Prognostic Significance of Stromal Platelet-Derived Growth Factor β-Receptor Expression in Human Breast Cancer

Abstract

This study systematically analyzes platelet-derived growth factor (PDGF) receptor expression in six types of common tumors as well as examines associations between PDGF β-receptor status and clinicopathological characteristics in breast cancer. PDGF receptor expression was determined by immunohistochemistry on tumor tissue microarrays. Breast tumor data were combined with prognostic factors and related to outcome endpoints. PDGF α- and β-receptors were independently expressed, at variable frequencies, in the tumor stroma of all tested tumor types. There was a significant association between PDGF β-receptor expression on fibroblasts and perivascular cells in individual colon and prostate tumors. In breast cancer, high stromal PDGF β-receptor expression was significantly associated with high histopathological grade, estrogen receptor negativity, and high HER2 expression. High stromal PDGF β-receptor expression was correlated with significantly shorter recurrence-free and breast cancer-specific survival. The prognostic significance of stromal PDGF β-receptor expression was particularly prominent in tumors from premenopausal women. Stromal PDGF α- and β-receptor expression is a common, but variable and independent, property of solid tumors. In breast cancer, stromal PDGF β-receptor expression significantly correlates with less favorable clinicopathological parameters and shorter survival. These findings highlight the prognostic significance of stromal markers and should be considered in ongoing clinical development of PDGF receptor inhibitors. This study systematically analyzes platelet-derived growth factor (PDGF) receptor expression in six types of common tumors as well as examines associations between PDGF β-receptor status and clinicopathological characteristics in breast cancer. PDGF receptor expression was determined by immunohistochemistry on tumor tissue microarrays. Breast tumor data were combined with prognostic factors and related to outcome endpoints. PDGF α- and β-receptors were independently expressed, at variable frequencies, in the tumor stroma of all tested tumor types. There was a significant association between PDGF β-receptor expression on fibroblasts and perivascular cells in individual colon and prostate tumors. In breast cancer, high stromal PDGF β-receptor expression was significantly associated with high histopathological grade, estrogen receptor negativity, and high HER2 expression. High stromal PDGF β-receptor expression was correlated with significantly shorter recurrence-free and breast cancer-specific survival. The prognostic significance of stromal PDGF β-receptor expression was particularly prominent in tumors from premenopausal women. Stromal PDGF α- and β-receptor expression is a common, but variable and independent, property of solid tumors. In breast cancer, stromal PDGF β-receptor expression significantly correlates with less favorable clinicopathological parameters and shorter survival. These findings highlight the prognostic significance of stromal markers and should be considered in ongoing clinical development of PDGF receptor inhibitors. Platelet-derived growth factor (PDGF) α- and β-tyrosine kinase receptors exert important control functions in mesenchymal cells, such as pericytes, fibroblasts and vascular smooth muscle cells during development.1Betsholtz C Insight into the physiological functions of PDGF through genetic studies in mice.Cytokine Growth Factor Rev. 2004; 15: 215-228Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar PDGF receptor activation has also been shown to be involved in multiple dimensions of cancer growth.2Ostman A Heldin CH PDGF Receptors as targets in tumor treatment.Adv Cancer Res. 2007; 97: 247-274Crossref PubMed Scopus (180) Google Scholar The clinical relevance of these findings is enhanced by the recent approval of tyrosine kinase inhibitors with PDGF receptor inhibitory activity, eg, imatinib, sunitinib, and sorafenib. PDGF receptor-dependent growth stimulation is well documented in malignant cells of some solid tumors, such as glioblastomas,3Joensuu H Puputti M Sihto H Tynninen O Nupponen NN Amplification of genes encoding KIT: PDGFRalpha and VEGFR2 receptor tyrosine kinases is frequent in glioblastoma multiforme.J Pathol. 2005; 207: 224-231Crossref PubMed Scopus (123) Google Scholar, 4Fleming TP Saxena A Clark WC Robertson JT Oldfield EH Aaronson SA Ali IU Amplification and/or overexpression of platelet-derived growth factor receptors and epidermal growth factor receptor in human glial tumors.Cancer Res. 1992; 52: 4550-4553PubMed Google Scholar, 5Hagerstrand D Hesselager G Achterberg S Wickenberg Bolin U Kowanetz M Kastemar M Heldin CH Isaksson A Nister M Ostman A Characterization of an imatinib-sensitive subset of high-grade human glioma cultures.Oncogene. 2006; 25: 4913-4922Crossref PubMed Scopus (74) Google Scholar, 6Kilic T Alberta JA Zdunek PR Acar M Iannarelli P O'Reilly T Buchdunger E Black PM Stiles CD Intracranial inhibition of platelet-derived growth factor-mediated glioblastoma cell growth by an orally active kinase inhibitor of the 2-phenylaminopyrimidine class.Cancer Res. 2000; 60: 5143-5150PubMed Google Scholar, 7Strawn LM Mann E Elliger SS Chu LM Germain LL Niederfellner G Ullrich A Shawver LK Inhibition of glioma cell growth by a truncated platelet-derived growth factor-beta receptor.J Biol Chem. 1994; 269: 21215-21222Abstract Full Text PDF PubMed Google Scholar dermatofibrosarcoma protuberans8Shimizu A O'Brien KP Sjoblom T Pietras K Buchdunger E Collins VP Heldin CH Dumanski JP Ostman A The dermatofibrosarcoma protuberans-associated collagen type Ialpha1/platelet-derived growth factor (PDGF) B-chain fusion gene generates a transforming protein that is processed to functional PDGF-BB.Cancer Res. 1999; 59: 3719-3723PubMed Google Scholar, 9Sjoblom T Shimizu A O'Brien KP Pietras K Dal Cin P Buchdunger E Dumanski JP Ostman A Heldin CH Growth inhibition of dermatofibrosarcoma protuberans tumors by the platelet-derived growth factor receptor antagonist STI571 through induction of apoptosis.Cancer Res. 2001; 61: 5778-5783PubMed Google Scholar and a subset of gastrointestinal stromal tumors.10Heinrich MC Corless CL Duensing A McGreevey L Chen CJ Joseph N Singer S Griffith DJ Haley A Town A Demetri GD Fletcher CD Fletcher JA PDGFRA activating mutations in gastrointestinal stromal tumors.Science. 2003; 299: 708-710Crossref PubMed Scopus (2003) Google Scholar, 11Corless CL Schroeder A Griffith D Town A McGreevey L Harrell P Shiraga S Bainbridge T Morich J Heinrich MC PDGFRA mutations in gastrointestinal stromal tumors: frequency, spectrum and in vitro sensitivity to imatinib.J Clin Oncol. 2005; 23: 5357-5364Crossref PubMed Scopus (678) Google Scholar Also, in hematological malignancies such as chronic myelomonocytic leukemia and idiopathic eosinophilic syndrome, PDGF α- or β-receptor signaling has been shown to be activated through translocations or deletions of the PDGF receptor genes.12Golub TR Barker GF Lovett M Gilliland DG Fusion of PDGF receptor beta to a novel ets-like gene, tel, in chronic myelomonocytic leukemia with t(5;12) chromosomal translocation.Cell. 1994; 77: 307-316Abstract Full Text PDF PubMed Scopus (1085) Google Scholar, 13Cools J DeAngelo DJ Gotlib J Stover EH Legare RD Cortes J Kutok J Clark J Galinsky I Griffin JD Cross NC Tefferi A Malone J Alam R Schrier SL Schmid J Rose M Vandenberghe P Verhoef G Boogaerts M Wlodarska I Kantarjian H Marynen P Coutre SE Stone R Gilliland DG A tyrosine kinase created by fusion of the PDGFRA and FIP1L1 genes as a therapeutic target of imatinib in idiopathic hypereosinophilic syndrome.N Engl J Med. 2003; 348: 1201-1214Crossref PubMed Scopus (1510) Google Scholar, 14Apperley JF Gardembas M Melo JV Russell-Jones R Bain BJ Baxter EJ Chase A Chessells JM Colombat M Dearden CE Dimitrijevic S Mahon FX Marin D Nikolova Z Olavarria E Silberman S Schultheis B Cross NC Goldman JM Response to imatinib mesylate in patients with chronic myeloproliferative diseases with rearrangements of the platelet-derived growth factor receptor beta.N Engl J Med. 2002; 347: 481-487Crossref PubMed Scopus (568) Google Scholar However, in most common solid tumors PDGF receptor signaling appears to be most important for the pericytes of the tumor vessels, and for the fibroblasts of the tumor stroma. Concerning the role of PDGF β-receptor signaling in pericytes, a series of experimental studies have demonstrated that stimulation of PDGF receptors on pericytes increases pericyte coverage of vessels in a manner that is associated with increased vessel function and, in some cases, also increased tumor growth.15Furuhashi M Sjoblom T Abramsson A Ellingsen J Micke P Li H Bergsten-Folestad E Eriksson U Heuchel R Betsholtz C Heldin CH Ostman A Platelet-derived growth factor production by B16 melanoma cells leads to increased pericyte abundance in tumors and an associated increase in tumor growth rate.Cancer Res. 2004; 64: 2725-2733Crossref PubMed Scopus (153) Google Scholar, 16Abramsson A Lindblom P Betsholtz C Endothelial and nonendothelial sources of PDGF-B regulate pericyte recruitment and influence vascular pattern formation in tumors.J Clin Invest. 2003; 112: 1142-1151Crossref PubMed Scopus (436) Google Scholar, 17Guo P Hu B Gu W Xu L Wang D Huang HJ Cavenee WK Cheng SY Platelet-derived growth factor-B enhances glioma angiogenesis by stimulating vascular endothelial growth factor expression in tumor endothelia and by promoting pericyte recruitment.Am J Pathol. 2003; 162: 1083-1093Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar Furthermore, vascular endothelial growth factor receptor-targeted antiangiogenic approaches in experimental tumor models appear to be most efficient on immature pericyte-poor vessels.18Benjamin LE Golijanin D Itin A Pode D Keshet E Selective ablation of immature blood vessels in established human tumors follows vascular endothelial growth factor withdrawal.J Clin Invest. 1999; 103: 159-165Crossref PubMed Scopus (1049) Google Scholar Finally, combinations of vascular endothelial growth factor receptor- and PDGF-receptor inhibitors have been demonstrated to exert synergistic antiangiogenic effects.19Bergers G Song S Meyer-Morse N Bergsland E Hanahan D Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors.J Clin Invest. 2003; 111: 1287-1295Crossref PubMed Scopus (1226) Google Scholar, 20Erber R Thurnher A Katsen AD Groth G Kerger H Hammes HP Menger MD Ullrich A Vajkoczy P Combined inhibition of VEGF and PDGF signaling enforces tumor vessel regression by interfering with pericyte-mediated endothelial cell survival mechanisms.FASEB J. 2004; 18: 338-340PubMed Google Scholar Studies in experimental tumor models have demonstrated that paracrine activation of PDGF receptors on fibroblasts acts as a potent signal for tumor stroma recruitment.21Forsberg K Valyi-Nagy I Heldin CH Herlyn M Westermark B Platelet-derived growth factor (PDGF) in oncogenesis: development of a vascular connective tissue stroma in xenotransplanted human melanoma producing PDGF-BB.Proc Natl Acad Sci USA. 1993; 90: 393-397Crossref PubMed Scopus (205) Google Scholar, 22Skobe M Fusenig NE Tumorigenic conversion of immortal human keratinocytes through stromal cell activation.Proc Natl Acad Sci USA. 1998; 95: 1050-1055Crossref PubMed Scopus (197) Google Scholar Other studies with PDGF antagonists have also demonstrated direct antitumoral effects of stromal PDGF receptor inhibition,23Pietras K Pahler J Bergers G Hanahan D Functions of paracrine PDGF signaling in the proangiogenic tumor stroma revealed by pharmacological targeting.PLoS Med. 2008; 5: e19Crossref PubMed Scopus (351) Google Scholar, 24Kitadai Y Sasaki T Kuwai T Nakamura T Bucana CD Fidler IJ Targeting the expression of platelet-derived growth factor receptor by reactive stroma inhibits growth and metastasis of human colon carcinoma.Am J Pathol. 2006; 169: 2054-2065Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar as well as beneficial effects on tumor drug uptake.25Pietras K Ostman A Sjoquist M Buchdunger E Reed RK Heldin CH Rubin K Inhibition of platelet-derived growth factor receptors reduces interstitial hypertension and increases transcapillary transport in tumors.Cancer Res. 2001; 61: 2929-2934PubMed Google Scholar, 26Pietras K Rubin K Sjoblom T Buchdunger E Sjoquist M Heldin CH Ostman A Inhibition of PDGF receptor signaling in tumor stroma enhances antitumor effect of chemotherapy.Cancer Res. 2002; 62: 5476-5484PubMed Google Scholar, 27Pietras K Stumm M Hubert M Buchdunger E Rubin K Heldin CH McSheehy P Wartmann M Ostman A STI571 enhances the therapeutic index of epothilone B by a tumor-selective increase of drug uptake.Clin Cancer Res. 2003; 9: 3779-3787PubMed Google Scholar, 28Baranowska-Kortylewicz J Abe M Pietras K Kortylewicz ZP Kurizaki T Nearman J Paulsson J Mosley RL Enke CA Ostman A Effect of platelet-derived growth factor receptor-beta inhibition with STI571 on radioimmunotherapy.Cancer Res. 2005; 65: 7824-7831PubMed Google Scholar The biological effects of PDGF receptors in tumor fibroblasts and pericytes, together with the advent of drugs with PDGF receptor-inhibitory activity thus motivates a systemic characterization of the expression pattern of PDGF α- and β-receptors in human tumors. In this study we have characterized the fibroblast and pericyte expression of PDGF α- and β-receptors in lymphomas and in colon, ovarian, prostate, lung and breast cancers. Furthermore the relationship between stromal PDGF β-receptor status and prognostic parameters and survival was analyzed in breast cancer. Porcine aortic endothelial (PAE) cells transfected with the PDGF α- or β-receptor (PAE/PDGFαR and PAE/PDGFβR cells) were kept in F12 (Sigma-Aldrich, Stockholm, Sweden) media containing 10% fetal calf serum Sigma-Aldrich, Stockholm, Sweden 1% glutamine, and antibiotics (penicillin, 100 units/ml, Sigma-Aldrich) and streptomycin (100 μg/ml, Sigma-Aldrich). PAE/PDGFαR and PAE/PDGFβR cells were starved overnight in medium containing 1% fetal calf serum following stimulation with or without 100 ng/ml PDGF-BB (Peprotech, London, UK) on ice for 1 hour. Cells were then washed in cold phosphate-buffered saline, (Medicago, Uppsala, Sweden) removed from the plate, and centrifuged at 2000 rpm for 10 minutes. The phosphate-buffered saline was discarded and cell pellets incubated in 4% phosphate-buffered paraformaldehyde (WWR, Stockholm, Sweden) solution overnight. The pellet was placed in a tissue embedding box in 70% ethanol and then placed in higher grades of alcohol for dehydration before being embedded in paraffin, sectioned, and put on Superfrost Plus slides (Menzel-Gläser, Braunschweig, Germany). A tumor TMA (TARP 4) containing 450 tumor biopsies were obtained from the National Cancer Institute Tissue Array Research Program, National Institutes of Health. The TARP 4 comprises 0.6 mm core punch biopsies of normal tissues, glioblastoma multiforme, malignant melanomas, lymphomas, breast, colon, lung, ovarian, and prostate adenocarcinomas with 25 to 75 samples of each tumor type. Clinical data were not available for these specimens. The breast cancer TMA was made from 512 consecutive cases of primary breast cancer diagnosed between 1988 and 1992 at the Department of Pathology, Malmö University Hospital. Median age at diagnosis was 64.2 years (range, 27–96) and median follow-up time from diagnosis until first breast cancer event was 106 months (range, 0–207). Follow-up data regarding recurrence-free survival and death was available for 507 patients. This cohort is described in detail elsewhere.29Borgquist S Holm C Stendahl M Anagnostaki L Landberg G Jirstrom K Oestrogen receptors alpha and beta show different associations to clinicopathological parameters and their co-expression might predict a better response to endocrine treatment in breast cancer.J Clin Pathol. 2008; 61: 197-203Crossref PubMed Scopus (57) Google Scholar Complete information regarding adjuvant systemic treatment was available for 389 patients. Of these, 157 had received adjuvant tamoxifen, 19 adjuvant chemotherapy, 209 had not received any adjuvant treatment, and 4 patients had received a combination of tamoxifen and chemotherapy. Before TMA construction, all breast cancer cases were histopathologically re-evaluated on hematoxylin and eosin-stained slides. Areas representative of cancer were then marked and TMAs constructed as described previously.30Kononen J Bubendorf L Kallioniemi A Barlund M Schraml P Leighton S Torhorst J Mihatsch MJ Sauter G Kallioniemi OP Tissue microarrays for high-throughput molecular profiling of tumor specimens.Nat Med. 1998; 4: 844-847Crossref PubMed Scopus (3536) Google Scholar PDGF receptor immunohistochemistry was done as described31Nupponen NN Paulsson J Jeibmann A Wrede B Tanner M Wolff JE Paulus W Ostman A Hasselblatt M Platelet-derived growth factor receptor expression and amplification in choroid plexus carcinomas.Mod Pathol. 2008; 21: 265-270Crossref PubMed Scopus (29) Google Scholar using either anti-PDGF β-receptor rabbit monoclonal (3169, Cell Signaling Technology, Danvers, MA) (2 μg/ml)) or anti-PDGF α-receptor rabbit polyclonal (3164, Cell Signaling Technology) (1:50) antibodies. Nonimmune rabbit serum was used as a negative control. The PDGF receptor staining was scored independently in fibroblasts and pericytes as negative (0), weak (1), moderate (2), or strong (3). The fibroblast PDGF β-receptor expression in breast tumors were dichotomized (0–2 and 3) for all statistical analyses. Analyses of PDGF α- and β-receptor expression dependency in fibroblasts and pericytes were done in STATISTICA by the Yates corrected χ2 test using 2 × 2 contingency tables (StatSoft Inc., Tulsa, OK). The χ2 test and Spearman’s correlation test were used for comparison of PDGF β-receptor expression and relevant patient and tumor characteristics in breast tumors. The Kaplan-Meier method and log rank test were used to compare recurrence-free survival and overall survival in different strata. Recurrence-free survival considered loco-regional and distant metastasis or breast cancer specific death as primary event, whereas contralateral events and non-breast cancer deaths were excluded. A Cox proportional hazards model was used for estimation of relative risks in both univariate and multivariate analyses including other relevant risk and therapy predictive factors as age, size, nodal status, histopathological grade, estrogen receptor (ER), progesterone receptor (PgR), and HER2 status in the model. All statistical tests were two-sided and P values <0.05 considered significant. Calculations were performed with the statistical package SPSS 15.0 (SPSS Inc.). A screen was performed on a series of in-house and commercial PDGF α- and β-Receptor antibodies to characterize their specificity and sensitivity. For this screen, sections of paraformaldehyde-fixed paraffin-embedded cultured cells of known PDGF receptor status were used. One pair of PDGF α- and β-receptor antibodies was selected for further use. The two selected antibodies showed an isoform-specific staining of PDGF α- and β-receptors (see Supplemental Figure S1 at http://ajp.amjpathol.org). Furthermore, the staining pattern in non-stimulated or ligand-stimulated cells was similar (see Supplemental Figure S1 at http://ajp.amjpathol.org), demonstrating that antibody-recognition was not affected by the activation status of receptors. For an initial description of the pattern of PDGF α- and β-receptor expression in human malignancies, immunohistochemistry analyses were performed on a TMA of lymphomas, ovarian, colon, lung and prostate cancers with the two antibodies. For each sample the staining in malignant cells, tumor fibroblasts and perivascular cells were scored separately as being negative or positive. In general, PDGF β-receptor was found on perivascular cells and on stromal fibroblasts but not on the malignant cells (Table 1 and Figure 1A). However, the frequency of tumors with PDGF receptor positive tumor fibroblasts varied largely between tumor groups (Table 1). Among these malignancies, PDGF β-receptor expression in fibroblasts was most common in lung and colon cancer, where 58% to 68% of tumors were positive (Table 1 and Figure 1, A and B). In contrast, only 11% of ovarian cancers had PDGF β-receptor positive fibroblasts (Table 1). Lymphomas and prostate cancers showed an intermediate frequency with around 20% of cases being positive (Table 1). The frequencies of positivity for perivascular PDGF β-receptor staining were in general higher, but again differences between tumor types were noted (Table 1). For example, 80% of colon tumors showed perivascular PDGF β-receptor staining, whereas only 31% of prostate cancers were positive.Table 1PDGF α- and β-Receptor Expression in Fibroblasts and Pericytes of Different Tumor TypesPDGFαRPDGFβRTumor typeFibroblasts (%)Pericytes (%)Fibroblasts (%)Pericytes (%)Lymphoma (n = 19–42)16251950Ovarian (n = 31–40)351158Colon (n = 53–59)65536880Lung (n = 39–43)24335874Prostate (n = 58–62)202131 Open table in a new tab PDGF α-receptor expression was not as frequent as the PDGF β-receptor in tumor fibroblasts, with frequencies ranging from 2% in prostate tumors to 65% in colon tumors (Table 1). Also, perivascular PDGF α-receptor expression was less common than perivascular PDGF β-receptor and ranged between 0% in prostate tumors to 53% in colon cancers. Analyses of the PDGF α- and β-receptors in the different tumor types failed to identify a significant association between the PDGF α- and β-receptor expression in tumor fibroblasts. Among all of the different tumor types analyzed, individual tumors were found that expressed none of the receptors, the β-receptor alone, or the β-receptor together with the α-receptor. Subsequent analyses focused on the relationship between PDGF β-receptor status of pericytes and fibroblasts in individual tumors. Both in colon and prostate tumors significant positive associations were observed between the PDGF β-receptor status of perivascular cells and stromal fibroblasts in individual tumors (Table 2).Table 2Significant Associations of PDGF β-Receptor Expression in Pericytes and Fibroblasts of Colon and Prostate Tumors Analyzed by the χ2 TestPericytesColon (n = 57)PDGFβR−PDGFβR+Fibroblasts PDGFβR−15.8%17.5% PDGFβR+5.3%61.4%P = 0.0019PericytesProstate (n = 61)PDGFβR−PDGFβR+Fibroblasts PDGFβR−62.3%16.4% PDGFβR+6.6%14.8%P = 0.0027 Open table in a new tab These observations thus demonstrate that PDGF α- and β-receptors are independently regulated. Furthermore, the relationship between the expression of PDGF β-receptors in fibroblasts and perivascular cells suggest a functional link between the PDGF receptor status of fibroblasts and perivascular cells. In breast cancer, we performed an extended analysis on TMAs with tumor samples linked to clinicopathological and outcome data. In this material 34.6% of samples were scored as strongly positive for fibroblast expression of PDGF β-receptor (Figure 2A and Supplemental Table S1 at http://ajp.amjpathol.org). Analyses of PDGF α-receptor identified only 1% of cases with moderate or strong staining (see Figure 2A and Supplemental Table S1 at http://ajp.amjpathol.org). Significant positive associations were observed between strong PDGF β-receptor expression in the tumor fibroblasts and high histological grade, ER and progesterone receptor negativity, HER2 expression, proliferation rate and tumor size (Table 3 and Figure 2B). Together these analyses thus clearly demonstrated a previously unrecognized relation between PDGF β-receptor expression in tumor fibroblasts and factors associated with an impaired prognosis.Table 3Associations between Stromal Fibroblast PDGF β-Receptor Expression and Clinicopathological Parameters in Breast CancerNumber (PDGFβR data) PDGFβR expression n (%)n = 512 (289)P value0–2 189 (65.4)3 100 (34.6)Age ⩽5024 (8.3)9 (3.1)0.07 >50165 (57.1)70 (24.2)Tumor size ⩽20 mm127 (43.9)57 (19.7)0.09 >20 mm62 (21.5)43 (14.9) Missing0 (0)0 (0)Nottingham histological grade I53 (18.3)17 (5.9)0.001**P < 0.01. II82 (28.4)35 (12.1) III53 (18.3)48 (16.6) Missing1 (0.4)0 (0)Node status Negative109 (37.7)53 (18.3)0.29 Positive59 (20.4)39 (13.5) Missing21 (7.3)9 (3.1)ER status Negative20 (6.9)19 (6.6)0.05*P < 0.05. Positive165 (57.1)81(28.0) Missing4 (1.4)0 (0)Progesterone receptor status Negative11 (3.8)16 (5.5)0.007**P < 0.01. Positive168 (58.1)84 (29.1) Missing10 (3.5)0 (0)HER2 immunohistochemistry 0136 (47.1)47 (16.3)0.01**P < 0.01. 139 (13.5)24 (8.3) 219 (6.6)12 (4.2) 312 (4.2)15 (5.2) Missing13 (4.5)2 (0.7)Ki67 0–10%81 (28.0)31 (10.7)0.001**P < 0.01. 11–25%68 (23.5)25 (8.7) >25%35 (12.1)39 (13.5) Missing5 (1.7)5 (0)χ2 test for linear trend. Hormone receptor status was assessed by immunohistochemistry using a cutoff at 10% positive nuclei, according to current clinical guidelines in Sweden.* P < 0.05.** P < 0.01. Open table in a new tab χ2 test for linear trend. Hormone receptor status was assessed by immunohistochemistry using a cutoff at 10% positive nuclei, according to current clinical guidelines in Sweden. The results from the analyses of stromal fibroblast expression of PDGF β-receptor were used together with clinical data to investigate possible prognostic significance of PDGF β-receptor expression. As shown in Figure 3, high PDGF β-receptor expression was associated with a significantly shorter recurrence-free survival (P = 0.019) and breast cancer specific survival (P = 0.019). In a Cox proportional hazards model high PDGF β-receptor expression in fibroblasts increased the risk of recurrence (relative risk = 1.67 [1.08–2.58] 95% confidence interval; P = 0.02) and breast cancer specific death (relative risk = 2.02 [1.16–3.49] 95% confidence interval; P = 0.01). This did not remain significant in multivariate analysis (see Supplemental Table S2 at http://ajp.amjpathol.org). To investigate if the prognostic significance was more pronounced in certain patient subsets, survival analyses were performed in strata according to menopausal status, node status, HER2 status, histological grade or endocrine treatment. As shown in Figure 3 and Supplemental Table S2 at http://ajp.amjpathol.org, the prognostic significance of PDGF β-receptor expression in fibroblasts was particularly prominent in premenopausal women. In this subset, PDGF β-receptor expression was also a significant predictor of recurrence-free survival in multivariate analysis (relative risk = 5.49 [1.33–22.74] 95% confidence interval; P = 0.02) (Supplemental Table S2 at http://ajp.amjpathol.org). These analyses thus identified a prognostic significance of stromal fibroblast PDGF β-receptor expression in an unselected breast cancer cohort, which was most prominent in the subset of premenopausal women. The analyses of PDGF receptor expression in stromal fibroblasts of different types of solid tumors and malignant lymphoma revealed large differences between tumor types (Table 1 and Figure 1). It is noteworthy that PDGF α-receptor expression was most common in lung and colon cancer, since previous studies have demonstrated particularly important roles for PDGF α-receptor signaling in mesenchymal cells of these organs.32Bostrom H Gritli-Linde A Betsholtz C PDGF-A/PDGF alpha-receptor signaling is required for lung growth and the formation of alveoli but not for early lung branching morphogenesis.Dev Dyn. 2002; 223: 155-162Crossref PubMed Scopus (103) Google Scholar, 33Karlsson L Lindahl P Heath JK Betsholtz C Abnormal gastrointestinal development in PDGF-A and PDGFR-(alpha) deficient mice implicates a novel mesenchymal structure with putative instructive properties in villus morphogenesis.Development. 2000; 127: 3457-3466PubMed Google Scholar This finding is thus compatible with the notion that stromal fibroblasts of solid tumors are derived from local mesenchymal cells, and share their particular pattern of growth factor receptor dependency. Additional studies should be done to further explore this issue. PDGF receptor expression in human tumors has been analyzed in previous studies with some conflicting results; some of these studies have reported common epithelial expression of PDGF receptors.34Fudge K Bostwick DG Stearns ME Platelet-derived growth factor A and B chains and the alpha and beta receptors in prostatic intraepithelial neoplasia.Prostate. 1996; 29: 282-286Crossref PubMed Scopus (78) Google Scholar, 35Donnem T Al-Saad S Al-Shibli K Andersen S Busund LT Bremnes RM Prognostic impact of platelet-derived growth factors in non-small cell lung cancer tumor and stromal cells.J Thorac Oncol. 2008; 3: 963-970Crossref PubMed Scopus (95) Google Scholar, 36Apte SM Bucana CD Killion JJ Gershenson DM Fidler IJ Expression of platelet-derived growth factor and activated receptor in clinical specimens of epithelial ovarian cancer and ovarian carcinoma cell lines.Gynecol Oncol. 2004; 93: 78-86Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar However, most of these earlier studies have not subjected antibodies to specificity controls of the same stringency as in the present study. 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