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Large‐scale genomic sequencing of colorectal cancers has been reported mainly for Western populations. Differences by stage and ethnicity in the genomic landscape and their prognostic impact remain poorly understood. We investigated 534 Japanese stage III colorectal cancer samples from the Phase III trial, JCOG0910. Targeted‐capture sequencing of 171 potentially colorectal cancer‐associated genes was performed, and somatic single‐nucleotide variants and insertion–deletions were determined. Hypermutated tumors were defined as tumors with MSIsensor score >7 and ultra‐mutated tumors with POLE mutations. Genes with alterations associated with relapse‐free survival were analyzed using multivariable Cox regression models. In all patients (184 right‐sided, 350 left‐sided), mutation frequencies were TP53, 75.3%; APC, 75.1%; KRAS, 43.6%; PIK3CA, 19.7%; FBXW7, 18.5%; SOX9, 11.8%; COL6A3, 8.2%; NOTCH3, 4.5%; NRAS, 4.1%; and RNF43, 3.7%. Thirty‐one tumors were hypermutated (5.8%; 14.1% right‐sided, 1.4% left‐sided). Modest associations were observed: poorer relapse‐free survival was seen with mutant KRAS (hazard ratio 1.66; p = 0.011) and mutant RNF43 (2.17; p = 0.055), whereas better relapse‐free survival was seen with mutant COL6A3 (0.35; p = 0.040) and mutant NOTCH3 (0.18; p = 0.093). Relapse‐free survival tended to be better for hypermutated tumors (0.53; p = 0.229). In conclusion, the overall spectrum of mutations in our Japanese stage III colorectal cancer cohort was similar to that in Western populations, but the frequencies of mutation for TP53, SOX9, and FBXW7 were higher, and the proportion of hypermutated tumors was lower. Multiple gene mutations appeared to impact relapse‐free survival, suggesting that tumor genomic profiling can support precision medicine for colorectal cancer. We demonstrated that the overall spectrum of mutations in our Japanese stage III colorectal cancer cohort (534 patients) was largely similar to that in Western populations, but the frequencies of mutation for TP53 (75.3%), SOX9 (11.8%), and FBXW7 (18.5%) were higher, and the proportion of hypermutated tumors (5.8%) was lower. We also revealed that poorer relapse‐free survival was seen with mutant KRAS (hazard ratio 1.66; p = 0.011) and mutant RNF43 (2.17; p = 0.055) as well as hypermutated tumors (0.53; p = 0.229), whereas better relapse‐free survival was seen with mutant COL6A3 (0.35; p = 0.040) and mutant NOTCH3 (0.18; p = 0.093), suggesting that tumor genomic profiling has the potential to support precision medicine for patients with colorectal cancer.

IntroductionIn locally recurrent rectal cancer (LRRC), surgery is a standard treatment for resectable disease. However, short-term and long-term outcomes are unsatisfactory due to the invasive nature of surgical procedures and the high proportion of local recurrence. Consequently, the identification of reliable prognostic and predictive biomarkers to guide treatment decisions may improve outcomes. The presence of circulating tumour DNA (ctDNA) in plasma after surgery may signify the presence of minimal residual disease (MRD) in various cancers. Therefore, we have launched a multi-institutional prospective observational study of ctDNA for MRD detection in conjunction with JCOG1801, a randomised, controlled phase III trial evaluating the efficacy of preoperative chemoradiotherapy (pre-CRT) compared with up-front surgery for LRRC (jRCTs031190076, NCT04288999).Methods and analysisJCOG1801A1 is the first correlative study that assesses ctDNA in LRRC patients enrolled in JCOG1801. Patients randomised to up-front surgery will provide whole blood samples at three time points (prior to surgery, after surgery and after postoperative chemotherapy); those to pre-CRT will provide at five time points (prior to pre-CRT, after pre-CRT, prior to surgery, after surgery and after postoperative chemotherapy). Cell-free DNA will be extracted from plasma and analysed by Guardant Reveal, a tumour tissue–agnostic assay that assesses both genomic alterations and methylation patterns to determine the presence or absence of ctDNA. We will compare the prognosis and treatment response of patients according to their ctDNA status after surgery and at other time points.Ethics and disseminationThe study protocol received approval from the Institutional Review Board of National Cancer Center Hospital East on behalf of the participating institutions in February 2023. The study is conducted in accordance with the precepts established in the Declaration of Helsinki and Ethical Guidelines for Medical and Biological Research Involving Human Subjects. Written informed consent will be obtained from all eligible patients prior to registration.

We undertook genomic analyses of Japanese patients with stage I esophageal squamous cell carcinoma (ESCC) to investigate the frequency of genomic alterations and the association with survival outcomes. Biomarker analysis was carried out for patients with clinical stage T1bN0M0 ESCC enrolled in JCOG0502 (UMIN000000551). Whole‐exome sequencing (WES) was performed using DNA extracted from formalin‐fixed, paraffin‐embedded tissue of ESCC and normal tissue or blood sample. Single nucleotide variants (SNVs), insertions/deletions (indels), and copy number alterations (CNAs) were identified. We then evaluated the associations between each gene alteration with a frequency of 10% or more and progression‐free survival (PFS) using a Cox regression model. We controlled for family‐wise errors at 0.05 using the Bonferroni method. Among the 379 patients who were enrolled in JCOG0502, 127 patients were successfully analyzed using WES. The median patient age was 63 years (interquartile range, 57‐67 years), and 78.0% of the patients ultimately underwent surgery. The 3‐year PFS probability was 76.3%. We detected 20 genes with SNVs, indels, or amplifications with a frequency of 10% or more. Genomic alterations in FGF19 showed the strongest association with PFS with a borderline level of statistical significance of P = .00252 (Bonferroni‐adjusted significance level is .0025). Genomic alterations in FGF4, MYEOV, CTTN, and ORAOV1 showed a marginal association with PFS (P < .05). These genomic alterations were all CNAs at chromosome 11q13.3. We have identified new genomic alterations associated with the poor efficacy of ESCC (T1bN0M0). These findings open avenues for the development of new potential treatments for patients with ESCC. According to whole‐exome sequencing of patients with stage I esophageal squamous cell carcinoma, amplification of FGF19 showed the strongest association with progression‐free survival.

We carried out a phase I/II trial of chemoradiotherapy concurrent with S‐1 and cisplatin to determine the maximum tolerated dose and recommended dose and to evaluate the efficacy and safety of this treatment in patients with esophageal carcinoma. Thoracic esophageal cancer patients with clinical stage II/III disease, excluding T4, were eligible. Chemotherapy consisted of S‐1 at a dose of 60–80 mg/m2/day on days 1–14, and cisplatin at 75 mg/m2 on day 1, repeated twice every 4 weeks. Single daily radiation of 50.4 Gy was given in 28 fractions concurrently starting on day 1. Patients achieving an objective response after chemoradiotherapy underwent two additional cycles of chemotherapy. Patient accrual was terminated early due to slow enrolment after 44 patients were accrued. In the phase I part, two of six patients experienced dose‐limiting toxicities at each level of S‐1 (S‐1 60 or 80 mg/m2/day). Considering treatment compliance, the recommended dose was determined to be S‐1 60 mg/m2/day. The complete response rate, the primary endpoint of phase II, was 59.5% (22/37; 90% confidence interval, 44.6–73.1%; weighted threshold, 57.2%; P = 0.46 by the exact binomial test) on central review. In the phase II part, 3‐year progression‐free survival was 48.4%, with a 3‐year overall survival of 61.9%. Grade 3 or 4 toxicity in phase II included leukopenia (57.9%), neutropenia (50%), hyponatremia (28.9%), anorexia (21.1%), anemia (18.4%), thrombocytopenia (18.4%), and febrile neutropenia (2.6%). No treatment‐related deaths were observed. Although this combination showed acceptable toxicity and favorable 3‐year survival, the study did not meet its primary endpoint. This trial was registered at the UMIN Clinical Trials Registry as UMIN000000710. This combination showed acceptable toxicity and favorable 3‐year survival.

Expression of the Wilms' tumor gene WT1 in primary astrocytic tumors was examined using a quantitative real‐time reverse transcriptase‐polymerase chain reaction (RT‐PCR) or immunohistochemistry. Real‐time RT‐PCR showed that WT1 mRNA was expressed at various levels in all of the 25 astrocytic tumors examined. Immunohistochemical analysis showed that WT1 protein was expressed in 5 of 6 low‐grade astrocytic tumors (grade l‐ll) and all of 18 high‐grade ones (grade III‐IV), and that expression levels of WT1 protein in high‐grade tumors were significantly higher than those in low‐grade ones. WT1 protein was not detected in the normal glial cells contained in the tumor specimens. Furthermore, treatment with WT1 antisense oligomers specifically inhibited growth of glioblastoma cell lines, U87‐MG, A172, and T‐98G. These results may indicate that the WT1 gene plays an important role in tumorigenesis of primary astrocytic tumors.

To define the optimal chemotherapy regimen for each patient we therefore used tissue from patients to identify molecular prognostic or predictive biomarkers. Endoscopic biopsy specimens from primary lesions and surgical specimens on a phase III trial in patients with unresectable advanced or recurrent gastric cancer treated with docetaxel with cisplatin plus S-1 (DCS) or cisplatin plus S-1 (CS), were collected. We measured the mRNA expression of ERCC1 and analyzed SNPs in GSTP1 and ERCC1. Low ERCC1 expression was associated with favorable prognosis for overall survival, OS by multivariable analysis (P = 0.001). There were significant interactions between the two treatment arms of DCS and CS, and ERCC1 mRNA expression. In patients with low ERCC1 expression of a favorable prognosis, DCS therapy was inferior to CS (P = 0.046). In addition to GSTP1 rs1695 (HR 1.728), ERCC1 rs3212980, ERCC1 rs2298881, ERCC1 rs3212964 with high expression of ERCC1 mRNA were associated with significantly worse prognosis with regard to OS. ERCC1 mRNA is an independent prognostic factor and predictive marker that can be used to guide the addition of docetaxel. The SNPs of ERCC1 and GSTP1 could be also prognostic or predictive factors.

In the thymi of WT1-transgenic (Tg) mice with the 17AA+/KTS- spliced form of the Wilms tumor gene WT1 driven by the lck promoter, the frequencies of CD4-CD8- double-negative (DN) thymocytes were significantly increased relative to those in normal littermates. Of the 4 subsets of CD4-CD8- DN thymocytes, the DN1 (CD44+CD25-) subset increased in both frequency and absolute cell number, whereas the DN2 (CD44+CD25+) and DN3 (CD44-CD25+) subsets decreased, indicating the blocking of thymocyte differentiation from the DN1 to the DN2 subsets. Furthermore, CD4-CD8+ T-cell receptor (TCR) -gammadelta T-cells increased in both frequency and absolute cell number in the spleen and peripheral blood of the WT1-Tg mice relative to those of normal littermates. The CD8 molecules of these CD4-CD8+ TCRgammadelta T-cells were CD8alphabeta, suggesting that they originated from the thymus. These results are the first direct evidence demonstrating that the WT1 gene is involved in the development and differentiation of T-lineage cells.

Leukemia-specific promoters and enhancers for gene therapy had never been reported. Since the Wilms' tumor gene WT1 is overexpressed in almost all types of leukemia, WT1 is an ideal target of leukemia-specific therapy. To explore the possibility of gene therapy for leukemia using WT1 promoter and enhancer, their activities in several kinds of cells were analyzed by using the enhanced green fluorescent protein (EGFP) gene as a reporter. First, we identified the best combination (654P/EGFP/int3- enh/3'-enh vector) of the 654-bp WT1 promoter and the two WT1 enhancers located in intron 3 and at the 3' end of the WT1 gene for inducing EGFP expression in K562 cells, which endogenously expressed WT1. When this was transfected into WT1-expressing leukemia cells (K562, HEL), WT1-nonexpressing hematopoietic cells (Daudi, U937), and WT1-expressing nonhematopoietic cells (TYK-nu-CPr, SW480, 293 T), 19.8, 22.9, 1.47, 1.43, 4.50, 4.16, and 1.09 times EGFP expression was induced, respectively, compared to that by the promoter-less EGFP vector. These results showed that the 654P/EGFP/int3-enh/3'-enh vector specifically induced high levels of EGFP expression in WT1-expressing leukemia cells. 654P/int3- enh/3'-enh vector containing transgenes such as suicide genes might become useful tools for leukemia-specific gene therapy.