MbShea@PWP@English@UMCP

Abstract:
This laboratory manual documents a Sanger sequencing assay and helpful databases used to detect the FLT3 F691L mutation and classify its clinical significance to promote personalized therapy in cancer patients. The FLT3 F691L mutation has been identified in acute myeloid leukemia (AML) and been shown in clinical studies to confer resistance to gilteritinib, a recently FDA approved FLT3 inhibitor. Mutations that confer resistance to certain drug treatments interfere with the ability of cancer patients to respond well to treatments and/or increase their chance of relapsing. The assay portion of this manual includes the laboratory procedures necessary to determine if a sample has the FLT3 F691L mutation. The procedures detailed include cell-counting, running an IC50 assay, and Sanger sequencing. The analysis tools, or database, portion of this manual demonstrates the advantages of three human genetic databases in terms of analyzing the clinical significance or contribution of the mutation to a disease. VarSome provides pathogenic prediction scores for determining the potential pathogenicity of a variant; COSMIC documents the number of patient samples reported with a mutation and the disease linked to the mutation; gnomAD calculates the frequency of a specific mutation in the general population and for individual ethnic groups. Future work will include validating this Sanger sequencing assay in a clinical lab for patient samples, and this laboratory manual can be adapted to fit other mutations and/or other cancers.

Word Count: 228

Reader’s profile:
Since this is a lab manual that other interns or beginner researchers can use as a guide, the difficult-to-reach-reader would be an incoming intern or beginner researcher lacking confidence in the lab setting, whether it be in carrying out laboratory procedure or conducting/understanding research and databases.

Reader’s response:
Although this manual presents a lot of information in one sitting, I appreciated the easy-to-follow break down of the procedure and database systems. There was a lot of detail in the background section that went over my head, but the key points that I needed to know for the rest of the paper were made clear. The procedure appears very complex and intimidating but the picture diagraming the order of dilutions really helps simplify the IC50 procedure. In the description of the databases, the links were provided and the author took me step-by-step through where I needed to click and what sections of the database are most meaningful in this context. The inclusion of real data and database queries helped me see how the data from the procedure and information from the databases can be interpreted and analyzed. I am provided a greater sense of confidence by having this document in front of me while in lab.

C, perhaps a brief cover note can address this? That over time in the lab, the background material will become clearer? That this material gives a vocabulary with which to ask questions. Perhaps that is a way to address this.

Do you define Sanger sequencing? Might be helpful for students to know that this is am important innovation in sequencing. We keep building more powerful ways to sequence.

Great plan for your real project. Also, I love that you are helping address the suffering of this cancer.