Microsatellite instability is inversely associated with type 2 diabetes mellitus in colorectal cancer

Background Microsatellite instability (MSI) is a clonal change in the number of repeated DNA nucleotide units in microsatellites. High-frequency MSI (MSI-H) colorectal cancers (CRCs) are known to have different clinicopathological features compared with microsatellite stable (MSS) CRCs. In addition, previous studies have shown that type2 diabetes mellitus (T2DM) is a risk factor for malignant tumors including CRCs. The aim of this study was to investigate the relationship between T2DM and MSI-H colorectal cancer. Methods The study design is a single center, cross-sectional study. Data from a series of 936 patients with CRCs were collected and MSI status was assessed. Results In total, 29 (3.1%) and 907 (96.9%) tumors were classified as having MSI-H and low-frequency microsatellite instability or being MSS (MSS), respectively. Of the 936 patients, 275 (29.6%) were associated with T2DM. One (3.4%) of the 29 MSI-H patients and 274 (30.2%) of the 907 MSS patients had T2DM. Thus, the incidence of T2DM was significantly less frequent in MSI-H compared with MSS patients (Fisher’s exact test: p = 0.0007). Conclusions We conclude that MSS tumors are significantly more common than MSI-H tumors among individuals with T2DM.


Introduction
Colorectal cancer (CRC) is one of the most common solid tumors, and details associated with its carcinogenesis have been intensively studied. Some colorectal cancers are microsatellite stable (MSS). The typical development of MSS tumors proceeds stepwise by the inactivation of increasing numbers of tumor suppressor genes, including the APC and p53 genes, through mutation as well as loss of heterozygosity (LOH) and the activation of oncogenes such as KRAS [1,2].
On the other hand, microsatellite instability (MSI) is a phenotype resulting from a defect in mismatch repair genes such as MSH2 [3,4], MLH1 [4], and MSH6 [5,6]. No MSI tumor shows LOH at these tumor suppressor genes [7], and target genes for frameshift mutations in CRCs are different from those in MSS tumors. Well-known target genes for MSI tumors include TGFβRII [8], IGFIIR [9], and BAX [10]. Testing colorectal cancers for MSI is an effective method of screening for Lynch syndrome because approximately 90% of Lynch syndrome tumors have high microsatellite instability (MSI-H) [11]. Although MSI is also observed in sporadic CRC, CRCs with MSI-H, irrespective of whether they are hereditary or not, have important therapeutic and diagnostic characteristics. CRCs with MSI-H are generally associated with a better prognosis [12], but their prognosis is less favorable with 5-FU based chemotherapy [13]; in addition, these tumors have less metastasis, and are more likely to be a right sided or metachronous multiple CRCs [12,14].
Type 2 diabetes mellitus (T2DM) is associated with malignant tumors, including CRC [15]. It has been reported that CRC incidences in diabetic patients were 1.27-1.40 in colon and 1.19-1.36 in rectum [16][17][18]. Jiang Y et al. reported that relative risk of CRC incidence for T2DM were 1.27 (95% CI: 1.21-1.34) by meta-analysis of cohort studies [19]. However, the relationship between microsatellite instability of CRC and T2DM has not been clear yet. In this study, we investigated a relationship between T2DM and MSI in CRC.

Patients
This study is a single center, retrospective cross-sectional study. We selected 936 consecutive colorectal cancer patients who underwent surgical resection at the Tokyo Metropolitan Cancer and Infectious diseases Center Komagome Hospital from January 2008 to January 2014 after obtaining their informed consent. The study was performed after approval of the Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital Ethical Committee (ID: 612). Patients with inflammatory bowel disease or a known history of familial adenomatous polyposis and Lynch syndrome were excluded. If the patient had two or more colorectal tumors resected, then the tumor that was most advanced was selected for analysis.
We defined patients with T2DM as those who had already been diagnosed with T2DM or those who had a high concentration of glycated hemoglobin (HbA1c > 6.5%) which was measured before the first colorectal surgery. The criteria for T2DM diagnosis depends on Standards of Medical Care in Diabetes 2018 by American Diabetes Association [20].
Body weight (kg) and height (m) were measured immediately before surgery. The body mass index (BMI) was calculated as weight in kilograms divided by the square of the height in meters (kg/m 2 ).
Microsatellite instability analysis and mutation analysis. Colorectal cancers and corresponding normal tissues were obtained with informed consent and were stored at −80˚C immediately after resection. Genomic DNA samples were extracted using the QIAamp DNA mini kit (QIAGEN, Valencia, CA,USA). Methods to determine microsatellite instability and KRAS and BRAF mutations were describe previously. Briefly, polymerase chain reaction (PCR) was performed to amplify at least five repetitive sequence loci from the tumor and normal tissue samples: BAT25, BAT26, D2S123, D5S346, and D17S250. Microsatellite instability status was defined as MSI-H (2-5 of the 5 markers used were unstable) and MSS (none or only 1 of the 5 markers was unstable), as described in the National Cancer Institute guidelines for MSI testing (22). All samples were analyzed to identify any BRAF (V600E) and KRAS (codons 12 and 13) mutations by direct sequencing.

Statistical analysis
The Fisher's exact test was used to evaluate the relationship between two discrete and dichotomous variables. The analysis of association between categorical variables was performed using logistic regression method. An optimal cut-off value for predicting MSI status was analyzed using receiver operating characteristic (ROC) curves. Areas under the curve (AUC) were also calculated. All statistical tests were 2-sided, and P values of �0.05 were considered to indicate statistical significance. All statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University, Japan), which is a graphical-user interface for R (The R Foundation for Statistical Computing, Vienna, Austria, version 3.5.1). This interface is a modified version of R commander (version 2.5-1) that includes statistical functions that are frequently used in biostatistics.

Results
Total of 936 CRC patients were enrolled during study period in this study. All patients underwent surgical resection of the primary tumor and the diagnosis of adenocarcinoma was made pathologically. None received neo-adjuvant chemotherapy or radiotherapy. The clinical and pathological characteristics of the patients are shown in Table 1. Of the 936 colorectal cancers, 29 (3.1%) and 907 (96.9%) tumors were classified as MSI-H and MSS, respectively. Significant differences between MSI-H and MSS cancers were observed with respect to sex (p = 0.022), age (p = 0.007), tumor location (p < 0.0001) and histology (p < 0.0001); whereas, there were no significant differences with respect to UICC classification (p = 0.57). One hundred seventyfive (18.7%) patients had T2DM and no patient had type 1 diabetes mellitus. The incidence of T2DM was significantly less frequent in MSI-H than MSS patients (p = 0.0007). However, there was no significant difference in BMI between MSI-H and MSS patients (p = 0.65). Of the 936 CRC patients, 277 (29.6%) had KRAS mutation and 49 (5.2%) had BRAF mutation. None of the 29 MSI-H patients were a KRAS mutation, whereas 277 (30.5%) of the 907 MSS patients had a KRAS mutation, and frequency of KRAS mutation was significantly different between MSI-H and MSS (p < 0.0001). Inversely, frequency of BRAF mutation was significantly high in MSI-H patients than in MSS patients (p < 0.0001). According to logistic regression analysis, age, tumor location, histology and T2DM status were independent factors in MSI-H tumor ( Table 2). Table 3 showed clinicopathological features according to T2DM status. T2DM+ was significantly more frequent in male patients, elderly patients and obesity patients; however, no correlation was seen between T2DM+ and tumor location, histology or UICC classification. BRAF gene mutation was significantly more frequent in T2DM-patients, while KRAS gene mutation was not correlated with T2DM status.

Discussion
Our study findings indicated that T2DM is significantly less common among MSI-H patients compared with MSS patients. This is the first report to indicate that T2DM may be associated with MSS CRC. There have been no previous reports of a relationship between T2DM and MSI status in CRC, even though it is well known that the incidence of malignant tumors is increased in patients with T2DM [7,21]. Our study revealed the frequency of MSI-H tumors to be 3.1%, which reported as well as in previous studies in Asian countries is lower than that reported in Western countries [22,23]. Asaka et al reported on the frequency of MSI in 940 Japanese CRC patients and found that 5.9% were MSI-H and 94.1% were MSS/MSI-L [24]. The incidence of rectal cancer is higher in Japanese individuals (approximately 40% of CRCs) compared with individuals from Western countries (approximately 20% of CRCs), which would reflect a lower rate of MSI-H CRC because rectal cancer is less likely to show MSI-H than colon cancer. In addition, as we reported previously, MSI-H is less frequent even in right colon cancer in Japanese individuals than in Western. [25]. Race may thus affect MSI status. T2DM has been shown to be a risk factor for malignant tumors including CRC [15]. T2DM and CRC are major causes of morbidity and mortality in the United States, Western countries, and increasingly in Japan [26,27]. Dietary and lifestyle risk factors for developing insulin resistance and T2DM, such as a Western diet, physical inactivity, and obesity, have also been linked to an increased risk of CRC [28][29][30]. Furthermore, an association between metabolic syndrome and CRC is now supported by a large number of epidemiological studies [16,17,[31][32][33]. In this study, the frequency of obesity was almost identical between the MSS and MSI-H groups, and thus, we were able to compare the incidence of T2DM as an independent risk factor for colorectal cancer.
Previous reports have shown that in patients who suffer from T2DM, hyperinsulinemia, or factors related to insulin resistance, such as hyperglycemia or hypertriglyceridemia, are associated with colorectal carcinogenesis [16]. IGF-1, which is suggested to stimulate cell proliferation by its activation, is reported to be associated with CRC both epidemiologically and experimentary [34]. Moreover, biologic interactions among insulin, IGF-1, and IGFBPs may increase the risk of CRC through diet and associated factors, including Wnt pathway and PI3K/Akt pathway [35][36][37][38][39][40].
The current study had some limitations as follows: (1) selection bias caused by retrospective nature of the study; (2) a single-center study; (3) our study revealed that T2DM was associated with MSS colorectal cancer; however, we could not provide the mechanism. Nonetheless, considering that there are only a few publications on association with T2DM and MSS colorectal cancer, we believe that our findings will help researchers and physicians clarify the nature of colorectal cancer. And, we also know that further studies are required to overcome these limitations.

Conclusions
We conclude that MSS tumors are significantly more common than MSI-H tumors among individuals with T2DM. Methodology: Yujiro Nakayama, Tatsuro Yamaguchi.

Supporting information
Project administration: Yujiro Nakayama.