Molecular Pathology Projects

Molecular Pathology Projects - Research at Peter Mac

Identifying the patients likely to respond to neoadjuvant radiotherapy of the rectum

Supervisor: Professor Stephen Fox

Hypothesis:

Expression of hypoxia and downstream signalling pathways will identify patients with rectal carcinoma likely to respond to primary chemo-radiation.

Clinical issues:

Rectal carcinoma is a significant cause of mortality and morbidity in Australia. It is treated by radiotherapy before resection to try and shrink the tumour to improve survival and reduce local recurrence. In some patients complete tumor regression is achieved allowing sphincter preservation. The ability to predict tumor response from such treatments would therefore improve the selection of patients for such treatments. The efficacy of radiotherapy is determined by the degree of tissue hypoxia (oxygen tension) and hypoxia is associated with an aggressive tumour phenotype.

The transcriptional complex hypoxia inducible factor (HIF) has emerged as a key regulator mediating many cellular responses necessary to adapt to changes in oxygen tension. The HIF complex is composed of a heterodimer of HIF-1a (or HIF-2a) and HIF-b (also known as aryl-hydrocarbon nuclear translocator). HIF-b is constitutively expressed and is involved in several transcriptional systems whereas the two HIF-a subunits are specific to the hypoxic pathway. In normoxic conditions the HIF-a units are unstable since two prolyl residues within the oxygen-dependent degradation domains of HIF-a subunits are hydroxylated by prolyl hydroxylases and di-oxygen as a co-substrate. This results in one oxygen incorporated into the prolyl residue of HIF-a, allowing rapid targeting and degradation by the proteasome pathway via the von Hippel-Lindau (VHL) protein and the ubiquitin E3 ligase complex.

However, in hypoxia, as frequently occurs within tumours, there is insufficient oxygen to allow this process resulting in HIF-a stabilisation, translocation to the nucleus where it is able to bind HIF-b. The complex then recruits co-activators that bind specific DNA hypoxia response elements (HREs) resulting in increased mRNA transcription.

A further level of control is achieved through hydroxylation of an asparagine residue by factor inhibitor of HIF (FIH) at the C terminus of HIF-1a that interferes with CBP binding and thus transcription.

For more information about this project contact:

Professor Stephen Fox

+61 (0)3 9656 1529

stephen.fox@petermac.org

Distinguishing recurrent breast tumours from de novo primary breast carcinomas

Supervisor: Professor Stephen Fox and Assoc. Professor Alexander Dobrovic

Profiling tumours for a series of cancer specific changes will allow the ready determination of the relationships between primary and recurrent/new primary breast cancers.

Clinical issues:

Patients with breast cancer are at increased risk of a second breast cancer and also from local relapses in the breast. Currently we do not have the ability to accurately identify whether a new breast tumour arising in the breast is a new primary breast cancer or a recurrence of a previous tumour. This is essential since their treatments are very different.
Recurrent breast cancer is considered a metastasis but not all recurrences have similar biological behaviour and therefore a "recurrence" breast tumour classification system on which to base treatment decisions is needed. This is likely to become more important with the further molecular characterisation of breast cancer for targeted treatment.
The true frequency of multifocal / multicentric breast cancers is unknown. Morphology alone is not adequate in distinguishing between truly synchronous tumours and multiple deposits of the same tumour. This issue is of importance since staging and treatment decisions are based on these observations.

Tumour specific markers based on mutation and methylation will be used to compare primary and ipsilateral second presentation. These markers have been selected because the readout is only minimally affected by the presence of contaminating normal tissue. The technique for screening is the novel methodology of high resolution melting analysis which has been recently developed in this Laboratory and allows rapid and sensitive 'in-tube" typing within a few hours.

For more information about this project contact:

Professor Stephen Fox

+61 (0)3 9656 1529

stephen.fox@petermac.org

Assoc Professor Alexander Dobrovic

+61 (0)3 9656 1807

alex.dobrovic@petermac.org

Understanding the molecular mechanism of long term survival after radiotherapy for lung cancer

Supervisor: Assoc Professor Alexander Dobrovic and Dr. Michael McManus

We seek to understand why about 1% of patients with apparently-incurable non-small cell lung cancer have experienced long-term survival and even cure after low dose palliative radiotherapy at Peter Mac. This initiative builds on a highly successful research project. (MacManus MP et al., Cancer. 2006 106:1110-6). That study attained widespread publicity in the medical and lay press and media, both internationally and in Australia, including the Health Report with Norman Swann, and was discussed in articles in the Lancet and in Nature Clinical Reviews.

Hypothesis:

The tumours in these patients are likely to have a defect with in DNA repair or in DNA damage sensing pathways which underlies their extraordinary radiosensitivity. This defect/these defects will also occur in some patients treated with conventional radiotherapy.

Experimental plan: Methylation profiling for methylated DNA repair genes: As the amount of DNA from the tumours will be limited and the DNA undergoes further degradation during bisulphite modification, a whole genome amplification method specific for bisulphite modified DNA will be used. Methylation of genes involved in the detection and repair of damage caused by ionising radiation will be assessed. These include ATM, CHK2, BRCA1, and FANCF.

Mutation status of genes determining sensitivity to radiation: DNA will be screened for p53 (exons 5-8), PTEN (exons 5-8), and PIK3Ca (exons 9, 20) mutations using high resolution melting. Inactivating mutations at any of these genes is likely to lead to radioresistance and would be more likely to be found in the control population.

Profiling expression: We will use a panel of 190 genes including all known DNA repair genes, selected anti- and pro-apoptotic genes, HIF1 alpha to assess hypoxia and genes that Ramaswamy reported as being associated with metastasis in adenocarcinoma, particularly lung adenocarcinoma.

For more information about this project contact:

Assoc Professor Alexander Dobrovic

+61 (0)3 9656 1807

alex.dobrovic@petermac.org