Yuta Muto

DNA methylation alterations of AXIN2 in serrated adenomas and colon carcinomas with microsatellite instability

BackgroundRecent work led to recognize sessile serrated adenomas (SSA) as precursor to many of the sporadic colorectal cancers with microsatellite instability (MSI). However, comprehensive analyses of DNA methylation in SSA and MSI cancer have not been conducted.MethodsWith an array-based methylation sensitive amplified fragment length polymorphism (MS-AFLP) method we analyzed 8 tubular (TA) and 19 serrated (SSA) adenomas, and 14 carcinomas with (MSI) and 12 without (MSS) microsatellite instability. MS-AFLP array can survey relative differences in methylation between normal and tumor tissues of 9,654 DNA fragments containing all NotI sequences in the human genome.ResultsUnsupervised clustering analysis of the genome-wide hypermethylation alterations revealed no major differences between or within these groups of benign and malignant tumors regardless of their location in intergenic, intragenic, promoter, or 3′ end regions. Hypomethylation was less frequent in SSAs compared with MSI or MSS carcinomas. Analysis of variance of DNA methylation between these four subgroups identified 56 probes differentially altered. The hierarchical tree of this subset of probes revealed two distinct clusters: Group 1, mostly composed by TAs and MSS cancers with KRAS mutations; and Group 2 with BRAF mutations, which consisted of cancers with MSI and MLH1 methylation (Group 2A), and SSAs without MLH1 methylation (Group 2B). AXIN2, which cooperates with APC and β-catenin in Wnt signaling, had more methylation alterations in Group 2, and its expression levels negatively correlated with methylation determined by bisulfite sequencing. Within group 2B, low and high AXIN2 expression levels correlated significantly with differences in size (P = 0.01) location (P = 0.05) and crypt architecture (P = 0.01).ConclusionsSomatic methylation alterations of AXIN2, associated with changes in its expression, stratify SSAs according to some clinico-pathological differences. We conclude that hypermethylation of MLH1, when occurs in an adenoma cell with BRAF oncogenic mutational activation, drives the pathway for MSI cancer by providing the cells with a mutator phenotype. AXIN2 inactivation may contribute to this tumorigenic pathway either by mutator phenotype driven frameshift mutations or by epigenetic deregulation contemporary with the unfolding of the mutator phenotype.

Clinical and molecular assessment of regorafenib monotherapy.

Regorafenib has shown survival benefits in metastatic colorectal cancer patients who were exacerbated after all standard therapies. Some patients, however, exhibit severe adverse events (AEs) resulting in treatment discontinuation. Therefore, the selection of patients likely to benefit from regorafenib is crucial. Twenty patients were treated with regorafenib for metastatic colorectal cancer; 122 plasma samples were taken from 16 of these patients for monitoring of circulating tumor DNA (ctDNA) in the blood. The treatment response, AEs, overall survival (OS), progression-free survival (PFS) and tumor morphologic changes on CT images were evaluated. KRAS mutant ctDNA was determined using digital PCR. Median PFS and OS were 2.5 and 5.9 months, respectively. Treatment was discontinued because of disease progression (PD) in 10 patients, and AEs in another 10 patients. AEs included hyperbilirubinemia, severe fatigue and skin rash. Hyperbilirubinemia was seen in two patients with multiple bilateral liver metastases, and severe fatigue in another 2 patients with poor performance status (PS). These severe AEs resulted in treatment discontinuation. Ten patients had a median PFS of 2.1 months with AE related discontinuation; PD occurred at 3.5 months (p=0.00334). Four patients exhibited a morphologic response, achieving better PFS times of 3.5, 5.3, 5.6 and 14.2 months. Emergence of the KRAS mutation in ctDNA was observed during anti-EGFR antibody treatment in 3 patients among 11 with KRAS wild-type tumors; it was detectable in the blood prior to radiographic detection of PD. Moreover, the KRAS mutation declined in two patients during regorafenib monotherapy. These patients were re-challenged with anti-EGFR antibody. Patients with extensive multiple liver metastases or poor PS are unlikely to benefit from regorafenib. Patients with a morphologic response will probably benefit from regorafenib with adequate management of other AEs. KRAS monitoring in ctDNA could be useful regarding treatment response and in determining treatment strategy.

Monitoring circulating tumor DNA revealed dynamic changes in KRAS status in patients with metastatic colorectal cancer

KRAS mutated circulating tumor DNA (MctDNA) can be monitored in the blood of patients with metastatic colorectal cancer (mCRC), but dynamic changes have not been determined. Four hundred and fifty-seven plasma samples were collected prospectively from 85 mCRC patients who underwent chemotherapy. MctDNA in plasma was detected by droplet digital PCR, and the percentage of MctDNA in total circulating cell-free DNA was calculated. KRAS assessment in tumor tissues showed 29 patients with the mutant-type (MT) and 56 patients with the wild-type (WT). Twenty-three of 29 MT patients (79.3%) and 28 of 56 WT patients (50.0%) showed MctDNA. Emergence of MctDNA was recognized during treatments with various drugs. Regardless of KRAS status in tumor tissues, patients with MctDNA in blood showed poor progression-free survival with first-line treatment. Median percentage of MctDNA accounted for 10.10% in MT patients and 0.22% in WT patients. These differences between MT and WT likely affected patterns of changes in MctDNA. KRAS monitoring identified dynamic changes in MctDNA, such as continuous, intermittent, and transient changes (quick elevation and disappearance). Emergence of MctDNA involved drug resistance, except for transient changes, which were seen in WT patients and likely corresponded with the drug response. Transient changes could be involved in recovery of sensitivity to anti-EGFR antibody in WT patients. Monitoring MctDNA during various treatments showed dynamic changes in KRAS status and could provide useful information for determining treatments for patients with mCRC.

Morphological response contributes to patient selection for rescue liver resection in chemotherapy patients with initially un-resectable colorectal liver metastasis

Morphological response is considered an improved surrogate to the Response Evaluation Criteria in Solid Tumors (RECIST) model with regard to predicting the prognosis for patients with colorectal liver metastases. However, its use as a decision-making tool for surgical intervention has not been examined. The present study assessed the morphological response in 50 patients who underwent chemotherapy with or without bevacizumab for initially un-resectable colorectal liver metastases. Changes in tumor morphology between heterogeneous with uncertain borders and homogeneous with clear borders were defined as an optimal response (OR). Patients were also assessed as having an incomplete response (IR), and an absence of marked changes was assessed as no response (NR). No significant difference was observed in progression-free survival (PFS) between complete response/partial response (CR/PR) and stable disease/progressive disease (SD/PD), according to RECIST. By contrast, PFS for OR/IR patients was significantly improved compared with that for NR patients (13.2 vs. 8.7 months; P=0.0426). Exclusion of PD enhanced the difference in PFS between OR/IR and NR patients (15.1 vs. 9.3 months; P<0.0001), whereas no difference was observed between CR/PR and SD. The rate of OR and IR in patients treated with bevacizumab was 47.4% (9/19), but only 19.4% (6/31) for patients that were not administered bevacizumab. Comparison of the survival curves between OR/IR and NR patients revealed similar survival rates at 6 months after chemotherapy, but the groups exhibited different survival rates subsequent to this period of time. Patients showing OR/IR within 6 months appeared to be oncologically stable and could be considered as candidates for surgical intervention, including rescue liver resection. Comparing the pathological and morphological features of the tumor with representative optimal response, living tumor cells were revealed to be distributed within the area of vascular reconstruction induced by bevacizumab, resulting in a predictive value for prognosis in the patients treated with bevacizumab. The present findings provided the evidence for physicians to consider patients with previously un-resectable metastatic colorectal cancer as candidates for surgical treatment. Morphological response is a useful decision-making tool for evaluating these patients for rescue liver resection following chemotherapy.

Tumor size is an independent risk predictor for metachronous colorectal cancer.

Non-hereditary colorectal cancer (CRC) patients are at higher risk of developing independent metachronous CRC than cancer-naïve individuals, but the reason is unknown. We studied metachronous CRC risk factors among one thousand five Japanese CRC patients who underwent surgery for CRC. Relative hazard risk of clinical and pathological features was assessed by univariate and multivariate Coxs proportional hazard regression analysis. Observed metachronous CRC incidence was also compared with the expected cancer incidence of the general population in Japan. Twenty-seven metachronous CRCs developed in 24 patients (2.4%) during a follow-up period of 3,676 person-years. Multivariate analysis revealed two factors associated with a high metachronous CRC risk: synchronous CRC (HR = 6.13; p = 1.3×10−4) and tumor size ≥ 6.5 cm (HR = 4.34; p = 1×10−3). Patients with either synchronous or large solitary tumors exhibited a higher risk for metachronous CRC than patients with solitary small tumors (HR = 7.3; p = 4.3×10−6) and that the general Japanese population (SIR = 7.01; p = 3.5×10−9), while patients with solitary small tumors did not (SIR = 1.07; p = 0.8). If patients younger than 60 years were excluded, the observations remained unchanged, with tumor size becoming stronger predictor (HR = 5.67; p = 1.7×10−4) than the presence of synchronous CRC (HR = 5.34; p = 9.6×10−4). Our novel finding that primary tumor size is a strong independent risk factor for metachronous CRC increases the sensitivity of prediction more than twice the presence of synchronous CRC. Our data provides new insights to assess the risk for metachronous lesions that should improve the surveillance regimen for CRC.

Heterogeneous expression of zinc-finger E-box-binding homeobox 1 plays a pivotal role in metastasis via regulation of miR-200c in epithelial-mesenchymal transition.

Although epithelial-mesenchymal transition (EMT) has been implicated as the pivotal event in metastasis, there is insufficient evidence related to EMT in clinical settings. Intratumor heterogeneity may lead to underestimation of gene expression representing EMT. In the present study, we investigated the expression of EMT-associated genes and microRNAs in primary colorectal cancer while considering intratumor heterogeneity. One-hundred and thirty-three multiple spatially separated samples were obtained from 8 patients with metastatic colorectal cancers and 8 with non-metastatic colorectal cancers, from the tumor center (TC), invasive front (IF) and metastasis. Differences in gene and microRNA expression were investigated by microarray and quantitative reverse-transcription PCR. Gene expression microarray analysis detected 7920 sites showing differing levels of gene expression among the TC, IF and metastasis. Expression of the EMT-associated gene zinc-finger E-box-binding homeobox 1 (ZEB1) significantly increased in the IF (p<0.01). To exclude individual differences, the expression ratio between TC and IF in each tumor was applied to analysis. This approach enabled recognition of the activation of the VEGF and Wnt signaling pathways, which were involved in metastasis via promotion of EMT. While no activation of these pathways was seen at the TC, regardless of whether tumors were metastatic or non-metastatic, they were preferentially activated at the IF in metastatic tumors, where high ZEB1 expression was seen in connection with decreased miR-200c expression. Multiple sampling in a tumor revealed that heterogeneous ZEB1 expression induced by EMT-associated signaling pathways played a pivotal role in metastasis via regulation of miR-200c.

Sequential administration of XELOX and XELIRI is effective, feasible and well tolerated by patients with metastatic colorectal cancer

Sequential administration of the chemotherapy regimes capecitabine and oxaliplatin (XELOX) and capecitabine and irinotecan (XELIRI) in the first- to second-line treatment setting would allow patients to be managed more easily in an outpatient unit. However, a small number of studies have raised concerns of cumulative adverse events as a consequence of the continuous use of capecitabine. To investigate this, the present study conducted a retrospective review of 81 consecutive metastatic colorectal cancer (mCRC) patients treated with the oxaliplatin, fluorouracil and leucovorin-irinotecan, fluorouracil and leucovorin (FOLFOX-FOFIRI/F-F) regimen (n=40) or the XELOX-XELIRI (X-X) regimen (n=41) in first- to second-line chemotherapy in Saitama Medical Center between 2006 and 2012. The disease control rate (DCR), the progression free survival (PFS), the overall survival (OS) and the time to failure of strategy (TFS) from first to second-line chemotherapy, as well as adverse events, were assessed and compared between patients receiving X-X or F-F. A total of 10 and 20 patients were additionally treated with bevacizumab in the F-F and X-X regimens, respectively, during first or second-line chemotherapy. There was no significant difference in DCR and the median PFS between the two regimens for first or second-line chemotherapy. There was no significant difference in the median OS and TFS between the two regimens (OS=24.5 and TFS=14 months in the F-F vs. 23.2 and 12.0 months in the X-X). Regarding adverse events, 45.0% of patients (18/40) exhibited grade 3-4 neutropenia throughout treatment with F-F. Whilst, 15.0% of patients (6/41) exhibited grade 3 hypertension throughout treatment with X-X, which was effectively controlled by a single antihypertensive drug. The results show that sequential administration of X-X is as effective and feasible as F-F treatment, while additionally reducing the frequency of infusion visits and eliminating the need for a central venous access device or home infusion pump, thereby offering a more convenient treatment option to patients with mCRC.

Significance of the Difference in the Size of Liver Tumors in the Managementof Patients with Colorectal Liver Metastases

Background: The combination of chemotherapy and surgery is currently accepted for the treatment of patients with technically resectable colorectal liver metastases. It is, however, hard to determine which of these modalities should be the forward treatment. In this study, we assessed the usefulness of the difference in tumor size assessed in pretherapeutic imaging in the selection of chemotherapy in these patients. Methods: We present a retrospective review of 80 consecutive colorectal liver metastases without extrahepatic tumors. The relapse-free survival (RFS), progression-free survival (PFS) and overall survival (OS) were evaluated and compared between patients who underwent surgery (n=66) and chemotherapy (n=14) according to clinical features. In particular, we addressed pretherapeutic imaging studies including the distribution and number of metastatic liver tumors. In addition, the ratio of tumor size (largest to smallest tumor) was calculated; two groups classified as R<5 (ratio <5) and R ≥ 5 (ratio ≥ 5) were compared. Results: Univariate analysis was performed in the surgery group; significant differences in RFS were found regarding time of occurrence, the number of tumors and the ratio of tumor diameters. Multivariate analysis showed that the ratio of tumor size, R ≥ 5, was the only independent prognostic risk factor concerning both RFS and OS. We then compared the outcome of patients with prognostic risk factors between surgery and chemotherapy. Surgery achieved significantly better OS than chemotherapy, with the exception of the R ≥ 5 group. No difference in OS, in addition to RFS and PFS, was seen in the R ≥ 5 groups regardless of treatment. Conclusion: CRC patients with resectable liver metastases with R ≥ 5 showed no significant difference in outcome using surgery or chemotherapy. Chemotherapy could be used as an alternative to forward surgery to address oncological concerns such as the presence of latent metastases or poor treatment outcome in these patients.