Gillanders, W.

Muenst, S.

Soysal, S.

Tzankov, A.

RERO DOC Digital Library

REBUTTAL LETTERAuthor’s response to ‘‘Letter to the editor: unvalidated antibodiesand misleading results’’S. Muenst • A. Tzankov • W. E. Gillanders •S. D. SoysalPublished online: 1 August 2014 Springer Science+Business Media New York 2014To the Editor,Rimm et al. challenge our study demonstrating an associ-ation between PD-L1 expression and poor prognosis inhuman breast cancer [1] raising a concern about the anti-body we used for immunohistochemistry.Rimm et al. refer to ab58810 (Abcam) as an unvalidatedantibody. We share Rimm et al.’s concern about the use ofunvalidated antibodies in immunohistochemistry studies,and we performed validation studies of the ab58810 anti-body prior to using it in our study. Specifically, wescreened several anti-PD-L1 antibodies for tissue speci-ficity using human term placenta and lymphoid tissue, asmentioned in our paper [1]. Human term placenta iscommonly used for the validation of anti-PD-L1 antibodiesand was used by Rimm et al. for validation of anti-PD-L1antibodies in their manuscript on PD-L1 expression in non-small cell lung cancer (NSCLC) [2]. We tested severalantigen retrieval and staining protocols until we were sat-isfied with the specificity of tissue staining. With an opti-mized protocol using ab58810, we observed strong stainingof the trophoblast layer in human term placenta but theabsence of staining in the stromal cells and vessels of thechorionic villi, as can be appreciated in Figs. 1–3. In ton-sils, strong granular cytoplasmic staining of PD-L1 wasobserved in follicular dendritic cells (Fig. 4). It is impor-tant to recognize the fact that Rimm et al. report a lack oftissue specificity with ab58810 in their study [2] does notpreclude the possibility that a positive result can beobtained with a different staining protocol. The ab58810antibody also recognizes the PD-L1 antigen in Westernblot analysis, as demonstrated on the Abcam website(http://www.abcam.com/cd274-antibody-ab58810.html).Rimm et al. also comment on nuclear staining observedin some breast cancer cells in Fig. 1 of our manuscript [1].Of note, the majority of breast cancer cells in Fig. 1 clearlyshow cytoplasmic staining. We have attached additionalimages from our breast cancer tissue micro array (TMA),confirming that the staining was predominantly cytoplas-mic (Figs. 5–7). We acknowledge that staining is observedin some apoptotic nuclei, but this nuclear staining was notconsidered for the evaluation of PD-L1 expression. Asstated in the Material & Methods section of our paper [1],only cytoplasmic and membranous staining was consideredspecific.Of note, we have previously evaluated our TMA for thepresence of PD-1? tumor-infiltrating lymphocytes (TIL) [3].We observed a statistically significant association betweenPD-L1 expression by tumor cells and the presence of PD-1?TIL in the same tumors (p \ 0.001). This observation pro-vides additional evidence to support the integrity of immu-nohistochemical staining in our manuscript.In preparation of this response, we stained our breastcancer TMA with an additional, newly developed anti-PD-L1 antibody (clone E1L3 N, Cell Signaling), which has beenvalidated by Rimm et al. according to their recent ASCOThis rebuttal letter refers to the article availbale at doi:10.1007/s10549-014-3061-0.S. Muenst (&)  A. TzankovInstitute of Pathology, University Hospital Basel,Scho¨nbeinstrasse 40, 4032 Basel, Switzerlande-mail: simone.muenst@usb.chW. E. GillandersDepartment of Surgery, Washington University School ofMedicine, St. Louis, MO, USAS. D. SoysalDepartment of Surgery, University Hospital Basel, Basel,Switzerland123Breast Cancer Res Treat (2014) 147:459–462DOI 10.1007/s10549-014-3064-xposter (Domain-specific PD-L1 protein measurement in non-small cell lung cancer; http://meetinglibrary.asco.org/content/132010-144). With this clone, we observed a strongassociation between PD-L1 expression and poor tumor dif-ferentiation (G3), estrogen receptor (ER) negativity andworse survival. These findings further support the notion thatPD-L1 protein expression is associated with poor prognosisin breast cancer.We acknowledge that our results are not consistent withSchalper’s et al. findings that PD-L1 mRNA expression isassociated with better prognosis in breast cancer [4].However, our study is consistent with the results of Ghebehet al. who reported an association in breast cancer betweenPD-L1 expression and higher tumor grade, HER2 expres-sion, absence of ER expression [5], and high Ki-67expression [6], which are all known negative prognosticfactors. Furthermore, there are numerous reports in theliterature that expression of PD-L1 is associated with poorprognosis in melanoma [7], NSCLC [8], renal cancer [9],ovarian cancer [10], esophageal cancer [11], pancreaticcancer [12], and gastric cancer [13]. Importantly, the studyabout renal cancer was performed utilizing the PD-L1clone M5H1, which Rimm et al. claim to be the onlyvalidated antibody [2], and the study on esophageal cancerwas performed analyzing PD-L1 gene expression [11].There are important differences between our study andthat of Schalper et al. [4] that may explain the disparateresults. First, our study evaluated PD-L1 protein expressionby immunohistochemistry, while Schalper et al. evaluatedPD-L1 mRNA expression. Although PD-L1 mRNAexpression appears to be associated with PD-L1 proteinexpression, additional studies will need to be performed toconfirm this observation. Second, there is a significantdifference in the percentage of patients who are positive forPD-L1 protein expression (23.1 % in our study) vs. PD-L1mRNA expression (55.7, and 59.5 % in Schalper et al.).The significantly different thresholds for consideringtumors positive for PD-L1 expression could substantiallyFigs. 1–3 Human placental tissue stained with the anti-PD-L1antibody clone ab58810, demonstrating intense staining of thetrophoblast layer and the absence of staining in the stromal andvascular regionsFig. 4 Human tonsil stained with the anti-PD-L1 antibody cloneab58810, demonstrating strong granular cytoplasmic staining of thefollicular dendritic cells and the absence of staining in the surround-ing lymphocytes460 Breast Cancer Res Treat (2014) 147:459–462123affect the results observed. Third, there is a significantdifference in the cohorts studied. Our cohort appeared toinclude patients with more aggressive disease as 72 % hadprimary tumors that were pT2 and above (compared to18 % in Schalper et al.), and 44 % had lymph nodemetastases (compared to 19.6 % in Schalper et al.).Although the lack of proliferation rate and other bio-markers in the cohort of Schalper et al. precludes anapproximation of intrinsic subtype, it appears that thiscohort has a greater representation of smaller, ER-positiveand HER2-negative tumors (most probably Luminal type).Of note, we observed that there was no association betweenPD-L1 expression and survival in Luminal A breast cancers(p = 0.132), while it was highly significant in the HER2-(p = 0.013) and basal-like subtypes (p \ 0.001).We agree that the absence of a validated standardizedstaining and analysis protocol for PD-L1 has contributed toconcerns about the reliability of immunohistochemistrystaining for PD-L1. We were aware of this limitation whenwe initiated our study and openly discussed this issue inour paper [1]. We also acknowledge that we failed tosufficiently explain the antibody validation in the Materials& Methods section of our paper.The role of PD-L1 expression in various human cancersis still not completely understood, and conflicting resultshave been reported. The impact of PD-L1 expression oncancer biology may depend on the level of PD-L1expression, tissue localization, and clinical context. It istherefore clear that additional studies need to be performedin order to validate preliminary results and define theclinical significance. This is particularly important giventhe promising results to date with novel therapeutics tar-geting the PD-1/PD-L1 pathway.References1. Muenst S, Schaerli AR, Gao F, Daster S, Trella E, Droeser RA,Muraro MG, Zajac P, Zanetti R, Gillanders WE, Weber WP,Soysal SD (2014) Expression of programmed death ligand 1 (PD-L1) is associated with poor prognosis in human breast cancer.Breast Cancer Res Treat. doi:10.1007/s10549-014-2988-52. Velcheti V, Schalper KA, Carvajal DE, Anagnostou VK, SyrigosKN, Sznol M, Herbst RS, Gettinger SN, Chen L, Rimm DL (2014)Programmed death ligand-1 expression in non-small cell lung can-cer. Lab Invest 94(1):107–116. doi:10.1038/labinvest.2013.1303. Muenst S, Soysal SD, Gao F, Obermann EC, Oertli D, GillandersWE (2013) The presence of programmed death 1 (PD-1)-positivetumor-infiltrating lymphocytes is associated with poor prognosisin human breast cancer. Breast Cancer Res Treat 139(3):667–676.doi:10.1007/s10549-013-2581-34. Schalper KA, Velcheti V, Carvajal D, Wimberly H, Brown J,Pusztai L, Rimm DL (2014) In situ tumor PD-L1 mRNAexpression is associated with increased TILs and better outcomein breast carcinomas. Clin Cancer Res 20(10):2773–2782. doi:10.1158/1078-0432.CCR-13-27025. Ghebeh H, Mohammed S, Al-Omair A, Qattan A, Lehe C, Al-Qudaihi G, Elkum N, Alshabanah M, Bin Amer S, Tulbah A,Ajarim D, Al-Tweigeri T, Dermime S (2006) The B7-H1 (PD-L1) T lymphocyte-inhibitory molecule is expressed in breastcancer patients with infiltrating ductal carcinoma: correlationwith important high-risk prognostic factors. Neoplasia 8(3):190–198. doi:10.1593/neo.05733Figs. 5–7 Breast cancer TMA punches stained with the anti-PD-L1antibody clone ab58810, demonstrating cytoplasmic staining andaccumulation of antibody in apoptotic cell nuclei, the latter not beingconsidered in our study [1]Breast Cancer Res Treat (2014) 147:459–462 4611236. Ghebeh H, Tulbah A, Mohammed S, Elkum N, Bin Amer SM,Al-Tweigeri T, Dermime S (2007) Expression of B7-H1 in breastcancer patients is strongly associated with high proliferative Ki-67-expressing tumor cells. Int J Cancer 121(4):751–758. doi:10.1002/ijc.227037. Hino R, Kabashima K, Kato Y, Yagi H, Nakamura M, Honjo T,Okazaki T, Tokura Y (2010) Tumor cell expression of pro-grammed cell death-1 ligand 1 is a prognostic factor for malignantmelanoma. Cancer 116(7):1757–1766. doi:10.1002/cncr.248998. Mu CY, Huang JA, Chen Y, Chen C, Zhang XG (2011) Highexpression of PD-L1 in lung cancer may contribute to poorprognosis and tumor cells immune escape through suppressingtumor infiltrating dendritic cells maturation. Med Oncol28(3):682–688. doi:10.1007/s12032-010-9515-29. Thompson RH, Kuntz SM, Leibovich BC, Dong H, Lohse CM,Webster WS, Sengupta S, Frank I, Parker AS, Zincke H, BluteML, Sebo TJ, Cheville JC, Kwon ED (2006) Tumor B7-H1 isassociated with poor prognosis in renal cell carcinoma patientswith long-term follow-up. Cancer Res 66(7):3381–3385. doi:10.1158/0008-5472.CAN-05-430310. Hamanishi J, Mandai M, Iwasaki M, Okazaki T, Tanaka Y,Yamaguchi K, Higuchi T, Yagi H, Takakura K, Minato N, HonjoT, Fujii S (2007) Programmed cell death 1 ligand 1 and tumor-infiltrating CD8? T lymphocytes are prognostic factors of humanovarian cancer. Proc Natl Acad Sci USA 104(9):3360–3365.doi:10.1073/pnas.061153310411. Ohigashi Y, Sho M, Yamada Y, Tsurui Y, Hamada K, Ikeda N,Mizuno T, Yoriki R, Kashizuka H, Yane K, Tsushima F, OtsukiN, Yagita H, Azuma M, Nakajima Y (2005) Clinical significanceof programmed death-1 ligand-1 and programmed death-1 ligand-2 expression in human esophageal cancer. Clin Cancer Res11(8):2947–2953. doi:10.1158/1078-0432.CCR-04-146912. Nomi T, Sho M, Akahori T, Hamada K, Kubo A, Kanehiro H,Nakamura S, Enomoto K, Yagita H, Azuma M, Nakajima Y(2007) Clinical significance and therapeutic potential of theprogrammed death-1 ligand/programmed death-1 pathway inhuman pancreatic cancer. Clin Cancer Res 13(7):2151–2157.doi:10.1158/1078-0432.CCR-06-274613. Wu C, Zhu Y, Jiang J, Zhao J, Zhang XG, Xu N (2006) Immu-nohistochemical localization of programmed death-1 ligand-1(PD-L1) in gastric carcinoma and its clinical significance. ActaHistochem 108(1):19–24. doi:10.1016/j.acthis.2006.01.003462 Breast Cancer Res Treat (2014) 147:459–462123

Author's response to "Letter to the editor: unvalidated antibodies and misleading results”

http://doc.rero.ch/record/326402/files/10549_2014_Article_3064.pdf

Author's response to "Letter to the editor: unvalidated antibodies and misleading results”

Abstract

Similar works

Full text

Available Versions

RERO DOC Digital Library