Thymoma

Results are based in part upon data generated by TCGA Research Network (http://cancergenome.nih.gov/). We aggregated TCGA transcriptomic and RPPA data from public repositories, listed in the “Data availability” section. RNA-seq expression data were processed by TCGA at the gene level, rather than at the transcript level. Tumors spanned 32 different TCGA projects, each project representing a specific cancer type, listed as follows: LAML, acute myeloid leukemia; ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; LGG, lower grade glioma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; CHOL, cholangiocarcinoma; CRC, colorectal adenocarcinoma (combining COAD and READ projects); ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; MESO, mesothelioma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumors; THYM, thymoma; THCA, thyroid carcinoma; UCS, uterine carcinosarcoma; UCEC, uterine corpus endometrial carcinoma; UVM, uveal melanoma. Cancer molecular profiling data were generated through informed consent as part of previously published studies and analyzed per each original study’s data use guidelines and restrictions.

Aligned RNA-seq bam files were analyzed using the ISB Cancer Genomics Cloud (https://isb-cgc.appspot.com/). These 33 cancer types included in this study are adrenocortical carcinoma [ACC], bladder urothelial carcinoma [BLCA], brain lower grade glioma [LGG], breast invasive carcinoma [BRCA], cervical squamous cell carcinoma and endocervical adenocarcinoma [CESC], cholangiocarcinoma [CHOL], colon adenocarcinoma [COAD], esophageal carcinoma [ESCA], glioblastoma multiforme [GBM], head and neck squamous cell carcinoma [HNSC], kidney chromophobe [KICH], kidney renal clear cell carcinoma [KIRC], kidney renal papillary cell carcinoma [KIRP], acute myeloid leukemia [LAML], liver hepatocellular carcinoma [LIHC], lung adenocarcinoma [LUAD], lung squamous cell carcinoma [LUSC], lymphoid neoplasm diffuse large B cell lymphoma [DLBC], mesothelioma [MESO], ovarian serous cystadenocarcinoma [OV], pancreatic adenocarcinoma [PAAD], pheochromocytoma and paraganglioma [PCPG], prostate adenocarcinoma [PRAD], rectum adenocarcinoma [READ], sarcoma [SARC], skin cutaneous melanoma [SKCM], stomach adenocarcinoma [STAD], testicular germ cell tumors [TGCT], thymoma [THYM], thyroid carcinoma [THCA], uterine carcinosarcoma [UCS], uterine corpus endometrial carcinoma [UCEC], and uveal melanoma [UVM]. The sample set consists of 10,337 total TCGA samples, 9,624 tumor samples, and 713 normal samples.
Level-3 gene expression (RSEM) and segment-based copy number data were downloaded from Broad GDAC firehose (https://gdac.broadinstitute.org) (version: 2016_01_28). Gene-based copy number data were obtained by intersecting with RefSeq gene annotation bed file (version: 2013-07-27). Mutation calls were provided by the Multi-Center Mutation Calling in Multiple Cancers (MC3) working group within TCGA (Key Resources Table; Ellrott et al., 2018 ).

Frozen specimens of thymomas and normal tissues were slightly shredded with scissors and total mRNA was extracted using TRIzol RNA Isolation Reagent (Gibco, #10296028). Expression level of GGT1 was analyzed by one-step probe qPCR using Luna Universal Probe One-Step RT-qPCR Kit (NEB, #E3006) and IDT PrimeTime^® qPCR Assays composed of Forward/Reverse primer pair and PrimeTime Probe. Primers were hGGT1 for GGT1 expression, and hGAPDH and hHPRT1 as internal standards. Primer and probe information is described in Supplementary Table S2. The mixture information is shown in Supplementary Table S3. The samples were centrifuged and each sample was measured by triplicate using a LightCycler^®480 System II (F. Hoffmann-La Roche Ltd, Basel, Switzerland). The mRNA expression levels of GGT1 were expressed relative to internal standards and compared between tumor and normal.

22 patients with thymoma or thymic carcinoma who underwent surgery between February 2013 and January 2021 at the University of Tokyo were included in the study. These are relatively rare diseases and only a few cases are operated on each year. In addition, only tumors larger than 2 cm were selected in this study to avoid compromising pathological assessment. Twenty patients were enrolled for the ex vivo fluorescence imaging study (Figs. 1, 2, 3, 4 and Supplementary Table S1), one patient was enrolled for the experiment involving application to serial sections of tumor and normal tissue (Fig. 5) and one patient was enrolled for the chemical inhibition experiment (Fig. 6). Some patients were included in plural analyses. Written informed consent was obtained from all patients, and this study was approved by the ethics committee of The University of Tokyo and the local ethics committees. All experiments were performed in accordance with guidelines and regulations approved by the ethics committees. All specimens were taken intraoperatively. Fluorescence images, except for the inhibition experiment and the experiment comparing HMRG only and gGlu-HMRG, were collected within a day after resection. For the inhibition experiment, frozen specimens were thawed at room temperature and used within 6 months. With regard to the experiment comparing HMRG only and gGlu-HMRG, data from frozen and raw specimens are mixed.

Manifests containing fragments per kilobase per million (FPKM) normalized RNA-seq data from 34 TCGA cohorts – Acute Myeloid Leukemia - (TCGA-LAML), Adrenocortical carcinoma (TCGA-ACC), Bladder Urothelial Carcinoma (TCGA-BLCA), Glioblastoma multiforme and Brain Lower Grade Glioma and (TCGA-GBMLGG), Breast Invasive Carcinomas (TCGA-BRCA), Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma (TCGA-CESC), Cholangiocarcinoma (TCGA-CHOL), Chronic Myelogenous Leukemia (TCGA-LCML), Colon Adenocarcinoma (TCGA-COAD), Esophageal Carcinoma (TCGA-ESCA), Head and Neck Squamous Cell Carcinoma (TCGA-HNSC), pan-Kidney Cancer (TCGA-KIPAN), Liver Hepatocellular Carcinoma (TCGA-LIHC), Lung Adenocarcinoma (TCGA-LUAD), Lung Squamous Cell Carcinoma (TCGA-LUSC), Lymphoid Neoplasm Diffuse Large B-cell Lymphoma (TCGA-DLBC), Mesothelioma (TCGA-MESO), Ovarian Serous Cystadenocarcinoma (TCGA-OV), Pancreatic Adenocarcinoma (TCGA-PAAD), Pheochromocytoma and Paraganglioma (TCGA-PCPG), Prostate Adenocarcinoma (TCGA-PRAD), Rectum Adenocarcinoma (TCGA-READ), Sarcoma (TCGA-SARC), Skin Cutaneous Melanoma (TCGA-SKCM), Stomach Adenocarcinoma (TCGA-STAD), Testicular Germ Cell Tumors (TCGA-TGCT), Thymoma (TCGA-TGCT), Thyroid Carcinoma (TCGA-THCA), Uterine Carcinosarcoma (TCGA-USC), Uterine Corpus Endometrial Carcinoma (TCGA-UCES), Uveal Melanoma (TCGA-UVM) were downloaded from the Broad Institute GDAC (TCGA data version 20150601). Patients were then sorted by increasing B7-H3 expression in each cohort.