Research ArticleExpression of CAF-Related Proteins Is Associated withHistologic Grade of Breast Phyllodes Tumor

Hye Min Kim Yu Kyung Lee and Ja Seung Koo

Department of Pathology Yonsei University College of Medicine Seoul Republic of Korea

Correspondence should be addressed to Ja Seung Koo kjs1976yuhsac

Received 18 September 2016 Accepted 10 October 2016

Academic Editor Gad Rennert

Copyright copy 2016 Hye Min Kim et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Purpose The purpose of this study was to investigate the expression of cancer-associated fibroblast- (CAF-) related proteins andthe implications in breast phyllodes tumor (PT)Methods Tissue microarrays of 194 PT cases (151 benign PT 27 borderline PT and16 malignant PT) were constructed We performed immunohistochemical staining for CAF-related proteins (podoplanin prolyl4-hydroxylase FAP120572 S100A4 PDGFR 120572120573 and NG2) and analyzed the results according to clinicopathologic parameters ResultsExpression of PDGFR120572 and PDGFR120573 in the stromal component increased with increasing histologic grade of PT (119901 = 0003 and119901 = 0034 resp) Among clinicopathologic parameters only expression of FAP120572 in stroma was associated with distant metastasis(119901 = 0002) In univariate analysis stromal expression of PDGFR120572 was associated with shorter overall survival (119901 = 0002) In Coxmultivariate analysis stromal overgrowth and PDGFR120572 stromal positivity were associated with shorter overall survival (119901 = 0006and 119901 = 0050 resp) Furthermore expression of PDGFR120573 in stroma was associated with shorter overall survival in patientswith malignant PT (119901 = 0041) Conclusion Stromal expression of PDGFR120572 and PDGFR120573 increased with increasing histologicgrade of PT In addition PDGFR stromal positivity was associated with shorter overall survival These results suggest that CAFsare associated with breast PT progression

1 Introduction

Progress in cancer research has increasingly revealed theclinical significance of the tumormicroenvironment Amongvarious components of the tumormicroenvironment cancer-associated fibroblasts (CAFs) one of the most impor-tant elements have been widely studied [1] CAFs arelocated near the cancer cells and have been reported to beinvolved in tumor initiation tumor-stimulatory inflamma-tion metabolism metastasis drug response and immunesurveillance [2] Despite their significant effect on cancercells the exact cell origin of CAFs is not completely under-stood and there is even controversy concerning the definitionof CAF [1 2] Various proteins have been suggested asmarkers for CAFs including 120572-smooth muscle actin (SMA)[3] tenascin-C [4] chondroitin sulfate proteoglycan (NG2)[5] platelet-derived growth factor receptor (PDGFR)120572120573 [6]fibroblast activation protein (FAP) [7] podoplanin [8] prolyl4-hydroxylase [9] and fibroblast-specific protein- (FSP-) 1[5] CAFs have been suggested to show various functional

subtypes that exhibit different characteristics [10] supportingthe hypothesis that CAFs have various phenotypes

Phyllodes tumor (PT) is a relatively rare biphasic breasttumor that accounts for only 03ndash15 of all breast tumors[11] It is composed of atypical spindle cell stroma andusually benign epithelium [12] Pathologically differentialdiagnosis is difficult because several histologic findings ofPT overlap with those of fibroadenoma which is also afibroepithelial tumor and PT shows heterogeneous histologicfeatures within the tumor [11 13] In addition some casesof PT show several clinical features of malignancy such asrelapse or distant metastasis [14]The histologic classificationof PT varies according to authors but PT has been classifiedas benign borderline and malignant according to the WorldHealthOrganization classification of tumors of the breast [11]A higher histologic grade of PT is associated with increasedrisk of tumor recurrence or distant metastasis indicationsof tumor aggressiveness Furthermore among malignant PT9ndash36 of cases experience local recurrence 9ndash40 of casesprogress with metastasis to the lungs brain or liver [15]

Hindawi Publishing CorporationDisease MarkersVolume 2016 Article ID 4218989 10 pageshttpdxdoiorg10115520164218989

2 Disease Markers

Table 1 Source clone and dilution of antibodies used for immunohistochemical staining

Antibody Company Clone DilutionCAF phenotype-related proteins

Podoplanin Abcam Cambridge UK 18H5 1 100Prolyl 4-hydroxylase Abcam Cambridge UK Polyclonal 1 200FAP120572 Abcam Cambridge UK Polyclonal 1 100S100A4 Abcam Cambridge UK Polyclonal 1 100PDGFR120572 Abcam Cambridge UK Polyclonal 1 100PDGFR120573 Abcam Cambridge UK Y92 1 100NG2 Abcam Cambridge UK NG2 1 50

and some cases suffer death due to recurrence and distantmetastasis [16 17] The fibroblast is an important componentof PT [18 19] therefore variations in tumor biology ofPT could be accounted for by the phenotype of associatedfibroblasts However the role of CAF-related proteins inbreast PT is poorly understoodThe purpose of this study wasto investigate the expression of CAF-related proteins and itsimplications in breast PT

2 Materials and Methods

21 Patient Selection Tissue samples from patients who werediagnosed with breast PT from 2000 to 2010 in Severancehospital were selected All tissues were fixed in 10 bufferedformalin and embedded in paraffin Archival hematoxylinand eosin- (HampE-) stained slides for each case were reviewedby a single pathologist (Ja Seung Koo) The PT histologicgrade was based on the World Health Organization classi-fication of tumors of the breast [11] The histologic gradewas evaluated with HampE-stained slides and clinical dataincluding patient age recurrence distant metastasis andpatient survival were obtained by reviewing the patientsrsquomedical records The study was approved by the InstitutionalReview Board of Yonsei University Severance Hospital

22 Tissue Microarray Construction of tissue microarraywas performed as previously described Briefly a representa-tive area was selected on the HampE-stained slide of the tumorand a corresponding spot was marked on the surface of theparaffin block Using a biopsy needle the selected area waspunched out and the 5 mm tissue core was placed in a 5 times6 recipient block To minimize extraction bias we extractedtwo tissue cores for every PT case Finally each separate tissuecore was assigned a unique tissue microarray location num-ber linked to the database including other clinicopathologicdata and was used for immunohistochemical staining

23 Immunohistochemistry The antibodies used for immun-ohistochemical staining in this study are shown in Table 1 Allimmunostaining was performed using the constructed tissuemicroarray For immunohistochemistry 5 120583m sections wereobtained with a microtome transferred onto adhesive slidesand dried at 62∘C for 30min After incubation with primary

antibodies immunodetection was performed with biotiny-lated antimouse immunoglobulin followed by peroxidase-labeled streptavidin using a labeled streptavidin biotinkit with 331015840-diaminobenzidine chromogen as substrateFor the negative control the primary antibody incubationstep was omitted Harris hematoxylin was used for tissuecounterstaining All immunohistochemical markers wereassessed using light microscopy and evaluation of stainingwas performed by calculating the proportion of stained cellsand immunostaining intensity The proportion of stainedcells was defined as follows 0 negative 1 less than 30positive and 2 more than 30 positiveThe immunostainingintensity was defined as follows 0 negative 1 weak 2 mod-erate and 3 strongThe scores for proportion of stained cellsand immunostaining intensity were multiplied and stainingwas defined as positive when the final score was gt1 [20]

24 Statistical Analysis Data were analyzed using SPSS forWindows Version 210 (SPSS Inc Chicago IL USA) Con-tinuous variableswere compared using two-tailed Studentsrsquo 119905-test and categorical data were compared using the Chi squaretest To evaluate the time to tumor recurrence and comparethe survivals between groups Kaplan-Meier survival curvesand the log-rank test were used Multivariate survival analy-ses using a Coxrsquos proportional hazard model were performedto characterize the prognostic factors in PT A two-tailed 119901value lt 005 was considered statistically significant

3 Results

31 Basal Characteristics of Phyllodes Tumors This studyincluded 194 cases of breast PT including 151 cases of benignPT 27 cases of borderline PT and 16 cases of malignant PTThe basal characteristics of the patients are shown in Table 2Greater patient age and larger tumor sizewere associatedwithhigher PT histologic grade (119901 = 0017 and 119901 = 0001 resp)Higher rate of tumor recurrence and distant metastasis werealso associated with higher PT histologic grade (119901 lt 0001)Eight cases with distant metastasis showed lung metastasis

32 Expression of CAF-Related Proteins according to PhyllodesTumor Histologic Grade There were no significant differ-ences in immunohistochemical staining in the epithelialcomponent according to PT histologic grade for any of the

Disease Markers 3

Table 2 Clinicopathologic characteristics of patients with phyllodes tumor

Parameters Total 119899 = 196 (100) PT benign 119899 = 153(100)

PT borderline 119899 = 27(100)

PT malignant 119899 = 16(100) 119901 value

Age (years mean plusmn SD) 401 plusmn 123 389 plusmn 122 423 plusmn 115 476 plusmn 129 0017Tumor size (cm mean plusmnSD) 40 plusmn 26 36 plusmn 21 43 plusmn 25 67 plusmn 46 lt0001

Stromal cellularity lt0001Mild 121 (617) 120 (784) 1 (37) 0 (00)Moderate 63 (321) 33 (216) 23 (852) 7 (438)Marked 12 (61) 0 (00) 3 (111) 9 (562)

Stromal atypia lt0001Mild 156 (796) 151 (987) 5 (185) 0 (00)Moderate 30 (153) 2 (13) 20 (741) 8 (500)Marked 10 (51) 0 (00) 2 (74) 8 (500)

Stromal mitosis lt00010ndash410 HPFs 154 (786) 153 (1000) 1 (37) 0 (00)5ndash910 HPFs 31 (158) 0 (00) 26 (963) 5 (312)ge1010 HPFs 11 (56) 0 (00) 0 (00) 11 (688)

Stromal overgrowth lt0001Absent 179 (913) 153 (1000) 24 (889) 2 (125)Present 17 (87) 0 (00) 3 (111) 14 (875)

Tumor margin lt0001Circumscribed 176 (898) 150 (980) 20 (741) 6 (897)Infiltrative 20 (102) 3 (20) 7 (259) 10 (625)

Tumor recurrence 18 (92) 5 (33) 6 (222) 7 (438) lt0001Distant metastasis 8 (41) 0 (00) 1 (37) 7 (438) lt0001PT phyllodes tumor HPF high-power fields

proteins analyzed (Table 3) For the stromal componentthe expression of PDGFR120572 and PDGFR120573 increased withincreasing PT histologic grade (119901 = 0003 and 119901 = 0034resp) (Table 4 and Figure 1)

33 Correlation between the Expression of CAF-Related Pro-teins in Phyllodes Tumor and Clinicopathologic ParametersWe investigated the correlation between the expression ofCAF-related proteins in PT and clinicopathologic parame-ters Only expression of FAP120572 in the stromal cells was associ-ated with distant metastasis (119901 = 0002) (Figure 2) Otherclinicopathologic parameters including age tumor sizestromal cellularity stromal atypia stromal mitosis stromalovergrowth tumor margin and tumor recurrence were notassociated with the expression of CAF-related proteins in PT

34 Impact of Expression of CAF-Related Proteins on PatientPrognosis Univariate analysis showed that stromal expres-sion of PDGFR120572 was associated with shorter overall survival(119901 = 0002) (Table 5 and Figure 3(a)) Furthermore inCoxmultivariate analysis higher PT histologic grade (hazardratio 7990 95 CI 2196ndash2907 and 119901 = 0002) and stromalovergrowth (hazard ratio 7288 95 CI 1225ndash4335 and119901 = 0029) were associated with shorter disease-free survivalRegarding overall survival stromal overgrowth (hazard ratio

5810 95 CI 3116ndash1083 and 119901 = 0006) and PDGFR120572positivity (hazard ratio 5486 95 CI 1003ndash3001 and 119901 =0050) were associated with shorter overall survival (Table 6)Furthermore stromal expression of PDGFR120572 (119901 = 0052)(Figure 3(b)) and PDGFR120573 (119901 = 0041) (Figure 3(c)) wasassociated with shorter overall survival in malignant PTalthough statistical significance was not reached regardingPDGFR120572 expression

4 Discussion

In this study we investigated the expression of CAF-relatedproteins in breast PT according to histologic grade with theaim of identifying a new therapeutic target for PT

Recently there have been many studies on the tumormicroenvironment as a novel therapeutic target The tumormicroenvironment includes nontumor cells with nontrans-formed elements including immune system elements (suchas macrophages and lymphocytes) blood vessel cells fibrob-lasts myofibroblasts mesenchymal stem cells adipocytesand extracellular matrix in close proximity to tumor cellsPT is a representative fibroepithelial tumor characterizedby fibrous stroma composed of fibroblasts surrounding theepithelium Previous studies reported that CD34-expressingfibroblasts exist in mammary stroma Because CD34-expressing fibroblasts were observed in both fibroadenoma

4 Disease Markers

Table 3 Expression of CAF-related proteins in epithelial component of phyllodes tumor according to histologic gradelowast

Parameters Total 119899 = 179 (100) PT benign 119899 = 151(100)

PT borderline 119899 = 23(100)

PT malignant 119899 = 5(100) 119901 value

Podoplanin 0329Negative 35 (196) 27 (179) 6 (261) 2 (400)Positive 144 (804) 124 (821) 17 (739) 3 (600)

Prolyl 4-hydroxylase 0621Negative 174 (972) 146 (967) 23 (1000) 5 (1000)Positive 5 (28) 5 (33) 0 (00) 0 (00)

FAP120572 0794Negative 166 (927) 140 (927) 21 (913) 5 (1000)Positive 13 (73) 11 (73) 2 (87) 0 (00)

S100A4 0571Negative 93 (520) 81 (536) 10 (435) 2 (400)Positive 86 (480) 70 (464) 13 (565) 3 (600)

PDGFR120572 0738Negative 145 (810) 121 (801) 20 (870) 4 (800)Positive 34 (190) 30 (199) 3 (130) 1 (200)

PDGFR120573 NANegative 179 (1000) 151 (1000) 23 (1000) 5 (1000)Positive 0 (00) 0 (00) 0 (00) 0 (00)

NG2 0216Negative 60 (335) 52 (344) 5 (217) 3 (600)Positive 119 (665) 99 (656) 18 (783) 2 (400)

lowastSeventeen tumors without an epithelial component were excludedPT phyllodes tumor

and PT [19] efficient treatment for fibroepithelial tumorsmight be achieved by targeting the fibrous stroma Howeverthere are no studies on the expression of CAF-related pro-teins in PT and as PT shows heterogeneous tumor stromalfeatures the expression of CAF-related proteins is predictedto vary in each type

In this study we performed immunohistochemical stain-ing for CAF-related proteins focusing on the stromal com-ponent Our results showed that the expression of PDGFR120572and PDGFR120573 increased with increasing PT histologic gradePlatelet-derived growth factor is a major mitogen for severalcell types including connective tissue cells that is activatedby binding to two protein tyrosine kinase receptors (PDGFR120572and PDGFR120573) PDGF signaling in tumor cells induces pointmutations amplification and translocations which stimulateautocrine stimulatory loops [6 21] For example PDGFR120572and 120573 are two well-known receptors that participate in breastcancer progression [22] In addition in breast cancer thedesmoplastic response appears to be mediated by PDGF-AAsignaling in PDGFR120572 type CAFs [23] Desmoplasia refers tothe growth of fibrous or connective tissue near the tumorand is increased in tumors with aggressive properties Theresults from our study revealed that higher grade PT withmore active fibroblasts showed higher expression of PDGFR120572and 120573 suggesting that fibroblasts with a CAF phenotype areassociated with PT progression

Stromal PDGFR120572 expression was related to the prognosisof patients with PT Furthermore subgroup analysis inmalignant PT showed that stromal PDGFR120573 expression wasassociated with shorter overall survival and although statis-tical significance was not noted there was also a trend forincreased stromal PDGFR120572 expression In previous studies ofglioma [24] squamous cell carcinoma of the head and neck[25] colorectal cancer [26] pancreatic cancer [27] and T celllymphoma [28] PDGFR activation induced the intracellularsignaling pathway and promoted cell migration invasionsurvival and proliferation [29] Similarly PDGFR activationis related to lymphatic metastasis in pancreatic cancer [30]and gastric cancer [31] suggesting that PDGFR might beused as a prognostic marker in malignant PT This should bevalidated in future studies

We found that expression of FAP120572 in stroma was associ-ated with distant metastasis Fibroblast activation protein isexpressed by reactive CAFs in tumor stroma or granulationtissue and is known to be involved in wound healing Theexpression of FAP in CAFs is reported in various carcinomasand is used as an important marker of CAF [32 33] In ameta-analysis that analyzed the clinical implication of FAPoverexpression in solid tumors of colorectal cancer pan-creatic adenocarcinoma non-small cell lung cancer breastcancer medullary thyroid carcinoma and oral squamous cellcarcinoma high FAP expression was related to the risk of

Disease Markers 5

Benign Borderline Malignant

Podoplanin

FAP120572

S100A4

NG2

HampE stain

E S E

SE

S

Prolyl 4-hydroxylase

PDGFR120572

PDGFR120573

Figure 1 Expression of CAF-related proteins according to the histologic grade of phyllodes tumorThe stromal expression of PDGFR120572 and 120573increased with increasing phyllodes tumor histologic grade The yellow circle indicates epithelium and the white circle indicates the stromaCAF cancer-associated fibroblast HampE hematoxylin and eosin stain FAP fibroblast activation protein PDGFR platelet-derived growthfactor receptor NG2 chondroitin sulfate proteoglycan

6 Disease Markers

Table 4 Expression of CAF-related proteins in the stromal component of phyllodes tumor according to histologic grade

Parameters Total 119899 = 196 (100) PT benign 119899 = 153(100)

PT borderline 119899 = 27(100)

PT malignant 119899 = 16(100) 119901 value

Podoplanin 0728Negative 52 (265) 41 (268) 8 (296) 3 (188)Positive 144 (735) 112 (732) 19 (704) 13 (812)

Prolyl 4-hydroxylase 0563Negative 192 (980) 149 (974) 27 (1000) 16 (1000)Positive 4 (20) 4 (26) 0 (00) 0 (00)

FAP120572 0105Negative 188 (959) 149 (974) 25 (926) 14 (875)Positive 8 (41) 4 (26) 2 (74) 2 (125)

S100A4 0104Negative 65 (332) 56 (366) 7 (259) 2 (125)Positive 131 (668) 97 (634) 20 (741) 14 (875)

PDGFR120572 0003Negative 179 (913) 142 (928) 26 (963) 11 (688)Positive 17 (87) 11 (72) 1 (37) 5 (312)

PDGFR120573 0034Negative 80 (408) 69 (451) 5 (185) 6 (375)Positive 116 (592) 84 (549) 22 (815) 10 (625)

NG2 0695Negative 93 (474) 75 (490) 11 (407) 7 (438)Positive 103 (526) 78 (510) 16 (593) 9 (562)

PT phyllodes tumor

182

66 20

50

100

150

200

No distant metastasis Distant metastasis

FAP120572 (S) negativeFAP120572 (S) positive

p = 0002

Figure 2 Correlation between stromal expression of fibroblastactivation protein (FAP)120572 in phyllodes tumor anddistantmetastasis

distant metastasis (OR 256) [34] showing similar results toour study FAP regulates proteolysis of the extracellular cell

matrix in tumor stroma causing stromal cell proliferationand invasiveness Therefore it could be speculated thattumors with high expression of FAP120572 are prone to metastasisto distant organs [35 36]

The clinical significance of this study is the role of CAFsas a potential therapeutic target Sunitinib a US Food andDrug Administration- (FDA-) approved PDGFR inhibitor iscurrently used in the treatment of advanced renal cell carci-noma [37] advanced progressive pancreatic neuroendocrinetumor [38] and advanced radioiodine refractory thyroidcarcinoma [39] Furthermore ongoing preclinical trials aretesting the therapeutic effect of another PDGFR inhibitorimatinib mesylate in gastrointestinal stromal tumor [40 41]and in vitro and in vivo studies of malignant peripheral nervesheath tumors have shown promising results [42] Thereforeinhibition of PDGFR in PT might be a potential therapeuticstrategy However further investigation regarding the effectof these inhibitors in PT is required

Taken together our data show that expression ofPDGFR120572 and PDGFR120573 in the stromal component increasedwith increased PT histologic grade Also stromal expressionof PDGFR was associated with shorter overall survivalOverall the expression of CAF-related proteins is related tothe histologic grade of breast PT and PDGFR inhibitors inparticular have potential as novel treatments for PT

Disease Markers 7

Table 5 Univariate analysis of the impact of CAF-related proteins in the stromal component of phyllodes tumor on patient prognosis usingthe log-rank test

Parameters Number of patientsTotalrecurrencemetastasis

Disease-free survival Overall survivalMedian survival (95

CI) months 119901 value Median survival(95 CI) months 119901 value

Podoplanin 0936 0494Negative 5253 160 (146ndash174) 166 (155ndash178)Positive 144135 166 (158ndash175) 177 (171ndash182)

Prolyl 4-hydroxylase na naNegative 192188 na naPositive 400 na na

FAP120572 0099 0233Negative 188167 164 (157ndash171) 172 (167ndash177)Positive 821 131 (71ndash191) 162 (123ndash200)

S100A4 0607 0792Negative 6553 163 (152ndash174) 168 (159ndash177)Positive 131135 165 (155ndash174) 176 (170ndash182)

PDGFR120572 0698 0002Negative 179165 167 (159ndash174) 177 (173ndash182)Positive 1723 121 (102ndash140) 113 (90ndash136)

PDGFR120573 0394 0087Negative 8061 164 (154ndash174) 174 (170ndash178)Positive 116127 163 (153ndash173) 172 (164ndash179)

NG2 0830 0216Negative 9382 145 (136ndash154) 155 (151ndash160)Positive 103106 165 (155ndash175) 172 (164ndash180)

Table 6 Multivariate analysis of disease-free and overall survival in patients with phyllodes tumors

Included factor Disease-free survival Overall survivalHazard ratio 95 CI 119901 value Hazard ratio 95 CI 119901 value

Histologic grade 0002 NABenign versus

borderlinemalignant 7990 2196ndash2907 NA NA

Stromal cellularity 0597 0467Mild versus

moderatemarked 0625 0109ndash3569 2862 0169ndash4860

Stromal atypia 0886 0708Mild versus

moderatemarked 0881 0158ndash4925 0630 0056ndash7049

Stromal mitosis 0854 04370ndash410 HPF versusgt410 HPF 0782 0057ndash1081 0210 0004ndash1070

Stromal overgrowth 0029 0006Absent versus present 7288 1225ndash4335 5810 3116ndash1083

Tumor margin 0275 0187Circumscribed versus

infiltrative 0468 0120ndash1830 0321 0060ndash1732

PDGFR120572 (stromal) 0867 0050Negative versus

positive 0879 0193ndash4008 5486 1003ndash3001

HPF high-power field

8 Disease Markers

PDGFR120572 (S) negative

PDGFR120572 (S) positive

Phyllodes tumor

p = 0002

0

02

04

06

08

10

Cum

ulat

ive o

vera

ll su

rviv

al

100 150 200500Months

PDGFR120572 (S) negative

(a)

PDGFR120572 (S) negative

PDGFR120572 (S) positive

Malignant phyllodes tumor

p = 0052

0

02

04

06

08

10

Cum

ulat

ive o

vera

ll su

rviv

al

100 1257525 500Months(b)

Malignant phyllodes tumor

p = 0041

100 1257525 500Months

0

02

04

06

08

10

Cum

ulat

ive o

vera

ll su

rviv

al

PDGFR120573 (S) negative

PDGFR120573 (S) positive

(c)Figure 3 Overall survival curves according to the status of CAF-related proteins in stromal component of phyllodes tumor (a) Univariateanalysis showed that stromal expression of PDGFR120572 was associated with shorter overall survival in phyllodes tumor (b) Stromal expressionof PDGFR120572 was associated with shorter overall survival in malignant phyllodes tumor although statistical significance was not reachedregarding PDGFR120572 expression (c) Stromal expression of PDGFR120573was associated with shorter overall survival inmalignant phyllodes tumorCAF cancer-associated fibroblast PDGFR platelet-derived growth factor receptor

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was supported by a grant from the NationalRampD Program for Cancer Control Ministry of Healthamp Welfare Republic of Korea (1420080) Additionally it

was supported by Basic Science Research Program throughthe National Research Foundation of Korea (NRF) fundedby the Ministry of Science ICT and Future Planning(2015R1A1A1A05001209)

References

[1] O E Franco A K Shaw D W Strand and S W HaywardldquoCancer associated fibroblasts in cancer pathogenesisrdquo Seminarsin Cell and Developmental Biology vol 21 no 1 pp 33ndash39 2010

Disease Markers 9

[2] A Ostman ldquoCancer-associated fibroblasts recent develop-ments and emerging challengesrdquo Seminars in Cancer Biologyvol 25 pp 1ndash2 2014

[3] A Desmouliere C Guyot and G Gabbiani ldquoThe stromareaction myofibroblast a key player in the control of tumor cellbehaviorrdquo International Journal of Developmental Biology vol48 no 5-6 pp 509ndash517 2004

[4] O De Wever Q-D Nguyen L Van Hoorde et al ldquoTenascin-C and SFHGF produced by myofibroblasts in vitro provideconvergent pro-invasive signals to human colon cancer cellsthrough RhoA and Racrdquo FASEB Journal vol 18 no 9 pp 1016ndash1018 2004

[5] H Sugimoto TMMundelMWKieran andRKalluri ldquoIden-tification of fibroblast heterogeneity in the tumor microenvi-ronmentrdquo Cancer Biology and Therapy vol 5 no 12 pp 1640ndash1646 2006

[6] K Pietras T Sjoblom K Rubin C-H Heldin and A OstmanldquoPDGF receptors as cancer drug targetsrdquo Cancer Cell vol 3 no5 pp 439ndash443 2003

[7] M Kraman P J Bambrough J N Arnold et al ldquoSuppressionof antitumor immunity by stromal cells expressing fibroblastactivation protein-120572rdquo Science vol 330 no 6005 pp 827ndash8302010

[8] A Kawase G Ishii K Nagai et al ldquoPodoplanin expression bycancer associated fibroblasts predicts poor prognosis of lungadenocarcinomardquo International Journal of Cancer vol 123 no5 pp 1053ndash1059 2008

[9] Y Kojima A Acar E N Eaton et al ldquoAutocrine TGF-120573 andstromal cell-derived factor-1 (SDF-1) signaling drives the evo-lution of tumor-promoting mammary stromal myofibroblastsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 107 no 46 pp 20009ndash20014 2010

[10] E Cortez P Roswall and K Pietras ldquoFunctional subsetsof mesenchymal cell types in the tumor microenvironmentrdquoSeminars in Cancer Biology vol 25 pp 3ndash9 2014

[11] F A Tavassoli and P Devilee World Heath OrganizationClassification of Tumors IARC Press Lyon France 2003

[12] S Dacic S Kounelis E Kouri andMW Jones ldquoImmunohisto-chemical profile of cystosarcoma phyllodes of the breast a studyof 23 casesrdquo Breast Journal vol 8 no 6 pp 376ndash381 2002

[13] B Anderson T Lawton C Lehman and R Moe ldquoPhyllodestumorrdquo in Disease of the Breast M Morrow and C OsborneEds pp 991ndash1006 Lippincott Williams amp Wilkins Philadel-phia 2004

[14] J Ben hassouna T Damak A Gamoudi et al ldquoPhyllodestumors of the breast a case series of 106 patientsrdquo AmericanJournal of Surgery vol 192 no 2 pp 141ndash147 2006

[15] J Mitus M Reinfuss J W Mitus et al ldquoMalignant phyllodestumor of the breast treatment and prognosisrdquo Breast Journalvol 20 no 6 pp 639ndash644 2014

[16] P-H Tan T Jayabaskar K-L Chuah et al ldquoPhyllodes tumors ofthe breast the role of pathologic parametersrdquo American Journalof Clinical Pathology vol 123 no 4 pp 529ndash540 2005

[17] A V Barrio B D Clark J I Goldberg et al ldquoClinicopathologicfeatures and long-term outcomes of 293 phyllodes tumors of thebreastrdquo Annals of Surgical Oncology vol 14 no 10 pp 2961ndash2970 2007

[18] F I Aranda J B Laforga and J I Lopez ldquoPhyllodes tumorof the breast An immunohistochemical study of 28 caseswith special attention to the role of myofibroblastsrdquo PathologyResearch and Practice vol 190 no 5 pp 474ndash481 1994

[19] J S Silverman and A Tamsen ldquoMammary fibroadenomaand some phyllodes tumour stroma are composed of CD34+fibroblasts and factor XIIIa+ dendrophagesrdquo Histopathologyvol 29 no 5 pp 411ndash419 1996

[20] K Y Won G Y Kim Y W Kim J Y Song and S-J LimldquoClinicopathologic correlation of beclin-1 and bcl-2 expressionin human breast cancerrdquo Human Pathology vol 41 no 1 pp107ndash112 2010

[21] J Andrae R Gallini and C Betsholtz ldquoRole of platelet-derived growth factors in physiology and medicinerdquo Genes andDevelopment vol 22 no 10 pp 1276ndash1312 2008

[22] F Meng C L Speyer B Zhang et al ldquoPDGFR120572 and 120573 playcritical roles in mediating Foxq1-driven breast cancer stemnessand chemoresistancerdquo Cancer Research vol 75 no 3 pp 584ndash593 2015

[23] Z-M Shao M Nguyen and S H Barsky ldquoHuman breastcarcinoma desmoplasia is PDGF initiatedrdquo Oncogene vol 19no 38 pp 4337ndash4345 2000

[24] I Nazarenko S-M Hede X He et al ldquoPDGF and PDGFreceptors in gliomardquoUpsala Journal of Medical Sciences vol 117no 2 pp 99ndash112 2012

[25] J D Schultz S Rotunno F Riedel et al ldquoSynergistic effectsof imatinib and carboplatin on VEGF PDGF and PDGF-R120572120573expression in squamous cell carcinoma of the head and neck invitrordquo International Journal of Oncology vol 38 no 4 pp 1001ndash1012 2011

[26] E J A Steller D A Raats J Koster et al ldquoPDGFRB pro-motes liver metastasis formation of mesenchymal-like colorec-tal tumor cellsrdquoNeoplasia (United States) vol 15 no 2 pp 204ndash217 2013

[27] H Kurahara K Maemura Y Mataki M Sakoda H Shinchiand S Natsugoe ldquoImpact of p53 and PDGFR-120573 expression onmetastasis and prognosis of patients with pancreatic cancerrdquoWorld Journal of Surgery vol 40 no 8 pp 1977ndash1984 2016

[28] P P Piccaluga M Rossi C Agostinelli et al ldquoPlatelet-derivedgrowth factor alpha mediates the proliferation of peripheralT-cell lymphoma cells via an autocrine regulatory pathwayrdquoLeukemia vol 28 no 8 pp 1687ndash1697 2014

[29] C-H Heldin and B Westermark ldquoMechanism of action andin vivo role of platelet-derived growth factorrdquo PhysiologicalReviews vol 79 no 4 pp 1283ndash1316 1999

[30] S Weissmueller E Manchado M Saborowski et alldquoMutant p53 drives pancreatic cancer metastasis throughcell-autonomous PDGF receptor 120573 signalingrdquo Cell vol 157 no2 pp 382ndash394 2014

[31] M Kodama Y Kitadai T Sumida et al ldquoExpression ofplatelet-derived growth factor (PDGF)-B and PDGF-receptor120573 is associated with lymphatic metastasis in human gastriccarcinomardquo Cancer Science vol 101 no 9 pp 1984ndash1989 2010

[32] M Meng W Wang J Yan et al ldquoImmunization of stromal celltargeting fibroblast activation protein providing immunother-apy to breast cancer mouse modelrdquo Tumor Biology vol 37 no8 pp 10317ndash10327 2016

[33] H Wang Q Wu Z Liu et al ldquoDownregulation of FAPsuppresses cell proliferation and metastasis throughPTENPI3KAKT and Ras-ERK signaling in oral squamouscell carcinomardquo Cell Death and Disease vol 5 no 4 Article IDe1155 2014

[34] F Liu L Qi B Liu et al ldquoFibroblast activation proteinoverexpression and clinical implications in solid tumors ameta-analysisrdquo PLoS ONE vol 10 no 3 Article ID e01166832015

10 Disease Markers

[35] D Yuan B Liu K Liu G Zhu Z Dai and Y Xie ldquoOverexpres-sion of fibroblast activation protein and its clinical implicationsin patients with osteosarcomardquo Journal of Surgical Oncologyvol 108 no 3 pp 157ndash162 2013

[36] M A Goscinski Z Suo V A Floslashrenes L Vlatkovic J MNesland and K-E Giercksky ldquoFAP-120572 and uPA show differentexpression patterns in premalignant and malignant esophageallesionsrdquo Ultrastructural Pathology vol 32 no 3 pp 89ndash962008

[37] R C Coelho T Reinert F Campos et al ldquoSunitinib treatmentin patients with advanced renal cell cancer the BrazilianNational Cancer Institute (INCA) experiencerdquo InternationalBrazilian Journal of Urology vol 42 no 4 pp 694ndash703 2016

[38] ldquoEfficiency of sunitinib in Chinese patients with advancedprogressive pancreatic neuroendocrine tumorrdquo Zhongguo YiXue Ke Xue Yuan Xue Bao vol 38 no 3 pp 300ndash304 2016

[39] V Atallah A Hocquelet C Do Cao et al ldquoActivity and safetyof sunitinib in patients with advanced radioiodine refractorythyroid carcinoma a retrospective analysis of 57 patientsrdquoThyroid vol 26 no 8 pp 1085ndash1092 2016

[40] E Y Moawad ldquoPredicting effectiveness of imatinib mesylatein tumors expressing platelet-derived growth factors (PDGF-AA PDGF-BB) stem cell factor ligands and their respectivereceptors (PDGFR-120572 PDGFR-120573 and c-kit)rdquo Journal of Gas-trointestinal Cancer vol 46 no 3 pp 272ndash283 2015

[41] Y Hayashi M R Bardsley Y Toyomasu et al ldquoPlatelet-derived growth factor receptor-120572 regulates proliferation ofgastrointestinal stromal tumor cells with mutations in KIT bystabilizing ETV1rdquo Gastroenterology vol 149 no 2 pp 420ndash432e16 2015

[42] J Ohishi M Aoki K Nabeshima et al ldquoImatinib mesylateinhibits cell growth of malignant peripheral nerve sheathtumors in vitro and in vivo through suppression of PDGFR-120573rdquoBMC Cancer vol 13 article 224 2013

Disease Markers 3

Table 2 Clinicopathologic characteristics of patients with phyllodes tumor

Parameters Total 119899 = 196 (100) PT benign 119899 = 153(100)

PT borderline 119899 = 27(100)

PT malignant 119899 = 16(100) 119901 value

Age (years mean plusmn SD) 401 plusmn 123 389 plusmn 122 423 plusmn 115 476 plusmn 129 0017Tumor size (cm mean plusmnSD) 40 plusmn 26 36 plusmn 21 43 plusmn 25 67 plusmn 46 lt0001

Stromal cellularity lt0001Mild 121 (617) 120 (784) 1 (37) 0 (00)Moderate 63 (321) 33 (216) 23 (852) 7 (438)Marked 12 (61) 0 (00) 3 (111) 9 (562)

Stromal atypia lt0001Mild 156 (796) 151 (987) 5 (185) 0 (00)Moderate 30 (153) 2 (13) 20 (741) 8 (500)Marked 10 (51) 0 (00) 2 (74) 8 (500)

Stromal mitosis lt00010ndash410 HPFs 154 (786) 153 (1000) 1 (37) 0 (00)5ndash910 HPFs 31 (158) 0 (00) 26 (963) 5 (312)ge1010 HPFs 11 (56) 0 (00) 0 (00) 11 (688)

Stromal overgrowth lt0001Absent 179 (913) 153 (1000) 24 (889) 2 (125)Present 17 (87) 0 (00) 3 (111) 14 (875)

Tumor margin lt0001Circumscribed 176 (898) 150 (980) 20 (741) 6 (897)Infiltrative 20 (102) 3 (20) 7 (259) 10 (625)

Tumor recurrence 18 (92) 5 (33) 6 (222) 7 (438) lt0001Distant metastasis 8 (41) 0 (00) 1 (37) 7 (438) lt0001PT phyllodes tumor HPF high-power fields

proteins analyzed (Table 3) For the stromal componentthe expression of PDGFR120572 and PDGFR120573 increased withincreasing PT histologic grade (119901 = 0003 and 119901 = 0034resp) (Table 4 and Figure 1)

33 Correlation between the Expression of CAF-Related Pro-teins in Phyllodes Tumor and Clinicopathologic ParametersWe investigated the correlation between the expression ofCAF-related proteins in PT and clinicopathologic parame-ters Only expression of FAP120572 in the stromal cells was associ-ated with distant metastasis (119901 = 0002) (Figure 2) Otherclinicopathologic parameters including age tumor sizestromal cellularity stromal atypia stromal mitosis stromalovergrowth tumor margin and tumor recurrence were notassociated with the expression of CAF-related proteins in PT

34 Impact of Expression of CAF-Related Proteins on PatientPrognosis Univariate analysis showed that stromal expres-sion of PDGFR120572 was associated with shorter overall survival(119901 = 0002) (Table 5 and Figure 3(a)) Furthermore inCoxmultivariate analysis higher PT histologic grade (hazardratio 7990 95 CI 2196ndash2907 and 119901 = 0002) and stromalovergrowth (hazard ratio 7288 95 CI 1225ndash4335 and119901 = 0029) were associated with shorter disease-free survivalRegarding overall survival stromal overgrowth (hazard ratio

5810 95 CI 3116ndash1083 and 119901 = 0006) and PDGFR120572positivity (hazard ratio 5486 95 CI 1003ndash3001 and 119901 =0050) were associated with shorter overall survival (Table 6)Furthermore stromal expression of PDGFR120572 (119901 = 0052)(Figure 3(b)) and PDGFR120573 (119901 = 0041) (Figure 3(c)) wasassociated with shorter overall survival in malignant PTalthough statistical significance was not reached regardingPDGFR120572 expression

4 Discussion

In this study we investigated the expression of CAF-relatedproteins in breast PT according to histologic grade with theaim of identifying a new therapeutic target for PT

Recently there have been many studies on the tumormicroenvironment as a novel therapeutic target The tumormicroenvironment includes nontumor cells with nontrans-formed elements including immune system elements (suchas macrophages and lymphocytes) blood vessel cells fibrob-lasts myofibroblasts mesenchymal stem cells adipocytesand extracellular matrix in close proximity to tumor cellsPT is a representative fibroepithelial tumor characterizedby fibrous stroma composed of fibroblasts surrounding theepithelium Previous studies reported that CD34-expressingfibroblasts exist in mammary stroma Because CD34-expressing fibroblasts were observed in both fibroadenoma

4 Disease Markers

Table 3 Expression of CAF-related proteins in epithelial component of phyllodes tumor according to histologic gradelowast

Parameters Total 119899 = 179 (100) PT benign 119899 = 151(100)

PT borderline 119899 = 23(100)

PT malignant 119899 = 5(100) 119901 value

Podoplanin 0329Negative 35 (196) 27 (179) 6 (261) 2 (400)Positive 144 (804) 124 (821) 17 (739) 3 (600)

Prolyl 4-hydroxylase 0621Negative 174 (972) 146 (967) 23 (1000) 5 (1000)Positive 5 (28) 5 (33) 0 (00) 0 (00)

FAP120572 0794Negative 166 (927) 140 (927) 21 (913) 5 (1000)Positive 13 (73) 11 (73) 2 (87) 0 (00)

S100A4 0571Negative 93 (520) 81 (536) 10 (435) 2 (400)Positive 86 (480) 70 (464) 13 (565) 3 (600)

PDGFR120572 0738Negative 145 (810) 121 (801) 20 (870) 4 (800)Positive 34 (190) 30 (199) 3 (130) 1 (200)

PDGFR120573 NANegative 179 (1000) 151 (1000) 23 (1000) 5 (1000)Positive 0 (00) 0 (00) 0 (00) 0 (00)

NG2 0216Negative 60 (335) 52 (344) 5 (217) 3 (600)Positive 119 (665) 99 (656) 18 (783) 2 (400)

lowastSeventeen tumors without an epithelial component were excludedPT phyllodes tumor

and PT [19] efficient treatment for fibroepithelial tumorsmight be achieved by targeting the fibrous stroma Howeverthere are no studies on the expression of CAF-related pro-teins in PT and as PT shows heterogeneous tumor stromalfeatures the expression of CAF-related proteins is predictedto vary in each type

In this study we performed immunohistochemical stain-ing for CAF-related proteins focusing on the stromal com-ponent Our results showed that the expression of PDGFR120572and PDGFR120573 increased with increasing PT histologic gradePlatelet-derived growth factor is a major mitogen for severalcell types including connective tissue cells that is activatedby binding to two protein tyrosine kinase receptors (PDGFR120572and PDGFR120573) PDGF signaling in tumor cells induces pointmutations amplification and translocations which stimulateautocrine stimulatory loops [6 21] For example PDGFR120572and 120573 are two well-known receptors that participate in breastcancer progression [22] In addition in breast cancer thedesmoplastic response appears to be mediated by PDGF-AAsignaling in PDGFR120572 type CAFs [23] Desmoplasia refers tothe growth of fibrous or connective tissue near the tumorand is increased in tumors with aggressive properties Theresults from our study revealed that higher grade PT withmore active fibroblasts showed higher expression of PDGFR120572and 120573 suggesting that fibroblasts with a CAF phenotype areassociated with PT progression

Stromal PDGFR120572 expression was related to the prognosisof patients with PT Furthermore subgroup analysis inmalignant PT showed that stromal PDGFR120573 expression wasassociated with shorter overall survival and although statis-tical significance was not noted there was also a trend forincreased stromal PDGFR120572 expression In previous studies ofglioma [24] squamous cell carcinoma of the head and neck[25] colorectal cancer [26] pancreatic cancer [27] and T celllymphoma [28] PDGFR activation induced the intracellularsignaling pathway and promoted cell migration invasionsurvival and proliferation [29] Similarly PDGFR activationis related to lymphatic metastasis in pancreatic cancer [30]and gastric cancer [31] suggesting that PDGFR might beused as a prognostic marker in malignant PT This should bevalidated in future studies

We found that expression of FAP120572 in stroma was associ-ated with distant metastasis Fibroblast activation protein isexpressed by reactive CAFs in tumor stroma or granulationtissue and is known to be involved in wound healing Theexpression of FAP in CAFs is reported in various carcinomasand is used as an important marker of CAF [32 33] In ameta-analysis that analyzed the clinical implication of FAPoverexpression in solid tumors of colorectal cancer pan-creatic adenocarcinoma non-small cell lung cancer breastcancer medullary thyroid carcinoma and oral squamous cellcarcinoma high FAP expression was related to the risk of

Disease Markers 5

Benign Borderline Malignant

Podoplanin

FAP120572

S100A4

NG2

HampE stain

E S E

SE

S

Prolyl 4-hydroxylase

PDGFR120572

PDGFR120573

Figure 1 Expression of CAF-related proteins according to the histologic grade of phyllodes tumorThe stromal expression of PDGFR120572 and 120573increased with increasing phyllodes tumor histologic grade The yellow circle indicates epithelium and the white circle indicates the stromaCAF cancer-associated fibroblast HampE hematoxylin and eosin stain FAP fibroblast activation protein PDGFR platelet-derived growthfactor receptor NG2 chondroitin sulfate proteoglycan

6 Disease Markers

Table 4 Expression of CAF-related proteins in the stromal component of phyllodes tumor according to histologic grade

Parameters Total 119899 = 196 (100) PT benign 119899 = 153(100)

PT borderline 119899 = 27(100)

PT malignant 119899 = 16(100) 119901 value

Podoplanin 0728Negative 52 (265) 41 (268) 8 (296) 3 (188)Positive 144 (735) 112 (732) 19 (704) 13 (812)

Prolyl 4-hydroxylase 0563Negative 192 (980) 149 (974) 27 (1000) 16 (1000)Positive 4 (20) 4 (26) 0 (00) 0 (00)

FAP120572 0105Negative 188 (959) 149 (974) 25 (926) 14 (875)Positive 8 (41) 4 (26) 2 (74) 2 (125)

S100A4 0104Negative 65 (332) 56 (366) 7 (259) 2 (125)Positive 131 (668) 97 (634) 20 (741) 14 (875)

PDGFR120572 0003Negative 179 (913) 142 (928) 26 (963) 11 (688)Positive 17 (87) 11 (72) 1 (37) 5 (312)

PDGFR120573 0034Negative 80 (408) 69 (451) 5 (185) 6 (375)Positive 116 (592) 84 (549) 22 (815) 10 (625)

NG2 0695Negative 93 (474) 75 (490) 11 (407) 7 (438)Positive 103 (526) 78 (510) 16 (593) 9 (562)

PT phyllodes tumor

182

66 20

50

100

150

200

No distant metastasis Distant metastasis

FAP120572 (S) negativeFAP120572 (S) positive

p = 0002

Figure 2 Correlation between stromal expression of fibroblastactivation protein (FAP)120572 in phyllodes tumor anddistantmetastasis

distant metastasis (OR 256) [34] showing similar results toour study FAP regulates proteolysis of the extracellular cell

matrix in tumor stroma causing stromal cell proliferationand invasiveness Therefore it could be speculated thattumors with high expression of FAP120572 are prone to metastasisto distant organs [35 36]

The clinical significance of this study is the role of CAFsas a potential therapeutic target Sunitinib a US Food andDrug Administration- (FDA-) approved PDGFR inhibitor iscurrently used in the treatment of advanced renal cell carci-noma [37] advanced progressive pancreatic neuroendocrinetumor [38] and advanced radioiodine refractory thyroidcarcinoma [39] Furthermore ongoing preclinical trials aretesting the therapeutic effect of another PDGFR inhibitorimatinib mesylate in gastrointestinal stromal tumor [40 41]and in vitro and in vivo studies of malignant peripheral nervesheath tumors have shown promising results [42] Thereforeinhibition of PDGFR in PT might be a potential therapeuticstrategy However further investigation regarding the effectof these inhibitors in PT is required

Taken together our data show that expression ofPDGFR120572 and PDGFR120573 in the stromal component increasedwith increased PT histologic grade Also stromal expressionof PDGFR was associated with shorter overall survivalOverall the expression of CAF-related proteins is related tothe histologic grade of breast PT and PDGFR inhibitors inparticular have potential as novel treatments for PT

Disease Markers 7

Table 5 Univariate analysis of the impact of CAF-related proteins in the stromal component of phyllodes tumor on patient prognosis usingthe log-rank test

Parameters Number of patientsTotalrecurrencemetastasis

Disease-free survival Overall survivalMedian survival (95

CI) months 119901 value Median survival(95 CI) months 119901 value

Podoplanin 0936 0494Negative 5253 160 (146ndash174) 166 (155ndash178)Positive 144135 166 (158ndash175) 177 (171ndash182)

Prolyl 4-hydroxylase na naNegative 192188 na naPositive 400 na na

FAP120572 0099 0233Negative 188167 164 (157ndash171) 172 (167ndash177)Positive 821 131 (71ndash191) 162 (123ndash200)

S100A4 0607 0792Negative 6553 163 (152ndash174) 168 (159ndash177)Positive 131135 165 (155ndash174) 176 (170ndash182)

PDGFR120572 0698 0002Negative 179165 167 (159ndash174) 177 (173ndash182)Positive 1723 121 (102ndash140) 113 (90ndash136)

PDGFR120573 0394 0087Negative 8061 164 (154ndash174) 174 (170ndash178)Positive 116127 163 (153ndash173) 172 (164ndash179)

NG2 0830 0216Negative 9382 145 (136ndash154) 155 (151ndash160)Positive 103106 165 (155ndash175) 172 (164ndash180)

Table 6 Multivariate analysis of disease-free and overall survival in patients with phyllodes tumors

Included factor Disease-free survival Overall survivalHazard ratio 95 CI 119901 value Hazard ratio 95 CI 119901 value

Histologic grade 0002 NABenign versus

borderlinemalignant 7990 2196ndash2907 NA NA

Stromal cellularity 0597 0467Mild versus

moderatemarked 0625 0109ndash3569 2862 0169ndash4860

Stromal atypia 0886 0708Mild versus

moderatemarked 0881 0158ndash4925 0630 0056ndash7049

Stromal mitosis 0854 04370ndash410 HPF versusgt410 HPF 0782 0057ndash1081 0210 0004ndash1070

Stromal overgrowth 0029 0006Absent versus present 7288 1225ndash4335 5810 3116ndash1083

Tumor margin 0275 0187Circumscribed versus

infiltrative 0468 0120ndash1830 0321 0060ndash1732

PDGFR120572 (stromal) 0867 0050Negative versus

positive 0879 0193ndash4008 5486 1003ndash3001

HPF high-power field

8 Disease Markers

PDGFR120572 (S) negative

PDGFR120572 (S) positive

Phyllodes tumor

p = 0002

0

02

04

06

08

10

Cum

ulat

ive o

vera

ll su

rviv

al

100 150 200500Months

PDGFR120572 (S) negative

(a)

PDGFR120572 (S) negative

PDGFR120572 (S) positive

Malignant phyllodes tumor

p = 0052

0

02

04

06

08

10

Cum

ulat

ive o

vera

ll su

rviv

al

100 1257525 500Months(b)

Malignant phyllodes tumor

p = 0041

100 1257525 500Months

0

02

04

06

08

10

Cum

ulat

ive o

vera

ll su

rviv

al

PDGFR120573 (S) negative

PDGFR120573 (S) positive

(c)Figure 3 Overall survival curves according to the status of CAF-related proteins in stromal component of phyllodes tumor (a) Univariateanalysis showed that stromal expression of PDGFR120572 was associated with shorter overall survival in phyllodes tumor (b) Stromal expressionof PDGFR120572 was associated with shorter overall survival in malignant phyllodes tumor although statistical significance was not reachedregarding PDGFR120572 expression (c) Stromal expression of PDGFR120573was associated with shorter overall survival inmalignant phyllodes tumorCAF cancer-associated fibroblast PDGFR platelet-derived growth factor receptor

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was supported by a grant from the NationalRampD Program for Cancer Control Ministry of Healthamp Welfare Republic of Korea (1420080) Additionally it

was supported by Basic Science Research Program throughthe National Research Foundation of Korea (NRF) fundedby the Ministry of Science ICT and Future Planning(2015R1A1A1A05001209)

References

[1] O E Franco A K Shaw D W Strand and S W HaywardldquoCancer associated fibroblasts in cancer pathogenesisrdquo Seminarsin Cell and Developmental Biology vol 21 no 1 pp 33ndash39 2010

Disease Markers 9

[2] A Ostman ldquoCancer-associated fibroblasts recent develop-ments and emerging challengesrdquo Seminars in Cancer Biologyvol 25 pp 1ndash2 2014

[3] A Desmouliere C Guyot and G Gabbiani ldquoThe stromareaction myofibroblast a key player in the control of tumor cellbehaviorrdquo International Journal of Developmental Biology vol48 no 5-6 pp 509ndash517 2004

[4] O De Wever Q-D Nguyen L Van Hoorde et al ldquoTenascin-C and SFHGF produced by myofibroblasts in vitro provideconvergent pro-invasive signals to human colon cancer cellsthrough RhoA and Racrdquo FASEB Journal vol 18 no 9 pp 1016ndash1018 2004

[5] H Sugimoto TMMundelMWKieran andRKalluri ldquoIden-tification of fibroblast heterogeneity in the tumor microenvi-ronmentrdquo Cancer Biology and Therapy vol 5 no 12 pp 1640ndash1646 2006

[6] K Pietras T Sjoblom K Rubin C-H Heldin and A OstmanldquoPDGF receptors as cancer drug targetsrdquo Cancer Cell vol 3 no5 pp 439ndash443 2003

[7] M Kraman P J Bambrough J N Arnold et al ldquoSuppressionof antitumor immunity by stromal cells expressing fibroblastactivation protein-120572rdquo Science vol 330 no 6005 pp 827ndash8302010

[8] A Kawase G Ishii K Nagai et al ldquoPodoplanin expression bycancer associated fibroblasts predicts poor prognosis of lungadenocarcinomardquo International Journal of Cancer vol 123 no5 pp 1053ndash1059 2008

[9] Y Kojima A Acar E N Eaton et al ldquoAutocrine TGF-120573 andstromal cell-derived factor-1 (SDF-1) signaling drives the evo-lution of tumor-promoting mammary stromal myofibroblastsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 107 no 46 pp 20009ndash20014 2010

[10] E Cortez P Roswall and K Pietras ldquoFunctional subsetsof mesenchymal cell types in the tumor microenvironmentrdquoSeminars in Cancer Biology vol 25 pp 3ndash9 2014

[11] F A Tavassoli and P Devilee World Heath OrganizationClassification of Tumors IARC Press Lyon France 2003

[12] S Dacic S Kounelis E Kouri andMW Jones ldquoImmunohisto-chemical profile of cystosarcoma phyllodes of the breast a studyof 23 casesrdquo Breast Journal vol 8 no 6 pp 376ndash381 2002

[13] B Anderson T Lawton C Lehman and R Moe ldquoPhyllodestumorrdquo in Disease of the Breast M Morrow and C OsborneEds pp 991ndash1006 Lippincott Williams amp Wilkins Philadel-phia 2004

[14] J Ben hassouna T Damak A Gamoudi et al ldquoPhyllodestumors of the breast a case series of 106 patientsrdquo AmericanJournal of Surgery vol 192 no 2 pp 141ndash147 2006

[15] J Mitus M Reinfuss J W Mitus et al ldquoMalignant phyllodestumor of the breast treatment and prognosisrdquo Breast Journalvol 20 no 6 pp 639ndash644 2014

[16] P-H Tan T Jayabaskar K-L Chuah et al ldquoPhyllodes tumors ofthe breast the role of pathologic parametersrdquo American Journalof Clinical Pathology vol 123 no 4 pp 529ndash540 2005

[17] A V Barrio B D Clark J I Goldberg et al ldquoClinicopathologicfeatures and long-term outcomes of 293 phyllodes tumors of thebreastrdquo Annals of Surgical Oncology vol 14 no 10 pp 2961ndash2970 2007

[18] F I Aranda J B Laforga and J I Lopez ldquoPhyllodes tumorof the breast An immunohistochemical study of 28 caseswith special attention to the role of myofibroblastsrdquo PathologyResearch and Practice vol 190 no 5 pp 474ndash481 1994

[19] J S Silverman and A Tamsen ldquoMammary fibroadenomaand some phyllodes tumour stroma are composed of CD34+fibroblasts and factor XIIIa+ dendrophagesrdquo Histopathologyvol 29 no 5 pp 411ndash419 1996

[20] K Y Won G Y Kim Y W Kim J Y Song and S-J LimldquoClinicopathologic correlation of beclin-1 and bcl-2 expressionin human breast cancerrdquo Human Pathology vol 41 no 1 pp107ndash112 2010

[21] J Andrae R Gallini and C Betsholtz ldquoRole of platelet-derived growth factors in physiology and medicinerdquo Genes andDevelopment vol 22 no 10 pp 1276ndash1312 2008

[22] F Meng C L Speyer B Zhang et al ldquoPDGFR120572 and 120573 playcritical roles in mediating Foxq1-driven breast cancer stemnessand chemoresistancerdquo Cancer Research vol 75 no 3 pp 584ndash593 2015

[23] Z-M Shao M Nguyen and S H Barsky ldquoHuman breastcarcinoma desmoplasia is PDGF initiatedrdquo Oncogene vol 19no 38 pp 4337ndash4345 2000

[24] I Nazarenko S-M Hede X He et al ldquoPDGF and PDGFreceptors in gliomardquoUpsala Journal of Medical Sciences vol 117no 2 pp 99ndash112 2012

[25] J D Schultz S Rotunno F Riedel et al ldquoSynergistic effectsof imatinib and carboplatin on VEGF PDGF and PDGF-R120572120573expression in squamous cell carcinoma of the head and neck invitrordquo International Journal of Oncology vol 38 no 4 pp 1001ndash1012 2011

[26] E J A Steller D A Raats J Koster et al ldquoPDGFRB pro-motes liver metastasis formation of mesenchymal-like colorec-tal tumor cellsrdquoNeoplasia (United States) vol 15 no 2 pp 204ndash217 2013

[27] H Kurahara K Maemura Y Mataki M Sakoda H Shinchiand S Natsugoe ldquoImpact of p53 and PDGFR-120573 expression onmetastasis and prognosis of patients with pancreatic cancerrdquoWorld Journal of Surgery vol 40 no 8 pp 1977ndash1984 2016

[28] P P Piccaluga M Rossi C Agostinelli et al ldquoPlatelet-derivedgrowth factor alpha mediates the proliferation of peripheralT-cell lymphoma cells via an autocrine regulatory pathwayrdquoLeukemia vol 28 no 8 pp 1687ndash1697 2014

[29] C-H Heldin and B Westermark ldquoMechanism of action andin vivo role of platelet-derived growth factorrdquo PhysiologicalReviews vol 79 no 4 pp 1283ndash1316 1999

[30] S Weissmueller E Manchado M Saborowski et alldquoMutant p53 drives pancreatic cancer metastasis throughcell-autonomous PDGF receptor 120573 signalingrdquo Cell vol 157 no2 pp 382ndash394 2014

[31] M Kodama Y Kitadai T Sumida et al ldquoExpression ofplatelet-derived growth factor (PDGF)-B and PDGF-receptor120573 is associated with lymphatic metastasis in human gastriccarcinomardquo Cancer Science vol 101 no 9 pp 1984ndash1989 2010

[32] M Meng W Wang J Yan et al ldquoImmunization of stromal celltargeting fibroblast activation protein providing immunother-apy to breast cancer mouse modelrdquo Tumor Biology vol 37 no8 pp 10317ndash10327 2016

[33] H Wang Q Wu Z Liu et al ldquoDownregulation of FAPsuppresses cell proliferation and metastasis throughPTENPI3KAKT and Ras-ERK signaling in oral squamouscell carcinomardquo Cell Death and Disease vol 5 no 4 Article IDe1155 2014

[34] F Liu L Qi B Liu et al ldquoFibroblast activation proteinoverexpression and clinical implications in solid tumors ameta-analysisrdquo PLoS ONE vol 10 no 3 Article ID e01166832015

10 Disease Markers

[35] D Yuan B Liu K Liu G Zhu Z Dai and Y Xie ldquoOverexpres-sion of fibroblast activation protein and its clinical implicationsin patients with osteosarcomardquo Journal of Surgical Oncologyvol 108 no 3 pp 157ndash162 2013

[36] M A Goscinski Z Suo V A Floslashrenes L Vlatkovic J MNesland and K-E Giercksky ldquoFAP-120572 and uPA show differentexpression patterns in premalignant and malignant esophageallesionsrdquo Ultrastructural Pathology vol 32 no 3 pp 89ndash962008

[37] R C Coelho T Reinert F Campos et al ldquoSunitinib treatmentin patients with advanced renal cell cancer the BrazilianNational Cancer Institute (INCA) experiencerdquo InternationalBrazilian Journal of Urology vol 42 no 4 pp 694ndash703 2016

[38] ldquoEfficiency of sunitinib in Chinese patients with advancedprogressive pancreatic neuroendocrine tumorrdquo Zhongguo YiXue Ke Xue Yuan Xue Bao vol 38 no 3 pp 300ndash304 2016

[39] V Atallah A Hocquelet C Do Cao et al ldquoActivity and safetyof sunitinib in patients with advanced radioiodine refractorythyroid carcinoma a retrospective analysis of 57 patientsrdquoThyroid vol 26 no 8 pp 1085ndash1092 2016

[40] E Y Moawad ldquoPredicting effectiveness of imatinib mesylatein tumors expressing platelet-derived growth factors (PDGF-AA PDGF-BB) stem cell factor ligands and their respectivereceptors (PDGFR-120572 PDGFR-120573 and c-kit)rdquo Journal of Gas-trointestinal Cancer vol 46 no 3 pp 272ndash283 2015

[41] Y Hayashi M R Bardsley Y Toyomasu et al ldquoPlatelet-derived growth factor receptor-120572 regulates proliferation ofgastrointestinal stromal tumor cells with mutations in KIT bystabilizing ETV1rdquo Gastroenterology vol 149 no 2 pp 420ndash432e16 2015

[42] J Ohishi M Aoki K Nabeshima et al ldquoImatinib mesylateinhibits cell growth of malignant peripheral nerve sheathtumors in vitro and in vivo through suppression of PDGFR-120573rdquoBMC Cancer vol 13 article 224 2013

4 Disease Markers

Table 3 Expression of CAF-related proteins in epithelial component of phyllodes tumor according to histologic gradelowast

Parameters Total 119899 = 179 (100) PT benign 119899 = 151(100)

PT borderline 119899 = 23(100)

PT malignant 119899 = 5(100) 119901 value

Podoplanin 0329Negative 35 (196) 27 (179) 6 (261) 2 (400)Positive 144 (804) 124 (821) 17 (739) 3 (600)

Prolyl 4-hydroxylase 0621Negative 174 (972) 146 (967) 23 (1000) 5 (1000)Positive 5 (28) 5 (33) 0 (00) 0 (00)

FAP120572 0794Negative 166 (927) 140 (927) 21 (913) 5 (1000)Positive 13 (73) 11 (73) 2 (87) 0 (00)

S100A4 0571Negative 93 (520) 81 (536) 10 (435) 2 (400)Positive 86 (480) 70 (464) 13 (565) 3 (600)

PDGFR120572 0738Negative 145 (810) 121 (801) 20 (870) 4 (800)Positive 34 (190) 30 (199) 3 (130) 1 (200)

PDGFR120573 NANegative 179 (1000) 151 (1000) 23 (1000) 5 (1000)Positive 0 (00) 0 (00) 0 (00) 0 (00)

NG2 0216Negative 60 (335) 52 (344) 5 (217) 3 (600)Positive 119 (665) 99 (656) 18 (783) 2 (400)

lowastSeventeen tumors without an epithelial component were excludedPT phyllodes tumor

and PT [19] efficient treatment for fibroepithelial tumorsmight be achieved by targeting the fibrous stroma Howeverthere are no studies on the expression of CAF-related pro-teins in PT and as PT shows heterogeneous tumor stromalfeatures the expression of CAF-related proteins is predictedto vary in each type

In this study we performed immunohistochemical stain-ing for CAF-related proteins focusing on the stromal com-ponent Our results showed that the expression of PDGFR120572and PDGFR120573 increased with increasing PT histologic gradePlatelet-derived growth factor is a major mitogen for severalcell types including connective tissue cells that is activatedby binding to two protein tyrosine kinase receptors (PDGFR120572and PDGFR120573) PDGF signaling in tumor cells induces pointmutations amplification and translocations which stimulateautocrine stimulatory loops [6 21] For example PDGFR120572and 120573 are two well-known receptors that participate in breastcancer progression [22] In addition in breast cancer thedesmoplastic response appears to be mediated by PDGF-AAsignaling in PDGFR120572 type CAFs [23] Desmoplasia refers tothe growth of fibrous or connective tissue near the tumorand is increased in tumors with aggressive properties Theresults from our study revealed that higher grade PT withmore active fibroblasts showed higher expression of PDGFR120572and 120573 suggesting that fibroblasts with a CAF phenotype areassociated with PT progression

Stromal PDGFR120572 expression was related to the prognosisof patients with PT Furthermore subgroup analysis inmalignant PT showed that stromal PDGFR120573 expression wasassociated with shorter overall survival and although statis-tical significance was not noted there was also a trend forincreased stromal PDGFR120572 expression In previous studies ofglioma [24] squamous cell carcinoma of the head and neck[25] colorectal cancer [26] pancreatic cancer [27] and T celllymphoma [28] PDGFR activation induced the intracellularsignaling pathway and promoted cell migration invasionsurvival and proliferation [29] Similarly PDGFR activationis related to lymphatic metastasis in pancreatic cancer [30]and gastric cancer [31] suggesting that PDGFR might beused as a prognostic marker in malignant PT This should bevalidated in future studies

We found that expression of FAP120572 in stroma was associ-ated with distant metastasis Fibroblast activation protein isexpressed by reactive CAFs in tumor stroma or granulationtissue and is known to be involved in wound healing Theexpression of FAP in CAFs is reported in various carcinomasand is used as an important marker of CAF [32 33] In ameta-analysis that analyzed the clinical implication of FAPoverexpression in solid tumors of colorectal cancer pan-creatic adenocarcinoma non-small cell lung cancer breastcancer medullary thyroid carcinoma and oral squamous cellcarcinoma high FAP expression was related to the risk of

Disease Markers 5

Benign Borderline Malignant

Podoplanin

FAP120572

S100A4

NG2

HampE stain

E S E

SE

S

Prolyl 4-hydroxylase

PDGFR120572

PDGFR120573

Figure 1 Expression of CAF-related proteins according to the histologic grade of phyllodes tumorThe stromal expression of PDGFR120572 and 120573increased with increasing phyllodes tumor histologic grade The yellow circle indicates epithelium and the white circle indicates the stromaCAF cancer-associated fibroblast HampE hematoxylin and eosin stain FAP fibroblast activation protein PDGFR platelet-derived growthfactor receptor NG2 chondroitin sulfate proteoglycan

6 Disease Markers

Table 4 Expression of CAF-related proteins in the stromal component of phyllodes tumor according to histologic grade

Parameters Total 119899 = 196 (100) PT benign 119899 = 153(100)

PT borderline 119899 = 27(100)

PT malignant 119899 = 16(100) 119901 value

Podoplanin 0728Negative 52 (265) 41 (268) 8 (296) 3 (188)Positive 144 (735) 112 (732) 19 (704) 13 (812)

Prolyl 4-hydroxylase 0563Negative 192 (980) 149 (974) 27 (1000) 16 (1000)Positive 4 (20) 4 (26) 0 (00) 0 (00)

FAP120572 0105Negative 188 (959) 149 (974) 25 (926) 14 (875)Positive 8 (41) 4 (26) 2 (74) 2 (125)

S100A4 0104Negative 65 (332) 56 (366) 7 (259) 2 (125)Positive 131 (668) 97 (634) 20 (741) 14 (875)

PDGFR120572 0003Negative 179 (913) 142 (928) 26 (963) 11 (688)Positive 17 (87) 11 (72) 1 (37) 5 (312)

PDGFR120573 0034Negative 80 (408) 69 (451) 5 (185) 6 (375)Positive 116 (592) 84 (549) 22 (815) 10 (625)

NG2 0695Negative 93 (474) 75 (490) 11 (407) 7 (438)Positive 103 (526) 78 (510) 16 (593) 9 (562)

PT phyllodes tumor

182

66 20

50

100

150

200

No distant metastasis Distant metastasis

FAP120572 (S) negativeFAP120572 (S) positive

p = 0002

Figure 2 Correlation between stromal expression of fibroblastactivation protein (FAP)120572 in phyllodes tumor anddistantmetastasis

distant metastasis (OR 256) [34] showing similar results toour study FAP regulates proteolysis of the extracellular cell

matrix in tumor stroma causing stromal cell proliferationand invasiveness Therefore it could be speculated thattumors with high expression of FAP120572 are prone to metastasisto distant organs [35 36]

The clinical significance of this study is the role of CAFsas a potential therapeutic target Sunitinib a US Food andDrug Administration- (FDA-) approved PDGFR inhibitor iscurrently used in the treatment of advanced renal cell carci-noma [37] advanced progressive pancreatic neuroendocrinetumor [38] and advanced radioiodine refractory thyroidcarcinoma [39] Furthermore ongoing preclinical trials aretesting the therapeutic effect of another PDGFR inhibitorimatinib mesylate in gastrointestinal stromal tumor [40 41]and in vitro and in vivo studies of malignant peripheral nervesheath tumors have shown promising results [42] Thereforeinhibition of PDGFR in PT might be a potential therapeuticstrategy However further investigation regarding the effectof these inhibitors in PT is required

Taken together our data show that expression ofPDGFR120572 and PDGFR120573 in the stromal component increasedwith increased PT histologic grade Also stromal expressionof PDGFR was associated with shorter overall survivalOverall the expression of CAF-related proteins is related tothe histologic grade of breast PT and PDGFR inhibitors inparticular have potential as novel treatments for PT

Disease Markers 7

Table 5 Univariate analysis of the impact of CAF-related proteins in the stromal component of phyllodes tumor on patient prognosis usingthe log-rank test

Parameters Number of patientsTotalrecurrencemetastasis

Disease-free survival Overall survivalMedian survival (95

CI) months 119901 value Median survival(95 CI) months 119901 value

Podoplanin 0936 0494Negative 5253 160 (146ndash174) 166 (155ndash178)Positive 144135 166 (158ndash175) 177 (171ndash182)

Prolyl 4-hydroxylase na naNegative 192188 na naPositive 400 na na

FAP120572 0099 0233Negative 188167 164 (157ndash171) 172 (167ndash177)Positive 821 131 (71ndash191) 162 (123ndash200)

S100A4 0607 0792Negative 6553 163 (152ndash174) 168 (159ndash177)Positive 131135 165 (155ndash174) 176 (170ndash182)

PDGFR120572 0698 0002Negative 179165 167 (159ndash174) 177 (173ndash182)Positive 1723 121 (102ndash140) 113 (90ndash136)

PDGFR120573 0394 0087Negative 8061 164 (154ndash174) 174 (170ndash178)Positive 116127 163 (153ndash173) 172 (164ndash179)

NG2 0830 0216Negative 9382 145 (136ndash154) 155 (151ndash160)Positive 103106 165 (155ndash175) 172 (164ndash180)

Table 6 Multivariate analysis of disease-free and overall survival in patients with phyllodes tumors

Included factor Disease-free survival Overall survivalHazard ratio 95 CI 119901 value Hazard ratio 95 CI 119901 value

Histologic grade 0002 NABenign versus

borderlinemalignant 7990 2196ndash2907 NA NA

Stromal cellularity 0597 0467Mild versus

moderatemarked 0625 0109ndash3569 2862 0169ndash4860

Stromal atypia 0886 0708Mild versus

moderatemarked 0881 0158ndash4925 0630 0056ndash7049

Stromal mitosis 0854 04370ndash410 HPF versusgt410 HPF 0782 0057ndash1081 0210 0004ndash1070

Stromal overgrowth 0029 0006Absent versus present 7288 1225ndash4335 5810 3116ndash1083

Tumor margin 0275 0187Circumscribed versus

infiltrative 0468 0120ndash1830 0321 0060ndash1732

PDGFR120572 (stromal) 0867 0050Negative versus

positive 0879 0193ndash4008 5486 1003ndash3001

HPF high-power field

8 Disease Markers

PDGFR120572 (S) negative

PDGFR120572 (S) positive

Phyllodes tumor

p = 0002

0

02

04

06

08

10

Cum

ulat

ive o

vera

ll su

rviv

al

100 150 200500Months

PDGFR120572 (S) negative

(a)

PDGFR120572 (S) negative

PDGFR120572 (S) positive

Malignant phyllodes tumor

p = 0052

0

02

04

06

08

10

Cum

ulat

ive o

vera

ll su

rviv

al

100 1257525 500Months(b)

Malignant phyllodes tumor

p = 0041

100 1257525 500Months

0

02

04

06

08

10

Cum

ulat

ive o

vera

ll su

rviv

al

PDGFR120573 (S) negative

PDGFR120573 (S) positive

(c)Figure 3 Overall survival curves according to the status of CAF-related proteins in stromal component of phyllodes tumor (a) Univariateanalysis showed that stromal expression of PDGFR120572 was associated with shorter overall survival in phyllodes tumor (b) Stromal expressionof PDGFR120572 was associated with shorter overall survival in malignant phyllodes tumor although statistical significance was not reachedregarding PDGFR120572 expression (c) Stromal expression of PDGFR120573was associated with shorter overall survival inmalignant phyllodes tumorCAF cancer-associated fibroblast PDGFR platelet-derived growth factor receptor

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was supported by a grant from the NationalRampD Program for Cancer Control Ministry of Healthamp Welfare Republic of Korea (1420080) Additionally it

was supported by Basic Science Research Program throughthe National Research Foundation of Korea (NRF) fundedby the Ministry of Science ICT and Future Planning(2015R1A1A1A05001209)

References

[1] O E Franco A K Shaw D W Strand and S W HaywardldquoCancer associated fibroblasts in cancer pathogenesisrdquo Seminarsin Cell and Developmental Biology vol 21 no 1 pp 33ndash39 2010

Disease Markers 9

[2] A Ostman ldquoCancer-associated fibroblasts recent develop-ments and emerging challengesrdquo Seminars in Cancer Biologyvol 25 pp 1ndash2 2014

[3] A Desmouliere C Guyot and G Gabbiani ldquoThe stromareaction myofibroblast a key player in the control of tumor cellbehaviorrdquo International Journal of Developmental Biology vol48 no 5-6 pp 509ndash517 2004

[4] O De Wever Q-D Nguyen L Van Hoorde et al ldquoTenascin-C and SFHGF produced by myofibroblasts in vitro provideconvergent pro-invasive signals to human colon cancer cellsthrough RhoA and Racrdquo FASEB Journal vol 18 no 9 pp 1016ndash1018 2004

[5] H Sugimoto TMMundelMWKieran andRKalluri ldquoIden-tification of fibroblast heterogeneity in the tumor microenvi-ronmentrdquo Cancer Biology and Therapy vol 5 no 12 pp 1640ndash1646 2006

[6] K Pietras T Sjoblom K Rubin C-H Heldin and A OstmanldquoPDGF receptors as cancer drug targetsrdquo Cancer Cell vol 3 no5 pp 439ndash443 2003

[7] M Kraman P J Bambrough J N Arnold et al ldquoSuppressionof antitumor immunity by stromal cells expressing fibroblastactivation protein-120572rdquo Science vol 330 no 6005 pp 827ndash8302010

[8] A Kawase G Ishii K Nagai et al ldquoPodoplanin expression bycancer associated fibroblasts predicts poor prognosis of lungadenocarcinomardquo International Journal of Cancer vol 123 no5 pp 1053ndash1059 2008

[9] Y Kojima A Acar E N Eaton et al ldquoAutocrine TGF-120573 andstromal cell-derived factor-1 (SDF-1) signaling drives the evo-lution of tumor-promoting mammary stromal myofibroblastsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 107 no 46 pp 20009ndash20014 2010

[10] E Cortez P Roswall and K Pietras ldquoFunctional subsetsof mesenchymal cell types in the tumor microenvironmentrdquoSeminars in Cancer Biology vol 25 pp 3ndash9 2014

[11] F A Tavassoli and P Devilee World Heath OrganizationClassification of Tumors IARC Press Lyon France 2003

[12] S Dacic S Kounelis E Kouri andMW Jones ldquoImmunohisto-chemical profile of cystosarcoma phyllodes of the breast a studyof 23 casesrdquo Breast Journal vol 8 no 6 pp 376ndash381 2002

[13] B Anderson T Lawton C Lehman and R Moe ldquoPhyllodestumorrdquo in Disease of the Breast M Morrow and C OsborneEds pp 991ndash1006 Lippincott Williams amp Wilkins Philadel-phia 2004

[14] J Ben hassouna T Damak A Gamoudi et al ldquoPhyllodestumors of the breast a case series of 106 patientsrdquo AmericanJournal of Surgery vol 192 no 2 pp 141ndash147 2006

[15] J Mitus M Reinfuss J W Mitus et al ldquoMalignant phyllodestumor of the breast treatment and prognosisrdquo Breast Journalvol 20 no 6 pp 639ndash644 2014

[16] P-H Tan T Jayabaskar K-L Chuah et al ldquoPhyllodes tumors ofthe breast the role of pathologic parametersrdquo American Journalof Clinical Pathology vol 123 no 4 pp 529ndash540 2005

[17] A V Barrio B D Clark J I Goldberg et al ldquoClinicopathologicfeatures and long-term outcomes of 293 phyllodes tumors of thebreastrdquo Annals of Surgical Oncology vol 14 no 10 pp 2961ndash2970 2007

[18] F I Aranda J B Laforga and J I Lopez ldquoPhyllodes tumorof the breast An immunohistochemical study of 28 caseswith special attention to the role of myofibroblastsrdquo PathologyResearch and Practice vol 190 no 5 pp 474ndash481 1994

[19] J S Silverman and A Tamsen ldquoMammary fibroadenomaand some phyllodes tumour stroma are composed of CD34+fibroblasts and factor XIIIa+ dendrophagesrdquo Histopathologyvol 29 no 5 pp 411ndash419 1996

[20] K Y Won G Y Kim Y W Kim J Y Song and S-J LimldquoClinicopathologic correlation of beclin-1 and bcl-2 expressionin human breast cancerrdquo Human Pathology vol 41 no 1 pp107ndash112 2010

[21] J Andrae R Gallini and C Betsholtz ldquoRole of platelet-derived growth factors in physiology and medicinerdquo Genes andDevelopment vol 22 no 10 pp 1276ndash1312 2008

[22] F Meng C L Speyer B Zhang et al ldquoPDGFR120572 and 120573 playcritical roles in mediating Foxq1-driven breast cancer stemnessand chemoresistancerdquo Cancer Research vol 75 no 3 pp 584ndash593 2015

[23] Z-M Shao M Nguyen and S H Barsky ldquoHuman breastcarcinoma desmoplasia is PDGF initiatedrdquo Oncogene vol 19no 38 pp 4337ndash4345 2000

[24] I Nazarenko S-M Hede X He et al ldquoPDGF and PDGFreceptors in gliomardquoUpsala Journal of Medical Sciences vol 117no 2 pp 99ndash112 2012

[25] J D Schultz S Rotunno F Riedel et al ldquoSynergistic effectsof imatinib and carboplatin on VEGF PDGF and PDGF-R120572120573expression in squamous cell carcinoma of the head and neck invitrordquo International Journal of Oncology vol 38 no 4 pp 1001ndash1012 2011

[26] E J A Steller D A Raats J Koster et al ldquoPDGFRB pro-motes liver metastasis formation of mesenchymal-like colorec-tal tumor cellsrdquoNeoplasia (United States) vol 15 no 2 pp 204ndash217 2013

[27] H Kurahara K Maemura Y Mataki M Sakoda H Shinchiand S Natsugoe ldquoImpact of p53 and PDGFR-120573 expression onmetastasis and prognosis of patients with pancreatic cancerrdquoWorld Journal of Surgery vol 40 no 8 pp 1977ndash1984 2016

[28] P P Piccaluga M Rossi C Agostinelli et al ldquoPlatelet-derivedgrowth factor alpha mediates the proliferation of peripheralT-cell lymphoma cells via an autocrine regulatory pathwayrdquoLeukemia vol 28 no 8 pp 1687ndash1697 2014

[29] C-H Heldin and B Westermark ldquoMechanism of action andin vivo role of platelet-derived growth factorrdquo PhysiologicalReviews vol 79 no 4 pp 1283ndash1316 1999

[30] S Weissmueller E Manchado M Saborowski et alldquoMutant p53 drives pancreatic cancer metastasis throughcell-autonomous PDGF receptor 120573 signalingrdquo Cell vol 157 no2 pp 382ndash394 2014

[31] M Kodama Y Kitadai T Sumida et al ldquoExpression ofplatelet-derived growth factor (PDGF)-B and PDGF-receptor120573 is associated with lymphatic metastasis in human gastriccarcinomardquo Cancer Science vol 101 no 9 pp 1984ndash1989 2010

[32] M Meng W Wang J Yan et al ldquoImmunization of stromal celltargeting fibroblast activation protein providing immunother-apy to breast cancer mouse modelrdquo Tumor Biology vol 37 no8 pp 10317ndash10327 2016

[33] H Wang Q Wu Z Liu et al ldquoDownregulation of FAPsuppresses cell proliferation and metastasis throughPTENPI3KAKT and Ras-ERK signaling in oral squamouscell carcinomardquo Cell Death and Disease vol 5 no 4 Article IDe1155 2014

[34] F Liu L Qi B Liu et al ldquoFibroblast activation proteinoverexpression and clinical implications in solid tumors ameta-analysisrdquo PLoS ONE vol 10 no 3 Article ID e01166832015

10 Disease Markers

[35] D Yuan B Liu K Liu G Zhu Z Dai and Y Xie ldquoOverexpres-sion of fibroblast activation protein and its clinical implicationsin patients with osteosarcomardquo Journal of Surgical Oncologyvol 108 no 3 pp 157ndash162 2013

[36] M A Goscinski Z Suo V A Floslashrenes L Vlatkovic J MNesland and K-E Giercksky ldquoFAP-120572 and uPA show differentexpression patterns in premalignant and malignant esophageallesionsrdquo Ultrastructural Pathology vol 32 no 3 pp 89ndash962008

[37] R C Coelho T Reinert F Campos et al ldquoSunitinib treatmentin patients with advanced renal cell cancer the BrazilianNational Cancer Institute (INCA) experiencerdquo InternationalBrazilian Journal of Urology vol 42 no 4 pp 694ndash703 2016

[38] ldquoEfficiency of sunitinib in Chinese patients with advancedprogressive pancreatic neuroendocrine tumorrdquo Zhongguo YiXue Ke Xue Yuan Xue Bao vol 38 no 3 pp 300ndash304 2016

[39] V Atallah A Hocquelet C Do Cao et al ldquoActivity and safetyof sunitinib in patients with advanced radioiodine refractorythyroid carcinoma a retrospective analysis of 57 patientsrdquoThyroid vol 26 no 8 pp 1085ndash1092 2016

[40] E Y Moawad ldquoPredicting effectiveness of imatinib mesylatein tumors expressing platelet-derived growth factors (PDGF-AA PDGF-BB) stem cell factor ligands and their respectivereceptors (PDGFR-120572 PDGFR-120573 and c-kit)rdquo Journal of Gas-trointestinal Cancer vol 46 no 3 pp 272ndash283 2015

[41] Y Hayashi M R Bardsley Y Toyomasu et al ldquoPlatelet-derived growth factor receptor-120572 regulates proliferation ofgastrointestinal stromal tumor cells with mutations in KIT bystabilizing ETV1rdquo Gastroenterology vol 149 no 2 pp 420ndash432e16 2015

[42] J Ohishi M Aoki K Nabeshima et al ldquoImatinib mesylateinhibits cell growth of malignant peripheral nerve sheathtumors in vitro and in vivo through suppression of PDGFR-120573rdquoBMC Cancer vol 13 article 224 2013

Disease Markers 5

Benign Borderline Malignant

Podoplanin

FAP120572

S100A4

NG2

HampE stain

E S E

SE

S

Prolyl 4-hydroxylase

PDGFR120572

PDGFR120573

Figure 1 Expression of CAF-related proteins according to the histologic grade of phyllodes tumorThe stromal expression of PDGFR120572 and 120573increased with increasing phyllodes tumor histologic grade The yellow circle indicates epithelium and the white circle indicates the stromaCAF cancer-associated fibroblast HampE hematoxylin and eosin stain FAP fibroblast activation protein PDGFR platelet-derived growthfactor receptor NG2 chondroitin sulfate proteoglycan

6 Disease Markers

Table 4 Expression of CAF-related proteins in the stromal component of phyllodes tumor according to histologic grade

Parameters Total 119899 = 196 (100) PT benign 119899 = 153(100)

PT borderline 119899 = 27(100)

PT malignant 119899 = 16(100) 119901 value

Podoplanin 0728Negative 52 (265) 41 (268) 8 (296) 3 (188)Positive 144 (735) 112 (732) 19 (704) 13 (812)

Prolyl 4-hydroxylase 0563Negative 192 (980) 149 (974) 27 (1000) 16 (1000)Positive 4 (20) 4 (26) 0 (00) 0 (00)

FAP120572 0105Negative 188 (959) 149 (974) 25 (926) 14 (875)Positive 8 (41) 4 (26) 2 (74) 2 (125)

S100A4 0104Negative 65 (332) 56 (366) 7 (259) 2 (125)Positive 131 (668) 97 (634) 20 (741) 14 (875)

PDGFR120572 0003Negative 179 (913) 142 (928) 26 (963) 11 (688)Positive 17 (87) 11 (72) 1 (37) 5 (312)

PDGFR120573 0034Negative 80 (408) 69 (451) 5 (185) 6 (375)Positive 116 (592) 84 (549) 22 (815) 10 (625)

NG2 0695Negative 93 (474) 75 (490) 11 (407) 7 (438)Positive 103 (526) 78 (510) 16 (593) 9 (562)

PT phyllodes tumor

182

66 20

50

100

150

200

No distant metastasis Distant metastasis

FAP120572 (S) negativeFAP120572 (S) positive

p = 0002

Figure 2 Correlation between stromal expression of fibroblastactivation protein (FAP)120572 in phyllodes tumor anddistantmetastasis

distant metastasis (OR 256) [34] showing similar results toour study FAP regulates proteolysis of the extracellular cell

matrix in tumor stroma causing stromal cell proliferationand invasiveness Therefore it could be speculated thattumors with high expression of FAP120572 are prone to metastasisto distant organs [35 36]

The clinical significance of this study is the role of CAFsas a potential therapeutic target Sunitinib a US Food andDrug Administration- (FDA-) approved PDGFR inhibitor iscurrently used in the treatment of advanced renal cell carci-noma [37] advanced progressive pancreatic neuroendocrinetumor [38] and advanced radioiodine refractory thyroidcarcinoma [39] Furthermore ongoing preclinical trials aretesting the therapeutic effect of another PDGFR inhibitorimatinib mesylate in gastrointestinal stromal tumor [40 41]and in vitro and in vivo studies of malignant peripheral nervesheath tumors have shown promising results [42] Thereforeinhibition of PDGFR in PT might be a potential therapeuticstrategy However further investigation regarding the effectof these inhibitors in PT is required

Taken together our data show that expression ofPDGFR120572 and PDGFR120573 in the stromal component increasedwith increased PT histologic grade Also stromal expressionof PDGFR was associated with shorter overall survivalOverall the expression of CAF-related proteins is related tothe histologic grade of breast PT and PDGFR inhibitors inparticular have potential as novel treatments for PT

Disease Markers 7

Table 5 Univariate analysis of the impact of CAF-related proteins in the stromal component of phyllodes tumor on patient prognosis usingthe log-rank test

Parameters Number of patientsTotalrecurrencemetastasis

Disease-free survival Overall survivalMedian survival (95

CI) months 119901 value Median survival(95 CI) months 119901 value

Podoplanin 0936 0494Negative 5253 160 (146ndash174) 166 (155ndash178)Positive 144135 166 (158ndash175) 177 (171ndash182)

Prolyl 4-hydroxylase na naNegative 192188 na naPositive 400 na na

FAP120572 0099 0233Negative 188167 164 (157ndash171) 172 (167ndash177)Positive 821 131 (71ndash191) 162 (123ndash200)

S100A4 0607 0792Negative 6553 163 (152ndash174) 168 (159ndash177)Positive 131135 165 (155ndash174) 176 (170ndash182)

PDGFR120572 0698 0002Negative 179165 167 (159ndash174) 177 (173ndash182)Positive 1723 121 (102ndash140) 113 (90ndash136)

PDGFR120573 0394 0087Negative 8061 164 (154ndash174) 174 (170ndash178)Positive 116127 163 (153ndash173) 172 (164ndash179)

NG2 0830 0216Negative 9382 145 (136ndash154) 155 (151ndash160)Positive 103106 165 (155ndash175) 172 (164ndash180)

Table 6 Multivariate analysis of disease-free and overall survival in patients with phyllodes tumors

Included factor Disease-free survival Overall survivalHazard ratio 95 CI 119901 value Hazard ratio 95 CI 119901 value

Histologic grade 0002 NABenign versus

borderlinemalignant 7990 2196ndash2907 NA NA

Stromal cellularity 0597 0467Mild versus

moderatemarked 0625 0109ndash3569 2862 0169ndash4860

Stromal atypia 0886 0708Mild versus

moderatemarked 0881 0158ndash4925 0630 0056ndash7049

Stromal mitosis 0854 04370ndash410 HPF versusgt410 HPF 0782 0057ndash1081 0210 0004ndash1070

Stromal overgrowth 0029 0006Absent versus present 7288 1225ndash4335 5810 3116ndash1083

Tumor margin 0275 0187Circumscribed versus

infiltrative 0468 0120ndash1830 0321 0060ndash1732

PDGFR120572 (stromal) 0867 0050Negative versus

positive 0879 0193ndash4008 5486 1003ndash3001

HPF high-power field

8 Disease Markers

PDGFR120572 (S) negative

PDGFR120572 (S) positive

Phyllodes tumor

p = 0002

0

02

04

06

08

10

Cum

ulat

ive o

vera

ll su

rviv

al

100 150 200500Months

PDGFR120572 (S) negative

(a)

PDGFR120572 (S) negative

PDGFR120572 (S) positive

Malignant phyllodes tumor

p = 0052

0

02

04

06

08

10

Cum

ulat

ive o

vera

ll su

rviv

al

100 1257525 500Months(b)

Malignant phyllodes tumor

p = 0041

100 1257525 500Months

0

02

04

06

08

10

Cum

ulat

ive o

vera

ll su

rviv

al

PDGFR120573 (S) negative

PDGFR120573 (S) positive

(c)Figure 3 Overall survival curves according to the status of CAF-related proteins in stromal component of phyllodes tumor (a) Univariateanalysis showed that stromal expression of PDGFR120572 was associated with shorter overall survival in phyllodes tumor (b) Stromal expressionof PDGFR120572 was associated with shorter overall survival in malignant phyllodes tumor although statistical significance was not reachedregarding PDGFR120572 expression (c) Stromal expression of PDGFR120573was associated with shorter overall survival inmalignant phyllodes tumorCAF cancer-associated fibroblast PDGFR platelet-derived growth factor receptor

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was supported by a grant from the NationalRampD Program for Cancer Control Ministry of Healthamp Welfare Republic of Korea (1420080) Additionally it

was supported by Basic Science Research Program throughthe National Research Foundation of Korea (NRF) fundedby the Ministry of Science ICT and Future Planning(2015R1A1A1A05001209)

References

[1] O E Franco A K Shaw D W Strand and S W HaywardldquoCancer associated fibroblasts in cancer pathogenesisrdquo Seminarsin Cell and Developmental Biology vol 21 no 1 pp 33ndash39 2010

Disease Markers 9

[2] A Ostman ldquoCancer-associated fibroblasts recent develop-ments and emerging challengesrdquo Seminars in Cancer Biologyvol 25 pp 1ndash2 2014

[3] A Desmouliere C Guyot and G Gabbiani ldquoThe stromareaction myofibroblast a key player in the control of tumor cellbehaviorrdquo International Journal of Developmental Biology vol48 no 5-6 pp 509ndash517 2004

[4] O De Wever Q-D Nguyen L Van Hoorde et al ldquoTenascin-C and SFHGF produced by myofibroblasts in vitro provideconvergent pro-invasive signals to human colon cancer cellsthrough RhoA and Racrdquo FASEB Journal vol 18 no 9 pp 1016ndash1018 2004

[5] H Sugimoto TMMundelMWKieran andRKalluri ldquoIden-tification of fibroblast heterogeneity in the tumor microenvi-ronmentrdquo Cancer Biology and Therapy vol 5 no 12 pp 1640ndash1646 2006

[6] K Pietras T Sjoblom K Rubin C-H Heldin and A OstmanldquoPDGF receptors as cancer drug targetsrdquo Cancer Cell vol 3 no5 pp 439ndash443 2003

[7] M Kraman P J Bambrough J N Arnold et al ldquoSuppressionof antitumor immunity by stromal cells expressing fibroblastactivation protein-120572rdquo Science vol 330 no 6005 pp 827ndash8302010

[8] A Kawase G Ishii K Nagai et al ldquoPodoplanin expression bycancer associated fibroblasts predicts poor prognosis of lungadenocarcinomardquo International Journal of Cancer vol 123 no5 pp 1053ndash1059 2008

[9] Y Kojima A Acar E N Eaton et al ldquoAutocrine TGF-120573 andstromal cell-derived factor-1 (SDF-1) signaling drives the evo-lution of tumor-promoting mammary stromal myofibroblastsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 107 no 46 pp 20009ndash20014 2010

[10] E Cortez P Roswall and K Pietras ldquoFunctional subsetsof mesenchymal cell types in the tumor microenvironmentrdquoSeminars in Cancer Biology vol 25 pp 3ndash9 2014

[11] F A Tavassoli and P Devilee World Heath OrganizationClassification of Tumors IARC Press Lyon France 2003

[12] S Dacic S Kounelis E Kouri andMW Jones ldquoImmunohisto-chemical profile of cystosarcoma phyllodes of the breast a studyof 23 casesrdquo Breast Journal vol 8 no 6 pp 376ndash381 2002

[13] B Anderson T Lawton C Lehman and R Moe ldquoPhyllodestumorrdquo in Disease of the Breast M Morrow and C OsborneEds pp 991ndash1006 Lippincott Williams amp Wilkins Philadel-phia 2004

[14] J Ben hassouna T Damak A Gamoudi et al ldquoPhyllodestumors of the breast a case series of 106 patientsrdquo AmericanJournal of Surgery vol 192 no 2 pp 141ndash147 2006

[15] J Mitus M Reinfuss J W Mitus et al ldquoMalignant phyllodestumor of the breast treatment and prognosisrdquo Breast Journalvol 20 no 6 pp 639ndash644 2014

[16] P-H Tan T Jayabaskar K-L Chuah et al ldquoPhyllodes tumors ofthe breast the role of pathologic parametersrdquo American Journalof Clinical Pathology vol 123 no 4 pp 529ndash540 2005

[17] A V Barrio B D Clark J I Goldberg et al ldquoClinicopathologicfeatures and long-term outcomes of 293 phyllodes tumors of thebreastrdquo Annals of Surgical Oncology vol 14 no 10 pp 2961ndash2970 2007

[18] F I Aranda J B Laforga and J I Lopez ldquoPhyllodes tumorof the breast An immunohistochemical study of 28 caseswith special attention to the role of myofibroblastsrdquo PathologyResearch and Practice vol 190 no 5 pp 474ndash481 1994

[19] J S Silverman and A Tamsen ldquoMammary fibroadenomaand some phyllodes tumour stroma are composed of CD34+fibroblasts and factor XIIIa+ dendrophagesrdquo Histopathologyvol 29 no 5 pp 411ndash419 1996

[20] K Y Won G Y Kim Y W Kim J Y Song and S-J LimldquoClinicopathologic correlation of beclin-1 and bcl-2 expressionin human breast cancerrdquo Human Pathology vol 41 no 1 pp107ndash112 2010

[21] J Andrae R Gallini and C Betsholtz ldquoRole of platelet-derived growth factors in physiology and medicinerdquo Genes andDevelopment vol 22 no 10 pp 1276ndash1312 2008

[22] F Meng C L Speyer B Zhang et al ldquoPDGFR120572 and 120573 playcritical roles in mediating Foxq1-driven breast cancer stemnessand chemoresistancerdquo Cancer Research vol 75 no 3 pp 584ndash593 2015

[23] Z-M Shao M Nguyen and S H Barsky ldquoHuman breastcarcinoma desmoplasia is PDGF initiatedrdquo Oncogene vol 19no 38 pp 4337ndash4345 2000

[24] I Nazarenko S-M Hede X He et al ldquoPDGF and PDGFreceptors in gliomardquoUpsala Journal of Medical Sciences vol 117no 2 pp 99ndash112 2012

[25] J D Schultz S Rotunno F Riedel et al ldquoSynergistic effectsof imatinib and carboplatin on VEGF PDGF and PDGF-R120572120573expression in squamous cell carcinoma of the head and neck invitrordquo International Journal of Oncology vol 38 no 4 pp 1001ndash1012 2011

[26] E J A Steller D A Raats J Koster et al ldquoPDGFRB pro-motes liver metastasis formation of mesenchymal-like colorec-tal tumor cellsrdquoNeoplasia (United States) vol 15 no 2 pp 204ndash217 2013

[27] H Kurahara K Maemura Y Mataki M Sakoda H Shinchiand S Natsugoe ldquoImpact of p53 and PDGFR-120573 expression onmetastasis and prognosis of patients with pancreatic cancerrdquoWorld Journal of Surgery vol 40 no 8 pp 1977ndash1984 2016

[28] P P Piccaluga M Rossi C Agostinelli et al ldquoPlatelet-derivedgrowth factor alpha mediates the proliferation of peripheralT-cell lymphoma cells via an autocrine regulatory pathwayrdquoLeukemia vol 28 no 8 pp 1687ndash1697 2014

[29] C-H Heldin and B Westermark ldquoMechanism of action andin vivo role of platelet-derived growth factorrdquo PhysiologicalReviews vol 79 no 4 pp 1283ndash1316 1999

[30] S Weissmueller E Manchado M Saborowski et alldquoMutant p53 drives pancreatic cancer metastasis throughcell-autonomous PDGF receptor 120573 signalingrdquo Cell vol 157 no2 pp 382ndash394 2014

[31] M Kodama Y Kitadai T Sumida et al ldquoExpression ofplatelet-derived growth factor (PDGF)-B and PDGF-receptor120573 is associated with lymphatic metastasis in human gastriccarcinomardquo Cancer Science vol 101 no 9 pp 1984ndash1989 2010

[32] M Meng W Wang J Yan et al ldquoImmunization of stromal celltargeting fibroblast activation protein providing immunother-apy to breast cancer mouse modelrdquo Tumor Biology vol 37 no8 pp 10317ndash10327 2016

[33] H Wang Q Wu Z Liu et al ldquoDownregulation of FAPsuppresses cell proliferation and metastasis throughPTENPI3KAKT and Ras-ERK signaling in oral squamouscell carcinomardquo Cell Death and Disease vol 5 no 4 Article IDe1155 2014

[34] F Liu L Qi B Liu et al ldquoFibroblast activation proteinoverexpression and clinical implications in solid tumors ameta-analysisrdquo PLoS ONE vol 10 no 3 Article ID e01166832015

10 Disease Markers

[35] D Yuan B Liu K Liu G Zhu Z Dai and Y Xie ldquoOverexpres-sion of fibroblast activation protein and its clinical implicationsin patients with osteosarcomardquo Journal of Surgical Oncologyvol 108 no 3 pp 157ndash162 2013

[36] M A Goscinski Z Suo V A Floslashrenes L Vlatkovic J MNesland and K-E Giercksky ldquoFAP-120572 and uPA show differentexpression patterns in premalignant and malignant esophageallesionsrdquo Ultrastructural Pathology vol 32 no 3 pp 89ndash962008

[37] R C Coelho T Reinert F Campos et al ldquoSunitinib treatmentin patients with advanced renal cell cancer the BrazilianNational Cancer Institute (INCA) experiencerdquo InternationalBrazilian Journal of Urology vol 42 no 4 pp 694ndash703 2016

[38] ldquoEfficiency of sunitinib in Chinese patients with advancedprogressive pancreatic neuroendocrine tumorrdquo Zhongguo YiXue Ke Xue Yuan Xue Bao vol 38 no 3 pp 300ndash304 2016

[39] V Atallah A Hocquelet C Do Cao et al ldquoActivity and safetyof sunitinib in patients with advanced radioiodine refractorythyroid carcinoma a retrospective analysis of 57 patientsrdquoThyroid vol 26 no 8 pp 1085ndash1092 2016

[40] E Y Moawad ldquoPredicting effectiveness of imatinib mesylatein tumors expressing platelet-derived growth factors (PDGF-AA PDGF-BB) stem cell factor ligands and their respectivereceptors (PDGFR-120572 PDGFR-120573 and c-kit)rdquo Journal of Gas-trointestinal Cancer vol 46 no 3 pp 272ndash283 2015

[41] Y Hayashi M R Bardsley Y Toyomasu et al ldquoPlatelet-derived growth factor receptor-120572 regulates proliferation ofgastrointestinal stromal tumor cells with mutations in KIT bystabilizing ETV1rdquo Gastroenterology vol 149 no 2 pp 420ndash432e16 2015

[42] J Ohishi M Aoki K Nabeshima et al ldquoImatinib mesylateinhibits cell growth of malignant peripheral nerve sheathtumors in vitro and in vivo through suppression of PDGFR-120573rdquoBMC Cancer vol 13 article 224 2013

6 Disease Markers

Table 4 Expression of CAF-related proteins in the stromal component of phyllodes tumor according to histologic grade

Parameters Total 119899 = 196 (100) PT benign 119899 = 153(100)

PT borderline 119899 = 27(100)

PT malignant 119899 = 16(100) 119901 value

Podoplanin 0728Negative 52 (265) 41 (268) 8 (296) 3 (188)Positive 144 (735) 112 (732) 19 (704) 13 (812)

Prolyl 4-hydroxylase 0563Negative 192 (980) 149 (974) 27 (1000) 16 (1000)Positive 4 (20) 4 (26) 0 (00) 0 (00)

FAP120572 0105Negative 188 (959) 149 (974) 25 (926) 14 (875)Positive 8 (41) 4 (26) 2 (74) 2 (125)

S100A4 0104Negative 65 (332) 56 (366) 7 (259) 2 (125)Positive 131 (668) 97 (634) 20 (741) 14 (875)

PDGFR120572 0003Negative 179 (913) 142 (928) 26 (963) 11 (688)Positive 17 (87) 11 (72) 1 (37) 5 (312)

PDGFR120573 0034Negative 80 (408) 69 (451) 5 (185) 6 (375)Positive 116 (592) 84 (549) 22 (815) 10 (625)

NG2 0695Negative 93 (474) 75 (490) 11 (407) 7 (438)Positive 103 (526) 78 (510) 16 (593) 9 (562)

PT phyllodes tumor

182

66 20

50

100

150

200

No distant metastasis Distant metastasis

FAP120572 (S) negativeFAP120572 (S) positive

p = 0002

Figure 2 Correlation between stromal expression of fibroblastactivation protein (FAP)120572 in phyllodes tumor anddistantmetastasis

distant metastasis (OR 256) [34] showing similar results toour study FAP regulates proteolysis of the extracellular cell

matrix in tumor stroma causing stromal cell proliferationand invasiveness Therefore it could be speculated thattumors with high expression of FAP120572 are prone to metastasisto distant organs [35 36]

The clinical significance of this study is the role of CAFsas a potential therapeutic target Sunitinib a US Food andDrug Administration- (FDA-) approved PDGFR inhibitor iscurrently used in the treatment of advanced renal cell carci-noma [37] advanced progressive pancreatic neuroendocrinetumor [38] and advanced radioiodine refractory thyroidcarcinoma [39] Furthermore ongoing preclinical trials aretesting the therapeutic effect of another PDGFR inhibitorimatinib mesylate in gastrointestinal stromal tumor [40 41]and in vitro and in vivo studies of malignant peripheral nervesheath tumors have shown promising results [42] Thereforeinhibition of PDGFR in PT might be a potential therapeuticstrategy However further investigation regarding the effectof these inhibitors in PT is required

Taken together our data show that expression ofPDGFR120572 and PDGFR120573 in the stromal component increasedwith increased PT histologic grade Also stromal expressionof PDGFR was associated with shorter overall survivalOverall the expression of CAF-related proteins is related tothe histologic grade of breast PT and PDGFR inhibitors inparticular have potential as novel treatments for PT

Disease Markers 7

Table 5 Univariate analysis of the impact of CAF-related proteins in the stromal component of phyllodes tumor on patient prognosis usingthe log-rank test

Parameters Number of patientsTotalrecurrencemetastasis

Disease-free survival Overall survivalMedian survival (95

CI) months 119901 value Median survival(95 CI) months 119901 value

Podoplanin 0936 0494Negative 5253 160 (146ndash174) 166 (155ndash178)Positive 144135 166 (158ndash175) 177 (171ndash182)

Prolyl 4-hydroxylase na naNegative 192188 na naPositive 400 na na

FAP120572 0099 0233Negative 188167 164 (157ndash171) 172 (167ndash177)Positive 821 131 (71ndash191) 162 (123ndash200)

S100A4 0607 0792Negative 6553 163 (152ndash174) 168 (159ndash177)Positive 131135 165 (155ndash174) 176 (170ndash182)

PDGFR120572 0698 0002Negative 179165 167 (159ndash174) 177 (173ndash182)Positive 1723 121 (102ndash140) 113 (90ndash136)

PDGFR120573 0394 0087Negative 8061 164 (154ndash174) 174 (170ndash178)Positive 116127 163 (153ndash173) 172 (164ndash179)

NG2 0830 0216Negative 9382 145 (136ndash154) 155 (151ndash160)Positive 103106 165 (155ndash175) 172 (164ndash180)

Table 6 Multivariate analysis of disease-free and overall survival in patients with phyllodes tumors

Included factor Disease-free survival Overall survivalHazard ratio 95 CI 119901 value Hazard ratio 95 CI 119901 value

Histologic grade 0002 NABenign versus

borderlinemalignant 7990 2196ndash2907 NA NA

Stromal cellularity 0597 0467Mild versus

moderatemarked 0625 0109ndash3569 2862 0169ndash4860

Stromal atypia 0886 0708Mild versus

moderatemarked 0881 0158ndash4925 0630 0056ndash7049

Stromal mitosis 0854 04370ndash410 HPF versusgt410 HPF 0782 0057ndash1081 0210 0004ndash1070

Stromal overgrowth 0029 0006Absent versus present 7288 1225ndash4335 5810 3116ndash1083

Tumor margin 0275 0187Circumscribed versus

infiltrative 0468 0120ndash1830 0321 0060ndash1732

PDGFR120572 (stromal) 0867 0050Negative versus

positive 0879 0193ndash4008 5486 1003ndash3001

HPF high-power field

8 Disease Markers

PDGFR120572 (S) negative

PDGFR120572 (S) positive

Phyllodes tumor

p = 0002

0

02

04

06

08

10

Cum

ulat

ive o

vera

ll su

rviv

al

100 150 200500Months

PDGFR120572 (S) negative

(a)

PDGFR120572 (S) negative

PDGFR120572 (S) positive

Malignant phyllodes tumor

p = 0052

0

02

04

06

08

10

Cum

ulat

ive o

vera

ll su

rviv

al

100 1257525 500Months(b)

Malignant phyllodes tumor

p = 0041

100 1257525 500Months

0

02

04

06

08

10

Cum

ulat

ive o

vera

ll su

rviv

al

PDGFR120573 (S) negative

PDGFR120573 (S) positive

(c)Figure 3 Overall survival curves according to the status of CAF-related proteins in stromal component of phyllodes tumor (a) Univariateanalysis showed that stromal expression of PDGFR120572 was associated with shorter overall survival in phyllodes tumor (b) Stromal expressionof PDGFR120572 was associated with shorter overall survival in malignant phyllodes tumor although statistical significance was not reachedregarding PDGFR120572 expression (c) Stromal expression of PDGFR120573was associated with shorter overall survival inmalignant phyllodes tumorCAF cancer-associated fibroblast PDGFR platelet-derived growth factor receptor

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was supported by a grant from the NationalRampD Program for Cancer Control Ministry of Healthamp Welfare Republic of Korea (1420080) Additionally it

was supported by Basic Science Research Program throughthe National Research Foundation of Korea (NRF) fundedby the Ministry of Science ICT and Future Planning(2015R1A1A1A05001209)

References

[1] O E Franco A K Shaw D W Strand and S W HaywardldquoCancer associated fibroblasts in cancer pathogenesisrdquo Seminarsin Cell and Developmental Biology vol 21 no 1 pp 33ndash39 2010

Disease Markers 9

[2] A Ostman ldquoCancer-associated fibroblasts recent develop-ments and emerging challengesrdquo Seminars in Cancer Biologyvol 25 pp 1ndash2 2014

[3] A Desmouliere C Guyot and G Gabbiani ldquoThe stromareaction myofibroblast a key player in the control of tumor cellbehaviorrdquo International Journal of Developmental Biology vol48 no 5-6 pp 509ndash517 2004

[4] O De Wever Q-D Nguyen L Van Hoorde et al ldquoTenascin-C and SFHGF produced by myofibroblasts in vitro provideconvergent pro-invasive signals to human colon cancer cellsthrough RhoA and Racrdquo FASEB Journal vol 18 no 9 pp 1016ndash1018 2004

[5] H Sugimoto TMMundelMWKieran andRKalluri ldquoIden-tification of fibroblast heterogeneity in the tumor microenvi-ronmentrdquo Cancer Biology and Therapy vol 5 no 12 pp 1640ndash1646 2006

[6] K Pietras T Sjoblom K Rubin C-H Heldin and A OstmanldquoPDGF receptors as cancer drug targetsrdquo Cancer Cell vol 3 no5 pp 439ndash443 2003

[7] M Kraman P J Bambrough J N Arnold et al ldquoSuppressionof antitumor immunity by stromal cells expressing fibroblastactivation protein-120572rdquo Science vol 330 no 6005 pp 827ndash8302010

[8] A Kawase G Ishii K Nagai et al ldquoPodoplanin expression bycancer associated fibroblasts predicts poor prognosis of lungadenocarcinomardquo International Journal of Cancer vol 123 no5 pp 1053ndash1059 2008

[9] Y Kojima A Acar E N Eaton et al ldquoAutocrine TGF-120573 andstromal cell-derived factor-1 (SDF-1) signaling drives the evo-lution of tumor-promoting mammary stromal myofibroblastsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 107 no 46 pp 20009ndash20014 2010

[10] E Cortez P Roswall and K Pietras ldquoFunctional subsetsof mesenchymal cell types in the tumor microenvironmentrdquoSeminars in Cancer Biology vol 25 pp 3ndash9 2014

[11] F A Tavassoli and P Devilee World Heath OrganizationClassification of Tumors IARC Press Lyon France 2003

[12] S Dacic S Kounelis E Kouri andMW Jones ldquoImmunohisto-chemical profile of cystosarcoma phyllodes of the breast a studyof 23 casesrdquo Breast Journal vol 8 no 6 pp 376ndash381 2002

[13] B Anderson T Lawton C Lehman and R Moe ldquoPhyllodestumorrdquo in Disease of the Breast M Morrow and C OsborneEds pp 991ndash1006 Lippincott Williams amp Wilkins Philadel-phia 2004

[14] J Ben hassouna T Damak A Gamoudi et al ldquoPhyllodestumors of the breast a case series of 106 patientsrdquo AmericanJournal of Surgery vol 192 no 2 pp 141ndash147 2006

[15] J Mitus M Reinfuss J W Mitus et al ldquoMalignant phyllodestumor of the breast treatment and prognosisrdquo Breast Journalvol 20 no 6 pp 639ndash644 2014

[16] P-H Tan T Jayabaskar K-L Chuah et al ldquoPhyllodes tumors ofthe breast the role of pathologic parametersrdquo American Journalof Clinical Pathology vol 123 no 4 pp 529ndash540 2005

[17] A V Barrio B D Clark J I Goldberg et al ldquoClinicopathologicfeatures and long-term outcomes of 293 phyllodes tumors of thebreastrdquo Annals of Surgical Oncology vol 14 no 10 pp 2961ndash2970 2007

[18] F I Aranda J B Laforga and J I Lopez ldquoPhyllodes tumorof the breast An immunohistochemical study of 28 caseswith special attention to the role of myofibroblastsrdquo PathologyResearch and Practice vol 190 no 5 pp 474ndash481 1994

[19] J S Silverman and A Tamsen ldquoMammary fibroadenomaand some phyllodes tumour stroma are composed of CD34+fibroblasts and factor XIIIa+ dendrophagesrdquo Histopathologyvol 29 no 5 pp 411ndash419 1996

[20] K Y Won G Y Kim Y W Kim J Y Song and S-J LimldquoClinicopathologic correlation of beclin-1 and bcl-2 expressionin human breast cancerrdquo Human Pathology vol 41 no 1 pp107ndash112 2010

[21] J Andrae R Gallini and C Betsholtz ldquoRole of platelet-derived growth factors in physiology and medicinerdquo Genes andDevelopment vol 22 no 10 pp 1276ndash1312 2008

[22] F Meng C L Speyer B Zhang et al ldquoPDGFR120572 and 120573 playcritical roles in mediating Foxq1-driven breast cancer stemnessand chemoresistancerdquo Cancer Research vol 75 no 3 pp 584ndash593 2015

[23] Z-M Shao M Nguyen and S H Barsky ldquoHuman breastcarcinoma desmoplasia is PDGF initiatedrdquo Oncogene vol 19no 38 pp 4337ndash4345 2000

[24] I Nazarenko S-M Hede X He et al ldquoPDGF and PDGFreceptors in gliomardquoUpsala Journal of Medical Sciences vol 117no 2 pp 99ndash112 2012

[25] J D Schultz S Rotunno F Riedel et al ldquoSynergistic effectsof imatinib and carboplatin on VEGF PDGF and PDGF-R120572120573expression in squamous cell carcinoma of the head and neck invitrordquo International Journal of Oncology vol 38 no 4 pp 1001ndash1012 2011

[26] E J A Steller D A Raats J Koster et al ldquoPDGFRB pro-motes liver metastasis formation of mesenchymal-like colorec-tal tumor cellsrdquoNeoplasia (United States) vol 15 no 2 pp 204ndash217 2013

[27] H Kurahara K Maemura Y Mataki M Sakoda H Shinchiand S Natsugoe ldquoImpact of p53 and PDGFR-120573 expression onmetastasis and prognosis of patients with pancreatic cancerrdquoWorld Journal of Surgery vol 40 no 8 pp 1977ndash1984 2016

[28] P P Piccaluga M Rossi C Agostinelli et al ldquoPlatelet-derivedgrowth factor alpha mediates the proliferation of peripheralT-cell lymphoma cells via an autocrine regulatory pathwayrdquoLeukemia vol 28 no 8 pp 1687ndash1697 2014

[29] C-H Heldin and B Westermark ldquoMechanism of action andin vivo role of platelet-derived growth factorrdquo PhysiologicalReviews vol 79 no 4 pp 1283ndash1316 1999

[30] S Weissmueller E Manchado M Saborowski et alldquoMutant p53 drives pancreatic cancer metastasis throughcell-autonomous PDGF receptor 120573 signalingrdquo Cell vol 157 no2 pp 382ndash394 2014

[31] M Kodama Y Kitadai T Sumida et al ldquoExpression ofplatelet-derived growth factor (PDGF)-B and PDGF-receptor120573 is associated with lymphatic metastasis in human gastriccarcinomardquo Cancer Science vol 101 no 9 pp 1984ndash1989 2010

[32] M Meng W Wang J Yan et al ldquoImmunization of stromal celltargeting fibroblast activation protein providing immunother-apy to breast cancer mouse modelrdquo Tumor Biology vol 37 no8 pp 10317ndash10327 2016

[33] H Wang Q Wu Z Liu et al ldquoDownregulation of FAPsuppresses cell proliferation and metastasis throughPTENPI3KAKT and Ras-ERK signaling in oral squamouscell carcinomardquo Cell Death and Disease vol 5 no 4 Article IDe1155 2014

[34] F Liu L Qi B Liu et al ldquoFibroblast activation proteinoverexpression and clinical implications in solid tumors ameta-analysisrdquo PLoS ONE vol 10 no 3 Article ID e01166832015

10 Disease Markers

[35] D Yuan B Liu K Liu G Zhu Z Dai and Y Xie ldquoOverexpres-sion of fibroblast activation protein and its clinical implicationsin patients with osteosarcomardquo Journal of Surgical Oncologyvol 108 no 3 pp 157ndash162 2013

[36] M A Goscinski Z Suo V A Floslashrenes L Vlatkovic J MNesland and K-E Giercksky ldquoFAP-120572 and uPA show differentexpression patterns in premalignant and malignant esophageallesionsrdquo Ultrastructural Pathology vol 32 no 3 pp 89ndash962008

[37] R C Coelho T Reinert F Campos et al ldquoSunitinib treatmentin patients with advanced renal cell cancer the BrazilianNational Cancer Institute (INCA) experiencerdquo InternationalBrazilian Journal of Urology vol 42 no 4 pp 694ndash703 2016

[38] ldquoEfficiency of sunitinib in Chinese patients with advancedprogressive pancreatic neuroendocrine tumorrdquo Zhongguo YiXue Ke Xue Yuan Xue Bao vol 38 no 3 pp 300ndash304 2016

[39] V Atallah A Hocquelet C Do Cao et al ldquoActivity and safetyof sunitinib in patients with advanced radioiodine refractorythyroid carcinoma a retrospective analysis of 57 patientsrdquoThyroid vol 26 no 8 pp 1085ndash1092 2016

[40] E Y Moawad ldquoPredicting effectiveness of imatinib mesylatein tumors expressing platelet-derived growth factors (PDGF-AA PDGF-BB) stem cell factor ligands and their respectivereceptors (PDGFR-120572 PDGFR-120573 and c-kit)rdquo Journal of Gas-trointestinal Cancer vol 46 no 3 pp 272ndash283 2015

[41] Y Hayashi M R Bardsley Y Toyomasu et al ldquoPlatelet-derived growth factor receptor-120572 regulates proliferation ofgastrointestinal stromal tumor cells with mutations in KIT bystabilizing ETV1rdquo Gastroenterology vol 149 no 2 pp 420ndash432e16 2015

[42] J Ohishi M Aoki K Nabeshima et al ldquoImatinib mesylateinhibits cell growth of malignant peripheral nerve sheathtumors in vitro and in vivo through suppression of PDGFR-120573rdquoBMC Cancer vol 13 article 224 2013

Disease Markers 7

Table 5 Univariate analysis of the impact of CAF-related proteins in the stromal component of phyllodes tumor on patient prognosis usingthe log-rank test

Parameters Number of patientsTotalrecurrencemetastasis

Disease-free survival Overall survivalMedian survival (95

CI) months 119901 value Median survival(95 CI) months 119901 value

Podoplanin 0936 0494Negative 5253 160 (146ndash174) 166 (155ndash178)Positive 144135 166 (158ndash175) 177 (171ndash182)

Prolyl 4-hydroxylase na naNegative 192188 na naPositive 400 na na

FAP120572 0099 0233Negative 188167 164 (157ndash171) 172 (167ndash177)Positive 821 131 (71ndash191) 162 (123ndash200)

S100A4 0607 0792Negative 6553 163 (152ndash174) 168 (159ndash177)Positive 131135 165 (155ndash174) 176 (170ndash182)

PDGFR120572 0698 0002Negative 179165 167 (159ndash174) 177 (173ndash182)Positive 1723 121 (102ndash140) 113 (90ndash136)

PDGFR120573 0394 0087Negative 8061 164 (154ndash174) 174 (170ndash178)Positive 116127 163 (153ndash173) 172 (164ndash179)

NG2 0830 0216Negative 9382 145 (136ndash154) 155 (151ndash160)Positive 103106 165 (155ndash175) 172 (164ndash180)

Table 6 Multivariate analysis of disease-free and overall survival in patients with phyllodes tumors

Included factor Disease-free survival Overall survivalHazard ratio 95 CI 119901 value Hazard ratio 95 CI 119901 value

Histologic grade 0002 NABenign versus

borderlinemalignant 7990 2196ndash2907 NA NA

Stromal cellularity 0597 0467Mild versus

moderatemarked 0625 0109ndash3569 2862 0169ndash4860

Stromal atypia 0886 0708Mild versus

moderatemarked 0881 0158ndash4925 0630 0056ndash7049

Stromal mitosis 0854 04370ndash410 HPF versusgt410 HPF 0782 0057ndash1081 0210 0004ndash1070

Stromal overgrowth 0029 0006Absent versus present 7288 1225ndash4335 5810 3116ndash1083

Tumor margin 0275 0187Circumscribed versus

infiltrative 0468 0120ndash1830 0321 0060ndash1732

PDGFR120572 (stromal) 0867 0050Negative versus

positive 0879 0193ndash4008 5486 1003ndash3001

HPF high-power field

8 Disease Markers

PDGFR120572 (S) negative

PDGFR120572 (S) positive

Phyllodes tumor

p = 0002

0

02

04

06

08

10

Cum

ulat

ive o

vera

ll su

rviv

al

100 150 200500Months

PDGFR120572 (S) negative

(a)

PDGFR120572 (S) negative

PDGFR120572 (S) positive

Malignant phyllodes tumor

p = 0052

0

02

04

06

08

10

Cum

ulat

ive o

vera

ll su

rviv

al

100 1257525 500Months(b)

Malignant phyllodes tumor

p = 0041

100 1257525 500Months

0

02

04

06

08

10

Cum

ulat

ive o

vera

ll su

rviv

al

PDGFR120573 (S) negative

PDGFR120573 (S) positive

(c)Figure 3 Overall survival curves according to the status of CAF-related proteins in stromal component of phyllodes tumor (a) Univariateanalysis showed that stromal expression of PDGFR120572 was associated with shorter overall survival in phyllodes tumor (b) Stromal expressionof PDGFR120572 was associated with shorter overall survival in malignant phyllodes tumor although statistical significance was not reachedregarding PDGFR120572 expression (c) Stromal expression of PDGFR120573was associated with shorter overall survival inmalignant phyllodes tumorCAF cancer-associated fibroblast PDGFR platelet-derived growth factor receptor

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was supported by a grant from the NationalRampD Program for Cancer Control Ministry of Healthamp Welfare Republic of Korea (1420080) Additionally it

was supported by Basic Science Research Program throughthe National Research Foundation of Korea (NRF) fundedby the Ministry of Science ICT and Future Planning(2015R1A1A1A05001209)

References

[1] O E Franco A K Shaw D W Strand and S W HaywardldquoCancer associated fibroblasts in cancer pathogenesisrdquo Seminarsin Cell and Developmental Biology vol 21 no 1 pp 33ndash39 2010

Disease Markers 9

[2] A Ostman ldquoCancer-associated fibroblasts recent develop-ments and emerging challengesrdquo Seminars in Cancer Biologyvol 25 pp 1ndash2 2014

[3] A Desmouliere C Guyot and G Gabbiani ldquoThe stromareaction myofibroblast a key player in the control of tumor cellbehaviorrdquo International Journal of Developmental Biology vol48 no 5-6 pp 509ndash517 2004

[4] O De Wever Q-D Nguyen L Van Hoorde et al ldquoTenascin-C and SFHGF produced by myofibroblasts in vitro provideconvergent pro-invasive signals to human colon cancer cellsthrough RhoA and Racrdquo FASEB Journal vol 18 no 9 pp 1016ndash1018 2004

[5] H Sugimoto TMMundelMWKieran andRKalluri ldquoIden-tification of fibroblast heterogeneity in the tumor microenvi-ronmentrdquo Cancer Biology and Therapy vol 5 no 12 pp 1640ndash1646 2006

[6] K Pietras T Sjoblom K Rubin C-H Heldin and A OstmanldquoPDGF receptors as cancer drug targetsrdquo Cancer Cell vol 3 no5 pp 439ndash443 2003

[7] M Kraman P J Bambrough J N Arnold et al ldquoSuppressionof antitumor immunity by stromal cells expressing fibroblastactivation protein-120572rdquo Science vol 330 no 6005 pp 827ndash8302010

[8] A Kawase G Ishii K Nagai et al ldquoPodoplanin expression bycancer associated fibroblasts predicts poor prognosis of lungadenocarcinomardquo International Journal of Cancer vol 123 no5 pp 1053ndash1059 2008

[9] Y Kojima A Acar E N Eaton et al ldquoAutocrine TGF-120573 andstromal cell-derived factor-1 (SDF-1) signaling drives the evo-lution of tumor-promoting mammary stromal myofibroblastsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 107 no 46 pp 20009ndash20014 2010

[10] E Cortez P Roswall and K Pietras ldquoFunctional subsetsof mesenchymal cell types in the tumor microenvironmentrdquoSeminars in Cancer Biology vol 25 pp 3ndash9 2014

[11] F A Tavassoli and P Devilee World Heath OrganizationClassification of Tumors IARC Press Lyon France 2003

[12] S Dacic S Kounelis E Kouri andMW Jones ldquoImmunohisto-chemical profile of cystosarcoma phyllodes of the breast a studyof 23 casesrdquo Breast Journal vol 8 no 6 pp 376ndash381 2002

[13] B Anderson T Lawton C Lehman and R Moe ldquoPhyllodestumorrdquo in Disease of the Breast M Morrow and C OsborneEds pp 991ndash1006 Lippincott Williams amp Wilkins Philadel-phia 2004

[14] J Ben hassouna T Damak A Gamoudi et al ldquoPhyllodestumors of the breast a case series of 106 patientsrdquo AmericanJournal of Surgery vol 192 no 2 pp 141ndash147 2006

[15] J Mitus M Reinfuss J W Mitus et al ldquoMalignant phyllodestumor of the breast treatment and prognosisrdquo Breast Journalvol 20 no 6 pp 639ndash644 2014

[16] P-H Tan T Jayabaskar K-L Chuah et al ldquoPhyllodes tumors ofthe breast the role of pathologic parametersrdquo American Journalof Clinical Pathology vol 123 no 4 pp 529ndash540 2005

[17] A V Barrio B D Clark J I Goldberg et al ldquoClinicopathologicfeatures and long-term outcomes of 293 phyllodes tumors of thebreastrdquo Annals of Surgical Oncology vol 14 no 10 pp 2961ndash2970 2007

[18] F I Aranda J B Laforga and J I Lopez ldquoPhyllodes tumorof the breast An immunohistochemical study of 28 caseswith special attention to the role of myofibroblastsrdquo PathologyResearch and Practice vol 190 no 5 pp 474ndash481 1994

[19] J S Silverman and A Tamsen ldquoMammary fibroadenomaand some phyllodes tumour stroma are composed of CD34+fibroblasts and factor XIIIa+ dendrophagesrdquo Histopathologyvol 29 no 5 pp 411ndash419 1996

[20] K Y Won G Y Kim Y W Kim J Y Song and S-J LimldquoClinicopathologic correlation of beclin-1 and bcl-2 expressionin human breast cancerrdquo Human Pathology vol 41 no 1 pp107ndash112 2010

[21] J Andrae R Gallini and C Betsholtz ldquoRole of platelet-derived growth factors in physiology and medicinerdquo Genes andDevelopment vol 22 no 10 pp 1276ndash1312 2008

[22] F Meng C L Speyer B Zhang et al ldquoPDGFR120572 and 120573 playcritical roles in mediating Foxq1-driven breast cancer stemnessand chemoresistancerdquo Cancer Research vol 75 no 3 pp 584ndash593 2015

[23] Z-M Shao M Nguyen and S H Barsky ldquoHuman breastcarcinoma desmoplasia is PDGF initiatedrdquo Oncogene vol 19no 38 pp 4337ndash4345 2000

[24] I Nazarenko S-M Hede X He et al ldquoPDGF and PDGFreceptors in gliomardquoUpsala Journal of Medical Sciences vol 117no 2 pp 99ndash112 2012

[25] J D Schultz S Rotunno F Riedel et al ldquoSynergistic effectsof imatinib and carboplatin on VEGF PDGF and PDGF-R120572120573expression in squamous cell carcinoma of the head and neck invitrordquo International Journal of Oncology vol 38 no 4 pp 1001ndash1012 2011

[26] E J A Steller D A Raats J Koster et al ldquoPDGFRB pro-motes liver metastasis formation of mesenchymal-like colorec-tal tumor cellsrdquoNeoplasia (United States) vol 15 no 2 pp 204ndash217 2013

[27] H Kurahara K Maemura Y Mataki M Sakoda H Shinchiand S Natsugoe ldquoImpact of p53 and PDGFR-120573 expression onmetastasis and prognosis of patients with pancreatic cancerrdquoWorld Journal of Surgery vol 40 no 8 pp 1977ndash1984 2016

[28] P P Piccaluga M Rossi C Agostinelli et al ldquoPlatelet-derivedgrowth factor alpha mediates the proliferation of peripheralT-cell lymphoma cells via an autocrine regulatory pathwayrdquoLeukemia vol 28 no 8 pp 1687ndash1697 2014

[29] C-H Heldin and B Westermark ldquoMechanism of action andin vivo role of platelet-derived growth factorrdquo PhysiologicalReviews vol 79 no 4 pp 1283ndash1316 1999

[30] S Weissmueller E Manchado M Saborowski et alldquoMutant p53 drives pancreatic cancer metastasis throughcell-autonomous PDGF receptor 120573 signalingrdquo Cell vol 157 no2 pp 382ndash394 2014

[31] M Kodama Y Kitadai T Sumida et al ldquoExpression ofplatelet-derived growth factor (PDGF)-B and PDGF-receptor120573 is associated with lymphatic metastasis in human gastriccarcinomardquo Cancer Science vol 101 no 9 pp 1984ndash1989 2010

[32] M Meng W Wang J Yan et al ldquoImmunization of stromal celltargeting fibroblast activation protein providing immunother-apy to breast cancer mouse modelrdquo Tumor Biology vol 37 no8 pp 10317ndash10327 2016

[33] H Wang Q Wu Z Liu et al ldquoDownregulation of FAPsuppresses cell proliferation and metastasis throughPTENPI3KAKT and Ras-ERK signaling in oral squamouscell carcinomardquo Cell Death and Disease vol 5 no 4 Article IDe1155 2014

[34] F Liu L Qi B Liu et al ldquoFibroblast activation proteinoverexpression and clinical implications in solid tumors ameta-analysisrdquo PLoS ONE vol 10 no 3 Article ID e01166832015

10 Disease Markers

[35] D Yuan B Liu K Liu G Zhu Z Dai and Y Xie ldquoOverexpres-sion of fibroblast activation protein and its clinical implicationsin patients with osteosarcomardquo Journal of Surgical Oncologyvol 108 no 3 pp 157ndash162 2013

[36] M A Goscinski Z Suo V A Floslashrenes L Vlatkovic J MNesland and K-E Giercksky ldquoFAP-120572 and uPA show differentexpression patterns in premalignant and malignant esophageallesionsrdquo Ultrastructural Pathology vol 32 no 3 pp 89ndash962008

[37] R C Coelho T Reinert F Campos et al ldquoSunitinib treatmentin patients with advanced renal cell cancer the BrazilianNational Cancer Institute (INCA) experiencerdquo InternationalBrazilian Journal of Urology vol 42 no 4 pp 694ndash703 2016

[38] ldquoEfficiency of sunitinib in Chinese patients with advancedprogressive pancreatic neuroendocrine tumorrdquo Zhongguo YiXue Ke Xue Yuan Xue Bao vol 38 no 3 pp 300ndash304 2016

[39] V Atallah A Hocquelet C Do Cao et al ldquoActivity and safetyof sunitinib in patients with advanced radioiodine refractorythyroid carcinoma a retrospective analysis of 57 patientsrdquoThyroid vol 26 no 8 pp 1085ndash1092 2016

[40] E Y Moawad ldquoPredicting effectiveness of imatinib mesylatein tumors expressing platelet-derived growth factors (PDGF-AA PDGF-BB) stem cell factor ligands and their respectivereceptors (PDGFR-120572 PDGFR-120573 and c-kit)rdquo Journal of Gas-trointestinal Cancer vol 46 no 3 pp 272ndash283 2015

[41] Y Hayashi M R Bardsley Y Toyomasu et al ldquoPlatelet-derived growth factor receptor-120572 regulates proliferation ofgastrointestinal stromal tumor cells with mutations in KIT bystabilizing ETV1rdquo Gastroenterology vol 149 no 2 pp 420ndash432e16 2015

[42] J Ohishi M Aoki K Nabeshima et al ldquoImatinib mesylateinhibits cell growth of malignant peripheral nerve sheathtumors in vitro and in vivo through suppression of PDGFR-120573rdquoBMC Cancer vol 13 article 224 2013

8 Disease Markers

PDGFR120572 (S) negative

PDGFR120572 (S) positive

Phyllodes tumor

p = 0002

0

02

04

06

08

10

Cum

ulat

ive o

vera

ll su

rviv

al

100 150 200500Months

PDGFR120572 (S) negative

(a)

PDGFR120572 (S) negative

PDGFR120572 (S) positive

Malignant phyllodes tumor

p = 0052

0

02

04

06

08

10

Cum

ulat

ive o

vera

ll su

rviv

al

100 1257525 500Months(b)

Malignant phyllodes tumor

p = 0041

100 1257525 500Months

0

02

04

06

08

10

Cum

ulat

ive o

vera

ll su

rviv

al

PDGFR120573 (S) negative

PDGFR120573 (S) positive

(c)Figure 3 Overall survival curves according to the status of CAF-related proteins in stromal component of phyllodes tumor (a) Univariateanalysis showed that stromal expression of PDGFR120572 was associated with shorter overall survival in phyllodes tumor (b) Stromal expressionof PDGFR120572 was associated with shorter overall survival in malignant phyllodes tumor although statistical significance was not reachedregarding PDGFR120572 expression (c) Stromal expression of PDGFR120573was associated with shorter overall survival inmalignant phyllodes tumorCAF cancer-associated fibroblast PDGFR platelet-derived growth factor receptor

Competing Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This study was supported by a grant from the NationalRampD Program for Cancer Control Ministry of Healthamp Welfare Republic of Korea (1420080) Additionally it

was supported by Basic Science Research Program throughthe National Research Foundation of Korea (NRF) fundedby the Ministry of Science ICT and Future Planning(2015R1A1A1A05001209)

References

[1] O E Franco A K Shaw D W Strand and S W HaywardldquoCancer associated fibroblasts in cancer pathogenesisrdquo Seminarsin Cell and Developmental Biology vol 21 no 1 pp 33ndash39 2010

Disease Markers 9

[2] A Ostman ldquoCancer-associated fibroblasts recent develop-ments and emerging challengesrdquo Seminars in Cancer Biologyvol 25 pp 1ndash2 2014

[3] A Desmouliere C Guyot and G Gabbiani ldquoThe stromareaction myofibroblast a key player in the control of tumor cellbehaviorrdquo International Journal of Developmental Biology vol48 no 5-6 pp 509ndash517 2004

[4] O De Wever Q-D Nguyen L Van Hoorde et al ldquoTenascin-C and SFHGF produced by myofibroblasts in vitro provideconvergent pro-invasive signals to human colon cancer cellsthrough RhoA and Racrdquo FASEB Journal vol 18 no 9 pp 1016ndash1018 2004

[5] H Sugimoto TMMundelMWKieran andRKalluri ldquoIden-tification of fibroblast heterogeneity in the tumor microenvi-ronmentrdquo Cancer Biology and Therapy vol 5 no 12 pp 1640ndash1646 2006

[6] K Pietras T Sjoblom K Rubin C-H Heldin and A OstmanldquoPDGF receptors as cancer drug targetsrdquo Cancer Cell vol 3 no5 pp 439ndash443 2003

[7] M Kraman P J Bambrough J N Arnold et al ldquoSuppressionof antitumor immunity by stromal cells expressing fibroblastactivation protein-120572rdquo Science vol 330 no 6005 pp 827ndash8302010

[8] A Kawase G Ishii K Nagai et al ldquoPodoplanin expression bycancer associated fibroblasts predicts poor prognosis of lungadenocarcinomardquo International Journal of Cancer vol 123 no5 pp 1053ndash1059 2008

[9] Y Kojima A Acar E N Eaton et al ldquoAutocrine TGF-120573 andstromal cell-derived factor-1 (SDF-1) signaling drives the evo-lution of tumor-promoting mammary stromal myofibroblastsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 107 no 46 pp 20009ndash20014 2010

[10] E Cortez P Roswall and K Pietras ldquoFunctional subsetsof mesenchymal cell types in the tumor microenvironmentrdquoSeminars in Cancer Biology vol 25 pp 3ndash9 2014

[11] F A Tavassoli and P Devilee World Heath OrganizationClassification of Tumors IARC Press Lyon France 2003

[12] S Dacic S Kounelis E Kouri andMW Jones ldquoImmunohisto-chemical profile of cystosarcoma phyllodes of the breast a studyof 23 casesrdquo Breast Journal vol 8 no 6 pp 376ndash381 2002

[13] B Anderson T Lawton C Lehman and R Moe ldquoPhyllodestumorrdquo in Disease of the Breast M Morrow and C OsborneEds pp 991ndash1006 Lippincott Williams amp Wilkins Philadel-phia 2004

[14] J Ben hassouna T Damak A Gamoudi et al ldquoPhyllodestumors of the breast a case series of 106 patientsrdquo AmericanJournal of Surgery vol 192 no 2 pp 141ndash147 2006

[15] J Mitus M Reinfuss J W Mitus et al ldquoMalignant phyllodestumor of the breast treatment and prognosisrdquo Breast Journalvol 20 no 6 pp 639ndash644 2014

[16] P-H Tan T Jayabaskar K-L Chuah et al ldquoPhyllodes tumors ofthe breast the role of pathologic parametersrdquo American Journalof Clinical Pathology vol 123 no 4 pp 529ndash540 2005

[17] A V Barrio B D Clark J I Goldberg et al ldquoClinicopathologicfeatures and long-term outcomes of 293 phyllodes tumors of thebreastrdquo Annals of Surgical Oncology vol 14 no 10 pp 2961ndash2970 2007

[18] F I Aranda J B Laforga and J I Lopez ldquoPhyllodes tumorof the breast An immunohistochemical study of 28 caseswith special attention to the role of myofibroblastsrdquo PathologyResearch and Practice vol 190 no 5 pp 474ndash481 1994

[19] J S Silverman and A Tamsen ldquoMammary fibroadenomaand some phyllodes tumour stroma are composed of CD34+fibroblasts and factor XIIIa+ dendrophagesrdquo Histopathologyvol 29 no 5 pp 411ndash419 1996

[20] K Y Won G Y Kim Y W Kim J Y Song and S-J LimldquoClinicopathologic correlation of beclin-1 and bcl-2 expressionin human breast cancerrdquo Human Pathology vol 41 no 1 pp107ndash112 2010

[21] J Andrae R Gallini and C Betsholtz ldquoRole of platelet-derived growth factors in physiology and medicinerdquo Genes andDevelopment vol 22 no 10 pp 1276ndash1312 2008

[22] F Meng C L Speyer B Zhang et al ldquoPDGFR120572 and 120573 playcritical roles in mediating Foxq1-driven breast cancer stemnessand chemoresistancerdquo Cancer Research vol 75 no 3 pp 584ndash593 2015

[23] Z-M Shao M Nguyen and S H Barsky ldquoHuman breastcarcinoma desmoplasia is PDGF initiatedrdquo Oncogene vol 19no 38 pp 4337ndash4345 2000

[24] I Nazarenko S-M Hede X He et al ldquoPDGF and PDGFreceptors in gliomardquoUpsala Journal of Medical Sciences vol 117no 2 pp 99ndash112 2012

[25] J D Schultz S Rotunno F Riedel et al ldquoSynergistic effectsof imatinib and carboplatin on VEGF PDGF and PDGF-R120572120573expression in squamous cell carcinoma of the head and neck invitrordquo International Journal of Oncology vol 38 no 4 pp 1001ndash1012 2011

[26] E J A Steller D A Raats J Koster et al ldquoPDGFRB pro-motes liver metastasis formation of mesenchymal-like colorec-tal tumor cellsrdquoNeoplasia (United States) vol 15 no 2 pp 204ndash217 2013

[27] H Kurahara K Maemura Y Mataki M Sakoda H Shinchiand S Natsugoe ldquoImpact of p53 and PDGFR-120573 expression onmetastasis and prognosis of patients with pancreatic cancerrdquoWorld Journal of Surgery vol 40 no 8 pp 1977ndash1984 2016

[28] P P Piccaluga M Rossi C Agostinelli et al ldquoPlatelet-derivedgrowth factor alpha mediates the proliferation of peripheralT-cell lymphoma cells via an autocrine regulatory pathwayrdquoLeukemia vol 28 no 8 pp 1687ndash1697 2014

[29] C-H Heldin and B Westermark ldquoMechanism of action andin vivo role of platelet-derived growth factorrdquo PhysiologicalReviews vol 79 no 4 pp 1283ndash1316 1999

[30] S Weissmueller E Manchado M Saborowski et alldquoMutant p53 drives pancreatic cancer metastasis throughcell-autonomous PDGF receptor 120573 signalingrdquo Cell vol 157 no2 pp 382ndash394 2014

[31] M Kodama Y Kitadai T Sumida et al ldquoExpression ofplatelet-derived growth factor (PDGF)-B and PDGF-receptor120573 is associated with lymphatic metastasis in human gastriccarcinomardquo Cancer Science vol 101 no 9 pp 1984ndash1989 2010

[32] M Meng W Wang J Yan et al ldquoImmunization of stromal celltargeting fibroblast activation protein providing immunother-apy to breast cancer mouse modelrdquo Tumor Biology vol 37 no8 pp 10317ndash10327 2016

[33] H Wang Q Wu Z Liu et al ldquoDownregulation of FAPsuppresses cell proliferation and metastasis throughPTENPI3KAKT and Ras-ERK signaling in oral squamouscell carcinomardquo Cell Death and Disease vol 5 no 4 Article IDe1155 2014

[34] F Liu L Qi B Liu et al ldquoFibroblast activation proteinoverexpression and clinical implications in solid tumors ameta-analysisrdquo PLoS ONE vol 10 no 3 Article ID e01166832015

10 Disease Markers

[35] D Yuan B Liu K Liu G Zhu Z Dai and Y Xie ldquoOverexpres-sion of fibroblast activation protein and its clinical implicationsin patients with osteosarcomardquo Journal of Surgical Oncologyvol 108 no 3 pp 157ndash162 2013

[36] M A Goscinski Z Suo V A Floslashrenes L Vlatkovic J MNesland and K-E Giercksky ldquoFAP-120572 and uPA show differentexpression patterns in premalignant and malignant esophageallesionsrdquo Ultrastructural Pathology vol 32 no 3 pp 89ndash962008

[37] R C Coelho T Reinert F Campos et al ldquoSunitinib treatmentin patients with advanced renal cell cancer the BrazilianNational Cancer Institute (INCA) experiencerdquo InternationalBrazilian Journal of Urology vol 42 no 4 pp 694ndash703 2016

[38] ldquoEfficiency of sunitinib in Chinese patients with advancedprogressive pancreatic neuroendocrine tumorrdquo Zhongguo YiXue Ke Xue Yuan Xue Bao vol 38 no 3 pp 300ndash304 2016

[39] V Atallah A Hocquelet C Do Cao et al ldquoActivity and safetyof sunitinib in patients with advanced radioiodine refractorythyroid carcinoma a retrospective analysis of 57 patientsrdquoThyroid vol 26 no 8 pp 1085ndash1092 2016

[40] E Y Moawad ldquoPredicting effectiveness of imatinib mesylatein tumors expressing platelet-derived growth factors (PDGF-AA PDGF-BB) stem cell factor ligands and their respectivereceptors (PDGFR-120572 PDGFR-120573 and c-kit)rdquo Journal of Gas-trointestinal Cancer vol 46 no 3 pp 272ndash283 2015

[41] Y Hayashi M R Bardsley Y Toyomasu et al ldquoPlatelet-derived growth factor receptor-120572 regulates proliferation ofgastrointestinal stromal tumor cells with mutations in KIT bystabilizing ETV1rdquo Gastroenterology vol 149 no 2 pp 420ndash432e16 2015

[42] J Ohishi M Aoki K Nabeshima et al ldquoImatinib mesylateinhibits cell growth of malignant peripheral nerve sheathtumors in vitro and in vivo through suppression of PDGFR-120573rdquoBMC Cancer vol 13 article 224 2013

Disease Markers 9

[2] A Ostman ldquoCancer-associated fibroblasts recent develop-ments and emerging challengesrdquo Seminars in Cancer Biologyvol 25 pp 1ndash2 2014

[3] A Desmouliere C Guyot and G Gabbiani ldquoThe stromareaction myofibroblast a key player in the control of tumor cellbehaviorrdquo International Journal of Developmental Biology vol48 no 5-6 pp 509ndash517 2004

[4] O De Wever Q-D Nguyen L Van Hoorde et al ldquoTenascin-C and SFHGF produced by myofibroblasts in vitro provideconvergent pro-invasive signals to human colon cancer cellsthrough RhoA and Racrdquo FASEB Journal vol 18 no 9 pp 1016ndash1018 2004

[5] H Sugimoto TMMundelMWKieran andRKalluri ldquoIden-tification of fibroblast heterogeneity in the tumor microenvi-ronmentrdquo Cancer Biology and Therapy vol 5 no 12 pp 1640ndash1646 2006

[6] K Pietras T Sjoblom K Rubin C-H Heldin and A OstmanldquoPDGF receptors as cancer drug targetsrdquo Cancer Cell vol 3 no5 pp 439ndash443 2003

[7] M Kraman P J Bambrough J N Arnold et al ldquoSuppressionof antitumor immunity by stromal cells expressing fibroblastactivation protein-120572rdquo Science vol 330 no 6005 pp 827ndash8302010

[8] A Kawase G Ishii K Nagai et al ldquoPodoplanin expression bycancer associated fibroblasts predicts poor prognosis of lungadenocarcinomardquo International Journal of Cancer vol 123 no5 pp 1053ndash1059 2008

[9] Y Kojima A Acar E N Eaton et al ldquoAutocrine TGF-120573 andstromal cell-derived factor-1 (SDF-1) signaling drives the evo-lution of tumor-promoting mammary stromal myofibroblastsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 107 no 46 pp 20009ndash20014 2010

[10] E Cortez P Roswall and K Pietras ldquoFunctional subsetsof mesenchymal cell types in the tumor microenvironmentrdquoSeminars in Cancer Biology vol 25 pp 3ndash9 2014

[11] F A Tavassoli and P Devilee World Heath OrganizationClassification of Tumors IARC Press Lyon France 2003

[12] S Dacic S Kounelis E Kouri andMW Jones ldquoImmunohisto-chemical profile of cystosarcoma phyllodes of the breast a studyof 23 casesrdquo Breast Journal vol 8 no 6 pp 376ndash381 2002

[13] B Anderson T Lawton C Lehman and R Moe ldquoPhyllodestumorrdquo in Disease of the Breast M Morrow and C OsborneEds pp 991ndash1006 Lippincott Williams amp Wilkins Philadel-phia 2004

[14] J Ben hassouna T Damak A Gamoudi et al ldquoPhyllodestumors of the breast a case series of 106 patientsrdquo AmericanJournal of Surgery vol 192 no 2 pp 141ndash147 2006

[15] J Mitus M Reinfuss J W Mitus et al ldquoMalignant phyllodestumor of the breast treatment and prognosisrdquo Breast Journalvol 20 no 6 pp 639ndash644 2014

[16] P-H Tan T Jayabaskar K-L Chuah et al ldquoPhyllodes tumors ofthe breast the role of pathologic parametersrdquo American Journalof Clinical Pathology vol 123 no 4 pp 529ndash540 2005

[17] A V Barrio B D Clark J I Goldberg et al ldquoClinicopathologicfeatures and long-term outcomes of 293 phyllodes tumors of thebreastrdquo Annals of Surgical Oncology vol 14 no 10 pp 2961ndash2970 2007

[18] F I Aranda J B Laforga and J I Lopez ldquoPhyllodes tumorof the breast An immunohistochemical study of 28 caseswith special attention to the role of myofibroblastsrdquo PathologyResearch and Practice vol 190 no 5 pp 474ndash481 1994

[19] J S Silverman and A Tamsen ldquoMammary fibroadenomaand some phyllodes tumour stroma are composed of CD34+fibroblasts and factor XIIIa+ dendrophagesrdquo Histopathologyvol 29 no 5 pp 411ndash419 1996

[20] K Y Won G Y Kim Y W Kim J Y Song and S-J LimldquoClinicopathologic correlation of beclin-1 and bcl-2 expressionin human breast cancerrdquo Human Pathology vol 41 no 1 pp107ndash112 2010

[21] J Andrae R Gallini and C Betsholtz ldquoRole of platelet-derived growth factors in physiology and medicinerdquo Genes andDevelopment vol 22 no 10 pp 1276ndash1312 2008

[22] F Meng C L Speyer B Zhang et al ldquoPDGFR120572 and 120573 playcritical roles in mediating Foxq1-driven breast cancer stemnessand chemoresistancerdquo Cancer Research vol 75 no 3 pp 584ndash593 2015

[23] Z-M Shao M Nguyen and S H Barsky ldquoHuman breastcarcinoma desmoplasia is PDGF initiatedrdquo Oncogene vol 19no 38 pp 4337ndash4345 2000

[24] I Nazarenko S-M Hede X He et al ldquoPDGF and PDGFreceptors in gliomardquoUpsala Journal of Medical Sciences vol 117no 2 pp 99ndash112 2012

[25] J D Schultz S Rotunno F Riedel et al ldquoSynergistic effectsof imatinib and carboplatin on VEGF PDGF and PDGF-R120572120573expression in squamous cell carcinoma of the head and neck invitrordquo International Journal of Oncology vol 38 no 4 pp 1001ndash1012 2011

[26] E J A Steller D A Raats J Koster et al ldquoPDGFRB pro-motes liver metastasis formation of mesenchymal-like colorec-tal tumor cellsrdquoNeoplasia (United States) vol 15 no 2 pp 204ndash217 2013

[27] H Kurahara K Maemura Y Mataki M Sakoda H Shinchiand S Natsugoe ldquoImpact of p53 and PDGFR-120573 expression onmetastasis and prognosis of patients with pancreatic cancerrdquoWorld Journal of Surgery vol 40 no 8 pp 1977ndash1984 2016

[28] P P Piccaluga M Rossi C Agostinelli et al ldquoPlatelet-derivedgrowth factor alpha mediates the proliferation of peripheralT-cell lymphoma cells via an autocrine regulatory pathwayrdquoLeukemia vol 28 no 8 pp 1687ndash1697 2014

[29] C-H Heldin and B Westermark ldquoMechanism of action andin vivo role of platelet-derived growth factorrdquo PhysiologicalReviews vol 79 no 4 pp 1283ndash1316 1999

[30] S Weissmueller E Manchado M Saborowski et alldquoMutant p53 drives pancreatic cancer metastasis throughcell-autonomous PDGF receptor 120573 signalingrdquo Cell vol 157 no2 pp 382ndash394 2014

[31] M Kodama Y Kitadai T Sumida et al ldquoExpression ofplatelet-derived growth factor (PDGF)-B and PDGF-receptor120573 is associated with lymphatic metastasis in human gastriccarcinomardquo Cancer Science vol 101 no 9 pp 1984ndash1989 2010

[32] M Meng W Wang J Yan et al ldquoImmunization of stromal celltargeting fibroblast activation protein providing immunother-apy to breast cancer mouse modelrdquo Tumor Biology vol 37 no8 pp 10317ndash10327 2016

[33] H Wang Q Wu Z Liu et al ldquoDownregulation of FAPsuppresses cell proliferation and metastasis throughPTENPI3KAKT and Ras-ERK signaling in oral squamouscell carcinomardquo Cell Death and Disease vol 5 no 4 Article IDe1155 2014

[34] F Liu L Qi B Liu et al ldquoFibroblast activation proteinoverexpression and clinical implications in solid tumors ameta-analysisrdquo PLoS ONE vol 10 no 3 Article ID e01166832015

10 Disease Markers

[35] D Yuan B Liu K Liu G Zhu Z Dai and Y Xie ldquoOverexpres-sion of fibroblast activation protein and its clinical implicationsin patients with osteosarcomardquo Journal of Surgical Oncologyvol 108 no 3 pp 157ndash162 2013

[36] M A Goscinski Z Suo V A Floslashrenes L Vlatkovic J MNesland and K-E Giercksky ldquoFAP-120572 and uPA show differentexpression patterns in premalignant and malignant esophageallesionsrdquo Ultrastructural Pathology vol 32 no 3 pp 89ndash962008

[37] R C Coelho T Reinert F Campos et al ldquoSunitinib treatmentin patients with advanced renal cell cancer the BrazilianNational Cancer Institute (INCA) experiencerdquo InternationalBrazilian Journal of Urology vol 42 no 4 pp 694ndash703 2016

[38] ldquoEfficiency of sunitinib in Chinese patients with advancedprogressive pancreatic neuroendocrine tumorrdquo Zhongguo YiXue Ke Xue Yuan Xue Bao vol 38 no 3 pp 300ndash304 2016

[39] V Atallah A Hocquelet C Do Cao et al ldquoActivity and safetyof sunitinib in patients with advanced radioiodine refractorythyroid carcinoma a retrospective analysis of 57 patientsrdquoThyroid vol 26 no 8 pp 1085ndash1092 2016

[40] E Y Moawad ldquoPredicting effectiveness of imatinib mesylatein tumors expressing platelet-derived growth factors (PDGF-AA PDGF-BB) stem cell factor ligands and their respectivereceptors (PDGFR-120572 PDGFR-120573 and c-kit)rdquo Journal of Gas-trointestinal Cancer vol 46 no 3 pp 272ndash283 2015

[41] Y Hayashi M R Bardsley Y Toyomasu et al ldquoPlatelet-derived growth factor receptor-120572 regulates proliferation ofgastrointestinal stromal tumor cells with mutations in KIT bystabilizing ETV1rdquo Gastroenterology vol 149 no 2 pp 420ndash432e16 2015

[42] J Ohishi M Aoki K Nabeshima et al ldquoImatinib mesylateinhibits cell growth of malignant peripheral nerve sheathtumors in vitro and in vivo through suppression of PDGFR-120573rdquoBMC Cancer vol 13 article 224 2013

10 Disease Markers

[35] D Yuan B Liu K Liu G Zhu Z Dai and Y Xie ldquoOverexpres-sion of fibroblast activation protein and its clinical implicationsin patients with osteosarcomardquo Journal of Surgical Oncologyvol 108 no 3 pp 157ndash162 2013

[36] M A Goscinski Z Suo V A Floslashrenes L Vlatkovic J MNesland and K-E Giercksky ldquoFAP-120572 and uPA show differentexpression patterns in premalignant and malignant esophageallesionsrdquo Ultrastructural Pathology vol 32 no 3 pp 89ndash962008

[37] R C Coelho T Reinert F Campos et al ldquoSunitinib treatmentin patients with advanced renal cell cancer the BrazilianNational Cancer Institute (INCA) experiencerdquo InternationalBrazilian Journal of Urology vol 42 no 4 pp 694ndash703 2016

[38] ldquoEfficiency of sunitinib in Chinese patients with advancedprogressive pancreatic neuroendocrine tumorrdquo Zhongguo YiXue Ke Xue Yuan Xue Bao vol 38 no 3 pp 300ndash304 2016

[39] V Atallah A Hocquelet C Do Cao et al ldquoActivity and safetyof sunitinib in patients with advanced radioiodine refractorythyroid carcinoma a retrospective analysis of 57 patientsrdquoThyroid vol 26 no 8 pp 1085ndash1092 2016

[40] E Y Moawad ldquoPredicting effectiveness of imatinib mesylatein tumors expressing platelet-derived growth factors (PDGF-AA PDGF-BB) stem cell factor ligands and their respectivereceptors (PDGFR-120572 PDGFR-120573 and c-kit)rdquo Journal of Gas-trointestinal Cancer vol 46 no 3 pp 272ndash283 2015

[41] Y Hayashi M R Bardsley Y Toyomasu et al ldquoPlatelet-derived growth factor receptor-120572 regulates proliferation ofgastrointestinal stromal tumor cells with mutations in KIT bystabilizing ETV1rdquo Gastroenterology vol 149 no 2 pp 420ndash432e16 2015

[42] J Ohishi M Aoki K Nabeshima et al ldquoImatinib mesylateinhibits cell growth of malignant peripheral nerve sheathtumors in vitro and in vivo through suppression of PDGFR-120573rdquoBMC Cancer vol 13 article 224 2013