위암 환자와 건강 대조군 사이의 미생물총 및 세균유래 나노 소포체 분포의 차이 분석 : 이를 통한 위암 진단 알고리즘의 개발

Background and Objectives Viral and bacterial infections facilitate carcinogenesis in certain organs. For example, the secretion of virulence factors by Helicobacter pylori causes oxidative stress, chronic inflammation, and host DNA damage, resulting in carcinoma development. Recent studies indicated that changes of gut microbiota contribute to the development of chronic diseases including cancer, and bacteria-derived extracellular vesicles (EVs) are important for host-microbe communication. The aims of this study were to evaluate the composition of the microbiota between a gastric cancer group and control group and to develop a diagnostic algorithm for gastric cancer. Materials and Methods As the case group, the gastric cancer group included patients diagnosed with gastric cancer at Elwha Womans University Mokdong Hospital and who were admitted for surgery between September 2016 and September 2017. As a control group, patients without underlying disease who came to Haeundae Paik Hospital for medical checkup were evaluated. After matching with age and sex, finally 118 healthy control patients and 94 gastric cancer patients for urine samples, 163 healthy control patients and 73 gastric cancer patients for plasma samples, 120 healthy control patients and 67 gastric cancer patients for stool-EVs samples, and 116 healthy control patients and 63 gastric cancer patients for stool-bacteria samples were analyzed. Microbiota from the stools and EVs from the urine, plasma, and fecal samples were analyzed by 16S rDNA amplicon sequencing. The marker selection criteria were a sequencing depth greater than 1500, average greater than 0.005, t-test result less than 0.05, and fold-change of more than 2 among the markers found to differ from the control group. The final markers were selected using a stepwise selection method. We developed an algorithmic model by logistic regression and verified this model by internal validation. Results The composition of the microbiota differed significantly between the healthy control and gastric cancer groups. For urine samples, Proteobacteria (p) EVs were decreased (29.4 ± 13.5% vs. 38.3 ± 17.4% in the case vs. control groups, p 〈 0.01) and Firmicutes (p) EVs were increased in the cancer group (34.7 ± 29.4% vs. 29.4 ± 8.5%, p = 0.02). Particularly, Proteus (g) and Morganella (g) in the Proteobacteria (p) showed significant changes and were selected as markers. For plasma samples, Actinobacteria (p) EVs were decreased in the cancer group (9.3 ± 5.2% vs. 11.3 ± 7.3%, p 〈 0.01). Additionally, Micrococcus (g) in Actinobacteria was selected as a marker. For EVs from fecal samples, Proteobacteria (p) were decreased in the cancer group (9.4 ± 9.8% vs. 19.0 ± 17.2%, p 〈 0.01). Proteus (g) and Cupriavidus (g) in the Proteobacteria (p) were selected as markers. For bacteria from fecal samples, Cyanobacteria (p) were decreased in the cancer group (0.06 ± 0.4% vs. 0.4 ± 0.8%, p 〈 0.01). Particularly, YS2 (o) in the Cyanobacteria (p) was selected as a marker. For internal validation, the proposed algorithm using the selected markers was accurate based on the AUC values of 0.9324, 0.7672, 0.8878, and 0.8889 in the urine, plasma, EVs of the stool, and bacteria of the stool, respectively. Conclusions Taken together, these data revealed that there were significant differences in the distribution of microbiota and EVs between the healthy control and gastric cancer groups. The diagnostic algorithm developed using these results was particularly accurate when urine and stool samples were used. Further studies including external validation are needed.;배경 및 목적 각종 바이러스 및 세균성 감염은 특정 장기에서 암의 발생을 촉진한다. 예를 들면, 헬리코박터 균이 분비하는 독성 인자는 위 점막에 산화 스트레스, 만성 염증 상태를 야기하고, 결국 숙주인 인체의 DNA 손상을 유발하여 암을 일으키게 된다. 최근 연구에서는 이러한 인체내 미생물총의 변화가 암을 포함한 만성 질환의 발병에 기여하고, 특히 세균에서 유래한 나노 소포체는 숙주인 인체와 미생물 간의 신호 전달에 중요한 역할을 한다고 보고되고 있다. 따라서 본 연구에서는 위암 환자와 건강 대조군 사이의 미생물총 및 세균유래 나노 소포체의 분포를 비교 분석하고, 이를 통해 위암 진단을 위한 알고리즘을 개발해 보고자 하였다. 재료 및 방법 위암 환자군은 이화여대 목동병원에서 위암으로 진단받고 2016년 9월부터 2017년 9월까지 수술을 받기 위해 입원한 환자를 대상으로 하였으며, 건강 대조군은 해운대 백병원에서 건강 검진을 위해 내원한 동반 질환이 없는 환자를 대상으로 하였다. 최소한의 비뚤림 보정을 위해 나이와 성별이 차이 없도록 두 그룹을 짝지은 뒤, 최종적으로 소변 시료는 118명의 대조군과 94명의 위암 환자군에서, 혈장 시료는 163명의 대조군과 73명의 위암 환자군에서, 대변-소포체 분석 시료로는 120명의 대조군과 67명의 위암 환자군에서, 대변-세균 분석 시료로는 116명의 대조군과 63명의 위암 환자군에서 수집하였다. 미생물총은 대변 시료에서, 세균유래 나노 소포체는 소변, 혈장, 대변 시료에서 분석하였고, 16S rDNA를 염기서열 분석하는 기법으로 메타게놈 분석을 시행하였다. 한편, 알고리즘 개발을 위한 표지자 선택 기준으로는 시퀀싱 깊이가 1500 이상, 평균 분포 비율이 0.5% 이상, t 검정 결과 0.05 미만 및 대조군과의 차이가 2배 이상인 것으로 정하였고, 최종 표지자는 단계적 선택법을 사용하여 선택하였다. 로지스틱 회귀 분석을 이용하여 알고리즘 모델을 개발하고, 내부 검증을 통해 이 모델을 검증하였다. 결과 미생물총의 조성은 건강 대조군과 위암 환자 사이에 유의한 차이가 있었다. 소변 시료의 경우, 위암 환자군에서 Proteobacteria (p) 소포체의 감소 (위암 29.4 ± 13.5% 대 대조군 38.3 ± 17.4%, p < 0.01), Firmicutes (p) 소포체의 증가 (위암 34.7 ± 29.4% 대 대조군 29.4 ± 8.5%, p = 0.02)가 관찰되었다. 특히, Proteobacteria (p)의 Proteus (g)와 Morganella (g)는 뚜렷한 변화를 보여, 표지자로 선택되었다. 혈장 시료의 경우, 위암 환자에서 Actinobacteria (p) 소포체가 감소하였고 (위암 9.3 ± 5.2% 대 대조군 11.3 ± 7.3%, p < 0.01), 이 중 Micrococcus (g)가 표지자로 선택했다. 대변 시료에서 얻은 소포체의 경우, 위암 환자에서 Proteobacteria (p)가 감소하여 (위암 9.4 ± 9.8% 대 대조군 19.0 ± 17.2 %, p < 0.01), 이 중 Proteus (g)와 Cupriavidus (g)를 표지자로 선택했다. 대변 시료에서 분석한 미생물총의 경우, 위암 환자에서 Cyanobacteria (p)가 감소했고 (위암 0.06 ± 0.4% 대 대조군 0.4 ± 0.8%, p < 0.01), 그 중 YS2 (o)가 표지자로 선택되었다. 개발된 알고리즘을 내부 검증한 결과, 소변, 혈장, 대변의 소포체, 대변의 미생물총에서 각각 0.9324, 0.7672, 0.8878, 0.8889의 AUC 값을 보였다. 결론 본 연구에서는 건강한 대조군과 위암 환자군 사이의 미생물총과 세균유래 나노 소포체의 분포는 유의한 차이가 있음을 보여주었다. 이러한 결과를 사용하여 개발한 위암 진단 알고리즘은 소변과 대변 시료를 사용했을 때 특히 정확했으며, 향후 외부 검증을 포함한 추가 연구가 필요하다.I. Introduction 1 II. Materials 3 III. Methods 5 A. Bacteria and bacterial EVs separation 5 1. EVs from the urine, plasma, stool 5 2. Bacteria from the stool 5 B. DNA extraction and quantification 5 C. PCR amplification of the V3-V4 hypervariable regions of the 16S ribosomal RNA genes 6 D. Index PCR 7 E. Pre-sequencing quality control 8 F. Library Denaturing and Misses Sample Loading 9 G. Analysis of bacterial composition in the microbiota 10 H. Development of a predictive diagnostic model 10 I. Statistical analysis 11 IV. Results 12 A. Differences in distribution of microbiota and their secreted EVs between patients with gastric cancer and healthy controls 12 1. Metagenomic results of urine EVs 14 2. Metagenomic results of plasma EVs 18 3. Metagenomic results of stool EVs 22 4. Metagenomic results of stool bacteria 26 B. Development of diagnostic algorithm 30 1. Marker selection and proposal of algorithm 30 2. Results of internal validation 34 V. Discussion 39 VI. Conclusion 46 References 47 Abstract in Korean 5

위절제 범위 및 장문합 길이에 따른 당뇨 조절 효과 및 기전에 관한 동물실험

학위논문 (석사)-- 서울대학교 대학원 : 의학과 외과학 전공, 2016. 2. 이혁준.목적. 본 연구의 목적은 동물모델에서 위절제 범위 및 장문합 길이에 따른 당뇨 조절 효과 및 기전을 알아봄으로써 궁극적으로 당뇨를 동반한 위암 환자에서의 변형 위절제술의 가능성을 평가해 보고자 함이다. 방법. Sprague-Dawley 쥐에게3개월 간 고지방식이를 통해 비만형 포도당 불내성을 유발하였다. 총 네 그룹으로 나누어 1) 95%의 위절제 및 roux-limb(15cm), 담췌 limb(40cm)에 문합을 시행한 원거리 루와이 위공장문합술(long-limb RYGB, conventional RYGB, cRYGB) (n = 9), 2) 95%의 위절제 및 roux-limb(8cm), 담췌 limb(4cm)에 문합을 시행한 단거리 루와이 위공장문합술(short-limb RYGB, sRYGB) (n=9), 3) 70%의 위절제 및 roux-limb(8cm), 담췌 limb(4cm)에 문합을 시행한 기저부 보존 단거리 위공장문합술(fundus-sparing RYGB, fRYGB) (n=9) 또는 4) 가짜 수술 (sham operation) (n = 9)을 시행하였다. 수술 후 6주 뒤, 경구 당부하 검사를 시행하였고, 비만형 당뇨에 영향을 주는 장관 호르몬인 인슐린, 글루카곤-유사 펩티드-1 (GLP-1), 포도당-의존성 인슐린 펩티드 (GIP)와 그렐린을 분석하였다. 결과. sRYGB 군에서는 cRYGB 군과 비교하였을 때 섭취량은 차이가 없었으나 고혈당의 호전은 보이지 않았다. cRYGB 군은 통계학적으로 유의하게 섭취량 감소, 체중감소, 혈당 감소를 보였다 (p<0.05). 경구 당부하 검사를 하는 동안cRYGB 와 sRYGB 군에서, 통계적으로 유의하게 혈당이 30분까지 급격하게 증가하였고, 인슐린은 30분까지 급격하게 증가하였으나 통계적 유의성은 없었다. 총 GLP-1은 cRYGB군에서 30분에서 다른 군에 비해 높은 경향을 보였다. fRYGB 군은 가짜 수술군과 유사한 체중 변화와 혈당 변화를 보였고, 경구 당부하 검사와 GLP-1은 서서히 지속적으로 증가하는 모습을 보였으며, 인슐린과 GIP는 음식물의 위 배출 지연에 기인한 것으로 추정되는 낮은 최고점을 보였다. 결론. 본 연구에서 체중 감소 뿐만 아니라 당뇨의 호전을 이루기 위해서는 95%의 위 절제를 동반한 원거리 루와이 위공장문합술을 시행해야 함을 알 수 있었다. 이는 글루카곤 유사 펩티드-1의 급격한 증가와 관련되었을 것으로 추정된다.Purpose. The aim of this study was to examine the effect and mechanism of roux-en Y resectional gastric bypass (RYGB) on improvement of diabetes according to the length of anastomosis and the range of gastric resection in animal model. Methods. Sprague-Dawley rats were induced to obese glucose intolerance via high fat diet for 3 months. They were underwent long-limb RYGB (conventional RYGB, cRYGB) which was about 95% gastric resection with 15cm of roux-limb and 40cm of biliopancreatic (BP) limb (n = 9), short-limb RYGB which was about 95% gastric resection with 8cm of roux-limb and 4cm of BP limb (sRYGB) (n=9), fundus-sparing RYGB which was about 70% gastric resection with 8cm of roux-limb and 4cm of BP limb (fRYGB) (n=9) or sham operation (n = 9). After 6 weeks, oral glucose tolerance tests (OGTT) were performed, and gut hormone which can contribute to the obese diabetes including insulin, glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotrophic peptide (GIP), and ghrelin were analyzed. Results. In sRYGB group, there was no difference in food intake compared with cRYGB group, but improvement of hyperglycemia was not shown. The cRYGB group showed significantly decreased food intake, body weight, and random glucose (p<0.05). In the cRYGB and sRYGB groups, glucose was significantly steeply increased till 30 min and insulin was sharply increased till 30 min without significance during OGTT. Total GLP-1 was higher at 30 in the cRYBG than other groups without significance. fRYGB group showed similar change of body weight and random glucose compared with sham group and showed slowly increased pattern in OGTT and GLP-1 and lowest peak point in insulin and GIP due to time-lag caused by gastric stasis. Conclusion. We could identify that long-limb roux-en Y bypass with 95% gastric resection was needed to achieve not only the loss of body weight, but also improvement of diabetes through this experiment. This could be related with increase of GLP-1.Introduction 1 Materials and Methods 3 Results 9 Discussion 22 References 28 Abstract (Korean) 32Maste

Natural History of Gastric Cancer: Observational Study of Gastric Cancer Patients Not Treated During Follow-Up

Background: Understanding the natural progression of untreated gastric cancer is critical for determining the disease prognosis as well as treatment options and timing. The aim of this study is to analyze the natural history of gastric cancer. Patients and Methods: We included patients with gastric cancer who had not received any treatment and were staged using endoscopy/endoscopic ultrasonography and computed tomography on at least two follow-up visits during intervals of nontreatment. Tumor volumes were also measured in addition to the staging. Survival of each stage at diagnosis was also analyzed. Results: A total of 101 patients were included. The mean follow-up period was 35.1 ± 34.4 months. The gastric cancer doubling time was 11.8 months for T1 and 6.2 months for T4. The progression time from early gastric cancer to advanced gastric cancer was 34 months. It decreased as the stages advanced: from 34 months between tumor-nodes-metastasis stage I and II to 1.8 months between stage III and IV. No variable was identified as a risk factor for cancer progression. The 5-year survival rates of untreated patients were 46.2% in stage I and 0% in stage II, stage III, and stage IV. Conclusions: The progression and doubling times of gastric cancer shorten as the stages advance. Objective data reported in this study can be a critical factor in determining treatment timing and screening interval. © 2019, Society of Surgical Oncology

Inguinal hernia in preterms in neonatal intensive care units: Optimal timing of herniorrhaphy and necessity of contralateral exploration in unilateral presentation

Background: We sought to determine the optimal timing of IH repair in preterms and the need for routine contralateral exploration. Methods: Medical records of 3690 pediatric patients who underwent unilateral IH repair between January 1998 and December 2009 were reviewed. We assessed medical record review and telephone interviews. In total, 1990 patients were enrolled in the study. Early, early-delayed, and late repair were defined as herniorrhaphy performed within 7 days of diagnosis, later than 7 days of diagnosis, and after discharge from the NICU, respectively. Results: Of 1990 patients, 90 preterms and 1900 full-terms were included. Among these, 7, 11 and 72 preterm patients received early, early-delayed and late IH repairs, respectively. Preoperative incarceration and postoperative complication rates were not different, but the recurrence rate was higher in the early repair group. Two group analysis of early and early-delayed vs. late repairs indicated similar results. The rates of synchronous and metachronous bilateral IH (SBIH, MBIH) were observed to be higher and the diagnostic interval of MBIH was shorter in pre terms than in full-terms (35.6% vs. 15.9%, P &amp;amp;lt; 0.001; 12.2% vs. 6.3%, P &amp;amp;lt; 0.001; 5.2 vs. 41.8 months, P = 0.003). Conclusion: Our results indicate that IH repair is safe to perform in preterm babies in the NICU at a delayed or late stage since the preoperative incarceration and recurrence rates were not different. Contralateral exploration could be considered in preterms because the rates of SBIH and MBIH were significantly higher and the MBIH diagnosis interval was shorter than in full-terms. (C) 2018 Published by Elsevier Inc

Bariatric/Metabolic Surgery Induces Noticeable Changes of Microbiota and Their Secreting Extracellular Vesicle Composition in the Gut

Introduction: Microbial ecology is reported to be an important regulator of energy homeostasis and glucose metabolism. Microbes secrete extracellular vesicles (EVs) during their proliferation and death to communicate with other cells. To investigate the roles of gut microbiota in glucose metabolism, we analyzed serial changes of gut microbe and microbial EV composition before and after bariatric/metabolic surgery (BMS). Methods: Twenty-eight Wistar rats were fed on high-fat diet (HFD) to induce obesity and diabetes. Five of them compared with 5 rats fed on regular chow diet (RCD). Among the remaining 23 rats, Roux-en-Y gastric bypass (RYGB) (n = 10), sleeve gastrectomy (SG) (n = 10), or sham operation (n = 3) was randomly performed. Gut microbiota and EVs from fecal samples were analyzed by 16S rDNA amplicon sequencing. Results: The present study showed that microbial diversity was decreased in HFD-fed rats versus RCD-fed rats. In addition, BMS reversed glucose intolerance and microbial richness which were induced by HFD. In terms of microbiota and microbial EV composition, both RYGB and SG enhance the composition of phyla Proteobacteria, Verrucomicrobia, and their secreting EVs, but decrease phylum Firmicutes and its EVs. We tried to demonstrate specific genera showed a significant compositional difference in obesity/diabetes-induced rats compared with normal rats and then restored similarly toward normal rats’ level after BMS. At the genus level, Lactococcus, Ruminococcus, Dorea in Firmicutes(p), Psychrobacter in Proteobacteria(p), and Akkermansia in Verrucomicrobia(p) fit these conditions after BMS. Conclusion: We suggest that these genera are the candidates contributing to obesity and diabetes improvement mechanism after BMS. © 2019, Springer Science+Business Media, LLC, part of Springer Nature

Efficacy of Assessing Intraoperative Bowel Perfusion with Near-Infrared Camera in Laparoscopic Gastric Cancer Surgery

Background: Anastomotic leakage is a severe complication after gastric cancer surgery. Inadequate blood supply is regarded as an important risk factor. The aim of the study was to evaluate the feasibility and usefulness of intraoperative assessment of anastomotic vascular perfusion in gastric cancer surgery using near-infrared (NIR) camera imaging with indocyanine green (ICG)-enhanced fluorescence technique. Materials and Methods: From March 2015 to 2016, 30 patients undergoing laparoscopic gastrectomy for gastric cancer were prospectively evaluated. After completing the anastomosis, 2.5-5.0 mg of ICG was injected via peripheral veins. All anastomoses and resection margins were investigated using NIR camera to assess anastomotic perfusion. The assessment was performed using the adopted perfusion score of fluorescence activity, which ranged from 1 to 5 (1 = no uptake, and 5 = iso-fluorescent to all other segments). Results: Twenty-six distal gastrectomy (20 gastroduodenostomies, 6 gastrojejunostomies), 3 total gastrectomies (TG), and 1 pylorus-preserving gastrectomy were performed. The gap of visualization was 4.1 +/- 3.2 minutes (range, 2-15) after ICG injection. Twenty-three of 30 patients (76.7%) showed technically successful ICG visualization. Among gastroduodenostomies, the average scores for gastric and duodenal sides were 3.5 and 3.7. Among gastrojejunostomies, the average scores for gastric, jejunal, and duodenal stump sides were 3.5, 4.0, and 3.8 (jejunojejunostomy, 3.5). Among TG, the average scores for esophagojejunostomy, duodenal stump, and jejunojejunostomy were 3.7, 4.0, 4.0, and 4.7. One case of leakage occurred in this study. Other complications included fluid collection and stenosis in 1 patient each. Conclusions: This study showed intraoperative ICG angiography using NIR camera is feasible and provides imaging of anastomotic blood flow. Further studies are needed for practice

Prediction of Postoperative Mortality in Patients with Organ Failure After Gastric Cancer Surgery

Background: Scarce data are available on the characteristics of postoperative organ failure (POF) and mortality after gastrectomy. We aimed to describe the causes of organ failure and mortality related to gastrectomy for gastric cancer and to identify patients with POF who are at a risk of failure to rescue (FTR). Methods: The study examined patients with POF or in-hospital mortality in Seoul National University Hospital between 2005 and 2014. We identified patients at a high risk of FTR by analyzing laboratory findings, complication data, intensive care unit records, and risk scoring including Acute Physiology and Chronic Health Evaluation (APACHE) IV, Sequential Organ Failure Assessment (SOFA) score, and Simplified Acute Physiology Score (SAPS) 3 at ICU admission. Results: Among the 7304 patients who underwent gastrectomy, 80 (1.1%) were identified with Clavien–Dindo classification (CDC) grade ≥ IVa. The numbers of patients with CDC grade IVa, IVb, and V were 48 (0.66%), 11 (0.15%), and 21 (0.29%), respectively. Pulmonary failure (43.8%), surgical site complication (27.5%), and cardiac failure (13.8%) were the most common causes of POF and mortality. Cancer progression (100%) and cardiac events (45.5%) showed high FTR rates. In univariate analysis, acidosis, hypoalbuminemia, SOFA, APACHE IV, and SAPS 3 were identified as risk factors for FTR (P &amp;amp;lt; 0.05). Finally, SAPS 3 was identified as an independent predictive factor for FTR. Conclusions: Cancer progression and acute cardiac failure were the most lethal causes of FTR. SAPS 3 is an independent predictor of FTR among POF patients after gastrectomy. © 2020, Société Internationale de Chirurgie

Multicenter results of long-limb bypass reconstruction after gastrectomy in patients with gastric cancer and type II diabetes

Background/Objective: The number of gastric cancer and type II diabetes mellitus is increasing in Korea. Metabolic surgery could be extended to gastric cancer patients with type II diabetes, especially those who are expected to achieve long-term survival. This study aimed to investigate change of diabetic status in patients undergoing long-limb Roux-en-Y bypass reconstruction compared with conventional Billroth II after curative gastrectomy. In total, 130 patients from five university hospital centers underwent long-limb Roux-en Y reconstruction after radical distal gastrectomy. Methods: In the long-limb group, the length of biliopancreatic limbs was more than 80 cm, and the length of the Roux limb was more than 80 cm. The control group comprised 96 patients who underwent conventional Billroth II reconstruction after distal gastrectomy. Follow-up data at three, six, nine, and 12 months were compared between the two groups. Results: Fasting blood sugar (FBS) and hemoglobin (Hb) A1c levels decreased more significantly in the long-limb Roux-en-Y group (FBS: 28.8 mg/dL; HbA1c: 0.72%). However, decreases in body mass index, albumin, and hemoglobin did not differ significantly between the two groups. Diabetes control significantly improved in the long-limb group. In multivariate analysis, long-limb bypass reconstruction was the significant factor for glycemic outcomes. Conclusion: Roux-en-Y bypass with increased length of limbs after gastrectomy shows a favorable glycemic control for gastric cancer patients with type II diabetes without nutritional deficit and anemia. To obtain future perspectives, large-scale prospective studies with long-term outcomes are needed. (C) 2019 Asian Surgical Association and Taiwan Robotic Surgery Association. Publishing services by Elsevier B.V