Hiromitsu Komiyama

Inhibition of PAI-1 induces neutrophil-driven neoangiogenesis and promotes tissue regeneration via production of angiocrine factors in mice

Plasminogen activator inhibitor-1 (PAI-1), an endogenous inhibitor of a major fibrinolytic factor, tissue-type plasminogen activator, can both promote and inhibit angiogenesis. However, the physiologic role and the precise mechanisms underlying the angiogenic effects of PAI-1 remain unclear. In the present study, we report that pharmacologic inhibition of PAI-1 promoted angiogenesis and prevented tissue necrosis in a mouse model of hind-limb ischemia. Improved tissue regeneration was due to an expansion of circulating and tissue-resident granulocyte-1 marker (Gr-1(+)) neutrophils and to increased release of the angiogenic factor VEGF-A, the hematopoietic growth factor kit ligand, and G-CSF. Immunohistochemical analysis indicated increased amounts of fibroblast growth factor-2 (FGF-2) in ischemic gastrocnemius muscle tissues of PAI-1 inhibitor-treated animals. Ab neutralization and genetic knockout studies indicated that both the improved tissue regeneration and the increase in circulating and ischemic tissue-resident Gr-1(+) neutrophils depended on the activation of tissue-type plasminogen activator and matrix metalloproteinase-9 and on VEGF-A and FGF-2. These results suggest that pharmacologic PAI-1 inhibition activates the proangiogenic FGF-2 and VEGF-A pathways, which orchestrates neutrophil-driven angiogenesis and induces cell-driven revascularization and is therefore a potential therapy for ischemic diseases.

Tissue type plasminogen activator regulates myeloid-cell dependent neoangiogenesis during tissue regeneration.

Ischemia of the heart, brain, and limbs is a leading cause of morbidity and mortality worldwide. Treatment with tissue type plasminogen activator (tPA) can dissolve blood clots and can ameliorate the clinical outcome in ischemic diseases. But the underlying mechanism by which tPA improves ischemic tissue regeneration is not well understood. Bone marrow (BM)-derived myeloid cells facilitate angiogenesis during tissue regeneration. Here, we report that a serpin-resistant form of tPA by activating the extracellular proteases matrix metalloproteinase-9 and plasmin expands the myeloid cell pool and mobilizes CD45(+)CD11b(+) proangiogenic, myeloid cells, a process dependent on vascular endothelial growth factor-A (VEGF-A) and Kit ligand signaling. tPA improves the incorporation of CD11b(+) cells into ischemic tissues and increases expression of neoangiogenesis-related genes, including VEGF-A. Remarkably, transplantation of BM-derived tPA-mobilized CD11b(+) cells and VEGFR-1(+) cells, but not carrier-mobilized cells or CD11b(-) cells, accelerates neovascularization and ischemic tissue regeneration. Inhibition of VEGF signaling suppresses tPA-induced neovascularization in a model of hind limb ischemia. Thus, tPA mobilizes CD11b(+) cells from the BM and increases systemic and local (cellular) VEGF-A, which can locally promote angiogenesis during ischemic recovery. tPA might be useful to induce therapeutic revascularization in the growing field of regenerative medicine.

Glasgow Prognostic Score as a Prognostic Factor in Patients Undergoing Curative Surgery for Colorectal Cancer

Background/Aims: Systemic inflammatory responses have been reported to be independent predictors of cancer-specific survival in colorectal cancer. The Glasgow Prognostic Score (GPS), which is an inflammation-based prognostic factor, is defined by the presence of elevated C-reactive protein and hypoalbuminemia. The purpose of this study was to estimate whether GPS can be a prognostic factor in patients undergoing curative surgery for colorectal cancers. Methods: We studied 166 patients with stage II (TNM classification) and 200 patients with stage III who had undergone curative surgery for colorectal cancer between 1999 and 2004. Univariate and multivariate analyses were performed to evaluate the relationship between clinicopathological factors and prognosis. Results: Among patients with stage II, location and GPS were independent factors on multivariate analysis. In particular, GPS was revealed to be the strongest factor in cancer-specific survival (HR: 7.43, 95% confidence interval, CI: 2.86–19.30, p < 0.0001). On the other hand, among patients with stage III, the number of metastatic lymph nodes was the only independent factor on multivariate analysis (HR: 1.14, 95% CI: 1.07–1.20, p < 0.0001). GPS was not a prognostic factor in cancer-specific survival in stage III. Conclusion: Among patients with stage II, GPS was predictive of cancer-specific survival.

The Validity of Predicting Prognosis by the Lymph Node Ratio in Node-Positive Colon Cancer

Background/Aims: Because the TNM system disregards the number of lymph nodes dissected and inter-individual differences exist in the number of regional lymph nodes, the lymph node ratio (LNR), which is estimated by dividing the number of metastatic lymph nodes by the number of dissected lymph nodes, has been proposed as a prognostic factor in recent years. The purpose of the present study is to examine the validity of predicting prognosis using the LNR in node-positive colon cancer. Methods: Three hundred and eleven patients with lymph node metastases who underwent curative surgery for colon cancer at our department between 1992 and 2005 were enrolled. Univariate and multivariate analyses were performed to evaluate the relationship between clinicopathological factors and prognosis. Results: Among the patients with ≥12 dissected lymph nodes, differentiation, invasion depth and TNM N category were found to be significant independent prognostic factors. On the other hand, among the patients with ≤11 dissected lymph nodes, differentiation and the LNR were found to be significant independent prognostic factors. Conclusion: Among the patients with ≤11 dissected lymph nodes, LNR was a significant independent prognostic factor.

Impact of chromosome 17q deletion in the primary lesion of colorectal cancer on liver metastasis

Colorectal cancer is a prevalent malignancy worldwide, and investigations are required to elucidate the underlying carcinogenic mechanisms. Amongst these mechanisms, de novo carcinogenesis and the adenoma to carcinoma sequence, are the most understood. Metastasis of colorectal cancer to the liver often results in fatality, therefore, it is important for any associated risk factors to be identified. Regarding the treatment of the disease, it is important to manage not only the primary colorectal tumor, but also the liver metastases. Previously, through gene variation analysis, chromosomal loss has been indicated to serve an important role in liver metastasis. Such analysis may aid in the prediction of liver metastasis risk, alongside individual responses to treatment, thus improving the management of colorectal cancer. In the present study, we aimed to clarify a cause of the liver metastasis of colorectal cancer using comparative genomic hybridization analysis. A total of 116 frozen samples were analyzed from patients with advanced colorectal cancer that underwent surgery from 2004 to 2011. The present study analyzed mutations within tumor suppressor genes non-metastatic gene 23 (NM23), deleted in colorectal carcinoma (DCC) and deleted in pancreatic carcinoma, locus 4 (DPC4), which are located on chromosomes 17 and 18 and have all been reported to affect liver metastasis of colorectal cancer. The association between chromosomal abnormalities (duplication and deletion) and liver metastasis of colorectal cancer was evaluated using comparative genomic hybridization. Cluster analysis indicated that the group of patients lacking the long arm of chromosome 17 demonstrated the highest rate of liver metastasis. No significant association was observed between the frequency of liver metastases for synchronous and heterochronous colorectal cancer cases and gene variation (P=0.206). However, when these liver metastasis cases were divided into the synchronous and heterochronous types, the ratio of each was significantly different between gene variation groups, classified by the existence of the 17q deletion (P=0.023). These results indicate that the deletion of 17q may act as a predictive marker of liver metastasis in postoperative states.

Laparoscopic intersphincteric resection using needlescopic instruments

Intersphincteric resection (ISR) is a procedure designed to preserve anal function in cases with very low rectal cancer. We report our clinical experience with laparoscopic ISR (Lap ISR) performed using needlescopic instruments. First, a camera port is created at the umbilicus. Two 5-mm ports are then inserted at the right upper and lower quadrants. Two needlescopic forceps (Endo-Relief™ Hope Denshi Co., Chiba, Japan) are inserted at the left upper and lower quadrants. We then perform the following procedures; ligation of the inferior mesenteric artery and vein, total mesorectal excision and dissection of the intersphincteric space. After the transanal intersphincteric dissection, the specimen is extracted through the anus and a hand —sewn coloanal anastomosis is performed. The covering ileostomy is finally created at the right upper port. We performed Lap ISR using needlescopic forceps in two patients with very low rectal cancer. In both cases, we were able to perform this procedure without insertion of an additional port or to change the needlescopic forceps to conventional 5-mm forceps. Lap ISR with needlescopic instruments is a feasible procedure for minimally invasive surgery.

Investigation of free cancer cells in peripheral blood using CEA mRNA expression in perioperative colorectal cancer patients

The aim of this study was to evaluate the impact of laparoscopic surgery (Lap) on circulating free tumor cells in colorectal cancer patients. In this study, we selected carcinoembryonic antigen (CEA) mRNA expression in peripheral blood as the marker of the circulating tumor cells and compared this marker between Lap and open colectomy (OC), to investigate differences due to surgical approach. A total of 50 patients underwent curative surgery for solitary colorectal cancer at our department, between June, 2008 and February, 2011. The patients were divided into OC and Lap groups (25 patients each). Total RNA was extracted subsequent to peripheral blood collection prior to surgery, immediately following surgery and 1, 3 and 7 days after surgery. CEA mRNA was detected with reverse transcription polymerase chain reaction (RT-PCR) and the association between peripheral blood CEA mRNA-positive rate, surgical findings and clinicopathological characteristics was investigated. The peripheral blood CEA mRNA-positive rate was significantly increased immediately after surgery, compared to the preoperative rate (P=0.001), but decreased over time. No significant differences were observed at any blood-sampling time point after postoperative day 1. The positive rate was significantly increased in the OC group immediately after surgery, compared to the preoperative rate (P=0.004). However, there were no significant differences between the rates prior to and immediately after surgery in the Lap group. The patients were then divided into those who were peripheral blood CEA mRNA-positive and -negative after surgery (postoperative positive and negative groups, respectively) and the clinicopathological characteristics were compared. Significant differences were identified between the groups in lower rectal cancer patients and patients with a large intraoperative blood loss (P=0.001 and P=0.01, respectively). In conclusion, in colorectal cancer patients, there were no significant differences in the perioperative peripheral blood CEA mRNA-positive rate or its short-term changes between patients undergoing OC and Lap surgery. It was suggested that Lap is equivalent to OC with regard to free cancer cells.

An effective 5-fluorouracil, levofolinate, and oxaliplatin therapy for recurrent breast cancer: a case report

IntroductionTherapy comprising 5-fluorouracil, levofolinate, and oxaliplatin is currently the most common chemotherapy for colorectal cancer. We experienced a successful case of advanced colon cancer and recurrent breast cancer with 5-fluorouracil, levofolinate, and oxaliplatin therapy.Case presentationA 43-year-old Japanese woman who had already undergone surgery three times for bilateral breast cancer was admitted to our hospital for the treatment of advanced transverse colon cancer. Preoperative computed tomography demonstrated a swollen lymph node at her right upper clavicle, and fine-needle aspiration biopsy of the lymph node showed that it was a metastasis from the breast cancer. A laparoscopic-assisted colectomy was performed and the pathology demonstrated that the final stage was IIIC (T4aN2aM0, Union for International Cancer Control, 7th edition). The pathological findings and immunohistochemistry showed that the transverse colon tumor was not a metastatic lesion from the breast cancer, but was a de novo colon cancer. Chemotherapy was necessary for both the recurrent breast cancer and the Stage IIIC colon cancer. Therapy of 5-fluorouracil, levofolinate, and oxaliplatin was administered; the therapy included 5-fluorouracil, which is considered to be effective for both colon and breast cancer. After two courses of 5-fluorouracil, levofolinate, and oxaliplatin, the lymph node began to shrink and almost completely disappeared after eight courses of 5-fluorouracil, levofolinate, and oxaliplatin.ConclusionWe surmise that 5-fluorouracil, levofolinate, and oxaliplatin have the potential to provide a good response for tumors that are sensitive to fluorinated pyrimidine and platinum-containing anticancer drugs such as breast cancer.

T1 colorectal cancer with synchronous liver metastasis.

The patient was a 68-year-old man who was admitted to our hospital with a liver tumor. Abdominal ultrasonography and computed tomography revealed a liver tumor 30 mm in diameter. On colonoscopy, a pedunculated tumor with a central depression (20 mm in diameter) was observed in the ascending colon, and this tumor was considered to be invading deeply into the submucosal layer. Right hemicolectomy with D3 lymphadenectomy and partial hepatectomy were performed simultaneously. On histopathological examination of the resected specimen, the tumor was a well-differentiated tubular adenocarcinoma with 3,000 μm invasion of the submucosal layer. The liver tumor showed histological findings similar to those of the primary colorectal carcinoma. The pathological stage according to the 7th edition of the TNM classification was stage IV (T1N0M1). Nine months after the operation, computed tomography revealed hepatic hilar lymph node metastases and a great deal of ascites. The patient ultimately died 14 months after the operation.

High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor

Despite current advances in human colorectal cancer (CRC) treatment, few radical therapies are effective for the late stages of CRC. To overcome this clinical challenge, tumor xenograft mouse models using long-established human carcinoma cell lines and many transgenic mouse models with tumors have been developed as preclinical models. They partially mimic the features of human carcinomas, but often fail to recapitulate the key aspects of human malignancies including invasion and metastasis. Thus, alternative models that better represent the malignant progression in human CRC have long been awaited. We herein show generation of patient-derived tumor xenografts (PDXs) by subcutaneous implantation of small CRC fragments surgically dissected from a patient. The colon PDXs develop and histopathologically resemble the CRC in the patient. However, few spontaneous micrometastases are detectable in conventional cross-sections of affected distant organs in the PDX model. To facilitate the detection of metastatic dissemination into distant organs, we extracted the tumor organoid cells from the colon PDXs in culture and infected them with GFP lentivirus prior to injection into highly immunodeficient NOD/Shi-scid IL2Rγnull (NOG) mice. Orthotopically injected PDX-derived CRC organoid cells consistently form primary tumors positive for GFP in recipient mice. Moreover, spontaneously developing micrometastatic colonies expressing GFP are notably detected in the lungs of these mice by fluorescence microscopy. Moreover, intrasplenic injection of CRC organoids frequently produces hepatic colonization. Taken together, these findings indicate GFP-labelled PDX-derived CRC organoid cells to be visually detectable during a multistep process termed the invasion-metastasis cascade. The described protocols include the establishment of PDXs of human CRC and 3D culture of the corresponding CRC organoid cells transduced by GFP lentiviral particles.