ndividualized oncology, also termed “personalized,” “precision,” “patient-centric,” and “targeted” oncology, is now front and center in both cancer drug development and standard-of-care (SOC) therapy for cancer patients. Individualization is focused on the potential molecular target(s) in each patient’s cancer, with the target used as a biomarker for selection of a drug for that target, or avoidance of its use due to absence of the target. Biomarkers can also serve as indicators of resistance pathways that will render drugs ineffective.

The approach of using a targeted treatment, rather than a next-up tryout of another line of generic, albeit evidence-based therapy, has the intent of improving outcome for patients. The major theoretical advantages are the use of efficacious drugs with better outcomes, fewer adverse events from use of ineffective drugs, and lower cost with higher value for patients. In targeted drug development, identification of a biomarker as a companion diagnostic for each agent has been a required part of the process for many years.

Biomarker Quality

The quality of biomarker testing is crucial, and multiple issues in translation of basic science findings and molecular pathology into clinical use have emerged in efforts directed at individualized oncology. A variety of methodologies are applied in biomarker use, most commonly immunohistochemistry and next-generation sequencing of tumor DNA and cDNA for mRNA expression. These methodologies are affected by both common and distinct issues, but common issues often relate to sample procurement and handling, or “pre-analytic” considerations. For example, discordant testing results for the same tumor with different biomarker assays are well-described. The explanations include intratumor heterogeneity of the target analyte, but specimen characteristics, such as sampling and different performance characteristics of methodologies can also contribute.

This methodological problem is obvious when discordant sequencing results are obtained from identical aliquots of the same starting material analyzed with different methods or with the same method in different laboratories. In this situation, characteristics including differences in informatics pipelines with alignment and variant-calling differences in sequence analysis contribute to the discordance. Further, post-analytic decision support using identical sequencing results can be complicated by discordance in interpretation due to differences in reference databases for determining actionability and variable algorithms for assigning levels of evidence.

The rapid emergence of newly identified molecular abnormalities is occurring with the widespread application of sequencing, posing challenges to curating the findings. In addition, time and research are often required to determine if newly recognized variants of unknown significance translate into meaningful differences in tumor biology.

Efforts in Biomarker Quality Improvement

Numerous organizations and professional societies have undertaken improvement of molecular pathology processes to improve assay quality. For example, the ACCE model from the Office of Public Health Genomics at the Centers for Disease Control and Prevention has emphasized the evaluation of analytical validity in the laboratory, clinical validity relative to the disease of interest, and clinical utility in improving patient outcomes along with the overlay of ethical, legal and social implications of the use of assays. SOC integral biomarkers can contribute to the estimation of prognosis for a treat/no-treat decision. When treatment is warranted, agent selection can be influenced by tumor biomarker status.

Numerous potential biomarkers are emerging from basic and translational science, and the highest level of evidence for their use in clinical practice continues to derive from clinical trials directed by integral markers to assign patients to treatment arms. These results also influence payer coverage decisions. The efforts at standardization of methodologies, development of control analytes, and use of proficiency testing have paid dividends in improving assay performance and quality of testing for patients.

In the laboratory development of molecular assays for regulatory-accredited clinical laboratories that perform SOC as well as integral marker testing for biomarker-directed clinical trials, pre-analytics, analytics, and post-analytics are considered. Great attention has been paid to the analytics by regulatory agencies and payers. Considerations include content actionability related to therapeutic agents under consideration, the number and coverage of genes in panels, the nature of analytes that are to be used for analysis, the quality of those analytes, and turnaround times. Actionability of the assay results, defined as the ability to be done or acted upon and having practical value, is crucial, but the entire process begins with the specimens to be analyzed. The processes and procedures used to obtain specimens and prepare the analytes for testing (i.e., the pre-analytics) can make or break assays, especially when the methodologies are less robust than is optimal and influenced by the specimen characteristics.

Factors Important in Pre-Analytics

As shown in the table below, a wide variety of considerations are important in pre-analytics.

Pre-analytical considerations begin with patient selection. The most common usage of individualized oncology is in patients with advanced disease or participating in integral-marker clinical trials. The fitness of the patient and extent and location of tumor burden influence the suitability of an individual patient for an invasive biopsy procedure. Second is the timing of the testing during the course of the disease. Tumor biology factors that influence specimens include the occurrence of neoplastic progression over time due to the well-known genomic instability of most cancers, the selective effects of therapy in eliminating susceptible tumor cell populations and leaving resistant cells behind, and the tumor microenvironment in which the cells reside, including different organ sites.

There is continuing controversy regarding the need for obtaining prospective biopsy specimens for therapeutic decisions. The proximate tumor that threatens the patient is the logical target, but the potential harms from obtaining a new biopsy specimen are a consideration. As a consequence, archived specimens, often separated by years and multiple lines of therapy from the current clinical status of the patient, are commonly used. Another consideration is the nature of the tumor tissue available for analysis. Formalin-fixed paraffin-embedded tissue is the standard in pathology practice, despite years of attempts to change to less damaging procedures that maintain the tissue characteristics critical to diagnostic pathology. Assay procedures that require frozen tumor or tumor stabilized in buffers or expensive proprietary fixatives to preserve labile protein or RNA analytes create a major impediment to routine clinical application of the associated assay and the conduct of clinical trials at multiple accrual sites.

The quantity and the quality of tissue required for assay is important. In the case of core biopsy specimens, the fewer number of needle passes and the smaller size of the needle used in parenchymal organ biopsies are important to lessen the risk of adverse events. These potential harms must be balanced against the amount of material required for assays to be performed. Fit-for-purpose considerations drive assay selection and the specimens that are needed. The selection and timing of assays during the clinical course for an individual patient will vary depending upon the information needed for a management decision. The specimens must be fit-for-purpose for the assay as well as the patient, and they must provide sufficient material of appropriate quality for successful testing. Unsuccessful testing that provides no results for the patient who has undergone an invasive procedure is obviously an unsatisfactory outcome, but exceeding an 80 percent success rate is difficult in current medical practice, and re-biopsy subjects the patient to additional risk.

The fit-for-purpose aspects of the specimens themselves include both the quality and quantity of the tissue. Tissue quality describes the status of the tumor, including presence of viable tumor cells with minimized necrosis (which affects labile analytes to a far greater extent than DNA) and the presence of non-neoplastic tissue that always accompanies solid tumor. The detrimental effects of formalin, even with buffering, as a nearly universal fixative are well-known in molecular laboratories. The quantity of tumor tissue and percentage of tumor cells relative to the “contaminating,” non-neoplastic cells influence assay outcomes, as different testing methodologies have different requirements. Microdissection to enrich for tumor cells is used commonly. The amount of starting tumor analyte and the limit of detection of the assay also influence the results for different types of biomarkers. Mutations can often be detected in specimens with very low percentages of tumor DNA, but copy number variation is heavily influenced by the presence of diluting non-neoplastic cells. Cytology specimens are attractive due to the smaller needles used for fine needle aspiration and the extraction of tumor cells from stroma, which often leads to an enriched specimen but provides relatively lower tumor quantities.

Improving Pre-Analytics

Collaboration with the physicians who obtain specimens is key. Interventional radiologists need to know the purpose for which the specimen is being obtained. Accurate diagnosis can usually be achieved on small specimens, including fine needle aspirations, but often will not yield sufficient material for additional molecular testing. Selection of the lesion to be biopsied needs to take into account the topography as well as characteristics of the tumor. Sampling plays an important role in addressing tumor heterogeneity in that taking specimens from multiple areas of a tumor or from multiple tumors has advantages. The logistics of handling the specimens in the interventional radiology suite adds complexity to obtaining high-quality and sufficient specimens, and the use of pre-prepared kits with all required fixatives (neutral buffered formalin and cytology fixation fluid) as well as instructions for the specimens to be obtained are helpful. Protocols and procedures for the processing of the obtained specimens and extraction of analytes are especially important for individualized oncology due to the resultant decisions for patient management.

A Glimpse of the Future?

As is apparent, there is substantial room for improvement in pre-analytics. Methodologies for real-time assessment of core and fine needle aspiration specimens at collection currently rely on touch preps or smears for staining and light microscopy. Methods for imaging tissue cores by optical light-based microscopy are under development. Automated microdissection instruments have become available. “Liquid biopsies” that provide assays of tumor cells or their constituents in plasma obtained by venipuncture or from other body fluids hold great promise for minimally invasive sequential evaluation of tumor status, if and when clinical utility is demonstrated.

Conclusions

There is no question that the quality of results generated for use in individualized oncology begins with optimized pre-analytic collection and processing of specimens. The rapid advances in technologies and methodologies available for use for integral markers in clinical trials and SOC molecular testing in the clinical laboratory environment complicate this important aspect of laboratory testing. Pathologists, as the physicians responsible for clinical laboratories, have now been given the remarkable opportunity to become Chief Assay Quality Officers and provide great benefit to oncology patients in the era of individualized oncology.