Plasma and wound fluid levels of eight proangiogenic proteins are elevated after colorectal resection

ARTICLE HIGHLIGHTS

Research background

Colorectal resection (CRR) has been previously shown to be associated with elevations in plasma levels of 11 proteins with proangiogenic effects [including vascular endothelial growth factor (VEGF)] that persist for 3 to 5 wk. Further, plasma from the second and third postop weeks has also been shown to promote endothelial cell proliferation, migration, and invasion which are critical to neovascularization. The noted persistent proangiogenic blood protein alterations might stimulate the growth of residual metastases that remain after resection of the primary tumor. The etiology of these elevations is unknown. The time course of the healing process and the fact that angiogenesis plays a critical role in wound healing makes the surgical wounds a possible source of the added protein in the blood. This study was done to simultaneously measure the levels of 8 proteins (VEGF, placental growth factor, angiopoetin-2, monocyte chemotactic protein-1, chitinase 3 like 1 protein, osteopontin, matrix metallo-proteinase-2 and matrix metallo-proteinase-3), all previously shown to be persistently elevated after CRR, in both the blood and fluid from the surgical wounds at multiple postop time points. The significance of this study is that it might demonstrate potentially important, heretofore unknown, systemic manifestations of wound healing.

Research motivation

The main topics of this study were: (1) Determination of the impact of CRR on blood levels of 8 proteins during the first 2 to 3 wks; and (2) To measure, at the same time points, the levels of the same 8 proteins in fluid from either pelvic or abdominal wall wounds. This is the first study to determine the perioperative levels of 8 proteins in the same population of patients and also the first to assess a population of benign pathology (cancer free) patients in addition to a group of cancer patients. If similar blood protein elevations were noted in the benign and cancer groups then it would be clear that the noted blood compositional changes were not related to the cancer diagnosis. A key motivation for this study was to determine the wound fluid (WFL) levels of 8 proangiogenic proteins as this would provide insight into wound healing, in general, and also might reveal a source of the plasma protein increases. Another motivation for this study was the desire to determine if the makeup of WFL from the pelvis in patients who had rectal resections would be similar to that obtained from the abdominal wall wounds. Therefore, this data should provide insight into wound healing in 2 different locations. Determining that wound levels of the proteins in question were notably higher than blood levels (which are also elevated from their baseline) at the same time points would establish that the healing wounds transiently but significantly alter the blood composition. This information would make clear the importance of minimizing the overall surgical trauma incurred in cancer patients. Also, this knowledge, by confirming the proangiogenic nature of the blood for 1 mo post-surgery, may compel doctors to look for anti-cancer agents that could be given during the early postop period in an effort to negate these potentially tumor stimulatory conditions.

Research objectives

The main objectives of this study were the determination of plasma and WFL levels of the 8 proteins in question, simultaneously, at multiple postop time points. The hypothesis was that WFL levels of these proteins would be greater than blood levels because of the angiogenesis occurring in the healing wounds. Another objective was to confirm that after CRR the blood levels of the 8 proteins were persistently elevated for the first 3 wk. Yet other objectives were to determine if similar postop plasma increases were noted in cancer and benign colon disease patients and to ascertain if the protein concentrations in fluid from pelvic and abdominal wounds were similar or different. As mentioned, demonstrating that blood levels of these proangiogenic proteins remain elevated for 3 wk after surgery would confirm that surgery has long lasting systemic manifestations that have the potential to impact growth in residual cancer postop. If true, these results may motivate researchers to look for new anti-cancer agents that could be used early after surgery. Establishing that wound levels are higher than the corresponding blood levels would show that there is a concentration gradient between the healing wounds and the circulation; this would also suggest that the wounds may be a source of the additional protein in the blood. Determination of the similarity or difference between pelvic and abdominal wall WFL will provide insight into wound healing and will also guide future studies.

Research methods

This study concerned patients who underwent CRR for cancer or for benign colorectal pathology. This study was carried out under the auspices of two separate IRB protocols, one that called for obtaining multiple perioperative blood samples and clinical data for research purposes and the second that concerns harvesting of WFL samples from patients in whom Jackson Pratt drains were placed in either the pelvis or the main abdominal incision (consent obtained post-surgery). Preop blood samples were obtained before surgery from all patients. Blood and WFL samples were simultaneously obtained by research personnel on postop day (POD) 1, 3, and at least 1 late post-discharge time point provided the wound drain remained in place. The late samples, by necessity, were bundled into 2 “time points” (POD 7-13, POD 14-20). Post discharge late samples were obtained in only a fraction of the overall populations due to drain removal and the timing of the first office visit. WFL and blood samples were processed and aliquots of plasma and WFL frozen in a timely fashion. This is one of a small number of studies to collect fluid samples from both abdominal wall and pelvic wounds. WFL and plasma protein levels were determined in duplicate via highly specific commercially available Enzyme-Linked ImmunoSorbent Assays. This is the first study to assess perioperative blood levels of 8 proteins at multiple postop time points and the first, to our knowledge, to assess WFL levels for this number of proteins. Demographic, clinical, perioperative, and pathology data were obtained prospectively and entered into the IRB approved above mentioned data bank. The Wilcoxon signed-rank match paired test was used for the pre vs postop plasma comparison while the Mann-Whitney test was utilized for the plasma vs WFL comparisons.

Research results

A total of 35 cancer and 31 benign disease patients were studied. The vast majority underwent minimally invasive procedures; 11% of the cancer group and 6% of the benign disease group had open procedures. The majority of the cancer cases were rectal resections (60%) whereas the majority of the benign patients had either a sigmoid or right colectomy (67%). As regards the location of the Jackson Pratt drains, in the cancer group there were 23 pelvic and 12 subcutaneous abdominal wound drains whereas in the benign group there were 8 pelvic and 23 subcutaneous drains. As regards the preop vs postop plasma comparisons, there were a total of 32 points of comparison (8 proteins × 4 postop sampling points). The postop median plasma levels were significantly elevated from preop baseline at all 32 cancer time points and at 29 of 32 of the benign group time points. Of note, the range of the percent change from baseline values for the cancer and benign pathology groups were similar. This assessment of 8 proteins in the two populations verifies and substantiates the results of previous studies that each concerned 1 or, at most, 2 proteins. The results demonstrate that CRR is associated with plasma elevations that persist for at least 3 wk post-surgery. Further, these results prove that the elevations are related to the surgical procedure itself and not the indication for surgery (cancer vs benign pathology). These results also make clear the need to determine the oncologic consequences of the 3 to 5 wk long period when the blood is decidedly proangiogenic. New anti-cancer treatments that can be given during the first post month should be considered. Of note, when the pelvic and subcutaneous WFL results were compared, for all 8 proteins, at the great majority of time points, there was no statistical difference in protein levels between the 2 locations, thus, for the following analysis the WFL results from the 2 drain locations were combined. As regards the WFL vs plasma level comparisons for the 8 proteins, the median WFL levels were significantly greater than the corresponding plasma level at all 32 time points in both groups. The WFL median level was at least 3 × higher than plasma levels in 90%-91%, 5 × higher (or greater) in 68%-69%, and 30 × greater in 29% of patients in both groups. Of note, the highest WFL levels were noted at the POD 7-13 or 14-20 time points for 6 of the 8 proteins in both groups. These results prove that median wound levels of these proangiogenic proteins are notably greater than the corresponding plasma levels and that there is a substantial gradient between the wounds and circulation. Also, these results strongly support the hypothesis that the healing wounds are the source of the added protein in the blood. Similar studies that assessed different groups of proteins or different operations (gastrectomy, hepatectomy, pneumonectomy, etc.,) would increase our understanding of surgery’s systemic impact and perhaps lead to attempts to block, in some way, the deleterious systemic manifestations of major surgical trauma. Larger studies of this type would also allow a more detailed comparison between WFL from the pelvic and subcutaneous locations.

Research conclusions

There are 4 new findings of this study. The first is that WFL levels of the 8 proteins assessed are notably higher than the corresponding plasma levels which, in turn, are elevated from their preop baselines. These results support the hypothesis that the wounds are a major source of the added protein in the blood. These results also suggest that angiogenesis plays a prominent role in wound healing during the first month after surgery. The second new finding is the demonstration in this population of CRR patients that the plasma levels of all 8 proteins were significantly elevated for at least 3 wk after surgery (prior studies considered only 1-2 proteins per population). The third new finding is that plasma protein elevations similar to those found in cancer populations are found following surgery for benign pathology; thus the changes are related to the surgery and not the indication. The fourth new finding is that the make-up of WFL from 2 different locations are similar, as regards the levels of the proteins in question. This aspect needs further study and verification since the numbers of samples from each location limited the ability to detect differences at the later time points. The plasma results, by proving that long duration proangiogenic protein increases are present, raises the fear that these changes may promote tumor growth postoperatively in patients with residual disease. This realization should logically prompt studies to verify this hypothesis as well as to search for ways to limit these deleterious oncologic effects. The plasma and WFL results regarding 8 proteins in both cancer and benign pathology patients makes clear the fact that major surgery results in systemic blood compositional changes that last far longer than previously imagined; further, there is the potential that these changes may negatively impact cancer patients with residual disease. The new methods and study approaches put forth in this study are the simultaneous obtaining of blood and WFL samples at multiple time points during the first 3 wk after surgery and the assessment of 8 different proteins in a single population. As stated above, these results support the main hypothesis that the surgical wounds are the source of the added protein in the blood which significantly elevates plasma levels for weeks after surgery. The results also verify that long lasting plasma protein changes occur after surgery done for benign indications (as is the case for cancer populations).

Research perspectives

The results of this study add further evidence and support for the concept that CRR (and likely major surgery, in general) results in significant changes in the plasma levels of a substantial number of proteins that persist for at least 3 wk after surgery. Prior studies regarding the 8 proteins assessed in the present study have demonstrated that the full duration of the significant elevations is 3 to 5 wks. Documenting that all 8 proteins are persistently increased after surgery in patients with cancer or benign problems proves that these effects are related to the surgical procedure and not the presence of a cancer. The finding of much higher levels of these proteins in WFL than in the blood makes clear that wound healing is an involved and lengthy process in which that angiogenesis plays a central role. It also strongly suggests that the wounds are the source of the added protein. The fact that major surgery (tissue trauma) and the process of healing that follows alters the blood composition so that it is decidedly proangiogenic for a month’s time has important implications. These systemic changes may accelerate the growth of residual tumor metastases by stimulating tumor angiogenesis. Future studies are needed to determine the clinical ramifications of the demonstrated plasma changes. Also, perioperative levels of other proteins that may influence tumor growth and establishment are indicated to better define surgery’s effects. A better understanding of surgery’s effects may lead to modifications in technique that will ameliorate the deleterious transient effects. Finally, the authors believe that anti-cancer drugs that can be used early after surgery should be sought so as to provide protection against accelerated tumor growth during the early postop period.