Tumor-infiltrating B cells are an important component in the microenvironment but have unclear anti-tumor effects. We enhanced our previous computational algorithm TRUST to extract the B cell immunoglobulin hypervariable regions from bulk tumor RNA-sequencing data. TRUST assembled more than 30 million complementarity-determining region 3 sequences of the B cell heavy chain (IgH) from The Cancer Genome Atlas. Widespread B cell clonal expansions and immunoglobulin subclass switch events were observed in diverse human cancers. Prevalent somatic copy number alterations in the MICA and MICB genes related to antibody-dependent cell-mediated cytotoxicity were identified in tumors with elevated B cell activity. The IgG3–1 subclass switch interacts with B cell–receptor affinity maturation and defects in the antibody-dependent cell-mediated cytotoxicity pathway. Comprehensive pancancer analyses of tumor-infiltrating B cell–receptor repertoires identified novel tumor immune evasion mechanisms through genetic alterations. The IgH sequences identified here are potentially useful resources for future development of immunotherapies.
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The data sets generated during the study are available from FireCloud and with dbGap permission to retrieve restricted TCGA data. The RNA-seq data set generated for validating TRUST performance is available in the SRA repository (PRJNA492301), and the matched iRepertoire data are available at https://bitbucket.org/liulab/ng-bcr-validate/src/master/iRep/.
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We thank F. Alt, M. Shipp, and G. Freeman for helpful discussions during manuscript preparation, and T. Wu for suggesting the Single Instruction Multiple Data (SIMD) acceleration. We also acknowledge the following funding sources for supporting this work: NCI, grants U01 CA226196 (X.S.L.) and U24 CA224316 (X.S.L.), Chinese Scholarship Council Funding (Jian Zhang), CPRIT RR170079 (B.L.), the Breast Cancer Research Foundation (X.S.L.), and the National Natural Science Foundation of China, 81702701 (T.L.).
X.S.L. is a cofounder and board member of GV20 Oncotherapy, SAB of 3DMed Care, consultant for Genentech, and stock holder of BMY, TMO, WBA, ABT, ABBV, and JNJ. J.S.L. is a cofounder and board member of Beijing Neoantigen Biotechnology Co. Ltd, and shareholder of BGNE, DVAX, CELG, NKTR, CRSP, and EDIT. K.W.W. serves on the SAB of Nextech, TCR2, and T-scan. He is also a consultant for Novartis. His laboratory has sponsored research agreements with Novartis, BMS, and Astellas. N.H. is a founder and SAB member of Neon Therapeutics and SAB member of IFM Therapeutics.
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A correction to this article is available online at https://doi.org/10.1038/s41588-019-0437-4.
Integrated supplementary information
a, Precision and sensitivity of modified TRUST to call B cell IgH CDR3 sequences with 20 replicates for different coverages (see Methods). Box plots show the lower, median, and upper quartiles of values, and lines extend to 1.5 times the interquartile range. b,c, Clonal frequency and overlap of IgH CDR3s from BCR-seq and TRUST. Six lung tumor samples were sequenced by both BCR-seq and RNA-seq. b, Pearson’s correlations of clonal frequencies from TRUST and BCR-seq. The sample sizes in the 6 cases were 441, 810, 738, 452, 631, and 398, respectively. c, Jaccard index of overlapped CDR3s in B cell repertoires. B cell repertoires from BCR-seq and TRUST have much higher overlaps for matched samples than unmatched samples.
a, IgH CDR3 length distributions and sequence motifs. Histograms show the distributions of CDR3 amino acid sequence length by each immunoglobulin isotype. The sequence logos are for all of the CDR3s with the highest frequency in length (14 aa) in TCGA samples. b, Ratio of synonymous and missense mutations in CDR3s. We summarized all of the SHMs within the CDR3 region and classified them by the effect on amino acid sequences. Synonymous mutations are the nucleotide mutations with no effect on the protein sequence. Missense mutations are the ones that change the amino acid in the protein sequence. c, Correlation between the SHM rate and AID expression. Patient samples (n = 4,708) were grouped by the SHM rates and displayed the distribution of AID expression levels. The boxes in violin plots show the lower, median, and upper quartiles of values, and the curves show the density of values.
Supplementary Figure 3 Grid search on the optimal parameters for clustering B cell CDR3s and the differences among clones.
a–c, Heatmaps show the values of FDR (a), cluster ratio (b), and final score (c) for ranking the clustering results of 500 randomly sampled pairs. The best solution with the highest final score was marked in red boxes. d, CDR3 amino acid length comparison for expanded (n = 2,687,374) and non-expanded (n = 32,631) B cell clones. Expanded clones are complete IgH CDR3s found in any B cell cluster (see Methods), and the remaining complete CDR3s are non-expanded ones. Statistical significance was evaluated by Student’s t-test. The red dots and the numbers show the mean values and the bars extend to 2 s.d. on both sides. The curves in the violin plots show the value distribution. e, Overlap of B cell clonotypes for tumor samples and adjacent normal tissues. We chose matched TCGA tumor and adjacent normal tissue samples with at least 500 clonotypes called by TRUST. As a comparison, we downloaded the top 100 B cell clones of paired normal and tumor samples published by Katoh et al.22. We observed a similar Jaccard index of tumor–normal clonotype overlap between data from TCGA (n = 26) and from Katoh et al.22 (n = 30). The two-sided P value was from Wilcoxon signed-rank test.
a, IgG3–1 switch level is the number of B cell clusters with both IgG1 and IgG3 divided by the total number of unique CDR3s. The group of top 25% samples with high IgG3–1 switch was compared with the bottom 25% samples in terms of the overall survival (see Supplementary Table 3 for sample sizes). Hazard ratios and statistical significance from Wald test were evaluated using Cox proportional hazard regressions corrected for tumor purity and patient age. FDR correction was performed using the Benjamini–Hochberg procedure for testing on multiple cancer types. b, Kaplan–Meier curves for significant tumor types were shown with raw P values at the bottom.
a, SHM high and low were split by the median SHM rate in all patient samples. IgG3–1 switch high or low was split by the median IgG3–1 switch level among patient samples (see Supplementary Table 3 for sample sizes). Hazard ratios and statistical significance from Wald test were evaluated using Cox proportional hazard regressions corrected for tumor purity and patient age. FDR correction was performed using Benjamini–Hochberg procedure for testing on multiple cancer types. b, Kaplan–Meier curves for significant tumor types were shown with raw P values at the bottom.
Supplementary Figure 6 MICA amplification in TCGA samples and the associations with gene expression, IgG1/3 level, and patient survival.
a, MICA amplification in paired normal and tumor samples. The table summarizes the number of patients with different MICA amplification status in paired tumor and adjacent normal tissues. MICA AMP means amplification in the MICA/MICB genes, and MICA WT means no amplification. b, MICA amplification correlated with gene expression differences (n = 9,846). Box plots show the differential expression of MICA, ADAM17, and MMP14 in tumors with or without MICA amplifications, where the boxes show the lower, median, and upper quartiles of values, and lines extend to 1.5 times the interquartile range. Statistical significance was evaluated using Wilcoxon rank sum test. c, IgG1/3 level in tumor samples with or without MICA amplification (n = 5,104). The boxplot shows the distribution of the IgG1/3 switch level in tumor samples with or without MICA amplification. Statistical significance was evaluated using Wilcoxon rank sum test (two-sided P value). Box plots show the lower, median, and upper quartiles of values, and lines extend to 1.5 times the interquartile range. d, Kaplan–Meier curve shows the survival differences in all TCGA patients with or without MICA amplification (n = 7,147). e, For each tumor type, hazard ratios and statistical significance from Wald test were evaluated using Cox proportional hazard regressions corrected for tumor purity and patient age. f, High- or low-IgG1/3 groups were split by the median value of IgG1/3 levels in each MICA group (see Supplementary Table 3 for sample sizes). The IgG1/3 level was the number of IgG1 or IgG3 B cell clusters normalized by the total number of unique CDR3s. Hazard ratios and statistical significance were evaluated using Cox proportional hazard regressions corrected for tumor purity and patient age. FDR correction was performed using the Benjamini–Hochberg procedure for testing on multiple cancer types.
Supplementary Figures 1–6
Summary of all TCGA RNA-seq samples involved in this study by disease types
Summary information for patients involved in this study, including survival, disease type, number of B cell clusters, IgG subclass switch events, somatic hypermutation rate, copy number alterations in MICA, and tumor purity
Sample sizes and P values
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Hu, X., Zhang, J., Wang, J. et al. Landscape of B cell immunity and related immune evasion in human cancers. Nat Genet 51, 560–567 (2019) doi:10.1038/s41588-018-0339-x
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