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Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts
Chapters
Summary July 25th, 2020
Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients’ tumors into mice and testing responses to corresponding drugs.
Transcript
The protocol described here is aimed to demonstrate an algorithm for testing treatment efficacy using in vivo cancer models. The protocol consists of a combination of several techniques. Whole genome bio-sequencing of human tumor specimens is used to identify genomic alterations.
These include both gene rearrangements and gene copy number changes. So the analysis of identified alterations is performed in order to select potentially druggable changes. The drugs selected based on genomic analysis are then used for in vivo treatment of corresponding tumors grown in immunocompromised mice.
The developed algorithm represents a promising approach to aid treatment decisions for the care of cancer patients. Use the Panda tool or an analogous software to identify targetable alterations. We can list the genes which were identified by microsequencing resulted as a simple tab delineated file using standard accepted gene symbols.
Add the pound sign to the headerline of the list to ensure that the table header is transferred to the pathway level U of the software. Upload the file by clicking on the corresponding navigation tab. Assign a single icon to represent the underlying data by clicking on the icon of choice, and then clicking on the finalize tab.
Once then a patient files are uploaded, preview the page to identify a column that displays the number of annotated genes per pathway. This is the last column on the right. Use a pathway filter in the upper left of the main window to restrict the number of pathways displayed to the ones that contain the genes of interest.
To identify pathways that have more genes annotated than would be expected by chance, use a function located under the enrichment tab. A column then is added to the main table that shows corresponding p-value from a Fisher's exact test. Select the database to display potential druggable genes from preset annotation by checking an appropriate icon on the left of the main window.
To select a pathway for visualization, click on its name displayed in the pathway viewer page. Icons representing each annotation set are displayed next to the associated gene. Clicking on any gene in the pathway will open the corresponding gene cards page.
Select the pathways that showed annotated genes of interest and hits for potential drugs for further analysis. Perform the tissue work in a laminar flow hood to maintain sterile conditions. Lay the tumor tissue in a dish containing cold PBS or tissue culture media, such as RPMI, DMEM, containing antibiotics.
Identify and isolate viable tumor material from adjacent normal and necrotic tissue with the help from a pathologist. Use sterile forceps and scalpel to remove necrotic material pointed out by a pathologist. To perform subcutaneous engraftment, cut the tumor tissue with sterile forceps, scalpel or surgical scissors into small fragments, roughly 2 x 2 x 2 millimeters in size.
Transfer the fragmented tissue to a pre-chilled petri dish on ice. Let cold matrigel into the dish with the fragmented tissue, approximately 200 microliters per 10 pieces of tissue. Mix well and let the tissue fragments soak in the matrigel for 10 minutes.
Use sterile surgical scissors and forceps to make a 5-10 millimeters vertical skin incision on both flanks of a mouse. Insert straight forceps gently into the subcutaneous space to create a pocket large enough for a tumor fragment to be placed under the fat pad. Use sterile straight forceps to insert tumor fragments in the previously prepared pocket in each of five mice.
Close the skin incision using tissue glue. After implantation, to inhibit lymphocyte proliferation, inject each mouse with 100 microliters of rituximab. Prepare the mouse for oral gavage by pinching the skin of the back and bending it backward so that the head and lips of the mouse are immobilized.
Insert the gavage probe down the back of the throat of the mouse until the probe reaches the esophagus. Make sure that the probe is not inserted too far as the lungs of the mouse may perforate causing death. The representative genome float illustrates the landscape of genomic changes in one tumor.
Typical for high-grade serosubtype tumors, multiple gains blue lines and losses red lines, were identified indicating high levels of genomic instability. The top trend alteration for therapeutic intervention in the OC101 tumor was an amplification at chromosome 17, involving ERBB2, a gene which codes for HER2 receptor. To validate the results on the DNA level, several sets of specific primers were designed for the edges of the amplified region.
And PCR was carried out. No amplification product was absorbed when control human DNA was used. Specific bands were identified for OC101 tumor DNA.
In another variant tumor, T14, numerous DNA gains were observed. Those included AKT2 in RICTOR genes. Validation carried out on the protein level, using immunoblotting showed high levels of AKT in RICTOR.
A significant reduction in the tumor burden was observed in the chemotherapy treated group by the end of week six. There was an extra benefit over chemotherapy alone in the groups which received a combination treatment. Tumor tissue was collected for molecular analysis of treatment response at the end of the treatment trial.
Total and phosphorylated levels of S6, AKT, and mTOR were determined using immunoblotting. Comparison of the levels of these proteins in untreated, chemotherapy-treated, and treated with AKT or mTOR inhibitor showed a marked decrease for the latter two. The presented approach is very useful to conduct the clinical trial in PDX models.
It takes advantage of molecular characteristics of the tumor as obtained by genomic profiling to determine the best choice of drugs for testing.
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