ANTICANCER RESEARCH 33: 3759-3764 (2013)

PTK7 Expression in Triple-negative Breast Cancer

BEYHAN ATASEVEN1,2, REGINA ANGERER1, RONALD KATES3, ANGELA GUNESCH1, PJOTR KNYAZEV4, BERNHARD HÖGEL5, CLEMENS BECKER5, WOLFGANG EIERMANN6 and NADIA HARBECK7

1Department of Gynecology and Obstetrics, Red Cross Women’s Hospital, Munich, Germany; 2Department of Gynecology and Gynecologic Oncology, Kliniken Essen-Mitte, Evangelische Huyssens-Stiftung, Essen, Germany; 3Breast Center, Department of Gynecology and Obstetrics Maistrasse Campus, Ludwig Maximilian University Munich, Munich, Germany; 4Department of Molecular Biology, Max Planck Institute of Biochemistry, Martinsried, Germany; 5Department of Pathology, Red Cross Women’s Hospital Munich, Munich, Germany; 6Department of Gynecology and Oncology, Interdiscipilnary Oncology Center Munich, Germany; 7Breast Center, Department of Gynecology and Obstetrics, Großhadern Campus, Ludwig Maximilian University Munich, Munich, Germany

Abstract. Background: Protein -7 (PTK7) immunoglobulin-like loops, a transmenbrane domain and an plays an important role in cancer. Our aim was to evaluate inactive catalytic tyrosine kinase domain (2, 3). PTK7 seems PTK7 in triple-negative breast cancer (TNBC). Materials to be highly involved in the WNT (named after the Drosophilia and Methods: PTK7 Expression was assessed by Wingless (Wg) and the mouse Int-1 )-pathways (4), which immunohistochemistry (IHC) in 133 patients with TNBC. again represent key pathways for epithelial mesenchymal Expression levels were correlated with clinicopathological transition (EMT) and play important roles in cancer (5-8). A features and survival, taking chemotherapy into account. potential impact of PTK7 expression has been studied in Results: Positive PTK7 expression was detected in 28.6% of several malignancies, including colon, lung, gastric and breast tumors. In the total population, no significant difference was cancer, acute myeloid leukemia and liposarcoma (9-15). The detected in disease-free survival (DFS) or overall survival observed impact of PTK7 overexpression on patient outcome (OS) related to PTK7 status. However, in patients treated by (prognosis) varies among malignancies. Interestingly, patients chemotherapy, patients with PTK7-negative tumors seemed with AML with PTK7 overexpression seemed to be resistant to have better DFS than those with PTK7-positive tumors, to anthracycline-based chemotherapy with a reduced leukemia- particularly for patients treated with only anthracycline- free survival compared to those with PTK7-negative AML (11). therapy drugs. In patients receiving other chemotherapy Triple-negative breast cancer (TNBC) is a clinically defined regimens, PTK7 status had no significant impact on DFS or subtype of breast cancer with lack of expression of estrogen OS. Conclusion: Our results support earlier findings that receptor (ER), progesterone receptor (PR), and human PTK7 may be associated with resistance to anthracycline- epidermal growth factor-2 (HER2). In comparison to patients based chemotherapy. with receptor-positive tumors, TNBC is associated with younger age, poorer differentiation, larger tumor size, and Protein tyrosine kinase-7 (PTK7), a pseudokinase, also known worse outcome (e.g. shorter DFS and OS, higher incidence of as colon carcinoma kinase (CCK4), was originally identified visceral and brain metastasis, shorter time to death after as a protein overexpressed in colon cancer cell lines (1). recurrence) (16). In terms of chemotherapy, anthracyclines are Structurally, it contains an extracellular domain with seven central components of standard neo- and adjuvant regimes. However, there are controversial data addressing the question of anthracycline benefit in breast cancer (17). Despite this controversial discussion, anthracyclines are an integral Correspondence to: Beyhan Ataseven, Department of Gynecology and component of chemotherapy in TNBC in current guidelines. Gynecologic Oncology, Kliniken Essen-Mitte, Evangelische There have not been any prospective trials defining the role of Huyssens-Stiftung, Henricistrasse 92, 45136 Essen, Germany. Tel: +49 anthracyclines specifically in TNBC. The biological 20117434001, Fax: +49 20117434001, e-mail: [email protected] significance of PTK7 in TNBC and its clinical impact on Key Words: PTK7, triple-negative breast cancer, anthracycline anthracycline-based chemotherapy have, therfore, not yet been resistance, prognosis. investigated. Due to a lack of targeted-therapy options and its

0250-7005/2013 $2.00+.40 3759 ANTICANCER RESEARCH 33: 3759-3764 (2013) unfavorable biology, TNBC continues to be associated with chromogen, resulting in brown staining. Finally, the sections were poor outcome. Novel biomarkers and potential new therapy counterstained with hematoxylin, dehydrated and mounted in Eukitt approaches are therefore urgently needed. The aim of our study (VWR, Darmstadt, Germany). Immunohistochemical staining of was for the first time (i) to investigate PTK7 expression in PTK7 was independently evaluated by two pathologists in a double- blinded setting with negative controls. The level of expression was TNBC by IHC, (ii) to correlate PTK7 expression with scored according to the criteria for IHC HER2 assessment. We clinicopathological features, (iii) to evaluate the impact of defined a binary score from HER2 DAKO IRS by classifying PTK7 expression according to (neo-)adjuvant therapy and (iv) DAKO-negative and borderline staining (0, 1+ and 2+) as PTK7- to identify a possible predictive role of PTK7 regarding negative, and highly positive staining (3+) as PTK7-positive. resistance to anthracycline-based chemotherapy. Statistics. For associations of categorical parameters, Chi-square and Materials and Methods Fisher’s exact test were performed. Continuous variables were compared in two groups by the t-test or Mann-Whitney test where appropriate. Survival estimates (OS and DFS) were obtained using Patient population and data collection. For this retrospective study, the Kaplan Meier method and compared by the log-rank test. Cox we searched consecutive patient records (1999 to 2010) at our regression and time-varying survival analysis (non-proportional institution (Red Cross Women’s Hospital, Munich, Germany); 133 hazards) were performed. Significance is reported for two-sided p- patients with primary TNBC were included. This study was approved values below 0.05 DFS and OS were defined in months from the date by the local Ethics Committee (aaproval number 422-11). TNBC was of initial surgery until progression (local/distant) and death from any defined as negative ER and PR status, as assessed by an cause, respectively. Patients lost to follow-up or without documented immunoreactive score (IRS) (18) and negative HER2 (according to events were censored at last follow-up. Calculations were performed American ASCOCAP Guidelines) (19). Stage-adapted breast/axillary using SPSS (version 20, IBM Corporation, New York, USA). surgery was performed. Patients were treated by radiotherapy according to current national guidelines. Clinicopathological features, type of chemotherapy and follow-up were recovered from patient Results records. The chemotherapy regimen was determined according to guidelines at the time of diagnosis. Taxanes were more frequently Patients’ characteristics and outcome. Clinical and included in recent years: Out of the 133 patients, 101 (75.9%) histopathological patients’ characteristics are summarized in received chemotherapy [16 neoadjuvant (NACT), 85 adjuvant]; 32 Table I. The median age at diagnosis was 56.5 years (24.1%) patients did not receive chemotherapy based either on patient (range=27-87 years). At a median follow-up of 48.1 months, preferences or due to old age or early stage of disease. Typical there were 29 deaths and 17 recurrences. Cohort C patients chemotherapy regimes were either anthracycline-based who did not receive any chemotherapy were older (median (Epirubicin/Cyclophosphamide, Doxorubicin/ Cyclophosphamide, 5- age=68.0 years) than patients in cohort A (median age=54.9 FU/Epirubicin/Cyclophosphamide, Fluorouracil/Doxorubicin/ Cyclo- phosphamide, 4-6 cycles), or taxane-based (Docetaxel/ years) and cohort B (median age=49.5 years). Regarding Doxorubicin/Cyclophosphamide, Docetaxel/Carboplatin, 4-6 cycles), tumor size, there were no significant differences between the or 6 cycles of Cyclophosphamide/Methotrexate/Fluorouracil (CMF). cohorts. Comparing nodal status, cohort B patients more In patients receiving NACT, PTK7 expression was measured both frequently had node-positive disease than did cohort A and C before and after NACT. In 18 cases, PTK7 expression was obtained in patients (p<0.001). In cohort C, significantly fewer patients primary tumors and recurrent disease. In order to identify potential had grade 3 tumors than in cohorts A and B (p=0.003). interactions between PTK7-expression and resistance to Estimated 5-year DFS and OS in patients with pT1 tumors anthracyclines, we defined cohort A as patients receiving exclusively anthracycline-based chemotherapy (n= 62, 46.6%), cohort B as those was higher than in those with larger tumors (DFS 71.7% vs. receiving chemotherapy including agents other than anthracyclines 51.7%, p=0.031 and OS 91.3% vs. 57.0%, p<0.001), (e.g. taxane-based and others; n=39, 29.3%); and cohort C as patients respectively. 5-Year DFS/OS rates in patients treated with receiving no chemotherapy (n=32, 24.1%). chemotherapy were 66.6 %/75.3% versus 50.3%/74.4% without chemotherapy (p=0.184/0.854), respectively. Immunohistochemistry. For our standardized PTK7 antibody staining procedure, we obtained 2-3 μm slices of formalin-fixed, PTK7 expression and association with clinicopathological paraffine-embedded tumor tissue. These sections were deparaffinized in xylene and rehydrated through graded features. PTK7-positive expression, as defined above, was concentrations of ethanol to distilled water. Endogenous peroxidase detected in 28.6% (38/133) of tumors. Expression of PTK7 activity was blocked by incubating the sections in 3% hydrogen was predominantly located in the cytoplasm. Membranous peroxide for 10 min at room temperature. Immunodetection of staining was also observed, particularly at the transition PTK7 was performed using the primary rabbit polyclonal antibody between normal and malignant tissue, and in tumors with against PTK7 (Atlas Antibodies, Stockholm, Sweden) at a dilution high expression (Figure 1). of 1:200 and heat antigen retrieval (100˚C, 6 min) in 10 mmol/l Comparing PTK7 expression with clinicopathological citrate buffer (pH 6.0) as a pre-treatment. Sections were then incubated with the horseradish peroxidase (HRP) one-step polymer features, higher levels of PTK7 occurred more frequently in (Zytochem, Berlin, Germany) and were visualized using 3,3’ smaller tumors (≤2 cm); no further significant associations diaminobenzidine (DAB, Biogenex, Fremont CA, USA) as were detected (Table I).

3760 Ataseven et al: PTK7 Expression in TNBC

Table I. Clinicopathological parameters of patients and Protein tyrosine kinase 7 (PTK7) expression.

PTK7 expression

Parameter N Negative Positive Chi p-Value (%) (%) square (2-sided)

Age ≤50 years 43 28 (65.1) 15 (34.9) Age >50 years 90 67 (74.4) 23 (25.6) 1.241 0.265 Tumor size pT ≤2cm (T1)a 71 49 (69.0) 22 (31.0) pT >2cm (T2-4)a 46 40 (87.0) 6 (13.0) 4.936 0.026 Grade pG 1a 3 3 (100.0) 0 (0.0) pG 2a 35 25 (71.4) 10 (28.6) pG 3a 79 61 (77.2) 18 (22.8) 1.415 0.493 Invasive ductal 101 73 (72.3) 28 (27.7) Invasive medullary 23 18 (78.3) 5 (21.7) Other histological type 9 4 (44.4) 5 (55.6) 3.773 0.152 Node-negative (pN)a 81 64 (79.0) 17 (21.0) Node-positive (pN)a 36 25 (69.4) 11 (30.6) 1.253 0.263 Patients in Cohort A 62 44 (71.0) 18 (29.0) n.a. n.a. Cohort B 39 28 (71.8) 11 (28.1) n.a. n.a. Cohort C 32 23 (71.9) 9 (28.1) n.a. n.a.

aPatients treated with preoperative chemotherapy (n=16) were excluded from statistical analyses; n.a. Not applicable.

PTK7 expression differences before and after NACT and in recurrent disease. In 16 patients, we assessed PTK7 expression before and after NACT. In four patients after NACT, Figure 1. A: Protein tyrosine kinase 7 (PTK7)-positive sample, pathological complete remission (pCR) was achieved; out of representing specific staining of tumor cells. Staining was these, two were primarily PTK7-positive. In the remaining predominantly located in the cytoplasm and membrane. Adjacent cases, PTK7 expression after NACT was either comparable to connective tissue shows negative PTK7 staining. B: Weak expression the pre-therapeutic staining or weaker. No increase in staining leading to PTK7-negative staining. Magnification ×200. was seen (data not shown). In the 18 cases with primary tumor and recurrence, no systematic trend of PTK7 expression change between primary tumor and recurrence was seen (PTK7 PTK7 Expression and outcome. In the collective as a whole, no expression: 13 cases with no difference; one case with weaker significant impact of PTK7 expression on DFS or OS was seen. PTK7 expression; 4 cases with stronger staining). To evaluate potential interactions of PTK7 with chemotherapy, we first compared DFS in cohort A vs. B (systemically-treated Discussion patients). As illustrated in Figure 2A, in time-varying survival analysis, DFS was significantly better in cohort A than B PTK7 is up-regulated in various malignancies including (p=0.025) within the first two years after initial surgery. These gastric cancer (12), lung cancer (15), and AML. PTK7 findings suggest a particular benefit from anthracyclines for knockdown or silencing assays in different cancer cell-lines prevention of early relapse in our TNBC cohort. DFS in resulted in growth inhibition, reduced cell migration, reduced patients receiving chemotherapy was investigated according to proliferation and invasiveness, and increased apoptosis (9, 11, PTK7 status: A non-significant trend towards better DFS for 14). These data offer a rationale for targeted therapy in PTK7- patients with PTK7-negative tumors was observed (p=0.452) overexpressing cancer. In our study, IHC PTK7 expression in (Figure 2B). To evaluate the hypothesis of anthracycline TNBC was evaluated for the first time. We correlated PTK7 resistance in patients with PTK7-positive tumors, we assessed expression with clinicopathological parameters and patient survival times of patients treated by exclusively anthracycline- outcome and investigated whether PTK expression was based chemotherapy (n=62) according to PTK7 expression. A related to anthracycline resistance. The only significant substantial though non-significant difference in 5-year DFS association between PTK7 expression and clinicopathological between PTK7-positive (n=18) and PTK7-negative cases parameters was more frequent PTK7-positivity in patients (n=44) (53.8% vs. 76.0%) was observed (Figure 2C). with smaller tumors (≤2 cm). Whereas no impact of PTK7

3761 ANTICANCER RESEARCH 33: 3759-3764 (2013)

Figure 2. A: Comparing disease-free survival (DFS) with respect to applied chemotherapy regimen. Exclusively anthracycline-based scheme (cohort A) versus taxane-based or other schemes (cohort B). In time-varying survival analysis, DFS was significantly better in Cohort A than B (p=0.025) within the first two years after initial surgery. These findings suggest a particular benefit from anthracyclines for prevention of early relapse in our triple negative breast cancer cohort. B: Comparing DFS with respect to Protein tyrosine kinase 7 (PTK7) expression inpatients treated with chemotherapy (cohort A+B). Patients with PTK7-negative tumor tended to have better DFS (p=0.452). C: In patients treated exclusively with anthracycline-based therapy (cohort A), PTK7 expression seemed to be associated with worse DFS, although not significantly (p=0.203).

AML patients was significantly reduced in cases of PTK7 overexpression, resistance induction was hypothesized for PTK7. In PTK7-positive cell lines, this clinical discovery was substantiated showing higher cell survival and resistance to apoptosis induced by doxorubicin (11). We observed a (non- expression was found in the TNBC collective as a whole, we significant) trend towards poorer survival in patients with detected a trend towards poorer survival in patients with PTK7-positive tumors treated with anthracycline-based PTK7-positive disease treated by chemotherapy. chemotherapy, i.e. consistent with this hypothesis. If PTK7 was In the literature, a multifaceted picture has emerged regarding indeed able to induce anthracycline resistance, this would have the clinical relevance of PTK7. For example, PTK7 expression major clinical consequences, because anthracyclines currently was associated with good prognosis in gastric and pulmonary represent the backbone of breast cancer chemotherapy, adenocarcinoma. In gastric cancer, PTK7 expression assessed especially in TNBC. Even though a significant difference was by IHC was an independent prognostic factor for favorable DFS not identified, our results provide the first evidence for PTK7 and OS (12). Endoh et al. (15) calculated a prognostic risk in breast cancer, particularly TNBC. However, as existing index in pulmonary adenocarinoma as a weighted combination literature shows, an important role of PTK7 in breast cancer, of expression levels. Interestingly, the PTK7 coefficient especially TNBC, is likely and therefore further investigations was negative, i.e. high PTK7 gene expression was associated are warranted. Identification of anthracycline resistance in breast with good prognosis. In liposarcoma, high PTK7 expression cancer will require either larger sample sizes or longer follow- was related to shorter 3-year distant relapse-free survival (14). up. In particular, future studies should be powered to examine In AML, PTK7 was also associated with poorer leukemia-free subgroups, due to the heterogenous nature of TNBC. The survival and OS (11) in univariate and multivariate analysis. optimal determination method and scoring for PTK7 expression Moreover, since leukemia-free survival in anthracycline-treated also deserve further investigation. To our knowledge, this is the

3762 Ataseven et al: PTK7 Expression in TNBC first study reporting on PTK7-expression in breast cancer 10 Saha S, Bardelli A, Buckhaults P, Velculescu VE, Rago C, St focusing on TNBC as well as the clinical implications of PTK7. Croix B, Romans KE, Choti MA, Lengauer C, Kinzler KW and Due to having a small collective of only 133 TNBC cases, our Vogelstein B: A phosphatase associated with metastasis of colorectal cancer. Science 294(5545): 1343-1346. 2001. findings can only be regarded as hypothesis-generated. Yet, 11 Prebet T, Lhoumeau AC, Arnoulet C, Aulas A, Marchetto S, considering the multi-faceted role of PTK7 in WNT signaling, Audebert S, Puppo F, Chabannon C, Sainty D, Santoni MJ, our results underline the fact that further studies elucidating the Sebbagh M, Summerour V, Huon Y, Shin WS, Lee ST, Esterni B, clinical impact of PTK7 are needed. Vey N and Borg JP: The cell polarity PTK7 receptor acts as a modulator of the chemotherapeutic response in acute myeloid Conflicts of Interest leukemia and impairs clinical outcome. Blood 116(13): 2315- 2323, 2010. P. Knyazev is listed as inventor on the European Patent Application No. 12 Lin Y, Zhang LH, Wang XH, Xing XF, Cheng XJ, Dong B, Hu 10075169.2 with the title "PTK-7 protein involved in breast cancer". Y, Du H, Li YA, Zhu YB, Ding N, Du YX, Li JY and Ji J: PTK7 Much of this work was conducted at our hospital in collaboration as a novel marker for favorable gastric cancer patient survival. J with the Ludwig Maximilian University Munich; some of the results Surg Oncol 106(7): 880-886, 2012. reported here are included in the doctoral thesis of Regina Angerer 13 Gorringe KL, Boussioutas A and Bowtell DD: Novel regions of (LMU). chromosomal amplification at 6p21, 5p13, and 12q14 in gastric cancer identified by array comparative genomic hybridization. Acknowledgements Genes Chrom Cancer 42(3): 247-259, 2005. 14 Gobble RM, Qin LX, Brill ER, Angeles CV, Ugras S, O’Connor We thank all patients for participating in this trial and donating their RB, Moraco NH, Decarolis PL, Antonescu C and Singer S: cancer tissue. Statistical analysis (REK) was supported by a grant Expression profiling of liposarcoma yields a multigene predictor from the University of Munich (LMU excellence initiative) to NH. of patient outcome and identifies genes that contribute to liposarcomagenesis. Cancer Res 71(7): 2697-705, 2011. References 15 Endoh H, Tomida S, Yatabe Y, Konishi H, Osada H, Tajima K, Kuwano H, Takahashi T and Mitsudomi T: Prognostic model of pulmonary adenocarcinoma by expression profiling of eight genes 1 Mossie K, Jallal B, Alves F, Sures I, Plowman GD and Ullrich A: as determined by quantitative real-time reverse transcriptase Colon carcinoma kinase-4 defines a new subclass of the receptor polymerase chain reaction. J Clin Oncol 22(5): 811-819, 2004. tyrosine kinase family. Oncogene 11(10): 2179-2184, 1995. 16 Dent R, Trudeau M, Pritchard KI, Hanna WM, Kahn HK, Sawka 2 Park SK, Lee HS and Lee ST: Characterization of the human CA, Lickley LA, Rawlinson E, Sun P and Narod SA: Triple- full-length PTK7 cDNA encoding a receptor protein tyrosine negative breast cancer: clinical features and patterns of kinase-like molecule closely related to chick KLG. J Biochem recurrence. Clin Cancer Res 13(15 Pt 1): 4429-34, 2007. 119(2): 235-239, 1996. 17 Gennari A, Sormani MP, Pronzato P, Puntoni M, Colozza M, 3 Jung JW, Ji AR, Lee J, Kim UJ and Lee ST: Organization of the Pfeffer U and Bruzzi P: HER2 status and efficacy of adjuvant human PTK7 gene encoding a receptor protein tyrosine kinase- anthracyclines in early breast cancer: A pooled analysis of like molecule and alternative splicing of its mRNA. Biochim randomized trials. J NatCancer Inst 100(1): 14-20, 2008. Biophys Acta 1579(2-3): 153-163, 2002. 18 Remmele W, Stegner HE. Recommendation for uniform definition 4 Lhoumeau AC, Puppo F, Prebet T, Kodjabachian L and Borg JP: of an immunoreactive score (IRS) for immunohistochemical PTK7: A cell polarity receptor with multiple facets. Cell Cycle, estrogen receptor detection (ER-ICA) in breast cancer tissue. Der 10(8): 1233-1236, 2011. Pathologe 8(3): 138-140, (in German), 1987. 5 Takemaru KI, Ohmitsu M and Li FQ: An oncogenic hub: Beta- 19 Wolff AC, Hammond ME, Schwartz JN, Hagerty KL, Allred DC, catenin as a molecular target for cancer therapeutics. Handb Exp Cote RJ, Dowsett M, Fitzgibbons PL, Hanna WM, Langer A, Pharmacol 186: 261-284, 2008. McShane LM, Paik S, Pegram MD, Perez EA, Press MF, Rhodes 6 Khramtsov AI, Khramtsova GF, Tretiakova M, Huo D, Olopade A, Sturgeon C, Taube SE, Tubbs R, Vance GH, van de Vijver M, OI, Goss KH: WNT/β-Catenin Pathway Activation Is Enriched Wheeler TM, Hayes DF: American Society of Clinical in Basal-Like Breast Cancers and Predicts Poor Outcome. Am J Oncology/College of American Pathologists guideline Pathol 176(6): 2911-2920, 2010. recommendations for human epidermal 2 7 Katoh M: WNT/PCP signaling pathway and human cancer testing in breast cancer. J Clin Oncol 25(1): 118-145, 2007. (review). Oncol Rep 14(6): 1583-1588, 2005. 8 Geyer FC, Lacroix-Triki M, Savage K, Arnedos M, Lambros MB, MacKay A, Natrajan R and Reis-Filho J: β-Catenin pathway activation in breast cancer is associated with triple- negative phenotype but not with CTNNB1 mutation. Mod Pathol 24(2): 209-231, 2011. 9 Speers C, Tsimelzon A, Sexton K, Herrick AM, Gutierrez C, Culhane A, Quackenbush J, Hilsenbeck S, Chang J and Brown P: Identification of novel kinase targets for the treatment of Received June 27, 2013 estrogen receptor-negative breast cancer. Clin Cancer Res Revised July 12, 2013 15(20): 6327-6340, 2009. Accepted July 15, 2013

3763