he high prevalence of gene fusions involving the ERG gene (50% to 70%) and the unprecedented specificity of ERG oncoprotein overexpression for prostate cancer1 has led to a body of research on clinical applications for prostate cancer diagnosis and treatment. Research suggests that the ERG oncoprotein overexpression has oncogenic functions in prostate cancer, increasing tumor cell invasion, abrogating prostate cell differentiation, and acting as a surrogate marker of androgen receptor signaling defects.2,3
The first monoclonal antibody (clone 9FY) for ERG overexpression in immunohistochemistry (IHC) was developed by the Center for Prostate Disease Research of the Uniformed Services University of the Health Sciences (CPDR) in 2009. Subsequent research using the CPDR clone found that ERG oncoprotein overexpression by immunohistochemistry was 99.9% specific for prostate cancer.1
As to how this data can be used, Shah says, “ERG positive HGPIN may be an indication for re-biopsy while ERG positive and morphologically suspicious atypical glands are indicative of cancer.” Similar clinical benefits of multiplex IHC techniques have been established for difficult clinical challenges such as differentiation of usual ductal hyperplasia and atypical ductal hyperplasia in breast cancer,4 and differentiation of adeno- vs. squamous-cell carcinoma in non-small cell lung cancer.5 A further result of this seminal research effort was that ERG positive foci of prostatic intraepithelial neoplasia (PIN) were strongly correlated with clonally related ERG positive cancer elsewhere in the prostate.
Other applications for ERG
Several non-prostate diagnostic applications are based on native ERG protein expression persisting in malignant vascular endothelial cells, such as angiosarcomas and Kaposi sarcomas. ERG is also expressed in non-vascular tumors such as acute myeloid leukemia (AML) and subsets of Ewing sarcomas.
Increased ERG utility with multiplex IHC: Additional diagnostic information can be obtained through the use of multiplex IHC. Two critical histological diagnoses that are predictive of an underlying prostatic adenocarcinoma are atypical small acinar proliferations (ASAP), also referred to as ATYP, and high-grade prostatic intraepithelial neoplasia (HGPIN). Today, a multiplex IHC cocktail has been developed which combines ERG with a basal cell marker (CK5) to help with these critical diagnoses.
Figure 1. ERG-CK5-stain-of-prostate-cancer.
Rajal B. Shah, MD, director, Urologic Pathology at Caris Life Sciences, explains the clinical usefulness of the multiplex IHC test, “CK5 and ERG in combination give the pathologist the ability to determine how aggressively the patient should be followed up, or even if a re-biopsy may be required. CK5 helps to define the integrity of the basal cell layer, so the pathologist can confirm the existence of high-grade prostatic intraepithelial neoplasia (HGPIN); even if cancer is not found in the biopsy, ERG positive HGPIN gives strong support for the existence of prostate adenocarcinoma within a few millimeters of the biopsy due to a 'field affect.'”
Table 1. Throughput per shift.
Regarding diagnostic of ASAP, Shaw comments, “The CK5 and ERG multiplex IHC cocktail also increases confidence in diagnosis of ASAP or ATYP, since ERG positive samples are almost certain to be cancer.”
Multiplex IHC benefits laboratory productivity: This technique also provides significant benefits to the laboratory, including increased throughput as well as reduced labor and reagent costs. The conversation about overall productivity IHC instrumentation has often been limited, even trivialized, to a single question related to one portion of the workflow: Does an IHC staining instrument automate the deparaffinization/antigen retrieval step in the IHC workflow? While this is an important aspect to consider, from a LEAN perspective, this particular step may not comprise the most labor intensive step in the IHC process; the actual hands-on time to do this portion of the process is about 90 seconds to four minutes, depending on the batch size, amounting to only a few seconds per slide. It must be noted that the total time, including incubation, for manual depar/antigen retrieval process is about 40 minutes to 50 minutes. This is often faster, however, than when done on automated instruments and does not require the laboratorian's attention once the run is going.
If slide labels are required that are very large and require careful or even agonizing placement on the slide to avoid overlapping with large tissues, this can take longer per slide than depar/antigen retrieval. Re-entering demographic and test data for each patient can take in the range of a half minute per slide, depending on the data fields to be input. Importantly, this can also result in patient identification errors with potential for legal liability.
Consideration of all these factors in combination can result in equivalent hands-on time or, with some laboratory workflows, significant reductions in actual hands-on-time when comparing IHC systems that automate depar/antigen retrieval, but require minute label adjustment and hand entry of patient data vs. an instrument that does not automate the depar/antigen retrieval step, but does provide easy label application and automation of patient data entry through an efficient and flexible LIS interface.
Further, use of a different instrument to automate the depar/antigen retrieval step can significantly decrease run times and increase throughput (see Figure 1). This is somewhat like having both a washer and dryer in your workflow instead of having only one of these perform both processes. The financial impact of this can be large since, for some labs, it may mean the difference between having to budget for an additional large capital purchase, as well as maintain and service an additional instrument. Finally, running multiple antibodies on a single slide means the laboratory only has to pay for one set of reagents, resulting in significant cost savings for the laboratory.
ERG is an emerging marker with enormous specificity for prostate cancer that can also assist with other diagnoses, such as AML and endothelial tumors. Multiplex IHC can increase the clinical value that ERG delivers, enabling more accurate diagnosis of HGPIN and ASAP (ATYP) and potentially impacting aggressiveness of treatment, while increasing laboratory productivity.
Mark Cross is senior director of sales and marketing, and establishes biomarker and companion diagnostics partnerships for Biocare Medical in Concord, CA, which produces multiplex IHC products such as ADH-5 multiplex IHC cocktail and the PulmoPanel for NSCLC differentiation. He can be contacted at mcross < at > biocare.netbiocare.net.
- Furusato B, Tan SH, Young D, et al. ERG Oncoprotein expression in prostate cancer: clonal progression of ERG-positive tumor cells and potential for ERG based stratifi cation. Prostate Cancer PD. 2010;13:228-237.
- Kumar-Sinha C, Tomlins SA, Chinnaiyan AM. Recurrent gene fusions in prostate cancer. Nat Rev Cancer. 2008;8:497-511.
- Klezovitch O, Risk M, Coleman I, et al. A causal role for ERG in neoplastic transformation of prostate epithelium. Proc Natl Acad Sci USA. 2008; 105:2105-2110.
- Jain RK, Mehta R, Dimitrov R, et al. Atypical ductal hyperplasia: interobserver and intraobserver variability. Mod Pathol. Accessed June 10, 2011. Advance online publication, April 29, 2011;doi:10.1038/modpathol.2011.66 (also appearing in July 2011 Mod Pathol).
- Tacha D, Yu C, Haas T. TTF-1, Napsin A, p63, TRIM 29, Desmoglein-3 and CK5: An Evaluation of Sensitivity and Specifi city, and Correlation of Tumor Grade for Lung Squamous Cell Carcinoma vs. Lung Adenocarcinoma. Mod Pathol. 2011;(24) Supp 1, Abstract 1808:425A.