Central Nervous System Infiltration Research Articles

Neuroinflammation in amyotrophic lateral sclerosis: pathogenic insights and therapeutic implications.

Neuroinflammation appears to be an important pathogenic process in amyotrophic lateral sclerosis (ALS). Dysfunction of central immune pathways, including activation of microglia and astrocytes, and peripherally derived immune cells, initiate noncell autonomous inflammatory mechanisms leading to degeneration. Cell autonomous pathways linked to ALS genetic mutations have been recently identified as contributing mechanism for neurodegeneration. The current review provides insights into the pathogenic importance of central and peripheral inflammatory processes in ALS pathogenesis and appraises their potential as therapeutic targets. ALS is a multistep process mediated by a complex interaction of genetic, epigenetic, and environmental factors. Noncell autonomous inflammatory pathways contribute to neurodegeneration in ALS. Activation of microglia and astrocytes, along with central nervous system infiltration of peripherally derived pro-inflammatory innate (NK-cells/monocytes) and adaptive (cell-mediated/humoral) immune cells, are characteristic of ALS. Dysfunction of regulatory T-cells, elevation of pro-inflammatory cytokines and dysbiosis of gut microbiome towards a pro-inflammatory phenotype, have been reported as pathogenic mechanisms in ALS. Dysregulation of adaptive and innate immunity is pathogenic in ALS, being associated with greater disease burden, more rapid disease course and reduced survival. Strategies aimed at modulating the pro-inflammatory immune components could be of therapeutic utility.

Intracranial residual lesions following early intensification in a patient with T-cell acute lymphoblastic leukemia: a case report

BackgroundT-cell acute lymphoblastic leukemia (T-ALL) tends to involve central nervous system (CNS) infiltration at diagnosis. However, cases of residual CNS lesions detected at the end of induction and post early intensification have not been recorded in patients with T-ALL. Also, the ratio and prognosis of patients with residual intracranial lesions have not been defined.Case presentationA 9-year-old boy with T-ALL had multiple intracranial tumors, which were still detected post early intensification. To investigate residual CNS lesions, we used 11C-methionine (MET)-positron emission tomography. Negative MET uptake in CNS lesions and excellent MRD status in bone marrow allowed continuing therapies without hematopoietic cell transplantation.ConclusionsIn cases with residual lesions on imaging studies, treatment strategies should be considered by the systemic response, direct assessment of spinal fluid, along with further development of noninvasive imaging methods in CNS. Further retrospective or prospective studies are required to determine the prognosis and frequency of cases with residual intracranial lesions after induction therapy.

Treatment of secondary CNS lymphoma using CD19-targeted chimeric antigen receptor (CAR) T cells

BackgroundAggressive B cell lymphoma with secondary central nervous system (CNS) involvement (SCNSL) carries a dismal prognosis. Chimeric antigen receptor (CAR) T cells (CAR-T) targeting CD19 have revolutionized the treatment for B cell lymphomas; however, only single cases with CNS manifestations successfully treated with CD19 CAR-T have been reported.MethodsWe prospectively enrolled 4 patients with SCNSL into our study to assess clinical responses and monitor T cell immunity.ResultsTwo of four SNCSL patients responded to the CD19-targeted CAR-T. Only one patient showed a substantial expansion of peripheral (PB) CAR-T cells with an almost 100-fold increase within the first week after CAR-T. The same patient also showed marked neurotoxicity and progression of the SNCSL despite continuous surface expression of CD19 on the lymphoma cells and an accumulation of CD4+ central memory-type CAR-T cells in the CNS. Our studies indicate that the local production of chemokine IP-10, possibly through its receptor CXCR3 expressed on our patient’s CAR-T, could potentially have mediated the local accumulation of functionally suboptimal anti-tumor T cells.ConclusionsOur results demonstrate expansion and homing of CAR-T cells into the CNS in SNCSL patients. Local production of chemokines such as IP-10 may support CNS infiltration by CAR-T cells but also carry the potential of amplifying local toxicity. Future studies investigating numbers, phenotype, and function of CAR-T in the different body compartments of SNSCL patients receiving CAR-T will help to improve local delivery of “fit” and highly tumor-reactive CAR-T with low off-target reactivity into the CNS.

Patterns and prognostic impact of CNS infiltration in adults with newly diagnosed acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is highly associated with central nervous system (CNS) infiltration and can be associated with higher risk of relapse. Conventional cytology (CC) is the traditional method for diagnosing CNS infiltration, although the use of immunophenotyping by flow cytometry (FC) has gained prominence in recent years due to its higher sensitivity. Also, some authors have proposed that CSF contamination by a traumatic lumbar puncture (TLP) could have a clinical impact. This retrospective study accessed the impact of CNS infiltration by CC or FC on overall survival, event-free survival, and relapse rate. In a cohort of 105 newly diagnosed ALL patients, CNS1, CNS2, and CNS3 status were found in 84%, 14%, and 2%, respectively. We found that extramedullary disease at the diagnosis, higher leukocyte counts, and higher blast percentage were associated with a positive CC. Sensitivity and specificity of CC were 53% and 98%, respectively. Three-year overall survival was 42.5%. Conversely, TLP was not associated with a positive CC nor had an impact on relapse rates. In multivariate analysis, a positive CC was associated with an increased relapse rate (HR 2.074, p = 0.037), while its detection by FC did not associate with this endpoint. Survival rates seem to be increasing over the last years by the adoption of a stratified CNS prophylaxis risk strategy. CSF contamination does not represent a major concern according to our report, as it did not increase CNS involvement or relapse rates.

SARS-CoV-2-associated T-cell infiltration in the central nervous system.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Although an acute SARS-CoV-2 infection mainly presents with respiratory illness, neurologic symptoms and sequelae are increasingly recognised in the long-term treatment of COVID-19 patients. The pathophysiology and the neuropathogenesis behind neurologic complications of COVID-19 remain poorly understood, but mounting evidence points to endothelial dysfunction either directly caused by viral infection or indirectly by inflammatory cytokines, followed by a local immune response that may include virus-specific Tcells. However, the type and role of central nervous system-infiltrating T cells in COVID-19 are complex and not fully understood. We analysed distinct anatomical brain regions of patients who had deceased as a result of COVID-19-associated pneumonia or complications thereof and performed T cell receptor Vβ repertoire sequencing. Clonotypes were analysed for SARS-CoV-2 association using public TCR repertoire data. Our descriptive study demonstrates that SARS-CoV-2-associated T cells are found in almost all brain areas of patients with fatal COVID-19 courses. The olfactory bulb, medulla and cerebellum were brain regions showing the most SARS-CoV-2 specific sequence patterns. Neuropathological workup demonstrated primary CD8+ T-cell infiltration with a perivascular infiltration pattern. Future research is needed to better define the relationship between T-cell infiltration and neurological symptoms and its long-term impact on patients' cognitive and mental health.

Ursolic acid derivative UAOS-Na treats experimental autoimmune encephalomyelitis by immunoregulation and protecting myelin.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Ursolic acid (UA) can be used in the MS treatment with anti-inflammatory and neuroprotective activities. However, UA is insoluble in water, which may affect its medication effectiveness. In our previous study, UAOS-Na, a water-soluble derivative of UA was obtained. In this study, we evaluated the pharmacological effects and explored its underlying mechanism of UAOS-Na on experimental autoimmune encephalomyelitis (EAE). Firstly, the pharmacodynamics of UAOS-Na was investigated in EAE and Cuprizone-induced mice. And then the possible mechanisms were investigated by TMT proteomics and verified by in vitro and in vivo experiments. UAOS-Na (30 mg/kg/d) delayed the onset time of EAE from 11.78 days post immunization (dpi) to 14.33 dpi, reduced the incidence from 90.0% to 42.9%. UAOS-Na (60 mg/kg/d) reduced the serum levels of IFN-γ, IL-17A, TNF-α and IL-6, reduced the mononuclear cell infiltration of spinal cord, and inhibited the overexpression of key transcription factors T-bet and ROR-γt of EAE mouse spinal cord. In addition, UAOS-Na attenuated demyelination and astrogliosis in the CNS of EAE and cuprizone-induced mice. Mechanistically, proteomics showed that 96 differential expression proteins (DEPs) were enriched and 94 were upregulated in EAE mice compared with normal group. After UAOS-Na treatment, 16 DEPs were enriched and 15 were downregulated, and these DEPs were markedly enriched in antigen processing and presentation (APP) signaling pathway. Moreover, UAOS-Na downregulated the protein levels of Tapbp and H2-T23 in MHC-I antigen presentation pathway and reduced the proliferation of splenic CD8 T cells, thereby inhibiting the CNS infiltration of CD8 T cells. Our findings demonstrated that UAOS-Na has both myelin protective and anti-inflammatory effects. And it could reduce the inflammation of MS by downregulating the expression of Tapbp and H2-T23 in the MHC-I antigen presentation pathway.

Phase I Open-Label Single-Arm Study of Sscart-19 in Adult Patients with Relapsed/Refractory B-Cell Acute Lymphoblastic Leukemia in China

Background: Although CAR-T therapy has shown amazing efficacy in the treatment of hematologic malignancies in recent years, it is also accompanied by high-frequency of cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS), especially in acute lymphoblastic leukemia patients. Therefore, it is not recommended to enroll patients with active CNS involvement in CAR-T therapy. ssCART-19 is an autologous CD19-specific chimeric antigen receptor T-cell with short hairpin RNA-IL-6 gene silencing element. ssCART-19 can simultaneously express CAR structure and IL-6 silencing element. CAR structure can recognize and bind CD19 positive tumor cells, enhance T cell amplification, and finally exert T cell killing activity, such as releasing cytokines and perforin. Post-transcriptional expression of short hairpin RNA-IL-6 gene silencing element can recognize mRNA of IL-6, inhibit the expression of endogenous IL-6 in CAR-T cells through RNA interference mechanism, and then reduce the stimulation or activation intensity of downstream immune cells (mainly monocytes), with the control of the overall release intensity of cytokines, it reduces the incidence of severe CRS and ICANS, at the end reduces the clinical risk of CAR-T therapy. Methods: The trial of ssCART-19 in adult patients with R/R B-cell ALL (NCT04825496) is a single-arm, open-label Phase 1 study conducted in China. Patients underwent leukapheresis to obtain T cells for ssCART-19 manufacturing. Patients received fractionated infusions split over 3 days (10%; 30%; 60%) after lymphodepleting chemotherapy (3 days fludarabine and cyclophosphamide). Adverse events (AEs) were graded according to CTCAE, v5.0; CRS and ICANS were graded according to ASTCT Consensus Grading. The overall response rate(ORR) of complete response (CR) and CR with incomplete hematological recovery (CRi) within 3 months, duration of remission, relapse-free survival, and OS were assessed as secondary endpoints. Results : ssCART-19 was administered at 3 doses levels (DL) of 1×10 6/kg, 5×10 6/kg, or 10×10 6/kg. As of June 30th 2023 data cutoff (the median follow-up 15.9 months), 13 patients enrolled and underwent leukapheresis, 12 patients were received ssCART-19 treatment, 5 patients in DL1, 6 patients in DL2, and 1 patients in DL3. ssCART-19 was manufactured using serum-free culture system and no manufacture failure was reported. All 12 patients diagnosed with B-cell ALL were relapsed (75.0%) and refractory (25.0%) to multiple lines of prior therapy (100.0% patients had received 2 or more therapies). Five patients (41.7%) had more than 50% blasts in the bone marrow. All 12 patients did not have DLT events within 28 days. The most common ssCART-19-related AEs were transient neutropenia (100.0%), lymphopenia (100.0%), leukopenia (100.0%), anaemia (100.0%) and thrombocytopenia (83.3%). Adverse events of special interest (AESI) were CRS and ICANS. Among the 12 patients, 9 patients (75.0%) developed CRS, and 3 patients (25.0%) were grade 3 CRS. Notably, none of the 12 patients were occurred ICANS. No death cases were reported due to CRS or ICANS. A 83.3% overall response rate was achieved in efficacy-evaluable patients (n=12), with 7 complete response (CR, 58.3%), and 3 CR with incomplete hematological recovery (CRi,25.0%). The median duration of remission, relapse-free survival and OS have not been reached. Among them, 6 patients were in ongoing remission for over 12 months. Notably, one of the 12 patients (S012) had central nervous system infiltration with grade 2 before ssCART-19 infusion(Figure 1). After ssCART-19 therapy, the patient had no ICANS and got continuous remission. Robust CAR T-cell expansion occurred in all patients with a median time to T max of 12.5 days (range 9-14d), and peak copy number (C max) of 52495 (194-348000) copies/μg DNA. Conclusions ： ssCART-19 CAR-T cell therapy can effectively reduce the incidence of severe CRS and ICANS, especially control the occurrence of ICANS. And ssCART-19 achieved a high rate of remission in adult patients with R/R B-cell ALL. The ORR within 3 months were 83.3%. And, ssCART-19 shows good clinical efficacy in the treatment of leukemia patients with central nervous system infiltration. Thus, with the combination of short hairpin RNA-IL-6 gene silencing element, ssCART-19 can greatly improve the safety of CAR-T therapy, especially in central nervous system infiltration patients.

Deciphering Transcriptomic and (Epi)Genetic Signatures of Central Nervous System Infiltration in Patients Diagnosed with Diffuse Large B-Cell Lymphoma

Introduction Central Nervous System (CNS) involvement in Diffuse Large B Cell Lymphoma (DLBCL) is a rare complication with bad prognosis. The CNS Prognostic Index is employed to categorize patients at a high risk of CNS relapse; however, its effectiveness is not accurate. The discovery of precise biomarkers for predicting CNS involvement in DLBCL patients remains unmet. Our objective was to characterize the immune, transcriptomic, genomic and epigenomic hallmarks of systemic tumors at diagnosis of patients who later experienced relapse in the CNS. By exploring these biological profiles, we sought to gain valuable insights into the underlying mechanisms and potential biomarkers associated with CNS relapse in DLBCL. Methods We conducted a retrospective study including 38 patients diagnosed with nodal DLBCL who experienced relapse within the first two years after diagnosis. Among these patients, 12 individuals relapsed specifically in the CNS, while 26 relapsed in systemic sites. As a control group, we included 21 patients who did not relapse. DNA and RNA were extracted from FFPE tumor samples collected at diagnosis. To evaluate immune cell infiltration, we utilized the nCounter PanCancer Immune Profiling Panel (Nanostring); cell-of-origin (COO) was determined using the Lymph2Cx assay. Transcriptomic profiles were generated by bulk RNA sequencing and mutational analysis was performed using a custom targeted NGS panel, which included the most frequently mutated genes observed in lymphoid neoplasms. Methylation profiles were obtained using the Illumina Infinium MethylationEPIC Array 850K. Results Immune profiling analysis showed cell composition was not significantly different between groups; however, we observed distinct immune gene expression patterns at diagnosis in patients who experienced CNS relapse. Multiple genes were overexpressed in the CNS-relapsed group compared to non-relapsed group, which encompassed various functional categories including chemokine signaling such as IL7R (FC=2; P=0.05) and CCR6 (FC=3.4; P=0.01); adhesion like VCAM1 (FC=1.6; P=0.03) and ITGA4 (FC=2.2; P=0.006); and immune regulation such as CD1A (FC=4.8; P=0.001), LTB (FC=3.3; P=0.009) and BTLA (FC=2.3; P=0.02) (Fig.1A). The ITGA4 gene also presented higher expression levels in the CNS-relapsed group when compared to both systemic-relapsed and non-relapsed groups combined (FC=1.7; P=0.03). No significant differences were observed in the COO classification between groups. Whole-genome transcriptomic data revealed enrichment in gene sets associated with proliferation and survival in the CNS-relapsed group (Fig.1B). Analysis of genetic alterations indicated that patients with CNS relapse exhibited an increased mutation load and frequency in PIM1, IGLL5, and KMT2D genes. Finally, analysis of the CNS relapse-related epigenome showed 343 differentially hypomethylated CpG sites compared to both systemic and non-relapsed groups together, affecting a total of 211 genes. Pathway enrichment analysis of these genes revealed a link between CNS infiltration and cell migration and locomotion. Conclusions Tumors from DLBCL patients that will relapse in the CNS present a higher chemokine, adhesion and proliferation signatures at diagnosis. Of notice, BTLA, CCR6 and IL7R proteins have been previously reported to be implicated in B cell entry into the CNS in B-cell malignancies. Interestingly, patients with CNS relapse demonstrated significant overexpression of the ITGA4 gene, which encodes the integrin α4 subunit (CD49d) of the Very Late Antigen-4. Engagement between CD49d on B cells and its endothelial ligand VCAM-1 is required for migration of these cells across the blood-brain barrier. In fact, Natalizumab (anti-CD49d mAb) is administered in multiple sclerosis to impede entrance and accumulation of B cells in the brain. Initial experiments in mice have shown increased expression of CD49d in lymphoma B cells that have infiltrated the brain, compared to those in the spleen. Herein, we have demonstrated that DLBCL cells at diagnosis already own specific transcriptomic and (epi)genetic hallmarks that could confer a higher capacity to infiltrate the CNS. Validation of these findings in larger cohorts of patients could improve selection of patients with an increased risk of CNS involvement in need of prophylactic CNS-directed therapies at diagnosis.

High Efficacy and Safety of Nanobody Based Anti-BCMA CAR-T Cell Therapy in Treating Patients with Relapsed or Refractory Multiple Myeloma

Introduction Refractory or relapsed (R/R) multiple myeloma (MM) is often associated with a poor prognosis following chemotherapy or even transplantation. BCMA-targeted CAR-T therapy has demonstrated notable efficacy in R/R MM. In the past, the overall response rate (ORR) was relatively low at around 60%, but in recent years, advancements in the manufacturing of BCMA-CAR-T products have led to a substantial improvement in efficacy. Here we explore the efficacy and safety of nanobody-based anti-BCMA CAR-T cell therapy in R/R MM from a clinical trial (https://clinicaltrials.gov NCT04447573). Methods Autologous peripheral blood lymphocytes were collected from patients, who subsequently underwent intravenous fludarabine (30mg/m 2/d) and cyclophosphamide (300mg/m 2/d) lymphodepletion chemotherapy from day-5 to day -3. The SL-BCMA CAR molecule, an anti-BCMA CAR-T cell product generated by Senlang Biotechnology, features a structure that includes dual nanobody VHHs targeting BCMA (dVHHs), CD8α hinge region, CD8α transmembrane domain (TMD), co-stimulatory domain (4-1BB), and CD3 zeta domain (CD3ζ). Among them, dVHHs consist of two different VHHs connected by short peptides. The CD8α hinge region provides an effective spatial architecture for the binding of dVHHs and BCMA protein. The first signal of CAR-T cell activation is completed along with the intracellular CD3ζ domain. The 4-1BB intracellular co-stimulatory domain provides co-stimulatory signals for the activation of CAR-T cells. Under the dual signal stimulation, CAR-T cells are activated and expanded. After the BCMA CAR-T cells infusion on day 0, patients underwent evaluations, including immunoglobulin fixed electrophoresis, free light chains in blood and urine, bone marrow penetration, and whole-body PET CT to evaluate the efficacy at the first month, second and third month, and every three months thereafter. Results From April 2021 to May 2023, 21 R/R MM patients in our hospital were treated with SL-BCMA CAR-T, including 10 males and 11 females. The median age is 57 years (30-73 years). Among them, there were 2 cases with Plasma cell leukemia, 10 had extramedullary multiple lesions, and 2 had paralysis caused by central nervous system infiltration. Pre-CAR-T, 11 patients had a prior transplant history including 10 who relapsed after autologous transplantation and 1 who relapsed after allogeneic transplantation. One patient had a rare type of anaplastic Plasma cell tumor. Seven patients presented high-risk factors characterized by FISH abnormalities such as t (4; 14) (p16; q32), t (14; 16) (q32; q23), and 3 had TP53 mutations. The median BCMA expression rate among 19 patients was 53.3% (range: 18.5% -100%), while the remaining 2 patients displayed non-BCMA expression. The median BCMA CAR-T cell dose was 1*10 6/Kg (0.3-2*10 6/Kg). One month following BCMA-CAR-T cell infusion, the ORR was 95%(20/21), and the CR rate was 38% (8/21). The patient who initially showed no response achieved a partial response following a second infusion of BCMA-CAR-T cells. One died at about 2.5 months due to lung infection. Evaluations conducted at the three-month mark showed an ORR of 95% (19/20) and a CR rate of 80% (16/20). One patient relapsed at the second month and underwent salvage allogeneic hematopoietic stem cell transplantation. For the long follow-up data, the median duration of remission was 11 months. Of the 7 deaths, 2 were attributed to infection during disease control, and 5 were due to disease progression. (Figure 1) In terms of side effects, 6 patients developed grade 2 cytokine release syndrome (CRS), and only 1 patient developed grade 3 CRS. Only one patient had grade 1 neurotoxicity. Conclusions The clinical trial demonstrated that BCMA CAR-T therapy, composed of dual nanobody VHHs targeting BCMA (dVHHs), exhibits a high ORR with a manageable safety profile in treating R/R MM patients. This extends even to high-risk patients, such as those with extramedullary lesions, cytogenetics high-risk groups, and patients with plasma cell leukemia or anaplastic plasma cell tumor.

Lncrna Famlf-2 Promotes Leukemogenesis Via the DHX9/c-Myc Axis in Acute Lymphoblastic Leukemia

Long noncoding RNAs (lncRNAs), defined as the transcripts longer than 200 nt without protein-coding capacity, have been found to be aberrantly expressed in diverse human diseases including leukemia. Our previous study identified Familial acute myelogenous leukemia related factor ( FAMLF) in an acute myeloblastic leukemia (AML) pedigree. Two splice variants of FAMLF, FAMLF-1 and FAMLF-2 were detected. FAMLF-1 is upgraded in AML patients and associated with poor prognosis. However, the full cDNA sequence of FAMLF-2 is not verified and its role in leukemogenesis remain unclear. Herein, we aim to investigate the functional and mechanistic roles of FAMLF-2 in leukemia. Using real-time quantitative RT-PCR assay, we found that the expression of FAMLF-2 was at higher levels in Acute Lymphoblastic Leukemia(ALL) cell lines when compared to AML cell lines. Bone marrow cells were obtained from de novo ALL patients. qRT-PCR showed FAMLF-2 was up-regulated in ALL patients (N=115) compared to healthy controls (N=72). The FAMLF-2 expression was positively correlated with the poor prognosis in ALL patients by Kaplan-Meier analysis. These data demonstrated that FAMLF-2 is overexpressed in ALL patients and is associated with poor prognosis. To verify the full cDNA sequence of FAMLF-2, 5‘ and 3‘ rapid amplification of the cDNA ends (RACE) was performed, and we identified the 2393 nt of FAMLF-2. FAMLF-2 is predicted to not have protein-coding potential. Further subcellular analysis showed FAMLF-2 is mainly localized in the nucleus of Jurkat cells by lncRNA FISH and subcellular fractionation assay. To further verify the role of FAMLF-2 in ALL leukemogenesis, a xenograft model was established in NCG mice by subcutaneously or intravenously injection of engineered Jurkat cells (knockdown group: shNC and sh FAMLF-2, overexpression group: pCDH and FAMLF-2). We found that the size and weight of tumors in FAMLF-2 knockdown group were significantly decreased compared with the empty vector in subcutaneous ALL xenograft tumor model. and the mice in the sh FAMLF-2 groups survived longer than those in the shNC groups in introvenous ALL xenograft tumor model (sh FAMLF-2 vs. shNC: 55 days vs. 43 days, P<0.01). These data suggested that downregulation of FAMLF-2 inhibits leukemia development. Although no significant difference was found in the overall survival of mice overexpressed FAMLF-2. We observed the accelerated central nervous system(CNS) infiltration of leukemia cells when FAMLF-2 was overexpressed (clinical score at 32th day from Vector vs. FAMLF-2: 2.7 scores vs. 1.2 scores, p < 0.01 ). In vitro study revealed knockdown of FAMLF-2 inhibited cell proliferation and promoted cell apoptosis in ALL cells, and vice versa. Taken together, our clinical, mouse model, and AML cell data demonstrated that FAMLF-2 promotes leukemogenesis. RNA sequencing was performed to study how FAMLF-2 promotes ALL development. Gene Set Enrichment Analysis (GSEA) of genes downregulated by FAMLF-2 showed “Myc targets” with the highest normalised enrichment score (NES) in Jurkat cells. C-Myc is a well-recognized transcription factor that involves in leukemogenesis. To investigate whether FAMLF-2 performed direct interaction with c-Myc, we performed RNA pull-down followed by mass spectrometry analysis. Instead of c-Myc, DExH-Box Helicase 9 (DHX9) has the highest abundance. The Western blot and RNA immunoprecipitation (RIP) assay were further performed to validated the direct interaction between FAMLF-2 and DHX9. DHX9 is an RNA helicase that participates in c-Myc mRNA stability. Inhibition of FAMLF-2 suppressed DHX9 and c-Myc expression. Reduced interaction between DHX9 protein and c-Myc mRNA were observed when FAMLF-2 were downregulated. Furthermore, RNA stability assays suggested that knockdown of FAMLF-2 leads to a declined half-life of c-Myc mRNA. Collectively, these results demonstrated that FAMLF-2 regulates the stability of c-Myc mRNA through direct binding of DHX9, thus involved in the ALL development. In summary, our study revealed the previous undefined cDNA sequence of FAMLF-2, and provides new insights of FAMLF-2 in leukemogenesis via the DHX9/c-Myc axis, which might act as a prospective prognostic biological marker in ALL.

Selinexor With Anti-PD-1 Antibody as a Potentially Effective Regimen for Patients With Natural Killer/T-Cell Lymphoma Failing Prior L-Asparaginase and PD-1 Blockade.

Natural killer/T-cell lymphoma (NKTCL) is a rare and heterogeneous tumor type of non-Hodgkin's lymphoma (NHL) with a poor clinical outcome. There is no standardized salvage treatment failing l-asparaginase-based regimens. Here we report our retrospective results of the combined use of selinexor and PD-1 blockade (tislelizumab) in 5 patients with NKTCL who had exhausted almost all available treatments. A total of 5 patients with relapsed/refractory(R/R) NK/T-cell lymphomas failing prior l-asparaginase and anti-PD-1 antibody were retrospectively collected. They were treated with at least one cycle of XPO1 inhibitor plus the same anti-PD-1 antibody. Anti-PD-1 antibody (Tislelizumab) was administrated at 200 mg on day 1 every 3 weeks and selinexor doses and schedules ranged from 40 mg weekly for 2 weeks per 21-day cycle to 60 mg weekly per cycle. Five patients with relapsed NKTCL with extensive organ involvement including 4 central nervous system (CNS) infiltration patients were included. Four patients achieved objective responses including 3 complete responses (CR) and 1 partial response (PR). After a median follow-up time of 14.5 (range, 5-22) months, 1 patient was still in remission with CR, and the other 4 patients discontinued due to disease progression with a median progression-free survival (PFS) of 6 months and median overall survival (OS) of 12 months. Four patients with CNS involvement achieved a median OS of 8 months. Our data suggest that selinexor in combination with an anti-PD-1 antibody is a promising small molecule and immunotherapy combination regimen for patients with relapsed or refractory NKTCL.

Combination Treatment of Locoregionally Aggressive Granulomatosis with Polyangiitis and Cranial Base Infiltration.

To present a personalized approach in three cases of treatment-resistant, locoregionally aggressive forms of cANCA-positive granulomatosis with polyangiitis (GPA) and skull base involvement. Three patients with GPA and skull base involvement were described alongside a critical review of the current literature. All presented patients suffered from GPA with an inflammatory tumor at the skull base, alongside cerebellopontine angle involvement, cranial nerve palsies, cerebellar disorders, concomitant hearing loss, and severe otalgia. Symptoms were associated with progressive granulomatous destruction of the temporal bone, laryngopharynx, and central nervous system infiltration. Treatment with cyclophosphamide and high doses of glucocorticoid steroids were ineffective but subsequent therapy with rituximab was successful in the presented cases. The literature review showed that the course of the disease with skull base involvement is associated with poorer clinical and radiological responses to standard pharmacotherapies. Granulomatous inflammation localized in the skull base is associated with a more aggressive disease progression and is less likely to respond to pharmacotherapy. Standard induction therapy with cyclophosphamide and glucocorticoid steroids may be ineffective. A better response may be achieved by using rituximab and concomitant local treatment with glucocorticoid steroid injections.

Correlation Analysis between Cerebrospinal Fluid Status and Prognosis in Childhood with Acute Lymphoblastic Leukemia by Flow Cytometry

To study the cerebrospinal fluid (CSF) status and prognosis value in patients with newly diagnosed acute lymphoblastic leukemia (ALL) by flow cytometry (FCM). The clinical features of the 75 newly diagnosed ALL patients from September 2020 to December 2021 in our centre were retrospective analyzed, as well as the bone marrow (BM) and CSF minimal residual disease (MRD) data, and the CSF conventional cytology data. Central nervous system infiltration(CNSI) positive was as CSF MRD positive by FCM or leukemia cells detected by conventional cytology. The status of CSF were compared and analyzed by FCM and conventional cytology, the clinical features and the prognosis value of different CNSI status in these patients were analyzed. Among 75 newly diagnosed ALL, 16 cases (21%) with CNSI positive (CNSI+) were detected by FCM, while only 2 positive cases (3%) were detected by conventional cytology. The CNSI+ rate detected by FCM was significantly higher than conventional cytology(P<0.05). Compared with CNSI- ALL patients, the median age of CNSI+ ALL patients was significantly younger, and the median platelet count was significantly lower, the difference was statistically significant (P<0.05). Up to follow-up time (August 31, 2022), four ALL patients were died, including 3 patients were CNSI- and 1 patient was CNSI+. Furthermore, three cases were primary disease relapse, including 1 case was CNSI+. There was no significant difference in overall survival (OS) rate and relapse-free survival (RFS) rate of the patients with different CNSI status. Compared with conventional cytology, FCM is a more sensitive assay to evaluate the central nervous system status in ALL patients. After active treatment, there was no significant difference in OS and RFS between patients with different CNSI status at diagnosis.

EFFICACY AND SAFETY OF BTK INHIBITORS IN VITREORETINAL LYMPHOMA: A SINGLE‐CENTER RETROSPECTIVE ANALYSIS OF 24 PATIENTS

Purpose: Vitreoretinal lymphoma (VRL) is a rare subtype of lymphoma. The optimal treatment strategy is not yet defined. 60%–90% of VRL will progress to PCNSL. Therefore, central nervous system (CNS) prophylaxis for VRL patients is critical. Our previous studies have shown that BTK inhibitors monotherapy can replace repeated intravitreal injection of low-dose methotrexate, with rapid effect and a certain preventive value for the CNS. This study aimed to expand the sample size and extend the follow-up time to retrospectively analyze the efficacy and safety of BTK inhibitors in VRL. Method: A total of 24 VRL patients admitted to the Department of Hematology in Beijing Tongren Hospital from May 2020 to December 2022 were included. The clinical characteristics, treatment methods, treatment outcomes, survival status and treatment-related adverse reactions were retrospectively analyzed. Results: The median age was 62 years. There were 18 primary VRLs (PVRLs). The IL-10 in aqueous humor at baseline was significantly increased, and the IL-10/IL-6 ratio was greater than 1. There was no evidence of CNS involvement in 23 patients. All patients were treated with a BTK inhibitors (zanubrutinib 160 mg bid, or orelabrutinib 150 mg qd), and nine of them were also treated with intraocular injection of methotrexate. After 1 month of treatment, 21 patients had CR (87.5%) and 2 patients had PR (8.3%). The IL-10/IL-6 levels of aqueous humor were lower than 1 in all patients. The median duration of BTK inhibitors was 10 months. At a median follow-up of 17 months, 15 patients (62.5%) developed disease progression, including 11 with CNS infiltration and 4 with intraocular recurrence. The median PFS was 11.9 months, and the median OS was not reached. The 1-year PFS rate was 34% and the 1-year OS rate was 96%. The recurrence rate was 33.3% in the combined treatment group and 80.0% in the monotherapy group. Three patients died, all from CNS progression. BTK inhibitors were well tolerated, with grade 1-2 hypertension of 16.7%, grade 1 hypotension of 4.2%, grade 1 joint pain of 8.3%, grade 1 subcutaneous bleeding and ecchymosis of 25.0%. Four patients developed COVID-19 infection and BTK inhibitor medication was suspended. All patients had negative COVID-19 test within 2 weeks and did not develop significant pneumonia. Conclusion: BTK inhibitors showed rapid local control of VRL and were well tolerated. However, monotherapy with BTK inhibitors does not provide adequate central nervous system prophylaxis, and the combination of BTK inhibitors with other drugs, such as high-dose methotrexate, should be explored. The research was funded by: This work was supported by grants from the National Natural Science Foundation of China (grant No. 81873450, 82170181), Beijing Hospitals Authority Youth Programme (code: QML20200201), and Beijing Natural Science Foundation (No.7222027) to Liang Wang. Keywords: aggressive B-cell non-Hodgkin lymphoma, molecular targeted therapies No conflicts of interests pertinent to the abstract.

Clinical Analysis of Patients with Blastic Plasmacytoid Dendritic Cell Neoplasm

To explore the clinical characteristics, treatment, and prognosis of patients with blastic plasmacytoid dendritic cell neoplasm(BPDCN). Clinical data of 5 patients diagnosed with BPDCN in Wuhan First Hospital and Wuhan Tongji Hospital from June 2016 to November 2021 were retrospectively analyzed. Among the 5 patients, 3 were male and 2 were female, with a median age of 28(10-52) years old. Four patients showed obvious skin damage at the initial diagnosis; the other one showed clinical manifestations of acute leukemia rather than obvious skin damage at the initial diagnosis, but infiltrated skin when the disease relapsed after treatment. Other infiltration sites of lesions included bone marrow (2/5), peripheral blood (2/5), lymph nodes (3/5), liver and spleen (2/5). All patients had no clinical manifestation of central nervous system infiltration. Tumor cell specific immune markers CD4, CD56, CD123 were all positive, and the median Ki-67 index was 70%. TET2, ASXL1 and NRAS gene mutations were found respectively in 3 patients by next-generation sequencing technique (NGS). ALL-like, AML-like and invasive NK/T cell lymphoma-like first-line induction chemotherapy regimens were used for the patients. One patient died of severe complications during the early stage of chemotherapy, 3 patients were evaluated as CR, and 1 patient was evaluated as PR. 2 patients were recurred and progressed after induction of chemotherapy, and one of them was evaluated as CR after re-treatment. One patient received autologous hematopoietic stem cell transplantation (auto-HSCT) and got long-term survival (OS 87 months). 3 patients received allogeneic hematopoietic stem cell transplantation (allo-HSCT), of which one died of transplantation related complications, and 2 cases survived. The median follow-up time of 4 patients with evaluable efficacy was 28.5(9-84) months, the median OS time was 31.5(10-87) months. BPDCN is a highly heterogeneous malignant tumor with a poor prognosis. HSCT, especially allo-HSCT can significantly improve the prognosis of BPDCN patients.

Indolent T-cell lymphoma with pleural effusion and neurological signs in a cat: A case report.

This case report describes a 10-year-old castrated male Russian Blue cat who was referred to the hospital for respiratory distress and coughing. Tests for FeLV and FIV infection were negative. Pleural fluid was removed and subjected to cytological and lymphocyte clonality analysis. Thoracic radiography and ultrasonography confirmed atelectasis in some pulmonary lobes. The cat was diagnosed with stage III small- to medium-cell T cell lymphoma, and was treated with prednisolone (5 mg/head) and L-asparaginase (10000 units/m2). With this treatment, the pleural effusion gradually subsided and respiratory signs improved. However, the cat developed decreased motor function of the hindlimbs, forelimbs, and tongue, leading to the administration of 1-(2-Chloroethyl)3-cyclohexyl-1-nitrosourea (CCNU) (10mg/head or 38 mg/m2) for suspected central nervous system infiltration of lymphoma. Neurological signs dramatically improved within seven days, and CCNU (15mg/head or 54 mg/m2) was administered five more times until day 284, after which it was replaced with chlorambucil (2mg/head every four days). On day 393, weight loss and abdominal lymphadenopathy were observed, and CCNU (10mg/head) was administered again on the day 407 but was ineffective. Unfortunately, the cat eventually died on the day 466 with marked abdominal lymphadenopathy.

Neuro-ophthalmic Complications in Pediatric Leukemia.

Optic neuropathy in childhood leukemia occurs through multiple direct and indirect mechanisms, including leukemic infiltration of the optic nerve, infection, blood dyscrasias, or adverse effects of treatment. We aimed to characterize visual outcomes in pediatric patients with leukemia-associated neuro-ophthalmic manifestations. We retrospectively identified patients with leukemia and optic nerve pathology over 13 years by diagnostic billing codes. We collected information on demographics, presentation, treatment course, and visual outcomes directly from medical records. Of the 19 patients who met inclusion criteria, 17 (89.5%) had pseudotumor cerebri and 2 had direct optic nerve infiltration. Causes of increased intracranial pressure included central nervous system infiltration (6 of 17), hyperviscosity/leukemia (2 of 17), venous sinus thrombosis (3 of 17), medication induced (5 of 17), and bacterial meningitis (1 of 17). 47.1% (8 of 17) had papilledema at the time of leukemia diagnosis, and 94.1% (16 of 17) of patients with pseudotumor cerebri were treated with acetazolamide. At presentation, 3 patients had decreased vision secondary to macular ischemia, subhyaloid vitreous hemorrhage, or steroid induced glaucoma. Following treatment of pseudotumor cerebri, binocular visual acuity was ≥20/25 in all patients. One patient with optic nerve infiltration had a final visual acuity of count fingers in the affected eye. In our chart review, the most common mechanism of neuro-ophthalmic involvement in pediatric leukemia was elevated intracranial pressure from a myriad of causes. Visual outcomes from patients with elevated intracranial pressure were excellent. Understanding the mechanisms by which leukemia can cause optic nerve disease in pediatric patients can facilitate earlier diagnosis and treatment and potentially improve visual outcomes.

The contribution of the peripheral immune system to neurodegeneration.

Microglial cells are the major immune cells of the central nervous system (CNS), and directly react to neurodegeneration, but other immune cell types are also able to react to pathology and can modify the course of neurodegenerative processes. These mainly include monocytes/macrophages and lymphocytes. While these peripheral immune cells were initially considered to act only after infiltrating the CNS, recent evidence suggests that some of them can also act directly from the periphery. We will review the existing and emerging evidence for a role of peripheral immune cells in neurodegenerative diseases, both with and without CNS infiltration. Our focus will be on amyotrophic lateral sclerosis, but we will also compare to Alzheimer's disease and Parkinson's disease to highlight similarities or differences. Peripheral immune cells are easily accessible, and therefore may be an attractive therapeutic target for neurodegenerative diseases. Thus, understanding how these peripheral immune cells communicate with the CNS deserves deeper investigation.

Bystander CD8+ T cells are Important Antiviral Effectors in the Brain

Abstract The central nervous system (CNS) presents a unique immunological environment with restricted infiltration of immune cells from the periphery. Since much is unknown about CNS-infiltrating immune cells during infection, we utilized the attenuated Venezuelan equine encephalitis virus vaccine strain TC83 (VEEV-TC83) to study T cell responses to this viral infection. VEEV-TC83 intracranial (IC) infection of wild-type mice produces high viral titers in the brain without lethality, enabling the study of the complete antiviral immune response in the CNS. Many T cells rapidly (5dpi) infiltrate the CNS, in a seemingly antigen-independent manner, upon IC VEEV-TC83 infection. During infection, CD8+ T cells (~40% of CNS-infiltrating T cells) in the brain express high levels of innate-memory markers. These bystander CD8+ T cells can produce IFNγ when treated with IL-12 and IL-18, cytokines known to induce innate signaling. IL-12, IL-18, and IL-15 (another innate-memory T cell supporter) are upregulated upon IC VEEV-TC83 infection. IC infection of IL-15 knockout (KO) mice results in decreased CNS infiltration by bystander CD8+ T cells. CD8+ T cells that do infiltrate the brains of IL-15 KO mice express fewer activation markers and cytokines, and are therefore likely not fully functional. Additionally, microglia express innate-memory cytokines during infection and likely support the recruitment and function of bystander CD8+ T cells in the CNS during infection. Data deposited in GEO indicate that infection with many neurotropic viruses induces Il15, Il12, and Il18 expression in CNS cells and tissues. Bystander CD8+ T cells are likely an important first line of defense during viral infection of the CNS. Supported by grants from the NIH (Training grant T32 AI07405 and RO1 NS123431)

B cells Regulation by Interferon-gamma in a Neuromyelitis Optica Spectrum Disorder animal model

Abstract Neuromyelitis Optica Spectrum Disorder (NMOSD) is an autoimmune disease of the central nervous system (CNS) mediated by Th17 and auto-antibody response against the water channel protein Aquaporin-4 (AQP4). In NMOSD, B cells plays a central role regulating autoreactive T and antibody secreting cells, and the use of anti-B cells therapy is a common practice for patients. Despite this, the mechanisms of B cells response against AQP4 are poorly understood. We have demonstrated that the immune tolerance against the immunodominant AQP4 201–220peptide in mice is maintained by IFN-g, controlling disease incidence, severity, and chronicity. Actively immunization of C57BL/6 (WT) treated with neutralizing anti-IFN-g antibodies, IFN-g knockout (IFNGKO) and IFN-g receptor KO (IFNRKO) mice was induced using the AQP4 201–220peptide, showing an ascendant paralysis starting from the tail to complete hind limb paralysis. CNS histological analysis showed an increased presence of T and B cells, and the complement system in lesion sites, associated with CNS damage. CNS Th17 profile was also observed in the anti-IFN-g treated group. Conversely, IFN-g treatment in IFNGKO mice decrease disease severity with a reduction of CNS CD4 +and B cells. Remarkably, IFN-g regulates the IL-6 expression by B cells in an antigen specific fashion. B-cell depletion significantly reduced the disease incidence, T cells CNS infiltration and anti-AQP4 201–220serum antibodies. Finally, using anti-IL-6R treatment, we showed that IL-6 signaling is necessary for the B cell activation and their induction of Th17 cells. In conclusion, AQP4 201–220tolerance is strictly regulated by IFN-g and its signaling, restraining B cells pathogenicity through IL-6 signaling modulation. This work was supported by National Institutes of Health grants R21AI151438 and R01 NS099334