B cells are associated with survival and immunotherapy response in sarcoma

Soft-tissue sarcomas represent a heterogeneous group of cancer, with more than 50 histological subtypes1,2. The clinical presentation of patients with different subtypes is often atypical, and responses to therapies such as immune checkpoint blockade vary widely3,4. To explain this clinical variability, here we study gene expression profiles in 608 tumours across subtypes of soft-tissue sarcoma. We establish an immune-based classification on the basis of the composition of the tumour microenvironment and identify five distinct phenotypes: immune-low (A and B), immune-high (D and E), and highly vascularized (C) groups. In situ analysis of an independent validation cohort shows that class E was characterized by the presence of tertiary lymphoid structures that contain T cells and follicular dendritic cells and are particularly rich in B cells. B cells are the strongest prognostic factor even in the context of high or low CD8+ T cells and cytotoxic contents. The class-E group demonstrated improved survival and a high response rate to PD1 blockade with pembrolizumab in a phase 2 clinical trial. Together, this work confirms the immune subtypes in patients with soft-tissue sarcoma, and unravels the potential of B-cell-rich tertiary lymphoid structures to guide clinical decision-making and treatments, which could have broader applications in other diseases.

section_content: Data was analysed with R software (version 3. 4. 4 ) and packages gplots, survival and FactoMineR. Custom code was produced in R for the analysis. 

For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e. g. GitHub). See the Nature Research guidelines for submitting code & software for further information.
section_name: Data analysis

section_content: All manuscripts must include a data availability statement. This statement should provide the following information, where applicable:

-Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability

The transcriptomic datasets analysed in this study can be accessed on the GDC Portal (TCGA SARC) and the Gene Expression Omnibus repository (accession numbers GSE21050, GSE21122, GSE30929). Immunohistochemistry, gene expression and clinical-related to NTUH cohorts (Fig. 3, Extended Data Figs. 7 and 8 ) are available from the corresponding author on reasonable request. The data that support the findings related to Fig. 4 are available from SARC but restrictions apply to the availability of these data, which were used under license for the study. Data are however available upon reasonable request to HAT (HTawbi@mdanderson. org) and with permission of SARC. All code used in this study is available from the authors upon reasonable request. 

The specificity of anti-CD3, anti-CD4, anti-CD8, anti-CD20, anti-CD21, anti-CD23, anti-CD34, anti-CXCR5 and anti-DC-Lamp antibodies, and MECA-79 (PNAd) was validated on FFPE tonsil sections as positive control. For anti-CD20, certified manufacturing facilities from the company guarantee full quality control including western blot and studies using COS-1 cells transfected with cDNA encoding the CD20 molecule indicate that the antibody labels an intracytoplasmic epitope localized on the CD20 molecule. For anti-CD8, certified manufacturing facilities from the company guarantee full quality control including western blot and indicate that the antibody recognizes the cd8alpha chain. For anti-CD34, certified manufacturing facilities from the company guarantee full quality control. For anti-CD21, certified manufacturing facilities from the company guarantee full quality control including western blotting of the immunogen, and that the antibody labels cells or cell lines known to express CD21 (Raji, NC 37, tonsil cells), whereas no labeling is observed in the CD21-negative Jurkat cells (T-cell line) and human erythrocytes. For anti-CD23, certified manufacturing facilities from the company guarantee full quality control including western blotting, IHC on human tonsils and flow cytometry on Raji cells. For anti-CXCR5, certified manusfacturing facilities from the company guarantee full quality control using human CXCR5 transfectants by flow cytometry and lack of cross reactivity with human CXCR2, CXCR3, or CXCR4 transfectants. For PNAd, certified manufacturing facilities from the company guarantee full quality control including western blotting, IHC and flow cytometry. For anti-PD-L1, specificity was validated by the company using immunohistochemical analysis of paraffin-embedded human placenta using PD-L1 (E1L3N®) XP® Rabbit mAb in the presence of control peptide or antigen-specific peptide. Specificity was verified by using FPE sections from placenta as positive control and cerebral cortex tissue as negative control. Anti-PD-1 (Freeman GJ and col. ) was obtained from CoStim Pharmaceuticals and validated as described in Fig. S1 of Giraldo et al., Clinical Cancer Research, 2015. Tonsil, placenta and cerebral cortex slides were obtained from Geneticist Inc.
section_name: Data Policy information about availability of data

section_name: Ethics oversight

section_content: The research was approved by the Research Ethics Committee of NTUH (201605061RINA). 

Note that full information on the approval of the study protocol must also be provided in the manuscript.
section_name: Ethics oversight