Techniques for measuring minimal residual disease in leukemia Dr. Nibras Saleem Al-Ammar

Minimal residual disease (MRD) Refers to leukemic cells which have survived in the blood of cancer patients during or after treatment.

MRD Diagnosis The diagnosis of Minimal Residual Disease is far from straightforward. The number of harmful cells which remain following treatment by chemotherapy for example, is often exceptionally small and therefore very difficult to detect.

Microscopic examination: ■ This is still used to detect presence of cancerous cells in tissue samples. ■ However, in MRD, this method was simply not accurate enough and not reliable because: 1. the number of potentially leukemic cells is so small. 2. leukemic cells look virtually identical to normal, healthy, immature WBC

DNA - based tests These tests were designed to detect a leukemic specific DNA sequence and use a chain reaction technique to generate cells. If the resulting cells were cancerous, MRD was successfully diagnosed.

■ The markers used for DNA-based testing are often chromosomal translocations. ■ Other genes utilized for MRD detection include: microsatellites immunoglobulin T cell receptor.

RNA- based tests ►RNA transcription is a key element in the process of cell division and more suitable for testing than DNA in some circumstances. ►These tests are very sensitive and DNA is a fragile molecule (RNA even more so) requiring careful handling during the testing process.

Immunological tests Another option which came about for pathologists to identify the level of MRD. Detecting unusual groups of cell-surface proteins which can be an indicator of MRD.

■ These proteins can be stained with fluorescent dye labeled antibodies and detected using Flow Cytometry. ■ The limit of detection of immunological tests is generally about 1 in 10,000 cells and cannot be used on leukemia that don’t have an identifiable and stable leukemic phenotype.

Next-Generation Sequencing: Some new techniques use Next-Generation Sequencing to detect MRD. ■ Detect DNA mutations found uniquely in cancer cells in cell-free plasma of a patient’s blood.

■ Chronix, Biomedical has labs at the Goettingen University in Germany ■ Chronix, Biomedical has labs at the Goettingen University in Germany. Their minimal residual disease detection test has achieved 100% sensitivity, whereas close competitors have only reached about 70% to 80%.

■ Chronix has discovered multi-cancer biomarkers that relate to the earliest events in genomic instability and appear to be present in most major cancers regardless of the cell of origin.

■ These biomarkers will be the basis for detecting the real-time presence in blood of specific cancer DNA. ■ Also the Company has focused on the use of its technology as a companion diagnostic in breast cancer.

■ The protocol using next-generation sequencing technology will determine a patient’s personal cancer biomarkers.

■ The identity of these personal biomarkers will determine in real-time measurement with cost-effective PCR tests whether the cancer treatment was successful in eliminating the cancer and, in turn, whether or not further treatment is necessary.