Seiji Ito

Quantitative detection of disseminated free cancer cells in peritoneal washes with real-time reverse transcriptase-polymerase chain reaction: a sensitive predictor of outcome for patients with gastric carcinoma.

ObjectiveTo evaluate the feasibility of real-time reverse transcriptase–polymerase chain reaction (RT-PCR) detection of free cancer cells in the peritoneal washes as a prognostic indicator for patients with gastric carcinoma. Summary Background DataPeritoneal lavage cytology (CY) is an excellent prognostic determinant but lacks sensitivity. This can be improved by using RT-PCR to quantitate carcinoembryonic antigen (CEA) mRNA in peritoneal washes. MethodsPeritoneal washes were obtained from 189 patients with gastric carcinoma during laparotomy. CEA mRNA levels and CEA/GAPDH mRNA ratios were quantified using a real-time PCR system with fluorescent hybridization probes. Receiver-operating characteristic plots were used to determine which of these parameters should be used as a marker for the intraperitoneal cancer cells. The prognostic significance of its positivity was then evaluated by Kaplan-Meier curves, and its value as an independent prognostic factor was evaluated by multivariate analysis. ResultsThe sensitivity and specificity of real-time RT-PCR with an optimal cutoff value were 80% and 94%; those for conventional cytology were 56% and 91%. The survival of 16 patients who were CY-PCR+ was poor and approached that of 35 CY+ patients. Recurrence as peritoneal carcinomatosis was frequent among PCR+ patients but rare for their PCR- counterparts. PCR+ was a significant independent prognostic factor, along with the presence of node metastasis and serosal invasion, but CY+ was not. ConclusionsQuantitative RT-PCR of peritoneal washes can replace cytologic examination as a tool for the sensitive evaluation of the risk of intraperitoneal recurrence in patients with gastric carcinoma.

Rapid quantitative detection of carcinoembryonic antigen–expressing free tumor cells in the peritoneal cavity of gastric‐cancer patients with real‐time RT‐PCR on the lightcycler

Detection of free cancer cells in the peritoneal cavity by RT‐PCR using carcinoembryonic antigen (CEA) as a target gene is a more sensitive predictor of peritoneal dissemination than conventional cytology in gastric‐cancer patients. Difficulties with this method are the lack of quantitative assessment of free cancer cells and the length of time before completion. To overcome these problems, we have established a rapid and quantitative detection method using a novel real‐time fluorescence PCR system (LightCycler). Using this device with hybridization probes as fluorophores, we detected CEA mRNA in peritoneal washes during surgery (within 3 hr) without any post‐PCR procedure. This method could reproducibly quantitate 10 to 106 CEA‐expressing colon carcinoma cells per 107 peripheral blood leukocytes, a comparable sensitivity to conventional RT‐PCR with a wide dynamic range. Analysis of peritoneal washes from 109 gastric‐cancer patients with this assay revealed relative values of CEA transcripts that correlated well with the depth of tumor invasion (p < 0.01). Average values of CEA transcript in peritoneal washes in patients with cytology (−)/RT‐PCR(−), cytology (−)/RT‐PCR(+) and cytology (+)/RT‐PCR(+) results were 0.64, 1,525 and 6,715, respectively. Moreover, CEA transcripts in peritoneal washes in patients with synchronous peritoneal metastasis were more than 50‐fold higher than in those without metastasis. These results suggest a positive correlation between CEA mRNA levels in peritoneal washes and prognosis. We conclude that real‐time RT‐PCR with hybridization probes is a sensitive, quantitative, specific and rapid method to detect free cancer cells in peritoneal washes. This clinically relevant system is a powerful technique to evaluate the risk of peritoneal recurrence in patients with gastric cancer. Int. J. Cancer 89:411–417, 2000.

Real-time observation of micrometastasis formation in the living mouse liver using a green fluorescent protein gene-tagged rat tongue carcinoma cell line

Initial arrest, attachment, extravasation and subsequent extravascular growth of tumor cells in the secondary organs are believed to be crucial events for hematogenous metastasis, but the actual processes in living animals remain unclear. For the present study, we established green fluorescent protein (GFP)–expressing rat tongue carcinoma cell lines (RSC3) that permit real‐time analysis of micrometastasis formation in combination with intravital video microscopy (IVVM). With this system, GFP‐expressing metastatic (LM‐EGFP) and non‐metastatic (E2‐EGFP) cell lines could be visualized at the cellular level in live mice for more than 1 month. Real‐time IVVM analysis of liver metastases after intraportal injection of cells via a mesenteric vein revealed that both LM‐EGFP and E2‐EGFP tumor cells arrest similarly in sinusoidal vessels near terminal portal venules within 0.4 sec, during which time no evidence of a “rolling”‐like movement along endothelial cell surfaces was observed. Quantitative analysis of GFP‐positive foci showed that E2‐EGFP cells were completely sheared from the liver sinusoid within 3 days, with no solitary dormant cells, whereas a substantial number of LM‐EGFP cells remained in the liver, probably due to stable attachment to the sinusoidal wall. Confocal laser scanning microscopic study in combination with laminin immunohistochemistry revealed that only LM‐EGFP cells started growth at 3 to 4 days after inoculation and that most of the growing foci were surrounded by subsinusoidal basement membrane. Our results suggest that micrometastasis formation by LM‐EGFP cells consists of initial tumor cell arrest due to size constraints of the vessel, stable attachment to subsinusoidal basement membrane and subsequent intravascular growth before extravasation. The difference in metastatic potential between the 2 lines may reside in their capacity to attach stably to the vessel wall rather than their potential for initial cell arrest or subsequent growth. The system used in the present study may be a powerful tool for analyzing targets for various anti‐metastatic agents in the sequential process of metastasis.

Quantitative detection of CEA expressing free tumor cells in the peripheral blood of colorectal cancer patients during surgery with real-time RT-PCR on a LightCycler

We applied novel real-time reverse transcriptase-polymerase chain reaction (RT-PCR) with a LightCycler for quantitative detection of carcinoembryonic antigen (CEA) mRNA expressing tumor cells in the peripheral blood of colorectal cancer patients. Analysis of peripheral blood samples from 99 potentially curative colorectal cancer patients revealed a significantly higher mean CEA mRNA value in post-operative bloods (18.71) than in pre-operative blood (1.03) (P=0.003). Kaplan-Meier analysis demonstrated disease free survival of patients with positive CEA mRNA in post-operative blood to be significantly shorter than in cases negative for CEA mRNA (P=0.03). These results suggest that tumor cells could be shed into the bloodstream during surgical procedures, and these free tumor cells are accompanied by a poor patient outcome. Real-time quantitative RT-PCR is a useful technique for quantitative assessment of free tumor cells in the peripheral blood of colorectal cancer patients.

A Phase III Study of Laparoscopy-assisted Versus Open Distal Gastrectomy with Nodal Dissection for Clinical Stage IA/IB Gastric Cancer (JCOG0912)

A Phase III study was started in Japan to evaluate the non-inferiority of overall survival of laparoscopy-assisted distal gastrectomy with open distal gastrectomy in patients with clinical IA (T1N0) or IB [T1N1 or T2(MP)N0] gastric cancer. This study followed the previous Phase II study to confirm the safety of laparoscopy-assisted distal gastrectomy (JCOG0703) and began in March 2010. A total of 920 patients will be accrued from 33 institutions within 5 years. The primary endpoint is overall survival. The secondary endpoints are relapse-free survival, proportion of laparoscopy-assisted distal gastrectomy completion, proportion of conversion to open surgery, adverse events, short-term clinical outcomes, postoperative quality of life. Only a credentialed surgeon can be responsible for both open distal gastrectomy and laparoscopy-assisted distal gastrectomy.

Follow-up surveillance for recurrence after curative gastric cancer surgery lacks survival benefit.

AbstractBackground: Although routine follow-up to detect asymptomatic recurrence after surgery for gastric cancer is recommended, the effect of such reassessment on survival has not been evaluated. Methods: Clinical records of patients developing recurrent disease after potentially curative resection between 1985 and 1996 were retrieved. Among these patients, 197 were in our follow-up program. We analyzed survival in these patients according to the presence or absence of cancer-related symptoms when recurrent disease was diagnosed. Results: Of all patients with recurrent disease, 50% were diagnosed within 1 year and 75% within 2 years of surgery. Asymptomatic recurrence, detected in 88 patients (45%), frequently represented distant metastasis. Although early detection significantly improved survival after detection of recurrent disease, disease-free survival for this subset was shorter. Thus, no significant difference in overall survival was observed. Conclusions: Early detection of asymptomatic gastric cancer recurrence did not improve overall survival of patients with recurrence after curative resection. Until development of more effective treatment for this disease, close follow-up may offer no survival benefit.

Clinicopathological significance of fibrotic capsule formation around liver metastasis from colorectal cancer

Purpose: The fibrous capsule around hepatocellular carcinoma is well known to be an indicator of a good prognosis. However, the fibrotic stromal response in the liver to a metastatic tumor remains unclear. Patients and methods: In order to clarify the prevalence of fibrotic capsular formation around liver metastases as well as the prognostic and biological significance of the fibrotic capsule, 69 colorectal cancer patients, who underwent radical hepatectomy due to liver metastases, were investigated using immunohistochemical methods. Results: Encapsulated metastases as defined by a thick fibrotic band surrounding the entire surface of a metastasis were detected in 20% of the cases. The rate of initial recurrence in the remnant liver, which is a strong indicator for poor prognosis of colorectal liver metastasis, was significantly lower in the encapsulated metastasis group as compared with the non-encapsulated metastasis group. Proliferating fibroblastic cells in the capsule were myofibroblasts positively stained for α-smooth muscle actin (α-SMA) and they deposited dense extracellular matrices rich in collagen Type 1 in the layer of the inner half and secreted MMP-1, MMP-2, and TIMP-1 in the layer of the outer half of the capsule. Activation of α-SMA positive hepatic stellate cells (HSC) was also observed in the liver parenchyma adjacent to metastases. Conclusions: The results indicate that fibrotic capsular formation is associated with a lower rate of initial local recurrence in the remnant liver, and that the capsule may serve as a mechanical and chemical barrier to local invasion by metastatic tumor cells. Proliferating stromal cells in the capsule are myofibroblasts, probably derived from HSC activated by colorectal liver metastasis in the liver parenchyma.

Laparoscopic Wedge Resection for Gastrointestinal Stromal Tumors of the Stomach: Initial Experience

PurposeSurgery for gastrointestinal stromal tumors (GIST) of the stomach is now frequently performed using a laparoscopic approach. We investigated the feasibility and effectiveness of laparoscopy in the management of GIST of the stomach.MethodsWe reviewed the records of 12 consecutive patients who underwent laparoscopic surgery for GIST between April 2000 and April 2004, and compared their short-term outcomes with those of patients who underwent open surgery. All laparoscopic wedge resections were done using stapling devices and 3–4 trocars, often with the aid of intraoperative gastroscopy. We examined all patients preoperatively using various diagnostic modalities, including endoscopic ultrasonography-guided fine-needle aspiration (EUS-FNA). A laparoscopic approach was not indicated if the tumor was located near the cardia or pylorus or if it was ≧5 cm in diameter.ResultsA specific diagnosis of GIST was obtained preoperatively by EUS-FNA in 10 of the 12 patients. The median diameter of the lesion was 2.7 cm (range, 1.5–4.8 cm). Although intraoperative complications were encountered in two patients, conversion to open surgery was not required, and we were able to perform complete tumor excision with negative surgical margins in all patients. The median operative time was 100 min (range, 65–180 min), similar to that for open surgery. First flatus was passed earlier, and the interval to resuming oral intake was shorter than after open surgery. No major postoperative complications such as leakage developed, and the median postoperative hospital stay was 7 days (range, 5–12 days). All diagnoses made by EUS-FNA were confirmed by immunohisto-pathological evaluation of resected specimens.ConclusionLaparoscopic wedge resection is a feasible treatment option for GISTs of the stomach if the lesion is <5 cm in diameter.

ABO Genotype and the Risk of Gastric Cancer, Atrophic Gastritis, and Helicobacter pylori Infection

Background: Although several studies have investigated the association between ABO blood type and risk of gastric cancer (GC), atrophic gastritis (AG), and Helicobacter pylori (HP) infection, no study has investigated these associations by using ABO genotype. Methods: We conducted a case–control study in 703 patients with GC and 1,465 noncancer patients. We also conducted a cross-sectional study by using 1,406 of these 1,465 controls, who were examined for pepsinogens and anti-HP IgG antibody levels in serum. ABO genotype was determined from single nucleotide polymorphisms in ABO gene. We used rs8176719 to mark the O allele, and rs8176746 and rs8176747 to mark the B allele. ORs and 95% CIs were calculated by a multivariate logistic model. Results: We observed significant associations between ABO genotype and GC, AG, and HP infection. ORs (95% CIs) of GC were 0.70 (0.50–0.99) for OO and 0.53 (0.36–0.77) for BO relative to AA genotype. An increased risk of GC was observed with addition of the A allele (Ptrend < 0.001), and a decreased risk with that of the B allele (Ptrend = 0.023). An OR of AG was 0.73 (95% CI, 0.53–0.99) for blood type B relative to blood type A, and an OR of HP infection was 0.39 (95% CI, 0.17–0.87) for BB relative to AA genotype. Conclusion: This study identified a statistically significant association between ABO genotype and GC risk. In addition, ABO gene locus may influence AG prevalence and HP infection. Impact: Further studies are necessary to confirm these findings. Cancer Epidemiol Biomarkers Prev; 20(8); 1665–72. ©2011 AACR.

Association of prostate stem cell antigen gene polymorphisms with the risk of stomach cancer in Japanese

A recent whole‐genome association study identified a strong association between polymorphisms in the prostate stem cell antigen (PSCA) gene and stomach cancer risk. In this case‐control study, we aimed to validate this association, and further to explore environmental factors possibly interacting with PSCA polymorphisms in 708 incident stomach cancer cases and 708 age–sex matched controls. The association between PSCA polymorphisms and Helicobacter pylori infection was also examined. We found that rs2294008 and rs2976392, which were strongly linked to each other (D′ = 1.00), were significantly associated with stomach cancer risk. Per allele odds ratio for rs2994008 was 1.40 (95% confidence interval: 1.19–1.65; p = 3.7 × 10−5). We found significant interaction with a family history of stomach cancer in first‐degree relatives (p‐heterogeneity = 0.009). Similar to originally reported association, we found significant heterogeneity between diffuse and intestinal type (p‐heterogeneity = 0.007). No association was seen between PSCA polymorphisms and H. pylori infection. In conclusion, PSCA polymorphisms are associated with stomach cancer risk in Japanese. A possible interaction with family history warrants further evaluation.