Professional associations collaborate to define standards for molecular testing of colorectal cancer tissues
Interview by Steve Halasey
A new, evidence-based clinical practice guideline on molecular biomarker testing for patients with colorectal cancer (CRC) identifies opportunities for improving patient outcomes. Compiled by a multidisciplinary panel of experts appointed by the American Society for Clinical Pathology (ASCP), the American Society of Clinical Oncology (ASCO), the Association for Molecular Pathology (AMP), and the College of American Pathologists (CAP), the guideline was jointly published this February in the American Journal of Clinical Pathology, Archives of Pathology & Laboratory Medicine, Journal of Clinical Oncology, and Journal of Molecular Diagnostics.1–4
Antonia R. Sepulveda, MD, PhD, FASCP, FCAP, Columbia University, AMP.
The objective of the collaborative project was to develop standard practices for molecular biomarker testing of CRC tissues, in order to guide epidermal growth factor receptor (EGFR) therapies as well as conventional chemotherapy regimens. The expert panel conducted a systematic review of more than 4,000 articles in the field, and compiled 21 guideline statements focused on mutational testing of EGFR signaling pathway genes for targeted therapies, prognostic biomarkers, and laboratory approaches for operationalizing molecular testing for CRC. To find out more about the new guidelines, CLP recently spoke with Antonia R. Sepulveda, MD, PhD, FASCP, FCAP, professor of pathology and cell biology and vice chair for translational research at Columbia University, and project cochair on behalf of AMP.
CLP: How did the collaborating organizations determine to take on this project to establish guidelines for molecular testing of colorectal cancer tissues—and why now?
Antonia R. Sepulveda, MD, PhD, FASCP, FCAP: To benefit CRC patients, the organizations recognized the need to create this guideline and make biomarker testing recommendations using new discoveries in the field. We convened a panel of experts to develop the guideline and publish it in the organizations’ respective journals.
This pivotal guideline addresses a wide range of molecular biomarkers in patients with early and advanced colorectal cancer to help establish standard molecular biomarker testing, guide targeted therapy decisions, and advance personalized care for patients with colorectal cancer, which is the second leading cause of cancer-related death in the United States for women and men combined.
Hopefully, widespread application of these recommendations will result in improved outcomes for patients with colorectal cancer, including those with metastatic disease.
CLP: The body of research on molecular CRC testing is quite large—the expert panel considered more than 4,000 articles—why has it not previously led to guidelines for testing?
Sepulveda: Molecular diagnostics is a rapidly evolving field of medicine. The post-genome era and the emphasis on precision genomic-based medicine are providing enormous amounts of new data. Many promising new molecular cancer biomarkers are emerging as tools that can be used to enhance successful treatment of patients with CRC and other cancers. This is apparent in the large number of articles identified for the CRC project.
There are multiple aspects that need to be considered when developing clinical practice guidelines. We followed the Institute of Medicine standards for developing clinical practice guidelines. Before clinical guidelines are issued, the strength of evidence needs to be considered, and this is based on systematic literature review approaches. The review of such a large body of literature required the contribution of a panel consisting of experts from various areas that deal with CRC patients and their clinical workup. Assembling the broad expertise available in the CRC expert panel was possible due to the collaboration of four leading medical societies. So, it must be recognized that bringing a project of this scale to completion with the publication of the guidelines is a complex operation.
In the end, 21 guideline statements were established (eight recommendations, 10 expert consensus opinions, and three “no recommendation” statements) based on evidence from a comprehensive literature review that included more than 4,000 articles.
CLP: The new guideline focuses mostly on testing of clinically actionable genes in the EGFR signaling pathway. How do such test results inform a physician’s therapeutic decisions?
Sepulveda: Monoclonal antibody therapies that target the epidermal growth factor receptor (EGFR) bind the EGFR extracellular domain, blocking EGFR signaling pathways in colorectal cancer cells. The first guideline recommendation is that “patients with CRC being considered for anti-EGFR therapy must receive RAS mutational testing. Mutational analysis should include KRAS and NRAS codons 12 and 13 of exon 2, 59 and 61 of exon 3, and 117 and 146 of exon 4 (“expanded” or “extended” RAS),” because the literature shows that only patients with non-mutated, “wild-type” RAS genes derive improved outcomes from the anti-EGFR monoclonal antibody therapies cetuximab and panitumumab. Since about 50% of CRC cases will be positive for RAS mutation, this information has clinical utility, not only so that the treatment can be given to non-mutated-RAS patients who are likely to respond, but also so that potential toxicity and unnecessary costs can be avoided for those patients with mutated RAS genes in their tumors.
We also concluded that there is insufficient evidence at this time to recommend BRAF V600, PIK3CA mutational status, or PTEN IHC as predictive molecular biomarkers for response to anti-EGFR inhibitors.
CLP: The guideline offers very specific recommendations for testing of several genes (eg, RAS, BRAF), but finds insufficient evidence to make recommendations in other cases. What are the key factors that give some biomarkers greater support than others?
Sepulveda: There are several reasons why there was insufficient evidence to make recommendations for some of the biomarkers. Fundamentally, this happens in the systematic review process when there is little confidence that available evidence reflects a true effect, so an estimate of effect is very uncertain and may change with additional evidence. While the systematic review captured published data for these biomarkers, the outcomes might show minimal differences between the treatment and non-treatment arms, or the results of different studies might be contradictory. In contrast, a recommendation could be made for biomarkers that had high-quality studies, including large patient groups and prospective randomized trials, so that additional research is very unlikely to change the confidence in the estimate of effect.
CLP: Will the guideline likely impel researchers to look again at biomarkers for which evidence has so far been somewhat weak?
Sepulveda: The guideline highlights several biomarkers that require further evidence to establish their clinical utility—or lack thereof. For example, PIK3CA and BRAF V600E mutation status will need further studies to evaluate their utility as predictive biomarkers of response to anti-EGFR monoclonal antibody therapy. New methodological analyses, including sub-group analyses, and evaluation of clinical utility of molecular testing in response to novel therapies that continue to emerge, may shed light on the clinical actionability of these biomarkers.
CLP: Are researchers learning about other biomarkers of CRC that might be handled similarly? How long will it take for such research to come to fruition?
Sepulveda: Our systematic review identified several CRC molecular biomarkers that showed either prognostic or treatment predictive characteristics; however, these were reported only in single studies. Most of these molecular biomarkers were tested for expression using immunohistochemistry or RNA techniques. In order for these biomarkers to be considered at the level of a recommendation for testing in routine clinical practice, it would be necessary for the initial results to be confirmed by other studies. This may take years, since specific clinical trials are generally not ongoing. Interestingly, some of the gene expression biomarkers showed promise as predictive of response to radiochemotherapy in rectal cancers; but establishing their clinical utility will require larger prospective studies.
CLP: The guideline’s strongest recommendations have less to do with identifying and testing particular biomarkers than with validation and quality improvement processes that labs should adopt when conducting molecular testing. How did the systematic literature review inform these recommendations?
Sepulveda: Laboratory testing of molecular biomarkers involves the selection of assays, type of specimens to be tested, timing for the ordering of tests, and turnaround time for testing results. In recent years we have seen a plethora of technical approaches for mutational testing that can effectively be used as long as test specificity and sensitivity meet the clinical needs. While earlier testing approaches were focused on one or a few testing targets, the current need for multiple molecular markers from potentially minute tumor samples is leading to greater use of gene panels such as targeted next-generation sequencing (NGS) cancer panels, which can assay from a few to hundreds of genes and amplicons with known mutational hotspots in cancer.
The systematic literature review captured a good number of studies that evaluated the laboratory and clinical performance of assays for mutation detection. Most of the studies were done to detect KRAS exon 2 mutations, with fewer studies including other RAS mutations or alterations in BRAF or PIK3CA. The data extracted from these studies were used to support the recommendations.
That being said, many studies evaluating the performance of laboratory tests used relatively small sample sizes, limiting their use in the systematic review. So there is a need for larger studies of biomarker testing assays that should be designed as prospective, rather than retrospective studies, to address their laboratory as well as their clinical performance. This points to the need for making substantially more resources available to the laboratory to support appropriately powered large-scale assay evaluation.
CLP: Are the guideline’s recommendations about laboratory procedures focused on CRC-specific testing, or are they generalizable to testing in other areas?
Sepulveda: The evidence-based recommendations for this guideline focused on molecular biomarkers identified as most useful in patients with colorectal cancer, in order to select patients who can benefit from treatment with targeted or conventional therapies. Technically, these same underlying principles can generally be applied to the testing of other tumors.
CLP: The guideline acknowledges the distinction between commercial tests with FDA clearance or approval and laboratory-developed procedures. How do the guideline’s recommendations differ in these cases?
Sepulveda: The guideline includes a strong recommendation stating that “performance of molecular biomarker testing for colorectal carcinoma must be validated in accordance with best laboratory practices.”
For non-waived, non-FDA-approved assays (laboratory-developed procedures or LDPs), validation must be performed. Validation design must include the required elements of analytical accuracy (specificity and sensitivity), precision, and analytical sensitivity (limit of detection), interfering substances, and reportable range, as applicable. Clinical sensitivity and specificity, as well as positive and negative predictive value, should be considered additions.
For FDA-cleared/approved assays (without any modification), only verification of test specifications, including accuracy, precision, reportable range, and reference range, needs to be done.
CLP: The guideline anticipates the future development of new biomarkers as well as the adoption of new testing technologies, including “liquid biopsy” and next-generation sequencing approaches. How do you expect such innovations to advance toward adoption?
Sepulveda: Regarding new testing technologies, the literature available at the time of our systematic review already had strong evidence showing that next-generation sequencing (NGS) targeted gene panels are able to meet the sensitivity of detection used in CRC clinical trials (detecting at least 5% mutant alleles), with otherwise adequate performance characteristics, and permitting simultaneous testing of hundreds of mutations, including those in extended RAS, BRAF, and PIK3CA mutation testing. So in many laboratories we are seeing adoption of NGS mutational testing of tumors, including CRC.
On the other hand, there were very limited published data on liquid biopsy utilization for colorectal cancer monitoring. There is of course great interest in the use of noninvasive testing, primarily to detect mutations in circulating tumor DNA (ctDNA), and we recognize that this approach offers great potential for clinical use. Clearly, more data are needed, specifically reporting on patient outcomes of studies where ctDNA testing (liquid biopsy) is being used to monitor response to therapy.
Additionally, as the guideline was in the final stages of preparation, data emerged from a study indicating that deficient DNA mismatch repair or microsatellite instability may have predictive value in patients with advanced CRC given anti-programmed cell death protein-1 (PD-1) immune checkpoint inhibitor therapy. The results from additional studies will be needed to provide sufficiently strong evidence to raise this potential application of CRC tissue testing for consideration as a recommendation in the guidelines.
CLP: The collaborating organizations have committed to updating this guideline 4 years from now—or sooner if new research warrants such an effort. What kinds of changes do you think the next iteration of the guideline will need to address?
Sepulveda: Yes, we will continue to make additional recommendations that are intended to further streamline molecular testing processes and contribute to improving patient outcomes. Based on the emerging testing technologies, I would anticipate that more refined testing recommendations for specific platforms such as NGS and ctDNA/liquid biopsy may appear in future updates.
In terms of emerging clinical applications, it appears that recommendations regarding testing of CRC for the identification of those patients who are likely to respond to immune therapy may be needed. However, we need sufficient published evidence to become available, and it is difficult to predict a timeline
Steve Halasey is chief editor of CLP.
References
- Sepulveda AR, Hamilton SR, Allegra CJ, et al. Molecular biomarkers for the evaluation of colorectal cancer; guideline from the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology. Am J Clin Pathol. 2017;147(3):221–260; doi: 10.1093/ajcp/aqw209.
- Sepulveda AR, Hamilton SR, Allegra CJ, et al. Molecular biomarkers for the evaluation of colorectal cancer; guideline from the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology. Arch Pathol Lab Med. Early online release, February 2017; doi: 10.5858/arpa.2016-0554-CP.
- Sepulveda AR, Hamilton SR, Allegra CJ, et al. ASCO special article: molecular biomarkers for the evaluation of colorectal cancer; guideline from the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology. J Clin Oncol. Published online before print, 6 February 2017; doi: 10.1200/jco.2016.71.9807.
- Sepulveda AR, Hamilton SR, Allegra CJ, et al. Special article: molecular biomarkers for the evaluation of colorectal cancer; guideline from the American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology, and American Society of Clinical Oncology. J Mol Diagn. 2017;19(2):187–225; doi: 10.1016/j.jmoldx.2016.11.001.
