Intratumoural immune cell landscape in germinoma reveals multipotent lineages and exhibits prognostic significance
Division of Brain Tumor Translational Research, National Cancer Center Research Institute, Tokyo, Japan
Department of Neurosurgery, Faculty of Medicine, The University of Tokyo Hospital, Tokyo, Japan
Search for more papers by this authorDivision of Brain Tumor Translational Research, National Cancer Center Research Institute, Tokyo, Japan
Search for more papers by this authorDivision of Gene and Immune Medicine, National Cancer Center Research Institute, Tokyo, Japan
Search for more papers by this authorDivision of Brain Tumor Translational Research, National Cancer Center Research Institute, Tokyo, Japan
Department of Pathology and Clinical Laboratories, National Cancer Center Hospital, Tokyo, Japan
Search for more papers by this authorDivision of Gene and Immune Medicine, National Cancer Center Research Institute, Tokyo, Japan
Search for more papers by this authorDivision of Cancer Genomics, National Cancer Center Research Institute, Tokyo, Japan
Search for more papers by this authorDivision of Brain Tumor Translational Research, National Cancer Center Research Institute, Tokyo, Japan
Department of Neurosurgery and Neuro‐Oncology, National Cancer Center Hospital, Tokyo, Japan
Search for more papers by this authorDivision of Brain Tumor Translational Research, National Cancer Center Research Institute, Tokyo, Japan
Division of Pediatric Neuro‐Oncology, Saitama Medical University International Medical Center, Saitama, Japan
Search for more papers by this authorDivision of Brain Tumor Translational Research, National Cancer Center Research Institute, Tokyo, Japan
Department of Pediatrics, Osaka City General Hospital, Osaka, Japan
Search for more papers by this authorDivision of Brain Tumor Translational Research, National Cancer Center Research Institute, Tokyo, Japan
Department of Neurosurgery, Graduate School of Medicine, Yokohama City University, Kanagawa, Japan
Search for more papers by this authorDepartment of Neurosurgery, Faculty of Medicine, The University of Tokyo Hospital, Tokyo, Japan
Department of Neurosurgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
Search for more papers by this authorDepartment of Neurosurgery, Faculty of Medicine, The University of Tokyo Hospital, Tokyo, Japan
Search for more papers by this authorDepartment of Neuro‐Oncology/Neurosurgery, Saitama Medical University International Medical Center, Saitama, Japan
Search for more papers by this authorDivision of Pediatric Neuro‐Oncology, Saitama Medical University International Medical Center, Saitama, Japan
Search for more papers by this authorDepartment of Neurosurgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
Department of Neurosurgery, Kurume University, Fukuoka, Japan
Search for more papers by this authorDepartment of Neurosurgery, Faculty of Medicine, Hiroshima University, Hiroshima, Japan
Search for more papers by this authorDepartment of Neurosurgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
Search for more papers by this authorDepartment of Neurosurgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
Search for more papers by this authorDepartment of Neurosurgery, Graduate School of Medical Science, Kanazawa University, Ishikawa, Japan
Search for more papers by this authorDepartment of Neurosurgery, Kansai Medical University Hospital, Osaka, Japan
Search for more papers by this authorDepartment of Neurosurgery, Kansai Medical University Hospital, Osaka, Japan
Search for more papers by this authorDepartment of Neurosurgery, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan
Search for more papers by this authorDepartment of Neurosurgery, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan
Search for more papers by this authorDepartment of Neurosurgery, Chiba Cancer Center, Chiba, Japan
Search for more papers by this authorDepartment of Neurosurgery, Osaka National Hospital, National Hospital Organization, Osaka, Japan
Department of Biomedical Research and Innovation, Institute for Clinical Research, Osaka National Hospital, National Hospital Organization, Osaka, Japan
Search for more papers by this authorDepartment of Neurosurgery, Faculty of Medicine, Kyorin University, Tokyo, Japan
Search for more papers by this authorDepartment of Neurosurgery, Faculty of Medicine, Kyorin University, Tokyo, Japan
Search for more papers by this authorDepartment of Neurological Surgery, Dentistry, and Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Okayama, Japan
Search for more papers by this authorDepartment of Neurosurgery, Kyusyu University Hospital, Fukuoka, Japan
Search for more papers by this authorDepartment of Neurosurgery, University of Tsukuba Hospital, Ibaraki, Japan
Search for more papers by this authorDepartment of Neurosurgery, University of Tsukuba Hospital, Ibaraki, Japan
Search for more papers by this authorDepartment of Neurosurgery, Fujita Health University Hospital, Aichi, Japan
Search for more papers by this authorDepartment of Neurosurgery, Hamamatsu University Hospital, Shizuoka, Japan
Search for more papers by this authorDepartment of Neurosurgery, Kitasato University, Kanagawa, Japan
Search for more papers by this authorDepartment of Neurosurgery, Dokkyo Medical Univeristy, Tochigi, Japan
Search for more papers by this authorDepartment of Neurosurgery and Neuro‐Oncology, National Cancer Center Hospital, Tokyo, Japan
Search for more papers by this authorDepartment of Neurosurgery and Neuro‐Oncology, National Cancer Center Hospital, Tokyo, Japan
Search for more papers by this authorDivision of Cancer Genomics, National Cancer Center Research Institute, Tokyo, Japan
Search for more papers by this authorDivision of Cancer Genomics, National Cancer Center Research Institute, Tokyo, Japan
Search for more papers by this authorDepartment of Pathology, Hidaka Hospital, Gunma, Japan
Search for more papers by this authorDepartment of Neurosurgery, Graduate School of Medicine, Yokohama City University, Kanagawa, Japan
Search for more papers by this authorDepartment of Neurosurgery, Graduate School of Medicine, Yokohama City University, Kanagawa, Japan
Search for more papers by this authorCorresponding Author
Division of Brain Tumor Translational Research, National Cancer Center Research Institute, Tokyo, Japan
Correspondence: Koichi Ichimura, Division of Brain Tumor Translational Research, National Cancer Center Research Institute, 5‐1‐1, Tsukiji, Chuo‐ku, Tokyo, Japan 104‐0045. Tel: 81 3 3542 2511 ext 3838; Fax: 81 3 3542 8170; E‐mail: kichimur@ncc.go.jpSearch for more papers by this authorDivision of Brain Tumor Translational Research, National Cancer Center Research Institute, Tokyo, Japan
Department of Neurosurgery, Faculty of Medicine, The University of Tokyo Hospital, Tokyo, Japan
Search for more papers by this authorDivision of Brain Tumor Translational Research, National Cancer Center Research Institute, Tokyo, Japan
Search for more papers by this authorDivision of Gene and Immune Medicine, National Cancer Center Research Institute, Tokyo, Japan
Search for more papers by this authorDivision of Brain Tumor Translational Research, National Cancer Center Research Institute, Tokyo, Japan
Department of Pathology and Clinical Laboratories, National Cancer Center Hospital, Tokyo, Japan
Search for more papers by this authorDivision of Gene and Immune Medicine, National Cancer Center Research Institute, Tokyo, Japan
Search for more papers by this authorDivision of Cancer Genomics, National Cancer Center Research Institute, Tokyo, Japan
Search for more papers by this authorDivision of Brain Tumor Translational Research, National Cancer Center Research Institute, Tokyo, Japan
Department of Neurosurgery and Neuro‐Oncology, National Cancer Center Hospital, Tokyo, Japan
Search for more papers by this authorDivision of Brain Tumor Translational Research, National Cancer Center Research Institute, Tokyo, Japan
Division of Pediatric Neuro‐Oncology, Saitama Medical University International Medical Center, Saitama, Japan
Search for more papers by this authorDivision of Brain Tumor Translational Research, National Cancer Center Research Institute, Tokyo, Japan
Department of Pediatrics, Osaka City General Hospital, Osaka, Japan
Search for more papers by this authorDivision of Brain Tumor Translational Research, National Cancer Center Research Institute, Tokyo, Japan
Department of Neurosurgery, Graduate School of Medicine, Yokohama City University, Kanagawa, Japan
Search for more papers by this authorDepartment of Neurosurgery, Faculty of Medicine, The University of Tokyo Hospital, Tokyo, Japan
Department of Neurosurgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
Search for more papers by this authorDepartment of Neurosurgery, Faculty of Medicine, The University of Tokyo Hospital, Tokyo, Japan
Search for more papers by this authorDepartment of Neuro‐Oncology/Neurosurgery, Saitama Medical University International Medical Center, Saitama, Japan
Search for more papers by this authorDivision of Pediatric Neuro‐Oncology, Saitama Medical University International Medical Center, Saitama, Japan
Search for more papers by this authorDepartment of Neurosurgery, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan
Department of Neurosurgery, Kurume University, Fukuoka, Japan
Search for more papers by this authorDepartment of Neurosurgery, Faculty of Medicine, Hiroshima University, Hiroshima, Japan
Search for more papers by this authorDepartment of Neurosurgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
Search for more papers by this authorDepartment of Neurosurgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan
Search for more papers by this authorDepartment of Neurosurgery, Graduate School of Medical Science, Kanazawa University, Ishikawa, Japan
Search for more papers by this authorDepartment of Neurosurgery, Kansai Medical University Hospital, Osaka, Japan
Search for more papers by this authorDepartment of Neurosurgery, Kansai Medical University Hospital, Osaka, Japan
Search for more papers by this authorDepartment of Neurosurgery, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan
Search for more papers by this authorDepartment of Neurosurgery, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan
Search for more papers by this authorDepartment of Neurosurgery, Chiba Cancer Center, Chiba, Japan
Search for more papers by this authorDepartment of Neurosurgery, Osaka National Hospital, National Hospital Organization, Osaka, Japan
Department of Biomedical Research and Innovation, Institute for Clinical Research, Osaka National Hospital, National Hospital Organization, Osaka, Japan
Search for more papers by this authorDepartment of Neurosurgery, Faculty of Medicine, Kyorin University, Tokyo, Japan
Search for more papers by this authorDepartment of Neurosurgery, Faculty of Medicine, Kyorin University, Tokyo, Japan
Search for more papers by this authorDepartment of Neurological Surgery, Dentistry, and Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Okayama, Japan
Search for more papers by this authorDepartment of Neurosurgery, Kyusyu University Hospital, Fukuoka, Japan
Search for more papers by this authorDepartment of Neurosurgery, University of Tsukuba Hospital, Ibaraki, Japan
Search for more papers by this authorDepartment of Neurosurgery, University of Tsukuba Hospital, Ibaraki, Japan
Search for more papers by this authorDepartment of Neurosurgery, Fujita Health University Hospital, Aichi, Japan
Search for more papers by this authorDepartment of Neurosurgery, Hamamatsu University Hospital, Shizuoka, Japan
Search for more papers by this authorDepartment of Neurosurgery, Kitasato University, Kanagawa, Japan
Search for more papers by this authorDepartment of Neurosurgery, Dokkyo Medical Univeristy, Tochigi, Japan
Search for more papers by this authorDepartment of Neurosurgery and Neuro‐Oncology, National Cancer Center Hospital, Tokyo, Japan
Search for more papers by this authorDepartment of Neurosurgery and Neuro‐Oncology, National Cancer Center Hospital, Tokyo, Japan
Search for more papers by this authorDivision of Cancer Genomics, National Cancer Center Research Institute, Tokyo, Japan
Search for more papers by this authorDivision of Cancer Genomics, National Cancer Center Research Institute, Tokyo, Japan
Search for more papers by this authorDepartment of Pathology, Hidaka Hospital, Gunma, Japan
Search for more papers by this authorDepartment of Neurosurgery, Graduate School of Medicine, Yokohama City University, Kanagawa, Japan
Search for more papers by this authorDepartment of Neurosurgery, Graduate School of Medicine, Yokohama City University, Kanagawa, Japan
Search for more papers by this authorCorresponding Author
Division of Brain Tumor Translational Research, National Cancer Center Research Institute, Tokyo, Japan
Correspondence: Koichi Ichimura, Division of Brain Tumor Translational Research, National Cancer Center Research Institute, 5‐1‐1, Tsukiji, Chuo‐ku, Tokyo, Japan 104‐0045. Tel: 81 3 3542 2511 ext 3838; Fax: 81 3 3542 8170; E‐mail: kichimur@ncc.go.jpSearch for more papers by this authorAbstract
Aims
Alterations in microenvironments are a hallmark of cancer, and these alterations in germinomas are of particular significance. Germinoma, the most common subtype of central nervous system germ cell tumours, often exhibits massive immune cell infiltration intermingled with tumour cells. The role of these immune cells in germinoma, however, remains unknown.
Methods
We investigated the cellular constituents of immune microenvironments and their clinical impacts on prognosis in 100 germinoma cases.
Results
Patients with germinomas lower in tumour cell content (i.e. higher immune cell infiltration) had a significantly longer progression‐free survival time than those with higher tumour cell contents (P = 0.03). Transcriptome analyses and RNA in‐situ hybridization indicated that infiltrating immune cells comprised a wide variety of cell types, including lymphocytes and myelocyte‐lineage cells. High expression of CD4 was significantly associated with good prognosis, whereas elevated nitric oxide synthase 2 was associated with poor prognosis. PD1 (PDCD1) was expressed by immune cells present in most germinomas (93.8%), and PD‐L1 (CD274) expression was found in tumour cells in the majority of germinomas examined (73.5%).
Conclusions
The collective data strongly suggest that infiltrating immune cells play an important role in predicting treatment response. Further investigation should lead to additional categorization of germinoma to safely reduce treatment intensity depending on tumour/immune cell balance and to develop possible future immunotherapies.
Supporting Information
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| nan12570-sup-0001-FigS1-S7.pptxapplication/mspowerpoint, 6.6 MB | Figure S1. (A) A comparison of tumour cell content (%) determined by methylation data from Infinium Human Methylation450 BeadChips (Illumina) and by microscopic inspection of haematoxylin and eosin stained tumour cells in formalin‐fixed paraffin‐embedded (FFPE) specimens is plotted (n = 36). The concordance rate between the two data sets was P < 0.0001 by linear regression analysis. (B) Kaplan–Meier curves generated by a linear regression analysis of the progression‐free survival according to tumour cell content, using cut‐off levels (60% and 70%) that were different from the main figure (Figure 1B). These analyses also demonstrated that cases rich in tumour cells showed worse prognosis than those cases with scarce tumour cells (P = 0.02 and 0.005 respectively).Figure S2. Gene expression (log2) was compared with the tumour‐void rate (1‐tumour cell content rate, which represents tumour microenvironment), and R coefficient together with the slope value was plotted and analysed by linear correlation regression for 480 immune‐related genes, which are arranged on the X‐ and Y‐axes respectively. Genes exhibiting the highest correlation were selected with the threshold of R coefficient > 0.6 and the slope > 3 (n = 108) and analysed using the Database for Annotation, Visualization and Integrated Discovery for gene ontology (inset table). The results suggested that the most dominant cellular activity in the tumour tissue in correlation to tumour microenvironment was cellular immune activity.Figure S3. CIBERSORT was applied to messenger RNA expression data by RNA sequence in 35 cases. The immune cells were categorized into nine types, and cases were arranged in ascending order of immune cell burden by the absolute scores of CIBERSORT. There was a tendency of higher rate of B cells in tumour cell‐rich cases, while higher rate of macrophage was seen in tumour cell‐scarce cases (A). The absolute signature gene scores are displayed for the nine types of immune cells. B cells, T cells and macrophage were the most dominant constituents in the tumour microenvironment, each accounting for approximately 30% (B).Figure S4. Representative RNA scope images for eight probes for two cases. Intensity scores and proportions are indicated in insets (see Materials and methods). Bars indicate 25 μm. A: GCT72, B: GCT79.Figure S5. Kaplan–Meier curves of progression‐free survival (PFS) in cases above (Red line) and below (Blue line) the median values of expression are shown for each of 11 representative immune‐related genes. Statistically significant differences were found for CD4 and nitric oxide synthase 2 (NOS2) expression, and high expression of CD4 was associated with longer PFS, while high expression of NOS2 was associated with shorter PFS.Figure S6. The top portion shows a heatmap generated by unsupervised hierarchical clustering of expression of nine immune checkpoint genes, including PD‐1 (PDCD1) and PD‐L1 (CD274), using RNA sequencing data from 35 cases. The bottom portion shows tumour cell content plotted in a bar graph. Cases were separated into three clusters with significantly different degrees of tumour cell contents (P = 0.0002): tumour cell ‘rich’, ‘intermediate’ and ‘scarce’ groups. Those germinomas with lower tumour cell contents (i.e. a higher degree of immune cell infiltration) showed higher expression of immune checkpoint genes. Of note, 31 of 35 cases in this clustering analysis were categorized in the same groups as in the clustering using 480 immune‐related genes in Figure 2A.Figure S7. The figure shows a linear correlation between the number of somatic mutations and tumour cell contents (%) calculated with methylation analysis for 12 cases (R2 = 0.41). One outlier (GCT27), which had an exceptionally large number of somatic mutations (286) and very low tumour cell content (14% by methylation analysis and < 5% by haematoxylin and eosin staining), was omitted from this figure (see Discussion). |
| nan12570-sup-0002-TableS1-S9.docxWord document, 1.3 MB | Table S1. Clinical data of 100 germinoma and 1 normal brain/testis tissues used for quantitative reverse transcription polymerase chain reaction (qRT‐PCR), RNA scope, RNA‐seq analyses.Table S2. Breakdown of the number of cases which underwent each analysis out of 100 cases in the study.Table S3. Primer sequences for quantitative reverse transcriptase polymerase chain reaction.Table S4. Result of analyses on the relationship between tumour content and progression‐free survival using multiple cut‐off values of tumour content percentage.Table S5. Immune‐related 480 genes used for the analysis of expression.Table S6. Upregulated genes in tumour cell‐scarce group in 480 immunity‐related genes.Table S7. Gene ontology terms enriched in tumour cell‐scarce group.Table S8. Result of CIBERSORT (absolute values) using RNA sequence data by permutation of 1000 for 35 cases.Table S9. Total results for RNA in situ hybridization. |
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