Using the Power of Flow Cytometry to Measure Minimal Residual Disease (MRD)

Using the Power of Flow Cytometry to Measure Minimal Residual Disease (MRD):      Next Generation Flow Cytometry (NSG) 

Minimal Residual Disease (MRD)  is the name given to the small number of leukemia or cancer cells in the bone marrow (BM)  that remain in a patient during their treatment, or after treatment when the patient is in remission (Figure 1).   MRD is the main cause of relapse in the hematologic cancers, Multiple Myeloma (MM), Lymphoma and Leukemia.   Thus a way to monitor MRD is needed to evaluate efficacy of treatment, prognosis, and identify patients at higher risk of relapse.  An ideal method has to have broad application and high sensitivity, for example, the ability to detect 1 cancer cell in 1 million bone marrow cells sampled (10-6).

 

Figure 1. Minimal Residual Disease is the presence of a very small number of cancer cells that remain after treatment. MRD can be present even in patients who have had a complete response (CR), and is a negative predictor of clinical outcome. Graphic courtesy of Jiseong Kim.

Only in the past 10 years have tests been developed that have this sensitivity.  For example, Allele-Specific oligonucleotide quantitative PCR (ASOqPCR), next generation sequencing (NGS), and conventional 4-8 color flow cytometry methods have been used to monitor MRD in bone marrow in Multiple Myeloma (MM) patients.     Early evaluation showed that conventional “first generation” 4-8 color flow cytometry had broad application (>95% of patients), while the molecular methods (ASOqPCR and NGS) had a more restricted application (50-90% of cases, due to the high numbers of somatic hypermutations, resulting in variable primer annealing/unpredictable amplification).  But, in early assessments, the sensitivity of the first generation conventional flow method had a limit of detection (LOD) of 10-4, and was  less sensitive than the molecular methods (10-4– 10-6), although steady improvement increased the sensitivity of conventional flow LOD to 10-5 (Paiva, 2016).   Flow methods have a number of advantages over PCR-based methods including high speed, clinical applicability, world wide availability, and huge sampling power, but until recently lacked standardization, and had lower sensitivity than NGS.    But, all that changed as newer flow methods evolved to meet that challenge.

In 2017, Flores-Montero et al. reported the development of a Next Generation Flow (NGF) method that standardized reagents (optimized fluorochomes, antibody clones, enhanced tissue preparation to enable sampling up to 10-7 BMC, and automated gating strategy) and further increased the sensitivity of the flow approach for MRD detection in MM.    The authors compared and validated their 2- tube, 8-color, EuroFlow NGF method (Table 1) (median BM volume 1.5 mL) to a conventional 8-color flow method (single 8-color panel, 300 μL of BM).  The study looked at 110 follow-up BM samples from MM patients who had either Complete Response (CR) or Very Good Partial Response (VGPR) to treatment.   The positive rate of MRD detection for NGF was 47 vs 33 for conventional 8-colorflow (p=0.003).  Notably, 25% of patients that were classified as MRD-negative by conventional flow were MRD positive by NGF.   When MRD assessment by NGF was compared head to head with NSG in a subset of samples (n=31), NGF had superior sensitivity (p=0.06).    Overall, the EuroFlow NGF method had increased sensitivity (10-6), and significantly improved the predictive clinical value over conventional Flow and NGS.  Additionally, the authors point out the multiple advantages of NGF over NSG; NGF is faster (< 4 hours), is standardized and reproducible, and does not require patient specific probes like NSG.

Table 1. NGF Optimized Flow Cytometry panels to detect MRD in MM (see Flores-Montero 2017 Supplemental Information for specific clone information).

 

Although we are highlighting the advances in MRD detection in MM in this blog, the prognostic value of MRD has been demonstrated  in other hematologic malignancies, including  chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and acute myeloid leukemia (AML) (Van Dongen, 2015, Theunnisen, 2017).   Other recent advances using flow cytometry include the use of liquid biopsy approaches based on the detection of very small numbers of circulating tumor cells (CTCs) which now enable MRD detection in patients with various malignancies (Pantel, 2019, Bhagwat 2018).  It’s a very exciting time, as Flow cytometry continues to make multiple contributions to improving and advancing oncology diagnostics and prognostics.

FCSL is always looking at innovators in the field of flow cytometry. One of our objectives is to promote innovations and medical applications. EuroFlow is a consortium launched in 2006 by the European Scientific Foundation for Laboratory HematoOncology.  It is an initiative to bring consensus to the immunophenotyping markers used for testing on all blood-borne cancers.    EuroFlow experts focus on developing fast, accurate and very sensitive methods of testing samples with flow cytometry one of these being the MRD assay.

References: 

Bhagwat, N Dulmage, K, Pletecher CH, et al 2018, An integrated flow cytometry-based

platform for isolation and molecular characterization of circulating tumor single cells and

clusters, Nature, Scientific Reports, 8:  5035 1-14.  

Flores-Montero J, Sanoa-Flores L, Paiva, B, Puig N, Garcia-Sanchez O, et al.  2017 Next Generation Flow for highly sensitive and standardized detection of minimal residual disease, Leukemia, 31, 2094-2103.

Moreua, P and Zamangi, E, 2017, MRD in Multiple Myeloma; More Questions than Answers?  Blood Cancer Journal 7:639, 1-4.

Paiva B, Dedena, MT, Puig, N, Arara, P, Vidriales MB, Cordon L et al. 2016, Minimal Residual Disease monitoring and  immune profiling in multiple myeloma in elderly patients, Blood, 127: 3165-3174.

Pantel K, and Alix-Panabieres C, 2019, Liquid biopsy and minimal residual disease — latest advances and implications for cure, Nat Rev Clin Oncol. Review.

Theunnisen P et al., 2017, Standardized flow cytometry for highly sensitive MRD measurements in B-cell acute lymphoblastic leukemia, Blood, 129:347-357.

Van Dongen, JM, Van der Valden, VJH, Burggerman, M, Orfao, A , 2015,  Minimal residual disease diagnostics in acute lymphoblastic leukemia: need for sensitive, fast, and standardized technologies, Blood 125:3996-4009.