Sanger Sequencing and Fragment Analysis Help Improve Clinical Outcomes of Acute Lymphoblastic Leukemia (ALL) in Pediatric Patients

Article by Cleta D’sa and Caroline McMahon

Great Ormond Street Hospital for Children NHS Foundation Trust in London is England’s leading center for treating sick children and is closely associated with University College London, Institute of Child Health (UCL, ICH). Stuart Adams, PhD, is the joint Lead Health Care Scientist for the Trust as well as the Principal Clinical Scientist in the Specialist Integrated Haematology and Malignancy Diagnostic Service (SIHMDS) Molecular Department of Haematology. Dr Adams runs a clinical service, and also has an honorary contract with UCL, where he carries out translational research in the area of hematopoietic stem cell transplants (HSCT), gene therapy (GT) and immune reconstitution.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children, with around 300 new cases per year in the UK. As head of clinical lab service, Adams and his team test pediatric patients who have been identified as having either B-cell or T-cell ALL [1,2]. Because these diseases are aggressive and progress quickly, obtaining test results fast is key to helping patients receive the right therapies that may improve clinical outcomes. Currently, all diagnostic and some relapse samples need Sanger sequencing as part of their workup. Out of the other estimated 6,000 to 7,000 samples tested each year at the SIHMDS lab, about three-quarters are tested using Sanger sequencing (monogenic inborn errors of the immune system) [3] or fragment analysis (post HSCT or GT) [4,5,6] for clinical routine diagnostics.

Capillary electrophoresis: Rapid analysis enables faster decisions

Capillary electrophoresis (CE) is a separation technique in which charged species are separated based on charge and size, and by different rates of migration in an electric field. Offering an effective and fast approach for the separation and quantification of a large variety of therapeutic substances in biological samples, CE can be used to assess tissue well-being and for monitoring success of treatment. CE is highly automated, and as such allows for fast turnaround time. CE is a powerful technique that allows for the detection of a target sequence using Sanger sequencing or fragment analysis.

Sanger sequencing helps to effectively monitor and predict treatment

The SIHMDS lab uses Sanger sequencing for DNA mutation testing on individual genes. Although much work has moved over to the NGS platform, Sanger sequencing is needed to identify gene mutations in a timely fashion to enable gene therapy for inborn errors of the immune system and to ensure a patient is placed on the appropriate treatment pathway. Sanger sequencing is also used for minimal residual disease (MRD) testing to identify clonal rearrangements of the T-cell receptor genes or the B-cell IGH gene. This personalized medicine approach means that every child has their own assay designed based on the unique specific clonal rearrangement associated with their leukemia. A Sanger sequencing result is therefore required before a child is eligible for treatment.

According to Adams, these personalized Sanger sequencing MRD clonality tests have “dramatically improved outcomes compared to before we developed these methods and before these trials were running.”

Adams explains: “[Before these methods] every child got treated with the same dosing treatment of chemotherapy. This meant that many of the children were over-treated as they did not have high-risk disease. Most children are low-risk, so the first round of chemotherapy clears disease and no more chemotherapy is needed. They were all getting more treatment with all the secondary risk. Now we stop treatment completely or much earlier based on the results.” Sanger sequencing testing is helping to effectively design patient-specific assays used downstream to monitor and predict treatment.

Fragment analysis rapid result turnaround leads to informed clinical decisions

Some children with relapse or refractory leukemia or with inborn errors of the immune system receive HSCT. Fragment analysis is used for chimerism testing using short tandem repeats (STR) PCR multiplexing with different dyes to track chimerism levels in patients who have had HSCT [6]. The lab had been using restriction fragment led polymorphisms (RFLP), which required a lot of DNA and wasn’t suitable for working on fractionated samples that were sorted into different T-cell or B-cell populations. This process was slow and clunky, with results that were not fully quantitative. “In every way, fragment analysis has revolutionized the way we can provide results,” says Adams. “We have [a] much better understanding now on the meaning of a lot of children’s results, and how long-term recovery and reconstitution is likely to be, which we didn’t know 10 years ago.” The new analysis only requires 10ng of DNA, which means a lot less blood is needed – this is essential when dealing with small children or babies. In addition, turnaround time can be within a single day.

“A big advantage of using fragment analysis is the fact that we can run many samples in one go and we can turn around results really quickly, which is needed. We do try to turn results around in 48 hours as standard, but within 24 hours in urgent cases,” Adams says. Rapid result turnaround is imperative to making effective and informed clinical decisions when it comes to determining whether HSCT is successful, or if patience is the necessary course of action because cells are just taking a little longer to recover. If a patient has completely lost their graft, the team must quickly plan a second transplant as soon as possible because the patient has been left without a functioning immune system. Similarly, they can predict the graft relapse. When monitoring a patient after HSCT, they can see if a patient is gradually losing their graft and can rapidly get that result to the clinical team to withdraw immune suppression to give that graft the chance to flourish. Fragment analysis is also used for research purposes permitting better understanding of immune recovery following treatment [7,8].

Outpatient clinics take place only on Wednesdays; therefore, there is a large volume of patients being tested on a single day. Not only does rapid turnaround time help with clinical decision-making, it also enables the team to test the large patient volumes. Being able to prioritize a large volume of results means the team is quickly able to bring relief to parents of patients whose results are good and who have been in agony for up to a month waiting to hear the good news that their child’s HSCT has worked.

While he’s a health care scientist by day, Adams also applies his strong research skills at night to a very different craft: beer brewing. After hearing a friend’s disgruntled feedback about the lack of quality beer in his country and then visiting and discovering that the beer indeed tasted like “liquid cardboard,” Adams decided it was time to experiment with craft beer brewing. While not yet an expert, Adams is already seeing and tasting some positive results with his recipes. Always striving to improve upon results, Adams is a constant scientist – in and out of the lab.

References

1. Wright, G., Watt, E., Inglott, S., Brooks, T., Bartram, J., and Adams, S. (2019). Clinical benefit of a high throughput sequencing approach for MRD in acute lymphoblastic leukemia. Pediatric Blood & Cancer 66(8) e27787.
2. Bartram, J., Goulden, N., Wright, G., Adams, S., Brooks, T., Edwards, D., Inglott, S., Yousafzai, Y., Hubank, M., and Halsey, C. (2018). High throughput sequencing in acute lymphoblastic leukemia reveals clonal architecture of central nervous system and bone marrow compartments. Haematologica 103(3):e110-114.
3. Adams, S.P., Wilson, M., Harb, E., Fairbanks, L., Xu-Bayford, J., Brown, L., Kearney, L., Madkaikar, M., and Gaspar, H.B. (2015). Spectrum of mutations in a cohort of UK patients with ADA deficient SCID: segregation of genotypes with specific ethnicities. Clinical Immunology 161:174-179.
4. Elfeky, R., Shah, R., Unni, M., Rao, K., Chiesa, R., Amrolia, P., Worth, A., Flood, T., Abinun, M., Nademi, Z., Hambleton, S., Cant, A., Gilmour, K., Adams, S., Ahsan, G., Barge, D., Gennery, A., Qasim, W., Slatter, M., and Veys, P. (2019). New graft manipulation strategies improved outcome of mismatched stem cell transplantation in children with primary immunodeficiencies. Journal of Allergy and Clinical Immunology 144(1):280-293.
5. Elfeky, R., Silva, J., Chiesa, R., Rao, K., Amrolia, P., Lucchini, G., Gilmour, K., Adams, S., Bibi, S., Worth, A., Thrasher,A., Qasim, W., and Veys, P. (2018). 100% survival after transplantation of 34 Wiskott Aldrich Syndrome patients over 20 years. Journal of Allergy and Clinical Immunology 142(5):1654-1656.
6. Kricke, S., Mhaldien, S., Fernandes, R., Villanueva, C., Shaw, A., Veys, P., and Adams, S. (2018). Chimerism analysis in the paediatric setting – Direct PCR from bone marrow, whole blood and cell fractions. Journal of Molecular Diagnostics 20(3):381-388.
7. Gkazi, A., Margetts, B., Attenborough, T., Mhaldien, S., Standing, J., Oakes, T., Heather, J., Booth, J., Pasquet, M., Chiesa, R., Veys, P., Klein, N., Chain, B., Callard, R., and Adams, S.P. (2018). Clinical T cell receptor repertoire deep sequencing and analysis: An application to monitor immune reconstitution following cord blood transplantation. Frontiers in Immunology 22(9):2547.
8. Giardino, G., Radwan, N., Koletsi, P., Adams, S., Ip, W., Worth, A., Jones, A., Meyer-Parsonson, I., Gaspar, H.B., Gilmour, K., Davies, E.G., and Ladomenou, F. (2019). Clinical and immunological features in a cohort of patients with partial DiGeorge syndrome followed at a single centre. Blood 133(24):2586-2596.