Hepatocellular carcinoma(HCC) is the fifth most common cancer and the second leading cause of cancer-related deaths worldwide. Although the prognosis of patients with HCC is generally poor, the5-year ...survival rate is > 70% if patients are diagnosed at an early stage. However, early diagnosis of HCC is complicated by the coexistence of inflammation and cirrhosis. Thus, novel biomarkers for the early diagnosis of HCC are required. Currently, the diagnosis of HCC without pathological correlation is achieved by analyzing serum α.fetoprotein levels combined with imaging techniques. Advances in genomics and proteomics platforms and biomarker assay techniques over the last decade have resulted in the identification of numerous novel biomarkers and have improved the diagnosis of HCC. The most promising biomarkers,such as glypican-3, osteopontin, Golgi protein-73 and nucleic acids including microRNAs, are most likely to become clinically validated in the near future. These biomarkers are not only useful for early diagnosis of HCC, but also provide insight into the mechanisms driving oncogenesis. In addition, such molecular insight creates the basis for the development of potentially more effective treatment strategies. In this article,we provide an overview of the biomarkers that are currently used for the early diagnosis of HCC.
Clinical successes demonstrated by chimeric antigen receptor T-cell immunotherapy have facilitated further development of T-cell immunotherapy against wide variety of diseases. One approach is the ...development of "off-the-shelf" T-cell sources. Technologies to generate T-cells from pluripotent stem cells (PSCs) may offer platforms to produce "off-the-shelf" and synthetic allogeneic T-cells. However, low differentiation efficiency and poor scalability of current methods may compromise their utilities. Here we show improved differentiation efficiency of T-cells from induced PSCs (iPSCs) derived from an antigen-specific cytotoxic T-cell clone, or from T-cell receptor (TCR)-transduced iPSCs, as starting materials. We additionally describe feeder-free differentiation culture systems that span from iPSC maintenance to T-cell proliferation phases, enabling large-scale regenerated T-cell production. Moreover, simultaneous addition of SDF1α and a p38 inhibitor during T-cell differentiation enhances T-cell commitment. The regenerated T-cells show TCR-dependent functions in vitro and are capable of in vivo anti-tumor activity. This system provides a platform to generate a large number of regenerated T-cells for clinical application and investigate human T-cell differentiation and biology.
The use of allogeneic, pluripotent stem‐cell‐derived immune cells for cancer immunotherapy has been the subject of recent clinical trials. In Japan, investigator‐initiated clinical trials will soon ...begin for ovarian cancer treatment using human leukocyte antigen (HLA)‐homozygous‐induced pluripotent stem cell (iPSC)‐derived anti–glypican‐3 (GPC3) chimeric antigen receptor (CAR)‐expressing natural killer/innate lymphoid cells (NK/ILC). Using pluripotent stem cells as the source for allogeneic immune cells facilitates stringent quality control of the final product, in terms of efficacy, safety and producibility. In this paper, we describe our methods for the stable, feeder‐free production of CAR‐expressing NK/ILC cells from CAR‐transduced iPSC with clinically relevant scale and materials. The average number of cells that could be differentiated from 1.8‐3.6 × 106 iPSC within 7 weeks was 1.8‐4.0 × 109. These cells showed stable CD45/CD7/CAR expression, effector functions of cytotoxicity and interferon gamma (IFN‐γ) production against GPC3‐expressing tumor cells. When the CAR‐NK/ILC cells were injected into a GPC3‐positive, ovarian‐tumor‐bearing, immunodeficient mouse model, we observed a significant therapeutic effect that prolonged the survival of the animals. When the cells were injected into immunodeficient mice during non–clinical safety tests, no acute systemic toxicity or tumorigenicity of the final product or residual iPSC was observed. In addition, our test results for the CAR‐NK/ILC cells generated with clinical manufacturing standards are encouraging, and these methods should accelerate the development of allogeneic pluripotent stem cell‐based immune cell cancer therapies.
This translational study aimed to develop anti–GPC3 CAR‐expressing NK/ILC cells derived from HLA‐homozygous iPSC clone as an effective cell therapy against disseminated ovarian tumors and to assess the clinical cell manufacturing and pre–clinical aspects of the therapy, including safety and efficacy. Those aspects of the therapy clarified in the study provide perspective for the planned clinical trial.
Glypican-3 (GPC3), a 65 kD protein consisting of 580 amino acids, is a heparan sulfate proteoglycan bound to the cell membrane by glycosylphosphatidylinositol. This protein is expressed in the liver ...and the kidney of healthy fetuses but is hardly expressed in adults, except in the placenta. Contrarily, GPC3 is specifically expressed in hepatocellular carcinoma (HCC), ovarian clear cell carcinoma, melanoma, squamous cell carcinoma of the lung, hepatoblastoma, nephroblastoma (Wilms tumor), yolk sac tumor, and some pediatric cancers. Although the precise function of GPC3 remains unclear, it has been strongly suggested that it is related to the malignant transformation of HCC. We identified GPC3 as a promising target for cancer immunotherapy and have been working on the development of cancer immunotherapeutic agents targeting it through clinical trials. In some trials, it was revealed that the GPC3 peptide vaccines we developed using human leukocyte antigen-A24- and A2-restricted GPC3-derived peptides could induce GPC3-specific cytotoxic T cells in most vaccinated patients and thereby improve their prognosis. To further improve the clinical efficacy of cancer immunotherapy targeting GPC3, we are also developing next-generation therapeutic strategies using T cells engineered to express antigen-specific T-cell receptor or chimeric antigen receptor. In addition, we have successfully monitored the levels of serum full-length GPC3 protein, which is somehow secreted in the blood. The utility of GPC3 as a biomarker for predicting tumor recurrence and treatment efficacy is now being considered. In this review article, we summarize the results of clinical trials carried out by our team and describe the novel agent targeting the cancer-specific shared antigen, GPC3.
It is becoming recognized that screening of oncology drugs on a platform using two-dimensionally (2D)-cultured cell lines is unable to precisely select clinically active drugs; therefore ...three-dimensional (3D)-culture systems are emerging and show potential for better simulating the in vivo tumor microenvironment. The purpose of this study was to reveal the differential effects of chemotherapeutic drugs between 2D- and 3D-cultures and to explore their underlying mechanisms. We evaluated differences between 2D- and 3D-cultured breast cancer cell lines by assessing drug sensitivity, oxygen status and expression of Ki-67 and caspases. Three cell lines (BT-549, BT-474 and T-47D) developed dense multicellular spheroids (MCSs) in 3D-culture, and showed greater resistance to paclitaxel and doxorubicin compared to the 2D-cultured cells. An additional three cell lines (MCF-7, HCC-1954, and MDA-MB-231) developed only loose MCSs in 3D, and showed drug sensitivities similar to those found in the 2D-culture. Treatment with paclitaxel resulted in greater increases in cleaved-PARP expression in the 2D-culture compared with the 3D-culture, but only in cell lines forming dense 3D-MCSs, suggesting that MCS formation protected the cells from paclitaxel-induced apoptosis. Hypoxia was observed only in the dense 3D-MCSs. BT-549 had fewer cells positive for Ki-67 in 3D- than in 2D-culture, suggesting that the greater G0-dormant subpopulation was responsible for its drug resistance in the 3D-culture. BT-474 had a lower level of caspase-3 in the 3D- than in the 2D-culture, suggesting that the 3D-environment was anti-apoptotic. Finally, we compared staining for Ki-67 and caspases in the 2D- and 3D-primary-cultured cells originating from a patient-derived xenograft (PDX), fresh PDX tumor, and the patient's original tumor; 2D-cultured cells showed greater proportions of Ki-67-positive and caspase-3-positive cells, in agreement with the view that 3D-primary culture better represents characteristics of tumors in vivo. In conclusion, 3D-cultured cells forming dense MCSs may be better than 2D-cultured cells in simulating important tumor characteristics in vivo, namely hypoxia, dormancy, anti-apoptotic features and their resulting drug resistance.
This study performed a comprehensive molecular characterization of microsatellite instability-high (MSI-H)/mismatch repair-deficient (dMMR) gastrointestinal (GI) tumors to elucidate predictors of ...response to PD-1 blockade.
Forty-five patients with MSI-H/dMMR GI tumors, including gastric cancer, colorectal cancer, cholangiocarcinoma, small intestine cancer, pancreatic cancer, and duodenal cancer, receiving PD-1 blockade were analyzed. We conducted the genomic profiling of GI tumors by whole-exome sequencing or targeted next-generation sequencing. The tumor microenvironment was evaluated by transcriptomic analysis and multiplex fluorescence IHC.
Patients with low tumor mutational burdens (TMBs) had lower objective response rates (ORRs; 0% vs. 48.8%) and a significantly shorter progression-free survival (PFS; 2.3 vs. 15.6 months; HR, 6.20;
= 0.002) than those with high TMBs. Among common gene alterations in GI tumors, only
mutations, which were mutually exclusive with a low TMB, were significantly associated with a lower ORRs than wild-type
(21.4 vs. 54.8%; odds, 4.45;
= 0.045). Compared with wild-type
mutations in the phosphatase domain were associated with significantly lower ORRs (12.5 vs. 54.8%;
= 0.049), shorter PFS (2.6 vs. 15.6 months; HR, 5.04;
< 0.001), lower intratumoral CD8
T-cell levels, higher intratumoral CD204
macrophage levels, and PI3K/AKT/mTOR pathway enrichment, whereas
mutations in the C2 domain were not.
Low TMBs and
mutations, especially mutations in the phosphatase domain associated with an immunosuppressive environment, were mutually exclusive and might be negative predictors of PD-1 blockade responses in patients with MSI-H/dMMR GI tumors.
Early detection of pancreatic ductal adenocarcinoma (PDAC) is essential for improving patient survival rates, and noninvasive biomarkers are urgently required to identify patients who are eligible ...for curative surgery. Here, we examined extracellular vesicles (EVs) from the serum of PDAC patients to determine their ability to detect early‐stage disease. EV‐associated proteins purified by ultracentrifugation and affinity columns underwent proteomic analysis to identify novel PDAC markers G protein‐coupled receptor class C group 5 member C (GPRC5C) and epidermal growth factor receptor pathway substrate 8 (EPS8). To verify the potency of GPRC5C‐ or EPS8‐positive EVs as PDAC biomarkers, we analyzed EVs from PDAC patient blood samples using ultracentrifugation in two different cohorts (a total of 54 PDAC patients, 32 healthy donors, and 22 pancreatitis patients) by immunoblotting. The combination of EV‐associated GPRC5C and EPS8 had high accuracy, with area under the curve values of 0.922 and 0.946 for distinguishing early‐stage PDAC patients from healthy controls in the two cohorts, respectively, and could detect PDAC patients who were negative for CA19‐9. Moreover, we analyzed 30 samples taken at three time points from 10 PDAC patients who underwent surgery: before surgery, after surgery, and recurrence as an early‐stage model. These proteins were detected in EVs derived from preoperative and recurrence samples. These results indicated that GPRC5C‐ or EPS8‐positive EVs were biomarkers that have the potential to detect stage I early pancreatic cancer and small recurrent tumors detected by computed tomography.
Early detection of pancreatic ductal adenocarcinoma (PDAC) is essential for improving patient survival rates. This study demonstrated that PDAC patients who were negative for CA19‐9 could be detected by extracellular vesicles (EV)‐associated GPRC5C or EPS8, and these EV‐associated proteins showed high accuracy for distinguishing patients with stage I early pancreatic cancer from healthy controls. EV‐associated GPRC5C and EPS8 will contribute to the early detection and help in the design of potential curative surgical options.
There is no established postoperative adjuvant therapy for hepatocellular carcinoma (HCC), and improvement of patient prognosis has been limited. We conducted long‐term monitoring of patients within ...a phase II trial that targeted a cancer antigen, glypican‐3 (GPC3), specifically expressed in HCC. We sought to determine if the GPC3 peptide vaccine was an effective adjuvant therapy by monitoring disease‐free survival and overall survival. We also tracked GPC3 immunohistochemical (IHC) staining, CTL induction, and postoperative plasma GPC3 for a patient group that was administered the vaccine (n = 35) and an unvaccinated patient group that underwent surgery only (n = 33). The 1‐y recurrence rate after surgery was reduced by approximately 15%, and the 5‐y and 8‐y survival rates were improved by approximately 10% and 30%, respectively, in the vaccinated group compared with the unvaccinated group. Patients who were positive for GPC3 IHC staining were more likely to have induced CTLs, and 60% survived beyond 5 y. Vaccine efficacy had a positive relationship with plasma concentration of GPC3; high concentrations increased the 5‐y survival rate to 75%. We thus expect GPC3 vaccination in patients with HCC, who are positive for GPC3 IHC staining and/or plasma GPC3 to induce CTL and have significantly improved long‐term prognosis.
This phase II study demonstrated that postoperative adjuvant vaccination may reduce the 1‐y recurrence rate and prolong overall survival in patients who are positive for GPC3 IHC staining. The GPC3 peptide vaccine used induces specific CTLs and can be expected to improve patient prognosis.
Cancer immunotherapy has a long developmental history, beginning with William Coley's first bacterial mixture ('Coley's toxin') in 1891, which led to the development of nonspecific immunotherapy. ...After the research team of Thierry Boon succeeded in isolating the first melanoma antigen gene (MAGE-1) and identifying its major histocompatibility complex-restricted peptide in 1991, many kinds of cancer antigens were successively identified and so-called cancer vaccines were clinically tested. Although cancer vaccine therapy is expected to be the new cancer immunotherapy, it is currently unable to yield sufficient therapeutic effects when used alone and has thus not yet been approved as a drug. Meanwhile, various types of cell therapies, including tumor-infiltrating lymphocyte therapy, T-cell receptor-engineered T-cell therapy, and chimeric antigen receptor T-cell therapy, have shown remarkable clinical efficacy. Additionally, the discovery of immune checkpoint molecules has led to the success of immune checkpoint inhibitors, and cancer immunotherapy has now become a major pillar of cancer treatment. Currently, there are high expectations for the development of personalized neoantigen vaccines and T-cell therapies. The era of personalized cancer immunotherapy combined with immune checkpoint inhibitors is expected to arrive circa 2030.
Long-read sequencing of full-length cDNAs enables the detection of structures of aberrant splicing isoforms in cancer cells. These isoforms are occasionally translated, presented by HLA molecules, ...and recognized as neoantigens. This study used a long-read sequencer (MinION) to construct a comprehensive catalog of aberrant splicing isoforms in non-small-cell lung cancers, by which novel isoforms and potential neoantigens are identified.
Full-length cDNA sequencing is performed using 22 cell lines, and a total of 2021 novel splicing isoforms are identified. The protein expression of some of these isoforms is then validated by proteome analysis. Ablations of a nonsense-mediated mRNA decay (NMD) factor, UPF1, and a splicing factor, SF3B1, are found to increase the proportion of aberrant transcripts. NetMHC evaluation of the binding affinities to each type of HLA molecule reveals that some of the isoforms potentially generate neoantigen candidates. We also identify aberrant splicing isoforms in seven non-small-cell lung cancer specimens. An enzyme-linked immune absorbent spot assay indicates that approximately half the peptide candidates have the potential to activate T cell responses through their interaction with HLA molecules. Finally, we estimate the number of isoforms in The Cancer Genome Atlas (TCGA) datasets by referring to the constructed catalog and found that disruption of NMD factors is significantly correlated with the number of splicing isoforms found in the TCGA-Lung Adenocarcinoma data collection.
Our results indicate that long-read sequencing of full-length cDNAs is essential for the precise identification of aberrant transcript structures in cancer cells.
PDF
