A novel approach for rapid screening of mitochondrial D310 polymorphism

BACKGROUND: Mutations in the mitochondrial DNA (mtDNA) have been reported in a wide variety of human neoplasms. A polynucleotide tract extending from 303 to 315 nucleotide positions (D310) within the non-coding region of mtDNA has been identified as a mutational hotspot of primary tumors. This region consists of two polycytosine stretches interrupted by a thymidine nucleotide. The number of cytosines at the first and second stretches are 7 and 5 respectively, according to the GeneBank sequence. The first stretch exhibits a polymorphic length variation (6-C to 9-C) among individuals and has been investigated in many cancer types. Large-scale studies are needed to clarify the relationship between cytosine number and cancer development/progression. However, time and money consuming methods such as radioactivity-based gel electrophoresis and sequencing, are not appropriate for the determination of this polymorphism for large case-control studies. In this study, we conducted a rapid RFLP analysis using a restriction enzyme, BsaXI, for the single step simple determination of 7-C carriers at the first stretch in D310 region. METHODS: 25 colorectal cancer patients, 25 breast cancer patients and 41 healthy individuals were enrolled into the study. PCR amplification followed by restriction enzyme digestion of D310 region was performed for RFLP analysis. Digestion products were analysed by agarose gel electrophoresis. Sequencing was also applied to samples in order to confirm the RFLP data. RESULTS: Samples containing 7-C at first stretch of D310 region were successfully determined by the BsaXI RFLP method. Heteroplasmy and homoplasmy for 7-C content was also determined as evidenced by direct sequencing. Forty-one percent of the studied samples were found to be BsaXI positive. Furthermore, BsaXI status of colorectal cancer samples were significantly different from that of healthy individuals. CONCLUSION: In conclusion, BsaXI RFLP analysis is a simple and rapid approach for the single step determination of D310 polymorphism of mitochondrial DNA. This method allows the evaluation of a significant proportion of samples without the need for sequencing- and/or radioactivity-based techniques

Prognostic significance of metastatic lymph node ratio in gastric cancer: a Western-center analysis

Abstract Background Tumor-node-metastasis (TNM) staging is the central gastric cancer (GC) staging system, but it has some disadvantages. However, the lymph node ratio (LNR) can be used regardless of the type of lymphadenectomy and is considered an important prognostic factor. This study aimed to evaluate the relationship between LNR and survival in patients who underwent curative GC surgery. Methods All patients who underwent radical gastric surgery between January 2014 and June 2022 were retrospectively evaluated. Clinicopathological features of tumors, TNM stage, and survival rates were analyzed. LNR was defined as the ratio between metastatic lymph nodes and total lymph nodes removed. The LNR groups were classified as follows: LNR0 = 0, 0.01  0.25. Tumor characteristics and overall survival (OS) of the patients were compared between LNR groups. Results After exclusion, 333 patients were analyzed. The mean age was 62 ± 14 years. According to the LNR classification, no difference was found between groups regarding age and sex. However, TNM stage III disease was significantly more common in LNR3 patients. Most patients (43.2%, n = 144) were in the LNR3 group. In terms of tumor characteristics (lymphatic, vascular, and perineural invasion), the LNR3 group had significantly poorer prognostic factors. The Cox regression model defined LNR3, TNM stage II—III disease, and advanced age as independent risk factors for survival. Patients with LNR3 demonstrated the lowest 5-year OS rate (35.7%) (estimated mean survival was 30 ± 1.9 months) compared to LNR 0–1–2. Conclusion Our study showed that a high LNR was significantly associated with poor OS in patients who underwent curative gastrectomy. LNR can be used as an independent prognostic predictor in GC patients