Efficacious Combination Drug Treatment for Colorectal Cancer That Overcomes Resistance to KRAS G12C Inhibitors.

Recent advances in combinatorial chemistry led to the discovery of inhibitors targeting the KRAS G12C-mutant protein. However, efficacy of its monotherapy on colorectal cancer is limited. Thus, effective combination drugs should be explored for applicable patients with colorectal cancer to fully benefit from the KRAS G12C inhibitor treatment. Here we used a patient-derived colorectal cancer stem cell (PD-CRC-SC) spheroid culture and showed that three-drug combination of inhibitors against KRAS G12C, EGFR, and FGFR synergistically suppressed the growth of colorectal cancer cells carrying the KRAS G12C mutation. Likewise, a combination of KRAS G12C and SHP2 inhibitors was also effective. Importantly, activation of the PI3K/AKT pathway in heregulin-responsive colorectal cancer cells canceled out the effect of KRAS G12C inhibition, which was largely overcome by PI3K inhibitors. These results reveal that evaluating efficacy of combination therapies with PD-CRC-SC spheroids can be a promising strategy to find the best regimen for patients with colorectal cancer.

Single-incision laparoscopic cholecystectomy with the right accessory hepatic duct diagnosed preoperatively: A case report.

Accessory hepatic duct or gallbladder duplication is considered to be a risk factor for bile duct injuries and open conversion during laparoscopic cholecystectomy (LC). A 32-year-old woman with epigastric pain was referred to our department. Gallstone disease in the gallbladder was diagnosed by ultrasonography and magnetic resonance cholangiopancreatography. The involvement of an accessory hepatic duct was suspected during endoscopic retrograde cholangiography. Drip infusion cholangiography with computed tomography showed that the cystic duct merged with the accessory right hepatic duct. Single-incision LC (SILC) was successfully performed without bile duct injury. The operative time and intraoperative blood loss were 145 min and 1 mL, respectively. The patient was discharged 3 days' postoperatively, without complications. The involvement of the accessory right hepatic duct is a rare anomaly and is considered to be a risk factor for bile duct injuries. However, obtaining pre-operative images enabled us to perform SILC successfully.

Induction of human pluripotent stem cell-derived natural killer cells for immunotherapy under chemically defined conditions.

Natural killer (NK) cells are innate lymphocytes and show cytotoxicity against tumor cells without prior antigen specific stimulation. Because of their innate properties, NK cells are being considered for immunotherapies against various malignancies or leukemia. Human pluripotent stem cells (hPSCs) are capable of inducing enough NK cells for allogeneic transplantation. However, current induction protocols require feeder cells or human or bovine serum for the differentiation and expansion of NK cells, which incurs potential risk for contamination and may cause lot dependency in the cells. To address these issues, here we established a differentiation protocol for developing functional NK cells from hPSCs under a completely chemically-defined condition. The resultant PSC-derived NK cells show comparable phenotypes to those produced under serum-containing condition, exerting strong killing potential against a leukemia cell line in vitro and resistance to tumor growth in vivo. Our protocol can be a useful tool for applying PSC-derived NK cells to future cellular cancer immunotherapies.

A Novel Three-Step Approach for Secure Splenectomy During Laparoscopic Total Gastrectomy for Gastric Cancer.

BACKGROUND: Splenectomy during total gastrectomy increases operative morbidity (Nakata et al. in Surgical endoscopy 7:1817-1822, 2015). Establishing a safe approach to laparoscopic splenectomy is one of the most urgent issues in the treatment of proximal advanced gastric cancer, which invades to the greater curvature (Kawamura et al. in Gastric Cancer 3:662-668, 2015). We developed a novel three-step procedure for splenectomy during laparoscopic total gastrectomy (LTG). METHODS: Splenectomy consisted of three steps. Step 1 (dorsal approach): The pancreatic tail and spleen were mobilized. This step delineates the dissection plane and the anatomy around the pancreatic tail. Step 2 (suprapancreatic approach): The suprapancreatic peritoneum was incised to fenestrate to the mobilized space. The no. 11d station was dissected. The inferior branch of the splenic artery was exposed. Step 3 (splenic hilum approach): The spleen was lifted up to straighten the splenic hilum. The aim was to prolong the splenic vasculature and enable the surgeon to transect splenic vasculatures easily despite their anatomical diversity. Division of the splenic branches promotes mobility of the pancreatic tail, enabling precise dissection and preservation of its blood supply. RESULTS: Of 45 patients with gastric cancer who underwent LTG, seven underwent concurrent splenectomy. In all cases, splenectomy was successfully accomplished. The median operation time, duration of splenectomy, blood loss, number of total retrieved lymph nodes, lymph node counts from stations 10 and 11d, and drain amylase levels on the third postoperative day were 382 min, 94 min, 30 ml, 51, 5, 5, and 158 IU/L, respectively. Postoperative morbidity more severe than Clavien-Dindo grade 2 occurred in one case, with no pancreas-related morbidity. No mortality or conversion occurred. CONCLUSIONS: This laparoscopic procedure allows adequate nodal dissection and safe splenectomy.

Laparoscopic transhiatal lymphadenectomy in the lower mediastinum for adenocarcinoma of the esophagogastric junction.

Laparoscopic transhiatal esophagogastrectomy is difficult because the lower mediastinum is so deeply located that the operative field is narrow and restricted by surrounding organs. Therefore, we performed lymphadenectomy with opening of the bilateral mediastinal pleura to maintain safety and obtain better exposure of lymph nodes and important organs. We will present our technique for laparoscopic lower mediastinal lymphadenectomy and reconstruction for cancer of the esophagogastric junction. Five abdominal ports were used. Retraction of the left lobe of the liver exposed the esophageal hiatus. A long, narrow gastric tube (3 cm wide) was formed, and regional abdominal lymph nodes (No. 1, 2, 3a, 7, 8a, 9, 19, and 20) were resected. The diaphragmatic hiatus was widely split and the opened bilateral mediastinal pleura enabled better exposure for lymph node dissection and reconstruction. The level where the inferior vena cava passed through the diaphragm into the chest was used as a landmark to identify supradiaphragmatic (No. 111) and lower thoracic paraesophageal nodes (No. 110), which were completely retrieved with this procedure. The posterior mediastinal nodes (No. 112pulR, 112pulL, and 112aoA) were also retrieved with bilateral opening of the mediastinal pleura and dissection of the inferior pulmonary ligaments. An esophagogastric tube anastomosis with pseudo-fornix was made with a no-knife linear stapler to prevent postoperative reflux esophagitis. This approach enabled safe and accurate laparoscopic lower mediastinal nodal dissection. With the advantage of a narrow gastric tube, the good working space made tension-free anastomosis possible.

Safety and usefulness of needle-guided resection of levator muscles in laparoscopic abdominoperineal resection for low rectal cancer.

INTRODUCTION: During laparoscopic abdominoperineal resection (APR) for low rectal cancer, it is difficult to resect the levator muscles and remove a cylindrical specimen without venturing close to the rectal wall to ensure negative circumferential resection margins (CRM). To solve this problem, we developed a needle-guided, laparoscopic, abdominoperineal resection (LAPR) technique. AIM: To present the safety and superiority of our technique, "needle-guided LAPR". MATERIAL AND METHODS: In 2015, we performed needle-guided LAPR in 5 patients. In brief, the procedure is performed as follows. After total mesorectum excision to the level of the levator muscles, a needle is inserted through the perineum from the dorsal side of the internal aspect of the anus toward the sacral tip. The levator muscles and fat tissue are resected laparoscopically by following the needle. After the levator muscles have been resected, the needle is followed in a similar manner to resect the specimen from the perineum, enabling easy access to the intra-abdominal space and removal of the specimen. No position change is required during the perineal operation or pelvic floor reconstruction. RESULTS: Mean age was 68 years and 3 patients were male. There were no intraoperative complications or conversions to open surgery. The mean operation time and intraoperative blood loss were 319 min and 131 ml, respectively. All specimens were cylindrical in shape and had negative CRM. There were no postoperative complications. CONCLUSIONS: Needle-guided LAPR was easily and safely performed to achieve accurate resection of the levator muscles. This technique could contribute to standardization of LAPR.

A portable platform for stepwise hematopoiesis from human pluripotent stem cells within PET-reinforced collagen sponges.

Various systems for differentiating hematopoietic cells from human pluripotent stem cells (PSCs) have been developed, although none have been fully optimized. In this report, we describe the development of a novel three-dimensional system for differentiating hematopoietic cells from PSCs using collagen sponges (CSs) reinforced with poly(ethylene terephthalate) fibers as a scaffold. PSCs seeded onto CSs were differentiated in a stepwise manner with appropriate cytokines under serum-free and feeder-free conditions. This process yielded several lineages of floating hematopoietic cells repeatedly for more than 1 month. On immunohistochemical staining, we detected CD34+ cells and CD45+ cells in the surface and cavities of the CS. Taking advantage of the portability of this system, we were able to culture multiple CSs together floating in medium, making it possible to harvest large numbers of hematopoietic cells repeatedly. Given these findings, we suggest that this novel three-dimensional culture system may be useful in the large-scale culture of PSC-derived hematopoietic cells.

Longterm retention rate and risk factor for discontinuation due to insufficient efficacy and adverse events in Japanese patients with rheumatoid arthritis receiving etanercept therapy.

OBJECTIVE: Assessing retention rate and risk factor for drug discontinuation is important for drug evaluation. We examined a 3-year retention rate and the risk factor for discontinuation due to insufficient efficacy (IE) and adverse events (AE) in Japanese patients with rheumatoid arthritis (RA) who are receiving etanercept (ETN). METHODS: Data were collected from 588 patients treated with ETN as a first biologic from the Tsurumai Biologics Communication Registry. Baseline characteristics for the incidence of both IE and AE were analyzed using the Cox proportional-hazards regression model. Patients were divided into groups based on age and concomitant methotrexate (MTX). Drug retention rates were calculated using the Kaplan-Meier method and compared among groups using the log-rank test. RESULTS: ETN monotherapy without concomitant MTX [MTX(-)] was significantly related to a higher incidence of discontinuation due to IE [hazard ratio (HR) = 2.226, 95% CI 1.363-3.634]. Older age and MTX(-) were significantly related to a higher incidence of discontinuation due to AE [HR = 1.040, 1.746, 95% CI 1.020-1.060, 1.103-2.763, respectively]. The MTX(-)/≥ 65 years group had the lowest retention rate (p < 0.001). The discontinuation rate due to IE was lower in the MTX(+)/< 65 years group compared to < 65 years/MTX(-), ≥ 65 years/MTX(-) group (p = 0.006, p < 0.001, respectively). The discontinuation rate due to AE was highest in the MTX(-)/≥ 65 years group (p < 0.001). CONCLUSION: Our findings suggest that the risk of discontinuation due to IE was high in the patients who did not use concomitant MTX and that the risk of discontinuation due to AE was high in elderly patients who did not use concomitant MTX.

[Hepatic resection for local recurrence after radiofrequency ablation therapy for colorectal liver metastases].

Patients who underwent hepatic resection of locally recurrent tumors after radiofrequency ablation(RFA)for colorectal liver metastases (CRLM) were retrospectively investigated. Among 12 patients who underwent RFA as first-line treatment for CRLM, 7 experienced local recurrence, 5 of whom (6 nodules) underwent hepatic resection. The mean diameter (range) of the tumors was 9.5(5-16) mm, and they were located at S2, S7 (adjacent to the right hepatic vein), S5/6 (between the root of the anterior and the posterior Glisson's pedicle), S1r (right paracaval portion), S6, and S3. No local recurrence was observed after hepatic resection. In conclusion, hepatic resection must be the initial therapeutic strategy for CRLM, and the indication for RFA must be considered carefully.

Monitoring C-reactive protein levels to predict favourable clinical outcomes from tocilizumab treatment in patients with rheumatoid arthritis.

OBJECTIVES: The inflammatory cytokine interleukin-6 (IL-6) directly stimulates C-reactive protein (CRP) expression. The present study aimed to examine how clinical treatment outcomes of rheumatoid arthritis (RA) with tocilizumab (TCZ), a humanised monoclonal anti-IL-6 receptor antibody, are related to CRP levels monitored for 52 weeks. METHODS: One hundred and twenty-two RA patients who underwent TCZ treatment between May 2008 and September 2009 were registered in the Tsurumai Biologics Communication Registry. Data were collected at initiation of treatment (baseline) and over 52 weeks for Disease Activity Score 28-ESR (DAS28-ESR), Boolean core measurements, serum CRP levels and matrix metalloproteinase-3 levels. To compare clinical results, patients were divided into three groups based on treatment time required to achieve normal CRP levels. RESULTS: Multivariate analysis using the Cox proportional-hazards regression model found that higher CRP levels at baseline was a significant and independent factor in predicting normal CRP levels over 52 weeks (hazard ratio 0.86 per 1 mg/dL). In contrast, disease duration, concomitant methotrexate use and previous tumour necrosis factor inhibitor failure were not significant factors. Patients with normal CRP levels at 12 weeks of TCZ treatment achieved better clinical outcomes, including remission based on DAS28-ESR criteria, compared to patients with elevated CRP levels at 12 weeks. CONCLUSIONS: Adequate suppression of pathological IL-6 signalling during TCZ treatment improves clinical outcomes and can be monitored with serum CRP levels, a readily available biomarker in clinical practice.

A selective estrogen receptor modulator inhibits tumor necrosis factor-α-induced apoptosis through the ERK1/2 signaling pathway in human chondrocytes.

Tumor necrosis factor α (TNF-α) is a pleiotropic cytokine mediating inflammatory as well as cell death activities, and is thought to induce chondrocytic chondrolysis in inflammatory and degenerative joint diseases. Selective estrogen receptor modulators (SERMs), such as raloxifene, which are commonly used in clinical settings act as estrogen agonists or antagonists. It is assumed that estrogens have a potential role in cartilage protection; however, the precise molecular mechanism for the protective effects of estrogens is unclear. This study was designed to examine whether raloxifene inhibits TNF-α-induced apoptosis in human chondrocytes and to clarify the mechanisms involved. We also investigated the signaling pathways responsible for the anti-apoptotic effect of raloxifene. Apoptosis in chondrocytes was determined by DNA fragmentation assay and caspase-3 activation. Raloxifene significantly inhibited TNF-α-induced caspase-3 activation and cell DNA fragmentation levels in chondrocytes. The inhibitory effect of raloxifene was abolished by the estrogen receptor antagonist ICI 182,780. Extracellular signal-regulated kinase 1/2 (ERK1/2) regulates apoptosis, acting as an apoptotic or anti-apoptotic signal. TNF-α-induced apoptosis was significantly enhanced by the ERK1/2 pathway inhibitor PD98059. Raloxifene stimulated a further increase in ERK1/2 phosphorylation in TNF-α-treated chondrocytes. Furthermore, the anti-apoptotic effects of raloxifene were inhibited by PD98059. In addition, the anti-apoptotic effects of raloxifene were completely abolished in ERK1/2 siRNA-treated chondrocytes. These results suggest that raloxifene prevents caspase-3-dependent apoptosis induced by TNF-α in human chondrocytes by activating estrogen receptors and the ERK1/2 signaling pathway.

Study protocol of a multicenter registry of patients with rheumatoid arthritis starting biologic therapy in Japan: Tsurumai Biologics Communication Registry (TBCR) study.

Biologic agents have proven to be effective against rheumatoid arthritis (RA) in clinical trials and post-marketing surveillance (PMS) studies. However, limited follow-up periods and strict criteria for recruitment might lead to an underestimation of adverse events. To document the long-term course of patients with RA treated with biologics in clinical settings, we established the Tsurumai Biologics Communication Registry (TBCR). First, we retrospectively collected data of patients registered for any biologic PMS study or clinical trial at participating institutes. Thus far, thirteen institutes have joined the registry and 860 patients have been identified. Comparing baseline characteristics by age and initiation year of biologics, young patients had significantly less joint damage and dysfunction and a higher dose of concomitant methotrexate (MTX) compared to older patients. Older age and functional class were significantly related to the incidence of adverse events that resulted in discontinuation of the 1st biologic treatment. The TBCR is in its initial stages, and information on all patients newly starting biologic therapy at participating institutes is being collected prospectively. Differences in baseline characteristics by age and initiation year of biologics need to be carefully evaluated in order to report on drug-related survival and long-term prognosis, using follow-up data in the near future.

Effect of total arthroplasty combined with anti-tumor necrosis factor agents in attenuating systemic disease activity in patients with rheumatoid arthritis.

We assessed the effect of total large-joint arthroplasty combined with anti-tumor necrosis factor (TNF) therapy for rheumatoid arthritis (RA). We studied 45 RA patients (age 57.91 ± 12.74 years, RA duration 13.43 ± 8.28 years) who underwent total arthroplasty (35 knees, 19 hips, 3 elbows, and 1 ankle) between August 2002 and November 2009. All patients received anti-TNF agents (infliximab, 22; etanercept, 33; adalimumab, 3) during the period of the study (that is, they were being treated with the agents when operated on and postoperatively). The disease activity score 28 (DAS28)-erythrocyte sedimentation rate (mean ± standard deviation) in all patients improved significantly from baseline (just before the operation; 4.32 ± 0.99) to 1 year after the operation (3.35 ± 0.93) in contrast with the finding that the mean DAS28-ESR values had remained unchanged from 1 year before the operation to the baseline. Changes in clinical variables in the 58 cases were investigated at baseline, and at 4, 12, and 52 weeks after the operation. The patients were divided by a median split of baseline demographics into 2 groups for further evaluation. Compared with the high-value groups, those with low C-reactive protein and matrix metalloproteinase-3 values showed better results and had lower disease activity. Overall, the DAS28-ESR in both groups had improved 1 year after the operation. In RA patients who are being treated with anti-TNF agents, large-joint arthroplasty may be beneficial, not only for the relief of pain arising from joint destruction, but also for the systemic reduction of RA activity.

Crystal structures of protein glutaminase and its pro forms converted into enzyme-substrate complex.

Protein glutaminase, which converts a protein glutamine residue to a glutamate residue, is expected to be useful as a new food-processing enzyme. The crystal structures of the mature and pro forms of the enzyme were refined at 1.15 and 1.73 Å resolution, respectively. The overall structure of the mature enzyme has a weak homology to the core domain of human transglutaminase-2. The catalytic triad (Cys-His-Asp) common to transglutaminases and cysteine proteases is located in the bottom of the active site pocket. The structure of the recombinant pro form shows that a short loop between S2 and S3 in the proregion covers and interacts with the active site of the mature region, mimicking the protein substrate of the enzyme. Ala-47 is located just above the pocket of the active site. Two mutant structures (A47Q-1 and A47Q-2) refined at 1.5 Å resolution were found to correspond to the enzyme-substrate complex and an S-acyl intermediate. Based on these structures, the catalytic mechanism of protein glutaminase is proposed.

Potential for HER-2/neu molecular targeted therapy for invasive bladder carcinoma: comparative study of immunohistochemistry and fluorescent in situ hybridization.

Analysis of HER-2/neu expression in invasive bladder carcinoma was performed in order to evaluate the potential for molecular targeted therapy targeting HER-2. The subjects were 40 patients who were pathologically diagnosed with invasive transitional cell carcinoma of the bladder (pT2 to pT4). A Hercep test kit was used to detect HER-2 expression, and a Path Vysion kit was used for gene amplification. On immunohistochemical (IHC) staining, the primary tumors were HER-2 positive in 17 patients (17/40, 42.5%). According to the classification of grade, one Grade 2 patient (1/3) and 16 Grade 3 patients (16/37) were positive (P=0.99). According to the classification of stage, 12 pT2 patients (12/22, 54.5%), 2 pT3 patients (2/13, 15.3%), and 3 pT4 patients (3/5, 60%) were positive (P=0.55). Lymph node metastasis was found in 10 patients, and 3 pN2 patients were HER-2 positive (3/6, 50%) (P=0.32). A statistically significant difference was observed between HER-2-positive primary tumors and metastatic lymph nodes (P=0.02). In fluorescent in situ hybridization (FISH), HER-2/neu gene amplification was detected in the primary tumors in 5 patients (5/40, 12.5%). In all these patients, IHC staining was determined as 3+. Lymph node metastasis was found in 3 pN2 patients (3/6) (P=0.32), and in these patients with HER-2/neu gene-amplified metastatic lymph nodes, the primary tumors were also positive for gene amplification (P=0.02). In these cases, IHC staining was 3+ as well. The concordance rate of IHC-positive cases with cases positive for HER-2/neu gene amplification in FISH was 12.5% (5/40), and the concordance rate of IHC 3+ and gene amplification was 71%. This result suggests that, at present, patients who may potentially benefit from molecular targeted therapy targeting HER-2/neu for invasive bladder carcinoma should be identified by gene amplification analysis using FISH in IHC 3+ patients. In addition, it suggested that efficacy of molecular targeted therapy can be expected even for patients with metastatic lymph nodes as long as the primary tumors are positive for HER-2 expression.

An immunohistochemical study of chromogranin A and human epidermal growth factor-2 expression using initial prostate biopsy specimens from patients with bone metastatic prostate cancer.

OBJECTIVES: To investigate, using prostate needle-biopsy specimens at diagnosis from patients with bone metastatic prostate cancer, whether the relationship between neuroendocrine (NE) cell differentiation and human epidermal growth factor-2 (HER-2) expression is a prognostic factor for outcome. PATIENTS AND METHODS: The study included 50 patients diagnosed as having bone metastatic prostate cancer between January 1998 and December 2001. We tested for NE cell differentiation by using immunohistochemical (IHC) staining for chromogranin A (CgA), and for HER-2, using a commercial test for IHC staining. RESULTS: Eleven patients (22%) were positive for CgA; there was a significant difference in the time to recurrence (P = 0.025) but no significant differences in cause-specific survival rate or survival rate after recurrence. In all, 21 patients (42%) were positive for HER-2; the cause-specific survival rate, time to recurrence and survival rate after recurrence were all significantly more favourable in the HER-2-negative group (P = 0.008, 0.049 and 0.025, respectively). In the 49 patients for whom both factors could be determined, there was no significant correlation between CgA and HER-2 positivity. CONCLUSIONS: NE cell differentiation of the primary tumour in patients with bone metastatic prostate cancer does not reflect the prognosis, whereas HER-2 overexpression is a prognostic factor for an unfavourable outcome. These results suggest that NE cell differentiation is not induced by HER-2.

Is neuroendocrine cell differentiation detected using chromogranin A from patients with bone metastatic prostate cancer a prognostic factor for outcome?

We evaluated the usefulness of overexpression of neuroendrocrine (NE) cell differentiation determined by immunohistochemical staining for chromogranin A (Cg A) in diagnostic needle biopsy specimens of bone metastatic prostate cancers. A total of 50 patients diagnosed as having bone metastatic prostate cancer were studied. The period of observation was between 6.9 and 79.4 months (median 48.7 months). Cg A was detected by immunostaining using the labeled streptavidin biotin method. Cg A-positivity was defined as the presence of immunostained cells in 10% or more of the tumor. All statistical analyses were carried out using the Statistical Package for Social Sciences Software, version 10.0 for Windows. Eleven patients (22%) were classified into the Cg A-positive group. There were no significant differences in clinical data between the Cg A-positive and Cg A-negative groups. The 5-year cause-specific survival rate was 34.1% for the Cg A-positive group and 55.2% for the Cg A-negative group (p=0.3763). The 3-year non-recurrence rate was 9.1% for the Cg A-positive group and 35.9% for the Cg A-negative group, and this difference was significant (p=0.0253). The 3-year cause-specific survival rates after recurrence were 38.4% and 42.3% respectively (p=0.8125). We consider that NE cell differentiation of the primary tumor in cases of bone metastatic prostate cancer is not a prognostic factor for outcome.

Endoscope-assisted minilaparotomic radical retropubic prostatectomy.

OBJECTIVE: To assess the clinical efficacy of endoscope-assisted minilaparotomic radical retropubic prostatectomy (EAM-RRP) compared with conventional radical retropubic prostatectomy (cRRP). METHODS: From September 2001 to December 2003, 30 patients with localized prostate cancer were treated by EAM-RRP. The surgical manipulation was performed through the wound with thoracoscopic assistance, using standard surgical instruments. In all cases, 800 mL of blood was collected from the patient for autotransfusion. For both EAM-RRP and cRRP, the internal iliac and obturator lymph nodes were dissected before the prostate removal. Clinical indicators such as operation time, blood loss, and duration of postoperative urine incontinence were analysed in the two groups. RESULTS: The postoperative period before ambulation and the duration of postoperative urine incontinence were significantly shorter after EAM-RRP than after cRRP, while no significant difference was found in operation time, blood loss, and duration of urethral catheterization. None of the cases required allotransfusion. CONCLUSION: EAM-RRP, which had a shorter postoperative period before ambulation and continence, is considered a safe and useful technique for radical prostatectomy.