Paul Rooney

Inverse relationship between hyaluronan and collagens in development and angiogenesis

The extracellular matrix plays a vital role in regulating normal tissue development and function--largely via the specific arrangement of macromolecules such as collagens, proteoglycans, glycosaminoglycans and glycoproteins. Previous reports have concentrated on associations between combinations of collagens/proteoglycans, collagens/glycoproteins and proteoglycans/glycosaminoglycans whilst little information is available on associations between collagens and free glycosaminoglycans. In this review, we discuss possible associations between collagens and the glycosaminoglycan hyaluronan; macromolecules which are known to exhibit changes in amount and composition during development and under pathological conditions. We demonstrate two types of collagen/hyaluronan association in vivo: the first, during the formation of extracellular matrix structures where neither collagens nor hyaluronan are degraded, resulting in the regulation of collagen fibrillogenesis, and the second, involving an inverse correlation between collagen synthesis and hyaluronan degradation and vice versa. We suggest that associations between collagens and hyaluronan play an important role in the initiation and maintenance of angiogenesis and put forward a model of cartilage vascularisation which relies on these associations.

Low molecular weight heat shock protein HSP27 is a prognostic indicator in rectal cancer but not colon cancer

Objective There are currently no biomarkers in routine clinical use for determining prognosis in rectal cancer. In a preliminary proteomic study, variation in the levels of heat shock protein 27 (HSP27) in colorectal cancer samples was observed. The expression of HSP27 in a cohort of 404 patients with colorectal cancer with a predominantly poor prognosis was characterised and an investigation was undertaken of whether the differences were related to clinical outcome. HSP27 levels in diagnostic rectal biopsies were compared with matched surgical samples to determine whether changes in expression occurred in the time between biopsy and surgery and to investigate whether preoperative radiotherapy affected expression. Finally, the relationship between HSP27 expression and outcome was examined in an independent cohort of 315 patients with a predominantly good prognosis. Methods HSP27 levels were determined using combined two-dimensional gel electrophoresis and tandem mass spectrometry (12 cases) and by immunohistochemistry using tissue microarrays of colorectal cancers sampled at surgery and 80 diagnostic rectal biopsies. Results HSP27 overexpression was strongly associated with poor cancer-specific survival in rectal cancer (n=205, p=0.0063) but not colon cancer (n=199, p=0.7385) in the cohort with a poor prognosis. Multivariate Cox regression confirmed nodal metastases (p=0.0001) and HSP27 expression (p=0.0233) as independent markers of survival in rectal cancer. HSP27 levels remained unchanged in the majority of cases (65/80, 81%) between diagnostic biopsies and matched surgical samples, regardless of whether patients had undergone preoperative radiotherapy. In the cohort with a good prognosis the association between HSP27 and survival was not observed in patients with either rectal (n=115; p=0.308) or colon cancer (n=200; p=0.713). Conclusion In a large cohort of patients with a poor prognosis, HSP27 is an independent marker of poor outcome in rectal cancer; its expression is not altered by neoadjuvant radiotherapy. This finding requires validation in an independent similar cohort of patients with rectal cancer. HSP27 levels merit evaluation as a stratification factor for treatment of rectal cancer.

Mast cells in the pathogenesis of chronic back pain: a hypothesis.

The pathophysiology of chronic low back pain is poorly understood, mainly because it is difficult to study experimentally or objectively. Recently it has been found that there is a relationship between neovascularization and innervation of the usually avascular and aneural intervertebral disc at the sites of discogenic pain. These data, together with the recognized involvement of mast cells in tissue repair, in the induction of angiogenesis, and in the production of and response to neurotrophic stimuli such as nerve growth factor, has suggested the hypothesis that mast cells may have a causative role in chronic low back pain. If so, the mast cell may represent an attractive therapeutic target. Copyright

Hyaluronan, angiogenesis and malignant disease.

Dear Sir, We congratulate Dr. Lokeshwar’s group for another landmark paper in this journal, wherein they demonstrated that the saliva of patients with high-grade head-and-neck squamous cell carcinoma contained both high and low m.w. HA.1 The latter, i.e., o-HA, is generated when HAase, an endoglycosidase, degrades HA. Although the authors did allude to the issue of HA and its relevance to angiogenesis, we highlight here its pathologic relevance in human malignancy and present our supporting unpublished data. The angiogenic activity of HA is dependent on its molecular mass. High m.w. nHA is antiangiogenic, whereas almost 2 decades ago we reported that HA degradation products of a specific size (3–25 disaccharide units) induce angiogenesis in vivo.2 Subsequently, we have shown that o-HA fragments of 3–10 disaccharides were angiogenic in several in vivo assays and stimulated EC migration, proliferation and sprout formation.3–6 The biological activities of HA are mediated through interaction with cell surface receptors such as CD44, resulting in activation of intracellular signalling events. In collaboration with Dr. R. Montesano’s group, we found that HA degradation products influence EC invasion of a 3-D ECM.7 o-HA acted synergistically with vascular endothelial growth factor but not basic fibroblast growth factor. We reported that o-HA induces phosphorylation and activation of mitogen-activated protein kinase in bovine ECs, as well as upregulating the early response genes c-fos, c-jun and jun-B, which control the expression of other genes, including those of matrix-degrading proteases.8 Because HA acts via its receptor, CD44, it is conceivable that o-HA promotes cell invasion and tube formation by activating intracellular signalling pathways that result in modulation of pericellular proteolysis. Also, o-HA induced multiple signalling pathways involved in EC migration, proliferation and wound healing.9,10 More relevant to Lokeshwar’s publication is our finding that sera of children with renal tumours contain o-HA. Briefly, renal tumours are one of the major groups of childhood solid malignancy,Wilms’ tumour (nephroblastoma) being the most frequent in this group. Another kidney tumour BMRTC has been distinguished from Wilms’ tumour.11 BMRTC comprises about 4% of all primary renal neoplasms in childhood. In the Manchester region, with a population of 1 million children, we expect to encounter approximately one BMRTC case every 5 years. BMRTC has been associated with poor prognosis, unlike Wilms’ tumour.11 Furthermore, in contrast to Wilms’ tumour, BMRTC occurs predominantly in boys and, as the name implies, frequently metastasises to bone (in 60% of cases compared to 1% for Wilms’ tumour). HA is a normal component of human serum. Its half-life in the circulation is 2.5–5.5 min and its mean serum concentration, 42 25 g/l in healthy adults.12 In contrast to patients with Wilms’ tumour and mesothelioma, patients with many other types of cancer have serum HA levels within the normal range.13,14 It is possible that the m.w. of HA, rather than its level in serum, may be pathognomonic for certain cancers. Indeed, we found that the HA level in the sera of normal children was barely detectable and had a m.w. of 1 to 5 x 10.5 In both Wilms’ and BMRTC patients, very high levels of HA were found in preoperative serum samples; these fell markedly following surgical excision of tumours.15 We also demonstrated the presence of low m.w. HA (similar to the angiogenic fragment of HA) in the sera of BMRTC patients. In contrast, high m.w. HA (which is not angiogenic) was found in the sera of Wilms’ patients. Following surgery in BMRTC patients, not only did serum HA levels fall to a value within normal ranges but also the HA which remained was of high m.w. We now have unpublished data to show that tissue culture medium from BMRTC cells, in contrast to Wilms’ tumours, contains low m.w. o-HA. Furthermore, FPLC separation of HAase-digested n-HA (kindly provided by Dr. I. Scott, Unilever, London, UK) has enabled us to narrow down the HA size to between 5 and 7 disaccharides, which continue to retain their angiogenic potential. None of these disaccharides individually was able to induce angiogenesis in chicken CAM assay. However, in combination, they had the ability to induce angiogenesis in vivo and EC migration in vitro. Future studies should be directed toward ascertaining whether the size of o-HA is a hallmark of a certain type of malignancy. Yours sincerely, Mark SLEVIN, David WEST, Pat KUMAR, Paul Rooney and Shant KUMAR

Sterilisation of skin allograft with gamma irradiation

The primary surgical requirement of skin allografts within the UK is for cryopreserved viable allografts as these engraft to the wound bed and gain a vascular supply, thus providing true wound closure and a superior clinical performance. Consequently the only disinfection treatment the skin receives is exposure to an antibiotic cocktail. However, antibiotic treatment does not reliably decontaminate skin allografts and 22% of cryopreserved skin fails microbial acceptance criteria and cannot be used clinically. We describe here a study which was carried out to determine a means of saving and using the microbiologically failed skin. Four different treatment regimens were investigated; treatment with 20%, 50% and 85% glycerol followed by 25 kGy irradiation at -80 degrees C, and treatment with 85% glycerol at ambient (30-40 degrees C) temperature and irradiation. Following treatment, the grafts were evaluated for their histological structure, in vitro cytotoxicity and handling properties. The radioprotective effects of the different glycerol concentrations and temperatures on microorganisms were also determined. The data indicate that 25 kGy irradiation of deep-frozen skin in 20% glycerol sterilised the tissue without any histological, cytotoxicological or physical alterations compared to normal cryopreserved skin. In contrast, irradiation of all other glycerol concentrations elicited some cytotoxicity and/or histological effect. These non-viable grafts can be made available for surgical use when cryopreserved viable grafts are not available or required.

Colorectal cancer – a prospective evaluation of symptom duration and GP referral patterns in an inner city teaching hospital

Objective  A high percentage of colorectal cancer patients (CRC) present as an emergency. Our aim was to evaluate delays in referral based on patient and general practitioner (GP) factors to see if there was any difference between elective and emergency patients.

The unfolded protein response regulator GRP78 is a novel predictive biomarker in colorectal cancer.

Adjuvant fluoropyrimidine‐based (5‐FU) chemotherapy is a mainstay of treatment for colorectal cancer (CRC), but only provides benefit for a subset of patients. To improve stratification we examined (for the first time in CRC), whether analysis of GRP78 expression provides a predictive biomarker and performed functional studies to examine the role of GRP78 in sensitivity to 5‐FU. 396 CRC patient samples were collected in a prospective uniform manner and GRP78 expression was determined by immunohistochemistry on tissue microarrays using a well‐validated antibody. Expression was correlated with clinicopathological parameters and survival. The role of GRP78 in 5‐FU sensitivity was examined in CRC cells using siRNA, drug inhibition and flow cytometry. GRP78 expression was significantly elevated in cancer tissue (p < 0.0001), and correlated with depth of invasion (p = 0.029) and stage (p = 0.032). Increased overall 5‐year survival was associated with high GRP78 expression (p = 0.036). Patients with stage II cancers treated by surgery alone, with high GRP78 also had improved survival (71% v 50%; p = 0.032). Stage III patients with high GRP78 showed significant benefit from adjuvant chemotherapy (52% vs. 28%; p = 0.026), whereas patients with low GRP78 failed to benefit (28% vs. 32%; p = 0.805). Low GRP78 was an independent prognostic indicator of reduced overall 5‐year survival (p = 0.004; HR = 1.551; 95%CI 1.155–2.082). In vitro, inhibition of GRP78 reduces apoptosis in response to 5‐FU in p53 wild‐type cells. GRP78 expression may provide a simple additional risk stratification to inform the adjuvant treatment of CRC and future studies should combine analysis with determination of p53 status.

Outcomes in 2748 patients referred to a colorectal two-week rule clinic.

Objective  To assess the 3‐year outcomes of a nurse‐led, one‐stop, 2‐week rule (TWR) clinic for suspected colorectal cancer (CRC) in a large teaching hospital.

Potential benefits of fast-track concepts in paediatric colorectal surgery

BACKGROUND Structured care pathways optimising peri-operative care have been shown to significantly enhance post-operative recovery. We aim to determine if enhanced recovery after surgery (ERAS) principles could provide benefit for paediatric patients undergoing major colorectal resection for inflammatory bowel disease (IBD). METHODS Children undergoing elective bowel resection for IBD at a regional paediatric unit using standard methods of peri-operative care were matched to adult cases from an associated tertiary referral university hospital already using an ERAS program. Cases were matched for disease type, gender, operative procedure, and ASA grade. RESULTS Forty-four children undergoing fifty procedures were identified. Thirty-four were matched to adult cases. Total length of stay in the paediatric group was significantly longer than in the adult group (6 vs. 9 days; P=0.001). Paediatric patients were slower to start solid diet (1 vs. 4 days; P<0.0001) and were slower to mobilize post-operatively (1 vs. 4 days; P<0.0001). No difference was seen in time to restoration of bowel function (2 vs. 3 days; P=0.49). Thirty day readmissions and total in-hospital morbidity were not significantly different between the groups. CONCLUSION Potentially, application of ERAS in paediatric surgery could accelerate recovery and reduce length of post-operative stay thereby improving quality and efficiency of care.

Management of iatrogenic recto‐urethral fistula by transanal rectal flap advancement

Aim  Recto‐urethral fistulas are an uncommon, but devastating complication following rectal or urinary tract surgery. Repair is often difficult, and the optimal approach is unclear. We report our recent experience using an endorectal advancement flap.