As our understanding of tumor development and its microenvironment has improved, it has become increasingly clear that the heterogeneous nature of cancer biology means there is no “one size fits all” approach for treating cancer patients. Meanwhile, omics studies and other techniques now allow us to assess biomarkers, sequence DNA, and examine gene and protein expression profiles. With these insights, the focus has shifted to precision oncology – where treatments can be tailored to each patient’s immune profile to improve efficacy.

As researchers and clinicians delve into precision oncology, they need methods for assessing the potential outcomes of these treatments. One such method is to measure Minimal Residual Disease (MRD). MRD refers to a small number of cancerous cells that may remain in the body following treatment for diseases like acute myeloid leukemia (AML). Given that these cells remain at such low frequencies and typically do not cause patients to exhibit symptoms, it can be difficult to detect them without advanced and highly sensitive techniques.

Research has shown that patients with low or undetectable MRD response have a lower risk of relapse and better survival outcomes compared to those who test positive¹. In order to properly integrate MRD testing into clinical trials, it is critically important to have this testing standardized and comparable across multiple centers of study. In a recent correspondence with the journal Leukemia, Scott et al. discussed a series of recommendations made by an advisory board on AML and MRD².

Building the council

Previously, the European LeukemiaNet (ELN) group established technical and reporting aspects using RT-PCR and flow cytometry staining to assess AML MRD testing. In 2021, the group listed next generation sequencing analysis as an additional validating factor and allowed the use of MRD as a surrogate marker in clinical trials². To further standardize MRD testing, an advisory board comprised of several experts was established to make recommendations on future initiatives to be undertaken.

Recommendations of the board

The board prioritized the development of higher order, primary reference materials (RM), by which other quality control materials can be calibrated. In order to confirm the RMs have been produced to a high standard, they should follow international guidelines set forth by the European Union In Vitro Diagnostics Regulation (EU-IVDR). Subsequently, it would be ideal to develop Research Use Only (RUO) and quality control materials calibrated to RMs.

The board suggested that current manufacturing practices should utilize ISO regulatory systems be used to certify RMs (similar to the ISO:13485 system that oversees Mimix reference standards production). In the future, they advise it would be ideal to follow World Health Organization (WHO) guidelines, which contain recommendations for clinical practice or public health policy. WHO guidelines are developed through a rigorous process in order to provide trustworthy international standards³ and would be particularly helpful in AML MRD testing where RMs are not yet available. Certified RMs for RT-PCR should be cell-based like Revvity’s 100% cell-derived Mimix controls. This helps to remove uncertainty regarding RNA extraction and cDNA synthesis when generating normalized ratio results.

Lastly, for RT-PCR based MRD assays, any values generated should be in reference to the ABL1 gene for standardization. To this point, there are a fraction of labs that utilize other genes like GUSB, B2M, or HMBS, which can conflate data or lead to misinterpretation.

Mimicking genomic complexity

The board agreed standardization of AML MRD is beneficial, but more importantly, that this process should be easy to implement and cost effective. Of the markers to be standardized, particular emphasis was placed on RUNX1::RUNX1T1, CBFB::MYH11 type A, and NPM1 type A testing by RT-PCR. These markers represent some of the most common somatic changes in AML patients.

Revvity provides Mimix™ Reference Standards derived from cell lines in order to maintain genomic complexity and mimic patient material from sample preparation steps to downstream analysis. These standards are produced under ISO:13485 regulations. Suitable for next generation sequencing, droplet-digital, and Real-Time PCR as well as Sanger sequencing, Revvity Mimix reference materials are platform-agnostic for implementation into RUO quality control workflows.

For AML research, Revvity specifically offers Myeloid DNA Reference Standard and Myeloid Cancer Panel cfDNA Reference Standard, the latter of which uses cell-free DNA to support verification of liquid biopsy-based MRD assays. The Myeloid DNA Reference Standard contains 22 variants across 19 genes relevant to myeloid cancer, while the Myeloid Panel cfDNA Reference Standard contains 15 clinically-relevant variants across 14 genes. Both products include ABL1 and NPM1, which were cited as high value targets by the board.

Mimix cell line-derived reference standards can offer users reliable controls with patient-like performance for confirmation of their myeloid sequencing assays. Revvity also provides Myeloid Cancer Panel cfDNA Negative Control Reference Standard. This control can be used to further dilute allelic frequencies, enabling users to challenge their test methodology, which is a critical step in MRD set up.

In order to make advancements in healthcare for conditions like AML, it cannot be understated how important the international standards and guidelines will be to ensure results can be corroborated and compared consistently. Read the full article to learn more about the board’s recommendations and the efforts being made to standardize AML MRD testing.