Case Study: DiaCarta – Translational Genomics Powered by Molecular Clamp Technology
1. The challenges that our clients faced
Acute myeloid leukemia (AML) is an aggressive hematopoietic neoplasm characterized by an accumulation of myeloid cells in the bone marrow because of impaired differentiation and proliferation, resulting in hematopoietic insufficiency which is associated with significant mortality and morbidity. The presence of minimal residual disease, that is, small numbers of neoplastic cells which persist after cancer therapy has been shown in independent cohorts to correlate with disease relapse, even in patients with the very limited disease. Minimal residual disease detection is particularly important in individuals treated with myeloablative hematopoietic stem cell transplantation, as often, these patients are younger.
Nucleophosmin 1 (NPM1) is one of the most commonly mutated genes in AML. Activating insertion mutations in exon 12 of NPM1 are frequent and recurrently observed in nearly one-third of all acute myeloid leukemia patients and approximately 60% of patients with normal karyotype. The most common mutation subtype, Type A, is a 4-base pair insertion of TCTG (p.Trp288Cysfs*10, NM_002520.4:c.956_959dup). Type A mutations are frequently seen in adults, representing approximately 75 to 80% of NPM1 mutations in acute myeloid leukemia.
Our client wanted to utilize a molecular test for detection of minimal residual disease (MRD) in acute myeloid leukemia (AML) patients and desired to develop a real-time PCR based molecular test for detection of the most common NPM1 genetic variant for detection of MRD. The client had previously been attempting to develop the real-time qPCR assay but was having problems in achieving the sensitivity that they needed for use in a clinically relevant assay. They had tried many different approached with very little success including allele-specific PCR (ASPCR) and locked nucleic acid (LNA) wild-type blockers with little success.
2. The solution that we provided and the methodology and approach undertook by us
We provided a synthetic xenonucleic acid (XNA) oligomer specific for the clients’ wild-type sequence of their target gene of interest that completely blocked the amplification of wild-type templates in a real-time TaqMan based qPCR cDNA that was reverse transcribed from target RNA from AML patient samples. Genetic variations in cDNA templates containing the target site were selectively amplified in the presence of a large excess of wild-type templates as these were not bound by the XNA oligomer the principle of XNA based PCR clamping (QClampTM) is shown in Fig. 1.
Fig 1. Principle of XNA blocker PCR
Thus only mutant templates containing the 4-base pair insertion mutation found in AML are amplified during the PCR process, and this allowed the client to develop a highly sensitive real-time fluorescent hydrolysis probe (TaqMan) based PCR assay for detection of mutant NPM1 templates in patient samples.
3. The benefits that our clients reaped in terms of advantages and the impact of their ROI
The client was able to establish a rapid, highly sensitive screening assay to monitor AML patients who had received myeloablative hematopoietic stem cell transplantation therapy for a minimal residual disease which could be run on a standard real-time qPCR instrument that was already in place at their facility. This significantly reduced the turnaround time, sample throughput and costs for them for patient monitoring as previously they had been employing the multi-parametric flow cytometric identification of minimal residual disease which is the clinical standard of care for minimal residual disease detection in acute lymphoblastic leukemia and is used commonly for posttreatment monitoring of acute myeloid leukemia.
However, flow cytometry requires detection of leukemia-associated immunophenotypes in the context of regenerative hematopoiesis, as well as complex instrumentation and analysis of high-dimensional data, making it challenging to perform routine or on a standardized basis.
Further, as no universal immunophenotype for relapsed blasts is observed in acute myeloid leukemia, it is necessary to employ a relatively large antibody panel to identify specific markers that can be used to monitor individual patients. Additionally, the posttherapy immunophenotypic drift of leukemic blasts may confound consistent detection of minimal residual disease by flow cytometry, particularly in the context of marrow regeneration.
Considerable time and cost savings and more robust and efficient management of AML patients’ therapy at the client’s institution were achieved.
Next-generation sequencing (NGS) is a powerful tool that has seen a fast increase in clinical labs although only a few NGS tests have been approved by the FDA. However, there have been a lot of debate on if variants from NGS sequencing should be confirmed either by Sanger sequencing, the gold standard, or other techniques such as quantitative PCR, or the combination, or other methods.
Eighty five percent of the lung cancer patients are non-small cell lung cancer (NSCLC) patients. Among this population, patients with exon 19 deletion and L858R mutations respond well to the first (such as erlotinib and gefitinib) and second generation (such as afatinib and dacomitinib) of tyrosine kinase inhibitors (TKIs). However, all the respondents develop resistance after 9 to 14-month period and more than 50% of the resistance cases are due to the single point mutation at exon 20, T790M.
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