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Supplementary Figure from Combination Neoantigen-Based Dendritic Cell Vaccination and Adoptive T-Cell Transfer Induces Antitumor Responses Against Recurrence of Hepatocellular Carcinoma

A high rate of recurrence after curative therapy is a major challenge for the management of hepatocellular carcinoma (HCC). Currently, no effective adjuvant therapy is available to prevent HCC recurrence. We designed a personalized neoantigen-loaded dendritic cell vaccine and neoantigen-activated T-cell therapy, and used it as adjuvant therapy to treat 10 patients with HCC who had undergone curative resection or radiofrequency ablation in the first stage of a phase II trial (NCT03067493). The primary outcomes were safety and neoantigen-specific immune response. Disease-free survival (DFS) was also evaluated. The immunotherapy was successfully administered to all the patients without unexpected delay and demonstrated a reasonable safety profile with no grade ≥3 treatment-related side effects reported. Seventy percent of patients generated de novo circulating multiclonal neoantigen-specific T-cell responses. Induced neoantigen-specific immunity was maintained over time, and epitope spreading was observed. Patients who generated immune responses to treatment exhibited prolonged DFS compared with nonresponders (P = 0.012), with 71.4% experiencing no relapse for 2 years after curative treatment. High expression of an immune stimulatory signature, enhanced immune-cell infiltration (i.e., CD8+ T cells), and upregulated expression of T-cell inflammatory gene profiles were found in the primary tumors of the responders. In addition, neoantigen depletion (immunoediting) was present in the recurrent tumors compared with the primary tumors (7/9 vs. 1/17, P = 0.014), suggesting that immune evasion occurred under the pressure of immunotherapy. Our study indicates that neoantigen-based combination immunotherapy is feasible, safe, and has the potential to reduce HCC recurrence after curative treatment.

A high rate of recurrence after curative therapy is a major challenge for the management of hepatocellular carcinoma (HCC). Currently, no effective adjuvant therapy is available to prevent HCC recurrence. We designed a personalized neoantigen-loaded dendritic cell vaccine and neoantigen-activated T-cell therapy, and used it as adjuvant therapy to treat 10 patients with HCC who had undergone curative resection or radiofrequency ablation in the first stage of a phase II trial (NCT03067493). The primary outcomes were safety and neoantigen-specific immune response. Disease-free survival (DFS) was also evaluated. The immunotherapy was successfully administered to all the patients without unexpected delay and demonstrated a reasonable safety profile with no grade ≥3 treatment-related side effects reported. Seventy percent of patients generated de novo circulating multiclonal neoantigen-specific T-cell responses. Induced neoantigen-specific immunity was maintained over time, and epitope spreading was observed. Patients who generated immune responses to treatment exhibited prolonged DFS compared with nonresponders (P = 0.012), with 71.4% experiencing no relapse for 2 years after curative treatment. High expression of an immune stimulatory signature, enhanced immune-cell infiltration (i.e., CD8+ T cells), and upregulated expression of T-cell inflammatory gene profiles were found in the primary tumors of the responders. In addition, neoantigen depletion (immunoediting) was present in the recurrent tumors compared with the primary tumors (7/9 vs. 1/17, P = 0.014), suggesting that immune evasion occurred under the pressure of immunotherapy. Our study indicates that neoantigen-based combination immunotherapy is feasible, safe, and has the potential to reduce HCC recurrence after curative treatment.

Table S5 shows detailed information of structure variations in the six HCC patients with WGS analysis.

Fig.S1 shows flow chart of enrollment for multifocal HCC patients with anti-PD-1 therapy. Fig.S2 shows CT images of the six HCC patients with two tumors in the liver. Fig.S3 shows purity of tumor content of the six HCC patients with two tumors in the liver. Fig.S4 shows HBV integration sites in the six HCC patients with two tumors in the liver. Fig.S5 shows expression level of TERT in the six HCC patients with two tumors in the liver. Fig.S6 shows landscape of inter-chromosomal translocations and copy number alterations in small and large tumors of four cases. Fig.S7 shows number and types of chromosomal rearrangement in the six HCC patients with two tumors in the liver. Fig.S8 shows copy number aberrations in the six HCC patients with two tumors in the liver. Fig.S9 shows mutational landscape of the six HCC patients with two tumors in the liver. Fig.S10 shows non-silent mutations in the six HCC patients with two tumors in the liver. Fig.S11 shows profile of mutational cancer cell fractions of small and large tumors in the six HCC patients. Fig.S12 shows unsupervised clustering of RNA expression in tumors and adjacent normal liver tissues of 12 multifocal HCC patients. Fig.S13 shows expression level of immune functional genes in small and large tumors of 12 multifocal HCC patients. Fig.S14 shows expression level of immune stimulators in small and large tumors of 12 multifocal HCC patients. Fig.S15 shows expression level of immune inhibitors in small and large tumors of 12 multifocal HCC patients. Fig.S16 shows mutations of immune-related genes in the six HCC patients with two tumor nodules in the liver. Fig.S17 shows images of the small and large tumors before and after anti-PD-1 therapy.

Purpose:Targeted therapy and immunotherapy are transforming the treatment approach for intrahepatic cholangiocarcinoma (ICC). However, little is known about the intertumor heterogeneity (ITH) of multifocal ICC and its impacts on patient response to these treatments. We aimed to characterize the immunogenomic and epigenomic heterogeneity of multifocal ICC to guide treatment decision making.Experimental Design:We obtained 66 tumor samples from 16 patients with multifocal ICC and characterized the tumor and immune heterogeneity using whole-exome sequencing, bulk and single-cell RNA sequencing, methylation microarray, and multiplex immunostaining. Patients were divided into high- or low-ITH groups according to the median ITH index. Two independent cohorts were used to validate findings. Responses to anti-PD-1 therapy were assessed.Results:Multifocal ICC presented considerable intertumor genomic, transcriptional, and epigenomic heterogeneity within a patient in high ITH group. The immune profile among multiple tumors within a patient was relatively less heterogeneous in high- or low-ITH group, and consistent responses of multiple tumors to anti-PD-1 immunotherapy were observed. Unsupervised clustering of immune markers identified one low and one high immune subtype, with higher immune cell infiltration, closer tumor–immune cell interactions, and upregulated IFN-signature expression in high-immune subtype. Determining expression levels of CD8B and ICOS facilitated this immune classification and prediction of patient prognosis. Finally, promoter DNA methylation contributed to different immune profiles of two subtypes by regulating immune-gene expression.Conclusions:There is comprehensive heterogeneity in the genome, transcriptome, and epigenome of multifocal ICC. On the basis of the less heterogeneous immune profile of ICC, we suggest an immune classification that stratifies patients' prognosis and may support personalized immunotherapy.

Supplementary Data from Multiomic Analysis Reveals Comprehensive Tumor Heterogeneity and Distinct Immune Subtypes in Multifocal Intrahepatic Cholangiocarcinoma

Table S9 shows potentially functional mutations of immune-related genes in the six HCC patients with WGS analysis.

Table S3 shows summary of whole genome sequencing in the six patients with two tumor nodules in the liver.

Table S1 shows clinicopathologic information of 12 multifocal HCC patients. Table S2 shows PCR primers used for validation of HBV integration sites. Table S4 shows HBV integration analysis in HCC patients with WGS data. Table S8 shows P-value of pathways enrichment analysis in the small and large tumors of 12 multifocal HCC patients. Table S10 shows correlation between the number of rearrangements and immune activity. Table S11 shows detailed clinical data of eight patients with anti-PD-1 therapy.

Table S6 shows detailed information of copy number alterations in the six HCC patients with WGS analysis.

Purpose:Immune checkpoint inhibitor therapy is emerging as the promising option for patients with advanced hepatocellular carcinoma. We aimed to investigate the heterogeneity of different tumor nodules of the same patient with multifocal hepatocellular carcinomas in response to immunotherapy and its molecular mechanisms.Experimental Design:We attained 45 surgical tumor samples including 33 small and 12 large nodules from 12 patients with multifocal hepatocellular carcinoma and evaluated genomic and immune heterogeneity among tumors through whole-genome sequencing and RNA sequencing. IHC was performed to validate the expression of immune markers. The responses to anti–programmed cell death protein-1 (PD-1) therapy in patients with multifocal hepatocellular carcinoma were evaluated.Results:The small and large tumors within the same patient presented with similar genomic characteristics, indicating their same genomic origin. We further found the small tumors had higher immune cell infiltration including more CD8+ T cells, M1 macrophages, and monocytes as compared with large tumors. Besides, the expression of interferon signature predictive of response to anti–PD-1 therapy was significantly upregulated in the small tumors. Moreover, the immune pathways were more vigorous along with less active proliferation pathways in the small tumors. In keeping with this, we found that small nodules were more sensitive to anti–PD-1 therapy than large nodules in patients with multifocal hepatocellular carcinoma.Conclusions:The small tumors in patients with multifocal hepatocellular carcinoma had higher immune cell infiltration and upregulation of immune pathways as compared with the large tumors, which can partially explain the different responses of small and large tumors in the same case to anti–PD-1 therapy.

Table S7 shows detailed information of candidate somatic mutations in the six HCC patients with WGS analysis.