Advanced Staining (IHC, ISH) in Clinical Diagnostics’ Genomic Age

For more information regarding product trends and market development in molecular testing – whether in histology, molecular assays or molecular microbiology and virology – please consult Kalorama Information’s authoritativeThe Worldwide Market for In Vitro Diagnostic Tests, 10th Edition.

The least glamorous and most labor intensive segment of clinical molecular diagnostic testing, histology and advanced stains such as in situ hybridization (ISH) and immunohistochemistry (IHC) endure even as their markets shift to other lab workflows and emerging IVD technologies. Automation has been a crucial trend for advanced stains that place substantial demands on clinical labs in terms of personnel training and hands-on time. Upgrades in terms of throughput and analysis for ISH and IHC may not counter the strengths of alternative workflows in PCR and sequencing, but ensure growth for advanced staining amid competitive pressures. In situ hybridization and IHC tests are expected to maintain share in their core applications and leverage unique capabilities in niche areas of histology diagnostics.

Reagent cost is an issue in the adoption of new diagnostic platforms, particularly molecular diagnostic tests at the point of care (POC) or incorporating sequencing. In this aspect, IHC will maintain an advantage in cancer testing as antibody reagents cost less than nucleic acid probes, PCR mixes, and next-generation sequencing (NGS) reagents. Immunohistochemical stains remain a preferred first line screening tool for companion diagnostic testing such as HER2 and ALK due to lower cost and easier interpretation. Fluorescent ISH (FISH) in these cases is often reserved for inconclusive results or confirmation of positive IHC results.

Nucleic acid amplification tests and sequencing are increasingly used for many pharmacogenetic and prognostic gene markers in oncology, but other tests still require scoring of in situ gene amplification in cells and pathologist interpretation of ISH-marked gene translocations. In situ hybridization remains standard in HER2 and ALK testing as well as testing of hematopoietic neoplasms (leukemia, lymphomas, multiple myeloma, and myelodysplasia). Outside of these core cancer testing areas, FISH testing is threatened by test migration to other formats. Market leader Abbott Laboratories’ FISH revenue has deteriorated along with FISH reagent markets in oncology and cytogenetics. Companion assays are moving towards liquid biopsy formats or testing of circulating tumoral DNA from patient blood samples. Prenatal testing has also shifted rapidly away from traditional karyotyping and chromosomal staining. Future markets in cytogenetics will be claimed by NGS and microarray-based comparative genomic hybridization (aCGH).

How can the ISH market sustain itself? The test form will continue to be favored in select applications for the morphological context it provides pathologists. The transition of advanced staining to digital pathology platforms also enables multispectral imaging or the overlay of fluorescent and bright-field microscopy. Overlaid images allow for multiplex analysis using basic stains, IHC and FISH over the same tissue section. Dual-probe chromogenic ISH (CISH) has also been introduced to areas of FISH testing such as HER2. The alternative probe design removes the need expensive fluorescent microscopy with results viewed under bright-field illumination. The potential next generation of clinical ISH technology is RNA ISH. The direct assaying of RNA overcomes the limited availability of IHC antibodies for some protein targets; lack of protein biomarkers for non-encoding genes; high costs for antibody development and validation; relative lack of antibody specificity compared to oligonucleotide probes; poor antibody sensitivity for secreted or scarce proteins; and non-linear correlation between certain proteins and their corresponding RNA sequences. RNA ISH technology is a prospective format for commercial companion diagnostic assays. Advanced Cell Diagnostics’ RNAscope is the leading RNA ISH probe technology in the research market and may emerge as the basis for companion assays following its use in clinical trials for companion therapies.