rearrangements. A bespoke bioinformatic pipeline was developed to identify IG/TCR rearrangements in the WGS samples. SOC targeted HTS analysis was performed using the open source Vidjil bioinformatic software. A cut-off of 5% (clone proportion of total clonotypes) was used to define an acceptable disease-specific clone for monitoring. Results All 62 samples had a greater number of rearrangements detected by WGS than targeted HTS. The mean number of clones &amp;gt;5% detected was 2.7 by targeted HTS and 3.1 by WGS. For rearrangements identified by targeted HTS at a 5% cut-off, 97% (185 out of 190) were identified by WGS. All targets were identified by WGS using a 10% cut-off (Table 1).Despite the lower depth of coverage of the WGS assay, it retrieved 89% of sequences seen in SOC down to a level of 1%, as well as additional re-arrangements not seen by the SOC assay (Figure 1). As well as additional rearrangements being discovered by WGS, we observed novel rearrangements (dd3/ja rearrangements) not previously identified using a targeted approach. To establish if the additional sequences identified by WGS were clinically applicable as MRD targets, we designed allele specific oligonucleotides for a representative 32 rearrangements for testing by qPCR. Over half (n=16) were able to be used to track MRD in follow-up samples. New primer/probe sets were designed for the novel dd3/ja rearrangements identified by WGS, these were also tested by qPCR and performed well enough to be clinically applicable for MRD monitoring. Conclusions WGS approach outperformed targeted HTS, identifying more IG/TCR rearrangements suitable for molecular MRD monitoring in all patients. This is of particularly benefit in cases where targeted HTS only identifies 1 or 2 potential MRD targets. WGS also has the advantage of identifying other key gene rearrangements (BCR-ABL, KMT2A, SIL-TAL, IKZF1) that make excellent targets for MRD monitoring. Beyond this, WGS delivers all diagnostic information, germline and additional somatic findings that aid diagnosis. For ALL, in one test WGS can now replace all molecular diagnostics. In England all patients with leukemia are eligible for centralised WGS. With continued improvements in turnaround times, and reducing costs, we propose that WGS should be the new diagnostic standard for pediatric ALL

Antigen Receptor Gene Repertoire Profiling in Kidney Transplant Recipients Post-Sars-Cov-2 Vaccination

Kidney transplant recipients (KTRs) are highly susceptible to viral infections, while also exhibiting a higher risk of developing severe COVID-19. Recent studies have demonstrated a suboptimal humoral response to current COVID-19 vaccines in this cohort, necessitating multiple vaccine doses. Nevertheless, low rates of seroconversion have been observed even after booster vaccinations, highlighting the need for the in-depth characterization of both humoral and cellular immune responses towards the development of innovative and effective immunoprophylactic strategies tailored for KTRs. Here, we investigated the immunogenetic features of the T cell receptor (TR) and B cell receptor immunoglobulin (BcR IG) gene repertoires in KTRs using targeted Next Generation Sequencing (NGS). In more detail, we studied 19 KTRs at two consecutive timepoints, after the 2 nd (T1) and multiple vaccination doses (T2), respectively. We included as a control group 19 healthy individuals (HIs) sampled either after two mRNA vaccination doses or after a documented SARS-CoV-2 infection. Seroconversion (&amp;gt;1300 AU/ml of anti-RBD IgG) was measured by the Abbott SARS-CoV-2 IgG assay: all HIs tested positive, in sharp contrast to KTRs who failed to exhibit humoral responses after the 2 nd dose (T1). Immunogenetic characterization of TR/BcR IG gene repertoires was performed by paired-end NGS; resultant data were processed through the TRIP software. Only productive V(D)J gene rearrangements were considered for the computation of clonotypes (i.e. TR/BcR IG gene rearrangements with identical V gene usage and complementarity-determining region 3 amino acid sequence-CDR3 aa). Starting with the TR gene repertoire, diversity significantly increased at T2 (average Shannon diversity: KTR-T1=638 vs KTR-T2=3212, p&amp;lt;0.0005| KTR-T2=3212 vs HI=2146, p&amp;lt;0.05). TR clonality (mean cumulative frequency of the 10 dominant TR clonotypes/sample) was significantly increased at T1 in KTRs vs HIs (KTR-T1=37% vs HI=25%, p&amp;lt;0.05), eventually decreasing at T2, reaching comparable levels to those measured in HIs (KTR-T2=27%). At T1, a median of 7.6% of TR clonotypes/sample in KTRs were SARS-specific (high-affinity TR clonotypes to SARS-specific peptide-MHC complexes, &amp;gt;0.9 binding score determined by the ERGO-II algorithm), almost identical to what we found for HIs (7.7%). Although the TR gene repertoire was renewed in T2, the percentage of SARS-specific clonotypes remained essentially identical (median of 7.8%). Turning to the BcR IG gene repertoire, despite the absence of a serological response at T1, the percentage of the predicted SARS-specific BcR IG clonotypes (high-homology with SARS-specific CDR3 aa sequences published at CoV-AbDab, &amp;gt;0.9 identity score) did not differ between KTRs (0.72% at T1 and 0.65% at T2) vs HIs (0.63%), raising the intriguing possibility of B cell anergy in the former. Overtime assessment revealed that BcR IG clonotype diversity significantly increased at T2 vs T1 in KTRs (average Shannon diversity: KTR-T1=12006 vs KTR-T2=38576 p&amp;lt;0.05) and strongly correlated with the presence of SARS-specific BcR IG clonotypes (r=0.65, p&amp;lt;0.001). Nevertheless, the cumulative fraction of SARS-specific BcR IG clonotypes at KTR-T2 decreased (median cumulative frequency of SARS-specific clonotypes: KTR-T2=0.45% vs HI=0.67%, p&amp;lt;0.05). Of note, multiple immunizations resulted to SARS-specific BcR IG clonotypes with a significantly increased number of somatic hypermutations (SHM) compared to either HIs (p&amp;lt;0.0005) or T1 (p&amp;lt;0.05). In conclusion, our findings suggest that the lack of seroconversion at T1 in KTRs is potentially counteracted by cellular sensitization, likely mediated by SARS-specific T cells, that is sustained by booster vaccination. The latter also drives the accumulation of SHM in BcR IG clonotypes, suggesting distinct differentiation trajectories (germinal center-independent at T1 vs germinal center-dependent at T2) that could give rise to new B cell clones with increased neutralizing potency.

Hematological Activity in Primary Antiphospholipid Syndrome: A Common Manifestation Outside the Classification Criteria

Introduction: Thrombocytopenia and autoimmune hemolytic anemia (AIHA) are extra-criteria manifestations of antiphospholipid syndrome (APS). They have a higher incidence in secondary cases, and there is little information on primary antiphospholipid syndrome (pAPS). Aim: To evaluate the clinical characteristics and profile of antibodies in pAPS with hematological activity. Material and methods: A retrospective study included patients with pAPS, diagnosed according to the Sapporo criteria with follow-up at third level of care in Mexico City from 1990 to 2021. Descriptive statistics were used, and chi-squared test, Mann-Whitney U test, and logistic regression were performed. Results: A total of 97 patients with pAPS were included; 73.2% (71) were women with a mean age of 33.9 + 11.5 years and a mean follow-up of 132 months (24-360). We found that 77% (75) presented thrombotic and 37% (36) obstetric manifestations. In 68% (66) of patients, hematological manifestations (non-exclusive groups) were found. Further, 63.9% had thrombocytopenia, the most common kind being severe, and a minority had AIHA in 13.4%. Table 1 describes the clinical manifestations. Patients with thrombocytopenia were younger, with 32 + 10.3 years vs 37 + 13 years ( p = 0.046). A total of 59.7% (37) had a triple marker. In comparison with patients without thrombocytopenia, 69.4% (43) presented positive lupus anticoagulant vs 48.6% ( p = 0.043) and OR 2.5 (CI 95% 1.06-6.1). An 83.9% (52) had positive IgG anticardiolipin vs 62.9% ( p = 0.019), OR 3.21 (CI 95% 1.19-8.63) with a higher titer of 19.4 vs 6.1 ( p = 0.017) and OR 1.01 (CI 95% 0.99-1.04). They had higher titers of antiβ2 glycoprotein-I IgM of 6.8 vs 5.2 ( p = 0.019) with OR 1.03 (CI 95% 0.99-1.06) and of antiphosphatidyl serine/prothrombin IgM of 52.8 vs 16.8 ( p = 0.004), OR 1.01 (CI 95% 1- 1.02). In patients with AHAI, anti-β2 glycoprotein-I IgM antibody titers of 7.1 vs 5.9 were identified, which were higher compared with those who did not present it ( p = 0.026). Those without hematological activity had more thrombosis ( p &amp;lt; 0.001). When comparing the groups with and without thrombosis, we observed that those with thrombosis had lower titers of antiβ2 glycoprotein-I IgM in 5.8 vs. 7.4, p = 0.036, OR 0.97 (CI 95% 0.94-1) and higher anti-phosphatidyl serine/prothrombin titers in 61.2 vs 12.1, p = 0.021 with OR 1.02 (CI 95% 1-1.04). A total of 76.3% (74) received anticoagulant and 24.7% (24) received antiplatelet. Immunosuppression was prescribed in 38.1% (37). Those with severe thrombocytopenia received: prednisone in 54.5% (18) of cases, methylprednisolone in 36.4% (12), rituximab in 3% (1), and splenectomy in 36.4% (12). In patients with AHAI, 84.6% (11) received immunosuppressants and 46.2% (6) underwent splenectomy. Conclusion: A higher incidence of hematological activity was identified in pAPS, mostly in young women. The most common kind was severe thrombocytopenia, as opposed to what was described in literature as mild to moderate. Differences were found regarding the titer and positivity of antibodies. With thrombocytopenia, there was a higher frequency of positive lupus anticoagulant, anticardiolipin IgG and antiβ2 glycoprotein-I IgM with higher titers. Patients had anti-phosphatidyl serine/prothrombin antibodies as an extended panel and a correlation with hematological activity was identified. Knowing the serology of pAPS is useful to identify in good time patients who may present severe hematological manifestations. No patient died of a hematologic complication; however splenectomy was required in some cases. To our knowledge this is one of the few studies that evaluate hematological manifestations in pAPS.

Design of a Superior Factor VIIIa Mimetic By Coupling a Membrane Binding Domain to a Factor IXa Binding Antibody Fragment

Emicizumab is a bispecific antibody that augments Factor X (FX) activation by factor IXa (FIXa) in the absence of factor VIIIa (FVIIIa) by binding and approximating the substrate FX and protease FIXa. Clinical use of this FVIIIa mimetic significantly reduces bleeding episodes in Hemophilia A (HA) patients. Membrane dependent assembly of FVIIIa-FIXa-FX is an absolute requirement for the intrinsic tenase activity. In contrast, emicizumab lacks any membrane binding ability itself and exhibits significant FXa generation even in the absence of membranes. This major distinction likely allows FVIIIa to function at sub-nanomolar concentrations to greatly accelerate the activation of FX while only much lower enhancements of rate are achieved at several 100-fold higher concentrations of emicizumab. We sought to test whether the addition of a membrane binding feature in the monospecific FIXa binding fragment of emicizumab could alone approximate the cofactor activity obtained with the bispecific antibody. We first generated a single chain antibody fragment (scFv) of the FIXa binding arm of emicizumab by joining the variable heavy chain (V H) with the variable light chain (V L) to produce V H9V L. In line with previous observations that FIXa one arm antibodies of emicizumab that can only bind FIXa and impart a modest cofactor mimetic activity, V H9V L, facilitated FIXa-mediated FX activation at a much lower rate in comparison to bispecific emicizumab. The scFv, V H9V L, was used as a template to introduce membrane binding properties. The Factor V C2-domain, which binds membrane, was fused at the C-terminus of V H9V L via a repeating Gly 4Ser flexible linker of ~ 5.7 nm to allow the V H and V L domains of the scFv to potentially orient correctly at the membrane surface. The resulting construct V H9V LC2 showed a substantial amplification in cofactor mimetic activity as compared to V H9V L. In vitro kinetic analyses with purified proteins and synthetic phospholipid vesicles containing phosphatidylcholine (PC) and phosphatidylserine (PS) (PC:PS, 75:25 %) showed approximately 22-fold faster activation of FX by V H9V LC2 as compared to V H9V L. Remarkably, despite its inability to bind FX, cofactor mimetic activity of V H9V LC2 was about two-fold higher than that of emicizumab. Potent enhancement of FX activation by V H9V LC2 observed with purified proteins was also reflected in thrombin generation assays (TGA). V H9V LC2 added at 100 nM brought thrombin generation into the reference range in congenital FVIII-deficient HA plasma supplemented with 4 µM PC:PS and triggered with either low tissue factor (0.1 pM), or factor XIa (0.1 nM). The role of C2 domain-mediated increase in cofactor activity of V H9V LC2 was further assessed by using an antibody fragment (E 9scFv) that inhibits the binding of factor V to membranes. Increasing concentrations of E 9scFv caused a dose-dependent reduction in the rate of FXa formation catalyzed by the complex of V H9V LC2: FIXa (20nM:25nM). Inhibition saturated at ~ 85% at 100 nM E 9scFv, indicating a substantial contribution of membrane binding to the cofactor mimetic function of V H9V LC2. To evaluate whether V H9V LC2 can restore clotting in HA plasma with FVIII inhibitors, normal pooled plasma was supplemented with a FVIII neutralizing antibody to mimic FVIII inhibitor plasma. Addition of V H9V LC2 to this plasma restored clotting time to the levels shown by normal pooled plasma, suggesting that V H9V LC2 can bypass FVIII inhibitor activity. The engineered scFv with a factor V C2 domain fusion, V H9V LC2, demonstrates that mere addition of a membrane binding feature to the FIXa arm of the emicizumab surpasses the mimetic activity achieved by a bispecific antibody that exploits both bridging and allostery to be an effective cofactor. Such a construct also avoids the burden of productive assembly from a combination of three separate polypeptide chains, as in a functional bispecific antibody. Replicating the membrane binding feature into bispecific FVIIIa mimetic antibodies could further amplify function and more closely resemble FVIIIa in both activity and membrane dependent regulation. Our results provide surprising insights into how a single chain, monospecific, membrane-anchored FVIIIa mimetic enhances the catalytic activity of FIXa.

Should We Screen All the Immune Thrombocytopenia Patients for Helicobacter Pylori Infection in High-Prevalence Countries?

Background: Immune thrombocytopenia (ITP) is an autoimmune disease mediated by anti-platelet autoantibodies. Helicobacter pylori ( H. pylori) may modulate the Fcγ-receptor balance of monocytes/macrophages in favor of activating Fcγ receptors, and its components may mimic the molecular makeup of platelet antigens. In Mexico, the percentage of seroprevalence is 66%. There is not enough evidence available to determine the impact of the screening and eradication of H. pylori in ITP patients in the context of a region with a high prevalence of this infection. Study design and methods: This retrospective cohort study included all adult patients older than 18 years, newly diagnosed with ITP in a reference center in Mexico City. We evaluated the frequency and impact of H. pylori screening and eradication in patients diagnosed between 2016 and 2023. The H. pylori screening was made by urea breath test. Response criteria were based on the 2019-American Society of Hematology guidelines. Results: After a median follow-up of 38 months (interquartile range (IQR) 16.7 - 58.2 months), 106 patients with ITP were identified, of whom 73 (69%) were women. Most of them (92.3%) required first line treatment, all with corticosteroids: 44.9% dexamethasone, 44.9% prednisone, and 10.2% methylprednisolone followed by prednisone. Only 3 patients (3%) received intravenous immune globulin. Overall response rate (ORR) was observed in 76% with complete response (CR) of 47%, and partial response (PR) of 29%. During follow-up, 28 patients (26.5%) developed chronic ITP. Screening for H. pylori was performed in 82 patients (77.5%): 35.8% at diagnosis, 21.9% at relapse and 19.8% at a different time point. A comparison was made between the patients who underwent H. pylori test at diagnosis (N=38) vs. those who didn't (N=67) and the only baseline difference was a younger age in patients untested: 38 vs. 44 years (p=0.026) (see Table 1). Only 2/38 patients tested for H. pylori at diagnosis had gastrointestinal symptoms and both were negative for H. pylori. All the patients with a positive result for H. pylori infection received treatment and in all the patients eradication was confirmed. There was no significant difference in responses to the first line treatment between patients with and without H. Pylori infection: CR 30.0% vs. 44.4%, PR 50.0% vs. 27.7% and non-response 20.0% vs. 27.7% (p=0.387). Patients who underwent an H. Pylori test at diagnosis and eradication treatment if positive, had a lower relapse rate: 88.4% vs. 62.1%, p=0.009 (OR 0.213; 95% CI 0.069-0.664). Median relapse free survival was higher in patients who were screened for H pylori at diagnosis vs. those who were not: 15 months (95% CI 2.1-27.9 months) vs. 5 months (95% CI 2.5-7.5 months), p=0.001) (see Figure 1). Conclusions: These results suggest that screening and eradication of H pylori since diagnosis can be associated with a lower risk of relapse and a higher relapse-free survival in the context of a country with a high-prevalence of H. pylori infection. We found no relation with the presence of H. pylori and the platelet count or gastrointestinal symptoms, so the screening should be considered regardless of these aspects. The impact of treating H. pylori on ITP-responses cannot be assessed because all the patients receive eradication treatment, but the strategy of screening and treating if positive is associated with lower relapse-rates.

Factor V Inhibitor in COVID-19 an Unusual Presentation

Introduction : A huge variety of haematological manifestations and derangements have been frequently published with COVID-19 infections, however Factor V deficiency and COVID-19 infection has not been documented. We are reporting a case of acquired Factor V deficiency due to inhibitor caused by COVID 19 infection . Factor V deficiency is a rare bleeding disorder having either inherited or acquired forms. Acquired factor V deficiency (AFVD) can present at any age, with the manifestations ranging from abnormal deranged PT/PTT to serious bleeding and quite often challenging to diagnose. The risk factors for development of inhibitors include surgical procedures, Hepatitis, antibiotic, Urinary tract infections, blood transfusions, haemodialysis, malignancies like multiple myeloma, amyloidosis and autoimmune disorders. Around 30% of cases of AFVI are found to be idiopathic. Studies suggest that there is a temporal Connection between SARS-CoV-2 infection and the development of coagulopathy. This could be possible since COVID -19 infection is well associated with autoimmune coagulation abnormalities such as lupus anticoagulant, antiphospholipid antibodies, and venous thromboembolism. The precise mechanisms behind the relationship between Covid19 infection and development of Factor V deficiency is not fully understood. SARS-CoV-2 infection induces a proinflammatory state causing endothelial damage, platelet activation and hyperviscosity. We suggest that the factor V inhibitor was formed in the setting of immune dysregulation from the underlying COVID-19 as coagulation profile was normal before admission to hospital. Result and method: A 62 year old gentleman with background of dyslipidemia and IBS was admitted to the hospital with asymptomatic COVID pneumonia. The patient was started on Lopinavir-Ritonivir 500mg BID and enoxaparin 40mg OD. Upon routine testing patient was found to have a deranged coagulation profile (PT: 41.9sec/APTT: 104.7sec/INR: 3.5/Fibrinogen: 3.04gm/L/ D-dimer: &amp;lt;0.30mg/L FEU). Daily results kept showing a steady rise of coagulation parameters (PT: 46.5sec/APTT: 122.0sec/INR: 3.9) however the patient was asymptomatic. Results demonstrated severe factor V deficiency (&amp;lt;5.7%) with mildly reduced Factor VII (53.8%) and a mildly elevated factor VIII (195.4%). PT and APTT mixing studies showed no correction of the prolonged clotting times before and after 2 hours incubation, hence the failure of correction suggested the presence of an inhibitor for which Bethesda test for Inhibitor's titer and autoimmune screening were sent. Considering the Factor V inhibitor deficiency with background of active COVID-19 infection and to prevent bleeding complications, the patient was started on Inhibitor eradication therapy with Dexamethasone 20 mg, and Immune globulin intravenous 45 gm. Inhibitor titers came back positive for an inhibitor level of 9 BU/ml. Autoimmune screen for ANA and anti-B2 glycoprotein came back negative. Malignancy was ruled out. Throughout the hospital stay , we did not encounter any bleeding manisfestions or drop in haemoglobin. Since the patient had a previous record of normal coagulation profile in 2019, a diagnosis of COVID-19 infection-related Factor V deficiency with inhibitor was made. After a four-days course of inhibitors eradication, PT INR and aPTT started coming down with no significant complications, the patient was discharged on oral prednisolone and vitamin K. Follow up in the clinic after 3 weeks showed complete normalization of coagulation profile. Conclusion: A wide range of haematological manifestation have been reported with COVID-19 infection, but the association of Factor V deficiency due to inhibitors and COVID-19 infection have not been reported in the literature earlier. Treatment will be focusing on treating the primary cause and eradication of the inhibitors.

A Phase 3 Study of Eltrombopag Vs. Standard First-Line Management for Newly Diagnosed Immune Thrombocytopenia in Children: Trial in Progress Update

Background: Immune thrombocytopenia (ITP) causes low platelet counts, variable bleeding symptoms, and reductions in health-related quality of life (HRQoL). Despite this, novel therapies for newly diagnosed ITP have not been introduced in over 30 years. Eltrombopag, a thrombopoietin receptor agonist (TPO-RA), was FDA approved for children with chronic ITP in 2015. The use of eltrombopag for adults with newly diagnosed ITP has been described in two small single-center trials (Tripathi et al, Int J Hematol, 2014; Gόmez-Almaguer et al, Blood, 2014), and pediatric hematologists do use TPO-RAs off-label in some cases of newly diagnosed ITP (Neunert et al, Pediatr Blood Cancer, 2016). TPO-RAs may be an efficacious and more durable first-line therapy for children with newly diagnosed ITP who require treatment. This abstract provides a 4-year interval update on the Pediatric ITP Newly diagnosed patients Eltrombopag vs Standard therapy (PINES) trial in progress. Study Design and Methods: The PINES trial, NCT03939637, is an investigator-initiated prospective, open label, randomized, multi-center trial sponsored by the ITP Consortium of North America (ICON) and funded by Novartis. The primary objective is to determine if the proportion of patients with a platelet response, defined as ≥3 of 4 platelet counts &amp;gt;50 x10 9/L during weeks 6-12 without rescue treatment, is significantly greater in patients with newly diagnosed ITP treated with eltrombopag compared to those treated with standard first-line therapy. Patients (n=162) from 30 ICON centers (Figure) are randomized 2:1, stratified by age and treatment status, to receive the experimental treatment, eltrombopag, or investigator's choice of 3 standard first-line therapies: prednisone, intravenous immune globulin or anti-D globulin at standardized doses. Eligible patients are ages 1 to &amp;lt;18 with primary ITP, within 3 months of diagnosis, with platelet count &amp;lt;30 x10 9/L who require pharmacologic treatment from the perspective of the treating clinician. Baseline treatment status is 1) upfront treatment, or patients within 10 days of ITP diagnosis with no prior treatment, and 2) treatment failure, or patients previously managed with observation or a first-line standard agent. Patients are ineligible if they can be observed without medication or have severe bleeding. A one-sided z-test, at alpha=0.025, will be used to compare the proportion of patients who have a platelet response between the two treatment arms. All randomized patients will be analyzed in the intention-to-treat analysis. Secondary objectives include treatment arm comparison of bleeding scores (WHO Bleeding Scale and Modified Buchanan Score), changes in HRQoL (measured by the Kids ITP Tool, Hockenberry Fatigue Scale, PROMIS, and Global Change Scale), and changes in percentage of CD4 +25 +Foxp3 + regulatory T (Treg) cells. Due to challenges related to recruitment and the logistics and safety of obtaining frequent assessments during the COVID-19 pandemic, the primary endpoint was re-defined in 2021 to what is considered a clinically equivalent definition of response, from ≥6 of 8 weekly to ≥3 of 4 biweekly platelet counts &amp;gt;50 x10 9/L during weeks 6-12. Baseline Characteristics: Since first site activation in 2019, 100 patients have been enrolled (Table). Subjects include 42 patients aged 1-&amp;lt;6 years, 34 patients aged 6-&amp;lt;12 years, and 24 patients aged 12-&amp;lt;18 years. Thirty-six patients received upfront treatment and 64 patients had treatment failure prior to enrollment. Median baseline platelet count at enrollment was 5 x10 9/L (range 1 - 28 x10 9/L). Median WHO Bleeding Scale at enrollment was 1 (range 0 - 3) and median modified Buchanan Overall Score was 3 (range 0 - 3). Baseline HRQoL surveys have been obtained for &amp;gt;80% of eligible subjects. Treg samples have been obtained for 95% of subjects, and 74% of subjects have consented to optional biology studies. Conclusion: Despite historical difficulty completing interventional trials in pediatric ITP and pandemic-related enrollment challenges, PINES trial enrollment is progressing successfully and will ultimately provide definitive data regarding the efficacy of eltrombopag in children with newly diagnosed ITP. There are no current data supporting the use of TPO-RAs for newly diagnosed ITP in children, or as monotherapy for newly diagnosed ITP in adults. Results from this trial therefore carry the potential to change management practices in ITP.

Infection Outcomes and Hypogammaglobulinemia in Patients with Non-Hodgkin Lymphoma Treated with Immunoglobulin Replacement Therapy

CONCLUSIONS Patients diagnosed with non-Hodgkin lymphoma (NHL) histologies often develop hypogammaglobulinemia (HGG), a secondary immunodeficiency (SID) that is associated with exposure to NHL-related treatments. Those with HGG frequently experience infections, which represent the leading cause of death in NHL (33% of cases). Patients with HGG who experience recurrent infections may benefit from immunoglobulin replacement therapy (IgRT). We aimed to assess real-world IgRT utilization and associated infection outcomes in patients with NHL. This retrospective, longitudinal, observational study evaluated HGG (immunoglobulin G [IgG] &amp;lt; 500 mg/dL) status and infection outcomes among adult patients diagnosed with NHL after 2010 who had ≥ 12 months of clinical data and ≥ 3 visits/year after NHL diagnosis and received immune globulin infusion (human), 10% (IG10% [ie, Gammagard Liquid]). De-identified clinical data from the Massachusetts General Brigham Research Patient Data Registry was used. IgG testing, infection outcomes, and antimicrobial use were compared 3 months before versus 3 months after initiation of IG10%. Generalized estimating-equation logistic regression models were used to obtain odds ratios (ORs), 95% confidence intervals (CIs), and P values. A total of 13,232 patients with NHL were identified. Among patients with NHL, 623 (4.7%) received ≥ 1 IgRT during the follow-up period, of whom, 563 (90.4%) received IG10%. The majority of patients with NHL treated with IG10% were male (61.3%), White (90.1%), with a median (interquartile range [IQR]) age of 62 (53-69) years, and median (IQR) follow-up of 4.8 (2.3-7.7) years. Median (IQR) administrations of IG10% across recipients were 2 (1-4) and over half of patients (55.6%) received ≥ 2 IG10% administrations. Median (IQR) time from initiation of IG10% to end of follow-up was 25.6 (6.8-55.2) months. Most patients had ≥ 1 IgG test in the follow-up period (n = 532 [94.5%]), median (IQR) number of IgG tests was 10 (4-27), and 84.2% of patients had ≥1 IgG test result available before initiation of IG10%. Of the patients treated with IG10% who had IgG test results available (n = 532), 81.6% were identified with HGG (&amp;lt; 500 mg/dL). Few patients had IgG testing at the time of first NHL diagnosis (13.3%), and median (IQR) time from diagnosis to first IgG test was 4.9 (0.8-17.4) months. Of the patients with ≥ 2 IG10% administrations (n = 313), median (IQR) time from the first administration of IG10% to subsequent IgG testing was 1.0 (0.3-2.8) month. Most patients (91.3%) who received ≥ 2 IG10% administrations and ≥ 1 IgG testing had an IgG test between first and second administrations of IG10%. Median (IQR) serum IgG levels were higher in the 3 months following initiation of IG10% versus 3 months before initiation of IG10% (520.0 mg/dL [413.0-670.0] vs 339.0 mg/dL [228.0-405.0]), and significantly fewer patients had HGG (34.7% vs 83.0%, P &amp;lt; 0.0001). In addition, significantly lower odds of infection (OR 0.60, 95% CI 0.47-0.75), severe infection (OR 0.40, 95% CI 0.32-0.50), and associated antimicrobial use were observed 3 months after IG10% initiation (Figures 1 and 2). In this real-world study of patients with NHL, treatment with IG10% was shown to be associated with significantly lower rates of HGG as well as lower odds of infection, severe infection, and antimicrobial use 3 months after IG10% initiation. Guidelines and consensus-based recommendations are needed to optimize the use of IgG testing, initiation of IgRT, and SID management in patients with NHL.

Avatrombopag Safe and Effective in Treatment of Severe Refractory Thrombocytopenia in a Pediatric Patient with Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia Following Allogeneic Hematopoietic Stem Cell Transplantation

Background: Persistent thrombocytopenia following allogeneic stem cell transplantation (alloHSCT) leads to an increase in morbidity and mortality. Causes of severe thrombocytopenia following HSCT include poor graft function, graft-versus-host disease (GVHD), infections, drug toxicities, underlying autoimmune diseases and relapse of underlying disease. Treatments can include thrombopoietin receptor agonists (TPO-RA), immunomodulators, and stem cell boost. Avatrombopag is a second generation TPO-RA approved for the treatment of primary chronic immune thrombocytopenia (ITP) in adult patients with poor response to standard treatments or in patients with chronic liver disease (CLD) associated thrombocytopenia requiring surgical interventions. Phase III clinical trials have shown greater platelet responses in chronic ITP and reduced transfusion needs in patients with CLD. Avatrombopag differs from other TPO-RA medications due to majority of elimination occurring in the gut, thus avoiding hepatic toxicity. Additionally, it does not require dietary restrictions. As there is limited literature in pediatric patients following alloHSCT, we describe the use of avatrombopag in a pediatric patient with chronic severe thrombocytopenia (&amp;lt;10 x10 3/uL) post HSCT. Objective: To describe the use of avatrombopag for severe refractory thrombocytopenia in a pediatric alloHSCT recipient. Case Presentation: We present a 14-year-old Hispanic male with Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL) who received a maternal haploidentical HSCT with myeloablative conditioning and post-transplant cyclophosphamide. His post HSCT course was complicated by adenoviremia, hepatic variant GVHD, Enterobacter cloacae bacteremia and poor graft function. Seven months following HSCT, patient was found to be severely pancytopenic. Bone marrow morphology demonstrated 20-30% cellularity without evidence of leukemia relapse or GVHD. Lab evaluation was not consistent with transplant associated thrombotic microangiopathy. He received a stem cell boost for presumed poor graft function. Patient demonstrated partial response by resolution of neutropenia and anemia; however, severe thrombocytopenia persisted. Multiple bone marrow evaluations remained negative for disease with full donor chimerism and negative TCR clonality. CXCL9 was noted to be elevated at 4270 and Immature Platelet fraction was elevated at 8.8. Platelet antibodies remained negative and all findings consistent with persistent severe ITP. Multiple treatments including romiplostim; eltrombopag; high dose immune globulin (IVIG); rituximab; methylprednisolone; and decitabine were not successful in improving platelet count (Figure 1). Evaluation for severe thrombocytopenia remained negative. During the course of severe thrombocytopenia, patient required several hospitalizations for symptomatic thrombocytopenia, requiring multiple platelet transfusions. Due to the severity of disease, he was granted a compassionate use investigational new drug (IND 165837) for avatrombopag and began on day +332. Dosing was initiated at 20 mg daily with a dosage adjustment to achieve platelet recovery to &amp;gt;50 x10 3/uL free from transfusion. The dose was titrated per manufacturer to maximum dose of 40 mg daily with response to platelet count &amp;gt;100 x10 3/uL in approximately 6 weeks time. By three months of treatment, dose was titrated down 40 mg three times a week and 20 mg on alternating days. Patient continues to maintain platelets above 100 x10 3/uL with no medication toxicities or adverse events noted. Patient has been able to return to his pre-alloHSCT lifestyle including contact sports and re-immunizations.

Clocking the Processes and Challenges of Urgent Red Blood Cell Exchange for Acute Complications of Sickle Cell Disease

Background: Red blood cell exchange transfusion (RBCX) is the therapy of choice for some acute complications of sickle cell disease (SCD), including stroke, severe acute chest syndrome and multi-organ failure. Rapid initiation of RBCX is critical to reduce morbidity and mortality in those with acute complications of SCD. A typical RBCX procedure requires anywhere from 6-10 units of packed red blood cells (RBC) and measures are taken to provide higher-fidelity RBC matches (key RHCE and KEL antigens at a minimum, ±suitable KIDD, FY, Ss profiles for the sensitized). Our recent review of urgent apheresis transfers to our tertiary-care centre, including 26 for RBCX, demonstrated that the median time per transfer was approximately 4 hours. Not yet characterized is the additional time required to start RBCX once a patient arrives. The time required to investigate antibody repertoires, and to procure sufficient quantities of blood that can bypass these specificities, is of interest in a specialty center with substantial but not unlimited investigative expertise and inventory. Methods: This was a retrospective database and chart review of all patients referred to our apheresis service for urgent RBCX for severe complications of SCD between Jan 1, 2013 to Dec 31, 2020. Time stamps for admission, blood bank, and apheresis processes were extracted from the electronic medical record and blood bank database. Categorical variables were analyzed using nonparametric tests. Results: Thirty-eight patients were included (65.8% males, 35.2% females) (Table 1). HbSS was the most common SCD genotype represented (81.6%). Most patients (71.1%) were transferred from another hospital, with the remaining patients arriving through the emergency department (26.3%) and one (2.6%) as a direct admission from clinic. ACS +/- additional complications constituted the most common indication for RBCX (78.9%). Two patients died due to complications of multi-organ failure, the remaining patients were discharged from hospital. Six patients (15.8%) had a positive antibody screen. The majority had RBC phenotype (42.1%) or genotype +/- phenotype (36.8%) on file to facilitate extended matching, and sufficient appropriate units were available in the blood bank in 82% of cases. Four (10.5%) patients required top-up transfusions prior to RBCX due to significant anemia. The median number of units issued per procedure was 9 (IQR 2). The median times from index group and screen (G&amp;S) collection to sample accession, and from accession to result were 23 minutes (IQR 26) and 46 minutes (IQR 31), respectively (Figure 1). The median time from G&amp;S result to RBC units issued was 348.5 minutes (IQR 479). The median total time from patient admission (or determination of RBCX indication) to RBC units issued was 569 minutes (IQR 464). A positive antibody screen, patient origin (ED vs. transfer), and the availability of sufficient RBC units in house were associated with turnaround time from accession of the index G&amp;S sample to the issuing of RBCs (p&amp;lt;0.01 for all). Patients having antibodies to high prevalence antigens (e.g. anti-U) or to multiple targets (demanding unique donor profiles) had some of the longest times to initiate treatment. These constraints necessitated orders and coordinated deliveries of units from across the geographic network of the national blood provider.Discussion: These data from the largest apheresis unit in the country characterize the time invested in blood preparation for an urgent RBCX for acute SCD complications. A positive antibody screen is predictive of increased time to procedure start, reflecting delays inherent to serologic investigations, product sourcing, and serologic crossmatch times. While the precise moment of a blood request was available for a minority of patients, the G&amp;S time stamp was used as a surrogate, thus potentially overestimating turnaround times in the blood bank. Nevertheless, the serologic demands inherent to a case present a tension between speed (treating the disease) and safety (averting immune rejection reactions).

Clinical Characterization of Fusion Genes in Acute Myeloid Leukemia without Recurrent Genetic Abnormalities

Introduction Acute myeloid leukemia (AML) is a biologically highly heterogeneous disease. An increasing number of genetic abnormalities responsible for its pathogenesis are being incorporated in latest classifications to refine AML diagnosis. Myelodysplasia-related AML (AML-MR) is a broad category defined by mutations in a significant number of genes and cytogenetic alterations. Besides, there are still patients that must be classified according to phenotypic characteristics (AML defined by differentiation, AML-DD). Both categories still constitute heterogeneous groups in which further investigation is warranted to better define their underlying pathogenesis. Transcriptome sequencing has proven to be useful for fusion genes identification. In this study, we aimed to identify fusion genes by RNAseq in AML without recurrent genetic abnormalities and correlate them with clinical and phenotypic data. Methods One hundred and nine patients diagnosed with AML without recurrent genetic alterations between 1998 and 2020 in hospitals from CETLAM cooperative group were included. RNA samples collected at diagnosis were sequenced following a paired-end Illumina protocol. Five additional RNA samples from healthy subjects were sequenced for filtering purposes. Fusions were detected using STAR-Fusion version 1.9.0, against a reference based on GRCh38 and annotation gencode 35. In 66 cases, mutational information was available from whole-exome or targeted next-generation sequencing. In the other 43 cases, RNAseq data were used for variant calling in 40 recurrently mutated genes. Statistical analysis was done with R (version 4.04). Results Fusion genes candidates were found in 53 of the 109 patients (49%). Likely artifacts were removed by ruling out fusions also found in healthy samples, fusions affecting immunoglobulin genes, fusions involving the same gene with multiple partners within the same sample or those found in more than one case without being a known fusion. After this process, 46 fusion candidates found in 29 different patients (27%) were manually classified as Tier 1 (well-known fusions, n=7), Tier 2 (fusions affecting recurrently mutated genes in AML without a functional validation, n=6), Tier 3 (fusions of unknown significance, n=25) and Tier 4 (fusions found in healthy tissues, n=8). Tier 1 and Tier 2 fusion genes are shown in Table 1. NSD1::NUP98 was the only fusion gene found twice, with the exact same breakpoint, in two male patients of young age (27 and 28 years). ETV6 was the gene more often involved in fusions (n=4), always with a different partner. Patient characteristics of Tier 1 or Tier 2 fusion carriers (FC) are detailed in Table 1. Diagnosis in this group consisted of AML-DD (n=7) and AML-MR (n=4). FC were younger at diagnosis (41 years vs 58 years, P=0.005) and showed a higher median bone marrow blast count (70% vs 54%, P=0.07). Four of the eleven FC (36%) had a normal karyotype, in contrast to 58% in the whole cohort ( P=0.18). No difference was found either in white blood cell count, peripheral blast count or the presence of morphological dysplasia. Ninety-three patients were eligible for intensive treatment. Two FC were primary refractory (75% vs 89%, P=0.18) and died without ever achieving a complete response (CR). Nine patients received transplantation in first CR (autologous, n=2; allogeneic, n=7), one of them dying of transplant-related complications. From the remaining six patients, four experienced relapse. Only one of these patients achieved a sustained remission after a second allogeneic transplantation. Two-year overall survival was similar between FC group and the rest of the cohort (46% vs 53%, P=0.6) Of note, Tier 1 fusions NUP188::PHYHD1 and ETV6::NTRK3 were the only driver events found in two cases. The latter patient presented with a refractory relapse and died 11 months after diagnosis, though nowadays he could have benefit of NTRK inhibitors. In another case, the identification of MLLT10::ATP5MG was useful as molecular marker during follow-up. Conclusions Transcriptome sequencing by RNAseq identifies a considerable amount of fusion genes in AML. Ten percent of our cohort of patients with AML without recurrent genetic abnormalities have at least one Tier 1 or Tier 2 fusion. Infrequent fusions may be driver events in AML with clinical implications in terms of diagnosis, prognosis and targeted therapy.

Single Nuclei Multiomics Analysis of Transcriptional and Chromatin Accessibility of Tumor Cells Uncovers Molecular Signatures and Regulatory Elements of Malignant Clonal Evolution in Multiple Myeloma

Category Multiple Myeloma and Plasma Cell Dyscrasias: Basic and Translational Introduction Multiple myeloma (MM) is a hematological cancer resulting from abnormal proliferation of plasma cells (PC) in bone marrow. The progression of the disease from Monoclonal Gammopathy of Undetermined Significance (MGUS), Smoldering Multiple Myeloma (SMM), to MM involves multiple drivers and dynamic interactions of premalignant/malignant cells. In the advancement of single-cell technologies, the same-cell readouts of nucleus RNA sequencing (snRNA-seq) and Assay for Transposase-Accessible Chromatin sequencing (snATAC-seq) provide a less biased measurement of gene expressions and chromatin accessible regions. Profiling and analyzing both omics data at different stages of MM may reveal essential molecular signatures and regulatory elements for microenvironmental modification and evolution. These insights could benefit MM patients as novel targets for early therapeutic intervention at different stages of progression. Methods Bone marrow aspirates were collected from 33 individuals: Healthy donors (n=3), patients with MGUS (n=2), SMM (n=5), MM (n=21), and Plasma Cell Leukemia (PCL; n=2). Viable mononuclear cells underwent CD138+ sorting and nuclei isolation prior to library preparation. Libraries were prepared with the Chromium Multiome kit (10X Genomics) and sequenced on NovaSeq S4 flow cells at UAMS. The clonal V(D)J arrangements of each sample and expression level from HD samples were used to distinguish malignant (MM) from normal plasma cells (PC). The MM data from snATAC-seq and snRNA-seq were integrated using Signac with the weighted nearest neighbor methods from Seurat. All inter- and intra-tumor clusters were evaluated for markers of high-risk disease, differential gene expression, differentially accessible regions, and transcription factor activities. Results We performed snRNA-seq and snATAC-seq sequencing to generate multi-stage, molecular classified-specific chromatin accessibility and transcriptional profiles of plasma cell neoplasms comprising 439,134 cells across 33 samples. The malignant plasma cells for each sample were extracted by the unique clonotypic signature of the immunoglobulin gene rearrangement. Of these, we detected the highest clonality in MGUS samples, suggesting clonal competition at the premalignant stage. Based on our primary microarray data, we detected the overexpression of molecular signatures for most cells in each sample, confirming the intratumor cell heterogeneity. Furthermore, we used the ratio of 70-gene signature of aggressive disease and proliferation signatures to investigate the clonal composition of MM cells corresponding to the risk of relapse. Surprisingly, the proportion of high-risk cells increased respectively from low to high-risk samples. The expansion of high-risk clonality might exhibit competition among malignant cell subpopulations regarding microenvironmental adaptation. The multiomics integration generated less distinct clusters between MGUS and SMM, indicating the gradual complex alterations of subclonal gene expression and chromatin accessibility in malignant transformation. Interestingly, most accessible regions were found in intronic/distal intergenic regions implying the long-range gene regulation from cis-regulatory elements. Based on the positive/negative correlation of binding motifs within differentially accessible regions, we identified unique enriched transcription factors for each disease stage, suggesting the mechanism patterns in the evolutionary process of malignant cells. Conclusion In the largest multiomics study to date of CD138+ cells, we demonstrate the malignant clonal evolution using gene expression and chromatin accessibility profiling from patients with early-stage disease to highly aggressive form. Throughout the time of disease progression, we found an increase in the proportion of malignant cells with high-risk signatures. Also, we uncover unique signatures of chromatin accessibility for each molecular classification that can further refine our understanding of functional heterogeneity in multiple myeloma. Collectively, our joint single nuclei profiling reveals additional insights into the malignant clonal evolution and investigates novel regulatory elements involved in disease progression.

Integrative Single-Cell RNA-Seq and Single-Cell ATAC-Seq Analysis of Human Plasma Cell Differentiation

Plasma cells (PC) are highly specialized cells representing the end stage of B cell differentiation. They play an important role in humoral immunity by synthesizing and secreting antibodies protecting the host against infections. We have shown that PC differentiation (PCD) can be reproduced in vitrofrom memory B cells (MBC). MBC differentiate into CD20 low/-CD38 - pre-plasmablasts (prePB), CD20 -CD38 +CD138 - plasmablasts (PB) and CD20 -CD38 +CD138 + PC. The molecular changes occurring during PCD are recapitulated in this in vitrodifferentiation model. However, a major challenge exists to decipher the spatiotemporal epigenetic and transcriptional programs that drives the early stages of PCD. We combined single-cell (sc) RNA-seq and scATAC-seq to decipher the trajectories involved in PCD. scRNA-seq experiments on the four populations (MBC, prePB, PB and PC) generated during normal PCD revealed a highly specific transcriptomic profile for MBC and PC, and a strong heterogeneity of the prePB and PB. The prePB stage presented the most differentially expressed genes (DEG) with almost 2000 DEG showing that the most important changes take place during this stage. Epigenetic analysis using scATAC-seq technology showed that prePB, PB and PC stages were clearly separated from the MBC highlighting chromatin remodeling induced by B cell activation and differentiation. As observed on the transcriptomic level, the number of differentially accessible peaks was higher in prePB (4660) than in other stages (MBC: 641; PB: 44; PC: 105). Among genes that were commonly identified using scATAC-seq and RNA-seq, we identified several TF such as BATF and BATF3 in prePB. Interestingly, differentially accessible peaks characterizing the prePB stage were enriched in motifs of BATF3, FOS and BATF belonging to the TF AP-1 family and among these differentially accessible peaks, 38% of peaks presented BATF3 motif. Since BATF3 TF was previously identified operating in short impulse manner at prePB stage, BATF3 target genes may represent a key transcriptional node involved in PCD. The integration of the chromatin accessibility and transcriptomic data revealed a more mature population of prePB cells characterized by open chromatin in PC genes without significant expression suggesting that a population of prePB were already committed to become antibody-secreting cells. To focus on the understanding of the processes occurring during the transition from prePB to PB which remains largely unknown, we computationally clustered and ordered cells. Pseudotemporal analyses underlined maturation trajectories in prePB with early prePB characterized by downregulation of B cell markers ( CD19, CD22, CD83, CCR7, CCL17 and CCL22) and B cell TF (SPIB, PAX5, STAT5A, RUNX3 and BATF3) together with upregulation of PC markers, adhesion molecules and growth factor receptors ( CD27, CD38, SLAMF7, BCMA, ITGA4, IL-6R, IL-6ST and INSR). The transition from early prePB to more mature prePB is associated with downregulation of AICDA and PRC2 complex ( EZH2, EED). In the transitional prePB, a first wave of UPR activation is observed, associated with mTORC1 pathway activation. In these prePB, HSAP5, also called binding immunoglobulin protein (BiP) is overexpressed together with ERN1, leading to the activation of the Ire1 pathway, known to splice XBP-1 (sXBP-1) producing a highly active TF. Then, prePB overexpress EGR1, FOS, CXCR4 and TFRC. Mature prePB overexpress KLF2 that participate in bone marrow PC homing. Then, PB overexpress gene involved in conventional UPR and protein secretion associated to immunoglobulin gene expression. Altogether, these data underlined that prePB present already UPR priming through mTORC1 pathway activation to prepare for PC function. XBP1 driven UPR activation will be coordinated in PB related to antibody synthesis increase. Integration of transcriptomic and epigenetic data at the single cell level revealed that a population of prePB already undergone epigenetic remodeling related to PC profile together with UPR activation and are committed to differentiate in PC. These results and the supporting data generated with our PCD model provide a resource for the identification of molecular circuits that are crucial for early and mature PC biological function and survival. These data thus provide critical insights into epigenetic- and transcriptional-mediated reprogramming events that sustain PCD.

Light Chain Amyloidosis Plasma Cells Show Specific Chromatin Accessibility and Transcriptome

Immunoglobin light chain amyloidosis (AL) is a monoclonal gammopathy characterized by the accumulation of malignant plasma cells (PC) in the bone marrow. These PCs secrete misfolded immunoglobulin light chains fibrils, which deposit in various organs, leading to organ dysfunction. Unlike other monoclonal gammopathies such as Multiple Myeloma, both its rarity and the lower BM PC burden has limited the deep study of the tumor population. The aim of this study was to analyze the transcriptome and epigenome of the PC from patients with AL amyloidosis to identify the role of chromatin modifications in the pathogenesis of the disease. Highly purified clonal PCs were obtained from bone marrow aspirates samples of 58 AL amyloidosis patients at diagnosis, 19 monoclonal gammopathy of undetermined significance (MGUS), 149 MM and 17 healthy donors. RNA was obtained from PCs and Illumina Stranded Total RNA-seq protocol was performed according to manufacturer's specifications. Accessible chromatin mapping was performed using FAST-ATACseq (Corces et al, 2016) with minor modifications. The data obtained were aligned with the GRCh38 version of the human genome and custom pipelines were used for computational analyses. Expression of immunoglobulin genes was removed for RNAseq analysis. Unsupervised principal component analysis of the RNAseq and ATACseq data showed a clear distinction between PCs from AL patients, MM and HD. To obtain a reliable measure of the magnitude of epigenomic and transcriptome changes in AL, we compared the RNAseq and ATACseq data of AL with MM or HD. The RNAseq analysis showed some clear transcriptional programs (TPs) related to AL and different from MM and HD (Figure 1): TP1 showed 89 genes upregulated and associated with positive regulation of protein kinases and TP2 showed 274 genes downregulated and correlated with immunoregulatory interactions and mitotic regulation. On the other hand, TP3 included 560 downregulated genes associated with glutamate pathway and glycosylation of proteins and TP4 revealed 362 upregulated genes linked with B cell activation. Both patterns were identified in AL patients and HD in comparison with MM patients. Regarding chromatin accessibility, we detected a similar number of ATAC peaks between the AL and MGUS samples and with a significant difference compared to the MM samples (p=0.00014), suggesting that AL and PCs from MGUS have a lower chromatin accessibility (Figure 2). These results provide evidence that the biological perturbations in the chromatin accessibility and transcriptome of PCs from AL patients are highly specific. Although the results depicted in Figure 1 demonstrate that AL patients clustered together by relatively homogenous chromatin accessibility and transcriptome, compared with MM patients, we further investigated whether there were any discernible differences among AL patients themselves. To address this, we conducted an analysis of chromatin accessibility and transcriptome in relation to various factors, including age, gender, organ affected, light chain isotype, percentage of tumoral PCs, presence of t(11,14) or amp1q. Remarkably, we identified differences in the transcriptome between AL patients with and without t(11;14), identifying, 46 upregulated genes linked with T cell and lymphocyte apoptosis and inflammatory cytokines production and 257 downregulated genes linked with FGFR family ligand binding and activation and protein ubiquitination and phosphorylation, in t(11,14) positive AL patients. These results suggest that t(11;14) may exert a significant modulation on the transcriptome of PCs of AL patients. Finally, scRNAseq was performed to address the heterogeneity of PCs. Preliminary analysis has shown a remarkable homogeneity in malignant PCs from AL patients with very limited transcriptional differences between PCs clusters. In summary, in this study we have characterized the PCs of AL patients revealing a homogeneous and specific chromatin accessibility and transcriptome that could contribute to explain the differences between AL and other gammopathies, such as MM. In addition, the identification of these epigenetic and transcriptomic alterations in AL will allow us to better understand the aberrant biology driving this disease, such as those AL with t(11;14). Moreover, these findings open new avenues for the development of innovative therapeutic strategies to improve the outcomes for AL patients.