SPINK2 Protein Expression Is an Independent Adverse Prognostic Marker in AML and Is Potentially Implicated in the Regulation of Ferroptosis and Immune Response.

There is an urgent need for the identification as well as clinicopathological and functional characterization of potent prognostic biomarkers and therapeutic targets in acute myeloid leukemia (AML). Using immunohistochemistry and next-generation sequencing, we investigated the protein expression as well as clinicopathological and prognostic associations of serine protease inhibitor Kazal type 2 (SPINK2) in AML and examined its potential biological functions. High SPINK2 protein expression was an independent adverse biomarker for survival and an indicator of elevated therapy resistance and relapse risk. SPINK2 expression was associated with AML with an NPM1 mutation and an intermediate risk by cytogenetics and European LeukemiaNet (ELN) 2022 criteria. Furthermore, SPINK2 expression could refine the ELN2022prognostic stratification. Functionally, an RNA sequencing analysis uncovered a potential link of SPINK2 with ferroptosis and immune response. SPINK2 regulated the expression of certain P53 targets and ferroptosis-related genes, including SLC7A11 and STEAP3, and affected cystine uptake, intracellular iron levels and sensitivity to erastin, a specific ferroptosis inducer. Furthermore, SPINK2 inhibition consistently increased the expression of ALCAM, an immune response enhancer and promoter of T-cell activity. Additionally, we identified a potential small-molecule inhibitor of SPINK2, which requires further characterization. In summary, high SPINK2 protein expression was a potent adverse prognostic marker in AML and might represent a druggable target.

Acute promyelocytic leukaemia: population-based study of epidemiology and outcome with ATRA and oral-ATO from 1991 to 2021.

BACKGROUND: The epidemiology and treatment of acute promyelocytic leukaemia (APL) are changing. We have incorporated oral arsenic trioxide (oral-ATO) into induction/maintenance. METHODS: Newly-diagnosed APL from 1991 to 2021 divided into three 10-year periods were studied to define its epidemiology and how oral-ATO impacted on its outcome. Primary endpoints included APL incidence, early deaths (ED, first 30 days), and overall survival (OS). Secondary endpoints included post-30-day OS, relapse-free survival (RFS), and incidence of second cancers. RESULTS: APL occurred in 374 males and 387 females at a median age of 44 (1-97) years. Annual incidences increased progressively, averaging 0.32 per 100,000 people. All-trans retinoic acid (ATRA)-based and oral-ATO-based regimens were used in 469 and 282 patients. There were 144 EDs, occurring almost exclusively in ATRA-based inductions (N = 139), being more with males, age > 50 years, leucocyte > 10 × 109/L, diagnosis during 1991-2009 and fewer with oral-ATO-based regimens. After a median of 75 (interquartile range: 14-161) months, 5-year and 10-year OS were 68.1% and 63.3%, inferior with males, age > 50 years, leucocyte > 10 × 109/L, high-risk Sanz score and superior with oral-ATO-based regimens. Factoring out EDs, 5-year and 10-year post-30-day OS were 84.0% and 78.1%, inferior with males and superior with oral-ATO-based regimens. In 607 CR1 patients, the 5-year RFS was 83.8%, superior with diagnosis in 2010-2021 and oral-ATO-based regimens. Second cancers developed in 21 patients, unrelated to oral-ATO-based regimens. CONCLUSIONS: There was an increasing incidence of APL, and all survivals were superior with the use of oral-ATO-based regimens. This study formed part of the Acute Promyelocytic Leukaemia Asian Consortium Project (ClinicalTrials.gov identifier: NCT04251754).

Topologic Analysis of Plasma Mitochondrial DNA Reveals the Coexistence of Both Linear and Circular Molecules.

BACKGROUND: Cellular mitochondrial DNA (mtDNA) is organized as circular, covalently closed and double-stranded DNA. Studies have demonstrated the presence of short mtDNA fragments in plasma. It is not known whether circular mtDNA might concurrently exist with linear mtDNA in plasma. METHODS: We elucidated the topology of plasma mtDNA using restriction enzyme BfaI cleavage signatures on mtDNA fragment ends to differentiate linear and circular mtDNA. mtDNA fragments with both ends carrying BfaI cleavage signatures were defined as circular-derived mtDNA, whereas those with no cleavage signature or with 1 cleavage signature were defined as linear-derived mtDNA. An independent assay using exonuclease V to remove linear DNA followed by restriction enzyme MspI digestion was used for confirming the conclusions based on BfaI cleavage analysis. We analyzed the presence of BfaI cleavage signatures on plasma DNA ends in nonhematopoietically and hematopoietically derived DNA molecules by sequencing plasma DNA of patients with liver transplantation and bone marrow transplantation. RESULTS: Both linear and circular mtDNA coexisted in plasma. In patients with liver transplantation, donor-derived (i.e., liver) mtDNA molecules were mainly linear (median fraction, 91%; range, 75%-97%), whereas recipient-derived (i.e., hematopoietic) mtDNA molecules were mainly circular (median fraction, 88%; range, 77%-93%). The proportion of linear mtDNA was well correlated with liver DNA contribution in the plasma DNA pool (r = 0.83; P value = 0.0008). Consistent data were obtained from a bone marrow transplantation recipient in whom the donor-derived (i.e., hematopoietic) mtDNA molecules were predominantly circular. CONCLUSIONS: Linear and circular mtDNA molecules coexist in plasma and may have different tissue origins.

Methylation analysis of plasma DNA informs etiologies of Epstein-Barr virus-associated diseases.

Epstein-Barr virus (EBV) is associated with a number of diseases, including malignancies. Currently, it is not known whether patients with different EBV-associated diseases have different methylation profiles of circulating EBV DNA. Through whole-genome methylation analysis of plasma samples from patients with nasopharyngeal carcinoma (NPC), EBV-associated lymphoma and infectious mononucleosis, we demonstrate that EBV DNA methylation profiles exhibit a disease-associated pattern. This observation implies a significant potential for the development of methylation analysis of plasma EBV DNA for NPC diagnostics. We further analyse the plasma EBV DNA methylome of NPC and non-NPC subjects from a prospective screening cohort. Plasma EBV DNA fragments demonstrate differential methylation patterns between NPC and non-NPC subjects. Combining such differential methylation patterns with the fractional concentration (count) and size of plasma EBV DNA, population screening of NPC is performed with an improved positive predictive value of 35.1%, compared to a count- and size-based only protocol.

Prevalence and Clinicopathologic Significance of BRAF V600E Mutation in Chinese Multiple Myeloma Patients.

BACKGROUND: Previous studies in Western countries demonstrated BRAF V600E mutation only in a small subset of multiple myeloma (MM) patients. However, the prevalence and clinicopathologic significances of this mutation remain unclear in Chinese MM patients. PATIENTS AND METHODS: We studied diagnostic bone marrow samples from 205 Chinese MM patients by allele-specific PCR to detect BRAF V600E mutation and by high-resolution melting assay to detect KRAS and NRAS mutations. The mutations were confirmed by independent assays. RESULTS: BRAF V600E mutation was found in 9.3% of the cases, the highest prevalence hitherto reported. In addition, the mutation was significantly associated with hypercalcemia and a male predominance but not with aggressive extramedullary diseases or a high serum creatinine level as reported in Western studies. Importantly, BRAF V600E mutation was an adverse prognostic factor for overall survival in younger MM patients by subgroup analysis. Concurrent analysis of RAS mutations highlighted differential alteration spectrum of RAS signaling between Chinese and Western MM, which may suggest a unique myeloma-related genetic profile in Chinese patients. CONCLUSION: Our study revealed a higher prevalence of BRAF V600E mutation in Chinese MM patients. The associated prognostic impacts on younger patients could be beneficial to risk stratification and potential application of BRAF-targeted therapies in Chinese MM management. This is the first large-scale study revealing the prevalence and clinicopathologic significances of BRAF V600E mutation in Chinese myeloma.

Stimulation of adrenergic activity by desipramine enhances hematopoietic stem and progenitor cell mobilization along with G-CSF in multiple myeloma: A pilot study.

Hematopoietic stem cell (HSC) release is positively regulated by the sympathetic nervous system through the ß3 adrenergic receptor. Preclinical studies have demonstrated that the combination of desipramine and G-CSF resulted in improved HSC mobilization. Here, we present the results of an open-label single-arm pilot study in patients with multiple myeloma undergoing autologous stem cell transplantation (ASCT) to assess the safety and efficacy of desipramine combined with G-SCF to induce HSC mobilization. The primary endpoint was safety of the combination including engraftment kinetics. The secondary endpoint was the proportion of patients who collected ≥5 × 106 CD34+ cells/kg. Outcomes were compared with historical matched controls during the same time period with multiple myeloma mobilized with G-CSF. All study patients received desipramine 100 mg daily for 7 days, starting 4 days prior to G-CSF administration (D-3) and continued taking it along with G-CSF for a total of 7 days. Six of ten patients enrolled completed the protocol with minimal side effects. All of them achieved the target collection of 5 × 106 CD34 cells/kg in a median of 1.5 apheresis session with two patients needing additional plerixafor (16%), while 11 out of 13 patients (85%) achieved the target of 5 × 106 CD34 cells/kg in the historical control group in a median of 2 apheresis procedures and seven patients needed plerixafor (54%). The combination of desipramine and G-CSF is safe and signals improved mobilization over G-CSF alone, providing a possible alternative means of mobilization that needs further investigation.

Retrospective study of the prevalence and progression of monoclonal gammopathy in HIV positive versus HIV negative patients.

The significance of HIV associated paraproteins and their risk of progression to hematological malignancies remains unclear. We compared the development of hematological malignancies among HIV+ (n = 266) and HIV- (n = 537) patients with monoclonal gammopathies. HIV+ and HIV- patients with a positive serum protein electrophoresis test (SPEP) were studied. HIV+ SPEP+ were more likely to have faint and oligoclonal paraproteins (F-SPEP) and less likely to have discrete bands (D-SPEP) compared to HIV- SPEP+. The incidence of hematological malignancies was significantly lower in the HIV+ compared to the HIV- (6.4% vs 15.4%, p < 0.0002). Upon subgroup analysis, the lower incidence of hematological malignancies was noted for HIV+ patients with F-SPEP but not for those with D-SPEP. Copyright © 2015 John Wiley & Sons, Ltd.

MRI of diffuse large B-cell non-Hodgkin's lymphoma of the head and neck: comparison of Waldeyer's ring and sinonasal lymphoma.

To document the magnetic resonance imaging (MRI) features of diffuse large B-cell lymphoma (DLBCL) in Waldeyer's ring (WR) and the sinonasal (SN) region, and to identify any differences between lymphatic and extra-lymphatic DLBCLs, and predictors of disease beyond the neck. Primary, nodal, and multifocal sites on head and neck MRI were compared between 31 WR and 15 SN DLBCL, and between 27 patients with disease confined to the head and neck and 16 patients with disease beyond the neck, using logistic regression. Compared to SN, WR DLBCLs had significantly smaller primary tumour volumes (p = 0.009), less deep invasion (p = 0.001), and more nodal disease (p = 0.016). Tumour site (WR vs. SN) was an independent predictor of deep invasion (p = 0.007). Nodal and multifocal diseases were predictors of disease beyond the neck (p = 0.027 and 0.011, respectively). Lymphatic WR DLBCLs were less locally aggressive but had greater propensity to nodal spread than extra-lymphatic SN DLBCLs. Nodal and multifocal diseases predicted disease beyond the neck.

Viral co-infections and paraproteins in HIV: effect on development of hematological malignancies.

The role of viral co-infections and paraproteins in the development of hematological malignancies (HMs) in HIV remains unclear. Using our large database of HIV+ patients, we investigated whether co-infection and paraproteinemia increase the risk of HM. Data on demographics, hepatitis B (HBV) and hepatitis C virus (HCV) co-infections, paraproteinemia, HIV characteristics, and biopsy proven malignant hematological disorders for HIV+ patients were collected over a 10-year period in a large urban hospital setting. We identified 10,293 HIV+ patients who were followed for a median duration of 53 months. Of the 10,293 patients with HIV, 229 (2.2 %) were diagnosed with a HM. Over 85 % of patients in both groups were tested; no significant difference in the prevalence of chronic HBV or HCV was noted between the HM positive (n = 229) and HM negative (n = 9992) patients. The serum protein electrophoresis test was performed for 1371 of the 10,221 patients. HM positive patients, compared to HM negative, were more likely to be tested for paraproteins (OR 3.3, 95 % CI 2.5-4.4) and more likely to have a discrete paraprotein band (OR 3.3, 95 % CI 1.2-8.9). Discrete paraproteins exclusively correlated with the development of plasma cell malignancies. Faint or oligoclonal protein bands were seen in high grade B cell lymphomas but did not show a significant correlation with HM development. Chronic hepatitis B or C infections did not correlate with the development of HM in HIV; however, viral influence on host gene transformation may have been impacted by anti-viral therapy limiting the duration of high viremic states.

Intermediaries of branched chain amino acid metabolism induce fetal hemoglobin, and repress SOX6 and BCL11A, in definitive erythroid cells.

High levels of fetal hemoglobin (HbF) can ameliorate human ß-globin gene disorders. The short chain fatty acid butyrate is the paradigmatic metabolic intermediary that induces HbF. Inherited disorders of branched-chain amino acid (BCAA) metabolism have been associated with supranormal HbF levels beyond infancy, e.g., propionic acidemia (PA) and methylmalonic acidemia (MMA). We tested intermediaries of BCAA metabolism for their effects on definitive erythropoiesis. Like butyrate, the elevated BCAA intermediaries isovalerate, isobutyrate, and propionate, induce fetal globin gene expression in murine EryD in vitro, are associated with bulk histone H3 hyperacylation, and repress the transcription of key gamma globin regulatory factors, notably BCL11A and SOX6. Metabolic intermediaries that are elevated in Maple Syrup Urine Disease (MSUD) affect none of these processes. Percent HbF and gamma (γ) chain isoforms were also measured in non-anemic, therapeutically optimized subjects with MSUD (Group I, n=6) or with Isovaleric Acidemia (IVA), MMA, or PA (Group II, n=5). Mean HbF was 0.24 ± 0.15% in Group I and 0.87 ± 0.13% in Group II (p=.01); only the Gγ isoform was detected. We conclude that a family of biochemically related intermediaries of branched chain amino acid metabolism induces fetal hemoglobin during definitive erythropoiesis, with mechanisms that mirror those so far identified for butyrate.

Epstein-Barr virus as a paradigm in nasopharyngeal cancer: from lab to clinic.

Nasopharyngeal carcinoma (NPC) of the undifferentiated subtype remains endemic in southern China, with a peak incidence in this region approaching 30 cases per 100,000 population per year. Despite advances in chemotherapy and radiation delivery techniques in localized disease, distant metastasis is still common and NPC remains the seventh leading cause of cancer death in the region. There is great need for early diagnosis, developing novel therapies, and identifying patients with localized disease at higher risk of future recurrence or metastasis to appropriately tailor their treatment and improve outcomes. Knowledge of the integral involvement of Epstein-Barr virus (EBV) in the pathogenesis of undifferentiated NPC has been of seminal importance in developing strategies to optimize disease management. The close association with EBV is being evaluated in multiple settings including screening of at-risk populations, disease prognostication, development of targeted therapies, optimizing adjuvant treatment, and early recurrence detection. These translational studies are likely to have an enormous effect on management of undifferentiated NPC and significantly improve the landscape of the disease in years to come.

Response of paroxysmal nocturnal hemoglobinuria clone with aplastic anemia to rituximab.

Paroxysmal nocturnal hemoglobinuria is caused by expansion of a hematopoietic stem cell clone with an acquired somatic mutation in the PIG-A gene. This mutation aborts the synthesis and expression of the glycosylphosphatidylinositol anchor proteins CD55 and CD59 on the surface of blood cells, thereby making them more susceptible to complement-mediated damage. A spectrum of disorders occurs in PNH ranging from hemolytic anemia and thrombosis to myelodysplasia, aplastic anemia and, myeloid leukemias. Aplastic anemia is one of the most serious and life-threatening complications of PNH, and a PNH clone is found in almost a third of the cases of aplastic anemia. While allogeneic bone marrow transplantation and T cell immune suppression are effective treatments for aplastic anemia in PNH, these therapies have significant limitations. We report here the first case, to our knowledge, of PNH associated with aplastic anemia treated with the anti-CD20 monoclonal antibody rituximab, which was associated with a significant reduction in the size of the PNH clone and recovery of hematopoiesis. We suggest that this less toxic therapy may have a significant role to play in treatment of PNH associated with aplastic anemia.

Current and emerging therapies in primary myelofibrosis.

Primary myelofibrosis is a clonal hematopoietic disorder characterized by ineffective hematopoiesis and progressive bone marrow fibrosis. Patients with high risk myelofibrosis as determined by their advanced age, degree of anemia, leukocytosis, constitutional symptoms and high percentage of circulating blasts have a very short median survival of 2 years. In addition quality of life is significantly compromised due to cytokine induced constitutional symptoms, frequent transfusion for cytopenias and bulky splenomegaly. Progression to myelogenous leukemia occurs in about 20% of patients within 10 years of diagnosis and is often fatal. Allogeneic hematopoietic transplantation is the only curative therapy but is limited by patient eligibility, transplant related mortality and graft versus host disease. Androgens, erythropoietin analogues, hydroxyurea, alkylators and spleen directed therapies have all been used with limited efficacy and no curative potential. The discovery of mutations in the hematopoietic progenitors of patients with myelofibrosis, including the JAK2 V617F mutation and others has greatly improved our understanding of the disease and facilitated development of newer targeted therapies. Our article will review new discoveries in the pathogenesis of myelofibrosis and focus on emerging targeted treatments. These novel therapies including oral JAK2 inhibitors, immunomodulators, as well as inhibitors of HDAC and mTOR, in isolation and in combination are likely to improve outcomes in management of this disease.

Induction of fetal/embryonic globin gene expression depends on intact cell signaling in definitive erythroid cells.

OBJECTIVE: The induction of fetal hemoglobin during definitive erythropoiesis is a major therapeutic goal in ß-globin gene disorders. Butyrate induces fetal hemoglobin, and p38 phosphorylation has been implicated in this process. We studied p38 and the effect of its inhibitors in a physiologic primary cell model of fetal/embryonic globin gene induction during definitive erythropoiesis. METHODS: p38 phosphorylation was evaluated in a short-term culture of definitive erythroid precursor (EryD) cells following butyrate induction, absent prolonged exposure to cytokines. The impact of p38 inhibitors on embryonic/fetal globin gene induction by butyrate and on normal erythroid processes, including proliferation, differentiation, cell cycle occupancy, and RNA transcription, was also examined. RESULTS: p38 phosphorylation, maximal at harvest of murine fetal liver-derived EryD (FL EryD), when minimal embryonic/fetal globin gene expression is seen, is suppressed by EPO (as reported by others). Butyrate initially delays EPO-mediated suppression of p38 phosphorylation, but p38 phosphorylation thereafter, at 30 minutes to 48 hours, is equivalent and at low levels in EPO-treated FL EryD, with or without butyrate. Inhibitors of p38, at 10-50 µM, prevent butyrate-mediated induction of embryonic/fetal globin gene expression. We found that p38 inhibitors, which also disrupt non-p38 signaling pathways, perturb cell division, erythroid differentiation, transit through the cell cycle, and RNA transcription in primary EryD. CONCLUSION: p38 inhibitors interrupt normal erythropoiesis and the capacity for embryonic/fetal globin gene induction. However, p38 signaling is maximal in primary EryD at harvest, when embryonic globin genes are minimally expressed, and diminishes thereafter. We conclude that p38 inhibitors disrupt cellular pathways that are essential to butyrate-induced embryonic/fetal globin gene expression. However, levels of p38 phosphorylation are not coordinate with embryonic/fetal globin gene expression in EryD, and increased signaling through p38 may not be the sine qua non for embryonic/fetal globin gene induction.

The OnControl bone marrow biopsy technique is superior to the standard manual technique for hematologists-in-training: a prospective, randomized comparison.

The purpose of this study was to compare a novel bone marrow device with the standard marrow needle in a prospective, randomized study in a teaching hospital employing hematologists-in-training. The new device, the OnControl Bone Marrow (OBM) Biopsy System, utilizes a battery-powered drill to insert the needle. Fifty-four bone marrows (27 standard and 27 OBM) were performed by 11 fellows under the observation and supervision of 3 attending hematologists and 1 research technologist. The primary endpoint of the study, the mean length of the marrow biopsy specimens, a surrogate for marrow quality, was determined by a pathologist in a blinded manner. The mean length of the marrow biopsy specimens was significantly longer (56%) for the OBM group (15.3 mm) than for the standard bone marrow (SBM) group (9.8 mm), P<0.003. An objectively determined secondary endpoint; mean procedure time, skin-to-skin; also favored the OBM group (175 s) versus the SBM group (292 s), P<0.007. Several subjective secondary endpoints also favored the OBM group. Only minor adverse events were encountered in the OBM and SBM study groups. It was concluded that bone marrow procedures (BMPs) performed by hematologists-in-training were significantly faster and superior in quality when performed with the OBM compared to the SBM. These data suggest that the OBM may be considered a new standard of care for adult hematology patients. OBM also appears to be a superior method for training hematology fellows.

Iron overload in sickle cell disease.

In sickle cell disease transfusions improve blood flow by reducing the proportion of red cells capable of forming sickle hemoglobin polymer. This limits hemolysis and the endothelial damage that result from high proportions of sickle polymer-containing red cells. Additionally, transfusions are used to increase blood oxygen carrying capacity in sickle cell patients with severe chronic anemia or with severe anemic episodes. Transfusion is well-defined as prophylaxis (stroke) and as therapy (acute chest syndrome and stroke) for major complications of sickle cell disease and has been instituted, based on less conclusive data, for a range of additional complications, such as priapism, vaso-occlusive crises, leg ulcers, pulmonary hypertension, and during complicated pregnancies. The major and unavoidable complication of transfusions in sickle cell disease is iron overload. This paper provides an overview of normal iron metabolism, iron overload in transfused patients with sickle cell disease, patterns of end organ damage, diagnosis, treatment, and prevention of iron overload.