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Molecular Pathology
of these pathways could be used clinically, we conducted a randomized phase II trial based on letrozole (LET arm) with or Laboratory
without ‘metronomic’ oral cyclophosphamide. PI3K, AKT, and mTOR were assessed on tumour specimens collected before and after treatment in patients randomized in this trial. The primary aim was to explore the changes of these molecular targets before and after treatment. Secondary aims were to Development and implementation of personalised
evaluate the relationship between these targets and conventional medicine biomarkers into the clinic
clinical and biological prognostic variables and to correlate the The era of personalised medicine has now spread from its changes of these targets with clinical response and patient beginnings in haematology (imatinib) and breast cancer outcome. We observed that basal expression of the pathway (tamoxifen) to encompass a wide number of tumour types and was not significantly correlated with response or patient targets. Personalised medicine involves the choice of drug outcome. Both letrozole alone and letrozole with treatment as a consequence the presence of a biomarker (often cyclophosphamide resulted in a significant reduction of PI3K a defined mutation) that indicates the susceptibility or resistance expression (P = 0.02 and P < 0.005, respectively) and of a patient to a specific drug. We have been working with a phospho-mTOR expression (P = 0.0001 and P = 0.0001, number of our medical oncology colleagues to develop and respectively). pAKT showed no change in the letrozole arm, implement appropriate methodology to detect these whereas it was significantly decreased in the letrozole plus predictive biomarkers using blood or tumour biopsies from cyclophosphamide arm (P < 0.005). pAKT expression patients. Our expertise in this area is now nationally and reduction was associated with a greater response rate (P = 0.05) internationally recognized with the result that our diagmostic lab and greater reduction in Ki67 expression (P = 0.05). has become the Australian centre of choice to offer statewide Phospho-mTOR expression reduction was associated with or national testing. Tests currently on offer include KRAS, BRAF, a significantly longer disease-free survival in a multivariate EGFR, NRAS, JAK2, NPM and TP53 together with S/FISH assays for ALK, MET and HER2. In addition, we are also offering a comprehensive portfolio of testing for familial cancer genes We conclude that Letrozole inhibits key molecules in the PI3K including BRCA1 and BRCA2 as well as clonality testing and pathway. Changes in these molecules may have prognostic translocation identification for haematological malignancies.
significance. These results should be taken into account when planning prospective trials testing up-front aromatase inhibitor Predicting the response of hormonal therapy on breast
with drugs targeting the PI3K/AKT/mTOR signalling pathway.
cancer subsets
Developing assays for personalised medicine
Endocrine therapies that interfere with estrogen receptor (ER) function have contributed to a dramatic reduction in breast The personalisation of medicine will depend on being able to cancer mortality. To date, aromatase inhibitors have been shown rapidly perform screening assays for markers that will predict to be the most effective endocrine treatment in postmenopausal response to therapy. High resolution melting (HRM) is a rapid women with ER-positive breast cancer. The results obtained and efficient method of screening that relies on the precise with the third-generation aromatase inhibitor letrozole showed an monitoring of the melting of a DNA duplex. We have developed improvement in patient outcome compared to results based on sensitive HRM screening assays for multiple changes in cancer, tamoxifen as an endocrine treatment. This benefit translates into notably mutations in the KRAS, TP53, BRAF and KIT genes and disease-free survival (DFS) improvement for adjuvant treatment epigenetic changes in the MGMT and BRCA1 genes. Some of and overall survival in patients with metastatic disease. However, these assays are now being used diagnostically, particularly the not all ER-positive breast cancers respond to endocrine KRAS mutation assay that is being used to determine patients’ manipulation and many initially responding tumours develop resistance to therapy with EGFR inhibitors. However, mutation resistance. Currently we cannot identify patients likely to respond detection in clinical tumour samples is challenging when the to such therapies, which leads to overtreatment, exposure of proportion of tumour cells, and thus mutant alleles, is low. patients to potential drug toxicity and inefficient use of limited Recently, a number of highly sensitive techniques have been developed but cannot be validated by sequencing due to its limited sensitivity. In addition, it is important to discriminate false The growth factor family of epidermal growth factor (EGFR and positives due to PCR errors or template degradation from true HER2) are recognized to be implicated in such endocrine mutations. We therefore have developed an adaptation of HRM, resistance through activation of mitogen-activated protein limited copy number HRM (LCN-HRM) which utilises the kinase/extracellular signal-related kinase and/or the ability of HRM to detect heteroduplexes when variant sequences phosphatidylinositol 3’-kinase (PI3K)/AKT/molecular target of are present. Multiple replicate reactions with a limited number of rapamycin (mTOR) pathway. In vitro studies have shown that target sequences per reaction readily allow low frequency after long-term estrogen deprivation, i.e., during long-term mutations to be detected by their aberrant melting patterns. aromatase inhibitor administration, breast tumour cells exhibit LCN-HRM is an effective and rapid single step method to an activation of the PI3K/AKT/mTOR pathway as an adaptive enable levels of sequence variation below the normal sensitivity phenomenon of breast cancer cells to the low estrogen of dideoxynucleotide sequencing to be detected in a way that environment. To address whether measurement of members then allows identification by sequencing.
Molecular Pathology Laboratory: Created by Cancer Research, July 2013.
The role of SNPs in the predisposition to somatic
Methylation of the CpG island in the MGMT promoter region
is a frequent event in several cancer types, including colorectal cancer, lung cancer, lymphoma and glioblastoma. A correlation between methylation and the T allele of the SNP rs16906252 in colorectal carcinomas has previously been reported. As aberrant MGMT methylation can be an early event in tumour development, we tested the hypothesis that normal individuals possessing the T allele may be predisposed to somatic methylation at the MGMT promoter. Peripheral blood monononuclear cell DNA from 89 healthy individuals was genotyped at rs1690625 and assessed for the methylation status of the MGMT promoter region using independent quantitative methodologies capable of detecting low level methylation. There was a strong association between presence of the T allele and detectable methylation (p=0.00005) in the peripheral blood DNA. Furthermore, when a MSP assay flanking the SNP was used to amplify methylated sequences in heterozygotes, only the T allele was methylated. Thus, detectable somatic methylation of the MGMT promoter in normal individuals is strongly associated with the T allele of the rs16906252 MGMT promoter SNP. We are currently examining the involvement of this SNP in cancer predisposition.
Hypoxia in prostate cancer
Hypoxia profoundly influences tumour behaviour conferring
a poor prognosis and resistance to chemo and radiotherapy. BNIP3 is a hypoxia-induced protein involved in cell death and survival but its role in human tumours is unclear. We investigated the role of BNIP3 in prostate cancer. The expression of BNIP3, the androgen receptor (AR), hypoxia inducible factor (HIF)-1a, HIF-2a and another hypoxia regulated gene GLUT1 were assessed in tissue microarrays constructed from 149 archival radical prostatectomy specimens. Statistical analyses compared expression of these factors between each other, conventional clinicopathological parameters and PSA recurrence. Since an association between BNIP3 and AR and the HIFs was observed, the influence of hypoxia, dihydrotestosterone and the AR blocker, Casodex, was also investigated in prostate cell lines. BNIP3 was expressed in both the nucleus and cytoplasm. There was a significant correlation between cytoplasmic BNIP3 expression and Gleason score, age, AR and GLUT1. There was a significant correlation between nuclear BNIP3 expression and HIF-1α expression and HIF-2α expression but no correlation between BNIP3 and pre-operative PSA, tumour volume, margin positivity or capsular invasion. There was an increase in BNIP3 expression under conditions of hypoxia (0.1% 02) but not with dihydrotestosterone stimulation or with Casodex treatment. Our findings suggest that BNIP3 is directly regulated by hypoxia but that there may be a hormonal independent mechanism coordinating the expression of BNIP3 in prostate tumours.
For more information contact: Professor Stephen Fox Phone: 03 9656 1529 Email: Molecular Pathology Laboratory: Created by Cancer Research, July 2013.



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