Abstract
Tadayoshi Yamaura1,2,*, Hiroyuki Miyoshi1,4,*, Hisatsugu Maekawa1,2, Tomonori Morimoto1,2, Takehito Yamamoto1,2, Fumihiko Kakizaki1, Koichiro Higasa3,5, Kenji Kawada2, Fumihiko Matsuda3, Yoshiharu Sakai2 and M. Mark Taketo1,4
1Division of Experimental Therapeutics, Graduate School of Medicine, Kyoto University, Yoshida-Konoé-cho, Sakyo-ku, Kyoto 606-8501, Japan
2Department of Surgery, Graduate School of Medicine, Kyoto University, Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
3Center of Genomic Medicine, Graduate School of Medicine, Kyoto University, Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
4Office of Society-Academia Collaboration for Innovation, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan
5Present address: Department of Genome Analysis, Institute of Biomedical Science, Kansai Medical University, Hirakata, Osaka 573-1010, Japan
*These authors contributed equally to this work
Correspondence to:
M. Mark Taketo, email: taketo@mfour.med.kyoyo-u.ac.jp
Keywords: colorectal cancer; spheroid; cancer stem cell; molecular oncology; immunotherapy
Received: June 30, 2018 Accepted: December 10, 2018 Published: December 25, 2018
ABSTRACT
Mismatch repair (MMR)-deficient or microsatellite instability (MSI) colorectal cancer includes two subtypes; Lynch syndrome and sporadic MSI cancer, both of which generate multiple neoantigens due to unrepaired mutations. Although such patients respond very well to immune checkpoint therapy, their diagnosis can be confused by low quality DNA samples owing to formalin fixation and/or low cancer cell content. Here we prepared high-quality DNA samples from in vitro-cultured cancer spheroids that consisted of the pure cell population. We evaluated their diagnostic power by on-chip electrophoresis, mutational burden assessment, and direct sequencing. Because formalin-fixed paraffin-embedded (FFPE) tissues are widely used as the DNA source, we compared such samples with spheroid DNA. Additionally, we performed immunohistochemistry (IHC) for MMR proteins on spheroids as well as primary tumor sections. Of 111 cases of colorectal cancer patients, we found seven MSI-high cases in which all diagnostic results agreed on spheroid-based assays, whereas the results with the FFPE DNA were less reliable though analyzable. Importantly, there was an MSS case that appeared as MSI by IHC on primary tumor sections. Based on these results, we propose to employ cultured cancer spheroids as the source of both DNA and IHC specimens for more reliable clinical diagnosis.