Patent Description:
Effective cancer management depends on early diagnosis, accurate tumor staging, and consistent monitoring. While many methods have been developed for the detection and quantification of nucleic acids, e.g., NanoDrop Microvolume Spectrophotomer and Fluorometer, many current diagnostic procedures are invasive, expensive and unpleasant. In multiple recent published studies, circulating cell-free DNA (cfDNA) concentration and integrity (fragmentation pattern) has shown promise as a highly sensitive and specific, minimally invasive blood biomarker for multiple cancer types (see, e.g., <NPL>;<NPL>);<NPL>)). A number of these studies have indicated the utility of a highly sensitive assay to measure cfDNA integrity (fragmentation pattern) and concentration based on quantitation of an ALU element, the most common type of retrotransposable elements (RE) in the human genome (Table <NUM>). RE-based methods for quantitating DNA are attractive due to their superior sensitivity (multi-copy representation in the genome) and robustness. The sequence of ALU Yb8 and other ALU Yb subfamilies are known in the art, see e.g., <NPL>incorporated herein by reference in its entirety.

The most commonly employed cfDNA integrity/concentration assessment method, the ALU <NUM>/<NUM> bp index, targets sequences of a single ALU element, and thus the two fragments analyzed are not independent. This precludes use of these targets in a single multiplexed assay for maximum accuracy, efficiency and practical clinical use. This prior art method poses several particular problems. First, evaluating the first sequence and the second sequence in conventional single-plex polymerase chain reactions (PCR) wherein a single target is amplified in a single reaction well rather than multiplexing the two sequences into a single reaction mixture introduces well-to-well variability into the results. Every PCR reaction is somewhat different from every other PCR reaction, and experimental variation in set-up steps, such as variation in pipetting volumes, introduces error and can impact the results. Secondly, it has been shown (see U. Patent Application Publication No. <CIT>, incorporated herein in its entirety) that the primers used in prior art studies to amplify these specific <NUM> bp/<NUM> bp sequences show poor primer specificity, with false signals being generated from non-template controls. Thirdly, single-plex amplification prohibits the incorporation of an internal PCR control. The use of an internal PCR control is useful for confirming the success of the reaction and for providing confidence that other experimental factors such as the presence of PCR inhibitors in the sample have not interfered with sample integrity. Additionally, single-plex amplification of each target is cumbersome, more labor-intensive and less cost effective than is running a multiplexed amplification.

One of the cancer types studied using cell free DNA integrity is colorectal cancer (CRC). The current gold-standard for CRC diagnosis and staging is colonoscopy and subsequent histological examination. While specific and accurate, colonoscopy is invasive, expensive, and poses some risks; all of which decrease patient compliance to screening recommendations and discourage routine monitoring. In CRC and a few other cancer types, tissue biopsy is supplemented with detection of cancer protein biomarkers in blood serum, e.g. carcinoembryonic antigen (CEA). Such assays have the significant advantage of being minimally invasive and also do not require immediate localization of the tumor. Nevertheless, these assays suffer from limited sensitivity. CEA, one component of the current standard of care for CRC post-treatment monitoring, has relatively low sensitivity and specificity for early (stages I and II) and late (stages III and IV) disease (early: <NUM>% sensitivity and <NUM>% specificity; late: <NUM>% sensitivity and <NUM>% specificity) (<NPL>)). Given this performance, CEA is not recommended for CRC diagnosis according to the National Comprehensive Cancer Network guidelines for CRC (<NPL>).

Furthermore, imaging tests such as computerized tomography (CT) scans, bone scan, magnetic resonance imaging (MRI), positron emission tomography (PET) scan, ultrasound, and x-ray, among other radiological imaging, may be used to monitor disease progression and therapeutic effectiveness. Such tests have the downside of exposing patients to large amounts of radiation over the course of their treatment and while they are in remission, are costly, and may be burdensome.

Characterization of cell-free DNA (cfDNA), DNA found in circulation in human blood plasma and serum, has emerged as an exciting prospect for a new generation of blood-based tools for cancer detection, monitoring and surveillance. It is also an exciting prospect for monitoring minimum residual disease, therapeutic effectiveness, and disease progression. Nucleic acid circulation in human blood plasma was first reported in <NUM> (<NPL>)). Leon, et al. , (<NUM>) were the first to report that mean cfDNA levels were significantly higher in the serum of patients with malignant cancers versus healthy patients (<NPL>). In the past two decades, many details of cfDNA biology, and the relationship between cfDNA and disease, have been elucidated. A brief primer of these studies is provided below.

Circulating cfDNA is derived from both the nuclear and mitochondrial genomes of normal and tumor cells (Mandel and Matais <NUM>, referenced supra; <NPL>)). Both coding and noncoding portions of the genome are represented among circulating cfDNA (<NPL>on). Although several mechanisms are believed to contribute to the circulating cfDNA pool, including spontaneous release of free, exosome encapsulated, and microvesicle-encapsulated DNA into the bloodstream, cell death is the major generator of circulating cfDNAs (<NPL>)). Cell turnover in normal cells is ordinarily due to apoptosis, which results in stereotyped sized fragments of DNA: a monomeric form composed of ~ <NUM> bp fragments of DNA and associated nucleosomes, and reduced amounts of oligomeric forms. Alternatively, tumor cells turn over using a diversity of cell death pathways, not only apoptosis, but also necrosis, autophagy, and mitotic catastrophe (<NPL>)). Non-apoptotic pathways non-specifically and incompletely degrade DNA, generating substantially longer DNA fragments, up to <NUM> kilo bases in the case of necrosis (Jahr, S. , cited supra). Differences in the rate of cell death and type of cell death pathway utilized between normal and cancer cells lead to distinct characteristics of cfDNA pools that distinguish patients with and without cancer. cfDNAs have variable half-life within the body, ranging from minutes to hours (<NPL>); <NPL>); <NPL>). Short half-life implies that circulating cfDNA levels provide a dynamic measure of the physiological and pathological state of an individual. Finally, there is evidence that a small fraction of circulating cfDNA from blood is able to pass the kidney barrier and enter urine. These cfDNAs are called 'trans-renal' cfDNAs (<NPL>);<NPL>)). The specific physiology of transrenal cfDNAs awaits detailed exploration.

Circulating cfDNAs from patients with and without cancer differ in a number of ways. Tumor genomes harbor specific genetic and epigenetic alterations that distinguish them from normal genomes, and these differences are reflected in cfDNAs. Nonspecific characteristics of cfDNA, such as concentration and integrity, differ between cancer patients and control subjects due to the specific mechanisms of cfDNA release into the blood by normal versus tumor cells. cfDNA concentration and integrity have often been found to be elevated in patients with cancer due to high rate of tumor cell death (reviewed in <NPL>;<NPL>)). However, absolute cfDNA concentration significantly varies among currently employed assays, significantly hampering the ability to compare results across studies. There is currently no standardized, validated, commercially available cfDNA concentration and integrity assay. There are no reports in the prior art of using a multiplexed quantitative polymerase chain reaction (qPCR) system of the kind described herein for accurate simultaneous measurement of concentration and integrity of cfDNA.

Efforts in cell-free DNA (cfDNA) testing using blood samples focus almost exclusively on mutations which are not tumor-type agnostic and lack the analytic sensitivity required for therapy monitoring. cfDNA was detected in blood as early as the <NUM> by Madel and Metais (<NPL>. ) and since this time, many studies have shown that the presence of elevated levels of cfDNA in the blood of patients with several cancer types including colorectal cancer (CRC) have poor prognosis (<NPL>. The elevation in cfDNA levels originates through the release of DNA from cellular necrosis and apoptosis of tumor cells. In healthy individuals, the main source of cfDNA in circulating blood is through necrosis. Necrosis generates a spectrum of DNA fragments of different sizes, due to random digestion by DNases (up to several kbp). Apoptosis generates small and uniform DNA fragments (less than <180bp) (<NPL>). A recent study of fragment length of cfDNA shows mutant KRAS alleles tend to be significantly shorter when compared with DNA fragments bearing wild-type allele by densitometry in pancreatic cancer (<NPL>). Older publications often reached unreliable conclusions about correlation of cfDNA fragment size concentration and cancer progression due to inadequate cell free DNA purification protocols for small DNA fragments, as well as sample collection, plasma separation and storage protocols causing contamination of large DNA fragments from whole cell degradation. However, with the availability of new sample collection tubes with preservatives to prevent cellular contamination, such as Streck tubes, as well as availability of improved cfDNA extraction methods, several recent published studies have clearly established that cfDNA from cancer cells are smaller in fragment size as compared to cfDNA produced by noncancerous cells (<NPL>).

In clinical oncology, cfDNA has been suggested as a new surrogate marker for therapy response, disease progression and/or detecting early relapse. More recent studies have begun to explore the potential use of circulating tumor DNA (ctDNA) and oncogene biomarkers for prognostic uses (<NPL>. Although ctDNA markers can provide great information in cancer biology, challenges and limitations have arisen when working with it as ctDNA can be as little as <NUM>% of the entire cfDNA in plasma (<NPL>). Due to heterogeneity intratumorally as well as between tumors and metastatic lesions make it difficult to detect the cancer progression within individual patients. Additionally, the use of oncogene biomarkers may only represent a subpopulation of patients expressing these genes resulting in the missed opportunity to monitor an entire population of patients undergoing treatment. For ctDNA analysis, the use of sophisticated instrumentation by highly trained personnel, high blood volume requirements and cost are prohibitive factors especially in low resource areas and for economically disadvantaged patients. On the contrary, cfDNA is circulating in every individual's blood while elevated in cancer patients. A recent study comparing RECIST results to Carcinoembryonic Antigen (CEA), cfDNA or ctDNA levels demonstrated cfDNA had the highest correlation compared to RECIST for tumor burden and tumor volume of the main lesion (<NPL> ). This study highlights a great potential use of cfDNA for cancer monitoring, which can track the change in tumor burden.

Retrotransposable Elements (REs) are mobile element insertion polymorphisms that are essentially homoplasy-free characters, identical by descent and easy to genotype (reviewed in <NPL>). ALUs are REs that are approximately <NUM> bp insertions and are distributed throughout the human genome in large copy number. In addition to the major retrotransposon families, REs include smaller families of transposons such as SVA or long interspersed element ("LINE"). SVA elements, named after its main components, short interspersed element ("SINE"), variable number tandem repeat ("VNTR") and Alu element ("ALU"), contain the hallmarks of retrotransposons, in that they are flanked by target site duplications ("TSDs"), terminate in a poly(A) tail and they are occasionally truncated and inverted during their integration into the genome (<NPL>); <NPL>). Long-interspersed Elements (LINE) are similar to ALU and SVA in that they also contain the hallmarks of retrotransposons and are high copy number, but differ in size, being up to several kilo bases in length (<NPL>).

RE-based quantitation methods are advantageous when compared to current, commercially available systems due to the presence of a large number of fixed insertions. With a high copy number of subfamily-specific RE repeats within the human genome, these human-specific DNA assays have a very sensitive dynamic range of <NUM> pg to <NUM> ng (<NPL>)). For example, the ALUYb lineage contains approximately <NUM> copies per genome and SVA contains approximately <NUM> full length element copies per genome (Wang, H. , referenced supra; <NPL>)). This large copy number minimizes the effect of variation between individuals, resulting in highly reproducible quantitation values.

<CIT>, is entitled "Development of a Highly Sensitive Quantification System for Assessing DNA Degradation and Quality in Forensic Samples" and describes the detection of DNA quality with a multiplex reaction using ALU and SVA for human DNA quantification. Though very useful for forensic purposes, the described method does not detail specific application to cell free DNA from plasma and/or serum. The amplicon sizes needed for a cfDNA assay are different from those needed for forensic applications, and other details of the two methods such as amplification conditions and primer/probe concentrations differ as well.

Exemplary embodiments of prior art methods and systems for multiplex determination of cfDNA in a sample are disclosed in <CIT>, <CIT> and <CIT>.

A majority of healthy (non-cancer) human cfDNA fragment sizes are around <NUM>-180bp long. Cell free DNA released from cancer cells (often called circulating tumor DNA or ctDNA) are shorter than the cfDNA released from normal cells. In contrast to cfDNA in samples from cancer patients, the majority of cfDNA fragments from non-cancer humans are generated from apoptotic cells, generating around <NUM> bp-long (or <NUM>-180bp) fragments equivalent to the length of DNA that wraps around <NUM> nucleosome, and sometimes accompanied by DNA fragments with sizes in multiples of <NUM> bp. The qPCR method of measurement of any retrotransposable element (RE) target sequence quantitates cfDNA fragments equal to or longer than the size of the RE target sequence. For example, an qPCR measurement of Yb8 ALU target sequence of 80bp quantitates cfDNA fragments of >80bp in length, including both short and long cfDNA fragments that comprise the <NUM> bp RE target sequence. On the other hand, qPCR measurement of a 265bp SVA target sequence quantitates cfDNA fragments of >265bp, those comprising the <NUM> bp RE target sequence, which does not include cfDNA fragments of less than 265bp in length.

The present invention is directed to subject matter as defined in the appended claims.

Herein we discuss the development and evaluation of a retrotransposable interspersed element (RE) based multiplexed qPCR assays to robustly quantitate and distinguish cfDNA integrity and concentration in test and control subjects' bodily fluids, e.g., plasma and serum. The assays provide accurate, minimally invasive, rapid, high-throughput, and cost-effective methods with the potential to complement for characterizing minimum residual disease, therapeutic effectiveness, and disease, e.g., cancer, progression in humans, thereby improving patient outcomes.

The methods described herein for assessing the cfDNA and ctDNA integrity and concentration and thereby assessing the presence of cancer cells do not depend on a clonal mutation being present in the cancer cells. As such, the methods are "agnostic" in that the methods can be applied to samples from patients having many different types of cancers. Moreover, the sensitivity of the methods described herein is far greater than other cfDNA and ctDNA assays as the levels of cfDNA and ctDNA above a normal threshold are detected in virtually all cancer patients tested. In addition, the methods described herein have low Cost of Goods Sold, are based on commonly used qPCR lab methodology and have a fast Turnaround Time (TAT), e.g., the DNA integrity and concentration can be assayed and a conclusion as to the presence of cancer cells or if a cancer therapy is ineffective and whether a patient has progressive disease can be completed quickly, e.g., in less than <NUM> hours, less than <NUM> hours, less than <NUM> hours, or less than about <NUM> hours.

An embodiment of the invention is a method whereby RE targets are simultaneously assayed in a single, highly sensitive qPCR reaction, wherein a single RE target is amplified in a single qPCR reaction vessel, e.g., a well, (singleplex qPCR) or wherein multiple RE targets are amplified in a single qPCR reaction vessel, e.g., a well (multiplex qPCR) , optionally including an internal positive control to monitor the presence of PCR inhibitors potentially present in the sample of blood serum, plasma, urine, or other biological fluid. This method enables development of an accurate, rapid, affordable, minimally invasive, high throughput, cost effective clinical test to complement or replace existing procedures and improve characterizing minimum residual disease, therapeutic effectiveness, and disease progression in humans.

Accordingly, one embodiment of the invention is a qPCR method that accurately quantitates cfDNA in a patient's biological fluids including, e.g., blood plasma or serum as an indication of cancer cells present in the patient or as an indication of the ineffectiveness of a neoadjuvant or a cancer therapy or as an indication the patient has progressive disease. The method may be singleplex wherein a single RE target is amplified in a single qPCR reaction well or the method may be multiplex wherein multiple RE targets are amplified in a single qPCR reaction well.

Another embodiment of the invention is a qPCR method that accurately provides a determination of the extent of fragmentation or integrity of cfDNA in biological fluids including, e.g., blood plasma or serum, as an indication in the level of "minimum residual disease" ("MRD"). The method may be singleplex wherein a single RE target is amplified in a single qPCR reaction well or the method may be multiplex wherein multiple RE targets are amplified in a single qPCR reaction well.

It is also disclosed a three RE target (a first "short" RE target, a second "short" RE target, and one "long" RE target) multiplex RE-qPCR assay to accurately and robustly obtain cfDNA concentration, a determination of fragmentation and integrity, and DNA integrity index ("DII" or "DI") of biological samples from normal controls and patients having cancer, e.g. colorectal cancer (CRC), by direct qPCR from plasma or serum samples with or without DNA purification. The assay may also include one internal positive control synthetic target. The short RE targets are preferably about 60bp to about 135bp in length, about <NUM> bp to about 130bp, or about <NUM> to about <NUM> bp in length with the proviso that the short RE targets differ sufficiently, e.g., in length and sequence, so that their amplification products generated in the qPCR assay can be distinguished from each other, e.g., the short RE targets may differ at least by about 10bp, at least by about <NUM> bp, or at least by about <NUM> bp in length. The third long RE target is preferably about <NUM> bp to <NUM> bp in length, or <NUM> bp to <NUM> bp, e.g., about <NUM> bp to <NUM> bp. DII indicates a level of cfDNA fragmentation and is a ratio of long target quantities to a short target quantity. DII as used herein is a ratio of the long RE target, e.g., <NUM> base-pairs to the short RE targets, e.g., <NUM> base-pairs (<NUM> bp/<NUM> bp). When DII (<NUM> bp/<NUM> bp) is lower than <NUM>, it indicates the major source of cfDNA is from apoptotic cells. When DII (<NUM> bp/<NUM> bp) is above <NUM>, cfDNA are also generated through necrosis. Information on DNA integrity and DII, is found in <NPL>) and<NPL>) incorporated herein in their entirety by reference.

An embodiment of the invention is a multiplexed method to quantitate the integrity of circulating cell free human DNA in a test subject, comprising providing a sample of serum, plasma, urine, or other biological fluid from the test subject, the sample comprising cell free human DNA, and the cell free human DNA comprising a first and second short RE target each having a length of between about <NUM> and 135bp, about <NUM> to about <NUM> bp or about <NUM> and <NUM> base pairs, then using a multiplex quantitative polymerase chain reaction (qPCR) method to quantitate the short RE targets, obtaining for the quantitated RE targets a threshold cycle number, comparing each threshold cycle number with a standard curve to determine a quantity of the RE targets that was present in the sample, and determining the quantity of each of the RE targets is higher in the test subject's sample as compared to a control sample, e.g. a sample from a healthy subject, concluding the test subject should receive a treatment and administering the treatment to the test subject. For example, the cfDNA concentration measured for a first short RE target of <NUM> bp (Yb-<NUM>-80bp), and a second RE target of <NUM> bp (Yb-<NUM>-120bp), and a third RE target of <NUM> bp (SVA <NUM>), in plasma samples from <NUM> healthy controls and <NUM> cancer patients is set forth in Table <NUM>. The RE targets were amplified using the primer pairs for Yb-<NUM>-80bp, Yb-<NUM>-120bp and SVA <NUM> set forth in Tables 2A and 2B. The data in Table <NUM> demonstrate that while the absolute levels of the retrotransposable element targets are all different in each sample the amount of cfDNA in cancer patients is greater than that in control subjects. The concentration of the shortest <NUM> bp target is consistently higher than the longer the <NUM> bp target and the <NUM> bp target indicating that the cfDNA is highly degraded (apoptotic cell death). The method may further comprise the step of concluding the subject is in need of a cancer therapy or has progressive disease based on the difference in the amount of the short targets being above the threshold amount in a control sample, and optionally also based on the DII of the sample, and then administering the treatment to the subject.

It is further disclosed a multiplexed method to quantitate the integrity of circulating cell free human DNA, comprising providing a sample of serum, plasma, urine, or other biological fluid, preferably a plasma sample, the sample comprising cell free human DNA, the cell free human DNA comprising two retrotransposable element (RE) targets, a short RE target sequence between 60bp and <NUM> base pairs or between, 60bp and <NUM> base pairs, or about 70bp to about 130bp, and a long RE target sequence between 200bp-300bp, about 207bp to about 270bp, or about <NUM> bp to about <NUM> base pairs, the retrotransposable element genomic targets are preferably independent of each other, using a multiplex quantitative polymerase chain reaction (qPCR) method to separately and simultaneously quantitate the short and long RE targets, obtaining for each quantitated RE target a threshold cycle number, comparing each threshold cycle number with a standard curve to determine for each quantitated RE target a quantity of the RE targets that were present in the sample, and (i) calculating a ratio of the quantity of the long RE target to the quantity of the short RE target, and concluding based on the long RE target/short RE target ratio the subject should receive a treatment and administering the treatment to the subject.

It is further disclosed a multiplexed method to identify a subject who has cancer or MRD comprising,.

It is further disclosed a multiplexed method to identify a neoadjuvant or cancer therapy as ineffective or identify a subject who has a progressive cancer, is in remission, or has MRD comprising,.

In the multiplexed method to identify a cancer therapy as ineffective or identify a subject who has a progressive cancer, the difference between the quantity of the short RE targets in each of the first and second samples may be determined by determining the value of the amount of the shorter of the short targets minus the amount of the other short RE target in the first sample (said value = Frag <NUM>) and determining the value of the amount of the shorter of the short targets minus the amount of the other short RE target in the second sample (said value = Frag <NUM>) and determining the value of Frag1 minus Frag2 (said value = FragDiff) wherein a FragDiff of greater than a threshold value identifies the cancer therapy as ineffective or the subject as having progressive cancer or MRD.

In certain embodiments of the multiplexed method of the present invention, the retrotransposable element genomic targets may be an interspersed ALU, SVA or LINE1 element. In certain embodiments of the multiplexed method of the present invention, the retrotransposable element genomic targets may be each independently an interspersed ALU, SVA, or LINE element. In certain embodiments, these retrotransposable element genomic targets may each have a copy number in excess of <NUM> copies per genome.

Some embodiments of the multiplexed method of the present invention further comprise a step of adding a synthetic DNA sequence to the sample as an internal positive control (IPC) prior to the using step/ qPCR quantitation step, quantitating the internal positive control in the using step, and utilizing the quantitative internal positive control result in the comparing step to improve the accuracy and reliability of the comparing step to determine the amounts of the RE targets.

In embodiments of the multiplexed method of the present invention, the use of an internal positive control enables a determination of the concentration of cell free DNA in the sample.

In some embodiments of the multiplexed method of the present invention, the sample of serum, plasma, urine, or other biological fluid may be placed in a single tube, and the qPCR reactions for quantitation of the nucleic acid fragments may be carried out in that same single tube. Alternatively, each nucleic acid fragment may be separately and simultaneously amplified in separate tubes.

In some embodiments of the multiplexed method disclosed, the ratio of the quantity of the longer RE target to the quantity of a shorter RE target may serve as the DII of circulating cell free DNA for diagnostic and therapeutic applications. These diagnostic applications may include one or more of the characterizing minimum residual disease, therapeutic effectiveness, and disease progression in human patients, and treating such patients.

In certain embodiments, the multiplexed method of the present invention may include a step of deactivating or eliminating proteins that bind to the short nucleic acid fragment or the long nucleic acid fragment. This may be done by mixing the sample with a buffer including a surfactant and chelating agent, enzymatically digesting the protein, then using heat to deactivate and inactivate the digested protein, followed by centrifugation. Alternatively, dilution of the sample using <NUM> parts sterile water to one part sample by volume may have the effect of deactivating or eliminating these proteins.

In some embodiments, the multiplexed methods of the present invention may include a step of separating amplification products obtained from the qPCR reaction using electrophoresis. In some embodiments of this invention, the amplification products of the qPCR method used in the methods of this invention may be detected and/or quantified using electrical biosensors (see <NPL>).

In some embodiments, the multiplexed methods of the present invention may include a step of determining an optimum temperature for the qPCR reaction.

The multiplexed methods of the present invention may include a sample that comes from an individual who is suffering from cancer, is in remission from cancer, or who is at risk for developing cancer, who has received a treatment for cancer, e.g., a targeted therapy, chemotherapy, immunotherapy, targeted-immunotherapy, surgery to remove a tumor, or a radiotherapy. Targeted therapy is a type of cancer treatment that uses drugs or other substances to precisely identify and attack certain types of cancer cells. A targeted therapy can be used by itself or in combination with other treatments, such as traditional or standard chemotherapy, surgery, or radiation therapy.

In certain embodiments, the present invention may take the form of a multiplexed system for evaluating the effectiveness or ineffectiveness of a cancer therapy or for characterizing cancer in humans, the system including a sample of serum, plasma, urine, or other biological fluid, preferably a plasma sample, the sample comprising cell free DNA. The cell free DNA comprises one short retrotransposable element targets, or two short retrotransposable element targets, and optionally a long retrotranspoable element target. The short retrotransposable element targets may have a length in the range of about <NUM> base pairs to about <NUM> base pairs, about <NUM> to about 130bp, or <NUM> base pairs to about <NUM> base pairs, and two short retrotransposable element targets, may each with a length of <NUM> base pairs to <NUM> base pairs, or about <NUM> bp to about <NUM> bp, or about <NUM> base pairs to about <NUM> base pairs, preferably the two short RE targets differ in size and sequence sufficiently to distinguish their amplification products generated in the qPCR assay, e.g., the short RE targets differ in size by at least about <NUM> bp, at least about <NUM> bp, or at least about <NUM>. The third RE target being a fragment of another multi-copy retrotransposon with a length of about <NUM> bp to about 300bp, e.g., <NUM> bp to <NUM> base pairs. In an embodiment, the retrotransposable element targets are independent of one another. The system may further comprise an internal positive control (IPC) comprising synthetic DNA, a TaqMan® probe corresponding to each RE target and IPC, each probe comprising a detectable label that is distinct from the labels incorporated into the other probes, a forward primer and a reverse primer pair for amplifying each RE target and IPC, a DNA standard for generating standard curves for each RE target and IPC, a qPCR system for simultaneously amplifying each RE target and IPC and for producing a threshold cycle number for each RE target and IPC, and a qPCR data analysis system for producing DNA quantitation values for each RE target by interpolation using threshold cycle numbers and linear standard curves and for using the DNA quantitation values to produce an indication of the integrity of the cell free DNA and for characterizing cancer in a human.

In one embodiment, one or more of the retrotransposable element targets used in the methods and systems of the invention described herein, are an ALU (e.g. ALU-Yb8) target or an SVA. The ALU target may be, for example, a <NUM> bp target, a <NUM> bp target, a 71bp target, an <NUM> bp target, a <NUM> bp target, a <NUM> bp target. The targets may be amplified with forward and reverse primers. In embodiments comprising multiple RE targets from the same RE, e.g., ALU Yb-<NUM>, , PCR blockers/PNA clamping may be included to limit extension from the primers beyond the position of the blockers, thus limiting the extension from a primer pair used to amplify one RE target into the other RE target and thereby enhancing the specificity by limiting the production of extraneous or overlapping products. Preferably the PCR blockers are peptide nucleic acid (PNA) oligos and bind to the retrotransposable element between the targets to be amplified. See e.g., <FIG> depicting the position of the forward and reverse primers used to amplify an <NUM> base pair target and <NUM> bp target on an ALU, e.g., ALU-Yb8, and the position of the <NUM> bp blocker that limits extension from the <NUM> bp forward primer, and a <NUM> bp blocker that limits extension from the <NUM> bp blocker. In this embodiment, during qPCR the <NUM> base pair and <NUM> base pair specific forward primer and reverse primer hybridize to their respective sites on the ALU, but extension from the primers is limited by the presence of the PCR blocker at their respective sites.

There is a clear need in cancer management, and colorectal cancer (CRC) treatment specifically, for a standardized and validated blood test to sensitively and robustly quantitate cfDNA integrity and concentration. The present application addresses this need by creating a multiplex qPCR assay for quantitating cfDNA integrity and concentration based on retrotransposable element targets to identify, characterize and/or appropriately treat the patient having cancer, e.g., progressive disease, or MRD. The assays are also useful in indentifying a cancer therapy's effectiveness or ineffectiveness.

The most commonly employed method conducted by others in the field of cfDNA integrity and concentration assessment for cancer detection and monitoring is qPCR using the ALU <NUM>/<NUM> index. The methods described herein for assessing integrity and concentration of cfDNA and ctDNA quantitates "short" retrotransposable element targets having lengths between 60bp and 135bp, <NUM> bp to 130bp, or between 60bp and <NUM> bp to reliably indicate therapy effectiveness or ineffectiveness. The ranges between 60bp and 135bp, between <NUM> to about 130bp, e.g., 71bp to 132bp, or between 60bp and <NUM> bp ranges of ALU, SVA and LINE1 retrotransposable elements targets are also useful for discriminating between normal (non-cancer) human and humans with cancer, particularly progressive disease, or the presence of MRD. Preferably the retrotransposable elements are ALU, e.g., Yb-<NUM> ALU, SVA, or LINE1.

MRD refers to the small number of malignant cancer cells that remain in the body during or after treatment (see NCI Dictionary of Cancer Terms, https://www. gov/publications/dictionaries/ cancer-terms/def/<NUM>). Even when a patient is in remission from cancer and the solid tumor has shrunk beyond detection, the patient may still have MRD. The MRD assessment is used to determine if additional treatment is necessary, if a treatment already administered has been effective in reducing tumor load, or to select and administer a particular treatment of the subject. MRD assessment is mainly used in blood cancers (leukemia, lymphoma and myeloma), but is being studied in other solid cancers. MRD assessment has been used in guiding the treatment of cancer patients in cases of, e.g., resected hepatoma, resection of mastectomy, esophageal cancer, rectal cancer, anal cancer, head and neck cancer, colon cancer, lung cancer, breast cancer, neu metastatic breast cancer.

Cancer patients in remission must undergo quarterly imaging (e.g. MRI, x-ray, CT scan, or other radiology studies) to determine whether the cancer has returned. However, some patients in remission may not have a solid tumor that is detectable by imaging studies, but may still have MRD. The methods described herein for quantitating the integrity and concentration of cfDNA by using short retrotrasposable elements target(s) having a length between 60bp and 135bp, 70bp to 130bp, or <NUM> to 120bp, may be used to characterize cancer or MRD. The change in the amount of the quantitated short RE target sequence between 60bp and 135bp, <NUM> bp to 130bp or <NUM> bp to <NUM> bp over time may be used alone or in conjunction with standard assays to reliably identify subjects who have MRD or cancer progression or evaluate the ineffectiveness of a cancer therapy. Based upon a determination that the subject has MRD or progressive cancer, or the ineffectiveness of a therapy, additional rounds of therapy or another therapy may be administered to the subject. We demonstrate herein that cfDNA comprising elevated or increasing amounts of short ALU Yb8 targets of <NUM> base pair to <NUM> base pair, about 70bp to about 130bp, or 60bp to about 120bp sequence as compared to the amount of long RE targets, e.g., SVA or LINE targets, between <NUM> bp and about <NUM> bp, or between <NUM> bp and about <NUM> bp, between 260bp and 265bp, e.g., 265bp or <NUM> to be highly effective in discriminating between normal humans (non-cancer) and humans with cancer (see e.g., <FIG> and <FIG>). And because the methods herein do not rely on detecting CEA, the methods are "agnostic" and can be applied to samples from patients having or suspected of having any type of cancer, e.g., colorectal cancer (CRC), hepatoma, esophageal cancer, rectal cancer, anal cancer, head and neck cancer, colon cancer, lung cancer, e.g., non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC) breast cancer, and blood cancers, e.g., leukemia.

The methods described herein for assessing cfDNA integrity and concentration, using a sample from a subject, e.g., a plasma or serum sample or another bodily fluid sample, and RE targets, are useful in detecting, measuring, or monitoring cancer and are an additional parameter for use in the assessment of tumor load, cancer progression, therapy ineffectiveness and or MRD such that an appropriate treatment is administered to the subject. The methods described herein allow for detection of cancer cells in patients who have a nearly undetectable level as determined by standard clinical tests, such as imaging assays, e.g., CT scans or Xrays, or detection of cancer cells in a blood or tissue sample. The patients may be a patient suspected of having or treated for hepatoma, esophageal cancer, rectal cancer, anal cancer, head and neck cancer, colon cancer, colorectal cancer (CRC), lung cancer, e.g., non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC) breast cancer, and blood cancers, e.g., leukemia. Thus, the methods described herein are an improvement over existing methods because they reduce patients' exposure to radiation from imaging studies.

Patients diagnosed with cancer, including patients receiving a cancer therapy, may be categorized based on their disease progression, e.g., following a cycle of chemotherapy or immunotherapy or other therapeutic regime. A "complete response" ("CR") patient is one where there is no evidence of the disease due to a disappearance of all target lesions as determined by standard methods, e.g., such as CT scans or detection of cancer cells in a blood or tissue sample. A "stable disease" ("SD") patient is one where there is neither sufficient shrinkage of cancer lesion size to qualify for partial response ("PR") nor sufficient increase in lesion size to qualify for "progressive disease" ("PD") using as a reference the smallest sum of diameter of target lesions. A PR patient is one who demonstrates at least a <NUM>% decrease in the sum of the diameters of target lesions vs. the baseline sum of the diameters of the target lesions. Additionally, the sum of the diameters of the target lesions must demonstrate an absolute increase of at least <NUM> or one or more new lesions have been detected to be considered PR. A PD patient is one where there is at least a <NUM>% increase in the sum of the diameters of the target lesions vs. the smallest sum of target lesions, which may be the baseline sum.

The present invention is non-invasive and may also be used for screening high risk patients for onset of cancer, e.g., hepatoma, esophageal cancer, rectal cancer, anal cancer, head and neck cancer, colon cancer, colorectal cancer, lung cancer, breast cancer, neu metastatic breast cancer and blood cancers, e.g., leukemia. Patients may be considered "high risk" for a variety of reasons including past family history of cancer, environmental exposure, and lifestyle. However, it is not feasible, highly wasteful, and harmful for patients to be exposed to radiological scans to screen them for cancer.

The present invention may be used to distinguish between therapy ineffectiveness or futility and therapies that are partially ineffective. Current methods make it burdensome, costly, and inefficient to determine whether a therapy is ineffective in a patient or the patient experience a partial response to a therapy. The present invention allows clinical providers to detect noninvasively and quickly whether the therapy is entirely ineffective or partially ineffective. This allows providers to make quicker and better informed clinical decisions about patient therapy and administer an appropriate therapy.

One drawback of many currently available methods is the inability to identify cell necrosis. One method for identifying cell necrosis is the DII. DII is a ratio of long fragments quantities to short fragment quantities. DII indicates a level of cfDNA fragmentation. When the DII using the ratio of 265bp to 80bp targets is calculated and determined to be lower than <NUM>, it indicates the major source of cfDNA is from apoptotic cells. When the DII using the ratio of 265bp to 80bp targets is calculated and determined to be above <NUM>, cfDNA are also generated through necrosis. This DII may be used in the methods of this invention to assess cell necrosis.

Described herein are methods and systems for quantitating the integrity of circulating cell free human DNA and implementing a treatment of a patient. Disclosed herein is a method for quantitating the integrity of circulating cell free human DNA and implementing a treatment of a patient comprising:.

The bodily fluid samples used in the methods of this invention should be treated so as to remove cells. Suitable bodily fluids include, e.g., serum, plasma, urine, saliva, tears or other biological fluid. Preferably the sample used in the methods and system of this invention is a plasma sample.

In the methods of this invention two or more short retrotransposable element targets of between <NUM> to 135bp, about <NUM> to about <NUM> bp or <NUM> bp to about <NUM> bp, may be subjected to the quantitative polymerase chain reaction (qPCR) method to quantitate the targets.

The methods of this invention may further comprise a step of adding a synthetic DNA sequence to the sample as an internal positive control (IPC) and quantitating the retrotransposable element targets and the IPC, and utilizing the quantitative IPC result in the step of comparing the qPCR threshold cycle numbers to a standard curve to improve the accuracy and reliability of the comparing step. The IPC also enables a determination of a concentration of cell free DNA in the sample when quantitating the RE targets by qPCR in a single tube.

The methods of this invention may further comprise a step of adding a hybridization probe that hybridizes to the RE targets to detect the targets. The probe may be added to the sample before the target(s) are subject to q-PCR or thereafter. The probe may include an observable label. Any observable label routinely used in the art for labeling nucleic acid probes could be used to label the probe, e.g., a fluorescent label. Suitable fluorescent probes include, e.g., FAM, Cy5, Hex, or Cy3). The observable label may be detected using a microfluidic device.

The retrotransposable elements of the methods of this invention include e.g., an ALU, particularly ALU Yb8, an SVA, or a LINE element. The retrotransposable element may have a copy number in excess of <NUM> copies per genome.

In the methods of this invention the short retrotransposable element targets may have a length from about <NUM> base pairs to about <NUM> base pairs, about <NUM> base pairs to about <NUM> base pairs, about <NUM> base pairs to about <NUM> base pairs, and about <NUM> bp to about <NUM> bp,. For example, the retrotransposable element target may have a length of e.g. 60bp, 65bp, <NUM> bp, <NUM> bp, <NUM> bp, <NUM> bp, or <NUM> bp. The RE targets may be amplified with the forward and reverse primer pairs set forth in Table 2A, and/or 2B:.

The samples used in the methods of this invention may be from a patient has been diagnosed as having a has stage I, stage II, stage III or stage IV cancer, is suffering from cancer, is in remission from cancer, is at risk for developing cancer, has had surgery to remove a tumor, has undergone a neoadjuvant therapy, a targeted therapy, a chemotherapy, immunotherapy and/or radiotherapy to treat a cancer.

The methods of this invention are also useful in further evaluating the patient having a minimum residual disease diagnosis to implement a disease treatment. For example, in an embodiment of this invention a determination is made that the quantity of the short RE targets as compared to the long Re targets is higher in the sample from the patient than that of a control sample, e.g., a sample from a healthy subject, and in view of that determination an appropriate treatment of the patient is instituted, e.g., a targeted therapy, cancer chemotherapy, immunotherapy, or radiotherapy is administered. Such treatment might include e.g., antineoplastic agents, alkylating agents, topoisomerase inhibitors, mitotic inhibitors, methotrexate, vinca alkaloids, antimetabolites, antifolates, pyrimidine antagonists, purine analogs, purine antagonists, proteasome inhibitors, tyrosine kinase inhibitors, nitrogen mustards, or another cancer therapy. Alternatively, a determination of a threshold cycle number of the quantitated nucleic acid fragment, is made and based on that number the clinical provider administers the treatment to the patient.

It is disclosed a method to quantitate the integrity of circulating cell free human DNA and optionally to implement a treatment of a subject, comprising: providing a sample from a subject, preferably a sample that has been treated to remove cells, the sample comprising cell free human DNA comprising a first RE target being <NUM> base pairs and the second RE target having a length between <NUM> and <NUM> base pairs, e.g., <NUM> bp; using a quantitative polymerase chain reaction (qPCR) method to quantitate the first and second RE targets; obtaining for the quantitated RE targets a threshold cycle number; comparing the threshold cycle number with a standard curve to determine a quantity of each of the RE targets that was present in the sample; calculating a ratio of the quantity of the <NUM> RE target to the quantity of the between <NUM> and <NUM> base pair nucleic acid fragment; and using the quantitated nucleic acid fragment to quantitate the integrity of the circulating cell free human DNA and optionally to implement treatment of a patient. The subj ect's sample may be serum, plasma, urine, or other biological fluid from a human, preferably the sample is a plasma sample. The targets may be amplified in singleplex qPCR wherein a single target is amplified in a single reaction well or the targets may be amplified in a multiplex qPCR wherein all the targets are amplified in a single reaction well.

It is further disclosed a method to quantitate the integrity of circulating cell free human DNA and optionally to implement a treatment of a subject, comprising: providing a sample from a subject, preferably a sample that has been treated to remove cells, , the sample comprising cell free human DNA comprising a first short RE nucleic acid target having a length between <NUM> and <NUM> base pairs, 70bp and about 130bp, e.g., <NUM> and <NUM> base pairs, or between <NUM> and 120bp (the first RE target), and the second RE nucleic acid target having a length between <NUM> to <NUM> base pairs, between about <NUM> and <NUM> bp, or between <NUM> and <NUM> base pairs; using a quantitative polymerase chain reaction (qPCR) method to quantitate the first and second RE targets; obtaining for the quantitated RE nucleic acid targets a threshold cycle number; comparing the threshold cycle number with a standard curve to determine a quantity of each of the RE nucleic acid targets that was present in the sample; calculating a ratio of the quantity of the short RE target to the quantity of second RE target; and using the quantitated nucleic acid targets to quantitate the integrity of the circulating cell free human DNA and to implement treatment of a patient. The subject's sample may be serum, plasma, urine, or other biological fluid from a human, preferably the sample is a plasma sample. The first and second RE target may be a target of the same retrotransposable element or may be different retrotransposable elements. If they are from the same retrotransposable element then PCR blockers may be included to limit extension from the primers beyond the position of the blockers, thus limiting the extension from a primer pair used to amplify one RE target into the other RE target and thereby enhancing the specificity by limiting the production of extraneous or overlapping products. In an embodiment the first and second RE targets are targets of an ALU, an SVA or a LINE1 target. In an embodiment the first and second RE targets are targets of an ALU or SVA target or a LINE1 target. Preferably the short RE target is an ALU or an SVA target, e.g., a Yb8 ALU target, and the long RE element is an SVA or LINE1 target. In an embodiment the prime pairs used in the qPCR to quantitate the RE targets are selected from the primer pairs of Table 2A and 2B and 2C. The targets may be amplified in singleplex qPCR wherein a single target is amplified in a single reaction well or the targets may be amplified in a multiplex qPCR wherein all the targets are amplified in a single reaction well.

An embodiment of this invention is a method to quantitate the integrity of circulating cell free human DNA and optionally to implement a treatment of a subject, comprising:.

The RE targets may be amplified in singleplex qPCR wherein a single target is amplified in a single reaction well or the targets may be amplified in a multiplex qPCR wherein all the targets are amplified in a single reaction well.

The methods of this invention are contemplated to be useful in identifying a subject having progressive disease or MRD. Accordingly, an embodiment of this invention is a method for identifying a subject having progressive cancer or MRD, said method comprising:.

The targets may be amplified in singleplex PCR wherein a single target is amplified in a single reaction well or the targets may be amplified in a multiplex PCR wherein all the targets are amplified in a single reaction well.

A subject identified as having progressive disease or MRD may be administered a cancer therapy or MRD therapy. The method may further comprise the step of determining the DNA integrity index (DII) of the cfDNA in the sample.

In an embodiment of this method, an increase in Frag2 as compared to Frag1 may be determined by subtracting Frag1 from Frag2 to generate a value, FragDiff, that is compared to a threshold value and based on that comparison it is concluded that the ctDNA has increased and identifies the subject as having progressive disease or MRD and an appropriate therapy may be administered.

Neoadjuvant therapies, which include, e.g., chemotherapy, hormone therapy, immunotherapy, radiation therapy, and targeted therapy are delivered to a subject before the main treatment is administered to help reduce the size of a tumor or kill cancer cells that have spread. Neoadjuvant therapies are recommended when a patient with early-stage cancer, stage I, stage II or stage III, undergoes surgery or radiation therapy. The methods of this invention may be applied to a sample of subject having a stage I, stage II, stage III or stage IV cancer wherein the samples are obtained from the subject before and after the neoadjuvant therapy to quantitate the integrity of circulating cell free human DNA and to implement a treatment of a subject. The methods of this invention may also be applied to samples from a subject who has had a therapy for hepatoma, esophageal cancer, rectal cancer, anal cancer, head and neck cancer, colon cancer, colorectal cancer, lung cancer, breast cancer, neu metastatic breast cancer or a blood cancer, e.g., leukemia, and the first sample was taken from the subject before administering the a first cycle of therapy and the second sample was taken from the subject after administering the first therapy cycle, but before the administration of another cycle of therapy, and as such the first and second samples may be obtained from the subject at least <NUM> week apart, at least <NUM> weeks apart, at least <NUM> weeks apart, at least <NUM> weeks apart, e.g. <NUM> to <NUM> days apart. The therapy may be a targeted therapy, a chemotherapy, immunotherapy or radiotherapy. The therapy may be treatment with an antineoplastic agents, alkylating agents, topoisomerase inhibitors, mitotic inhibitors, methotrexate, vinca alkaloids, antimetabolites, antifolates, pyrimidine antagonists, purine analogs, purine antagonists, proteasome inhibitors, tyrosine kinase inhibitors, nitrogen mustards, immunotherapy, or another cancer therapy.

In the methods described herein for identifying the patient as having progressive disease or MRD, The short and long retrotransposable elements may have a copy number in excess of <NUM> copies per genome, e.g., the short retrotransposable interspersed element may be an ALU or an SVA and the long RE may be an ALU, SVA or LINE.

The short RE targets may be from about <NUM> base pairs to about <NUM> base pairs, or from about <NUM> base pairs to about <NUM> base pairs, or from about <NUM> base pairs to about <NUM> base pairs, or from about <NUM> base pairs to about <NUM> base pairs. The long RE target may be about <NUM> bp to about <NUM> bp or about <NUM> bp to about <NUM> bp, or about <NUM> bp to about <NUM> bp in length.

The forward and reverse primer pairs used to amplify the short and long target sequences in the qPCR may be selected from the following forward and reverse primer pairs of Tables 2A, 2B, or 2C.

The samples used in the methods described herein may be a sample of serum, plasma, urine, or other biological fluid, preferably the sample is a plasma sample.

The method may further comprise a step of adding a synthetic DNA sequence as an internal positive control (IPC) to the samples prior to quantitating each of the short and long RE targets in the first and second samples by qPCR, and then quantitating the IPC and utilizing the quantitative IPC result in the step of comparing the threshold cycle number of each quantitated RE target with a standard curve to improve the accuracy and reliability of the comparing step. For example, the use of the IPC enables a determination of a concentration of cell free DNA in the sample.

It is specifically contemplated that the quantitation of the short and long retrotransposable interspersed elements of each sample by qPCR may be carried out in a single tube or well.

The amplified RE targets may be detected with one or more hybridization probes that hybridize specifically to the RE targets sequences. The probes may comprise an observable label, e.g., a fluorescent label, e.g., FAM, Cy5, Hex, or Cy3. The observable label could be detected using a microfluidic device. In some embodiments of this invention, the amplification products of the qPCR method used in the methods of this invention may be detected and/or quantified using electrical biosensors (see <NPL>).

Also an embodiment of this invention is a system for characterizing cancer or MRD in a patient, the system comprising:.

The system may be a singleplex system wherein a single target is amplified in a single reaction well or the system may be a multiplex system where multiple target are amplified in a single reaction well.

In the system of this invention for characterizing cancer or MRD in a patient the patient may be a patient who is suffering from a cancer, e.g., is diagnosed as having a stage <NUM>, stage II or stage III cancer, is in remission from cancer, is at high risk for developing cancer, has been categorized by another method as having a complete response ("CR"), a stable disease ("SD"), a partial response ("PR"), or progressive disease ("PD"), or has had a neoadjuvant therapy, has had surgery to remove a tumor, or has undergone chemotherapy, immunotherapy or radiotherapy to treat the cancer or MRD. The cfDNA in the multiplex system may further comprise cfDNA comprising a long retrotransposable element target having a length of between <NUM> bp and <NUM> bp, or <NUM> bp to <NUM> bp, e.g., <NUM>-<NUM> bp, a TaqMan probe corresponding to the long RE target, and forward and reverse primers for amplifying the long RE target. In the system, the forward primer and reverse primer pair for amplifying the RE targets are selected from Table 2A, 2B or 2C.

The method of this invention are contemplated for allow for the quantitated RE target amounts to be correlated to one cancer cell in <NUM>,<NUM> total cells or greater, one cancer cell in <NUM>,<NUM>,<NUM> total cells or greater, one cancer cell in <NUM>,<NUM>,<NUM> cells or greater.

Serum and plasma separation were performed according to the standard protocol and within four hours of collection, and stored at -<NUM> until they were processed. Care was taken to avoid freeze-thaw cycles. For serum specimens, whole blood is collected in the commercially available red-topped test tube Vacutainer (Becton Dickinson). For plasma specimens, whole blood is collected in the commercially available anticoagulant-treated tubes e.g., EDTA-treated or citrate-treated.

Two separate protocols have previously been described for direct DNA quantification from either human serum (<NPL>, incorporated herein in its entirety) or plasma (<NPL>, incorporated herein in its entirety). We used both of these methods on serum and plasma and compared the amplification efficiency from both methods. The first method includes deactivation or elimination of proteins that bind to template DNA or DNA polymerase and might invalidate qPCR results. Briefly, a volume of <NUM>µL of each serum or plasma sample was mixed with <NUM>µL of a preparation buffer that contains <NUM>/L Tween <NUM>, <NUM> Tris, and <NUM> EDTA. This mixture was then digested with <NUM>µg of proteinase K solution (Qiagen) at <NUM>. for <NUM>, followed by <NUM> of heat deactivation and inactivation at <NUM>. After subsequent centrifugation at <NUM>,<NUM> for <NUM>, <NUM>µL of the supernatant (containing <NUM>-µL equivalent volume of serum or plasma) was used as a template for each direct RE-qPCR reaction. The second method bypasses the protein removal step and only requires <NUM>:<NUM> dilution of the serum/plasma sample with sterile H<NUM>O.

For comparison to and validation of direct quantification of cfDNA, RE-qPCR has been performed on isolated, purified cfDNA. cfDNA was purified by magnetic bead extraction or by using the silica based membrane QIAamp DNA Investigator Kit (Qiagen).

In general primers and labeled probes used in the qPCR reactions may be obtained from Eurofins MWG/Operon, Integrated DNA Technologies, or a variety of other vendors.

Short ALU primer sets were designed to produce amplicon lengths of <NUM> bp, <NUM> bp, 105bp, <NUM> bp, and <NUM> bp among others, were developed for use in the assays of the present invention. The primer sequences are shown in Table 2A. Primer pairs to produce amplicon lengths from SVA of 100bp, 104bp, <NUM> bp, 116bp, 118bp, 126bp, 132bp, <NUM> bp, <NUM> bp, <NUM> bp, <NUM> bp, or to produce amplicon lengths of 252bp, 257bp, 262bp, and 267bp from LINE1, among others are set forth in Table 2B and 2C. The primer pairs were developed using Primer <NUM> software and an SVA or LINE1 (genebank ID: AH005269 (PUBMED <NUM>) retrotransposon sequence. Because the SVA and LINE1 sequences are truncated in many individuals and also have sequence similarities with ALU sequences in certain regions, the target SVA sequences were selected from the SVA-R region, and the target LINE1 sequence was selected from the LINE1 ORF2 region, which have no or minimal sequence similarity as compared with the ALU sequence. The primer sequences and probes that hybridize to the amplified targets are shown in Table 2A, 2B and 2C.

Additional primer design based on ALU Yb8, SVA and LINE1 may be done using Primer software (<NPL>; <NPL>).

The qPCR assays were run on an Applied Biosystems <NUM> Real Time PCR instrument and/or the Biorad CFX, but useful instrument platforms are not limited thereto. The qPCR assays of the present invention may be adapted to work on most Real-Time PCR instruments. To assess the concentration and integrity index of serum and plasma circulating cfDNA, both short and long fragments may be amplified and quantified. The short fragment primer sets may amplify the short (apoptotic) DNA fragments, whereas the long fragment primer sets may amplify the long (non-apoptotic) DNA fragments. The RE-qPCR multiplex reaction may contain three targets in a Taqman based assay: a short RE target, a long RE target, and a synthetic IPC sequence. The hybridization probes detecting each target may be labeled with different fluorophores (e.g. FAM, Cy5, Hex, or Cy3) to enable simultaneous detection. The following PCR conditions may be used, but they can be modified as necessary: <NUM> <NUM> denaturation cycle, followed by <NUM> cycles of <NUM>-step qPCR (<NUM> at <NUM> and <NUM> at <NUM> combined annealing/extension time) at maximum ramp speed. Additional PCR parameters (i.e. cycle number, denaturation and annealing/extension times and temperatures) are investigated to obtain a robust, sensitive qPCR multiplex.

Short Yb8 and long SVA primer pairs selected from those shown in Table 2A and 2B were combined into eight different multiplex sets (Yb8-<NUM> & SVA-<NUM>, Yb8-<NUM> & SVA-<NUM>, Yb8-<NUM> & SVA-<NUM>, Yb8-<NUM> & SVA-<NUM>, Yb8-<NUM> & SVA-<NUM>, Yb8-<NUM> & SVA-<NUM>, Yb8-<NUM> & SVA-<NUM>, and Yb8-<NUM> & SVA-<NUM>). The optimal temperature for each multiplex was determined by a temperature gradient ranging from <NUM> to <NUM>. The concentration of primers and additives including DMSO and additional MgCh were optimized for each multiplex set.

The reaction mixture of each multiplex Yb8-SVA-qPCR included a template, forward primer and reverse primer pairs, fluorescent probe, Brilliant Multiplex QPCR Master Mix (Agilent) and the additives bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), and magnesium chloride (MgCh). Real-time PCR amplification was performed with pre-cycling heat activation of DNA polymerase at <NUM> for <NUM> followed by <NUM> cycles of denaturation at <NUM> for <NUM> sec and extension at <NUM>-<NUM> (adapted to the multiplex set) in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories). The quantification of DNA in each sample was determined by use of a calibration curve with serial dilutions (20ng/ul to <NUM>.

The qPCR assays may be run on an Applied Biosystems <NUM> Real Time PCR instrument and/or the Biorad CFX, but useful instrument platforms are not limited thereto. The qPCR assays of the present invention may be adapted to work on most Real-Time PCR instruments. To assess the concentration of a 97bp target of ALU Y8b in plasma circulating cfDNA in control healthy subjects compared to cancer patients, plasma samples of control (healthy subjects) and test (samples from patients with metastatic colorectal cancer (mCRC)) containing cfDNA was combined with the <NUM> bp forward primer (GTGGCTCACGCCTGTAAT)(SEQ ID NO: <NUM>) , <NUM> bp reverse primer (GGGTTTCACCTTGTTAGCCA) (SEQ ID NO: <NUM>), a fluorescent probe comprising TGGATCATGAGGTCAGGAGAT (SEQ ID NO: <NUM>), Brilliant Multiplex QPCR Master Mix (Agilent) and the additives bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), and magnesium chloride (MgCh). Real-time PCR amplification was performed with pre-cycling heat activation of DNA polymerase at <NUM> for <NUM> followed by <NUM> cycles of denaturation at <NUM>-<NUM> for <NUM>-<NUM> sec and extension at <NUM>-<NUM> (depending on the multiplex set) in an ABI <NUM> Instrument (ThermoFisher Scientific). The quantification of DNA in each sample was determined by use of a calibration curve with serial dilutions (<NUM> ng/ul to <NUM> pg/ul).

To assess the concentration of plasma circulating cfDNA, both 80bp and 97bp fragments of ALU Yb8 are amplified and quantified. The RE-qPCR multiplex reaction contains three targets in a TaqMan® based assay: <NUM> bp ALU Yb8 forward and reverse primers (GGAAGCGGAGCTTGCAGTGA (SEQ ID NO:<NUM>) and AGACGGAGTCTCGCTCTGT CGC (SEQ ID NO: <NUM>)) , the <NUM> bp ALU Yb8 RE forward and reverse primers GTGGCTCACGCCTGTAAT (SEQ ID NO: <NUM>) and GGGTTTCACCTTGTTAGCCA(SEQ ID NO:<NUM>)), an 80R- blocker, peptide nucleic acid (PNA) oligo which binds to the <NUM> bp ALU Yb8 fragment, a 97R-PNA blocker a PNA which binds to the <NUM> bp ALU Yb8 fragment and probes that hybridize to the <NUM> bp and the <NUM> bp sequences (e.g., Fluorophore-TGAGGTCAGGAGATCGAGACCATCC-Quencher)(SEQ ID NO: <NUM>), and in some instances a synthetic IPC sequence. PNA oligo mimics DNA. In PNA, the negatively-charged sugar phosphate backbone of DNA is replaced with an uncharged pseudo-peptide backbone. The two strands of a PNA/DNA hybrid therefore lack the electrostatic repulsion as observed for DNA/DNA duplexes, giving rise to thermal stability. Hybridization probes are also included in some instances for detecting each target and the probes are labeled with different fluorophores (e.g. FAM, Cy5, Hex, or Cy3) to enable simultaneous detection. The reaction mixture includes the forward primers, reverse primers, the blockers, the fluorescent probe, Brilliant Multiplex QPCR Master Mix (Agilent) and the additives bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), and magnesium chloride (MgCh). Real-time PCR amplification is performed with pre-cycling heat activation of DNA polymerase at <NUM> for <NUM> followed by <NUM> cycles of denaturation at <NUM> for <NUM> sec and extension at <NUM>-<NUM> (depending on the multiplex set) in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories). The quantification of DNA in each sample is determined by use of a calibration curve with serial dilutions (20ng/ul to <NUM>.

<FIG> shows the two PCR target regions of 80bp and 97bp on the Yb8 sequence. In order to amplify the 97bp and 80bp separately, we use two peptide nucleic acid (PNA) oligos to block PCR extension beyond the target regions. PNA oligos are used as sequence specific PCR blockers because PNA probes have strong binding affinity and specificity to its target DNA and are not recognized by DNA polymerase as primer. In the diagram 97F-Blocker binds the complement sequence of Alu-Yb8 between 97bp and 80bp target regions and prevents DNA elongation from the 97bp forward primer beyond the region where the 97bp reverse primer binds. In a similar way, 80R-Blocker binds Alu-Yb8 sequence between 97bp and 80bp target regions and prevents DNA elongation from the 80bp reverse primer beyond the region where the 80bp forward primer binds. By incorporating these two PCR blockers, it is possible to prevent PCR amplification of nearly entire Alu-Yb8 sequence that can occur with the 97F/80R primer pair. We are then able to compare the amounts of the <NUM> bp and <NUM> bp fragments in the plasma of control samples from healthy subjects and the plasma from cancer patients.

Plasma from <NUM> control subjects, healthy subjects without cancer and <NUM> subjects having cancer were subjected to qPCR assays to assess the level of ctDNA. The qPCR assays were run on an Applied Biosystems <NUM> Real Time PCR instrument and/or the Biorad CFX, but useful instrument platforms are not limited thereto. The qPCR assays of the present invention may be adapted to work on most Real-Time PCR instruments. To assess the concentration and integrity index of serum circulating cfDNA in the samples from control and cancer subject, a first ALU Yb8 target of <NUM> bp, and a second ALU Yb8 target of <NUM> bp and an SVA target of <NUM> bp were amplified and quantified in a RE-qPCR multiplex reaction. The RE-qPCR multiplex reaction contained three targets in a Taqman based assay: the first ALU Yb8 target of <NUM> bp , the second ALU Yb8 target of <NUM> bp, a third SVA target of <NUM> bp, and a synthetic internal positive control (IPC) sequence. The hybridization probes detecting each amplified target were labeled with different fluorophores (FAM, Cy5, or Hex) to enable simultaneous detection. The following PCR conditions are used: <NUM> <NUM> denaturation cycle, followed by <NUM> cycles of <NUM>-step qPCR (<NUM> at <NUM> and <NUM> at <NUM> combined annealing/extension time) at maximum ramp speed.

The reaction mixture of each multiplex Yb8-qPCR included the forward primers and reverse primers for the first Yb8-<NUM> target and for the second ALU Yb8-<NUM> target, and the long SVA <NUM> target (see Table 2A and 3B for primer pair sequences). the fluorescent probes for detecting the amplified fragments, Brilliant Multiplex QPCR Master Mix (Agilent) and the additives bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), and magnesium chloride (MgCl<NUM>). Real-time PCR amplification was performed with pre-cycling heat activation of DNA polymerase at <NUM> for <NUM> followed by <NUM> cycles of denaturation at <NUM> for <NUM> sec and extension at <NUM>-<NUM> (depending on the multiplex set) in a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories). The quantification of the targets in each sample was determined by use of a calibration curve with serial dilutions (20ng/ul to <NUM>. Table <NUM> sets forth the quantitated amounts of the 80bp target, the <NUM> bp target and the <NUM> bp target in each sample and the difference in the quantitated amount of <NUM> bp Yb8 target and the quantitated amount of <NUM> bp Yb8 target.

Data analysis was performed utilizing the respective AB <NUM>, QuantStudio-<NUM> or BioRad CFX instrument software. Melt curve analysis was generated using Qiagen's QuantiTect <NUM> SYBR1 Green PCR Kit (Cat#<NUM>) and operated using the Applied Biosystems <NUM> Real Time PCR instrument. For each experiment, a freshly prepared <NUM>-fold serial dilution of high molecular weight standard DNA (ranging from <NUM> ng/µL to <NUM> ng/µL) was run in duplicate on each plate to generate standard curves for the long and short targets. The standard curves are plotted CT vs. Delta Rn (the fluorescence emission intensity of the reporter dye divided by the fluorescence emission intensity of the passive reference dye). Resultant DNA quantitation values are interpolated from the resulting linear standard curves. At least one negative No Template Control (NTC) was run on each plate.

In experiments where a ratio between DNA concentration of a 265bp SVA long target and 80bp ALU short target were calculated, the DNA concentration of the long target divided by DNA concentration of the short target provides an indication as to the degree of DNA integrity for the quantified sample. DNA integrity index is calculated as the ratio of concentrations ([concentration of long RE marker]/[concentration of short RE marker]). Quality metrics, including PCR efficiencies (i.e. slope) of both short and long targets, Y-intercept values, and verification of no true amplification in negative controls was assessed.

Blinded samples of plasma from <NUM> cancer patients were subjected to a qPCR to assess the level of ctDNA using the methods described herein and identify patients as having progressive disease. The plasma samples were from patients who had been previously diagnosed as having either colorectal cancer, non-small cell lung cancer, small cell lung cancer or breast cancer and had received either chemotherapy, targeted therapy, immunotherapy, or a combination of therapies.

A first plasma sample was obtained from the patients before receiving a cycle of therapy and a second plasma sample was obtained <NUM> days to <NUM> days after the cycle of therapy and before receiving another cycle of therapy. The qPCR assays were run on an Applied Biosystems <NUM> Real Time PCR instrument and/or the Biorad CFX, but useful instrument platforms are not limited thereto and the qPCR assays of the present invention may be adapted to work on most Real-Time PCR instruments.

To assess the concentration and integrity index of the cfDNA in the samples from control and cancer patients, a first ALU Yb8 target of <NUM> bp, and a second ALU Yb8 target of <NUM> bp and an SVA target of <NUM> bp were amplified and quantified in a RE-qPCR multiplex reaction. The sequence of the primer pairs used to amplify yb-<NUM>-<NUM>, yb-<NUM><NUM> and SVA <NUM> are set forth in Table <NUM>. The RE-qPCR multiplex reaction was a Taqman® based assay comprising Brilliant Multiplex QPCR Master Mix (Agilent), bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), and magnesium chloride (MgCh), and comprised the plasma sample, a primer pair for amplifying the first ALU yb-<NUM> target of <NUM> bp, a primer pair for amplifying the second ALU Yb8 target of <NUM> bp, and a primer pair for amplifying a third SVA target of <NUM> bp, and a synthetic internal positive control (IPC). The amplification products were detected with hybridization probes for the Yb-<NUM>80bp target, the Yb8-105bp target and the SVA <NUM> bp target, each labeled with a different fluorophore to enable simultaneous detection of the different amplified targets.

Real-time PCR amplification was performed with pre-cycling heat activation of DNA polymerase in a QuantStudio-<NUM> (Thermofisher Scientifics). The quantification of the RE targets in each sample was determined by use of a calibration curve with serial dilutions (20ng/ul to <NUM>. 6pg/ul) and the difference between the amount of the first Yb8 80bp target and the amount of the second Yb8 105bp target in the first sample (Frag1) and the amount of the first Yb8 80bp target and the amount of the second Yb8 105bp target in the second samples of plasma (Frag2) were calculated. The difference between Frag2 and Frag1 (Frag Diff) was also calculated. An increase in the amount of the 80bp yb-<NUM> target as compared to the 105bp yb-<NUM> target in the second sample as compared to the first sample indicated an increase in the ctDNA.

<FIG> depicts the FragDiff of the <NUM> patients, who had been classified as having progressive disease (triangles), or having non-progressive disease (circles). <FIG> demonstrates that the method disclosed herein rapidly assesses cfDNA integrity. <FIG> also demonstrates that based upon the FragDiff being above a threshold level, the method rapidly and reliably identifies a patient as having progressive disease see also <FIG>. Thus, the method described herein can also be used as a factor for rapidly concluding the patient has progressive disease or the cancer treatment was not effective. This is in contrast to other standard assays, e.g., CT scans, Xrays, and CEA measurements, that require weeks, if not months, before it is determined the patient has progressive disease and a therapy can be identified as ineffective.

Claim 1:
A method to quantitate the integrity of circulating cell free human DNA and implement a treatment of a patient, said method comprising:
(a) providing a first and second sample of serum, plasma, urine, or other biological fluid from a subject wherein the first and second samples are obtained at least one week apart, at least <NUM> weeks apart, at least <NUM> weeks apart or at least <NUM> weeks apart, the first and second samples comprising cell free human DNA (cfDNA), the cfDNA comprising a first and second short retrotransposable interspersed element (RE) target sequence having a length of between about <NUM> base pairs to about <NUM> base pairs, wherein the first and second short targets differ in length;
(b) quantitating each of the first and second short RE targets in the first and second samples using a quantitative polymerase chain reaction (qPCR) method;
(c) obtaining for each of the quantitated RE targets in the first and second samples a threshold cycle number;
(d) comparing the threshold cycle number of each quantitated RE target with a standard curve to determine an amount of each of the quantitated RE targets that were present in the samples, wherein the amount of short RE targets in the second samples is indicative of the integrity of the circulating cell free DNA;
(e) determining an increase in the amount of short RE targets in the second sample as compared to the first sample above a threshold level, and
(f) determining a treatment for a patient having an increase in the amount of short RE targets in the second sample as compared to the first sample above a threshold level.