Masahisa Ohtsuka

Micrometastasis Volume in Lymph Nodes Determines Disease Recurrence Rate of Stage II Colorectal Cancer: A Prospective Multicenter Trial

Purpose: We reported in a retrospective study that the presence of micrometastasis in lymph nodes, when assessed by carcinoembryonic antigen (CEA)-specific RT-PCR, is a significant prognostic factor in stage II colorectal cancer. The aim of this study was to clarify the clinical value of micrometastasis in a prospective multicenter trial. Experimental Design: From November 2001 to December 2005, a total of 419 colorectal cancer cases were preoperatively registered at a central data center. Of them, 315 node-negative stage II colorectal cancer cases were enrolled. After RNA quality check, 304 colorectal cancer cases were analyzed for CEA mRNA in lymph nodes by both conventional RT-PCR (a band method) and quantitative RT-PCR. Long-term prognosis of the patients was determined by each method. Results: A positive band for CEA mRNA was detected in 73 (24.0%) of 304 patients. Postoperative adjuvant chemotherapy was applied in 31 CEA band-positive cases with an oral 5-fluorouracil derivative HCFU (1-hexylcarbamoyl-5-fluorouracil) for 1 year, whereas chemotherapy was not administered to CEA band-negative group. Multivariate Cox regression analyses revealed that a high micrometastasis volume (high MMV, n = 95) was an independent poor prognostic factor for 5-year disease-free survival (DFS; P = 0.001) and 5-year overall survival (OS; P = 0.016). Conclusions: This prospective clinical trial demonstrates that micrometastasis volume is a useful marker in identifying patients who are at high or low risk for recurrence of stage II colorectal cancer. Clin Cancer Res; 22(13); 3201–8. ©2016 AACR.

Concurrent expression of C4.4A and Tenascin-C in tumor cells relates to poor prognosis of esophageal squamous cell carcinoma

C4.4A is a glycolipid-anchored membrane protein expressed in several human malignancies. We recently found that C4.4A expression was associated with poor prognosis of esophageal squamous carcinoma cells (ESCCs), but the underlying mechanism is unknown. To uncover this, we performed PCR array analysis using the HCT116 cell line, a positive control for C4.4A expression and we found that Tenascin-C (TNC) among the many adhesion molecules and extracellular matrix proteins was the best candidate for C4.4A molecule induction. Based on in vitro studies using the TE8 esophageal cancer cells, we examined by immunohistochemistry TNC expression in 111 ESCCs. We found that the TNC-positive group (24.3%) had significantly poorer prognosis than the TNC-negative group in 5-year overall survival. We also found there was a significant correlation between TNC and C4.4A in ESCC tissues (P=0.007). Finally, we found that only the double-positive group for C4.4A and TNC had a significantly worse prognosis (P=0.005). Our data suggest that TNC expression in ESCC may in part explain why C4.4A is associated with a poor prognosis of ESCC since TNC can promote invasion and metastasis.

Impact of stereotactic body radiotherapy on colorectal cancer with distant metastases

Stereotactic radiotherapy is a minimally invasive technique for delivering highly focused ionizing radiation with extreme precision. This technique was initially developed in neurosurgical practice and applied to extracranial lesions in the 1990s, and was termed stereotactic body radiotherapy (SBRT). Studies have reported that the resection of distant metastases from colorectal cancer (CRC) contributes to relatively long-term survival. However, the resection of pulmonary and liver metastases is not possible for various reasons. SBRT offers a therapeutic alternative to unresectable metastatic lesions. The present study describes three cases of distant metastasis from CRC that exhibited a complete response (CR) to SBRT. Case 1 is a 70-year-old man with recurrent liver metastases after surgery for rectal cancer with liver metastasis (S3: diameter 1.8 cm and volume 3.0 ml; S6: diameter 1.3 cm and volume 1.2 ml). Cases 2 and 3 were 65-year-old and 70-year-old men, respectively. Both patients had pulmonary metastasis after surgery for rectal and cecum cancer (Case 2: diameter 1.2 cm and volume 0.9 ml; Case 3: diameter 0.8 cm and volume 0.27 ml). All cases were moderately differentiated adenocarcinomas. No serious adverse side-effects were observed during the therapy. CR was obtained in all patients on the basis of computed tomography 15-33 months after radiotherapy. Our experience supports that SBRT is a safe and alternative technique for resection in patients with distant metastasis from CRC who have small metastatic tumor volume.

Inhibition of angiopoietin 2 attenuates lumen formation of tumour-associated vessels in vivo

Anti-angiogenic therapy, inhibition of a co-operative process with vascular endothelial cells and pericytes could be an effective strategy to treat malignant tumours. Apart from vascular endothelial growth factor (VEGF), angiopoietin 2 (Ang2) is a promising target of anti-angiogenic therapy. Although inhibition of Ang2 has been shown to decrease tumour size in preclinical and phase I trials, its mechanisms of action remain largely unknown. To elucidate the mechanisms of Ang2 inhibition, we have focused on differentiation of the vessels as well as on growth of the vessels, especially in vivo. L1-10, a selective Ang2 inhibitor was used. The in vitro effects of Ang2 inhibition or addition of Ang2 using HUVECs were also examined. Growth and differentiation of tumour-associated vessels were investigated in xenografts derived from a colon cancer treated by L1-10. Effects of VEGF inhibition were also examined to discriminate Ang2-specific action on the tumour-associated vessels. In vitro studies showed that VEGF enhanced proliferation and tube formation of HUVECs, and caused a significant increase in Rac1 and CDC42 expression when cultured in the collagen matrix gel, whereas neither Ang2 nor L1-10 affected in vitro behaviour of HUVECs or levels of the proteins. In vivo, on the other hand, we found that Ang2 inhibition with treatment of L1-10 dose‑dependently decreased tumour growth. Furthermore, we found that L1-10 treatment extends the tumour-associated vessels whilst it suppressed a sound lumen formation. Histological analysis on xenografts suggests that Ang2 inhibition could have disturbed in vivo vascular differentiation. Our data provide a novel aspect that Ang2 may play an essential role in in vivo vascular differentiation, thus supporting a rationale for Ang2-targeted therapy against colon cancer.

Distinct expression of C4.4A in colorectal cancer detected by different antibodies.

The metastasis-associated gene C4.4A encodes a glycolipid-anchored membrane protein expressed in several human malignancies. The present study aimed to perform a detailed assessment of C4.4A expression in colorectal cancer tissues, in terms of intra-cellular localization, intra-tumoral location and difference in molecular weight. To advance this goal, we developed three new antibodies against the C4.4A protein (two polyclonal Abs: C4.4A-119 and C4.4A-277 and one monoclonal Ab: C4.4A GPI-M) to use in addition to the two previously produced polyclonal Abs (C4.4A-81, C4.4A GPI-P). Antibody specificities were confirmed by absorption tests. Western blot analysis and immunohistochemistry showed that the C4.4A-119 and C4.4-277 Abs detected 70-kDa C4.4A, mainly in the cytoplasm, irrespective of intra-tumoral location. The C4.4A GPI-P and C4.4A GPI-M Abs reacted with the membranous ~40-kDa C4.4A, exclusively at the tumor invasive front, and each detected an identical tumor cell population. The tested antibodies showed varied C4.4A detection rates in 33 CRC tissues. The C4.4A-277 Ab yielded the highest positive rate in 29 of 33 CRC tissues (87.9%), while the C4.4A GPI-P and C4.4A GPI-M Abs each only showed 33.3% positivity. The present findings suggest that the GPI anchor signaling sequence may be essential for detecting membranous C4.4A at the invasive front of CRC tissues.