Masaru Shinozaki

Incidence of BRAF Oncogene Mutation and Clinical Relevance for Primary Cutaneous Melanomas

Purpose: The purpose of the study was to clarify the incidence of B-raf oncogene (BRAF) mutations in primary cutaneous melanomas, their relation to tumor progression, and effect on disease outcome. Somatic mutations of BRAF kinase, a component of the Ras-mitogen-activated protein/extracellular signal-regulated kinase kinase-mitogen-activated protein kinase pathway, are frequently reported (>65%) in nevi and malignant melanomas. Experimental Design: We assessed BRAF mutation frequency in exons 11 and 15 in primary (n = 59) and metastatic (n = 68) melanomas. Direct sequencing of PCR products was performed on DNA isolated and purified from microdissected tumors. Results: Eighteen mutations (31%) at exon 15 were detected in primary melanoma with a significantly (P = 0.001) higher frequency in patients < 60 years old. Incidence of BRAF mutation did not correlate with Breslow thickness. Presence of BRAF mutation of primary tumors did not effect overall disease-free survival. BRAF mutation frequency in metastatic lesions was 57% and significantly (P = 0.0024) higher than primary melanomas. Conclusions: The study suggests that BRAF mutation may be acquired during development of metastasis but is not a significant factor for primary tumor development and disease outcome.

Aberrant hypermethylation of ID4 gene promoter region increases risk of lymph node metastasis in T1 breast cancer

ID4 gene is a member of the inhibitor of DNA-binding (ID) family, which inhibits DNA binding of basic helix–loop–helix transcription factors. Certain human primary breast cancers reportedly have low or no expression of ID4 protein, but its role in carcinogenesis and cancer progression is unknown. To determine its possible role, we examined epigenetic inactivation of ID4 gene by promoter hypermethylation in human breast cell lines and T1 breast cancer tissues. Methylation status of ID4 promoter CpG island was assessed by methylation-specific PCR (MSP); ID4 mRNA level was assessed by quantitative real-time RT–PCR. Of eight cell lines, two were fully methylated, four were partially methylated, and two were not methylated. ID4 mRNA level was suppressed in fully methylated cell lines. ID4 hypermethylation was observed in 16 of 24 (67%) node-positive and seven of 36 (19%) node-negative T1 primary breast cancers matched by patient age and tumor diameter. It was a significant risk factor for nodal metastasis (OR 13.1, P=0.0004). ID4 mRNA level was suppressed in hypermethylated cancer specimens (P=0.014). ID4 may play an important suppressive role in tumor progression, and its silencing by hypermethylation may increase the risk of regional lymph node metastasis.

Epigenetic Inactivation of ID4 in Colorectal Carcinomas Correlates with Poor Differentiation and Unfavorable Prognosis

Purpose: ID4 gene is a member of the inhibitor of DNA binding (ID) family proteins that inhibit DNA binding of basic helix-loop-helix transcription factors. The epigenetic inactivation of ID4 gene on colorectal cancer (CRC) development and its clinical significance was assessed. Experimental Design: In CRC cell lines, ID4 methylation status of the promoter region was assessed by methylation-specific PCR and bisulfite sequencing. The mRNA expression level was assessed by quantitative real-time reverse transcription-PCR. The methylation status of 9 normal epithelia, 13 adenomas, 92 primary CRCs, and 26 liver metastases was assessed by methylation-specific PCR. ID4 protein expression was assessed by immunohistochemistry analysis of tissue specimen. Results: CRC cell lines were shown to be hypermethylated, and mRNA expression was suppressed and could be restored by 5-aza–cytidine treatment. In clinical specimens from normal epithelia, adenomas, primary CRCs, and liver metastases, the frequency of ID4 hypermethylation was 0 of 9 (0%), 0 of 13 (0%), 49 of 92 (53%), and 19 of 26 (73%), respectively, with a significant elevation according to CRC pathological progression. Methylation status of primary CRCs significantly correlated with histopathological tumor grade (P = 0.028). Immunohistochemistry analysis showed ID4 expression of normal colon epithelia, adenomas, and unmethylated primary CRCs but not hypermethylated CRC specimens. Among 76 American Joint Committee on Cancer stage I to IV patients who had undergone curative surgical resection, overall survival was significantly poorer in patients with hypermethylated ID4 bearing tumors (P = 0.0066). Conclusions: ID4 gene is a potential tumor suppressor gene for which methylation status may play an important role in the CRC progression.

Estrogen receptor and HER2/neu status affect epigenetic differences of tumor-related genes in primary breast tumors

IntroductionEstrogen receptor (ER)-positive breast cancers are considered prognostically more favorable than ER-negative tumors, whereas human epidermal growth factor receptor (HER)2/neu-positive breast cancers are associated with worse prognosis. The objective of the present study was to determine whether ER-positive and ER-negative status relates to epigenetic changes in breast cancer-related genes. To evaluate epigenetic differences in tumor-related genes relating to ER and HER2/neu status of primary tumors, we examined the promoter methylation status of the promoter region CpG islands of eight major breast tumor-related genes (RASSF1A, CCND2, GSPT1, TWIST, APC, NES1, RARβ2, and CDH1).MethodsPaired ER-positive (n = 65) and ER-negative (n = 65) primary breast tumors (n = 130) matched for prognostic factors were assessed. DNA was extracted from paraffin-embedded tumor tissue after microdissection, and methylation-specific PCR and capillary-array electrophoresis analysis were performed.ResultsIn early stages of tumor progression (T1 and N0), RASSF1A and CCND2 were significantly (P < 0.05) more methylated in ER-positive than in ER-negative tumors. GSTP1 hypermethylation was more frequent in the lymph node metastasis positive group than in the negative group. Double negative (ER-negative, HER2/neu-negative) breast cancers had significantly lesser frequencies of RASSF1A, GSTP1, and APC methylation (P < 0.0001, P < 0.0001, and P = 0.0035, respectively). Both ER and HER2/neu status correlated independently with these epigenetic alterations.ConclusionWe demonstrated significant differences in tumor-related gene methylation patterns relevant to ER and HER2/neu status of breast tumors. This may be of significance in the assessment of targeted therapy resistance related to ER and HER2/neu status in breast cancer patients.

Patients with extraintestinal manifestations have a higher risk of developing pouchitis in ulcerative colitis: multivariate analysis

Background: Restorative proctocolectomy and ileal pouch‐anal anastomosis (IPAA) have been the surgical treatments of choice for ulcerative colitis (UC). However, the IPAA is sometimes complicated with pouchitis. Furthermore, the cumulative risk and risk factors for developing pouchitis in patients with UC undergoing IPAA have not been reported in any Asian population. The aim of our study is to clarify the cumulative risk and risk factors for developing pouchitis in Japanese patients with UC undergoing IPAA. Methods: Fifty‐eight patients with UC undergoing IPAA were retrospectively evaluated for the presence of pouchitis, gender, age of onset, history of smoking, presence of extraintestinal manifestations (EIMs) and type of operation. The diagnosis of pouchitis was based on the pouchitis disease activity index. The cumulative risk and risk factors for developing pouchitis were assessed. Results: The cumulative risks for developing pouchitis at 1 and 5 years were 9.0% and 14.4%, respectively. The estimated risks of pouchitis at 5 years was 48.1% in patients with EIMs and 4.6% in those without. Both univariate and multivariate analyses revealed that the presence of EIMs is a significant risk factor (hazard ratio 16.85, 95% confidence interval 3.12–91.00; P = 0.001). Conclusions: The presence of EIMs is a significant risk factor for the development of pouchitis in Japanese patients with UC undergoing IPAA.

Genetic Alterations in Ulcerative Colitis-associated Neoplasia Focusing on APC, K-ras Gene and Microsatellite Instability

The status of genetic alterations in ulcerative colitis (UC)‐associated neoplasia (UCAN) was investigated focusing on microsatellite instability (MSI) which is seen in a certain fraction of colorectal carcinomas, and adenomatous polyposis coli (APC) gene and K‐ras gene, in which mutations occur in the early stage of sporadic colorectal tumorigenesis. Thirty‐one UCAN from 15 UC patients who had undergone colorectal resection at our institution were investigated. There were 8 lesions of invasive carcinoma, 15 high‐grade dysplasia (HGD) and 8 low‐grade dysplasia (LGD). DNA was extracted from each neoplastic lesion and corresponding non‐neoplastic tissue by a microdissection method. MSI status at 9 microsatellite loci, loss of heterozygosity (LOH) at the APC locus, and K‐ras codon 12 point mutation were examined. As for MSI, 4/31 (13%) UCAN (carcinoma: 1/8 (13%), HGD: 2/15 (13%), LGD: 1/8 (13%)) were MSI‐high (3 or more unstable loci) and 12/31 (39%) UCAN (carcinoma: 3/8 (38%), HGD: 6/15 (40%), LGD: 3/8 (38%)) were MSI‐low (1 or 2 unstable loci). LOH at the APC locus was not found in 9 UCAN from 6 informative (heterozygous) cases. The K‐ras mutation rate of UCAN was 3/31 (9.7%) (carcinoma: 2/8 (25%), HGD: 1/15 (7%) and LGD: 0/8). MSI is relatively common in UCAN and is present at the early stage of tumorigenesis of UCAN, while the involvement of genetic alterations of the APC gene and K‐ras gene is small. MSI may be one of the mechanisms of the increased neoplastic risk in UC, and UCAN may develop through a different carcinogenic pathway from sporadic carcinomas.

Cytomegalovirus infection in ulcerative colitis

Objective. Cytomegalovirus (CMV) infection has been reported as an exacerbating factor in inflammatory bowel disease but the relationship between CMV infection and ulcerative colitis (UC) remains unclear. There has been no detailed research to elucidate the clinicopathologic features of CMV infection in UC using surgical specimens. The aim of this study was to investigate the clinicopathologic features of CMV infection in UC patients who had undergone colectomy. Material and methods. Surgical specimens taken from UC patients were examined for CMV infection. The patients were divided into three groups: severe, refractory, and UC-associated dysplasia or cancer according to the operative indications. CMV infection rates were evaluated and a comparison of clinical parameters was made between CMV-positive and CMV-negative patients, and the risk factors for CMV infection were analyzed using multivariate analyses. Results. It was found that 25% of 32 patients were positive for CMV in the severe UC group; 8.3% of 72 patients were positive for CMV in the refractory UC group. None of the 22 patients was positive for CMV in the UC-associated dysplasia or cancer group. The CMV-positive rate in the severe UC group was significantly higher than that in the other groups (p<0.05). Patients’ age at the time of operation was higher in the CMV-positive group than in the CMV-negative group among the patients with severe UC (p<0.01), and age at operation was an independent risk factor for CMV infection. Conclusions. CMV is found more frequently in severe UC than refractory UC and UC-associated cancer or dysplasia. Higher age can be a risk factor for CMV infection in patients with severe UC. However, a high steroid dose may not always be a risk factor for CMV infection.

Allelic imbalance of APAF-1 locus at 12q23 is related to progression of colorectal carcinoma.

APAF-1 gene, located at chromosome locus 12q23, is a key factor in the mitochondrial apoptotic pathway downstream of p53, and is a potential tumor suppressor gene. We hypothesized that APAF-1 gene dysfunction due to allelic imbalance (AI) contributes to the development and progression of colorectal carcinoma (CRC). AI at APAF-1 locus and microsatellite instability (MIN) in CRCs and adenomas were assessed by multiple microsatellite markers. The frequency of AI significantly increased with tumor progression; 0 of 33 (0%) adenomas, 14 of 49 (29%) primary CRCs, and 18 of 34 (53%) liver metastases had AI. A total of 12 metastases were matched with corresponding primary CRCs; in 11 of 12 (92%) pairs, the metastasis had same AI status as the corresponding primary tumor. APAF-1 mRNA transcription level was significantly decreased with AI in liver metastases (P=0.009). Promoter hypermethylation was found in three of 35 (9%) primary CRCs and one of 15 (7%) liver metastases by methylation-specific PCR but was not correlated with AI. MIN was observed in 11 of 49 (23%) primary CRCs and was a favorable prognostic factor. Our results suggest that APAF-1 gene haploinsufficiency caused by AI increases with tumor progression, and relates to hepatic metastasis.

The incidence and outcome of pelvic sepsis following handsewn and stapled ileal pouch anal anastomoses.

The incidence and outcome of pelvic sepsis was analyzed in 210 patients who underwent restorative proctocolectomy for ulcerative colitis (UC) in 197 patients, and for familial adenomatous polyposis (FAP) in 13 patients. Pelvic sepsis developed in 18 patients (8.6%) and a significantly higher incidence was seen in men than in women, at 13.6% vs 3.7%, respectively (P < 0.05). The incidence of pelvic sepsis in patients with UC complicated by toxic megacolon and/or fulminant colitis was significantly higher that in those without any preoperative complications, at 36.4% vs 7.4% (P < 0.05). The incidence of pelvic sepsis following handsewn anastomosis was significantly higher than that following stapled anastomosis, at 15.6% vs 5.5% (P < 0.05). The outcome of pelvic sepsis in patients with a stapled anastomosis was better than that in those with a handsewn anastomosis. The prognosis of women who developed pelvic sepsis was better than that of men who developed pelvic sepsis. The risk factors predisposing to pelvic sepsis were UC, especially when complicated by toxic megacolon and/or fulminant colitis, and male sex, while a handsewn anastomosis was more vulnerable than a stapled anastomosis.

High proliferative activity is associated with dysplasia in ulcerative colitis

PURPOSE: Ulcerative colitis is associated with an increased risk of colorectal neoplasia. Markers of proliferation are reported to be valuable in the diagnosis of dysplasia in ulcerative colitis. However, it is not known whether dysplastic change or proliferative change occurs first. Whether abnormal proliferation is present in normal-seeming mucosa in ulcerative colitis was investigated. METHODS: Eighteen cancer or high-grade dysplasia specimens and 9 low-grade dysplasia specimens from 5 patients and 51 specimens from 31 patients without neoplasia were studied. Immunostaining with anti-Ki 67 antibody was used to evaluate proliferative activity. Labeling index (in the superficial one-half of crypt) was calculated. Crypts with labeling index more than 0.3 were determined to have abnormal proliferation. RESULTS: The mean ± standard error of the mean labeling index in specimens negative for dysplasia (0.056±0.004) was significantly lower than that in low-grade dysplasia specimens (0.418±0.024) and that in high-grade dysplasia specimens (0.503±0.027;P<0.0001). In specimens negative for dysplasia, only 4 (4 cases) of 339 (1.2 percent) crypts had abnormal proliferation, whereas the ratio of crypts with abnormal proliferation was 76 percent (54/71) in low-grade dysplasia and 92.1 percent (35/38) in high-grade dysplasia. The labeling index in background mucosa was 0.139±0.009, which was significantly higher than that in specimens negative for dysplasia (P<0.001). In background mucosa 15.7 percent of crypts showed abnormal proliferation. A follow-up study revealed that two of four cases developed cancer or high-grade dysplasia one and seven years after proliferative abnormality was detected in nondysplastic specimens. CONCLUSION: Ki-67 immunostaining can be an aid in the diagnosis of dysplasia. High proliferating activity in background mucosa suggests that proliferating activity change precedes dysplasia detected with hematoxylin-and-eosin staining.