Thank you very much for your participation in the feasibility study of neoantigen DC therapy. We are currently conducting the analysis of neoantigens as described in the information sheet to which you gave your consent. As the study has progressed, it has become necessary to perform protein-level analysis of the neoantigens. This analysis will be outsourced to the Division of Rare Cancer Research at the National Cancer Center, and part of the samples and data related to neoantigens will also be used by that division. We kindly ask for your understanding regarding this provision. The title, significance, objectives, and methods of this study are outlined below.
If you do not wish your samples or information to be used or provided for this study, or if you initially agreed but later change your mind, please contact us by email at the address provided at the end of this document. From the time we receive your request, we will promptly discontinue the use and provision of your samples and data. Please note, however, that if it is no longer feasible to remove your information from results that have already been published or from existing datasets, we will inform you of that fact, and we ask for your understanding in such cases.
Title of the study:
Proteogenomic Identification of Neoantigens in Circulating Tumor Cells
Purpose and significance of the study:
The purpose of this study is to identify neoantigens in circulating tumor cells using proteogenomic analysis. Since the clinical introduction of immune checkpoint inhibitors, immunotherapy for cancer has drawn considerable attention. However, it is known that these agents are ineffective in many cases of pancreatic and colorectal cancer, and even in cancer types where they are considered effective, reported response rates are only about 5–30%. Various developmental therapies are being explored to further advance cancer immunotherapy, and among these, neoantigen-based immunotherapy has attracted particular interest. Neoantigens are antigens that are expressed only in tumor tissue and are believed to act synergistically with immune checkpoint inhibitors.
To identify neoantigens, we analyze the entire set of genes in both tumor tissue and normal tissue, identify gene mutations expressed only in the tumor tissue, and, from among them, select those relevant to antitumor immunity. This type of analysis conventionally requires tumor tissue. In the present study, however, we used circulating tumor cells (CTCs) obtained by apheresis as a minimally invasive alternative method of sampling tumor material. The number of CTCs collected was sufficient for neoantigen identification.
In this study, our goal is to identify neoantigens from CTCs. The outline of the study is as follows: DNA and RNA are extracted from CTCs, and whole-exome sequencing and RNA sequencing are performed. In parallel, proteins are extracted and subjected to mass spectrometry. Using the genomic data obtained, we first predict candidate neoantigens. At the same time, we use proteogenomics software developed by the Division of Rare Cancer Research at the National Cancer Center to construct a patient-specific amino acid sequence database based on each patient’s genomic data. Using this database, we then identify neoantigens that are actually expressed and specific to the CTCs. By establishing a method to identify CTC-specific neoantigens, we expect to obtain insights that may contribute to the development of immunotherapies aimed at preventing cancer metastasis, and we consider this work to be of significant importance.
Methods:
In this study, in addition to the whole-exome sequencing and candidate neoantigen identification already performed by Novacellum Inc., we will newly conduct mass spectrometric analysis. Whole-exome sequencing is carried out on CTCs collected during treatment as well as on normal peripheral blood cells, in order to detect genetic alterations present only in the CTCs. From these mutations, candidate neoantigens are selected in silico using dedicated computer software. Separately from these analyses, mass spectrometry is performed on frozen, stored CTC samples.
Subsequently, to identify neoantigens that are specifically expressed in CTCs, we conduct proteogenomic analysis. Using the CTC whole-exome sequencing data, we create a CTC-specific amino acid sequence database with sample-specific proteome database–construction software developed by the Division of Rare Cancer Research at the National Cancer Center Research Institute. Predicted neoantigen amino acid sequences from existing data are then added to this database. Using DIA-NN, a software platform for the analysis of mass spectrometry data, we perform database searches of existing mass spectrometry datasets against the constructed amino acid sequence database and thereby identify neoantigens that are actually expressed at the protein level.
Name of participating research institution:
Division of Rare Cancer Research, National Cancer Center
Contact for those who do not wish to have their samples/information provided to the Division of Rare Cancer Research at the National Cancer Center, or for other inquiries and complaints:
Novacellum Inc.
Naohide Yamashita
email：n-yamashita@novacellum.com
