Fang-Ming Chen

Molecular detection of APC, K- ras, and p53 mutations in the serum of colorectal cancer patients as circulating biomarkers.

Early detection of tumor DNA in serum/plasma prior to the development of recurrence or metastases could help improve the outcome of patients with colorectal cancer (CRC) after tumor resection. Recent advances in the detection of tumor DNA in the serum/plasma has opened up numerous new areas for investigation and new possibilities for molecular diagnosis. APC and K-ras mutations are considered to be early-stage developments of CRCs, whereas p53 mutations are thought to be relatively late events in the tumorigenesis of CRCs. The aim of this study was to search for the presence of genetic mutations in the DNA extracted from the serum of CRC patients and healthy subjects. We simultaneously evaluate the significance of APC, K-ras, and p53 gene mutations in cancer tissues and their paired serum samples of 104 CRC patients by polymerase chain reaction-single strand conformation polymorphism analysis (PCR-SSCP) followed by direct sequencing. Additionally, analysis was carried out to detect the serum carcinoembryonic antigen (CEA) levels in CRC patients. Overall, we found at least one of the gene mutations in tumor tissues from 75% (78/104) of the CRC patients. Comparison of the three molecular markers showed that the detection rates in the serum were 30.4%, 34.0%, and 34.2% for APC, K-ras, and p53 genes, respectively. Of these patients, 46.2% (36/78) were identified as having positive serum results, whereas all healthy controls remained negative. The overall positive tumor DNA detection rates in the serum were 0% (0/7) for Dukes’ A classification, 22.4% (11/49) for Dukes’ B, 48.7% (19/39) for Dukes’ C, and 66.7% (6/9) for Dukes’ D. The detection rate increased as the tumor stage progressed (p = 0.012). Concurrently, a significant difference was observed between lymph node metastases and positive serum tumor DNA detection (p < 0.001). A significantly higher postoperative metastasis/recurrence rate in patients harboring gene mutations with serum tumor DNA than those without serum tumor DNA was also demonstrated (p < 0.001). However, no significant correlation between the postoperative metastasis/recurrence and serum CEA levels was observed (p = 0.247). These data suggest that the identification of circulating tumor DNA using the molecular detection of APC, K-ras, and p53 gene mutations is a potential tool for early detection of postoperative recurrence/metastases. Moreover, these genes may be potential molecular markers of poor clinical outcome in CRC patients.

Randomized Controlled Trial of LigaSure with Submucosal Dissection versus Ferguson Hemorrhoidectomy for Prolapsed Hemorrhoids

IntroductionThe aim of this study was to compare the outcomes between the LigaSure vessel sealing system and the conventional closed Ferguson hemorrhoidectomy procedure performed by diathermy.MethodsA series of 84 patients with grades III and IV hemorrhoids were randomized into two groups: (1) LigaSure hemorrhoidectomy with submucosal dissection (42 patients) and (2) Ferguson hemorrhoidectomy (42 patients). The patient demographics, operative details, parenteral analgesic requirement, postoperative pain score (assessed by an independent assessor), operating time, intraoperative blood loss, hospital stay, early and delayed complications, and time off from work or normal activity were recorded. The patients were regularly followed up at 1, 2, 4, 6, and 8 weeks after surgery.ResultsThere were no statistically significant differences between the two groups in terms of age, gender, duration of symptoms, grade of the hemorrhoid(s), or number of hemorrhoids resected. The mean operating time for LigaSure hemorrhoidectomy with submucosal dissection was significantly shorter than that for the Ferguson hemorrhoidectomy (11.3 ± 0.4 vs. 34.2 ± 0.7 minutes; P < 0.0001). Patients treated with the LigaSure method had significantly less blood loss (P < 0.0001), a better pain score (P < 0.0001), less parenteral analgesic requirement (P < 0.0001), shorter hospital stay (P < 0.0001), and less time off from work or normal activity (P < 0.0001). There was no difference in the early and delayed postoperative complications between the two groups.ConclusionsLigaSure hemorrhoidectomy with submucosal dissection is a safe, effective procedure for grade III and IV hemorrhoids. Patients derive greater short-term benefits: reduced intraoperative blood loss, operating time, and postoperative pain as well as earlier resumption of work or normal activity. Long-term follow-up with a larger number of patients is required to confirm the long-term results of this procedure.

Combination of multiple mRNA markers (PTTG1, survivin, UbcH10 and TK1) in the diagnosis of taiwanese patients with breast cancer by membrane array

Objective: Early detection is a prerequisite to the effective reduction of morbidity and mortality from breast cancer. The present study intended to employ a high-throughput membrane array to detect a panel of mRNA markers expressed by circulating tumor cells (CTCs) in the peripheral blood of female patients with breast cancer. Methods: Peripheral blood was sampled from 92 breast cancer patients and 100 normal persons. CTCs were detected by using a membrane array technique. The markers used included the pituitary tumor transforming gene 1, survivin, UbcH10 and thymidine kinase 1. Results: The results showed that the membrane array could positively detect 5 cancer cells per 1 ml of peripheral blood in breast cancer cell dilution experiments. For the panel of 4 mRNA markers, sensitivity and specificity were elevated up to 86 and 88%, respectively. Furthermore, it was found that the patients’ clinicopathological characteristics tumor size (p = 0.006), histologic grade (p = 0.012), lymph node metastasis (p = 0.001) and TNM stage (p = 0.006) significantly correlated with the positive detection rate of the multimarker panel. Conclusions: These findings demonstrated that our multimarker membrane array method could detect CTCs in the circulation of breast cancer patients with considerably high sensitivity and specificity.

Sarco/endoplasmic reticulum calcium-ATPase 2 expression as a tumor marker in colorectal cancer.

Maintaining a high calcium concentration in the endoplasmic reticulum through the action of sarco/endoplasmic reticulum calcium-ATPases (SERCAs) is crucial in many cell functions involved in intracellular signal transduction, control of proliferation, programmed cell death, or the synthesis of mature proteins. Recent studies have found that many SERCAs have altered expression patterns in various malignancies. The purpose of the current study was to quantify the expression of SERCA2 in colorectal cancer (CRC) tissues and the corresponding noncancerous tissues, and to statistically analyze whether the SERCA2 expression levels correlate with the clinico-pathologic features and prognosis of CRC patients. Paired colorectal tissue samples from cancerous and the corresponding noncancerous tissues were obtained from 50 patients who underwent surgical resection. Semiquantitative measurements of SERCA2 messenger RNA (mRNA) expression were done using the multiplex reverse transcriptase-polymerase chain reaction. CRC tissues were analyzed through immunohistochemistry for the SERCA2 protein. SERCA2 mRNA overexpression in cancerous tissues compared with normal counterparts was observed in 45 of 50 (90%) patients. The mean expression level of SERCA2 mRNA in cancerous tissues was significantly higher than that in noncancerous tissues (P=0.01). Increased SERCA2 protein expression was significantly correlated with serosal invasion (P=0.012), lymph node metastasis (P=0.009), and advanced tumor stage (P=0.004). Furthermore, patients with high SERCA2 expression had a significantly poorer overall survival rate than patients with low SERCA2 (P=0.032). Multivariate analyses indicated that tumor stage (P=0.015) and SERCA2 expression were independently correlated with overall survival (P=0.018). The result of this study indicated that SERCA2 may be a molecular determinant in the development and progression of CRC. The molecular mechanisms underlying the SERCA-dependent calcium accumulation and CRC tumorigenesis are worthy of further investigations.

Mutation analysis of the putative tumor suppressor gene PTEN/MMAC1 in advanced gastric carcinomas

A novel tumor suppressor gene, PTEN/MMAC1, located on chromosome band 10q23.3, encodes a 403-amino acid, dual-specificity protein phosphatase. The defects in this gene are responsible for the development of some advanced cancers. Inactivating alterations, including mutations and deletions, in the PTEN/MMAC1 gene have been identified in several types of human cancers and cancer cell lines. To clarify the participation of the PTEN/MMAC1 gene in advanced gastric carcinogenesis, we screened their frequency of mutations in primary advanced gastric adenocarcinoma tissues. Cancer specimens and their corresponding normal tissues were obtained surgically from 60 patients with pathologically proven advanced gastric carcinoma at the Department of Surgery of Kaohsiung Medical University Hospital. All nine exons of the PTEN/MMAC1 gene were amplified using polymerase chain reaction and screened for mutations by single-strand conformation polymorphism analysis and followed by direct sequencing. After neutral polyacrylamide gel electrophoresis, 17 patients (28.3%) showed an apparent electrophoretic mobility shift between the cancer and its paired normal tissue. These results from direct sequencing indicated that mutations consisted of eight cases (47.1%) of missense mutation, five silent mutations (29.4%), two nonsense mutations (11.8%), a 12-bp deletion (5.9%), and a mutation within the splice donor site of intron 6 (5.9%). The mutation hot spots at codons 45, 66, 82 and 204 in advanced gastric cancer have not been observed previously. Based on the present analysis, our study implicated that the mutations of the PTEN/MMAC1 gene do not occur at a significant rate in human advanced gastric carcinoma, but the rare clustered mutation site (exons 2–6) perhaps suggested that PTEN/MMAC1 might contribute to the gastric carcinogenesis and its progression.

A Double-Blind Randomized Study Comparing the Efficacy and Safety of a Composite vs a Conventional Intravenous Fat Emulsion in Postsurgical Gastrointestinal Tumor Patients

BACKGROUND Composite intravenous fat emulsion, a fat emulsion composed of soybean oil, medium-chain triglycerides (MCT), olive oil, and fish oil, was evaluated for metabolic efficacy, immune modulation, clinical efficacy, safety, and tolerance in surgical gastrointestinal (GI) tumor patients. METHODS In a prospective, randomized, double-blind study, 40 patients were randomized after elective digestive surgery to receive isonitrogenous, isoenergetic parenteral nutrition for 5 days postoperatively with either composite 20% IVFE (composed of soybean, MCT, olive, and fish oils) or a conventional long-chain triglyceride (LCT)/MCT 20% IVFE (LCT/MCT IVFE); IVFE was dosed at 1-2 g/kg body weight. Safety and efficacy parameters were assessed on operation day (day 0) and at the end of study (day 6). Adverse events were documented daily and clinical outcomes were recorded and compared between the groups. RESULTS Metabolic parameters, laboratory parameters, proinflammatory cytokine levels, adverse events, and clinical outcomes did not differ between the 2 groups, with the exception that postoperative low-density lipoprotein levels decreased significantly in the composite IVFE group (93.2 ± 24.3 vs 110.5 ± 26.4 mg/dL, P = .038). CONCLUSIONS composite IVFE was comparable with conventional LCT/MCT IVFE in efficacy, safety, tolerance, and clinical outcomes in surgical GI tumor patients.

Laparoscopic assisted placement of peritoneal dialysis catheters for selected patients with previous abdominal operation.

Peritoneal dialysis is an established alternative method for the management of patients with end-stage renal disease. Recently, laparoscopy has been utilized in assisting the insertion of catheters under direct vision. The efficacy of the laparoscopic approach for patients with a history of abdominal surgery remains largely unknown. The purpose of this study is to evaluate laparoscopy in the placement of peritoneal dialysis catheters for selected patients with previous abdominal operation. Laparoscopic assisted placement of peritoneal dialysis catheters was performed in 20 patients, who were carefully selected preoperatively and who also underwent previous abdominal operation between April 1999 and July 2001. Previous abdominal operation included appendectomy, ovarian resection, hysterectomy, cesarean section, open cholecystectomy, segmental resection of the small intestine, and truncal vagotomy with pyloroplasty. The procedure was performed using two 10-mm and one 5-mm abdominal trocar. All of the patients tolerated this procedure without significant surgical complications. However, 3 patients developed temporary hemoperitoneum, and 1 patient developed dialysate leakage. The overall success rate of catheter function (> 30 days after laparoscopy) was 90%, except in 2 cases where the catheter functioned poorly due to severe intra-abdominal adhesions. Simultaneous laparoscopic adhesiolysis was successfully performed in 5 cases. Laparoscopic implantation of peritoneal dialysis catheters appears to be a straightforward procedure, even for patients with previous abdominal operation. We believe that this technique may extend the application of peritoneal dialysis treatment in patients with previous abdominal surgery after discreet evaluation preoperatively.

Comparison of clinical outcome of breast cancer patients with T1-2 tumor and one to three positive nodes with or without postmastectomy radiation therapy.

OBJECTIVE The value of postmastectomy radiation therapy for breast cancer patients with T1-2 tumor and one to three positive nodes remains controversial. The purpose of this retrospective study was to compare the clinical outcomes of breast cancer patients with T1-2 and one to three positive nodes with and without postmastectomy radiation therapy. METHODS Between May 1990 and June 2008, of 318 breast cancer patients with T1-2 and one to three positive nodes who had undergone modified radical mastectomy, 163 received postmastectomy radiation therapy and 155 did not. The clinico-pathologic characteristics were analyzed for clinical outcomes including loco-regional recurrence, distant metastasis, disease-free survival and overall survival. RESULTS During the median follow-up period of 102 months, the clinical outcomes in postmastectomy radiation therapy versus no-postmastectomy radiation therapy groups were as follows: loco-regional recurrence rate (3.1 versus 11.0%, P= 0.006); distant metastasis rate (20.9 versus 27.7%, P= 0.152); 10-year disease-free survival rate (73.8 versus 61.3%, P= 0.001); and 10-year overall survival rate (82.1 versus 76.1%, P= 0.239). Through a multivariate analysis, a positive nodal ratio of ≥25% (hazard ratio= 4.571, P= 0.003) and positive lymphovascular invasion (hazard ratio= 2.738, P= 0.028) were found to be independent poor prognostic predictors of loco-regional recurrence. The reduction in loco-regional recurrence (hazard ratio= 0.208, P= 0.004) by postmastectomy radiation therapy was found to be significant. CONCLUSIONS On the basis of our results, postmastectomy radiation therapy is highly recommended for breast cancer patients with T1-2 and one to three positive nodes, especially for high-risk subgroups with a positive nodal ratio of ≥25% and positive lymphovascular invasion, not only for reducing loco-regional recurrence but also for improving disease-free survival.

High frequency of activated K‐ras codon 15 mutant in colorectal carcinomas from Taiwanese patients

Colorectal carcinogenesis is regarded as a multistep process resulting from accumulation of genetic alterations, including activation of protooncogenes and inactivation of tumor suppressor genes via signal transduction trigger the stage‐wise progression to malignancy. The reported incidence of K‐ras mutation detected in general tissue samples ranges from 21–60% in primary colorectal cancers (CRC). To assess the prevalence and spectrum of K‐ras mutations in Taiwanese patients with CRC, we analyzed 65 CRC patients by polymerase chain reaction‐single strand conformation polymorphism analysis, followed by direct sequencing. K‐ras mutations were detected in 43.1% (28 of 65) of the tumors. The mutational hot spots were located at codons 12, 13, 15 and 20, especially with the highest frequency at codon 15. To understand whether the codon 15 mutations in CRC were associated with activation of K‐ras oncogene and the alterations of its biocharacteristics, mutant K‐ras genes were cloned from tumor tissues and then inserted into expression vector pBKCMV to construct the prokaryotic expression plasmid pK15MCMV. Mutant K‐ras genes were expressed at high levels in E. coli and the mutant K‐ras proteins were shown to be functional with respect to their well‐known specific, high‐affinity, GDP/GTP binding. The purified K‐ras protein from E. coli was then measured for its intrinsic GTPase activity and the extrinsic GTPase activity in the presence of GTPase‐activating protein for ras. We found that the extrinsic GTPase activity of the codon 15 mutant K‐ras proteins (p21K‐ras15M) in the presence of GAP is much lower than that of the wild‐type K‐ras protein (p21 BN), whereas the intrinsic GTPase activity is nearly the same as that of the wild‐type K‐ras protein. The results indicated that mutation at the codon 15 of K‐ras gene indeed decreased GTPase activity in CRC, however, its association with tumorigenesis of CRC needs be clarified by further studies.

CLINICAL CHARACTERISTICS OF PATIENTS WITH SPORADIC COLORECTAL CANCER AND PRIMARY CANCERS OF OTHER ORGANS

Most cancer patients often neglect the possibility of secondary cancer. Colorectal cancer (CRC) is the third leading cause of cancer death in Taiwan. It is important to be aware of the clinical characteristics of double cancer in CRC patients for early diagnosis and treatment. We retrospectively analyzed 1,031 CRC patients who underwent surgical treatment at the Department of Surgery of Kaohsiung Medical University Hospital between January 1998 and December 2004. Among these patients, CRC was accompanied by cancer of other organs in 17 patients (1.65%), either synchronously or metachronously. Therefore, we describe our experience regarding the location of CRC, the clinical symptoms and signs of these patients, the TNM stage, histology, phase, association with other malignancies, interval between cancers and clinical outcomes. Of the 17 patients in whom CRC was accompanied by primary cancer of other organs, there were four synchronous and 13 metachronous multiple cancer patients. Our patient group comprised six men and 11 women with ages ranging from 47 to 88 years (median age, 66 years). The most common location of CRC was the sigmoid colon. Six gastric cancers (35.2%) and six breast cancers (35.2%) were associated with primary CRC. The remaining six second primary cancers were one lung cancer, one thyroid cancer, one cervical cancer, one ovarian cancer, one skin cancer, and one urinary bladder cancer. Of the 13 metachronous multiple cancer patients, eight patients developed subsequent CRC after primary cancers of other organs, whereas two patients developed a subsequent second primary cancer after CRC. The intervals between the development of metachronous multiple cancers ranged from 2 to 19 years. In this retrospective analysis, breast and gastric cancer patients were at increased risk of developing subsequent secondary CRC. Careful attention should always be paid to the possibility of secondary CRC in treating these cancer patients. Cancer patients with hematochezia or gastrointestinal symptoms/signs should be evaluated for the possibility of second primary CRC during their regular follow‐up.