US-20260125768-A1 - METHODS OF USING GIANT CELL NUCLEIC ACID CHARACTERIZATION IN CANCER SCREENING, DIAGNOSTICS, TREATMENT AND RECURRENCE

Abstract

The characterization of nucleic acids obtained from cancer-associated cells circulating in the blood of a subject, and the use of such characterizations in cancer screening, diagnostics, treatment, and recurrence, are disclosed.

Inventors

• Daniel Adams
• Cha-Mei Tang

Assignees

• CREATV MICROTECH, INC.

Dates

Publication Date

20260507

Application Date

20260102

Claims (16)

1. 1 . A method of assaying for cancer-associated molecular changes in giant cells, or giant naked nuclei, or both, said method comprising: subjecting a biological sample obtained from a subject to a size exclusion methodology, wherein cells and naked nuclei measuring 8 microns or larger are collected, wherein the collected cells and naked nuclei include giant cells, or giant naked nuclei, or both, and analyzing the collected cells and/or naked nuclei for cancer-associated molecular changes, thereby analyzing cancer-associated molecular changes in giant cells, or giant naked nuclei, or both.
2. 2 . The method of claim 1 , wherein the cancer-associated molecular changes are mutations, epigenetic modifications, or both.
3. 3 . The method of claim 1 , wherein the collected cells and naked nuclei include one or more of CTCs, naked nuclei of CTCs, CECs, naked nuclei of CECs, EMTs, and naked nuclei of EMTs.
4. 4 . The method of claim 1 , wherein all collected cells and naked nuclei are analyzed for cancer-associated molecular changes.
5. 5 . The method of claim 1 , wherein when cancer-associated molecular changes are identified, further comprising making a prognosis based on the identity of the cancer-associated molecular changes.
6. 6 . The method of claim 1 , wherein when cancer-associated molecular changes are identified, further comprising making a prediction of treatment response based on the identity of the cancer-associated molecular changes.
7. 7 . The method of claim 1 , wherein when cancer-associated molecular changes are identified, further comprising diagnosing the subject as having cancer.
8. 8 . The method of claim 1 , wherein when cancer-associated molecular changes are identified, further comprising diagnosing cancer recurrence in the subject.
9. 9 . A method for determining resistance to a cancer treatment, said method comprising: isolating giant cells, or giant naked nuclei, or both, from a biological sample of a subject, analyzing the giant cells and/or giant naked nuclei to obtain a second set of cancer-associated molecular changes, comparing the second set of cancer-associated molecular changes to a first set of cancer-associated molecular changes obtained from giant cells and/or giant naked nuclei in a similar biological sample obtained from the subject earlier in time, wherein when the identity of the first and second sets of cancer-associated molecular changes are different, the subject is determined to be resistant to the cancer treatment.
10. 10 . The method of claim 9 , wherein the cancer-associated molecular changes are mutations, epigenetic modifications, or both.
11. 11 . The method of claim 9 , wherein the giant cells and/or giant naked nuclei are isolated from the biological sample using one or more of (i) size exclusion methodology, (ii) an analyte capture element, (iii) red blood cell lysis, and (iv) white blood cell depletion.
12. 12 . The method of claim 11 , wherein the size exclusion methodology is a filter that retains cells of 8 microns or larger.
13. 13 . The method of claim 11 , wherein the analyte capture element is an antibody that recognizes cell surface or intracellular markers of the giant cells and/or giant naked nuclei.
14. 14 . The method of claim 9 , wherein the nucleic acids are intact or undegraded nucleic acids.
15. 15 . The method of claim 9 , wherein the analysis is performed on a single giant cell, a group of two or more giant cells, a single giant naked nuclei, group of two or more giant naked nuclei, or a combination thereof.
16. 16 . The method of claim 9 , further comprising assaying for cancer-associated molecular changes in one or more of circulating tumor cells (CTCs), circulating endothelial cells (CECs), and epithelial mesenchymal transition cells (EMTs), wherein said CTCs, CECs, and EMTs are isolated from the biological sample in conjunction with isolating the giant cells and/or giant naked nuclei from the biological sample.

Description

FIELD OF THE INVENTION The present invention generally relates to characterization of nucleic acids obtained from cancer-associated cells circulating in the blood of a subject, and the use of such characterizations in cancer screening, diagnostics, treatment, and recurrence. BACKGROUND OF THE INVENTION Driver mutations in cancer, generally defined as mutations within a gene that confer a selective growth advantage on a cell, and thus promote cancer development, are typically found through analysis of tumor tissue. The analysis for mutations can use many available molecular techniques (i.e. PCR, sequencing, in situ hybridization, etc.). Driver mutations can be difficult to study because of cell heterogeneity and the ability of resistance in subpopulations to exist. In the case of tumor heterogeneity, different cell populations with different mutations exist spatially within tumors. Therefore when a small piece of tumor tissue is used in the analysis of mutations, the mutations may only represent a small portion of the mutations actually present in the total tumor population. In the case of resistant subpopulations, over time and treatments, subpopulations of tumor cells with drug resistant mutations begin to propagate in the tumor area. Because resistance happens temporally, the original tissue tested for mutations may not have the resistant mutations, which appears later in development of the disease. Multiple methods can be used to test for heterogeneity and temporally for resistant tumor subpopulations. First, tumor tissue can be obtained from biopsy or from surgically removed tumor after an operation. The advantage of obtaining tumor tissue is that it provides an adequate number of tumor cells from which to obtain accurate mutational analysis. However, there are a number of potential problems. A tumor might change after tissue is obtained. Obtaining tumor biopsies can be difficult, might not be possible, might be dangerous, costly and painful. Additionally, a re-biopsy may only isolate a single subpopulation while numerous heterogeneous populations may exist. Tissue samples cannot cover all areas of the tumor. Second, circulating tumor DNA (ctDNA) is tumor-derived, fragmented DNA found in the bloodstream that is not associated with cells. ctDNA is just a small fraction of cell-free DNA (cfDNA) found in the blood, where cfDNA accounts for all DNA in the blood and it is not limited to DNA of tumor origin. Currently there are a number of research, development and commercialization efforts utilizing ctDNA for a spectrum of clinical utilities. Blood plasma is used as the source of ctDNA for the tumor analysis. The advantages of ctDNA is that it is possible to obtain plasma in real time. However, there are a number of disadvantages. ctDNA analysis often misidentifies non-malignant background mutations not associated with the tumor and may not identify the rarer tumor mutations from the more common background nucleic acids from normal tissue. As a person ages, mutations not associated with tumors begin occurring in the body naturally that will be mistakenly identified in the ctDNA. Another cause of concern using ctDNA is that the concentration of ctDNA is low compared to DNA from cells from the rest of the body. Early release of a special article by Merker, J D, et al., Circulating Tumor DNA Analysis in Patients with Cancer, American Society of Clinical Oncology and College of American Pathologists Joint Review, 2018 (doi: 10.5858/arpa.2018-0901-SA) discusses the issues of applications utilizing ctDNA. At this time, their conclusion of ctDNA for solid tumors and the analysis of sequence or copy number variants in DNA are as follow: “Some ctDNA assays have demonstrated clinical validity and utility with certain types of advanced cancer; however, there is insufficient evidence of clinical validity and utility for the majority of ctDNA assays in advanced cancer. Evidence shows discordance between the results of ctDNA assays and genotyping tumor specimens and supports tumor tissue genotyping to confirm undetected results from ctDNA tests. There is no evidence of clinical utility and little evidence of clinical validity of ctDNA assays in early-stage cancer, treatment monitoring, or residual disease detection. There is no evidence of clinical validity and clinical utility to suggest that ctDNA assays are useful for cancer screening, outside of a clinical trial.” Third, a commonly recognized potential sources of samples of tumor associated cells are circulating tumor cells (CTCs) in the blood of patients with solid tumors. However, CTCs are able to provide real time tumor samples only if there are sufficient number of CTCs collected. Currently, there is a push for sequencing analysis from single cells by commercial companies and researchers. The advantage of sequencing CTCs is the ability to provide real time mutational analysis. Again, there are also many disadvantages. (i) For the majority of solid tumors, CTCs are