Prognostic value of microvessel density in stage II and III colon cancer patients: a retrospective cohort study.

BACKGROUND: Microvessel density (MVD), as a derived marker for angiogenesis, has been associated with poor outcome in several types of cancer. This study aimed to evaluate the prognostic value of MVD in stage II and III colon cancer and its relation to tumour-stroma-percentage (TSP) and expression of HIF1A and VEGFA. METHODS: Formalin-fixed paraffin-embedded (FFPE) colon cancer tissues were collected from 53 stage II and 54 (5-fluorouracil-treated) stage III patients. MVD was scored by digital morphometric analysis of CD31-stained whole tumour sections. TSP was scored using haematoxylin-eosin stained slides. Protein expression of HIF1A and VEGFA was determined by immunohistochemical evaluation of tissue microarrays. RESULTS: Median MVD was higher in stage III compared to stage II colon cancers (11.1% versus 5.6% CD31-positive tissue area, p < 0.001). High MVD in stage II patients tended to be associated with poor disease free survival (DFS) in univariate analysis (p = 0.056). In contrast, high MVD in 5FU-treated stage III patients was associated with better DFS (p = 0.006). Prognostic value for MVD was observed in multivariate analyses for both cancer stages. CONCLUSIONS: MVD is an independent prognostic factor associated with poor DFS in stage II colon cancer patients, and with better DFS in stage III colon cancer patients treated with adjuvant chemotherapy.

Lymphangiogenic Gene Expression Is Associated With Lymph Node Recurrence and Poor Prognosis After Partial Hepatectomy for Colorectal Liver Metastasis.

OBJECTIVES: To investigate the relevance of lymphangiogenic gene expression in primary and liver metastasis of colorectal cancer (CRC) and identify determinants of lymphatic invasion. BACKGROUND: Lymphatic development promoting vascular endothelial growth factor C (VEGFC) is associated with poor outcome in primary CRC. For colorectal liver metastasis (CRLM), intrahepatic lymph invasion and lymph node metastasis are poor prognostic factors. Exact biological factors promoting lymphatic involvement remain elusive, just as the association with molecular subtypes of CRC. METHODS: We designed a lymphangiogenic gene set (VEGFC, Nrp-2, PDPN, LYVE-1, MRC1, CCL-21) and applied it to large datasets of CRC. Gene expression of the lymphangiogenic signature was assessed in resected CRLM specimens by Rt-QPCR. In vitro experiments were performed with colon cancer cell line Colo320 (high Nrp-2 expression) and human dermal microvascular lymphatic endothelial cells (LECs). RESULTS: Lymphangiogenic gene expression was associated with poor prognosis in both primary and liver metastasis of CRC. CRLM with high expression of consensus molecular subtype-4 identifier genes also exhibited high lymphangiogenic gene expression. Lymph node recurrence following CRLM resection was associated with high expression of VEGFC and Nrp-2. Blocking Nrp-2 significantly reduced invasion of Colo320 cells through an LEC monolayer. CONCLUSIONS: Lymphangiogenic gene expression is correlated with worse prognosis and consensus molecular subtype-4 in both primary and liver metastatic CRC. VEGFC and Nrp-2 expression may be predictive of lymph node involvement in recurrence after resection of CRLM. Nrp-2, expressed on both tumor and LECs, may have a mechanistic role in lymphatic invasion and is a potential novel target in CRC.

Inhibition of RAF1 kinase activity restores apicobasal polarity and impairs tumour growth in human colorectal cancer.

BACKGROUND AND AIM: Colorectal cancer (CRC) remains one of the leading causes of cancer-related death. Novel therapeutics are urgently needed, especially for tumours with activating mutations in KRAS (∼40%). Here we investigated the role of RAF1 in CRC, as a potential, novel target. METHODS: Colonosphere cultures were established from human tumour specimens obtained from patients who underwent colon or liver resection for primary or metastatic adenocarcinoma. The role of RAF1 was tested by generating knockdowns (KDs) using three independent shRNA constructs or by using RAF1-kinase inhibitor GW5074. Clone-initiating and tumour-initiating capacities were assessed by single-cell cloning and injecting CRC cells into immune-deficient mice. Expression of tight junction (TJ) proteins, localisation of polarity proteins and activation of MEK-ERK pathway was analysed by western blot, immunohistochemistry and immunofluorescence. RESULTS: KD or pharmacological inhibition of RAF1 significantly decreased clone-forming and tumour-forming capacity of all CRC cultures tested, including KRAS-mutants. This was not due to cytotoxicity but, at least in part, to differentiation of tumour cells into goblet-like cells. Inhibition of RAF1-kinase activity restored apicobasal polarity and the formation of TJs in vitro and in vivo, without reducing MEK-ERK phosphorylation. MEK-inhibition failed to restore polarity and TJs. Moreover, RAF1-impaired tumours were characterised by normalised tissue architecture. CONCLUSIONS: RAF1 plays a critical role in maintaining the transformed phenotype of CRC cells, including those with mutated KRAS. The effects of RAF1 are kinase-dependent, but MEK-independent. Despite the lack of activating mutations in RAF1, its kinase domain is an attractive therapeutic target for CRC.

Surgical and Oncologic Outcomes After Major Liver Surgery and Extended Hemihepatectomy for Colorectal Liver Metastases.

PURPOSE: To determine the surgical and oncologic outcomes after major liver surgery for colorectal liver metastases (CRLM) at a Dutch University Hospital. PATIENTS AND METHODS: Consecutive patients with CRLM who had undergone major liver resection, defined as ≥ 4 liver segments, between January 2000 and December 2015 were identified from a prospectively maintained database. RESULTS: Major liver surgery was performed in 117 patients. Of these, 26 patients had undergone formal extended left or right hemihepatectomy. Ninety-day postoperative mortality was 8%. Major postoperative complications occurred in 27% of patients; these adverse events were more common in the extended hemihepatectomy group. Median disease-free survival was 11 months and median overall survival 44 months. CONCLUSION: Major liver surgery, including formal extended hemihepatectomy, is associated with significant operative morbidity and mortality but can confer prolonged overall survival for patients with CRLM.

SIRT1/PGC1α-Dependent Increase in Oxidative Phosphorylation Supports Chemotherapy Resistance of Colon Cancer.

PURPOSE: Chemotherapy treatment of metastatic colon cancer ultimately fails due to development of drug resistance. Identification of chemotherapy-induced changes in tumor biology may provide insight into drug resistance mechanisms. EXPERIMENTAL DESIGN: We studied gene expression differences between groups of liver metastases that were exposed to preoperative chemotherapy or not. Multiple patient-derived colonosphere cultures were used to assess how chemotherapy alters energy metabolism by measuring mitochondrial biomass, oxygen consumption, and lactate production. Genetically manipulated colonosphere-initiated tumors were used to assess how altered energy metabolism affects chemotherapy efficacy. RESULTS: Gene ontology and pathway enrichment analysis revealed significant upregulation of genes involved in oxidative phosphorylation (OXPHOS) and mitochondrial biogenesis in metastases that were exposed to chemotherapy. This suggested chemotherapy induces a shift in tumor metabolism from glycolysis towards OXPHOS. Indeed, chemotreatment of patient-derived colonosphere cultures resulted in an increase of mitochondrial biomass, increased expression of respiratory chain enzymes, and higher rates of oxygen consumption. This was mediated by the histone deacetylase sirtuin-1 (SIRT1) and its substrate, the transcriptional coactivator PGC1α. Knockdown of SIRT1 or PGC1α prevented chemotherapy-induced OXPHOS and significantly sensitized patient-derived colonospheres as well as tumor xenografts to chemotherapy. CONCLUSIONS: Chemotherapy of colorectal tumors induces a SIRT1/PGC1α-dependent increase in OXPHOS that promotes tumor survival during treatment. This phenomenon is also observed in chemotherapy-exposed resected liver metastases, strongly suggesting that chemotherapy induces long-lasting changes in tumor metabolism that potentially interfere with drug efficacy. In conclusion, we propose a novel mechanism of chemotherapy resistance that may be clinically relevant and therapeutically exploitable.

Paired image- and FACS-based toxicity assays for high content screening of spheroid-type tumor cell cultures.

Novel spheroid-type tumor cell cultures directly isolated from patients' tumors preserve tumor characteristics better than traditionally grown cell lines. However, such cultures are not generally used for high-throughput toxicity drug screens. In addition, the assays that are commonly used to assess drug-induced toxicity in such screens usually measure a proxy for cell viability such as mitochondrial activity or ATP-content per culture well, rather than actual cell death. This generates considerable assay-dependent differences in the measured toxicity values. To address this problem we developed a robust method that documents drug-induced toxicity on a per-cell, rather than on a per-well basis. The method involves automated drug dispensing followed by paired image- and FACS-based analysis of cell death and cell cycle changes. We show that the two methods generate toxicity data in 96-well format which are highly concordant. By contrast, the concordance of these methods with frequently used well-based assays was generally poor. The reported method can be implemented on standard automated microscopes and provides a low-cost approach for accurate and reproducible high-throughput toxicity screens in spheroid type cell cultures. Furthermore, the high versatility of both the imaging and FACS platforms allows straightforward adaptation of the high-throughput experimental setup to include fluorescence-based measurement of additional cell biological parameters.

Robot-assisted laparoscopic myomectomy compared with standard laparoscopic myomectomy.

OBJECTIVE: To compare surgical outcomes of laparoscopic myomectomy and robot-assisted laparoscopic myomectomy. METHODS: Retrospective cohort study of 115 consecutive laparoscopic myomectomy and 174 consecutive robot-assisted laparoscopic myomectomy performed at Brigham and Women's Hospital over a period of 31 months. Uterine incisions were closed in multiple layers (running barbed suture was used for most cases in the laparoscopic myomectomy group). Surgical outcomes measured included operative time, estimated intraoperative blood loss, length of hospital stay, and perioperative complications. Odds ratios and 95% confidence intervals were calculated from multivariable logistic regression models; adjusted geometric means were estimated from linear regression models on logged outcomes because of skewed distributions. RESULTS: Surgical groups were similar in age, body mass index, and leiomyoma characteristics. Robot-assisted laparoscopic myomectomy had significantly longer operative time than laparoscopic myomectomy (adjusted geometric mean of 195.1 compared with 118.3 minutes, P<.001) and higher estimated blood loss (adjusted geometric mean of 110.0 compared with 85.9 mL, P=.04), but postoperative complications were similar. CONCLUSION: Robot-assisted laparoscopic myomectomy and laparoscopic myomectomy have similar operative outcomes in a high-volume surgical practice. Operative time and intraoperative estimated blood loss were significantly greater in the robot-assisted laparoscopic myomectomy group, but the level of statistical significance for intraoperative estimated blood loss was marginal and the clinical significance was undetermined. Use of barbed suture in the laparoscopic myomectomy group may account for these differences. LEVEL OF EVIDENCE: II.

Prospective evaluation of quality of life in total versus supracervical laparoscopic hysterectomy.

STUDY OBJECTIVE: To evaluate and compare recovery times and quality of life (QOL) in patients undergoing a total laparoscopic hysterectomy (TLH) and laparoscopic supracervical hysterectomy (LSH). DESIGN: Patients underwent either a TLH or LSH. After surgery, patients maintained a daily log documenting pain, nausea, use of pain medications, and return to daily activities. They also completed a QOL questionnaire (SF-36) before and after surgery. DESIGN CLASSIFICATION: Prospective cohort study (Canadian Task Force Classification II-1). SETTING: University teaching hospital. PATIENTS: A total of 122 women undergoing laparoscopic hysterectomy. MEASUREMENTS AND MAIN RESULTS: A total of 122 women underwent TLH (n = 71) or LSH (n = 51) for benign indications from February 2008 to January 2010. There was a significantly higher postoperative improvement of QOL scores in the LSH group in 6 of 10 questionnaire categories and summary scores, including physical functioning (p =.03), role physical (p =.002), and bodily pain (p =.03). There were no significant differences in use of pain medications, level of pain, level of nausea, or return to normal activities. CONCLUSION: LSH appears to provide greater improvement in short-term postoperative QOL compared with TLH. No significant differences were found in postoperative pain or return to daily activities.

Small bowel obstruction associated with use of a gelatin-thrombin matrix sealant (FloSeal) after laparoscopic gynecologic surgery.

We report 2 cases of laparoscopic gynecologic procedures, complicated by small bowel obstruction possibly related to use of a hemostatic agent. The cause was most likely from excess material not incorporated in the hemostatic clot at the site of application. Gentle irrigation and removal of excess material from the site of application is recommended by the manufacturer of FloSeal and may reduce the risk of postoperative adhesion formation and small bowel obstruction.

Bidirectional barbed suture: an evaluation of safety and clinical outcomes.

OBJECTIVE: To evaluate the safety and efficacy of using bidirectional barbed suture in laparoscopic myomectomy (LM) and total laparoscopic hysterectomy (TLH). METHODS: This was a case series of clinical outcomes following 172 consecutive LM and TLH cases over a 1-year period conducted at a university teaching hospital. It included 172 women (ages 17 to 81), requiring a myomectomy or hysterectomy for symptomatic uterine fibroids, pelvic pain, or abnormal uterine bleeding; 117 women underwent TLH and 55 women underwent LM. Patients were contacted over the phone 6 months after surgery to inquire about number of days of postoperative vaginal bleeding, visits to the hospital due to bleeding, dyspareunia, and other potential complications. RESULTS: For TLH, the average duration of surgery was 109 minutes, average uterine weight was 256 grams (range, 18 to 1242), and average blood loss was 71 mL. In LM, average duration of surgery was 125 minutes, average weight of fibroids was 252 g, average number of fibroids removed was 4.0, and average blood loss was 159 mL. Seven percent of patients and 8% of their partners had persistent dyspareunia after surgery. There were no conversions to laparotomy. CONCLUSIONS: The use of bidirectional barbed suture appears to be safe for closing the vaginal cuff in a TLH and for closing the hysterotomy site during a laparoscopic myomectomy.

Laparoscopic entry: the modified alwis method and more.

Despite the rapid advances in laparoscopic surgery in the past 2 decades, the initial entry still accounts for approximately 40% to 50% of laparoscopic complications and should be considered the most dangerous step of a laparoscopic procedure. In this review, the authors share a technique for initial umbilical entry, and provide alternative entry sites in cases where umbilical entry is contraindicated.