Burt Cagir

Guanylyl Cyclase C Messenger RNA Is a Biomarker for Recurrent Stage II Colorectal Cancer

Stage at diagnosis is the most important prognostic determinant for patients with colorectal cancer (1-6), and it dictates the role of adjuvant chemotherapy in this disease (7-10). Given the prognostic and therapeutic importance of staging, accurate histopathologic evaluation of lymph nodes to detect invasion by tumor cells is crucial. However, conventional microscopic lymph node examination has methodologic limitations (6, 11). It can be difficult to differentiate single or even small clumps of tumor cells from other types of cells, which limits sensitivity. The standard practice of examining only a limited number of tissue sections from each lymph node can omit from review more than 99% of each specimen and can introduce sampling error. These limitations are evident when the frequency of disease recurrence in patients with stage I and stage II disease is considered. By definition, such patients do not have extraintestinal disease at the time of curative resection. However, recurrence rates of 10% to 30% have been reported for lesions confined to the mucosa (stage I disease), and rates of 30% to 50% have been reported for lesions confined to the bowel wall (stage II disease) (12, 13). Alternate methods of detecting small numbers of tumor cells have been used for staging, including intensive review of serial tissue sections, immunohistochemical analysis to detect tumor-associated antigens, polymerase chain reaction (PCR) to detect tumor-specific mutations, and reverse transcriptase (RT) PCR to detect the expression of tumor-associated biomarkers (6, 11). In some studies of colorectal cancer, staging by these sensitive methods has been correlated with disease. However, the fact that serial sectioning is labor- and cost-intensive, the lack of uniform association between mutations and neoplastic transformation, and the nonspecificity of many biomarkers limit the applicability of these methods. An easily detected biomarker that is specifically expressed by all colorectal tumors would be useful for disease staging. Guanylyl cyclase C is expressed in normal intestinal mucosal cells, adenomatous polyps, and primary and metastatic colorectal tumors but not in extraintestinal tissues or tumors (14-17). Expression of guanylyl cyclase C has been detected by RT-PCR in all of the histologically confirmed colorectal tumors and colorectal cancer cell lines that have been examined (14-17). Therefore, guanylyl cyclase C may be a specific biomarker for metastasis of extraintestinal colorectal cancer (16, 17). We examined whether expression of guanylyl cyclase C messenger RNA (mRNA) in lymph nodes was associated with disease recurrence in patients with stage II colorectal cancer who had presumably been cured by surgical resection. Methods Patients and Tissues We examined the tumor registry database at Thomas Jefferson University (Philadelphia, Pennsylvania) for patients who had been treated for colorectal cancer between 1989 and 1995, an interval that permitted adequate follow-up (Figure). The initial search was designed to exclude patients who developed recurrent disease more than 3 years after the index surgery. In this way, we avoided inadvertent inclusion of patients who had metachronous rather than recurrent cancer. Our search yielded 445 patients with invasive colon or rectal cancer and no evidence of metastases (tumor, node, metastasis [TNM] classification, N0 M0) at surgery. Of these 445 patients, 260 had surgery at Thomas Jefferson University that yielded lymph nodes. Subsequently, 167 patients were excluded because they had stage I or less severe disease (T0-T2 N0 M0), because they developed recurrent disease locally or at unspecified sites, or because they received neoadjuvant chemotherapy or radiation therapy. Fifty-six patients with no evidence of recurrence were excluded because they had less than 6 years of follow-up. Of 18 patients who had had no evidence of disease for 6 or more years after surgery and were considered clinically cured, 16 had pathologic specimens available for further analysis; these patients formed the control group. Of 19 patients who developed metastases up to 3 years after surgery, 12 had pathologic specimens available for further analysis; these patients formed the case-patient group. The remaining 9 patients were excluded from analysis. Two controls (12.5% [patients 9 and 16]) and 1 case-patient (8.3% [patient 24]) received 5-fluorouracil-based adjuvant chemotherapy after surgery. Figure. Algorithm for selecting patient biopsy samples for analysis. Reverse Transcriptase Polymerase Chain Reaction In our study, lymph nodes were obtained for analysis under an institutional review board-approved protocol that maintained patient anonymity. Preliminary tests showed that mRNA isolated from single 10-m sections of individual lymph nodes yielded insufficient RNA for RT-PCR analysis. Consequently, at least five 10-m sections of representative lymph nodes for each patient were pooled and deparaffinized, and the total RNA was isolated (17). Reverse transcriptase polymerase chain reaction was performed by using the RNA PCR Kit, version 2 (Takara Shuzo Co., Ltd., Kyoto, Japan) (16, 17). We used only total RNA that yielded amplicons after -actin-specific RT-PCR was used (16, 17). Guanylyl cyclase C-specific RT-PCR and nested carcinoembryonic antigen-specific RT-PCR were performed as described elsewhere (16-18). Reactions from RT-PCR were separated by electrophoresis on 4% NuSieve 3:1 agarose (FMC Bioproducts, Rockland, Maine) and by amplification products visualized by ethidium bromide. We included positive control specimens, consisting of RNA isolated from human colorectal cancer cells that expressed guanylyl cyclase C and carcinoembryonic antigen (Caco2 cells [American Type Culture Collection, Rockville, Maryland]), and negative control specimens, consisting of RNA from lymph nodes without colorectal cancer and incubations in which no template was added. Amplicon identity was confirmed by sequencing (16, 17). Production of guanylyl cyclase C-specific amplicons was confirmed by Southern blot analysis, which used a 32P-labeled antisense probe that complemented a sequence found within the amplicon (19). Statistical Analysis Results are expressed as the mean SD, except for disease-free and overall survival, which are expressed as the median (range). We calculated P values by using the Fisher exact test. The odds ratios, with exact 95% CIs, were calculated by using the StatXact 4.0 statistical software package (Cytel Software Corp., Cambridge, Massachusetts). Role of the Funding Source Targeted Diagnostics and Therapeutics, Inc., which provided a portion of the grant support for this study, was not involved in the design of the study or in the collection and analysis of the data; it also had no role in the decision to submit the paper for publication. Results Characteristics of Patients Evaluated by Reverse Transcriptase Polymerase Chain Reaction Patients ranged in age from 37 to 85 years (mean, 68.1 9.5 years). Women and men were similar in age (range, 52 to 85 years [mean, 64.5 10.5 years] and 37 to 82 years [mean, 70.9 7.8 years], respectively). The ratio of men to women was 8:9 among controls and 5:7 among case-patients. One woman was African-American; all other patients were white. The ratio of cases of T3 to T4 disease was 3:13 among controls and 4:8 among case-patients. Patients were followed for 9 to 105 months (mean, 67.4 30.7 months). Controls were followed for 73 to 105 months (mean, 89.9 7.8 months), and case-patients were followed for 9 to 78 months (mean, 37.3 22.6 months). In the control group, 1 patient (6.3%) developed a new primary colonic lesion 96 months after initial diagnosis, 1 patient (6.3%) died of causes unrelated to colorectal cancer, and the remaining 14 patients (87.5%) were alive and free of disease 88 months after diagnosis (range, 73 to 97 months). In the case-patient group, 8 patients (66.7%) died of recurrent colorectal cancer after 13 months of disease-free survival (range, 3 to 35 months) and after 19 months of overall survival (range, 9 to 64 months). Four patients (33%) were alive with metastases after 12 months of disease-free survival (range, 2 to 36 months) and 52 months of overall survival (range, 17 to 78 months). Analysis by Reverse Transcriptase Polymerase Chain Reaction of RNA Expression in Lymph Nodes For all 28 patients, 524 lymph nodes (mean, 18.4 12.5 lymph nodes per patient) collected at surgery were reported to be free of tumor in the original histologic review. The number of lymph nodes obtained from each patient at the time of initial operative staging was similar in the control group (mean, 19.9 13.2 lymph nodes per patient) and the case-patient group (mean, 17.2 12.7 lymph nodes per person). Lymph nodes were omitted from RT-PCR analysis because they were not available from the pathology department (326 lymph nodes from 28 patients [62.2% of 524 lymph nodes obtained at surgery]). Of the 198 lymph nodes that were available for RT-PCR analysis, 19.7% (39 lymph nodes from 7 patients [7.4% of 524 lymph nodes obtained at surgery]) did not yield RNA. The number of lymph nodes available for RT-PCR analysis was similar in the control group (mean, 6.4 3.0 lymph nodes) and the case-patient group (mean, 8.1 6.3 lymph nodes). Twenty-one patients (75%) yielded 159 paraffin-embedded lymph nodes (mean, 7.6 5.2 lymph nodes per patient) that could be adequately evaluated by RT-PCR. In 5 case-patients (41.7%) and 2 controls (16.7%), -actin-specific amplicons (an indicator of intact RNA) were not detected in the total RNA from pooled sections of lymph nodes; these 7 patients were excluded from further analysis. Total RNA extracted from the pooled lymph node sections of the remaining 21 patients was analyzed by RT-PCR using guanylyl cyclase C-specific primers. Guanylyl cyclase C-specific amplicons were not detected in any reaction that used RNA from lymph nodes of control

Changing epidemiology of anorectal melanoma.

PURPOSE: We reviewed 117 cases of anorectal melanoma to better define epidemiologic and survival characteristics of this rare neoplasm. METHODS: The National Cancer Institute Surveillance, Epidemiology, and End Results database covering the period 1973 through 1992 was used. This represents 9.5 percent of the United States population. Melanoma arising in the anorectum was identified using International Classification of Diseases for Oncology codes. Two-tailed Studentst-test, chi-squared, and Wilcoxons tests were used for comparisons of means, proportions, and actuarial survival rates, respectively. RESULTS: One hundred seventeen cases of anorectal melanoma were identified, representing 0.048 percent of all colorectal malignancies in the database. The male-to-female ratio was 1:1.72. The mean age was 66±16 years. Mean age by gender, however, was lower for males (57 years) then for females (71 years;P<0.001). The age difference represents an increased incidence of anorectal melanoma in males younger than the age of 45 years. Furthermore, the incidence of anorectal melanoma in young males ages between 25 to 44 years tripled in the San Francisco area when compared with all other locations (14.4vs. 4.8 per 10 million population;P=0.06). Males have a survival advantage over females (62.8 percentvs. 51.4 percent 1-year and 40.6 percentvs. 27.7 percent 2-year;P<0.01). CONCLUSIONS: The overall incidence of anorectal melanoma continues to rise and survival rates remain poor. A new trend toward bimodal age distribution was observed. There is indirect evidence that implicates human immunodeficiency virus infection as a risk factor. Survival rate is better in young patients aged 25 to 44 years.

Strictureplasty for Crohn's disease

PURPOSE: The objective of this study was to analyze patient outcome after strictureplasty for management of intestinal stricture caused by Crohns disease based on differences in surgical procedures. METHODS: A MEDLINE search was performed using a medical subject heading analysis for strictureplasty in Crohns disease. Meta-analysis of multiple variables for outcome was performed using random-effects models. RESULTS: Five hundred six patients underwent 1,825 strictureplasties for Crohns disease with minimal morbidity and zero mortality. Ninety percent of strictures were less than 10 cm in length. Approximately 85 percent of these procedures used the Heineke-Mikulicz technique and 13 percent used Finney strictureplasty. Forty-four percent of procedures included concurrent bowel resection. Recurrence rate of Crohns disease after strictureplasty was increased in patients with longer study duration after surgery (P=0.04), who showed symptoms of active disease (P=0.02), who experienced preoperative weight loss (P=0.02), or who received the Heineke-Mikulicz procedure (P=0.008). The proportion of patients requiring additional surgery was increased with longer study duration (P=0.006), with properative weight loss (P=0.001), or with the Heineke-Mikulicz procedure (P=0.005). The proportion of patients requiring additional surgery was decreased when a Finney strictureplasty was used (P=0.008) as compared with those treated by the Heineke-Mikulicz procedure. CONCLUSION: Although the Heineke-Mikulicz technique is most often used for Crohns strictureplasty, outcome analysis revealed the Finney strictureplasty may reduce the reoperation rate.

Use of guanylyl cyclase C for detecting micrometastases in lymph nodes of patients with colon cancer

INTRODUCTION: Guanylyl cyclase C appears to be expressed only in colorectal cancer cells in extraintestinal tissues. Thus, guanylyl cyclase C may be useful as a marker to detect colorectal cancer micrometastases not detectable by histopathology in lymph nodes of patients. METHODS: Twelve patients with colon adenocarcinoma, Dukes Stages A through C2, and one patient with a tubulovillous adenoma were included in this study. Forty-two lymph nodes were collected from fresh surgical specimens, and each was examined by histopathology and reverse transcription followed by polymerase chain reaction using guanylyl cyclase C-specific primers. Histopathology identified colon cancer cells in 6 of 16 lymph nodes from five Dukes Stage C patients but not in lymph nodes from the patient with a tubulovillous adenoma, the Dukes Stage A patient, or six Dukes Stage B patients. Reverse transcription followed by polymerase chain reaction using guanylyl cyclase C-specific primers was performed on all 42 lymph nodes. RESULTS: Guanylyl cyclase C messenger RNA was not detected by reverse transcription followed by polymerase chain reaction in lymph nodes from the patient with the tubulovillous adenoma or the patient with Dukes Stage A colon carcinoma. Seven lymph nodes from Dukes Stage C patients revealed guanylyl cyclase C messenger RNA including six lymph nodes containing histopathologically confirmed metastases. Of significance, guanylyl cyclase C messenger RNA was detected in 6 of 21 lymph nodes from Dukes Stage B patients. Indeed, clinical staging of two patients could be upgraded from B to C using reverse transcription followed by polymerase chain reaction and guanylyl cyclase C-specific primers. CONCLUSION: Reverse transcription followed by polymerase chain reaction using guanylyl cyclase C-specific primers might be useful to more accurately assess micrometastases in lymph nodes of colorectal cancer patients undergoing disease staging.

Adenosquamous carcinoma of the colon, rectum, and anus: epidemiology, distribution, and survival characteristics.

PURPOSE: There have been 49 cases of adenosquamous carcinoma of the colon, rectum, and anus reported in the English literature. We have reviewed 145 cases of adenosquamous carcinoma to better define epidemiologic and survival characteristics of this extremely rare colon carcinoma. METHODS: The National Cancer Institutes Surveillance, Epidemiology, and End Results program public use CD-ROM file for the years 1973 through 1992 were reviewed. This represents approximately 9.5 percent of the United States population. Adenosquamous carcinomas arising in the colon, rectum, and anus were identified using the International Classification of Diseases-O codes. The Astler-Coller tumor classification was used for staging. Two-tailed Studentst-test, Mantel-Haenszel chi-squared tests, and generalized Wilcoxons tests were used for comparisons of means, proportions, and actuarial survival rates, respectively. Survival curves were calculated by the Kaplan-Meier method. RESULTS: One hundred forty-five cases of adenosquamous carcinoma were identified, representing 0.06 percent of all colorectal malignancies. The mean age of patients was 67 years. Eighty-four percent of patients were Caucasians, 15 percent were Afro-Americans, and 1 percent were other races. Afro-Americans were diagnosed at a significantly younger age (median age, 62 years;P=0.03). Fifty-three percent of the carcinomas were located in the sigmoid colon, rectum, and anus, 28 percent in the right colon, and the rest in the middle segment. Seventy-four percent of distal cases were staged A through C, compared with 44 percent of proximal cases. Patients with adenosquamous carcinoma of the sigmoid colon, rectum, and anus survived longer than all other patients (P=0.001). Patients with adenosquamous carcinoma Stages A and B1 had survival rates similar to patients with comparably staged adenocarcinomas. Fifty percent of the patients, including most of the patients with D stage, died in the first year. Patients with Stages B2, C, and D adenosquamous carcinomas had a significantly shorter survival than the comparably staged adenocarcinomas (P≤0.02). The overall adjusted five-year survival rate was 30.7 percent. In those patients who survived more than 24 months, the five-year survival was 84 percent. CONCLUSIONS: The survival rates for patients with adenosquamous carcinoma Stages A and B1 are similar to patients with comparably staged colorectal adenocarcinomas. However, we found that patients with colorectal and anal adenosquamous carcinomas staged B2 through D have significantly poorer survival than patients with comparably staged adenocarcinomas, supporting the previous reports of a poor prognosis associated with adenosquamous carcinomas.

Comparison of conventional and nonconventional strictureplasties in Crohn's disease: a systematic review and meta-analysis.

BACKGROUND: The Heineke-Mikulicz and Finney techniques are conventional strictureplasties that have been used to manage short (<10 cm) and medium-length (>10 cm and <20 cm) strictures from Crohn’s disease. Nonconventional strictureplasty techniques have emerged to facilitate bowel conservation for atypical strictures. These techniques include the modified Finney, combined Heineke-Mikulicz and Finney, modified Heineke-Mikuliczs, Michelassi, and modifications of it and others. OBJECTIVE: The aim of this study is to compare conventional vs nonconventional strictureplasties with respect to short-term complications and long-term results. DATA SOURCES AND STUDY SELECTION: A MEDLINE search was performed using “Crohn’s disease,” “surgical therapy,” “strictureplasty,” “complications,” “reoperation,” and “recurrence” as medical subject headings. Studies conducted between 1975 and June 31, 2010 were found via PubMed, Ovid, Embase, and Cochrane databases and categorized into 3 groups. These groups consist of centers performing conventional strictureplasties, nonconventional strictureplasties, or both. Studies with at least 3 patients were reviewed. INTERVENTIONS: A mixed-effects meta-analysis for each outcome was performed by use of Supermix software by SSI Scientific Software International. MAIN OUTCOME MEASURES: We focused on immediate and long-term complication rates among the groups. The 6 immediate complications include small-bowel obstructions, sepsis, other infections, reoperations, early postoperative GI bleeds, and other early complications. The 5 long-term complications include recurrent strictures, small-bowel obstructions, reoperations, carcinoma, and deaths. RESULTS: We reviewed 32 studies with 1616 patients who underwent 4538 strictureplasties. One thousand one hundred fifty-seven patients underwent conventional strictureplasties with an early complication rate of 15%; 459 patients underwent nonconventional strictureplasties with an early complication rate of 8%. A late complication rate of 29% for the conventional strictureplasty group and 17% for the nonconventional strictureplasty group was noted. LIMITATIONS: We are limited by the data published with the inherent risk of finding and analyzing mostly articles with positive results. CONCLUSION: The nonconventional strictureplasty techniques were noninferior to the conventional strictureplasty procedures with respect to all prespecified outcomes.

Colorectal cancer staging and adjuvant chemotherapy

Colorectal cancer is a significant cause of morbidity and mortality in Western populations. The standard of care for staging patients with colorectal cancer to determine prognosis and identify patients who will receive adjuvant therapy continues to be histopathology of regional lymph nodes. However, the significant variability in survival within each staging category likely reflects the heterogeneity of detecting micrometastatic disease employing this technique. Novel molecular markers of micrometastases currently in development will permit more accurate staging of patients with colorectal cancer. These advances in staging will distinguish patients who will maximally benefit from adjuvant therapy from those who have an especially good prognosis in whom chemotherapy can be avoided. In addition, new adjuvant chemotherapeutic agents, novel combinations of those agents and creative dosing schedules currently being investigated will offer considerable advantages with respect to ease of administration, safety and tolerability, quality of life and efficacy. Ultimately, it is anticipated that advances in molecular diagnostics will define unique biochemical characteristics of patients’ tumours, permitting individualisation of chemotherapeutic regimens employing novel agents that specifically exploit those characteristics.

Ventral hernia repair: outcomes change with long-term follow-up.

Most ventral hernia recurrences happen within 2 years of surgery. There appears to be a continued although low subsequent yearly rate of recurrence for open repairs.

Full-thickness skin graft anoplasty: novel procedure.

We describe a novel technique to treat anal stenosis by reconstructing the anal canal by the use of a full-thickness skin graft from the abdominal wall. This treatment was successfully applied in our institution and showed positive results.