GSDMB is increased in IBD and regulates epithelial restitution/repair independent of pyroptosis.

Gasdermins are a family of structurally related proteins originally described for their role in pyroptosis. Gasdermin B (GSDMB) is currently the least studied, and while its association with genetic susceptibility to chronic mucosal inflammatory disorders is well established, little is known about its functional relevance during active disease states. Herein, we report increased GSDMB in inflammatory bowel disease, with single-cell analysis identifying epithelial specificity to inflamed colonocytes/crypt top colonocytes. Surprisingly, mechanistic experiments and transcriptome profiling reveal lack of inherent GSDMB-dependent pyroptosis in activated epithelial cells and organoids but instead point to increased proliferation and migration during in vitro wound closure, which arrests in GSDMB-deficient cells that display hyper-adhesiveness and enhanced formation of vinculin-based focal adhesions dependent on PDGF-A-mediated FAK phosphorylation. Importantly, carriage of disease-associated GSDMB SNPs confers functional defects, disrupting epithelial restitution/repair, which, altogether, establishes GSDMB as a critical factor for restoration of epithelial barrier function and the resolution of inflammation.

Clearing the Fog surrounding Chemobrain.

With the advent of more successful chemotherapies, the number of cancer survivors continues to increase. Unfortunately, many of these patients will exhibit long-term sequelae from their treatments, including serious cognitive deficits that impair daily function. In this issue of Cell, Gibson et al. (2019) demonstrate that chemotherapy-related cognitive impairment is orchestrated by microglia.

Methotrexate Chemotherapy Induces Persistent Tri-glial Dysregulation that Underlies Chemotherapy-Related Cognitive Impairment.

Chemotherapy results in a frequent yet poorly understood syndrome of long-term neurological deficits. Neural precursor cell dysfunction and white matter dysfunction are thought to contribute to this debilitating syndrome. Here, we demonstrate persistent depletion of oligodendrocyte lineage cells in humans who received chemotherapy. Developing a mouse model of methotrexate chemotherapy-induced neurological dysfunction, we find a similar depletion of white matter OPCs, increased but incomplete OPC differentiation, and a persistent deficit in myelination. OPCs from chemotherapy-naive mice similarly exhibit increased differentiation when transplanted into the microenvironment of previously methotrexate-exposed brains, indicating an underlying microenvironmental perturbation. Methotrexate results in persistent activation of microglia and subsequent astrocyte activation that is dependent on inflammatory microglia. Microglial depletion normalizes oligodendroglial lineage dynamics, myelin microstructure, and cognitive behavior after methotrexate chemotherapy. These findings indicate that methotrexate chemotherapy exposure is associated with persistent tri-glial dysregulation and identify inflammatory microglia as a therapeutic target to abrogate chemotherapy-related cognitive impairment. VIDEO ABSTRACT.

Mitochondrial One-Carbon Pathway Supports Cytosolic Folate Integrity in Cancer Cells.

Mammalian folate metabolism is comprised of cytosolic and mitochondrial pathways with nearly identical core reactions, yet the functional advantages of such an organization are not well understood. Using genome-editing and biochemical approaches, we find that ablating folate metabolism in the mitochondria of mammalian cell lines results in folate degradation in the cytosol. Mechanistically, we show that QDPR, an enzyme in tetrahydrobiopterin metabolism, moonlights to repair oxidative damage to tetrahydrofolate (THF). This repair capacity is overwhelmed when cytosolic THF hyperaccumulates in the absence of mitochondrially produced formate, leading to THF degradation. Unexpectedly, we also find that the classic antifolate methotrexate, by inhibiting its well-known target DHFR, causes even more extensive folate degradation in nearly all tested cancer cell lines. These findings shed light on design features of folate metabolism, provide a biochemical basis for clinically observed folate deficiency in QDPR-deficient patients, and reveal a hitherto unknown and unexplored cellular effect of methotrexate.

Methotrexate suppresses psoriatic skin inflammation by inhibiting muropeptide transporter SLC46A2 activity.

Cytosolic innate immune sensing is critical for protecting barrier tissues. NOD1 and NOD2 are cytosolic sensors of small peptidoglycan fragments (muropeptides) derived from the bacterial cell wall. These muropeptides enter cells, especially epithelial cells, through unclear mechanisms. We previously implicated SLC46 transporters in muropeptide transport in Drosophila immunity. Here, we focused on Slc46a2, which was highly expressed in mammalian epidermal keratinocytes, and showed that it was critical for the delivery of diaminopimelic acid (DAP)-muropeptides and activation of NOD1 in keratinocytes, whereas the related transporter Slc46a3 was critical for delivering the NOD2 ligand MDP to keratinocytes. In a mouse model, Slc46a2 and Nod1 deficiency strongly suppressed psoriatic inflammation, whereas methotrexate, a commonly used psoriasis therapeutic, inhibited Slc46a2-dependent transport of DAP-muropeptides. Collectively, these studies define SLC46A2 as a transporter of NOD1-activating muropeptides, with critical roles in the skin barrier, and identify this transporter as an important target for anti-inflammatory intervention.

Methotrexate recognition by the human reduced folate carrier SLC19A1.

Folates are essential nutrients with important roles as cofactors in one-carbon transfer reactions, being heavily utilized in the synthesis of nucleic acids and the metabolism of amino acids during cell division1,2. Mammals lack de novo folate synthesis pathways and thus rely on folate uptake from the extracellular milieu3. The human reduced folate carrier (hRFC, also known as SLC19A1) is the major importer of folates into the cell1,3, as well as chemotherapeutic agents such as methotrexate4-6. As an anion exchanger, RFC couples the import of folates and antifolates to anion export across the cell membrane and it is a major determinant in methotrexate (antifolate) sensitivity, as genetic variants and its depletion result in drug resistance4-8. Despite its importance, the molecular basis of substrate specificity by hRFC remains unclear. Here we present cryo-electron microscopy structures of hRFC in the apo state and captured in complex with methotrexate. Combined with molecular dynamics simulations and functional experiments, our study uncovers key determinants of hRFC transport selectivity among folates and antifolate drugs while shedding light on important features of anion recognition by hRFC.

Deep mutational scanning of Pneumocystis jirovecii dihydrofolate reductase reveals allosteric mechanism of resistance to an antifolate.

Pneumocystis jirovecii is a fungal pathogen that causes pneumocystis pneumonia, a disease that mainly affects immunocompromised individuals. This fungus has historically been hard to study because of our inability to grow it in vitro. One of the main drug targets in P. jirovecii is its dihydrofolate reductase (PjDHFR). Here, by using functional complementation of the baker's yeast ortholog, we show that PjDHFR can be inhibited by the antifolate methotrexate in a dose-dependent manner. Using deep mutational scanning of PjDHFR, we identify mutations conferring resistance to methotrexate. Thirty-one sites spanning the protein have at least one mutation that leads to resistance, for a total of 355 high-confidence resistance mutations. Most resistance-inducing mutations are found inside the active site, and many are structurally equivalent to mutations known to lead to resistance to different antifolates in other organisms. Some sites show specific resistance mutations, where only a single substitution confers resistance, whereas others are more permissive, as several substitutions at these sites confer resistance. Surprisingly, one of the permissive sites (F199) is without direct contact to either ligand or cofactor, suggesting that it acts through an allosteric mechanism. Modeling changes in binding energy between F199 mutants and drug shows that most mutations destabilize interactions between the protein and the drug. This evidence points towards a more important role of this position in resistance than previously estimated and highlights potential unknown allosteric mechanisms of resistance to antifolate in DHFRs. Our results offer unprecedented resources for the interpretation of mutation effects in the main drug target of an uncultivable fungal pathogen.

Post-Transplantation Cyclophosphamide-Based Graft-versus-Host Disease Prophylaxis.

BACKGROUND: In patients undergoing allogeneic hematopoietic stem-cell transplantation (HSCT), a calcineurin inhibitor plus methotrexate has been a standard prophylaxis against graft-versus-host disease (GVHD). A phase 2 study indicated the potential superiority of a post-transplantation regimen of cyclophosphamide, tacrolimus, and mycophenolate mofetil. METHODS: In a phase 3 trial, we randomly assigned adults with hematologic cancers in a 1:1 ratio to receive cyclophosphamide-tacrolimus-mycophenolate mofetil (experimental prophylaxis) or tacrolimus-methotrexate (standard prophylaxis). The patients underwent HSCT from an HLA-matched related donor or a matched or 7/8 mismatched (i.e., mismatched at only one of the HLA-A, HLA-B, HLA-C, and HLA-DRB1 loci) unrelated donor, after reduced-intensity conditioning. The primary end point was GVHD-free, relapse-free survival at 1 year, assessed in a time-to-event analysis, with events defined as grade III or IV acute GVHD, chronic GVHD warranting systemic immunosuppression, disease relapse or progression, and death from any cause. RESULTS: In a multivariate Cox regression analysis, GVHD-free, relapse-free survival was significantly more common among the 214 patients in the experimental-prophylaxis group than among the 217 patients in the standard-prophylaxis group (hazard ratio for grade III or IV acute GVHD, chronic GVHD, disease relapse or progression, or death, 0.64; 95% confidence interval [CI], 0.49 to 0.83; P = 0.001). At 1 year, the adjusted GVHD-free, relapse-free survival was 52.7% (95% CI, 45.8 to 59.2) with experimental prophylaxis and 34.9% (95% CI, 28.6 to 41.3) with standard prophylaxis. Patients in the experimental-prophylaxis group appeared to have less severe acute or chronic GVHD and a higher incidence of immunosuppression-free survival at 1 year. Overall and disease-free survival, relapse, transplantation-related death, and engraftment did not differ substantially between the groups. CONCLUSIONS: Among patients undergoing allogeneic HLA-matched HSCT with reduced-intensity conditioning, GVHD-free, relapse-free survival at 1 year was significantly more common among those who received cyclophosphamide-tacrolimus-mycophenolate mofetil than among those who received tacrolimus-methotrexate. (Funded by the National Heart, Lung, and Blood Institute and others; BMT CTN 1703 ClinicalTrials.gov number, NCT03959241.).

A phase 2 trial of CD24Fc for prevention of graft-versus-host disease.

ABSTRACT: Patients who undergo human leukocyte antigen-matched unrelated donor (MUD) allogeneic hematopoietic stem cell transplantation (HSCT) with myeloablative conditioning for hematologic malignancies often develop acute graft-versus-host disease (GVHD) despite standard calcineurin inhibitor-based prophylaxis in combination with methotrexate. This trial evaluated a novel human CD24 fusion protein (CD24Fc/MK-7110) that selectively targets and mitigates inflammation due to damage-associated molecular patterns underlying acute GVHD while preserving protective immunity after myeloablative conditioning. This phase 2a, multicenter study evaluated the pharmacokinetics, safety, and efficacy of CD24Fc in combination with tacrolimus and methotrexate in preventing acute GVHD in adults undergoing MUD HSCT for hematologic malignancies. A double-blind, placebo-controlled, dose-escalation phase to identify a recommended dose was followed by an open-label expansion phase with matched controls to further evaluate the efficacy and safety of CD24Fc in preventing acute GVHD. A multidose regimen of CD24Fc produced sustained drug exposure with similar safety outcomes when compared with single-dose regimens. Grade 3 to 4 acute GVHD-free survival at day 180 was 96.2% (95% confidence interval [CI], 75.7-99.4) in the CD24Fc expansion cohort (CD24Fc multidose), compared with 73.6% (95% CI, 63.2-81.4) in matched controls (hazard ratio, 0.1 [95% CI, 0.0-0.6]; log-rank test, P = .03). No participants in the CD24Fc escalation or expansion phases experienced dose-limiting toxicities (DLTs). The multidose regimen of CD24Fc was well tolerated with no DLTs and was associated with high rates of severe acute GVHD-free survival after myeloablative MUD HSCT. This trial was registered at ClinicalTrials.gov as #NCT02663622.

Olokizumab versus Placebo or Adalimumab in Rheumatoid Arthritis.

BACKGROUND: The cytokine interleukin-6 is involved in the pathogenesis of rheumatoid arthritis. Olokizumab, a humanized monoclonal antibody targeting the interleukin-6 cytokine directly, is being tested for the treatment of rheumatoid arthritis. METHODS: In a 24-week, phase 3, multicenter, placebo- and active-controlled trial, we randomly assigned (in a 2:2:2:1 ratio) patients with rheumatoid arthritis and an inadequate response to methotrexate to receive subcutaneous olokizumab at a dose of 64 mg every 2 or 4 weeks, adalimumab (40 mg every 2 weeks), or placebo; all patients continued methotrexate therapy. The primary end point was an American College of Rheumatology 20 (ACR20) response (≥20% fewer tender and swollen joints and ≥20% improvement in three of five other domains) at week 12, with each olokizumab dose tested for superiority to placebo. We also tested the noninferiority of each olokizumab dose to adalimumab with respect to the percentage of patients with an ACR20 response (noninferiority margin, -12 percentage points in the lower boundary of the 97.5% confidence interval for the difference between groups). RESULTS: A total of 464 patients were assigned to receive olokizumab every 2 weeks, 479 to receive olokizumab every 4 weeks, 462 to receive adalimumab, and 243 to receive placebo. An ACR20 response at week 12 occurred in 44.4% of the patients receiving placebo, in 70.3% receiving olokizumab every 2 weeks (difference vs. placebo, 25.9 percentage points; 97.5% confidence interval [CI], 17.1 to 34.1), in 71.4% receiving olokizumab every 4 weeks (difference vs. placebo, 27.0 percentage points; 97.5% CI, 18.3 to 35.2), and in 66.9% receiving adalimumab (difference vs. placebo, 22.5 percentage points; 95% CI, 14.8 to 29.8) (P<0.001 for the superiority of each olokizumab dose to placebo). Both olokizumab doses were noninferior to adalimumab with respect to the percentage of patients with an ACR20 response at week 12 (difference, 3.4 percentage points [97.5% CI, -3.5 to 10.2] with olokizumab every 2 weeks and 4.5 percentage points [97.5% CI, -2.2 to 11.2] with olokizumab every 4 weeks). Adverse events, most commonly infections, occurred in approximately 70% of the patients who received olokizumab. Antibodies against olokizumab were detected in 3.8% of the patients receiving the drug every 2 weeks and in 5.1% of those receiving it every 4 weeks. CONCLUSIONS: In patients with rheumatoid arthritis who were receiving maintenance methotrexate, olokizumab was superior to placebo and noninferior to adalimumab in producing an ACR20 response at 12 weeks. Larger and longer trials are required to determine the efficacy and safety of olokizumab in patients with rheumatoid arthritis. (Supported by R-Pharm; CREDO2 ClinicalTrials.gov number, NCT02760407.).

Central nervous system status is prognostic in T-cell acute lymphoblastic leukemia: a Children's Oncology Group report.

To determine the prognostic significance of central nervous system (CNS) leukemic involvement in newly diagnosed T-cell acute lymphoblastic leukemia (T-ALL), outcomes on consecutive, phase 3 Children's Oncology Group clinical trials were examined. AALL0434 and AALL1231 tested efficacy of novel agents within augmented-Berlin-Frankfurt-Münster (aBFM) therapy. In addition to testing study-specific chemotherapy through randomization, the AALL0434 regimen delivered cranial radiation therapy (CRT) to most participants (90.8%), whereas AALL1231 intensified chemotherapy to eliminate CRT in 88.2% of participants. In an analysis of 2164 patients with T-ALL (AALL0434, 1550; AALL1231, 614), 1564 had CNS-1 (72.3%), 441 CNS-2 (20.4%), and 159 CNS-3 (7.3%). The 4-year event-free-survival (EFS) was similar for CNS-1 (85.1% ± 1.0%) and CNS-2 (83.2% ± 2.0%), but lower for CNS-3 (71.8% ± 4.0%; P = .0004). Patients with CNS-1 and CNS-2 had similar 4-year overall survival (OS) (90.1% ± 0.8% and 90.5% ± 1.5%, respectively), with OS for CNS-3 being 82.7% ± 3.4% (P = .005). Despite therapeutic differences, outcomes for CNS-1 and CNS-2 were similar regardless of CRT, intensified corticosteroids, or novel agents. Except for significantly superior outcomes with nelarabine on AALL0434 (4-year disease-free survival, 93.1% ± 5.2%), EFS/OS was inferior with CNS-3 status, all of whom received CRT. Combined analyses of >2000 patients with T-ALL identified that CNS-1 and CNS-2 status at diagnosis had similar outcomes. Unlike B-ALL, CNS-2 status in T-ALL does not impact outcome with aBFM therapy, without additional intrathecal therapy, with or without CRT. Although nelarabine improved outcomes for those with CNS-3 status, novel approaches are needed. These trials were registered at www.clinicaltrials.gov as #NCT00408005 (AALL0434) and #NCT02112916 (AALL1231).

Rheumatology: What You May Have Missed in 2023.

Many patients with rheumatologic conditions receive care from physicians other than rheumatologists. Here we note key findings from 6 studies in rheumatology published in 2023 that offer valuable insights for internal medicine specialists and subspecialists outside of rheumatology. The first study investigated the effect of low-dose glucocorticoids on patients with rheumatoid arthritis (RA) over 2 years and challenged existing perceptions about the risks of glucocorticoids in this setting. The second study focused on the updated guideline for preventing and treating glucocorticoid-induced osteoporosis. With the chronic and widespread use of glucocorticoids, the American College of Rheumatology emphasized the importance of assessing fracture risk and initiating pharmacologic therapy when appropriate. The third study explored the potential use of methotrexate in treating inflammatory hand osteoarthritis, suggesting a novel approach to managing this challenging and common condition. The results of the fourth article we highlight suggest that sarilumab has promise as an adjunct treatment of polymyalgia rheumatica relapse during glucocorticoid dosage tapering. The fifth study evaluated sublingual cyclobenzaprine for fibromyalgia treatment, noting both potential benefits and risks. Finally, the sixth article is a systematic review and meta-analysis that assessed the therapeutic equivalence of biosimilars and reference biologics in the treatment of patients with RA. Knowledge of this recent literature will be useful to clinicians regardless of specialty who care for patients with these commonly encountered conditions.

Perioperative dose-dense methotrexate, vinblastine, doxorubicin, and cisplatin in muscle-invasive bladder cancer (VESPER): survival endpoints at 5 years in an open-label, randomised, phase 3 study.

BACKGROUND: The optimal perioperative chemotherapy for patients with muscle-invasive bladder cancer is not defined. The VESPER (French Genito-Urinary Tumor Group and French Association of Urology V05) trial reported improved 3-year progression-free survival with dose-dense methotrexate, vinblastine, doxorubicin and cisplatin (dd-MVAC) versus gemcitabine and cisplatin (GC) in patients who received neoadjuvant therapy, but not in the overall perioperative setting. In this Article, we report on the secondary endpoints of overall survival and time to death due to bladder cancer at 5-year follow-up. METHODS: VESPER was an open-label, randomised, phase 3 trial done at 28 university hospitals or comprehensive cancer centres in France, in which adults (age ≤18 years and ≤80 years) with primary bladder cancer and histologically confirmed muscle-invasive urothelial carcinoma were randomly allocated (1:1; block size four) to treatment with dd-MVAC (every 2 weeks for a total of six cycles) or GC (every 3 weeks for a total of four cycles). Overall survival and time to death due to bladder cancer (presented as 5-year cumulative incidence of death due to bladder cancer) was analysed by intention to treat (ITT) in all randomly assigned patients. Overall survival was assessed by the Kaplan-Meier method with the treatment groups compared with log-rank test stratified for mode of administration of chemotherapy (neoadjuvant or adjuvant) and lymph node involvement. Time to death due to bladder cancer was analysed with an Aalen model for competing risks and a Fine and Gray regression model stratified for the same two covariates. Results were presented for the total perioperative population and for the neoadjuvant and adjuvant subgroups. The trial is registered with ClinicalTrials.gov, NCT01812369, and is complete. FINDINGS: From Feb 25, 2013, to March 1, 2018, 500 patients were randomly assigned, of whom 493 were included in the final ITT population (245 [50%] in the GC group and 248 [50%] in the dd-MVAC group; 408 [83%] male and 85 [17%] female). 437 (89%) patients received neoadjuvant chemotherapy. Median follow-up was 5·3 years (IQR 5·1-5·4); 190 deaths at the 5-year cutoff were reported. In the perioperative setting (total ITT population), we found no evidence of association of overall survival at 5 years with dd-MVAC treatment versus GC treatment (64% [95% CI 58-70] vs 56% [50-63], stratified hazard ratio [HRstrat] 0·79 [95% CI 0·59-1·05]). Time to death due to bladder cancer was increased in the dd-MVAC group compared with in the GC group (5-year cumulative incidence of death: 27% [95% CI 21-32] vs 40% [34-46], HRstrat 0·61 [95% CI 0·45-0·84]). In the neoadjuvant subgroup, overall survival at 5 years was improved in the dd-MVAC group versus the GC group (66% [95% CI 60-73] vs 57% [50-64], HR 0·71 [95% CI 0·52-0·97]), as was time to death due to bladder cancer (5-year cumulative incidence: 24% [18-30] vs 38% [32-45], HR 0·55 [0·39-0·78]). In the adjuvant subgroup, the results were not conclusive due to the small sample size. Bladder cancer progression was the cause of death for 157 (83%) of the 190 deaths; other causes of death included cardiovascular events (eight [4%] deaths), deaths related to chemotherapy toxicity (four [2%]), and secondary cancers (four [2%]). INTERPRETATION: Our results on overall survival at 5 years were in accordance with the primary endpoint analysis (3-year progression-free survival). We found no evidence of improved overall survival with dd-MVAC over GC in the perioperative setting, but the data support the use of six cycles of dd-MVAC over four cycles of GC in the neoadjuvant setting. These results should impact practice and future trials of immunotherapy in bladder cancer. FUNDING: French National Cancer Institute.

Osteosarcoma cell death induced by innovative scaffolds doped with chemotherapeutics.

Osteosarcoma (OS) cancer treatments include systemic chemotherapy and surgical resection. In the last years, novel treatment approaches have been proposed, which employ a drug-delivery system to prevent offside effects and improves treatment efficacy. Locally delivering anticancer compounds improves on high local concentrations with more efficient tumour-killing effect, reduced drugs resistance and confined systemic effects. Here, the synthesis of injectable strontium-doped calcium phosphate (SrCPC) scaffold was proposed as drug delivery system to combine bone tissue regeneration and anticancer treatment by controlled release of methotrexate (MTX) and doxorubicin (DOX), coded as SrCPC-MTX and SrCPC-DOX, respectively. The drug-loaded cements were tested in an in vitro model of human OS cell line SAOS-2, engineered OS cell line (SAOS-2-eGFP) and U2-OS. The ability of doped scaffolds to induce OS cell death and apoptosis was assessed analysing cell proliferation and Caspase-3/7 activities, respectively. To determine if OS cells grown on doped-scaffolds change their migratory ability and invasiveness, a wound-healing assay was performed. In addition, the osteogenic potential of SrCPC material was evaluated using human adipose derived-mesenchymal stem cells. Osteogenic markers such as (i) the mineral matrix deposition was analysed by alizarin red staining; (ii) the osteocalcin (OCN) protein expression was investigated by enzyme-linked immunosorbent assay test, and (iii) the osteogenic process was studied by real-time polymerase chain reaction array. The delivery system induced cell-killing cytotoxic effects and apoptosis in OS cell lines up to Day 7. SrCPC demonstrates a good cytocompatibility and it induced upregulation of osteogenic genes involved in the skeletal development pathway, together with OCN protein expression and mineral matrix deposition. The proposed approach, based on the local, sustained release of anticancer drugs from nanostructured biomimetic drug-loaded cements is promising for future therapies aiming to combine bone regeneration and anticancer local therapy.

Dynamic genomic changes in methotrexate-resistant human cancer cell lines beyond DHFR amplification suggest potential new targets for preventing drug resistance.

BACKGROUND: Although DHFR gene amplification has long been known as a major mechanism for methotrexate (MTX) resistance in cancer, the early changes and detailed development of the resistance are not yet fully understood. METHODS: We performed genomic, transcriptional and proteomic analyses of human colon cancer cells with sequentially increasing levels of MTX-resistance. RESULTS: The genomic amplification evolved in three phases (pre-amplification, homogenously staining region (HSR) and extrachromosomal DNA (ecDNA)). We confirm that genomic amplification and increased expression of DHFR, with formation of HSRs and especially ecDNAs, is the major driver of resistance. However, DHFR did not play a detectable role in the early phase. In the late phase (ecDNA), increase in FAM151B protein level may also have an important role by decreasing sensitivity to MTX. In addition, although MSH3 and ZFYVE16 may be subject to different posttranscriptional regulations and therefore protein expressions are decreased in ecDNA stages compared to HSR stages, they still play important roles in MTX resistance. CONCLUSION: The study provides a detailed evolutionary trajectory of MTX-resistance and identifies new targets, especially ecDNAs, which could help to prevent drug resistance. It also presents a proof-of-principal approach which could be applied to other cancer drug resistance studies.

NLRP3 (rs10754558) gene polymorphism and tumor necrosis factor alpha as predictors for disease activity and response to methotrexate and adalimumab in psoriasis.

BACKGROUND: Psoriasis has a global prevalence of 1-3%, with variations observed across different ethnic groups and geographical areas. Disease susceptibility and response to anti-tumor necrosis factor-α (TNFα) drugs suggest different genetic regulatory mechanisms which may include NLR family pyrin domain containing 3 (NLRP3) polymorphism. Evaluation of the NLRP3 gene polymorphism, the serum level of CRP and TNFα in psoriasis patients and assessment of the NLRP3 (rs10754558) gene polymorphism, CRP and TNFα with disease severity and their role as biomarkers for response to Methotrexate and Adalimumab in psoriasis. The study had a total of 75 patients diagnosed with psoriasis vulgaris, who were compared to a control group of 75 healthy individuals. RESULTS: There was a highly significant difference in NLRP3 genotypes and alleles distribution between psoriasis patients and controls (P = 0.002,0.004). The heterozygote genotype GC (OR = 3.67,95%CI:1.75-7.68, P = 0.0006), was linked with increased risk of psoriasis. Additionally, The GC genotype was significantly associated with nonresponse to psoriasis therapy (OR = 11.7,95%CI:3.24-42.28, P = 0.0002). Regarding serum CRP and TNFα levels, there was a highly statistically significant difference between psoriasis patients and controls (P < 0.0001), and there was also a highly statistically significant difference between responders and non-responders in psoriasis patients regarding PASI 50 (P < 0.0001). CONCLUSIONS: The NLRP3 (rs10754558) genotypes GC was associated with the severe form of psoriasis and with nonresponse to psoriasis medication. Therefore, NLRP3 (rs10754558) gene polymorphism is an important prognostic biomarker in psoriasis patients. The serum TNFα can be used as a predictor for response to therapy in psoriasis patients. More research for evaluation of role of the NLRP3 gene polymorphism in the genetic risks and treatment outcomes associated with psoriasis is still required.

High-dose methotrexate pharmacokinetics and its impact on prognosis of paediatric acute lymphoblastic leukaemia patients: A population pharmacokinetic study.

This study delivers a comprehensive evaluation of the efficacy and pharmacokinetics of high-dose methotrexate (HDMTX) in a large cohort of Chinese paediatric acute lymphoblastic leukaemia patients. A total of 533 patients were included in the prognostic analysis. An association was observed between lower steady-state MTX concentrations (<56 µmol/L) and poorer outcomes in intermediate-/high-risk (IR/HR) patients. Subgroup analysis further revealed that this relationship between concentrations and prognosis was even more pronounced in patients with MLL rearrangements. In contrast, such an association did not emerge within the low-risk patient group. Additionally, utilizing population pharmacokinetic modelling (6051 concentrations from 815 patients), we identified the significant impact of physiological maturation, estimated glomerular filtration rate, sex and concurrent dasatinib administration on MTX pharmacokinetics. Simulation-based recommendations include a reduced dosage regimen for those with renal insufficiency and a specific 200 mg/kg dosage for infants under 1 year. The findings underscore the critical role of HDMTX in treating IR/HR populations and call for a reassessment of its application in lower-risk groups. An individualized pharmacokinetic dosage regimen could achieve the most optimal results, ensuring the largest proportion of steady-state concentrations within the optimal range.

Older age, CNS leukaemic involvement and induction tumour lysis increases the risk of methotrexate (MTX)-induced neurotoxicity in childhood acute lymphoblastic leukaemia/lymphoma: Experience from a tertiary care centre in South India.

Methotrexate (MTX), although an indispensable part of contemporary treatment protocols for childhood acute lymphoblastic leukaemia (ALL)/lymphomas (LBL) in improving outcomes, can lead to serious neurotoxicity with long-term consequences. The aetiopathogenesis, predisposing factors and treatment for MTX-induced neurotoxicity are not yet well defined. The aim of our study was to detect the incidence, risk factors and to assess the overall outcomes of MTX-induced neurotoxicity among large cohort of paediatric ALL/LBL patients treated on a uniform protocol. We conducted retrospective audit of medical records of 622 consecutive children (≤14 years) diagnosed with ALL and LBL between January 2018 and December 2022 and treated on modified BFM-95 protocol at the Department of Pediatric Oncology, Regional Cancer Centre, Thiruvananthapuram. Risk factors predisposing to MTX-induced neurotoxicity were identified using binary logistic regression analysis. Forty-three children were diagnosed with MTX-induced neurotoxicity with an incidence rate of 6.9%. More than two-thirds of them had high-grade MTX-induced neurotoxicity CTCAE v5.0 with a median age of 9 years (range: 9 months to 14 years). Almost half of them developed MTX neurotoxicity during Protocol M followed by Phase-Ib consolidation (15%). Majority of these patients (84%, 36/43) were challenged again with MTX, with 11% (4/36) developing recurrence. Fifteen per cent had persistent neurological deficits at last follow-up. Univariate analysis found older age (age > 5 years) (p < 0.001), T-cell phenotype (p = 0.040), tumour lysis syndrome during induction (p < 0.001), baseline renal problems prior to MTX exposure (p < 0.001) and CNS leukaemic involvement (p < 0.003) to be significantly associated with MTX neurotoxicity. On multivariate analysis, older age (>5 years), tumour lysis during induction and CNS leukaemia retained statistical significance (p < 0.05). Methotrexate-induced neurotoxicity during paediatric acute lymphoblastic leukaemia/lymphoma therapy is a transient phenomenon in majority and re-challenge with MTX is generally safe. Older age children who develop tumour lysis during induction and CNS leukaemic involvement are at increased risk for MTX-induced neurotoxicity during ALL/LBL treatment.

Clinical factors for central nervous system progression and survival in primary vitreoretinal lymphoma.

Primary vitreoretinal lymphoma (PVRL) is a rare subtype of malignant lymphoma with a poor prognosis because of high frequency of central nervous system (CNS) progression. Identification of factors associated with CNS progression is essential to improve the prognosis of patients with PVRL. We conducted a retrospective study of 54 patients diagnosed with PVRL and treated at our hospital to identify factors associated with CNS progression and prognosis. All patients were treated with intravitreal methotrexate (MTX) injections in the affected eyes until lesion resolution. Twenty-four patients were treated with systemic administration of high-dose MTX (systemic HD-MTX) every other week for a total of five cycles following intravitreal MTX injection. Of 24 patients, 20 completed five cycles of systemic HD-MTX. The 5-year cumulative incidence of CNS progression and overall survival (OS) rate were 78.0% and 69.0% respectively. By univariate and multivariate analyses, bilateral disease and the detection of B-cell clonality confirmed by flow cytometric analysis were risk factors associated with CNS progression. Moreover, systemic HD-MTX completion reduced the risk of CNS progression and was identified as a factor affecting OS. In this study, factors for CNS progression identified may potentially contribute to the optimized therapeutic stratification to improve the survival of patients with PVRL.

Rheumatoid arthritis: prediction of future clinically-apparent disease, and prevention.

PURPOSE OF REVIEW: This review discusses updates in the prediction and prevention of future rheumatoid arthritis (RA). RECENT FINDINGS: In individuals with musculoskeletal symptoms and elevated antibodies to citrullinated proteins (ACPA) without clinical inflammatory arthritis (IA), a 'simple' score has a positive predictive value (PPV) of ∼28% for clinical IA/RA within 1 year, and a comprehensive score (including ultrasound) has a PPV of ∼71% for clinical RA within 5 years. Controlled clinical trials in individuals at-risk for future RA have been performed using corticosteroids, rituximab, atorvastatin, methotrexate, hydroxychloroquine and abatacept. Abatacept modestly reduced rates of incident clinical RA and imaging inflammation within the trials, rituximab delayed clinical IA, and methotrexate improved function, symptoms and imaging inflammation. Vitamin D with or without omega 3 fatty acids reduced incidence of autoimmune diseases, including RA. While not proven in controlled clinical trials, observational studies suggest exercise, weight loss and smoking cessation may reduce progression to clinical RA. SUMMARY: Prediction and prevention of RA is advancing although there are no currently approved interventions for prevention. Future studies should include deeper evaluation of the pathophysiology of RA development to improve prediction and identify key pathways to target in future clinical trials, as well as develop infrastructure to support prevention-related research.