Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2015 Sep 1.
Published in final edited form as: Breast Cancer Res Treat. 2014 Aug 3;147(2):457–458. doi: 10.1007/s10549-014-3061-0

Unvalidated antibodies and misleading results

David Rimm 1,✉, Kurt Schalper 2, Lajos Pusztai 3
PMCID: PMC4216678  NIHMSID: NIHMS618666  PMID: 25086631

To the Editor

We would like to raise caution regarding the results of a recent publication in the July issue of Breast Cancer Research and Treatment by Muenst et al. This article entitled “Expression of programmed death ligand 1 (PD-L1) is associated with poor prognosis in human breast cancer” appears to have arrived at an unreliable conclusion due to technical reasons. The investigators used an unvalidated polyclonal antibody to assess the expression of PD-L1. They have not provided validation to prove that the antibody binds to PD-L1. It is concerning that in Fig. 1, they show clear-cut, predominantly nuclear localization for a cytoplasmic and membrane protein. In a recent paper [10] we test four commercially available antibodies to PD-L1 that were all marketed as potentially suitable for immunohistochemistry but in our hands, only 1 validated using FFPE preparations from cell line PD-L1 transfectants and human term placenta as control for endogenous/native PD-L1 expression. We have expanded this effort in a recent ASCO poster showed only 2 of 7 PD-L1 antibodies validated when tested for specificity. In another publication, we show that both the protein and the mRNA are associated with better prognosis in two independent breast cancer cohorts [8]. We have also recently submitted an assessment of PD-L1 in a neo-adjuvant breast cancer cohort where high PD-L1 is associated with PathCR. The results by Muenst et al. are also contrary to numerous reports in the literature that show expression is associated with good prognosis in Melanoma [9]; colorectal carcinomas [5], and Merkel Cell carcinomas [6]; rather than the poor prognosis.

We believe that it is imperative to first rigorously validate any antibody for the purpose that it is used. Poorly validated antibodies may provide misleading information and contribute to the much larger issue of significant non-reproducibility of published research results [2, 7]. Our group has highlighted this problem in several publications [1], and we have described series of assays that can be easily done to show antibody sensitivity, specificity and reproducibility (validation) [3]. There are some published guidelines for IHC and FISH (called MISFISHIE [4]), they are not widely used. More recently the Global Biological Standards Institute (see www.gbsi.org) focused a task force on developing more rigorous standards for IHC and analytical reporting, but they have not yet produced a document.

Thus, the problem may be clearer than the solution. This letter is written in hopes of minimizing future erroneous publications by drawing attention to the necessity of in-house validation of reagents before reporting results.

Footnotes

This letter to the editor refers to the article available at 10.1007/ s10549-014-2988-5 and a rebuttal letter to this letter to the editor is available at doi: 10.1007/s10549-014-3064-x.

Contributor Information

David Rimm, Email: david.rimm@yale.edu, Department of Pathology, School of Medicine, Brady Memorial Laboratory, 310 Cedar Street, P.O. Box 208023, New Haven, CT, USA.

Kurt Schalper, Department of Pathology, School of Medicine, Brady Memorial Laboratory, 310 Cedar Street, P.O. Box 208023, New Haven, CT, USA.

Lajos Pusztai, Department of Medicine (Oncology), School of Medicine, Brady Memorial Laboratory, 310 Cedar Street, P.O. Box 208023, New Haven, CT, USA.

References

  • 1.Anagnostou VK, Welsh AW, Giltnane JM, Siddiqui S, Liceaga C, Gustavson M, Syrigos KN, Reiter JL, Rimm DL. Analytic variability in immunohistochemistry biomarker studies. Cancer Epidemiol Biomark Prev. 2010;19(4):982–991. doi: 10.1158/1055-9965.EPI-10-0097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Begley CG, Ellis LM. Drug development: raise standards for preclinical cancer research. Nature. 2012;483(7391):531–533. doi: 10.1038/483531a. [DOI] [PubMed] [Google Scholar]
  • 3.Bordeaux J, Welsh A, Agarwal S, Killiam E, Baquero M, Hanna J, Anagnostou V, Rimm D. Antibody validation. Biotechniques. 2010;48(3):197–209. doi: 10.2144/000113382. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Deutsch EW, Ball CA, Berman JJ, Bova GS, Brazma A, Bumgarner RE, Campbell D, Causton HC, Christiansen JH, Daian F, Dauga D, Davidson DR, Gimenez G, Goo YA, Grimmond S, Henrich T, Herrmann BG, Johnson MH, Korb M, Mills JC, Oudes AJ, Parkinson HE, Pascal LE, Pollet N, Quackenbush J, Ramialison M, Ringwald M, Salgado D, Sansone SA, Sherlock G, Stoeckert CJ, Jr, Swedlow J, Taylor RC, Walashek L, Warford A, Wilkinson DG, Zhou Y, Zon LI, Liu AY, True LD. Minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE) Nat Biotechnol. 2008;26(3):305–312. doi: 10.1038/nbt1391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Droeser RA, Hirt C, Viehl CT, Frey DM, Nebiker C, Huber X, Zlobec I, Eppenberger-Castori S, Tzankov A, Rosso R, Zuber M, Muraro MG, Amicarella F, Cremonesi E, Heberer M, Iezzi G, Lugli A, Terracciano L, Sconocchia G, Oertli D, Spagnoli GC, Tornillo L. Clinical impact of programmed cell death ligand 1 expression in colorectal cancer. Eur J Cancer. 2013;49(9):2233–2242. doi: 10.1016/j.ejca.2013.02.015. [DOI] [PubMed] [Google Scholar]
  • 6.Lipson EJ, Vincent JG, Loyo M, Kagohara LT, Luber BS, Wang H, Xu H, Nayar SK, Wang TS, Sidransky D, Anders RA, Topalian SL, Taube JM. PD-L1 expression in the Merkel cell carcinoma microenvironment: association with inflammation, Merkel cell polyomavirus and overall survival. Cancer Immunol Res. 2013;1(1):54–63. doi: 10.1158/2326-6066.CIR-13-0034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Pusztai L, Hatzis C, Andre F. Reproducibility of research and preclinical validation: problems and solutions. Nat Rev Clin Oncol. 2013;10(12):720–724. doi: 10.1038/nrclinonc.2013.171. [DOI] [PubMed] [Google Scholar]
  • 8.Schalper KA, Velcheti V, Carvajal D, Wimberly H, Brown J, Pusztai L, Rimm DL. In situ tumor PD-L1 mRNA expression is associated with increased TILs and better outcome in breast carcinomas. Clin Cancer Res. 2014;20(10):2773–2782. doi: 10.1158/1078-0432.CCR-13-2702. [DOI] [PubMed] [Google Scholar]
  • 9.Taube JM, Klein AP, Brahmer JR, Xu H, Pan X, Kim JH, Chen L, Pardoll DM, Topalian SL, Anders RA. Association of PD-1, PD-1 ligands, and other features of the tumor immune microenvironment with response to anti-PD-1 therapy. Clin Cancer Res. 2014 doi: 10.1158/1078-0432.CCR-13-3271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Velcheti V, Schalper KA, Carvajal DE, Anagnostou VK, Syrigos KN, Sznol M, Herbst RS, Gettinger SN, Chen L, Rimm DL. Programmed death ligand-1 expression in non-small cell lung cancer. Lab Invest. 2014;94(1):107–116. doi: 10.1038/labinvest.2013.130. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES