Yasuaki Enokida

SNP (–617C>A) in ARE-Like Loci of the NRF2 Gene: A New Biomarker for Prognosis of Lung Adenocarcinoma in Japanese Non-Smoking Women

Purpose The transcription factor NRF2 plays a pivotal role in protecting normal cells from external toxic challenges and oxidative stress, whereas it can also endow cancer cells resistance to anticancer drugs. At present little information is available about the genetic polymorphisms of the NRF2 gene and their clinical relevance. We aimed to investigate the single nucleotide polymorphisms in the NRF2 gene as a prognostic biomarker in lung cancer. Experimental Design We prepared genomic DNA samples from 387 Japanese patients with primary lung cancer and detected SNP (c.–617C>A; rs6721961) in the ARE-like loci of the human NRF2 gene by the rapid genetic testing method we developed in this study. We then analyzed the association between the SNP in the NRF2 gene and patients’ overall survival. Results Patients harboring wild-type (WT) homozygous (c.–617C/C), SNP heterozygous (c.–617C/A), and SNP homozygous (c.–617A/A) alleles numbered 216 (55.8%), 147 (38.0%), and 24 (6.2%), respectively. Multivariate logistic regression models revealed that SNP homozygote (c.–617A/A) was significantly related to gender. Its frequency was four-fold higher in female patients than in males (10.8% female vs 2.7% male) and was associated with female non-smokers with adenocarcinoma. Interestingly, lung cancer patients carrying NRF2 SNP homozygous alleles (c.–617A/A) and the 309T (WT) allele in the MDM2 gene exhibited remarkable survival over 1,700 days after surgical operation (log-rank p = 0.021). Conclusion SNP homozygous (c.–617A/A) alleles in the NRF2 gene are associated with female non-smokers with adenocarcinoma and regarded as a prognostic biomarker for assessing overall survival of patients with lung adenocarcinoma.

Eprobe-mediated screening system for somatic mutations in the KRAS locus

Activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) loci are largely predictive of resistance to epidermal growth factor receptor (EGFR) therapy in colorectal cancer (CRC). A highly sensitive detection system for the KRAS gene mutations is urgently needed; however, conventional methods have issues with feasibility and cost performance. Here, we describe a novel detection system using a fluorescence ‘Eprobe’ capable of detecting low level KRAS gene mutations, via real-time PCR, with high sensitivity and simple usability. We designed our Eprobes to be complementary to wild-type (WT) KRAS or to the commonly mutated codons 12 and 13. The WT Eprobe binds strongly to the WT DNA template and suppresses amplification by blocking annealing of the primer during PCR. Eprobe-PCR with WT Eprobe shows high sensitivity (0.05–0.1% of plasmid DNA, 1% of genomic DNA) for the KRAS mutation by enrichment of the mutant type (MT) amplicon. Assay performance was compared to Sanger sequencing using 92 CRC samples. Discrepancies were analyzed by mutation genotyping via Eprobe-PCR with full match Eprobes for 7 prevalent mutations and the next generation sequencing (NGS). Significantly, the Eprobe system had a higher sensitivity for detecting KRAS mutations in CRC patient samples; these mutations could not be identified by Sanger sequencing. Thus, the Eprobe approach provides for highly sensitive and convenient mutation detection and should be useful for diagnostic applications.

Rapid Detection of SNP (c.309T>G) in the MDM2 Gene by the Duplex SmartAmp Method

Background Genetic polymorphisms in the human MDM2 gene are suggested to be a tumor susceptibility marker and a prognostic factor for cancer. It has been reported that a single nucleotide polymorphism (SNP) c.309T>G in the MDM2 gene attenuates the tumor suppressor activity of p53 and accelerates tumor formation in humans. Methodology In this study, to detect the SNP c.309T>G in the MDM2 gene, we have developed a new SNP detection method, named “Duplex SmartAmp,” which enabled us to simultaneously detect both 309T and 309G alleles in one tube. To develop this new method, we introduced new primers i.e., nBP and oBPs, as well as two different fluorescent dyes that separately detect those genetic polymorphisms. Results and Conclusions By the Duplex SmartAmp method, the genetic polymorphisms of the MDM2 gene were detected directly from a small amount of genomic DNA or blood samples. We used 96 genomic DNA and 24 blood samples to validate the Duplex SmartAmp by comparison with results of the conventional PCR-RFLP method; consequently, the Duplex SmartAmp results agreed totally with those of the PCR-RFLP method. Thus, the new SNP detection method is considered useful for detecting the SNP c.309T>G in the MDM2 gene so as to judge cancer susceptibility against some cellular stress in the clinical setting, and also to handle a large number of samples and enable rapid clinical diagnosis.

Highly sensitive detection of a HER2 12-base pair duplicated insertion mutation in lung cancer using the Eprobe-PCR method

Somatic mutation in human epidermal growth factor receptor-related 2 gene (HER2) is one of the driver mutations in lung cancer. HER2 mutations are found in about 2% of lung adenocarcinomas (ADCs). Previous reports have been based mainly on diagnostic screening by Sanger sequencing or next-generation sequencing (NGS); however, these methods are time-consuming and complicated. We developed a rapid, simple, sensitive mutation detection assay for detecting HER2 12 base pair-duplicated insertion mutation based on the Eprobe-mediated PCR method (Eprobe-PCR) and validated the sensitivity of this assay system for clinical diagnostics. We examined 635 tumor samples and analyzed HER2 mutations using the Eprobe-PCR method, NGS, and Sanger sequencing. In a serial dilution study, the Eprobe-PCR was able to detect mutant plasmid DNA when its concentration was reduced to 0.1% by mixing with wild-type DNA. We also confirmed amplification of the mutated plasmid DNA with only 10 copies per reaction. In ADCs, Eprobe-PCR detected the HER2 mutation in 2.02% (9/446), while Sanger sequencing detected it in 1.57% (7/446). Eprobe-PCR was able to detect the mutation in two samples that were undetectable by Sanger sequencing. All non-ADC samples were wild-type. There were no discrepancies between frozen and formalin-fixed paraffin-embedded tissues in the nine samples. HER2 mutations detected by NGS data validated the high sensitivity of the method. Therefore, this new technique can lead to precise molecular-targeted therapies.

Impact of the Bim Deletion Polymorphism on Survival Among Patients With Completely Resected Non–Small-Cell Lung Carcinoma

Purpose A deletion polymorphism of the Bim gene has been reported to be a prognostic factor for patients with non–small-cell lung cancer (NSCLC) treated with epidermal growth factor receptor-tyrosine kinase inhibitors in the Asian population. We investigated the impact of the Bim deletion polymorphism on survival among patients with completely resected NSCLC. Patients and Methods The Bim polymorphism was detected by polymerase chain reaction analysis. We measured overall survival (OS) and recurrence-free survival rates in 411 patients and postrecurrence survival (PRS) in 94 patients who experienced recurrence and received additional anticancer therapy. Results The Bim deletion polymorphism was detected in 61 patients (14.8%). OS rates were significantly lower for patients with the Bim deletion polymorphism than for those with the wild-type sequence. On multivariable analysis, the Bim deletion polymorphism was identified as an independent prognostic factor for OS (hazard ratio, 1.98; 95% CI, 1.17 to 3.36; P = .011). Among the 94 patients who experienced recurrence and were treated with anticancer therapy, patients with the Bim deletion polymorphism showed significantly poorer PRS than those with the wild-type sequence (median, 9.8 months v 26.9 months, respectively; P < .001). Multivariable analysis revealed that the Bim deletion polymorphism was an independent predictor of PRS (hazard ratio, 3.36; 95% CI, 1.75 to 6.47; P < .001). This trend remained apparent in subgroup analyses stratified by EGFR status, histology, and therapeutic modality. Conclusion The Bim deletion polymorphism is a novel indicator of shortened PRS among patients with recurrent NSCLC treated with anticancer therapy in the Asian population.

A Case of Splenic Metastasis of Ovarian Cancer Treated with CompleteLaparoscopic Splenectomy and Transvaginal Specimen Extraction

A 61-year-old woman was diagnosed with right inguinal lymph node and splenic metastasis of ovarian serous cystadenocarcinoma. We performed right inguinal lymph node dissection and total laparoscopic splenectomy in the supine position followed by transvaginal specimen extraction (TVSE). First, using three ports, we extracted the right inguinal lymph node. We repaired the posterior wall of the inguinal canal using a mesh plug. We added two ports and displaced the spleen from the retroperitoneum and lifted it using a snake retractor, disconnecting the hilum using an automatic suturing device. Next, the posterior wall of the vagina was intraperitoneally incised and an Alexis® laparoscopic system was inserted into the vagina. The cap maintained aeroperitoneum, a collection bag was inserted in the abdominal cavity via the vagina, and the spleen was collected. When the spleen was removed from the body, partial fragmentation of the organ was required in the bag. Organ fragmentation was performed only within the bag, and we made sure not to tear the bag. The vaginal wound was laparoscopically sutured. The patient had no operative complications and was able to actively ambulate at first day after surgery due to a slight postoperative pain. Total laparoscopic splenectomy with TVSE in the supine position may be a safe and feasible method for selected female patients. This technique enables minimally invasive surgery for female patients with splenic disease.

A case of splenic metastasis of ovarian cancer treated with complete laparoscopic splenectomy and transvaginal specimen extraction

A 61-year-old woman was diagnosed with right inguinal lymph node and splenic metastasis of ovarian serous cystadenocarcinoma. We performed right inguinal lymph node dissection and total laparoscopic splenectomy in the supine position followed by transvaginal specimen extraction (TVSE). First, using three ports, we extracted the right inguinal lymph node. We repaired the posterior wall of the inguinal canal using a mesh plug. We added two ports and displaced the spleen from the retroperitoneum and lifted it using a snake retractor, disconnecting the hilum using an automatic suturing device. Next, the posterior wall of the vagina was intraperitoneally incised. And an Alexis® laparoscopic system was inserted into the vagina. The cap maintained aeroperitoneum, a collection bag was inserted in the abdominal cavity via the vagina, and the spleen was collected. When the spleen was removed from the body, partial fragmentation of the organ was required in the bag. Organ fragmentation was performed only within the bag, and we made sure not to tear the bag. The vaginal wound was laparoscopically sutured. The patient had no operative complications and was able to actively ambulate at the first day after surgery due to a slight postoperative pain. Total laparoscopic splenectomy with TVSE in the supine position may be a safe and feasible method for selected female patients. This technique enables minimally invasive surgery for female patients with splenic disease.

Transvaginal Hybrid NOTES Procedure for Treatment of Gallstone Ileus.

Gallstone ileus is a rare mechanical bowel obstruction, and previously reported cases have been treated laparoscopically with good results. Although transvaginal hybrid NOTES without a minilaparotomy has been reported to decrease the incidence of surgical wound complications, to our knowledge, this procedure has not been used previously to treat gallstone ileus. We present a case of a 63-year-old woman who underwent transvaginal hybrid NOTES procedure for treatment of gallstone ileus. This case was admitted to our hospital following acute-onset abdominal pain and vomiting. We diagnosed gallstone ileus with cholecystoduodenal fistula by computed tomography and performed totally laparoscopic surgery using only three 5 mm abdominal ports with transvaginal specimen extraction and enterectomy. The patients postoperative course was uneventful, and laparoscopic cholecystectomy and fistula repair were performed 8 months after the initial surgery. The patient experienced additional pain relief and good cosmetic outcomes. In conclusion, using transvaginal hybrid NOTES may become a future option to minimize the invasiveness of other laparoscopic procedures.

Prognostic potential of the MDM2 309T>G polymorphism in stage I lung adenocarcinoma

The MDM2 protein plays an important role in the regulation of cell proliferation and apoptosis via ubiquitination and proteasome‐mediated degradation of p53. The genetic polymorphism rs2279744 (c.309T>G) of the MDM2 gene is reportedly associated with susceptibility and/or prognosis in various cancers. In this study, we investigated the risk factors for worse survival in patients with lung adenocarcinoma (AC). We examined the association between c.309T>G and the prognosis of lung cancer by retrospectively reviewing 453 lung cancer patients. We studied both, clinicopathological and genetic characteristics, including the c.309T>G, p53 Arg72Pro, EGFR, KRAS, and p53 mutations. Associations between these factors and survival outcome were analyzed using Cox proportional hazards models. The frequencies of MDM2 polymorphisms were T/T, 20.8%; T/G, 48.6%, and G/G, 30.7%. The overall survival (OS) of AC patients with pathological stage I disease and the MDM2 T/T genotype was significantly shorter than that of those with the T/G or G/G genotypes (P = 0.02). Multivariate analysis revealed that the MDM2 T/T genotype was an independent, significant prognostic factor (hazard ratio [HR] = 2.23; 95% confidence interval [CI]: 1.07–4.65; P = 0.03). The MDM2 T/T genotype was predictive of poorer survival in a Japanese population. Genotyping for this polymorphism might predict the clinical outcomes of stage I AC patients.