Elevated WBP2 Expression in HER2-Positive Breast Cancers Correlates with Sensitivity To

Author Manuscript Published OnlineFirst on December 28, 2018; DOI: 10.1158/1078-0432.CCR-18-3228 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Elevated WBP2 expression in HER2-positive breast cancers correlates with sensitivity to

trastuzumab-based neo-adjuvant therapy: A Retrospective and Multicentric Study

Shin-Ae Kang1, Jye Swei Guan1, Hock Jin Tan1, Tinghine Chu1,2, Aye Aye Thike3, Cristina

Bernadó4, Joaquín Arribas4, Chow Yin Wong5, Puay Hoon Tan3, Mihir Gudi6, Thomas Choudary

Putti7, Joohyuk Sohn8, Swee Ho Lim9, Soo Chin Lee10 and Yoon Pin Lim1,2

1) Department of Biochemistry, Yong Loo Lin School of Medicine, National University of

Singapore, Singapore 117545

2) NUS Graduate School for Integrative Sciences and Engineering, National University of

Singapore, Singapore 119077

3) Division of Pathology, Singapore General Hospital, Singapore 169856

4) Preclinical Research Program, Vall d’Hebron Institute of Oncology (VHIO) and CIBERONC,

Barcelona, Spain 08035

5) Department of General Surgery, Singapore General Hospital, Singapore 169608

6) Department of Pathology and Laboratory Medicine, KK Women's and Children's Hospital,

Singapore 229899

7) Department of Pathology, Yong Loo Lin school of medicine, National University of

Singapore, Singapore 119074

8) Department of Medical Oncology, Yonsei Cancer Center, Yonsei University College of

Medicine, Seoul, Korea 03722

9) KK Breast Department, KK Women's and Children's Hospital, Singapore 229899

10) Department of Haematology-Oncology, National University Cancer Institute, Singapore 119074

Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on December 28, 2018; DOI: 10.1158/1078-0432.CCR-18-3228 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Running title: WBP2 level correlates with trastuzumab neoadjuvant therapy

Key words: HER2-positive breast cancer / neoadjuvant therapy/ trastuzumab / WBP2

Corresponding Author: Yoon Pin Lim, Ph.D., Assistant Professor

Department of Biochemistry, Yong Loo Li School of Medicine, National University of

Singapore, MD4, Level 1, 5 Science Drive 2, Singapore 117545

Phone: Office +65 6601 1891, Email: [email protected]

Conflict of interest

The authors have declared that no conflict of interest exists.

Acknowledgments

The study is funded by Exploit Technologies, Agency for Science, Technology and Research

(F09/X/037_c) and the National Medical Research Council, Ministry of Health

(NMRC/OFIRG/0034/2017), Singapore.

J Arribas is supported by grants of the Breast Cancer Research Foundation (BCRF-17-008) and

Instituto de Salud Carlos III (PI16/00253) for establishing and maintaining the resistant patient- derived xenografts (PDXs).

Translational relevance

Not all HER2-positive breast cancer patients respond well to trastuzumab-based chemotherapy.

Preclinical clinical studies involving in vitro, PDX and animal models revealed that WBP2 augmented the inhibitory effects of trastuzumab in HER2-positive breast cancer cells by enhancing trsatuzumab’s effect on cell cycle arrest. Patients with tumor expressing high WBP2 level had higher pathologic complete response (pCR) of 67.19% compared to those with low

WBP2 (26.58%). The pCR was even higher in subgroups of patients whose tumors had high

WBP2 and aged below 50 years (77.78%) or were pre-menopausal in status (73.33%).

Retrospective analysis revealed WBP2 to have sensitivity of 80 to 81% and specificity of 76.5 to

80% in discriminating between patients showing pCR and non-pCR. WBP2 is a potential

companion diagnostics for the management of HER2-positve breast cancer with trastuzumab-

based therapies.

Abstract

Purpose: Trastuzumab-based chemotherapy has shown remarkable clinical benefits for HER2-

positive breast cancer patients. However, treatment regimens involving trastuzumab had little or

no effect for a subset of patients. Preliminary studies revealed WW-binding protein 2 (WBP2),

an oncogenic transcription co-activator, to be co-amplified with HER2 in 36% of HER2-positive

breast cancers. We hypothesize that WBP2 regulates and correlates with the response of HER2-

positive breast cancer to trastuzumab.

Experimental Design: The co-expression of WBP2 and HER2 in breast tumors was validated

using immunohistochemistry. The role and mechanism of WBP2 in regulating breast cancer

response to trastuzumab was elucidated using in vitro, patient-derived xenograft and murine xenograft models. A multi-center retrospective study involving 143 patients given neoadjuvant trastuzumab-based chemotherapy was conducted to determine if WBP2 expression correlates with pathologic complete response.

Results: Elevated expression of WBP2 significantly enhanced breast cancer’s response to trastuzumab by augmenting trastuzumab-induced downregulation of HER2 and arrest of cell cycle via inhibition of cyclin D expression. High level of WBP2 correlated with better pathologic

complete response (67.19%) compared to low WBP2 level (26.58%). The highest response was

observed in subgroups of patients with high WBP2-expressing tumors and also aged below 50

years (77.78%) or were pre-menopausal in status (73.33%). Retrospectively, WBP2

demonstrated sensitivity of 80 to 81% and specificity of 76.5 to 80% in discriminating between

patients showing pCR and non-pCR.

Conclusions: WBP2 expression correlates with the response of HER2-positive breast cancer to trastuzumab-based neoadjuvant chemotherapy.

Introduction

Breast cancer is the second leading cause of cancer-related deaths in women, especially

among women aged 40-59 years (1). Breast cancer can be molecularly sub-classified into four

groups depending on the Estrogen (ER)/Progesterone (PR) and human epidermal growth factor

receptor 2 (HER2) status: luminal A (ER and/or PR positive with low Ki67), luminal B (ER

and/or PR positive and either HER2 positive or HER2 negative with high Ki67), HER2 (HER2

positive), and basal-like (usually but not always ER/PR/HER2 negative/triple negative) (2).

HER2 (also known as ErbB2/neu) is amplified and overexpressed in 15-20% of breast cancer.

Patients with HER2-positive tumors have poorer prognosis and significantly lower overall

survival compared to patients with HER2-negative tumors (3,4).

Trastuzumab humanized monoclonal antibody is the first HER2-targeted therapy that was

approved by the United States Food and Drug Administration (US FDA) for metastatic HER2-

overexpressing breast cancer as the first-line therapy often in combination with chemotherapy (5-

7). HER2 or EGFR2 is one of the 4 members of the EGF receptor (EGFR) family of tyrosine

kinases. It is an orphan receptor and can either dimerize with itself (especially when

overexpressed) or with other members such as EGFR (HER1) to initiate downstream signaling

events. Trastuzumab inhibits HER2-positive breast cancer cells by binding to the extracellular

domain of HER2. The exact mechanism is unknown although trastuzumab-induced receptor

endocytosis among others has been implicated (8). Historically, when trastuzumab was used

alone in non-selected patients with metastatic breast cancer, only about 10–15% of patients had a

partial response (7). The objective response rate improved to 26% when patients with HER2-

overexpressing metastatic disease receiving first-line treatment was selected (9). In the current setting, the response rate of patients to neoadjuvant trastuzumab in combination with chemotherapy ranges between 30 to 53% (10,11). These results reveal an important gap in

clinical oncology – HER2 is the only approved biomarker for anti-HER2-based therapy and the administration of anti-HER2 drugs based on HER2 status alone is insufficient in achieving a good response rate. Better and/or complementary predictors are required.

WW-domain binding protein 2 (WBP2) was first identified to be a cognate ligand of the

WW domain of Yes kinase-associated protein (YAP) (12,13). It was subsequently shown to be an adapter protein for Pax8 thyroid specific transcription factor (14) and a transcription coactivator in ER/PR signaling (15). The function of WBP2 in cancer was not known until its discovery as a novel breast cancer-associated protein that is post-translationally modified by

EGFR through tyrosine phosphorylation (16). Eventually, WBP2 was demonstrated to be an oncogene whose overexpression transformed normal mammary epithelial cells and conferred aggressive traits to cancer cells (17). At the same time, WBP2 was shown to be required for the oncogenic and growth promoting function of TAZ (18) and YAP (19). Recently, it was found that Wnt signaling promotes breast cancer by blocking ITCH E3 ligase-mediated degradation of

WBP2 (20). In the same study, analysis of >400 clinical specimens revealed that WBP2 is overexpressed in breast tumors and high WBP2 expression level correlated with disease aggression and poor patient survival (20). These studies highlighted the important role of WBP2 to breast cancer.

Two observations led to the hypothesis that WBP2 is a molecular determinant/predictor for anti-HER2-based therapies: first, WBP2 is within the EGFR signaling network in which

HER2 plays an intimate role; second, WBP2 is a prognostic factor and potent oncogene that regulates cancer cell growth. In this study, we established WBP2 as a downstream substrate of

EGFR/HER2 signaling and investigated the role of WBP2 in regulating the response of breast cancer cells to trastuzumab and the mechanism through which this occurs in preclinical studies.

Finally, we conducted a retrospective study to determine the correlation of WBP2 levels to response of HER2-positive breast tumors to neoadjuvant trastuzumab-based chemotherapy.

Materials and Methods

Reagents. In-house WBP2 polyclonal antibody was generated as previously described (17).

Monoclonal anti-WBP2 (MABS441-clone 4C8H10) was obtained from EMD Millipore. Anti- phosphotyrosine antibody-HRP (PY20) (MA1-12445) and anti-HER2 antibody (554299) were purchased from BD Biosciences. Anti-phospho-HER2 (PA5-17696) and anti-β-tubulin (MA5-

16308-1MG) antibodies were purchased from Thermo Scientific. Anti-phospho-EGFR (2231), anti-EGFR (2232), anti-phospho-AKT (9271), and anti-AKT (9272) antibodies were purchased from Cell Signaling Technology. Anti-cyclin D1 antibody (sc-20044) was purchased from Santa

Cruz Biotechnology. Trastuzumab (Herceptin®) was obtained from Roche. HER2 siRNAs,

WBP2 siRNAs, and luciferase siRNA were purchased from Thermo Scientific. WBP2 overexpression plasmid and knock down (shRNAs) constructs were previously described (17,20).

Cell lines and culture conditions. The human breast cancer cell lines, BT-474 and SK-BR-3, were purchased from American Type Cell Culture (ATCC). ZR-75-30 was a kind gift from Dr.

Boon Tin Chua (Institute of Molecular and Cell Biology, Singapore). All cell lines were tested using MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza) and verified to be free of

Mycoplasma and used after two - five passages from thawing. BT-474, SK-BR-3, and ZR-75-30 were maintained in RPMI1640 containing 10% FBS (Thermo Scientific) and 100U penicillin/streptomycin (Invitrogen). For transient expression, cells were transfected with HER2 siRNA or WBP2 siRNA using jetPRIME transfection reagent (Polyplus Transfection), according to the manufacturer’s recommendations. For stable expression, cells were transduced with each expression lentivirus, followed by selection with 400µg/ml hygromycin B (Invitrogen) for BT-

474 or 0.5µg/ml puromycin (Invitrogen) for SK-BR-3. After 2-3 weeks, selected colonies were pooled and expanded.

Cell proliferation assay. Cells were plated in 96-well plates for 2D culture or 96-well ultra-low

attachment plates for 3D culture (Corning) at 10,000 cells per well. After 3 or 5 days of

incubation with trastuzumab, the viability of cells was measured using CellTiter 96® Aqueous

Non-Radioactive Cell Proliferation Assay (Promega).

Cell cycle analysis. Cells were collected and fixed in 70% ethanol at 4˚C overnight. After

washing with PBS, the cells were stained with PI/RNase staining buffer (PBS containing 0.1%

triton X-100, 0.2 mg/ml RNase A, 20 ug/ml PI) for 15 min at room temperature. The DNA

content of cells was measured by flow cytometry (LSR Fortessa Flow Cytometry Analyser, BD

Biosciences). Proportions of cells in G1, S, and G2/M phases were analyzed using FACSDiva

Software (BD Biosciences).

Subcellular fractionation. Membrane and cytoplasmic extracts were prepared using the Mem-

PERTM Plus Membrane Protein Extraction Kit (Thermo Scientific), as per manufacturer's

instructions.

Western blot analyses and immunoprecipitation assays. Cell lysis, Western blot analysis, and

immunoprecipitation assay were performed as described previously (17,20). The detail methods

can be found in Supplementary Information.

Reverse transcription and real-time polymerase chain reaction. Total RNA was isolated using

PureLink RNA Mini Kit (Thermo Scientific) and reverse transcribed using random hexamer

primers and RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). qPCR reactions

were performed using QuantiFast Probe PCR Kit (Qiagen) as per manufacturer's instructions.

Predesigned primers/probes were obtained from IDT.

Bioinformatics analysis. Copy Number Variation (CNV) data and RNA-Sequencing (RNA-Seq)

data from TCGA-BRCA (21) was downloaded from the GDC Data Portal (22). The data were

cleaned to remove samples with incomplete data. The HER2 expression status were assessed

from the RNA-Seq data. The differences in CNV profile between Primary Tumor and Solid

Tissue Normal were then computed as described in Supplementary Information. Gene list

analysis was performed using PANTHER (23). A custom Python script was developed to query

PubMed® (Supplementary Methods). The word cloud was then generated using the Text

Analytics Toolbox of MATLAB® R2018b (MathWorks®, 2018).

Xenograft model. All animal experiments were performed in accordance with the institutional

guidelines of and were approved by Institutional Animal Care and Use Committee of National

University of Singapore. For the xenograft model, 5-week-old female athymic nude mice (n= 6-7,

In Vivos) were implanted with 0.72 mg 60 day release 17β-estradiol pellets (Innovative Research) and after 2 days, BT-474 control (vector) or WBP2 overexpressing cells (1 × 107 in 200 μl of

DPBS and Matrigel 1:1 mixture) were injected subcutaneously into a mouse mammary fat pad.

When the tumors reached the size of 150 – 200 mm3, the mice were divided into groups, keeping

average tumor size similar between groups, and treated with trastuzumab (Herceptin®) (10mg/kg,

Roche) or PBS (control) by intraperitoneally (IP) twice weekly for three weeks. The tumor size

was measured twice weekly with calipers and tumor volumes were calculated as follow: volume

= (width2 × length)/2.

Patient-Derived Xenografts (PDX) Trastuzumab-sensitive breast cancer PDX (PDX118) and

trastuzumab-resistant breast cancer PDXs (PDX118TRs) established by Arribas’ group as

previously described (24).

Human specimens. The study was conducted in accordance with the Declaration of Helsinki and

written informed consent was obtained from all participants. All specimens obtained after

protocol approval by Institutional Review Boards.

Retrospective study cohort 1: Correlation of WBP2 and HER2 to patients’ outcomes

Breast tumor tissues from patients who received surgery between 1997 and 2007 as well as the

clinical/histopathological information (including HER2 IHC scores) were obtained from

National Cancer Centre, Singapore (NCCs) (n=52), National University Hospital, Singapore

(NUH) (n=205) and Singapore General Hospital (SGH) (n=126). The information for some cases

are not available. Therefore, the total number of cases analyzed may not be the same as the

original number available. Patients received the treatment and surgery in accordance with each

institution’s standard treatment, but the treatment information was not available for this cohort.

The samples in this cohort were randomized and include HER2-positive and –negative breast

cancer cases.

Retrospective study cohort 2: Correlation of WBP2 with pathologic complete response to

trastuzumab-based neo-adjuvant therapy

The cohort here includes patients with stage I - III HER2-positive breast cancer who received

neoadjuvant trastuzumab-based chemotherapy between 2005 and 2017 and from whom archival

tumor specimens and clinical information were available. Patients received anthracycline or non-

anthracycline-containing regimens in combination with trastuzumab as part of each institution’s

standard treatment. Neoadjuvant trastuzumab with anthracycline-containing chemotherapy

regimens included AC-TH regimen (doxorubicin/cyclophosphamide followed by paclitaxel or docetaxel/trastuzumab) and FEC-TH regimen (fluorouracil/epirubicin/cyclophosphamide

followed by paclitaxel or docetaxel/trastuzumab). Neoadjuvant trastuzumab with non-

anthracycline-containing chemotherapy regimens included TCH regimen (paclitaxel or

docetaxel/carboplatin combined with trastuzumab) and TH regimen (paclitaxel or docetaxel

combined with trastuzuamb). Specimens comprised FFPE biopsies that were collected before

neoadjuvant treatment. A total of 143 cases complete with clinical information such as

pathologic response information were obtained from NUH, Kandang Kerbau Women's and

Children's Hospital (KKH), and Yonsei Cancer Center (YCC).

Immunohistochemical analysis. The detailed protocol for immunohistochemistry was

previously described (17,20). Immunohistochemistry of pre-treatment biopsy specimens was done in the NUH pathology department core using BenchMark ULTRA IHC autostainer (Roche

Ventana). All IHC slides were scored in a blinded fashion by pathologists. A scale of 0 to 3 was

used for the stain intensity - 0 (no staining), 1+ (mild), 2+ (moderate), and 3+ (strong staining).

Statistical analysis. All in vitro experiments were repeated at least three times and the results

were presented as mean ± SD. The comparisons between each group were determined by

Student’s t test. P-values of <0.05 were considered statistically significant and expressed as *P <

0.05; **P < 0.01; ***P < 0.001. For in vivo and retrospective study, the data represents mean ±

SEM and the significance of differences or association was evaluated using Mann-Whitney U

test. Kaplan-Meier method was used to plot the overall survival (OS) and disease-free survival

(DFS) curves. DFS and OS were defined as time from date-of-diagnosis to date-of-first-

recurrence and death or last follow-up date, respectively. Survival between groups was compared

using Log-rank test. The associations between pCR and WPB2 expression or/with clinical

characteristics were investigated by contingency tables and analyzed by Fisher’s exact test. All

statistical analyses were performed using SPSS and GraphPad Prism.

Results

HER2 amplification-associated gene network in breast cancer. The up-regulation of HER2 in

breast cancer is usually associated with an amplification of the HER2 locus (25). Inadvertently,

other genes along chromosome 17q (C17q) arm are co-amplified with HER2 (26). It is conceivable that HER2 and its associated genes along C17q not only form a molecular network but also cooperatively and functionally contribute to the phenotype of HER2-positive breast cancer. The HER2-associated genes may also regulate the response of HER2-positive breast cancer to drugs and are therefore potential companion diagnostics for HER2-based therapeutics.

Hence, we attempted to map the genes that are co-amplified with HER2, performed pre-clinical

tests to investigate their interactions with HER2 in the presence of trastuzumab and validate if

the expression of these candidate biomarkers correlate with response of breast tumors to

trastuzumab-based treatment via a retrospective study.

First, we obtained the copy number variation (CNV) and RNA-Seq data from the TCGA-

BRCA project (21), and employed the workflow shown in Fig. 1A to identify regions of

chromosome 17 that were amplified in HER2 up-regulated breast cancer. RNA-Seq data was

used to identify cases with HER2 up-regulated, while CNV data was used to identify regions of

chromosome 17 that were amplified or deleted. As shown in Fig. 1B, the p-arm of chromosome

17 experienced a significant deletion, while 3 regions along q-arm of chromosome 17 were significantly amplified in HER2 overexpressing breast cancer. 1145 genes were identified to reside in these 3 significantly amplified regions. Gene list analysis using the PANTHER

Classification System (23) was performed to provide a general understanding of the genes co- amplified with HER2. Interestingly, cellular processes (GO: 0009987) and metabolic processes

(GO: 0008152) accounted for about half of the hits (Fig. 1C). Amplification of genes involved in

these critical processes is likely to result in dysregulation and cancer phenotype.

To shortlist candidates that play role in regulating HER2 in breast cancer, we queried

PubMed® and found that 862 of the 1145 amplified genes are not implicated in breast cancer.

We further reasoned that candidates that influence the response of HER2-positive breast cancer to anti-HER2 drugs are likely to be proteins that are associated with EGFR and/or HER2 signaling. From the remaining 283 genes, only 101 genes are related to either EGFR signaling or

HER2 signaling. Out of the 101 genes associated with EGFR/HER2 and breast cancer, 55 of them have 2 or more publications and are represented in a word cloud. The size of the words reflects the number of publications (in a Log2 scale) that relate the gene to EGFR or HER2 in breast cancer (Fig. 1D). As expected, HER2 (ERBB2) was the top hit, with 9947 publications (as of September 2018). Found in this HER2-network of genes is WW-domain Binding Protein 2

(WBP2), an emerging oncogene recently implicated in breast cancer development (17,20) and a phosphotyrosine substrate of EGFR signaling pathway (16). Our data revealed that WBP2 and

HER2 are co-amplified in 36% of the HER2-positive breast cancers. It is conceivable that tyrosine phosphorylation of WBP2 by EGFR is HER2-dependent and aberrant WBP2 expression may be a novel mechanism that regulates cancer cellular response to anti-HER2 drugs.

WBP2 and HER2 expression in combination correlate with a worse prognosis in breast cancer patients than either alone. Since WBP2 and HER2 oncogenes are demonstrated to be co-amplified in C17q, we hypothesized that WBP2 co-express with HER2 in clinical breast cancers at the protein level and in combination they give rise to poorer prognosis. To test these hypotheses, a retrospective study was performed. Immunohistochemistry (IHC) of HER2 and

WBP2 was performed on 296 resected breast tumor tissues. Consistent with previous studies, patients with HER2-positive tumors had worse overall survival (p=0.011) and disease free survival (p=0.137) than the HER2-negative group (Fig. 1E) (4,27). Next, we analyzed the

correlation of WBP2 with HER2 status. Both nuclear and cytoplasmic WBP2 expression was

significantly higher in HER2-positive breast tumors compared to HER2-negative tumors (Fig.

1F). Since WBP2 expression and HER2 status were positively correlated, we proceeded to

examine if patients’ outcome is associated with WBP2 and HER2 in combination. We found that patients whose tumors showed high nuclear WBP2 and HER2-positive expression had the worst overall (HR: 4.49, p<0.001) and disease free survival (HR: 2.58, p=0.004) than other groups

(Fig.1G). WBP2 expression in combination with HER2 appears to be more powerful than either alone for breast cancer prognosis. It suggests that WBP2 can be used to further prognosticate

HER2-positive breast cancer patients.

HER2 is required for EGF-induced WBP2 tyrosine phosphorylation. The association of

WBP2 with HER2 in clinical specimens, coupled to the previous finding that WBP2 is a

downstream target of EGF signaling led to the postulation that tyrosine phosphorylation of

WBP2 by EGFR requires HER2, since EGFR and HER2 are known to heterodimerize in the

presence of EGF (16). To test this hypothesis, HER2 expression was knocked down using two

different siRNAs in HER2+/WBP2+ breast cancer cell lines. Knock down of HER2 abolished

EGF induced-tyrosine phosphorylation of WBP2 in SK-BR-3 and ZR-751 breast cancer cells

(Fig. 1H and 1I). This indicates that HER2 is required for tyrosine phosphorylation and activation of WBP2 by EGF.

WBP2 expression enhances trastuzumab-response in breast cancer cells. Given that WBP2

is downstream of HER2, aberrant expression of oncogenic WBP2 (20) may influence cancer

cellular response to trastuzumab. To investigate the effect of WBP2 expression on the response

of HER2-positive breast cancer cells to trastuzumab as a single agent, SK-BR-3, ZR-75-30 and

BT-474 breast cancer cell lines were selected since they are HER2-positive and trastuzumab

sensitive as shown by our preliminary study and other reports (28). While trastuzumab is rarely

used as a single agent in the clinic, a clean/simplistic system involving only trastuzumab was needed to prove a direct link and to study the interactions between WBP2, HER 2 and trastuzumab. As would be evident later, this “reductionist” approach in the lab would be

complemented with an actual and more complex clinical setting in which the correlation of

WBP2 expression with breast tumor response to trastuzumab with chemotherapy treatment

would be tested.

WBP2 expression was silenced using two different shRNAs in SK-BR-3 and two different

siRNAs in ZR-75-30, while the overexpression of WBP2 was achieved using a lentiviral system

in BT-474. Each cell line was subsequently treated with increasing doses of trastuzumab (1, 10,

100 μg/ml) and the cell growth analyzed for 3 days (for SK-BR-3) or 5 days (for ZR-75-30 and

BT-474) following incubation in 2D or 3D culture condition. In WBP2-overexpressing BT-474

cells, the response to trastuzumab (100 μg/ml) increased by 2.1 fold for 2D culture (p=0.001) and

2.2 fold for 3D culture (p=0.003) compared to vector control (Fig.2A and 2B). On the other

hand, knock down of WBP2 significantly reduced the response to trastuzumab in SK-BR-3 (by

~37 %) (Fig. 2C and 2D) and ZR-75-30 cells (by ~45%) (Fig. 2E and 2F) compared to the

controls. These data provide evidence that WBP2 expression level influences the response of

breast cancer cells to trastuzumab.

WBP2 enhances the modulation of EGFR/HER2 signaling in the presence of trastuzumab.

The mechanism of trastuzumab as an antitumor agent remains an area of active research. Some groups have shown that trastuzumab induced down-regulation of HER2 level (29-31). To determine whether WBP2 affects trastuzumab-induced downregulation of HER2 expression,

WBP2-overexpressing BT-474 cells and WBP2 knocked-down SK-BR-3 or ZR-75-30 cells were

treated with increasing doses of trastuzumab (1, 10, 100 μg/ml) and HER2 protein expression

analyzed using Western blot analysis. In WBP2-overexpressing BT-474 cells, both HER2 and

EGFR levels were substantially decreased when exposed to a low dose of trastuzumab, compared

to control cells (Fig 2G). Consistently, knock down of WBP2 inhibited trastuzumab-dependent

down-regulation of HER2 and EGFR levels in SK-BR-3 and ZR-75-30 breast cancer cells. In

contrast, HER2 levels were decreased by trastuzumab in a concentration-dependent manner in

control cells (scrambled shRNA or Luc siRNA) (Fig. 2H and 2I). Like HER2 and EGFR,

inactivation of AKT by trastuzumab, which has been reported by previous studies (32-34), was enhanced by WBP2 overexpression and diminished by WBP2 knock down. Figure 2J and 2K demonstrated that it was the cell-surface species of HER2 that was affected by WBP2 in the

presence of trastuzumab. Collectively, our data suggests that WBP2 enhances the inhibitory

effect of trastuzumab on cancer cell proliferation by augmenting trastuzumab-induced down-

regulation of HER2/EGFR signaling.

WBP2 promotes trastuzumab-induced G1 arrest by enhancing downregulation of Cyclin

D1 expression. Previous studies showed that trastuzumab treatment induced G1 cell cycle arrest,

leading to inhibition of cell proliferation (35). To investigate the involvement of WBP2 in

trastuzumab-induced G1 arrest, we performed cell-cycle FACS analysis of WBP2 knocked-down

vs. control ZR-75-30 cells. Trastuzumab increased the number of cells at G0/G1 phase by 14.5 %

compared to control (no trastuzumab treatment; p=0.0003) while trastuzumab decreased the

number of cells at S and G2/M phase by 52.4% and 37.0%, respectively. Knock down of WBP2

using two different WBP2 siRNAs abolished the effects of trastuzumab on cell cycle (Fig. 2L).

We further examined WBP2’s involvement in trastuzumab-mediated G1 arrest by analyzing

cyclin D expression. Trastuzumab decreased cyclin D protein level by 87.6% (p<0.001) in ZR-

75-30 cells as compared to untreated cells (Luc si); This effect was only 44.6% to 56.2% when

WBP2 was silenced with two separate siRNAs (Fig. 2M). Similarly, the decrease in cyclin D

mRNA level by trastuzumab was abolished when WBP2 was knocked down (Fig. 2N).

Collectively, these results suggest that WBP2 promotes trastuzumab-induced G1 arrest via

down-regulation of cyclin D.

WBP2 overexpression sensitizes breast tumor to Trastuzumab in vivo. Our in vitro

observations indicated that cancer cellular response to trastuzumab depended on WBP2 level. To

confirm observation, an in vivo tumor xenograft model was used. BT-474 cells stably expressing

WBP2 were injected into the mammary fat pad of athymic nude mice. When the size of tumors

reached 150 – 200 mm3, the mice were divided into 2 groups and treated with trastuzumab (10

mg/kg) or PBS by intraperitoneal (IP) injection twice weekly for three weeks. The size of

WBP2-overexpressing tumor after trastuzumab treatment was reduced to 5.23% as compared to

no treatment (100%), whereas the size of tumor with vector control was reduced only to 38.33%

(Fig. 3A and 3B). As the difference between the two trastuzmab-treated conditions appeared small due to the scale used in the plot shown in Fig 3A and 3B, separate plots are provided in

Supplementary Figure 1 to show their differences more clearly. Hence, overexpression of WBP2

increased tumor response to trastuzumab by 7.32 times (p = 0.0047). The results support the

notion that high WBP2 level sensitized breast cancer to trastuzumab.

WBP2 expression correlates with trastuzumab response in Patient-Derived Xenograft

(PDX) model. To obtain more evidence that high WBP2 expression is associated with positive

response to trastuzumab treatment, we examined WBP2 and HER2 expression in a Patient-

Derived Xenograft (PDX) model of drug resistance to trastuzumab. Tumor samples from

trastuzumab-sensitive breast cancer PDX (PDX118) and trastuzumab-resistant breast cancer

PDXs (PDX118TRs) established by Arribas’ group were used (24). The IHC staining shows that

trastuzumab-sensitive sample (PDX118) was HER2-positive and WBP2-positive. Interestingly, 3

(TR2-4) out of 5 tumors that acquired trastuzumab resistance had lower HER2 and WBP2

expression (Fig. 3C). Pearson’s correlation test revealed that HER2 and WBP2 expression had a

correlation coefficient (Pearson r) of r=0.8944 (p=0.0161), indicating strong association between

HER2 and WBP2 expression (Fig. 3D). The data suggest that WBP2 in combination with HER2

expression is a determinant of response to trastuzumab.

High WBP2 expression is associated with pathologic complete response of HER2-positive

breast cancer patients to neoadjuvant trastuzumab-based treatment. To investigate whether

WBP2 expression correlates with the response of HER2-positive breast cancer to trastuzumab in

the clinical setting where chemotherapies are used in conjunction, a retrospective study was

conducted. One hundred and forty three cases of pre-treatment biopsy specimens from breast

cancer patients who received trastuzumab-based neo-adjuvant chemotherapy were collected from

3 different cohorts; NUH, KKH, and YCC. The characteristics of the patients before treatment

are provided in Table 1. Overall, 64 (44.76%) patients achieved pCR after trastuzumab-based

neoadjuvant chemotherapy which is similar to previous studies (30%-53%) (10,11). The ER- or

PR-negative group showed higher pCR than the ER- or PR-positive group, which is also

consistent with previous reports (10). No statistically significant relationship between pCR and other clinical factors was obtained (Supplementary Table 1).

To analyze WBP2 expression in pre-treatment biopsy specimens, immunohistochemistry (IHC)

of WBP2 was performed using a WBP2 polyclonal antibody (17,20). The specificity of the

WBP2 polyclonal antibody for IHC was validated previously (20). The IHC score for WBP2 was

higher in the pCR group than non-pCR group (mean WBP2 IHC score, 2.63 vs 2.05, p<0.0001)

(Fig. 4A). Next, the breast tumors were segregated into high (=3) and low WPB2 IHC score

(<3). The high- WBP2 expressing group had significantly higher pCR of 2.5 times more than the

low WBP2- expressing group (67.19% vs 26.58%, respectively) (Fig. 4B and Table 2). We further examined whether there were clinical factors that might be associated with pCR and

WBP2 expression. When the patients were first stratified by age (<50 and ≥ 50 years old) and menopausal status and the breast tumors further stratified based on WBP2 IHC score (=3 and <3), the pCR of patients with high WBP2 and age below 50 years was 77.78% while patients with high WBP2 and pre-menopausal was 73.33%, both of which are higher than the pCR of 67.19% for tumors with high WBP2 alone (Fig 4C and 4D).

Retrospective receiver operating characteristic (ROC) curve analysis was performed to determine

the optimal sensitivity and specificity of WBP2 expression in discriminating pCR from non-pCR

cases. The areas under the curve (AUC) was 0.72 with the sensitivity and specificity at 67.19%

and 73.42% respectively when a WBP2 IHC cut-off score of 2.5 was used (Fig. 4E -black line and Table 2). Both the sensitivity and specificity were higher in patients aged below 50 years

(80.77% and 80.00%) and those in the pre-menopausal group (81.48% and 76.47%) (Table 2).

The AUC for the pre-menopausal patients (0.80) and patients aged below 50 years (0.81) were

higher compared to overall group (0.72) (Fig. 4E - red line and blue line, respectively). No

significant correlation was observed for other clinical factors including ER/PR status, tumor

grade, TNM stage, chemotherapy regimen (data not shown). Collectively, the retrospective study

indicates that high WBP2 expression in pre-treatment biopsy is positively correlated with

pathologic complete response, especially for those aged below 50 years group and in the pre-

menopausal group.

Discussion

Chromosome 17 aneusomy often occurs in breast cancer and is associated with poor prognosis in invasive breast carcinoma (36). Clinical trials showed that polysomy of chromosome 17 might be associated with trastuzumab response in HER2 FISH-negative but

HER2 IHC-positive breast cancer (37,38). These studies indicate that other genes in chromosome

17 might be involved in trastuzumab response in HER2-positive breast cancer. Through bioinformatic analysis of chromosome 17q, WBP2 was found to be co-amplified with HER2 in human breast tumors. We previously reported that WBP2 is an oncogene that promotes cell growth, proliferation, and invasion via EGFR/Wnt pathway crosstalk in breast cancer (17,20).

WBP2 is phosphorylated by c-Src and c-Yes though EGF/EGFR signaling and phosphorylated/activated WBP2 translocates to the nucleus, acting as a transcriptional coactivator to promote growth of breast cancer cells. WBP2 is overexpressed in breast cancer and its expression is correlated with poor OS/DFS. In this study, we further discovered that patients with tumors that co-express WBP2 and HER2 have the worst prognosis compared to either alone. EGF-induced tyrosine phosphorylation of WBP2 was also found to be dependent on

HER2 in this study. Taking these observations into account, we tested the hypothesis that WBP2 expression regulates the response of breast cancer to trastuzumab-based treatment.

Trastuzumab is the first-line therapy for HER2-positive breast cancer patients. However, not all of these patients respond to treatments that involved trastuzumab (9). It is conceivable that breast cancer’s response to tratuzumab does not solely depend on HER2 expression, but also on other biomarkers that are molecularly networked with HER2. Several studies have reported potential biomarkers that are associated with positive or negative response to trastuzumab.

(39,40). For example, loss of PTEN and PIK3CA mutations have been observed in trastuzumab-

resistant breast cancers and both are linked to poor prognosis and lower overall survival (39).

HER2-positive breast cancer patients with PIK3CA mutation showed lower pCR (28.6%) to

HER2-targeting neoadjuvant therapies, as compared to wild-type PIK3CA (53.1%)(40). In this

study, we provide evidence that WBP2 is a potential predictor of trastuzumab response in HER2-

positive breast cancer.

Trastuzumab plus chemotherapy in the neoadjuvant setting is given to HER2-positive

breast cancer with locally advanced or inflammatory breast cancer. This treatment strategy has

shown significantly improved pathologic response compared to chemotherapy alone (41,42).

Pathologic complete response (pCR) is correlated with better prognosis and shows clinical

responsiveness to systemic therapy. The neoadjuvant treatment approach is becoming diverse

and the choice of treatment is best personalized, based on the individual’s molecular portrait.

Identifying novel biomarkers beyond HER2 status that could improve pCR would be helpful for patients and clinicians in choosing a treatment strategy with better clinical outcome. In this study, we observed that patients with HER2-positive breast cancer and high WBP2 expression

showed higher pCR (67.19%) to trastuzumab-based neoadjuvant chemotherapy, as compared to

the overall HER2-positive breast cancer cases (44.76%). Subgroup analysis showed that pCR

was higher in the group of patients aged below 50 years and whose tumors possessed high WBP2

level (77.78%) compared to those above 50 years old. Similarly pre-menopausal patients with

tumors that expressed elevated WBP2 level showed higher pCR (73.33%) than the non-

menopausal group. The sensitivity and specificity of WBP2 in discriminating between pCR and

non-pCR were also better in patient groups with age below 50 years (80.77% and 80.00%) and of pre-menopausal status (81.48% and 76.47%) compared to the overall group (67.19% and

73.42%). Since the average age of menopause is 49-52 years old (43), there would be significant

overlap of patients between the 2 groups. Given that menopausal status is sometimes not well

documented in the medical records while age is a more objective parameter, we recommend that

age is a more useful clinical factor in conjunction with the molecular determinants WBP2 and

HER2 for stratification of patients for precision medicine using trastuzumab. The ability to

identify patients who are likely to respond well to neoadjuvant trastuzumab allows clinicians to

better plan therapeutic interventions for patients. Patients can also be better counselled regarding

what to expect from the neoadjuvant therapy. It would also be possible to predict which patients

would attain successful downstaging of their tumors from neoadjuvant therapy, thus allowing for

breast conserving surgery, instead of mastectomy in some instances. This will have significant

impact on the patients’ cosmetic and psychological outcome.

We showed that WBP2 expression correlated with response to trastuzumab-based

neoadjuvant therapy. However, it should be noted that the retrospective study performed in this study involved patients who had received neo-adjuvant therapy comprising trastuzumab in combination with chemotherapy regimens that varied according to the physician in attendance and hospital practices. This therefore confounds the data generated and possibly the conclusions drawn. Recognizing this weakness, we performed an analysis to investigate whether WBP2 levels correlates with response to chemotherapy regimens. The chemotherapy regimens given

can be classified as 1) Anthracyclines-based regimens, such as Adriamycin (doxorubicin) and 2)

Non-Anthracyclines based. As shown in Supplementary Table 1, there was no significant

difference in the pCR rate between these groups of patients treated with Anthracyclines and non-

Anthracyclines regimens (45.36% and 44.44%, respectively) compared to the overall rate of

44.76% in the non-stratified population. Furthermore, we stratified tumors with high WBP2 level

into those treated with Anthracyclines and non-Anthracyclines-based regimens. The pCR of

patients with high WBP2-expressing tumors treated with Anthracyclines-based and non-

Anthracyclines-based regimens was 69.23% and 64.00%, respectively (Supplementary Fig. 2A),

both of which were similar to the pCR of the group of samples stratified based on high WBP2

alone (67.19%) (Supplementary Fig. 2A and Table 2).

These results above indicate that the chemotherapy regimens do not influence pCR of

patients to trastuzumab-based neo-adjuvant therapy. It also supports the notion that WBP2 does

not correlate with response to chemotherapy and strengthens the link between WBP2 and

trastuzumab targeted therapy. This is further corroborated by preliminary in vitro data which argues that WBP2 does not regulate breast cancer response to chemotherapy since overexpression or knock down of WBP2 did not affect cellular viability upon doxorubicin treatment (Supplementary Fig. 2B-2D). Nevertheless, it would be necessary to confirm and establish the value of WPB2 in predicting response to trastuzumab-based chemotherapy via a more tightly controlled prospective study. Taken together, the molecular basis for the role of

WBP2 as a companion diagnostics for trastuzumab precision medicine is putatively through its

interaction with HER2 in breast cancer cells.

Neoadjuvant therapy for HER2-positive breast cancer has improved significantly over

time with clinical trials supporting the combinatorial use of different anti-HER2 inhibitors,

including lapatinib (tykerb) and pertuzumab (44,45). Since our data shows that WBP2 regulates

HER2 and its downstream signaling, it is conceivable that WBP2 also enhances response of

HER-positive breast cancer to other HER2-targetring agents such as pertuzumab and lapatinib.

Further studies will be needed to test these hypothesis.

In conclusion, there is a clinical need to improve the response rate of HER2-positive breast cancer patients to HER2-targeted therapeutics especially in the neoadjuvant setting. Our study posits that trastuzumab is more effective in HER2-positive breast cancers that express high levels of WBP2. WBP2 is a potential companion diagnostic for further stratification of HER2- positive breast cancer patients for more effective anti-HER2 therapies.

Author contributions

Conception and design: S-AK, YPL

Development of methodology: S-AK, JSG, HJT, TC, YPL

Acquisition of data (including xenograft model, PDX model, clinical samples): S-AK, JSW, HJT,

TC, CB, JA, CYW, PHT, MG, TCP, JS, SHL, SCL, YPL

Analysis and interpretation of data (including statistical analysis, biostatistics, computational analysis): S-AK, HJT, TC, TCP, YPL

Writing, review, and/or revision of the manuscript: S-AK, JSG, HJT, TC, AAT, JA, CYW, PHT,

MG, TCP, JS, SHL, SCL, YPL

Study supervision: YPL

All authors read and approved the manuscript for publication.

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Figure legends

Figure 1. (A-D) Bioinformatic analysis of TCGA-BRCA (Breast Invasive Carcinoma) dataset. (A) Flow chart describing the analysis pipeline of copy number variation (CNV) profile of HER2 overexpressing breast cancers. (B) Differences in CNV profile between Primary Tumor and Non-Cancer Solid Tissue. Positive values indicate amplification in Primary Tumor, while negative values indicate deletion in Primary Tumors. Colormap indicates the statistical significance (p-value) of the difference between Primary Tumor and Solid Tissue Normal. (C) Pie chart showing biological processes associated with genes co-amplified with HER2. Panther Classification System was used for the analysis. (D) Word cloud showing the frequency of association between each co-amplified genes with breast cancer, and EGFR or HER2. (E-G) Immunohistochemistry and correlation analyses of WBP2 and HER2 expression in human breast tumors to survival outcomes. (E) Kaplan-Meier survival analysis of the overall (OS) and disease free (DFS) survival of patients segregated into HER2+ and HER2- groups (n=221). (F) Box plot of WBP2 expression in HER2+ and HER2- breast tumors (n=296). (G) Kaplan-Meier analysis of overall survival and disease-free survival of patients segregated based on WBP2 and HER2 expression status (n=221). High WBP2 expression is defined as nuclear IHC score of more than 1. Statistical significance was determined by Matt-Whitney U test and Log rank test (*** P < 0.001). (H-I) Phosphorylation of WBP2 is dependent on HER2. HER2 was knocked down in human breast cancer cells, SK-BR-3(H) and ZR-751(I) via HER2-specific siRNAs. Luciferase siRNA was used as negative control. Cells were treated with 50ng/ml EGF for 10min after 24hr serum starvation. Cell lysates were immunoprecipitated (IP) with anti-WBP2 antibody and phosphorylation of endogenous WBP2 were analyzed by Western blot (IB) using anti- phosphotyrosine (PY20) and anti-WBP2 antibodies. HER2 was analyzed by Western blot (IB) with indicated antibodies. β-tubulin was used as protein loading control.

Figure 2. (A-F) Dose-dependent effect of trastuzumab on the proliferation of HER2-postive breast cancer cells with WPB2 overexpressed or knocked down. WBP2 was overexpressed in BT-474 using lentivirus (A and B) or knocked down using two different WBP2-specific shRNA in SK-BR-3 (C and D) or two different WBP2-specific siRNAs in ZR-75-30 (E and F). Cells were plated in 96-well plates for 2D culture (A, C, E) or 96-well ultra-low attachment plates for 3D culture (B, D, F) at 10,000 cells per well. After 3 days (SK-BR-3) or 5 days (BT-474 and ZR- 75-30) of incubation with trastuzumab, cell viability was measured using CellTiter 96 aqueous non-radioactive cell proliferation assay. Cell viability was calculated as fold change compared to trastuzumab-untreated control cells. The data represent mean ± SD (n=3). Statistical significance was determined by Student’s t-test (* or +P < 0.05; ** or ++P < 0.01; *** or +++P < 0.001 vs. vector or control). (G-I) Dose-dependent effect of trastuzumab on HER2 and its downstream signaling pathway. WBP2 was overexpressed in BT-474 using WBP2 expressing lentivirus (G) or knock down using two different shRNAs targeting WPB2 in SK-BR-3 (H) or two different siRNAs targeting WBP2 in ZR-75-30 (I). Cells were treated with different concentrations of trastuzumab (0, 1, 10, 100 µg/ml) for 3 days (SK-BR-3) or 5 days (BT-474 and ZR-75-30).

Expression and phosphorylation of proteins were analyzed by Western blot using the indicated antibodies. (J-K) Effect of WBP2 on cell surface HER2 level in the absence or presence of trastuzumab. BT-474 cells expressing WBP2 or vector (J) and SK-BR-3 cells expressing WBP2 shRNA or scramble shRNA (K) were treated with different concentrations of trastuzumab (0, 1, 10, 100 µg/ml). After 3 days (SK-BR-3) or 5 days (BT-474) of treatment, cells were separated into membrane and cytosol fraction using Mem-PERTM Plus Membrane Protein Extraction Kit. HER2 level in the membrane and cytosol fractions were analyzed by Western blot. (L-N) Effect of WBP2 on trastuzumab-induced G1 arrest. Two different siRNAs targeting WBP2 were transfected into ZR-75-30 cells that were subsequently treated with 100 µg/ml of trastuzumab for 5 days. (L) Cells were stained with Propidium iodide (PI) and cell cycle distribution was analyzed by flow cytometry. (M and N) Cyclin D1 protein level (M) and mRNA level (N) were analyzed. Protein expression level of cyclin D1 was quantitated and normalized to β-tubulin using ImageJ software and cyclin D1 expression in control (Luc siRNA or Vector) without trastuzumab was defined as 1. WBP2 or cyclin D1 mRNA expression level was analyzed by qRT-PCR and normalized to 18s rRNA. The data represent mean ± S.D. from three independent experiments (n=3). Statistical significance was determined by Student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.001 vs. control).

Figure 3. (A-B) Effect of WBP2 on tumor response to trastuzumab in vivo. 5-week-old female athymic nude mice (n= 6-7) were implanted with 0.72 mg 60 day release 17β-estradiol pellets. After 2 days, BT-474 control (vector) or WBP2 overexpressing cells (1 × 107 of cells in 200 μl of DPBS and Matrigel 1:1 mixture) were injected subcutaneously into a mouse mammary fat pad. When the tumor size reached 150 – 200 mm3, the mice were divided into groups, keeping average tumor size similar between groups, and treated with trastuzumab (10mg/kg, Roche) or PBS (control) intraperitoneally (IP) twice weekly for three weeks. (A and B) The tumor size was measured twice weekly with calipers and tumor volumes calculated as follow: volume = (width2 × length)/2. Tumor growth was presented in time-course line plot (A) and end-point dot plot (B). The data represent mean ±SEM. Statistical significance was determined by Mann– Whitney test. (C-D) HER2 and WBP2 expression in PDX model of isogenic trastuzumab- sensitive and –resistant breast cancer cells. (C) HER2 and WBP2 expression in tumor from trastuzumab-sensitive or resistant Patient-Derived Xenografts (PDXs) that were established in Dr. Joaquín Arribas’s research group (24) were analyzed by immunohistochemistry. The scale bars represent 250 μm. (D) Statistical correlation studies of the IHC scores for HER2 and WBP2 for each tumor was performed using the Pearson correlation test.

Figure 4. Correlation between WBP2 expression and pathologic complete response. (A-D) Analyses of WBP2 expression (based on IHC) in breast tumors of neoadjuvant trastuzumab+chemotherapy-treated patients segregated into those that showed pathologic complete response (pCR) and partial/non-pathologic complete response (non-pCR) (n=143). (A) WBP2 IHC scores in pCR or non-pCR groups. The data represent mean ±SEM. Statistical significance was determined by Mann–Whitney test. (B-D) WBP2 expression was segregated

into IHC score of 3 or <3 and pCR or non-pCR in the overall group (B), in the age <50 years group (C), and pre-menopausal group (D). Statistical significance was determined by Fisher’s exact test. (E) ROC curves analysis was conducted for overall group, pre-menopausal group, and aged below 50 years group. Area under the curve (AUC) of overall, pre-menopausal group, and aged below 50 years group was calculated (***, p<0.0001).

Table 1. Patients Characteristics

Characteristics N (%) Overall population 143 Complete pathological response pathological response 64 44.8 (pCR) Median age [range] (years) 53 [25 - 76] <50 56 39.2 Age ≥50 86 60.1 NA 1 0.7 Pre-Menopause 61 42.7 Post-Menopause 71 49.7 Menopausal status Peri-Menopause 10 7.0 NA 1 0.7 Negative 77 53.8 ER status Postive 63 44.1 Null (NA) 3 2.1 Negative 83 58.0 PR status Postive 57 39.9 Null (NA) 3 2.1 1 - - 2 49 34.3 Tumour Grade 3 79 55.2 NA 15 10.5 T1 19 13.3 T2 80 55.9 T stage T3 29 20.3 T4 13 9.1 NA 2 1.4 N0 40 28.0 N1 22 15.4 N stage N2 64 44.8 N3 13 9.1 NA (NX) 4 2.8 I 8 5.6 II 84 58.7 Overall TNM stage III 49 34.3 NA 2 1.4

Table 2. Association between WBP2 IHC score and pCR

WBP2 pCR Non-pCR Sensitivity Specificity Characteristics p-valueA 95% CI 95% CI IHC score N % N % (%) (%) Overall 64 44.76 79 55.24 population 54.31% to 62.28% to =3 43 67.19 21 32.81 67.19 73.42 ***, 78.41% 82.73% <0.0001 <3 21 26.58 58 73.42 Age 60.65% to 61.43% to <50 =3 21 77.78 6 22.22 80.77 80.00 ***, 93.45% 92.29% <0.0001 <3 5 17.24 24 82.76 40.82% to 53.75% to ≥50 =3 22 59.46 15 40.54 57.89 68.75 *, 0.017 73.69% 81.34% <3 16 32.65 33 67.35 Menopausal

status 61.92% to 58.83% to Pre-menopause =3 22 73.33 8 26.67 81.48 76.47 ***, 93.70% 89.25% <0.0001 <3 5 16.13 26 83.87 33.06% to 53.47% to =3 16 57.14 12 42.86 51.61 70.00 Post-menopause n.s., 0.088 69.85% 83.44% <3 15 34.88 28 65.12

A, Fisher’s exact test

Elevated WBP2 expression in HER2-positive breast cancers correlates with sensitivity to trastuzumab-based neo-adjuvant therapy:A Retrospective and Multicentric Study

Shin-Ae Kang, Jye Swei Guan, Hock Jin Tan, et al.

Clin Cancer Res Published OnlineFirst December 28, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-18-3228

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