Zheming Lu

T cell receptor β-chain repertoire analysis reveals intratumour heterogeneity of tumour-infiltrating lymphocytes in oesophageal squamous cell carcinoma.

Oesophageal squamous cell carcinoma (ESCC) has a generally poor prognosis, due to the lack of effective treatment methods. Immunotherapeutic approaches based on tumour‐infiltrating lymphocytes (TILs) have demonstrated that durable responses are produced in some patients with solid tumours, which suggests the potential feasibility of clinical application of immunotherapy for ESCC. However, many of the basic characteristics of TILs in ESCC are poorly understood, including clonality, specificity and spatial heterogeneity of the response of TILs, which depends on the interaction between antigens and T cell receptors (TCRs). We used ultra‐deep sequencing of rearranged genes in TCR β‐chain (TCRβ) to profile the basic characteristics of T cells in tumour tissues (four to six regions from each tumour) as well as matched adjacent normal tissue and peripheral blood from seven patients diagnosed with primary ESCC. We found that T cell clones within ESCCs were quite different from those of the peripheral blood and even the adjacent normal tissues in general. Although there was a relatively higher degree of overlap of intratumoural TCRβ repertoires than those between the tumour and other tissues, intratumoural TCRβ repertoires were spatially heterogeneous. Due to the restricted sampling, high‐throughput TCRβ sequencing could characterize the diversity and composition of a limited (compartment‐dependent) fraction of the respective T cell clones in any individual ESCC, expanding our understanding of immune behaviour and immune response and shedding more light on ESCC immunotherapy. Copyright

Clonality analysis of multifocal papillary thyroid carcinoma by using genetic profiles.

Papillary thyroid carcinoma (PTC) is the most common adult thyroid malignancy and often presents with multiple anatomically distinct foci within the thyroid, known as multifocal papillary thyroid carcinoma (MPTC). The widespread application of the next‐generation sequencing technologies in cancer genomics research provides novel insights into determining the clonal relationship between multiple tumours within the same thyroid gland. For eight MPTC patients, we performed whole‐exome sequencing and targeted region sequencing to identify the non‐synonymous point mutations and gene rearrangements of distinct and spatially separated tumour foci. Among these eight MPTCs, completely discordant mutational spectra were observed in the distinct cancerous nodules of patients MPTC1 and 5, suggesting that these nodules originated from independent precursors. In another three cases (MPTC2, 6, and 8), the distinct MPTC foci of these patients had no other shared mutations except BRAF V600E, also indicating likely independent origins. Two patients (MPTC3 and 4) shared almost identical mutational spectra amongst their separate tumour nodules, suggesting a common clonal origin. MPTC patient 7 had seven cancer foci, of which two foci shared 66.7% of mutations, while the remaining cancer foci displayed no common non‐synonymous mutations, indicating that MPTC7 has multiple independent origins accompanied by intraglandular disease dissemination. In this study, we found that 75% of MPTC cases arose as independent tumours, which supports the field cancerization hypothesis describing multiple malignant lesions. MPTC may also arise from intrathyroidal metastases from a single malignant clone, as well as multiple independent origins accompanied by intrathyroidal metastasis. Copyright

T cell receptor β-chain repertoire analysis reveals the association between neoantigens and tumour-infiltrating lymphocytes in multifocal papillary thyroid carcinoma

To explore whether a few nonsynonymous somatic mutations could induce activation and proliferation of neoantigen‐specific tumour‐infiltrating lymphocytes (TILs) in tumours with low mutation rates, we analysed a patient with multifocal papillary thyroid carcinoma (seven noncontiguous cancer foci) to investigate the relationship between neoantigens and TILs. These seven foci had a few or no nonsynonymous somatic mutations; moreover, multiple loci had similar or different spectra of mutations. We used high‐throughput sequencing of the rearranged genes in T cell receptor β‐chain (TCRβ) to reveal the basic characteristics of T cells in seven tumour foci and matched adjacent normal tissue. We found that in multifocal papillary thyroid carcinoma the number of nonsynonymous somatic mutations was positively associated with oligoclonal TCRβ repertoire, and tumour foci with similar spectra of mutations had higher overlap of TCRβ repertoire. In conclusion, the number of nonsynonymous somatic mutations is small in tumours with low mutation rates but these mutations still play an important role in activating neoantigen‐specific TILs.

Whole-Genome Analysis of Human Papillomavirus Types 16, 18, and 58 Isolated from Cervical Precancer and Cancer Samples in Chinese Women

Human papillomavirus (HPV) types 16, 18 and 58 are ranked the top three high-risk HPV types for cervical intraepithelial neoplasia (CIN) and invasive carcinoma. We aimed to evaluate the diversity of HPV16, HPV18, and HPV58 genetic variants by HPV capture technology combined with next generation sequencing. 295, 73, and 148 variations were observed in 51 HPV16, 7 HPV18, and 11 HPV58 genomes, respectively. HPV16 isolates were predominantly of the A variant lineage, and sublineage A4 (Asian) was the most common. However, there were no significant differences in the distribution of HPV16 A1–3 and A4 variants between CIN1-, CIN2/3, and cervical cancer groups. The 7 HPV18 genomes were assigned to the A3/A4 and A1 sublineages. Of the 11 HPV58 genomes, the most predominant variant sublineages were A2, followed by A1 and B2. The majority of HPV16/18 samples containing contiguous genomic deletions were found to harbor HPV integration. Some T-cell epitope sequences in HPV16 E6 and E7 showed considerable divergence from the prototype NC_001526, suggesting their importance in immunotherapy of HPV-associated carcinomas. In conclusion, sequence diversity and phylogenies of HPV16, 18, and 58 provide the basis for future studies of discrete viral evolution, epidemiology, pathogenicity, and the differences in response to vaccines.

Targeted next generation sequencing identifies somatic mutations and gene fusions in papillary thyroid carcinoma

138 papillary thyroid carcinoma (PTC) samples were assessed for somatic mutation profile and fusion genes by targeted resequencing using a cancer panel (ThyGenCapTM) targeting 244 cancer-related genes and 20 potential fusion genes. At least one genetic alteration (including mutations and fusion genes) was observed in 118/138 (85.5%) samples. The most frequently mutated gene was BRAF V600E (57.2%). Moreover, we identified 11 fusion genes including eight previously reported ones and three novel fusion genes, UEVLD-RET, OSBPL9-BRAF, and SQSTM1-NTRK3. Alterations affecting the mitogen-activated protein kinase (MAPK) signaling pathway components were seen in 69.6% of the PTC cases and all of these driver mutations were mutually exclusive. Univariate analysis ascertained that the fusion genes were strongly associated with distinct clinicopathological characteristics, such as young age, local invasion, extensive metastasis, and disease stage. In conclusion, our approach facilitated simultaneous high-throughput detection of gene fusions and somatic mutations in PTC samples.

Genome-wide profiling of the human papillomavirus DNA integration in cervical intraepithelial neoplasia and normal cervical epithelium by HPV capture technology

HPV integration plays an important role in cervical carcinogenesis. HPV genotypes and the exact integration sites were investigated using HPV capture technology combined with next generation sequencing in 166 women. Three, one and six integration sites were verified in 7 HPV-positive ‘normal cervical epithelium’, 6 HPV-positive CIN2 and 15 HPV-positive CIN 3 samples, respectively. Of the 10 integrations, one and nine were involved with HPV33 and HPV16, respectively. Our study accurately evaluated HPV integration level in CINs and normal cervical tissues using high-throughput viral integration detection method providing basic evidence for HPV integration-driven cervical carcinogenesis.

Clonal distribution and intratumour heterogeneity of the B-cell repertoire in oesophageal squamous cell carcinoma: Intratumour heterogeneity of the BCR repertoire in ESCC

Recent successes in tumour immunotherapies have highlighted the importance of tumour immunity. However, most previous studies to date have focused on T‐cell immune response, although B cells are key players in the core immune network and are associated with T‐cell immune response. Based on our previous study delineating T‐cell receptor (TCR) repertoire in seven patients with oesophageal squamous cell carcinoma (ESCC), this study profiled the B‐cell receptor (BCR) repertoire of multiple tumour regions, adjacent normal tissue, and blood from the same seven patients to reveal the characteristics of B‐cell immunity and the relationship to TCR repertoire in ESCC patients. We found that intratumour BCR repertoire was significantly more oligoclonal than matched adjacent normal tissue or peripheral blood and, moreover, clonal amplification of B cells in multiple tumour regions was significantly heterogeneous, although clonal amplification of the TCR repertoire across different tissue compartments and regions of the same tumour was similar. However, both BCR and TCR repertoires in the tumour microenvironment were distinct from those in adjacent normal tissues and blood, and thus represented a group of B and T cells that were spatially confined to the tumour microenvironment and could react to tumour antigens. Additionally, B‐ and T‐cell clones varying between different tumour regions showed intratumour heterogeneity of B‐ and T‐cell immune response. Thus, multiple tumour biopsies could be essential to comprehensively delineate the adaptive immune response to an individual ESCC. These findings expand our understanding of adaptive anti‐tumour immunity and shed more light on ESCC immunotherapy. This study provides insights into the intratumour heterogeneity of the BCR repertoire as well as the difference and relationship between the BCR and TCR repertoire in ESCC, expanding our understanding of adaptive anti‐tumour immunity and ESCC immunotherapy. Copyright

TCR repertoire intratumor heterogeneity of CD4+ and CD8+ T cells in centers and margins of localized lung adenocarcinomas: TCR heterogeneity of CD4+ and CD8+ TILs in lung cancer

Intratumor heterogeneity (ITH) of T cell receptor (TCR) repertoire in different T‐cell subsets and locations in lung adenocarcinomas was unclear. Here, we investigated percentages and TCR repertoire of freshly isolated CD4+ and CD8+ tumor infiltrating lymphocytes (TILs) in tumor centers and margins by flow cytometry on 80 tumor samples from 20 patients and high‐throughput TCR sequencing on 27 and 25 samples of CD4+ and CD8+ TILs from seven patients. Our results demonstrated that amount and TCR repertoire diversity of CD4+ TILs were significantly higher than those of CD8+ TILs and moreover substantial ITH regarding amount and TCR repertoire of CD4+ and CD8+ TILs were observed. Additionally, ITH of CD4/CD8 T‐cell ratio and CD8+ TIL repertoire across center regions was lower than that across margin regions. The amount and TCR repertoire ITH of CD4+ and CD8+ TILs and mean clonality of CD8+ TILs in tumor centers were associated with relapse. Our study provides insights into amount and TCR repertoire ITH of CD4+ and CD8+ TILs in tumor centers and margins as well as corresponding association with prognosis in lung adenocarcinoma patients, suggesting potential clinical significance of TCR repertoire.

Mutation analysis of the EBV-lymphoblastoid cell line cautions their use as antigen-presenting cells

Lymphoblastoid cell lines (LCLs) have been widely used as professional antigen‐presenting cells (APCs). However, neoantigen‐loaded LCLs could induce nonspecific T‐cell response, which could be due to expression of both Epstein–Barr virus (EBV) antigens and nonsynonymous mutations arising in LCLs. Since the number of passages could influence mutational characteristics of LCLs, and moreover extensive proliferation of LCLs in vitro is necessary to activate T cells for immunotherapy, we comprehensively profiled mutational characteristics by comparing eight sets of B cells and matched high‐passage LCLs using whole‐exome sequencing in order to assess the effect of nonsynonymous mutations arising in LCLs on nonspecific T‐cell response. We found 315 nongermline mutations (approximately 40mut/subject) randomly distributed across all chromosomes including 18 mutations in immunoglobulin V and J genes in eight LCLs, of which 137 candidate neoantigens (approximately 17mut/subject) were identified. The underlying mutational processes linked to EBV‐transformed LCLs could be attributed to activation induced cytidine deaminase gene expression which contributes to cytosine mutation clusters in LCLs through cytosine deamination. Pathways significantly enriched by nonsilent mutations of each LCL were totally different among all LCLs. In conclusion, high‐passage LCLs may not be suitable to serve as APCs due to random nonsilent mutations, particularly for presentation of neoantigens of low immunogenicity, although further experimental proofs are needed.

Differential DNA methylation profiles of human B lymphocytes and Epstein-Barr virus-immortalized B lymphocytes

Objective This study aimed to comprehensively assess Epstein-Barr virus (EBV)-induced methylation alterations of B cell across whole genome. Methods We compared DNA methylation patterns of primary B cells and corresponding lymphoblastoid cell lines (LCLs) from eight participants. The genome-wide DNA methylation profiles were compared at over 850,000 genome-wide methylation sites. Results DNA methylation analysis revealed 87,732 differentially methylated CpG sites, representing approximately 12.41% of all sites in LCLs compared to primary B cells. The hypermethylated and hypomethylated CpG sites were about 22.75% or 77.25%, respectively. Only 0.8% of hypomethylated sites and 4.5% of hypermethylated sites were located in CpG islands, whereas 8.0% of hypomethylated sites and 16.3% of hypermethylated sites were located in shore (N_shore and S_shore). Using principal component analysis of the DNA methylation profiles, primary B cells and LCLs could be accurately predicted. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differently methylated genes revealed that most of the top GO biological processes were related to cell activation and immune response, and some top enrichment pathways were related with activation and malignant transformation of human B cells. Conclusions Our study demonstrated genome-wide DNA methylation variations between primary B cells and corresponding LCLs, which might yield new insight on the methylation mechanism of EBV-induced immortalization.