Minimally invasive colorectal resection for benign pathology is associated with persistent proangiogenic plasma compositional changes.

BACKGROUND: Minimally invasive colorectal resection for cancer is associated with increased plasma levels of numerous proangiogenic proteins for 3 to 4 weeks postoperatively, and plasma from postoperative weeks 2 and 3 stimulates proangiogenic endothelial cell behavior in vitro. It is unknown if similar plasma changes occur after minimally invasive colorectal resection for benign pathology. OBJECTIVE: The aim of this study is to assess 1) plasma levels of angiopoetin-2, placental growth factor, and soluble vascular cell adhesion molecule-1 after minimally invasive colorectal resection for benign pathology and 2) postoperative plasma's effects on in vitro endothelial cell proliferation (branch point formation), migration, and invasion. DESIGN: Prospectively gathered plasma samples taken from patients undergoing colorectal resection who consented to participate in an institutional review board-approved plasma and data bank were used for ELISAs and in vitro endothelial cell studies. SETTINGS: The plasma and clinical data used were collected at 3 hospitals. PATIENTS: Patients undergoing minimally invasive colorectal resection for benign indications who were enrolled in a plasma/data bank and for whom adequate samples and volumes of plasma were available were included in the study. MAIN OUTCOME MEASURES: Perioperative plasma levels of angiopoetin-2, placental growth factor, and soluble vascular cell adhesion molecule-1 were the primary outcomes measured. In vitro rates of endothelial cell branch point formation, migration, and invasion were determined after the addition of preoperative and postoperative plasma samples to endothelial cell cultures. RESULTS: Plasma from 86 patients undergoing minimally invasive colorectal resection for benign indications was assessed (diverticulitis, 30; benign polyps, 56). Plasma levels of angiopoetin-2, placental growth factor, and soluble vascular cell adhesion molecule-1 were significantly increased for 3 to 4 weeks postoperatively compared with preoperative levels. In regard to the endothelial cell culture assays, significantly increased endothelial cell branch point formation, invasion, and migration results were noted with plasma from the second and third weeks postoperatively in comparison with preoperative culture results. LIMITATIONS: The weaknesses of this study are the limited numbers of late postoperative plasma samples and the need to bundle late samples into 7- to 12-day time blocks. CONCLUSIONS: Minimally invasive colorectal resection for benign pathology is associated with persistent proangiogenic plasma alterations similar to those found in patients who have cancer. Surgical trauma and not the indication is the likely cause.

Minimally invasive colorectal resection is associated with significantly elevated levels of plasma matrix metalloproteinase 3 (MMP-3) during the first month after surgery which may promote the growth of residual metastases.

INTRODUCTION: MMP-3, a member of the matrix metalloproteinase (MMP) family, is involved in the breakdown of the extracellular matrix in tissue remodeling and may also play a role in cancer progression and metastasis. Minimally invasive colorectal resection (MICR) may increase plasma MMP-3 levels directly via surgical trauma or indirectly due to surgery-associated elevations in TNF-α and IL1 which are regulators of MMP-3. This study's purpose was to evaluate plasma MMP-3 levels during the first month after MICR for colorectal cancer. METHODS: Patients enrolled in an IRB approved data/plasma bank who underwent elective MICR for CRC. Blood plasma samples had been collected preoperatively, on postoperative day (POD) 1, 3 and at varying postoperative time points and were stored at -80 °C. The late samples (POD 7-41) were bundled into 7 day time blocks and considered as single time points. MMP-3 levels were analyzed in duplicate via ELISA and the results reported as mean ± SD. The paired t test was used for analysis (significance, p < 0.008 after Bonferroni's correction). RESULTS: A total of 73 CRC patients who underwent MICR met the inclusion criteria. The mean PreOp MMP-3 level was 14.9 ± 7.8 ng/ml (n = 73). Significantly elevated mean plasma levels were noted on POD 1 (21.4 ± 14.7 ng/ml, n = 73, p < 0.0001), POD 3 (37.9 ± 21.5 ng/ml, n = 72, p < 0.0001), POD 7-13 (22.0 ± 13.0 ng/ml, n = 56, p < 0.0001), POD 14-20 (21.9 ± 10.3 ng/ml, n = 20, p = 0.003), and on POD 21-27 (21.9 ± 11.43 ng/ml, n = 20, p = 0.002) when compared to PreOp levels. Plasma levels returned to the PreOp baseline at the POD 28-41 time point (n = 16, p = 0.07). CONCLUSION: Plasma MMP-3 levels remained significantly elevated from baseline for 4 weeks after MICR for CRC. The early postoperative increase in MMP-3 levels may be due to the surgery-related acute inflammatory response; the elevation noted during weeks 2-3 may be related to wound healing. Increased MMP-3 levels may promote metastases or the growth of residual cancer.

Perioperative polyphenon E- and siliphos-inhibited colorectal tumor growth and metastases without impairment of gastric or abdominal wound healing in mouse models.

INTRODUCTION: Perioperative anticancer therapy that does not impair wound healing is needed to counter the persistent proangiogenic plasma compositional changes that occur after colorectal resection. Polyphenon E (PolyE), a green tea derivative (main component EGCG), and Siliphos (main component silibinin), from the milk thistle plant, both have antitumor effects. This study assessed the impact of PolyE/Siliphos (PES) on wound healing and the growth of CT-26 colon cancer in several murine models. METHODS: One wound healing and three tumor studies were performed. Tumor Study (TS)1 assessed the impact of PES on subcutaneous tumor growth, whereas TS2 assessed PES's impact on subcutaneous growth when given pre- and post-CO(2) pneumoperitoneum (pneumo), sham laparotomy, or anesthesia alone. TS3 determined the ability of PES to limit hepatic metastases (mets) after portal venous injection of tumor cells. In the final study, laparotomy and gastrotomy wound healing were assessed several ways. BALB/c mice were used for all studies. The drugs were given via drinking water (PolyE) and gavage (Siliphos), daily, for 7-9 days preprocedure and for 7-21 days postoperatively. Tumor mass, number/size of hepatic mets, and proliferation and apoptosis rates were assessed. The abdominal breaking strength and energy to failure were measured postmortem as was gastric bursting pressures. RESULTS: PES significantly inhibited subcutaneous growth in the nonoperative setting. PES also significantly decreased the number/size of liver mets when given perioperatively. Abdominal wound breaking strength, energy to wound failure, and collagen content were not altered by PES; gastrotomy bursting strength also was not affected by PES. Neither drug alone had a significant impact on tumor growth. CONCLUSIONS: The PES combination inhibited subcutaneous and hepatic tumor growth yet did not impair wound healing. PES holds promise as a perioperative anticancer therapy.

Plasma soluble vascular adhesion molecule-1 levels are persistently elevated during the first month after colorectal cancer resection.

INTRODUCTION: Plasma from the second and third weeks after minimally invasive colorectal resection (MICR) has high levels of the proangiogenic proteins VEGF and angiopoietin 2 and also stimulates, in vitro, endothelial cell (EC) proliferation and migration, which are critical to wound and tumor angiogenesis. Soluble vascular cell adhesion molecule-1 (sVCAM-1) stimulates EC chemotaxis and angiogenesis. The impact of MICR on blood levels of sVCAM-1 is unknown. This study's purpose was to determine plasma sVCAM-1 levels after MICR in colorectal cancer (CRC) patients. METHODS: Blood samples from 90 patients (26% rectal, 74% colon) were obtained preoperatively, on postoperative days (POD) 1 and 3, and at other points during the next 2 months. The late samples were bundled into 7-day time blocks. sVCAM-1 levels were determined in duplicate via ELISA and reported as ng/ml. Student's t test was used for data analysis (significance, P < 0.008 after Bonferroni correction). RESULTS: The mean incision length was 7.3 ± 3.1 cm, and the conversion rate was 3%. Compared with preoperative (PreOp) levels (811.3 ± 233.2), the mean plasma sVCAM-1 level was significantly higher on POD 1 (905.7 ± 292.4, P < 0.001) and POD 3 (977.7 ± 271.8, P < 0.001). Levels remained significantly elevated for the POD 7-13, POD 14-20, POD 21-27, and POD 28-67 time blocks. CONCLUSIONS: MICR for CRC is associated with a persistent increase in plasma sVCAM-1 levels during the first month. This sustained increase may promote angiogenesis and stimulate the growth of residual tumor cells early after surgery.

Plasma from the second and third weeks after open colorectal resection for cancer stimulates in vitro endothelial cell growth, migration, and invasion.

INTRODUCTION: Angiogenesis is central to wound healing and tumor growth. Postoperative (postop) plasma from weeks 2 and 3 after minimally invasive colorectal resection (MICR) stimulates endothelial cell (EC) migration (MIG), invasion (INV), and proliferation (all vital to angiogenesis) compared with preoperative (preop) plasma results and may promote postop tumor growth. The purpose of this study was to determine whether plasma from open colorectal resection (OCR) patients has similar proangiogenic EC effects in vitro. METHODS: OCR cancer patient plasma from institutional review board-approved banks was used; patients with preop and one postop sample from postoperative days (POD) 7-33 were eligible. Samples were bundled into 7- to 13-day periods and considered as single time points. In vitro cultures of human umbilical venous ECs were used for the EC proliferation (BPF, Branch Point Formation), INV, and MIG assays performed with preop, POD 7-13, POD 14-20, and POD 21-33 plasma. Data were analyzed by paired t test and were reported as mean ± standard deviation (significance, P < 0.05). RESULTS: Plasma from 53 cancer patients (25 rectal and 28 colon) was used. Because of limited postop samples, the number for each time point varies: POD 7-13, n = 30; POD 14-20, n = 26; and POD 21-33, n = 17. In vitro EC BPF was significantly greater at the POD 7-13 (P < 0.0001) and POD 14-20 (P < 0.0001) time points versus preop results. Significantly greater EC INV and MIG were noted on POD 7-13 and POD 14-20 versus the preop plasma results (P < 0.0001). In regards to POD 21-33, a significantly greater result was noted only for the INV assay versus preop. CONCLUSIONS: Plasma from weeks 2 and 3 after OCR stimulates in vitro EC BPF, INV, and MIG. A significant difference from preop baseline was noted only for the INV assay in week 4. The OCR and previous MICR results were largely similar. Tumor angiogenesis may be stimulated after OCR and MICR for 3 weeks. Further studies are warranted.

Minimally invasive colon resection is associated with a persistent increase in plasma PlGF levels following cancer resection.

BACKGROUND: Minimally invasive colorectal resection (MICR) is associated with persistently elevated plasma VEGF levels that may stimulate angiogenesis in residual tumor foci. Placenta growth factor (PlGF) stimulates neovascularization in tumors by modulating VEGF's effects. This study's purpose was to determine the impact of MICR on blood PlGF levels in cancer patients (Study A) and to compare PreOp levels in patients with cancer and benign (BEN) disease (Study B). METHODS: Blood samples were collected preoperatively, on postoperative day (POD) 1, POD 3, and at various time points 2-4 weeks after surgery. Samples from 7-day periods after POD 6 were bundled to allow analysis. Plasma PlGF levels were determined via ELISA, results reported as mean±SD, and data analyzed via t test. Significance was set at p<0.008 after Bonferroni correction. RESULTS: Study A: 76 colorectal cancer (CRC) patients had MICR (laparoscopic, 59%; hand-assisted, 41%). The mean length of stay was 5.8±2.1 days. The mean PreOp PlGF level was 15.4±4.3 pg/ml. Significantly increased levels were noted on POD 1 (25.8±7.7 pg/ml, p<0.001), POD 3 (22.9±6.7, p<0.001), POD 7-13 (19.2±5.1, p<0.001), and POD 14-20 (19.5±6.7, p<0.002). The mean POD 21-27 level was not significantly different from baseline. Study B included 126 CRC and 111 BEN patients. PreOp levels were higher in the CRC patients (15.6±5.3 pg/ml) than in the BEN group (13.5±5.5 pg/ml, p=0.001). CONCLUSIONS: PlGF levels are elevated for 3 weeks after MICR and PreOp plasma levels are higher in CRC patients than in BEN disease patients. The cause of the postoperative increase is unclear. The persistently higher blood levels of PlGF and VEGF after MICR may stimulate angiogenesis in residual tumor foci. Further studies regarding late blood protein alterations after surgery appear to be indicated.

The cancer testis antigens CABYR-a/b and CABYR-c are expressed in a subset of colorectal cancers and hold promise as targets for specific immunotherapy.

INTRODUCTION: Calcium-binding tyrosine phosphorylation-regulated protein (CABYR) is expressed in the human germ line but not in adult human tissues, thus, it is considered a cancer testis protein. The aim of this study is to evaluate the CABYR isoforms: a/b and c mRNA expression in colorectal cancer (CRC) and to determine if these proteins hold promise as vaccine targets. MATERIALS AND METHODS: CABYR mRNA expression in a set of normal human tissues, including the testis, were determined and compared using semi-quantitative PCR. As regards the tumor and normal mucosal samples from study patients, RNA was extracted and cDNA generated after which quantitative PCR was carried out. Analysis of CABYR protein expressions by immunohistochemistry in tumor and normal colon tissues was also performed. RESULTS: A total of 47 paired CRC and normal tissue specimens were studied. The percent of patients with a relative expression ratio of malignant to normal (M/N) tissues over 1 was 70% for CABYR a/b and 72% for CABYR c. The percent with both a M/N ratio over 1 and expression levels over 0.1% of testis was 23.4% for CABYR-a/b and 25.5% for CABYR c. CABYR expression in tumors was further confirmed by immunohistochemistry. CONCLUSIONS: CABYR a/b and c hold promise as specific immunotherapy targets, however, a larger and more diverse group of tumors (Stage 1-4) needs to be assessed and evaluation of blood for anti-CABYR antibodies is needed to pursue this concept.

P-Cadherin (CDH3) is overexpressed in colorectal tumors and has potential as a serum marker for colorectal cancer monitoring.

INTRODUCTION: Placental-Cadherin (CDH3) is a cell adhesion molecule vital to cellular localization and tissue integrity. It is highly expressed in the placenta (PLC)and is overexpressed by many types of cancer. P-cadherin levels in colorectal cancer (CRC) remains poorly characterized. This study's purpose was to determine P-cadherin expression in CRC and normal tissues and to assess plasma CDH3 levels in order to determine the relationship, if any, between cancer stage, P-cadherin expression and plasma CDH3 levels. METHODS: An IRB approved plasma, tumor, and prospective data bank was utilized. CRC patients for whom tumor and normal colon tissue samples were available were enrolled. Tumor samples were OCT embedded and stored at -80C°. Total purified RNA was isolated from tissue samples and cDNA synthesized. CDH3 expression was analyzed by quantitative PCR (QPCR) using the SYBR Green platform. Tumor expression levels were determined and compared to levels in normal colonic tissue and PLC. Expression in 22 different normal tissues was also assessed by RT-PCR. Plasma CDH3 levels were determined via ELISA in patients for whom preoperative blood samples were available. Results: A total of 77 paired CRC and normal colon specimens (36 M/ 41 F, age 67.3±14.5) were assessed (82% colon, 18% rectal; Cancer Stage 2, 44; Stage 3, 33). All tested tumors had CDH3 expression levels over 0.1% of the PLC level and tumor to normal colon ratios greater than 1. CDH3 expression was noted in 14/22 normal organ tissues. There was a positive correlation between tumor CDH3 QPCR and preoperative CDH3 blood levels (n=57, P= 0.038). Expression levels were significantly higher in rectal vs. colon tumors (p=0.019). Conclusion: CDH3 expression was elevated in CRC tumors as compared to normal tissue. CDH3 was expressed by numerous normal organs and, thus, is not a viable vaccine target, however, the correlation between plasma and tumor CDH3 levels suggests CDH3 may have value as a diagnostic or prognostic marker.