The ability to achieve pathologic downstaging after neoadjuvant chemoradiotherapy (NCRT) is correlated with improved survival in locally advanced rectal cancer (LARC). However, there is no effective predictive markers. In this study, the expression of angiogenic markers was evaluated in pre-treatment biopsies and corresponding post-treatment resection specimens, and were correlated to histopathological tumour characteristics and response. Fifty-five patients with stage II/III rectal cancer treated with 5-fluorouracil (5-FU)-based NCRT were studied. All patients were administered NCRT followed by surgical resection. Immunohistochemical staining for angiogenic markers [hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), stromal cell-derived factor 1α (SDF-1α) and placental growth factor (PlGF)] was performed on specimens obtained before NCRT and after surgery. Expression of VEGF, PlGF and HIF-1α protein was downregulated after NCRT in the rectal cancer tissues (P<0.001, P=0.001 and P=0.044, respectively). However, SDF-1α was upregulated after NCRT (P<0.001). Moreover, upregulated expression of SDF-1α (P=0.016) and positive PlGF staining (P=0.001) after NCRT were significantly associated with resistance to NCRT. On multivariate analysis, positive PlGF staining after NCRT was found to be independently associated with resistance to NCRT (P=0.013). Our data suggest that SDF-1α and PlGF should be evaluated as new targets for NCRT in LARC.
Rectal cancer (RC) is a major health issue and is one of the leading causes of cancer-related death worldwide (1). Neoadjuvant chemoradiotherapy (NCRT) followed by surgical resection is the current standard treatment for locally advanced rectal cancer (LARC). It offers improved local control, reduced toxicity and higher rates of sphincter preservation without compromising oncological outcome compared with post-operative treatment (2,3). A pathologic complete response (pCR) is one of the best predictive markers of a favourable prognosis. However, approximately 15–30% of patients experience a pCR, whereas the majority of patients have some degree of residual disease after NCRT (4). Thus, if patients with tumours that are responsive to NCRT could be identified at the time of diagnosis, then NCRT could be administered in a more individualised manner.
Recent studies have attempted to identify predictive biomarkers such as Ki-67, p53, p21, p27, bax, BCL-2, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR), survivin and thymidylate synthase (5,6). However, clinical use of these biomarkers requires further evaluation in prospective clinical trials (7).
Angiogenesis is necessary for tumour growth and malignant progression, with VEGF being a key angiogenic factor. High VEGF expression was found to be associated with poor survival in colorectal cancers (8). In particular, bevacizumab, a humanised monoclonal antibody inhibiting VEGF-A, in combination with standard chemotherapy regimens was beneficial both in terms of response rate and survival as first-and second-line treatment of patients affected by metastatic colorectal cancer. In patients affected by LARC who underwent radical surgery and adjuvant chemoradiation, tumour VEGF overexpression was found to be associated with a statistically higher risk of local recurrence and metastasis (9). Not only VEGF, but placental growth factor (PlGF) are potentially useful predictive factors in rectal cancer (10).
Hypoxia is one of the key stimuli for the release of angiogenic markers (AMs) necessary for angiogenesis and tumour growth. Hypoxic tumours not only have a more aggressive nature (11,12), but studies in cervical and head and neck cancer demonstrated that tumour hypoxia decreases the response to radiation treatment (13). The effect of tumour hypoxia on the response to radiation therapy is relevant to the management of rectal cancer.
Hypoxia-inducible factor 1α (HIF-1α) is a protein involved in the cellular response to hypoxia centrally. It activates a variety of downstream genes involved in anaerobic metabolism and angiogenesis (14–16). These downstream gene protein products, which include VEGF, stromal cell-derived factor 1α (SDF-1α) and PlGF, promote cell survival under hypoxic conditions. Among patients with colorectal cancer, expression of these 4 AMs has been shown to correlate with rates of lymph node or liver metastasis, disease-free survival, and overall survival (17–20). However, the effects of pre-operative treatment on expression of AMs in rectal cancer remain unclear. The 4 AMs chosen for this study include HIF-1α, VEGF, SDF-1α and PlGF.
The aims of this exploratory study were to a) characterise expression of AMs in LARC before NCRT, b) investigate the change in AM expression after NCRT and c) evaluate the relationship between AM expression and tumour response.
Patients and methodsPatients and neoadjuvant chemoradiotherapy regimen
Between March 2005 and August 2009 at Soonchunhyang University Hospital, a total of 55 patients with non-metastatic, locally advanced (radiological T3–T4 or N+ and/or clinically bulky) and biopsy-proven primary rectal cancer received NCRT. The whole pelvic field received 25 fractions of 180 cGy/day over 5 weeks, for a total of 4,500–5,040 cGy, using a four-field box technique. Chemotherapy was administered intravenously and consisted of 5-fluorouracil (5-FU; 425 mg/m2/day) and leucovorin (20 mg/m2/day) during the first and fifth weeks of radiotherapy. Surgical resection at 6–8 weeks was performed following the completion of NCRT. All data were collected and recorded prospectively, and the clinical and pathological features were reviewed retrospectively. The patients were classified according to the 6th edition of the American Joint Committee on Cancer staging system (21). Surgical specimens were evaluated for histopathologic staging as well as for pathologic response to NCRT. The detailed characteristics of the patients are listed in Table I. Our study was approved by the Clinical Ethics Review Committee of the Soonchunhyang University Hospital, Cheonan, Republic of Korea. Clinical consent was obtained from all patients.
Tissue microarray (TMA) construction
Areas representative of cancer were marked on haematoxylin and eosin-stained slides and TMAs were constructed. TMAs were created from formalin-fixed by 10% neutral buffered formalin, paraffin-embedded tissues using a 2-mm-diameter punch (Unitma; Unitech Science, Seoul, Korea). TMA blocks were assembled by obtaining duplicate cores from one patient block and re-embedding the two cores in an arrayed recipient block (Unitma; Unitech Science). A TMA block contains 60 cores from 30 samples.
Tumour response
Clinical stage was performed by an independent review conducted by a radiologist, and pathologic stage was reviewed by two independent pathologists. Downstaging was defined as staging reduction from pre-treatment stage (cStage) to pathologic stage (ypStage) (i.e. cIII to ypII, ypI or yp0; cII to ypI or yp0). Pathologic response (tumour regression) to NCRT was semiquantitatively determined by the amount of viable tumour versus the amount of fibrosis, ranging from no evidence of any NCRT effect to a complete response with no viable tumour identified, as described by Dworak et al (22). The following were characteristics of each grade: grade 0, no regression; grade 1, minor regression (dominant tumour mass with obvious fibrosis in 25% or less of the tumour mass); grade 2, moderate regression (dominant tumour mass with obvious fibrosis in 26–50% of the tumour mass); grade 3, good regression (dominant fibrosis outgrowing the tumour mass; i.e. >50% tumour regression); and grade 4, total regression (no viable tumour cells, only fibrotic mass). Patients with tumour regression grade (TRG) of 3 or 4 were considered as the responder group in our study.
Immunohistochemical (IHC) staining
The TMAs were sectioned at 4-μm intervals, deparaffinised three times in xylene for 30 min and rehydrated with graded alcohols (100% ethyl alcohol for 5 min, 95% ethyl alcohol for 3 min and 75% ethyl alcohol for 3 min) and then heated in antigen retrieval solution (sodium citrate, pH 6.0) in a microwave for 20 min. Sections were incubated in H2O2 for 10 min at room temperature. Furthermore, the sections were incubated with 150 ml of the primary antibodies, VEGF (1:200; Millipore, USA), PlGF (1:200; R&D system, USA), HIF-1α (1:50; Proteintech, USA) and SDF-1α (1:100; Novus Biologicals, USA) at 4°C overnight. Subsequently, the sections were washed in PBS buffer three times for 3 min, treated with 150 ml secondary antibody for 1 h at room temperature and stained with DAB solution (Dako, USA). The sections were then washed in PBS buffer for 10 min. Finally, the sections were counterstained with hematoxylin for 3 min at room temperature, washed in distilled water 3 times for 3 min and mounted with coverslips.
IHC analysis
The VEGF, PlGF, HIF-1α and SDF-1α stained tissue cores were examined by 2 independent pathologists and a consensus score was determined for each specimen. A positive reaction for both antibodies was scored into 4 grades, according to the intensity of the staining: 0, 1+, 2+ and 3+. The percentages of positive cells were also scored into 4 categories: 0 (0%), 1 (1–33%), 2 (34–66%), and 3 (67–100%). The final score, calculated as the product of the intensity score multiplied by the percentage score, was classified as follows: 0 for negative; 1–3 for weak; 4–6 for moderate; and 7–9 for strong.
Statistical analysis
The correlations between expression levels of hypoxia-related proteins and pathologic response to NCRT were evaluated by the χ2 or Fisher’s exact test. The univariate and multivariate analyses between response to NCRT and clinical or histopathologic parameters were performed by binary logistic regression model. All P-values quoted were two-sided, and P<0.05 was considered to indicate a statistically significant difference. All the analyses were performed using SPSS v. 17.0 (SPSS, Inc., Chicago, IL, USA).
ResultsAssociation between AM expression and clinicopathological variables
The mean age of the 55 patients with LARC was 56 years (range, 18–82 years). In regards to gender, 43 (78.2%) were male, and 12 (21.8%) were female. Regarding the stage of disease, 11 (20.0%) were at stage II, and 44 (80.0%) were at stage III. Concerning the T stage, 44 (80.0%) were T3 and 11 (20.0%) were T4. The number of negative lymph node metastases was 11 (20.0%); N1 was 22 (40.0%), and N2 was 22 (40.0%). A pCR was obtained in 9.1% cases (5 patients). Patient characteristics are summarised in Table I. As shown in Table I, expression levels of AMs were not statistically correlated to the clincopathological variables.
Change in AM expression in LARC before NCRT and after surgery
The positive expression rate of HIF-1α, VEGF, PlGF and SDF-1α was 47.3% (26/55), 56.4% (31/55), 65.5% (36/55) and 70.9% (39/55) before NCRT, respectively. Weak, moderate and strong staining intensity of AMs is illustrated in Fig. 1. The expression rate of HIF-1α, VEGF, SDF-1α and PlGF was increased by 1.8% (1/55), 3.6% (2/55), 30.9% (17/55) and 7.3% (4/55) after NCRT, respectively. Expression of VEGF, PlGF and HIF-1α protein was downregulated after NCRT in the rectal cancer tissues (P<0.001, P=0.001 and P=0.044, respectively). However, SDF-1α was upregulated after NCRT (P<0.001; Table II, Fig. 2).
Relationship between tumour response to NCRT and clinicopathological variables
Upregulated expression of SDF-1α (P<0.016) and positive PlGF staining (P=0.001) after NCRT were significantly associated with resistance to NCRT. However, other clinicopathologic variables showed no correlation with tumour response (Table III). In multivariate analyses, positive PlGF staining after NCRT was found to be associated with resistance to NCRT [P=0.013; OR=0.197, 95% confidence interval (CI), 0.055–0.705]. Only low pre-treatment tumour lymph node staging was associated with pCR (P=0.002; Table IV).
Relationship with AM expression
Before NCRT, an association was identified between HIF-1α expression and SDF-1α (P=0.034). HIF-1α was not correlated with VEGF and PlGF. However, SDF-1α had an association with PlGF (P=0.005). After surgery, HIF-1α expression was not correlated with SDF-1α (P=0.621), and SDF-1α tended to be associated with PlGF (P=0.052).
Discussion
Recently, studies have attempted to identify predictive biomarkers, yet various studies only compared pre-treatment and post-treatment changes in biomarker expression (5). In this study, we investigated the predictive relevance of AM expression both in pre-treatment biopsies and in corresponding surgical specimens of 55 patients with LARC treated with standadised 5-FU-based NCRT. Comparing pre-treatment biopsies and surgical specimens, we observed a downregulation of VEGF, PlGF and HIF-1α. However, SDF-1α was upregulated after NCRT. In addition, upregulated SDF-1α after NCRT was significantly associated with resistance to NCRT. Our findings suggest that SDF-1α is one of the important targets for resistance to NCRT and this finding is significant.
SDF-1α, also known as chemokine ligand 12 (CXCL12), and its receptor CXCR4, play important roles in the onset and progression of primary or metastatic cancer from various organs (23–26). In colorectal cancer (CRC), elevated SDF-1α expression is associated with metastasis and poor prognosis (27,28). In our investigation, upregulation of SDF-1α in surgical specimens was related to resistance to NCRT. Thus, SDF-1α appears to be a predictive marker to chemoradiation treatment. In an in vitro study using a CRC cell line, the results indicate that CXCR4 antagonistic therapy might prevent tumour cell dissemination and metastasis in CRC patients, consequently improving survival (29). Therefore, the targeting of SDF-1α represents an attractive adjuvant treatment to eradicate cancer cells and induce anti-angiogenic effects in highly hypoxic tumours. Further study evaluating the distinctive value of SDF-1α expression in LARC patients receiving NCRT is warranted. However, we did not observe a relationship between expression of AMs before NCRT and tumour reponse. Therefore, it is not possible to choose the ‘right’ patients who may require additional therapeutics (such as anti-angiogenesis), except NCRT, by analhysis of the specimen before treatment. These findings are difficult for clinical application.
We also found that positive expression of PlGF after NCRT was correlated with resistance to NCRT in multivariate analyses. PlGF is a cytokine in the VEGF family of growth factors, with 53% homology to VEGF (30). It primarily regulates the angiogenic switch under pathologic states (31). PlGF recruits smooth muscle precursors that envelop developing vessels in tumours and together with VEGF produces more stable and mature vessels. PlGF may also facilitate metastasis by increasing the motility and invasion of malignant cells (32). Tumour overexpression of PlGF and VEGF together is associated with increased tumour angiogenesis and cancer growth (33,34). However, in general, there was no correlation between elevated VEGF expression and survival (35,36). Our results suggest that PlGF, than VEGF, is also an important target for resistance to NCRT. It would be worthwhile to determine whether or not PlGF is a predictive biomarker for patients with LARC receiving NCRT.
As shown in this study, an association was identified between HIF-1α and SDF-1α (P=0.034). HIF-1α was not correlated with VEGF and PlGF. However, SDF-1α had an association with PlGF (P=0.005). Although HIF-1α expression is known to drive expression of downstream proteins, differences in individual protein half-lives may not allow for a direct relationship between HIF-1α and other proteins (37). Downstream proteins may have been influenced by other signaling pathways independent of HIF-1α, making their expression levels somewhat variable in relation to HIF-1α. The limited sample size and the heterogeneity of intratumoural oxygenation may also be responsible for these findings.
In summary, SDF-1α and PlGF are relevant for resistance to NCRT. By comparison of pre-therapeutic and post-therapeutic intratumoural SDF-1α and PlGF, our results suggest that therapeutic strategies to downregulate expression of SDF-1α and PlGF during pre-operative treatment or to inhibit SDF-1α/PlGF mediated signaling pathways may further increase the individual tumour response and, as a consequence, improve patient prognosis. Based on our results, patients with increased expression of SDF-1α or positive expression of PlGF after NCRT might benefit from additional anti-SDF-1α/PlGF therapeutics.
Acknowledgements
The authors would like to thank Kim Hyung-Joo and Park So-Young for providing excellent technical assistance.
ReferencesSiegelRNaishadhamDJemalACancer statistics, 2012CA Cancer J Clin6210292012SauerRBeckerHHohenbergerWRodelCWittekindCFietkauRMartusPTschmelitschJHagerEHessCFKarstensJHLierschTSchmidbergerHRaabRGerman Rectal Cancer Study GroupPreoperative versus postoperative chemoradiotherapy for rectal cancerN Engl J Med351173117402004RohMSColangeloLHO’ConnellMJYothersGDeutschMAllegraCJKahlenbergMSBaez-DiazLUrsinyCSPetrelliNJWolmarkNPreoperative multimodality therapy improves disease-free survival in patients with carcinoma of the rectum: NSABP R-03J Clin Oncol27512451302009MaasMNelemansPJValentiniVDasPRödelCKuoLJCalvoFAGarcía-AguilarJGlynne-JonesRHaustermansKMohiuddinMPucciarelliSSmallWJrSuárezJTheodoropoulosGBiondoSBeets-TanRGBeetsGLLong-term outcome in patients with a pathological complete response after chemoradiation for rectal cancer: a pooled analysis of individual patient dataLancet Oncol118358442010SprengerTRödelFBeissbarthTConradiLCRotheHHomayounfarKWolffHAGhadimiBMYildirimMBeckerHRödelCLierschTFailure of downregulation of survivin following neoadjuvant radiochemotherapy in rectal cancer is associated with distant metastases and shortened survivalClin Cancer Res17162316312011EddenYWexnerSDBerhoMThe use of molecular markers as a method to predict the response to neoadjuvant therapy for advanced stage rectal adenocarcinomaColorectal Dis145555612012TrakarnsangaAIthimakinSWeiserMRTreatment of locally advanced rectal cancer: controversies and questionsWorld J Gastroenterol18552155322012ChinKFGreenmanJGardinerEKumarHToppingKMonsonJPre-operative serum vascular endothelial growth factor can select patients for adjuvant treatment after curative resection in colorectal cancerBr J Cancer83142514312000CascinuSGrazianoFCatalanoVStaccioliMPRossiMCBaldelliAMBarniSBrennaASecondinoSMurettoPCatalanoGAn analysis of p53, BAX and vascular endothelial growth factor expression in node-positive rectal cancer. Relationships with tumour recurrence and event-free survival of patients treated with adjuvant chemoradiationBr J Cancer867447492002WillettCGDudaDGdi TomasoEBoucherYAncukiewiczMSahaniDVLahdenrantaJChungDCFischmanAJLauwersGYShellitoPCzitoBGWongTZPaulsonEPoleskiMVujaskovicZBentleyRChenHXClarkJWJainRKEfficacy, safety, and biomarkers of neoadjuvant bevacizumab, radiation therapy, and fluorouracil in rectal cancer: a multidisciplinary phase II studyJ Clin Oncol27302030262009BrizelDMScullySPHarrelsonJMLayfieldLJBeanJMProsnitzLRDewhirstMWTumor oxygenation predicts for the likelihood of distant metastases in human soft tissue sarcomaCancer Res569419431996NordsmarkMAlsnerJKellerJNielsenOSJensenOMHorsmanMROvergaardJHypoxia in human soft tissue sarcomas: adverse impact on survival and no association with p53 mutationsBr J Cancer84107010752001KoukourakisMIBentzenSMGiatromanolakiAWilsonGDDaleyFMSaundersMIDischeSSivridisEHarrisALEndogenous markers of two separate hypoxia response pathways (hypoxia inducible factor 2 alpha and carbonic anhydrase 9) are associated with radiotherapy failure in head and neck cancer patients recruited in the CHART randomized trialJ Clin Oncol247277352006SowterHMRatcliffePJWatsonPGreenbergAHHarrisALHIF-1-dependent regulation of hypoxic induction of the cell death factors BNIP3 and NIX in human tumorsCancer Res61666966732001LalAPetersHSt CroixBHaroonZADewhirstMWStrausbergRLKaandersJHvan der KogelAJRigginsGJTranscriptional response to hypoxia in human tumorsJ Natl Cancer Inst93133713432001ZhongHDe MarzoAMLaughnerELimMHiltonDAZagzagDBuechlerPIsaacsWBSemenzaGLSimonsJWOverexpression of hypoxia-inducible factor 1 alpha in common human cancers and their metastasesCancer Res59583058351999IshigamiSIAriiSFurutaniMNiwanoMHaradaTMizumotoMMoriAOnoderaHImamuraMPredictive value of vascular endothelial growth factor (VEGF) in metastasis and prognosis of human colorectal cancerBr J Cancer78137913841998RajaganeshanRPrasadRGuillouPJPostonGScottNJayneDGThe role of hypoxia in recurrence following resection of Dukes’ B colorectal cancerInt J Colorectal Dis23104910552008WeiSCLiangJTTsaoPNHsiehFJYuSCWongJMPreoperative serum placenta growth factor level is a prognostic biomarker in colorectal cancerDis Colon Rectum52163016362009SaigusaSToiyamaYTanakaKYokoeTOkugawaYFujikawaHMatsusitaKKawamuraMInoueYMikiCKusunokiMCancer-associated fibroblasts correlate with poor prognosis in rectal cancer after chemoradiotherapyInt J Oncol386556632011GreeneFLPageDLFlemingIDFritzAGBalchCMHallerDGAJCC Cancer Staging Handbook - TNM Classification of Malignant Tumors6th editionSpringer-VerlagNew York2002DworakOKeilholzLHoffmannAPathological features of rectal cancer after preoperative radiochemotherapyInt J Colorectal Dis1219231997MüllerAHomeyBSotoHGeNCatronDBuchananMEMcClanahanTMurphyEYuanWWagnerSNBarreraJLMoharAVerasteguiEZlotnikAInvolvement of chemokine receptors in breast cancer metastasisNature41050562001OrimoAGuptaPBSgroiDCArenzana-SeisdedosFDelaunayTNaeemRCareyVJRichardsonALWeinbergRAStromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretionCell1213353482005SchraderAJLechnerOTemplinMDittmarKEMachtensSMengelMProbst-KepperMFranzkeAWollensakTGatzlaffPAtzpodienJBuerJLauberJCXCR4/CXCL12 expression and signalling in kidney cancerBr J Cancer86125012562002TaichmanRSCooperCKellerETPientaKJTaichmanNSMcCauleyLKUse of the stromal cell-derived factor-1/CXCR4 pathway in prostate cancer metastasis to boneCancer Res62183218372002MatsusueRKuboHHisamoriSOkoshiKTakagiHHidaKNakanoKItamiAKawadaKNagayamaSSakaiYHepatic stellate cells promote liver metastasis of colon cancer cells by the action of SDF-1/CXCR4 axisAnn Surg Oncol16264526532009YoshitakeNFukuiHYamagishiHSekikawaAFujiiSTomitaSIchikawaKImuraJHiraishiHFujimoriTExpression of SDF-1 alpha and nuclear CXCR4 predicts lymph node metastasis in colorectal cancerBr J Cancer98168216892008HeckmannDLaufsSMaierPZucknickMGiordanoFAVeldwijkMREcksteinVWenzFZellerWJFruehaufSAllgayerHA Lentiviral CXCR4 overexpression and knockdown model in colorectal cancer cell lines reveals plerixafor-dependent suppression of SDF-1α-induced migration and invasionOnkologie345025082011WeiSCTsaoPNYuSCShunCTTsai-WuJJWuCHSuYNHsiehFJWongJMPlacenta growth factor expression is correlated with survival of patients with colorectal cancerGut546666722005CarmelietPMoonsLLuttunAVincentiVCompernolleVDe MolMWuYBonoFDevyLBeckHScholzDAckerTDiPalmaTDewerchinMNoelAStalmansIBarraABlacherSVandenDriesscheTPontenAErikssonUPlateKHFoidartJMSchaperWCharnock-JonesDSHicklinDJHerbertJMCollenDPersicoMGSynergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditionsNat Med75755832001FischerCJonckxBMazzoneMZacchignaSLogesSPattariniLChorianopoulosELiesenborghsLKochMDe MolMAutieroMWynsSPlaisanceSMoonsLvan RooijenNGiaccaMStassenJMDewerchinMCollenDCarmelietPAnti-PlGF inhibits growth of VEGF(R)-inhibitor-resistant tumors without affecting healthy vesselsCell1314634752007LandriscinaMCassanoARattoCLongoRIppolitiMPalazzottiBCrucittiFBaroneCQuantitative analysis of basic fibroblast growth factor and vascular endothelial growth factor in human colorectal cancerBr J Cancer787657701998AdiniAKornagaTFiroozbakhtFBenjaminLEPlacental growth factor is a survival factor for tumor endothelial cells and macrophagesCancer Res62274927522002LeeJCChowNHWangSTHuangSMPrognostic value of vascular endothelial growth factor expression in colorectal cancer patientsEur J Cancer367487532000KhoranaAARyanCKCoxCEberlySSahasrabudheDMVascular endothelial growth factor, CD68, and epidermal growth factor receptor expression and survival in patients with stage II and stage III colon carcinoma: a role for the host response in prognosisCancer979609682003Lee-KongSARubyJAChessinDBPucciarelliSShiaJRiedelERNittiDGuillemJGHypoxia-related proteins in patients with rectal cancer undergoing neoadjuvant combined modality therapyDis Colon Rectum559909952012
Immunoreactivity in the immunostained rectal carcinoma samples. (A) VEGF was weakly expressed in the cytoplasm. (B) HIF-1α was moderately expressed in the cytoplasm and nuclei. (C) PlGF was weakly expressed in the cytoplasm. (D) SDF-1α was strongly expressed in the cytoplasm and cytoplasmic membrane (magnification, ×400). Scale bar, 50 μm.
Change in staining status after neoadjuvant chemoradiation (NCRT). Expression of VEGF, PIGF and HIF-1α protein was downregulated after NCRT (P<0.001, P=0.001 and P=0.044, respectively). However, SDF-1α was upregulated after NCRT (P<0.001).
Association between AMs and clinicopathological parameters.