Global gene expression profiling of clinical response to therapy has provided a useful means for biomarker and novel target discovery in several solid tumours [5, 13]. The work described in this paper has used and extended this experimental approach to rectal adenocarcinomas. The data presented constitutes an analysis from gene expression profiling of prospectively collected pre- and post-treatment tumour specimens from patients with rectal adenocarcinomas receiving pre-operative therapy.
Since a small number of rectal adenocarcinomas have been profiled (n = 10), stringent and focussed analysis of the microarray data was applied to identify leads for further investigation. This included hypothesis-driven focus on cell death pathways and comparison with our previously published cell line work. The key candidate was subsequently validated a in larger independent set (n = 234) using a different technique (immunohistochemistry).
The biological validity of the experimental model and the data is confirmed by the finding of significant alterations in the gene expression of previously implicated molecules and pathways, for example p21 which has been implicated in numerous studies [20–25]. The biological pathways identified (information 3 and 4) suggest a co-ordinated transcriptional response to radiotherapy- and CRT- induced cellular stress, consistent with other reports involving gene expression profiling in cell lines and several different cancer types [2, 11, 13, 25–29]. We hypothesize that this reflects distinct biological effects of these two treatments. However, the possibility of effects due to time course differences in the tumour sampling in each group cannot be excluded.
A supervised analysis of cell death genes, reveals shared genes and pathways. The analysis supports the hypothesise that initiation of cell death is a common final pathway resulting from a multitude of upstream responses to the insult and resultant cellular stress of cytotoxic chemotherapy or radiotherapy thereby accounting for gene expression overlap seen.
The majority of the genes identified in our analysis represent genes and pathways that have not previously been implicated in clinical response of rectal adenocarcinoma or as mechanisms of action or resistance to radiotherapy or 5FU or 5FU-based CRT. This is consistent with the findings of other gene expression profiling studies in rectal adenocarcinoma or other tumour types for radiotherapy or 5FU [6, 8–11, 26, 28–30]. However, it is important to note that this discovery phase utilised a small sample cohort and the candidate gene expression changes require further validation in a lrger independent cohort.
APRIL/TNFSF13 was found to be upregulated following CRT but not radiotherapy alone in rectal cancers and was also up-regulated in 5FU resistant cell lines in our previous studies . The biological function of APRIL as a secreted molecule that has autocrine and paracrine functions to promote cell survival and proliferation and its previously documented expression in colorectal adenocarcinoma but not normal cells outside the immune system, supported it's further investigation as a novel mechanism of 5FU action and resistance, and as a predictive biomarker [15–19, 31–35].
This study found that expression of APRIL protein in colorectal tumour stroma was associated with worse survival, but only in those patient's treated with adjuvant 5FU chemotherapy. This relationship was also maintained in a multivariate analysis of 5FU chemotherapy treated Stage III colorectal adenocarcinoma patients (HR 6.25, 1.47-26.31, p = 0.013), in which the Hazard ratio compares favourably to other previously published putative 5FU predictive biomarkers in colorectal cancer [2–4]. Tumour cell expression of APRIL was correlated with stromal staining but was not significantly associated with survival. Overall, APRIL appears to have no therapy independent prognostic impact in colorectal adenocarcinoma in this analysis.
Within the limitations of a retrospective study, these results suggest that APRIL may have clinical utility as a predictive biomarker to select patients who would not benefit from adjuvant 5FU monotherapy. For example, currently adjuvant 5FU is used clinically in an empirical way without predictive biomarkers in stage III patients and in this paradigm the majority of patients with Stage III cancers will not benefit from 5FU. Therefore, the ability to identify some of these stage III patients who will not benefit from 5FU has clear potential clinical utility in optimising and individualising clinical use of 5FU in this setting. An important question is whether APRIL confers cross resistance to other active agents used to treat colorectal cancer, especially Oxaliplatin and Irinotecan, this would be potentially useful to guide 5FU combination adjuvant therapy in stage III patients, but especially in stage II patients where 5FU alone appears to have limited benefit.
The data allows us to hypothesise that APRIL may provide a useful novel therapeutic target. Morphological examination has suggested that positively staining stromal cells include lymphocytes and fibroblasts, but not endothelial cells. This is consistent with evidence indicating that APRIL is predominantly secreted and exerts it's effects via cell surface receptors, acting in a paracrine or autocrine fashion [15–19, 31–35].
Our data indicate that APRIL might be secreted by tumour cells or stromal cells within the tumour. The APRIL signalling mechanisms that may mediate tumour cell survival are not well characterised . However, in vitro work in glioma cell lines and ex vivo studies in BCLL, has shown that APRIL stimulates proliferation and inhibits apoptosis in response to a wide range of stimuli, including CD95L, TRAIL and cytotoxic drugs and survival in B-CLL cells involves NFκB activation [15–19, 31–34]. More recently it has been suggested that tumour infiltrating neutrophils may be an important source of APRIL production in solid tumours .
If APRIL is functional as an extracellular secreted molecule this makes it amenable to targeting with either a small molecule inhibitor or monoclonal antibody, as has been employed successfully for other targets in solid tumours e.g. bevacizumab against VEGF. An anti-APRIL targeted therapy may be useful in reversal of acquired 5FU resistance or in combination in patients whose tumours over-express the molecule.
The lack of therapy independent prognostic impact suggests that an anti-APRIL therapy may not have anticancer activity on it's own, but the cell survivalpromoting activity may be more generally applicable to other therapeutic cell death stresses. Therefore, combination of an anti-APRIL agent with agents other than 5FU may be active, and our cell line data also suggest that they may be active in other tumour types, such as breast cancer.