bluuebunny/crossref_metadata_2025 huggingface dataset:
Defining Hereditary Pyropoikilocytosis (HPP) in the Molecular Diagnostic Era
Authors: Jamalapur Sri Lakshmi (Sindura) , Gadgeel Manisha , Ozgonenel Bulent , Ravindranath Yaddanapudi
Published: December 2023

Misconceptions continue in distinguishing hereditary elliptocytosis (HE) with hemolysis in early perinatal period and hereditary pyropoikilocytosis (HPP). In 1975, Zarkowsky and colleagues described a group of children with transfusion dependent congenital hemolytic anemia with the unique morphology of “microspherocytes, measuring 2-3 microns in diameter, and cells with blunted projections or triangular in shape”, a morphology resembling the heat induced damage to normal erythrocytes - hence the name hereditary pyropoikilocytosis (HPP). Subsequently, similar cases were described with or without demonstrable heat sensitivity (Ravindranath & Johnson, 1985). Regardless, the common denominators were 1) transfusion dependency early in infancy - resolved with splenectomy, 2) severe microcytosis, often <60 fL, and 3) a decrease in membrane bound spectrin (decreased spectrin/band3 ratio). Family studies showed normal RBC morphology in both parents (Ravindranath & Johnson, 1985) or in some cases, one parent with HE (Zarkowsky et al., 1975). Protein chemistry studies showed decreased tetramer formation and tryptic digests showed mutations within the n-terminal alpha 1 domain of alpha spectrin - the dimer/dimer interaction site. Thus, homozygosity or double heterozygosity of mutations in the dimer/dimer interaction site of alpha spectrin (alpha 1 domain of alpha spectrin) defined HPP while classical HE cases showed heterozygous mutations in the alpha 1 domain. A fact unexplained was how this results in spectrin deficiency, specifically, alpha spectrin deficiency (Ravindranath & Johnson, 1985).

Using the molecular analytical tool AnemiaID (courtesy of Agios Pharmaceuticals, Inc.), we show that HPP is defined by the co-inheritance of two SPTA1 mutations and Alpha-LeLy polymorphism ( αLeLy rs28525570; SPTA1[c.5572C>G;c.6531-12C>T]) in one or both alleles, thus explaining the increased propensity for red cell fragmentation and the spectrin deficiency noted on acrylamide gels [Table: Clinical and molecular characteristics of our cases]. Of note, homozygosity for αLeLy polymorphism and one SPTA1 variant did not result in clinical HPP phenotype [HE-2 in Table]. At a clinical laboratory level, the extreme microcytosis in HPP is evident in the markedly diminished band3 content on eosin-5'-maleimide (EMA) binding test- mean channel fluorescence (MCF) of <275.0 (control: 473.0-549.0) while HE (including neonatal HE with hemolysis) show a double peak pattern in EMA (the minor peak representing the fragmented cells while the major peak had MCF higher than control MCF) and the characteristic trapezoid pattern on osmotic gradient ektacytometry (not shown).
Suppression of Microtubule Acetylation Mediates Theanti-Leukemic Effect of CDK9 Inhibition
Authors: Xie Xi , Ren Ruibao , Zhang Bao yuan , Liu Ping
Published: December 2023

Suppression of Microtubule Acetylation Mediates the Anti-Leukemic Effect of CDK9 Inhibition

Xi Xie1, BaoyuanZhang1, Ping Liu1, Ruibao Ren1,2

1. Shanghai Institute of Hematology, State Key Laboratory for Medical Genomics, National Research Center for Translational Medicine at Shanghai, International Center for Aging and Cancer, Collaborative Innovation Center of Hematology, Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China.

2. International Center for Aging and Cancer, Hainan Medical University, Haikou, Hainan Province, China.

Cyclin-dependent kinase 9 (CDK9) is a crucial component of transcription and potential target for anti-cancer therapies, particularly for hematological malignancies. However, the precise mechanisms underlying the therapeutic effects of CDK9 inhibitors remain not fully understood. Here, we found that inhibiting CDK9 either pharmacologically or through gene downregulation, significantly reduced the levels of α-tubulin protein in a time- and dose-dependent manner. We further discovered that CDK9 inhibition led to increased susceptibility of α-tubulin to proteasomal degradation due to reduced acetylation at lysine 40 (K40), an important modification for microtubule stability. An acetylation-mimicking mutant of α-tubulin mitigated the anti-tumor effects of CDK9 inhibition. Mechanically, we identified that CDK9 inhibition downregulated the expression of ATAT1, the acetyltransferase responsible for α-tubulin acetylation, further compromising microtubule stability. Our study unravels a novel molecular mechanism by which CDK9 inhibition disrupts α-tubulin stability and provides valuable insights for exploring effective treatment regimens involving CDK9 inhibitors.
Impaired Hemoglobin Clearance By Liver Sinusoidal Endothelium Promotes Liver Damage in Sickle Cell Disease
Authors: Li Ziming , Katoch Omika , Ungalara Ramakrishna , Pradhan-Sundd Tirthadipa
Published: December 2023

Sickle cell disease (SCD) is a monogenic disorder that affects 100,000 African Americans and millions of people worldwide. Intra-erythrocytic polymerization of sickle hemoglobin (HbS) promotes erythrocyte sickling, impaired rheology, ischemia and hemolysis, leading to the development of progressive liver injury in SCD. Liver resident macrophages and monocytes are known to enable the clearance of HbS, however, the role of liver sinusoidal endothelial cells (LSECs) in HbS clearance and liver injury in SCD remains unknown. Using real-time intravital ( in vivo) imaging of the liver as well as primary culture of human and mice LSECs, we show for the first time that liver injury is associated with accumulation of HbS and iron in the LSECs, leading to the development of senescence in LSECs. Hepatic monocytes were observed to attenuate LSEC-senescence by accelerating HbS clearance in the liver of SS mice, however, this protection was impaired in P-selectin-deficient SCD mice secondary to reduced monocyte recruitment in the liver. These findings are the first to suggest that LSECs contribute to HbS clearance and LSEC-senescence promotes progressive liver injury in SS mice. Our results reveal a novel insight into the pathogenesis of hemolysis induced chronic liver injury in SCD caused by LSEC senescence. Identifying the regulators of LSEC mediated HbS clearance may lead to new therapies to prevent the progression of liver and other organ injuries in SCD.
Transducing Force into Procoagulation: PIEZO1-TMEM16F Functional Coupling in Blood Lining Cells
Authors: Shan Ke Zoe , Liang Pengfei , Zhang Yang , Yang Huanghe
Published: December 2023

Thrombosis is a major global disease burden that accounts for 1 in 4 deaths worldwide. Hypertension and preeclampsia greatly increase thrombotic risks by imposing high blood pressure and abnormal hemodynamic shear stress on blood-lining cells, namely endothelial cells (ECs) and placental syncytiotrophoblasts (STBs) in pregnant women. Nonetheless, it remains elusive how abnormal mechanical forces under hypertensive conditions promote coagulation. PIEZO1 mechanosensitive ion channel is a major force sensor in a wide variety of cells, including red blood cells (RBCs) and endothelial cells (ECs), where it transduces mechanical forces into chemical signals through its ion permeability to calcium. On the other hand, TMEM16F is a calcium-activated lipid scramblase (CaPLSase) that translocates phosphatidylserine (PS), an important procoagulant factor, to the cell surfaces of blood cells and endothelial cells. We recently discovered that TMEM16F is the sole CaPLSase in placental trophoblasts and RBCs. Our preliminary results show that PIEZO1 and TMEM16F are functionally coupled in RBCs, and calcium entry through PIEZO1 activates TMEM16F, leading to PS exposure. Based on all these, we hypothesize that in the blood lining cells, PIEZO1-TMEM16F coupling is responsible for transducing mechanical forces into PS exposure, a critical procoagulant signal for blood coagulation.

By employing immunostaining, patch clamp electrophysiology, and a fluorescence microscopy-based scrambling assay, we show that PIEZO1 is functionally expressed in placental trophoblasts, including STBs. We further demonstrate that PIEZO1 activation, either stimulated by its agonist Yoda1 or by shear stress, induces PS exposure in BeWo human trophoblast cell line and human umbilical vein endothelial cell line (HUVEC) but not the TMEM16F deficient cell lines. In addition, using chromogenic tenase and prothrombinase, and fluorescence fibrin assays, we show that PIEZO1 activation by its agonist, Yoda1, triggered coagulation activities in a TMEM16F-dependent fashion in trophoblasts. Our study thus discovers the functional expression of the PIEZO1 mechanosensitive channel in placental trophoblasts and elucidates a novel force-induced procoagulant mechanism in blood-lining cells. In this mechanism, PIEZO1-TMEM16F coupling transduces mechanical forces into procoagulant PS exposure on the blood lining cells. Enhanced PIEZO1-TMEM16F coupling may contribute to hypercoagulable states in hypertension and preeclampsia, and breaking the coupling is therefore a potential therapeutic strategy to prevent thrombotic risks associated with these diseases.
Successful Use of Haploid Hematopoietic Stem Cell Transplantation for Severe Glanzmann's Thrombasthenia
Authors: Wang Shuran , Xiao Peifang , Li Jie , Lu Jun , Hu Yixin , Hu Shaoyan , Ling Jing
Published: December 2023

Objective: Glanzmann's thrombasthenia (GT) is a rare autosomal recessive disorder, which characterized by the clinical manifestations from purpura to severe bleeding. It was caused by mutations of the ITGA2B or ITGB3 gene on chromosome 17, which lead to deficits or reduced expression of the platelet membrane glycoprotein GPIIb/IIIa. We report 5 pediatric patients with severe Glanzmann's thrombasthenia who underwent allogeneic hematopoietic stem cell transplant, to observe the outcomes of allo-HSCT in children with severe Glanzmann's thrombasthenia.

Methods: We retrospectively summarized the clinical and genetic profiles of five patients with Glanzmann's thrombasthenia who underwent allo-HSCT from June 2021 to July 2023. All patients were reduced intensity conditioning (Bufulsan 3.2-6.4mg/kg, CTX 120mg/kg, FLU 180mg/m 2, ATG 10mg/kg). After conditioning chemotherapy, all the patients received stem cells collected via bone marrow harvest and/or peripheral blood apheresis (PBSC) with CD34 dose of 2.89-7.2×10 6/kg and received graft vs. host disease (GvHD) prophylaxis with cyclosporine (CSA) and methotrexate (MTX).

Results: There were five children were involved in this observation, one patient suffered from recurrent epistaxis bleeding which was life-threatening; the second suffered from gastrointestinal bleeding and could only accept liquid diet to 10-years-old; other patients also suffered from epistaxis bleeding, gum bleeding or gastrointestinal bleeding. For all the patients, regular red cell transfusions were required, platelet transfusions and recombinant factor VIIa were also used in severe and life-threatening bleeding events. Two of the five developed antiplatelet antibodies before HSCT. All the patients were successfully treated with allo-HSCT. One case received non-relative matched donor of 10/10 HLA, one case with sibling matched donor, and 3 cases with haploid transplantation of HLA 5-6/10 matched. The mean time to neutrophil engraftment was 11 days (11-12 days), and the mean time to platelet engraftment was 16 days (15-18 days). One case had CMV reactivation, one case had HHV-6b infection, one case had grade II acute GVHD of the skin which was controlled rapidly. There was no evidence of chronic GVHD for all the patients. The platelet disorder was corrected to a normal level after allo-HSCT, with no significant complication related to the transplantation. Comparing with HLA matched donor, either from sibling or non-related, the patients receiving haploid transplantation with 5-6/10 HLA matched got similar outcome.

Conclusions: We proposed haploid transplantation treated with the reduced intensity conditioning regimens produced similar outcomes as HLA matched did which could be recommended as an alternative donor for severe Glanzmann's thrombasthenia.
Identification of Novel Transcripts and Potential Therapeutic Targets for Acute Leukemia
Authors: Wada Fumiya , Kato Shoya , Bhagat Shruti , Hirabayashi Shigeki , Son Raku , Oguchi Akiko , Takeuchi Kazuhiro , Sekito Sho , Hirai Tomoya , Zhang Zhiwei , Horisawa Yoshihito , Iwasaki Makoto , Takeda June , Kanda Junya , Sakamoto Takashi , Shirakawa Kotaro , Takaori-Kondo Akifumi , Murakawa Yasuhiro
Published: December 2023

Acute leukemia is a heterogenous disease with many genomic scars. Recent advances in targeted therapy and immunotherapy have improved the prognosis of this disease. However, prognosis still remains poor, and the identification of novel leukemia-specific transcripts may provide new strategies for leukemia therapy. We developed a novel long-read transcriptome method for sequencing full-length RNAs by directly capturing the 5′-end cap structures and the 3′-end poly(A)-tails of individual RNA molecules. We applied this method to bone marrow samples with various acute leukemias, including acute myeloid leukemia, acute lymphoid leukemia, and other rare types of acute leukemia. We covered full-length poly(A)+ RNAs with an average length distribution of over 3,000 bp, a much longer size distribution than previously reported. This not only led to the discovery of a wide array of uncharacterized transcript isoforms of known genes, but also to the discovery of 1,903 novel genes that are not annotated in the current human gene database. We also found that more than 60% of these new human genes were single exon genes and that many of them emerged from primates. This implicates that these new genes may contribute to human-specific leukemia biology. In addition, among these new human genes, 485 genes were predicted to have the potential to encode putative proteins using a GeneMarkS-T program. A fraction of new genes identified in this study were highly leukemia specific as shown by bulk CAGE-seq analysis of over 100 blood tumor samples and by single-cell RNA sequencing analysis of more than 300,000 bone marrow cells from leukemia patients, highlighting their potential as useful biomarkers and novel therapeutic targets. In sum, we constructed a comprehensive atlas of full-length RNA molecules in acute leukemia and identified a large number of uncharacterized ones. Our study provides a versatile framework for exploring novel transcripts and future therapeutic strategies in human diseases.
Trend in Von Willebrand Factor Profile in Women With Von Willebrand Disease During Pregnancy and Postpartum
Authors: Albusoul Linda , Shahid Muhammad , Vuyyala Sowjanya , Otrock Zaher K , Kuriakose Philip
Published: December 2023

Introduction:

Pregnancy is considered a hypercoagulable state, with levels of multiple coagulation factors including Factor VIII (FVIII) and von Willebrand Factor (VWF), progressively increasing throughout, reaching their peak levels during the third trimester. However, in women with von Willebrand disease (VWD), these changes are variable and could be blunted or absent due to the wide heterogeneity of phenotypes and pathophysiological mechanisms linked to this disorder, posing a significant clinical challenge. This study aims to describe the trend in the VWF profile in women with VWD during each trimester of pregnancy and in the postpartum period.

Methods:

This is a retrospective single-center study that included adult pregnant women diagnosed with VWD, who were followed at Henry Ford Health between 03/2012 and 11/2022. Patients with other bleeding disorders were excluded from the study. Data on baseline characteristics, including age, race, age at childbirth, and parity were collected. Additional data collected included VWF antigen (VWF:Ag) level, FVIII activity level, and VWF ristocetin cofactor (VWF:Rco) activity during the first, second, and third trimesters, as well as in the postpartum period.

Results:

A total of 33 cases were included in our study. At baseline, the mean VWF:Ag level in our patients was 50.5% (reference range 50%-150%). Compared to baseline, the VWF:Ag level increased by 48.71% in the first trimester, 80.59% in the second trimester, and 168.71% in the third trimester. Postpartum, the VWF:Ag level decreased by 51.29% compared to the third trimester; however, it remained 30.89% higher than baseline levels. While the FVIII level (reference range 50%-150%) was an average of 64.8% at baseline, it increased by 21.91% in the first trimester, 74.23% in the second trimester, and 143.67% in the third trimester, compared to baseline. Postpartum, the FVIII level decreased by 48.45% from the third trimester but remained 25.62% higher than baseline. Additionally, the VWF:Rco level was an average of 44.3% (reference range 51%-215%) at baseline. Compared to baseline, the VWF:Rco level increased by 61.85% in the first trimester, 90.52% in the second trimester, and 142.66% in the third trimester. It decreased by 49.12% postpartum compared to the third trimester but remained 23.48% higher than baseline.

Conclusion:

In women with VWD, VWF:Ag level, FVIII level, and VWF:Rco levels steadily increase during pregnancy, reaching their maximum levels during the third trimester. All three levels decreased in the postpartum period; however, they remain higher compared to baseline at a median follow up of 105 days after delivery.
Novel Fusion Gene Aven-NUTM1 Induces Mice Myeloid Leukemia Vulnerable to HDAC Inhibitors
Authors: Dan Yuqing , Ren Ruibao , Li Donghe , Liu Ping
Published: December 2023

Chromosomal rearrangements involving the NUTM1 gene produce fusion genes commonly found in NUT carcinoma. While recurring NUTM1 fusions have been reported in other neoplasms, including AML, their roles in leukemogenesis remain unclear.

We identified the AVEN-NUTM1 (AN) fusion gene in an AML patient resistant to standard chemotherapy, and went to assess AN's leukemogenicity via bone marrow transduction and transplantation. Mice transplanted with AN-transduced hematopoietic stem/progenitor cells (HSPCs) developed transplantable myeloid leukemia with a median survival of 342 days. RNA sequencing revealed downregulation of genes related to hematopoietic cell lineage commitment and upregulation of genes related to early hematopoietic progenitors and MYC-targeted genes in AN-transformed leukemic cells.

Co-immunoprecipitation confirmed that AVEN-NUTM1 can interact with and activate EP300 as previously reported. To determine whether AN's interaction with EP300 is related to its ability to induce malignant transformation, we mutated the interaction site and constructed mutants of other structural domains. Colony-forming and replating assays showed that HSPCs expressing AN with mutations in the EP300 binding site lost their ability to self-renew and form colonies.

A CUT&RUN assay followed by sequencing showed that the most enriched motif in overlapping peaks between AN and EP300 were ETS motifs, suggesting the involvement of an ETS transcription factor family member in AN-activated EP300-induced leukemia.

We further found that both the patient's primary cells and bone marrow cells from leukemic mice were especially vulnerable to HDAC inhibitors.

These results demonstrate that the AVEN-NUTM1 fusion gene can drive the development of myeloid leukemia in mice, AN's ability to bind and activate P300 is closely linked to its leukemogenicity, and that HDAC inhibitors are effective in targeting AN leukemia cells. The study provids valuable insights for the targeted treatment of NUTM1-related leukemia patients.
Study on the Synthetic Lethal Mechanism of Selective Killing of ALDH2 Deficient Acute Myeloid Leukemia Cells By Inhibiting BRIP1
Authors: Wang Jishi , Hu Xiuying
Published: December 2023

Background and objective: Although targets related to DNA double strand damage repair have clear tumor therapeutic potential, few have identified DNA double strand damage repair dependence in AML cells through genetic screening to date. In our previous experiments, we found that ALDH2 showed low expression in most newly diagnosed AML, and their unique ALDH2 vulnerability may make them preferentially dependent on BRIP1 by the DNA double-strand damage repair pathway, and have more synthetic lethal effects than other normal cells. Therefore, this study explored whether blocking BRIP1-mediated DNA double-strand damage repair could selectively eliminate AlDH2-deficient AML cells while preserving AlDH2-expressing normal cells present in most human tissues.

Methods: We examined the dependence of ALDH2-expressing cells on BRIP1 and methylation levels of ALDH2 promoters in different AML cell lines. The expressions of BRIP1 and ALDH2 in 6 AML cell lines and clinical samples were detected by Real-time PCR and Western blot, and the reverse expression pattern between the two was studied, and the correlation between the expression of ALDH2 and DNA base excision repair was analyzed. By targeting the inhibition of ALDH2 and BRIP1, in vitro and in vivo models were constructed to observe the proliferation and apoptosis of different AML cell lines and primary cells.

Results: In the group of AML cells with low expression of ALDH2, we found that BRIP1 showed a certain high expression compared with other groups. Among the 46 newly diagnosed AML samples, we divided the clinical samples into high and low groups based on the difference in the expression level and methylation degree of ALDH2. Compared with the group with high expression of ALDH2, BRIP1 in the group with low expression of ALDH2 was significantly increased (P < 0.01). The proliferation level of AML cells was significantly decreased (P < 0.01). BRIP1 inhibition induced apoptosis of U937 and TPH1 cells (AML cell lines with low expression of ALDH2) in a dose - and time-dependent manner (P < 0.05). In addition, in AML cell lines with high expression of ALDH2, we inhibited the expression of both ALDH2 and BRIP1, and the growth of AML cells was significantly down-regulated (P < 0.01). Similarly, we observed the same results when constructing an in vivo AML infiltration model in mice using AML primary cells.

Conclusion: The dependence of ALDH2-deficient cells on BRIP1 and the effect of targeting BRIP1 on the proliferation of AML cells could provide new ideas for finding new molecular targets for the treatment of AML.
Zanubrutinib Combined with R-CHOP As the First-Line Treatment for Newly Diagnosed Diffuse Large B-Cell Lymphoma with Extranodal Involvement: A Prospective Phase II Trial in China
Authors: Hu Chongling , Yang Tao , Jun Li , Zhang Wenjun , Guo Bingling , Wang Chaoyu , Liu Jun , Xiao Chunyan , Huang Dehong , Yang Zailin , Liu Tingting , Li Jieping , Nan Yingyu , Liu Yao
Published: December 2023

BACKGROUND

About one-third of Diffuse large B-cell lymphoma (DLBCL) arises primarily from extranodal sites, patients with extranodal involvement indicates poor clinical outcomes.There is an urgent need to find new treatments for DLBCL patients with extranodal involvement.

METHODS

We conducted a single-arm, prospective phase II trial for newly diagnosed DLBCL with extranodal involvement to evaluate the efficacy and safety of the zanubrutinib combined with R-CHOP. Patients received therapies: R-CHOP intravenously Rituximab (375mg/m2 on Day0), Cyclophosphamide (750mg/m2on Day1), Doxorubicin (50mg/m2 on Day1), Vincristine(1.4mg/m2 on Day1), and oral Prednisone(50mg/day Day1-5) with Zanubrutinib (160mg bid Day1-21) regimen.This trail was registered at Chinese Clinical Trial Registry(ChiCTR2100053056).

RESULTS

31 patients received ZR-CHOP regimen treatments at least 2 cycles, 26 patients completed 6 cycles. Patients had a median age of 68 years(38-79). 18 patients (58%) had intermediate and high risk of IPI score, 20 patients (65%) had stage III~IV disease, 22 patients (71%) were Non-GCB types, 19 patients (61%) Ki-67 More than 80%. 7 patients (23%) were double-expression lymphoma，5 patients (16%) with positive CD5 and 4 patients (13%) TP53 more than 80%. The objective response rate(ORR) was 96%(26/27), 12 patients(44%) reached complete remission(CR). 1 patient(3%) died for progression disease(PD) and 4 patients(13%) withdrew the trial. The most common adverse events of hematological toxicity were neutropenia, thrombocytopenia and anemia with 19%，13% and 10% grade 3/4 AEs respectively. The most common non-hematological toxicity were pneumonia, nausea and fatigue with 16%，6% and 3% grade 3/4 AEs .

CONCLUSIONS

Our results showed that Zanubrutinib plus R-CHOP(ZR-CHOP) is a safe and effective scheme for DLBCL patients with extranodal involvement. It indicate that the addition of zanubrutinib to the standard first-line treatment can enable these patients to achieve remission in the early stage of treatment and without significantly increasing toxicity.
Hematologic Response to Steroid Use in Patients with Primary Immune Thrombocytopenia: A Research Study in a Salvadoran Population
Authors: Rivas Luis Fernando
Published: December 2023

Introduction. Thrombocytopenia Immune (ITP) is an acquired thrombocytopenia caused by autoantibodies against platelet antigens. It is one of the more common causes of thrombocytopenia in otherwise asymptomatic adults. The use of high-doses dexamethasone has proven efficacy and safety in previously untreated patients with immune thrombocytopenia even though these findings are not entirely clear yet. AIM. The primary goal to assess the global response (platelets >30 x 109/L) and complete response (platelets >100 x 109/L) within the different implemented regimes in previously untreated adult with primary immune thrombocytopenia. Further goals aimed to describe the clinical characteristic of patients with primary immune thrombocytopenia and the most frequent adverse events in the use of steroids. Results. A total of 44 patients were treated with steroids, 16 patients in the high-dose dexamethasone (HD-DXM) group, 23 patients received methylprednisolone (MTP) and 5 patients with prednisone (PDN). The female gender was the most affected in all the groups with 61.3% (n=27) presenting more cases than the female sex in the methylprednisolone group 65.2% (n=15), although without significant statistical difference (P = 0.384). The mean age of onset was 38, 44, and 40 years, respectively (P = 0.352). The most frequent clinical manifestations were purpura sicca (petechiae and bruising) with 75%, 91.3% and 60.0%, for the dexamethasone, methylprednisolone and prednisone groups, respectively. The mean platelet count in the groups was around the mean of 26 x 109/L, with the methylprednisolone group presenting the lowest values (3 x 109/L). The overall response was different for the three groups (87.5% vs 95.6% vs 100%, P = 0.43) with a complete response of 56.2% vs 60.8% vs 80.0% (P = 0.85). Early response was achieved in the methylprednisolone group with 3.5 days, versus dexamethasone with 4.0 days and prednisone with 7 days. The sustained response at 6 months was lower in the prednisone group 80.0%. The most frequent adverse events occurred in the HD-DXM group with fatigue in 31.2% (n = 5) followed by edema, hyperglycemia and insomnia with 18.7% (n = 3). Conclusions. Our study demonstrated that PDN provides greater overall responses as initial treatment in ITP. MTP provides the fastest responses followed by HD-DXM. The sustained response was maintained in the HD-DXM Group. The HD-DXM group presented 68.7% cases with at least one adverse event associated with the use of the steroid. The findings suggest continuing to perform more studies in our population.
Prognostication of Large Granular Lymphocytic Leukemia in Clinical Practice: Three New Cases and Literature Review
Authors: Lambert-Swainston Chalette , Varada Neilmegh , Gollard Russell
Published: December 2023

Large Granular Lymphocytic Leukemia (LGLL) is a rare lymphoproliferative disorder of either CD3+ cytotoxic T-lymphocytes or CD3− natural killer cells with chronic and ill-defined clinical progression. Presentation at diagnosis can include asymptomatic or symptomatic cytopenias, recurrent infections, splenomegaly and rheumatoid arthritis. Multiple cell survival pathways are involved in the pathogenesis of LGLL, and it has been found to be specifically associated with aberrant STAT3 signaling. An increased number of clonal LGL cells in the blood establishes the diagnosis. Treatment regimens historically have included cyclophosphamide, methotrexate, steroids, cyclosporine, and splenectomy, but there are no available targeted therapies or definitive cure available for LGLL. Here we present three cases with varying treatment responses and outcomes that highlight the need for targeted therapy development. All three patients were confirmed to have T-LGLL by flow cytometry analysis.

Patient one is a 64 year old female, presented for moderate recurrent symptoms of LGLL, initially diagnosed 17 years prior and treated with 6 months of cyclophosphamide. On the second presentation she was found to have STAT3 missense mutation and was treated for a second time with cyclophosphamide which improved symptoms. She additionally required subcutaneous immunoglobulin for treatment of recurrent infection.

Patient two is a 72 year old male, presented for recurrent aphthous ulcers with severe leukopenia and neutropenia, found to have STAT3 negative LGLL. He received multiple courses of cyclophosphamide and was refractory to treatment with persistent symptoms despite improvement in leukopenia.

Patient three is a 70 year old male, presented for symptomatic lymphocytosis. He was found on flow cytometry to have LGLL. He improved without further testing or treatment and has remained in remission for several years.

These cases show the variation in presentation and response to therapy for patients with T-LGLL. Previous research has demonstrated the heterogeneity of LGLL and has additionally shown that STAT3 mutation was associated with decreased overall survival. This may be as yet simplifying the pathogenesis for targeting as we can still see heterogeneous clinical response even within STAT3 positive patients. One study showed that the size of the mutated clone correlated with degree of clonal expansion and that immunosuppressive therapy may not be able to reduce the clonal burden at high degrees of mutation. Our cases show that STAT3 positive status is not enough to determine prognosis and treatment course. The degree and types of mutation may need to be quantified as well as additional targets identified. While pursuing targeted treatment therapies for LGLL we must also pursue testing to assess disease burden.
Primary Immune Regulatory Disorders (PIRDs) That Amplify mTOR Signaling Share a T Cell Exhaustion-like Process
Authors: Conrey Peyton E , Campos Jose , Mauracher Andrea , Sayed Samir , Walter Jolan E. , Su Helen , Barmettler Sara , Leiding Jennifer W , Cooper Megan , MacFarland Suzanne P , Ruffner Melanie A , Farmer Jocelyn R , Rao V. Koneti , Henrickson Sarah E
Published: December 2023

Background: Primary Immune Regulatory Disorders (PIRDs) are a complex and challenging-to-treat subset of Inborn Errors of Immunity (IEI), which are characterized by immune dysregulation leading to recurrent infections, lymphoproliferation, and autoimmunity including refractory cytopenias. Since many ultra-rare monogenic PIRDs exist, it is not feasible to design targeted therapies for each. An alternate strategy is to identify shared aspects of T cell dysfunction and strategies targeting them. We have focused our studies on PIRDs that chronically amplify T cell receptor (TCR) signaling, mimicking chronic infection. Under conditions of chronic inflammation and antigen presentation, seen during chronic infections, CD8 T cell exhaustion (Tex) can result and is characterized by increased inhibitory receptor expression, altered transcriptional networks, epigenetic poise, and impaired T cell functions including cytokine production. Here, we have evaluated CD8 T cell dysfunction in PIRDs, including the potential for Tex.

Methods: Deep immune phenotyping and T cell functional analyses were performed using CyTOF and spectral flow cytometry, in addition to single cell RNA-sequencing and CITE-seq from untreated PIRD and healthy control PBMCs. Finally, CRISPR/Cas9 editing in healthy control CD8 T cells was used to create a cellular disease model.

Results: We identified a Tex-like process in activated PI3 kinase delta syndrome (APDS), CTLA-4 haploinsufficiency, and Ras-associated Autoimmune Leukoproliferative Disease (RALD), which share increased mTOR activation. We identified increased PD-1, CD39, TIGIT and TOX expression on CD8 T cells consistent with a Tex-like phenotype. We also found impaired CD8 T cell cytokine and proliferation consistent with Tex. Lastly, a cellular model of CTLA-4 haploinsufficiency was created for perturbation studies to evaluate best available therapies and target novel therapeutics in these rare disorders.

Conclusion: By identifying shared patterns of CD8 T cell dysfunction in these ultra-rare disorders, we may both identify novel therapeutic strategies and increase our understanding of CD8 T cell function, including cytokine production.
Iron Overload in Dialysis Patients: Serum Ferritin Underestimates Liver Iron Measured By MRI
Authors: Grant Christopher , Juarez Dennis , Pakbaz Zahra
Published: December 2023

Background: Parenteral iron supplementation and erythropoietin-stimulating agents are the mainstay management for anemia in patients requiring dialysis. Traditionally, iron status in this patient population is determined by monitoring of widely available and inexpensive serum ferritin level. However, serum ferritin is an acute phase reactant and its usefulness is disease specific. Therefore, several non-invasive methods to evaluate body iron storage have been studied, with measurement of liver iron concentration (LIC) by MRI FDA approved. In this retrospective chart review, we explored the utility of serum ferritin for predicting LIC in a convenience sample of adults on dialysis who had LIC measured by MRI.

Methods: Seven adults on dialysis who had been referred to an academic hematology clinic between July 2020 to June 2023 for elevated ferritin and subsequently had completed LIC by MRI were included in this chart review. Liver iron was quantified by T2* Relaxometry in a 1.5 Tesla MRI . Patients age, gender, etiology of end stage renal disease, duration on dialysis, initial LIC, and the ferritin and hemoglobin levels within three month of LIC were collected from patient medical records. Clinical recommendation for holding parenteral iron or starting chelation and patient-follow through were also recorded. In 2 patients, repeat LIC was available. The clinical recommendation for all patients with elevated LICs was to hold parenteral iron and not to start chelation but repeat LIC in 6 month intervals.

Result: The median age was 56 years (min 26 years - max 73 years) and 4 of 7 patients were female. The median time of first LIC from initiation of dialysis was 88.5 months (min 19 months - max 130 months). The median of initial LIC was 6.6 mg/g (min 4.6 mg/g - max 12.5 mg/g). The median ferritin was 1120 ng/mL (min 736 ng/mL - max 2650 ng/mL). The median hemoglobin was 10.8 g/dL (min 8.4 g/dL - max 13.7 g/dL). Figure 1A demonstrates that despite similar ferritin levels (bolded group or underlined group), LICs were very variable. Additionally, it is visible in figure 1A that serum ferritin underestimates the LIC (italicized) in this patient population. Holding iron infusion for 2 years in patient 4, resulted in LIC trending down from 6.3 mg/g to 3.2 mg/g (Figure 1B) with minimal impact on hemoglobin. Despite clinical recommendation to hold iron infusion in patient 5 with an initial LIC of 6.8 mg/g, iron infusion was continued. Repeat LIC for this patient 6 months later remained unchanged at 7.0 mg/g, while serum ferritin trended down from 1084 to 843.

Conclusion: Ferritin underestimates liver iron concentration in dialysis patients. More investigation is required to determine if liver iron assessment by MRI improves iron management in dialysis patients and optimize serum ferritin utility. This data also suggests that, chelation is not required in iron overloaded dialysis patients, and holding IV iron can result in decline in liver iron concentration.
Multiple Myeloma and DALY in the USA: Insights of the Global Burden of Disease
Authors: Cuartas Maria , Pan Chun-Wei
Published: December 2023

Introduction:

In contrast to the global trends, which indicate an escalating incidence and mortality of Multiple Myeloma (MM), the United States presents an inverse scenario. The successful development and deployment of therapeutic interventions have led to an appreciable decrease in mortality by 18%. Among the diverse array of prognostic factors associated with MM, age stands out as a significant element, typically associated with poor prognosis. With these considerations in mind, our study aimed to undertake a systematic analysis of the incidence, mortality, and Disability-Adjusted Life Years (DALY) related to MM over a decade in the United States.

Methods:

The Global Burden of Disease (2019) offers extensive assessments of the incidence, mortality, and DALY in the United States from 1990 to 2019. The data was gathered using the Global Health Data Exchange query tool. We analyzed the annual percentage change (APC) over this period and compared different age groups to enhance the current trend of incidence, mortality, and DALY in the United States between 2009-2019.

Results:

Our analysis was structured around three distinct age groups-15-49 years, 50-74 years, and 75+ years, with each category further stratified by gender. For the 15-49 years cohort, males consistently exhibited higher average Disability-Adjusted Life Years (DALYs) at 15.60 per 100,000, as compared to their female counterparts, who averaged at 10.05. Intriguingly, the Annual Percentage Change (APC) displayed a downward trajectory for DALYs, Deaths, and Incidence rates over the period under study, with respective APC values of -1.79, -1.81, and -1.95. This trend indicates a consistent improvement in health outcomes for this age group.

On the other hand, the 50-74 years age group revealed a significantly ascending trend in all health measures. Here, females and males recorded average DALYs of 98.13 and 143.33 per 100,000, respectively. The APC values for this group underscored an increasing trajectory-0.72 for both DALYs and Incidence and 0.92 for Deaths-signifying a worsening in health outcomes over the duration of observation.

Lastly, in the oldest age group of 75+ years, the DALYs surged to 235.78 for females and 379.87 for males per 100,000. Although the APC values for this age group were positive, they were relatively lower than those for the 50-74 years group, with APC values of 0.62 for DALYs, 0.44 for Deaths, and 0.39 for Incidence.

Conclusion:

New therapies in MM have decreased the average of DALY in the US over the studied decade. Between the ages 15-49 years, the study's results align with the current data correlating younger age with a better prognosis. However, when divided into age categories, the subgroup from 50-74 years showed a possible rapid deterioration in health outcomes when compared with patients above 75 years old. Additional studies are needed to better understand this underlying pathophysiology to have better results and develop further therapies.
CRM1/XPO1-FOXO1 Pathway Is Associated with Clinical Outcome and Represents a Promising Target in B-Cell Acute Lymphocytic Leukemia
Authors: Deng Shiyu , Ou Jiawang , Zihong Cai , Huang Zicong , Li Jia , Liu Qifa , Zhou Hongsheng
Published: December 2023

Background:

Currently, a proportion of B-Cell Acute Lymphocytic Leukemia (B-ALL) patients still show no response to existing therapeutic drugs and existing therapeutic drugs cannot further improve the prognosis of refractory and relapsed B-ALL. Thus, the development of a new therapeutic target to battle this deadly disease is imperative. Exportin-1 (XPO1), also known as chromosomal region maintenance 1(CRM1), plays an important role in the progression of tumorigenesis. However, the prognostic and therapeutic values of XPO1 in B-ALL have not been reported.

Methods:

The difference in XPO1 and Foxo1 expression between normal B cells and leukemia cells was using the Human Protein Atlas (HPA) dataset and RT-qPCR results. Correlations between XPO1 expression level and clinical outcome were analyzed using the RNA-seq data of 110 newly diagnosed B−ALL patients in Nanfang Hospital. Cell proliferation assays, apoptosis assays, and cell cycle analysis were performed to analyze the anti-tumor effects of selinexor (KPT-330) in vitro. Bioinformatics analysis, western blot, and RT-qPCR were performed to explore the underlying mechanism.

Results ：

Higher XPO1 levels or Foxo1 levels were associated with poor prognosis in newly diagnosed B-ALL patients. Using bone marrow (BM)， peripheral blood (PB) cells from B-ALL and normal people as well as B-ALL cell lines, we found that XPO1 and Foxo1 expression was significantly increased in B-ALL patients and B-ALL cell lines. And notably, there was a positive correlation between the expression of Foxo1 and XPO1, which was verified by the data in the HPA database, and the results were consistent. Foxo1 is an important transcription factor in B-ALL, mainly localized in the nucleus, and is important for leukemia cell survival. KPT-330 (Selinexor), a selective Inhibitor of XPO1, mechanistically inhibited the expression of Foxo1, a leukemia transcription factor (reduced in both the nuclear and cytoplasmic compartments), and led to a strong repression of Foxo1 target genes associated with cell cycle, DNA replication, and transcriptional regulation. This induced growth inhibition, apoptosis, and cycle arrest in B-ALL cells. Moreover, in combination with Foxo1 Inhibitor (AS1842856), the proliferation inhibition and apoptosis-inducing effects of KPT-330 on B-ALL could be significantly enhanced. Furthermore, the KPT-330 strongly triggered apoptosis in leukemic but with minimal effects on normal cells implying that drugs of this class show promise for the targeted therapy of B-ALL leukemia cells.

Conclusions:

Our findings demonstrate that XPO1 is a promising prognostic indicator and a valid target for antitumor treatment with selective inhibitor KPT-330.

This research was supported by the National Natural Science Foundation of China(NFSC82170163, 81970147), Clinical Study of Nanfang Hospital(LC2016ZD009/2019CR012).
Cryoprecipitate-Poor Plasma Instead of Fresh Frozen Plasma As Replacement Therapy in Thrombotic Thrombocytopenic Purpura: A Systematic Review and Meta-Analysis
Authors: Mafra Marcela P. , Roca Mora Maria Meritxell , Godoi Amanda , Valenzuela Andres
Published: December 2023

Background: Thrombotic thrombocytopenic purpura (TTP) is a life-threatening condition characterized by the presence of small-vessel thrombi enriched with platelets, leading to thrombocytopenia, thrombotic microangiopathy (TMA), and on occasion, resulting in end-organ damage. The standard therapeutic plasma exchange (TPE) approach, initiated after suspicion of TTP, is done with fresh frozen plasma (FFP). Yet, the use of cryoprecipitate-poor plasma (CPP) has been suggested as alternative in recent decades given its absence of von Willebrand factor, which would benefit the pathophysiology of TTP. However, its efficacy over FFP remains unclear. Therefore, we aimed to conduct a meta-analysis to compare the safety and efficacy of CPP versus FFP in patients with TTP.

Methods: We systematically searched PubMed, Cochrane Central and Embase for clinical studies published up to June 2023, comparing CPP to FFP as replacement fluids in TPP or TMA patients. Outcomes assessed were overall mortality, relapse rate, response to treatment and number of TPE sessions. We pooled outcomes as odds ratios (OR) and mean differences (MD) with 95% confidence intervals (CI). We also performed a subgroup analysis with randomized controlled trials (RCTs). Statistical analysis was performed with Review Manager version 5.4.1. Heterogeneity was assessed with I 2 statistics. The protocol was registered in PROSPERO (ID: CRD42023440665).

Results: A total of eight articles met inclusion criteria, of which three were RCTs. The combined dataset involved 290 patients, of which 144 (49.7%) were in CPP group and 146 (50.3%) in the FFP group. CPP significantly reduced overall mortality compared to FFP (OR 0.27; 95% CI 0.13-0.59; p=0.001; I 2=0%; Figure 1A). However, our subgroup analysis of RCTs showed no significant difference in overall mortality between groups (OR 0.49; 95% CI 0.13-1.84; p=0.29; I 2=0%; Figure 1B). Additionally, there were no significant differences between groups regarding relapses (OR 0.81; 95% CI 0.37-1.77; p=0.60; I 2=0%; Figure 1C), response to treatment (OR 2.20; 95% CI 0.69-7.07; p=0.18; I 2=44%; Figure 1D) or number of TPE sessions (MD 1.63; 95% CI -2.83 - 6.08; p=0.47; I 2=55%; Figure 1E).

Conclusion: Our meta-analysis shows that the safety and efficacy of CPP in TTP patients is at least similar to FFP in reducing mortality, relapse rate, and improving response to treatment. The reduced mortality with CPP found in the whole pool of studies, along with the trend towards reduced mortality found in the RCT-only analysis indicates the potential benefit of CPP over FFP. However, larger population-based randomized trials should contribute to narrow confidence intervals and decrease bias of observational studies, providing clearer conclusions for treatment efficacy in the future.