Histology of metastatic colorectal cancer in a lymph node.

BACKGROUND: A primary colorectal cancer (CRC) tumor can contain heterogeneous cancer cells. As clones of cells with different properties metastasize to lymph nodes (LNs), they could show different morphologies. Cancer histologies in LNs of CRC remains to be described. METHODS: Our study enrolled 318 consecutive patients with CRC who underwent primary tumor resection with lymph node dissection between January 2011 and June 2016. 119 (37.4%) patients who had metastatic LNs (mLNs) were finally included in this study. Cancer histologies in LNs were classified and compared with pathologically diagnosed differentiation in the primary lesion. The association between histologies in lymph node metastasis (LNM) and prognosis in patients with CRC was investigated. RESULTS: The histologies of the cancer cells in the mLNs were classified into four types: tubular, cribriform, poorly differentiated, and mucinous. Same degree of pathologically diagnosed differentiation in the primary tumor produced various histological types in LNM. In Kaplan-Meier analysis, prognosis was worse in CRC patients with moderately differentiated adenocarcinoma who had at least some mLN also showing cribriform carcinoma than for those whose mLNs all showed tubular carcinoma. CONCLUSIONS: Histology in LNM from CRC might indicate the heterogeneity and malignant phenotype of the disease.

Laparoscopic distal gastrectomy for synchronous adenocarcinoma, diffuse large B cell lymphoma and gastrointestinal stromal tumor in the stomach: a case report.

BACKGROUND: Synchronous lymphoma and adenocarcinoma are occasionally detected in the stomach. Gastrointestinal stromal tumor (GIST) and adenocarcinoma are sometimes seen simultaneously in the stomach. However, we rarely observe synchronous adenocarcinoma, lymphoma, and GIST in the stomach, and there are few reports on cases with these three lesions. CASE PRESENTATION: This is a case report of a 71-year-old man who had a laparoscopic distal gastrectomy and lymphadenectomy for three gastric tumors. Preoperative diagnoses were early adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma in the stomach, but final diagnosis was synchronous adenocarcinoma, diffuse large B cell lymphoma (DLBCL), and GIST. Helicobacter pylori (H. pylori) is highly involved in the development of DLBCL and MALT lymphoma in the stomach. Gastric adenocarcinoma is partially involved in chronic gastritis with atrophy and intestinal metaplasia caused by H. pylori infection. Indeed, a rapid urease test was found positive in this case. Therefore, we prescribed medicine to eliminate H. pylori after gastrectomy. CONCLUSION: This is the first case report where a patient underwent minimally invasive laparoscopic gastrectomy for synchronous adenocarcinoma, DLBCL and GIST in the stomach, although one patient who underwent open gastrectomy for synchronous adenocarcinoma, MALT lymphoma and GIST was previously reported.

Tumor RNA transfected DCs derived from iPS cells elicit cytotoxicity against cancer cells induced from colorectal cancer patients in vitro.

Significant efficacy of induced pluripotent stem cells (iPSCs) in generating DCs for cancer vaccine therapy was suggested in our previous studies. In clinical application of DC vaccine therapy, however, few DC vaccine systems have shown strong clinical response. To enhance immunogenicity in the DC vaccine, we transfected patient-derived iPSDCs with in vitro transcriptional RNA (ivtRNA), which was obtained from tumors of three patients with colorectal cancer. We investigated iPSDCs-ivtRNA which were induced by transfecting ivtRNA obtained from tumors of three colorectal cancer patients, and examined its antitumor effect. Moreover, we analyzed neoantigens expressed in colorectal cancer cells and examined whether iPSDCs-ivtRNA induced cytotoxic T lymphocytes (CTLs) against the predicted neoantigens. CTLs activated by iPSDCs-ivtRNA exhibited cytotoxic activity against the tumor spheroids in all three patients with colorectal cancer. Whole-exome sequencing revealed 1251 nonsynonymous mutations and 2155 neoantigens (IC50 < 500 nM) were predicted. For IFN-γ ELISPOT assay, these candidate neoantigens were further prioritised and 12 candidates were synthesized. IFN-γ ELISPOT assay revealed that the CTLs induced by iPSDCs-ivtRNA responded to one of the candidate neoantigens. In vitro CTLs obtained by transfecting tumor-derived RNA into iPSDCs derived from three patients with colorectal cancer showed potent tumor-specific killing effect.

Adenovirus Vectors Expressing Eight Multiplex Guide RNAs of CRISPR/Cas9 Efficiently Disrupted Diverse Hepatitis B Virus Gene Derived from Heterogeneous Patient.

Hepatitis B virus (HBV) chronically infects more than 240 million people worldwide, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Genome editing using CRISPR/Cas9 could provide new therapies because it can directly disrupt HBV genomes. However, because HBV genome sequences are highly diverse, the identical target sequence of guide RNA (gRNA), 20 nucleotides in length, is not necessarily present intact in the target HBV DNA in heterogeneous patients. Consequently, possible genome-editing drugs would be effective only for limited numbers of patients. Here, we show that an adenovirus vector (AdV) bearing eight multiplex gRNA expression units could be constructed in one step and amplified to a level sufficient for in vivo study with lack of deletion. Using this AdV, HBV X gene integrated in HepG2 cell chromosome derived from a heterogeneous patient was cleaved at multiple sites and disrupted. Indeed, four targets out of eight could not be cleaved due to sequence mismatches, but the remaining four targets were cleaved, producing irreversible deletions. Accordingly, the diverse X gene was disrupted at more than 90% efficiency. AdV containing eight multiplex gRNA units not only offers multiple knockouts of genes, but could also solve the problems of heterogeneous targets and escape mutants in genome-editing therapy.

Construction of adenovirus vectors simultaneously expressing four multiplex, double-nicking guide RNAs of CRISPR/Cas9 and in vivo genome editing.

Simultaneous expression of multiplex guide RNAs (gRNAs) is valuable for knockout of multiple genes and also for effective disruption of a gene by introducing multiple deletions. We developed a method of Tetraplex-guide Tandem for construction of cosmids containing four and eight multiplex gRNA-expressing units in one step utilizing lambda in vitro packaging. Using this method, we produced an adenovirus vector (AdV) containing four multiplex-gRNA units for two double-nicking sets. Unexpectedly, the AdV could stably be amplified to the scale sufficient for animal experiments with no detectable lack of the multiplex units. When the AdV containing gRNAs targeting the H2-Aa gene and an AdV expressing Cas9 nickase were mixed and doubly infected to mouse embryonic fibroblast cells, deletions were observed in more than 80% of the target gene even using double-nicking strategy. Indels were also detected in about 20% of the target gene at two sites in newborn mouse liver cells by intravenous injection. Interestingly, when one double-nicking site was disrupted, the other was simultaneously disrupted, implying that two genes in the same cell may simultaneously be disrupted in the AdV system. The AdVs expressing four multiplex gRNAs could offer simultaneous knockout of four genes or two genes by double-nicking cleavages with low off-target effect.

Highly multiplex guide RNA expression units of CRISPR/Cas9 were completely stable using cosmid amplification in a novel polygonal structure.

BACKGROUND: Genome editing using the CRISPR/Cas9 system is now well documented in basic studies and is expected to be applied to gene therapy. Simultaneous expression of multiplex guide RNA (gRNA) and Cas9/Cas9 derivative is attractive for the efficient knockout of genes and a safe double-nicking strategy. However, such use is limited because highly multiplex gRNA-expressing units are difficult to maintain stably in plasmids as a result of deletion via homologous recombination. METHODS: Lambda in vitro packaging was used instead of transformation for the construction and preparation of large, cos-containing plasmid (cosmid). Polymerase chain reaction fragments containing multiplex gRNA units were obtained using the Four-guide Tandem method. Transfection was performed by lipofection. RESULTS: We constructed novel cosmids consisting of linearized plasmid-DNA fragments containing up to 16 copies of multiplex gRNA-expressing units as trimer or tetramer (polygonal cosmids). These cosmids behaved as if they were monomer plasmids, and multiplex units could stably be maintained and amplified with a lack of deletion. Surprisingly, the deleted cosmid was removed out simply by amplifying the cosmid stock using lambda packaging. The DNA fragments containing multiplex gRNA-units and Cas9 were transfected to 293 cells and were found to disrupt the X gene of hepatitis B virus by deleting a large region between the predicted sites. CONCLUSIONS: We present a simple method for overcoming the problem of constructing plasmids stably containing multiplex gRNA-expressing units. The method may enable the production of very large amounts of DNA fragments expressing intact, highly-multiplex gRNAs and Cas9/Cas9 derivatives for safe and efficient genome-editing therapy using non-viral vectors.

Neoadjuvant Chemotherapy with Docetaxel, Cisplatin and S-1 for Resectable Advanced Esophageal Cancer.

BACKGROUND/AIM: Although the efficacy is limited, standard therapy for Stage II/III esophageal cancer in Japan includes neoadjuvant chemotherapy with cisplatin plus 5-fluorouracil. A phase II trial was conducted on patients with resectable advanced esophageal cancer obtaining neoadjuvant chemotherapy with docetaxel, cisplatin plus S-1 (DCS). PATIENTS AND METHODS: A total of 40 patients were enrolled, each treated by the following DCS regimen: docetaxel 40 mg/m2, cisplatin 60 mg/m2 on day 1, and S-1 80 mg/m2 on days 1-14, repeated every four weeks, for a maximum of three cycles. RESULTS: Clinical response rate was 76% and the pathological response rate (Grade 2/3) was 33%. Hematological toxicities of Grade 3/4 were leukopenia 50%, neutropenia 68%, and febrile neutropenia 18%. CONCLUSION: Neoadjuvant chemotherapy with DCS is a feasible therapeutic strategy for patients with advanced thoracic esophageal squamous cell carcinoma.

Cancer Vaccine Therapy Using Carcinoembryonic Antigen - expressing Dendritic Cells generated from Induced Pluripotent Stem Cells.

Clinical application of dendritic cell (DC) vaccine therapy is hindered by the need for a large quantity of DCs generated from peripheral blood monocytes of the patient. We investigated whether genetically modified human induced pluripotent stem cell (iPSC)-derived dendritic cells (hiPSDCs) expressing carcinoembryonic antigen (CEA) could induce CEA-specific cytotoxic T cells in a human model and whether genetically modified mouse iPSDCs (miPSDCs) expressing CEA showed an actual antitumor effect using a CEA transgenic mouse model. We differentiated hiPSDCs from iPSCs of three healthy donors and transduced CEA cDNA into the hiPSDCs. The surface marker expression, cytokine secretion and migratory capacity of the hiPSDCs were equivalent to those of human monocyte-derived DCs (hMoDCs). Cytotoxic T cells activated by hiPSDCs-CEA exhibited CEA-specific cytotoxic activity against the target cells expressing CEA. Furthermore, in the CEA transgenic mouse model, cytotoxic T cells activated in mice immunized with miPSDCs-CEA displayed CEA-specific cytotoxic activity against MC38-CEA. In the subcutaneous tumour model, vaccination with miPSDCs-CEA achieved a significant growth inhibitory effect on MC38-CEA. No adverse events caused by the administration of miPSDCs were observed. Genetic modification of iPSDCs, inducing the expression of CEA, is a promising tool for clinical applications of vaccine therapy for treating gastrointestinal cancer patients.

Circular stapling versus triangulating stapling for the cervical esophagogastric anastomosis after esophagectomy in patients with thoracic esophageal cancer: A prospective, randomized, controlled trial.

BACKGROUND: Several studies have reported that the triangulating stapling method decreases the incidence of anastomotic stricture after esophagectomy, but no randomized, controlled trial has confirmed the efficacy of the triangulating stapling method for cervical esophagogastrostomy. We compared triangulating stapling and circular stapling for cervical esophagogastric anastomosis regarding the decrease in anastomotic stricture after esophagectomy for thoracic esophageal cancer. METHODS: Between August 2010 and April 2014, 100 patients enrolled in this randomized, controlled trial at the Wakayama Medical University Hospital were allocated randomly to either the circular stapling group (n = 49) or the triangulating stapling group (n = 51). The primary end point was the incidence of anastomotic stricture within 12 months postoperatively. This randomized, controlled trial was registered with the University Hospital Medical Information Network Clinical Trial Registry (UMIN000004848). RESULTS: There were no differences between the circular stapling and triangulating stapling groups in terms of clinical data. The amount of time required for esophagogastric anastomosis was slightly greater for the triangulating stapling group (22 minutes) than for the circular stapling group (18 minutes) (P = .028). Anastomotic stricture occurred in 8 patients (17%) in the circular stapling group and 9 patients (19%) in the triangulating stapling group (P = .935). The rate of anastomotic leakage was 11% for the circular stapling group and 2% for the triangulating stapling group (P = .073). CONCLUSION: The triangulating stapling method for cervical anastomosis for thoracic esophageal cancer does not decrease the incidence of anastomotic stricture compared with the circular stapling method within 12 postoperative months but may affect the rate of anastomotic leakage.

Neoadjuvant Chemotherapy with Divided-dose Docetaxel, Cisplatin and Fluorouracil for Patients with Squamous Cell Carcinoma of the Esophagus.

BACKGROUND: The aim of this phase II study was to evaluate the feasibility of a neoadjuvant chemotherapy regimen consisting of divided-dose docetaxel and cisplatin, with 5-fluorouracil (NAC-DCF), for treatment of patients with stage II/III squamous cell carcinoma of the esophagus (SCCE). PATIENTS AND METHODS: The NAC-DCF regimen, consisting of 2-h infusion of docetaxel at 35 mg/m(2) on days 1 and 8, 4-h infusion of cisplatin at 12 mg/m(2) on days 1-5, and continuous infusion of 5-fluorouracil at 600 mg/m(2) on days 1-5, was administered. We compared NAC-DCF with conventional NAC-CF. RESULTS: The DCF group comprised of 45 patients, and the CF group comprised of 28 patients. The incidence of grade 3/4 neutropenia was significantly higher in the DCF group (56%) than in the CF group (0%). Grade 2/3 pathological response was attained in a significantly higher percentage of patients in the DCF group (40%) than in the CF group (11%) (p=0.0153). CONCLUSION: This DCF regimen led to a high frequency of pathological responses among patients with advanced SCCE.