Jaw-Yuan Wang

Molecular Detection of Circulating Tumor Cells in the Peripheral Blood of Patients with Colorectal Cancer Using RT-PCR: Significance of the Prediction of Postoperative Metastasis

BackgroundApproximately 20%–45% of colorectal cancer (CRC) patients ultimately develop local recurrence or metastasis following curative surgical resection. The latter is caused by tumor cells shed from the primary carcinoma prior to or during operation, currently undetected by standard clinical staging. Fortunately, the presence of tumor cells in peripheral blood can be detected by molecular methods and is being regarded increasingly as a clinically relevant prognostic factor.Materials and MethodsTo detect the presence of circulating tumor cells and evaluate their relationship to postoperative metastatic relapse, we simultaneously examined human telomerase reverse transcriptase (hTERT), cytokeratin-19 (CK-19), cytokeratin-20 (CK-20), and carcinoembryonic antigen (CEA) mRNA (messenger RNA) in the peripheral blood of 72 CRC patients and 30 healthy individuals. Using a reverse-transcriptase polymerase chain reaction (RT-PCR), these tumor-related mRNAs were amplified; in addition, analyses were carried out for their correlation with patients’ clinicopathologic features, as well as the occurrence of postoperative metastasis.ResultsIn RT-PCR analysis of the peripheral blood, 69.4% (50 out of 72), 66.7% (48 out of 72), 52.8% (38 out of 72), and 72.2% (52 out of 72) of CRC patients were positive for hTERT, CK-19, CK-20, and CEA mRNA respectively. All 30 healthy individuals were negative for hTERT and CEA mRNA expression, while 2 were positive for either CK-19 mRNA or CK-20 mRNA expression. The detection of CEA mRNA was significantly correlated with depth of tumor invasion (P = 0.012), vessel invasion (P = 0.035), TNM stage (P < 0.0001), and postoperative metastasis (P < 0.0001), while positive hTERT mRNA was correlated with TNM stage (P = 0.037) and CK-19 was correlated with depth of tumor invasion (P = 0.039) and postoperative metastasis (P = 0.017). In addition, multivariate logistic regression showed that only CEA mRNA was an independent and significant predictor of postoperative metastasis (P = 0.006). Our findings suggest that CEA mRNA may be a more reliable marker than hTERT, CK-19, and CK-20 for the detection of circulating cancer cells in the peripheral blood of CRC patients.ConclusionsUsing RT-PCR for the detection of CEA mRNA is feasible and may be a promising tool for early detection of micrometastatic circulating tumor cells in CRC patients. CRC patients expressing positive CEA mRNA in peripheral blood have a significantly higher risk of postoperative metastasis. Nevertheless, confirmation of CEA mRNA as a prognostic predictive factor requires the continuation of patient follow-up.

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.

Detection of KRAS Oncogene in Peripheral Blood as a Predictor of the Response to Cetuximab Plus Chemotherapy in Patients with Metastatic Colorectal Cancer

Purpose: Previously we developed membrane-arrays as a promising tool to detect circulating tumor cells (CTC) with KRAS oncogene in patients with malignancies. This study was conducted to determinate the predictive values of CTCs with KARS mutation by membrane-arrays for metastatic colorectal cancer patients treated with cetuximab plus chemotherapy. Experimental Design: Seventy-six metastatic colorectal cancer patients receiving cetuximab plus FOLFIRI or FOLFOX-4 chemotherapy were enrolled. KRAS mutation status in the peripheral blood of these patients was analyzed using membrane-arrays, and KRAS mutation status in tumors was analyzed by DNA sequencing. Results: Among 76 metastatic colorectal cancer patients, KRAS mutations in tumors and in peripheral blood were identified in 33 (43.4%) and 30 (39.5%) patients, respectively. The detection sensitivity, specificity, and accuracy of membrane-arrays for CTCs with KRAS oncogene were 84.4%, 95.3%, and 90.8%, respectively, and indeed a highly significant correlation to KRAS mutations in tumors (P < 0.0001) was observed. Forty-five (59.2%) patients responded to cetuximab plus chemotherapy, and 41 and 40 were wild-type KRAS in tumors and peripheral blood, respectively (both P < 0.0001). Patients with tumors that harbor wild-type KRAS are more likely to have a better progression-free survival and overall survival when treated with cetuximab plus chemotherapy (P < 0.0001). Likewise, patients with CTCs of wild-type KRAS in peripheral blood express a better progression-free survival and overall survival when treated with cetuximab plus chemotherapy (P < 0.0001). Conclusions: These findings provide evidence that detection of KRAS mutational status in CTCs, by gene expression array, has potential for clinical application in selecting metastatic colorectal cancer patients most likely to benefit from cetuximab therapy.

Prognostic significance of multiple molecular markers for patients with stage II colorectal cancer undergoing curative resection.

Objective:The aim of this study was to determine whether our constructed high-sensitivity colorimetric membrane-array method could detect circulating tumor cells (CTCs) in the peripheral blood of stage II colorectal cancer (CRC) patients and so identify a subgroup of patients who are at high risk for relapse. Summary Background Data:Adjuvant chemotherapy is not routinely recommended in patients diagnosed with UICC stage II CRC. However, up to 30% of patients with stage II disease relapse within 5 years of surgery from recurrent or metastatic disease. The identification of reliable prognostic factors for high-risk stage II CRC patients is imperative. Methods:Membrane-arrays consisting of a panel of mRNA markers that included human telomerase reverse transcription (hTERT), cytokeratin-19 (CK-19), cytokeratin-20 (CK-20), and carcinoembryonic antigen (CEA) mRNA were used to detect CTCs in the peripheral blood of 194 stage II CRC patients who underwent potentially curative (R0) resection between January 2002 and December 2005. Digoxigenin (DIG)-labeled cDNA were amplified by RT-PCR from the peripheral blood samples, which were then hybridized to the membrane-array. All patients were followed up regularly, and their outcomes were investigated completely. Results:Overall, 53 of 194 (27.3%) stage II patients were detected with the expression of all 4 mRNA markers using the membrane-array method. After a median follow up of 40 months, 56 of 194 (28.9%) developed recurrence/metastases postoperatively. Univariately, postoperative relapse was significantly correlated with the depth of invasion (P < 0.001), the presence of vascular invasion (P < 0.001), the presence of perineural invasion (P = 0.048), the expression of all 4 mRNA markers (P < 0.001), and the number of examined lymph nodes (P = 0.031). Meanwhile, using a multivariate logistic regression analysis, T4 depth of tumor invasion (P = 0.013), the presence of vascular invasion (P = 0.032), and the expression of all 4 mRNA markers (P < 0.001) were demonstrated to be independent predictors for postoperative relapse. Combination of the depth of tumor invasion, vascular invasion, and all 4 mRNA markers as predictors of postoperative relapse showed that patients with any 1 positive predictor had a hazard ratio of about 27-fold to develop postoperative relapse (P < 0.001; 95% CI = 11.42–64.40). The interval between the detection of all 4 positive molecular markers and subsequently developed postoperative relapse ranged from 4 to 10 months (median: 7 months). Furthermore, the expression of all 4 mRNA markers in all stage II CRC patients, or either stage II colon or rectal cancer patients were strongly correlated with poorer relapse-free survival rates by survival analyses (all P < 0.001). Conclusions:The pilot study suggests that the constructed membrane-array method for the detection of CTCs is a potential auxiliary tool to conventional clinicopathological variables for the prediction of postoperative relapse in stage II CRC patients who have undergone curative resection.

The Prognostic Significance of Total Lymph Node Harvest in Patients with T2–4N0M0 Colorectal Cancer

In patients with radically resected colorectal carcinoma, lymph node involvement is particularly important for a good prognosis and adjuvant therapy. The number of such lymph node recoveries is still controversial, with recommendations ranging from 6 to 17 nodes. The aim of this study is to determine if a specified minimum number of lymph nodes examined per surgical specimen can have any effect on the prognosis of patients who have undergone curative resection for T2–4N0M0 colorectal carcinoma. Between September 1999 and January 2005, a total of 366 patients who underwent radical resection for T2–4N0M0 colorectal carcinoma were retrospectively analyzed in a single institution. All specimen segments were fixed, with node identification performed by sight and palpation. We excluded 186 patients who received postoperative adjuvant chemotherapy via oral or intravenous transmission to prevent possible chemotherapeutic effects on patients’ prognosis; therefore, a total of 180 patients with T2–4N0M0 colorectal carcinoma were enrolled into this study. After the pathological examination, a mean of 12 lymph nodes (range 0–66) was harvested per tumor specimen. No postoperative relapse was found in this group, where the number of examined lymph nodes was 18 or more. Univariate analysis identified the size of the tumor, depth of invasion, grade of tumor, and number of examined lymph nodes, which were significantly correlated with postoperative relapse (all P < 0.05). Meanwhile, both the depth of tumor invasion and the number of harvested lymph nodes were independent predictors for postoperative relapse (P < 0.05). The 5-year overall survival rate of T2–4N0M0 colorectal carcinoma patients who had 18 or more lymph nodes examined was significantly higher than those who had less than 18 nodes examined (P = 0.015). Nodal harvest in patients undergoing radical resection for colorectal carcinoma was highly significant in the current investigation. Our results suggest that harvesting and examining a minimum of 18 lymph nodes per surgical specimen might be taken into consideration for more reliable staging of lymph node-negative colorectal carcinoma.

Multiple Molecular Markers as Predictors of Colorectal Cancer in Patients with Normal Perioperative Serum Carcinoembryonic Antigen Levels

Purpose: In this study, a high-sensitivity colorimetric membrane array method was used to detect circulating tumor cells (CTC) in the peripheral blood of colorectal cancer (CRC) patients with normal perioperative serum carcinoembryonic antigen (CEA) levels. This membrane array method was evaluated as a potential diagnostic and postoperative surveillance tool. Study Design: Membrane arrays consisting of a panel of mRNA markers that include human telomerase reverse transcriptase, cytokeratin-19, cytokeratin-20, and CEA mRNA were used to detect CTCs in the peripheral blood of 157 postoperative CRC patients with normal perioperative serum CEA levels and in 80 healthy individuals. Digoxigenin-labeled cDNA were amplified by reverse transcription-PCR from the peripheral blood samples, which were then hybridized to the membrane array. The sensitivity, specificity, and accuracy of membrane arrays for the detection of CTCs were then calculated. Results: Using the four markers in combination, expression of any three markers or all the four markers in this panel was significantly correlated with the clinicopathologic characteristics, including depth of tumor invasion, lymph node metastasis, tumor-node-metastasis stage, and postoperative relapse (all P < 0.05). The interval between the detection of all four positive molecular markers and subsequent elevated CEA ranged from 3 to 8 months (median 6 months). The expression of all four mRNA markers was an independent predictor for postoperative relapse. CRC patients with all four mRNA markers expression showed a significantly poorer survival rate than those with less than four positive markers. Conclusions: The constructed membrane array method was helpful in the early prediction of postoperative relapse in CRC patients with normal perioperative serum CEA levels.

Preoperative serum carcinoembryonic antigen, albumin and age are supplementary to UICC staging systems in predicting survival for colorectal cancer patients undergoing surgical treatment

BackgroundThe aim of this study was to determine influence of prognostic factors in addition to UICC staging systems, on cancer-specific and overall survival rates for patients with colorectal cancer (CRC) undergoing surgical treatment.MethodsBetween January 1996 and December 2006, a total of 1367 CRC patients who underwent surgical treatment in Kaohsiung Medical University Hospital were analyzed. We retrospectively investigated clinicopathologic features of these patients. All patients were followed up intensively, and their outcomes were investigated completely.ResultsOf 1367 CRC patients, there were seven hundred and fifty-seven males (55.4%) and 610 (44.6%) females. The median follow-up period was 60 months (range, 3–132 months). A multivariate analysis identified that low serum albumin level (P = 0.011), advanced UICC stage (P < 0.001), and high carcinoembryonic antigen (CEA) level (P < 0.001) were independent prognostic factors of cancer-specific survival. Meanwhile, a multivariate analysis showed age over 65 years (P < 0.001), advanced UICC stage (P < 0.001), and high CEA level (P < 0.001) were independent prognostic factors of overall survival. Furthermore, combination of UICC stage, serum CEA and albumin levels as predictors of cancer-specific survival showed that the poorer the prognostic factors involved, the poorer the cancer-specific survival rate. Likewise, combination of UICC stage, age and serum CEA level as predictors of overall survival showed that the poorer the prognostic factors involved, the poorer the overall survival rate. Of these prognostic factors, preoperative serum CEA level was the only significant prognostic factor for patients with stage II and III CRCs in both cancer-specific and overall survival categories.ConclusionPreoperative serum albumin level, CEA level and age could prominently affect postoperative outcome of CRC patients undergoing surgical treatment. In addition to conventional UICC staging system, it might be imperative to take these additional characteristics of factors into account in CRC patients prior to surgical treatment.

Overexpression of the c-met protooncogene in human gastric carcinoma : correlation to clinical features

Overexpression of hepatocyte growth factor receptor (c-met) has been detected in many human tumors. To investigate the possible involvement of c-met in human gastric carcinogenesis, we examined c-met expression in 45 patients with gastric carcinoma using Northern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), and immunohistochemical staining. The c-met mRNA expression was increased twofold and sevenfold in gastric carcinoma tissues compared to the adjacent normal tissues by Northern blot analysis and RT-PCR, respectively. In the immunohistochemical study, c-met protein was detected in 32 of 45 (71.1%) patients, with marked overexpression in gastric carcinoma compared with matched normal gastric tissues. The c-met-positive immunoreactivities were more frequently encountered in serosa-exposed and serosa-infiltrating gastric cancer (p = 0.003). In addition, tumor stage was another statistically significant parameter that was observed between the two groups (p = 0.02). Multivariate analyses revealed that the tumor stage (p = 0.014) and c-met overexpression (p = 0.031) were independently correlated with survival. These data suggest that overexpression of c-met may play a part in gastric carcinogenesis and may represent a prognostic factor for gastric cancer.Overexpression of hepatocyte growth factor receptor (c-met) has been detected in many human tumors. To investigate the possible involvement of c-met in human gastric carcinogenesis, we examined c-met expression in 45 patients with gastric carcinoma using Northern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), and immunohistochemical staining. The c-met mRNA expression was increased twofold and sevenfold in gastric carcinoma tissues compared to the adjacent normal tissues by Northern blot analysis and RT-PCR, respectively. In the immunohistochemical study, c-met protein was detected in 32 of 45 (71.1%) patients, with marked overexpression in gastric carcinoma compared with matched normal gastric tissues. The c-met-positive immunoreactivities were more frequently encountered in serosa-exposed and serosa-infiltrating gastric cancer (p = 0.003). In addition, tumor stage was another statistically significant parameter that was observed between the two groups (p = 0.02). Multivariate analyses revealed that the tumor stage (p = 0.014) and c-met overexpression (p = 0.031) were independently correlated with survival. These data suggest that overexpression of c-met may play a part in gastric carcinogenesis and may represent a prognostic factor for gastric cancer.

Activating KRAS mutations and overexpression of epidermal growth factor receptor as independent predictors in metastatic colorectal cancer patients treated with cetuximab.

Objective:Cetuximab, a monoclonal antibody targeting epidermal growth factor receptor (EGFR), has been proven to be efficient in metastatic colorectal cancer (mCRC); however, the therapeutic response is variable and markers predictive of response are urgently required. This study was conducted to determinate the predictive values of KRAS mutation status and EGFR expression in mCRC patients treated with cetuximab plus chemotherapy. Summary Background Data:Clinical benefit with EGFR-targeting antibodies seems to be restricted to a particular subgroup of mCRC patients. Therefore, the identification of reliable predictive factors for mCRC patients is imperative before the introduction of targeted chemotherapy. Methods:Ninety-five mCRC patients receiving cetuximab plus the FOLFIRI or FOLFOX-4 chemotherapy were enrolled into the present study. KRAS mutation status/EGFR expression levels were analyzed using direct sequencing, immunohistochemistry (IHC), and reverse transcription-polymerase chain reaction (RT-PCR) assay, respectively. The association between clinical response, progression-free survival (PFS) and overall survival (OS) as well as KRAS mutation status/EGFR expression levels were evaluated. Results:Of 95 mCRC patients, KRAS mutations were identified in 41 cases, and EGFR overexpression (protein or mRNA levels) were observed in 78 patients. Among 41 tumors with KRAS mutation, 33 were found to be activating mutants at codons 12, 13, 15 or 18, while 8 were nonactivating mutants at codons 20, 30, or 31. Fifty-five patients responded to cetuximab plus chemotherapy, 49 were EGFR overexpression and 46 were wild-type KRAS tumor status. Patients with tumors that express high EGFR levels or harbor wild-type KRAS are more likely to have a better PFS and OS when treated with cetuximab plus chemotherapy (all P < 0.05). Furthermore, patients with nonactivating KRAS mutants in tumors had a significantly better PFS and OS than patients with activating KRAS mutants (both P < 0.05). However, for patients with wild-type KRAS tumor status, EGFR expression remains a relevant predictor of clinical response. Conclusions:The study suggests that activating KRAS mutants is a particularly important independent predictive marker in mCRC patients treated with cetuximab plus chemotherapy, of which combing activating KRAS mutants and EGFR could help to identify the subgroup of patients who are most likely to respond to cetuximab plus chemotherapy.

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.