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Supplementary Table Legends and Information, Supplementary Figures 1-11

Supplementary Tables 1-13

Supplementary document

Supplementary Figure S1-S7

Primary central nervous system lymphoma (PCNSL) is an isolated type of lymphoma of the central nervous system and has a dismal prognosis despite intensive chemotherapy. Recent genomic analyses have identified highly recurrent mutations of MYD88 and CD79B in immunocompetent PCNSL, whereas LMP1 activation is commonly observed in Epstein–Barr virus (EBV)-positive PCNSL. However, a lack of clinically representative preclinical models has hampered our understanding of the pathogenic mechanisms by which genetic aberrations drive PCNSL disease phenotypes. Here, we establish a panel of 12 orthotopic, patient-derived xenograft (PDX) models from both immunocompetent and EBV-positive PCNSL and secondary CNSL biopsy specimens. PDXs faithfully retained their phenotypic, metabolic, and genetic features, with 100% concordance of MYD88 and CD79B mutations present in PCNSL in immunocompetent patients. These models revealed a convergent functional dependency upon a deregulated RelA/p65-hexokinase 2 signaling axis, codriven by either mutated MYD88/CD79B or LMP1 with Pin1 overactivation in immunocompetent PCNSL and EBV-positive PCNSL, respectively. Notably, distinct molecular alterations used by immunocompetent and EBV-positive PCNSL converged to deregulate RelA/p65 expression and to drive glycolysis, which is critical for intracerebral tumor progression and FDG-PET imaging characteristics. Genetic and pharmacologic inhibition of this key signaling axis potently suppressed PCNSL growth in vitro and in vivo. These patient-derived models offer a platform for predicting clinical chemotherapeutics efficacy and provide critical insights into PCNSL pathogenic mechanisms, accelerating therapeutic discovery for this aggressive disease.Significance:A set of clinically relevant CNSL xenografts identifies a hyperactive RelA/p65-hexokinase 2 signaling axis as a driver of progression and potential therapeutic target for treatment and provides a foundational preclinical platform.

Supplementary Table S1-S12

Supplementary Table S1, Supplementary legends for Figures S1-S3, Supplementary Methods and References

EGFRvIII promotes glutamate release by glioma cells in a kinase-independent manner.

EGFR interacts with the central portion of xCT and thereby promotes surface expression of xCT.

Sulfasalazine does not affect intracellular ROS levels in Becker or U87MG cells.

Central nervous system (CNS) tumor patients commonly undergo multimodality treatment in the course of their disease. Adverse effects and complications from these interventions have not been systematically studied, but pose significant challenges in clinical practice and impact function and quality of life, especially in the management of long-term brain tumor survivors. Here, the European Association of Neuro-Oncology (EANO) has developed recommendations to prevent, diagnose, and manage adverse effects and complications in the adult primary brain CNS tumor (except lymphomas) patient population with a specific focus on surgery, radiotherapy, and pharmacotherapy. Specifically, we also provide recommendations for dose adaptations, interruptions, and reexposure for pharmacotherapy that may serve as a reference for the management of standard of care in clinical trials. We also summarize which interventions are unnecessary, inactive or contraindicated. This consensus paper should serve as a reference for the conduct of standard therapy within and outside of clinical trials.

Background CNS germ cell tumors (GCTs) predominantly develop in pediatric and young adult patients with variable responses to surgery, radiation, and chemotherapy. This study aimed to examine the complex and largely unknown pathogenesis of CNS GCTs. Methods We used a combined transcriptomic and methylomic approach in 84 cases and conducted an integrative analysis of the normal cells undergoing embryogenesis and testicular GCTs. Results Genome-wide transcriptome analysis in CNS GCTs indicated that germinoma had a transcriptomic profile representative of primitive cells during early embryogenesis with high meiosis/mitosis potentials, while nongerminomatous GCTs (NGGCTs) had differentiated phenotypes oriented toward tissue formation and organogenesis. Co-analysis with the transcriptome of human embryonic cells revealed that germinomas had expression profiles similar to those of primordial germ cells, while the expression profiles of NGGCTs were similar to those of embryonic stem cells. Some germinoma cases were characterized by extensive immune-cell infiltration and high expression of cancer-testis antigens. NGGCTs had significantly higher immune-cell infiltration, characterized by immune-suppression phenotype. CNS and testicular GCTs (TGCTs) had similar mutational profiles; TGCTs showed enhanced copy number alterations. Methylation analysis clustered germinoma/seminoma and nongerminoma/nonseminoma separately. Germinoma and seminoma were co-categorized based on the degree of the tumor microenvironment balance. Conclusions These results suggested that the pathophysiology of GCTs was less dependent on their site of origin and more dependent on the state of differentiation as well as on the tumor microenvironment balance. This study revealed distinct biological properties of GCTs, which will hopefully lead to future treatment development.

INTELLANCE‐J was a phase 1/2 study of a potent antibody‐drug conjugate targeting epidermal growth factor receptor (EGFR), depatuxizumab mafodotin (Depatux‐M), as a second‐ or first‐line therapy, alone or combined with chemotherapy or chemoradiotherapy in 53 Japanese patients with World Health Organization (WHO) grade III/IV glioma. In second‐line arms, patients with EGFR‐amplified recurrent WHO grade III/IV glioma received Depatux‐M plus chemotherapy (temozolomide) or Depatux‐M alone regardless of EGFR status. In first‐line arms, patients with newly diagnosed WHO grade III/IV glioma received Depatux‐M plus chemoradiotherapy. The study was halted following lack of survival benefit with first‐line Depatux‐M in the global trial INTELLANCE‐1. The primary endpoint was 6‐month progression‐free survival (PFS) in patients with EGFR‐amplified tumors receiving second‐line Depatux‐M plus chemotherapy. Common nonocular treatment‐emergent adverse events (TEAEs) with both second‐line and first‐line Depatux‐M included lymphopenia (42%, 33%, respectively), thrombocytopenia (39%, 47%), alanine aminotransferase increase (29%, 47%), and aspartate aminotransferase increase (24%, 60%); incidence of grade ≥3 TEAEs was 66% and 53%, respectively. Ocular side effects (OSEs) occurred in 93% of patients receiving second‐line Depatux‐M plus chemotherapy and all patients receiving second‐line Depatux‐M alone or first‐line Depatux‐M plus chemoradiotherapy. Most OSEs were manageable with dose modifications and concomitant medications. The 6‐month PFS estimate was 25.6% (95% confidence interval [CI] 11.4‒42.6), and median PFS was 2.1 months (95% CI 1.9‒3.9) with second‐line Depatux‐M plus chemotherapy in the EGFR‐amplified subgroup. This study showed acceptable safety profile of Depatux‐M alone or plus chemotherapy/chemoradiotherapy in Japanese patients with WHO grade III/IV glioma. The study was registered at ClinicalTrials.gov (NCT02590263)., INTELLANCE‐J was a phase 1/2 study of a potent antibody‐drug conjugate targeting epidermal growth factor receptor (EGFR), depatuxizumab mafodotin, as a second‐ or first‐line therapy, alone or combined with chemotherapy or chemoradiotherapy in Japanese patients with World Health Organization grade III/IV glioma. The results of this trial demonstrate an acceptable safety profile of depatuxizumab mafodotin, with ocular side effects being the most common adverse events that were mostly reversible. Second‐line depatuxizumab mafodotin in combination with temozolomide resulted in a 6‐month progression‐free survival estimate of 25.6% (95% confidence interval 11.4‒42.6) in patients with EGFR‐amplified tumors and showed encouraging antitumor activity in this subgroup of patients (NCT02590263).

Primary central nervous system lymphoma (PCNSL) is a rare extra-nodal non-Hodgkin's lymphoma confined to the central nervous system with a diffuse large B-cell lymphoma (DLBCL) histology and is highly prevelant in elderly patients. Whole brain radiotherapy (WBRT) does not provide considerable remission; rather it is highly involved in the development of leukoencephalopathy with delayed neurotoxicity, notably in elderly patients. Standard care for newly diagnosed patients with PCNSL comprised induction with high-dose methotrexate (HD-MTX)-based multi-agent immunochemotherapy, such as R-MPV (rituximab, MTX, procarbazine, vincristine) yielding 70-75% complete response rate, followed by HD-cytarabine consolidation. Consolidation high-dose chemotherapy with the key drug thiotepa supported by autologous stem cell transplant has recently been investigated to replace WBRT in multiple randomized trials, demonstrating non-inferiority to WBRT with less neurotoxicity. Comprehensive genetic analyses have revealed high rates of oncogenic mutations in CD79B and MYD88 genes, the hallmarks for MCD/C5 subtype of DLBCL, leading to constitutive activation of NF-κB signaling pathways in PCNSL. Bruton's tyrosine kinase (BTK), an intermediate kinase downstream to CD79B/MYD88, has emerged as a promising therapeutic target. Furthermore, tirabrutinib, a second-generation BTK inhibitor, has shown substantial activity against relapsed/refractory PCNSL, resulting in its approval in 2020 in Japan. Additionally, other new agents against PI3-kinase and immunotherapies including immunomodulatory agents, immune checkpoint blockade, and CAR-T have been actively tested in clinical trials.

The current standard of diagnosing central nervous system (CNS) lymphoma is stereotactic biopsy, however the procedure has a risk of surgical complication. Liquid biopsy of the CSF is a less invasive, non‐surgical method that can be used for diagnosing CNS lymphoma. In this study, we established a clinically applicable protocol for determining mutations in MYD88 in the CSF of patients with CNS lymphoma. CSF was collected prior to the start of chemotherapy from 42 patients with CNS lymphoma and matched tumor specimens. Mutations in MYD88 in 33 tumor samples were identified using pyrosequencing. Using 10 ng each of cellular DNA and cell‐free DNA (cfDNA) extracted from the CSF, the MYD88 L265P mutation was detected using digital PCR. The conditions to judge mutation were rigorously determined. The median Target/Total value of cases with MYD88 mutations in the tumors was 5.1% in cellular DNA and 22.0% in cfDNA. The criteria to judge mutation were then determined, with a Target/Total value of 0.25% as the cutoff. When MYD88 mutations were determined based on these criteria, the sensitivity and specificity were 92.2% and 100%, respectively, with cellular DNA; and the sensitivity and specificity were 100% with cfDNA. Therefore, the DNA yield, mutated allele fraction, and accuracy were significantly higher in cfDNA compared with that in cellular DNA. Taken together, this study highlights the importance of detecting the MYD88 L265P mutation in cfDNA of the CSF for diagnosing CNS lymphoma using digital PCR, a highly accurate and clinically applicable method., Development of a liquid biopsy for less invasive diagnosis of CNS lymphoma is in progress. In this study, the conditions for detecting the MYD88 L265P mutation by digital PCR were set, and extremely high accuracy was confirmed. This method is fully clinically feasible.

Background An open-label, non-comparative study assessed the efficacy and safety of nivolumab in Japanese patients with first recurrence glioblastoma. Methods Patients with first recurrence of histologically confirmed World Health Organization Grade IV glioma, after treatment with temozolomide and radiotherapy, received nivolumab 3 mg/kg every 2 weeks until confirmed disease progression (Response Assessment in Neuro-Oncology criteria) or toxicity. Primary endpoint was 1-year overall survival rate assessed by Bayesian approach. The prespecified efficacy criterion was that the Bayesian posterior probability threshold for exceeding the 1-year overall survival of bevacizumab (34.5%) from the Japanese phase 2 study (JO22506) would be 93%. Results Of the 50 enrolled patients, 44 (88.0%) had recurrent malignant glioma (glioblastoma, gliosarcoma), and of these, 26 (59.1%) had at least one measurable lesion at baseline. The Bayesian posterior mean 1-year overall survival (90% Bayesian credible intervals) with nivolumab was 54.4% (42.27–66.21), and the Bayesian posterior probability of exceeding the threshold of the 1-year overall survival rate of bevacizumab (34.5%) was 99.7%. Median (90% confidence interval) overall and progression-free survival was 13.1 (10.4–17.7) and 1.5 (1.4–1.5) months, respectively. One partial response was observed (objective response rate 1/26 evaluable patients [3.8%]). Treatment-related adverse event rates were 14.0% for Grade 3–4 and 2.0% for Grade 5; most adverse events resolved and were manageable. Conclusions The 1-year overall survival with nivolumab monotherapy in Japanese patients with glioblastoma met the prespecified efficacy criterion. The safety profile of nivolumab was consistent with that observed in other tumor types. Clinical Trial Registration JapicCTI-152967.

Background The goal was to determine whether the addition of temozolomide (TMZ) to the standard treatment of high-dose methotrexate (HD-MTX) and whole-brain radiotherapy (WBRT) for primary central nervous system lymphoma (PCNSL) improves survival. Methods An open-label, randomized, phase III trial was conducted in Japan, enrolling immunocompetent patients aged 20–70 years with histologically confirmed, newly diagnosed PCNSL. After administration of HD-MTX, patients were randomly assigned to receive WBRT (30 Gy) ± 10 Gy boost (arm A) or WBRT ± boost with concomitant and maintenance TMZ for 2 years (arm B). The primary endpoint was overall survival (OS). Results Between September 29, 2014 and October 15, 2018, 134 patients were enrolled, of whom 122 were randomly assigned and analyzed. At the planned interim analysis, 2-year OS was 86.8% (95% confidence interval [CI]: 72.5–94.0%) in arm A and 71.4% (56.0–82.2%) in arm B. The hazard ratio was 2.18 (95% CI: 0.95–4.98), with the predicted probability of showing the superiority of arm B at the final analysis estimated to be 1.3%. The study was terminated early due to futility. O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status was measured in 115 tumors, and it was neither prognostic nor predictive of TMZ response. Conclusions This study failed to demonstrate the benefit of concomitant and maintenance TMZ in newly diagnosed PCNSL.

Although adult diffuse gliomas, IDH-wildtype, histologically diagnosed as grade 2-3 have been regarded as clinically equivalents with glioblastomas, the prognosis of this population has scarcely been discussed with further consideration of treatment backgrounds. This retrospective study aimed to investigate the prognosis of adult patients with IDH-wildtype diffuse gliomas histologically diagnosed as WHO grade 2-3 using a cohort with homogenous treatment background. A total of 214 IDH-wildtype cases were extracted from the collected data of the previous study investigating the prognostic significance of molecular markers (Arita, Acta Neuropathol Commun 2016). The inclusion criteria were as follows: supratentorial tumor, pretreatment Karnofsky Performance status of 70 or higher, wildtype-H3.3 status, chemoradiation therapy with temozolomide after the initial surgery. Overall survival (OS) and progression free survival (PFS) were evaluated by Kaplan-Meier methods. The histological diagnosis in this study was made based on the WHO 2016 classification, and the histological criteria is compatible with the current classification (CNS WHO5).The mean age was 62.8, 59.8 and 60.2 years in grade 2 (n=8), 3 (n=45) and 4 (n=161) case, respectively. The higher ratio of biopsy cases was associated lower grades (50% in grade 2, 22% in grade 3 and 9% in grade 4 cases). Grade 2-3 cases showed short survival (OS 24.7 months and PFS 9.7months) despite the intensive treatment of chemoradiation at the time of diagnosis. In details, OS was 41.5, 23.1 and 17.8 months in grade 2, 3 and 4 cases, respectively. PFS was 12.6, 9.7 and 8.2 months in grade 2, 3 and 4 cases, respectively. Our results revealed that IDH-wildtype grade 2-3 cases showed dismal prognosis even after the chemoradiation at the time of diagnosis. Further development of treatments is needed in this population as well as glioblastomas, IDH-wildtype.

Advanced molecular and pathophysiologic characterization of primary central nervous system lymphoma (PCNSL) has revealed insights into promising targeted therapeutic approaches. Medical imaging plays a fundamental role in PCNSL diagnosis, staging, and response assessment. Institutional imaging variation and inconsistent clinical trial reporting diminishes the reliability and reproducibility of clinical response assessment. In this context, we aimed to: (1) critically review the use of advanced positron emission tomography (PET) and magnetic resonance imaging (MRI) in the setting of PCNSL; (2) provide results from an international survey of clinical sites describing the current practices for routine and advanced imaging, and (3) provide biologically based recommendations from the International PCNSL Collaborative Group (IPCG) on adaptation of standardized imaging practices. The IPCG provides PET and MRI consensus recommendations built upon previous recommendations for standardized brain tumor imaging protocols (BTIP) in primary and metastatic disease. A biologically integrated approach is provided to addresses the unique challenges associated with the imaging assessment of PCNSL. Detailed imaging parameters facilitate the adoption of these recommendations by researchers and clinicians. To enhance clinical feasibility, we have developed both “ideal” and “minimum standard” protocols at 3T and 1.5T MR systems that will facilitate widespread adoption.

While intracerebral hemorrhage (ICH) scoring systems provide mortality and morbidity prediction, the actual mortality rates seem to be lower than those predicted by scoring systems in our clinical impression. To assess the validity of the ICH score and the Surgical Swedish ICH (SwICH) score, we retrospectively reviewed surgically treated ICH patients between 2012 and 2019. Uni- and multivariate analyses were performed to identify variables in predicting 30-day mortality. We identified 203 patients (mean ICH score 2.7; mean SwICH score 2.0). The actual 30-day mortality was 7%, which was significantly lower than those predicted by the ICH and the SwICH scores (55% and 16%, respectively; p

Primary central nervous system lymphoma (PCNSL) is an isolated type of lymphoma of the central nervous system and has a dismal prognosis despite intensive chemotherapy. Recent genomic analyses have identified highly recurrent mutations of MYD88 and CD79B in immunocompetent PCNSL, whereas LMP1 activation is commonly observed in Epstein–Barr virus (EBV)-positive PCNSL. However, a lack of clinically representative preclinical models has hampered our understanding of the pathogenic mechanisms by which genetic aberrations drive PCNSL disease phenotypes. Here, we establish a panel of 12 orthotopic, patient-derived xenograft (PDX) models from both immunocompetent and EBV-positive PCNSL and secondary CNSL biopsy specimens. PDXs faithfully retained their phenotypic, metabolic, and genetic features, with 100% concordance of MYD88 and CD79B mutations present in PCNSL in immunocompetent patients. These models revealed a convergent functional dependency upon a deregulated RelA/p65-hexokinase 2 signaling axis, codriven by either mutated MYD88/CD79B or LMP1 with Pin1 overactivation in immunocompetent PCNSL and EBV-positive PCNSL, respectively. Notably, distinct molecular alterations used by immunocompetent and EBV-positive PCNSL converged to deregulate RelA/p65 expression and to drive glycolysis, which is critical for intracerebral tumor progression and FDG-PET imaging characteristics. Genetic and pharmacologic inhibition of this key signaling axis potently suppressed PCNSL growth in vitro and in vivo. These patient-derived models offer a platform for predicting clinical chemotherapeutics efficacy and provide critical insights into PCNSL pathogenic mechanisms, accelerating therapeutic discovery for this aggressive disease. Significance: A set of clinically relevant CNSL xenografts identifies a hyperactive RelA/p65-hexokinase 2 signaling axis as a driver of progression and potential therapeutic target for treatment and provides a foundational preclinical platform.

Primary central nervous system lymphoma(PCNSL)is an aggressive, extranodal non-Hodgkin lymphoma that arises from the central nervous system, eyes, leptomeninges, and the spinal cord. Most PCNSLs are a type of diffuse large B-cell lymphoma(DLBCL), and the majority are categorized as a non-germinal center B-cell(GCB)subtype. Recent genetic studies have revealed several common genetic abnormalities in PCNSL, such as

Backgrounds Mutations in the isocitrate dehydrogenase (IDH)1 gene are favourable prognostic factors in newly diagnosed diffuse gliomas, whereas it remains controversial in the recurrent glioblastoma setting. Methods A total of 171 patients with newly diagnosed glioblastoma, either ‘primary’ glioblastoma or ‘secondary’ glioblastoma, treated at Kyorin University Hospital or Japanese Red Cross Medical Center from 2000 to 2015 were included. Patients with confirmed IDH1 status and O6-methylguanine-DNA methyltransferase promoter methylation status were retrospectively analysed for overall survival from the initial diagnosis (n = 147) and after the first progression (n = 122). Results IDH1 mutation but not IDH2 was noted in 19 of 147 patients with glioblastoma (12.9%). In patients with ‘primary’ glioblastoma (n = 136), median overall survival after the first progression was 13.5 and 10.5 months for mutant IDH1 and wild-type IDH1 glioblastoma, respectively (P = 0.747). Multivariate analysis revealed O6-methylguanine-DNA methyltransferase promoter methylation, and Karnofsky Performance status 60 or higher, were independent prognostic factors for better overall survival after the first progression. When ‘primary’ glioblastoma and ‘secondary’ glioblastoma were combined, median overall survival from the first progression was not significantly different between the mutant IDH1 group (10.1 months) and wild-type IDH1 group (10.5 months) (P = 0.559), whereas median overall survival from the initial diagnosis was significantly different (47.5 months vs.18.3 months, respectively; P = 0.035). Conclusions These results suggest that IDH1 mutation may not be a prognostic factor for survival at the first progression of patients with ‘primary’ glioblastoma and pretreated ‘secondary’ glioblastoma, and further warrant investigation in prospective studies., Overall survival after first progression of patients with primary glioblastoma was not significantly different between those with mutant IDH1 and wild-type IDH1. O6-methylguanine-DNA methyltransferase status and Karnofsky Performance status were independent better prognosticators.

BackgroundThe safety, tolerability, efficacy, and pharmacokinetics of tirabrutinib, a second-generation, highly selective oral Bruton’s tyrosine kinase inhibitor, were evaluated for relapsed/refractory primary central nervous system lymphoma (PCNSL).MethodsPatients with relapsed/refractory PCNSL, Karnofsky performance status ≥70, and normal end-organ function received tirabrutinib 320 and 480 mg once daily (q.d.) in phase I to evaluate dose-limiting toxicity (DLT) within 28 days using a 3 + 3 dose escalation design and with 480 mg q.d. under fasted conditions in phase II.ResultsForty-four patients were enrolled; 20, 7, and 17 received tirabrutinib at 320, 480, and 480 mg under fasted conditions, respectively. No DLTs were observed, and the maximum tolerated dose was not reached at 480 mg. Common grade ≥3 adverse events (AEs) were neutropenia (9.1%), lymphopenia, leukopenia, and erythema multiforme (6.8% each). One patient with 480 mg q.d. had grade 5 AEs (pneumocystis jirovecii pneumonia and interstitial lung disease). Independent review committee assessed overall response rate (ORR) at 64%: 60% with 5 complete responses (CR)/unconfirmed complete responses (CRu) at 320 mg, 100% with 4 CR/CRu at 480 mg, and 53% with 6 CR/CRu at 480 mg under fasted conditions. Median progression-free survival was 2.9 months: 2.1, 11.1, and 5.8 months at 320, 480, and 480 mg under fasted conditions, respectively. Median overall survival was not reached. ORR was similar among patients harboring CARD11, MYD88, and CD79B mutations, and corresponding wild types.ConclusionThese data indicate favorable efficacy of tirabrutinib in patients with relapsed/refractory PCNSL.Trial registrationJapicCTI-173646.

Objective The optimal regimen for use of high dose-methotrexate-based chemotherapy in primary central nervous system lymphoma is still under debate. We conducted a retrospective study to evaluate the treatment outcome of a combination immunochemotherapy consisting of rituximab, methotrexate, procarbazine and vincristine followed by with or without whole brain radiotherapy and consolidation cytarabine, in comparison with high dose-methotrexate monotherapy followed by full dose whole brain radiotherapy. Methods Newly diagnosed primary central nervous system lymphoma patients treated with either rituximab, methotrexate, procarbazine and vincristine or high dose-methotrexate in Kyorin University Hospital were identified, and the response rates and survival were compared. Toxicities, post-treatment transition of Mini-Mental State Examination, Karnofsky performance status score, Fazekas scale and prognostic factors were analysed in the rituximab, methotrexate, procarbazine and vincristine group. Results Ninety-five patients treated with rituximab, methotrexate, procarbazine and vincristine (n = 39) or high dose-methotrexate (n = 56) were analysed. The complete response/complete response unconfirmed rate was significantly higher in the rituximab, methotrexate, procarbazine and vincristine group (74.4 vs. 15.4%, P Conclusions Rituximab, methotrexate, procarbazine and vincristine was apparently promising in comparison with high dose-methotrexate monotherapy with manageable toxicity in this retrospective study, and further investigation is warranted.

This study aimed to explore the genetic alterations and to identify good responders in the experimental arm in the tumor samples from newly diagnosed glioblastoma (GBM) patients enrolled in JCOG0911; a randomized phase II trial was conducted to compare the efficacy of interferonβ (IFNβ) plus temozolomide (TMZ) with that of TMZ alone. Of 122 tumors, we performed deep targeted sequencing to determine the somatic mutations, copy number variations, and tumor mutation burden; pyrosequencing for O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation; Sanger sequencing for the telomerase reverse transcriptase (TERT) promoter; and microsatellite instability (MSI) testing in 95, 91, 91 and 72 tumors, respectively. We performed a multivariable Cox regression analysis using backward stepwise selection of variables including clinical factors (sex, age, performance status, residual tumor after resection, tumor location) and genetic alterations. Deep sequencing detected an IDH1 mutation in 13 tumors (14%). The MGMT promoter methylation by quantitative pyrosequencing was observed in 41% of the tumors. A mutation in the TERT promoter was observed in 69% of the tumors. While high tumor mutation burden (> 10 mutations per megabase) was seen in four tumors, none of the tumors displayed MSI-high. The clinical and genetic factors considered as independent favorable prognostic factors were gross total resection (hazard ratio [HR]: 0.49, 95% confidence interval, 0.30–0.81, P = 0.0049) and MGMT promoter methylation (HR: 0.43, 0.21–0.88, P = 0.023). However, tumor location at the temporal lobe (HR: 1.90, 1.22–2.95, P = 0.0046) was an independent unfavorable prognostic factor. No predictive factors specific to the TMZ + IFNβ + Radiotherapy (RT) group were found. This additional sub-analytical study of JCOG0911 among patients with newly diagnosed GBM showed that tumor location at the temporal lobe, gross total resection, and MGMT promoter methylation were significant prognostic factors, although no factors specific to IFNβ addition were identified.

Background Central nervous system (CNS) germ cell tumors (GCTs) are neoplasms predominantly arising in pediatric and young adult populations. While germinomas generally respond to chemotherapy and radiation, non-germinomatous GCTs (NGGCTs) require more intensive treatment. This study aimed to determine whether 12p gain could predict the prognosis of CNS GCTs. Methods Eighty-two CNS GCTs were included in this study. The 12p gain was defined by an additional 12p in the background of potential polyploidy or polysomy. Cases were analyzed using an Illumina methylation 450K array for copy number investigations and validated by fluorescence in situ hybridization (FISH). Results A 12p gain was found in 25-out-of-82 cases (30%) and was more frequent in NGGCTs (12% of germinoma cases and 50% of NGGCT cases), particularly in cases with malignant components, such as immature teratoma, yolk sac tumor, choriocarcinoma, and embryonal carcinoma. 12p gain and KIT mutation were mutually exclusive events. The presence of 12p gain correlated with shorter progression-free (PFS) and overall survival (OS) (10-year OS: 59% vs. 94%, with and without 12p gain, respectively, P = 0.0002), even with histology and tumor markers incorporated in the multivariate analysis. Among NGGCTs, 12p gain still had prognostic significance for PFS and OS (10-year OS: 47% vs. 90%, respectively, P = 0.02). The 12p copy number status was shared among histological components in mixed GCTs. Conclusions 12p gain may predict the presence of malignant components of NGGCTs, and poor prognosis of the patients. It may be associated with early tumorigenesis of CNS GCT.

Management of primary central nervous system lymphoma (PCNSL) includes induction and consolidation therapies in newly diagnosed patients, as well as second-line therapy in relapsed or refractory patients. The current standard-of-care induction therapy involves methotrexate (MTX)-based multi-agent immunochemotherapy with rituximab, methotrexate, procarbazine, and vincristine. Deferral or dose reduction of radiation therapy is considered in consolidation therapy, especially in elderly patients who carry a high risk of radiation-induced delayed neurotoxicity. Since elderly patients comprise the main population of PCNSL, minimally toxic treatments that are effective and feasible for them are strongly needed. For second-line therapy, rechallenge using MTX-based chemotherapy (in patients with a prior durable response to MTX-based chemotherapy) or radiation therapy is considered. Bruton's tyrosine kinase inhibitor tirabrutinib (for relapsed and refractory PCNSL) and high-dose chemotherapy with autologous stem cell transplantation support using thiotepa and busulfan (BuTT) were approved by the Japanese Ministry of Health and Welfare in March 2020 and has recently become available for clinical practice. While these novel treatments seem promising, the optimal use of these treatments along with the standard-of-care therapy of PCNSL should be defined and investigated in clinical trials.

Primary central nervous system lymphoma (PCNSL) is an aggressive extranodal non-Hodgkin lymphoma confined to the brain, eyes, and the spinal cord. The mechanism of central nervous system (CNS) tropism in PCNSL has not been fully elucidated. Diffuse large B cell lymphomas (DLBCLs) occasionally present with distant recurrence, which can involve inside and outside the CNS. Secondary central nervous system lymphomas (SCNSLs) are CNS relapse of systemic lymphoma. PCNSLs also rarely present with systemic relapse. We have previously reported in our study of whole exome sequencing that PCNSLs harbor frequent mutations in genes of B cell receptor pathway members and aberrant somatic hypermutation (aSHM) target genes. Although several genetic alterations were identified as more frequent in PCNSLs compared with systemic lymphomas, specific genetic alterations which serve as the driver for CNS tropism in PCNSLs has not been identified. In order to search for mutations which might serve as driver mutations in the CNS, we have performed targeted sequencing in paired samples from patients with recurrent lymphomas, either SCNSLs or PCNSL systemic relapses, using Ion Torrent multiplex PCR. Mutational profiles were compared between the primary and recurrent tumor. Six cases (four SCNSL cases and two PCNSL systemic relapse cases) were analyzed. Of note, in the SCNSL cases, several de novo mutations were enriched only among the recurrent CNS tumors. Among these mutations, BTG2 mutations were observed in 3/4 (75%), and B2M and KLHL14 mutations were observed in 2/4 (50%) cases. In the two PCNSL systemic relapse cases, KMT2D mutations were enriched only in the recurrent systemic tumors. It is suggested that these de novo mutations in the recurrent CNS tumors might serve as driver mutations in the CNS. Further analysis in larger cohorts, and functional studies are required in order to validate these findings.

Primary central nervous system lymphoma (PCNSL) affects primarily elderly patients and comprises 5% of all primary brain tumors with predominant histology of diffuse large B-cell lymphoma (DLBCL). Whole brain radiotherapy (WBRT) does not provide durable remission, rather is deeply involved in development of leukoencephalopathy with delayed neurotoxicity, especially in elderly patients. BBB-penetrating high-dose methotrexate (HD-MTX)-based multiagent immunochemotherapies have been extensively explored and have shown high activities as induction regimens, followed by consolidation therapies such as high-dose chemotherapy with autologous stem-cell transplant (HDC/ASCT). Recent progress in understanding molecular pathogenesis of PCNSL has shed light on developing novel molecular targeted therapies against key signaling molecules such as Bruton’s tyrosine kinase (BTK) and pathways leading to constitutive activation of NF-kB signaling. In this lecture, standard of care for PCNSL with its molecular landscape and issues to be pursuit to further improve outcome of PCNSL treatment will be discussed.

Background Glioblastoma (GBM) is an aggressive primary brain tumor with poor prognosis. One strategy for overcoming resistance is developing a new therapy targeting the cancer stem/initiating cells. We have shown that the antidiabetic drug metformin (MF) can induce differentiation of stem-like glioma-initiating cells and suppress tumor formation through AMPK-FOXO3 activation. We conducted a phase I/II study to examine the clinical effect of MF combined with standard maintenance temozolomide (TMZ). Here, we report the result of phase I part and the current status of phase II part. Patients and Methods Patients between 20 and 74 years of age with supratentorial GBM histologically diagnosed according to the World Health Organization 2016 classification were eligible. MF dose-escalation was planned using a 3 + 3 design. Dose-limiting toxicities (DLTs) were assessed during the first 6 weeks after MF initiation. Results Between February 2021 and January 2022, the first three patients were treated with 1,500 mg/day MF and the next four patients were treated with 2,250 mg/day MF, which is the maximum dose approved in Japan. The median age of the patients was 41 years. Three tumors (42.9 %) were IDH1/2 mutants and 4 (57.1 %) were IDH1/2 wild-types. No DLTs were observed. The most common adverse effects were appetite loss, nausea, and diarrhea, which were observed in three patients. All of them were manageable, with grade 1 or 2. Only one grade 3 seizure was reported, which was likely related to the tumor. Two patients experienced tumor progression at 6.0 and 6.1 months, and one died 12.2 months after initial surgery. The other five patients remained stable at the last follow-up. Conclusion MF dose of up to 2,250 mg/day combined with maintenance TMZ appeared to be well tolerated, and we proceeded to phase II study with 2,250 mg/day MF.

Background Aggressive resection of recurrent glioblastoma has been reported to prolong survival and should be considered for resectable recurrences. In recent years, expanded resection for primary glioblastoma, especially removal of the FLAIR hyperintensity region beyond the contrast-enhancing lesion (FLAIRectomy; FR), has been reported, but there are no reports of FR for recurrent glioblastomas. In this study, we examined the efficacy and safety of FR for recurrent glioblastoma. Methods Recurrent glioblastoma operated on at our institution from January 2014 to July 2022 were included. The extent of resection (EOR) of contrast-enhancing lesions and FLAIR hyperintensity lesion was measured. Progression-free survival (PFS), overall survival (OS), surgical morbidity, KPS, and MMSE were analyzed retrospectively. Removal of at least 20% of the FLAIR hyperintensity lesion was defined as FR. Results Thirty-five surgeries in 28 patients (age at surgery 15-81 years; median 54 years, male 20/female 15) were included. The EOR of the contrast-enhancing lesion was 96.6± 7.4%, with GTR in 28 cases and non-GTR in 7. The EOR of the lesion with FLAIR hyperintensity was 33.2±36.5% and FR was achieved in 15 cases (42.9%). The median preoperative KPS was 90 (50-100; mean 83.3 ) in the FR group and 80 (60-100; mean 81) in the non-FR group. Both PFS and OS from surgery at recurrence were significantly prolonged in the FR group (median PFS; 8.1 months vs. 4.6 months, p=0.032, median OS; 36.1 months vs. 15.9 months, p=0.009); there was no difference in PFS or OS between the GTR and non-GTR groups (p=0.33, 0.79). There were significantly fewer patients in the FR group having a decrease in KPS of 20 or more at 30/90 days postoperatively (p=0.037, 0.020). Conclusion In our retrospective analysis, FR prolongs PFS and OS without decreasing KPS for resectable recurrent glioblastoma. Further validation in prospective studies is warranted.

Background Primary central nervous system lymphoma (PCNSL) is the second common primary malignant brain tumor. Compared to glioma, the treatment period is often longer, but there have been few research reports on QOL evaluation during the treatment yet. In this study, we retrospectively examined the QOL scores obtained during and following treatment of PCNSL to determine the association between changes in the QOL score and response to treatment, with particular interest in the issue in the evaluation over time. Methods Patients with newly-diagnosed PCNSL who were treated and evaluated for QOL by EORTC QLQ-C30/BN-20 at our facility since April 2016 were included. Results A total of 69 patients were included, with a median age of 69, and a male to female ratio of 41:32. The median KPS was 70, and the median MMSE score was 29 points, indicating that a majority of the patients who could undergo the testing retained adequate cognitive function. There were 38 patients (55.1%) who maintained CR during the QOL evaluation period, and their QOL function scores tended to be maintained above the baseline. In contrast, 31 patients (44.9%) who relapsed during the QOL evaluation period showed a tendency to decline in global health at the time of recurrence or evaluation immediately before recurrence, with the worsened symptom score reflecting the localization of recurrence. Any associations of changes in QOL score with therapeutic methods were identified. Changes in QOL scores were rather frequently observed with exacerbation of comorbidities, perhaps due to a high incidence of PCNSL in the elderly patients. Conclusions EORTC QLQ-C30/BN-20 enabled QOL evaluation that might reflect treatment effect in PCNSL. The reporting rate to the questionnaire was low in patients with substantial brain dysfunction, and QOL evaluation may also be affected by comorbidities. Further investigation of influencing factors on QOL evaluation is warranted.

BACKGROUND In the updated 2021 WHO classification, diffuse gliomas were strictly reclassified by molecular statuses. In glioblastoma (GBM), the prognostic significance of the resection has been shown, but the excision rate to be achieved in the new classification has not been defined. Moreover, the correlation between the extent of resection (EOR) and the prognosis is still controversial for other gliomas. METHODS IDH 1/2 mutations and TERT promoter mutations were analyzed by Sanger sequencing, 1p/19q co-deletion by microsatellite or MLPA, and EGFR amplification and CDKN2A deletions by MLPA. The correlation between the resection rate and prognosis of each tumor type in the 2021/2016 WHO classification was analyzed retrospectively. RESULTS According to the 2021 classification, 293 patients with GBM, IDH-wild type, 68 with astrocytoma, IDH-mutant (2/3/4 22/26/20), and 58 with oligodendroglioma, IDH-mutant and 1p/19q co-deleted were identified. Based on the 2016 classification, they were diagnosed as 314 GBM, 106 astrocytoma (Gd II/III 37/69), and 59 oligodendroglioma. For GBM, IDH-wild type (2021 classification), a prolonged OS benefit was observed with >25% removal (p=0.042), whereas, more than 20% of tumor removal for patients with GBM (2016 classification) resulted in OS prolongation. EOR was a significant favorable prognostic factor for grade II/III astrocytoma by the 2016 classification, but not for astrocytoma, IDH-mutant (grade 2/3) in the 2021 classification. When analyzed only in patients with astrocytoma, IDH-mutant by the 2021 classification, EOR was not significantly prognostic, suggesting that poorer prognosis of patients with unresectable IDH-wild type astrocytomas (2016 classification) might have a negative impact on the outcome. CONCLUSION There was a strong correlation between EOR and prognosis in patients with GBM, and >25% removal was considered significant according to the 2021 classification. For astrocytoma in the 2021 classification, EOR did not show a prognostic significance, but further validation including grading effect is warranted.

Background Germinoma preferentially occurs in pediatric and young adult age groups. Although they are responsive to treatment with chemotherapy and radiation, the treatment may cause long-term sequelae in their later lives. Here, we searched for clinical and histopathological features to predict the prognosis of germinoma and affect treatment response. Methods A total of 114 germinoma cases were included in the analysis. We investigated the association between clinical factors, tumor cell content, and progression-free survival (PFS). Results The tumor cell content was widely distributed from Conclusions We found that tumor cell content significantly affected the prognosis of germinomas. Although validation of these results using an independent and larger cohort is necessary, this potentially opens the possibility of leveraging this pathological factor in future clinical trials when stratifying the treatment intensity.

The Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy (cIMPACT-NOW) update 3 recommends that histologic grade II and III IDH-wildtype diffuse astrocytic gliomas that harbor EGFR amplification, the combination of whole chromosome 7 gain and whole chromosome 10 loss (7 + /10 -), or TERT promoter (pTERT) mutations should be considered as glioblastomas (GBM), World Health Organization grade IV. In this retrospective study, we examined the utility of molecular classification based on pTERT status and copy-number alterations (CNAs) in IDH-wildtype lower grade gliomas (LGGs, grade II, and III). The impact on survival was evaluated for the pTERT mutation and CNAs, including EGFR gain/amplification, PTEN loss, CDKN2A homozygous deletion, and PDGFRA gain/amplification. We analyzed 46 patients with IDH-wildtype/pTERT-mutant (mut) LGGs and 85 with IDH-wildtype/pTERT-wildtype LGGs. EGFR amplification and a combination of EGFR gain and PTEN loss (EGFR + /PTEN -) were significantly more frequent in pTERT-mut patients (p 0.0001). Cox regression analysis showed that the pTERT mutation was a significant predictor of poor prognosis (hazard ratio [HR] 2.79, 95% confidence interval [CI] 1.55-4.89, p = 0.0008), but neither EGFR amplification nor EGFR + /PTEN - was an independent prognostic factor in IDH-wildtype LGGs. PDGFRA gain/amplification was a significant poor prognostic factor in IDH-wildtype/pTERT-wildtype LGGs (HR 2.44, 95% CI 1.09-5.27, p = 0.03, Cox regression analysis). The IDH-wildtype LGGs with either pTERT-mut or PDGFRA amplification were mostly clustered with GBM by DNA methylation analysis. Thus, our study suggests that analysis of pTERT mutation status is necessary and sufficient to diagnose IDH-wildtype diffuse astrocytic gliomas with molecular features of glioblastoma. The PDGFRA status may help further delineate IDH-wildtype/pTERT-wildtype LGGs. Methylation profiling showed that IDH-wildtype LGGs without molecular features of GBM were a heterogeneous group of tumors. Some of them did not fall into existing categories and had significantly better prognoses than those clustered with GBM.