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Digestive and Liver Disease 42 (2010) 679–684

Contents lists available at ScienceDirect

Digestive and Liver Disease

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limentary Tract

issue microarray constructs to predict a response to chemoradiationn rectal cancer

ergio Huertaa,∗, John Hromb, Xiaohuan Gaoa, Debabrata Sahac, Thomas Anthonya,enry Reinharta, Payal Kapurd

Department of Surgery, University of Texas Southwestern Medical Center, United StatesDepartment of Medical Oncology, University of Texas Southwestern Medical Center, United StatesDepartment of Radiation Oncology, University of Texas Southwestern Medical Center, United StatesDepartment of Pathology, University of Texas Southwestern Medical Center, United States

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rticle history:eceived 24 November 2009ccepted 2 February 2010vailable online 15 March 2010

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Purpose: To identify, using tissue microarray (TMA), an immunohistochemical panel predictive ofresponse to ionizing radiation (IR) in rectal cancer.Methods: TMA constructs were prepared from archived stage II/III rectal tumors and matching adjacentmucosa (n = 38) from patients treated with pre-operative chemoradiation. Immunohistochemistry (IHC)was performed for MIB, Cyclin E, p21, p27, p53, survivin, Bcl-2, and BAX. Immunoreactivity along withclinical variables was subjected to univariate and forward stepwise logistic regression analyses.Results: Pathological complete response (pCR) was 23.9%. The number of positive lymph nodes obtained in

IBumber of lymph nodesathological complete response53

the resected specimen was associated with pCR. Immunoreactivity for MIB (Sn 15%, Sp 65%, OR 0.33), p53(Sn 3%, Sp 84%, OR 0.16), Bcl-2 (Sn 11%, Sp 74%, OR 0.35), and BAX (Sn 92%, Sp 80%, OR 46) was associatedwith pathological response (all p’s < 0.001). Forward stepwise logistic regression analysis demonstratedthat MIB was an independent predictor of a response to chemoradiation (p = 0.001).Conclusions: A combined panel of mediators of apoptosis alone or combined with clinical factors is a

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. Introduction

Pre-operative chemoradiotherapy for the management of stageI/III rectal cancer results in a wide spectrum in clinical response.urrent data demonstrate a reduction in local recurrence [1]; how-ver, survival advantage is uncertain [2–5]. Additionally, the abilityf neoadjuvant therapy to reduce tumor size, a major objectivef this pre-operative treatment, is extraordinarily unpredictable.ine to 37% of patients demonstrate a pathological complete

esponse (pCR) with the various chemoradiotherapeutic regimensurrently available (i.e. 5-FU, ironotecan, oxaliplatin, bevacizumab,nd cetuximab in combination with IR) [6]. On the other hand, up to% of these patients may not respond at all. Appropriate selection

f patients in either side of the spectrum has not yet been possi-le either clinically or at the molecular level. The identification ofatients at risk of being radioresistant would benefit clinicians andatients alike in determining individuals that would respond to this

∗ Corresponding author at: Dallas VA Medical Center, Department of Surgery112), 4500 S. Lancaster Road, Dallas, TX 75216, United States.el.: +1 214 857 1800; fax: +1 214 648 6700.

E-mail address: Sergio.Huerta@UTSouthwestern.edu (S. Huerta).

590-8658/$36.00 Published by Elsevier Ltd on behalf of Editrice Gastroenterologica Italioi:10.1016/j.dld.2010.02.003

applied to rectal tumor biopsies to predict a response to chemoradiation.BAX; while MIB independently predicted a response to chemoradiation.shed by Elsevier Ltd on behalf of Editrice Gastroenterologica Italiana S.r.l.

neoadjuvant modality. In the patients predicted to have radiosensi-tive tumors a selective and individualized form of chemoradiationcould be instituted. In patients predicted to have radioresistanttumors, it is not justifiable to subject them to the adverse sideeffects of chemoradiation and alternative options could be under-taken such as surgical intervention without chemoradiation orinclusion of patients in clinical trials. Identification of a panel ofspecific molecular phenotype could be the cornerstone of chemora-diotherapeutic interventions. However, to date, no markers areavailable to predict tumor response to radiotherapy.

Our current understanding of the mechanisms that lead to resis-tance to chemoradiation are limited. Various markers including p53[7], p21 [8], p27 [9], NF�B [10], survivin [11], Bcl-2 [12] and medi-ators of apoptosis [13], have been investigated either individuallyor in combination, with variable results as predictors of responseto chemoradiation [reviewed in [6]]. The major limitation of thesestudies has been the use of individual markers by immunohisto-chemistry (IHC) in a heterogeneous patient population.

In the present study, we have developed Tissue Microarray(TMA) constructs of tumor and corresponding normal mucosa fromrectal cancer specimens of 36 patients who received pre-operativechemoradiation. IHC was performed with eight different antibod-ies. The aims of this study were to correlate the expression of these

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arkers either individually or in combination with clinical andncologic outcome and to develop an immunohistochemical panelhat could be used as a predictor of clinical response to chemora-iation.

. Methods

.1. Patients

All studies were undertaken with the approval and institutionalversight of the Institutional Review Board (IRB) for the Protec-ion of Human Subjects at the Dallas VA Medical (DVAMC) Center,he University of Texas Southwestern Medical Center, and Parkland

emorial Hospital (PMH). Clinical data were collected retrospec-ively from 59 patents from the DVAMC and 58 from PMH. Onlyatients with stage II and stage III rectal cancer who were sub-

ected to neoadjuvant chemoradiation were included in this study.er protocol, all our patients received 50.4 Gy ionizing radiationiven in combination with oral capecitibine 825 mg/m2 two timesaily, Monday through Friday, over 6 weeks.

Pathological response was defined by comparing the pre-reatment tumor size (as determined by EUS, CT, flexibleigmoidoscopy, and/or colonoscopy) with post-resection tumorize (as assessed by the pathological report and review of all slidesrom each specimen).

.2. Tumor tissues

In order to provide uniformity of pre-operative treatment andissue handling, for this study, samples (n = 38) were collected fromatients treated at a single institution (PMH) between April 2000nd November 2008.

.3. Tissue microarray (TMA)

All Hematoxylin and eosin (H&E) stained section from eachpecimen were reviewed by a staff pathologist (PK) to select rep-esentative areas of the tumor from which to acquire cores foricroarray analysis. Two samples, for each patient, one from the

umor and one from normal colonic epithelium not adjacent to theancer, were identified and circled on the H&E stained slides. Tissueicroarrays were built using a semiautomatic arraying instrument

Beecher Instruments, Silver Spring, MD) that uses two separateore needles for punching the donor and recipient blocks and a

ig. 1. There is a high variability of a response to ionizing radiation in rectal cancer. Each besponse to pre-operative ionizing radiation (the size of the tumor at diagnosis was comp

Disease 42 (2010) 679–684

micrometer-precise coordinate system for assembling tissue sam-ples on a block. For each case, single 0.6 mm (diameter 0.6 mm,height 3–4 mm) core diameter samples were obtained from thecircled areas of tumor and/or normal colonic mucosa from each“donor” block and placed on separate a “recipient” TMA block(45 mm × 20 mm, 0.7 mm center). All samples were spaced 0.5 mmapart as previously described [14].

2.4. Immunohistochemistry

We performed immunohistochemical staining for p21 (mon-oclonal mouse, SX118, Dako; dilution 1:200), p53 (monoclonalmouse, DO-7, Dako; dilution 1:2200), p27 (monoclonal mouse,SX53G8, Dako; dilution 1:150), Bcl-2 (monoclonal mouse, 124,Dako; dilution 1:600), survivin (polyclonal rabbit, 8E 2, BioCare;dilution 1:300), Ki-67 (monoclonal mouse, MIB-1, Dako; dilu-tion 1:300), Cyclin E (monoclonal mouse, 870P110, Dako; dilution1:400) and BAX (monoclonal rabbit, A3533, Dako; dilution 1:200)using known positive control tissues. Appropriate positive and neg-ative controls were used. All immunostaining was performed as wehave previously described [14].

2.5. TUNEL

Terminal deoxynucleotidyltransferase-mediated UTP end label-ing (TUNEL) staining for apoptotic cells was done on paraffin-embedded TMA sections, according to the protocol supplied withPromega Apoptosis and as published before [15,16].

2.6. Statistical analysis

All data is expressed as means ±SE. PRISM statistical analysissoftware (GraphPad Software Inc., San Diego, CA) was used for con-tingency table analysis and Student’s T-test. SigmaPlot for windowsversion 11.0 (Systat Software Inc., San Jose, CA) was employed formultivariate analysis.

2.7. Univariate analysis

Univariate analysis was performed in 117 patients by dividingthe patient population into those who achieved a pCR (n = 28) andall others (n = 89). Categorical data was analyzed by Fisher’s ExactTest or Chi Square. Continuous data was subjected to Student’s T-test. For analysis of the TMA constructs immunoreactivity in the

ar on the X-axis represents an individual patient. The Y-axis represents the clinicalared to the pathological report size).

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Table 1Patient demographics of allpatients with stage II/III rectalcancers treated with neoadjuvantchemoradiation.

Demographicsn = 117CpR = 23.9% (n = 28)Age = 59.8 ± 1.1-year-oldPreop tumor size = 5.9 ± 0.3 cmPostop tumor size = 2.7 ± 0.2 cmWhite = 52%

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wo groups: good responders (>50% pathological response; n = 19)s. poor responders (<50% pathological response; n = 19) was alsonalyzed by contingency tables.

.8. Multivariate analysis model

Multivariate analysis was performed first in all clinical vari-bles in 117 patients with pCR as the dependent variable and all ofhe clinical factors as independent variables by forward multistepogistic regression analysis.

A second model was constructed between good responders andoor responders for analysis of immunoreactivity with pathologi-al response as the dependent variable. Independent variables werell factors with a p-value of ≤0.2 (i.e. MIB from tumor, p21 fromumor, Bcl-2 from tumor, p53 from tumor, BAX from tumor, as wells clinical variables including number of positive nodes and tumortage). This analysis was also performed by forward multistep logis-ic regression analysis.

. Results

A review of 117 patients who received pre-operative ionizingadiation at the DVAMC and PMH that had complete informationegarding pre-operative assessment of tumor size revealed a wideange of pathological response. Over 23% of patients achieved aathological complete response (n = 28). On the other side of thepectrum, 20.5% (n = 24) either had no response or the tumor con-inued to grow up to 200% of the original size. The rest of theatients had a pathological response in between (44.4%, n = 44;ig. 1). The demographics for this patient population are depictedn Table 1.

.1. Analysis of clinical variables

Univariate analysis with pCR as the dependent variable andndependent variables including: pre-operative CEA, race, gender,umber of positive nodes, size of the tumor (T), and overall stage

ig. 2. Tissue microarray constructs. The area of the tumor was examined by a pathologistn a paraffin-embedded block, where each dot represents a different patient. Thin section

Hispanic = 22%Male = 75%Stage II = 60.7% (n = 71)

demonstrated that the only clinical parameter predictive of pCRwas the number of positive nodes obtained from the resectedspecimen [0.07 ± 0.05 vs. 1.0 ± 0.24; p = 0.049 (T-test)]. While notstatistically significant, stage demonstrated a trend towards statis-tic significance in patients with pCR [stage III (25.0%) vs. StageII (43.2%), p = 0.07 (Chi square)]. None of these clinical variablesdemonstrated to be an independent predictor of pCR.

We then proceeded to analyze tissue collected from 46 subjectswho had complete information on the pre-operative size of thetumor as well as sufficient tissue from the tumor and from adjacentmucosa. Eight patients were eliminated from this analysis becausethey had achieved a pCR and there was no tumor available for theTMA. We performed TMA constructs as depicted in Fig. 2.

3.2. TMA analysis of normal mucosa

TMA analysis of normal mucosa demonstrated no apoptosis as

determined by TUNEL in either good or poor responders. Table 2shows the results of the antibodies from the IHC of the TMA derivedfrom normal mucosa. None of the antibodies investigated in thisTMA demonstrated a significant association with tumor responseby univariate analysis. Because the p-value of all of these molecules

(PK) and a pouch biopsy performed as described in Section 2. Samples were placeds were then cut and placed onto slides for IHC analysis.

682 S. Huerta et al. / Digestive and Liver Disease 42 (2010) 679–684

Table 2Results univariate analysis from the tissue microarray in tissue derived from normal mucosa adjacent to tumor tissue. MIB (i.e. MIB-1 = anti-Ki-67 antibody), Cyc E (i.e. CyclinE), Sn = sensitivity, Sp = specificity, PPV = positive predictive value, NPV = negative predictive value, OR = odds ratio.

TMA normal mucosa

MIB Cyc E p21 p27 p53 Survivin Bcl-2 BAX

Good responders 14.7 ± 8.1% 10.8 ± 1.4% 12.8 ± 2.4% 8.3 ± 1.1% 0.0 0.0 32.0 ± 14.3% 52.5 ± 6.0%Poor responders 15.6 ± 3.8% 13.2 ± 3.1% 19.0 ± 4.9% 6.9 ± 1.5% 0.0 0.0 23.5 ± 5.3% 59.3 ± 7.9%

p 1.000 0.828 0.215 1.000 1.000 1.000 .027 0.476Sn 15% 11% 13% 8% – – 32% 53%Sp 84% 79% 81% 93% – – 76% 41%PPV 48% 45% 40% 53% – – 57% 47%NPV 50% 49% 48% 50% – – 52% 47%OR 0.94 0.83 0.64 1.15 – – 1.49 0.78

Table 3Results univariate analysis from the tissue microarray in tissue derived from tumor tissue. MIB (i.e. MIB-1 = anti-Ki-67 antibody), Cyc E (i.e. Cyclin E), Sn = sensitivity,Sp = specificity, PPV = positive predictive value, NPV = negative predictive value, OR = odds ratio.

TMA tumor tissue

MIB Cyc E p21 p27 p53 Survivin Bcl-2 BAX

Good responders 14.8 ± 6.0% 18.2 ± 3.4% 7.5 ± 5.3% 21.4 ± 5.6% 2.9 ± 2.0% 41.6 ± 10.5% 10.9 ± 6.3% 91.6 ± 4.2%Poor responders 35.3 ± 8.1% 18.9 ± 4.3% 14.6 ± 3.4% 18.7 ± 4.3% 16.4 ± 3.8% 44.1 ± 10.1% 25.8 ± 5.9% 20.1 ± 4.6%

p <0.001 1.000 0.180 0.860 <0.001 .890 <0.001 <0.001Sn 15% 18% 8% 21% 3% 42% 11% 92%Sp 65% 81% 85% 81% 84% 56% 74% 80%

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as >0.2, they were not included in the multivariate analysis. Sensi-ivity (Sn), specificity (Sp), positive predictive value (PPV), negativeredictive value (NPV), and odds ratio (OR) was included in thisable for completion and to allow comparison of these values withhe TMA constructs from the tumor tissue.

.3. TMA analysis of tumor tissue

TUNEL analysis of the TMA constructs from tissue derived fromhe tumor demonstrated a rate of apoptosis 65.2 ± 2.0 in the goodesponse group compared to 60.5 ± 3.0 in the poor response groupp = 0.470). The results of univariate analysis for each individual

olecule with the respective Sn, Sp, PPV, NPV and the OR areepicted in Table 3. Univariate analysis demonstrated a positiveelationship between a poor response to IR and immunoreactivityith antibodies specific for BAX (4.6-fold). An inverse relationshipas found with antibodies specific for MIB (2.4-fold), p53 (5.7-fold)

nd Bcl-2 (2.4-fold). The highest sensitivity (92%) was for BAX withn OR of 46.

Forward stepwise logistic regression analysis with response toR as the dependent variable and immunoreactivity for MIB fromumor, p21 from tumor, Bcl-2 from tumor, p53 from tumor, BAXrom tumor, as well as clinical variables including: number of pos-tive nodes and tumor stage as the independent variables waserformed demonstrated that MIB-Tumor (p = 0.001) was the onlyignificant factor.

. Discussion

Factors that predict resistance to pre-operative chemoradia-ion remain at large. In the present study, we developed a TMA

odel with molecules classically associated with a good response

o chemoradiation as assessed by a review of the literature in inde-endent studies by IHC (for a review of these studies see Ref. [6]).he aim of this analysis was to determine whether we could uselinical variables along with TMA constructs derived directly fromectal tumors and normal adjacent mucosa to predict a response

16% 49% 30% 82%46% 49% 45% 90%

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to neoadjuvant treatment in patients receiving the same form ofneoadjuvant modality for the management of stage II and III rectalcancer. In contrast to other studies, our analysis included sev-eral molecules (8 different antibodies and TUNEL) in the sameTMA model along with clinical variables found to be associatedwith a pCR. Additionally, we investigated mucosa adjacent to thetumor and subjected this to statistical analysis within the samemodel.

A previously published study has undertaken a similar approachby TMA in patients with rectal cancer treated with chemoradi-ation to evaluate recurrence and survival [17]. In their analysis,Debucquoy et al. demonstrated that Cox-2 emerged as a predic-tor of survival. Their study also found that EGFR, proliferation andapoptosis was affected by chemoradiation [17]. Our approach dif-fered from this analysis in that our major goal was to predict tumorslikely to respond to chemoradiation. This is important because ouranalysis showed that up to 21% of patients will not respond to thisform of neoadjuvant treatment (Fig. 1).

Of the molecules most commonly associated with a response toIR, p53 and the cyclin kinase dependent inhibitors (CDKI’s) suchas p27 and p21 are commonly studied in rectal cancer. In ouranalysis, we combined p53 along with CDKIs in the same TMAfrom both normal mucosa and tissue derived from the tumor.Analysis from normal mucosa revealed no immunoreactivity forp53 and there was no difference with regards to the CDKIs inthis TMA. The CDKI’s were not differentially expressed in goodresponders compared to poor responders in tissue derived fromthe tumor either. Our results demonstrated that p53 was differ-entially expressed between patients who achieved at least 50% ofresponse to chemoradiation in the neoadjuvant setting in samplesderived directly from the tumor.

In ex vivo studies, p53 positivity by IHC signifies the mutated

status of the protein because conformational changes of the p53protein resulting from mutations lead to protein stability anda longer half-life, which allows for increased detection by theantibodies [18,19]. Previous studies have shown that nuclearexpression of p53 in rectal cancers predicted treatment failure

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nd expression of nuclear p53 protein by (IHC) correlated withesistance to pre-operative chemoradiation [20].

Multiple studies have indicated p53 to be pivotal in radiation-nduced apoptosis in colorectal cancer in vitro and in vivo [6],ut these finding have been in disagreement with other analy-es [21,22]. Furthermore, p53 mutations may render cells a moreadiosensitive phenotype [23]. For instance, mutant p53 indicatedesistance to apoptosis in rectal cancers compared to wild-typeectal tissues by IHC analysis [24].

Additionally, the mutational status of p53 has been shown toe an important factor to determine the functional status of thisrotein in terms of a response to the cytotoxic effects of chemora-iation. While most mutations are localized to exons 5–8 of the53 gene, it is the mutation of codon 288 in exon 8 that seems toffect rectal cancers and lead to a worse prognosis [25]. Thus, thesepecific mutations (but not all p53 mutations) may lead to a moreesistant phenotype in patients with rectal cancer.

Our results are in agreement with the studies that show an asso-iation between p53 mutated form (i.e. positive immunoreactivityy IHC) and resistance to chemoradiation in tissue derived directlyrom the tumor. However, stepwise logistic regression analysisailed to identify p53 as an independent predictor of a responseo chemoradiation.

We also studied the mitochondrial mediators of apoptosis: BAXnd Bcl-2 as well as the inhibitor of apoptosis (IAP): survivin.he relative ratio of pro-apoptotic BAX and anti-apoptotic Bcl-2etermine release of cytochrome c from the intermitochondrialembrane, potentially augmenting the apoptotic response [26].

tudies assessing the role of Bcl-2 and BAX in resistance to ionizingadiation remain unclear [6].

Our analysis demonstrated that tissue derived from the tumorshat had a good response to chemoradiation was associated withncreased levels of BAX and a concomitant decrease in the levels ofcl-2. The BAX to Bcl-2 ratio was 10.7-fold greater in the tumors thatchieved a good response to chemoradiation compared to thosehat did not. Theoretically, this should have been associated with anncreased rate of apoptosis in the good responder group. Apoptosiss a mechanism by which IR exerts its therapeutic response. Thus,efects in the apoptotic machinery render resistance to radiationherapy in rectal cancer [6]. For instance, Rodel’s group has shownhat spontaneous apoptosis in pre-treatment biopsies was a goodredictor of pathological response [27]. Similarly, a recent studyssessed the role of both intrinsic and radiation-induced apopto-is as markers for prognosis in rectal cancer. This analysis included198 tumor samples from the Dutch Total Mesorectal Excision trial.poptosis was assessed by TUNEL in TMA blocks. The rate of recur-ence in patients who received irradiation was 5% compared to 10%n patients who did not. Non-irradiated patients with high apopto-is had a decrease in local recurrence by 1.7-fold [28]. Because weid not study tumor tissue prior to chemoradiation, our analysisid investigate spontaneous apoptosis. We did not observe a sig-ificant difference in radiation-induced apoptosis in tissue derived

rom the tumors. This might be a reflection of the small sample sizef our cohort of patients.

Survivin is one of eight members of the IAPs, which are underhe regulation of NF�B and directly inhibit caspases 9, 3, and 726]. Survivin has been shown to act as a constitutive radioresis-ant factor in colorectal cancer cells [6]. Short interfering RNA ofhe survivin gene increased apoptosis in reduced survival in col-rectal cancer cells [6]. Survivin has been demonstrated to cause,itotic arrest, cell cycle redistribution, and increase DNA double

ond breaks [6]. Additionally, the 5-year survival of patients withurvivin positive stage II colon cancer tumors was 41% lower thanatients with survivin negative tumors by IHC studies [6]. In spitef this strong evidence regarding survivin, our TMA analysis did notnd a relationship between survivin and a response to IR.

Disease 42 (2010) 679–684 683

Rapidly proliferating cells typically respond more substantiallyto cell damage induced by ionizing radiation. Assessment of cellproliferation is possible with the Ki-67 equivalent MIB antibody,which identifies cycling cells in formalin fixed tissue. In pre-irradiated tumor biopsies from rectal cancer patients, tumor size,proliferating cell nuclear antigen (PCNA)/mitotic activity and Ki-67predicted pCR to chemoradiation [29]. Ki-67 has also been shown tobe reduced in tumors compared to biopsies of rectal specimens fol-lowing chemoradiation, which is consistent with the observationthat rapidly proliferating cells are more sensitive to chemoradiation[17]. Our analysis investigated MIB only in post-irradiated tumorsand found that poor responders to IR had retained a high prolifer-ating index. Furthermore, MIB was an independent predictor of aresponse to chemoradiation.

An obvious limitation of our study is the small sample size.However, the goal of a panel predictive of a response to IR shouldprovide information for individual patients to be clinically usefulsuch that a small number of patients is desirable in determiningstatistical significance in this setting. A second limitation is thatwe investigated irradiated archived tissue as tumor tissue wasmore easily available for TMA constructs. To overcome this lim-itation, we also included adjacent normal mucosa, which shouldhave received less pre-operative IR. However, tissue derived fromadjacent mucosa failed to identify any tumor markers predictiveof a response to IR. Pre-irradiated biopsies are required to over-come these limitations. A third limitation of our study is thatwe arbitrarily divided tumors that responded to ionizing radia-tion into those that had ≥50% response the those that had ≤50%response. However, a 30% response might still be clinically sig-nificant depending on the tumor size. This limitation could beovercome by TMA constructs in pre-irradiated biopsies that are ableto achieve a pCR compared to non-responders utilizing the samemolecular markers. The present report supports the feasibility forsuch analysis.

In summary, our analysis indicated that p53, Bcl-2, BAX, andMIB were associated with a good response to ionizing radia-tion. MIB emerged as an independent predictor of a responseto IR with a NPV of 43% and an OR of 0.33. While multi-ple studies have been undertaken to identify the clinical utilityof any of these factors independently by IHC, our analysisundertook eight classically involved variables to predict a goodresponse to IR by TMA. This approach allowed for a morestandardized treatment for all tissue samples. Our results pro-vide evidence for the feasibility of TMA constructs with severalmolecules to evaluate pre-irradiated tumor biopsies to predict aresponse to IR. Clinically, this information might be applicableto develop TMAs in tumor biopsies in patients prior to irradi-ation to select subjects that might respond to chemoradiationcompared to patients who are unlikely to benefit from this pre-operative chemotherapeutic intervention. Further, understandingpathways of chemoresistance will enhance the development ofnovel radiosensitizing modalities for the management of patientswho do not respond to conventional chemotherapeutic interven-tions.

Conflict of interest statementNone declared.

List of abbreviations

TMA, tissue microarray; IHC, immunohistochemistry; IR,ionizing radiation; NPV, negative predictive value; PPV, pos-itive predictive value; pCR, pathological complete response.

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cknowledgements

This work was made possible by funds from the Department ofurgery at the University of Texas Southwestern (Shannon Funds)nd a Veterans Affairs VISN17 New Investigator Award.

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