Effect of Surgical Humidification on Inflammation and Peritoneal Trauma in Colorectal Cancer Surgery: A Randomized Controlled Trial.

BACKGROUND: Pre-clinical studies indicate that dry-cold-carbon-dioxide (DC-CO2) insufflation leads to more peritoneal damage, inflammation and hypothermia compared with humidified-warm-CO2 (HW-CO2). Peritoneum and core temperature in patients undergoing colorectal cancer (CRC) surgery were compared. METHODS: Sixty-six patients were randomized into laparoscopic groups; those insufflated with DC-CO2 or HW-CO2. A separate group of nineteen patients undergoing laparotomy were randomised to conventional surgery or with the insertion of a device delivering HW-CO2. Temperatures were monitored and peritoneal biopsies and bloods were taken at the start of surgery, at 1 and 3 h. Further bloods were taken depending upon hospital length-of-stay (LOS). Peritoneal samples were subjected to scanning electron microscopy to evaluate mesothelial damage. RESULTS: Laparoscopic cases experienced a temperature drop despite Bair-HuggerTM use. HW-CO2 restored normothermia (≥ 36.5 °C) by 3 h, DC-CO2 did not. LOS was shorter for colon compared with rectal cancer cases and if insufflated with HW-CO2 compared with DC-CO2; 5.0 vs 7.2 days, colon and 11.6 vs 15.4 days rectum, respectively. Unexpectedly, one third of patients had pre-existing damage. Damage increased at 1 and 3 h to a greater extent in the DC-CO2 compared with the HW-CO2 laparoscopic cohort. C-reactive protein levels were higher in open than laparoscopic cases and lower in both matched HW-CO2 groups. CONCLUSIONS: This prospective RCT is in accord with animal studies while highlighting pre-existing damage in some patients. Peritoneal mesothelium protection, reduced inflammation and restoration of core-body temperature data suggest benefit with the use of HW-CO2 in patients undergoing CRC surgery.

Modes of carbon dioxide delivery during laparoscopy generate distinct differences in peritoneal damage and hypoxia in a porcine model.

BACKGROUND: Insufflation with CO2 can employ continuous flow, recirculated gas and/or additional warming and humidification. The ability to compare these modes of delivery depends upon the assays employed and opportunities to minimize subject variation. The use of pigs to train colorectal surgeons provided an opportunity to compare three modes of CO2 delivery under controlled circumstances. METHODS: Sixteen pigs were subjected to rectal resection, insufflated with dry-cold CO2 (DC-CO2) (n = 5), recirculated CO2 by an AirSeal device (n = 5) and humidification and warming (HW-CO2) by a HumiGard device (n = 6). Peritoneal biopsies were harvested from the same region of the peritoneum for fixation for immunohistochemistry for hypoxia-inducible factor 1 alpha (HIF-1α) and scanning electron microscopy (SEM) to evaluate hypoxia induction or tissue/cellular damage, respectively. RESULTS: DC-CO2 insufflation by both modes leads to significant damage to mesothelial cells as measured by cellular bulging and retraction as well as microvillus shortening compared with HW-CO2 at 1 to 1.5 h. DC-CO2 also leads to a rapid and significant induction of HIF-1α compared with HW-CO2. CONCLUSIONS: DC-CO2 insufflation induces substantive cellular damage and hypoxia responses within the first hour of application. The use of HW-CO2 insufflation ameliorates these processes for the first one to one and half hours in a large mammal used to replicate surgery in humans.

Modeling open surgery in mice to explore peritoneal damage, carbon dioxide humidification and desmoidogenesis.

BACKGROUND: The exposure of the peritoneum to desiccation during surgery generates lasting damage to the mesothelial lining which impacts inflammation and tissue repair. We have previously explored open abdominal surgery in mice subjected to passive airflow however, operating theatres employ active airflow. Therefore, we sought an engineering solution to recapitulate the active airflow in mice. Similarly, to the passive airflow studies we investigated the influence of humidified-warm carbon dioxide (CO2) on this damage in the context of active airflow. Additionally, we addressed the controversial role of surgery in exacerbating desmoidogenesis in a mouse model of familial adenomatous polyposis. METHODS: An active airflow mouse-operating module manufactured to produce the equivalent downdraft airflow to that of a modern operating theatre was employed. We quantified mesothelial cell integrity by scanning electron microscopy (SEM) sampled from the peritoneal wall that was subjected to mechanical damage or not, with and without the delivery of humidified-warm CO2. To explore the role of open and laparoscopic surgery in the process of desmoidogenesis we crossed Apcmin/ + C57Bl/6 mice with p53 +/- mice to generate animals that developed desmoid tumors with 100% penetrance. RESULTS: One hour of active airflow generates substantial damage to peritoneal mesothelial cells and their microvilli as measured at 24 h post intervention, which is significantly greater than that generated by passive airflow. Use of humidified-warm CO2 mostly protects the mesothelium that had not experienced additional mechanical (surgical) damage at 24 h. Maximal damage was evident in all treatment groups regardless of flow or use of gas. At day 10 mechanically-damaged peritoneum remains in mice but is essentially repaired in the gas-treated groups. Regarding desmoidogenesis, operating procedures did not increase the frequency of desmoid tumors but their frequency correlated with time following surgery but not age of mice. CONCLUSIONS: Active airflow generates more peritoneal damage than passive airflow and is reduced significantly by the use of humidified-warm CO2. Introduced peritoneal damage is largely repaired in mice by day 10 with gas. Desmoid tumor incidence is not increased substantially by surgery itself but rises over time following surgery compared to non-surgery mice.

Novel Vaccine Targeting Colonic Adenoma: a Pre-clinical Model.

BACKGROUND: Colorectal cancer (CRC) is the second leading cause of cancer-related mortality in the USA. Over 80% of CRC develop from adenomatous polyps. Hence, early treatment and prevention of adenomas would lead to a significant decrease of disease burden for CRC. MYB is a transcription factor that is overexpressed in both precancerous adenomatous polyps and colorectal cancer, and hence an ideal immunotherapeutic target. We have developed a cancer vaccine, TetMYB, that targets MYB and aim to evaluate its efficacy in the prophylactic and therapeutic management of adenomatous polyps. MATERIAL AND METHODS: Six- to eight-week-old Apcmin/+ (Familial Adenomatous Polyposis model) and Apc580S (sporadic model) C57BL/6 mice were used. The Apcmin/+ mice are carried a germline mutation of one Apc allele whereas the Apc580S model has an inducible silencing of one Apc allele, when exposed to tamoxifen, via the Cre-Lox recombination enzyme system. In the prophylactic treatment group, Apcmin/+ and Apc580S C57BL/6 mice were vaccinated and surveyed for clinical signs of distress. Number of adenoma and survival were measured. In the therapeutic cohort, Apc580S C57BL/6 mice were given tamoxifen-laced food to activate Cre-Lox recombinase mediated silencing of one Apc allele and thus inducing adenoma development. Following adenoma detection, mice were vaccinated with TetMYB and treated with anti-PD-1 antibody and were analyzed for overall survival. RESULTS: In both the prophylactic and therapeutic setting, mice vaccinated with TetMYB had a significantly improved outcome, with the vaccinated Apcmin/+ mice having a median survival benefit of 70 days (p = 0.008) and the vaccinated Apc580S mice having a mean survival benefit of 134 days (p = 0.01) over the unvaccinated mice. In the prophylactic cohort, immunofluorescence confirmed a stronger cytotoxic CD8+ T cell infiltrate in the vaccinated group, implying an anti-tumor immune response. In the therapeutic cohort, vaccinated Apc580S mice showed significantly reduced adenoma progression rate compared to the unvaccinated mice (p = 0.0005). CONCLUSION: TetMYB vaccine has shown benefit in a prophylactic and therapeutic setting in the management of colonic adenoma in a murine model. This will form the basis for a future clinical trial to prevent and treat colonic adenomatous polyps.

Immunomodulation by MYB is associated with tumor relapse in patients with early stage colorectal cancer.

The presence of tumor immune infiltrating cells (TILs), particularly CD8(+) T-cells, is a robust predictor of outcome in patients with colorectal cancer (CRC). We revisited TIL abundance specifically in patients with microsatellite stable (MSS) CRC without evidence of lymph node or metastatic spread. Examination of the density of CD8(+) T-cells in primary tumors in the context of other pro-oncogenic markers was performed to investigate potential regulators of TILs. Two independent cohorts of patients with MSS T2-4N0M0 CRC, enriched for cases with atypical relapse, were investigated. We quantified CD8(+) and CD45RO(+) -TILs, inflammatory markers, NFkBp65, pStat3, Cyclo-oxygenase-2 (COX2) and GRP78 as well as transcription factors (TF), ß-catenin and MYB. High CD8(+) TILs correlated with a better relapse-free survival in both cohorts (p = 0.002) with MYB and its target gene, GRP78 being higher in the relapse group (p = 0.001); no difference in pSTAT3 and p65 was observed. A mouse CRC (CT26) model was employed to evaluate the effect of MYB on GRP78 expression as well as T-cell infiltration. MYB over-expressing in CT26 cells increased GRP78 expression and the analysis of tumor-draining lymph nodes adjacent to tumors showed reduced T-cell activation. Furthermore, MYB over-expression reduced the efficacy of anti-PD-1 to modulate CT26 tumor growth. This high MYB and GRP78 show a reciprocal relationship with CD8(+) TILs which may be useful refining the prediction of patient outcome. These data reveal a new immunomodulatory function for MYB suggesting a basis for further development of anti-GRP78 and/or anti-MYB therapies.

Peritoneal Tumorigenesis and Inflammation are Ameliorated by Humidified-Warm Carbon Dioxide Insufflation in the Mouse.

BACKGROUND: Conventional laparoscopic surgery uses CO2 that is dry and cold, which can damage peritoneal surfaces. It is speculated that disseminated cancer cells may adhere to such damaged peritoneum and metastasize. We hypothesized that insufflation using humidified-warm CO2, which has been shown to reduce mesothelial damage, will also ameliorate peritoneal inflammation and tumor cell implantation compared to conventional dry-cold CO2. METHODS: Laparoscopic insufflation was modeled in mice along with anesthesia and ventilation. Entry and exit ports were introduced to maintain insufflation using dry-cold or humidified-warm CO2 with a constant flow and pressure for 1 h; then 1000 or 1 million fluorescent-tagged murine colorectal cancer cells (CT26) were delivered into the peritoneal cavity. The peritoneum was collected at intervals up to 10 days after the procedure to measure inflammation, mesothelial damage, and tumor burden using fluorescent detection, immunohistochemistry, and scanning electron microscopy. RESULTS: Rapid temperature control was achieved only in the humidified-warm group. Port-site tumors were present in all mice. At 10 days, significantly fewer tumors on the peritoneum were counted in mice insufflated with humidified-warm compared to dry-cold CO2 (p < 0.03). The inflammatory marker COX-2 was significantly increased in the dry-cold compared to the humidified-warm cohort (p < 0.01), while VEGFA expression was suppressed only in the humidified-warm cohort. Significantly less mesothelial damage and tumor cell implantation was evident from 2 h after the procedure in the humidified-warm cohort. CONCLUSIONS: Mesothelial cell damage and inflammation are reduced by using humidified-warm CO2 for laparoscopic oncologic surgery and may translate to reduce patients' risk of developing peritoneal metastasis.

Therapeutic DNA vaccination against colorectal cancer by targeting the MYB oncoprotein.

Cancers can be addicted to continued and relatively high expression of nuclear oncoproteins. This is evident in colorectal cancer (CRC) where the oncoprotein and transcription factor MYB is over expressed and essential to continued proliferation and tumour cell survival. Historically, targeting transcription factors in the context of cancer has been very challenging. Nevertheless, we formulated a DNA vaccine to generate a MYB-specific immune response in the belief MYB peptides might be aberrantly presented on the cell surface of CRC cells. MYB, like many tumour antigens, is weakly immunogenic as it is a 'self' antigen and is subject to tolerance. To break tolerance, a fusion vaccine was generated comprising a full-length MYB complementary DNA (cDNA) flanked by two potent CD4-epitopes derived from tetanus toxoid. Vaccination was achieved against tumours initiated by two distinct highly aggressive, syngeneic cancer cell lines (CT26 and MC38) that express MYB. This was done in BALB/c and C57BL/6 mouse strains respectively. We introduced multiple inactivating mutations into the oncogene sequence for safety and sub-cloned the cDNA into a Food and Drug Administration (FDA)-compliant vector. We used low dose cyclophosphamide (CY) to overcome T-regulatory cell immune suppression, and anti-program cell death receptor 1 (anti-PD-1) antibodies to block T-cell exhaustion. Anti-PD-1 administered alone slightly delayed tumour growth in MC38 and more effectively in CT26 bearing mice, while CY treatment alone did not. We found that therapeutic vaccination elicits protection when MC38 tumour burden is low, mounts tumour-specific cell killing and affords enhanced protection when MC38 and CT26 tumour burden is higher but only in combination with anti-PD-1 antibody or low dose CY, respectively.

MYB is essential for mammary tumorigenesis.

MYB oncogene upregulation is associated with estrogen receptor (ER)-positive breast cancer, but disease requirements for MYB function in vivo have not been explored. In this study, we provide evidence of a critical requirement for MYB functions in models of human and murine breast cancer. In human breast cancer, we found that MYB expression was critical for tumor cell growth both in vitro and in vivo in xenograft settings. In transgenic knockout mice, tissue-specific deletion of the murine MYB gene caused a transient defect in mammary gland development that was reflected in delayed ductal branching and defective apical bud formation. In mouse mammary tumor virus (MMTV)-NEU mice where tumors are initiated by activation of HER2, MYB deletion was sufficient to abolish tumor formation. In the more aggressive MMTV-PyMT model system, MYB deletion delayed tumorigenesis significantly. Together, the findings in these transgenic knockout models implied that MYB was critical during an early window in mammary development when it was essential for tumor initiation, even though MYB loss did not exert a lasting impact upon normal mammary function. Two important MYB-target genes that promote cell survival, BCL2 and GRP78/BIP, were each elevated compared with nontransformed mammary epithelial cells, thereby promoting survival as confirmed in colony formation assays in vitro. Taken together, our findings establish a role for MYB at the hub of ER- and HER2-dependent pathways in mammary carcinogenesis.