WO2009051842A2 - Detection of cancer by measuring genomic copy number and strand length in cell-free dna - Google Patents

Abstract

The present invention relates, e.g., to a method for diagnosing a cancer in a subject, comprising measuring in a cell-free body fluid of the subject a) the amount of DNA in the body fluid from an amplified cancer-associated chromosomal locus, compared to a positive and/or a negative reference standard, wherein the positive and negative standards reflect the amount of the DNA in the body fluid of a subject having the cancer, or not having the cancer, respectively, and b) the strand length integrity index of the DNA in the body fluid from the amplified cancer-associated chromosomal locus compared to a baseline value, wherein if the amount of the measured DNA is statistically significantly increased compared to the negative reference standard, and/or is statistically the same as the positive reference standard, and if the strand length integrity index is at least about 0.65, this is indicative that the subject has the cancer.

Description

DETECTION OF CANCER BY MEASURING GENOMIC COPY NUMBER AND STRAND LENGTH IN CELL-FREE DNA

This research was supported by grants from The U.S. Department of Defense (OC04-0060) and the National Cancer Institute (CAl 03937). The U.S. government thus has certain rights in the invention.

This application claims the benefit of the filing date of U.S. provisional application 60/999,414, filed October 18, 2007, which is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

This invention relates, e.g., to a diagnostic method for cancer detection.

BACKGROUND INFORMATION

The development of tumor biomarkers that can be clinically applicable in body fluids for cancer diagnosis is useful for clinical management of patients with cancer. It is well recognized that several solid malignant tumors release a significant amount of genomic DNA into body fluids including, e.g., blood, urine and saliva. Tumor-released DNA has been detected by virtue of specific genetic and epigenetic alterations including point mutations, microsatellite alterations, allelic imbalance, translocation, promoter methylation and the presence of viral sequences. In addition to those specific molecular genetic alterations, total elevated DNA concentration and increased DNA strand integrity in cell-free DNA have been reported in plasma samples from cancer patients. As compared to non-neoplastic controls, total cell-free DNA concentration was higher in ovarian (Chang et al. (2002) Clin Cancer Res 8, 2580-2585; Chang et al. (2002) J Natl Cancer Inst 94, 1697-1703), breast, prostate, esophageal and lung carcinomas as well as melanoma. Furthermore, increased DNA strand length (integrity) of total cell-free DNA was found in plasma samples in ovarian cancer (Wang et al. (2003) Cancer Res 63_, 3966-3968) and in the stool of colorectal tumors.

Increased DNA copy number or genomic amplification at certain loci have been reported in a variety of human cancers, but not in normal tissues. For example, a variety of chromosomal loci have been reported to be amplified in ovarian carcinomas, in particular the cyclin E locus (Nakayama et al. (2007) In t J Cancer 120, 2613-2617). DESCRIPTION OF THE DRAWINGS

Figure 1 shows a ROC curve comparison in the diagnosis of ovarian cancer cases versus benign controls. Using cyclin E copy number and a strand length integrity index ( — ), the AUC is 0.936; cyclin E copy number only ( ), AUC is 0.896; and DNA strand length integrity index only (— ), AUC is 0.698.

DETAILED DESCRIPTION

The inventors describe herein a method for diagnosing a cancer (a malignant tumor, neoplasm, malignancy) in a subject, comprising measuring in a body fluid (e.g., a cell-free preparation of a body fluid) both the amount (e.g., copy number) and the strand length (integrity) of DNA from a chromosomal locus that is amplified in the type of cancer being detected, compared to a reference standard and/or a baseline (control) value. For example, the inventors have found that the measurement of both cyclin E copy number and strand length (integrity) in a suitable body fluid provides a highly sensitive and specific approach for diagnosing ovarian cancer. This method is superior to the analysis of either of these two parameters individually, which cannot be used independently to detect ovarian cancer. Unexpectedly, and advantageously, a method of the invention is significantly superior to conventional diagnostic procedures, such as cytology analysis.

Other advantages of a method of the invention include that it is rapid, economical, simple, robust, highly sensitive, specific and accurate. A method of the invention can detect cancers at an earlier stage than can conventional cytology methods; for example, one can detect ovarian cancers which are at the curable stage (stage 1 or stage 2), whereas conventional cytology methods can not. Moreover, the use of liquid test samples allows for a relatively non-invasive sampling method, which avoids the unpleasantness and side-effects of invasive procedures (such as tissue biopsies) and is especially useful for the detection of a cancer in an inaccessible tissue.

One aspect of the invention is a method for diagnosing (detecting) a cancer in a subject, comprising measuring in a cell-free body fluid from the subject

(a) the amount of DNA in the body fluid from an amplified cancer-associated chromosomal locus, compared to a positive and/or a negative reference standard, wherein the positive and negative standards are representative of (e.g., reflect, are proportional to) the amount of the DNA in the body fluid of a population of subjects having the cancer, or not having the cancer, respectively, and

(b) the strand length integrity index of the DNA in the body fluid from the amplified cancer- associated chromosomal locus compared to a baseline value (denominator), wherein if the amount of the measured DNA is statistically significantly increased (e.g., at least about 5-fold greater, or at least about 30-fold greater) compared to the negative reference standard, and/or is statistically the same as the positive reference standard; and if the strand length integrity index is at least about 0.65, this is indicative that the subject very likely has the cancer. That is, the subject has at least about a 75% chance (e.g., at least about a 75%, 80%, 85%, 90%, 95% chance) of having the cancer.

The positive reference standard may be, e.g., (a) an aliquot of the DNA in a cell-free body fluid from the amplified cancer-associated chromosomal locus of a subject which has the cancer, or the average (e.g., mean) of such values from a population or pool of such subjects, or (b) a synthetically prepared DNA molecule in the amount, or proportional to the amount, of the DNA of (a); and the negative reference standard may be, e.g., (c) an aliquot of the DNA in a cell-free body fluid from the amplified cancer- associated chromosomal locus of a subject which does not have the cancer, or the average (e.g., mean) of such values from a population or pool of such subjects, or (d) a synthetically prepared DNA molecule in the amount, or proportional to the amount, of the PCR amplification product of (c), or an aliquot of the DNA in a cell-free body fluid which lacks DNA from the cancer-associated chromosomal locus. The DNA of these positive and/or negative reference standards can be amplified by PCR to provide a standard which reflects the amount of the DNA that has been PCR amplified from a test sample. In one embodiment of the invention, in which the amount of DNA from an amplified cancer- associated chromosomal locus in a cell-free body fluid from a subject has been measured by PCR amplification of the DNA in the body fluid, the positive reference standard may be, e.g., (a) a PCR amplification product of the DNA in a cell-free body fluid from the amplified cancer-associated chromosomal locus of a subject which has the cancer, or the average (e.g., mean) of such values from a population or pool of such subjects or (b) a synthetically prepared DNA molecule in the amount, or proportional to the amount, of the PCR amplification product of (a); and the negative reference standard may be, e.g., (c) a PCR amplification product of the DNA in a cell-free body fluid from the amplified cancer-associated chromosomal locus of a subject which does not have the cancer, or the average (e.g., mean) of such values from a population or pool of such subjects or (d) a synthetically prepared DNA molecule in the amount, or proportional to the amount, of the PCR amplification product of (c), or an aliquot of the DNA in a cell-free body fluid which lacks DNA from the cancer-associated chromosomal locus. In one embodiment of the invention, the length integrity index is determined by measuring the difference of the Cycle threshold (Ct) of a PCR product of at least about 300 bp (e.g., about 400 bp) which is PCR amplified from the cancer-associated chromosomal locus DNA in the body fluid, and the Ct of a PCR product of about 100 bp which is PCR amplified from a control portion of the chromosome (e.g., the p53 gene) in the body fluid.

The amount of the cancer-associated DNA from an amplified chromosomal locus and the length integrity of the cancer-associated DNA may be determined together, by conducting all of the PCR measurements in a single, multiplexed PCR reaction. In another embodiment, the amount of the cancer-associated DNA from an amplified chromosomal locus and the length integrity of the cancer-associated DNA are determined together, in a single PCR reaction, by measuring the amount of a PCR product of at least about 300 bp that is PCR amplified from the amplified cancer- associated chromosomal locus DNA in the body fluid, compared to a positive and/or negative reference standard.

The PCR amplification in a method of the invention can be real-time PCR. The cancer- associated amplified chromosomal locus can comprise, e.g., the Cyclin E, EGFR,

HER2/Neu, myc gene, HBXAP/Rsf-1 gene, RAS gene, AKT gene, PHGCA gene, Rsf-1 gene, and NOTCH (e.g. NOTCH 3) gene.

A variety of types of cancer can be diagnosed by a method of the invention, including, e.g., ovarian, breast, lung, prostate, colorectal, esophageal, pancreatic, prostate, gastrointestinal, bladder, kidney, liver, lung, head and neck (including oral cavity) or brain cancer.

A variety of body fluids (e.g., a cell-free body fluid generated from a pleural effusion, ascites sample, plasma, urine or sputum) can be tested in a method of the invention.

A method of the invention can be used in conjunction with another diagnostic method for the cancer, including, e.g., assaying for the presence of an alteration in the cancer-associated amplified locus which is associated with the cancer, such as a point mutation, a microsatellite alteration, an allelic imbalance, a translocation, a promoter methylation and/or the presence of a viral sequence; or performing a conventional cytology assay for the cancer.

Another aspect of the invention is a kit for diagnosing a cancer in a subject, using a method of the invention, comprising (a) a set of PCR primers for amplifying an about 400 bp fragment of Cyclin E; (b) a set of PCR primers for amplifying an about 100 bp fragment of p53: and (c) positive and negative standards for quantitating the amounts of the DNA products. Test samples (body fluids, such as blood, effusion, urine and sputum) for analysis can be obtained from any suitable source. Suitable subjects from which the body fluids can be collected include any animal which has, or is suspected of having, a cancer, such as vertebrate animals, e.g. mammals, including pets, farm animals, research animals (mice, rats, rabbits, guinea pigs, etc) and primates, including humans.

A variety of suitable cancers, examples of which will be evident to a skilled worker, can be detected by a method of the invention. These include, e.g., ovarian, breast, lung, prostate, colorectal, esophageal, pancreatic, prostate, gastrointestinal, bladder, kidney, liver, lung, head and neck (including oral cavity), or brain cancer, or melanomas. In one embodiment of the invention, the cancer is ovarian cancer, e.g., in a female human.

"Cell-free" body fluids used in a method of the invention are body fluids into which DNA from cancer cells, e.g. tumors, has been released, and from which all or substantially all particulate material in the preparation, such as cells or cell debris, has been removed. These samples are sometimes referred to herein as cell-free "effusion samples." It will be evident to a skilled worker that a cell-free body fluid generally contains only a few if any cells, but that a number of cells can be present in a "cell-free" body fluid, provided that those cells do not interfere with a method of the invention. A skilled worker will recognize how many cells can be present without interfering with the assay. For example, 1 ,000 or fewer cells (e.g., 1 , 10, 50, 100, 500 or 1 ,000 cells) can generally be present in a volume of one liter of body fluid without interfering with the assay. Suitable body fluids for analysis will be evident to a skilled worker. These include, e.g., blood (e.g., whole blood, plasma or serum), lymph fluid, serous fluid, a ductal aspirate sample, bronchoalveolar lavage, a lung wash sample, a breast aspirate, a nipple discharge sample, peritoneal fluid, duodenal juice, pancreatic duct juice, bile, an esophageal brushing sample, glandular fluid, amniotic fluid, cervical swab or vaginal fluid, ejaculate, semen, prostate fluid, cerebrospinal fluid, a spinal fluid sample, a brain fluid sample, lacrimal fluid, tears, conjunctival fluid, synovial fluid, saliva, stool, sperm, urine, sweat, fluid from a cystic structure (such as an ovarian cyst), nasal swab or nasal aspirate, or a lung wash sample.

It will be evident to a skilled worker what source of body fluid is suitable for the detection of a particular type of cancer. For example, for ovarian cancer, suitable cell-free body fluids can be generated from, e.g., a pleural effusion, ascites fluid (effusion in the abdominal cavity), plasma, urine or sputum. For the detection of pancreatic cancer, one can assay, e.g., pancreatic duct juice (sometimes referred to as "pancreatic juice" or "juice"), for example obtained during endoscopy, brushings of the pancreatic duct, bile duct or aspirates of cyst fluid. For the detection of lung cancer, sputum or bronchoalveolar lavage can be used. For head and neck cancer in the oral or pharyngeal cavity, sputum or wash from the mouth can be used. For colon cancer, prostate cancer, breast cancer and nasopharyngeal cancer, suitable body fluids include stool, prostate fluid, breast aspirate and nasal swab/wash, respectively.

Methods for preparing a DNA sample from a body fluid (e.g., a cell-free body fluid) are conventional and well-known in the art. It maybe desirable to include an agent in the sample which inhibits DNAase activity. For example, for the isolation of DNA from a plasma sample, anticoagulants contained in whole blood can inhibit DNAse activity. Suitable anti-coagulants include, e.g., chelating agents, such as ethylenediaminetetraacetic acid (EDTA), which prevents both DNAse-caused DNA degradation and clotting of whole blood samples.

In general, a body fluid sample is treated to remove cells, cellular debris and the like. For example, a urine sample, a pleural effusion or an ascites sample can be subjected to centrifugation, following conventional procedures, and the supernatant containing the DNA isolated; or a sample can be filtered to remove the cells or cell debris. Typical, non-limiting methods for isolating DNA from a pleural effusion or ascites fluid are described in the Examples herein.

In a method of the invention, DNAs are measured which are amplified from at least one cancer-associated locus (region) of a chromosome of the animal. Numerous examples have been described of chromosomal loci which are amplified in subjects having a cancer, but not in "normal' subjects (e.g., subjects which do not have the cancer, including subjects which have a benign tumor). Such genes are referred to herein as "cancer-associated" genes. Among the amplified loci which can be detected by a method of the invention are many oncogenes, including, e.g., the cyclin E locus, EGFR locus, HER2/Neu locus, myc locus, HBXAP/Rsf-1 locus, RAS locus, AKT locus, PIK3CA locus, Rsf-1 locus, and NOTCH (e.g. NOTCH 3) locus. The Cyclin E locus is a particularly useful marker, because it has been shown to amplified in a large number of cancers, including, e.g., ovarian, breast, lung, prostate, colorectal, esophageal, pancreatic, prostate, stomach, lung and brain cancer.

The analysis of the amount and strand integrity index of more than one amplified chromosomal locus (e.g., 2, 3, 4 or more loci) can improve the performance of a method of the invention. Any combination of cancer-associated amplified chromosomal loci can be assayed. For example, a panel comprising two or more (e.g., 2, 3, or all 4) of Cyclin E, Rsf-1 , NOTCH 3 and AKT 2 can be assayed. In one embodiment of the invention, the cancer to be detected is ovarian cancer, e.g., in a female human; and the chromosomal locus which is amplified is cyclin E, Notch 3, HBXAP/Rsf-1, AXT2, or P1K3CA, preferably cyclin E. (See, e.g., Nakayama et al. (2007) Int J Cancer 120, 1613- 2617.) Genomic DNA can be isolated by standard methods or with kits that are commercially available. Methods for isolating DNA and other molecular biology methods used in the invention can be carried out using conventional procedures. See, e.g., discussions in Sambrook, et al. (1989), Molecular Cloning, a Laboratory Manual, Cold Harbor Laboratory Press, Cold Spring Harbor, N. Y.; Ausubel et al. (1995). Current Protocols in Molecular Biology, N.Y., John Wiley & Sons; Davis et al. (1986), Basic Methods in Molecular Biology, Elseveir Sciences Publishing,, Inc., New York; Hames et al. (1985), Nucleic Acid Hybridization, IL Press; Dracopoli et al. (current edition) Current Protocols in Human Genetics, John Wiley & Sons, Inc.; and Coligan et al. (current edition) Current Protocols in Protein Science, John Wiley & Sons, Inc.

Methods for measuring the amount (copy number) of a DNA of interest are routine and conventional. For example, one can quantitate the amount of a DNA of interest with a nucleic acid microarray assay; by quantitative in situ hybridization [e.g., with one or more nucleic acid probes that are detectably labeled, and which hybridize specifically under selected hybridization conditions (e.g. under conditions of high stringency) to at least part of a chromosomal locus of interest]; or by Northern blotting. By hybridizing "specifically" is meant herein that two components (e.g. an amplified DNA and a nucleic acid probe) bind selectively to each other and not generally to other components unintended for binding to the subject components. The parameters required to achieve specific interactions can be determined routinely, using conventional methods in the art. For example, the hybridization can be carried out under conditions of high stringency. As used herein, "conditions of high stringency" or "high stringent hybridization conditions" means any conditions in which hybridization will occur when there is at least about 95%, preferably about 97 to 100%, nucleotide complementarity (identity) between the nucleic acids (e.g., a polynucleotide of interest and a nucleic acid probe). Generally, high stringency conditions are selected to be about 5°C to 20°C lower than the thermal melting point (T_m) for the specific sequence at a defined ionic strength and pH. Appropriate high stringent hybridization conditions include, e.g., hybridization in a buffer such as, for example, 6X SSPE-T (0.9 M NaCl, 60 mM NaH₂ PO₄, 6 mM EDTA and 0.05% Triton X-100) for between about 10 minutes and about at least 3 hours (in a preferred embodiment, at least about 15 minutes) at a temperature ranging from about 4°C to about 37°C. In one embodiment, hybridization under high stringent conditions is carried out in 5xSSC, 50% dionized Formamide, 0.1 % SDS at 42°C overnight.

In one embodiment of the invention, the amount of a DNA of interest is quantitated by binding it to a detectable agent, such as SYBR^{R ΓM} Green, and measuring the amount of the bound agent. Kits and reagents for detecting, for example, SYBR^R™ Green are commercially available.

The fluorescence intensity which reflects the concentration of DNA can be measured by a commercially available fluorescence plate reader.

In another embodiment of the invention, the amount of a DNA of interest in the body fluid is quantitated by performing a polymerase chain reaction (PCR) on that DNA, e.g. by real-time PCR. One PCR primer (e.g., an anchored primer) is selected which hybridizes under selected hybridization conditions to a portion of the sequence of interest, and a second PCR primer is selected which will amplify a segment of a nucleic acid of a desired size from the sequence. Typical amplicons range in size from between about 100 and 10,000 base pairs (bp), e.g. between about 100 and 1 ,000 bases pairs bp, e.g. about 200, 250, 500, 600, 700 or 800 bp. For a further discussion of lengths of DNA that can be amplified by PCR in a method of the invention, see the discussion below of "strand length (integrity)." For example, the Examples herein illustrate one embodiment of the invention in which a DNA of about 400 bp is generated by real time PCR. In this embodiment, the second PCR primer hybridizes under selected hybridization conditions to a portion of the sequence of the chromosomal locus of interest which lies about 400 bp downstream of the first primer. Variations of this method, such as the use of nested PCR primers, will be evident to a skilled worker and can also be employed.

By "about" a particular polynucleotide length is meant herein plus or minus 20%. Thus, "about" 400 bp includes a range of 320-480 bp. For entities that are indivisible, such as the number of base pairs, if "about" that entity is not an integer, it will be evident to a skilled worker that the nearest integer is meant. The endpoints of ranges, as used herein, are included within the range. For example, a range of 320-480 bp includes both 320 and 480 bp. "About" also means plus or minus 20% when applied to other numbers, such as the fold increase or decrease of an amount of DNA, a DNA length integrity index, etc. For example, a DNA length integrity index of about 0.8 includes a range of 0.65 to 0.96.

Without wishing to be bound by any particular mechanism, it is suggested that much of the genomic DNA released from tumors into body fluids (e.g., into the systemic circulation) in cancer patients results from cellular necrosis. As reported by some of the present inventors (Wang et al. (2003) Cancer Research 63, 3966-3968), tumor necrosis in solid malignant ovarian neoplasms produces a spectrum of DNA fragments with different strand lengths because of random and incomplete digestion of genomic DNA by a variety of DNAases. By contrast, cell death in normal tissues is primarily through apoptosis, which results in small and uniform DNA fragments of about 185-200 bp (Herrera et al. (2005) Clin Chem 51, 1 13-1 18; Taback et al. (2001) Cancer Res 61, 5723-5726). Therefore, the detection of a DNA molecule in an effusion sample which is greater than about 200 bp in length (e.g., greater than about 250, 300, 350, 400, 450, 500, or more bp) indicates that the DNA is likely to be from a tumor rather than from a non-cancerous tissue. The determination of DNA strand length in body fluid can be accomplished independently of, or simultaneously with, the determination of the amount of the DNA (copy number). As noted above, the minimum length of a DNA from a tumor in an effusion sample is likely to be greater than about 200 bp (e.g., greater than about 250, 300, 350, 400, 450, 500, or more bp). The maximum length that can be PCR amplified is considerably larger, e.g., as long as about 10,000 bp. In theory, if the conditions for handling DNA in the sample are gentle enough not to break the DNA, and if the thermostable polymerase used in the PCR reaction is processive enough to elongate sufficiently long sequences of DNA, one should be able to detect DNA molecules of about 10,000 bp, or even longer. Under the conditions used in the Examples herein, the optimal length for demonstrating strand length integrity of the DNA was shown to be about 400 bp. Longer DNA products were presumably degraded under the experimental conditions used.

When the DNA amount and strand integrity are determined independently, a "DNA strand integrity index" (sometimes referred to herein as a "DNA integrity index" or a "DNA length index") can be used. To determine a DNA integrity index, the amount of a DNA molecule of interest of a particular length in a cell-free effusion sample, as determined by PCR, is compared to the amount of a short sequence (e.g., about 100 bp) of a control DNA molecule, as determined by PCR. In one embodiment of the invention, as illustrated in the Examples herein, the length (integrity) of a DNA of interest is measured by comparing the difference between the Ct (cycle threshold) of an about 400 bp PCR product of a test DNA and the Ct of a 100 bp PCR product of either the test DNA, itself, or of a control DNA. The control DNA can be, e.g., from a constitutive gene, such as β-actin. Other suitable constitutive control genes will be evident to a skilled worker.

In one embodiment of the invention, the control DNA is from a tumor suppressor gene that is deleted in at least one of the alleles of some subjects with cancer. Suitable genomic regions harboring tumor suppressor control genes include, e.g., p53, PTEN, Rb locus, orpl6/pl4 locus. For example, the inventors have found that p53 DNA is a useful control. One of the alleles of this gene is deleted in many subjects having cancer (e.g., in ovarian cancer, lung cancer, head and neck cancer, gastrointestinal cancer, bladder cancer and renal cancer). Therefore, the total amount of this control DNA is decreased by a factor of about two compared to the test DNA in subjects having cancer, resulting in a two-fold higher ratio of the test DNA to the control. As shown in the Examples, the ratio of the AUC (area under the curve) of cyclin E of 400 bp compared to p53 of 100 bp was 0.698, which was higher than that of cyclin E of 400 bp compared to cyclin E of 100 bp (0.614).

In the experiment shown in the Examples, one might have expected the use of the longer cyclin E PCR products, such as 600 bp and 800 bp, to be more accurate for differentiating cancer cases from benign controls. However, as shown in Table 4, the longer the DNA strands, the lower their abundance in an effusion supernatant. This presumably reflects that fact that the longer DNAs were fragmented, which might have decreased the overall assay sensitivity. In other embodiments of the invention, gentler conditions can be used to prepare the DNA for analysis and/or a more processive DNA polymerase can be use, so longer PCR products can be used.

In a method of the invention, the DNA integrity index is a measure of the size of the DNA in the body fluid from the cancer-associated amplified chromosome locus. The value of an integrity index is a function of a number of variables, including, e.g., the size of the PCR DNA product, or the abundance of specific PCR products. As is shown in Table 4, when the strand integrity index is defined as the difference between the Ct (cycle threshold) of a 400 bp PCR product in a body fluid from a cancer-associated gene (in this case, Cyclin E) and the Ct of a 100 bp PCR product in the body fluid from the p53 gene, a DNA length index of about 0.9 is strongly correlated with a patient having ovarian cancer. In this case, the cut-off value to distinguish a person with ovarian cancer from an individual without neoplastic disease is based on ROC curve analysis to determine the optimal DNA index for this purpose. Areas under ROC curves provide a measure of the overall ability of a diagnostic test with multiple cutoffs to distinguish between diseased and non-diseased individuals. Using this index, one can achieve the best sensitivity and specificity.

A person of ordinary skill in the art will be able to determine a suitable cut-off value for the length integrity index for any marker (e.g., amplified chromosomal locus) of interest, having any size of interest (e.g., about 300, 400, 500, 600, 700, 800, 900, 1 ,000 or greater number of base pairs), without undue experimentation. For example, a strand length integrity index of at least about 0.65 (in conjunction with a determination that the amount of the DNA in the cell-free body fluid from the cancer-associated locus is statistically significantly increased compared to a negative reference standard) is indicative that the subject has the cancer. For example, the strand length integrity index can be at least about 0.7, 0.75, 0.8, 0.85, 0.9, or 0.95, or between about 0.8 and 0.9, or between about 0.8 and 1.0. As noted, the definition used in the Examples herein of the cyclin E DNA strand length

(integrity) index is the difference between the Ct of cyclin E (400 bp) and the Ct of p53 (100 bp). The raw data can be, e.g., from -4 to +6. The smaller the number, the longer the length of the cyclin E DNA. For a more convenient measure than using the raw data, one can normalize the raw data, using a 0 to 1.0 scale. Consider, for example, a case in which a sample from a test subject has an original index (based on the raw data) of -3. The rescaled index (within a range of 0 to 1.0) is then 0.9. This value is greater than a cut-off of 0.65, and thus indicates that the subject being tested has the cancer. The theoretical upper limit of the DNA integrity index is 1.0. In this example, the strand integrity index is defined as the difference between the cycle threshold number of 400 bp and 100 bp. Because the cycle number is the power of 2, the difference in cycle number is equal to the ratio of the absolute copy number of 400 bp to 100 bp.

Methods for designing PCR primers and for carrying out PCR reactions, including reaction conditions, such as the presence of salts, buffers, ATP, dNTPs, etc. and the times and temperature of incubation, are conventional and can be optimized readily by one of skill in the art. See, e.g., Innis et al., editors, PCR Protocols (Academic Press, New York, 1990); McPherson et al, editors, PCR: A Practical Approach, Volumes 1 and 2 (IRL Press, Oxford, 1991 , 1995); Barany( 1991)POJM^Ws and Applications J_, 5-16; Diffenbach et al., editors, PCR Primers, A Laboratory Manual (Cold Spring Harbor Press); etc. A variety of types of thermophilic DNA polymerases may be used, including high fidelity DNA polymerases.

Any of a variety of reaction chambers (e. g. , containers, wells of a plate, etc.) can be used, in any of a variety of formats. For example, one can employ 96 well PCR plates, 384 well PCR plates, 1536 well PCR plates, etc. Containers can be closed to form a leak -proof seal, in order to reduce or prevent cross-contamination of samples. Suitable formats for performing PCR reactions include computer-controlled thermal cyclers.

In one embodiment of the invention, quantitative or semi-quantitative real-time quantitative PCR (real-time PCR) is used to measure copy number. Real-time PCR is a technique that evaluates the level of PCR product accumulation during amplification. See, e.g., Gibson etal. (1996) Genome Research 6, 995-1001 , or Heid et al. (1996) Genome Research 6, 986-994). This technique permits quantitative evaluation of DNA levels from multiple loci, and/or in multiple samples.

Real-time PCR may, for example, be performed on the ABI 7700 Prism or on a GeneAmp^{R rM} 5700 sequence detection system (Applied Biosystems, Foster City, Calif). The 7700 system uses a forward and a reverse primer in combination with a specific probe with a 5' fluorescent reporter dye at one end and a 3' quencher dye at the other end (Taqman™). When the Real-time PCR is performed using Taq DNA polymerase with 5' -3' nuclease activity, the probe is cleaved and begins to fluoresce allowing the reaction to be monitored by the increase in fluorescence (Real-time). The 5700 system uses SYBR^{R ΓM} green, a fluorescent dye, that only binds to double stranded DNA, and the same forward and reverse primers as the 7700 instrument. Other conventional variants of these methods and devices may also be employed. Conventional methods are available for designing catching primers and fluorescent probes, e.g., the primer express program (Applied Biosystems, Foster City, Calif.). Optimal concentrations of primers and probes can be initially determined by those of ordinary skill in the art. Primers and probes, including control primers (e.g., for β-actin or p53) can be synthesized using conventional procedures or obtained commercially, e.g. from Perkin Elmer/ Applied Biosystems (Foster City, Calif.).

To quantitate the amount of specific amplified chromosomal locus, a standard curve is generated using a plasmid containing the gene of interest. In this way, one can generate samples with different concentrations of the reference sequence, and the test sample can be compared to the reference controls to determine the DNA concentrations. Standard curves are generated using the cycle threshold (Ct) values determined by the real-time PCR, which are related to the initial DNA concentration used in the assay. Standard dilutions ranging from 0-10⁶ copies of the gene of interest are generally sufficient. In addition, a standard curve is generated for the control sequence. This permits standardization of initial DNA content of a sample to the amount of control for comparison purposes. Several methods can be used to determine both the DNA strand length in the body fluid and the amount of the DNA (copy number) in the same reaction mixture. In one embodiment of the invention, all of the PCR measurements (e.g., using real time PCR) are conducted together in a single, multiplexed PCR reaction. In order to distinguish among the different PCR amplification products, conventional detectable markers which fluoresce to give different colors can be used. In another embodiment, an investigator takes advantage of the observation that the DNA from a cancer-related locus which is found in a body fluid from a patient having a cancer is generally at least about 200 bp in length. Therefore, one can determine the amount (copy number) of a DNA in a body fluid by PCR amplifying a PCR product from that DNA that is at least about 200 bp (e.g., at least about 250, 300, 350, 400, 450, 500, or more bp).

For any of the PCR amplification reactions used in a method of the invention, suitable controls will be evident to a skilled worker. For example, the PCR reactions can be normalized to a normalization control, such as the volume of the effusion sample, or the level of an abundant repetitive sequence dispersed in the genome, such as the Line-1 DNA element.

One aspect of the invention is a method for diagnosing (detecting) a cancer (e.g., ovarian cancer) in a subject, comprising screening an effusion sample (a body fluid, such as a cell-free body fluid) from the subject for both the amount of DNA from an amplified, cancer-associated chromosomal locus of interest, and the length integrity index of the DNA. A statistically significant increase in the amount of the DNA (having a high enough length integrity index), compared to a suitable negative reference standard (control), and/or an amount of the DNA that is statistically the same as a positive reference standard (control), is indicative of the cancer.

By "statistically significant" is meant a value that is reproducible or statistically significant, as determined using statistical methods that are appropriate and well-known in the art, generally with a probability value of less than five percent chance of the change being due to random variation. Some suitable statistical tests are described in Example I-C herein. For example, a significant increase in the amount of the DNA in an effusion sample from an cancer-associated amplified locus can be at least about 2-fold (e.g., at least about 5-fold, 10-fold, 15-fold, 20-fold, 25- fold, 30-fold, 100-fold, or more) higher than a negative reference standard. The degree of increase can be a factor of a number of variables, including the type and stage of the cancer, the age and weight of the subject, and the like.

A "positive reference standard," as used herein, reflects (represents, is proportional to) the amount of DNA from the cancer-associated chromosomal locus of interest in the same type of cell- free body fluid of a subject, or the average (e.g., mean) value for a population or pool of subjects, that have the cancer being tested for. In one embodiment of the invention, a value that is statistically the same as a positive reference standard is indicative of the cancer. A "negative reference standard," as used herein, reflects (represents, is proportional to) the amount of the DNA from the cancer- associated chromosomal locus of interest in the same type of cell-free body fluid of a subject, or the average (e.g., mean) value for a population or pool of subjects, that do not exhibit clinical evidence of the cancer of interest. Such "normal" controls do not have the cancer being tested for, or any type of cancer, or have a benign tumor of the type of cancer being assayed for. In one embodiment of the invention, the positive and negative reference standards are measured from subjects or pools of subjects, or are retrospective values from such subjects. Alternatively, and more conveniently, a positive or negative reference standard can comprise a defined amount of a DNA molecule of, e.g., the same or a different strand length (e.g., about 400 bp, 600 bp, 800 bp, 1000 bp, etc.), which reflects the amount of the DNA found in the body fluid of a subject as discussed above. Such a DNA can be prepared synthetically. In one embodiment, the DNA is in the same amount expected in a subject having the cancer being assayed for (positive reference standard), or not having the cancer being assayed for (negative reference standard). In another embodiment, the amount in the reference standard \s proportional to the amount expected in a subject having, or not having, the cancer being assayed, and the investigator applies a suitable multiple to convert the standard to the actual expected value.

A molecular method of the invention can be used in conjunction with other, secondary, methods for diagnosing a cancer. For example, one can evaluate allelic imbalance, e.g. by using digital SNP assays (as described, e.g, by Chang et al. (2002) Clin Cancer Res 8, 2580-2585); conventional cytology analysis (as described, e.g., by Motherby et al. (1999) Cytopathol 20, 350- 357; detection of mutations associated with the cancer (as described, e.g., by Parrella et al. (2003) Mod Pathol 16, 636-640), including a point mutation, a microsatellite alteration, a translocation, promoter methylation and/or the presence of a viral sequence; or determination of the amount of amplification of other amplified genomic loci [e.g., for ovarian cancer, the markers described by Nakayama et al. (2007) Int J Cancer 120, 2613-2617), or secretory tumor-associated markers (Borgono et al. (2004) MoI Cancer Res 2, 257-80; I. Shih (2007) Hum Immunol 68, 272-276; Shih et al. (2007) Gynecol Oncol 105, 501 -7)]. Other molecular assays will be evident to a skilled worker (see, e.g, Fiegl et al. (2004) J Clin Oncol 22, 474-83). In addition to PCR-based assays as discussed above, other molecular methods that can be used include, e.g., single molecule detection to measure the length of DNA based on the amount of DNA dye incorporated into the DNA fragments to be measured.

As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" point mutation, as used above, includes one or more point mutations. Secondary assays such as those discussed above can be carried out before a copy number/ length integrity assay of the invention (sometimes referred to herein as a "molecular" assay), as part of a preliminary screen; at the same time as the molecular assay is carried out; or after the molecular assay is carried out. As shown in the Examples herein, an assay for ovarian cancer which employed the detection of copy number of the cyclin E gene and determination of the DNA strand integrity, in conjunction with standard cytology analysis, provided 100% detection of ovarian cancer in the subjects tested. Another aspect of the invention is a diagnostic method for screening a sample from a cell- free body fluid (e.g., the circulation, or an effusion sample) of a subject, such as a non-symptomatic subject, or a subject having early stage cancer, for the presence of cancer in the subject, using a method of the invention. The detection in the sample of a statistically significantly elevated level of sufficiently long DNA from an amplified locus of interest would indicate a high probability of cancer and, in the case of an asymptomatic subject, necessitate a search for the cancer. In one embodiment, a method of the invention allows one to detect whether a subject has a curable form of the cancer, such a stage 1 or stage 2 cancer. It is generally difficult to detect cancer at such an early stage by using conventional cytology techniques. It is expected that one can "stage" a cancer with a method of the invention, since later stage cancers (stage 3 or 4) are expected to be characterized by increasing amounts of DNA from cancer-related chromosomal loci.

Another aspect of the invention is a diagnostic method for determining if a tumor in a subject is benign or malignant, comprising measuring DNA in a body fluid (e.g., a cell-free body fluid) from the subject by a method of the invention. A benign tumor will give rise to a lower amount of the tested DNA in the cell-free body fluid of than subject than will a malignant tumor. Another aspect of the invention is a method for monitoring the progress or prognosis of a cancer in a subject, comprising measuring DNA in a body fluid (e.g., a cell-free body fluid) from the subject by a method of the invention at various times during the course of the cancer.

Another aspect of the invention is a method for evaluating the efficacy of a cancer treatment of a subject (e.g., chemotherapy, radiation, biotherapy or surgical operation), comprising measuring DNA in a body fluid (e.g., a cell-free body fluid) from the subject by a method of the invention, at different times during the course of the treatment (e.g., before, during, and/or after the treatment).

Methods of the invention can be readily adapted to a high throughput format, using automated (e.g. robotic) systems, which allow many measurements to be carried out simultaneously.

Furthermore, the methods can be miniaturized. The order and numbering of the steps in the methods described herein are not meant to imply that the steps of any method herein must be performed in the order in which the steps are listed or in the order in which the steps are numbered. The steps of any method disclosed herein can be performed in any order which results in a functional method. Furthermore, the method may be performed with fewer than all of the steps, e.g., with just one step.

The phrase "a method for diagnosing" a cancer in a subject ..." is not meant to exclude tests in which no cancer is found. In a general sense, this invention involves assays to determine whether a subject has cancer, irrespective of whether or not such a cancer is detected.

Any combination of the materials useful in the disclosed methods can be packaged together as a kit for performing any of the disclosed methods. For example, reagents for performing PCR and for determining strand integrity can be packaged along with suitable PCR primers (e.g., a primer set designed to amplify a product of a desired size from a cancer-associated chromosomal locus of interest, such as a PCR product from the locus of about 250, 300, 350, 400, 450, 500, 600, 700, 800,

900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000 or longer bp). Primers for amplifying a panel of cancer-associated chromosomal loci can be included, as can primers for PCR amplifying positive and/or negative controls (for amount and/or size integrity). If desired, defined amounts of positive and negative standards {e.g., prepared synthetically) can be included. If desired, the reagents can be packaged in single use form, suitable for carrying one set of analyses.

Kits may supply reagents in pre-measured amounts so as to simplify the performance of the subject methods. Optionally, kits of the invention comprise instructions for performing the method. Other optional elements of a kit of the invention include suitable buffers, enzymes {e.g., a DNA polymerase for PCR amplifications, such as a Taq polymerase), packaging materials, etc. The kits of the invention may further comprise additional reagents that are necessary for performing the subject methods. The reagents of the kit may be in containers in which they are stable, e.g., in lyophilized form or as stabilized liquids.

In one embodiment of the invention, a kit comprises the primers shown in Table 2 for amplifying a 400 bp PCR product of cyclin E, and for amplifying a 100 bp PCR product of p53. Other optional primer pairs that can be included include PCR primer pairs for amplifying normalization controls, such as Line-1 elements. Synthetic DNAs that can serve as positive and/or negative standards can also be included.

In the foregoing and in the following example, all temperatures are set forth in uncorrected degrees Celsius; and, unless otherwise indicated, all parts and percentages are by weight. EXAMPLES Example I - Materials and Methods

A. Samples and genomic DNA isolation

The effusion samples were collected at the Innsbruck University Hospital in Austria and the National Norwegian Radium Hospital in Oslo, Norway. All specimens used were approved by the local institutional review boards or ethics committee and included a total of 268 anonymous effusions (140 ascites samples and 128 pleural effusions). The samples included 88 ovarian carcinoma effusions, 70 benign effusions (Table 1) and 110 effusions from other cancer types (34 lung carcinomas, 21 breast carcinomas, 10 endometrial carcinomas, 8 gastrointestinal carcinomas, 7 pancreatic carcinomas, 6 hepatocellular carcinomas, and 24 other miscellaneous cases).

Table 1. Clinical diagnosis of benign effusions

Diagnosis Number of patients

Cardiomyopathy 26

Liver cirrhosis 21

Pneumonia/Tuberculosis 15

Others

Renal failure 3

Pulmonary embolus 2

Meig's syndrome 1

Congenital chylothorax 1

Arthritis 1

Total 70

The diagnoses of effusions were based on clinical diagnosis and final pathology reports. There was no statistical significance in ages among the cancer and benign groups (p> 0.5, two-way ANOVA analysis). Effusion samples (3 ml - 2 L) were collected from patients, the samples were centrifuged for 5 minutes, and the supernatants were aliquoted and frozen immediately. Before genomic DNA extraction, the effusion supernatant was centrifuged again, and 200 μl of the supernatant, containing the cell-free DNA, was collected from tubes. The genomic DNA was extracted using a Qiagen DNA Blood Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. All samples were analyzed in a blinded fashion without prior knowledge of the specimen identity.

B. Primer selection and real-time PCR

The primers that amplified each genomic locus were designed based on the Santa Cruz website, genome,uscs.edu. The sequences of all PCR primers used in this study are listed in Table 2. Table 2. Primer nucleotide sequences used in the study.

The genomic loci were selected based on the frequency of amplification in ovarian serous carcinomas (Nakayama et al. (2007) Int J Cancer 120, 2613-17). Among them, the cyclin E locus was the most frequently amplified while the TP53 locus was commonly deleted (Kmet et al. (2003) Cancer 97, 389-404; Schuijer et al. (2003) Hum Mutat 21_, 285-291). Semi-quantitative real-time PCR was performed using locus-specific primers to obtain the cycle threshold and to calculate the DNA length integrity, which was defined as the ratio of the longer (400 bp) PCR products to the 100 bp PCR products. All samples were performed in duplicate. For semi-quantitative real-time PCR, an aliquot of 1 μl of the purified cell-free DNA was used in an 1 1.5 μl PCR mixture containing PCR buffer, 10 μM dNTP, and 0.125 U/ μl Platinum Taq (Invitrogen, Carlsbad, CA). The PCR protocol for iCycler was: denaturation for 1 minute at 94°C followed by 40 cycles of denaturation at 94°C for 30 seconds, annealing at 57⁰C for 30 seconds, and extension at 70⁰C for 5 minutes. The Bio-Rad iCycler software monitored the changes in fluorescence of SYBR^{R I M} Green I dye (Molecular Probe, Eugene, OR) in each cycle. The cycle threshold (Ct) value for each reaction was calculated by the iCycler software package. Alternatively, the real-time PCR was performed in ABI PRISM 7000 with Power SYBR^® Green PCR Master Mix (2X, Applied Biosystems) and the default program: 50 ⁰C for 2 minutes, 95°C for 10 minutes, and 40 cycles of the two-step reaction: 95 °C for 15 seconds and 60°C for 1 minute. Cyclin E DNA integrity index was modified from a previous report (Wang et al. (2003) Cancer Res 63, 3966-8) and was defined as Ct of cyclin E (400 bp) - Ct of p53 (100 bp).

C. Statistical Analysis

The results of the real-time PCR were analyzed using receiver operating characteristics (ROC) curves. ROC curves were constructed for cycle threshold of a given genomic locus and DNA integrity index as a diagnostic marker by plotting sensitivity versus 1 -specificity, followed by the calculation of the area under the curve (AUC) which was performed by a statistics program, MedCalc® version 8.1.1.0 (web site www.medcalc.be). Reproducibility of the real-time PCR assay was done by evaluating the coefficient of variation (CV) for each primer and disease subgroup. Linear regression was used to correlate the cyclin E copy number between ascites cell-free DNA and matched tumor cell pellets in 10 representative ovarian cancer effusions. The DNA copy number of a specific locus was normalized to the effusion volume or the level of the Line-1 DNA element because Line-1 represented the abundant repetitive sequences dispersed in the human genome and was thought to be a reliable reference marker to quantify specific genomic DNA fragments. Fisher exact test was used to determine the significant difference between cases and controls. Coefficient variation was used to determine assay reproducibility between two institutions.

Example II - Results

We first tested whether the DNA copy number was different between cancer cases and non- neoplastic controls using primers that amplified 100 bp of cyclin E, β-actin, p53, 2p24.1, 4pl5.31 and Line-1 (as the normalization control). We found that the copy number of all the genomic loci was higher in ovarian cancer effusions (n=88) than in benign samples (n=70) after normalization to effusion volume or Line-1 DNA (p<0.0001) (Table 3).

Table 3. DNA copy number (100 bp) of different loci in the diagnosis of ovarian cancer effusions.

Locus AUC (effusion volume*) AUC (Line-1 ^#) p-value

Cyclin E 0.832 0.847 < 0.0001 β-actin 0.816 0.81 1 < 0.0001 p53 0.815 0.807 < 0.0001

2p 0.693 0.685 < 0.0001 4p 0.657 0.703 < 0.0005

AUC: area under the ROC curve;

* Normalized to plasma volume; "normalized to Line-1 DNA copy number.

Receiver-operator characteristic (ROC) curves were used to compare the performance of the DNA copy number between cyclin E and non-amplified chromosomal loci in distinguishing ovarian cancer effusions from benign samples. As shown in Table 3, cyclin E primers demonstrated the highest performance in distinguishing ovarian cancer versus benign effusion samples with an area under ROC curve (AUC) of 0.832 [95% confidence interval (95% Cl), 0.762-0.889] when normalized to plasma volume and 0.847 (95% Cl, 0.738- 0.923) when normalized to Line-1 DNA.

We then assessed if a DNA length (integrity) index could improve the performance of cyclin E DNA copy number analysis in the differential diagnosis of malignant effusions. We tested the performance of different lengths of cyclin E DNA fragments using different reverse primers that were located 100 bp, 400 bp, 600 bp and 800 bp downstream from the anchored forward primer. The AUC of the abundance PCR product in distinguishing ovarian cancer versus benign effusions was highest using the 400 bp (AUC= 0.896) as compared to the 100 bp (AUC= 0.832), 600 bp (AUC= 0.813), and 800 bp (AUC= 0.825) (Table 4). Table 4. Results of the DNA copy number and DNA length index in the diagnosis of malignant effusions.

PCR Cycle DNA length AUC 95% CI p-value product threshold index

Cyclin E 100 31 0.832 0.762-0.889 O.0001

Cyclin E 100* 27.42 <0.35 0.879 0.781-0.944 O.0001

Cyclin E 400 30.93 0.896 0.838-0.939 O.0001

Cyclin E 400* 30.705 <0.90 0.936 0.868-0.975 O.0001

Cyclin E 600 31.865 0.813 0.741 -0.872 O.0001

Cyclin E 600* 30.82 <0.32 0.914 0.813-0.971 O.0001

Cyclin E 800 31.1 1 0.825 0.752-0.883 O.0001

Cyclin E 800* 31.1 1 <0.75 0.905 0.812-0.962 O.0001

*Combination of cyclin E copy number and DNA length index. Thus, we defined the cyclin E DNA strand length (integrity) index as the difference of Ct of cyclin E (400 bp) and Ct of p53 (100 bp). The reason to use the p53 genomic locus as the reference for the index is based on the fact that it is usually deleted in one of the alleles in ovarian serous carcinomas (Kmet et al. (2003) Cancer 97, 389-404; Schuijer et al. (2003) Hum Mutat 21_, 285-91), thereby increasing the index between cancer and benign cases. The cyclin E integrity index was significantly higher in malignant effusions than in benign effusions (p= 0.0014). As compared to the DNA length integrity index using β-actin (400 bp) - β-actin (100 bp) as we previously described (Wang et al. (2003) Cancer Res 63, 3966-3968), we found that the AUC of the cyclin E DNA integrity index defined in this study (cyclin E 400 bp - p53 100 bp) was higher (0.921) than the β-actin DNA integrity index (0.851). Furthermore, combining the cyclin E copy number and length index, we found the AUC for the cyclin E integrity index increased to 0.936 (95%C1: 0.868-0.975, pO.OOOl), with a sensitivity of 73% and a specificity of 97% (Fig. 1).

In the ovarian carcinoma group, 74 samples were obtained from ascites and 14 samples were from pleural effusions. There was no difference in cyclin E copy number and strand length index according to sample origins (p-value> 0.5). Similarly, in benign disease groups our results also showed no difference between ascites (n= 23) and pleural effusions (n= 47) (p-value> 0.3). Twenty- three effusion cases were selected in consecutive sequence of collection regardless of their cytology diagnoses and the remaining ovarian cancer cases were selected based on positive cytology. Therefore, only the former group of specimens was used to compare the results of the cyclin E assay to the traditional cytology. The cyclin E real-time PCR was able to identify 22 (95.6%) of 23 cases of ovarian carcinomas that were confirmed by clinical and surgical pathology findings using the cutoff value of 30.705 which was selected as the best value in distinguishing malignant cases versus benign controls based on ROC curve analysis. In the same group, cytology results were positive in 17 (73.9%) of 23 cases. Therefore, cyclin E assay was able to diagnose five additional cases of ovarian carcinoma which were not diagnosed by routine cytology. Combining the cyclin E assay with traditional cytology, all 23 cases of ovarian cancer were correctly identified, achieving 100% sensitivity in the diagnosis of malignant effusions.

Ten representative pairs of ascites samples and matched ascites tumor cell pellets from the same patients were compared to determine if there was a correlation in the cyclin E DNA copy number (normalized to Line-1 DNA copy number) between ascites cell-free DNA and the corresponding tumor cell DNA. Linear regression demonstrated a significant correlation (r = 0.946) of the cyclin E copy number between ascites cell-free DNA and the genomic DNA from tumor cell pellets of the matched cases. To determine whether the cyclin E assay was specific to ovarian carcinoma, the effusion specimens of ovarian carcinoma were compared to 1 10 malignant effusion samples of other cancer types. Using a Ct of cyclin E of 30.705, which was used as a cutoff to distinguish ovarian carcinoma from benign samples, the percentage of cases with Ct below the cutoff (i.e., higher copy number) was significantly higher in ovarian cancer cases (88.6%; 95% CI, 95.6%-81.6%) than in non-ovarian cancer cases (47.2%; 95% CI, 58.7%-35.7%) (p< 0.001). Reproducibility of the real-time PCR assay was assessed and the overall mean CV for the cyclin E PCR was 1.31% and for p53 PCR it was 1.02%. The reproducibility assay remained consistent and showed minimal variation between samples evaluated from day to day or batch to batch; CV for cyclin E ranged from 0.84% to 1.75% and CV for p53 from 0.59% to 1.52%. Furthermore, the realtime PCR data in representative specimens were analyzed in a separate institution (Development Center for Biotechnology, DCB) to assess the reproducibility of the assay for DNA integrity. A different experimental protocol was used for real-time PCR, including different primers, reagent composition, reaction program, and instruments. We found that the CV for cyclin E was within 3.19% ± 2.3%, whereas for Line-1 , it was 8.4% ± 4.8%.

Example HI - Discussion

In this study, we demonstrate that the measurement of quantity and quality of a specific amplified genomic region in cell-free DNA of body fluids can be used to distinguish a cancerous specimen from a benign one. We showed that the copy number of the cyclin E genomic fragments and its DNA length integrity were significantly higher in effusion samples of ovarian cancer than in benign effusions. This finding suggests that increased DNA levels and strand length (integrity) of a specific amplified genomic locus in body fluid are unique characteristics of tumor-released DNA, without regard to molecular genetic changes such as sequence mutations, microsatellite alterations and allelic imbalance. The measurement of cyclin E DNA fragments using real-time PCR provides a simple test to diagnose malignant effusions.

Effusions in the abdominal cavity (ascites) and the pleural compartment are associated with a variety of clinical conditions, including inflammatory disorders, infectious diseases, cardiac, liver and renal diseases as well as malignant neoplasms. Cytological examination is routinely performed to distinguish malignant from benign diseases. Although the sensitivity and specificity of cytology when combined with immunocytochemistry can be high in the diagnosis of malignant effusions, they can be variable (see, e.g., Davidson et al. (2003) Clin Lab Med Ti_., 729-54). This can be a result of small numbers of tumor cells in some of the effusion samples or the presence of a large amount of leukocytes, mesothelial cells and blood that obscure the detection of malignant cells. For example, inflammation which is commonly associated with a malignant effusion can result in reactive changes in mesothelial cells that make their morphological distinction from carcinoma cells difficult. Thus, the cyclin E real-time PCR assay described here provides an adjunct molecular test to distinguish malignant from benign effusions, providing increased clinical utility.

We first compared DNA copy number of cyclin E and other non-amplified chromosomal loci in effusion DNA samples using a quantitative real-time PCR assay of the 100 bp products. We demonstrated that measurement of the cyclin E DNA copy number has the best performance in the diagnosis of ovarian cancer effusions. Next, we focused on the cyclin E locus to improve its diagnostic efficacy by testing different lengths of PCR products and by combining DNA copy number and DNA integrity index of cyclin E. Without wishing to be bound by any particular mechanism, it appears that the significant correlation of cyclin E copy number between cell-free DNA from effusion and the DNA from the corresponding tumor cell pellets indicates that a significant amount of the cell-free DNA in body fluids was likely derived from tumor cells. The demonstration that the measurement of the cyclin E DNA copy number was better than other genomic loci that are rarely amplified in ovarian cancer suggests that the amplified genomic region was a better marker for cancer diagnosis, as it was released at higher levels from tumor cells into body fluids than the non-amplified chromosomal regions. The selection of the cyclin E locus also facilitated the differential diagnosis in malignant effusions that resulted from different types of cancer because cyclin E was most frequently amplified in ovarian cancer compared to other types of cancer analyzed in this study.

Example IV - Studies using different cancers and/or different amplified genomic loci and/or different body fluids The same experimental approach used in Example II, including the methods, reagents and primers provided in Example I, will be used to measure DNA copy number and length integrity in cell-free DNA in plasma, serum, effusion, urine and/or sputum samples. Although the cyclin E genomic locus is amplified (increase in DNA copy number) in many human cancers, we will also include other commonly amplified regions instead of, or in combination with, the cyclin E locus for cancer diagnosis. For example, we will use the EGFR locus for diagnosing head and neck (including oral cavity) and lung cancer, the HER2/Neu locus for diagnosing breast cancer, and the myc locus for diagnosing lung and/or gastrointestinal cancer. The PCR primer sequences to amplify those loci (400 bp or longer) will be 12-15 bp long sequences (both forward and reverse). It will be evident to a skilled worker how to identify and generate such PCR primers for each of these gene loci, using conventional procedures. It is expected that these approaches will allow one to diagnose the indicated types of cancer, as well as to achieve a higher performance (both sensitivity and specificity) of the described assays.

Example V - A prospective clinical study confirming that an assay of the invention can be used to diagnose cancer

A retrospective and/or prospective cohort of cases and controls will be initiated. The cases include 100 patients with malignant ovarian cancer (at different clinical stages), and the controls include 100 patients with benign adnexal mass and 100 (control) individuals without neoplastic diseases. Venous blood samples (10 cc) will be drawn using an EDTA tube upon informed consent and the samples will be labeled and grouped as experimental codes which then become anonymized.

The collection of blood samples will be in compliance to the institutional review board policies. Plasma samples will be prepared, aliquoted and genomic DNA will be purified from the equal amount of blood specimens using Qiagen blood kits. It is expected that our method will be able to distinguish patients with early stage ovarian cancer from those with benign adnexal masses and individuals without neoplastic diseases. We will use ROC analysis to determine the optimal cutoff of DNA concentration and length integrity for each locus in ovarian cancer types. Similar studies will also be performed in breast, lung and gastrointestinal cancer to determine the efficacy of our method in detecting early curable cancer in patients.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make changes and modifications of the invention to adapt it to various usage and conditions and to utilize the present invention to its fullest extent. The preceding preferred specific embodiments are to be construed as merely illustrative, and not limiting of the scope of the invention in any way whatsoever. The entire disclosure of all applications, patents, and publications cited above (including U.S. provisional application 60/999,414, filed October 18, 2007) and in the figures, are hereby incorporated in their entirety by reference.

Claims

We claim:

1. A method for diagnosing a cancer in a subject, comprising measuring in a cell-free body fluid from the subject a) the amount of DNA in the body fluid from an amplified cancer-associated chromosomal locus, compared to a positive and/or a negative reference standard, wherein the positive and negative reference standards are representative of the amount of the DNA in the body fluid of a population of subjects having the cancer, or not having the cancer, respectively, and b) the strand length integrity index of the DNA in the body fluid from the amplified cancer- associated chromosomal locus compared to a baseline value, wherein if the amount of the measured DNA is statistically significantly increased compared to the negative reference standard, and/or is statistically the same as the positive reference standard, and if the strand length integrity index is at least 0.65, this indicates that the subject has the cancer.

2. The method of claim 1, wherein the statistically significantly increased amount of the measured DNA from the amplified cancer-associated chromosomal locus is at least about 5-fold greater than the negative reference standard.

3. The method of claim 1, wherein the statistically significantly increased amount of the measured DNA from the amplified cancer-associated chromosomal locus is at least about 30-fold greater than the negative reference standard.

4. The method of claim 1 , wherein the amount of DNA from the amplified cancer-associated cchhrroommoossoommaall llooccuuss iiss mmeeaassuurreedd I by determining the amount of SYBR^{R I M} Green which binds to the DNA in the cell-free body fluid.

5. The method of claim 1 , wherein the amount of DNA in the body fluid from the amplified cancer- associated chromosomal locus is measured by real-time PCR of the DNA, and wherein the PCR amplification product is at least about 100 bp in length.

6. The method of claim 1 , wherein the positive reference standard is a) an aliquot of the DNA in a cell-free body fluid from the amplified cancer- associated chromosomal locus of a subject which has the cancer, or the average or mean of such values from a population or pool of such subjects, or b) a synthetically prepared DNA molecule in the amount, or proportional to the amount, of the DNA of a); and the negative reference standard is c) an aliquot of the DNA in a cell-free body fluid from the amplified cancer-associated chromosomal locus of a subject which does not have the cancer, or the average or mean of such values from a population or pool of such subjects, or d) a synthetically prepared DNA molecule in the amount, or proportional to the amount, of the DNA of c), or an aliquot of a cell-free body fluid which lacks DNA from the cancer-associated chromosomal locus.

7. The method of claim 1, wherein the positive reference standard is a) a PCR amplification product of the DNA in a cell-free body fluid from the amplified cancer-associated chromosomal locus of a subject which has the cancer, or the average or mean of such values from a population or pool of such subjects, or b) a synthetically prepared DNA molecule in the amount, or proportional to the amount, of the PCR amplification product of a); and the negative reference standard is c) a PCR amplification product of the DNA in a cell-free body fluid from the amplified cancer-associated chromosomal locus of a subject which does not have the cancer, or the average or mean of such values from a population or pool of such subjects, or d) a synthetically prepared DNA molecule in the amount, or proportional to the amount, of the PCR amplification product of c), or the PCR amplification product of an aliquot of a cell-free body fluid which lacks DNA from the cancer-associated chromosomal locus.

8. The method of claim 1 , wherein the length integrity index is determined by measuring the difference of the Cycle threshold (Ct) of a product of at least about 300 bp which is amplified by PCR from the cancer-associated chromosomal locus DNA in the body fluid, and the Ct of a product of about 100 bp which is which is amplified by PCR from a control portion of the chromosome in the body fluid.

9. The method of claim 8, wherein the PCR product which is amplified from the cancer-associated chromosomal locus DNA in the body fluid is about 400 bp.

10. The method of claim 8, wherein the control portion of the chromosome comprises the p53 gene.

1 1. The method of claim 8, wherein the integrity index is the difference of the Ct of an about 400 bp PCR product which is PCR amplified from the cancer-associated chromosomal locus DNA in the body fluid, and the Ct of an about 100 bp PCR product which is PCR amplified from the p53 gene DNA in the body fluid.

12. The method of claim 1, wherein the amount of the cancer-associated DNA from an amplified chromosomal locus and the length integrity of the cancer-associated DNA are determined together, wherein all of the PCR measurements are conducted together in a single, multiplexed PCR reaction.

13. The method of claim 1, wherein the amount of the cancer-associated DNA from an amplified chromosomal locus and the length integrity of the cancer- associated DNA are determined together, in a single PCR reaction, comprising, measuring the amount of a PCR product of at least about 300 bp that is PCR amplified from the amplified cancer-associated chromosomal locus DNA in the body fluid, compared to a positive and/or negative reference standard.

14. The method of claim 5, wherein the PCR reactions are normalized to a normalization control.

15. The method of claim 8, wherein the PCR reactions are normalized to a normalization control.

16. The method of any of claims 1 -15, wherein the cancer-associated amplified chromosomal locus comprises one or more of the Cyclin E, EGFR, HER2/Neu, myc, HBXAP/Rsf-1, RAS, AKT, PIK3CA, Rsf-1 , and/or NOTCH (e.g. NOTCH 3) genes.

17. The method of any of claims 1-15, wherein the cancer-associated amplified chromosomal locus comprises the Cyclin E gene.

18. The method of any of claims 1 -15, wherein the cancer is ovarian, breast, lung, prostate, colorectal, esophageal, pancreatic, prostate, head and neck, gastrointestinal, bladder, kidney, liver, lung, or brain cancer.

19. The method of any of claims 1-15, wherein the cancer is ovarian cancer.

20. The method of any of claims 1-15, wherein the body fluid is a cell-free body fluid generated from a pleural effusion, ascites sample, plasma, urine or sputum.

21. The method of any of claims 1-15, wherein the method is more accurate than a conventional cytology method for detecting the cancer.

22. The method of any of claims 1-15, further comprising assaying for the presence of an alteration in the cancer-associated amplified locus which is associated with the cancer.

23. The method of claim 22, wherein the alteration is a point mutation, a microsatellite alteration, an allelic imbalance, a translocation, a promoter methylation and/or the presence of a viral sequence.

24. The method of any of claims 1-15, further comprising performing a conventional cytology assay for the cancer.

25. The method of any of claims 1-15, which is a method for determining if a tumor in the subject is benign or malignant.

26. The method of any of claims 1-15, which is a method for detecting cancer at stage 1 or stage 2.

27. The method of any of claims 1 -15, which is a method for monitoring the progress or prognosis of a cancer in a subject, comprising measuring the DNA at various times during the course of the cancer.

28. The method of any of claims 1 -15, which is a method for evaluating the efficacy of a cancer treatment, comprising measuring the DNA at different times during the treatment.

29. The method of any of claims 1 -15, wherein in the method is high throughput.

30. The method of any of claims 1 -15, wherein the subject is human.

31. A method for diagnosing ovarian cancer in a subject, comprising measuring by real-time PCR, in a cell-free body fluid from a pleural effusion, ascites fluid, plasma, urine or sputum of the subject, a) the amount of DNA in the cell-free body fluid of Cyclin E DNA, compared to a positive and/or a negative reference standard, wherein the positive and negative standards are representative of the amount of the DNA in the cell-free body fluid of a population of subjects having the cancer, or not having the cancer, respectively, and b) the strand length integrity index of the Cyclin E DNA in the cell-free body fluid, wherein the strand length integrity index is the difference of the Ct of an about 400 bp fragment of Cyclin E which is PCR amplified from the body fluid and the Ct of an about 100 bp fragment of p53 which is PCR amplified from the body fluid, wherein if the amount of the measured Cyclin E DNA is statistically significantly increased compared to the negative reference standard, and/or is statistically the same as the positive reference standard, and if the strand length integrity index is at least 0.8, this indicates that the subject has ovarian cancer.

32. A kit for performing the method of claim 1 , comprising a) a set of PCR primers for amplifying an about 400 bp fragment of Cyclin E, b) a set of PCR primers for amplifying an about 100 bp fragment of p53, c) positive and negative standards for quantitating the amounts of the DNA products.