Need for speed in solid tumor molecular testing

Amy Carpenter

April 2024—As the call for fast turnaround of genetic testing results in tumor profiling grows louder, the need for rapid, reliable test methods becomes more pressing.

Meanwhile, with new genetic biomarkers emerging at a rapid pace, “everything has tipped the balance toward comprehensive next-generation sequencing analysis,” said Maria E. Arcila, MD, attending pathologist, molecular diagnostics and hematopathology services, Department of Pathology and Laboratory Medicine, Memorial Sloan Kettering Cancer Center. In the midst of this complexity, “the ability to provide rapid and simple results is lagging behind,” said Dr. Arcila, in addressing rapid molecular testing in solid tumors at the Association for Molecular Pathology meeting last year.

“The delivery of rapid and comprehensive molecular results remains a challenge and is a gap for all of us in clinical laboratories,” she said.

The windows for patient care are often narrow, Dr. Arcila noted, and every laboratory has “the pile of urgent cases that very soon become too late if the appropriate technology is not implemented. It’s disheartening to hear from our clinicians when results are urgently needed and we cannot provide those results.”

While recent advances in technology are addressing the needs for comprehensive assessment, she said, “rapid results get caught on complex testing, complex workflows, and often technical requirements for test batching.”

Dr. Arcila called lung adenocarcinoma “the poster child exemplifying the complexity of delivering both rapid and comprehensive results in solid tumors,” and the answer must come from more “elegant solutions,” she said, particularly given the need to provide results within 14 days of receipt of tissue despite operational inefficiencies that may make it difficult. In the context of lung cancer, she notes, “one must deal with very limited tumor material, while there is a need to assess a broad range of alterations, including point mutations, indels, and fusions.” Comprehensive NGS panels that can address this effectively could take many weeks. Sequential single-gene or low-multiplex assays may be faster, but they come at the risk of incomplete analysis and depleting the available tissue. The need for batching further complicates rapid results delivery: “It is commonly required for NGS technology or to facilitate workflows and cost-containment,” she notes.

Despite the existing guidelines for molecular testing in non-small cell lung carcinoma, in real-world practice many patients go untested or are tested but results cannot be provided in a clinically actionable time. In 2019, for instance, Gierman, et al., reported a review of 1,203 patients with advanced disease in a community oncology setting. This study found that less than 50 percent of patients with advanced NSCLC were tested for basic molecular markers and, of those tested, only 45 percent had documented evidence of treatment with the corresponding targeted therapies (Gierman HJ, et al. J Clin Oncol. 2019;37[15 suppl]:1585).

Things haven’t changed significantly since then, Dr. Arcila said.

In 2022, Sadik, et al., reported on their multisource U.S. commercial and Medicare claims and laboratory database analysis of patients with advanced NSCLC who could have, but did not, benefit from a personalized treatment because of various clinical practice gaps (Sadik H, et al. JCO Precis Oncol. 2022;6:e2200246). For every 1,000 patients in the study cohort, the authors reported, 497 are lost to precision oncology because of factors associated with getting biomarker test results. Of the 503 of 1,000 who did receive results from a biomarker test, 29 percent didn’t receive appropriate targeted treatment.

When choosing a technology to implement in a laboratory, numerous factors have to be considered, Dr. Arcila said, citing a list: the patient population and sample journeys, geographic location, institutional and laboratory workflows, the local expertise on the technology, and how it all fits into the laboratory in terms of its technical requirements, needs for batching, stop points, and bioinformatics support.

For rapid results, local testing is the No. 1 need, followed by instruments that are uncomplicated, compact, stable, durable, and ideally closed systems. For broad implementation, rapid testing technology has to be easy to implement, she said, and should be cost-effective and not labor-intensive. “Not everybody can implement Illumina or Genexus systems in their laboratory,” she said, and because instruments must be highly precise, repair and maintenance schedules require redundant backup systems to be in place to maintain clinical laboratory operations, adding to the overall cost. Rapid systems should also minimize manual processing steps and tube transfers, to eliminate possibilities for error and contamination, and should allow sample number flexibility so that one sample or 10 samples can be run, depending on case volume fluctuations.

“Batching requirements are a major issue in our laboratories,” Dr. Arcila noted, “because systems and workflows are generally designed for running multiple samples at a time. When rapid testing is needed, you may be forced to run one or a few samples, compromising the standard laboratory workflows and the cost-effectiveness.”

Rapid testing platforms must also allow robust performance across different sample types, she said, with minimal tissue requirements and the ability to multiplex several biomarkers. “By design, assays should allow easy updates to the content of the panels, to incorporate additional biomarkers that constantly emerge as clinically relevant.” The analysis process should be streamlined, ideally through remote access, for rapid review and interpretation and timely delivery of results.

At MSK, every lung adenocarcinoma is tested by the 505-gene MSK-IMPACT, a panel that requires complex workflows and extensive bioinformatic support. Despite the many benefits of such a comprehensive panel, Dr. Arcila said, providing rapid results is challenging.

“We deal with this by instituting reflex rapid, multiplex, and often non-NGS solutions that can take care of the most common genetic targets. Still, this further increases the overall complexity of testing very small samples and requires extensive optimization of every step to ensure maximal performance.”

For a non-NGS method, the MSK molecular diagnostics laboratory uses the cartridge-based approach from Idylla (Biocartis, Mechelen, Belgium). “This is a multiplex qPCR assay that allows testing of multiple mutations for single samples within a closed system without prior DNA extraction,” she said. All sample processing (liquefaction, cell lysis, extraction), amplification, mutation detection, and data analysis occur within the cartridge, and results are ready within two hours, with just a few minutes of hands-on time, Dr. Arcila said.

For lung adenocarcinoma, multiplex testing with the Idylla EGFR cartridge (51 mutations) allows for rapid screening of the most common targetable alterations. The Idylla KRAS cartridge (21 mutations) can be used as part of a sequential algorithm as KRAS mutations are mutually exclusive with other common drivers.

Idylla’s new assay, the GeneFusion cartridge, “is an elegant solution to screen for common fusions,” she said, referring to ALK, ROS1, RET, NTRK1/2/3 rearrangements, and MET exon 14 skipping. “The assay is RNA based and tests by targeting the gene partners with fusion-specific primers, but it also incorporates a more generic fusion detection approach, by expression imbalance between the three-prime and five-prime end of specific genes, as a surrogate for the presence of a fusion,” she explained. Because RNA can be abundant, one tissue section is often sufficient to perform the test.

Dr. Arcila and colleagues validated the GeneFusion assay for body fluids, brushes, washes, stained smear slides, and formalin-fixed, paraffin-embedded tissue (Chu Y-H, et al. J Mol Diagn. 2022,24[6]:642–654). They wrote: “The assay enables rapid screening for clinically actionable kinase alterations with quicker turnaround and lower tissue requirements compared with immunohistochemistry and molecular methods, while also circumventing the infrastructure dependencies associated with next-generation sequencing and fluorescence in situ hybridization.”

For the DNA-based assays (EGFR and KRAS), Dr. Arcila said, “we can also use aliquots of the NGS libraries from MSK-IMPACT to test on the Idylla cartridges while we’re waiting for NGS results.”

One of the drawbacks of the Idylla platform: “If you’re not extracting DNA or RNA, assessing the input and sufficiency of the material prior to testing may be challenging for new users, and it requires familiarity with the platform,” she said.

The ultimate assessment of suitability is made at the time the results are viewed. “As with other qPCR data, the larger the starting material—in this case the section of tissue—and proportion of tumor, the fewer cycles you need to be able to amplify the template and to reach the quantification thresholds [Cq]. Very limited material requires many more cycles to reach the Cq,” Dr. Arcila said. Interpreting these thresholds, the variability imparted by the different types of tissue loaded directly, and the potential artifacts are critical for a robust validation, she explained, adding, “Once this happens, it becomes a very usable platform.”