Molecular Phenotyping as a novel method of lymph node tracking in oesophageal cancer

Oesophago-gastric cancers (cancers of the gullet and stomach) have quadrupled in the last 30 years. Only 14% of patients with the disease survive for 5 years after their diagnosis. These cancers have devastating effects on physical and mental well beings of patients and their families. The most distressing symptom is probably the inability to swallow food, which has serious consequences on nutrition and on the patient's psychological state. Whereas extensive research has been carried out in other areas such as breast cancer, our knowledge of oesophago-gastric cancer remains limited. The OCCAMS collaboration has already led some ground-breaking research on oesophago-gastric cancers. Established work on cancer samples from patients has revealed that a combination of four specific genes was associated with the poorest patient survival. This research has enabled us to predict which patients will have worse outcome so that we can treat them accordingly and hence tailor cancer treatment. One of these four genes has been examined in the lymph nodes of these patients and has also been shown to be critical in predicting patient survival and ongoing work is unravelling how this gene is involved in the development and progression of cancer. Very little is still known about the way cancer spreads in the lymph nodes in oesophago-gastric cancer. Our present techniques of radiological imaging cannot accurately tell us which lymph nodes are involved in the cancer unless there are multiple cancer cells occupying most of the lymph node. Currently all lymph nodes in the field of the cancer are removed at the time of surgery to improve the chances of cure from the cancer. There is no established way of knowing which of these nodes are involved or not, until they are looked at under the microscope by our pathologist after the cancer has been removed surgically. Such extensive surgery is not without its risk of complications such as chest infections, heart attack, leaks from the join of the gullet and bowel, clots in the legs and lung. However, if we knew which lymph nodes to take out, as opposed to taking out all of them, then we could minimize the anaesthetic time and other complications that are associated with these operations. This would enable us to perform minimally invasive keyhole operations, but not at the expense of affecting cancer survival. Our aims are as below: 1) To look at the expression of certain genes in lymph nodes to help us understand how this affects survival. In the future some of these genes may also be targets for therapy. 2)To use state of the art molecular techniques to help us understand the pattern of lymph node spread in oesophago-gastric cancer, if we can understand this, we can block and prevent cancer spread. This information will also help us to do less extensive operations which will improve patient survival and experience.

Immunohistochemistry :Bond System(Leica Microsystems, Milton Keynes,UK).Antibodies will have specificity confirmed by Western blotting with positive and negative controls. Antibodies will be optimized with positive control cell blocks and oesophageal tissue before application to primary tumours and lymph nodes. PCR for oncogenic mutations:DNA will be extracted from serial sections from formalin fixed paraffin. Select gene mutations (single nucleotide variants, SNVs) which are frequently occurring and known to be relevant to lymph node metastases from our own ongoing work and from the literature. Bioinformatics support to robustly pick deleterious SNVs and verify these within the same cases as part of the ICGC project. PCR will be performed using standard techniques and high thoroughput systems using fluidigm platforms for analysis of multiple SNVs in parallel. Cell Lines:We have a panel of cell lines derived from oesophageal adenocarcinoma including 2 lines from metastases. These have been verified either in house or as part of an international effort to verify the lines for use in in vitro model systems. We will use standard sRNA methods to knockdown targets of interest or to overexpress them using standard transfection methods. Proliferation (promega Glo assay), apoptosis (FACS analysis, caspase expression) and invasion through matrigel will be determined using standard methods, already established in the laboratory for other targets. Site-directed mutagenesis may also be used to recapitulate the mutation of interest. Lymphangiogenesis: Human microvascular lymphatic endothelial cells will either be co-cultured with tumour cells or tumour conditioned medium for 24 hours. The ability of tumour cells to invade and home specifically to lymphatic vessels and lymph nodes rather than blood vessels will be determined using a combination of modified Boyden chamber and 3D co-culture models. Long-term imaging of cultures will be performed on a Leica SP5 confocal microsc

Currently we have a limited understanding of how cancer spreads to lymph nodes in oesophageal cancer. In addition established radiological techniques of detecting lymph node spread have limited specificity and sensitivity. During surgery, we currently remove all lymph nodes in the field of dissection to help improve patient survival. However this is often associated with increased post operative complications because of an increased anaesthetic time. The development of a method to accurately identify which lymph nodes are involved in cancer using novel molecular targets would revolutionize our understanding of oesophageal cancer. The main impact would be on patients with oesophageal cancer. If we could better delineate lymph node involvement, we could do smaller operations with less complications which would result in a significantly enhanced patient experience and a better overall quality of life. If patients have less invasive operations, and consequently have a shorter hospital stay, this will have benefit the National Health Service financially. Being able to predict the pattern of lymphatic spread would open doors to avenues of potential target therapy. This would have an impact in the longer term. Focused therapy against these molecular targets would result in personalised patient treatment. Patient survival would be improved and this would have a beneficial effect on patient's families. In addition, identification of molecular targets involved in lymph node spread would provide exciting opportunities for the development of novel imaging techniques to identifiy lymph node involvement at the time of presentation. The results of our projects would be published in conferences and in peer-reviewed journals. They would be available to other members of the MRC on the website, and may provide further avenues for research in different types of cancers. This work would add to the established achievements of the OCCAMS collaboration in this field of research. This would raise the profile of OCCAMS and generate new collaborations and possibilities for the future, in the aim of advancing research in the field of oesophageal cancer.