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www.nature.com/npjbcancer All rights reserved 2374-4677/15 ARTICLE OPEN Analyzing historical trends in breast cancer biomarker expression: a feasibility study (1947–2009) 1 2 1 3 4 2 2 Nancy Krieger , Laurel A Habel , Pamela D Waterman , Melina Shabani , Lis Ellison-Loschmann , Ninah S Achacoso , Luana Acton and Stuart J Schnitt BACKGROUND/OBJECTIVES: Determining long-term trends in tumor biomarker expression is essential for understanding aspects of tumor biology amenable to change. Limiting the availability of such data, currently used assays for biomarkers are relatively new. For example, assays for the estrogen receptor (ER), which are the oldest, extend back only to the 1970s. METHODS: To extend scant knowledge about the feasibility of obtaining long-term data on tumor biomarkers, we randomly selected 60 breast cancer cases (10 per decade) diagnosed between 1947–2009 among women members of the Kaiser Permanente Northern California health plan to obtain and analyze their formalin-ﬁxed parafﬁn-embedded (FFPE) tumor specimens. For each tumor specimen, we created duplicate tissue microarrays for analysis. RESULTS: We located tumor blocks and pathology reports for 50 of the 60 cases (83%), from which we randomly sampled 5 cases per decade for biomarker analysis (n = 30). All 30 cases displayed excellent morphology and exhibited biomarkers compatible with histologic type and grade. Test–retest reliability was also excellent: 100% for ER; 97% for human epidermal growth factor receptor 2 and epidermal growth factor receptor; 93% for progesterone receptor and cytokeratin 5/6; and 90% for Ki67 and molecular phenotype; the kappa statistic was excellent (40.9) for 4 of the 7 biomarkers, strong (0.6–0.8) for 2, and fair for only 1 (owing to low prevalence). CONCLUSIONS: These results indicate immunostaining for biomarkers commonly used to evaluate breast cancer biology and assign surrogate molecular phenotypes can reliably be employed on archival FFPE specimens up to 60 years old. npj Breast Cancer (2015) 1, 15016; doi:10.1038/npjbcancer.2015.16; published online 7 October 2015 INTRODUCTION spanning 6 decades (1947–2009). Favorable results would enhance interpretation of prior studies that have employed Determining long-term trends in tumor biomarkers is crucial for biomarker immunostains on old FFPE specimens, e.g., 30–40 understanding what aspects of tumor biology are amenable to 14,15 years old when analyzed, as well as encourage new research. change. Evidence of long-term trends critically complements cross-sectional comparisons, across geographical regions or social groups, because only long-term data can detect the impact MATERIALS AND METHODS of changing exogenous exposures. For example, the recent rise and fall in breast cancer incidence in many countries, linked to For our study, we analyzed FFPE specimens obtained from women 2–9 diagnosed with invasive breast cancer who were members of the rise and fall of postmenopausal hormone therapy use was Kaiser Permanente, Northern California (KPNC; institutional review board paralleled by a rise and fall in the incidence of estrogen receptor + 3,4 approval: Harvard School of Public Health/#CR-20929–02; KPNC/ positive (ER ) tumors. Of note, although breast cancers tumors + #CN-13LHabe-03-H). KPNC is an integrated healthcare delivery system are generally more often ER among US white compared with 16 17 established in the 1940s and whose cancer registry dates back to 1947. 10 + black women, the US white/black odds ratio for ER breast Since its inception as a health plan for workers employed in World War II tumors nevertheless exhibited a parallel rise and fall during this shipyards, KPNC’s membership has ranged from working class to same time period, a pattern likely attributable to changes in professional and has mirrored the well-known diversity of the San hormone therapy use. Francisco Bay Area and Central Valley, comprised of white, black, Hispanic, 17,18 Scant knowledge, however, exists about the feasibility of Asian and Paciﬁc Islander, and American Indian populations. Among the 60,904 breast cancer cases diagnosed between 1947 and locating and analyzing decades old, population-based archival 2009, 7,150 met our feasibility study’s eligibility criteria: 50–64 years old at formalin-ﬁxed parafﬁn-embedded (FFPE) tumor specimens. Con- diagnosis and invasive tumor⩾ 1 cm. We randomly selected 10 eligible tributing to the lack of such historical data is the relatively recent cases per each of the 6 time periods (hereafter referred to as decades: development of most currently used assays: in the case of breast 1947–1959; 1960–1969;…; 2000–2009), and used information available as cancer, for example, one of the ﬁrst such assays, for ER, became of 1987 to restrict sampling to cases with lymph node positive tumors. Our available only in the 1970s, and characterization of molecular rationale was to maximize the chance that biomarkers would be positive or phenotypes is an innovation of the 21st century CE. We credibly negative, given that advanced cases are more likely to be positive accordingly conducted a novel feasibility study, including assess- for human epidermal growth factor receptor 2 (HER2), epidermal growth − − 13,19 ment of test–retest reliability, for a series of breast cancer cases factor receptor (EGFR), high Ki67, ER , and PR . Thus, had the 1 2 Department of Social and Behavioral Sciences, Harvard T.H. Chan School of Public Health, Boston, MA, USA; Kaiser Permanente Division of Research, Oakland, CA, USA; 3 4 Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA and Centre for Public Health Research, Massey University, Wellington, New Zealand. Correspondence: N Krieger (firstname.lastname@example.org) Received 10 July 2015; revised 14 September 2015; accepted 17 September 2015 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited Historical trends in breast cancer biomarkers N Krieger et al npj Breast Cancer (2015) 15016 © 2015 Breast Cancer Research Foundation/Macmillan Publishers Limited Table 1. Immunostain assay results, based on two independent TMAs 1 and 2, for 30 randomly selected breast cancer tumor specimens (5 per decade; all FFPE) for: CK cocktail, ER, PR, HER2, CK 5/6, EGFR, Ki67, and molecular phenotype (Kaiser Permanente, Northern California, USA, 1947–2009) Year range ID Year of Histologic diagnosis Histologic CK cocktail ER PR HER2 CK 5/6 EGFR Ki67 Molecular phenotype diagnosis grade TMA1 TMA2 TMA1 TMA2 TMA1 TMA2 TMA1 TMA2 TMA1 TMA2 TMA1 TMA2 TMA1 TMA2 TMA1 TMA2 1947–1959 1–1 1955 Invasive carcinoma with ductal 2+ + + + −− −− −−− − 1.3% 0.3% Luminal A Luminal A and lobular features 1–2 1957 IDC 2 + + + + + + 3+ 3+ −−− − 9.0% 4.7% Luminal B Luminal B 1–3 1959 IDC 3 + + + + + + −− −−− − 2.7% 5.3% Luminal A Luminal A 1–4 1956 Invasive lobular carcinoma 2 + + + + + + −− −−− − 1.3% 2.0% Luminal A Luminal A 1–5 1956 IDC 3 − − − −−− −− −−− − 12.3% 12.7% Unclassiﬁable Unclassiﬁable 1960–1969 2–6 1968 Solid papillary carcinoma 2 + + + + + + −− −−− − 3.0% 3.3% Luminal A Luminal A 2–7 1969 IDC 3 + + − −−− 2+ 2+ −− + − 23.0% 18.0% Basal-like Unclassiﬁable 2–8 1969 IDC 2 + + + + + + 3+ 3+ −−− − 9.0% 5.7% Luminal B Luminal B 2–9 1968 Invasive mucinous carcinoma 2 −− + +++ −− −−− − 2.7% 4.0% Luminal A Luminal A 2–10 1965 IDC with medullary features 3 + + − −−− −− − + + + 7.0% 5.0% Basal-like Basal-like 1970–1979 3–11 1973 IDC 1 + + + + + + −− −−− − 6.0% 4.7% Luminal A Luminal A 3–12 1974 IDC 3 + + + + + + −− −−− − 6.0% 7.3% Luminal A Luminal A a a a a a a a a 3–13 1977 IDC 3 + + + − − − 16.3% Luminal B 3–14 1979 Solid papillary carcinoma 2 + + + + + + −− ++ −− 1.7% 2.5% Luminal A Luminal A 3–15 1974 IDC 3 + + − −−− 3+ 3+ + −− − 8.7% 8.0% HER2 HER2 1980–1989 4–16 1988 IDC 3 + + + + + + −− −−− − 2.0% 2.0% Luminal A Luminal A 4–17 1980 IDC 3 + + − −−− −− −−− − 9.7% 7.3% Unclassiﬁable Unclassiﬁable 4–18 1985 IDC 3 + + + + + −− −− − − − 10.0% 3.5% Luminal A Luminal A 4–19 1986 Tubular carcinoma 1 + + + + −− − −−− − 1.3% 1.0% Luminal A Unknown 4–20 1985 IDC 1 + + + + − + −− −−− − 1.0% 0.5% Luminal A Luminal A 1990–1999 5–21 1999 IDC 3 + + − −−− 3+ 3+ −−− − 12.7% 13.3% HER 2 HER 2 5–22 1994 Mixed IDC and mucinous 3 + + + +++ −− −−− − 9.3% 11.0% Luminal A Luminal A carcinoma 5–23 1996 Invasive carcinoma with ductal 2 + + + +++ −− −−− − 1.7% 3.5% Luminal A Luminal A and lobular features 5–24 1995 Invasive carcinoma with ductal 2 + + + +++ −− −−− − 16.0% 8.0% Luminal B Luminal A and lobular features 5–25 1998 Invasive carcinoma with ductal 2+ + + + −− −− −−− − 5.7% 7.7% Luminal A Luminal A and lobular features 2000–2009 6–26 2006 IDC 3 + + + + + + 3+ 3+ −−− − 17.7% 12.0% Luminal B Luminal B 6–27 2002 IDC 3 + + − −−− 3+ 3+ −−− − 13.3% 14.0% HER 2 HER 2 6–28 2000 Invasive carcinoma with ductal 2 + + + +++ − −−− − 5.7% 8.3% Luminal A Luminal A or and lobular features Luminal B 6–29 2002 IDC with medullary features 3 + + − −−− 2+ − + + + + 6.3% 7.7% Basal-like Basal-like 6–30 2000 IDC 3 + + + + + + −− −−− − 6.3% 6.7% Luminal A Luminal A Percent concordance 100% 100% 93% 97% 93% 97% 90% 90% Kappa (95% conﬁdence interval) 1.000 1.000 0.926 0.908 0.628 0.782 0.346 0.724 (1.000, 1.000) (1.000, 1.000) (0.705, 1.000) (0.731, 1.000) (0.155, 1.000) (0.372, 1.000) (−0.225, 0.916) (0.591, 0.976) Abbreviations: CK, cytokeratin; ER, estrogen receptor; EGFR, epidermal growth factor receptor; FFPE, formalin-ﬁxed parafﬁn-embedded; HER2, human epidermal growth factor receptor 2; ID, case identiﬁcation; IDC, invasive ductal carcinoma; PR, progesterone receptor; TMA, tissue microarray. 13,19–21 Molecular phenotype algorithm : + + − Luminal A—ER and/or PR , HER2 , Ki67o14%. + + + + + − Luminal B—ER and/or PR and HER2 OR ER and/or PR , HER2 , and Ki67 ⩾ 14%. − − + HER2—ER and PR , and HER2 . − − − + + Basal-like—ER ,PR , HER2 , CK 5/6 and/or EGFR . Results for case 3–13 were missing for TMA1. Results unknown owing to no tissue being present in all 3 cores obtained for the TMA. Kappa for Ki67 for dichotomous categorization as o14% vs. ⩾ 14%. Historical trends in breast cancer biomarkers N Krieger et al specimens included only early stage cancer, it would be less clear if In summary, our study provides novel evidence that current negative test results could be interpreted as truly negative—versus falsely immunostain techniques can feasibly and reliably be used on old negative—because the assay was not sensitive to biomarker expression in FFPE specimens, dating back 60 years. It accordingly suggests that 14,15 the older specimens. Among the 50 cases located with eligible blocks prior and future studies employing immunostains to char- containing tumor (as described below), and in accord with our a priori acterize long-term trends in tumor biomarker expression at the power calculations, we selected a random sample of 5 cases per decade population level can yield credible results. (total n = 30) for biomarker immunohistochemical analysis. To conduct the assays, we ﬁrst created tissue microarrays (TMAs) in duplicate, each employing 3, 0.6-mm cores per specimen. We assessed ACKNOWLEDGMENTS antigen preservation by using a cytokeratin (CK) “cocktail” immunostain We acknowledge, with permission (email: 8 February 2015), Dr Andrew Beck (consisting of antibodies AE1/AE3 and Cam5.2, which together recognize (Director, Molecular Epidemiology Research Laboratory, Pathology, Beth Israel a broad spectrum of CKs), and also performed immunostains for Deaconess Medical Center, Boston, MA, USA) and Dr Laleh Montaser-Kouhsari biomarkers routinely used to assess breast cancer biology and assign 13,19–22 (Post-doctoral Researcher, Pathology, Beth Israel Deaconess Medical Center, Boston, molecular phenotype based on surrogate markers : ER, progesterone MA, USA), for their assistance with the computer-assisted image analysis for the Ki67 receptor (PR), HER2, CK 5/6, EGFR, and Ki67. The Ki67 stains were quantitated assay results. by computer-assisted image analysis; for each case, the score equaled the average percentage of positive cells per core. Review of the biomarkers was blinded to tumor histologic type and grade, and review of each set of TMA results was independent and blinded to the other. For each TMA, if a CONTRIBUTIONS tumor marker was scored as positive for 1 or more cores, it received an NK conceived the study, designed and supervised the analyses, led interpretation of overall rating of positive for that marker. the results, and wrote the ﬁrst draft of the article. All authors designed the study and wrote the article. LAH led the work in identifying the eligible study participants and obtaining the tumor specimens, assisted by NA, and both contributed to interpreting RESULTS results. SJS led the work on tissue microarray construction and evaluation of biomarker expression, assisted by MS, and both contributed to interpreting the Among the random sample of 60 selected cases, we located results. PDW and LA assisted with study logistics and contributed to interpreting the pathology reports for 55 cases (92%), of which 50 (83% of the 60) results. LE-L contributed to interpreting results. had blocks that contained tumor tissue. Among the random sample of 5 cases per decade selected from these 50 cases, notably all 30 cases (100%) displayed excellent morphology and COMPETING INTERESTS the CK cocktail staining results (Table 1) indicated antigen The authors declare no conﬂict of interest. integrity was preserved for all but 2 of the cases (1 from the 1950s, 1 from the 1960s). Test–retest reliability was likewise excellent (Table 1): 100% for ER, 97% for HER2 and EGFR, 93% for FUNDING PR and CK 5/6, and 90% for Ki67 (scored as o14% vs. ⩾ 14% ). This work was supported by the National Cancer Institute (NCI) at the National The kappa statistic (taking into account chance agreement; Institutes of Health (NIH), grant number 1R21CA166115-01A1 (“Long-term trends in Table 1) was excellent (40.9) for 4 of the 7 biomarkers, moderate- breast cancer tumor proﬁles & disparities”), awarded to NK. The funder had no role in to-strong (0.6–0.8) for 2, and fair for 1 (0.346 for Ki67 as a the design and conduct of the study; collection, management, analysis, and dichotomous variable). On the basis of the biomarker results, interpretation of the data; and preparation, review, or approval of the article; and concordance for molecular phenotype was 90% (kappa 40.7): decision to submit the article for publication. classiﬁcation of 15 cases was concordant for Luminal A, 3 for Luminal B, 3 for basal-like, 3 for HER2, and 2 for “unclassiﬁed”; only REFERENCES 3 cases were discordant for classiﬁcation as either Luminal A or B. 1 Krieger N, Chen JT, Kosheleva A, Waterman PD. Shrinking, widening, reversing, Assay results indicated that ~ 80% of tumors were ER (i.e., 80% and stagnating trends in US socioeconomic inequities in cancer mortality: 1960- for all decades except for the 1960s, for which 3/5 (60%) of the + − + 2006. Cancer Causes Control 2012; 23: 297–319. cases were ER ), all ER tumors were grade 3, and virtually all ER 2 Krieger N, Chen JT, Waterman PD. Decline in US breast cancer rates after tumors were grade 1 or grade 2. 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