Time, Money, and Tissue

In the decades since Coons and colleagues published their revolutionary work on immunofluorescence detection of antigens in frozen tissue (1), immunohistochemistry (IHC) has become routine in the anatomic pathology laboratory. Each target antigen of interest has been individually identified within histological sections of formalin-fixed, paraffin-embedded (FFPE) tumors or other types of tissue (2). Single-marker IHC takes advantage of the labeling capabilities of horseradish peroxidase and alkaline phosphatase enzymes, in combination with their respective reactant chromogens, to produce a colorimetric stain for visualization under a light microscope (2,3). Alternatively, fluorescent reporters – fluorochromes – can visualize the antibody-antigen interaction, either by conjugation to the primary antibody (direct immunofluorescence) or via attachment to a secondary antibody that detects the species-specific primary (indirect immunofluorescence).

More recently, IHC users have shifted from the single-marker approach to multiplexed marker detection. Multiplex IHC methods can visualize multiple target antigens within a single tissue sample and can be further subcategorized as sequential or simultaneous (4). Generally, if the primary antibodies used are from the same host species, sequential multiplex IHC is required; otherwise, they can be cocktailed and incubated simultaneously.

But why use multiplex IHC at all? There are numerous advantages to visualizing multiple antigens simultaneously. Multiplex IHC maximizes the amount of data acquired from a single tissue sample – critical in conserving precious patient tissue. Unlike next-generation sequencing and mass spectrometry, in which the tissue sample is destroyed to test for individual target molecules, multiplex IHC also allows users to examine the spatial arrangements, interactions, and co-localizations of proteins of interest within the tissue architecture (5).

The complexity of the multiplex IHC protocol necessitates properly trained, highly skilled staff to achieve the most accurate and reproducible diagnoses. In clinical application, these technologically complex techniques require automation to achieve a simple, efficient, and easily understandable result for the clinical pathologist not well-versed in advanced multiplexing methods. Additionally, as the enhanced diagnostic utility of multiplex IHC is realized, the histology laboratory will experience increasing demand. Automation (and the associated standardization and reduction of variability) allows labs to achieve the quality, reproducibility, and speed necessary to meet this demand (6).

Multiplex IHC for diagnostics

One driving factor in the early adoption of multiplex IHC for clinical diagnostics was urologists’ collective move to use the smallest possible needle gauge to perform prostate biopsies. This caused pathologists great difficulty in reliably diagnosing (or ruling out) small foci of cancer; examining multiple minute tissue fragments was time-consuming and reproducibility of diagnosis was poor. The most glaring clinical need was the ability to differentiate between prostatic intraepithelial neoplasia (PIN) and carcinoma of the prostate. We also needed a way of clearly identifying, with high accuracy and specificity, any microinvasion or micrometastasis into adjacent prostate tissue. And, of course, this had to be done while conserving the limited amount of sample (prostate needle biopsies are thin filaments of tissue) and reducing the time to result.

With these constraints and needs in mind, a widely used antibody cocktail known as PIN-4 was developed (see Figure 1). It consists of one or two antibodies against high molecular weight cytokeratin (CK HMW), as well as antibodies to p63 and p504S (also known as AMACR enzyme). Studies have shown that combinations of CK HMW [34βE12], p63, and/or AMACR may be useful in differentiating normal prostate glands from PIN and prostatic adenocarcinoma (PCa) (7,8,9). In prostate tissue, CK HMW [34βE12] is a useful marker of basal cells of normal glands and PIN, a precursor lesion to prostatic adenocarcinoma; invasive prostatic adenocarcinoma, in contrast, typically lacks a basal cell layer (9,10,11).

p63, a homolog of the tumor suppressor p53, has been detected in nuclei of the basal epithelium in normal prostate glands; however, it is not expressed in malignant tumors of the prostate (12). α-methylacyl coenzyme A racemase (AMACR), also known as P504S, has been shown to be a specific marker of prostatic adenocarcinoma (13,14,15,16). Additionally, prostate glands involved in PIN have been found to express AMACR, whereas the enzyme was nearly undetectable in benign glands (16,17).

Pathologists encountered great difficulty in colocalizing the three individual antibody signals (CK HMW + p63 + AMACR) on three separate slides. In many cases, it proved impossible because the biopsies themselves were small, with the suspected PIN or PCa areas smaller or even absent as the tissue block was cut deeper for serial sectioning. However, applying these antibodies to an individual tissue section in a multiplex IHC cocktail format allows for the simultaneous pathological evaluation of each of these critical markers in the same focus or foci of interest, drastically improving reproducibility. It also improves the diagnostic accuracy of invasive prostatic adenocarcinoma to near certainty (18) – which is why the PIN-4 cocktail has become the standard of care in testing prostate needle biopsies.

The clinical application of multiplex IHC has since been expanded to other tissues to give enhanced differential diagnostic information. In breast, the ADH-5 (CK5/14 + p63 + CK7/18) multiplex cocktail (see Figure 2) can aid in the differential diagnoses of usual ductal hyperplasia (UDH), atypical ductal hyperplasia (ADH), and ductal carcinoma in situ (DCIS) (19). UDH carries minimal or no increased risk of breast cancer, and these patients do not undergo any additional procedures; however, ADH and DCIS progress to invasive carcinoma in 4–5 percent and 8–10 percent of cases, respectively. ADH and DCIS patients are advised to undergo excision surgery, with radiation treatment added for DCIS patients (20).

Histological differentiation between UDH, ADH, and DCIS has historically been difficult, with poor concordance among pathologists giving rise to potential misdiagnosis and improper treatment. The addition of the ADH-5 multiplex cocktail to routine histopathology testing significantly reduced overdiagnosis of ADH lesions, reclassifying those lesions as UDH. Studies have demonstrated that up to 40 percent of lesions diagnosed as ADH on core biopsies ended up reclassified as benign upon re-examination after surgical excision (21,22) – meaning that all of those patients could have avoided unnecessary surgery upon initial testing using the ADH-5 multiplex cocktail.

Multiplex IHC for therapeutic decisions

As the cancer treatment landscape has evolved, so have the diagnostic tools we use to make those critical therapeutic decisions. Now, immunotherapy is on the rise. A number of immune checkpoint inhibitors targeting either cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed cell death protein 1 (PD-1), or its ligand (PD-L1), have recently received FDA approval for the treatment of multiple cancer types. However, up to 60 percent of patients treated with these inhibitors see little to no benefit (23). A critical aspect in proper application of these newly designed immunotherapeutics is to establish valid predictive biomarkers to enhance patient selection.

One proposed approach to determining patient response to immune checkpoint inhibitors is to analyze the tumor microenvironment. An immune-active tumor microenvironment is critical to patient response to immunotherapy (24). We must not only understand the dynamic nature of the tumor, but also determine its interactions with its microenvironment to define the algorithm of biomarkers that will predict response to checkpoint inhibitors. Multiplex IHC is well-suited to resolve and define these elements and interactions (25,26).Profiling the tumor microenvironment within tissues requires evaluating multiple markers, including inflammatory cell subpopulations, tumor-infiltrating lymphocytes, and immune checkpoints (see Figure 3).

The next step forward

The automation of multiplex IHC is the next evolutionary step to maximizing its potential. The considerable throughput and performance demands placed on diagnostic laboratories for accurate, consistent, high-quality staining results will only increase as novel assays are developed. In turn, laboratories will demand more from their automated IHC staining platforms. New innovations that increase efficiency – such as simultaneous multiplex IHC technology capability, online deparaffinization, and energy-efficient, parallel-processing antigen retrieval – will allow laboratories to meet the throughput and performance demands and beyond, all while continuing to provide high-quality results. As we increasingly move toward multiplexing and automation in the anatomic pathology laboratory, we’ll save time, money, and precious patient tissue.

Disclosure: Jason Ramos is Vice President of Research and Development at Biocare Medical, Pacheco, USA.