Methods and Techniques to Examine Acute Lymphocytic Leukemia on a Molecular Level

By Ava Green | Hingham High School, Hingham, Massachusetts, United States

I. Introduction

With a 72.1% five-year survival rate, Acute Lymphocytic Leukemia was the sixth most common cause of cancer deaths in males in the U.S. from 2013 to 2017. (1) Acute Lymphocytic Leukemia, or ALL is a type of cancer in which stems cells from bone marrow produce too many lymphocytes, which are derived from blood stem cells.2 ALL is caused by a mutation in a single stem cell, which creates many mutated lymphocytes1. Lymphocytes are a type of white blood cell. (1) There are three types of lymphocytes: B lymphocytes, T lymphocytes, and natural killer cells. (2) When a patient has ALL, too many of one of these cell types are produced. Healthy white blood cells, platelets, and red blood cells do not have enough room in the bloodstream with all these extra cancerous cells, which can cause many other health issues. (2) Treatments for Acute Lymphocytic Leukemia include chemotherapy, radiation therapy, and targeted therapy. Chemotherapy uses drugs to kill cancerous cells or stop them from dividing and is injected into the bloodstream or taken by mouth. Additionally, patients may receive a stem cell transplant since chemotherapy can also kill stem cells. Radiation therapy uses X-rays and other forms of radiation to prevent cells from dividing. Targeted therapy is a drug that targets a specific type of cancer cells, and it usually causes less damage than chemotherapy and radiation therapy. (2) These treatments have been improved over the years as the medical field’s understanding of ALL has grown. (2) Some crucial developments in the understanding of ALL include when germline mutations were first associated with ALL in 2009 and in 2012 when a whole genome sequencing study identified mutations in T-Cells and studied how they could be linked to ALL. (3) Questions about ALL that are still under research today include how could a patient’s own immune cells be used to treat their ALL and what are the main causes of ALL. (1, 4).

Keywords: Acute Lymphocytic Leukemia; Germline Mutations in Pediatric ALL; T-Cell ALL Detection; Genetic Testing and Molecular Profiling in ALL Diagnosis  

II. Methods and Techniques

In the experiment “Predisposing germline mutations in high hyperdiploid acute lymphoblastic leukemia in children”, the researchers wanted to examine the many germline mutations suspected of causing Leukemia and determine which one could be a main cause. (5) These researchers hoped to make the genetics of ALL better understood, as ALL was the most common childhood cancer at the time of the study. (5) In order to achieve their goal, the researchers used Sanger sequencing to confirm that the samples they collected from patients with ALL contained a germline mutation. In order to perform Sanger sequencing, the gene being studied has to be PCR amplified. After a sufficient amount of the desired gene is produced through PCR, the template DNA is once again denatured. Primers bind to the gene, and DNA polymerase starts building the new, complementary strand. However, instead of only using dNTPs like in PCR, the DNA polymerase occasionally uses ddNTPs, which are nucleotides without the hydroxyl group that binds to the phosphate of the previous nucleotide. This ends the new DNA strand being made, resulting in many strands of the gene with varying lengths. These samples can be put through gel electrophoresis, which uses size to separate pieces of DNA. This gel can be observed and used to determine which genes are present in the sample. (6) In this experiment, PCR and Sanger sequencing was used to determine which mutations the patients had, and whether they were germline or not. The researchers determined through Sanger sequencing that three of their patients most likely had germline mutations. (5) From this entire lab, the researchers found many rare but predicted germline mutations and identified where these mutations were. This new data could help other scientists figure out where to do further testing on the human genome. The researchers concluded that further testing of the entire genome was needed since they only analyzed the known ALL-related genes, and many genes could have affected the development of ALL. (5) All of this new data and knowledge about the genome and its connection to ALL could aid in understanding ALL and other Leukemias as a whole, and help develop a more effective treatment for ALL patients. In the study titled “Comparison of Techniques for Detecting T-Cell Acute Lymphocytic Leukemia” by Susan L. Melvin, a large group of samples from patients with ALL soon after they were diagnosed were collected and tested using various detection methods. This study aimed to determine which of the tested techniques is the best for detecting ALL, specifically when the T lymphocytes are the cancerous cells. When it is T-Cell ALL, the disease is very aggressive, so improved treatments and faster diagnoses are needed. (7) The methods this study tested are the three assays of the E-Rosette Test, which were incubated at different temperatures and with different erythrocytes. (7) Additionally, the Indirect Immunofluorescence Test was used to determine how effective the other methods were in detecting T-Cell antigens. (7) The Indirect Immunofluorescence Test is when a primary antibody binds to the antigen, and then a secondary antibody flagged with a fluorescence protein binds to that primary antibody. (8) Direct Immunofluorescence is when the fluorescent protein is on the primary antibody, but it is not used as frequently, since Indirect Immunofluorescence is more sensitive and can detect multiple targets within a sample. (8) This technique allows scientists to see where an antigen is present and how much of it is present in a sample. The researchers concluded that the best methods were the rosetting with untreated sheep erythrocytes at 37 degrees C and 4 degrees C, since they gave the most consistent results. (7) They came to this conclusion after they performed the rosette test and then tested those results with the Indirect Immunofluorescence Test. The assays used in this study were already used to type malignant lymphoid diseases, (7) so their new application to ALL helped scientists gain a better understanding of T-Cell ALL and how it could be detected.

III. Conclusion

Both of these studies helped scientists gain a better understanding of ALL, and they both demonstrated real-life applications of different techniques students learn in textbooks. The results of both of these labs made sense, and there were no errors or points of weakness. There seemed to be very high standards in both of these laboratories, so their findings can be trusted. Both tests had realistic results, and there were no huge surprises in the data. In the “Predisposing germline mutations…” lab, there were questions about what other genes could affect ALL, and which ones have not been discovered. At the end of the discussion section, the researchers explained how further testing would have to be done to fully uncover all the information about germline mutations and ALL. (5) In the “Comparison of Techniques…” study, the discussion section touched on the possibility that patients whose stem cells produce fewer T-Cell-associated features will have difficulty being treated than those with the common ALL. This type of data would have to be obtained through further testing and follow-ups from the patients the samples were from. (7) Although Acute Lymphocytic Leukemia does not have a completely clear cause, (1) the past few decades have seen tremendous strides in understanding ALL on a molecular level.

IV. References

1. Cassaday, Ryan D. “Acute Lymphoblastic Leukemia.” Leukemia and Lymphoma Society, 2022. https://www.lls.org/leukemia/acute-lymphoblastic-leukemia.

2. Bethesda. “Adult Acute Lymphoblastic Leukemia Treatment.” National Cancer Institute, November 19, 2021. https://www.cancer.gov/types/leukemia/patient/adult-all-treatment-pdq.

3. Pui, Ching-Hon, and William E Evans. “A 50-Year Journey to Cure Childhood Acute Lymphoblastic Leukemia.” Seminars in hematology, July 1, 2014.

4. “Advances in Leukemia Research.” National Cancer Institute, February 10, 2023. https://www.cancer.gov/types/leukemia/research. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3771494/.

5. Smith, Adam J de, Geneviève Lavoie, Kyle M Walsh, et al.. “Predisposing Germline Mutations in High Hyperdiploid Acute Lymphoblastic Leukemia in Children.” Wiley Online Library, May 13, 2019. https://onlinelibrary.wiley.com/doi/full/10.1002/gcc.22765.

6. “Peer Reviewed Scientific Video Journal - Methods and Protocols.” JoVE. Accessed July 6, 2023. https://app.jove.com/science-education/12020/sanger-sequencing.

7. Melvin, Susan L. “Comparison of Techniques for Detecting T-Cell Acute Lymphocytic Leukemia.” American Society of Hematology, July 1, 1979. https://ashpublications.org/blood/article/54/1/210/161473/Comparison-of-techniques-for-detecting-T-cell.

8. Im, Kyuseok, Sergey Mareninov, M Fernando Palma Diaz, and William H Yong. “An Introduction to Performing Immunofluorescence Staining.” PubMed Central, January 1, 2020. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918834/.

The Scientific Review Article published, “Methods and Techniques to Examine Acute Lymphocytic Leukemia on a Molecular Level,” was received on July 17, 2023, and was reviewed and accepted on July 19, 2023. To contact editors and reviewers please click here.