Precision medicine based on surgical oncology in the era of genome‐scale analysis and genome editing technology

Abstract Accumulated evidence suggests that multiple molecular and cellular interactions promote cancer evolution in vivo. Surgical oncology is of growing significance to a comprehensive understanding of the malignant diseases for therapeutic application. We have analyzed more than 1000 clinical samples from surgically resected tissue to identify molecular biomarkers and therapeutic targets for advanced malignancies. Cancer stemness and mitotic instability were then determined as the essential predictors of aggressive phenotype with poor prognosis. Recently, whole genome/exome sequencing showed a mutational landscape underlying phenotype heterogeneity in caners. In addition, integrated genomic, epigenomic, transcriptomic, metabolic, proteomic and phenomic analyses elucidated several molecular subtypes that cluster in liver, pancreatic, biliary, esophageal and gastroenterological cancers. Identification of each molecular subtype is expected to realize the precise medicine targeting subtype‐specific molecules; however, there are obstacle limitations to determine matching druggable targets or synthetic lethal interactions. Current breakthroughs in genome editing technology can provide us with unprecedented opportunity to recapitulate subtype‐specific pathophysiology in vitro and in vivo. Given a great potential, on‐demand editing system can design actionable strategy and revolutionize precision cancer medicine based on surgical oncology.

intercommunications in the tumor microenvironments should contribute to in vivo evolution of malignant diseases, indicating essential and irreplaceable roles of clinical tissue samples resected surgically. 4,5 Recent advances of subtype stratification have been achieved by integrative studies of transcriptomics with genomics, epigenomics, metabolomics proteomics and phenomics using surgical specimens and clinicopathological data. 6 In this review, the current strategies for unparalleled challenge of subtype-guided treatment that links molecular properties to targeted therapy, and perspectives of the future of precision cancer medicine with genome editing technology, are discussed. As these potentials could be augmented by gastroenterological surgery, the concept of precision medicine based on surgical oncology is of importance in cancer treatment.

| CANCER STEMNESS REPROGRAMMING AS THERAPEUTIC RESISTANCE
A variety of phenotypic hallmarks of cancer is characterized as tumor heterogeneity in vitro and in vivo. 3 These heterogeneity patterns can be determined by molecular analyses, and transcriptomics using bulk tumor tissues are suitable for clustering to better understand the transcriptional networks that underpin the tumor microenvironment. 7,8 First, we carried out genome-wide transcriptome analysis on surgically resected samples using a microarray technique ( Figure 2). Subsequently, the stemness pathway 9 and mitotic abnormality 10 were identified as the main regulators of hepatocellular carcinoma (HCC) with poor prognosis.
F I G U R E 1 Hallmarks and transcriptomics of cancer. A, The hallmark catalog of cancer phenotypes is a manifestation of 10 essential alterations in cell physiology that collectively dictate malignant growth. 3 B, Genome-scale transcriptomic analysis with microarray identified the stemness pathway and mitotic abnormality as the main regulators of hepatocellular carcinoma with poor prognosis TANAKA | 107 The stemness phenotype comprises the essential component of intractable cancers. 11 Cancer cells with stem-like properties, called cancer stem cells (CSC), feature the ability of self-renewal and pluripotency to hierarchically organize tumor initiation and maintenance. 12 CSC lying at the apex of the hierarchy are intrinsically resistant to chemotherapeutic agents, and function as a source to metastasizing and relapsing. "Self-renewal" is theoretically based on asymmetric divisions of stem cells that give rise to one cell of the stem cell potency and another stimulated to differentiate further into non-stem cell types. 13 In our recent studies, the proteasomeindependent character of the stem cell fate (degron) was used for fluorescent visualization of CSC subpopulations in human HCC 14 as well as in pancreatic cancer 13 and colorectal cancer cells. 15 Noteworthy, this system to distinguish CSC from non-CSC showed asymmetric cell division, "self-renewal" sphere formation in a real-time manner, and over 1000-fold increase in tumorigenicity with heterogenic expansion in vivo. [14][15][16] As CSC might play a fundamental role in these awful malignant behaviors, investigations of the molecular targets of CSC may show particularly effective therapeutic approaches. 12 We showed EpCAM stemness marker as one of the therapeutic targets of human HCC in vitro and in vivo. 17 As stem cell features are addictive to p53 inactivation, 18 CSC-targeted therapy might be more effective on TP53-mutated subtype of HCC. 19 Chromatin dynamics play an essential role in stem cell fate determination. 20 We showed that metastatic potentials and gene expression profiles of CSC are regulated by histone modifications for openbivalent-closed chromatin statuses. 21 In our recent studies, sorafenib-resistant HCC was shown to acquire in vivo CSC features with histone modification. 22 We identified that H3K4me3 and H3K27ac

| MITOTIC INSTABILITY AND PUN CTUATED EQUILIBRIUM
One of the major difficulties in the treatment of HCC is the high frequency of tumor recurrence even after curative resection. According to our clinical studies, not the recurrence itself, but the rapid and lethal recurrence pattern has critical effects on prognosis of the patient with HCC. 24 In this regard, Aurora mitotic abnormality was shown as the essential pathway for such an aggressive phenotype of HCC. 7 Aurora kinases are serine/threonine kinases that play major roles in chromosomal alignment and segregation during mitosis and cytokinesis. 25  showing open chromatin states that are frequently coexistent with CpG demethylation in stemness-phenotype cells death by mitotic catastrophe. 28 In addition, sequential combination treatment with Aurora B inhibitor (barasertib) followed by Bcl2/xL inhibitor (navitoclax) significantly suppressed orthotopic liver tumors. 29 In the recent studies, Dauch et al 30 reported that TP53mutated human HCC cells were specifically sensitive to Aurora A inhibitor, thus suggesting a novel therapeutic strategy for this subtype of human HCC. These preclinical studies indicate that Aurora is a promising molecular target "Achilles' heel" for the treatment of aggressive HCC. 31 What is the critical role of the Aurora mitotic pathway in cancer progression? Our microarray-based comparative genomic hybridization (array-CGH) analysis on clinical samples showed that genomic instability was closely related to Aurora B overexpression in HCC. 10 The fraction of genome altered (FGA) in Aurora B-positive cases was significantly higher than that in Aurora B-negative cases (P = .009), suggesting that overexpression of Aurora B may contribute to genomic instability in HCC. Indeed, in vitro overexpression of Aurora A caused inactivation of the spindle assembly checkpoint during mitosis, leading to polyploidy and centrosome amplification. 32 Similarly, overexpression of Aurora B caused defective chromosome separation during mitosis, leading to aneuploidy with mitotic errors. 33 Amon's group and our collaborators clarified that poly-or aneuploidy is potentially critical for the fate of malignant evolution. 34,35 Chromosome segregation errors can lead to DNA damage and chromosomal aberrations such as poly-or aneuploidy which is linked to chromothripsis, a new class of complex catastrophic chromosomal rearrangement. 36 Chromothripsis is a dramatic event that results in the pulverization of one or a few select chromosomes followed by their highly error-prone re-stitching. This leads to extensive chromosome rearrangements, which often include deletions, non-balanced translocations, duplications, and inversions. Recently, chromothripsis associated with mitotic errors was identified as the principal evolutionary trajectory in aggressive cancer progression such as pancreatic adenocarcinoma. 37 The consequence of mitotic errors is not sequential but simultaneous, indicating "punctuated equilibrium", rather than "gradualism" in a subset of cases ( Figure 3B). The innovative investigations of malignant evolution will be essential to guide therapeutic strategies for lethal cancers.

| MOLECULAR SUBTYPES AND GENOME EDITING TECHN OLOGY
The genomic mutational landscape might contribute to practical comprehension of tumor heterogeneity. 38 Decades ago, manual DNA sequencing detected individual mutations in TP53 (30%~) and CTNNB1 encoding beta-catenin (~30%) in human HCC. 39 We have previously reported the closed relationship between TP53 mutations and HCC progression, 40 and the carcinogenic significance of Wnt/beta-catenin signaling pathways. 41,42 In recent years, nextgeneration sequencing for whole exome analysis elucidated that more than 60% of HCC carries aberrant activation of TERT (telomere reverse transcriptase) through promoter mutations, viral integrations or focal amplifications ( Figure 4A). 43 SWI/SNF chromatinremodeling complex was identified as another candidate of the major driver mutations in HCC. 10 Approximately 20%-30% of HCC carries genomic aberrations encoding SWI/SNF subunits such as ARID1A, ARID2, and PBRM1. 43,44 The SWI/SNF enzymatic complex functions as an ATP-dependent helicase to disrupt histone-DNA contacts to create a loop of DNA as the essential step F I G U R E 3 Mitotic instability and punctuated equilibrium. A, Aberrant expression of Aurora A and B induced poly-or aneuploidy with mitotic errors. 32,33 B, Evolution models for classical gradualism (blue) and alternatively punctuated equilibrium (red). 37 In the gradualism model, multiple transforming events are independently required for tumor development. In the punctuated equilibrium model, tumor development can be divided into two major events: the cancer-initiating event and then the revolutionary-chromothripsis event is triggered catastrophically by poly-or aneuploidy with mitotic errors 36 TANAKA | 109 F I G U R E 5 Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome-editing system. 48 Cas9 nuclease protein precisely cleaves the target DNA by use of short single-guide RNA (sgRNA), immediately followed by species-dependent protospacer adjacent motif (PAM). After generation of a doublestrand break (DSB), non-homologous end joining (NHEJ) repair pathway induces indel mutation, resulting in gene knock-out. Additionally, a homologous DNA template enables homology-directed repair (HDR) pathway, resulting in gene knock-in F I G U R E 4 Landscape of altered genes and clusters in hepatocellular carcinoma (HCC). A, Bar plot and main pathways indicating the major events for oncogenes (red) and tumor suppressor genes (blue) altered frequently in HCC. 43,44 B, Six mutation clusters and Kaplan-Meier plot of disease-free survival of HCC patients 46 required for DNA replication and transcription as well as DNA repair. 45 It is interesting that mutually exclusive patterns of gene mutations are recognized between TP53 and CTNNB1, or between ARID1A and ARID2. 43 Figure 5). Alternatively, in addition to Cas9 and sgRNA, a homologous DNA template enables a homology-directed repair (HDR) pathway that can introduce precise genetic modifications (e.g. knock-in mutations). 49 Although frequent inactivating mutations were detected in an aggressive subtype of HCC ( Figure 4B), it is still not understood how ARID2 plays tumor suppressor roles in cancer evolution.
Recently, we used CRISPR/Cas9 genome-editing technology to establish human HCC cells knocked out for the ARID2 gene. 50 ARID2 depletion attenuated nucleotide excision repair (NER) of DNA damage sites introduced by exposure to ultraviolet (UV) light as well as to chemical carcinogens, as XPG could not be accumulated without ARID2 ( Figure 6A). By using large-scale public data sets, we validated that ARID2 knock-out could lead to similar molecular changes in vivo and, moreover, observed a higher number of somatic mutations in ARID2-mutated subtypes than in the ARID2 wild-type across various types of cancers including HCC ( Figure 6B).
Our CRISPR-mediated knock-out for the ARID2 gene provided evidence that the NER process is disrupted through inhibition of the recruitment of XPG, resulting in susceptibility to carcinogens and potential hypermutation in the ARID2-mutated subtype of HCC.
These findings present far-reaching implications for therapeutic targets in cancers harboring ARID2 mutations. 51 The development of cancer immunotherapy has reached an important inflection point in the history of cancer therapy, 52 and the correlation of a higher mutational load and a higher rate of response to immune checkpoint F I G U R E 6 Disruption of DNA damage response and hypermutation in ARID2-mutated hepatocellular carcinoma (HCC). 50   and organoid models also provide potentially valuable information for estimating patient response to a given treatment, but there are some limitations to determine immunotherapy including checkpoint inhibitors. 64 Genome-scale analysis allows the identification of subtype clustering, and subtype-specific treatment could then be translated not only from PDX/organoids, but also from genome editing/ engineering models for targeted therapy (Figure 7). Precise characterization of the molecular subtypes encompassing tumor, stromal and immune components should uncover multi-molecular additions that promise future perspectives for the development of precision cancer medicine.