EP-4737594-A2 - DETECTION OF TUMOR-DERIVED DNA IN CEREBROSPINAL FLUID

Abstract

As cell-free DNA from brain and spinal cord tumors cannot usually be detected in the blood, we assessed the cerebrospinal fluid (CSF) that bathes the CNS for tumor DNA, here termed CSF-tDNA. The results suggest that CSF-tDNA could be useful for the management of patients with primary tumors of the brain or spinal cord.

Inventors

• BETTEGOWDA, CHETAN
• KINZLER, KENNETH W.
• VOGELSTEIN, BERT
• WANG, Yuxuan
• DIAZ, LUIS
• PAPADOPOULOS, NICKOLAS

Assignees

• The Johns Hopkins University

Dates

Publication Date

20260506

Application Date

20160712

Claims (15)

1. A method comprising: assaying nucleic acids in a Cerebral Spinal Fluid (CSF) sample from a human with a primary brain tumor for one or more mutations in the nucleic acids, wherein the nucleic acids are tumor-specific DNA shed from said primary brain tumor, wherein the mutations are present in the primary brain tumor but not in normal tissues of the human, and wherein the primary brain tumor has been determined by imaging to abut a cortical surface, ventricle, or basal cistern.
2. The method of claim 1 further comprising prior to the step of assaying nucleic acids in the CSF the step of assaying nucleic acids in the primary brain tumor tissue and determining the one or more mutations.
3. The method of claim 2 further comprising the step of assaying nucleic acids in a normal tissue of the human and determining absence of the one or more mutations.
4. The method of any one of claims 1-3 wherein the assaying determined one or more mutations in the nucleic acids in the sample.
5. The method of any one of claims 1-4 wherein the human has previously been subjected to surgical removal of the primary brain tumor.
6. The method of any one of claims 2-5 wherein the step of assaying the nucleic acids in the primary brain tumor tissue is performed using genome-wide screening.
7. The method of any one of claims 2-5 wherein the step of assaying the nucleic acids in the brain tumor tissue is performed using exome-wide screening.
8. The method of any one of claims 1-7 wherein the step of assaying employs a specific probe or specific primer to detect the one or more mutations, wherein the specific probe or specific primer hybridizes at or within 200 nucleotides of the mutation.
9. The method of any one of claims 1-8 wherein the primary brain tumor is a glioblastoma, a medulloblastoma, an anaplastic astrocytoma, or an ependymoma.
10. The method of any one of claims 1-9 wherein the CSF sample is from an intracranial space, a lumbar puncture, an implanted reservoir, or a cisternal puncture.
11. The method of any one of claims 1-10 wherein nested amplification reactions are used to detect the one or more mutations.
12. The method of any one of claims 1-11 wherein the mutation is selected from the group consisting of IDH1, TP53, TERT promoter, PIK3R1, PTEN, ZNF513, CLCA3P, ANKS3, HIST1H3C, TTC16, MARS, RGS 12, CC2D2A, CDH5, KDM6A, NF2, CASR, COL6A1, ITGA11, AQR, ADCK2, MTMR4, TENM2, and PTCH1.
13. The method of any one of claims 1-12 wherein the primary brain tumor has been determined by imaging to abut a ventricle.
14. The method of any one of claims 1-12 wherein the primary brain tumor has been determined by imaging to abut a basal cistern.
15. The method of any one of claims 1-14 further comprising the step of imaging the primary brain tumor to determine its location.

Description

This invention was made with government support under grants CA43460 and NS70024 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD OF THE INVENTION This invention is related to the area of nucleic acid assays. In particular, it relates to assays of small amounts of nucleic acid in body fluids. BACKGROUND OF THE INVENTION Approximately 25,000 individuals each year are diagnosed with a malignant brain or spinal cord tumor in the United States and more than half of these patients will die from their disease (1). While there are a number of different subtypes of primary central nervous system (CNS) cancers, nearly all are treated with maximal safe surgical resection followed by radiation and, in some cases, chemotherapy. Given the lack of clinically available biomarkers for CNS malignancies, the conventional method for disease monitoring in these patients is radiographic, using either computed tomography (CT) or magnetic resonance imaging (MRI) (2). Unfortunately, anatomic changes detected by these imaging modalities are often non-specific and slow to change even in the face of progressing or regressing disease. Moreover, it can be difficult to discriminate between treatment effect and cancer growth with imaging alone (3). Patients must, therefore, have additional surgeries for definitive tissue diagnosis or inappropriately wait for radiographic findings to change as their disease progresses. As a result, there is a great need for more sensitive and specific tumor biomarkers in neuro-oncology. The recent success of detecting circulating tumor cells (CTC's) in the peripheral blood of glioblastoma patients represents an important step towards this goal, with reported sensitivities between 21% and 39% (4-6). Circulating tumor-derived DNA (ctDNA) is found in the plasma of patients with most forms of malignancies (7-11). However, brain tumors, including high-grade gliomas and medulloblastomas, are an exception, with only a minority giving rise to detectable levels of ctDNA, perhaps because of the blood-brain barrier (8). There is a continuing need in the art to develop sensitive assays for difficult-to-detect analytes. SUMMARY OF THE INVENTION According to one aspect of the invention a method is provided. Nucleic acids in a Cerebral Spinal Fluid (CSF) sample from a human with a central nervous system (CNS) cancer are assayed for one or more mutations in the nucleic acids. The mutations are present in the cancer but not in normal tissues of the human. Another aspect of the invention is a method in which nucleic acids in a CNS cancer tissue of a human are assayed, and one or more mutations are determined. Nucleic acids in a normal tissue of the human are also assayed, and absence of the one or more mutations is determined. Nucleic acids in a Cerebral Spinal Fluid (CSF) sample from the human are assayed for the one or more mutations in the sample using a specific probe or specific primer to detect the one or more mutations. The specific probe or specific primer hybridizes at or within 200 nucleotides of the mutation. These and other embodiments which will be apparent to those of skill in the art upon reading the specification provide the art with BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1. Schematic showing the shedding of CSF-tDNA from Central Nervous System (CNS) malignancies. Tumor cells from primary brain and spinal cord tumors shed DNA into the cerebrospinal fluid (CSF) that bathes the CNS. DNA purified from the CSF is analyzed for tumor-specific mutations.Figs. 2A-2B. Representative MR images. (Fig. 2A) Example of tumor (red arrow) abutting a CSF space is shown. (Fig. 2B) Example of tumor not in contact with a CSF space is shown. Corresponding T2 images are provided for easier visualization of CSF.Fig. 3 (Table 1). Patient DemographicsFig. 4 (Table 2). Mutations detected in the CSF and tumor of each patientFig. 5A-5B (Tables 3A-3B). Fig. 5A. Associations between clinical characteristics and levels of CSF-tDNA. Fig. 5B. Associations between clinical characteristic and levels of CSF-tDNA.Fig. 6 (Table 4). Detection of CSF-tDNA using whole -exome sequencing (WWS)Fig. 7 (Table S1). Primers used for Mutations Detection. Forward primers (SEQ ID NO: 1-35). Reverse Primers (SEQ ID NO: 36-70).Fig. 8 (Table S1). Mutations identified in whole-exome sequencing A sequence listing is included as part of this application. DETAILED DESCRIPTION OF THE INVENTION The inventors have developed a way to avoid invasive surgical procedures to determine disease status and/or to avoid treatment delays when radiographic testing fails to show disease progression. Detection of tumor-specific DNA shed from primary CNS tumors into the surrounding cerebrospinal fluid (CSF-tDNA), can serve as a sensitive and exquisitely specific marker for quantifying tumor burden without invasive biopsies. CNS tumors which can be assessed include those of the brain and spinal cord. As discussed below