Periodic Reporting for period 1 - BASE6 (Targeted Microarrays for 5-hydroxymethylcytosine-based Diagnosis of Hematological Malignancies)
Periodo di rendicontazione: 2022-12-01 al 2024-05-31
5-Hydroxymethylcytosine (5hmC) is a critical marker in the study of dynamic epigenetic shifts, marking an initial phase in the DNA demethylation process. Its altered distribution in various cancers earmarks it as a valuable biomarker for disease detection and monitoring. The slow integration of 5hmC as a biomarker is primarily due to the high costs and complexity of current analysis methods, which are significant hurdles for its broad application. The lack of a method that can accurately and affordably map 5hmC in tumor or blood samples—where it has the most significant potential for clinical use yet presents the lowest levels—intensifies this challenge.
Our project is designed to overcome this obstacle by developing a specialized DNA microarray for 5hmC analysis. Utilizing a patented process for the direct fluorescent labeling of 5hmC in conjunction with commercially available microarrays, our goal is to generate detailed maps of 5hmC distribution in both healthy and disease states.
The initial phase of our experiment utilized a high-content custom microarray to investigate 5hmC at ~60,000 genomic sites with a potential epigenetic variation. The presence of 5hmC in a particular DNA sequence is reflected by varying fluorescence intensities across the array’s spots. The disparity in 5hmC distribution between healthy and diseased states acts as a biomarker for illness, and only the differential 5hmC loci will be featured on a condensed microarray, enabling disease identification at a significantly lower cost. Due to budgetary constraints, our proof of concept was limited to 30 samples. We initially planned to examine 10 samples each from Acute Myeloid Leukemia (AML), Follicular Lymphoma (FL), and healthy controls. However, upon commencing the experiment, we realized that a sample size of 10 for each condition would be insufficient and thus decided to concentrate on comparing AML with healthy controls, analyzing 15 samples from each group.
The anticipated impact of our project is not confined to scientific progress alone. By creating an economical platform for 5hmC analysis, we aim to democratize advanced diagnostic methods. This innovation has the potential to refine biomarker identification, inform personalized treatment choices, and ultimately enhance patient care. Our project is committed to bridging the gap between pioneering research and clinical practice, offering a tangible solution with significant implications for the future of healthcare.
Our project is designed to overcome this obstacle by developing a specialized DNA microarray for 5hmC analysis. Utilizing a patented process for the direct fluorescent labeling of 5hmC in conjunction with commercially available microarrays, our goal is to generate detailed maps of 5hmC distribution in both healthy and disease states.
The initial phase of our experiment utilized a high-content custom microarray to investigate 5hmC at ~60,000 genomic sites with a potential epigenetic variation. The presence of 5hmC in a particular DNA sequence is reflected by varying fluorescence intensities across the array’s spots. The disparity in 5hmC distribution between healthy and diseased states acts as a biomarker for illness, and only the differential 5hmC loci will be featured on a condensed microarray, enabling disease identification at a significantly lower cost. Due to budgetary constraints, our proof of concept was limited to 30 samples. We initially planned to examine 10 samples each from Acute Myeloid Leukemia (AML), Follicular Lymphoma (FL), and healthy controls. However, upon commencing the experiment, we realized that a sample size of 10 for each condition would be insufficient and thus decided to concentrate on comparing AML with healthy controls, analyzing 15 samples from each group.
The anticipated impact of our project is not confined to scientific progress alone. By creating an economical platform for 5hmC analysis, we aim to democratize advanced diagnostic methods. This innovation has the potential to refine biomarker identification, inform personalized treatment choices, and ultimately enhance patient care. Our project is committed to bridging the gap between pioneering research and clinical practice, offering a tangible solution with significant implications for the future of healthcare.
In the technical and scientific narrative of our project, we commenced with the collection of peripheral blood samples from the Hematology department at Tel Aviv Medical Center. The cohort consisted of 15 individuals with a new diagnosis of AML and 15 healthy control volunteers. Each participant provided informed consent, approved by the Helsinki Committee. We proceeded to extract genomic DNA from these samples and applied fluorescent labeling to 5hmC, adhering to our established epigenetic labeling protocol. The labeled DNA was then subjected to hybridization on a high-content microarray. Upon completion of the hybridization process, we conducted a scan of the array to ascertain the fluorescence intensities at each spot, which correspond to distinct genomic sequences and loci. These intensities are indicative of the 5hmC concentration at the respective genomic loci within the sample DNA.
The data analysis phase entailed identifying those spots on the array where fluorescence intensities varied between AML and healthy samples. We then evaluated whether these differential spots could serve as reliable classifiers for AML. Our analysis identified ~3,500 biomarkers with differential hydroxymethylation between AML patients and control samples. Validation tests based on these biomarkers yielded an accurate classification of 93% of AML cases and 100% of controls, translating to 100% specificity and 93% sensitivity.
Advancing to the second phase, we undertook a comprehensive literature review to gather existing data on genomic locations that exhibit epigenetic differences between AML and healthy controls. Integrating the differential markers identified from our high-content array with potential markers derived from the literature, we designed a smaller-scale custom microarray. This array is specifically tailored to include genomic locations pertinent to AML and has successfully reduced experimental costs threefold, showcasing the efficacy of our targeted approach.
The forthcoming step involves employing this streamlined microarray as the new discovery tool to retest AML and healthy samples. Our objective is to pinpoint the minimal number of differential biomarkers necessary to discriminate between the sample types. The reduced biomarker count on this new array facilitates the loading of more samples per slide, thereby accelerating the process and diminishing expenses. This strategic refinement is expected to further the project’s goal of enhancing diagnostic efficiency and cost-effectiveness.
The data analysis phase entailed identifying those spots on the array where fluorescence intensities varied between AML and healthy samples. We then evaluated whether these differential spots could serve as reliable classifiers for AML. Our analysis identified ~3,500 biomarkers with differential hydroxymethylation between AML patients and control samples. Validation tests based on these biomarkers yielded an accurate classification of 93% of AML cases and 100% of controls, translating to 100% specificity and 93% sensitivity.
Advancing to the second phase, we undertook a comprehensive literature review to gather existing data on genomic locations that exhibit epigenetic differences between AML and healthy controls. Integrating the differential markers identified from our high-content array with potential markers derived from the literature, we designed a smaller-scale custom microarray. This array is specifically tailored to include genomic locations pertinent to AML and has successfully reduced experimental costs threefold, showcasing the efficacy of our targeted approach.
The forthcoming step involves employing this streamlined microarray as the new discovery tool to retest AML and healthy samples. Our objective is to pinpoint the minimal number of differential biomarkers necessary to discriminate between the sample types. The reduced biomarker count on this new array facilitates the loading of more samples per slide, thereby accelerating the process and diminishing expenses. This strategic refinement is expected to further the project’s goal of enhancing diagnostic efficiency and cost-effectiveness.
This initiative establishes a groundbreaking approach for the early detection of diseases by employing specialized microarrays that scrutinize the 5hmC content within genomic DNA. It introduces an innovative biochemical method that achieves high sensitivity while maintaining affordability, presenting a promising avenue for clinical application.
The technology has garnered commercial attention, prompting plans for a more extensive project aimed at further validating and de-risking the concept.
The technology has garnered commercial attention, prompting plans for a more extensive project aimed at further validating and de-risking the concept.