Title: “Completion of the Molecular Pathology of the Runt family of genes in breast
cancer and breast cancer precursor lesions: biological and clinical implications”
(Grant Ref No: SGS 2011 10 13)
Name and address: Dr David P. Boyle, Centre for Cancer Research and Cell Biology,
Lisburn Rd, Belfast
Background:
We feel that an understanding of Runt-related transcription factors 1-3 (RUNX1, 2
and 3) and the regulation of their biological pathways will directly impact our
knowledge of these areas of human carcinogenesis. RUNX1-3 are a family of
transcription factors involved in multiple cancer types, namely leukaemias (RUNX1),
osteosarcoma (RUNX2) and other solid tumors (RUNX3). They are downstream
effectors of main molecular pathways and have critical roles in the regulation of cell
proliferation and cell death by apoptosis, and in angiogenesis, cell adhesion and
invasion. Our group has played a pivotal role in establishing our current
understanding of the molecular mechanisms of RUNX2 and RUNX3 in the context of
cancer development and progression [1]. In particular, our group has helped in the
mapping of RUNX3 and RUNX2 molecular changes in breast cancer and its preneoplastic stages. However, the mapping of all 3 genes in all stages and their clinicopathological relevance is incomplete.
Original Aims (copied from original application):
To establish a comprehensive analysis of the 3 members of the RUNX family in
relation to protein expression (IHC), gene expression (RNA), gene copy
number/amplification (FISH), methylation status (DNA) and sequencing (DNA), from
FFPE materials representing all aspects of the progression from normal breast ducts
to metastatic cancer. Detailed clinico-pathological information of all these samples
will be annotated to describe the diagnostic, prognostic and predictive value of these
biomarkers. In the process of developing this project, a comprehensive collection of
clinical materials will be created which, from this point onwards, will be available for
breast cancer studies in the Centre for Cancer Research and Cell Biology at any point
in time. It will also allow us to investigate the RUNX genes in cancer types
unrecorded to date.
Results:
The project started with 2 activities, namely a) to validate in our new lab (Northern
Ireland Molecular Pathology Laboratory) the techniques for analysis of the RUNX
family of genes that we had established in Singapore; b) to analyse baseline markers
of interest in pre-neoplastic lesions of the breast for correlation with RUNX status.
However, 2 main problems occurred. Firstly, we were not able to replicate the same
results that we had in our laboratory in Singapore, with the same degree of
biological and pathological stringency. Secondly, some studies at the time put into
question our original approach to analysis of RUNX status in human tissues [2,3]. As
a result, and after much trying to optimize and in view of the questioning of this
approach in the literature, we ceased to pursue this avenue. In parallel, however,
the analysis of other biomarkers in the study, some being putative druggable
biomarkers relating to growth and proliferative factors, the cell cycle, and apoptotic
pathways became interesting and are reviewed [4,5].
We have defined a general cohort of breast cancers in terms of putative actionable
and prognostic biomarkers, both singly across a general cohort and within intrinsic
molecular subtypes. We identified 293 patients treated with adjuvant
chemotherapy. Additional hormonal therapy and trastuzumab was administered
depending on hormonal and HER2 status respectively. We performed
immunohistochemistry for ER, PR, HER2, MM1, CK5/6, p53, TOP2A, EGFR, IGF1R,
PTEN, p-mTOR and e-cadherin. The cohort was classified into luminal (62%) and nonluminal (38%) tumours as well as luminal A (27%), luminal B HER2 negative (22%)
and positive (11%), HER2 enriched (13%) and triple negative (25%). Patients with
luminal tumours and co-overexpression of TOP2A or IGF1R loss displayed worse
overall survival (p=0.0251 and p=0.0008 respectively). Non-luminal tumours had
much greater heterogeneous expression profiles with no individual markers of
prognostic significance. Non-luminal tumours were characterised by EGFR and
TOP2A overexpression, IGF1R, PTEN and p-mTOR negativity and aberrant p53
expression.
Our results indicate that only a minority of intrinsic subtype tumours purely express
single novel actionable targets. This lack of pure biomarker expression is particular
prevalent in the triple negative subgroup and may allude to the mechanism of
targeted therapy inaction and myriad disappointing trial results. Utilising a
combinatorial biomarker approach may enhance studies of targeted therapies
providing additional information during design and patient selection while also
helping decipher disappointing trial results [6].
During this analysis we also noted a significant prognostic effect of extremes of p53
IHC expression and described a novel, reproducible scoring system and assessed the
relationship between differential p53 IHC expression patterns, TP53 mutation status
and patient outcomes for breast cancer. We found that patients with extreme p53
IHC expression have a worse OS compared to those with non-extreme expression.
Accounting for extremely negative as well as extremely positive p53 improves its
prognostic impact. Extreme expression positively correlates with nodal stage and
histological grade and negatively with hormone receptor status. Extreme expression
may relate to specific mutational status [7].
Following our analysis of invasive carcinoma, we sought to classify non-invasive
breast lesions for personalised therapy and chemoprevention strategies. Our main
findings to date are that benign and early neoplastic lesions display homogeneity in
their molecular profiles. Greatest diversity of biomarker expression occurs at the in
situ carcinoma phase of non-invasive lesions. p53 and Ki67 best predict the
association of DCIS with concurrent invasive disease. HER2 status is most likely to
show concordance between in situ and invasive disease. In non-HER2 expressing
DCIS, the next most frequently expressed biomarkers are ER and TOP2A [manuscript
in draft: Molecular classification of non-invasive breast lesions for personalised
therapy and chemoprevention].
Conclusions:
Human analysis of RUNX family is genes came into question during the process of
this research, both technically and conceptually, but the results have lead to
valuable research in understanding the biology of preneoplastic lesions of the breast.
This is important as non-invasive lesions are frequently encountered in diagnostic
breast specimens. Assays are required to further classify DCIS and its patient-specific
propensity to progress to invasion, to optimise management and avoid
overtreatment.
How Closely Have the Original Aims been met:
The research has lead to important insights in the nature of preneoplastic lesions of
the breast, but not in relation to the status of the RUNX family of genes in breast
cancer.
References
1. Subramaniam MM(1), Chan JY, Yeoh KG, Quek T, Ito K, Salto-Tellez M.
Molecular pathology of RUNX3 in human carcinogenesis. Biochim Biophys
Acta. 2009 Dec;1796(2):315-31.
2. Levanon D(1), Bernstein Y, Negreanu V, Bone KR, Pozner A, Eilam R, Lotem J,
Brenner O, Groner Y. Absence of Runx3 expression in normal gastrointestinal
epithelium calls into question its tumour suppressor function. EMBO Mol
Med. 2011 Oct;3(10):593-604.
3. Normile D. Dispute Over Tumor Suppressor Gene Runx3 Boils Over. Science
2011 Oct:442-443.
4. Boyle DP, Mullan P, Salto-Tellez M. Molecular mapping the presence of
druggable targets in preinvasive and precursor breast lesions: A
comprehensive review of biomarkers related to therapeutic interventions.
Biochim Biophys Acta. 2013; [Epub ahead of print].
5. Boyle DP, McCourt CM, Matchett KB, Salto-Tellez M. Molecular and
clinicopathological markers of prognosis in breast cancer. Expert Rev. Mol.
Diagn. 2013; 13(5).
6. Defining a therapeutic classification of breast cancer by actionable targets
(Presentation abstract AACR 2014, abstract number 1905)
7. Boyle DP, McArt DG, Irwin G, Wilhelm-Benartzi CS, Lioe TF, Sebastian E,
McQuaid S, Hamilton PW, James JA, Mullan PB, Catherwood MA, Harkin DP,
Salto-Tellez M. The prognostic significance of the aberrant extremes of p53
immunophenotypes in breast cancer. Histopathology; 2014 Feb 24. doi:
10.1111/his.12398. [Epub ahead of print]