Patients included in the present study were from a multicenter phase II clinical trial which is the first prospective evaluation of 90Y-RE in CRC patients with liver metastases who failed previous oxaliplatinum and irinotecan based chemotherapy regimen . It has been widely reported that alterations in genes, as survivin, p53 and Bcl-2, which regulate cell growth and apoptotic processes, are significantly associated to an unfavourable clinical outcome in CRC patients . In our series of 29 liver mCRC patients, we found that most tumors sampled prior to 90Y-RE were p53, survivin, and Bcl-2 highly positive and presented a high Ki-67 proliferation index. In contrast, we found a significant reduction in p53, survivin and Bcl-2 positive expression in liver metastasis sampled two months post-90Y-RE. There was also a trend towards a reduction in cells with a high proliferative index as measured by Ki-67. We have previously shown that colon cancers harboring p53 nuclear accumulation, as assessed by the DO7 anti-p53 antibody, represent a subset of tumors with a more aggressive clinical behaviour in patients with stage II tumors as well as in young patients [13, 16]. Furthermore, several studies have shown an increased incidence of p53 nuclear accumulation in liver metastases in comparison to the primary tumor, hypothesizing a role for p53 in CRC liver metastatization. In particular, the presence of ≥ 3 liver metastases identified a subset of patients with a very poor prognosis mainly when associated to p53 mutations . A number of studies have also shown that tumors that do not express detectable levels of Bcl-2, but which exhibited nuclear accumulation of p53, were associated with the shortest patient survival, while Bcl-2-positive and p53-negative tumors had the best prognosis [12, 17]. Studies conducted at our Institute showed that p53 positivity combined with Bcl-2 negativity and elevated Ki-67 score correlated with advanced tumor stage, poorly differentiated tumors and increased probability of relapse. Also elevated survivin expression levels in primary CRC are related to decreased survival [14, 15]. In resected liver tumors, altered expression of survivin, p53, Ki-67 and, more recently, KRAS mutations, have been shown to be independently predictive of hepatic recurrence and poor survival [13, 16, 18]. It is recently reported that defective mismatch repair predicts resistance to 5-fluorouracil (5FU) and KRAS mutation resistance to anti-EGFR antibody therapy . Nevertheless, no predictive markers of RE efficacy in mCRC have been identified up to now. In terms of the predictive response to radiotherapy, several studies have linked epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) expression to a lack of response to pre-operative radiotherapy in locally advanced rectal cancer [19–21]. Neither p53, Ki-67 and survivin expression appear to be correlated to pre-operative chemo-radiotherapy response and prognosis in locally advanced rectal cancer [22, 23]. To date, however, no study has evaluated the predictive value of molecular markers on radiosensitivity of CRC liver metastasis. In this context, our findings, although in a very limited number of patients, may be clinically relevant.
The rapid changes of biomarkers observed in our series post-90Y-RE may be due to clonal selection or to epigenetic changes, not previously recorded in this context. Such mechanisms are usually discussed in the context of cell adaption to chemotherapy and evolving resistance. Radio-sensitivity of colorectal cancer cells may be determined by p53 mutation [23, 24], whereas there is no evidence that chemotherapy per se cause changes in the cellular expression of p53 . This is the first time that we have recorded a down-staging in p53 protein expression after 90Y-RE.
It is likely that both disease progression and a prolonged prior chemotherapy affected the efficacy and tolerability of 90Y-RE in the liver. In fact, mild manifestations of non-alcoholic fatty liver disease (NAFLD) after 5FU , more serious non-alcoholic steatohepatitis after irinotecan and sinusoidal obstruction syndrome (SOS) after oxaliplatin-based treatment  have been recorded. Using the same biomarkers as in our study, Panasiuk and colleagues  showed that the intensification of inflammation in NAFLD may also impact on biomarker expression in human hepatocytes with the induction of pro-apoptotic protein p53 and the inhibition of anti-apoptotic Bcl-2.
There are clear limitations to our study, not least of which was the small patient numbers and limited tissue sampling. Nevertheless, we believe that our findings merit further investigation in prospective clinical trials. We are planning to evaluate this biomarker panel in a phase II randomized trial on 2nd line treatment. KRAS mutated CRC patients with unresectable liver metastasis will be randomized to receive systemic therapy vs systemic therapy plus 90Y-RE. The combined assessment of survivin, p53 and Bcl-2 pre and post-90Y-RE therapy may improve our ability to predict outcomes in the treatment paradigm of metastatic KRAS mutated CRC patients.