NEW YORK ─ A group of pathologists and scientists has formed the Consortium for Analytic Standardization in Immunohistochemistry (CASI) to build evidence for the integration of analytic standards into immunohistochemistry testing.
Funded by a $2 million grant from the National Cancer Institute, CASI's objective is to improve IHC assay accuracy and reproducibility by integrating reference materials with known analyte concentrations that enable assay comparability into routine IHC testing, Steve Bogen, CASI's principal investigator, said in an interview.
Immunohistochemistry is widely used in surgical pathology to evaluate cancers in tissue samples, and clinicians frequently seek out IHC-based companion diagnostics to identify patients for cancer treatments.
Nonetheless, "stratifying patients for a targeted anticancer drug requires a level of precision in protein expression that is difficult to achieve using immunohistochemistry," Bogen said.
Many issues can impact IHC assay performance, including the way tissue samples are processed and the subjectivity of an individual pathologist's interpretation of a test, but multiple studies have shown that the lack of calibration and standard reference materials are "the most significant contributors to variations in assay performance," Bogen said.
In a 2019 review article published in Applied Immunohistochemistry & Molecular Morphology, Bogen listed several IHC studies which showed that different labs testing the same tissue specimen received different answers from 10 to 30 percent of the time.
The studies adjusted for variability related to preanalytical issues and differences associated with slide interpretation, indicating that "there is substantial variability related to the lack of calibration of the assays," Bogen said.
One of the important goals of image analysis software is to minimize variabilities resulting from different pathologists interpreting assay results, he noted, adding that artificial intelligence algorithms being applied for this purpose are important to reducing variability. Nonetheless, these image analysis systems "will give you an incorrect image without calibrating the assay," he said.
The introduction of calibration and analytic standards have the potential to address reproducibility issues, Bogen said, giving pathologists certainty that their results are accurate and oncologists greater confidence in patient diagnosis and treatment selection.
Overall, CASI aims to contribute to greater IHC assay reproducibility by integrating industry standards in the form of calibrators and controls and traceable units of measure, he said.
The group is currently developing analytic sensitivity thresholds, including lower and upper limits of detection, for selected immunohistochemistry assays and sketching plans to create greater awareness of current analytic standards by describing why they are important and how they can be implemented, said Bogen, who is CEO of Boston Cell Standards, the Boston-based firm that is leading the CASI initiative and stands to benefit from its activities.
The company has patented technology for the development of immunohistochemistry assay calibration tools comprised of purified analytes conjugated to a clear cell-sized glass microbead at up to 10 defined analyte concentrations that are traceable to National Institute of Standards & Technology (NIST) Standard Reference Material 1934.
Measurement traceability to a single common primary standard ensures that laboratories worldwide can report the same results regardless of where they are located, Bogen said.
Immunohistochemistry staining of the calibrators is performed in the same way that tissue samples are stained and includes deparaffinization, hydration, and antigen retrieval, he said.
Calibrators are approximately the same size as cells, so pathologists examine them under the microscope just as they would examine patient tissue samples.
"The unique attribute of calibrators is that they are provided in a range of well-defined concentrations for the molecule being measured, the analyte," Bogen said. "High analyte concentrations lead to a dark color after processing in the assay. Those with low concentrations result in a faint color or none at all. Checking that the assay causes the expected color response across the various calibrator concentrations ensures that it’s working properly."
According to Keith Miller, a former professor of pathology at University College London, such tools are needed because "we generally have a poor ability to measure in histopathology."
Miller is also a former director of the UK National External Quality Assessment Scheme (UK NEQAS) for Immunohistochemistry & In-situ Hybridisation, a founding member of CASI.
"Immunohistochemistry is quite good at helping you detect cancer and telling you its type, but currently using IHC to quantify a target protein to inform treatment is quite challenging," he said.
Though technologies such as whole-slide imaging are making inroads and helping clinicians obtain more accurate results, the adoption of these systems is limited.
As a result, histopathologists looking to provide a recommendation for treatment generally have to evaluate and quantify tissue cells on a slide by viewing them through a microscope, Miller noted.
By comparison, using calibration tools validated for specific IHC assays, "testing laboratories around the world should be able to obtain the same results for the same type of assay and identify and discard those assays that are inaccurate," Miller said.
To implement its findings, CASI intends to publish validation data for assay calibration and provide analytic sensitivity recommendations for specific assays, sharing its findings with clinical laboratories, external quality assurance organizations, and pathology organizations.
Last August, the group published a validation of calibration standards for PD-L1 IHC assays in Modern Pathology. The researchers noted in their study that currently "basic analytic assay parameters such as lower limit of detection and dynamic range are unknown" to both CDx assay developers and end users.
However, they "solved this problem by developing completely new tools for IHC — calibrators with units of measure traceable to National Institute of Standards & Technology (NIST) Standard Reference Material (SRM) 1934," the researchers wrote.
Similar studies are being prepared, according to Bogen, for HER2, p53, and the BRAF V600E mutation IHC assays.
Last February, Bogen and his colleagues also published the results of a study in Clinical Chemistry, describing an estrogen receptor (ER) reference standard that enabled the precise interlaboratory alignment of ER immunohistochemistry test sensitivity for measuring cellular proteins in breast cancer. The "data demonstrate, for the first time, that the variable test results correlate with analytic sensitivity, which can now be measured quantitatively," the researchers wrote.
A description of CASI and its objectives has been accepted for publication to the Archives of Pathology in Laboratory Medicine.
"Right now, we are introducing the whole concept to pathologists, and very few of them know about it," Bogen said.
In the US, the College of American Pathologists, which conducts proficiency testing for pathology laboratories, is not a member. CAP did not respond to a request for comment for this article, and the American Clinical Laboratory Association said it did not have any comments about the CASI initiative.
Bogen said that other leading laboratory proficiency testing organizations including NordiQC in Aalborg, Denmark; UK NEQAS in London; and the Canadian Biomarker Quality Assurance based in Saskatoon are founding members of CASI.
That "increases the chances that pathologists will eventually adopt analytic assay standards," he said.