Immunomic, genomic and transcriptomic characterization of CT26 colorectal carcinoma

Background Tumor models are critical for our understanding of cancer and the development of cancer therapeutics. Here, we present an integrated map of the genome, transcriptome and immunome of an epithelial mouse tumor, the CT26 colon carcinoma cell line. Results We found that Kras is homozygously mutated at p.G12D, Apc and Tp53 are not mutated, and Cdkn2a is homozygously deleted. Proliferation and stem-cell markers, including Top2a, Birc5 (Survivin), Cldn6 and Mki67, are highly expressed while differentiation and top-crypt markers Muc2, Ms4a8a (MS4A8B) and Epcam are not. Myc, Trp53 (tp53), Mdm2, Hif1a, and Nras are highly expressed while Egfr and Flt1 are not. MHC class I but not MHC class…

Abstract A110: Mutant MHC class II epitopes drive therapeutic immune responses to cancer

Abstract Mutations are regarded as ideal targets for cancer immunotherapy. As neoepitopes with strict lack of expression in any healthy tissue, they are expected to be safe and could bypass the central tolerance mechanisms. Recent advances in nucleic acid sequencing technologies have revolutionized the field of genomics, allowing the readily targeting of mutated neoantigens for personalized cancer vaccination. We demonstrated in three independent murine tumor models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that unexpectedly the immunogenic mutanome is pre-dominantly recognized by CD4+ T cells. RNA vaccination with such MHC class II restricted immuno…

Bioinformatic methods for cancer neoantigen prediction

Tumor cells accumulate aberrations not present in normal cells, leading to presentation of neoantigens on MHC molecules on their surface. These non-self neoantigens distinguish tumor cells from normal cells to the immune system and are thus targets for cancer immunotherapy. The rapid development of molecular profiling platforms, such as next-generation sequencing, has enabled the generation of large datasets characterizing tumor cells. The simultaneous development of algorithms has enabled rapid and accurate processing of these data. Bioinformatic software tools encoding the algorithms can be strung together in a workflow to identify neoantigens. Here, with a focus on high-throughput sequen…

Enhanced protection of C57 BL/6 vs Balb/c mice to melanoma liver metastasis is mediated by NK cells.

ABSTRACT The B16F10 murine melanoma cell line displays a low expression of MHC class I molecules favoring immune evasion and metastases in immunocompetent C57 BL/6 wild-type mice. Here, we generated metastases to the liver, an organ that is skewed towards immune tolerance, by intrasplenic injection of B16F10 cells in syngeneic C57 BL/6 compared to allogeneic Balb/c mice. Surprisingly, Balb/c mice, which usually display a pronounced M2 macrophage and Th2 T cell polarization, were ∼3 times more susceptible to metastasis than C57 BL/6 mice, despite a much higher M1 and Th1 T cell immune response. The anti-metastatic advantage of C57 BL/6 mice could be attributed to a more potent NK-cell mediat…

A catalog of HLA type, HLA expression, and neo-epitope candidates in human cancer cell lines

Cancer cell lines are a tremendous resource for cancer biology and therapy development. These multipurpose tools are commonly used to examine the genetic origin of cancers, to identify potential novel tumor targets, such as tumor antigens for vaccine devel-opment, and utilized to screen potential therapies in preclinical studies. Mutations, gene expression, and drug sensitivity have been determined for many cell lines using next-generation sequencing (NGS). However, the human leukocyte antigen (HLA) type and HLA expression of tumor cell lines, characterizations necessary for the development of cancer vaccines, have remained largely incomplete and, such information, when available, has been …

Genomics Meets Cancer Immunotherapy

High-throughput cancer genomics and bioinformatics are revolutionizing our ability to profile tumor samples. With next-generation sequencing (NGS) and high-performance computing (HPC) platforms, we have developed the infrastructures to determine and characterize tumor genomes and transcriptomes within days. Now, we are integrating these platforms into both cancer immunology and patient therapy decision-making. Here, we briefly describe the technology platforms and highlight several emerging applications: profiling of tumor mutations and gene expression; determination of HLA type and tumor expression, enabling prediction of immunogenic tumor mutations; and identification of viruses present i…

MHC class I loaded ligands from breast cancer cell lines: A potential HLA-I-typed antigen collection.

Abstract To build a catalog of peptides presented by breast cancer cells, we undertook systematic MHC class I immunoprecipitation followed by elution of MHC class I-loaded peptides in breast cancer cells. We determined the sequence of 3196 MHC class I ligands representing 1921 proteins from a panel of 20 breast cancer cell lines. After removing duplicate peptides, i.e., the same peptide eluted from more than one cell line, the total number of unique peptides was 2740. Of the unique peptides eluted, more than 1750 had been previously identified, and of these, sixteen have been shown to be immunogenic. Importantly, half of these immunogenic peptides were shared between different breast cancer…

Mutated tumor alleles are expressed according to their DNA frequency

AbstractThe transcription of tumor mutations from DNA into RNA has implications for biology, epigenetics and clinical practice. It is not clear if mutations are in general transcribed and, if so, at what proportion to the wild-type allele. Here, we examined the correlation between DNA mutation allele frequency and RNA mutation allele frequency. We sequenced the exome and transcriptome of tumor cell lines with large copy number variations, identified heterozygous single nucleotide mutations and absolute DNA copy number and determined the corresponding DNA and RNA mutation allele fraction. We found that 99% of the DNA mutations in expressed genes are expressed as RNA. Moreover, we found a hig…

P0311 : Balb/c and C57/Bl6 mice exhibit differences in their susceptibility and anti-tumor response to B16F10 melanoma liver metastasis

In Silico HLA Typing Using Standard RNA-Seq Sequence Reads

Next-generation sequencing (NGS) enables high-throughput transcriptome profi ling using the RNA-Seq assay, resulting in billions of short sequence reads. Worldwide adoption has been rapid: many laboratories worldwide generate transcriptome sequence reads daily. Here, we describe methods for obtaining a sample’s human leukocyte antigen (HLA) class I and II types and HLA expression using standard NGS RNA- Seq sequence reads. We demonstrate the application using our algorithm, seq2HLA, and a publicly available RNA-Seq dataset from the Burkitt lymphoma cell line Raji.

Human Leukocyte Antigen Typing Using High-Throughput DNA and RNA Sequencing and Application for Cell Line Identification

CIMT 2014: Next waves in cancer immunotherapy - Report on the 12th annual meeting of the Association for Cancer Immunotherapy

More than 900 scientists around the world visited the 12th Annual Meeting of the Association for Cancer Immunotherapy (CIMT) in Mainz, Germany from 6–8 May, 2014. Recent advancements in various spe...

Mutanome directed cancer immunotherapy

Somatic mutations are important drivers of cancer development. Accumulating evidence suggests that a significant subset of mutations result in neo-epitopes recognized by autologous T cells and thus may constitute the Achilles' heel of tumor cells. T cells directed against mutations have been shown to have a key role in clinical efficacy of potent cancer immunotherapy modalities, such as adoptive transfer of autologous tumor infiltrating lymphocytes and immune checkpoint inhibitors. Whereas these findings strengthen the idea of a prominent role of neo-epitopes in tumor rejection, the systematic therapeutic exploitation of mutations was hampered until recently by the uniqueness of the reperto…

Definitive evidence for Club cells as progenitors for mutantKras/Trp53‐deficient lung cancer

Accumulating evidence suggests that both the nature of oncogenic lesions and the cell-of-origin can strongly influence cancer histopathology, tumor aggressiveness and response to therapy. Although oncogenic Kras expression and loss of Trp53 tumor suppressor gene function have been demonstrated to initiate murine lung adenocarcinomas (LUADs) in alveolar type II (AT2) cells, clear evidence that Club cells, representing the second major subset of lung epithelial cells, can also act as cells-of-origin for LUAD is lacking. Equally, the exact anatomic location of Club cells that are susceptible to Kras transformation and the resulting tumor histotype remains to be established. Here, we provide de…

In Silico Typing of Classical and Non-classical HLA Alleles from Standard RNA-Seq Reads

Next-Generation Sequencing (NGS) enables the rapid generation of billions of short nucleic acid sequence fragments (i.e., "sequencing reads"). Especially, the adoption of gene expression profiling using whole transcriptome sequencing (i.e., "RNA-Seq") has been rapid. Here, we describe an in silico method, seq2HLA, that takes standard RNA-Seq reads as input and determines a sample's (classical and non-classical) HLA class I and class II types as well as HLA expression. We demonstrate the application of seq2HLA using publicly available RNA-Seq data from the Burkitt's lymphoma cell line DAUDI and the choriocarcinoma cell line JEG-3.

HLA typing from RNA-Seq sequence reads.

We present a method, seq2HLA, for obtaining an individual's human leukocyte antigen (HLA) class I and II type and expression using standard next generation sequencing RNA-Seq data. RNA-Seq reads are mapped against a reference database of HLA alleles, and HLA type, confidence score and locus-specific expression level are determined. We successfully applied seq2HLA to 50 individuals included in the HapMap project, yielding 100% specificity and 94% sensitivity at a P-value of 0.1 for two-digit HLA types. We determined HLA type and expression for previously un-typed Illumina Body Map tissues and a cohort of Korean patients with lung cancer. Because the algorithm uses standard RNA-Seq reads and …

Mutant MHC class II epitopes drive therapeutic immune responses to cancer

Tumour-specific mutations are ideal targets for cancer immunotherapy as they lack expression in healthy tissues and can potentially be recognized as neo-antigens by the mature T-cell repertoire. Their systematic targeting by vaccine approaches, however, has been hampered by the fact that every patient's tumour possesses a unique set of mutations ('the mutanome') that must first be identified. Recently, we proposed a personalized immunotherapy approach to target the full spectrum of a patient's individual tumour-specific mutations. Here we show in three independent murine tumour models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that, unexpectedly, the …

Multi-Omics Characterization of the 4T1 Murine Mammary Gland Tumor Model

Background: Tumor models are critical for our understanding of cancer and the development of cancer therapeutics. The 4T1 murine mammary cancer cell line is one of the most widely used breast cancer models. Here, we present an integrated map of the genome, transcriptome, and immunome of 4T1. Results: We found Trp53 (Tp53) and Pik3g to be mutated. Other frequently mutated genes in breast cancer, including Brca1 and Brca2, are not mutated. For cancer related genes, Nav3, Cenpf, Muc5Ac, Mpp7, Gas1, MageD2, Dusp1, Ros, Polr2a, Rragd, Ros1, and Hoxa9 are mutated. Markers for cell proliferation like Top2a, Birc5, and Mki67 are highly expressed, so are markers for metastasis like Msln, Ect2, and P…