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αB-crystallin expression in breast cancer is associated with brain metastasis

αB-crystallin expression in breast cancer is associated with brain metastasis www.nature.com/npjbcancer All rights reserved 2374-4677/15 ARTICLE OPEN αB-crystallin expression in breast cancer is associated with brain metastasis 1 2 3 4 3 5 6 K David Voduc , Torsten O Nielsen , Charles M Perou , J Chuck Harrell , Cheng Fan , Hagen Kennecke , Andy J Minn , 7,9 8,9 Vincent L Cryns and Maggie CU Cheang BACKGROUND/OBJECTIVES: The molecular chaperone αB-crystallin is expressed in estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2 ‘triple-negative’ breast carcinomas and promotes brain and lung metastasis. We examined αB-crystallin expression in primary breast carcinomas with metastatic data to evaluate its association with prognosis and site-specific metastases. METHODS: αB-crystallin gene (CRYAB) expression was examined using publically available global-gene expression data (n = 855 breast tumors) with first site of distant metastasis information (‘855Met’). αB-crystallin protein expression was determined by immunohistochemistry using the clinically annotated British Columbia Cancer Agency (BCCA) tissue microarray (n = 3,987 breast tumors). Kaplan–Meier and multivariable Cox regression analyses were used to evaluate the prognostic value of αB-crystallin. Multivariable logistic regression analysis was used to evaluate risks of αB-crystallin and other markers for site of metastasis. RESULTS: In the 855Met data set, αB-crystallin gene (CRYAB) expression was an independent predictor of brain as the first distant site of relapse (hazards ratio, HR = 1.2, (95% confidence interval, CI 1.0–1.4), P = 0.021). In the BCCA series, αB-crystallin protein expression was an independent prognostic marker of poor breast cancer-specific survival (HR = 1.3, (95% CI 1.1–1.6), P = 0.014). Among patients with metastases, αB-crystallin was the strongest independent predictor of brain metastasis (odds ratio, OR = 2.99 (95% CI 1.83–4.89), Po0.0001) and the only independent predictor of brain as the first site of distant metastasis (OR = 3.15 (95% CI 1.43–6.95), P = 0.005). αB-crystallin was also associated with worse survival (3.0 versus 4.7 months, P = 0.007). CONCLUSIONS: αB-crystallin is a promising biomarker to identify breast cancer patients at high risk for early relapse in the brain, independent of estrogen receptor and human epidermal growth factor receptor-2 status. npj Breast Cancer (2015) 1, 15014; doi:10.1038/npjbcancer.2015.14; published online 21 October 2015 INTRODUCTION metastasis in this subtype could lead to biomarkers to help identify women at high risk for relapse at these sites. Over the past 15 years, gene expression studies in breast cancer αB-crystallin is a widely expressed member of the small heat have led to more accurate prognostic tools and to a greater 1–5 shock protein family that protects cells from stress by its dual understanding of the molecular heterogeneity of breast cancer. function as a molecular chaperone to preserve proteostasis and as Basal-like breast carcinomas have emerged as a distinctive a cell death antagonist that inhibits caspase-3 activation and molecular subtype defined by expression of genes characteristic 18–22 oxidative stress. αB-crystallin is commonly expressed in basal- of basal epithelial cells that overlap in large part with estrogen like and TNBCs and its expression correlates with resistance to receptor (ER), progesterone receptor (PR), and human epidermal 23–26 neoadjuvant chemotherapy and poor survival. More recently, growth factor receptor-2 (HER2) ‘triple-negative’ breast cancers 6,7 αB-crystallin has been demonstrated to inhibit extracellular matrix (TNBC). More recent studies point to molecular heterogeneity detachment-induced apoptosis (‘anoikis’), enhance penetration within the triple-negative subgroup with potential prognostic and 8–10 through an endothelial/astrocyte co-culture model of the blood– therapeutic implications. Overall, basal-like tumors are asso- brain barrier in vitro, and promote lung and brain metastasis in ciated with a higher risk of recurrence following conventional 27,28 4,11,12 orthotopic models of TNBC in immunodeficient mice. In adjuvant treatment and poor survival. Furthermore, basal-like addition, αB-crystallin is frequently expressed in brain metastases tumors have a characteristic pattern of distant metastasis to 13–15 the lungs and brain with infrequent relapse in bone. Although and its expression in primary breast carcinomas is associated with lung and brain metastasis gene signatures have been poor overall survival and poor survival after brain metastasis in a 16,17 cohort of 76 women with breast cancer brain metastasis. identified, the functional contribution of individual genes to site-specific metastasis in this subtype is poorly understood. Collectively, these findings point to a key pathogenic role of Clearly, the identification of pathogenic drivers of brain and lung αB-crystallin in lung and brain metastasis in TNBC. 1 2 Department of Radiation Oncology, University of British Columbia, Vancouver, BC, Canada; Department of Pathology and Laboratory Medicine, University of British Columbia, 3 4 Vancouver, BC, Canada; Department of Genetics, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Pathology, Virginia Commonwealth University, Richmond, VA, USA; Division of Medical Oncology, British Columbia Cancer Agency, University of British Columbia, Vancouver, BC, Canada; Department of Radiation Oncology, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; 7 8 Department of Medicine, University of Wisconsin Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA and Division of Clinical Studies, Clinical Trials and Statistics Unit (ICR-CTSU), Institute of Cancer Research, London, UK. Correspondence: VL Cryns or MCU Cheang (vlcryns@medicine.wisc.edu or maggie.cheang@icr.ac.uk) These authors contributed equally to this work. Received 22 May 2015; revised 17 July 2015; accepted 7 September 2015 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited αB-crystallin and brain metastasis in breast cancer KD Voduc et al Given the recent evidence supporting a prometastatic function Statistical analysis for αB-crystallin in TNBC, we postulated that αB-crystallin might Univariable survival estimates were calculated using the Kaplan–Meier method, and survival differences were estimated using log-rank tests. Due have utility as a prognostic biomarker to predict the risk of disease to the retrospective nature of the gene expression and IHC studies, the progression in TNBC patients. To this end, we examined the sample size for each study was not pre-specified. The primary endpoint for prognostic value of αB-crystallin (gene and protein) expression in the BCCA cohort is breast cancer-specific survival (BCSS). Multivariable Cox breast cancer using (1) publically available gene expression data regression analyses were used to test the independent prognostic value of from multiple breast cancer cohorts, which included sites of first αB-crystallin. Smoothed plots of weighted Schoenfeld residuals were distant metastasis, and (2) a large breast cancer tissue microarray used to test proportional hazard assumptions. The association of (TMA) linked to long-term outcomes, including metastatic sites. αB-crystallin expression with intrinsic subtypes was assessed using the Using these data sets, we confirmed the association between αB- χ -test. Multivariable logistic regression analysis was used to evaluate the association of αB-crystallin and other clinicopathological markers for site of crystallin protein expression and basal-like phenotype and poor metastasis. survival in univariable and multivariable models. Moreover, we observed that expression of the αB-crystallin gene (CRYAB) was associated with brain as a first site of distant relapse, while RESULTS αB-crystallin protein expression was associated with the develop- αB-crystallin gene (CRYAB) expression is associated with distant ment of brain metastasis (first site or any occurrence) and poor relapse in basal-like tumors survival after diagnosis of brain metastasis. Collectively, our To investigate whether expression of the αB-crystallin gene findings suggest that αB-crystallin (gene and/or protein) expres- (CRYAB) is associated with survival and site-specific metastasis, sion may be of clinical utility as a biomarker to identify women at we interrogated publicly available gene expression data of 855 high risk for brain metastasis. breast tumors from four studies with available information regarding the first site of distant metastasis (‘855Met’). Within the 855Met data set, there were 188 basal-like tumors defined by MATERIALS AND METHODS gene expression using the PAM50 classifier. Within this subset of Human breast tumor gene expression analysis basal-like tumors, CRYAB expression was significantly associated Publicly available global-gene expression profiles of 855 tumors were with an increased risk of distant relapse by multivariable Cox downloaded from four studies (GSE2034, GSE12276, GSE2603, and the regression analysis adjusted for ER status, age at diagnosis and NKI295), and combined using Distance Weighted Discrimination to remove the systematic biases present in different microarray data sets. The four nodal status (Table 1, hazards ratio = 1.1 (95% confidence interval, tumor data sets contained the sites of first distant relapses and time to CI 1.0–1.2), P = 0.045). This suggested that CRYAB gene expression relapse, and will be referred as ‘855Met’ in this current study. The could be a potential biomarker for metastatic behavior of basal- combined clinical data can be found at the University of North Carolina like tumors. microarray database. Gene expression data were row (gene) median centered and column (sample) standardized (s.d. = 1), respectively. Tumors CRYAB expression is associated with brain as the first site of distant were assigned into one of the intrinsic subtypes using the PAM50 relapse classifier. Given the recent study linking αB-crystallin to breast cancer brain metastasis, we examined the relationship between CRYAB British Columbia cancer agency cohort expression and brain metastasis as the first distant relapse. In The British Columbia cancer agency (BCCA) cohort includes 3,987 formalin- the 855Met data set, 376 patients developed distant relapse: 49 of fixed paraffin-embedded specimens from female patients with newly these patients had brain metastasis as the first site of relapse. In diagnosed, invasive breast cancer referred to BCCA from 1986 to 1992 that the multivariable Cox regression analysis for brain as a first site of were used to construct a large TMA linked to detailed clinicopathological 12,15 distant relapse of using all available cases in this cohort (852 data and long-term outcomes (median follow-up of 12 years). Most patients were treated with adjuvant systemic therapy according to tumors), CRYAB expression was significantly associated with earlier provincial cancer management guidelines. The BC Cancer Agency Research development of brain relapse, independent of standard clinico- Ethics Board approved all biomarker studies on tissue specimens, and pathological variables, including the PAM50-defined intrinsic associated clinical and outcomes data correlation. subtypes (Table 2, hazards ratio = 1.2 (95% CI 1.0–1.4), P = 0.021). Interestingly, although intrinsic subtypes were still significantly associated with distant relapse to brain, the basal-like subtype was Immunohistochemistry and TMAs TMAs were constructed from tissue blocks as described. αB-crystallin protein expression was evaluated by immunohistochemistry (IHC) using a mouse monoclonal antibody (1:200 dilution; ADI-SPA-222; Stressgen Biotechnologies/Enzo Life Sciences, Farmingdale, NY, USA) as described Table 1. Multivariable Cox regression analysis for the development of previously and was scored using an established, previously published any distant relapse in 188 patients with basal-like breast tumors in the cutoff, whereby tumors were considered positive for αB-crystallin if there 855Met cohort was any staining above background levels. αB-crystallin protein expression was scored by a pathologist (TON) who was blinded to clinical Basal-like tumors Hazard ratio to distant P-value outcomes. Samples with o50 tumor cells present in the TMA core were relapse (95% CI) excluded from the analysis. TMAs were digitally scanned, and the images CRYAB 1.1 (1.0–1.2) 0.045 are available for public access (http://www.gpecimage.ubc.ca/tma/web/ ER 1.0 (0.6–1.8) 0.96 viewer.php; username: CRYAB4000; password: CRYAB4000). Breast cancer positive (n= 29) vs. negative subtypes were defined using a previously validated immunopanel of six (n= 159) biomarkers including ER, PR, HER2, Ki67, epidermal growth factor receptor 8,31 Age (continuous variable) 1.0 (0.97–1.0) 0.43 (EGFR), and cytokeratin 5/6 (CK5/6). In brief, tumors were classified as Nodal status 0.5 (0.3–1.0) 0.066 Luminal A if ER and/or PR positive, and HER2 negative, and Ki67 low positive (n= 28) vs. negative (o14%); Luminal B if ER and/or PR positive, and HER2 negative, and Ki67 (n= 160) high (⩾14%); Luminal-HER2 if ER and/or PR positive, and HER2 positive; HER2 enriched if ER and PR negative and HER2 positive; Basal-like if Abbreviations: CI, confidence interval; ER, estrogen receptor. negative for all ER, PR, and HER2 but expressing EGFR and/or CK5/6; and as n= 188, events= 89. Triple-negative non-basal if negative for ER, PR, HER2, EGFR, and CK5/6. npj Breast Cancer (2015) 15014 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited αB-crystallin and brain metastasis in breast cancer KD Voduc et al Table 2. Multivariable Cox regression analysis for the development of Table 3. Clinicopathological characteristics and treatment regimens brain as the first site of distant relapse among 852 patients with of the 3,987 patients in the BCCA cohort complete clinicopathological data in the 855Met cohort N % Breast tumors Hazard ratio to brain mets P-value Age (years) as first site (95% CI) o40 294 7 40–49 843 21 CRYAB 1.2 (1.0–1.4) 0.021 50–65 1,423 36 ER 0.4 (0.2–0.9) 0.025 465 1,427 36 positive (n= 596) vs. negative (n= 256) Grade Nodal status 1.0 (0.5–2.0) 0.9 1 209 6 positive (n= 158) vs. 2 1,562 41 negative (n= 694) 3 2,037 54 Age (continuous) 0.98 (0.9–1.0) 0.078 NA 179 PAM50 0.036 LumA 1 Menstrual status at referral LumB 4.7 (1.7–13) 0.0031 Pre-menopausal 1,178 30 HER2 enriched 2.9 (0.9–9.3) 0.066 Post-menopausal 2,717 70 Basal like 1.3 (0.4–4.7) 0.66 NA 92 Normal like 1.7 (0.5–5.6) 0.39 Abbreviations: CI, confidence interval; ER, estrogen receptor; HER2, human Nodal status epidermal growth factor receptor-2; LumA, luminal A; LumB, luminal B. Negative 2,265 57 n= 852, brain metastasis events= 49. Positive 1,714 43 NA 8 Lymphovascular invasion not independently significant, when compared with Luminal A Negative 2,105 55 tumors, in the multivariable model including CRYAB. Analysis of Positive 1,709 45 the subgroup of 376 patients who developed distant recurrences NA 173 revealed that CRYAB expression was significantly associated with increased likelihood of developing brain metastasis (odds ratio: 1.2 Tumor size (cm) (95% CI 1.0–1.3), P = 0.016). However, this latter association was ≤ 2 2,077 52 not independent of gene expression-based intrinsic subtypes in 2–5 1,665 42 the multivariable logistic regression analysis (P = 0.08) of this 45 221 6 NA 24 smaller subset of patients. Collectively, these gene expression analyses indicate that CRYAB expression is associated with Initial Adjuvant Systemic Therapy increased risk of disease progression in TNBC and with brain None 1,676 42 metastasis as a first site of distant relapse in the entire cohort. Tamoxifen only 1,274 32 Chemotherapy only (AC, FAC or CMF) 725 18 αB-crystallin protein expression is associated with the basal-like Tamoxifen and chemotherapy 296 8 Other 16 phenotype and poor survival To validate and extend our observations from gene expression Abbreviations: AC, adriamycin and cyclophosphamide; BCCA, British studies, we interrogated the BCCA TMA of 3,987 breast tumors for Columbia Cancer Agency; CMF, cyclophosphamide, methotrexate, and 5- fluorouracil; FAC, 5-fluorouracil and AC; NA, not available. the expression of αB-crystallin protein by IHC. The clinicopatho- logical characteristics and treatment regimens for this cohort are indicated (Table 3). Scoring of αB-crystallin expression was possible for 3,235 cases. Of these, 359 (11%) were positive for prognosis. BCSS at 10 years was 64% in αB-crystallin-positive αB-crystallin expression. All the stained TMAs were digitally cases versus 75% in αB-crystallin-negative cases (Figure 1a, scanned, and primary image data are available for public access: Po0.0001 by log-rank test). Distant relapse-free survival at 10 http://www.gpecimage.ubc.ca/tma/web/viewer.php; (username: years was 62% in αB-crystallin-positive cases versus 70% in CRYAB4000; password: CRYAB4000). Using the six biomarker 8,31 αB-crystallin-negative cases (Figure 1b, P = 0.0016 by log-rank immunopanel to subclassify each breast tumor, a molecular test). αB-crystallin had independent prognostic significance for subtype was assigned to 3,009 of the 3,235 breast tumors with BCSS (Table 4, hazards ratio = 1.3 (95% CI 1.1–1.6), P = 0.014) in a αB-crystallin IHC data. αB-crystallin was infrequently expressed in multivariable Cox model adjusting for patient age, tumor grade, Luminal A tumors (54 of 1312, 4.1%), Luminal B tumors (33 of 766, + + systemic therapy, ER, and HER2 status. In this model, patient age, 4.4%), Luminal–HER2 tumors (8 of 199, 4.0%) or HER2 tumors (18 tumor grade, lymph node status, tumor size, and HER2 status were of 221, 8.1%). In contrast, αB-crystallin was highly expressed in also independently prognostic for BCSS. These findings validate basal-like tumors defined by triple-negative status and expression our prior results from a much smaller cohort of breast cancer of basal markers CK5/6 or EGFR (170 of 309, 55%) and to a lesser patients (n = 438) that αB-crystallin protein expression is an extent in triple-negative tumors that lacked expression of these independent prognostic biomarker of BCSS. basal markers (47 of 202, 23%). The differential distribution of αB-crystallin expression among the various molecular subtypes of αB-crystallin expression is associated with site-specific brain breast cancer was highly statistically significant (Po0.0001 by metastasis Χ -test). These findings are consistent with prior reports specifi- cally linking αB-crystallin expression to basal-like breast carcino- 1,054 (33%) of patients in the BCCA cohort with available 23–26,28,32,33 mas/TNBCs. αB-crystallin IHC staining developed metastatic breast cancer to In univariable analysis among all breast cancer subtypes, one or more organs, including 148 (4.5%) patients who developed αB-crystallin was a statistically significant marker of poor brain metastases. Among the patients with metastatic disease, © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited npj Breast Cancer (2015) 15014 αB-crystallin and brain metastasis in breast cancer KD Voduc et al Table 4. Cox multivariable analysis for breast cancer-specific survival (BCSS) in the BCCA cohort Variables HR 95% CI P-value Age at diagnosis, years 0.022 455 (n= 1,790) 1 o40 (n= 239) 1.2 0.91–1.5 0.22 40–55 (n= 946) 0.87 0.72–1.0 0.12 Grade 1.5 1.3–1.8 3(n= 1,643) vs. 2/1 (n= 1,332) o0.0001 Nodal status 2.6 2.1–3.0 Positive (n= 1,686) vs. negative o0.0001 (n= 1,289) Tumor size, cm o0.0001 ⩽2(n= 1,545) 1 2–5(n= 1,290) 1.6 1.4–1.8 o0.0001 45(n= 140) 2.2 1.6–2.8 o0.0001 Initial systemic therapy Any hormonal (n= 1,128) vs. none 0.88 0.74–1.1 0.16 (n= 1,847) Any chemo (n= 773) vs. none 0.86 0.69–1.1 0.14 (n= 2,202) Biomarkers ER-positive (n= 2,148) vs. negative 0.89 0.75–1.1 0.18 (n= 827) HER2-positive (n= 414) vs. negative 1.4 1.2–1.7 o0.0001 (n= 2,561) αB-crystallin-positive (n= 332) vs. 1.3 1.1–1.6 0.014 negative (n= 2,643) Abbreviations: BCCA, British Columbia Cancer Agency; CI, confidence interval; ER, estrogen receptor; HER2, human estrogen receptor-2; HR, hazards ratio. n= 2,972 cases with complete covariate data, also excluding 3 cases censored before the first event. αB-crystallin status was the only independent predictor for brain metastasis as the first site of distant relapse (odds ratio = 3.15 (95% CI 1.43–6.95), P = 0.005). αB-crystallin was also associated with a Figure 1. αB-crystallin expression is associated with worse breast shorter time interval between initial diagnosis of breast cancer cancer-specific survival (BCSS) and distant relapse-free survival (DFS) and the subsequent diagnosis of brain metastasis (median time to in univariable analyses. (a) Kaplan–Meier analysis of BCSS based on brain metastasis = 2.7 years versus 4.2 years, Po0.0001 by log- αB-crystallin expression. 10-year BCSS was 64% for αB-crystallin- rank test). Following the diagnosis of brain metastasis, patients positive cases (n = 359, number of events= 133) versus 75% for with αB-crystallin-positive metastatic breast cancer had worse αB-crystallin-negative cases (n = 2,870, number of events= 809), survival (median survival = 3.0 months versus 4.7 months, P = 0.007 Po0.0001 by log-rank test. (b) Kaplan–Meier analysis of DFS based by log-rank test). In addition, αB-crystallin expression was on αB-crystallin expression. 10-year DFS was 62% for αB-crystallin- associated with lower risk of liver metastasis (odds ratio = 0.53, positive cases (n = 359, number of events= 139) versus 70% for αB-crystallin-negative cases (n= 2876, number of events= 915), P = 0.009). However, there was no significant association between P= 0.0016 by log-rank test. αB-crystallin expression and lung or bone metastases. Collectively, these results indicate that αB-crystallin protein expression is an independent predictor for the development of brain metastasis (first site or any occurrence) and is associated with poor survival after diagnosis of brain metastasis relative to metastatic αB- αB-crystallin expression was significantly associated with brain crystallin-negative breast cancer. metastasis: 12% of patients with αB-crystallin-negative metastatic breast cancer had brain metastasis, in contrast to 33% of patients with αB-crystallin-positive cancer (Po0.0001 by Fischer's exact DISCUSSION test). In a multivariable logistic regression model including only HER2-positive and basal-like tumors/TNBC have a distinctive patients with metastatic breast cancer, αB-crystallin was the pattern of organ-specific distant metastasis that includes brain strongest independent predictor of brain metastasis (odds ratio = metastasis, a debilitating event that typically follows a rapidly fatal 2.99 (95% CI 1.83–4.89), Po0.0001); ER and HER2 status were also 13–15,34 course. Indeed, the prevalence of brain metastases among independent predictors of brain metastasis (Table 5). Multivariable patients with metastatic TNBC has been reported as high as 46% logistic regression, relating tumor size, nodal status, tumor grade, 35,36 systemic therapy, ER, HER, and αB-crystallin to the development of with median survival after diagnosis of 4.9–6.6 months. brain metastasis as the first site of distant relapse found that Moreover, the brain is the first site of distant metastasis in 4.7% npj Breast Cancer (2015) 15014 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited αB-crystallin and brain metastasis in breast cancer KD Voduc et al Table 5. Multivariable logistic regression analysis for the development of any brain metastasis and brain as the first site of relapse in the BCCA cohort Odds to any brain mets (95% CI), (events = 141) P-value Odds to brain as first distant site (95% CI), (events = 46) P-value Tumor size ⩽2cm (n= 375) 1 1 2–5cm (n= 525) 1.13 (0.76–1.69) 0.54 0.70 (0.37–1.33) 0.28 45cm (n= 69) 0.39 (0·13–1·18) 0.095 Nodal status Negative (n= 374) 1 1 Positive (n= 595) 0.53 (0·34–0·82) 0.005 0.55 (0.28–1.08) 0.082 Grade 1or 2 (n= 336) 1 1 3(n= 633) 1.2 (0·76–1·86) 0.441 1.19 (0.57–2.48) 0.65 Any initial chemo No (n= 648) 1 1 Yes (n= 321) 1.46 (0.92–2.30) 0.11 0.36 (0.19–0.71) 0.003 Any initial hormonal No (n= 556) 1 1 Yes (n= 413) 0.79 (0.50–1.24) 0.3 0.91 (0.45–1.84) 0.79 ER Negative (n= 316) 1 1 Positive (n= 653) 0.58 (0.37–0.91) 0.018 0.58 (0.28–1.24) 0.16 HER2 Negative (n= 783) 1 1 Positive (n= 186) 1.72 (1.08–2.73) 0.023 1.88 (0.89–3.96) 0.096 αB-crystallin Negative (n= 842) 1 1 Positive (n= 127) 2.99 (1.83–4.89) o0.0001 3.15 (1.43–6.95) 0.005 Abbreviations: BCCA, British Columbia Cancer Agency; CI, confidence interval; ER, estrogen receptor; HER2, human epidermal growth factor receptor-2. of patients with early-stage TNBC. Although HER2 has been We have also demonstrated that αB-crystallin protein expres- previously linked to the pathogenesis of brain metastasis, as sion is associated with a shorter time interval between the breast discussed further below, αB-crystallin has only recently been cancer diagnosis and the development of brain metastasis (2.7 reported to have a pathogenic role in brain metastasis in TNBC years versus 4.2 years, Po0.0001 by log-rank test) and shorter 28,38–40 models. Consistent with this pathogenic role, we have survival after diagnosis of brain metastasis (3.0 months versus demonstrated that αB-crystallin gene (CRYAB) expression is 4.7 months, P = 0.007 by log-rank test). These results are associated with distant relapse in TNBC patients and with earlier particularly striking given the poor survival of patients who development of brain relapse as a first distant site, independent of developed brain metastasis. Even among this subset of poor- standard clinicopathological variables and PAM50 subtyping in prognosis patients, αB-crystallin expression was associated with the entire cohort. Moreover, αB-crystallin protein expression in the significantly shorter survival after diagnosis of brain metastasis. primary breast tumor was an independent prognostic biomarker Our findings are consistent with a recent report from a cohort of of poor BCSS and was associated with a 33% risk of brain 76 patients with breast cancer who developed brain metastasis. metastasis among patients in the BCCA cohort who developed In that cohort, αB-crystallin expression was associated with inferior metastatic disease. This is comparable to the high incidence of survival after breast cancer diagnosis (1.4 vs. 4.7 years, P = 0.0002) brain metastasis observed in HER2-positive metastatic breast and survival after brain metastasis diagnosis (0.13 vs. 0.91 years, cancer (25–40%). Notably, both HER2 and αB-crystallin were P = 0.001). Collectively, these results point to αB-crystallin expres- independent predictors of brain metastasis (any occurrence) in sion in primary breast cancers as a promising biomarker to identify multivariate analyses. However, αB-crystallin expression was the patients at high risk for early relapse in the brain and poor survival strongest predictor of brain metastasis (any occurrence) and the after diagnosis. As such, these patients could be enrolled in clinical only independent predictor of brain metastasis as the first site of trials to evaluate preventive agents or modalities for brain distant relapse. These findings suggest that αB-crystallin (gene metastasis, particularly as less toxic options become available. and/or protein) expression in primary breast cancers may be a Indeed, with further validation, αB-crystallin expression testing useful biomarker to identify patients at greater risk for developing could be incorporated as one of the pre-planned biomarker tests brain metastasis. Because we observed that αB-crystallin is for contemporary biomarker-driven trials in patients with meta- preferentially expressed in basal-like tumors (55%) and rarely in static TNBC with extracranial disease to determine optimal HER2-positive tumors (8.1%), consistent with prior reports, surveillance and treatment approaches. The importance of our findings suggest that the αB-crystallin expression status validating αB-crystallin as a biomarker for brain metastasis is may be particularly informative among TNBC patients to help underscored by a recent study in which a promising three-gene identify individuals at greater risk for early or first-site brain signature for the development of early brain metastasis in HER2- 23–26,28,32,33,37 41 relapse. positive breast cancer failed subsequent validation. © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited npj Breast Cancer (2015) 15014 αB-crystallin and brain metastasis in breast cancer KD Voduc et al data and edited the manuscript. VLC conceptualized the project, analyzed the data, Intriguingly, the observed association of αB-crystallin expression wrote and edited the manuscript. MCUC conceptualized the project, assembled the in primary breast carcinomas and brain metastasis is biologically tissue microarray, generated the biomarker data, analyzed the data, wrote and edited the plausible in the light of recent data from cellular assays and manuscript. All authors read and approved the final manuscript. murine models pointing to a pathogenic role for αB-crystallin in breast cancer brain metastasis. αB-crystallin promotes adhesion to human brain microvascular endothelial cells, transendothelial COMPETING INTERESTS migration and penetration through an human brain microvascular CMP and TON are equity stock holders, and Board of Director Member of BioClassifier. endothelial cell or primary human astrocyte co-culture blood– 28 CMP, TON and MCUC are also listed as inventors on a patent application on the brain barrier model. Furthermore, overexpression of αB-crystallin PAM50 assay. 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(CMP), the Cancer Research UK (CRUK) Core grant C1491/A15955 (MCUC), and the 20 Mehlen P, Kretz-Remy C, Preville X, Arrigo AP. Human hsp27, Drosophila hsp27 Canadian Breast Cancer Foundation (BC/Yukon Division; KDV, TON, and HK). and human αB-crystallin expression-mediated increase in glutathione is essential for the protective activity of these proteins against TNFα-induced cell death. EMBO J 1996; 15: 2695–2706. CONTRIBUTIONS 21 Reddy VS, Reddy GB. Emerging role for αB-crystallin as a therapeutic agent: pros and cons. Curr Mol Med 2015; 15:47–61. KDV analyzed the data, wrote and edited the manuscript. TON assembled the tissue 22 Stegh AH, Kesari S, Mahoney JE, Jenq HT, Forloney KL, Protopopov A et al. microarray, generated the biomarker data, analyzed the data, and edited the manuscript. Bcl2L12-mediated inhibition of effector caspase-3 and caspase-7 via distinct CMP, JCH, and CF assembled the gene expression data and edited the manuscript. 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The images or other third party material in this 33 Won JR, Gao D, Chow C, Cheng J, Lau SY, Ellis MJ et al. A survey of immunohis- article are included in the article’s Creative Commons license, unless indicated tochemical biomarkers for basal-like breast cancer against a gene expression otherwise in the credit line; if the material is not included under the Creative Commons profile gold standard. Modern Pathol 2013; 26: 1438–1450. license, users will need to obtain permission from the license holder to reproduce the 34 Lin NU, Bellon JR, Winer EP. CNS metastases in breast cancer. J Clin Oncol 2004; material. To view a copy of this license, visit http://creativecommons.org/licenses/ 22: 3608–3617. by/4.0/ © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited npj Breast Cancer (2015) 15014 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png npj Breast Cancer Springer Journals

αB-crystallin expression in breast cancer is associated with brain metastasis

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Biomedicine; Biomedicine, general; Cancer Research; Oncology; Human Genetics; Cell Biology
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Abstract

www.nature.com/npjbcancer All rights reserved 2374-4677/15 ARTICLE OPEN αB-crystallin expression in breast cancer is associated with brain metastasis 1 2 3 4 3 5 6 K David Voduc , Torsten O Nielsen , Charles M Perou , J Chuck Harrell , Cheng Fan , Hagen Kennecke , Andy J Minn , 7,9 8,9 Vincent L Cryns and Maggie CU Cheang BACKGROUND/OBJECTIVES: The molecular chaperone αB-crystallin is expressed in estrogen receptor, progesterone receptor and human epidermal growth factor receptor-2 ‘triple-negative’ breast carcinomas and promotes brain and lung metastasis. We examined αB-crystallin expression in primary breast carcinomas with metastatic data to evaluate its association with prognosis and site-specific metastases. METHODS: αB-crystallin gene (CRYAB) expression was examined using publically available global-gene expression data (n = 855 breast tumors) with first site of distant metastasis information (‘855Met’). αB-crystallin protein expression was determined by immunohistochemistry using the clinically annotated British Columbia Cancer Agency (BCCA) tissue microarray (n = 3,987 breast tumors). Kaplan–Meier and multivariable Cox regression analyses were used to evaluate the prognostic value of αB-crystallin. Multivariable logistic regression analysis was used to evaluate risks of αB-crystallin and other markers for site of metastasis. RESULTS: In the 855Met data set, αB-crystallin gene (CRYAB) expression was an independent predictor of brain as the first distant site of relapse (hazards ratio, HR = 1.2, (95% confidence interval, CI 1.0–1.4), P = 0.021). In the BCCA series, αB-crystallin protein expression was an independent prognostic marker of poor breast cancer-specific survival (HR = 1.3, (95% CI 1.1–1.6), P = 0.014). Among patients with metastases, αB-crystallin was the strongest independent predictor of brain metastasis (odds ratio, OR = 2.99 (95% CI 1.83–4.89), Po0.0001) and the only independent predictor of brain as the first site of distant metastasis (OR = 3.15 (95% CI 1.43–6.95), P = 0.005). αB-crystallin was also associated with worse survival (3.0 versus 4.7 months, P = 0.007). CONCLUSIONS: αB-crystallin is a promising biomarker to identify breast cancer patients at high risk for early relapse in the brain, independent of estrogen receptor and human epidermal growth factor receptor-2 status. npj Breast Cancer (2015) 1, 15014; doi:10.1038/npjbcancer.2015.14; published online 21 October 2015 INTRODUCTION metastasis in this subtype could lead to biomarkers to help identify women at high risk for relapse at these sites. Over the past 15 years, gene expression studies in breast cancer αB-crystallin is a widely expressed member of the small heat have led to more accurate prognostic tools and to a greater 1–5 shock protein family that protects cells from stress by its dual understanding of the molecular heterogeneity of breast cancer. function as a molecular chaperone to preserve proteostasis and as Basal-like breast carcinomas have emerged as a distinctive a cell death antagonist that inhibits caspase-3 activation and molecular subtype defined by expression of genes characteristic 18–22 oxidative stress. αB-crystallin is commonly expressed in basal- of basal epithelial cells that overlap in large part with estrogen like and TNBCs and its expression correlates with resistance to receptor (ER), progesterone receptor (PR), and human epidermal 23–26 neoadjuvant chemotherapy and poor survival. More recently, growth factor receptor-2 (HER2) ‘triple-negative’ breast cancers 6,7 αB-crystallin has been demonstrated to inhibit extracellular matrix (TNBC). More recent studies point to molecular heterogeneity detachment-induced apoptosis (‘anoikis’), enhance penetration within the triple-negative subgroup with potential prognostic and 8–10 through an endothelial/astrocyte co-culture model of the blood– therapeutic implications. Overall, basal-like tumors are asso- brain barrier in vitro, and promote lung and brain metastasis in ciated with a higher risk of recurrence following conventional 27,28 4,11,12 orthotopic models of TNBC in immunodeficient mice. In adjuvant treatment and poor survival. Furthermore, basal-like addition, αB-crystallin is frequently expressed in brain metastases tumors have a characteristic pattern of distant metastasis to 13–15 the lungs and brain with infrequent relapse in bone. Although and its expression in primary breast carcinomas is associated with lung and brain metastasis gene signatures have been poor overall survival and poor survival after brain metastasis in a 16,17 cohort of 76 women with breast cancer brain metastasis. identified, the functional contribution of individual genes to site-specific metastasis in this subtype is poorly understood. Collectively, these findings point to a key pathogenic role of Clearly, the identification of pathogenic drivers of brain and lung αB-crystallin in lung and brain metastasis in TNBC. 1 2 Department of Radiation Oncology, University of British Columbia, Vancouver, BC, Canada; Department of Pathology and Laboratory Medicine, University of British Columbia, 3 4 Vancouver, BC, Canada; Department of Genetics, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Pathology, Virginia Commonwealth University, Richmond, VA, USA; Division of Medical Oncology, British Columbia Cancer Agency, University of British Columbia, Vancouver, BC, Canada; Department of Radiation Oncology, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; 7 8 Department of Medicine, University of Wisconsin Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA and Division of Clinical Studies, Clinical Trials and Statistics Unit (ICR-CTSU), Institute of Cancer Research, London, UK. Correspondence: VL Cryns or MCU Cheang (vlcryns@medicine.wisc.edu or maggie.cheang@icr.ac.uk) These authors contributed equally to this work. Received 22 May 2015; revised 17 July 2015; accepted 7 September 2015 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited αB-crystallin and brain metastasis in breast cancer KD Voduc et al Given the recent evidence supporting a prometastatic function Statistical analysis for αB-crystallin in TNBC, we postulated that αB-crystallin might Univariable survival estimates were calculated using the Kaplan–Meier method, and survival differences were estimated using log-rank tests. Due have utility as a prognostic biomarker to predict the risk of disease to the retrospective nature of the gene expression and IHC studies, the progression in TNBC patients. To this end, we examined the sample size for each study was not pre-specified. The primary endpoint for prognostic value of αB-crystallin (gene and protein) expression in the BCCA cohort is breast cancer-specific survival (BCSS). Multivariable Cox breast cancer using (1) publically available gene expression data regression analyses were used to test the independent prognostic value of from multiple breast cancer cohorts, which included sites of first αB-crystallin. Smoothed plots of weighted Schoenfeld residuals were distant metastasis, and (2) a large breast cancer tissue microarray used to test proportional hazard assumptions. The association of (TMA) linked to long-term outcomes, including metastatic sites. αB-crystallin expression with intrinsic subtypes was assessed using the Using these data sets, we confirmed the association between αB- χ -test. Multivariable logistic regression analysis was used to evaluate the association of αB-crystallin and other clinicopathological markers for site of crystallin protein expression and basal-like phenotype and poor metastasis. survival in univariable and multivariable models. Moreover, we observed that expression of the αB-crystallin gene (CRYAB) was associated with brain as a first site of distant relapse, while RESULTS αB-crystallin protein expression was associated with the develop- αB-crystallin gene (CRYAB) expression is associated with distant ment of brain metastasis (first site or any occurrence) and poor relapse in basal-like tumors survival after diagnosis of brain metastasis. Collectively, our To investigate whether expression of the αB-crystallin gene findings suggest that αB-crystallin (gene and/or protein) expres- (CRYAB) is associated with survival and site-specific metastasis, sion may be of clinical utility as a biomarker to identify women at we interrogated publicly available gene expression data of 855 high risk for brain metastasis. breast tumors from four studies with available information regarding the first site of distant metastasis (‘855Met’). Within the 855Met data set, there were 188 basal-like tumors defined by MATERIALS AND METHODS gene expression using the PAM50 classifier. Within this subset of Human breast tumor gene expression analysis basal-like tumors, CRYAB expression was significantly associated Publicly available global-gene expression profiles of 855 tumors were with an increased risk of distant relapse by multivariable Cox downloaded from four studies (GSE2034, GSE12276, GSE2603, and the regression analysis adjusted for ER status, age at diagnosis and NKI295), and combined using Distance Weighted Discrimination to remove the systematic biases present in different microarray data sets. The four nodal status (Table 1, hazards ratio = 1.1 (95% confidence interval, tumor data sets contained the sites of first distant relapses and time to CI 1.0–1.2), P = 0.045). This suggested that CRYAB gene expression relapse, and will be referred as ‘855Met’ in this current study. The could be a potential biomarker for metastatic behavior of basal- combined clinical data can be found at the University of North Carolina like tumors. microarray database. Gene expression data were row (gene) median centered and column (sample) standardized (s.d. = 1), respectively. Tumors CRYAB expression is associated with brain as the first site of distant were assigned into one of the intrinsic subtypes using the PAM50 relapse classifier. Given the recent study linking αB-crystallin to breast cancer brain metastasis, we examined the relationship between CRYAB British Columbia cancer agency cohort expression and brain metastasis as the first distant relapse. In The British Columbia cancer agency (BCCA) cohort includes 3,987 formalin- the 855Met data set, 376 patients developed distant relapse: 49 of fixed paraffin-embedded specimens from female patients with newly these patients had brain metastasis as the first site of relapse. In diagnosed, invasive breast cancer referred to BCCA from 1986 to 1992 that the multivariable Cox regression analysis for brain as a first site of were used to construct a large TMA linked to detailed clinicopathological 12,15 distant relapse of using all available cases in this cohort (852 data and long-term outcomes (median follow-up of 12 years). Most patients were treated with adjuvant systemic therapy according to tumors), CRYAB expression was significantly associated with earlier provincial cancer management guidelines. The BC Cancer Agency Research development of brain relapse, independent of standard clinico- Ethics Board approved all biomarker studies on tissue specimens, and pathological variables, including the PAM50-defined intrinsic associated clinical and outcomes data correlation. subtypes (Table 2, hazards ratio = 1.2 (95% CI 1.0–1.4), P = 0.021). Interestingly, although intrinsic subtypes were still significantly associated with distant relapse to brain, the basal-like subtype was Immunohistochemistry and TMAs TMAs were constructed from tissue blocks as described. αB-crystallin protein expression was evaluated by immunohistochemistry (IHC) using a mouse monoclonal antibody (1:200 dilution; ADI-SPA-222; Stressgen Biotechnologies/Enzo Life Sciences, Farmingdale, NY, USA) as described Table 1. Multivariable Cox regression analysis for the development of previously and was scored using an established, previously published any distant relapse in 188 patients with basal-like breast tumors in the cutoff, whereby tumors were considered positive for αB-crystallin if there 855Met cohort was any staining above background levels. αB-crystallin protein expression was scored by a pathologist (TON) who was blinded to clinical Basal-like tumors Hazard ratio to distant P-value outcomes. Samples with o50 tumor cells present in the TMA core were relapse (95% CI) excluded from the analysis. TMAs were digitally scanned, and the images CRYAB 1.1 (1.0–1.2) 0.045 are available for public access (http://www.gpecimage.ubc.ca/tma/web/ ER 1.0 (0.6–1.8) 0.96 viewer.php; username: CRYAB4000; password: CRYAB4000). Breast cancer positive (n= 29) vs. negative subtypes were defined using a previously validated immunopanel of six (n= 159) biomarkers including ER, PR, HER2, Ki67, epidermal growth factor receptor 8,31 Age (continuous variable) 1.0 (0.97–1.0) 0.43 (EGFR), and cytokeratin 5/6 (CK5/6). In brief, tumors were classified as Nodal status 0.5 (0.3–1.0) 0.066 Luminal A if ER and/or PR positive, and HER2 negative, and Ki67 low positive (n= 28) vs. negative (o14%); Luminal B if ER and/or PR positive, and HER2 negative, and Ki67 (n= 160) high (⩾14%); Luminal-HER2 if ER and/or PR positive, and HER2 positive; HER2 enriched if ER and PR negative and HER2 positive; Basal-like if Abbreviations: CI, confidence interval; ER, estrogen receptor. negative for all ER, PR, and HER2 but expressing EGFR and/or CK5/6; and as n= 188, events= 89. Triple-negative non-basal if negative for ER, PR, HER2, EGFR, and CK5/6. npj Breast Cancer (2015) 15014 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited αB-crystallin and brain metastasis in breast cancer KD Voduc et al Table 2. Multivariable Cox regression analysis for the development of Table 3. Clinicopathological characteristics and treatment regimens brain as the first site of distant relapse among 852 patients with of the 3,987 patients in the BCCA cohort complete clinicopathological data in the 855Met cohort N % Breast tumors Hazard ratio to brain mets P-value Age (years) as first site (95% CI) o40 294 7 40–49 843 21 CRYAB 1.2 (1.0–1.4) 0.021 50–65 1,423 36 ER 0.4 (0.2–0.9) 0.025 465 1,427 36 positive (n= 596) vs. negative (n= 256) Grade Nodal status 1.0 (0.5–2.0) 0.9 1 209 6 positive (n= 158) vs. 2 1,562 41 negative (n= 694) 3 2,037 54 Age (continuous) 0.98 (0.9–1.0) 0.078 NA 179 PAM50 0.036 LumA 1 Menstrual status at referral LumB 4.7 (1.7–13) 0.0031 Pre-menopausal 1,178 30 HER2 enriched 2.9 (0.9–9.3) 0.066 Post-menopausal 2,717 70 Basal like 1.3 (0.4–4.7) 0.66 NA 92 Normal like 1.7 (0.5–5.6) 0.39 Abbreviations: CI, confidence interval; ER, estrogen receptor; HER2, human Nodal status epidermal growth factor receptor-2; LumA, luminal A; LumB, luminal B. Negative 2,265 57 n= 852, brain metastasis events= 49. Positive 1,714 43 NA 8 Lymphovascular invasion not independently significant, when compared with Luminal A Negative 2,105 55 tumors, in the multivariable model including CRYAB. Analysis of Positive 1,709 45 the subgroup of 376 patients who developed distant recurrences NA 173 revealed that CRYAB expression was significantly associated with increased likelihood of developing brain metastasis (odds ratio: 1.2 Tumor size (cm) (95% CI 1.0–1.3), P = 0.016). However, this latter association was ≤ 2 2,077 52 not independent of gene expression-based intrinsic subtypes in 2–5 1,665 42 the multivariable logistic regression analysis (P = 0.08) of this 45 221 6 NA 24 smaller subset of patients. Collectively, these gene expression analyses indicate that CRYAB expression is associated with Initial Adjuvant Systemic Therapy increased risk of disease progression in TNBC and with brain None 1,676 42 metastasis as a first site of distant relapse in the entire cohort. Tamoxifen only 1,274 32 Chemotherapy only (AC, FAC or CMF) 725 18 αB-crystallin protein expression is associated with the basal-like Tamoxifen and chemotherapy 296 8 Other 16 phenotype and poor survival To validate and extend our observations from gene expression Abbreviations: AC, adriamycin and cyclophosphamide; BCCA, British studies, we interrogated the BCCA TMA of 3,987 breast tumors for Columbia Cancer Agency; CMF, cyclophosphamide, methotrexate, and 5- fluorouracil; FAC, 5-fluorouracil and AC; NA, not available. the expression of αB-crystallin protein by IHC. The clinicopatho- logical characteristics and treatment regimens for this cohort are indicated (Table 3). Scoring of αB-crystallin expression was possible for 3,235 cases. Of these, 359 (11%) were positive for prognosis. BCSS at 10 years was 64% in αB-crystallin-positive αB-crystallin expression. All the stained TMAs were digitally cases versus 75% in αB-crystallin-negative cases (Figure 1a, scanned, and primary image data are available for public access: Po0.0001 by log-rank test). Distant relapse-free survival at 10 http://www.gpecimage.ubc.ca/tma/web/viewer.php; (username: years was 62% in αB-crystallin-positive cases versus 70% in CRYAB4000; password: CRYAB4000). Using the six biomarker 8,31 αB-crystallin-negative cases (Figure 1b, P = 0.0016 by log-rank immunopanel to subclassify each breast tumor, a molecular test). αB-crystallin had independent prognostic significance for subtype was assigned to 3,009 of the 3,235 breast tumors with BCSS (Table 4, hazards ratio = 1.3 (95% CI 1.1–1.6), P = 0.014) in a αB-crystallin IHC data. αB-crystallin was infrequently expressed in multivariable Cox model adjusting for patient age, tumor grade, Luminal A tumors (54 of 1312, 4.1%), Luminal B tumors (33 of 766, + + systemic therapy, ER, and HER2 status. In this model, patient age, 4.4%), Luminal–HER2 tumors (8 of 199, 4.0%) or HER2 tumors (18 tumor grade, lymph node status, tumor size, and HER2 status were of 221, 8.1%). In contrast, αB-crystallin was highly expressed in also independently prognostic for BCSS. These findings validate basal-like tumors defined by triple-negative status and expression our prior results from a much smaller cohort of breast cancer of basal markers CK5/6 or EGFR (170 of 309, 55%) and to a lesser patients (n = 438) that αB-crystallin protein expression is an extent in triple-negative tumors that lacked expression of these independent prognostic biomarker of BCSS. basal markers (47 of 202, 23%). The differential distribution of αB-crystallin expression among the various molecular subtypes of αB-crystallin expression is associated with site-specific brain breast cancer was highly statistically significant (Po0.0001 by metastasis Χ -test). These findings are consistent with prior reports specifi- cally linking αB-crystallin expression to basal-like breast carcino- 1,054 (33%) of patients in the BCCA cohort with available 23–26,28,32,33 mas/TNBCs. αB-crystallin IHC staining developed metastatic breast cancer to In univariable analysis among all breast cancer subtypes, one or more organs, including 148 (4.5%) patients who developed αB-crystallin was a statistically significant marker of poor brain metastases. Among the patients with metastatic disease, © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited npj Breast Cancer (2015) 15014 αB-crystallin and brain metastasis in breast cancer KD Voduc et al Table 4. Cox multivariable analysis for breast cancer-specific survival (BCSS) in the BCCA cohort Variables HR 95% CI P-value Age at diagnosis, years 0.022 455 (n= 1,790) 1 o40 (n= 239) 1.2 0.91–1.5 0.22 40–55 (n= 946) 0.87 0.72–1.0 0.12 Grade 1.5 1.3–1.8 3(n= 1,643) vs. 2/1 (n= 1,332) o0.0001 Nodal status 2.6 2.1–3.0 Positive (n= 1,686) vs. negative o0.0001 (n= 1,289) Tumor size, cm o0.0001 ⩽2(n= 1,545) 1 2–5(n= 1,290) 1.6 1.4–1.8 o0.0001 45(n= 140) 2.2 1.6–2.8 o0.0001 Initial systemic therapy Any hormonal (n= 1,128) vs. none 0.88 0.74–1.1 0.16 (n= 1,847) Any chemo (n= 773) vs. none 0.86 0.69–1.1 0.14 (n= 2,202) Biomarkers ER-positive (n= 2,148) vs. negative 0.89 0.75–1.1 0.18 (n= 827) HER2-positive (n= 414) vs. negative 1.4 1.2–1.7 o0.0001 (n= 2,561) αB-crystallin-positive (n= 332) vs. 1.3 1.1–1.6 0.014 negative (n= 2,643) Abbreviations: BCCA, British Columbia Cancer Agency; CI, confidence interval; ER, estrogen receptor; HER2, human estrogen receptor-2; HR, hazards ratio. n= 2,972 cases with complete covariate data, also excluding 3 cases censored before the first event. αB-crystallin status was the only independent predictor for brain metastasis as the first site of distant relapse (odds ratio = 3.15 (95% CI 1.43–6.95), P = 0.005). αB-crystallin was also associated with a Figure 1. αB-crystallin expression is associated with worse breast shorter time interval between initial diagnosis of breast cancer cancer-specific survival (BCSS) and distant relapse-free survival (DFS) and the subsequent diagnosis of brain metastasis (median time to in univariable analyses. (a) Kaplan–Meier analysis of BCSS based on brain metastasis = 2.7 years versus 4.2 years, Po0.0001 by log- αB-crystallin expression. 10-year BCSS was 64% for αB-crystallin- rank test). Following the diagnosis of brain metastasis, patients positive cases (n = 359, number of events= 133) versus 75% for with αB-crystallin-positive metastatic breast cancer had worse αB-crystallin-negative cases (n = 2,870, number of events= 809), survival (median survival = 3.0 months versus 4.7 months, P = 0.007 Po0.0001 by log-rank test. (b) Kaplan–Meier analysis of DFS based by log-rank test). In addition, αB-crystallin expression was on αB-crystallin expression. 10-year DFS was 62% for αB-crystallin- associated with lower risk of liver metastasis (odds ratio = 0.53, positive cases (n = 359, number of events= 139) versus 70% for αB-crystallin-negative cases (n= 2876, number of events= 915), P = 0.009). However, there was no significant association between P= 0.0016 by log-rank test. αB-crystallin expression and lung or bone metastases. Collectively, these results indicate that αB-crystallin protein expression is an independent predictor for the development of brain metastasis (first site or any occurrence) and is associated with poor survival after diagnosis of brain metastasis relative to metastatic αB- αB-crystallin expression was significantly associated with brain crystallin-negative breast cancer. metastasis: 12% of patients with αB-crystallin-negative metastatic breast cancer had brain metastasis, in contrast to 33% of patients with αB-crystallin-positive cancer (Po0.0001 by Fischer's exact DISCUSSION test). In a multivariable logistic regression model including only HER2-positive and basal-like tumors/TNBC have a distinctive patients with metastatic breast cancer, αB-crystallin was the pattern of organ-specific distant metastasis that includes brain strongest independent predictor of brain metastasis (odds ratio = metastasis, a debilitating event that typically follows a rapidly fatal 2.99 (95% CI 1.83–4.89), Po0.0001); ER and HER2 status were also 13–15,34 course. Indeed, the prevalence of brain metastases among independent predictors of brain metastasis (Table 5). Multivariable patients with metastatic TNBC has been reported as high as 46% logistic regression, relating tumor size, nodal status, tumor grade, 35,36 systemic therapy, ER, HER, and αB-crystallin to the development of with median survival after diagnosis of 4.9–6.6 months. brain metastasis as the first site of distant relapse found that Moreover, the brain is the first site of distant metastasis in 4.7% npj Breast Cancer (2015) 15014 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited αB-crystallin and brain metastasis in breast cancer KD Voduc et al Table 5. Multivariable logistic regression analysis for the development of any brain metastasis and brain as the first site of relapse in the BCCA cohort Odds to any brain mets (95% CI), (events = 141) P-value Odds to brain as first distant site (95% CI), (events = 46) P-value Tumor size ⩽2cm (n= 375) 1 1 2–5cm (n= 525) 1.13 (0.76–1.69) 0.54 0.70 (0.37–1.33) 0.28 45cm (n= 69) 0.39 (0·13–1·18) 0.095 Nodal status Negative (n= 374) 1 1 Positive (n= 595) 0.53 (0·34–0·82) 0.005 0.55 (0.28–1.08) 0.082 Grade 1or 2 (n= 336) 1 1 3(n= 633) 1.2 (0·76–1·86) 0.441 1.19 (0.57–2.48) 0.65 Any initial chemo No (n= 648) 1 1 Yes (n= 321) 1.46 (0.92–2.30) 0.11 0.36 (0.19–0.71) 0.003 Any initial hormonal No (n= 556) 1 1 Yes (n= 413) 0.79 (0.50–1.24) 0.3 0.91 (0.45–1.84) 0.79 ER Negative (n= 316) 1 1 Positive (n= 653) 0.58 (0.37–0.91) 0.018 0.58 (0.28–1.24) 0.16 HER2 Negative (n= 783) 1 1 Positive (n= 186) 1.72 (1.08–2.73) 0.023 1.88 (0.89–3.96) 0.096 αB-crystallin Negative (n= 842) 1 1 Positive (n= 127) 2.99 (1.83–4.89) o0.0001 3.15 (1.43–6.95) 0.005 Abbreviations: BCCA, British Columbia Cancer Agency; CI, confidence interval; ER, estrogen receptor; HER2, human epidermal growth factor receptor-2. of patients with early-stage TNBC. Although HER2 has been We have also demonstrated that αB-crystallin protein expres- previously linked to the pathogenesis of brain metastasis, as sion is associated with a shorter time interval between the breast discussed further below, αB-crystallin has only recently been cancer diagnosis and the development of brain metastasis (2.7 reported to have a pathogenic role in brain metastasis in TNBC years versus 4.2 years, Po0.0001 by log-rank test) and shorter 28,38–40 models. Consistent with this pathogenic role, we have survival after diagnosis of brain metastasis (3.0 months versus demonstrated that αB-crystallin gene (CRYAB) expression is 4.7 months, P = 0.007 by log-rank test). These results are associated with distant relapse in TNBC patients and with earlier particularly striking given the poor survival of patients who development of brain relapse as a first distant site, independent of developed brain metastasis. Even among this subset of poor- standard clinicopathological variables and PAM50 subtyping in prognosis patients, αB-crystallin expression was associated with the entire cohort. Moreover, αB-crystallin protein expression in the significantly shorter survival after diagnosis of brain metastasis. primary breast tumor was an independent prognostic biomarker Our findings are consistent with a recent report from a cohort of of poor BCSS and was associated with a 33% risk of brain 76 patients with breast cancer who developed brain metastasis. metastasis among patients in the BCCA cohort who developed In that cohort, αB-crystallin expression was associated with inferior metastatic disease. This is comparable to the high incidence of survival after breast cancer diagnosis (1.4 vs. 4.7 years, P = 0.0002) brain metastasis observed in HER2-positive metastatic breast and survival after brain metastasis diagnosis (0.13 vs. 0.91 years, cancer (25–40%). Notably, both HER2 and αB-crystallin were P = 0.001). Collectively, these results point to αB-crystallin expres- independent predictors of brain metastasis (any occurrence) in sion in primary breast cancers as a promising biomarker to identify multivariate analyses. However, αB-crystallin expression was the patients at high risk for early relapse in the brain and poor survival strongest predictor of brain metastasis (any occurrence) and the after diagnosis. As such, these patients could be enrolled in clinical only independent predictor of brain metastasis as the first site of trials to evaluate preventive agents or modalities for brain distant relapse. These findings suggest that αB-crystallin (gene metastasis, particularly as less toxic options become available. and/or protein) expression in primary breast cancers may be a Indeed, with further validation, αB-crystallin expression testing useful biomarker to identify patients at greater risk for developing could be incorporated as one of the pre-planned biomarker tests brain metastasis. Because we observed that αB-crystallin is for contemporary biomarker-driven trials in patients with meta- preferentially expressed in basal-like tumors (55%) and rarely in static TNBC with extracranial disease to determine optimal HER2-positive tumors (8.1%), consistent with prior reports, surveillance and treatment approaches. The importance of our findings suggest that the αB-crystallin expression status validating αB-crystallin as a biomarker for brain metastasis is may be particularly informative among TNBC patients to help underscored by a recent study in which a promising three-gene identify individuals at greater risk for early or first-site brain signature for the development of early brain metastasis in HER2- 23–26,28,32,33,37 41 relapse. positive breast cancer failed subsequent validation. © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited npj Breast Cancer (2015) 15014 αB-crystallin and brain metastasis in breast cancer KD Voduc et al data and edited the manuscript. VLC conceptualized the project, analyzed the data, Intriguingly, the observed association of αB-crystallin expression wrote and edited the manuscript. MCUC conceptualized the project, assembled the in primary breast carcinomas and brain metastasis is biologically tissue microarray, generated the biomarker data, analyzed the data, wrote and edited the plausible in the light of recent data from cellular assays and manuscript. All authors read and approved the final manuscript. murine models pointing to a pathogenic role for αB-crystallin in breast cancer brain metastasis. αB-crystallin promotes adhesion to human brain microvascular endothelial cells, transendothelial COMPETING INTERESTS migration and penetration through an human brain microvascular CMP and TON are equity stock holders, and Board of Director Member of BioClassifier. endothelial cell or primary human astrocyte co-culture blood– 28 CMP, TON and MCUC are also listed as inventors on a patent application on the brain barrier model. Furthermore, overexpression of αB-crystallin PAM50 assay. 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