The Chemistry in Immunohistochemistry: A Reply From UK NEQAS

To the Editor.—We read with great interest and a growing sense of excitement the recently published editorial by Miller,¹  and the accompanying editorial by Magnani and Taylor.² 

As we are, respectively, the director and manager of the United Kingdom (UK) National External Quality Assessment Scheme for Immunocytochemistry and In-Situ Hybridisation (UK NEQAS ICC & ISH; also herein referred to as the “Scheme”), the subject of these editorials is obviously one which is of great professional interest to us.

The College of American Pathologists (CAP) immunohistochemistry (IHC) proficiency testing program is one of the largest, longest-running, and well-respected external quality assessment (EQA) programs in the world, as well as being one of the most influential. It was heartening, therefore, to read of its plans to disengage the interpretive element of immunohistochemical testing from its underlying technical aspects. And in doing so, to begin the process of putting the science, or, as Miller¹  terms it, the “chemistry,” back into IHC.

Why are we excited by this? Well, it is mainly because, like CAP’s committee review procedures, UK NEQAS ICC & ISH has been examining the methodologic aspects of IHC and their effects on quality since its inception, in our case, 1984. That was when the founding group of biomedical scientists and pathologists that latterly became UK NEQAS ICC & ISH first began to critically assess the quality of κ light chain demonstration in formalin-fixed tissues. This was the birth of EQA for immunohistochemical testing, and indeed the first time EQA was introduced into the discipline of cellular pathology as a whole, certainly within the UK.

Since then, UK NEQAS ICC & ISH has continued to extend its repertoire of EQA programs in an effort to keep pace with the ever-increasing use of immunohistochemical markers across the breadth of cellular pathology. We no longer offer a stand-alone program that solely requires its participants to demonstrate κ light chains. Some would argue that this is a shame, as success with the method required a masterful understanding of the science behind each step in the immunohistochemical methodology, such that it could be successfully manipulated to reliably demonstrate a protein that is present at both huge concentrations (in κ plasma cells) and at vanishingly small amounts (on the cell membranes of a subpopulation of B cells in the mantle zone).

But times have moved on, and being at the forefront of delivery of technical EQAs for IHC (and latterly also for in situ hybridization testing), the Scheme has needed to move on also and to explore and introduce innovative methods. Examples of which include the following:

1. The requirement for tissue controls that express the target protein at a range of concentrations around the critical clinical cutpoint. Important for diagnostic predictive markers. This was first introduced by UK NEQAS ICC & ISH into its estrogen receptors in breast cancer module, which started in early 1998 and continues to this day—more than 100 runs and 25 years later. (In passing, it should be noted that we are the only Scheme as far as we know that also assesses its participants’ in-house controls for adequacy in addition to the assessment of distributed materials.)

2. The utilization of multiple cell-line controls in which each cell line expresses the chosen protein at a clinically relevant level as a set of reference standards for use in EQA was first reported by the Scheme in its breast cancer human epidermal growth factor 2 (HER2) IHC module, which started in 2002,³  marking a further sea change by introducing the use of IHC as a predictive test for a single drug—trastuzumab (Herceptin)—and so beginning the era of companion diagnostics.

3. This was quickly followed by another first, with the introduction in 2004 of the ISH module for cellular pathology laboratories performing this technique to demonstrate HER2 gene copy number and its amplification status, once again as a predictive marker of response to trastuzumab in breast cancer. And our venture into the brave new world (for us) of interpretive as well as technical EQA.

4. We can skip forward nearly 20 years to the present day, missing as we do so the burgeoning growth in the use of IHC and in situ hybridization–based companion diagnostics in all areas of personalized medicine (not least in immuno-oncology, which didn’t even exist 20 years ago). And here once again we see UK NEQAS ICC & ISH responding to the need for a novel approach, this time in the introduction of a truly quantitative assessment method that uses digital image analysis to assess the quality of results produced by Ki-67 to measure proliferation,⁴  together with the use of purpose-designed cell-line arrays (Array Sciences LLC).

5. Which brings us right up to date with our most recently introduced module for low-level HER2 expression in breast cancer. Here we are addressing a critical educational need by combining expert peer-led visual assessment with a specially developed digital algorithm to give individually tailored feedback on technical quality and interpretive proficiency to our participants.⁵ 

Away from the focus on external quality, there have been significant advances made in addressing day-to-day internal quality control in the IHC laboratory, and in perhaps one area, the statement by Magnani and Taylor²  that “Of necessity, every IHC laboratory uses a different (nonstandardized) control tissue,” need no longer be true thanks to advances in the development of systems such as Qualitopix (Visiopharm A/S, Denmark) that monitor and audit day-to-day immunohistochemical staining consistency on standardized, validated test materials in real time.

We recognize that we are not the only EQA program that examines IHC at the methodologic level, and indeed we actively collaborate with many of our fellow schemes across the world, including CAP, through our association with the International Quality Network for Pathology (https://www.iqnpath.org/) organization. And we acknowledge that they, like us, have grown and developed novel tools and ways of assessing the quality of IHC.

By highlighting all these advances and developments, we are not intending to say to the CAP IHC committee “we got there first, now look how much catching up you need to do.” But rather, yes, you are right to do this now when we are at last beginning to have at our disposal all of the tools we need to objectively assess the effects that methodology has on immunohistochemical stain quality, which is the bedrock on which correct interpretation has its foundations.

We welcome this new development from the CAP IHC committee and are sure that the knowledge it will produce will greatly strengthen the science of IHC and the quality of its results, to the immeasurable benefit of the patients it serves.

It’s good to have you on board.