Biomarkers and Molecular Imaging as Predictors of Response to Neoadjuvant Chemoradiotherapy in Patients With Locally Advanced Rectal Cancer

Review

Biomarkers and Molecular Imaging as Predictors of Response to Neoadjuvant Chemoradiotherapy in Patients With Locally Advanced Rectal Cancer Chiara Molinari,1 Federica Matteucci,2 Paola Caroli,2 Alessandro Passardi3 Abstract Standard treatment of patients with locally advanced rectal cancer (LARC) includes neoadjuvant chemoradiotherapy (NCRT) followed by surgery. Tumor regression after NCRT varies substantially among individuals and pathological complete response is a known prognostic factor for LARC. The identification of a predictive model for response to chemoradiotherapy would help clinicians to identify patients who would probably benefit from multimodal treatment and to perform an early assessment of individual prognosis. Carcinoembryonic antigen has proven to be a good predictor of response in several clinical trials. Other widely studied predictive models in LARC include molecular biomarkers, analyzed at various levels and by different techniques, and molecular imaging, in particular magnetic resonance imaging and positron emission tomography/computed tomography. Although none of the studied markers have been approved in clinical practice, their evaluation in larger, prospective trials and in combined predictive models could be of use to define tailored therapeutic strategies. Clinical Colorectal Cancer, Vol. -, No. -, --- ª 2015 Elsevier Inc. All rights reserved. Keywords: LARC, MRI, NCRT, PET-CT, Predictive markers

Introduction A multimodal approach to locally advanced (clinical T3-4 and/or node-positive disease) rectal cancer (LARC), based on the use of concomitant radiotherapy (RT) and chemotherapy followed by radical surgery with total mesorectal excision, has led to a significant improvement in patient outcomes in recent decades.1,2 Trials that compared chemoradiotherapy before and after surgery have highlighted lower toxicity, reduced local recurrence rates, and prolonged disease-free survival (DFS) for the former, albeit with similar survival rates. This therapeutic approach also improves tumor resectability because of downstaging and increases the possibility of sphincter functionality preservation.3,4 For these reasons, neoadjuvant chemoradiotherapy (NCRT) followed by surgery has become the standard treatment for LARC. 1 Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy 2 Diagnostic Nuclear Medicine Unit, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy 3 Department of Medical Oncology, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy

Submitted: Feb 20, 2015; Accepted: May 29, 2015 Address for correspondence: Alessandro Passardi, MD, Department of Medical Oncology, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Via Maroncelli 40, 47014 Meldola, Italy Fax: þ39 0543 739151; e-mail contact: [email protected]

1533-0028/$ - see frontmatter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clcc.2015.05.014

Response to treatment varies substantially among patients but is recognized as an important prognostic factor. Different response indicators have been proposed, the most frequent being tumor regression grade (TRG), pathologic complete response (pCR), and downstaging. A pCR (no residual tumor cells) has been reported in 8% to 16% of patients who undergo NCRT5-8 and is associated with good DFS and 5-year overall survival (OS) rates. In particular, a meta-analysis of 14 studies reported better 5-year DFS in patients who obtained a pCR (83.3%) than in those who did not (65.6%; hazard ratio, 0.44; P < .0001). Similarly, TRG has been shown to be an independent prognostic factor for distant metastases and DFS.9-12 The determination of factors predicting tumor regression, and in particular pCR, is of considerable importance because it would permit tailored treatment strategies that include fewer invasive surgical approaches in cases of pCR, upfront surgery (thus avoiding ineffective toxic therapies), or intensified neoadjuvant treatment in patients with a lower likelihood of responding (eg, addition of irinotecan or oxaliplatin and/or biological agents, such as epidermal growth factor receptor [EGFR] and vascular endothelial growth factor [VEGF] inhibitors). Moreover, recent trials that evaluated a ‘wait and watch’ approach in patients with pCR have shown noteworthy long-term results.13,14 In this review we examine the tools available to predict response to NCRT in patients with LARC, with a focus in

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Response Predictive Markers in LARC particular on the role of biomarkers and molecular imaging. For our purposes, a systematic search of PubMed was performed using “rectal cancer,” “neoadjuvant,” “chemoradiotherapy,” “predictive,” “biomarkers,” and “molecular imaging” as keywords. We considered studies that evaluated patients with LARC and were treated with long-course RT combined with chemotherapy (mainly fluoropyrimidine-based, with or without oxaliplatin) followed by surgery. Studies in which neoadjuvant treatment consisted of single-modality therapy or used nonstandard cytotoxic agents were excluded or marginally evaluated. Accepted response evaluation markers were tumor downstaging, TRG, and pCR, and trials that evaluated only long-term outcomes such as OS and DFS were not considered.

Biomarkers DNA Alterations Chromosomal Instability. The amplification and deletion of entire chromosomes or chromosomal segments, defined as chromosomal instability (CIN), are well known features of most colorectal cancers. Genomic copy number changes are often responsible for altered gene transcription, and thus for cancer genetic and phenotype heterogeneity. Nevertheless, the relation between CIN and response to therapy in LARC is poorly understood. One of the first studies to analyze rectal cancer genomic imbalances in relation to response to NCRT used metaphase comparative genomic hybridization. Authors identified an association between 7q11-31, 7q32-36, and 20q11-13 and downstaging, although they concluded that the probability of detecting copy number changes by chance was high.15 In a study conducted by Molinari et al, higher resolution mapping of chromosomal copy number changes was achieved using array comparative genomic hybridization (aCGH). A number of specific chromosomal alterations that distinguish between responsive and nonresponsive tumors (eg, 2q21, 3q29, 7p22-21, 7q21, 7q36, 8q23-24, 10p14-13, 13q12, 13q31-34, 16p13, 17p13-12, and 18q23) were identified.16 The use of high-density whole genome oligonucleotide-based aCGH allowed Chen et al to identify copy number aberrations associated with response to NCRT. In particular, the analysis of 95 rectal cancers included in a prospective phase II study showed that loss of 15q11.1-q26.3 and of 12p13.31 were significantly associated with non-pCR and with pCR, respectively.17 An interesting study was recently published by Zaki et al on the role of CIN as a predictive marker of response to NCRT in LARC; the authors showed that errors in chromosome segregation led to downstream DNA damage and predicted enhanced pCR.18

2

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Mutations and Single-Nucleotide Polymorphisms. The presence of wild type (wt) or mutated tumor suppressor p53 in malignant cells seems to be related to sensitivity and resistance to DNA damage induced by radiation and/or chemotherapy. Despite its recognized role in the outcome of patients with LARC, the value of p53 mutations as a predictive marker remains unclear. However, recent data would seem to reinforce the role played by the wt p53 gene (not its protein expression) in determining complete response (CR) to NCRT. Conversely, its mutation, especially in combination with other gene mutations, is probably associated with resistance,19,20 a finding confirmed by the fact that

Clinical Colorectal Cancer Month 2015

p53-mutated tumor cells tend to accumulate during chemoradiotherapy.21 Because it has been shown that rat sarcoma viral oncogene enhances radioresistance in vitro, numerous groups have analyzed Kirsten RAS (KRAS) mutations in pretherapy rectal biopsies in relation to response to NCRT, and reported discordant results. In some studies, KRAS mutations were not found to be correlated with tumor regression,22,23 but grouping the mutations together on the basis of their respective amino acid exchange predicted different sensitivity to treatment. In contrast, Luna-Perez et al observed that wt KRAS tumors were more likely to be responsive than mutated tumors.24 Although these results should be interpreted with caution because of the many biases, other more recent studies came to similar conclusions, and confirmed an association between KRAS mutations and resistance to NCRT.20,25 In particular, Duldulao et al performed KRAS and p53 genotyping in 148 patients, and showed that tumors with any KRAS mutation were less likely to have a pCR than those with wt KRAS (P ¼ .006). Moreover, a concurrent p53 mutation was often identified in patients with codon 13 KRAS mutation, whereas mutations in other KRAS codons were associated with a lower frequency of p53 mutation.20 Specific haplotypes and genetic polymorphisms are associated with clinical phenotypes, toxicity, and different drug response. Thymidylate synthase (TS) has frequently been genotyped in an attempt to find a correlation between polymorphisms and response to NCRT in LARC. Although the number of tandem repeats within the TS enhancer, a region involved in the regulation of transcription and TS expression, has been shown to affect response, discordant results have been obtained.26-28 However, patients with the G>C substitution at the 12th nucleotide in the second repeat of 3R showed significantly greater downstaging, with only a trend toward a correlation with TRG.28 Of note, the role of TS genotyping in patient stratification was also validated in a prospective, single-institution phase II study with encouraging results. Patients characterized by specific TS single nucleotide polymorphisms (SNPs) associated with a probable response to treatment were given standard NCRT, and those with TS SNPs indicative of no response received intensified chemotherapy, and the latter achieved the same downstaging rate (also OS and DFS) as the former. In particular, a high rate of no residual pathological tumor size (ypT0) after neoadjuvant therapy was reported among the patients treated with the intensified schedule.29 Polymorphisms in the EGFR gene have also been investigated but not confirmed as possible regulators of gene expression and as predictive markers of response. Rather than focus on specific genes, Cecchin et al performed genotyping for 25 polymorphisms in 16 genes known to be involved in treatment-associated pathways. They identified 2 significant SNPs that discriminate between good and poor responders, 1 in human 8-oxoguanine DNA glycosylase, a gene affecting DNA repair, and another in methylenetetrahydrofolato reductase, a gene involved in the pharmacological action of fluoropyrimidines. A third SNP in adenosine triphosphate (ATP)-binding cassette B1, responsible for multidrug resistance, was found to have borderline significance.30 Interesting data were recently reported on the role of some DNA repair gene variants (amphiregulin and excision repair

Chiara Molinari et al cross-complementation group 1) in determining pCR in preoperatively treated LARC.31 Finally, Kim et al performed a genome-wide study and analyzed thousands of SNPs, and identified novel polymorphisms associated with response to NCRT in LARC.32

Gene Expression In addition to the large body of studies conducted on single or small numbers of markers, gene expression signature has also been evaluated to address this specific clinical problem in LARC. One of the earliest studies using cDNA microarray was performed on pretherapy biopsies from 30 homogeneously treated LARC patients. The set of 54 genes differentially expressed in responders and nonresponders (based on the downstaging system) identified in the training set (n ¼ 23) was confirmed in a validation set (n ¼ 7) using an alternative platform. This signature correctly predicted tumor behavior in 83% of patients (P ¼ .02).33 Subsequent studies also detected panels of differentially expressed genes for a therapeutic stratification of LARC with more than 80% predictive accuracy.34-37 However, each study included a relatively limited number of patients and also identified a different classifier, with a slight overlap between the gene lists38 even though common molecular pathways and cellular processes were identified, including DNA damage repair, microtubule organization, apoptosis, transcription, signal transduction, drug metabolism, and transport functions. Another agreement between signatures concerned genes related to matrix metalloproteinases, tumor necrosis factor/nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 molecular pathway, cell growth, and proliferation. For example, in a recent report by Palma et al on microarray analysis of LARC, patients who responded to treatment had tumors with v-myc avian myelocytomatosis viral oncogene homolog mRNA (but not protein) overexpression and increased proliferative capacity, known to be generally associated with higher sensitivity to chemotherapy.36 Finally, Watanabe et al analyzed 46 training samples using DNA array, and identified 24 differentially expressed probes among responders and nonresponders. They also validated their data using quantitative realtime polymerase chain reaction (qRT-PCR) in the same and independent sets, revealing a 4-gene signature (leucine-rich repeats and IQ motif containing 3, FERM domain containing 3, sterile alpha motif domain containing 5, and transmembrane channel-like 7) that accurately predicted response to NCRT. However, it must be underlined that the treatment schedule differed slightly from the standard one because of the use of tegafur-uracil and leucovorin as chemotherapeutic agents.39 In 2011, Brettingham-Moore et al reported that microarray predictors are not robust enough for clinical utility in rectal cancer. In fact, the authors not only obtained limited sensitivity and specificity in their cohort, but also failed to confirm the power of previously published predictive classifiers, possibly as a result of the different biological characteristics of the sample sets or of transcription changes that were too small to be detected.40 However, as highlighted by Watanabe et al, the use of qRT-PCR to construct a prediction model based on expression evaluation of a small number of significant genes selected in microarray studies is more feasible in clinical practice and allows for accurate and reproducible quantification of mRNA obtained

from even small amounts of fresh-frozen or paraffin-embedded tissue.39

Protein Expression A great deal of research has been performed to find molecular markers that differentiate between rectal cancer patients who respond or show resistance to combined treatment. Because a complex phenomenon such as chemoradiosensitivity cannot be attributed to the alteration of a single marker, high-throughput analyses offer the possibility of performing in-depth investigations, but generally require a high number of patients. For this reason, and despite frequently nonsignificant or discordant results, numerous studies have been conducted on a single or a small panel of biomarkers, mainly using immunohistochemistry (IHC), with the advantage of requiring only a small number of patients. Proliferation- and apoptosis-related proteins seem to be one of the most frequent categories analyzed (Ki-67, p53, p21, survivin, B-cell CLL/ lymphoma 2, BCL2-associated X protein) followed by evaluation of cytochrome c oxidase subunit II, EGFR, cancer stem cell markers (CD133, CD24, CD44) and tumor hypoxia and angiogenesis markers (hypoxia inducible factor 1, VEGF, stromal cell-derived factor 1 alpha, phosphatidylinositol glycan anchor biosynthesis, class F). TS, an important enzyme for proliferation and DNA synthesis, has also been extensively studied because it is the primary target of 5-fluorouracil (5-FU). A comprehensive summary of these findings is contained in a number of good reviews that highlight the frequently conflicting and inconclusive results obtained for most of the markers evaluated.41-44 Hur et al recently published a study on the tissue microarray evaluation of a panel of markers that have been shown to be significantly related to response in several studies (p53, p21, B-cell CLL/lymphoma 2, BCL2-associated X protein, EGFR, cytochrome c oxidase subunit II, mutL homolog 1, mutS homolog 2, Ku70, VEGF, TS, Ki-67). A predictive model and a scoring system based on the 4 most significant biomarkers (p53, p21, VEGF, and Ki-67) was subsequently developed, and showed that sufficient predictive accuracy can only be obtained by taking into account the expression level of several biomarkers.45 Increasing interest is also being shown in a number of new markers, including molecules involved in epithelial mesenchymal transition (EMT), in particular, E-cadherin and b-catenin, 2 integral adhesion molecules in epithelial surfaces. The intracellular distribution of the latter is of great importance because it is released from the cellular membrane after downregulation of E-cadherin, which increasing its nuclear concentration and thus its effect on EMT target genes. Drebber et al showed that patients with lower pretherapy levels of membranous b-catenin more often obtained a major histological response.46 Bhangu et al also highlighted a significant role of this protein in predicting response to NCRT, and showed that nucleic b-catenin expression together with reduced E-cadherin were significantly associated with no response in LARC treated with NCRT.47 Wang et al also showed a predictive value of nuclear b-catenin overexpression for radioresistance, and reported 83% accuracy, 65% sensitivity, and 88% specificity.48 Multidrug resistance-associated proteins (MRPs), also known as adenosine triphosphate-binding cassette (ABC) transporters, are another important class of molecules that might be associated with

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Response Predictive Markers in LARC response to irradiation and cytotoxic drugs.49 The significant association observed between sensitivity to neoadjuvant RT and downregulation of ABCC4 (MRP4) indicates a possible role of this protein as a predictive biomarker,50 and the correlation between high ABCC4 expression and shorter DFS makes it a good biomarker of poor outcome in LARC.51 Finally, the negative effect on pathological response of aberrant expression of MRP3, another member of this family, was recently shown by Yu et al in 144 pretreatment rectal biopsies from patients treated with NCRT.52 With regard to protein microarray technologies, Mammano et al published an interesting report in 2012 based on a new method for quantitative multiplexed analysis of protein signaling activation. Although the case series was very small (n ¼ 15) and submitted to heterogeneous chemotherapy, the authors evaluated a wellcontrolled clinical study set of tumor biopsies, and concluded that an activated state of the phosphatidylinositol 3-kinase-v-akt murine thymoma viral oncogene homolog pathway could be used to stratify patients.53 In fact, they showed that aberrant activation of b-catenin pathway members (ie, b-catenin, checkpoint kinase 2, glycogen synthase kinase-3a/b, and pyruvate dehydrogenase kinase, isozyme 1) correlated with response: in particular, the highest phosphorylation level of b-catenin in patients, which probably led to protein degradation, was associated with good response. This is in line with previous findings that linked b-catenin nucleic overexpression to radioresistance.47,48 The importance of tumor-infiltrating lymphocytes (TILs) also needs to be mentioned because it is known that radiosensitivity is greatly affected by the host’s immune function. CD4(þ) and CD8(þ) TIL numbers in pretherapy biopsies detected using IHC were strongly correlated with the tumor reduction ratio54 and Koelzer et al reported that high numbers of CD8i in biopsies predicted earlier pathological tumor size stages (P < .0001).55 Similar conclusions were reached by Anitei et al, who found a correlation between the densities of CD3(þ) and CD8(þ) TILs, evaluated using the immunoscore methodology and tumor downstaging.56

Epigenetic Changes

4

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Methylation Profile. Epigenetic alterations are known to play an important role in determining variability in treatment response. In particular, most studies that were carried out on the effects of DNA methylation on response to radiation have been performed on cell lines,57 with only a handful that focused on patients with LARC treated with NCRT. Jo et al assessed methylation levels of runt-related transcription factor 3, suppressor of cytokine signaling 1, neurogenin 1, insulin-like growth factor 2 and calcium channel, voltage-dependent, T type, alpha 1G subunit genes using methylation-specific polymerase chain reaction (PCR), and showed that cytosine-guanine (CpG) dinucleotides island methylator phenotype was significantly associated with worse DFS, despite a low frequency of high methylation levels. However, no significant correlation was found with response to therapy.58 In 2013 Molinari et al investigated the methylation profile of a panel of 24 tumor suppressor genes using methylationspecific multiplex ligation-dependent probe amplification in rectal cancer to assess their importance in determining resistance or sensitivity to NCRT. However, with the exception of the TIMP

Clinical Colorectal Cancer Month 2015

metallopeptidase inhibitor 3 gene, no significant correlation with TRG was noted.59 MicroRNA. Little is known about the role of microRNA (miR) in radiosensitivity and, despite several publications on in vitro analyses, very few have focused on specimens from patients, especially pretherapy biopsies from patients with LARC who received NCRT.60 However, a major limitation of these studies were the very small case series evaluated. In 2011 Drebber et al described a relation between miR-145 expression levels and pathological tumor regression in 40 patients. In particular, in pretherapeutic tissues, these authors unexpectedly found the highest miR-145 levels in Grade 3 tumor regression, unexpectedly followed by Grades 2, 1, and 4 (P ¼ not significant). However, the association was significant in the analysis when dichotomized response classes were used (minor response, Grades 1 and 2 and major response, Grades 3 and 4). Even in posttherapeutic samples, the low miR-145 expression correlated with regression grade divided into 4 classes (P ¼ .044), the same did not happened in the analysis with dichotomized response.61 Della Vittoria Scarpati et al performed a study on 38 pretherapy LARC biopsies using miR profiling and identified 14 miRs that were differentially expressed in patients who obtained a pCR (TRG 1) with respect to those who did not. In particular, miR-1183, miR-483-5p, miR-622, miR-125a-3p, miR-1224-5p, miR-188-5p, miR-1471, miR-671-5p, miR-1909, miR-630, and miR-765 were significantly upregulated in TRG 1 patients, and miR-1274b and miR-720 were downregulated. Of 14 miRs, 13 were also confirmed using qRT-PCR, of which miR-622 and miR-630, both involved in DNA repair regulation, showed 100% sensitivity and specificity in selecting TRG 1 patients.62 Svoboda et al studied the expression of a panel of numerous miRs in rectal cancer before starting NCRT and after 2 weeks of treatment, and highlighted that therapy-induced expression changes in miR-125b and miR-137 were significantly related to tumor regression.63 However, in their large-scale miR profiling study on untreated biopsies from 20 patients, the same miRs were not significantly associated with response, and overexpression of miR-215, miR-190b, miR-29b-2 in nonresponders and of let-7e, miR-196b, miR-450a, miR-450b-5p, and miR-99a* in responders was found to be significant. Notably, many of these are correlated with radio- or chemoresistance to TS inhibitors. Moreover, using this panel of miRs, the authors correctly classified 90% of responders and 90% of nonresponders.64 There was no overlap between this signature and that identified by Della Vittoria Scarpati et al,62 probably because of the different response class grouping and the somewhat different therapeutic approaches used. Conversely, a partial overlap was observed between the signature described by Svoboda et al64 and that obtained by Bandres et al, who reported that upregulation of miR-21*, miR-99*, miR-125b, miR-125b1*, let-7c, and miR-490 was significantly related to response and that downregulation of miR-21* and miR-125a-3p were associated with lack of response.65 Finally, Kheirelseid et al detected another specific predictive signature for response to NCRT, when they observed that miR-16, miR-590-5p, and miR-153 predicted CR and that miR-519c-3p and miR-561 predicted good response with a median accuracy of 100%. However the very small number of

Chiara Molinari et al patients (n ¼ 2) involved did not permit the authors to draw definitive conclusions.66

Potential New Circulating Biomarkers Circulating cell-free (cf) nucleic acids in the bloodstream have been identified and quantified. Because their levels are associated with tumor burden, malignant progression, and tumor response, they have been proposed as potential minimally invasive and costeffective biomarkers. Zitt et al analyzed the amount of cfDNA in plasma of 26 patients with conventionally treated LARC and observed a decrease in responders and a significant increase in nonresponders (P ¼ .006) at the end of therapy, which suggests that it might be more useful to monitor radiochemotherapy efficacy by analyzing the variation in cfDNA levels during treatment than simply before or after therapy.67 In line with these results, Agostini et al confirmed the importance of the decrease in the cfDNA and cfDNA integrity index (ie, the quantitative ratio between longer and shorter DNA fragments that reflects tumor status) after NCRT in the group of responders.68 Another promising marker for the minimally invasive monitoring of response to therapy is the catalytic protein of telomerase (ie, human telomerase reverse transcriptase [hTERT]), whose mRNA levels in plasma are significantly correlated with levels in colorectal tumor tissue.69 Pucciarelli et al investigated whether the plasma levels of total cfRNA and hTERT mRNA were related to pathological response to NCRT in 259 LARC patients. The authors showed that both markers were present at lower levels in samples taken before and after therapy from responders with respect to nonresponders, and only pre-hTERT was not significant in univariate analysis. Moreover, like the previously mentioned studies, the dynamic variations in these circulating markers during treatment strongly reflect therapy efficacy, with a significant reduction in hTERT levels only in responsive patients.70 Although recent findings highlight the correlation between circulating miR expression levels in some tumors, including colorectal cancer, and response to chemotherapy,71 to our knowledge no studies focused on LARC and NCRT have produced significant results. Blood also contains peptides and protein fragments released from tissues. Smith et al, in their study of 20 patients who underwent NCRT, performed proteomic analysis of serum at different time points using surface enhanced laser desorption ionization -time of flight- mass spectrometry (SELDI-TOF-MS), and identified 14 protein peaks that differentiated between good and poor responders, with 87.5% sensitivity and 80% specificity. These proteins were hypothesized to belong mainly to apoptotic signaling.72 Peripheral blood mononuclear cells (PBMCs) have also recently emerged as potential surrogate predictors of response to therapy in solid organs because of the role of immune response in tumor shrinkage induced by NCRT. Kitayama et al found a significant association between the number of circulating lymphocytes and CR to therapy, which supported the hypothesis that a lymphocyte-mediated immune reaction might be involved in the complete eradication of tumor cells.73 A microarray study of 35 LARC patients who received standard neoadjuvant treatments revealed that only a few genes among several thousand

were significantly differentially expressed in responder and nonresponder PBMCs, which indicated a possible role in antitumor immunity. However, only bromodomain PHD finger transcription factor, which encodes for a transcription factor that regulates apoptosis, was confirmed using qRT-PCR.74

Carcinoembryonic Antigen Carcinoembryonic antigen (CEA) is the most widely used tumor marker for colorectal cancer and the increase of preoperative CEA levels is considered an independent prognostic factor for DFS in colorectal carcinoma. Furthermore, CEA monitoring is indicated for the early detection of recurrence after radical surgery and for predicting response to chemotherapy in metastatic disease.75 The value of CEA as a predictive marker of response to the neoadjuvant treatment of LARC has been explored in several trials that focused mainly on the utility of CEA levels before and after NCRT. In the first retrospective trials, whether low levels of CEA before the start of preoperative NCRT were associated with higher tumor response was investigated. In the trials by Das et al76 and Moreno Garcia et al77 it was reported that patients with baseline CEA levels < 2.5 ng/mL had higher pCR rates than those with levels greater than the limit. Other trials explored the same hypothesis using 5 ng/mL as a cutoff value, and confirmed pretreatment CEA as a valuable predictor of tumor response.78-81 Wallin et al82 investigated a different cutoff value, 3.4 ng/mL, but again demonstrated the predictive value of low baseline CEA in terms of significantly higher pCR rates, especially in the group of nonsmokers (P ¼ .002). In a smaller trial Perez et al83 did not observe any correlation between pretreatment CEA levels and pCR or survival. The role of CEA posttreatment levels as a prognostic factor has also been widely investigated, the hypothesis being that a marker decrease could be associated with tumor response and affect survival. The previously mentioned trial by Perez et al83 showed that CEA levels after NCRT of < 5 ng/mL were associated with higher pCR rates and longer OS and DFS (P ¼ .01 and .03, respectively). Similarly, Jang et al84 showed that low CEA levels after NCRT (cutoff, 2.7 ng/mL) predicted tumor regression (P ¼ .001), ypT0-2 (P ¼ .002), and ypN0 (P ¼ .001). Yang et al85 observed a strong correlation between CEA levels after NCRT (cutoff 2.61 ng/mL) and pCR (P ¼ .011 in multivariate analysis), with a sensitivity of 87.5% and specificity of 76.7%. Taking into account the limitations of these trials because of their retrospective design and the relatively small cohorts of patients analyzed, the amount of data presented is consistent with a significant predictive role of CEA levels before and after NCRT. CEA grouping according to the reduction ratio of CEA levels from before to after NCRT was analyzed as an indicator of prognosis and response to treatment in some retrospective studies. Patients divided into different groups according to CEA modifications showed different prognoses in terms of DFS and OS, with a consistently high correlation across trials,84,86-89 but no differences were seen in terms of tumor response, which thus indicates that CEA modification was useful in predicting prognosis but not response.78,83 The trial by Yang et al85 was the only one whose results suggested a predictive role of CEA reduction, limited to patients with baseline CEA levels  6 ng/mL.

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Response Predictive Markers in LARC Molecular Imaging Positron Emission Tomography/Computed Tomography F-18 fluorodeoxyglucose (FDG)-positron emission tomography (PET)/computed tomography (CT) is used to evaluate increased glucose metabolism in cancer cells and can be used to assess CR to neoadjuvant chemotherapy. Metabolic changes in response to treatment occur before any structurally detectable change (eg, tumor shrinkage). In the neoadjuvant setting, serial FDG-PET/CT examinations might aid treatment planning in decisions on the appropriate length of neoadjuvant chemotherapy to maximize tumor response before surgical resection.90 Over the past few decades, several studies have used FDG-PET/CT to assess response to NCRT in patients with LARC. In a number of studies the efficacy of PET/CT in the early assessment of treatment was evaluated, with a comparison of this technique with histopathological findings and ultrasound imaging. In 1996, Findlay et al91 evaluated PET/CT in the assessment of response in LARC patients treated with 5-FU with or without interferon; a PET/CT scan was performed at baseline, 1 to 2 weeks after starting treatment, and again after 4 to 5 weeks. The results showed that PET/CT performed as early as 4 to 5 weeks after the start of therapy was capable of discriminating responders from nonresponders with 100% sensitivity and 90% specificity, thus improving patient management and eliminating the need for ineffective therapy. In 2000, Guillem et al, from the Memorial Sloan-Kettering Cancer Center, compared the results of FDG-PET/CT with histopathological data in 15 patients, and obtained 100% concordance.92 When pathological response was compared with standard uptake value (SUV), the authors reported perfect agreement in 5 of 15 cases (33%), an overestimation of the SUV in 3 of 15 patients (20%), and an underestimation in 7 of 15 patients (47%). The PET/CT scan was predictive of therapeutic response in 78% of patients. Subsequently, other studies correlated the metabolic response shown with the use of FDG-PET/CT with histopathological data from endorectal ultrasound. In particular, Amthauer et al93 studied a population of 22 consecutive patients with LARC who underwent neoadjuvant radiochemiotherapy and combined regional hyperthermia. Patients performed a FDG-PET/CT scan at baseline and 4 to 6 weeks after the end of treatment, and results showed a significantly greater decrease of maximum SUV

(SUVmax) in responders than in nonresponders evaluated using endorectal ultrasound and histopathology (60%  15% vs. 30%  18%, respectively; P ¼ .003; CI ¼ 95%). Other authors have hypothesized that a complete metabolic response might not correspond to a complete disappearance of histopathological disease. Tan et al94 reported that, despite an absence of metabolic activity detectable above background levels, viable tumor cells were still present in 85% of lesions. In a subset of 7 lesions in which neoadjuvant treatment obtained a complete metabolic response and a complete radiological response (according to Response Evaluation Criteria In Solid Tumors [RECIST]); histological analysis revealed viable tumor cells in 6 of the lesions. A reduction in the number of viable tumor cells to below the limit of detection could be the reason for their failure to be detected on FDG-PET/CT scans after treatment. It has also been hypothesized that an overestimation in the reduction of FDG uptake might be directly related to the effect of chemotherapeutic agents, which are known to alter the glucose metabolism of cancer cells, thereby reducing the uptake of 18F-FDG. Akhurst et al95 suggested that this variation in 18F-FDG uptake after cytotoxic therapy might be induced by a decrease in the glycolytic enzyme hexokinase. Conversely, another study that evaluated response to RT has shown that FDG-PET/CT is not capable of detecting the early therapeutic response of RT because of the high number of false positive results associated with a postactinic inflammatory effect.96 One of the most widely debated questions is the choice of the best parameter to use in semiquantitative analysis to assess response to therapy. In particular, Calvo et al97 analyzed the use of FDG-PET/CT to detect tumor changes induced by preoperative NCRT in 25 consecutive patients. A comparison between mean SUV values in tumors before and after treatment showed a statistically significant difference: 5.9 before versus 2.4 after (P < .001), and SUVmax after chemoradiation (cutoff of 2.5) was 3.3 for responders and 1.9 for nonresponders (P < .03). In addition, in most studies no correlation was found between the DSUV and pCR (Table 1).91,93,97-99 The largest study on the role of FDG-PET/CT in the staging and restaging workup of LARC treated with neoadjuvant NCRT and radical surgery included 81 patients.98 All patients underwent FDG-

Table 1 Prospective Studies of FDG-PET/CT as a Predictor of Outcome in Terms of PFS and OS After NCRT in LARC Patients Patients, n

Type of Study

Median Age, Years

Findlay et al

20

Prospective

59

Amthauer et al93

20

Prospective

53

Calvo et al97

64

Prospective

64

Capirci et al98 Martoni et al99

81 80

Prospective Prospective

58 65

91

6

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Treatment

Parameters

5-FU with interferonalpha 2b 5-FU, 45 Gy and hyperthermia Teg with 5-FU and FOLFOX-4, and 45-50 Gy 5-FU, and 50-56 Gy 5-FU with Oxa and Pan and 50.4 Gy

T:L ratio

100

90

DSUVmax

93

100

SUVmax

100

80

RI SUVmax after RI

Sensitivity, %

84.4 88 94

Specificity, %

80 34 31

Abbreviations: FDG-PET/CT ¼ F-18 fluorodeoxyglucose positron emission tomography-computed tomography; FOLFOX ¼ folinic acid, 5-fluorouracil and oxaliplatin; 5-FU ¼ 5-fluorouracil; LARC ¼ locally advanced rectal cancer; NCRT ¼ neoadjuvant chemoradiotherapy; OS ¼ overall survival; Oxa ¼ oxaliplatin; Pan ¼ panitumumab; PFS ¼ progression-free survival; RI ¼ response index; SUVmax ¼ maximum standard uptake value; Teg ¼ tegafur; T:L ratio ¼ F-18 fluorodeoxyglucose uptake in the tumor and normal liver.

Clinical Colorectal Cancer Month 2015

Chiara Molinari et al PET/CT scans at baseline and 5 to 6 weeks after the end of NCRT. SUV FDG uptake and the percentage of variation in SUV values (response index [RI]) between before and after NCRT were evaluated. The mean SUV and RI before NCRT were significantly greater than those after NCRT in responders compared with nonresponders (respectively, 15.8 vs. 5.9; P < .001; 71.3% vs. 38%; P ¼ .0038). Using an RI cutoff of 65% to define response to therapy, the authors reported 84.5% sensitivity, 80% specificity, a positive predictive value of 81.4% and a negative predictive value of 84.2%, with an overall accuracy of 81%. Martoni et al studied a group of 80 patients, and performed FDG-PET/CT scans at baseline (SUV-1) and after the end of treatment (SUV-2). All 3 parameters considered, SUV-1, SUV-2, and DSUV, showed high sensitivity (100%, 87.5%, and 93.7%, respectively) but very low specificity (10.9%, 34.4%, and 31.2%, respectively). Although the median baseline SUV was greater in nonresponders than in responders, the authors concluded that baseline FDG-PET/CT did not play a role in the standard staging workup as a predictor of either pCR or disease recurrence because of the low specificity. However, the achievement of complete metabolic response after NCRT, and in particular SUV normalization in responders was associated with better DFS.99 Another important element concerning FDG-PET/CT in LARC NCRT is the prediction of patient outcome in terms of progression-free survival (PFS) and OS. de Geus-Oei et al100 studied the predictive value of FDG-PET/CT in relation to OS. FDG-PET/ CT was performed at baseline and 2 (n ¼ 50 patients) and 6 months (n ¼ 19) after the start of treatment. The authors reported a significant increase in the mortality rate in metabolic nonresponders on FDG-PET/CT scans. Using European Organization for Research and Treatment of Cancer (EORTC) and RECIST criteria as morphofunctional parameters of metabolic response, the authors found significant differences in DSUV for PFS (P ¼ .002 and P ¼ .001 per EORTC and RECIST, respectively) and OS (P ¼ .064 and P ¼ .023 per EORTC and RECIST, respectively), although the delta metablic rate of glucose calculated using Patlak graphical analysis was predictive of PFS only when EORTC criteria was considered (P ¼ .036).101 Similar results were also reported by Dimitrakopoulou-Strauss et al102 in 28 patients treated with second-line FOLFOX (folinic acid, 5-fluorouracil, and oxaliplatin). The correlation between the result of the FDG-PET/CT scans after neoadjuvant therapy and the histological finding after surgery would seem to be as good an indicator of response to therapy as the differences between before and after NCRT SUV values, especially the %DSUV mean. The association between FDG-PET/CT findings and outcome in terms of PFS and OS can be considered as a reference element but requires further confirmation before this technique can be used in clinical practice. First, most studies carried out to date considered small case series. Furthermore, correct timing of the PET/CT scan after therapy is needed to avoid false negative results (too early after chemotherapy) and/or false positive results (too early after RT).

Magnetic Resonance Imaging Magnetic resonance imaging (MRI) plays a key role in staging and monitoring response to chemotherapy and RT in patients with colon cancer because of its characteristics of excellent

resolution and contrast definition between the lesion and the surrounding tissues.103,104 However, although the total size of the tumor can be accurately assessed using MRI, volume changes might not be immediately evident during NCRT.105 Hence, the availability of imaging techniques that permit the effects of treatment to be evaluated in the early stage of therapy is of fundamental importance. Dynamic contrast-enhanced MRI (DCE-MRI) is one of the most widely used methods of functional imaging because it provides information on vascular and tissue permeability.106-109 The usefulness of perfusion parameters derived from DCE-MRI to evaluate therapeutic response to neoadjuvant NCRT for LARC was studied by Kim et al,110 who reported that a significant decrease in tumor permeability media (Ktrans) was associated with good therapeutic response in LARC, and none of the other perfusion parameters differed significantly between responders and nonresponders. The authors also observed a decrease, albeit not significant, in the average Ktrans value after NCRT in both groups (pCR and non-pCR). In the study by George et al, patients considered responders because of the presence of a > 50% reduction in tumor volume after NCRT showed a marked decrease in the average Ktrans value at the end of NCRT.111 Thus, a notable reduction in mean Ktrans levels between before and after NCRT was associated with a good therapeutic response to NCRT in LARC. In other studies the use of diffusion weighted MRI (DW-MRI), which provides information on microscopic structures by detecting water proton mobility in tissues and is used in treatment monitoring, has been evaluated. The apparent diffusion coefficient (ADC) in DW-MRI provides a tool for quantitative analysis because it is linked to various factors including cellularity, tissue organization, extracellular space tortuosity, tumor proliferation, tumor grade, and tumor necrosis.112 This parameter has been used as a quantitative biomarker in rectal cancer after the promising results obtained for the monitoring and prediction of therapeutic response.105,113 With regard to the use of ADC before NCRT (pre-ADC) as a predictor of response, Sun et al observed that the mean pre-ADC value in the downstaged group was lower than that of the nondownstaged group (P ¼ .013) and that DADC was significantly higher in the T-downstaged patient group than in patients with no downstaging (P < .001).105 Despite these promising results, there is no general consensus about whether the lowest pre-ADC values can be considered as a predictor of response to NCRT because in one study the pre-ADC value did not accurately discriminate between CR and non-CR.114 Thus, pre-ADC values cannot be considered reliable enough to identify nonresponders who might be candidates for early surgery. With regard to ADC value after NCRT as a predictor of response, the same study showed that it differentiated between patients with and without pCR. In an evaluation of DADC dynamics during and after NCRT, Lambrecht et al115 observed a significantly greater value in patients with pCR than in those without, with 100% sensitivity and specificity for DADC during treatment and 100% and 93% after NCRT, respectively. Magnetic resonance imaging is currently used to assess and predict response to NCRT and although most studies have confirmed its predictive value and high sensitivity and specificity, they were

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Response Predictive Markers in LARC limited by small cases series and a lack of standardization of ADC acquisition. Thus, further confirmation of the usefulness of ADC and Ktrans in needed in larger studies before they can be used as predictive parameters in clinical trials.

Discussion

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The combination of RT and fluoropyrimidine-based chemotherapy is recommended as standard preoperative treatment in patients with LARC. Because of the high variability in response to treatment and the effect of the type of response on subsequent treatment strategy and survival, research has been focused on identifying markers that predict histological regression before surgery. Herein we analyzed the most important predictive models currently proposed. Carcinoembryonic antigen is a widely recognized marker of prognosis and recurrence for colorectal cancer patients, with the advantage of being an inexpensive, reproducible, and rapidly available determination compared with other biomarkers. Results of retrospective trials that evaluated different cutoff values have suggested that low CEA levels at baseline and after NCRT, and CEA reduction, might be useful in predicting pCR and better patient outcome. Although these data require validation in larger prospective trials, CEA is nevertheless routinely used in clinical practice to provide information on treatment efficacy. Molecular imaging is a promising technique for the early identification of responders to NCRT, even though studies that focused on the role of 18F-FDG PET/CT in predicting tumor response have obtained somewhat contradictory results. Several parameters have been considered, among which RI and %DSUV mean could prove to be valuable markers and are worthy of further evaluation. Some limitations are linked to low specificity, poor accuracy in assessing nodal status, and difficulty in distinguishing between residual tumor and physiologic mucosal activity uptake. Diffusion weighted MRI might be the best imaging tool to predict response to NCRT. Several studies have reported its accuracy in predicting tumor regression and, in particular, pCR, with high sensitivity and specificity. Moreover, ADC maps permit differentiation between residual tumor and radiation-induced fibrosis and inflammation. Although a plethora of biomarkers have been proposed as predictors of response to NCRT in LARC, their reliability remains uncertain. However, as shown in Figure 1, some important pathways would seem to be involved in response. Recent studies have hypothesized a central role of the mitogenactivated protein-kinase, phosphatidylinositol 3-kinase, and wingless-type MMTV integration site family-b-catenin pathways in determining response to NCRT, together with the most widely studied proliferative and apoptotic pathways. Moreover, further analyses of circulating and tumor infiltrating lymphocytes might clarify the mechanisms that underlie the responsiveness of LARC to NCRT. The study of these mechanisms could also help to identify biological response modifiers that recruit T cells into the tumor, and thus improve treatment efficacy, and new molecular targets for chemoradiosensitization. Because drug resistance or severe toxicity might be related to genetic variations in tumor cells and/or to the genetic background of each patient, polymorphisms are also promising predictive

Clinical Colorectal Cancer Month 2015

biomarkers but require higher levels of evidence before they can be considered clinically useful. Thus, retrospective studies on large numbers of samples with detailed clinical data, and prospective pharmacogenetic-guided clinical trials, albeit challenging, are needed. Results from the study by Tan et al on the use of TS genotyping for the choice of NCRT indicated the possibility to identify patients who require chemotherapy intensification to achieve downstaging and avoid the severe toxicity associated with this treatment for those predicted as good responders to standard therapy.29 There is growing evidence of the importance of evaluation of predictive markers over time rather than only before therapy. Some studies have focused on repeated biopsies during treatment, but this is, of course, a highly invasive approach. The role of ‘noninvasive’ markers such as PET-CT, MRI, CEA, or circulating biomarkers is important in this context because they provide the opportunity to monitor patients during NCRT and to predict early response to treatment. Finally, the combination of 2 or more of these parameters might offer increased predictive power.116,117 A number of frequently recurring limitations emerge from our analysis of available data. With regard to tumor biomarkers, some technical aspects, especially sample collection and handling, are a cause for concern. Frozen tissue is the material of choice for studies based on genome-wide profiling and gene signature but is not as easy to collect as formalin-fixed paraffin-embedded specimens. However, nucleic acids extracted from the latter are more degraded and are not always of sufficient quality for these analyses. Furthermore, the source and extraction method for circulating biomarkers might also bias results. The lack of reproducibility of assays between laboratories and the high variability in technical approaches represent another obstacle to the identification of clinically useful cancer biomarkers. Approaches based on the evaluation of single or small numbers of molecules might miss important biomarkers. The large-scale characterization of epigenetic changes, genome-wide profiling, or gene signatures could represent a solution to these problems because no previous assumptions have been made on which genes should be studied. However, multigene profile studies have shown that different approaches yield different gene sets that might be predictive of response. Nonhomogeneous results between studies might be due to differences in the resolution and type of spotted genes and to high data dimensionality, even when a limited number of patients are studied. In fact, array data generate interesting results, but their complexity and magnitude sometimes makes results difficult to interpret. For this reason, candidate genes must first be carefully validated in prospective trials before being used in clinical decision-making. The number of patients and treatment schedule might also be important drawbacks. Small sample sizes might be responsible for inadequate power of study conclusions. Moreover, differences in tumor staging and treatment among studies make direct comparisons of results extremely difficult. For example, the variability in chemotherapeutic regimens, radiation schedules and doses, and a variable interval between NCRT and surgery have led to different pCR rates in previously mentioned studies. Another important aspect to take into account is tumor response evaluation, which needs to be more standardized. Tumor, node, metastases downstaging, based on the comparison

Chiara Molinari et al Figure 1 Principal Pathways Involved in the Response to Chemoradiotherapy in Locally Advanced Rectal Cancer Patients

Abbreviations: TIL ¼ tumor-infiltrating lymphocyte; RAS/MAPK ¼ rat sarcoma viral oncogene homolog/mitogen-activated protein kinase; TNF/NF-kB ¼ tumor necrosis factor/nuclear factor of kappa light polypeptide gene enhancer in B-cells 1; WNT/TCF4 ¼ transcription factor 4.

between the preoperative clinical and postoperative pathologic stages, has poor accuracy and does not appear to be a suitable candidate for tumor response assessment.3 Five different TRG systems have been reported, based on the relationship between residual tumor and tumor-free fibrosis areas: Dowrak, Mandard, Dworak/Rodel, American Joint Commission on Cancer (AJCC), and Memorial Sloan Kettering Cancer Center.8,9 Trakarnsanga et al analyzed tumor specimens in 563 patients treated with

standard NCRT followed by total mesorectal excision at a single institution and scored them using all 5 of the TRG systems. A cross-comparison analysis on their association with recurrence and survival using a concordance index showed that all TRG systems were predictive of DFS, and that the AJCC was the most accurate.118 The major criticism of these systems is poor reproducibility among pathologists, and sometimes lymph node metastases are not included in the assessment. For this reason, some authors

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Response Predictive Markers in LARC considered results in extreme TRG classes separately from the often unclear intermediate response. CR according to the TRG system and pCR are more reproducible and standardized response categories, have a significant prognostic value,8-11 and could be considered as standard markers of tumor response for clinical trials performed in this context. In light of the mentioned limitations, larger cohort studies with a prospective and possibly multicentric design, long-term follow-up, homogeneous NCRT regimens, and validated response assessment are needed to confirm findings that have emerged from retrospective studies, and to obtain new results that might influence clinical practice. To our knowledge, the ongoing TransValid-KFO179/ GRCSG-Trials (TransValid A and B) are among the first biomarker-driven clinical trials for patients with LARC,119 together with the study conducted by Tan et al29 on the use of TS SNPs to stratify patients. These studies represent the much needed step toward tailored treatment. There is still insufficient evidence to include any of the proposed predictive models of response to NCRT in clinical practice. However, the integration of biomarkers and molecular imaging, and in particular, the possibility of the creation of combined models, could lead to a more personalized multimodal approach to the treatment of LARC.

Acknowledgments The authors thank Ursula Elbling for editing the manuscript.

Disclosure The authors have stated that they have no conflicts of interest.

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Response Predictive Markers in LARC 95. Akhurst T, Kates TJ, Mazumdar M, et al. Recent chemotherapy reduces the sensitivity of [18F]fluorodeoxyglucose positron emission tomography in the detection of colorectal metastases. J Clin Oncol 2005; 23:8713-6. 96. Konski A, Li T, Sigurdson E, et al. Use of molecular imaging to predict clinical outcome in patients with rectal cancer after preoperative chemotherapy and radiation. Int J Radiat Oncol Biol Phys 2009; 74:55-9. 97. Calvo FA, Domper M, Matute R, et al. 18F-FDG positron emission tomography staging and restaging in rectal cancer treated with preoperative chemoradiation. Int J Radiat Oncol Biol Phys 2004; 58:528-35. 98. Capirci C, Rubello D, Pasini F, et al. The role of dual-time combined 18-fluorodeoxyglucose positron emission tomography and computed tomography in the staging and restaging workup of locally advanced rectal cancer, treated with preoperative chemoradiation therapy and radical surgery. Int J Radiat Oncol Biol Phys 2009; 74:1461-9. 99. Martoni AA, Di Fabio F, Pinto C, et al. Prospective study on the FDG-PET/CT predictive and prognostic values in patients treated with neoadjuvant chemoradiation therapy and radical surgery for locally advanced rectal cancer. Ann Oncol 2011; 22:650-6. 100. de Geus-Oei LF, van Laarhoven HW, Visser EP, et al. Chemotherapy response evaluation with FDG-PET in patients with colorectal cancer. Ann Oncol 2008; 19:348-52. 101. Young H, Baum R, Cremerius U, et al. Measurement of clinical and subclinical tumour response using [18F]-fluorodeoxyglucose and positron emission tomography: review and 1999 EORTC recommendations. European Organization for Research and Treatment of Cancer (EORTC) PET study group. Eur J Cancer 1999; 35:1773-82. 102. Dimitrakopoulou-Strauss A, Strauss LG, Rudi J. PET-FDG as predictor of therapy response in patients with colorectal carcinoma. Q J Nucl Med 2003; 47: 8-13. 103. Beets-Tan RG, Beets GL, Vliegen RF, et al. Accuracy of magnetic resonance imaging in prediction of tumour-free resection margin in rectal cancer surgery. Lancet 2001; 357:497-504. 104. Gollub MJ, Schwartz LH, Akhurst T. Update on colorectal cancer imaging. Radiol Clin North Am 2007; 45:85-118. 105. Sun YS, Zhang XP, Tang L, et al. Locally advanced rectal carcinoma treated with preoperative chemotherapy and radiation therapy: preliminary analysis of diffusion-weighted MR imaging for early detection of tumor histopathologic downstaging. Radiology 2010; 254:170-8. 106. Jackson A, O’Connor JP, Parker GJ, et al. Imaging tumor vascular heterogeneity and angiogenesis using dynamic contrast-enhanced magnetic resonance imaging. Clin Cancer Res 2007; 13:3449-59.

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107. Goh V, Padhani AR, Rasheed S. Functional imaging of colorectal cancer angiogenesis. Lancet Oncol 2007; 8:245-55. 108. Figueiras RG, Goh V, Padhani AR, et al. The role of functional imaging in colorectal cancer. AJR Am J Roentgenol 2010; 195:54-66. 109. Devries AF, Griebel J, Kremser C, et al. Tumor microcirculation evaluated by dynamic magnetic resonance imaging predicts therapy outcome for primary rectal carcinoma. Cancer Res 2001; 61:2513-6. 110. Kim SH, Lee JM, Gupta SN, et al. Dynamic contrast-enhanced MRI to evaluate the therapeutic response to neoadjuvant chemoradiation therapy in locally advanced rectal cancer. J Magn Reson Imaging 2014; 40:730-7. 111. George ML, Dzik-Jurasz AS, Padhani AR, et al. Non-invasive methods of assessing angiogenesis and their value in predicting response to treatment in colorectal cancer. Br J Surg 2001; 88:1628-36. 112. Lambregts DM, Vandecaveye V, Barbaro B, et al. Diffusion-weighted MRI for selection of complete responders after chemoradiation for locally advanced rectal cancer: a multicenter study. Ann Surg Oncol 2011; 18:2224-31. 113. Genovesi D, Filippone A, Ausili Cefaro G, et al. Diffusion-weighted magnetic resonance for prediction of response after neoadjuvant chemoradiation therapy for locally advanced rectal cancer: preliminary results of a monoinstitutional prospective study. Eur J Surg Oncol 2013; 39:1071-8. 114. Kim SH, Lee JY, Lee JM, et al. Apparent diffusion coefficient for evaluating tumour response to neoadjuvant chemoradiation therapy for locally advanced rectal cancer. Eur Radiol 2011; 21:987-95. 115. Lambrecht M, Vandecaveye V, De Keyzer F, et al. Value of diffusion-weighted magnetic resonance imaging for prediction and early assessment of response to neoadjuvant radiochemotherapy in rectal cancer: preliminary results. Int J Radiat Oncol Biol Phys 2012; 82:863-70. 116. Lambrecht M, Deroose C, Roels S, et al. The use of FDG-PET/CT and diffusion-weighted magnetic resonance imaging for response prediction before, during and after preoperative chemoradiotherapy for rectal cancer. Acta Oncol 2010; 49:956-63. 117. Maffione AM, Ferretti A, Grassetto G, et al. Fifteen different 18F-FDG PET/CT qualitative and quantitative parameters investigated as pathological response predictors of LARC treated by neoadjuvant chemoradiation therapy. Eur J Nucl Med Mol Imaging 2013; 40:853-64. 118. Trakarnsanga A, Gonen M, Shia J, et al. Comparison of tumor regression grade systems for locally advanced rectal cancer after multimodality treatment. J Natl Cancer Inst 2014; 106:dju248. 119. Grade M, Wolff HA, Gaedcke J, Ghadimi BM. The molecular basis of chemoradiosensitivity in rectal cancer: implications for personalized therapies. Langenbecks Arch Surg 2012; 397:543-55.