Central Nervous System Lymphoma Research Articles

Nivolumab and Ibrutinib for Treatment of Patients with Refractory or Relapsed Central Nervous System Lymphoma

Background: Central Nervous System (CNS) lymphoma is a distinct entity of large B-cell lymphoma (LBCL) with a unique genomic profile with similarities to the activated B-cell (ABC) subtype, which may offer potential targets for therapy. These aberrancies commonly include MYD88 mutations, which enhances sensitivity to Bruton's tyrosine kinase (BTK) inhibitors, and translocation or copy number alterations of 9p24, which may associate with sensitivity with programmed death (PD)-targeted therapies. Expression of PD-L1 is common in CNS lymphoma and tumor infiltrating lymphocytes, and the majority of CNS lymphomas have a copy gain of 9p24.1. This suggests an attenuated immune response against CNS lymphoma. Ibrutinib in combination with nivolumab has shown safety and clinical activity in patients with relapsed non-Hodgkin lymphoma or chronic lymphocytic leukemia in a phase 1/2 trial, and therefore we hypothesized that this combination may be active in CNS lymphoma. Methods: We conducted an investigator-initiated, open label, single center, phase 2 clinical trial combining ibrutinib with nivolumab to treat patients with relapsed CNS lymphoma (NCT03770416). Adult patients were eligible if they had CNS lymphoma (primary or secondary) refractory to or relapsed after at least 1 prior CNS directed therapy, with adequate organ and bone marrow function, without prior ibrutinib or PD1 inhibitor therapy, and did not require persistent high dose steroids. The trial had two sequentially enrolled cohorts: Cohort A included ibrutinib 560mg oral daily for one cycle (28-days per cycle), followed by ibrutinib combined with nivolumab 240mg IV every 14 days. Cohort B included ibrutinib and nivolumab during the first cycle. Patients with a partial response or greater after 6 cycles of combined ibrutinib and nivolumab could continue therapy for up to 2 years or until progression of disease or unacceptable toxicity occurs. The primary objective was to determine the best overall response rate (ORR) of ibrutinib and nivolumab. Secondary objectives included the ORR of ibrutinib as a lead in prior to the combination, best complete response (CR) rate, landmark survival outcomes, and the safety of the combination. The trial planned to enroll 20 patients in both cohort A and B, but closed early due to slow accrual, due in part to the COVID19 pandemic. Results: The trial accrued 18 patients from February 2019 to June 2022, with all patients evaluable for response. The median age was 63 years (range 43-88), 33% were >70 years, and 78% were female. 89% had primary CNS lymphoma, 50% had non-GCB, 22% had GCB, and 28% were unable to be classified due to missing components of the Hans algorithm. The median number of prior lines of therapy was 2 (range, 1-4), 55% had refractory disease, 17% had prior stem cell transplant, and 11% had prior CAR T-cell therapy. 10 patients were enrolled to Cohort A (ibrutinib lead in), and 8 patients were enrolled to Cohort B (initial combination therapy). Adverse events included 50% with fatigue, 33% with nausea, and 28% with mucositis. For both cohorts, the best ORR was 77.8% (95% CI: 52~94%) and the best CR rate was 50% (95% CI: 26-74%). In cohort A, the ORR of ibrutinib as a lead in after 1 cycle was 90% (95% CI: 55.5-99.7%), and the CR rate was 60%. In GCB patients (n = 4), the ORR and CR rates were 25%. In non-GCB patients (n =9), the ORR and CR rates were 100% and 56%, respectively. With a median follow up time of 34.6 months, the median overall survival (OS) was 25.4 months (95% CI 12.5-NR), and the OS rate at 12 months was 70%. The median progression free survival (PFS) was 6.6 months (95% CI: 2.9 - NR), and the PFS rate at 18 months was 47%. 4 patients withdrew from protocol therapy in CR, including 2 who proceeded to stem cell transplant consolidation, and 2 who stopped due to fatigue. 3 patients withdrew from protocol therapy in PR, 2 due to mucositis and 1 due to arthralgias. 1 patient died of COVID19 infection with a CR after 4 cycles of therapy. Conclusion: Ibrutinib and Nivolumab resulted in significant clinical activity in patients with refractory/relapsed CNS lymphoma. Our trial is limited by small patient numbers but suggests the potential for targeted therapy combinations in future larger clinical studies.

Paired Circulating Tumor DNA in Cerebrospinal Fluid and Peripheral Blood: Reliable Technology for Diagnosing and Monitoring Primary and Secondary Central Nervous Lymphoma

Objective: This research delineates the temporal alterations of cell-free circulating tumor DNA (ctDNA) in the plasma and cerebrospinal fluid (CSF) of patients harboring primary and secondary central nervous system (CNS) lymphoma. The aim is to establish that CSF serves as a superior reservoir of ctDNA compared to plasma, thereby aiding in diagnostic processes and monitoring therapeutic efficacy. Concurrently, this study underscores the enhanced response and survival rates in patients administered Bruton's tyrosine kinase (BTK) inhibitors over an extended period exceeding three months. Methods: Data including cranial-enhanced magnetic resonance imaging (MRI) scans, leukocyte and protein concentrations in the CSF, exfoliated CSF cytology, flow cytometry, interleukin-10 to interleukin-6 ratios, ctDNA in plasma and CSF, were gathered from patients presenting primary and secondary CNS lymphoma（7 PCNSL,9 SCNSL）. The temporal ctDNA modifications in plasma and CSF during the patient's disease progression-initial onset, remission, and relapse-were scrutinized. Additionally, the implications of BTK inhibitor usage on patient response rates and survival rates were assessed. Results: Within the cohort of primary CNS lymphoma patients, ctDNA was detected in the CSF and plasma of 2 and 5 patients, respectively. Conversely, in patients with secondary CNS lymphoma, CSF and plasma ctDNA were discovered in 8 and 4 cases, respectively. The detection rate was contingent upon the central lesion's magnitude and position and the patient's surgical history related to the brain. Four patients, including two from each group, underwent cranial-enhanced MRI to appraise tumor burden at various disease stages, simultaneously with the monitoring of ctDNA in plasma and CSF. It was revealed that ctDNA levels in the CSF were in synchrony with the tumor burden, whereas plasma ctDNA levels remained relatively static. This indicates that CSF ctDNA may serve as a viable tool to assess therapeutic responses in CNS lymphoma patients. In two patients where surgical biopsy was unfeasible and both CSF exfoliation cytology and flow cytometry returned negative results, CSF ctDNA was positive, thereby underscoring its auxiliary role in diagnosing CNS lymphoma. Furthermore, the study indicated improved response and survival rates in patients subjected to BTK inhibitors for over three months. Conclusion: CSF ctDNA demonstrates potential in detecting CNS lymphoma and in the dynamic monitoring of therapeutic responses. Compared to cranial-enhanced MRI, exfoliated CSF cytology, and flow cytometry, CSF ctDNA testing offers superior sensitivity. For patients for whom brain surgical biopsy is not an option, CSF ctDNA testing emerges as a valuable supplementary diagnostic tool. CSF ctDNA allows for dynamic monitoring of therapeutic responses in CNS lymphoma patients, enabling the detection of residual lesions, and thereby guiding the formulation and duration of the treatment regimen. Patients administered oral BTK inhibitors for more than three months demonstrate higher treatment response and survival rates.

Efficacy and Safety of Orelabrutinib-Containing Regime (TORM) As First-Line Therapy in Primary Central Nervous System Lymphoma (PCNSL): A Retrospective Analysis

Introduction: Primary central nervous system lymphoma (PCNSL) is a rare, aggressive extranodal lymphoma with poor prognosis. Currently there is no standard therapeutic strategy for PCNSL, and high-dose methotrexate-based regimens remain the first option (Siegal et al. Acta Haematologica 2019). Recently, studies have shown that Bruton's tyrosine kinase (BTK) plays a crucial part in B cell receptor and Toll-like receptor signaling pathways, which are constitutively active in PCNSL (Shen et al. Front Oncol. 2022). BTK inhibitor has been proven effective in treating chronic lymphocytic leukemia and other B-cell lymphomas (Yoshitaka et al. Neuro-Oncology 2021). Orelabrutinib is a novel and highly selective BTK inhibitor with high cerebrospinal fluid (CSF) concentration, which has demonstrated efficacy and safety in PCNSL (Wu et al. Invest New Drugs. 2022). Thus, we conducted a retrospective study to evaluate the efficacy and safety of the orelabrutinib-containing regimens as first-line therapy for patients (pts) with PCNSL. Methods: This isa retrospective analysis of pts with PCNSL who received TORM regimens (temozolomide 150 mg/m 2 day 2-5; orelabrutinib 150 mg qd day 2-21; rituximab 375 mg/m 2 day 1; methotrexate 3.5 g/m 2 day 2; 3 weeks per cycle) as first-line induction therapy between June 2021 and April 2023. It was proposed to receive autologous stem cells transplantation (ASCT) as consolidation therapy or orelabrutinib monotherapy as maintenance after TORM therapy when deemed necessary. The primary endpoint was overall response rate (ORR) according to the International PCNSL Collaborative Group (IPCG) criteria. Secondary endpoints included progression-free survival (PFS), overall survival (OS), and safety. Results: Twelve pts (8 males) with PCNSL were included, with a median age of 63.5 years (range, 40-77). All pts had a histologically confirmed diffuse large B-cell lymphoma and a history of deep intracranial lesions. 8 pts (66.7%) had non-germinal center B-cell-like subtypes. 5 pts (41.7%) had an Eastern Cooperative Oncology Group performance status of ≥2, and 10 pts (83.3%) had International Extranodal Lymphoma Study Group scores ≥2. 4 pts (33.3%) underwent ASCT after induction therapy and 4 pts (33.3%) received orelabrutinib maintenance monotherapy. As of the cut-off date (July 1, 2023), the median follow-up was 11.7 (range, 1.9-23.0) months, and median treatment cycle was 6 cycles (range, 2-9). All 12 pts were evaluated for the treatment response. An interim evaluation after 3/4 cycles was available for 12 pts, showing complete remission (CR) rate of 83.3% (10/12), partial response (PR) rate of 16.7% (2/12), giving an ORR of 100.0% (12/12, Table 1). At data cutoff, of all 12 evaluable pts, the best ORR was 100.0% (12/12), with all pts achieved CR ( Table 1). The median time to response was 1.7 (range, 0.7-2.4) months. Of 12 responders, 9 had an ongoing response, 2 developed progressive disease, and 1 died of covid-19 infection ( Figure 1). Median PFS was 22.6 months, with a 95% CI of 10.4-not reached (NR). Based one OS event, median OS was 22.6 months (95% CI, NR-NR). The most common AEs were anemia. Reported AEs were generally manageable and resolved soon after supportive treatment. AEs associated with off-target activities such as atrial fibrillation, diarrhea, and major hemorrhage were not reported. Conclusions: TORM regimens demonstrated encouraging efficacy and well-tolerated safety profile among pts with PCNSL in this retrospective study. This orelabrutinib-containing regimens may provide a potential treatment strategy for pts with PCNSL.

Rituximab Ibrutinib Lenalidomide (R2I) Combination for Primary or Secondary Large B-Cell CNS Lymphoma: Last Resort or Bridge to Cellular Therapy?

Background: Primary central nervous system lymphoma (PCNSL) and secondary central nervous system lymphoma (SCNSL) each carry a poor prognosis. About 50% of patients with PCNSL will have refractory or relapsed disease correlating with dismal survival. SCNSL is associated with median overall survival (OS) of <6 months. (El-Galaly et al . Eur. J. Cancer 2018) Lenalidomide and ibrutinib each have single agent activity in PCNSL and SCNSL, albeit with limited duration of response (DOR). In combination, they have synergistic activity through modulation of NF-kappa B pathway. The regimen rituximab, lenalidomide, and ibrutinib (R2I) has demonstrated complete and durable responses in a subset of patients with good tolerability. (Houllier et al . Neurology 2021). We report our institutional outcomes with this regimen and its potential role as a bridge to autologous stem cell transplantation (ASCT) or CD19-targeted CAR T-cell therapy. Methods: This retrospective study included ten large B-cell lymphoma (LBCL) patients with PCNSL or SCNSL who received R2I regimen at Miami Cancer Institute from December 2020 to March 2023. Treatment response was assessed using International PCNSL Collaborative Group's criteria. (Abrey et al. J Clin Oncol 2005) Efficacy was evaluated from the start of R2I regimen. Progression free survival (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method. Results: The median age at R2I initiation was 71 (range, 57-85) years. Six of 10 (60%) patients were ≥65 years. Five of 10 (50%) patients had PCNSL. The median number of prior lines of therapy was 2 (range, 1-7). None of the patients had prior ASCT. The median number of cycles of R2I was 4 (range, 1-23). Four patients received a higher dose of rituximab (500mg/m 2). Overall response rates (ORR) and complete response (CR) rates were 80% (95% CI, 55.2 to 100) and 40% (95% CI, 9.6 to 70.3), respectively. The median time to best response was 1.5 (range, 1.0-13.0) months and the median DOR was 5.5 (range, 2.5-18.0) months. The 1-year PFS and OS were 38.8% (95% CI: 4.9 to 72.8) and 56% (95% CI: 22.5 to 89.4), respectively (Figure 1). In the PCNSL subgroup, the 1-year OS was 80% (95% CI, 44.9 to 100) and PFS was 60% (95% CI, 17 to 100). No differences in response rates to R2I between PCNSL and SCNSL were observed (p=0.9). There were no deaths or treatment discontinuations due to R2I toxicity. Two patients on R2I underwent subsequent cellular therapy. One PCNSL patient in CR received carmustine (BCNU) and thiotepa conditioning followed by autograft rescue and remains in remission 16 months post-ASCT. The second patient received CD19-targeted CAR T-cell therapy with axicabtagene ciloleucel for SCNSL. The patient expired 119 days after CAR T-cell therapy due to systemic disease relapse. Another patient, initially deemed ineligible for any cellular therapy due to poor performance status, has achieved a durable response with improvement of performance status and is currently awaiting ASCT. Conclusion: R2I is an effective and safe regimen in patients with PCNSL and SCNSL. Although responses without consolidation may be durable, R2I should be considered as an active drug combination and well-tolerated bridge to cellular therapy to achieve long term survival.

Association between Microenvironment-Related Genes and Prognosis of Primary Central Nervous System Lymphoma

Background: Comprehensive gene mutation profiling of primary central nervous system lymphoma (PCNSL) revealed genomic abnormalities associated with the NFκB signaling pathway and immune escape (Chapuy B, et al. Blood 2016). Although this has led to advances in targeted therapy, there are only a few candidate biomarkers for diagnosis and prediction of survival in PCNSL. Recurrence still occurs at high rate of over 60% and therapeutic resistance is a significant challenge in the management of recurrent PCNSL. Until now, the systemic profiling of tumor micro-environment (TME) was performed through gene expression analysis by whole transcriptome analysis (WTA) and single-cell RNA sequencing analysis (Heming M, et al. Genome medicine 2022), and stratification in PCNSL was attempted by using spatial transcriptome analysis (Xia Y, et al. Leukemia 2023). However, TME of PCNSL and their impact on prognosis remain uncertain. Objective: We performed this study to investigate the prognostic impact on survival and establish novel prognostic biomarkers in PCNSL. Methods: We analyzed the expression levels of 770 neuroinflammation-related (NFR) genes by the NanoString nCounter technology in tumor samples from 30 PNCSL patients. The clinical significance of genes and their association with prognosis were assessed. Genes related to “worse prognosis (WP)” or “better prognosis (BP)” were identified. We performed the univariable analysis using a cox proportional hazards model to evaluate the predictive value of expression of genes related to WP and clinical risk factors, such as age, sex, serum lactate dehydrogenase level, Karnofsky Performance Status (KPS), consciousness levels at diagnosis and lymphoma involvement of the deep brain structure. Gene expression data related to WP were subjected to multivariate analysis with clinical variables with p values less than 0.15 in the univariate analysis. The genes associated with WP were further validated using WTA of an independent PCNSL cohort (n=30, previously published by Fukumura K, et al. Acta neuropathologica 2016). Results: The median age at diagnosis was 69 years old (range, 32-83). The median follow-up period was 25 months (range, 1-110 months), with overall survival (OS) at 3 years of 39.7% and progression free survival at 3 years of 25.4%. Notably, 48 of 770 NFR genes were highly expressed in the WP group (3-year OS, 22.2%), compared with the BP group (3-year OS, 66.7%) (Figure 1). We found that oligodendrocyte and astrocyte-related signatures were enriched in the WP-associated gene set. Among clinical prognostic factors, KPS > 70 (p=0.0293), and consciousness levels at diagnosis (other than JCS 0-1) (p=0.104) were relatively associated with poor OS (p < 0.15) in univariate analysis. Multivariate analysis revealed that high expressions of TUBB4A (p=0.028, HR:3.88), S100B (p=0.046, HR:3.093) and SLC6A1 (p=0.034, HR:3.765) were significantly related to death independent from KPS and consciousness levels at diagnosis. Expression levels of these 3 genes were also significantly associated with poor OS in a validation cohort. Conclusion: Our observations suggest high expression of TUBB4A, S100B and SLC6A1 are the clinical indicators to predict poor prognosis in PCNSL patients. Furthermore, these data suggest that genes related to TME may play a crucial role in the pathogenesis of PCNSL, complementing the well-known involvement of the NF-kB signaling pathway. Therefore it is necessary to continue research focused on TME-targeted therapeutics to encounter drug resistance and refractoriness in PCNSL.

Methotrexate in Combination with Orelabrutinib, Thiotepa, and Rituximab (R-MTO) Followed By Autologous Stem-Cell Transplant for Primary Central Nervous System Lymphoma Treatment

Background: Primary central nervous system lymphoma (PCNSL) is a rare, aggressive B-cell malignancy. Despite the possibility of a cure, the prognosis remains poor. The standard treatment includes high-dose methotrexate and rituximab, followed by consolidation with either whole-brain radiation therapy or autologous stem cell transplantation (ASCT). However, there is still no clear consensus on the best treatment approach. To improve outcomes, we incorporated thiotepa chemotherapy, which has shown promising activity in inhibiting tumor growth by disrupting DNA bonds, into the regimen. Additionally, we introduced orelabrutinib, a novel and potent BTK inhibitor with high cerebrospinal fluid concentrations, which has demonstrated efficacy in treating PCNSL. In light of these findings, we conducted a clinical study to evaluate the efficacy and safety of a combination regimen of thiotepa, orelabrutinib, and methotrexate with rituximab (R-MTO) followed by autologous hematopoietic stem cell transplantation (HSCT) in patients with PCNSL. Methods: This is an ongoing, single-center, single-arm, open-label study conducted between April 2022 and July 2023. The study enrolled patients aged 18-80 years diagnosed with PCNSL. The patients received 5-8 cycles of R-MTO regimen as induction therapy, consisting of rituximab 375 mg/m 2 iv on day 0, MTX 3.5 g/m 2 iv on day1, thiotepa, 30-40 mg/m 2 on day 4 and orelabrutinib 150 mg qd po for 3 weeks per cycle). After completing the R-MTO therapy, patients were proposed to receive orelabrutinib monotherapy as maintenance treatment for up to 2 years. The efficacy was assessed by MRI/PET per the International PCNSL Collaborative Group (IPCG) criteria. Patients who did not experience disease progression subsequently underwent consolidation treatment with high-dose chemotherapy (HCT) plus ASCT. The HCT regimen involved busulfan 3.2 mg/kg on days -5, -4, thiotepa 250 mg/m 2 on days -7 and -6, cytarabine 2g/m 2 q12h on days -3 and -2, followed by reinfusion of stem cells on day 0. The primary endpoint of the study was progression-free survival (PFS) at 2 years, evaluated by intention-to-treat (ITT). Secondary endpoints included complete response (CR) rate, overall response rate (ORR), overall survival (OS) and toxicities. Results: Between April 2022 and July 2023, a total of 19 patients with PCNSL were enrolled in our study, including 16 who were newly diagnosed and had not received any prior treatment. The median age of the patients was 62 years, ranging from 31 to 72, with 9 out of 19 (47.37%) being male. By the data cutoff, all the patients had completed 4 cycles of treatment. Additionally, 8 out of 19 (42.11%) patients had completed six cycles of treatment, and 2 out of 19 (10.52%) patients had completed 7 to 8 cycles of treatment. The ORR was 94.73% (18/19), and the CR rate was 89.47% (17/19) after 4 cycles of treatment. However, one patient with a P53 mutation progressed after 2 cycles of treatment and unfortunately died 3.8 months after diagnosis. Among the six patients who underwent autologous HSCT, all of them (100%) remained in CR. One patient died due to a COVID-19 infection after 6 cycles of R-MTO and ASCT. The most common hematological adverse events associated with the R-MTO regimen were mild to moderate (grade 1-2) decreases in neutrophil count, which typically resolved within a few days. Conclusions: The R-MTO induction treatment has demonstrated notable efficacy in achieving higher response rates among patients with PCNSL, and the associated adverse events are manageable.

Macrophages Play a Key Role in Controlling Tumor Growth and Response to Immunotherapy in Primary Central Nervous System Lymphoma

Introduction Primary Central Nervous System Lymphoma (PCNSL) represents 5% of extranodal lymphomas. Current treatments include high-dose chemotherapy in combination with an anti-CD20 antibody, and whole-brain radiation, both of which are limited by their toxicity. Although some advances have been achieved during the last few years, the prognosis of patients with CNS lymphoma is substantially worse than those with other type of lymphomas, with 5-year survival rates around 30%. The highly aggressive nature of CNS lymphomas is in part due to the fact that the brain is an immunoprivileged organ; it presents very low levels of immunosurveillance, which contributes to inefficient immune responses against malignant cells. Of note, up to 70% of patients diagnosed with PCNSL also exhibit genetic alterations to avoid recognition by the immune system, like loss of MHC-I, that prevent presentation of tumor antigens, and amplifications in PD-L1, which increase the negative stimuli in cytotoxic T cells. The PD-1/PD-L1 interaction has mainly been considered to be a checkpoint that regulates T cells. However, it has recently been found that tumor-associated macrophages can also express PD-1 and become activated to attack tumor cells when it is blocked. Moreover, macrophages have also been found to be inhibited by MHC-I expression on cancer cells. Thus, cancer cells that downregulate MHC-I to avoid T cell surveillance are particularly vulnerable to macrophages phagocytosis, especially when other macrophage immune checkpoints are blocked, such as CD47. Against this background, we aimed to decipher the differential role of macrophages and T cells in the tumor growth and response to immunotherapy in primary CNS lymphoma. Methods Luciferase-expressing A20 murine B-cell lymphoma cells were genetically modified to knock-out MHCI or MHCII by CRISPR-Cas9 and injected into the brain of immunocompetent (IC) or macrophage-depleted (MD) mice. Once tumours were stablished, mice were treated intravenously with seven injections, twice a week, of anti-PD1, anti-CD47 or the combination of both. We monitored mice for survival and tumoral growth. Additionally, brains were collected from mice treated with three injections for analysis of macrophages, B cells and T cells using flow cytometry and IHC. Results In IC mice, all anti-PD1 treated mice showed significant differences in tumoral growth and survival compared to vehicle; however, MHCI- tumors had the lowest overall survival (Figure 1A). With anti-CD47 therapy, only MHCI- PCNSL mice had a significantly increased survival compared to vehicle. Combination was effective in all groups, but no drug synergy was seen. In MD mice, all tumors were more aggressive than in IC mice. With anti-PD1, MHCI+ PCNSL mice achieved a complete tumor regression and survived longer in comparison to vehicle. On the contrary, MHCI- PCNSL mice didn't respond to anti-PD1 therapy (Figure 1B). Of notice, MHCI+ PCNSL in both MD and IC models had a higher infiltration of active T cells when treated with anti-PD1. In MHCI- PCNSL, infiltrated T cells had less expression of activation markers in both models and macrophages were polarized to an M2 phenotype in IC mice. Conclusions Our study revealed a remarkable response to anti-PD1 therapy in MHCI+ CNS lymphoma tumors, regardless of the presence of macrophages in the tumor microenvironment. This indicates that T cells would be primarily responsible for the observed tumour regression. However, targeting CD47 in MHCI+ PCNSL was found to be insufficient in restoring macrophage phagocytosis. Despite observing positive effects of anti-PD1 and anti-CD47 drugs in our MHCI- immunocompetent mouse model, MHCI downregulation still induces a very aggressive, lethal disease. This suggests that these tumors can only be controlled by macrophages, as supported by the lack of treatment response in a MD microenvironment. Our findings highlight the critical role of macrophages in controlling the growth of lymphoma cells in the brain and their significance in the immunotherapy response for CNS lymphomas, particularly when MHCI expression is deficient. Blocking PD1 exhibits superior efficacy when macrophages are present, indicating T cells are not the only player in this tumoral scenario. As a result, our study suggests that a promising approach for treating primary CNS lymphomas would involve targeting both T cells and macrophages.

The Novel Biomarkers for Predicting Prognosis and Recurrence in Primary Central Nervous System Lymphoma: Exosomal Protein and Exosomal microRNA

Abstract Purpose Primary central nervous system lymphoma (PCNSL) is a highly heterogeneous tumor. Therefore, it is crucial to identify biomarkers that can dynamically monitor disease progression for individualized treatment and to improve prognosis. Exosomes carry a large amount of genetic and proteomic information and are stable and easy to obtain. They play important roles in disease diagnosis, treatment, and prognosis. This study aimed to evaluate the use of cerebrospinal fluid (CSF) exosomes in patients with PCNSL as biomarkers of tumor evolution. Methods From October 2021 to October 2022, patients diagnosed with PCNSL via pathology were selected from Beijing Tiantan Hospital affiliated to Capital Medical University. None of the patients had received any cancer-related treatment before diagnosis. Primary intraocular lymphoma and HIV-positive patients were excluded. Clinical information and laboratory findings of patients were recorded in detail. The responses were evaluated according to the response criteria formulated by the International Primary Nervous System Lymphoma Cooperation Group in 2005. According to the time of collecting CSF samples, the patients were divided into diagnosis group (before treatment, n=12), treatment group (after 2-4 treatment cycles, n=11) and complete remission (CR) group (achieved CR after 6 treatment cycles, n=11). We analyzed and compared the exosomal protein concentrations and microRNA expression in the CSF of patients in different groups using western blotting and quantitative reverse transcription PCR. Results There are CD20-positive exosomes in the CSF of patients with PCNSL (Figure 1). In the diagnosis group, the exosomal protein concentration of patients who achieved CR after 2 cycles induction treatment (early remission) was significantly lower than that of patients who did not achieve early remission (p=0.03). Higher exosomal protein concentrations (p=0.022) and exosomal miR-3681-3p expression (p=0.036) at diagnosis were significantly associated with shorter progression-free survival (Figure 2). The expression levels of miR-21-5p (p=0.001), miR-29b-3p (p=0.043), and miR-101-3p (p=0.007) in exosomes were significantly higher in the diagnosis group than in the CR group. Higher exosomal protein concentrations (p=0.012) and exosomal miR-101-3p expression (p=0.013) after CR were significantly associated with recurrence and shorter disease-free survival (Figure 2). The predictive efficacy of exosomal protein concentration and exosomal has-miR-101-3p in PCNSL was analyzed. The area under the receiver operator characteristic curve (AUC) was 0.833 (95% confidence interval 0.524-1.000; p<0.05) and 0.917 (95% confidence interval 0.731-1.000; p=0.033), respectively. Conclusions Exosomes in CSF may have the potential to evaluate prognosis and predict recurrence in PCNSL. It provides clinicians with selectable biomarkers for dynamic evaluation of disease progression.

Improving the Utilization and Tolerability of Thiotepa-Based Autologous Stem Cell Transplantation in Primary CNS Lymphoma

Background: Thiotepa-based autologous stem cell transplantation (ASCT) improves survival in primary central nervous system lymphoma (PCNSL) but up to 30-60% of patients are unable to receive ASCT after MATRix induction, frequently due to treatment-related adverse events. Common conditioning regimens such as thiotepa/BCNU or thiotepa/busulfan/cyclophosphamide have also been associated with risks of pulmonary toxicity or increased transplant-related mortality. Novel strategies to improve the utilization and tolerability of ASCT are therefore needed to maximize the curative potential of this therapy in PCNSL. Methods: This retrospective population-based study included all patients diagnosed with large B-cell lymphoma-type PCNSL who were intended for ASCT in the province of Alberta, Canada between 2011 and 2022. Provincial treatment guidelines recommended an abbreviated, low-intensity induction protocol to reduce toxicity and increase transplantation rates (Figure 1B). Step 1 included 2 cycles of rituximab/high-dose methotrexate (HD-MTX). Step 2 involved 1 cycle of rituximab/high-dose cytarabine (HDAC) for peripheral blood stem cell mobilization. Step 3 consisted of 2 cycles of rituximab/HD-MTX/HDAC and was made optional in July 2018 so that patients with sufficient disease response and performance status following Step 2 could proceed immediately to ASCT. Conditioning consisted of thiotepa 300 mg/m 2 ×2 days and busulfan 3.2mg/kg ×3 days (TT/Bu). Dose reductions of TT/Bu by 10% for every 5 years over the age of 60 were recommended as of May 2021. Progression-free survival (PFS) and overall survival (OS) were determined using the Kaplan-Meier method and compared between groups using the log-rank test. Results: The intention-to-transplant (ITT) cohort included 71 patients with median age 58 years (range 26-72), representing 81% of patients aged 18-60 and 55% of those aged 61-72 who were diagnosed with PCNSL in Alberta during the study period. The proportion of patients aged 61-72 who were treated with ITT increased from 42% before 2018 to 70% afterwards. Fifty-six (79%) ITT patients proceeded to ASCT while 15 (21%) did not due to poor performance status (n=6), treatment-related adverse events (n=5), or disease progression (n=4). There was 1 (1%) death due to infection-related non-relapse mortality (NRM) during induction. The routine omission of Step 3 starting in 2018 resulted in high transplantation rates at 29/35 (83%) compared to 27/36 (75%) in the preceding era. With median follow-up time 3.9 years, the 4-year PFS was 69% (95% CI 56-79%) and OS was 80% (95% CI 67-88%) in the ITT cohort (Figure 1A). For the 56 patients who received ASCT, 4-year PFS was 75% (95% CI 57-86%) and OS was 85% (95% CI 68-93%) from the time of ASCT. There was no significant difference in PFS between patients in complete response (CR)/unconfirmed CR versus partial response (PR) before ASCT (p=0.86); however, 5 patients with progressive disease before ASCT had inferior PFS compared to those with CR/CRu/PR (4-year PFS 40% vs 82%, p=0.0002). Relapse occurred in 8 (14%) patients at median 373 days (range 54-1411) after ASCT, including 7 with CNS relapse and 1 with systemic relapse. No patient in remission after ASCT received whole brain radiation therapy and most recovered without clinically significant neurologic deficits. There were no deaths due to transplant-related mortality. NRM occurred in 2 (4%) patients due to an unrelated cancer and stroke 1.1 and 4.3 years after ASCT, respectively. Among 27 patients >60 years old treated with ITT, 17 (63%) proceeded to ASCT, including 6 who received dose reductions of TT/Bu. The transplantation rate in this age group increased from 45% to 75% following the routine omission of Step 3. All 17 transplanted patients remain alive and in remission with median follow-up 2.5 years. Conclusion:In this real-world study of 71 patients with PCNSL, an abbreviated, low-intensity induction protocol resulted in high transplantation rates (~80%) and favorable long-term outcomes with 4-year OS 80%. Notably, TT/Bu conditioning achieved a low incidence of relapse with no transplant-related mortality observed, including among patients >60 years old. These data demonstrate that de-escalated induction therapy can result in improved rates of successful ASCT, thereby optimizing the outcomes of PCNSL.

Comparison of Methotrexate Maintenance Versus Autologous Stem Cell Transplant Consolidation after High-Dose Methotrexate-Based Induction Therapy in Primary Central Nervous System Lymphoma

Background: Primary central nervous system lymphoma (PCNSL) is a rare form of non-Hodgkin lymphoma with historically poor outcomes. Induction therapy with high-dose methotrexate (HD-MTX)-based regimens followed by consolidation with autologous stem cell transplant (auto-SCT) has become the mainstay of treatment with overall response rates (ORR) of 69-87% and two-year overall survival (OS) of 66-70% reported in prospective clinical trials. Limited data are available regarding a maintenance approach with single-agent HD-MTX, particularly in patients who are not auto-SCT candidates. Herein, we describe a comparison of outcomes in patients with PCNSL who underwent HD-MTX-based induction followed by either HD-MTX maintenance or auto-SCT consolidation at Mayo Clinic, Rochester. Methods: Patients with a diagnosis of PCNSL who received HD-MTX as part of induction therapy at Mayo Clinic between October 2010 and June 2022 were identified. Patients with prior or concurrent diagnosis of systemic lymphoma were excluded. Primary endpoints were progression-free survival (PFS) defined as time from post-induction treatment initiation to relapse, progression, or death due to any cause; and OS defined as time from post-induction treatment initiation to death due to any cause. We compared baseline characteristics by post-induction therapy using descriptive statistics. The primary endpoints PFS and OS were evaluated using Kaplan-Meier curves and compared by risk scores using a log-rank test. Results: A total of 148 patients were identified (51% female, 95% white) with median age at diagnosis of 66 years (range 29-85), with 48 patients (32%) age > 70. Most patients had multifocal disease (n=89, 60%) and deep brain involvement (n=100, 70%) at diagnosis. Few had vitreoretinal (n=15, 10%) or CSF involvement (n=9, 6%) at diagnosis. Using the Memorial Sloan Kettering (MSKCC) prognostic score for PCNSL, 35 patients (26%) scored class 3 (poor prognosis), 85 (62%) scored class 2, and 17 (12%) scored class 1. In total, 117 patients (79%) received methotrexate, rituximab, and temozolomide (MRT) induction therapy, and the remaining 31 patients (21%) received methotrexate and rituximab (MR) induction therapy (Table 1). A total of 70 patients (47%) underwent consolidation with auto-SCT and 37 patients (25%) received maintenance methotrexate. The patients who received maintenance methotrexate had higher median age of 72 versus 62 years, higher proportion of patients older than 70 (54% versus 16%, p<0.001), higher proportion of MSKCC prognostic score class 3 patients (33% versus 17%, p=0.026), and trend towards higher proportion of patients ECOG 2 or higher (41% versus 29%, p=0.109). A higher proportion of patients who underwent auto-SCT received MRT (vs MR) induction (n=64, 91%) compared to those who received maintenance methotrexate (n=24, 65%, p=0.004) (Table 1). The median follow-up for all patients was 4.5 years. At 1-, 3-, and 5-years post-induction treatment initiation, the PFS in the auto-SCT cohort was 85.8% (95% CI 77.7-94.9), 81.0% (95% CI 72.0-91.0), and 74.6% (95% CI 63.3-87.9), respectively; and the PFS in the HD-MTX maintenance cohort was 88.6% (95% CI 78.7-99.8), 72.6% (95% CI 58.8-89.8), and 72.6% (95% CI 58.8-89.8), respectively. At the same time points, the OS in the auto-SCT cohort was 94.0% (95% CI 88.4-99.9), 81.3% (95% CI 71.8-92.0), and 76.0% (95% CI 65.0-89.0), respectively; and the OS in the HD-MTX maintenance cohort was 94.3% (95% CI 86.9-100.0), 87.5% (76.8-99.8%), and 82.4% (69.0-98.4), respectively. Overall, there was no significant difference in PFS (p=0.64) or OS (p=0.95) based on post-induction treatment (Figure 1). Conclusions: Our study demonstrates comparable outcomes in PCNSL between a post-induction treatment approach with maintenance HD-MTX and consolidation auto-SCT, even though baseline characteristics indicate that patients who went on to receive auto-SCT were younger and had overall better performance status. This suggests that maintenance HD-MTX is a reasonable, time-limited strategy for patients with PCNSL responding to initial induction therapy, particularly in patients who are not candidates for auto-SCT.

Combination of Selinexor with BTK Inhibitor for Central Nervous System Diffuse Large B-Cell Lymphoma, Possible Mechanisms and Therapeutic Potential Exploration

Background: Primary central nervous system lymphoma (PCNSL) is a rare aggressive extra-nodal non-Hodgkin lymphoma (NHL) of which over 90% are diffuse large B-cell lymphoma (DLBCL). High-dose methotrexate (HD-MTX)-based regimens are the dominant therapy for newly diagnosed PCNSL, but the patients' complete remission rate (CRR) is merely about 50%. Bruton tyrosine kinase (BTK), a critical kinase in the B cell receptor (BCR) signaling pathway, contributes to the degradation of FOXO3a and promotes abnormal B cell proliferation. Recent studies revealed that BTK inhibitors (BTKi) play a role in various B cell lymphomas, including DLBCL, and have also shown promising efficacy in PCNSL. However, the clinical management of PCNSL is still challenging for its limited treatment options and poor prognosis, especially when patients developed acquired resistance to BTKi. Selinexor (KPT-330) is a first-ever oral small molecule Exportin-1 (nuclear output protein XPO1) inhibitor with a molecular weight of 443.31 Da, which can efficiently cross the blood-brain barrier. Currently Selinexor was found to promote the aggregation of FOXO3a and could synergistically inhibit PI3K/AKT and NF-κB pathways with BTKi to overcome acquired BTKi resistance. To investigate the therapeutic potential of Selinexor and BTKi in PCNSL, we examined the combination both in DLBCL cells and A20 transplantation mice, and further explored the clinical efficacy in PCNSL patients. Methods and Results: ABC-DLBCL cell lines (OCI-LY3 and OCI-LY10) were treated with Selinexor for 48 hours. Western blot analysis showed that Selinexor inhibited NF-κB, P-STAT3, and NFATc1 signaling pathways in DLBCL in vitro. Combining Selinexor(1μM) with ICP-022(2μM), a BTKi, resulted in more notable inhibition of the above tumor signaling pathways. A20 murine B-cell lymphoma transplantation models were divided into a control group, Selinexor monotherapy group, ICP-022 monotherapy group, and combination group. Tumor volume was measured after 30 days (Figures 1A), and the number of CD4+ T cells and CD8+ T cells and the corresponding cellular expression of PD-1 and TIM-3 in the spleen and tumor tissues of mice were detected. The results showed that the tumor volume in the combination group was minimized, and the number of CD4+ T cells and CD8+ T cells in spleens and tumors in this group was significantly increased, while PD-1 and TIM-3 expression was downregulated (Figures 1B-C). Based on these results, a prospective, open-label, single-arm, multicenter study was conducted with MSZ regimen (MTX, BTKi, Selinexor) for the treatment of PCNSL (Trial Registration Number: ChiCTR2200062154). Newly diagnosed primary and secondary central DLBCL patients (18-80 years old) received a 6-cycle MSZ regimen (HDMTX 3.5 g/m 2 3-h infusion d1, Zanubrutinib 160 mg Bid po, Selinexor 40 mg Biw po, 3 weeks per cycle) and evaluated by MRI/PET. 5 patients (2 GCB-DLBCL, 3 ABC-DLBCL) were enrolled, with a median age of 65 years (range: 54-73 years), 100% achieved metabolic remission (Figure 2A) and maintained long-term efficacy. 4 patients achieved early deep remissions of over 75% reduction with 1 cycle of treatment, and 1 patient had a complete remission after 2 cycles of treatment (Figures 2B). 3 patients chose radiation therapy as consolidation after the end of induction chemotherapy. 2 others were on maintenance therapy with Selinexor combined with Zanubrutinib, one of them has completed the two-year maintenance treatment and maintained sustained remission till now. Only 1 patient experienced several grade 3 adverse events (AEs) including anemia, decreased white blood cell and platelet counts, and elevated alanine aminotransferase.1 patient developed mild renal insufficiency, which recovered 6 months after the end of treatment. Patients were generally well-tolerated and there were no treatment-related deaths. Conclusion: This is the first report describing that Selinexor combined with BTKi can synergistically inhibit the DLBCL tumor signaling pathway and repairing the immune microenvironment. Correspondingly, in treatment of central DLBCL patients, the novel therapeutic strategy of MSZ regimen has achieved impressive results, with a CRR of 100%. Most patients attained early and rapid deep remissions with durable efficacy. This study provides theoretical support and preliminary clinical data for the combined application of Selinexor and BTK inhibitors.

Navigating the Blood-Brain Barrier: An Anti-CD19 Modified-Exosome Delivery System for Precise Targeting of CNS Lymphoma

High-dose methotrexate (HDMTX) serves as the cornerstone of central nervous system lymphoma (CNSL) treatment, but its efficacy is limited due to low Blood-Brain Barrier (BBB) penetration (2% to 20%) and adverse effects like myelosuppression and hepatotoxicity. Thus, this study focuses on exosome-based drug delivery to enhance BBB permeability, reducing methotrexate (MTX) dosage while targeting CNSL specifically (Figure 1A). Considering the recipient's compatibility and reduced risk of adverse reactions or rejection, exosomes derived from human adipose tissue-derived stem cells (ADSCs) were selected as drug delivery vehicles. ADSCs-Exo underwent purification through ultracentrifugation and were characterized using nanoparticle tracking analysis (NTA) and transmission electron microscope (TEM). CD19 is considered an excellent target for immunotherapy in DLBCL. To confer targeting capabilities, ADSCs were infected with a lentiviral vector encoding the anti-CD19 prior to the purification of exosomes. The anti-CD19 (patent number: ZL201811653085.8) was strongly expressed in ADSCs, integrated into ADSCs-derived exosomes, and localized on the external exosomal surface, as evidenced by western blots, colloidal gold immunoelectron microscopy, and flow cytometry. Based on the properties of MTX, we employed co-incubation to load MTX into anti-CD19-modified exosomes. The resulting exosomes, loaded with MTX, were then quantified for the encapsulated MTX using High Performance Liquid Chromatography. The anti-CD19-modified exosome-loaded methotrexate (anti-CD19-Exo-MTX) interacted with cerebrovascular endothelial cells and astrocytes of the BBB, leading to endocytosis and facilitating MTX transportation across the barrier. Additionally, anti-CD19-Exo-MTX crossed the BBB model without compromising its structure, as assessed by dextran's luminescent signal assay. Compared to drug-free formulations, anti-CD19-Exo-MTX exhibited enhanced BBB permeability, thereby improving its capability to inhibit CNSL cell proliferation. Liquid chromatography-tandem mass spectrometry identified and quantified proteins in exosomes, revealing significant enrichment of neuron projection and axon development-associated proteins. Subsequent neurite outgrowth assays, Nissl staining, and modified neurological severity scores confirmed the beneficial effects of ADSCs-Exo in mitigating MTX-induced neuron damage. To evaluate the efficacy of anti-CD19-Exo-MTX in vivo, intracranial orthotopic CNSL models in mice were established. Magnetic resonance imaging (MRI) analysis revealed a reduced disease burden in the anti-CD19-Exo-MTX group compared to both the MTX and Exo-MTX groups (Figure 1B), along with prolonged overall survival. Cerebrospinal fluid (CSF) drug concentration analysis demonstrated enhanced stability and longer-lasting drug levels for anti-CD19-Exo-MTX in the CSF, while showing no toxicity to vital organs, as evidenced by the absence of significant histological damage in the heart, liver, spleen, lung, and kidney tissues. To assess biodistribution and targeting of anti-CD19-Exo-MTX, exosomes were labeled with VivoTrack Dil (Fluorescence). Intracranial fluorescence signal was observed for anti-CD19-Exo-MTX, while Exo-MTX exhibited systemic distribution. Two hours post-injection, organs removal revealed weaker fluorescence signals in the liver and spleen for anti-CD19-Exo-MTX, confirming its specific targeting of MTX to CNSL in vivo. In conclusion, we developed anti-CD19-Exo-MTX, an exosome-based drug delivery platform, demonstrating enhanced BBB permeability and specific targeting for CNSL. This promising approach offers potential for effective CNSL treatment with reduced adverse effects.

Real-World Outcomes for Primary CNS Lymphoma from a Single Academic Institution: The Augusta Experience

Introduction: Primary central nervous system lymphoma (PCNSL) is a rare, aggressive, non-hodgkin lymphoma, characterized by exclusive confinement to the central nervous system. Outcomes are poor, with a 5 year overall survival of 30% with standard induction of high-dose-methotrexate (HD-MTX) based regimens followed by consolidation. Barring epidemiologic trends due to immunodeficiency disorders, PCNSL is a disease of the immunocompetent older patient (age >60 years). Incidence in those aged >70 years is 10 times higher than the general population. The overall survival hasn't significantly changed in 4 decades. We present retrospective data in a rural, elderly population in Georgia. Methods: We reviewed all patients diagnosed with PCNSL at our institution from January 1, 2013 to January 1, 2023. A retrospective chart review was conducted to assess for demographic factors, comorbidities, organ dysfunction, induction regimens, and consolidation treatments influencing patient care. Results: A total of 38 PCNSL patients were identified. 6 died and one was lost to follow-up prior to start of therapy and are not further analyzed here. All patients had DLBCL histology. The median age was 62.3 (21-82 years) with 54.8% male. Patients identified as non-Hispanic white (77.4%), African-American (9.68%), Asian (6.46%), Hispanic or Latino (3.23%), and multiracial (3.23%). The youngest patient aged 21 years had HIV/AIDS. 2 of 3 patients diagnosed in their 30s were on immunosuppression, for renal transplant and multiple sclerosis. No patients had hepatitis B or C. Medical comorbidities were common with 80.7% of patients having at least one which included HTN, TIIDM, history of CVA or CAD, and atrial fibrillation. 19.4% of patients had impaired renal function (CrCl <60 ml/min). Several patterns emerged when stratified by best treatment response at end of induction (EOI). Median follow-up was 12 mos (range: 0-66). Age, HIV status, gender, CrCl, positive CSF at baseline, or elevated LDH were not statistically significant predictors of response. This is probably due to the small sample size and missing data. Seizures at disease outset were associated with a statistically significant worse response to induction and all patients without seizures responded. This is likely a proxy for deep brain involvement and data are similar to CALGB 50202 where deep brain lesions independently correlated with a shorter progression-free-survival. Completion of ≥ 6 cycles of induction and a lower MTX level at discharge were predictors for response likely reflective of better organ function. Majority of patients with progressive disease received fewer than 6 cycles. A poorer EOI ECOG PS portended a worse response and all responders maintained an EOI PS of 0-2. Peak MTX level did not correlate with response. Sample size was too small to discriminate between discharge MTX thresholds of <0.05 vs 0.1 vs >0.15 however that will be an area of further investigation. Choice of induction regimen did not predict response statistically, however, all responders received HD-MTX-based combination chemotherapy (77.42%). 19.4% received HD-MTX alone and 3.2% received rituximab monotherapy. Only 8 (25.8%) of patients received consolidation. 7 received non-myeloablative therapy with 2 receiving additional WBRT and 1 patient received WBRT alone. Conclusion: PCNSL remains an area of high unmet need. Recently, IELSG43 and MARTA studies have shown that HD-MTX-based combination chemoimmunotherapy followed by autologous stem cell transplant leads to improved outcomes regardless of age; however, non-relapse mortality remains a concern. Our data comprising chiefly of an elderly cohort reiterate efficacy of combination chemo-immunotherapy and impact of number of induction cycles on response, despite age. Most common reasons for treatment discontinuation included performance status deterioration and death from disease or toxicity. A multidisciplinary approach with physical therapy, nutritionist, neurology, and geriatrics would be optimal, particularly for older and infirm patients. In addition, alternatives to consolidation such as targeted agent (e.g. Bruton tyrosine kinase inhibitor) maintenance should be considered to improve outcomes in the elderly. One patient at our center is receiving post-induction zanubrutinib with a complete remission to date (~16 mos). This will be a focus for a phase II clinical trial at our institution.

Impact of Folinic Acid Dosing on Efficacy and Toxicity of High-Dose Methotrexate in Central Nervous System Lymphoma

Treatment of primary central nervous system lymphoma (PCNSL) is centered around a backbone of high-dose methotrexate (HDMTX) with folinic acid (FA) rescue. Higher FA doses were associated with treatment failure in several retrospective studies in acute lymphatic leukemia (ALL), leading to the adoption of lower FA doses in ALL treatment protocols. In PCNSL, FA doses have varied widely, guidelines and consensus statements generally do not address FA dosing, and the effect of FA dosing on treatment outcomes has not been assessed. We retrospectively analyzed the medical records of patients treated at our institute for PCNSL between the years 2014 and 2022 to address this question. Patient characteristics are listed in table 1. Complete dosing data was available for 36 patients. The median number of HDMTX doses administered was 6.5 (range, 2-9;), with an average dose of 3.9 g/m 2 (SD +/- 0.74; range, 2.7-5.0). Per the local protocol, IV FA rescue began 24 hours after each MTX infusion at a fixed dose of 50 mg four times daily and continued until MTX clearance. The average cumulative dose of FA administered per treatment was 322 mg/m 2 (SD +/- 141; range, 140-700). 33 patients (92%) achieved either a partial or complete response on MRI following induction treatment. Three (8%) had progressive disease. 26 patients (72%) proceeded with consolidation treatment. Of these, 16 (44%) underwent autologous hematopoietic stem cell transplant. At a median follow up of 1.6 years (range, 0.3-8.1), 12 patients (33%) suffered progressive disease, disease relapse, or death. The 2-year PFS probability for the entire cohort was estimated at 64.5% (95% confidence interval [CI], 49.4-84.1%), and the 2-year OS as 70.7% (95% CI 56.0-89.2%). In univariate Cox regression analysis, average FA dose during MTX treatment was a significant predictor of both PFS and OS, with hazard ratios (HR) of 2.22 (95% CI 1.39-3.55; p < 0.001) and 2.07 (95% CI 1.31-3.29; p = 0.002), respectively, for each 100 mg/m 2 increase. The FA dose during the first treatment cycle, before assessment of response, was also significantly associated with worse PFS (HR 1.56; 95% CI 1.24-1.96; p < 0.001) and OS (HR 1.41; 95% CI 1.14-1.74; p = 0.001). A trend towards worse PFS and OS was seen with male gender, elevated serum LDH, and deep structure involvement. A Cox multivariate regression analysis using the variables age, gender, serum LDH, deep structure involvement, and the average MTX and FA doses, identified FA dose as an independent prognostic factor for PFS, with an estimated hazard ratio (HR) of 2.23 for each 100 mg/m 2 increase (95% CI, 1.25-3.97; p = 0.006). A smaller effect was seen when the FA dose during only the first treatment cycle was examined (HR 1.55; 95% CI 1.16-2.08; p = 0.003). Involvement of deep structures was also significantly associated with worse PFS, with an estimated HR of 8.59 (95% CI, 1.28-57.5; p = 0.027), and there was a borderline significant trend for worse PFS with male gender and elevated LDH. None of the examined variables were identified as statistically significant predictors of OS in the multivariate analysis. Logistic regression with 2-year PFS as the dependent variable was used to define a cutoff value of 350 mg/m 2 for FA dose. The group of patients who received FA doses above and below this threshold showed significantly different PFS curves (figure 1). The 2-year PFS probability was estimated at 56.6% (95% CI, 35.4-90.5%) for patients who received doses of FA above this threshold, and 70.5% (95% CI, 51.7%-96.2%) for patients who received doses below it. We identified no effect of FA dosing on hematologic, renal, hepatic, or gastrointestinal toxicity at the dose ranges examined. In summary, we found that high FA doses were associated with inferior PFS in PCNSL. Such high doses may be avoided through use of lower individual doses of FA, such as 15 mg/m 2 or 25 mg fixed doses as have been used in some protocols, rather than the 50 mg or higher doses used in our and other's practice. Our conclusions are moderated by the study's limitations, which include a small sample size limited to a single center and a retrospective design. However, they suggest that optimizing FA dosing in treatment of PCNSL might improve treatment efficacy without excess toxicity. Future larger and prospective studies will be required to better define the range of MTX and FA doses at which the balance between prevention of treatment toxicity and preservation of treatment efficacy is preserved.

Considering Older Patients As Candidates for Auto Stem Cell Transplants: A Comprehensive Study on Toxicities and Survival Analysis

Introduction: High-dose chemotherapy and autologous stem cell transplantation (ASCT) are commonly used for relapsed/refractory (R/R) Hodgkin (HL) and non-Hodgkin lymphoma (NHL). However, concerns about treatment-related mortality (TRM) and toxicity limit its use in older patients (pts). Existing evidence suggests higher rates of complications and inferior overall survival (OS) in older pts, but conflicting findings suggest that outcomes may be influenced by comorbidities rather than age ( Lahoud O, Curr Oncol Rep. 2015). Our study comparing ASCT outcomes and toxicities in lymphoma pts in younger and older age groups. Methods: In this retrospective study, we analyzed lymphoma pts who underwent ASCT at the Princess Margaret Cancer Centre from January 2015 to December 2019. After Institutional Review Board approval, clinical data was collected from institutional databases and patient charts. The hematopoietic cell transplantation comorbidity index (HCT-CI) and Charlson Comorbidity Index (CCI) were retrospectively calculated. Response assessment was done by Lugano 2014 classification. Grade 3-5 nonhematologic toxicities were collected from admission to day 100 using Common Terminology Criteria for Adverse Events version 5. Overall survival (OS) and progression-free survival (PFS) were calculated from date of transplant to death or disease progression. OS and PFS assessed via Kaplan-Meier; log rank tests and Cox regression performed. The patients were divided into two age groups: Group A (<65 years) and Group B (≥65 years). Results: There were 334 pts who underwent ASCT. 332 pts were analyzed; 2 pts were lost to follow-up and 16 pts were day 1 transfers, making acute toxicity data unavailable. The median age was 53 (range: 18-71), 17% of pts being aged ≥65. The majority of pts (69%) had NHL (table 1). The Eastern Cooperative Oncology Group (ECOG) performance status was ≤1 for 91%. Group B had a higher proportion of pts with ECOG score =2 (18% vs 7%, p < 0.001 ) and higher CCI (88% vs 27% , p < 0.001). NHL and mantle cell lymphoma (MCL) were more prevalent in Group B (93%, 23% vs 68%, 12% respectively, p< 0.001). Thirteen percent of NHL pts were transformed disease. Both groups had similar percentage of pts with central nervous system (CNS) lymphoma (~ 5%). Conditioning regimens varied based on lymphoma type: most of the HL/aggressive lymphomas: Etoposide+Melphalan (ML), CNS lymphoma: Thiotepa based regimen, and MCL Cytarabine + ML+/- Total Body Irradiation. The majority (83%) were treated for R/R disease, while 17% received it as first-line treatment. Prior to transplant, 76% of pts were in CR. Median follow-up was 38 months. Median OS was not reached for Group A, and 73 months for Group B. Five year OS were 82% and 74% respectively (p = 0.05). Median PFS was 75.2 months for Group A and 43.0 months for Group B, with 5-year PFS of 54% and 47% respectively (p = 0.2). Twenty percent of pts died during follow-up, with only 4% being NRM. From the entire cohort 3% of patients died within 100 days post-transplant, with no difference between groups. Three pts died within 100 days of ASCT with 2 deaths from sepsis (Group A) and 1 death from Respiratory failure (Group B). Common transplant-related toxicities did not differ significantly between groups (figure 1). Group B had significantly higher rates of Renal (7%vs 18%), Respiratory (16% vs 6%), and Metabolism abnormalities (14% vs 5%) (p = 0.021-0.028). Group B needed more blood product transfusions by 100 days post-transplant: Packed Cells (mean) 1.7 vs 1.1 units (p 0.08), Platelets (mean) 3 vs 1.9 units (p 0.002). Group B had a longer average length of stay 18 days vs. 14 days (p < 0.001). About 6% of pts required ICU admission, and 4% were discharged to rehabilitation/long-term care facilities, with no group differences. Conclusions: The study concludes that ASCT can be safely performed in appropriately selected elderly pts. TRM rates were not significantly higher in older compared to younger pts with lymphoma. However, older pts did experience higher rates of specific toxicities including renal failure, pulmonary complications, metabolism abnormalities, longer hospital stays, and increased transfusion requirements. In the multivariate analysis, older pts exhibited shorter OS, influenced by lymphoma type and treatment response. Further research is needed to develop risk stratification and geriatric assessments to improve toxicity prediction in elderly pts undergoing ASCT.

Germinal Center B-Cell-like (GCB) Phenotype Predicts Improved Outcomes in Central Nervous System Diffuse Large B-Cell Lymphoma Patients Receiving Stereotactic Radiosurgery (SRS)

Background: Diffuse large B cell lymphoma (DLBCL) constitutes most cases of both primary and secondary central nervous system lymphoma (CNSL). They represent a highly aggressive subset of non-Hodgkin lymphoma and prognosis varies depending on the cell of origin. Immunohistochemistry using antibodies for CD10, BCL-6, and MUM-1, also known as the Hans algorithm, is the most common method for determining the cell of origin and predicting outcomes in DLBCL. The study by Hans et al. found that the germinal center B-cell-like (GCB) group had a much better prognosis compared to the non-GCB group, with 5-year overall survival (OS) of 76% and 34% respectively. Despite chemotherapy, recurrence is common in these patients, and up to 40-50% do not achieve durable remission. It is important to identify who may benefit from more aggressive or experimental therapy at diagnosis. Stereotactic radiosurgery (SRS) is the administration of a localized, high dose of ionizing radiation and limited studies exist exploring SRS as a treatment modality in relapsed/refractory (r/r) CNSL or prognostic implications of the Hans algorithm in these patients. Aim/Objective: To perform a retrospective analysis of outcomes of patients with CNSL, phenotypes determined by the Hans algorithm, treated with SRS at a single comprehensive cancer center. Methods: We studied the demographic, clinical, pathological, and treatment characteristics of r/r CNSL patients (N=27), age ≥ 18 years, who received SRS at Roswell Park Comprehensive Cancer Center between 01/2000 and 01/2023. The Hans algorithm was used to group the patients into non-GCB (N=17) and GCB (N=10) cohorts. Overall survival (OS) and progression-free survival (PFS) were calculated and reported using Kaplan-Meier analyses and log-rank tests. Cox regression models were used to describe cross-cohort comparisons of survival. Results: The median Karnofsky performance status at diagnosis was 70 for both groups. The overall response rate (ORR) to SRS in GCB-type was 80% (7 complete response and 1 partial response; CR, PR). The ORR in non-GCB type was 58% (6 CR and 4 PR). Post-SRS, stem cell transplant (SCT) was avoided in 100% of the patients in the GCB group, while 17% of the patients in the non-GCB group received SCT. The 6-month overall survival (OS) rate was 100% and 46% for the GCB-type and non-GCB type respectively (p=0.096, Figure 1A). The 6-month progression-free survival (PFS) rate was 80% for the GCB-type and 33% for the non-GCB type (p=0.024, Figure 1B). In the GCB group, the median OS was 18.0 months while the median PFS was 13.8 months. In the non-GCB group, the median OS was 4.9 months while the median PFS was 3.6 months. The median time from chemotherapy completion to SRS administration was 8.0 months in the GCB-type and 2.0 months in the non-GCB type. Conclusion: Our data support that the GCB-type predicts a more favorable outcome in CNSL, with improved OS and PFS rates compared to the non-GCB type. In addition, the non-GCB type patients required earlier SRS administration and had a lower ORR to SRS. Classifying CNSL into GCB and non-GCB phenotypes using the Hans algorithm might help characterize patients into groups with different prognoses.

CNS Radiotherapy As Bridging Prior to CAR T-Cell Therapy for Non-Hodgkin B-Cell Lymphoma

Introduction: Up to 80% of patients who receive CAR T-cell therapy (CAR T) for central nervous system lymphoma (CNSL) require bridging therapy, yet optimal regimens remain undefined. Radiotherapy is an established strategy for extracranial lymphoma that provides beneficial cytoreduction. However, CNS bridging radiotherapy (CNS-BRT) prior to CAR T is controversial due to concerns of potential enhanced neurotoxicity. We explored the safety and response profiles in patients treated with CNS-BRT prior to CAR T. Methods: We identified patients with non-hodgkin B-cell lymphoma who received CNS-BRT at our institution prior to commercial CAR T infusion. CNS-BRT was defined as treatment delivered between apheresis and CAR T infusion or within 30 days prior to apheresis, targeting the CNS parenchyma, leptomeninges, or epidural spine. Patients with epidural spine disease were included since the adjacent spinal cord is part of the treatment field. Safety was evaluated by rate of cytokine release syndrome (CRS) and immune effector associated neurotoxicity syndrome (ICANS) after CAR T. Best CNS response post CAR T was evaluated using the International Primary CNS Lymphoma Collaborative Group (IPCG) or Response Assessment in Neuro-Oncology (RANO) for parenchymal or leptomeningeal lesions, respectively. Cytoreduction from CNS-BRT was estimated by calculating the change in lesion size (sum of product diameter) from before and after CNS-BRT, but before CAR T infusion. Risk of any CNS relapse after CAR T was estimated using the Gray's method with death as a competing risk. Patients with epidural spine disease were not evaluate for CNS response or relapse. Results: 12 patients received CNS-BRT with median follow up of 8.5 months (range: 3.2 - 30.2), median age of 60 (30-76), median Karnofsky Performance Status (KPS) of 80 (range: 50 - 90). Histologies included diffuse large B cell (DLBCL) (n = 9), mantle cell (n = 2), and Burkitt lymphoma (n = 1), with disease localizing to the brain parenchyma (n = 6), leptomeninges (n = 4), and epidural spine (n = 2). 9 patients had secondary CNSL and 1 patient had primary CNSL. Prior to CNS-BRT, 11/12 patients had progressive disease. 5/12 patients had disease outside of the CNS. 5/12 patients received prior autologous hematopoietic stem cell transplantation. 9/12 patients received prior high dose methotrexate with median 46-day interval to CNS-BRT (range: 0 - 393). RT targets included whole brain (n = 3), involved site (partial) brain (n = 4), involved site spine (n = 4), and orbits (n = 1) with median dose 24 Gy (range: 15 - 33). CAR T products included Tisagenlecleucel (n = 4), Lisocabtagene maraleucel (n = 7), and Axicabtagene ciloleucel (n = 1). Among 10 patients with available toxicity grading, 6/10 experienced CRS (n = 1 grade (G) 1, n = 4 G2, and n = 1 G3), and 3/10 experienced ICANS (n = 1 G1, n = 1 G3, n = 1 G4). 6/10 patients required tocilizumab and/or steroids. CNS-BRT achieved a 74.4% (95% CI, 62.9 - 85.9) mean reduction in lesion size prior to CAR T infusion. Best CNS response after CAR T infusion included 3 complete responses (CR), 6 partial responses (PR), and 1 progressive disease (PD). The patients who achieved PR remained in PR at last follow up. The 12-month estimated risk of CNS progression after CNS-BRT and CAR T infusion was 20.0% (95% CI, 3- 49). CNS progression was not observed in patients who received involved site brain RT. Conclusion: Preliminary data suggest CNS-BRT achieves rapid cytoreduction prior to CAR T therapy and is associated with a favorable CNS response profile. While overall numbers are limited to date, the rate of severe CRS and ICANS is similar to that of historical series of CNSL patients treated with CAR T. However, a larger cohort will be required to determine the safety of CNS-BRT. These data support further study of RT, and exploration of involved site brain RT, as an effective bridging modality for CAR T-cell therapy in CNSL.

Phase II, Single-Center, Prospective Study of Intrathecal Thiotepa for the Prophylaxis of Secondary Central Nervous System Involvement in Highly Aggressive B-Cell Lymphoma

Background Central nervous system (CNS) recurrence of systemic diffuse large B cell lymphoma (DLBCL) is a well-recognized and almost uniformly fatal complication that tends to occur within 2 years of initiating treatment. The probability of CNS relapse in DLBCL ranges from 4 to 7% overall but is increased to 20% in certain subgroups. Evidence supporting the use of intravenous methotrexate (MTX) or arabinoside (AraC) in high-risk patients is growing, however, the use of other agents remains to be defined. Thiotepa (TT) is a polyfunctional alkylating agent first developed in the 1950s. Both thiotepa and its metabolite tepa demonstrate good CNS bioavailability, making thiotepa a useful adjunctive chemotherapy for primary and secondary CNS lymphoma, particularly in the context of hematopoietic stem cell transplant (HSCT). Herein, we conduct this prospective trial to evaluate the efficacy and safety of prophylactic intrathecal thiotepa for the prevention of secondary CNS involvement in patients with DLBCL. Methods Patients The primary end point is 2-year cumulative incidence of CNS. The secondary end points are 2-year progression free survival (PFS), 2-year overall survival (OS), and safety. The main inclusion criteria include: adults patients ≥18 years old; histopathological confirmed DLBCL without previous antitumor therapy; high-risk CNS involvement (one of the following): primary breast or testicular lymphoma, involvement of any of the following organs: testis, breast, kidney, adrenal gland, paranasal sinus, epidural space or uterus; CNS International Prognostic Index (CNS-IPI)≥4; dual protein expression (DE) or double/triple hit (DH/TH) lymphoma. The main exclusion criteria include: Burkitt lymphoma, primary CNS lymphoma, or large B cell lymphoma transformed from indolent lymphoma; the presence of parenchymal or meningeal lymphoma; history of whole brain radiotherapy or craniospinal cord irradiation; obstructive hydrocephalus patients requiring neurosurgical intervention. Treatment protocol The systemic chemotherapy regimen is determined by investigator. All patients are need to receive IT thiotepa 10 mg and dexamethasone 5 mg on day 1 of each cycle of chemotherapy for at least 4 cycles. All patients are asked to lie flat for 4 to 6 hours after the lumbar puncture. Follow-up Routine biochemical and cytological analysis of cerebrospinal fluid is performed for each cycle. Imaging examination, such as brain magnetic resonance imaging (MRI) and body computed tomography (CT), is repeated every 2 cycles of chemotherapy, then every 3 months during follow-up. Results From February 2022 to June, 2023, 27 patients (female 14, male 13) received IT thiotepa. The clinical characteristics are shown in Table 1. The median age was 66 years old (range: 46-74). As first-line chemotherapy, 15 (55.6%) patients received rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone (RCHOP), 5 (18.5%) patients received RCHOP+BTK inhibitor, 3 (11.1%) patients received BTK inhibitor, lenalidomide with rituximab (ZR2) followed by RCHOP, and 4 (14.8%) patients received RCHOP with intravenous MTX. Thirteen (48.1%) patients had IPI score 4-6 and 17 (63%) patients had DE of c-myc and bcl-2. All patients received fluorescence in situ hybridization (FISH) cytogenetic testing for C-MYC, BCL-2, BCL-6 and TP53. Four (14.8%) patients had TP53 deletion, and 11 (40.7%) patients had sole rearrangement of C-MYC or BCL-2 or BCL-6, but no one were diagnosed with DH/TH lymphoma. All patient have received IT thiotepa for at least once, and 12 (44.4%) patients completed 4-6 cycles of IT, 84 cycles totally. IT thiotepa was a safe measure, for no one experienced post-LP headache, local infection, bleeding, or cerebral herniation. But there were 4 patients had five times of inability to identify direction and slow speech response. They could recover normally the next day without any treatment. Although the follow-up period was short, no patient developed CNS involvement. Conclusion Prophylactic intrathecal thiotepa is an effective and side-effects manageable measure for preventing secondary central nervous system involvement in high-risk DLBCL. Longer follow-up and larger-scale prospective trial is needed to testify this measure.

Real-World Efficacy and Safety of Rituximab, Lenalidomide and İbrutinib Combination in Patients with Relapsed and/or Refractory Non-Hodgkin Lymphoma

Background: Relapsed or refractory (R/R) Non-Hodgkin Lymphoma (NHL) is associated with poor outcomes and the therapic options are limited. Recently, the combination of rituximab, lenalidomide, and ibrutinib (RLI) suggest promising efficacy in some clinical studies. Consequently, we report outcome of patients with R/R HL treated with RLI in this retrospective study aiming at evaluating efficacy and safety of RLI in a real-life setting. Methods: This retrospective, single-center study analyzed the outcomes of RLI as an off label salvage theraphy in patients with R/R NHL between June 2020 and April 2022. All patients received Lenalidomide 10-25 mg po daily on days 1-21 every 28 days and Ibrutinib 560 mg po daily on days 1-28. Patients with diffuse large B-cell lymphoma (DLBCL), primary central nervous system lymphoma (PCNSL), marginal zone lymphoma (MZL) received Rituximab at a dose of 375 mg/m2 iv on day 1 of cycles 1-6 every 28 days, patients with mantle cell lymphoma (MCL) received the same dose of Rituximab once a week for 4 weeks during cycle 1, then on day 1 of cycles 2-12 every 8 weeks. Ibrutinib and/or lenalidomide was administered until progression or unacceptable toxicity. The response was assessed with according to the Lugano criteria. The primary end point was overall response rate (ORR). Secondary endpoints were complete response (CR), partial response (PR), time to response (TTR), duration of response (DoR), overall survival (OS), progression free survival (PFS) and safety. Results: We analyzed 21 patients including DLBCL (n = 16), PCNSL (n = 2), MCL (n = 2), MZL (n = 1). The median age was 60 years (range, 24-84). The majority of patients had advanced disease stage (53%) and were refractory to their last therapy (57%). The non-GCB type was more common (67%) in patients with DLBCL and PCNSL. Nine patients (56%) with DLBCL had MYC and BCL2 and/or BCL6 rearrangements. Median number of prior therapies was 3 (range, 2-6). Eleven patients had prior auto and/or allo stem cell transplantation. Median follow-up time was 6.6 months (range, 0.4-22.3). Eleven patients (52%), 6 patients (29%) and 2 patients (10%) received RLI for ≥ 3, 6 and 12 months respectively. Patients had received a median of 3 (range, 1-19) cycles of RLI. Progressive disease (PD) was the most common reason for treatment discontinuation (53%). All of the patients were evaluable for response. The median TTR was 2 months (range, 1.7-3.8). The ORR was 38% (95% CI 15%-61%) with 29% CR and 9% PR. At the time of the analysis, the median DoR was 12.8 monts (range, 4.4-19.5+). Deaths were reported for 13 (62%) patients [n = 10 PD, n = 3 adverse events (AEs)]. The median OS was 7.9 months (95% CI 2.2-13.5) and median PFS was 3.5 months (95% CI 0.0- 8.3). The most common non-hematologic AEs were fatigue (48%), infection (38%), diarrhea (29%). AEs were frequently reported at the 25 mg lenalidomise dose, but the majority (77%) of patients experienced a grade 1-2 AE. The hematologic AEs were neutropenia (48%) [grade 3-4 (100%)], thrombocytopenia (19%) and anaemia (19%). AEs led to discontinuation in 5% (n = 1) of patients (sepsis). Conclusions: Our results suggest that RLI is a viable therapeutic option with promising activity and favorable toxicity profile in patients with R/R NHL patients.

Efficacy and Safety of Selinexor-Based Combination Therapy in CNS Lymphoma: A Retrospective Experience

Introduction ： Primary central nervous system lymphoma (PCNSL) is a rare diffuse large B-cell lymphoma (DLBCL) with distinct clinical and biological features, characterized by an aggressive course and poor prognosis. Patients with relapsed/refractory (R/R) disease have an unmet medical need due to a median overall survival (OS) of only 6.8 months. Novel therapies are urgently required for patients with R/R CNSL. In June 2020, the U.S. Food and Drug Administration granted accelerated approval to selinexor for the treatment of adult patients with R/R DLBCL. Selinexor has demonstrated central nervous system (CNS) penetration and activity in secondary CNS lymphoma (SCNSL) patients. Thus, we conducted a retrospective review of PCNSL/SCNSL cases treated with a selinexor-based regimen to preliminarily evaluate its effectiveness and safety. Methods: We retrospectively analyzed the medical records of 13 patients with CNSL who received selinexor-based combination therapy between June 2021 and June 2023 at three research centers in China. Among these cases, 2 were previously untreated, and 11 were relapsed or refractory patients. All patients had histopathologically confirmed DLBCL at the time of initial diagnosis. Data collection included baseline characteristics, treatment efficacy and safety evaluation. Results: A total of 13 patients (10 PCNSL and 3 SCNSL) were included in the study, with a median age of 62 years (range, 28-73), including three male patients. The median international extranodal lymphoma study group (IELSG) score for PCNSL cases was 3 (range, 1-3). The median International Prognosis Index (IPI) score for the three systemic DLBCL cases was 5 (range, 3-5), and three cases were at Ann Arbor stage IV. Among the patients, 11 had relapsed or refractory disease with a median of 2 prior treatments (range, 1-4), and 10 of them were refractory to previous treatments. The main patient characteristics, responses, and toxicities of the selinexor-based combination regimen are summarized in Table 1. Figure 1 presents the response of all evaluable patients. The overall response rate (ORR) of the patients was 84.6% (11 /13), with a complete response (CR) rate of 76.9% (10/13). Both treatment-naïve patients achieved CR after four and six cycles of the selinexor-containing first-line regimen, respectively. They are currently experiencing sustained remission, with the longest duration reaching 13 months. For relapsed/refractory CNSL, the ORR and CR rate were 81.8% (9/11) and 72.7% (8/11), respectively. Two patients had stable disease (SD). Among the 9 patients who achieved remission, 4 patients are still in remission, with the longest duration reaching 20 months, 2 patients received CART bridging therapy, one received radiation therapy, and 2 patients experienced disease progression after 5 months, but all are currently still alive. These patients are currently under continuous observation. Due to the heterogeneity of the selinexor-based regimens, the categories and severity of adverse events (AEs) varied greatly among the different regimens. The median treatment cycle number was six (range, 2-22). One patient died due to pneumonia infection, one patient was lost to follow-up, three patients successfully bridged to CART/RT, and eight patients completed the treatment as planned. The common AEs included thrombocytopenia, infection, nausea, vomiting, and gastrointestinal bleeding(Table 1). Conclusion: This report provides encouraging evidence of the efficacy and tolerable safety of the selinexor-based regimen in the treatment of CNSL. However, further prospective studies with a larger sample size are needed to provide more comprehensive evidence regarding the efficacy of selinexor in central nervous system lymphoma and to determine the appropriate dosage and optimal combination strategies.v