Masamichi Yasuno

Higher frequency of smad4 gene mutation in human colorectal cancer with distant metastasis

We have previously detected an increased frequency of loss of heterozygosity (LOH) on chromosome 18q during progression of colorectal carcinomas. To clarify the target of 18qLOH, mutation of Smad4 and Smad2 genes was analysed in 176 colorectal tumors with different stages, including liver metastasis, from 111 sporadic, 52 familial adenomatous polyposis (FAP) and nine hereditary nonpolyposis colorectal cancer (HNPCC) patients. Mutation of other Smad gene families in the TGF-β signaling pathway was also examined. Twenty-one Smad4 mutations and one Smad2 mutation were detected, whereas mutation of Smad3, 6 and 7 genes was not detected. Smad4 mutations included seven frameshift, one inframe deletion, four nonsense and nine missense mutations, 95% of which resulted in alteration of Smad4 protein regions included in homo-oligomer and hetero-oligomer formation. Frequencies of tumors with Smad4 mutation were 0/40 (0%) in adenoma, 4/39 (10%) in intramucosal carcinoma, 3/44 (7%) in primary invasive carcinoma without distant metastasis, 6/17 (35%) in primary invasive carcinoma with distant metastasis, and 11/36 (31%) in distant metastasis (metastatic/non-metastatic: P=0.006 ∼0.01). Loss of the other allele was observed in 19 of 20 (95%) invasive and metastasized carcinomas with Smad4 mutations. In four cases both primary and metastasized carcinomas in the same patients showed the same mutations. The present results suggest that Smad4 gene is one of true targets of 18qLOH, and that its inactivation is involved in advanced stages, such as distant metastasis, in human colorectal carcinogenesis.

PIK3CA mutation is predictive of poor survival in patients with colorectal cancer

The PI3K‐AKT pathway is activated in a variety of human cancers, resulting in disturbance of cell growth, proliferation and survival. Among the factors affecting the pathway, the K‐Ras mutation and PIK3CA mutation are the most common oncogenic alterations in colorectal cancer. We hypothesized that these two mutations are important in activation of the PI3K pathway and colorectal carcinogenesis. In this study, we aimed to examine the influence of PIK3CA mutation and K‐Ras mutation on AKT activation, and to clarify whether PIK3CA mutation, K‐Ras mutation and p‐AKT expression may be used as parameters for predicting prognosis in colorectal cancer. Tissue samples from 158 colorectal cancer patients who underwent surgical resection were examined. The sequences of exon 1 of K‐Ras and exons 9 and 20 of PIK3CA were determined by direct sequencing using genomic DNA extracted from paraffin‐embedded blocks. Activation status of AKT was evaluated by immunohistochemical staining using phospho‐specific AKT antibody (Ser473). Correlation between these factors and clinicopathologic findings/patient survival were examined. As a result, PIK3CA mutation was significantly associated with shorter relapse‐free survival (RFS) in stage II/III patients (p = 0.0216) and shorter disease‐specific survival in all patients (p = 0.0357). In the multivariate analysis, PIK3CA mutation was the only independent and significant prognostic factor for RFS in stage II/III patients (p = 0.0433, HR 2.478). This study revealed the prognostic value of PIK3CA mutation in colorectal cancer patients. Patients with PIK3CA mutation should be followed up carefully. Moreover, our result suggests that inhibition of PIK3CA mutant may be a new molecular target therapy.

Effect of classification based on combination of mutation and methylation in colorectal cancer prognosis

Colorectal cancer (CRC) is caused by an accumulation of genetic alterations and epigenetic alterations. The molecular classification of CRCs based on genetic alterations and epigenetic alterations is evolving. Here, we examined mutations and methylation status in CRCs to determine if the combination of genetic and epigenetic alterations predicts prognosis. We examined 134 sporadic CRCs. We used the direct sequencing method to identify mutations in BRAF and AKT1, which are downstream of KRAS and PIK3CA, respectively, in the EGFR pathway. We used the Methylight method to determine the methylation status of hMLH1, p16, MINT1, MINT2 and MINT31. Both BRAF and AKT1 mutations were found in only one case (0.75%). Aberrant methylation of hMLH1, p16, MINT1, MINT2 and MINT31 was detected in 22.4, 35.1, 32.8, 59.7 and 41.0% of cases, respectively. The clinicopathological factors were not significantly correlated to mutation or methylation. Among the patients who had no mutation in the EGFR pathway, the overall survival was significantly shorter in the patients with methylation compared to the patients with no methylation in hMLH1 and p16 (p=0.0318). Methylation could play a key role in the prognosis of patients without mutations in the EGFR pathway. The combination of genetic and epigenetic alterations may be a good marker for the prognosis of CRC patients.

Lymph node ratio is a powerful prognostic index in patients with stage III distal rectal cancer: a Japanese multicenter study

PurposeThe present study aims to define the prognostic impact of the lymph node ratio (LNR) in patients with stage III distal rectal cancer.MethodsWe analyzed data from 501 patients who underwent curative resection (total mesorectal excision, TME) for stage III distal rectal cancer at 12 institutions between 1991 and 1998. Patients were divided into four groups according to quartiles based on LNR.ResultsAmong the 501 patients, 381 underwent TME with pelvic sidewall dissection (PSD). The median numbers of lymph nodes retrieved with and without PSD were 45 and 17, respectively (P < 0.0001). Forty-nine patients with lymph node retrieved less than 12 were excluded from further analyses. Among various clinicopathological parameters, univariate analysis identified age (P = 0.0059), histological grade (P < 0.0001), depth of tumor invasion (P = 0.0003), and number of positive nodes (P < 0.0001) and LNR (P < 0.0001) as prognostic factors. The Cox proportional hazards model revealed that age (P = 0.014), histological grade (P < 0.0001), depth of tumor invasion (P = 0.0002), and LNR (group 3, P = 0.0012; group 4, P < 0.0001) were independent prognostic factors. When the American Joint Committee on Cancer (AJCC) seventh staging system was added as a covariate, both AJCC stage (P < 0.0001) and LNR (P < 0.0001) were independent prognostic factors.ConclusionsAdding the LNR concept to the AJCC cancer staging system will improve accuracy in evaluating the nodal status of distal rectal cancer.

Evaluation of Aggressively Treated Patients With Unresectable Multiple Liver Metastases From Colorectal Cancer

AbstractPURPOSE: The aim of this study was to assess the value of aggressively treating patients with unresectable liver metastases from colorectal cancer and a poor prognosis. METHODS: From 1988 to 1999, 64 patients with unresectable multiple liver metastases from colorectal cancer who had received hepatic arterial infusion chemotherapy were investigated. All patients did not have synchronous extrahepatic metastases at the time of initiating our treatment. When liver metastases were suitable for resection after hepatic arterial infusion chemotherapy, we excised them and repeated prophylactic hepatic arterial infusion chemotherapy as long as possible. We evaluated the efficacy of hepatic arterial infusion chemotherapy by computed tomography and divided these patients into responders and nonresponders. We performed univariate analysis using the log-rank test to calculate predictive factors. In addition, the Cox proportional hazards model was used to perform multivariate analysis of factors related to survival. RESULTS: The survival rate of all patients was 67.8 percent after 1 year and 10 percent after 5 years. However, the survival rate for 16 patients who received hepatectomy after hepatic arterial infusion chemotherapy was 35.1 percent after five years. Multivariate analysis demonstrated that the response after hepatic arterial infusion chemotherapy was the most indicative prognostic factor. CONCLUSIONS: The prognosis of selected patients who responded to hepatic arterial infusion chemotherapy and received hepatectomy was improved. Applying aggressive treatment as outlined in our strategy may improve the chances of long-term survival.

Is Total Mesorectal Excision Always Necessary for T1–T2 Lower Rectal Cancer?

BackgroundThe goal of this multicenter study was to clarify the determinants of local excision for patients with T1–T2 lower rectal cancer.MethodsData from 567 consecutive patients who underwent radical resection for T1–T2 lower rectal cancer at 12 institutions between 1991 and 1998 were reviewed. Rates of lymph node metastasis were investigated using a tree analysis, which was hierarchized using independent risk factors for nodal involvement.ResultsThe independent risk factors for lymph node metastasis were female gender, depth of tumor invasion, histology other than well-differentiated adenocarcinoma, and lymphatic invasion. According to the first three parameters that can be obtained preoperatively, only 0.99% of the patients without risk factors had lymph node metastasis. On the other hand, even if the lower rectal cancer was at stage T1, women with histological types other than well-differentiated adenocarcinoma had an approximately 30% probability of having lymph node metastasis. Lymphatic invasion was most useful to predict nodal involvement among patients with T2 lower rectal cancer. The rates of lymph node metastasis in T2 patients with and without lymphatic invasion were 32.9% and 9.1%, respectively.ConclusionsGender is one of the most important predictors for lymph node metastasis in patients with early distal rectal cancer. Three parameters, including depth of tumor invasion, histology, and gender, are useful determinants for local excision. Additional studies are required to establish the minimum optimal treatment for T2 lower rectal cancer.

A new technique for endoscopic esophageal mucosectomy using a transparent overtube with intraluminal negative pressure (np-EEM)

Abstract: We devised a new technique for endoscopic esophageal mucosectomy using a transparent overtube (OT) with intraluminal negative pressure (np‐EEM) (Figs. 1,2). Basic studies were performed on six dogs. The following two methods were utilized in the np‐EEM technique in line with standard approaches used for mucosal resection: (1) A “snare” method was used in which the esophageal mucosa protruded, similar to a polyp, through the slit of the OT and was resected by an electro‐snare passing through the endoscope biopsy channel or the injection channel of the OT. A high‐frequency current was used for the resection (Figs. 2‐a 2‐b, Fig. 3, Color). (2) A “cutter” method was used in which the mucosal protrusion was resected by a recently developed electro‐cutter (Figs. 2‐a, 2‐c, 2‐d, Fig. 3, Color).

Alterations of repeated sequences in 5′ upstream and coding regions in colorectal tumors from patients with hereditary nonpolyposis colorectal cancer and Turcot syndrome

One of the characteristics of tumors from patients with germline mutations of DNA mismatch repair genes is instability at microsatellite regions (MSI). We analysed alterations at repeated sequences of coding regions, as well as those of 5′ upstream regions, in 29 MSI-High colorectal tumors from patients with hereditary nonpolyposis colorectal cancer (HNPCC) and Turcot syndrome. We found that repeated sequences in 5′ upstream regions were altered in these tumors, at considerable frequencies. The (A)10 repeat in the promoter region (position −178∼−169) of the GAPDH gene was altered in 17% of the tumors. The (A)10(TA)9 in the 5′ upstream region (position −318∼−291) of the mitochondrial isoleucyl tRNA synthetase gene (IleRS-A), coded in nuclear DNA, was altered in 59% of the tumors, whereas (A)9 in the 5′ upstream region (position −859∼−851) of cytoplasmic isoleucyl tRNA synthetase gene (IleRS-B) was not altered. Alteration at repeated sequences in the coding regions were 72% at TGFβRII(A)10, 24% at IGFIIR(G)8, 45% at BAX(G)8, 55% at E2F4(CAG)13, 66% at caspase-5 (A)10, 31% at MBD4(A)10, 55% at hMSH3(A)8 and 34% at hMSH6(C)8. The number of altered genes increased with the advancement of carcinoma according to Dukes categories: mean numbers of altered genes within these 10 genes were 2.6 for Dukes A, 4.7 for Dukes B and 7.8 for Dukes C. The mean number for adenomas was 2.0. These results suggest that the MSI phenotype also causes alteration of 5′ upstream regions which may affect apoptosis and some mitochondrial functions in HNPCC and Turcot tumors, and that accumulation of altered genes with repeated sequences is associated with the progression of HNPCC and Turcot colorectal tumors.

Histologic grade of metastatic lymph node and prognosis of rectal cancer

PURPOSE: It is important to identify cases with a high risk of recurrence to improve the prognosis of colorectal cancer. In this study the difference between the histology of the primary lesion and that of the metastatic lymph node was investigated in an attempt to identify the cases with a high risk of recurrence. METHODS: One-hundred eighty-five patients with Dukes C rectal cancer who had undergone curative resection were investigated. The histologic grade of the metastatic lymph node was determined and compared with other clinicopathologic factors to determine its significance as a prognostic factor. RESULTS: The histologic grade was the same between the primary lesion and the metastatic lymph node in 46.2 percent of all cases, although in the group with well-differentiated adenocarcinoma at the primary lesion the concordance was only 29.5 percent. In the group with well-differentiated adenocarcinoma at the primary lesion, the five-year survival rate was 75.3, 64, and 25 percent in the groups with well-differentiated, moderately differentiated, and poorly differentiated adenocarcinoma at the metastatic lymph node, respectively. The differences between the survival rates of well-differentiated and poorly differentiated adenocarcinoma at the metastatic lymph node were statistically significant (P<0.05). According to multivariate analysis the histologic grade of primary lesion was the most significant prognostic factor (hazard ratio: 2.2801,P=0.0008). However, in well-differentiated adenocarcinoma of patients with Dukes C rectal cancer at the primary lesion, the histology of metastatic lymph node was also an important prognostic factor. CONCLUSIONS: It is clear that the histologic grade between the primary lesion and metastatic lymph node was frequently different, especially in the group with well-differentiated adenocarcinoma at the primary lesion. The analysis of the metastatic lymph node was considered to have additional importance for the prediction of prognosis.

A Multicenter Phase II Trial of mFOLFOX6 Plus Bevacizumab to Treat Liver-Only Metastases of Colorectal Cancer that are Unsuitable for Upfront Resection (TRICC0808)

BackgroundA phase II clinical trial was conducted on colorectal cancer patients with only liver metastases (focal diameter exceeds 5 cm or the number of liver metastases is ≥5; H2·H3) to evaluate the liver resection rate and safety after 6 cycles of mFOLFOX6+bevacizumab (BV) therapy.MethodsmFOLFOX6+BV therapy was applied for 6 cycles to the patients with H2·H3 liver only metastasis. Hepatectomy was considered after the sixth cycle as a rule, and was performed if possible. The primary endpoint was the curative hepatectomy rate (R0 resection rate).ResultsForty-six patients were registered and 45 patients were included in the efficacy analysis. Of the 19 patients rated as unresectable before therapy, 18 completed 6 cycles of mFOLFOX6+BV therapy and subsequently underwent hepatectomy (16 were R0-resected). Of the 26 initially unresectable patients, 6 underwent hepatectomy (4 were RO-resected). The overall R0 resection rate was 44.4% (20/45). Chemotherapy-associated grade 3 or higher adverse events included neutrophil decreased (17.4%) and leukocyte decreased (8.7%), fatigue (6.5%) etc. Only hypertension (6.5%) and venous thromboembolism (2.2%) were BV-associated grade 3 or higher adverse events. Among the 25 patients who underwent hepatectomy, intraoperative/postoperative complications included grade 3 wound infections (2 cases), biloma, delayed wound healing and intraperitoneal abscess (each 1 case).ConclusionsIn colorectal cancer patients with liver-only metastases, mFOLFOX6+ BV therapy yielded a high hepatectomy rate and a high percentage of initially unresectable and subsequently resectable cases. The chemotherapy associated adverse events and hepatectomy complications were both within acceptable ranges.A phase II clinical trial was conducted on colorectal cancer patients with only liver metastases (focal diameter exceeds 5 cm or the number of liver metastases is ≥5; H2·H3) to evaluate the liver resection rate and safety after 6 cycles of mFOLFOX6+bevacizumab (BV) therapy. mFOLFOX6+BV therapy was applied for 6 cycles to the patients with H2·H3 liver only metastasis. Hepatectomy was considered after the sixth cycle as a rule, and was performed if possible. The primary endpoint was the curative hepatectomy rate (R0 resection rate). Forty-six patients were registered and 45 patients were included in the efficacy analysis. Of the 19 patients rated as unresectable before therapy, 18 completed 6 cycles of mFOLFOX6+BV therapy and subsequently underwent hepatectomy (16 were R0-resected). Of the 26 initially unresectable patients, 6 underwent hepatectomy (4 were RO-resected). The overall R0 resection rate was 44.4% (20/45). Chemotherapy-associated grade 3 or higher adverse events included neutrophil decreased (17.4%) and leukocyte decreased (8.7%), fatigue (6.5%) etc. Only hypertension (6.5%) and venous thromboembolism (2.2%) were BV-associated grade 3 or higher adverse events. Among the 25 patients who underwent hepatectomy, intraoperative/postoperative complications included grade 3 wound infections (2 cases), biloma, delayed wound healing and intraperitoneal abscess (each 1 case). In colorectal cancer patients with liver-only metastases, mFOLFOX6+ BV therapy yielded a high hepatectomy rate and a high percentage of initially unresectable and subsequently resectable cases. The chemotherapy associated adverse events and hepatectomy complications were both within acceptable ranges.