YCR annual science meeting

Tuesday 04 September 2007
Clarendon Lecture Theatre
and
Medical School Foyer
University of Leeds

Open Papers 1

Investigation of Chloromethylindolines as Tumour-selective Agents
Klaus Pors, Steve D Shnyder, Mark Sutherland, Paul Loadman, Jon Laye, Stephanie Guest, Nicola Harris, Patricia Cooper, Mark Searcey* and L H Patterson.
Institute of Cancer Therapeutics, University of Bradford, West Yorkshire, BD7 1DP, U.K; *School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich, Norfolk NR4 7TJ

Introduction
We are currently investigating extra-hepatic CYPs (CYP1A1, 1B1, 2J2 and 2W1) as targets to explore tumour-selective drug activation. Tumours expressing high levels of active CYPs could induce their own demise via the conversion of otherwise inert agents into potent cytotoxins. We here report on aryl-containing chloromethylindolines with tumour-selectivity in CYP1A1 expressing cancer cells.

Methods and Results
In order to evaluate the CYPs as potential drug targets we have designed specific primers for RT-PCR analysis and cloning of CYPs, and developed EJ-138 bladder sublines expressing CYP1A1, 1B1, 2J2 and 2W1. We have evaluated a library of inactivated (deshydroxylated, desOH) chloromethylindolines and shown that removal of a strategically placed hydroxyl group leads to loss of biological activity in vitro and in vivo. In contrast, activated chloromethylindolines (potential metabolites in tumour tissue) are ultrapotent in a panel of ovarian cancer cell lines. One agent from this novel class of compound has shown great than 1000-fold differential activity in CYP1A1-expressing vs wild-type cells in an in vitro hollow fibre assay.

Conclusion
We have identified a novel class of agent that possess potent activity in CYP1A1 over-expressing cancer cells.

MRI-Gel Dosimetry: State-of-the-art radiotherapy requires state-of-the-art verification
GP Liney, M Godber, AD Wilson, AW Beavis, JW Goodby, LW Turnbull

Introduction
State-of-the-art radiotherapy produces highly conformal doses, rendering routine quality assurance methods (e.g. 2D film) redundant. MRI-gel dosimetry offers a solution, whereby radiosensitive gel can be calibrated with respect to the induced changes in MR properties. This work demonstrates the long term stability, absolute accuracy and the efficacy of this approach using a head & neck cancer treatment plan.

Methods
Imaging was performed on a 3.0 Tesla GE Signa scanner. T2-weighted FSE images were acquired through irradiated gel containers for both calibration (between 0-25 Gy) and test distributions. A plot of R2 versus dose was obtained for subsequent dose mapping in test plans. One flask containing quadrants of 4,8,16 and 24 Gy was imaged on a weekly basis. Accuracy was assessed on a pixel-by-pixel basis in a further test flask.

A head & neck treatment plan was also delivered and comparison was made with the computer generated plan.
Results

Repeatability was excellent (< 1% % at room temperature.) Absolute accuracy was at least as good as film (1-3 %). The head & neck patient map demonstrated good agreement with the delivered intent.

Conclusions
MR-gel dosimetry is now a feasible method of providing quality assurance for modern-day treatment plans in 3D.

The Quality of Surgery in Colonic Cancer
West NP & Quirke P
Pathology & Tumour Biology, Leeds Institute of Molecular Medicine

Introduction
The importance of the plane of dissection in rectal cancer surgery was proven in the MRC CR07 trial. This led us to investigate whether the quality of colonic surgery could potentially have a similar impact.

Methods
We identified 313 colonic cancer resections performed between 1997 and 2001, which had adequate photographic images to retrospectively grade the plane of surgical dissection. We defined the quality of surgery as: dissection in the mesocolic plane with a high vascular tie, mesocolic plane, intramesocolic plane or muscularis propria plane. All cases were assessed by two independent observers.

Results
34.5% of cases were resected in the mesocolic plane, 42.8% in the intramesocolic plane and 22.7% in the muscularis plane. No cases showing a high vascular tie were seen. Overall interobserver variation of the grading was good with agreement in 86.3% of cases.

Discussion
There is wide variation in the quality of surgery for colonic cancer, although its relationship to outcome is currently unknown. This appears to be an exciting area for future research, which may have the potential to improve patient survival and local recurrence in a similar way to that seen for rectal cancer, if appropriate surgical education programs are developed.

Influence of tumour cell death mechanisms and immunoregulatory T cells on tumour growth and immunity
1,2SL Haywood-Small, 1K Hopkinson, 2MWR Reed, 2NJ Brown, 1AG Pockley.
1Immunobiology Research Unit and 2Microcirculation Research Group, School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield, S10 2JF, United Kingdom.

Tumour cell death might influence the induction of protective anti-tumour immunity, as necrosis is an inflammatory stimulus, whereas apoptosis can have anti-inflammatory consequences, at least some of which appear to be mediated via the induction of CD4+CD25+ regulatory T cells. The influence of tumour cell death on the development of protective anti-tumour immunity is being evaluated by administering viable (Annexin-/PI- cells sorted on a BD FACSAria™ flow cytometer), irradiated (5-10 Gy followed by sorting of Annexin-/PI- cells), apoptotic (10 mg/ml CD95 ligand for 48 hours; Annexin+/PI-) and necrotic (freeze-thaw treatment; Annexin+/PI+) EMT6 cells to Balb/c mice bearing a subcutaneous EMT6 mammary carcinoma. The EMT6 populations being used are greater than 90% pure. The administration of viable EMT6 cells into tumour bearing mice via the tail vein induces haemorrhage, a concomitant leukocytic infiltrate (n=4-6 mice per group) and reduces tumour growth. The influence of necrotic and apoptotic EMT6 cells (500-4000; irradiated and viable cells as controls) on tumour growth and the recruitment, expansion and regulatory function of CD4+CD25+ T cell populations is currently being evaluated. Overall, these findings will provide insight into immunoregulatory mechanisms controlling anti-tumour immunity and lead to more effective immunotherapeutic approaches. Funded by YCR.