EP4684036A2 - Systems, compositions, and methods for cancer dna detection - Google Patents

Abstract

The present disclosure provides methods, compositions, and systems related to the detection of cancer DNA. In particular, the present disclosure provides methods, compositions, and systems related to the electrochemical detection and/or quantification of cancer DNA in biological samples for monitoring of disease (e.g., cancer) status.

Description

SYSTEMS, COMPOSITIONS, AND METHODS FOR CANCER DNA DETECTION

FIELD

[0001] The present disclosure provides methods, compositions, and systems related to the detection of cancer DNA. In particular, the present disclosure provides methods, compositions, and systems related to the electrochemical detection and/or quantification of cancer DNA in biological samples for diagnosis, detection, prognosis, monitoring, and evaluation of disease (e.g., cancer) status.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63/491,154, filed March 20, 2023, the content of which is herein incorporated by reference in its entirety.

BACKGROUND

[0003] Tracking cancer progression is currently most frequently done using imaging methods. Imaging tracks tumor size, but requires large, expensive instrumentation, operated, and interpreted by highly trained individuals. Imaging is classically a lagging indicator, slow to indicate changes. To effectively treat patients with methods personalized to their response, an accessible tool is needed to quickly determine treatment efficacy and improve long-term outcomes.

SUMMARY

[0004] Embodiments of the present disclosure include methods for determining cancer status in a subject. In some embodiments, the methods comprise measuring cancer cell free DNA (cfDNA) in a first biological sample and a second biological sample, wherein the first and the second biological sample are obtained from the subject separated by a period of time and classifying the cancer as progressive, regressive, or unchanged based on a change in the amount, quantity, concentration and/or level of cancer cfDNA in the second biological sample as compared to the first biological sample.

[0005] In some embodiments, the methods further comprise: measuring cancer cell free DNA (cfDNA) in at least one additional biological sample separated from an immediately preceding biological sample by a period of time and classifying the cancer status based on the change of the amount, quantity, concentration and/or level of cancer cfDNA in the least one additional biological sample as compared to any preceding biological sample. [0006] In some embodiments, the cancer is progressive when the cancer cfDNA in the least one additional biological sample is increased as compared to any preceding biological sample. In some embodiments, the cancer is regressive when the cancer cfDNA in the least one additional biological sample is decreased as compared to any preceding biological sample.

[0007] In some embodiments, measuring cancer cfDNA comprises: adding an acidifying agent to a biological sample to form an acidic sample; contacting an electrode with the acidic sample; and detecting an electrochemical signal indicating the cancer cfDNA.

[0008] In some embodiments, detecting the electrochemical signal can include performing at least one of voltammetry, cyclic voltammetry, square wave voltammetry, differential pulse voltammetry, amperometry, chronoamperometry, potentiometry, chronopotentiometry, coulometry, chronocoulometry, conductometry, and impedometry.

[0009] In some embodiments, the electrochemical signal is a measure of the cancer cfDNA adsorbed to the electrode. In some embodiments, the electrochemical signal is a change in peak current magnitude following contacting the electrode with the acidic sample.

[0010] In some embodiments, a progressive cancer is characterized by a decreased peak current magnitude of a second biological sample compared to the first biological sample.

[0011] In some embodiments, the acidic sample is at a pH of less than about 4. In some embodiments, the acidic sample is at a pH from about 1 to about 3. In some embodiments, the acidic sample is at a pH of about 2. In some embodiments, the acidic sample comprises an acidifying agent.

[0012] In some embodiments, the methods further comprise: extracting cfDNA from the first biological sample, the second biological sample, and/or the at least one additional biological sample prior to measuring the amount, quantity, concentration and/or level of cancer cfDNA. In some embodiments, the methods further comprise: extracting cfDNA from the first biological sample, the second biological sample, and/or the at least one additional biological sample prior to forming an acidic sample. In some embodiments, the methods further comprise: extracting cfDNA from the first biological sample, the second biological sample, and/or the at least one additional biological sample prior to forming an acidic sample and prior to measuring the amount, quantity, concentration and/or level of cancer cfDNA.

[0013] In some embodiments, the first biological sample, the second biological sample, and the at least one additional biological sample are each individually selected from a whole blood sample, a plasma sample, a serum sample, and a urine sample.

[0014] In some embodiments, the cancer is progressive when the cancer cfDNA in the second biological sample is increased compared to the first biological sample. In some embodiments, the cancer is regressive when the cancer cfDNA in the second biological sample is decreased compared to the first biological sample.

[0015] In some embodiments, the period of time is at least one week. In some embodiments, the period of time is at least one month. In some embodiments, the period of time is less than 6 months.

[0016] In some embodiments, the methods further comprise treating, or changing the treatment of, the subject based on the cancer progression.

[0017] In some embodiments, the first biological sample predates the start of a treatment regimen and the second biological sample postdates the start of the treatment regimen. In some embodiments, the methods further comprise determining the clinical benefit of the treatment regimen based on if the cancer status was progressive, regressive, or unchanged. In some embodiments, the treatment regimen comprises surgery, administration of a chemotherapeutic agent, immunotherapy, radiotherapy, or combinations thereof. In some embodiments, the treatment regimen comprises immunotherapy. In some embodiments, the methods further comprise treating the subject based on the clinical benefit of the treatment regimen.

[0018] Embodiments of the present disclosure also include methods for detecting cancer in a subject. In some embodiments, the cancer is an advanced stage cancer.

[0019] In some embodiments, the methods comprise: measuring cancer cell free DNA (cfDNA) in a biological sample obtained from the subject and identifying the subject as having cancer based on: the cancer cfDNA as compared to: control, non-cancerous cfDNA; a cutoff value; or a cutoff value for a ratio of cancer cfDNA to total cfDNA.

[0020] In some embodiments, measuring cfDNA comprises: adding an acidifying agent to a biological sample to form an acidic sample; contacting an electrode with the acidic sample; and detecting an electrochemical signal indicating the cancer cfDNA.

[0021] In some embodiments, detecting the electrochemical signal can include performing at least one of voltammetry, cyclic voltammetry, square wave voltammetry, differential pulse voltammetry, amperometry, chronoamperometry, potentiometry, chronopotentiometry, coulometry, chronocoulometry, conductometry, and impedometry.

[0022] In some embodiments, the electrochemical signal is a measure of the cancer cfDNA adsorbed to the electrode. In some embodiments, the electrochemical signal is a change in peak current magnitude following contacting the electrode with the acidic sample.

[0023] In some embodiments, the acidic sample is at a pH of less than about 4. In some embodiments, the acidic sample is at a pH from about Ito about 3. In some embodiments, the acidic sample is at a pH of about 2. In some embodiments, the acidic sample comprises an acidifying agent.

[0024] In some embodiments, the methods further comprise: extracting cfDNA from the biological sample prior to forming an acidified sample, prior to measuring the cancer cfDNA, or both.

[0025] In some embodiments, the methods further comprise: extracting cfDNA from the biological sample, adjusting it to the desired concentration prior to measuring the cancer cfDNA.

[0026] In some embodiments, the biological sample is a whole blood sample, a plasma sample, a serum sample, and a urine sample.

[0027] Embodiments of the present disclosure also include methods for detecting cancer DNA in a sample. In some embodiments, the methods comprise: contacting an electrode with an acidic sample comprising or suspected of comprising cancer DNA and measuring an electrochemical signal indicating the cancer DNA.

[0028] In some embodiments, the acidic sample is at a pH of less than about 4. In some embodiments, the acidic sample is at a pH from about 1 to about 3. In some embodiments, the acidic sample is at a pH of about 2. In some embodiments, the acidic sample comprises an acidifying agent.

[0029] In some embodiments, the acidic sample comprises a biological sample. In some embodiments, the methods comprise adding the acidifying agent to the biological sample, thereby forming the acidic sample, prior to contacting the acidic sample with the electrode.

[0030] In some embodiments, wherein detecting the electrochemical signal can include performing at least one of voltammetry, cyclic voltammetry, square wave voltammetry, differential pulse voltammetry, amperometry, chronoamperometry, potentiometry, chronopotentiometry, coulometry, chronocoulometry, conductometry, and impedometry. In some embodiments, the electrochemical signal is a measure of the cancer DNA adsorbed to the electrode. In some embodiments, the electrochemical signal comprises a change in peak current magnitude following contacting the electrode with the acidic sample. In some embodiments, the electrode is a gold electrode.

[0031] In some embodiments, the acidic sample comprises less than 100 pg/uL DNA. In some embodiments, the acidic sample comprises less than 10 pg/uL DNA.

[0032] In some embodiments, the subject has cancer when the cancer cfDNA in the biological sample is greater than the control and/or greater than the cutoff value. In some embodiments, the subject has cancer when the ratio of cancer cfDNA to total cfDNA is greater than the cutoff value.

[0033] In some embodiments, the methods further comprise administering, or changing, a treatment regimen to the subject identified as having cancer. In some embodiments, the treatment regimen comprises surgery, administration of a chemotherapeutic agent, immunotherapy, radiotherapy, or combinations thereof.

[0034] Embodiments of the present disclosure also include systems for carrying out the disclosed methods. In some embodiments, the systems comprise: an electrochemical detection system comprising a gold electrode and an acidic sample comprising cancer DNA or a sample comprising cancer DNA and an acidifying agent. In some embodiments, the electrochemical detection system further comprises one or more of: a working electrode, a counter electrode, a reference electrode, a sample reservoir, a potentiostat and a data acquisition unit. In some embodiments, the system further comprises a sample purification system. In some embodiments, the sample purification system comprises filters, chromatography columns, chromatography beads, microfluidic devices, or combinations thereof.

[0035] Embodiments of the present disclosure also include compositions comprising cancer DNA and a buffer or acidifying agent. In some embodiments, the composition is at acidic pH. In some embodiments, the composition has a pH of less than about 4. In some embodiments, the composition has a pH from about 1 to about 3. In some embodiments, the composition has a pH of about 2.

[0036] Other embodiments and embodiments of the disclosure will be apparent in light of the following detailed description and related figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIG. 1 shows relative peak current values for cfDNA individual samples for healthy subjects (Normal), patients with colorectal cancer (CRC), and patients with non-small-cell lung cancer (NSCLC) following incubation of the cfDNA with the electrode at acidic (pH 2) and neutral (7.2).

[0038] FIGS. 2A-2E show relative peak current values for cfDNA individual samples for healthy subjects (Normal), patients with colorectal cancer (CRC), and patients with non-small- cell lung cancer (NSCLC). FIG. 2A shows the relative peak current values for each individual sample (Pl-6 Norm., Pl-5 CRC and Pl-4 NSCLC) on the SPEs, with error bars representing the standard deviation of n = 5 SPEs and 30, 25 and 20 total measurements for the three sample types, respectively. FIG. 2B shows the relative peak current values for each individual sample (Pl-6 Norm., Pl-5 CRC and Pl-4 NSCLC) on the thin-film gold electrodes (TFEs) with error bars representing the standard deviation of n = 4 TFEs and 24, 20 and 16 total measurements for the three sample types respectively. FIG. 2C shows the large separation between peak current values for cancer samples versus normal samples, red bars. Box and whisker plots corresponding to the relative peak current values in the bar graphs of FIGS. 2A and 3B show each individual measurement for the TFEs (FIG. 2D) and the SPEs (FIG. 2E). The p-values on the TFEs for Norm. vs. CRC and Norm. vs. NSCLC are 4.2E"⁴ and 1.5E"⁴ respectively. The p- values on the SPEs are 2.6E"⁵ and 4.8E"⁴ respectively.

[0039] FIGS. 3A-3C show relative peak current values for clinical cfDNA samples (10 pg/pL) obtained from patients with lymphoma (FIG. 3A, Patient 1), NSCLC (FIG. 3B, Patient 2) and NSCLC (FIG. 3C, Patient 3) at two different timepoints ranging from before diagnosis to after treatment. Error bars represent the standard deviation of n = 15 measurements using 5 different SPEs for each sample. The p-values indicate the statistical significance between the %ir values between T 1 and T2 for each patient.

[0040] FIGS. 4A and 4B show adsorption of clinical cfDNA pooled patient samples for healthy individuals (Norm., grey) and patients with colorectal (CRC, orange) or non-small-cell lung cancer (NSCLC, green). FIG. 4A is a graph of the relative peak current values as a function of cfDNA concentration (0.1 - 10 pg/pL). FIG. 4B shows the difference in relative peak current magnitude values between healthy and cancer cfDNA plotted as a function of concentration.

[0041] FIGS. 5A and 5B show cfDNA signals differentiate cancer patients responding to treatment (responders or cancer regression) and those patients in which the cancer is progressing (progressors or cancer progression) from two different sample sets: Scripps patient samples (FIG. 5A) and Drammen Hospital samples (FIG. 5B).

DETAILED DESCRIPTION

[0042] Disclosed herein are methods which electrochemically measure differences in cancer DNA based on its binding properties to electrodes, particularly gold electrodes. Analysis of the change in peak current magnitude before and after incubating an electrode with DNA correlated with a reduction in electrode surface area due to DNA adsorption. A significant decrease in the binding affinity of cancer cfDNA was observed compared to non-cancer cfDNA at pH 2, which resulted in a measurable difference in current.

[0043] Increases in circulating tumor DNA (ctDNA) abundance correlates with tumor progression and can be differentiated from other forms of circulating free DNA (cfDNA) by a number of methods, including changes in methylation status, but generally those methods are complex, expensive, and lack accuracy and specificity. Using electrochemical signal changes due to DNA adsorption to the electrode, cfDNA isolated from normal subjects was distinguished from cfDNA isolated from CRC/NSCLC patients. The methods provided herein facilitate accurate analysis of cancer progression/regression over time or and a measure of cancer treatment effectiveness. The disclosed methods are capable of determining cancer progression and treatment efficacy quickly, accurately, cost-effectively, before detection and monitoring using imagining techniques and without the lag-time associated with standard cfDNA analysis.

[0044J Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.

1. Definitions

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0046] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0047] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6- 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0048] The term “derived from” as used herein refers to cells or a biological sample (e.g., blood, tissue, bodily fluids, plants, etc.) and indicates that the cells or the biological sample were obtained from the stated source at some point in time. The term includes directly obtained from, isolated and cultured, or obtained, frozen, and thawed. The term “derived from” may also refer to a component or fragment of a cell obtained from a tissue or cell, including, but not limited to, a protein, a nucleic acid, a membrane or fragment of a membrane, and the like.

[0049] “Controls” as used herein generally refers to a reagent whose purpose is to evaluate the performance of a measurement system in order to assure that it continues to produce results within permissible boundaries (e.g., boundaries ranging from measures appropriate for a research use assay on one end to analytic boundaries established by quality specifications for a commercial assay on the other end). To accomplish this, a control should be indicative of patient results and optionally should somehow assess the impact of error on the measurement (e.g., error due to reagent stability, calibrator variability, instrument variability, and the like).

[0050] “Dynamic range” as used herein refers to range over which an assay readout is proportional to the amount of target molecule or analyte (e.g., cancer DNA) in the sample being analyzed. The dynamic range can be the range of linearity of the standard curve.

[0051] “Reference level” as used herein refers to an assay cutoff value that is used to assess diagnostic, prognostic, or therapeutic efficacy and that has been linked or is associated herein with various clinical parameters (e.g., presence of disease, stage of disease, severity of disease, progression, non-progression, or improvement of disease, etc.). However, it is well-known that reference levels may vary depending on the nature of the assay used and that assays can be compared and standardized. It further is well within the ordinary skill of one in the art to adapt the disclosure herein for other assays to obtain specific reference levels for those other assays based on the description provided by this disclosure. Whereas the precise value of the reference level may vary between assays, the findings as described herein should be generally applicable and capable of being extrapolated to other assays.

[0052] As used herein, the term “sample” is used in its broadest sense. In one sense, it is meant to include a specimen obtained from any source, including biological samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Such examples are not however to be construed as limiting the sample types. Preferably, a sample is a fluid sample such as a liquid sample. Examples of liquid samples that may be assayed include bodily fluids (e.g., blood, serum, plasma, saliva, urine, ocular fluid, semen, sputum, sweat, tears, pleural effusions, ascites, thin needle aspirates and spinal fluid. Viscous liquid, semisolid, or solid specimens may be used to create liquid solutions, eluates, suspensions, or extracts that can be samples. For example, throat or genital swabs may be suspended in a liquid solution to make a sample. Samples can comprise biological materials, such as cells, microbes, organelles, and biochemical complexes. Liquid samples can be made from solid, semisolid, or highly viscous materials, such as fecal matter, tissues, organs, biological fluids, or other samples that are not fluid in nature. For example, solid or semisolid samples can be mixed with an appropriate solution, such as a buffer, a diluent, and/or extraction buffer. The sample can be macerated, frozen and thawed, or otherwise extracted to form a fluid sample. Residual particulates may be removed or reduced using conventional methods, such as filtration or centrifugation.

[0053] “Test sample,” “sample from a subject,” “biological sample,” and “patient sample” as used interchangeably herein may be a sample of blood, such as whole blood (including for example, capillary blood, venous blood, dried blood spot, etc.), tissue, urine, serum, plasma, amniotic fluid, an anal sample (such as an anal swab specimen), lower respiratory specimens such as, but not limited to, sputum, endotracheal aspirate or bronchoalveolar lavage, nasal mucus, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes. The sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.

[0054] A variety of cell types, tissue, or bodily fluid may be utilized to obtain a sample. Such cell types, tissues, and fluid may include sections of tissues such as biopsy and autopsy samples, oropharyngeal specimens, nasopharyngeal specimens, nasal mucus specimens, frozen sections taken for histologic purposes, blood (such as whole blood, dried blood spots, etc.), plasma, serum, red blood cells, platelets, an anal sample (such as an anal swab specimen), interstitial fluid, cerebrospinal fluid, etc. Cell types and tissues may also include lymph fluid, cerebrospinal fluid, or any fluid collected by aspiration. A tissue or cell type may be provided by removing a sample of cells from a human and a non-human animal, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose). Archival tissues, such as those having treatment or outcome history, may also be used. Protein or nucleotide isolation and/or purification may not be necessary.

[0055] A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes, such a mouse models, prokaryotic models (e.g., bacteria), archea, and single-celled eukaryotes(e.g., yeast). Likewise, subject may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., humans and non-humans) that may benefit from the uses of compositions and methods contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment, the subject is a human.

[0056] As used herein, the term “treat,” “treating,” or “treatment” are each used interchangeably herein to describe reversing, alleviating, or inhibiting the progress of a disease and/or injury, or one or more symptoms of such disease, to which such term applies. Depending on the condition of the subject, the term also refers to preventing a disease, and includes preventing the onset of a disease, or preventing the symptoms associated with a disease (e.g., viral infection). A treatment may be either performed in an acute or chronic way. The term also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. Such prevention or reduction of the severity of a disease prior to affliction refers to administration of a treatment to a subject that is not at the time of administration afflicted with the disease.

[0057] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

2. Cancer DNA Detection

[0058] In accordance with the data described herein, embodiments of the present disclosure include methods for detecting cancer DNA (e.g., the presence or determining an amount, quantity, concentration and/or level of cancer DNA (e.g., cancer cfDNA, ctDNA)) in a sample. [0059] In some embodiments, the methods comprise contacting an electrode with an acidic sample comprising or suspected of comprising cancer DNA and measuring an electrochemical signal indicating the cancer DNA, e.g., the presence, amount, quantity, concentration and/or level of the cancer DNA. In some embodiments, the electrochemical signal increases with increasing presence, amount, quantity, concentration and/or level of the cancer DNA. In some embodiments, the electrochemical signal decreases with decreasing presence, amount, quantity, concentration and/or level of the cancer DNA.

[0060] In some embodiments, the acidic sample is at a pH of less than about 4. The acidic sample may be at a pH less that about 3.5, less than about 3, less than about 2.5, less than about 2, less than about 1.5, less than about 1. The acidic sample may be at a pH from about 1 to about 4, from about 1 to about 3, from about 1 to about 2, from about 2 to about 4, from about

2 to about 3, from about 3 to about 4. In select embodiments, the acidic sample is at a pH from about 1 to about 3.

[0061] In some embodiments, the acidic sample comprises an acidifying agent. The acidifying agent is any agent which acts to lower the pH of the comprising or suspected of comprising cancer DNA, such as an inorganic or organic acid. Suitable acidifying agents include, for example, organic acids such as ascorbic acid (vitamin C), salicylic acid, acetyl salicylic acid, acetic acid or a salt or a derivative thereof, ammonium or aluminum salts, phenol, inorganic acids such as hydrochloric acid, nitric acid or a salt or a derivative thereof. The acidifying agent may be present as a dissolved salt or in a liquid form. The acidifying agents may be part of a buffer or buffering system.

[0062] In some embodiments, the acidic sample comprises or is derived from a biological sample. For example, in some embodiments, the comprises adding the acidifying agent to the biological sample, thereby forming the acidic sample, prior to contacting the acidic sample with the electrode. The acidifying agent may be added as a dissolved salt, in a liquid form, in a buffer solution, or as a solid (e.g., powder or granulate). Alternatively, the acidic sample may be derived from a biological sample by other methods known in the art to adjust pH values, including but not limited to dialysis and column exchange.

[0063] Accordingly, embodiments herein provide compositions comprising cancer DNA and a buffer or acidifying agent, wherein the composition is at an acidic pH. In some embodiments, the composition is at a pH of less than about 4. The composition may be at a pH less that about 3.5, less than about 3, less than about 2.5, less than about 2, less than about 1.5, less than about 1. The composition may be at a pH from about 1 to about 4, from about 1 to about 3, from about 1 to about 2, from about 2 to about 4, from about 2 to about 3, from about

3 to about 4. In select embodiments, the composition is at a pH from about 1 to about 3.

[0064] The methods are not limited by the method for detecting an electrochemical signal. Measuring the electrochemical signal can include performing at least one of voltammetry, amperometry, potentiometry, coulometry, conductometry, impedometry, or other methods known in the art. In some embodiments, measuring the electrochemical signal can include performing at least one of cyclic voltammetry, square wave voltammetry, differential pulse voltammetry, chronoamperometry, chronopotentiometry, and chronocoulometry. In select embodiments, measuring the electrochemical signal can include performing at least one of differential pulse voltammetry and cyclic voltammetry. [0065] In some embodiments, the electrochemical signal is a measure of the presence, amount, quantity, concentration and/or level of cancer vs normal DNA adsorbed to the electrode. Any electrochemical signal altered by the adsorption of the cancer DNA to the electrode or the presence of cancer DNA in the sample may be measured and correlated to the amount or relative amount of cancer DNA. For example, standard or control values may be used to determine the relationship between the cancer DNA (e.g., the amount, quantity, concentration and/or level) to an electrochemical signal. These standard or control values may be determined prior to conducting the disclosed methods and may include determination of dynamic range, limit of detection, limit of quantitation, linearity, sensitivity, and other calibration values. In some embodiments, the electrochemical signal comprises a change in peak current magnitude following contacting the electrode with the acidic sample. In some embodiments, the electrochemical signal comprises a change in potential at peak following contacting the electrode with the acidic sample.

[0066] The correlation between electrochemical signal and cancer DNA may be dependent on the state of the sample (e.g., source, purification state, presence of interfering components, and the like), the quantity of sample, the type, size, or configuration of the electrode, and the methods used to measure the electrochemical signal.

[0067] The electrode may comprise any material which confers differential adsorption of cancer DNA compared to non-cancer DNA under acidic conditions. In some embodiments, the electrode comprises a metal (e.g., gold, platinum, palladium, silver, copper) or metal alloy. In some embodiments, the electrode is a gold electrode.

[0068] The electrode may be any size or form which facilitates contacting with the acidic sample. The electrode may be a screen-printed electrode, thin film or thick-film electrode. The electrode may be a thin-film electrode. The electrode may be modified to have a higher reactive surface, increase the mass transfer rate, and/or the electrocatalytic activity (e.g., with the use of nanoparticles, methods of forming electrode surface (e.g., by varying temperature at which the electrodes are screen-printed or type of ink used), type of substrate, thickness of film, and the like).

[0069] In some embodiments, the acidic sample comprises less than 300 pg/uL DNA (e.g., less than 250 pg/uL, less than 200 pg/uL, less than 150 pg/uL, less than 100 pg/uL, less than 90 pg/uL, less than 80 pg/uL, less than 70 pg/uL, less than 60 pg/uL, less than 50 pg/uL, less than 40 pg/uL, less than 30 pg/uL, less than 20 pg/uL, less than 10 pg/uL, less than 5 pg/uL, less than 2 pg/uL, less than 1 pg/uL, less than 0.1 pg/uL). In select embodiments, the acidic sample comprises less than 10 pg/uL DNA. The acidic sample may comprise 0.1 - 10 pg/pL DNA, e.g., about 0. 1 pg/pL DNA, about 0.5 pg/pL DNA, about 1 pg/pL DNA, about 1.5 pg/pL DNA, about 2 pg/pL DNA, about 2.5 pg/pL DNA, about 3 pg/pL DNA, about 3.5 pg/pL DNA, about 4 pg/pL DNA, about 4.5 pg/pL DNA, about 5 pg/pL DNA, about 5.5 pg/pL DNA, about 6 pg/pL DNA, about 6.5 pg/pL DNA, about 7 pg/pL DNA, about 7.5 pg/pL DNA, about 8 pg/pL DNA, about 8.5 pg/pL DNA, about 9 pg/pL DNA, about 9.5 pg/pL DNA, about 10 pg/pL DNA, or any ranges therein. Thus, in some embodiments, the method further comprises adjusting the concentration of the sample to a desired concentration prior to measuring the electrochemical signal.

3. Cancer Status and Diagnosis

[0070] Embodiments of the present disclosure also include methods for determining cancer status and diagnosing cancer in a subject. In some embodiments, the methods comprise measuring cancer cell free DNA (cfDNA) (e.g., an amount, quantity, concentration and/or level of cancer cfDNA) in one or more biological samples. The term “cell free DNA” or “cell-free DNA” or “cfDNA” refers to single and/or double-stranded deoxyribose nucleic acids (DNA) found free of cells, generally found in biological fluids including plasma, serum, urine, and the like.

[0071] In some embodiments, the sample comprises less than 300 pg/uL DNA (e.g., less than 250 pg/uL, 200 pg/uL, 150 pg/uL, 100 pg/uL, 90 pg/uL, 80 pg/uL, 70 pg/uL, 60 pg/uL, 50 pg/uL, 40 pg/uL, 30 pg/uL, 20 pg/uL, 10 pg/uL, 5 pg/uL, 2 pg/uL, 1 pg/uL, 0.1 pg/uL). In select embodiments, the acidic sample comprises less than 10 pg/uL DNA.

[0072] In some embodiments, the methods described herein measure total cancer cfDNA (e.g., the amount, quantity, concentration and/or level of total cancer cfDNA). For example, in some embodiments, the methods do not select any specific cfDNA sequences based for analysis. Rather, the methods query the cancer or non-cancer of the entire cfDNA in the sample, not a specific viral sequence, mutation, SNP, indel, or signature. Thus, in some embodiments, the methods do not comprise sequencing the cfDNA or associating the cancer status with any particular cfDNA sequence. In some embodiments, the methods do not comprise identifying the type or location of the methyl groups in the cancer cfDNA.

[0073] In some embodiments, the methods further comprise obtaining the biological sample(s) from the subject. In some embodiments, the biological sample(s) are each individually selected from a whole blood sample, a plasma sample, a serum sample, and a urine sample. The sample(s) can be obtained using techniques known to those skilled in the art, and the sample(s) may be used directly as obtained from the source or following a pretreatment to modify the character of the sample. Such pretreatment may include, for example, preparing plasma from blood, diluting viscous fluids, filtration, precipitation, dilution, distillation, mixing, concentration, inactivation of interfering components, the addition of reagents, lysing, and the like.

[0074] In some embodiments, the methods further comprise isolating nucleic acids, preferably DNA, from the sample prior to measuring the amount, quantity, concentration and/or level of cancer cfDNA. In select embodiments, the methods further comprise extracting cfDNA from a sample prior to measuring the amount, quantity, concentration and/or level of cancer cfDNA. cfDNA extraction may include the use of one or more of chromatography or affinity columns or magnetic beads, phenol-chloroform-based methods, and filtration-based methods.

[0075] In some embodiments, the methods further comprise analyzing the cfDNA and, if necessary, adjusting the concentration of the cfDNA prior to measuring the amount, quantity, concentration and/or level of cancer cfDNA. In some embodiments, analyzing the cfDNA is done in a manner which measures the total cfDNA (cancer and non-cancer related cfDNA). In some embodiments, analyzing the cfDNA is done in a manner which measures the non-cancer related cfDNA.

[0076] In some embodiments, the methods comprise measuring cancer cfDNA in a first biological sample and a second biological sample taken from the subject, wherein the second biological sample is separated from the first biological sample by a period of time. The period of time between taking the first biological sample and the second biological sample may be at least four hours, at least one day, at least one week, at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least nine months, at least twelve months, at least eighteen months, at least two years, or more. In some embodiments, the period of time between taking the first biological sample and the second biological sample is at least one week. In some embodiments, the period of time between taking the first biological sample and the second biological sample is at least one month.

[0077] The period of time between taking the first biological sample and the second biological sample may be less than one year, less than nine months, less than six months, less than five months, less than four months, less than three months, less than two months, or less than one month. In some embodiments, the period of time is less than about six months. The period of time may be less than the time necessary to detect changes in cancer or tumor burden by an imaging technique. In some embodiments, a second or subsequent biological sample is taken from the subject at the time of another bioassay which assesses change in cancer status. The bioassay may be used to confirm the results of the methods disclosed herein and/or assist in ruling out pseudo-progression (false positive apparent growth on imaging). For example, a second biological sample taken from the subject may be taken at the same time as imaging analysis of the cancer.

[0078] In some embodiments, the methods comprise classifying the cancer as progressive, regressive, or unchanged based on change of the cancer cfDNA (e.g., the amount, quantity, concentration and/or level of cancer cfDNA) in the second, or subsequent, biological sample as compared to the first biological sample. For example, the cancer is progressive, indicating an increased cancer burden, when the cancer cfDNA in the second biological sample is increased compared to the first biological sample. Alternatively, the cancer is regressive, indicating a decrease in cancer burden, when the cancer cfDNA in the second biological sample is decreased compared to the first biological sample. As such, the methods may evaluate the change in cancer status, e.g., the progression or regression of the cancer, over time.

[0079] In some embodiments, the first biological sample predates the start of a treatment regimen and the second biological sample postdates the start of the treatment regimen. In such instances, the methods may evaluate the progression or regression of the cancer after different interventions or treatment regimens (e.g., surgery, administration of a chemotherapeutic agent, immunotherapy, radiotherapy, or combinations thereof). In such instances, the methods may evaluate the growth of the cancer after different interventions or treatment regimens. Thus, the methods may allow short- or long-term longitudinal evaluation of single or multiple interventions or treatment regimens.

[0080] To evaluate the progression or regression of the cancer over time or after different interventions or treatment regimens, the methods may further comprise measuring cancer cell free DNA (cfDNA) (e.g., an amount, quantity, concentration and/or level of cancer cfDNA) in at least one additional biological sample separated from an immediately preceding, or any preceding, biological sample by a period of time. Thus, any number of biological samples taken at different timepoints during the duration of a cancer or during or after interventions or treatment regimens may be used in the disclosed methods with the cancer status or change in cancer status being determined between each sample or from the first sample to the most recent sample based on the change of the cancer cfDNA.

[0081] In some embodiments, the methods further comprise determining a growth rate of the cancer based on the cancer cell free DNA (cfDNA) in two or more biological samples or a change in slope of the cancer cell free DNA (cfDNA) in three or more biological samples. As such, the disclosed methods may evaluate the growth rate of the cancer over time before and/or after the start or change of a treatment regimen . The growth rate may be used to detect hyper progressive cancer, which is generally defined as a growth rate of over double the pre-treatment level.

[0082] Each of the samples may be separated by any period of time from weeks to years. In some embodiments, each sample is separated from the immediately preceding sample by at least one week, at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least nine months, at least twelve months, at least eighteen months, at least two years, or more. In some embodiments, each sample is separated from the immediately preceding sample by less than one year, less than nine months, less than six months, less than five months, less than four months, less than three months, less than two months, or less than one month.

[0083] The biological samples may be examined at the time or near the time of acquisition. Alternatively, biological samples may be taken at the desired timepoints and analyzed simultaneously at a single point in time. For example, one or more biological samples may be taken and stored prior to analysis at a later time point.

[0084] In some embodiments, the methods comprise identifying a subject as having or at risk of having a cancer based on the cancer cfDNA in a biological sample. The cancer may be identified by comparing the amount, quantity, concentration and/or level of cancer cfDNA in a biological sample to a control or reference, non-cancerous cfDNA amount, quantity, concentration, and/or level or previous samples from the same person. Further, the cancer may be identified based on a cutoff value for a ratio of the amount, quantity, concentration and/or level of cancer cfDNA to total cfDNA in the biological sample. For example, when the ratio of cancer cfDNA to total cfDNA is greater than a cutoff value, the subject may have cancer. Total cfDNA may be measured using any method known in the art, including a coordinating electrochemical method.

[0085] Alternatively, the cancer may be identified by comparing the amount, quantity, concentration and/or level of cancer cfDNA in a biological sample to a cutoff value for the amount, quantity, concentration and/or level of cancer cfDNA. For example, when the amount, quantity, concentration and/or level of cancer cfDNA in the biological sample is greater than the control and/or greater than the cutoff value, the subject may have cancer.

[0086] The methods are not limited by the manner in which the amount, quantity, concentration and/or level of cancer cfDNA in measured in the biological samples. In some embodiments, the methods used to measure cancer cfDNA are those described herein. In some embodiments, measuring an amount, quantity, concentration and/or level of cancer cfDNA comprises: adding an acidifying agent to a biological sample to form an acidic sample; contacting an electrode with the acidic sample and detecting an electrochemical signal indicating the amount, quantity, concentration and/or level of cancer cfDNA. Embodiments described above for the methods of measuring cancer DNA, including those directed to electrochemical methods, electrochemical signals, and acidic pH values are applicable to the measurements for cfDNA.

[0087] As described above, in some embodiments, the electrochemical signal comprises a change in peak current magnitude following contacting the electrode with the acidic sample. In some embodiments, a progressive cancer is characterized by a decreased peak current magnitude of a subsequent biological sample compared to a previous biological sample (e.g., second biological sample compared to a first biological sample). In some embodiments, a regressive cancer is characterized by an increased peak current magnitude of a subsequent biological sample compared to a previous biological sample (e.g., second biological sample compared to a first biological sample).

[0088] The methods described herein are not limited by type of cancer. The cancer may include carcinoma, sarcoma, lymphoma, leukemia, melanoma, mesothelioma, multiple myeloma, or seminoma. The cancer may be a cancer of the bladder, blood, bone, brain, breast, cervix, colon/rectum, endometrium, head and neck, kidney, liver, lung, lymph nodes, muscle tissue, ovary, pancreas, prostate, skin, spleen, stomach, testicle, thyroid, or uterus. In some embodiments, the cancer comprises a solid tumor. In some embodiments, the cancer is metastatic cancer. In some embodiments, the cancer is invasive and/or metastatic cancer (e.g., stage III cancer or stage IV cancer). In select embodiments, the cancer is a lymphoma. In select embodiments, the cancer is lung cancer or colorectal cancer.

[0089] In some embodiments, the cancer is invasive and/or metastatic (advanced stage) cancer (e.g., stage II cancer, stage III cancer or stage IV cancer). In some embodiments, the cancer is an early stage cancer (e.g., stage 0 cancer, stage I cancer), and/or is not invasive and/or metastatic cancer.

[0090] In some embodiments, the methods disclosed herein further comprise treating the subject. As such, the methods described herein may be integrated into a treatment regimen for a subject. For example, in some embodiments, the biological sample(s) is analyzed by the methods described herein and the subject is treated based on the results (e.g., commence a new treatment, continue existing treatment, change in treatment (e.g., change in intervention type, dose, timing, etc.), or stop treatment. [0091 ] In some embodiments, the treatment comprises administration of an anti-cancer agent or chemotherapeutic. “Anti-cancer agent” or “chemotherapeutic,” as used herein, refers to any small molecule or other drug used in cancer treatment or prevention, whether cytostatic, cytotoxic, kinase inhibitor, or other MOA. Chemotherapeutics include, but are not limited to, cyclophosphamide, methotrexate, 5 -fluorouracil, doxorubicin, docetaxel, daunorubicin, bleomycin, vinblastine, dacarbazine, cisplatin, paclitaxel, raloxifene hydrochloride, tamoxifen citrate, abemacicilib, afinitor, alpelisib, anastrozole, pamidronate, anastrozole, exemestane, capecitabine, epirubicin hydrochloride, eribulin mesylate, toremifene, fiilvestrant, letrozole, gemcitabine, goserelin, ixabepilone, emtansine, lapatinib, olaparib, megestrol, neratinib, palbociclib, ribociclib, talazoparib, thiotepa, toremifene, methotrexate, and tucatinib.

[0092] In some embodiments, the second therapy includes immunotherapy. Immunotherapies include chimeric antigen receptor (CAR) T-cell or T-cell transfer therapies, NK cell therapy, TIL cell therapy, other cellular therapy, cytokine therapy, immunomodulators, cancer vaccines, or administration of antibodies (e.g., monoclonal antibodies). In some embodiments, the immunotherapy comprises administration of antibodies. The antibodies may target antigens either specifically expressed by tumor cells or antigens shared with normal cells. In some embodiments, the immunotherapy may comprise an antibody targeting, for example, CD20, CD33, CD52, CD30, HER (also referred to as erbB or EGFR), VEGF, CTLA-4 (also referred to as CD 152), epithelial cell adhesion molecule (EpCAM, also referred to as CD326), and PD- 1 /PD-L 1. Suitable antibodies include, but are not limited to, rituximab, blinatumomab, trastuzumab, gemtuzumab, alemtuzumab, ibritumomab, tositumomab, bevacizumab, cetuximab, panitumumab, ofatumumab, ipilimumab, brentuximab, pertuzumab, and the like). In some embodiments, the additional therapeutic agent may comprise anti-PD-l/PD-Ll antibodies, including, but not limited to, pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, durvalumab, and ipilimumab. The antibodies may also be linked to a chemotherapeutic agent. Thus, in some embodiments, the antibody is an antibody-drug conjugate.

[0093] In some embodiments, the treating comprises active surveillance. As such, the methods described herein find use in classifying a patient as suitable for active surveillance. During active surveillance the subject is monitored with additional screenings or tests for changes in overall health or changes directly related to cancer progression. For example, in some embodiments, the methods comprise collecting or receiving a series of samples over a time period from the subject and detecting the cancer cfDNA (e.g. the amount, quantity, concentration and/or level) of cancer cfDNA in each of the series of samples and comparing any measurable change in the cancer cfDNA over the period of time. In some embodiments, each of the series of samples may be used for diagnosing or detecting cancer status or progression, as described in the methods herein.

[0094] The treatment may be administered to a subject by a variety of methods. In any of the uses or methods described herein, administration may be by various routes known to those skilled in the art, including without limitation oral, inhalation, intravenous, intramuscular, topical, subcutaneous, systemic, and/or intraperitoneal administration to a subject in need thereof. The treatment may be administered by parenteral administration (including, but not limited to, subcutaneous, intramuscular, intravenous, intraperitoneal, intracardiac and intraarticular injections).

4. Systems

[0095] The disclosure also provides a systems for measuring cancer DNA (e.g., the concentration, level, quantity, or amount of cancer DNA) in a sample, for example, as described in the above disclosed methods. The systems may comprise an electrochemical detection system comprising a gold electrode and an acidic sample comprising cancer DNA or a sample comprising cancer DNA and an acidifying agent.

[0096] In some embodiments, the system further comprises the electrochemical detection system further comprises one or more of: a working electrode, a counter electrode, a reference electrode a sample reservoir, and a data acquisition unit. One or more of the working electrode, counter electrode, reference electrode and sample reservoir may be integrated into a single device (e.g., a single electrode, an electrode array, a microfluidic device, an electrochemical sensor, or point-of -care device).

[0097] The sample reservoir may be configured to receive any type of sample, as described above, or may further comprise reagents/components (e.g., acidifying agent, purification reagent, buffers, and the like) used to process the sample prior to analysis. As described above, in some embodiments, the sample reservoir is integrated with one or more of the electrodes. The system may further comprise a sample receiving area in which the sample is initially provided prior to being loaded into the sample reservoir.

[0098] The system may further comprise a sample purification system. The purification system may comprise any of: chromatography columns or media, affinity columns or media, beads or particles, filters, and the like useful in purification of the sample (e.g., to remove contaminating materials or purify DNA). [0099] The system may further a flow management or dispensing device (e.g., a pump). The flow management or dispensing device may transfer the sample from the sample receiving area to the sample reservoir or to the electrodes.

[0100] The data acquisition unit may include a power supply, a potentiostat, a bipotentiostat, a galvanostat, an impedance analyzer, one or more processors (e.g., one or more computers or computer systems), and/or a computer-readable medium to perform any or all of: measuring the electrochemical signal, detecting the presence or determining an amount, quantity, concentration and/or level of cancer DNA, comparing the results between biological samples, comparing the results to cutoff values or reference values, classifying the cancer, or diagnosing the patient as having cancer. The data acquisition unit may be configured to communicate with the other components of the system or the data acquisition unit via wired or wireless communications.

[0101] In some embodiments, the system comprises an indicator or display configured to show the electrochemical signal or the presence, amount, quantity, concentration and/or level of cancer DNA. In some embodiments, the system comprises an indicator or display to show when the cancer DNA (e.g., cfDNA) (e.g., amount, quantity, concentration and/or level of cancer DNA (e.g., cfDNA)) is lower or higher than a reference amount, quantity, concentration and/or level. In some embodiments, the indicator or display is further configured to show the ratio of the amount, quantity, concentration and/or level of cancer DNA (e.g., cfDNA) to the amount, quantity, concentration and/or level of total DNA (e.g., total cfDNA) and/or when the ratio is lower or higher than a cutoff value.

[0102] The indicator or display may use any manner of visual or audible means to display the result including but not limited to text, graphs, charts, heat maps, other image based methods, color indications, beeping, and the like. The indicator or display may be configured to transmit the results to another device connected wirelessly or integral to the present system. For example, the indicator or display may transmit the result to a clinical device, a patient record, a patient device, a data storage repository, or similar.

[0103] The systems optionally may include disposable/consumable components that are utilized for the analysis or sample preparation. The system or kit may further contain additional containers or devices for use with the methods disclosed herein.

[0104] Also within the scope of the present disclosure are kits that any or all of the components of the systems. For example, in some embodiments, the kits include an electrochemical detection system comprising a gold electrode, an acidic sample comprising cancer DNA or a sample comprising cancer DNA and an acidifying agent, a working electrode, a counter electrode, a sample reservoir, a data acquisition unit, a sample purification system and/or a flow management or dispensing device (e.g., a pump). In some embodiments, the kits also include calibration and/or control samples.

[0105] Individual member components of the kits may be physically packaged together or separately. The components of the kits may be provided in bulk packages (e.g., multi-use packages) or single-use packages. The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like.

[0106] The kits can also comprise instructions for using the components of the kit. The instructions are relevant materials or methodologies pertaining to the kits. The materials may include any combination of the following: background information, list of components and their availability information (purchase information, etc.), brief or detailed protocols for using the compositions, troubleshooting, references, technical support, and any other related documents. Instructions can be supplied with the kits or as a separate member component, either as a paper form or an electronic form which may be supplied on a computer readable memory device or downloaded from an internet website, or as recorded presentation.

[0107] It is understood that the disclosed kits can be employed in connection with the disclosed methods.

5. Examples

[0108] It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present disclosure described herein are readily applicable and appreciable, and may be made using suitable equivalents without departing from the scope of the present disclosure or the aspects and embodiments disclosed herein. Having now described the present disclosure in detail, the same will be more clearly understood by reference to the following examples, which are merely intended only to illustrate some aspects and embodiments of the disclosure, and should not be viewed as limiting to the scope of the disclosure. The disclosures of all journal references, U.S. patents, and publications referred to herein are hereby incorporated by reference in their entireties.

[0109] The present disclosure has multiple aspects, illustrated by the following non- limiting examples.

Example 1

[0110] Multiple parameters were evaluated to improve electrochemical performance and adsorption magnitude of DNA to gold electrodes. The first parameter investigated was the pH of the incubation solution by monitoring the change in signal at pH 7.2 versus pH 2.0. A strong dependence on the adsorption of cancer cfDNA to gold was observed at pH 2, where the peak current magnitude of the electrode was significantly diminished for normal cfDNA in comparison to cancer cfDNA resulting in larger %iR for normal. At pH 7.2, no correlation on the adsorption of cancer versus non-cancer cfDNA was observed.

[0111] Evaluation of the adsorption of cfDNA samples to the electrode surface was performed using a series of differential pulse voltammetry (DPV) experiments. An initial baseline DPV measurement was performed first using 50 pL of 2.5 mM The electrode was then rinsed with distilled water and dried. Next, 10 pL of the sample containing cfDNA in either pH 2 or pH 7.2 buffer was dropped onto the working electrode surface and left to incubate for 10 minutes. The sample was then gently rinsed from the surface using PBS. Finally, the sample DPV measurement was performed using 50 pL of 2.5 mM K₃Fe(CN solution. The relative peak current (% ir) was calculated using peak current values for the baseline and measurement DPVs by the following equation: some embodiments, the results are displayed as 100- %iR.

[0112] Blood was collected in standard EDTA vacutainers by the University of Colorado BioRepository, plasma was harvested following centrifugation. DNA was isolated using UltraPrep DNA extraction methods as described in Raymond, Ramond, and Hill, 2020 (PLoS ONE; doi.org/10.1371/joumal.pone.0231854). The DNA was suspended in NaCl free TE buffer, then aliquoted and stored at 5° C.

[0113] Pools of cfDNA were made by mixing equal amounts of each cfDNA solution. The normal cfDNA pool was made by mixing 5 pL from each of six different healthy individual cfDNA samples, FIG. 1 (grey). The CRC cfDNA pool was made by mixing 5 pL from each of five different patient cfDNA samples diagnosed with CRC cancer, FIG. 1 (orange). The NSCLC cfDNA pool was made by mixing 5 pL from each of five different patient cfDNA samples diagnosed with NSCLC cancer, FIG. 1 (green).

[0114] DNA was tested for quality and nucleosomal quantity by Bioanalyzer capillary electrophoresis using an Agilent 2100 BioAnalyzer Instrument. The nucleosomal peaks of cfDNA were integrated using Agilent 2100 Expert software and this nucleosomal concentration was used going forward.

[0115] The DPV baseline measurement was conducted using a Palmsens benchtop potentiostat. The electrochemical window for the measurements was -0.5-0.5 V. The experimental parameters were E-step (0.01 V), E pulse (0.05 V), t pulse (0.05 s) and the scan rate (0.1 V/s). The baseline measurement was conducted using 50 pL of 2.5 mM

[0116] After the baseline measurement the electrode surface was rinsed thoroughly with distilled water and dried.

[0117] The working cfDNA samples were obtained by diluting the appropriate amount of cfDNA from each pool for each working concentration. The acidic samples were diluted in buffer to achieve pH 2. The neutral samples were diluted in 0. 1 M phosphate buffered saline solution. A 10 pL droplet of the cfDNA sample solution was then drop casted on the working electrode surface and was left to incubate for 10 minutes. The sample droplet was then gently rinsed from the electrode surface using approximately 5 drops of 0. 1 M PBS.

[0118] The DPV sample measurement was conducted using a Palmsens benchtop potentiostat. The electrochemical window for the measurements was -0.5-0.5 V. The experimental parameters were E-step (0.01 V), E pulse (0.05 V), t pulse (0.05 s) and the scan rate (0.1 V/s). The baseline measurement was conducted using 50 pL of 2.5 mM solution.

[0119] The relative peak current is defined as the change in the peak current magnitude of the baseline DPV after incubating cfDNA sample on the electrode surface and performing a second sample measurement DPV. The relative peak current (% ir) was calculated using peak current values for the baseline and sample DPV measurements by the following equation:

Example 2

[0120] Two types of commercially available gold electrodes were compared in the disclosed methods; screen-printed gold electrodes (SPEs) from Dropsens and thin-film gold electrodes (TFEs) from Micrux. These electrodes were chosen to compare the adsorption characteristics of patient cfDNA samples on different surface morphologies (e.g., rough vs smooth), and electrode compositions (e.g., screen-printed gold on a ceramic substrate and thin-film gold on a glass substrate).

[0121] Electrode characterization was performed using a variety of experimental techniques including optical profilometry (OP), electrochemical characterization (CVs) and scanning electron microscopy (SEM) to determine the surface roughness, electroactive surface area and the surface morphology of the commercial electrodes. Evaluation of the electrodes began with OP experiments to record a digital map of the roughness profiles for each electrode, while the electroactive surface areas were calculated using the Cottrell equation by performing a series of CVs at increasing scan rates. Data indicating the surface roughness, electroactive surface area, experimental throughput, and adsorption characteristics of patient cfDNA samples is shown for both the SPEs and the TFEs, Table 1. SEM images of the SPEs and the TFEs were recorded to observe differences in the surface morphology of each commercial electrode.

Table 1

Example 3

[0122] A study of individual patients was performed using cfDNA samples extracted from plasma using standard PCI extraction methods (25:24: 1 Phenol:Chloroform:Isoamyl) obtained from the University of Colorado Biorepository. cfDNA was obtained from 15 different subjects, 6 were from healthy individuals (Normal), 5 were from patients with colorectal (CRC) cancer and 4 were from patients with non-small-cell lung cancer (NSCLC). The relative adsorption of the patient cfDNA samples (each diluted to a working concentration of 1 pg/pL) were recorded on both the SPEs (FIG. 2A) and TFEs (FIGS. 2B and 2C) . The results are shown in FIGS. 2A-2C; the normal, CRC and NSCLC cfDNA samples are shown in grey (left), orange (middle), and green (right) respectively.

[0123] A clear distinction was observed between the relative peak current magnitude of cfDNA from healthy individuals and that of patients with either CRC or NSCLC. Not only is there no overlap but a region, as illustrated by the red lines in FIG. 2C, which shows ample separation between normal and cancer using TFEs, The cfDNA from healthy individuals (grey) showed a statistically significant signal difference compared to both cancer types; p<0.00005 and p<0.000002 for the SPEs and TFEs respectively, even at 1 pg/pL. In addition, higher overall adsorption of cfDNA, regardless of sample type, was observed for the TFEs compared to the SPEs. The sum of 30, 25 and 20 individual measurements across 15 different patient samples for the SPEs and 24, 20 and 16 individual measurements across 15 different patient samples for the TFEs highlighted the statistical significance in the difference in surface adsorption between the cfDNA of the healthy subjects and those with either cancer type. This distinction demonstrated the ability to differentiate electrochemical signal based on cancer status, a key milestone toward development of a POC platform for monitoring the efficacy of cancer treatment. The AUC is 1 representing, albeit in a small set, an 100% accurate separation. [0124] Evaluation of the adsorption of cfDNA samples to the electrode surface was performed using a series of differential pulse voltammetry (DPV) experiments as described in example 1.

[0125] Blood was collected in standard EDTA vacutainers, plasma was harvested following centrifugation. DNA was extracted using proteinase K and phenol:chloroform:isoamyl (25:24: 1) and suspended in no NaCl TE buffer.

[0126] The normal cfDNA samples were from each of the six different healthy individuals, FIGS. 2A-C (P1-P6, grey). The CRC cfDNA samples were from each of five different patient cfDNA samples diagnosed with CRC cancer, FIGS. 2A-C (P1-P5, orange). The NSCLC cfDNA samples were from each of four different patient cfDNA samples diagnosed with NSCLC cancer, FIGS. 2A-C (P1-P4, green). DNA was tested for quality and quantity as described in Example 1.

Example 4

[0127] Cancer burden of a diagnosed patient is expected to vary from time of diagnosis and throughout various stages of treatment reflecting progression and/or remission. Accordingly, the electrochemical signal derived from the cfDNA of a recovered patient in remission would more closely resemble the signal obtained from a healthy individual. Conversely, increased cancer burden resulting from ineffective therapy would yield an electrochemical response in the opposite direction. In this regard, the ability to monitor the efficacy of a patient specific cancer treatment would require longitudinal patient specific sample analysis. Sets of clinical cfDNA patient samples were obtained before and after cancer treatment and were electrochemically measured using SPEs, FIGS. 3A-3C. A plasma sample from a blood donor directly prior to diagnosis with aggressive B cell lymphoma (patient 1) was compared to a longitudinal sample from patient 1 after finishing treatment (1 year later). The samples were extracted using UltraPrep DNA extraction and normalized by AccuBlue High Sensitivity dsDNA kit concentration measurement (Biotium). The second set of plasma samples was obtained from a patient with NSCLC before the start of pembrolizumab and at day 34 after undergoing treatment (patient 2) where later imaging at 6 months determined the patient responded positively to treatment. Patient 2 samples were extracted using QIAamp MinElute cfDNA kit (Qiagen) and the concentration was measured using high sensitivity dsDNA Qubit kit (ThermoFisher). The third set of cfDNA was extracted using UltraPrep DNA extraction from urine obtained from PrecisionMed and normalized by Agilent 2100 BioAnalyzer. The patient had NSCLC. The first specimen was obtained June 30^th, 2020, at the time of therapy initiation; this was 3 weeks after the diagnosis. The second specimen was obtained July 29^th, 2021, after the patient progressed following treatment with nivolumab and ipilimumab.

[0128] Table 2 provides information on the cfDNA samples from all three patients, including the timepoints for sample collection and the expected cancer burden based upon treatment efficacy. The samples were diluted to 10 pg/pL in pH 2 buffer directly prior to incubation on the electrode. Dilution and measurement of the samples at each timepoint was performed 3 times, each time using 5 new SPEs (n = 15).

Table 2

* Remission was achieved for patient 2 roughly 6 months after obtaining the sample at timepoint 2.

[0129] The samples were analyzed as described above. The difference in relative peak current magnitude for the longitudinal cfDNA samples of the three patients tested (FIGS. 3A- 3C) were statistically significant (p=0.000004, p=0.017, p=0.013 respectively). At T1 when the expected cancer burden was highest for both patients 1 and 2, lower relative peak current value was observed; similar to the lower values obtained for patients with cancer (e.g., CRC or NSCLC) in FIGS. 1 & 2. However, the relative peak current magnitude increased for patients 1 and 2 when comparing T 1 and T2 respectively. These results corresponded to a more normal relative peak current value (FIG. 1 & 2) . Here, the lower signal obtained from cfDNA of cancer patients correlates to T1 (high expected cancer burden, Table 2) and the higher signal obtained from cfDNA of healthy subjects correlates to T2 (decreased cancer burden; or even remission, Table 2). The cfDNA specimens used in patient 2 were isolated from their urine, illustrating application to a variety of biofluids. The opposite effect was observed for patient 3, where the relative peak current was decreased when comparing T1 and T2. The cancer treatment for patient 3 was ineffective, and the sample acquired at T2 was correlated to a progressive disease profile. As a result, the lower relative peak current observed at T2 compared with T1 is in agreement with the expected adsorption of cfDNA for a high cancer burden. The time dependent significance of this study is highlighted for patient 2 where the increase in electrochemical signal, or return toward healthy cfDNA values, on day 34 (T2) presaged the clinical benefit detected by imaging 5 months later. In this case, the direction of the signal change not only correlated with response to treatment but detected it after just one month of successful treatment, and the benefit seen with the electrode at one month was confirmed via traditional imaging techniques at 6 months. The results from FIG. 3, highlight the potential of a liquid biopsy-based patient specific electrochemical diagnostic biosensor, with the ability to rapidly determine treatment efficacy on-site, prior to confirmation via imaging at a later date.

Example 5

[0130] To optimize the difference in adsorption magnitude between the cfDNA of healthy individuals (Norm.) and patients with either cancer type (CRC and NSCLC), the individual patient samples used in FIG. 2 were pooled and a series of measurements were taken to assess the effect of sample concentration on the cancer-dependent adsorption. A range of concentrations were investigated (e.g., 0. 1 - 10 pg/pL) at pH 2.0 for each of the pooled clinical patient cfDNA samples, FIG. 4A. The difference in adsorption between the normal and cancer cfDNA samples was calculated by taking the average of the relative peak current values for the normal cfDNA pool and subtracting the average of the values for both the CRC and NSCLC pools and was plotted as a function of concentration, FIG. 4B. The difference in adsorption between the cfDNA of healthy individuals and those with cancer (e.g., difference in average %ir values) at each concentration was quantified, and the p-values representing the statistical differences at each concentration are displayed in Table 3.

[0131] To identify the optimal cfDNA concentration with respect to the largest difference in adsorption between cfDNA from healthy individuals and those with either CRC or NSCLC cancer a range of concentrations (0.1 - 10 pg/pL) were investigated, FIG. 4A. Within the 0.1- 2 pg/pL range there is not a statistically significant separation between the relative peak current values for the normal and cancer cfDNA sample pools. However, at concentrations above 2 pg/pL statistically significant separation between the normal and cancer cfDNA pools was observed. FIG. 4B shows the difference in relative peak current (%ir) between the normal and an average of both cancer cfDNA pools as a function of increasing concentration. A correlation was observed; as the concentration of the pooled cfDNA sample incubated on the working electrode surface was increased, the difference in adsorption between the normal and cancer cfDNA pools was also increased. Table 3 shows the p-values generated from the relative peak current data in FIG. 4A, n = 8 SPEs per sample. Although there was not a linear trend between increasing concentration and the p-values for the difference in %ir, the most statistically significant differences in %ir were found at higher concentrations (e.g., 9 and 10 pg/pL). In addition, increasing the concentration more significantly (e.g., 25 pg/pL) resulted in a saturation of cfDNA at the electrode surface eliminating the ability of the sensor to distinguish between normal and cancer cfDNA as shown in FIG. 4B. As a result, a working concentration of 10 pg/pL was chosen for subsequent experiments investigating the ability to observe changes in %ir values for a single patient at different timepoints throughout the duration of an administered cancer treatment.

Table 3. Difference in adsorption of cfDNA from healthy individuals and those with both CRC and NSCLC cancer. The difference in adsorption was assessed through a correlation with the difference in %ir, or the reduction in peak current magnitude associated with the amount of Damount of DNA bound to the electrode surface. The p-values were calculated using a standard T-Test.

Example 6

[0132] Patient Samples from Scripps were run using the Dropsens electrochemical detection protocol. Each sample was diluted to a working concentration of 25 pg/pL in pH 2 incubation buffer prior to running the measurement.

[0133] The commercial gold screen-printed electrodes (SPE; C223BT model) with a gold working electrode diameter of 1.6 mm, gold counter electrode and a silver reference electrode were obtained from Dropsens and were stored at room temperature protected from light and humidity. The gold SPEs were fabricated on a ceramic substrate (33 x 10 x 0.5mm). [0134] Evaluation of the adsorption DNA samples to the electrode surface was performed using a series of differential pulse voltammetry (DPV) experiments conducted using a Palmsens benchtop potentiostat. The electrochemical window for the measurements was -0.5- 0.5 V. The experimental parameters were E-step (0.01 V), E pulse (0.05 V), t pulse (0.05 s) and the scan rate (0.1 V/s).

[0135] An adhesive barrier was applied to the SPE to avoid covering any of the three electrodes. The SPE was submerged in 50 pL of 2.5 mM F'₃Fe(ClV) ^_'^/4_solution. Two electrocleaning sweeps (DPV -1 - IV) were run followed by two baseline DPV (-0.5 - 0.5V) measurements, the second of which was used for calculations. A baseline was run for each SPE. The electrodes were rinsed with DI water, dried, and any electrodes with a reading below 23 pA with new electrodes were replaced and the above steps were repeated to confirm a reading above 23 pA.

[0136] The SPEs were stratified based on the baseline signal - ranking from highest to lowest peak current magnitude. All patient timepoints were exposed to the entire range of peak current magnitudes, for example alternate between patient timepoints for each electrode across the baseline spectrum of peak current magnitudes.

[0137] 2.5 pL of 25 pg/pL cfDNA solution was incubated inside the adhesive barrier. After

10 minutes electrode was rinsed with a drop of PBS solution. After fan-assisted drying 2.5 mM solution was incubated with the SPEs. Two DPV (-0.5 - 0.5V) measurements were run, with the second used for calculations.

[0138] Samples were measured using an n =8 via the Palmsens multiplexer. The relative peak current (% ir) was calculated using peak current values for the baseline and measurement DPVs by the following equation: i_r%

Example 7

[0139] Collection of samples from Drammen Hospital was conducted in Norway, the DNA was purified by a different group, and all of the patients had lung cancer. The analyzed group had 7 patients. In contrast to the previous set, most progressed. Thus, in many ways this group was different from the Scripps group (Example 6) and offered an opportunity to show the analysis is broadly applicable.

[0140] The samples were run using the BASi electrochemical detection protocol. Each sample was diluted to a working concentration of 25 pg/pL in pH 2 incubation buffer prior to running the measurement. [0141 ] The commercial gold screen-printed electrodes (SP1112) with a gold working electrode diameter of 2 mm, gold counter electrode and a silver reference electrode were obtained from BASi and were stored at room temperature protected from light and humidity. The gold SPEs were fabricated on a ceramic substrate.

[0142] Evaluation of the adsorption DNA samples to the electrode surface was performed using a series of differential pulse voltammetry (DPV) experiments conducted using a Palmsens benchtop potentiostat. The electrochemical window for the measurements was -0.5- 0.5 V. The experimental parameters were E-step (0.01 V), E pulse (0.05 V), t pulse (0.05 s) and the scan rate (0.1 V/s).

[0143] The BASi SPE was rinsed with PBS (pH 7.2) and dried. The SPE was submerged in 50 pL of 2.5 mM K₃Fe(CNy^ solution. Two baseline DPV (-0.5 - 0.5V) measurements were run, the second of which was used for calculations. A baseline was run for each SPE. The electrodes were rinsed with DI water, dried, and any electrodes with a reading below 23 p A with new electrodes were replaced and the above steps were repeated to confirm a reading above 23 p A.

[0144] The SPEs were stratified based on the baseline signal - ranking from highest to lowest peak current magnitude. All patient timepoints were exposed to the entire range of peak current magnitudes, for example alternated between patient timepoints for each electrode across the baseline spectrum of peak current magnitudes.

[0145] 6 pL of 25 pg/pL cfDNA solution was incubated with the working and reference electrodes by covering in a neat droplet. After 10 minutes electrode was rinsed with a drop of PBS solution. After fan-assisted drying 2.5 mM solution was incubated with the SPEs. Two DPV (-0.5 - 0.5V) measurements were run, with the second used for calculations.

[0146] Samples were measured using an n =8 via the Palmsens multiplexer. The relative peak current (% ir) was calculated using peak current values for the baseline and measurement DPVs by the following equation: i_r%

Materials and Methods

[0147] Materials PBS tablets (137 mM NaCl, 2.7 mM KC1 at pH 7.2), tris base, sodium chloride, Tween 20, and potassium hexacyanoferrate (III, IV) were purchased from Sigma Aldrich. Guanidinium isothiocyanate (GITC) was obtained from Chem-Impex. 100% ethanol and 0.5 M EDTA was obtained from ThermoFisher. All reagents used were of analytical grade without any cleaning or purification procedures. The un-methylated and methylated synthetic oligos (50bp dsDNA) were purchased from Integrated DNA Technologies (IDT) and were stored at 5°C in TBS buffer at pH 8.

[0148] Clinical Samples Clinical cfDNA samples for FIG. 2 were obtained from the University of Colorado Cancer Center Pathology Shared Resource Biorepository under the CU Anschutz IRB and following consent. Blood samples were collected in EDTA tubes, plasma processed, and processed by Phenol:Chloroform:Isoamyl (25:24: 1) extraction using standard methods. Clinical cfDNA samples were obtained from 16 different subjects, 6 healthy individuals (Normal), 5 colorectal cancer subjects (CRC), and 5 subjects with non-small-cell lung cancer (NSCLC), one of which was removed from individual analysis due to having no detectable DNA by Agilent BioAnalyzer high sensitivity dsDNA analysis. All patients were chosen for indicators of late-stage disease. All samples were obtained in TE Buffer, pH 8 (Fisher Scientific) and stored at -40°C. Concentrations were obtained using an Agilent BioAnalyzer 2100 Instrument and High Sensitivity dsDNA kit. Clinical cfDNA samples were defrosted and diluted to a working concentration using our incubation buffer upon use.

[0149] For FIG. 4, frozen 1 mL aliquots of plasma from all 16 subjects were obtained from the University of Colorado Cancer Center Pathology Shared Resource Biorepository and extracted using the UltraPrep procedure. In brief, each 1 mL sample was thawed and digested using 10 pL Protease K (ThermoFisher) and 650 pL digestion buffer (5 M GITC, 25% Tween 20, 25 mM EDTA, and 0.01 M Tris) for 1 hour at 56°C. The solution was mixed with 3.3 mL binding buffer (3.5 M GITC, 0.01 M Tris, 1 mM EDTA, 2.5% Tween 20, and 45% Isopropanol) and 40 pL 400 nm silica beads (Spherotech). After a 10-minute binding incubation, a magnetic separation rack was used to remove the solution and resuspend the beads in 500 uL wash buffer 1 (3 M GITC, 40 mM Bis-Tris buffer at pH 6, 2 mM EDTA, 5% Tween 20, and 30% Isopropanol) and then 500 pL wash buffer 2 (50 mM Tris pH 8, 0.5 pM EDTA, 80% ethanol). The beads were finally washed with 100 pL 100% ethanol. The beads were dried, then eluted into 30 pL TE buffer (10 mM Tris, 1 mM EDTA, pH 8).

[0150] FIG. 3 patient 1 plasma samples were obtained from PlasmaLab under their IRB. Blood was collected, processed to collect plasma, and stored at -80°C until use. The cfDNA was extracted using the UltraPrep protocol described above and analyzed concentration using Agilent BioAnalyzer 2100 and their High Sensitivity dsDNA kit. Patient 2 samples were obtained from collaborators Ramachandran (University of Colorado Anschutz) and Brustugun (University of Oslo) under their IRB, into EDTA tubes, plasma processed and stored at -80°C until use. Ramanchandran lab performed standard UltraPrep extraction methods and the concentration was analyzed using Qubit High Sensitivity dsDNA quantification kit. Patient 3 urine samples were obtained from PrecisionMed and were collected under their IRB. The urine was directly frozen at -80°C after collection. The cfDNA was extracted using the UltraPrep protocol described above and analyzed cfDNA concentration using Agilent BioAnalyzer 2100 and their High Sensitivity dsDNA kit (100 to 200 bp). All samples were stored at -20°C until electrochemical evaluation.

[0151] Electrode Materials The commercial gold screen-printed electrodes (C223BT model) with a gold working electrode diameter of 1.6 mm, gold counter electrode and a silver reference electrode were obtained from Dropsens and were stored at room temperature protected from light and humidity. The gold SPEs were fabricated on a ceramic substrate (33 x 10 x 0.5mm). The commercial thin-film gold electrodes (TPEs) were obtained from Micrux with a gold working electrode diameter of 1 mm and gold counter/reference electrodes. The three- electrode cell was fabricated on a glass substrate (10 x 6 x 0.75 mm) with an insulating polyimide layer used to delimit the electrochemical cell enabling the use of small sample volumes.

[0152] Electrochemical Cleaning Protocol TFEs Use of the thin-film gold electrodes from Micrux required an initial cleaning step. Electrode cleaning was conducted via cyclic voltammetry using 0.0 IM sulfuric acid. The electrochemical window used was (-1 - 1.2 V) and cycling was performed until the oxidation peak reached 0.2 mA. Further cycling beyond 0.2mA oxidation peak resulted in Au stripping at the working electrode surface.

[0153] DNA adsorption and Electrochemical Measurement Evaluation of the adsorption of DNA samples to the electrode surface was performed using a series of differential pulse voltammetry (DPV) experiments conducted using a Palmsens benchtop potentiostat. The electrochemical window for the measurements was -0.5 to 0.5 V. The experimental parameters were E-step (0.01 V), E pulse (0.05 V), t pulse (0.05 s) and the scan rate (0.1 V/s). An initial baseline DPV measurement was performed first using 50 pL of 2.5 mM K_3 Fe(CN)_6^A((3- /(4-))solution. The electrode was then rinsed with distilled water and dried. Next 10 pL of the sample containing cfDNA in either pH 2 or pH 7.2 buffer was dropped onto the working electrode surface and left to incubate for 10 minutes. The sample was then gently rinsed from the surface using PBS. Finally, the sample DPV measurement was performed using 50 pL of 2.5 mM K_3 Fe(CN)_6^A((3-/(4-))solution. Samples were measured using an n =8 via the Palmsens multiplexer. The relative peak current (% ir) was calculated using peak current values for the baseline and measurement DPVs by the following equation: i_r%=((i_Baseline- i_Sample))/i_Baseline * 100.

Claims

CLAIMS What is claimed is:

1. A method for determining cancer status in a subject, the method comprising: measuring cancer cell free DNA (cfDNA) in a first biological sample and a second biological sample, wherein the first and the second biological sample are obtained from the subject separated by a period of time; and classifying the cancer as progressive, regressive, or unchanged based on a change in cancer cfDNA in the second biological sample as compared to the first biological sample.

2. The method of claim 1, further comprising: measuring cfDNA in at least one additional biological sample separated from an immediately preceding biological sample by a period of time; and classifying the cancer status based on the change of the cancer cfDNA in the least one additional biological sample as compared to any preceding biological sample.

3. The method of claim 1 or 2, wherein measuring cancer cfDNA levels comprises: adding an acidifying agent to a biological sample to form an acidic sample; contacting an electrode with the acidic sample; and detecting an electrochemical signal indicating the cancer cfDNA.

4. The method of claim 3, wherein detecting the electrochemical signal can include performing at least one of voltammetry, cyclic voltammetry, square wave voltammetry, differential pulse voltammetry, amperometry, chronoamperometry, potentiometry, chronopotentiometry, coulometry, chronocoulometry, conductometry, and impedometry.

5. The method of claim 3 or 4, wherein the electrochemical signal is a measure of the cancer cfDNA adsorbed to the electrode.

6. The method of any one of claims 3-5, wherein the electrochemical signal is a change in peak current magnitude following contacting the electrode with the acidic sample.

7. The method of any one of claims 3-6, wherein a progressive cancer is characterized by a decreased peak current magnitude of second biological sample compared to the first biological sample.

8. The method of any one of claims 3-7, wherein the acidic sample has a pH of less than about 4.

9. The method of any one of claims 3-8, wherein the acidic sample has a pH from about 1 to about 3.

10. The method of any one of claims 3-9, wherein the acidic sample is at a pH of about 2.

11. The method of any one of claims 1-10, further comprising extracting cfDNA from the first biological sample, the second biological sample, and/or the at least one additional biological sample prior to forming an acidic sample, prior to measuring cancer cfDNA, or both.

12. The method of any one of claims 1-11, wherein the first biological sample, the second biological sample, and the at least one additional biological sample are each individually selected from a whole blood sample, a plasma sample, a serum sample, and a urine sample.

13. The method of any of claims 1-12, wherein the cancer is progressive when the cancer cfDNA in the second biological sample is increased compared to the first biological sample or the cancer cfDNA in the least one additional biological sample is increased as compared to any preceding biological sample.

14. The method of any of claims 1-12, wherein the cancer is regressive when the cancer cfDNA in the second biological sample is decreased compared to the first biological sample or the cancer cfDNA in the least one additional biological sample is decreased as compared to any preceding biological sample.

15. The method of any one of claims 1-14, wherein the period of time is at least one week.

16. The method of any one of claims 1-15, wherein the period of time is at least one month.

17. The method of any one of claims 1-16, wherein the period of time is less than 6 months.

18. The method of any one of claims 1-17, further comprising treating the subject based on the cancer progression.

19. The method of any one of claims 1-18, wherein the first biological sample predates the start of a treatment regimen and the second biological sample postdates the start of the treatment regimen.

20. The method of claim 19, wherein the method further comprises determining the clinical benefit of the treatment regimen based on if the cancer status was progressive, regressive, or unchanged.

21. The method of claim 19 or 20, wherein the treatment regimen comprises surgery, administration of a chemotherapeutic agent, immunotherapy, radiotherapy, or combinations thereof.

22. The method of any one of claims 19-21, wherein the treatment regimen comprises immunotherapy.

23. The method of any one of claims 19-22, further comprising treating the subject based on the clinical benefit of the treatment regimen.

24. A method for detecting cancer in a subject, the method comprising: measuring cancer cell free DNA (cfDNA) in a biological sample obtained from the subject; and identifying the subject as having cancer based on: the cancer cfDNA as compared to a control, non-cancerous cfDNA; the cancer cfDNA as compared to a cutoff value for the cancer cfDNA; or a cutoff value for a ratio of the cancer cfDNA to total cfDNA.

25. The method of claim 24, wherein measuring cancer cfDNA comprises: adding an acidifying agent to the biological sample to form an acidic sample; contacting an electrode with the acidic sample; and detecting an electrochemical signal indicating the cancer cfDNA in the biological sample.

26. The method of claim 25, wherein detecting the electrochemical signal can include performing at least one of voltammetry, cyclic voltammetry, square wave voltammetry, differential pulse voltammetry, amperometry, chronoamperometry, potentiometry, chronopotentiometry, coulometry, chronocoulometry, conductometry, and impedometry.

27. The method of claim 25 or 26, wherein the electrochemical signal is a measure of the cancer cfDNA adsorbed to the electrode.

28. The method of any one of claims 25-27, wherein the electrochemical signal is a change in peak current magnitude following contacting the electrode with the acidic sample.

29. The method of any one of claims 25-28, wherein the acidic sample has a pH of less than about 4.

30. The method of any one of claims 25-29, wherein the acidic sample has a pH from about 1 to about 3.

31. The method of any one of claims 25-30, wherein the acidic sample is at a pH of about 2.

32. The method of any of claims 25-31, further comprising extracting cfDNA from the biological sample prior to forming an acidic sample, prior to measuring the cancer cfDNA, or both.

33. The method of any one of claims 25-32, wherein the biological sample is a whole blood sample, a plasma sample, a serum sample, or a urine sample.

34. The method of any one of claims 25-33, wherein the subject has cancer when the cancer cfDNA in the biological sample is greater than the control and/or greater than the cutoff value for the cancer cfDNA.

35. The method of any one of claims 25-33, wherein the subject has cancer when the ratio of cancer cfDNA to total cfDNA is greater than the cutoff value.

36. The method of any one of claims 24-35, wherein the cancer is an advanced stage cancer.

37. The method of any one of claims 25-36, further comprising administering a treatment regimen to the subject identified as having cancer.

38. The method of claim 37, wherein the treatment regimen comprises surgery, administration of a chemotherapeutic agent, immunotherapy, radiotherapy, or combinations thereof.

39. A method for detecting cancer DNA in a sample, the method comprising: contacting an electrode with an acidic sample comprising or suspected of comprising cancer DNA; and measuring an electrochemical signal indicating cancer DNA.

40. The method of claim 39, wherein the acidic sample is at a pH of less than about 4.

41. The method of claim 39 or 40, wherein the acidic sample is at a pH from about 1 to about 3.

42. The method of any one of claims 39-41, wherein the acidic sample is at a pH of about 2.

43. The method of any one of claims 39-42, wherein the acidic sample comprises an acidifying agent.

44. The method of any one of claims 39-43, wherein the acidic sample comprises a biological sample.

45. The method of any one of claims 39-44, wherein the method comprises adding the acidifying agent to the biological sample, thereby forming the acidic sample, prior to contacting the acidic sample with the electrode.

46. The method of any one of claims 39-45, wherein detecting the electrochemical signal can include performing at least one of voltammetry, cyclic voltammetry, square wave voltammetry, differential pulse voltammetry, amperometry, chronoamperometry, potentiometry, chronopotentiometry, coulometry, chronocoulometry, conductometry, and impedometry.

47. The method of any one of claims 39-46, wherein the electrochemical signal is a measure of the cancer DNA adsorbed to the electrode.

48. The method of any one of claims 39-47, wherein the electrochemical signal comprises a change in peak current magnitude following contacting the electrode with the acidic sample.

49. The method of any one of claims 39-48, wherein the electrode is a gold electrode.

50. The method of any one of claims 39-49, wherein the acidic sample comprises less than 100 pg/uL DNA.

51. The method of any one of claims 39-50, wherein the acidic sample comprises less than 10 pg/uL DNA.

52. A system comprising: an electrochemical detection system comprising a gold electrode; and an acidic sample comprising cancer DNA or a sample comprising cancer DNA and an acidifying agent.

53. The system of claim 52, wherein the electrochemical detection system further comprises one or more of: a working electrode, a counter electrode, a sample reservoir, and a data acquisition unit.

54. The system of claim 52 or 53, further comprising a sample purification system.

55. The system of claim 54, wherein the sample purification system comprises filters, chromatography columns, chromatography beads, microfluidic devices, or combinations thereof.

56. A composition comprising cancer DNA and a buffer or acidifying agent, wherein the composition is at acidic pH.

57. The composition of claim 56, wherein the composition has a pH of less than about 4.

58. The composition of claim 56 or 57, wherein the composition has a pH from about 1 to about 3.

59. The composition of any one of claims 56-58, wherein the composition has a pH of about 2.