Tumor-associated macrophage infiltration is highly associated with PD-L1 expression in gastric adenocarcinoma.

BACKGROUND: Programmed death ligand 1 (PD-L1) is a key protein upregulated by tumor cells to suppress immune responses. Tumor-associated macrophages (TAMs) play a major role in this immunosuppression, but the relationship between PD-L1 expression and TAMs remains unclear in gastric adenocarcinoma (GAC). We simultaneously examined expression of PD-L1 and TAMs in GAC. METHODS: We performed immunohistochemical staining for PD-L1, CD68 (pan-macrophage), and CD163 (M2-like macrophage) in 217 GAC samples using a tissue microarray. Expression of PD-L1 and CD68- and CD163-positive cells was evaluated using the Cytoplasmic V2.0 algorithm in Aperio ImageScope software, and logistic regression analysis was used to compare expression patterns between groups. RESULTS: Thirty-one samples (14%) were positive for PD-L1 expression. The mean (± standard error) rates of infiltration were 6.83 ± 0.38% for CD68-positive cells and 6.16 ± 0.29% for CD163-positive cells. The mean rate of CD163-positive cell infiltration was significantly higher in diffuse GAC than in intestinal GAC (diffuse n = 111, 6.91%; intestinal n = 91, 5.26%; p = 0.006), but the mean rate of CD68-positive cell infiltration was similar between these types (p = 0.38). The mean infiltration rates of CD68- and CD163-positive cells in PD-L1-positive GAC were significantly higher than in PD-L1-negative GAC (CD68 p = 0.0002; CD163 p < 0.0001). In multivariate logistic regression analyses, CD163-positive cell infiltration was associated with PD-L1 expression (odds ratio 1.13; 95% confidence interval 1.02-1.25; p = 0.021). CONCLUSION: M2-like macrophage infiltration is highly associated with PD-L1 expression in GAC cells, suggesting that macrophage infiltration can serve as a potential therapeutic target.

A Prospective, Multicenter Phase II Study of the Efficacy and Feasibility of 15-minute Panitumumab Infusion Plus Irinotecan for Oxaliplatin- and Irinotecan-refractory, KRAS Wild-type Metastatic Colorectal Cancer (Short Infusion of Panitumumab Trial).

BACKGROUND: In some recently updated clinical guidelines, the fully humanized monoclonal antibody panitumumab, combined with irinotecan, has been recommended as an optional third-line chemotherapy for KRAS wild-type metastatic colorectal cancer (mCRC). The present prospective, multicenter phase II study evaluated the effectiveness and safety of short 15-minute panitumumab infusions. PATIENTS AND METHODS: From January 2011 to December 2011, patients with KRAS wild-type mCRC were enrolled at 8 centers. The key eligibility criteria were age ≥ 20 years and resistance or intolerance to irinotecan, fluoropyrimidine, and oxaliplatin. All patients received 6 mg/kg of panitumumab and 150 mg/m2 or the previous tolerated dose of irinotecan, biweekly, until disease progression or unacceptable toxicity. The initial panitumumab infusion was 60 minutes, followed by a 30-minute infusion and then 15-minute infusions. The primary endpoint was the confirmed response rate using Response Evaluation Criteria In Solid Tumors, version 1.0. The secondary endpoints were progression-free survival, overall survival, and toxicity. The trial is registered in the University Hospital Medical Information Network Clinical Trials Registry (UMIN no. 000004647). RESULTS: Of the 43 patients, the median age was 62 years (range, 32-75 years), 58% were male, and the Eastern Cooperative Oncology Group performance status was 0 to 1. The total response rate was 37.2% (95% confidence interval [CI], 23.0-53.3), and the confirmed response rate was 18.6% (95% CI, 8.4-33.4). The median progression-free and overall survival were 5.8 months (95% CI, 3.3-8.4 months) and 13.6 months (95% CI, 10.8-16.5 months), respectively. The most frequent grade 3/4 toxicities were anorexia (12%), leukopenia (9%), and neutropenia (9%). Nine patients did not reach the 15-minute infusion, primarily because of disease progression. No infusion-related reactions were observed. CONCLUSION: The short 15-minute panitumumab infusion regimen was well tolerated, without compromising safety or efficacy in patients with KRAS wild-type, oxaliplatin- and irinotecan-refractory mCRC.

Galectin-3 expression is prognostic in diffuse type gastric adenocarcinoma, confers aggressive phenotype, and can be targeted by YAP1/BET inhibitors.

BACKGROUND: Overexpression of Galectin-3 (Gal-3), a ß-galactoside binding protein, has been noted in many tumour types but its functional significance and clinical utility in gastric adenocarcinoma (GAC) are not well known. METHODS: We studied 184 GAC patients characterised by histologic grade, sub-phenotypes (diffuse vs intestinal), and ethnicity (Asians vs North Americans). Immunohistochemistry was performed to assess the expression of Gal-3 in human GACs and we correlated it to the clinical outcomes. Cell proliferation, invasion, co-immunoprecipitation and kinase activity assays were done in genetically stable Gal-3 overexpressing GC cell lines and the parental counterparts to delineate the mechanisms of action and activity of inhibitors. RESULTS: Most patients were men, Asian, and had a poorly differentiated GAC. Gal-3 was over-expressed in poorly differentiated (P=0.002) tumours and also in diffuse sub-phenotype (P=0.02). Gal-3 overexpression was associated with shorter overall survival (OS; P=0.026) in all patients. Although, Gal-3 over-expression was not prognostic in the Asian cohort (P=0.337), it was highly prognostic in the North American cohort (P=0.001). In a multivariate analysis, Gal-3 (P=0.001) and N-stage (P=<0.001) were independently prognostic for shorter OS. Mechanistically, Gal-3 induced c-MYC expression through increasing RalA activity and an enhanced YAP1/RalA/RalBP complex to confer an aggressive phenotype. YAP1/BET bromodomain inhibitors reduced Gal-3-mediated aggressive phenotypes in GAC cells. CONCLUSIONS: Gal-3 is an independent prognostic marker of shorter OS and a novel therapeutic target particularly in diffuse type GAC in North American patients.

Potentially functional variants in the core nucleotide excision repair genes predict survival in Japanese gastric cancer patients.

Functional genetic variants of DNA repair genes may alter the host DNA repair capacity, and thus influence efficiency of therapies. We genotyped eight potentially functional single nucleotide polymorphisms (SNPs) in genes (i.e. ERCC1, XPA, XPC, XPD and XPG) involved in the nucleotide excision repair (NER) pathway in 496 Japanese gastric cancer patients, and assessed overall survival and recurrence-free survival. The combined effects of risk genotypes of these eight SNPs in Japanese patients were further replicated in 356 North-American gastric cancer patients. In Japanese patients, we found that the XPC rs2228000 TT genotype was associated with shorter overall survival [hazards ratio (HR) = 1.75, 95% confidence interval (95% CI) = 1.07-2.86] and recurrence-free survival (HR = 2.17, 95% CI = 1.19-3.95), compared with CC/CT genotypes, and the XPG rs17655 CC genotype was associated with shorter overall survival (HR = 1.60, 95% CI = 1.08-2.36), compared with GG/CG genotypes. The number of observed risk genotypes in the combined analysis was associated with shorter overall survival and recurrence-free survival in a dose-response manner (P(trend) = 0.006 and P(trend) < 0.000) in Japanese patients; specifically, compared with those with ≤1 risk genotypes, those with ≥2 risk genotypes showed markedly shorter overall survival (HR = 1.79, 95% CI = 1.18-2.70) and recurrence-free survival (HR = 2.80, 95% CI = 1.66-4.73). The association between ≥2 risk genotypes and shorter overall survival was not significant (HR = 1.26, 95% CI = 0.82-1.94) in North-American patients, but the trends were similar in these two groups of patients. These data show that functional SNPs in NER core genes may impact survival in Japanese gastric cancer patients.

Significant changes in the intestinal environment after surgery in patients with colorectal cancer.

BACKGROUND: There have been very few detailed reports of the intestinal environment after surgical treatment for colorectal cancer (CRC). We analysed faecal microbiota, organic acids and pH to investigate the influence of colorectal surgery on the intestinal environment. METHODS: Faecal samples from 81 CRC patients were collected before the start of pre-operative preparation the day before surgery, as well as 7 days or more after surgery. Thirteen groups of intestinal microbiota, eight types of organic acids, and pH were measured using 16S rRNA-targeted reverse transcription-quantitative PCR, high-performance liquid chromatography and a pH meter, respectively. RESULTS: Total bacterial counts (10.3 ± 0.6 vs. 9.4 ± 1.2 log10 cells/g; p < 0.001) and the numbers of 6 groups of obligate anaerobes were significantly decreased after surgery. In contrast, the populations of Enterobacteriaceae, Enterococcus, Staphylococcus and Pseudomonas were significantly increased. Post-operatively, the concentration of total organic acids was lower (77.9 ± 40.1 vs. 50.1 ± 37.0 µmol/g; p < 0.001) than the pre-operative concentration, and a significant reduction in short-chain fatty acids (SCFAs) was observed. CONCLUSION: Significant changes in the intestinal environment, including marked decreases in obligate anaerobes, increases in pathogenic bacteria, and reductions in SCFAs, were detected after surgery for CRC.

Changes of the intestinal microbiota, short chain fatty acids, and fecal pH in patients with colorectal cancer.

BACKGROUND: New molecular biology-based methods of bacterial identification are expected to help elucidate the relationship between colorectal cancer (CRC) and intestinal microbiota. Although there is increasing evidence revealing the potential role of microbiota in CRC, it remains unclear whether microbial dysbiosis is the cause or the result of CRC onset. AIM: We investigated the changes of intestinal environments in CRC or adenoma. METHODS: We analyzed 13 groups of microbiota, 8 types of organic acids, and pH in feces obtained from the following 3 groups: individuals with CRC, adenoma, and non-adenoma. Ninety-three patients with CRC and 49 healthy individuals (22 with adenoma and 27 without adenoma) were enrolled. RESULTS: The counts of total bacteria (10.3 ± 0.7 vs. 10.8 ± 0.3 log10 cells/g of feces; p < 0.001), 5 groups of obligate anaerobe, and 2 groups of facultative anaerobes were significantly lower in the CRC group than in the healthy individuals. While the concentrations of short chain fatty acids (SCFAs) were significantly decreased in the CRC group, the pH was increased in the CRC group (7.4 ± 0.8 vs. 6.9 ± 0.6; p < 0.001). Comparison among the CRC, adenoma, and non-adenoma groups revealed that fecal SCFAs and pH in the adenoma group were intermediate to the CRC group and the non-adenoma group. Within the CRC group, no differences in microbiota or organic acids were observed among Dukes stages. CONCLUSIONS: CRC patients showed significant differences in the intestinal environment, including alterations of microbiota, decreased SCFAs, and elevated pH. These changes are not a result of CRC progression but are involved in CRC onset.

Pre-operative diagnosis and successful surgery of a strangulated internal hernia through a defect in the falciform ligament: a case report.

INTRODUCTION: Internal hernia within the falciform ligament is exceedingly rare. A literature search revealed only 14 cases of internal herniation of the small bowel through a congenital defect of the falciform ligament, most of which were found intra-operatively. CASE PRESENTATION: A 77-year-old Japanese woman presented to our emergency department with sudden hematemesis, occurring at least four to five times over a 12-hour period. No ulcer or gastrointestinal bleeding was detected on gastroendoscopy. A 40mm mass in the inferior lobe of the right lung was found on a chest X-ray, and our patient's symptoms were therefore initially ascribed to aspirated blood from lung tumor-associated hemoptysis. However, our patient continued to show signs of severe abdominal pain and decreased urine output despite aggressive hydration, leading her examining physicians to search for a possibly severe, occult abdominal pathology. On emergent computed tomography imaging, we found an acute strangulated internal hernia within the falciform ligament. Diagnosis was made by helical computed tomography, permitting rapid surgical intervention. CONCLUSIONS: Our findings on computed tomography imaging assisted with the pre-operative diagnosis and enabled us to make a rapid surgical intervention. Early diagnosis may help preclude significant strangulation with unnecessary resection.

Invariant natural killer T cells infiltrate intestinal allografts undergoing acute cellular rejection.

Immunological responses in human intestinal allografts are poorly understood and accurate diagnosis of acute cellular rejection remains difficult. Here, human intestinal allografts were analyzed by multi-color quantitative fluorescent immunohistochemical morphometry in order to monitor the clinical course of rejection. Morphometry gave two-dimensional plots based on size and circularity, and identified phenotypes of individual cells infiltrating the allograft by fluorescent staining. Using this method, invariant TCRVα24(+) NKT (iNKT) cells were observed in the intestinal allograft during rejection. Because these were not identified in the normal donor intestine before surgery, this finding was considered to be a signature of acute cellular rejection of the intestinal allograft. Infiltrating iNKT cells released IL-4 and IL-5, Th2-related cytokines that antagonize the Th1 responses that induce acute cellular rejection. Histological observation suggested eosinophilic enteritis in the mucosa with elevation of IL-4 and IL-5. In conclusion, iNKT cells were recruited to the intestine; however, because higher levels of IL-4 and IL-5 may contribute to eosinophilic enteritis, timely steroid administration is recommended for allograft injury due to enteritis, as well as acute cellular rejection.

Paraneoplastic neurological syndromes associated with gastric cancer: a case report and review of the literature.

Paraneoplastic neurological syndromes (PNS) are associated with small-cell lung cancer, breast and gynecological cancers. We describe a gastric neoplasm presented with neurological symptoms. A 74-year-old male presented with tonic-clonic seizures. Initial investigations were normal; however, brain magnetic resonance imaging showed abnormal signal intensity in the hippocampi. A diagnosis of PNS was suspected. The patient was then diagnosed with a gastric neuroendocrine carcinoma with N-type voltage-gated calcium channel antibodies. The neurological impairments improved after the primary was resected and the patient remains free of cancer and paraneoplastic syndrome. We reviewed 10 cases of PNS associated with gastric cancer and found several characteristics: (1) older men, (2) neuroendocrine component or predominance, (3) oncological outcome for patients with PNS is better than for patients without PNS, and (4) neurological impairment is diagnosed 6 months prior to the diagnosis of gastric malignancy. In conclusion, elderly men with symptoms suggestive of PNS should be investigated for a gastric neuroendocrine malignancy.

Functional outcome, quality of life, and efficacy of probiotics in postoperative patients with colorectal cancer.

PURPOSE: We investigated the functional outcome and health-related quality of life (QOL) of patients who underwent a surgical resection of colorectal cancer, and reviewed the efficacy of probiotics for improving bowel function. METHODS: A questionnaire was mailed to 193 patients. Questionnaires contained the Medical Outcomes Study Short-Form 36-Item Health Survey and the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire C30 as QOL scores, the Wexner incontinence score, and original questionnaire items about bowel functions. Probiotics, containing Bacillus natto and Lactobacillus acidophilus, were given to 77 patients for 3 months; after 3 months of treatment, the same questionnaire was administered. The results were analyzed by location of the resected cancer: rectal, colonic, right, and left. RESULTS: In the rectal group, defecation frequency, anal pain, and the Wexner score were significantly worse than in the colonic group. In the right group, the fecal form was looser and nighttime defecation frequency was higher than those of the left group. Three items in the QOL score of the right group were significantly worse compared with the left group. Functional outcome including defecation frequency, feeling of incomplete defecation, and five items in the QOL score were significantly improved after taking probiotics. Improvement in functional outcome and/or QOL was observed in all groups. CONCLUSIONS: Not only rectal resection but also rightside colectomy affected bowel dysfunction. Probiotics could be an effective treatment for improvement in functional outcome and QOL after colorectal resection.

A new laparoscopic surgical procedure to achieve sufficient mesorectal excision in upper rectal cancer.

Objective. Mesorectal excision corresponding to the location of a tumor, termed tumor-specific mesorectal excision (TSME), is commonly performed for resection of upper rectal cancer. We devised a new laparoscopic procedure for sufficient TSME with rectal transection followed by mesorectal excision. Operative Technique. After mobilization of the sigmoid colon and ligation of inferior mesenteric vessels, we dissected the mesorectum along the layer of the planned total mesorectal excision. The rectal wall was carefully separated from the mesorectum at the appropriate anal side from the tumor. After the rectum was isolated and transected using an endoscopic linear stapler, the rectal stump drew immediately toward the anal side, enabling the mesorectum to be identified clearly. In this way, sufficient TSME can be performed easily and accurately. This technique has been successfully conducted on 19 patients. Conclusion. This laparoscopic technique is a feasible and reliable procedure for achieving sufficient TSME.

Development of a simple model for predicting need for surgery in patients who initially undergo conservative management for adhesive small bowel obstruction.

BACKGROUND: Among patients with adhesive small bowel obstruction (ASBO) initially managed with a conservative strategy, predicting risk of operation is difficult. METHODS: We investigated ASBO patients at 2 different periods to derive and validate a clinical prediction model for risk of operation. RESULTS: One hundred fifty-four patients were enrolled into the derivation cohort and 96 into the validation cohort. Based on the derived scoring, including age > or =65 years, presence of ascites, and gastrointestinal drainage volume >500 mL on day 3, each patient was classified into 1 of 4 risk classes from low risk to high risk. When applied to the validation cohort, the positive predictive value (PPV) for operation in the high-risk class was 72%, while the negative predictive value (NPV) in the low-risk class was 100% with high sensitivity (100%) and specificity (96%). CONCLUSIONS: The prediction model performs well for risk stratification of need for surgical intervention following conservative strategy among ASBO patients.

All-trans-retinoic acid attenuates radiation-induced intestinal fibrosis in mice.

BACKGROUND: Intestinal fibrosis leading to severe bowel dysmobility or obstruction is a troublesome adverse effect of abdominal or pelvic radiation therapy. We have recently reported that all-trans-retinoic acid (ATRA) prevents radiation- or bleomycin-induced lung fibrosis. Here, we examined the impact of ATRA on the mouse model of radiation-induced intestinal fibrosis. MATERIALS AND METHODS: We evaluated the histology of late radiation fibrosis in surgical samples. We then performed histological examinations and quantitative measurements of mRNA of interleukin-6 and transforming growth factor-beta(1) in intestinal tissues of irradiated mice with or without intraperitoneal administration of ATRA and investigated the effect of ATRA on the transdifferentiation and the production of collagen of irradiated human intestinal fibroblasts. RESULTS: Human samples of late radiation enteritis showed thickened submucosa and serosa, consistent with mouse model. Administration of ATRA attenuated irradiation-induced intestinal fibrosis and reduced mRNA of interleukin-6 and transforming growth factor-beta(1). In vitro studies disclosed that ATRA suppressed the transdifferentiation of irradiated intestinal fibroblasts and diminished the production of collagen from the cells. CONCLUSIONS: Our findings indicate that ATRA ameliorates irradiation-induced intestinal fibrosis. ATRA could be a novel approach in the treatment of fibrosis associated with chronic radiation enteritis.

Long-term cure in surgery for extrarectal pelvic recurrence of rectal cancer.

PURPOSE: Survival benefit of radical surgery for locally recurrent rectal cancer depends on whether disease is cured rather than whether death is delayed. Cured patients gain decades of life and are spared from sufferings with recurrence. Unfortunately, the majority of patients undergoing surgery, particularly those with extrarectal pelvic recurrence, have poor outcomes with occult disseminated disease. This study was designed to identify which of these patients are curable. METHODS: Of 61 patients with pelvic recurrence treated by radical reexcision more than nine years before, 36 patients whose initial surgery was abdominoperineal resection were examined retrospectively. We used the logistic regression and Gamel-Boag regression models to estimate curability and identify predictors of cure. RESULTS: Ten patients survived five years and seven survived ten years. The cumulative disease-specific mortality curve leveled off 6.5 years after reexcision and remained at 74 percent (95 percent confidence interval, 60-89), indicating that the remaining 26 percent are curable. This value is comparable with the 23 percent curability estimated by the Gamel-Boag model, which also found that the disease-free interval from the initial surgery to the first recurrence is the best predictor of cure (P = 0.005). Of 11 patients with disease-free interval three years or more, 6 survived ten years, whereas 8 of 9 patients with disease-free interval less than one year died of second recurrence within three years of reexcision. CONCLUSIONS: Even patients with extrarectal pelvic recurrence may have isolated disease that is amenable to complete eradication. As a biologic marker, the disease-free interval serves to predict curability and may distinguish isolated disease from occult disseminated disease.

A new technique to achieve sufficient mesorectal excision in upper rectal cancer.

BACKGROUND: Appropriate partial mesorectal excision (PME) is extremely important for prevention of local recurrence even in upper rectal cancer. However, it is not always easy to conduct PME in the narrow pelvic cavity. We devised a new surgical technique that involves a rectal transection followed by PME. METHODS: After rectal mobilization in the layer targeted for total mesorectal excision, only the rectal wall was bluntly dissected at an appropriate distance from the tumor. Initial transection of the rectum allows drawing the rectum toward the anal side so that the mesorectum can be confirmed with a good visual field. Excision of the mesorectum was easy, and it could be resected in a short time. RESULTS: This technique was conducted on seven patients with upper rectal cancer and on four patients with rectosigmoid cancer. Separation of the rectal wall was comparatively easy, and we had no incidence of wall injury. The average distance from the rectal stump to the distal mesorectum in the freshly resected specimen was 15 mm, indicating satisfactory PME. CONCLUSIONS: This easily performed method is a promising procedure for achieving sufficient PME in upper rectal cancer.

The administration of naked plasmid DNA into the liver induces antitumor innate immunity in a murine liver metastasis model.

BACKGROUND: Gene therapy is a promising strategy against advanced cancer; however, the safety of viral vectors and the effectiveness of non-viral vectors have not yet been established. Recently, a hydrodynamics-based procedure was reported to be an effective and safe method to deliver and transduce DNA into the liver. Herein, we propose a strategy for liver metastasis by a hydrodynamics-based procedure to deliver naked non-coding plasmid DNA (pDNA) into the liver as an immunocompetent organ. METHODS AND RESULTS: Mice received a rapid intravenous (i.v.) injection of naked pDNA in a large volume of saline (0.1 ml/g body weight). The single administration of a naked non-coding pDNA by the hydrodynamics-based procedure before tumor cell inoculation strongly suppressed liver metastasis formation. However, the usual i.v. injection (200 microl/body) of the same dose of naked pDNA could not suppress liver metastasis formation. Following the methylation of CpG sequences within the pDNA using CpG methylase, injection of the methylated pDNA by the hydrodynamics-based procedure could not suppress liver metastasis formation. Gadolinium chloride pretreatment did not interfere with this antitumor effect, but anti-asialo GM1 antiserum treatment did. These findings indicated that natural killer (NK) cells, not Kupffer cells, were involved in this antitumor effect. The NK cytotoxic activities of liver mononuclear cells were strongly enhanced after receiving a naked pDNA by the hydrodynamics-based procedure. CONCLUSIONS: These observations suggest that unmethylated CpG motifs in pDNA stimulated immune cells, resulting in the activation of NK cells in the liver to suppress liver metastases in a murine model.

Pancreas-sparing tumor enucleation for pancreatic mucinous cystic neoplasms: experience with two patients.

BACKGROUND: Because mucinous cystic neoplasms (MCNs) occur in the body and tail of the pancreas, distal pancreatectomy has been conventionally performed. However, enucleation can be adopted in selected patients, preserving the pancreatic parenchyma. METHODS: We experienced two patients with MCN who underwent pancreatic tumor enucleation. Case 1 involved a very large MCN, 23 cm across. Connective tissues between the tumor and the pancreatic parenchyma were not dense, so it was relatively easy to perform pancreatic cyst resection. Case 2 involved a MCN, 5 cm across, located next to the body of the pancreas. Fibrotic changes were so dense that it was difficult to separate the tumor from the pancreatic parenchyma. Careful and gentle dissection enabled pancreas-sparing enucleation without injury to the cyst wall. RESULTS: Enucleation of MCNs were performed successfully, preserving the pancreatic parenchyma. No complications were observed in either case. CONCLUSIONS: It is important to adopt the appropriate surgical procedure for MCN, considering the balance between radical resection and preservation of pancreatic function. Although careful attention should be paid to the assessment of malignant potential in each case of MCN, pancreas-sparing tumor enucleation can be considered as one of the treatment options in selected patients.