Bristol-Myers Squibb.

SUMMARY Liver cancer has the second highest worldwide cancer mortality rate and has limited therapeutic options. We analyzed 363 hepatocellular carcinoma (HCC) cases by whole exome sequencing and DNA copy number analyses, and 196 HCC also by DNA methylation, RNA, miRNA, and proteomic expression. DNA sequencing and mutation analysis identified significantly mutated genes including LZTR1, EEF1A1, SF3B1, and SMARCA4. Significant alterations by mutation or down-regulation by hypermethylation in genes likely to result in HCC metabolic reprogramming (ALB, APOB, and CPS1) were observed. Integrative molecular HCC subtyping incorporating unsupervised clustering of five data platforms identified three subtypes, one of which was associated with poorer prognosis in three HCC cohorts. Integrated analyses enabled development of a p53 target gene expression signature correlating with poor survival. Potential therapeutic targets for which inhibitors exist include WNT signaling, MDM4, MET, VEGFA, MCL1, IDH1, TERT, and immune checkpoint proteins CTLA-4, PD-1, and PD-L1.

IntroductionAdaptive immunity plays a crucial role in tumor immunosurveillance. [1][2][3] It has been shown that tumor-infiltrating effector T cells are associated with improved prognoses in multiple human cancers, 4-6 whereas tumor-infiltrating regulatory T (Treg) cells are negatively associated with patient outcome. 6,7 Th17 cells are newly identified effector CD4 ϩ T cells. Th17 cells and interleukin-17 (IL-17) play an active role in inflammation and autoimmune diseases. [8][9][10][11][12][13][14][15] Th17 cells are found in both mouse and human tumors. 16,17 However, the biologic role of Th17 cells is poorly understood in the tumor microenvironment. In this report, we examined the phenotype, cytokine profile, generation, functional relevance, and immunologic and clinical predictive values of Th17 cells in 201 patients with ovarian cancers. We provide novel insight into the nature of Th17 cells in the tumor microenvironment in patients with cancer. This information may be useful for designing more effective cancer immunotherapies. Methods Human subjectsWe studied previously untreated patients with 201 ovarian carcinomas. Survival data were available for 85 patients (supplemental Table 1, available on the Blood website; see the Supplemental Materials link at the top of the online article). Patients gave written, informed consent in accordance with the Declaration of Helsinki. The study was approved by the University of Michigan Institutional Review Board. Cells and tissuesCells and tissues were obtained from ascites, blood, lymph nodes, and tumors as we described. 16,18,19 Immune cells, including monocytes, macrophages, myeloid dendritic cells, plasmacytoid dendritic cells, and T-cell subsets, were enriched using paramagnetic beads (StemCell Technologies) and sorted with FACSAria (Becton Dickinson) as we described. 16,18,19 Cell purity was more than 98% as confirmed by flow cytometry (LSR II; Becton Dickinson). FACSFor cytokine detection, the cells were stimulated with phorbol myristate acetate (50 ng/mL; Sigma-Aldrich), ionomycin (1 M; Sigma-Aldrich) for 4 hours before staining. Cells were first stained extracellularly with specific antibodies against human CD3, CD4, CD8, CD11b, CD11c, CD14, CD15, CD16, CD19, CD25, CD39, CD45, CD45RO, CD49a, CD49c, CD49d, CD49e, CD56, CD123, CD161, PD-1, CCR4, CCR6, CCR7, CXCR4, HLA-DR, and annexin V (BD Biosciences), CCR2, CXCR3, and CCR5 (R&D Systems), EpCam (StemCell Technologies), then were fixed and permeabilized with Perm/Fix solution (eBioscience), and finally were stained intracellularly with anti-IL-2, anti-IL-10, anti-IL-17, anti-tumor necrosis factor-␣, anti-interferon-␥ (IFN-␥), anti-Granzyme A, anti-Ki-67, and anti-FOXP3 (all from BD Biosciences, except anti-IL-17, eBioscience). Samples were acquired on a LSR II (BD Biosciences), and data were analyzed with DIVA software (BD Biosciences). Th17 induction and suppressionFresh peripheral blood and tumor-associated CD14 ϩ macrophages were sorted 19 and cocultured with T cells as indicated for 3 to 5 days in the An In...

Human tumors progress despite the presence of tumor associated antigen (TAA)-specific T cells. Many different molecular and cellular mechanisms contribute to the failure of T cells to eradicate the tumor. These include immune suppressive networks that impair ongoing T cell function and enable tumor escape. Recent studies have started to reveal the nature of effector T cells in the tumor microenvironment. In this article we discuss T cell anergy, exhaustion, senescence and stemness, and review the phenotype of dysfunctional T cell subsets and the underlying molecular mechanisms in the tumor microenvironments. We suggest that targeting T cell dysfunctional mechanisms and introducing/promoting T cell stemness are important approaches to treat patients with cancer.

Background & Aims Cancer stem cells (CSCs) can contribute to hepatocellular carcinoma (HCC) progression and recurrence following therapy. The presence of tumor-associated macrophages (TAMs) in patients with HCC is associated with poor outcomes. It is not clear whether TAMs interact with CSCs during HCC development. We investigated whether TAMs affect the activities of CSCs in the microenvironment of human HCCs. Methods HCCs were collected from 17 patients during surgical resection and single cell suspensions were analyzed by flow cytometry. CD14+ TAMs were isolated from the HCC cell suspensions and placed into co-culture with HepG2 or Hep3B cells, and CSC functions were measured. The interleukin 6 (IL6) receptor was blocked with a monoclonal antibody (tocilizumab), and STAT3 was knocked down with small hairpin RNAs in HepG2 cells. Xenograft tumors were grown in NOD-SCID/Il2Rgnull mice from human primary HCC cells or HepG2 cells. Results CD44+ cells from human HCCs and cell lines formed more spheres in culture and more xenograft tumors in mice than CD44− cells, indicating that CD44+ cells are CSCs. Incubation of the CD44+ cells with TAMs promoted expansion of CD44+ cells, and increased their sphere formation in culture and formation xenograft tumors in mice. In human HCC samples, numbers of TAMs correlated with numbers of CD44+ cells. Of all cytokines expressed by TAMs, IL6 was increased at the highest level in human HCC co-cultures, compared with TAMs not undergoing co-culture. IL6 was detected in the microenvironment of HCC samples and induced expansion of CD44+ cells in culture. Levels of IL6 correlated with stages of human HCCs and detection of CSC markers. Incubation of HCC cell lines with tocilizumab or knockdown of STAT3 in HCC cells reduced the ability of TAMs to promote sphere formation by CD44+ cells in culture and growth of xenograft tumors in mice. Conclusions CD44+ cells isolated from human HCC tissues and cell lines have CSC activities in vitro and form a larger number of xenograft tumors in mice than CD44− cells. TAMs produce IL6, which promotes expansion of these CSCs and tumorigenesis. Levels of IL6 in human HCC samples correlate with tumor stage and markers of CSCs. Blockade of IL6 signaling with tocilizumab, a drug approved by the Food and Drug Administration for treatment of rheumatoid arthritis, inhibits TAM-stimulated activity of CD44+ cells. This drug might be used to treat patients with HCC.