Lymphoma is a common hematopoietic malignancy in canines. Current diagnostic techniques to diagnose lymphoma are often invasive and expensive. Additionally, tumor heterogeneity complicates the accurate classification and diagnosis of specific subtypes, hindering the development of targeted therapy and prognostic assessments. We propose a minimally invasive liquid biopsy technique involving blood collection to detect cell-free DNA from tumors using Next-generation sequencing. We hypothesize that identical tumor aberrations can be found in matching plasma and tumor DNA.
Five dogs diagnosed with B-cell lymphoma through flow cytometry or PAAR were enrolled in the study. Samples collected included: (1) blood for plasma (cfDNA), (2) tumor tissue fine-needle aspirates (tumor DNA), and (3) buccal swabs (genomic DNA, germline control). Whole Genome Sequencing was performed using Illumina NovaSeq 6000, and the sequenced output was analyzed with bioinformatics tools to detect somatic variants in plasma and tumor samples.
Our results revealed many shared somatic variants between matched cfDNA and tumor DNA samples, with 1.7-49% of tumor variants also found in corresponding plasma samples. Shared variants constituted only 0.5-9% of all plasma somatic variants. Specific B-cell lymphoma mutations were identified in cfDNA, including MYC, POT1, and TRAF3, alongside other cancer-related genes. Tumor samples showed mutations in genes associated with canine and human B-cell lymphoma. This study suggests that tumor-specific genomic mutations can be detected in plasma, supporting the potential of liquid biopsy as a less invasive diagnostic tool. However, cfDNA may not capture the full genetic heterogeneity of tumors due to low tumor-derived DNA content in limited plasma volumes. / Master of Science / Lymphoma is a type of blood cancer affecting white blood cells. Canine lymphoma is a common neoplasia, with an incidence rate of 20 to 100 cases per 100,000 dogs, making it a significant research focus. Current diagnostic methods are invasive and costly. Additionally, the wide variety of tumor types in lymphoma makes it challenging to determine the exact subtypes, which is crucial for selecting the best treatment approach.
To overcome these challenges, we proposed a less invasive method known as "liquid biopsy". This technique involves taking a blood sample of a dog to find cell-free DNA from tumor cells using Next-Generation Sequencing technologies. We aimed to see if blood DNA could provide the same information as tumor DNA. In our study, we worked with five dogs diagnosed with B-cell lymphoma through traditional methods. We collected blood, tissue from needle biopsies, and buccal swabs from each dog. We then performed DNA extraction and sequencing on these samples.
Our findings showed that 1.7-50% of the mutations in tumor DNA were also detected in matched blood DNA, though these represented only a small fraction of all changes found in blood samples. Additionally, the blood samples also revealed mutations related to canine B-cell lymphoma in genes like MYC, POT1, and TRAF3. In conclusion, our study supports the use of liquid biopsy as a feasible and less invasive method to diagnose lymphoma in dogs. However, they might not show all genetic variations of the tumor due to limited tumor DNA content.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/120910 |
| Date | 12 August 2024 |
| Creators | Vadlamudi, Sai Navya |
| Contributors | Biomedical and Veterinary Sciences, da Silva Serpa, Priscila Beatriz, Xie, Hehuang David, Klahn, Shawna L. |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | ETD, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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