WO2007133725A1 - Biomarkers for detection and diagnosis of head and neck squamous cell carcinoma - Google Patents
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Classifications
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
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- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Definitions
- the invention relates to a panel of biomarkers and methods for diagnosis of head and neck squamous cell carcinoma (HNSCC).
- HNSCC head and neck squamous cell carcinoma
- sensitive, specific and reliable detection and identification of biomarkers that are uniquely produced in head and neck squamous cell carcinoma (HNSCC) are provided.
- HNSCC Head and neck squamous cell carcinoma
- UDT upper aerodigestive tract
- HNSCC head and neck squamous cell carcinoma
- a panel of biomarkers for the detection/diagnosis of head and neck squamous cell carcinoma are described.
- CD44, hyaluronic acid (HA) and hyaluronidase (HAase) comprise a related group of molecules with distinct roles in tumorigenesis that are detectable in saliva, and have been found to be useful for early detection of HNSCC.
- the panel of biomarkers are useful for distinguishing between patients with benign conditions and those with malignant disease.
- the above-mentioned biomarkers can be considered indicators of the presence of HNSCC or increased risk thereof in a subject.
- it is one object to provide a method of detecting/diagnosing HNSCC in a subject comprising assaying for the presence of at least one biomarker in a subject sample, and correlating a detection of the biomarker(s) with a diagnosis of, or indication of increased risk of, developing HNSCC, wherein the correlation takes into account the detection of one or more biomarker in the subject sample, as compared to the frequency or level of occurrence of the biomarker(s) in normal subjects, wherein the biomarker(s) is selected from: hyaluronic acid (HA); hyaluronidase (HAase) and CD44.
- HA hyaluronic acid
- HAase hyaluronidase
- the method of detecting HNSCC comprises comparing hyaluronic acid; hyaluronidase, CD44 and/or total protein values obtained from a patient with values from normal subjects. Total protein values in the saliva of patients with HNSCC have been found to be higher than total protein values in normal subjects.
- increased CD44, HA, and HAase levels in saliva or an oral rinse with normalization to protein values are diagnostic of HNSCC, or increased risk of future development thereof.
- increased absolute levels of CD44, HA, and HAase in saliva or an oral rinse are diagnostic of HNSCC, or increased risk of future development thereof.
- increased values of protein in saliva or an oral rinse are diagnostic of HNSCC, or increased risk of future development thereof.
- Levels of HA, HAase, CD44, and/or total protein are also indicative of tumor stage. For example, low levels of elevation are indicative of early stage cancer; higher levels are indicative of later stage cancer.
- detection of at least one biomarker is diagnostic of tumor stage.
- the type of biomarker detected can also be indicative of tumor stage.
- a method of monitoring effectiveness of treatment of HNSCC comprising measuring at least one of: HA; HAase, CD44, and/or total protein in a biological sample obtained from a patient, wherein decreased levels of at least one of HA; HAase, CD44, and/or total protein compared to levels detected prior to treatment in the same patient, is indicative of effective treatment.
- a method of predicting the course of HNSCC in a subject comprising measuring at least one of HA, HAase, CD44 and/or total protein in a biological sample obtained from said subject, wherein the degree of elevation of HA, hyaluronidase, CD44, and/or total protein compared to normal subjects, or in a population of subjects with HNSCC, is indicative of the severity of HNSCC, with a greater degree of elevation being indicative of more severe disease, and/or a less favorable prognosis. It is noted that in the case of CD44 very low levels may also be indicative of poorer prognosis since in very severe cases some genes including CD44 get turned off by promoter hypermethylation.
- a method of predicting recurrence of HNSCC in a subject comprising measuring at least one of HA, HAase, CD44, and/or total protein in a biological sample obtained from said subject, wherein an elevated level of HA, HAase, CD44, and/or total protein compared to normal subjects is predictive of increased likelihood of recurrence (i.e. of the same or an additional new tumor) of HNSCC.
- the presence or level of the biomarker(s) can be compared with prior values obtained from the subject (e.g. following treatment)
- the biomarkers can be detected, for example, using a protein assay, binding assay or an immunoassay. Exemplary assays are described in detail in the examples which follow. For a positive diagnosis, the biomarkers and/or total protein detected are elevated as compared to values in normal healthy controls.
- the subject sample may be selected, for example, from the group consisting of oral rinse, saliva, blood, blood plasma, serum, urine, tissue, cells, and liver.
- the sample is an oral rinse.
- a kit for diagnosing/detecting HNSCC or elevated risk thereof in a subject is provided. The kit may also be used for measuring treatment success or predicting recurrence of HNSCC.
- the kit comprises at least one means of detecting a biomarker selected from the group consisting of HA, HAase and total protein.
- the kit comprises means for detecting HA, HAase and total protein.
- the kit may also contain means for detecting CD44, in particular solCD44.
- the kit may contain one or more of: a substrate or container for holding a biological sample (e.g. of saliva or an oral rinse), reference standard(s) of biomarker(s) in solution or solid form, one or more antibodies specific for the biomarkers, and directions for carrying out detection assay(s) for the biomarkers with the contents of the kit.
- the kit comprises means for detecting HA, HAase, solCD44 and total protein.
- the kit comprises: (a) a substrate or container for holding a biological sample isolated from a human subject suspected of having HNSCC, or of being at risk thereof; (b) a fluorogenic agent that detects at least one biomarker; (c) a panel of biomarkers; and optionally, (d) printed instructions for reacting the agent with the biological sample or a portion of the biological sample to detect the presence or amount of at least one biomarker in the biological sample.
- the kit comprises a panel of biomarkers of any one or more of: HA and hyaluronidase to be used as standards, along with means of detecting HA and HAase and total protein.
- the kit may also contain a standard for and/or means for detecting CD44, in particular solCD44.
- the kit comprises antibodies specific for any one or more of biomarkers: HA 3 HAase, and CD44. [0022] Other aspects are described infra.
- Figure 1 is a scan of a Western blot comparing CM of HNSCC cell lines, SolCD44 positive control saliva, HNSCC saliva, and solCD44 negative control saliva.
- Figures 2A-2B are a scan of Western blots showing results following CD44s overexpression in SCC-25 transfectants versus wild-type.
- Figure 3 is a scan of a Western blot showing expression of CD44 in SCC-25 cells following transient transfection with CD44s is identical to expression following stable transfection. To confirm that the CD44 expression pattern seen in the SCC-25 transfectant pool is a result of overexpression of CD44 and not an artifact of incorporation into the host genome, transient transfection of SCC-25 using CD44s was performed. The band at 130 IeDa is also identical to the 13OkDa band seen in HNSCC saliva sample 2. ELISA confirmed that the transient transfectant contains 4 times more CD44 than the nontransfected cells. [0027J
- Figure 4 is a graph showing overexpression of CD44s results in significantly increased cell growth.
- Figure 6 is a graph showing a representative standard curve for solCD44 ELISA
- Figure 7 is a graph showing salivary solCD44 levels are elevated in HNSCC patients compared to normal nonsmokers and patients with benign disease. Data was transformed to log 2 solCD44 level to aide visualization of differences. Differences between HNSCC patients and the two groups without cancer both were highly statistically significant p ⁇ 0.0001.
- HNSCC includes cancers involving the oral cavity, pharynx, and larynx.
- Primary treatment modalities entail combinations of surgery, radiation, and chemotherapy. Because these tumors are often diagnosed in late stage, the necessary multimodality treatment results in disfigurement, severe speech, swallowing, and breathing problems and substantial healthcare costs. Since early detection of HNSCC could increase survival rates from 40% to 80%, a simple and inexpensive screening test would be useful. Currently physical exam, though inadequate, is the only screening tool available for this oppressive disease. Saliva provides an optimum medium for screening since it bathes the tumor and is convenient to obtain noninvasively. Several markers including solCD44, HA, and HAase have been identified in saliva and are overexpressed in HNSCC patients compared to controls. A panel of markers are described for the identification of early HNSCC with high sensitivity and specificity.
- Marker or “biomarker” are used interchangeably herein, and in the context of the present invention refer to a polypeptide (of a particular apparent molecular weight, or, in the case of HA, a molecule made of repeating disaccharide units) which is differentially present in a sample taken from patients having head and neck squamous cell carcinoma (HNSCC) as compared to a comparable sample taken from control subjects (e.g., a person with a negative diagnosis, normal or healthy subject).
- HNSCC head and neck squamous cell carcinoma
- a marker can be a polypeptide which is present at an elevated level or at a decreased level in samples of patients with head and neck squamous cell carcinoma (HNSCC) compared to samples of control subjects.
- HNSCC head and neck squamous cell carcinoma
- a marker can be a polypeptide which is detected at a higher frequency or at a lower frequency in samples of patients compared to samples of control subjects.
- a marker can be differentially present in terms of quantity, frequency or both.
- a marker, compound, composition or substance is differentially present between the two samples if the amount of the marker, compound, composition or substance in one sample is statistically significantly different from the amount of the marker, compound, composition or substance in the other sample.
- a compound is differentially present between the two samples if it is present at least about 120%, at least about 130%, at least about 150%, at least about 180%, at least about 200%, at least about 300%, at least about 500%, at least about 700%, at least about 900%, or at least about 1000% greater than it is present in the other sample, or if it is detectable in one sample and not detectable in the other.
- a marker, compound, composition or substance is differentially present between the two sets of samples if the frequency of detecting the polypeptide in samples of patients' suffering from head and neck squamous cell carcinoma (HNSCC), is statistically significantly higher or lower than in the control samples.
- HNSCC head and neck squamous cell carcinoma
- a biomarker is differentially present between the two sets of samples if it is detected at least about 120%, at least about 130%, at least about 150%, at least about 180%, at least about 200%, at least about 300%, at least about 500%, at least about 700%, at least about 900%, or at least about 1000% more frequently or less frequently observed in one set of samples than the other set of samples.
- Diagnostic means identifying the presence or nature of a pathologic condition and includes identifying patients who are at risk of developing HSCC. Diagnostic methods differ in their sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals who test positive (percent of "true positives").
- a "test amount” of a marker refers to an amount of a marker present in a sample being tested.
- a test amount can be either in absolute amount (e.g., ⁇ g/ml) or a relative amount (e.g., relative intensity of signals).
- a “diagnostic amount” of a marker refers to an amount of a marker in a subject's sample that is consistent with a diagnosis of head and neck squamous cell carcinoma (HNSCC).
- a diagnostic amount can be either in absolute amount (e.g., ⁇ g/ml) or a relative amount (e.g., relative intensity of signals).
- a "control amount" of a marker can be any amount or a range of amount which is to be compared against a test amount of a marker.
- a control amount of a marker can be the amount of a marker in a person without head and neck squamous cell carcinoma (HNSCC).
- a control amount can be either in absolute amount (e.g., ⁇ g/ml) or a relative amount (e.g., relative intensity of signals).
- the terms "polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of ⁇ -amino acid residues, in particular, of natural ly-occuring ⁇ -amino acids.
- polypeptides can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins.
- polypeptide include glycoproteins, as well as non-glycoproteins.
- Detectable moiety refers to a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
- useful labels include 32 P, 35 S, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin-streptavidin, dioxigenin, haptens and proteins for which antisera or monoclonal antibodies are available, or nucleic acid molecules with a sequence complementary to a target.
- the detectable moiety often generates a measurable signal, such as a radioactive, chromogenic, or fluorescent signal, that can be used to quantify the amount of bound detectable moiety in a sample. Quantitation of the signal is achieved by, e.g., scintillation counting, densitometry, or flow cytometry.
- Antibody refers to a polypeptide ligand substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, which specifically binds and recognizes an epitope (e.g., an antigen).
- the recognized immunoglobulin genes include the kappa and lambda light chain constant region genes, the alpha, gamma, delta, epsilon and mu heavy chain constant region genes, and the myriad immunoglobulin variable region genes.
- Antibodies exist, e.g., as intact immunoglobulins or as a number of well characterized fragments produced by digestion with various peptidases. This includes, e.g., Fab' and F(ab) ! 2 fragments.
- antibody also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies. It also includes polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, or single chain antibodies. "Fc" portion of an antibody refers to that portion of an immunoglobulin heavy chain that comprises one or more heavy chain constant region domains, CHi, CH 2 and CH3, but does not include the heavy chain variable region. [0045] By “binding assay” is meant a biochemical assay wherein the biomarkers are detected by binding to an agent, such as an antibody, through which the detection process is carried out. The detection process may involve radioactive or fluorescent labels, and the like.
- immunoassay is an assay that uses an antibody to specifically bind an antigen (e.g., a marker).
- the immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.
- the phrase “specifically (or selectively) binds" to an antibody or “specifically (or selectively) immunoreactive with,” when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies.
- the specified antibodies bind to a particular protein at least two times the background and do not substantially bind in a significant amount to other proteins present in the sample.
- Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
- a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein.
- solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).
- sample generally refers to a human, although the methods of the invention are not necessarily limited to humans, and should be useful in other mammals.
- sample is used herein in its broadest sense.
- a sample comprising polynucleotides, polypeptides, peptides, antibodies fragments and derivatives thereof may comprise a bodily fluid; a soluble fraction of a cell preparation, or media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA.
- RNA, or cDNA polypeptides, or peptides in solution or bound to a substrate; a cell; a tissue; a tissue print; a fingerprint, skin or hair; fragments and derivatives thereof.
- at risk of is intended to mean at increased risk of, compared to a normal' " subject, or compared to a control group, e.g. a patient population.
- a subject "at risk of developing HNSCC is at increased risk compared to a normal population, and a subject "at risk of a recurrence of HNSCC may be considered at increased risk of having a recurrence as compared to the risk of a recurrence among all treated HNSCC patients
- "Increased risk” or “elevated risk” mean any statistically significant increase in the probability, e.g., that the subject will develop HNSCC, or a recurrence thereof.
- the risk is preferably increased by at least 10%, more preferably at least 20%, and even more preferably at least 50% over the control group with which the comparison is being made.
- "CD44 marker” is intended to include soluble CD44 and isoforms thereof.
- HNSCC Head and Neck Squamous Cell Carcinoma
- a method of detecting and diagnosing head and neck squamous cell carcinoma comprises assaying for at least one biomarker in a subject sample, and correlating the detection of the biomarker(s) with a diagnosis of HNSCC or with increased risk of development of HNSCC, wherein the correlation takes into account the number of and level of biomarker(s) in the sample, as compared to normal values.
- the biomarker(s) is selected from HA, HAase, and CD44.
- at least one of the biomarkers is detected, more preferably, a plurality of the biomarkers are detected.
- one or more isoforms of solCD44 is detected.
- the subject sample may be selected, for example, from the group consisting of saliva, an oral rinse, blood, blood plasma, serum, urine, tissue, cells, and liver.
- the sample is saliva or an oral rinse.
- Saliva can be collected using many methods. One common method is whole saliva collection. Saliva is collected, often over a set period of time, from the anterior oral cavity, where the majority is released under resting conditions. Oral rinses involve use of a set amount of a fluid, often saline, that is manipulated in the mouth and helps release substances adherent to the lining of the oral cavity, larynx and pharynx. It is theorized that whole saliva may reflect systemic expression of substances while oral rinses are more reflective of local expression of substances.
- the CD44 marker is intended to include soluble CD44 and isoforms thereof.
- the biomarkers can be detected using a protein assay, binding assay, an immunoassay, or any other suitable assay known to those of skill in the art. Exemplary assays are described in detail in the examples which follow. For a positive diagnosis of
- the biomarkers and/or total protein detected are elevated as compared to a normal healthy control.
- detection of at least one biomarker is diagnostic of tumor staging. It has been shown, for example, in bladder cancer that certain biomarkers are indicative of aggressive tumors (Lokeshwar et al., 2000, J. Urol. 163:348-56), and it is expected that the presence/amount of the biomarkers disclosed herein will be indicative of tumor staging in HNSCC.
- Saliva as a screening medium: Saliva is becoming a well-accepted screening medium for various disease processes. It has an advantage over blood because it is readily accessible and noninvasive.
- the average daily production of whole saliva varies between 1 and 1.5 liters.
- Components of whole saliva include blood and blood derivatives from intraoral bleeding and gingival crevicular fluid, extrinsic substances such as food, epithelial lining cells, microbes, bronchial, nasal, salivary gland secretions.
- the majority of saliva, in the unstimulated state originates from submandibular glands (65%) with 20% from the parotid gland and the remainder from sublingual and minor salivary glands located throughout the upper aerodigestive tract (UADT).
- saliva contains a variety of electrolytes, immunoglobulins, proteins, enzymes, mucins, and nitrogenous products and is hypotonic especially in the unstimulated state.
- Normal pH ranges from 6-7.
- the salivary flow rate is influenced by the size of the salivary glands, hydration status, nutritional state, stimulus, and gender. Total protein concentrations of whole saliva in the unstimulated state give an accurate indication of the hydration state of an individual.
- Saliva is typically assayed as the product of an oral rinse, as described, for example, below.
- CD44 comprises a family of isoforms expressed in many cell types.
- CD44 CD44 epithelial
- CD44v CD44v3-l 0 in keratinocytes.
- CD44v CD44 variant isoforms
- CD44 proteins are also released in soluble form (solCD44) via proteases and are detectable in normal circulation and saliva. Detection methods using solCD44 are described, e.g., in U.S. Appl. No. 11/090,705, filed March 28, 2005, which is incorporated herein by reference. [0060] Overexpression of normally expressed isoforms also promotes oncogenesis. CD44 transfection increases migration and confers metastatic potential to some cell types, while blocking cell surface CD44 binding to HA reduces tumor cell growth and migration. CD44 associates with other molecules to mediate oncogenic signaling. These include members of the ERBB family of receptor tyrosine kinases such as ERBB 1 and ERBB2.
- CD44 also functions as a platform for growth factors and members of the matrix metalloproteinase (MMP) family of enzymes, further contributing to signaling events.
- MMP matrix metalloproteinase
- Hyaluronic Acid HA is a nonsulfated glycosaminoglycan (GAG) 5 overexpressed in certain cancers.
- HA is synthesized by hyaluronan synthase on the surface of cells and is comprised of repeating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine. It is present in body fluids, tissues, and extracellular matrix. It interacts with cell surface receptors (e.g., CD44, RHAMM, etc.) and, through these interactions, regulates cell adhesion, migration, and proliferation.
- HA may be synthesized by stromal cells, tumor cells or both. In tumor tissues, HA supports metastasis by promoting tumor cell migration, offering protection against immune surveillance and causing a partial loss of contact-medicated inhibition of cell growth and migration.
- HA small fragments of HA are angiogenic and have been isolated from urine of bladder cancer patients, prostate cancer tissue, and saliva from HNSCC patients. Concentrations of HA are elevated in several cancers, including colon, breast, prostate, bladder and lung. Tissue expression of HA in tumors such as colon and breast, indicates a poor prognosis.
- Hyaluronidase is an endoglycosidase that degrades HA into small angiogenic HA fragments. HA and HA fragments stimulate endothelial cell proliferation, adhesion and migration by activating the focal adhesion kinase and MAP kinase pathways. HAase alters the expression of CD44 isoforms and is associated with increased tumor cell cycling. Of the 6 human HAases encoded by different genes, three are characterized at the protein level.
- kits Such a kit may comprise a carrier means being compartmentalized to receive in close confinement there with one or more container means such as vials, tubes and the like, each of said container means comprising the separate elements of the immunoassay.
- container means such as vials, tubes and the like
- each of said container means comprising the separate elements of the immunoassay.
- container means may contain standard solutions comprising serial dilutions of the HNSCC biomarkers to be detected, or appropriate quantities of the biomarkers in dry or concentrated form to be made up into standard solutions by the end user.
- the standard solutions of HNSCC biomarkers may be used to prepare standard curves with the concentration of each HNSCC biomarker plotted on the abscissa and the detection signal on the ordinate.
- the results obtained from a sample containing any one of the HNSCC biomarkers may be interpolated from such a plot to give the concentration of each detected biomarker.
- kits for diagnosing HNSCC or elevated risk thereof in a subject comprising a panel of biomarkers
- the kit comprising (a) a substrate for holding a biological sample isolated from a human subject suspected of having HNSCC or of having elevated risk thereof, (b) one or more fluorogenic agents that detect biomarkers; (c) a panel of biomarkers; and, (d) printed instructions for reacting the agent with the biological sample or a portion of the biological sample to detect the presence or amount of at least one marker in the biological sample.
- the kit comprises a panel of biomarkers of any one or more of hyaluronic acid (HA); hyaluronidase, and CD44.
- the kit further comprises antibodies specific for any one or more biomarkers: HA, HAase, and CD44, and means for determining total protein.
- HA HA
- HAase HAase
- CD44 means for determining total protein.
- the kit can provide both a panel of HNSCC biomarkers, e.g. to be used for standard curves, and antibodies thereto if desired.
- the kit will detect biomarkers using antibodies or other suitable detection methods.
- Example 1 Detection of HA, HAase, CD44 and lnterteukin-8 (IL-8) [0068] solCD44 appears to be a robust marker for HNSCC.
- a panel of markers was examined, to attempt to improve those parameters. Twelve HNSCC saliva specimens and 12 matched control saliva specimens were studied. HNSCC samples were taken in consecutive order from a randomly generated database. Subjects were excluded if they had a limited ability to gargle. There were no significant differences between the HNSCC and control groups with regard to gender, age, race, ethnicity, smoking history, alcohol consumption or oral health. HNSCC subjects were taken in consecutive order froma randomly generated database.
- HA concentration was measured using the ELISA-like assay described by Fosang et al (Matrix. 1990; 10: 306-13) and modified by Lokeshwar er ⁇ / (Cancer Res. 1997; 57: 773-77). Using the competitive binding principle, serial duplicate dilutions of saliva of cell lines were incubated in HA-coated microtiter plates with biotinylated HA binding protein. Plates were washed, HA binding protein was quantitated with an avidin-biotin detection system, and HA concentration was determined via standard graph.
- HAase levels were measured using an ELISA-like assay similar to that by Stern and Stern ⁇ Matrix 1992; 12: 397-03) with modifications by Lokeshwar et al (Cancer Res. 1997; 57: 778-83).
- Microtiter wells coated with HA were incubated with duplicate serial dilutions of saliva for 16 hours in assay buffer. HA remaining on the wells was determined using the same biotinylated HA- binding protein and avidin-biotin detection system as the HA test.
- HAase concentration was determined via standard graph.
- samples were run in triplicate and the test performed according to the manufacturers instructions. After determining average marker levels for each sample, the optimal sensitivity, specificity and accuracy was calculated for each marker. Then sensitivity, specificity and accuracy were evaluated for combinations of markers.
- Freeze-thaw cycles and stability For each marker, 3-5 samples were aliquoted to determine whether significant changes in marker levels occur with multiple freeze-thaw cycles or after storage for 8 hours on ice. Our results show that all three markers are stable after multiple freeze-thaw cycles and storage on ice for 8 hours.
- CD44 is a robust marker for PINSCC 5 however, a pane! of markers may improve specificity and sensitivity of HNSCC screening.
- HA and HAase are elevated in saliva of HNSCC patients compared to controls.
- Samples are collected from patients at the clinic or screening site. For collection, five milliliters of normal saline is placed in the subject's mouths. Patients are asked to- swish for five seconds, gargle for five seconds and then spit into a specimen cup. Saliva is placed on ice for transport and stored at -80 degrees. As recommended, subjects are asked to refrain from oral hygiene procedures, smoking, eating and drinking for at least 1 hour prior to collection. Samples are stored on ice for transport since solCD44, HA and HAase levels are stable on ice for 8 hours prior to freezing at -80 0 C. The samples may be fractioned to permit multiple investigations without freeze-thaw cycles and stored at -80 0 C.
- SoICD44, HA andHAase assays The solCD44 ELISA is carried out according to the instructions supplied by the manufacturer (Bender MedSystems, Vienna, Austria) with modifications. This assay recognizes all CD44 normal and variant isoforms. Samples may be tested in batches of approximately 30 samples per plate and measured at full concentration. For the rare sample whose level exceeds that of the highest standard, a repeat measurement is made at 1 A concentration. The HA and HAase tests are performed as described. Samples may be tested in batches of approximately 10 per plate at various concentrations to ensure that that the measured levels fall on the standard curve. A separate aliquot of saliva for each of the 3 assays; solCD44, HA and HAase can be prepared.
- Quality Control Since quality control is an ongoing process, full quality control measures for each test (CD44, HA and HAase) should be applied, with reproducibility between duplicates on the same plate and duplicates on separate plates being assessed. Further, two analysts can test the same 30 samples on the same day and the same analysts assess the same 30 samples on separate days to quantitate variation introduced by different analysts, and day-to-day variation. To account for day-to-day, batch-to-batch and performer- to-performer differences, the following daily consistency testing as recommended by the
- EORTC-NCI working group may be used.
- a calibration curve in duplicate, a precision profile, and run a high and low concentration control sample on each plate will be performed. Stability of the control samples by analyzing plots of absorbance versus dilution factor over time should be verified.
- Comparing levels ofsolCD44, HA andHAase between HNSCC patients and controls Preferably, cancer patients and control subjects should be characterized by demographic data (e.g., age, gender, race, and socioeconomic status) and risk factors such as tobacco and alcohol use and oral health. The characteristics of the two groups can be compared by Student's t-test for continuous variables and Chi-square test or Fisher's exact test for categorical data.
- the mean level for each marker can be calculated separately for HNSCC patients and control subjects (with and without benign disease), with corresponding confidence limits, and compared with Student's t-test. Subjects with benign disease may also be analyzed separately using the same methods, to determine if any marker is elevated with benign disease. Multiple regression will determine whether there is a significantly higher expression of each marker in HNSCC after adjusting for risk factors.
- multivariate classification models can be constructed to determine the best combination of biomarkers for cancer prediction.
- logistic regression the association of each biomarker on the dependent variable (cancer/ ⁇ on-cancer) singly and in combination while controlling for potential covariates, such as age, gender, and smoking history is calculated.
- Backward stepwise regression will be used to find the best final model.
- Leave-one-out cross-validation can be used to validate the final logistic regression model.
- a final ROC curve can then be computed from the final logistic model using the fitted probabilities from the model as possible cutpoints for computation of sensitivity and specificity.
- cluster analysis can be used to produce a classification tree for the entire group (cancers and controls) using the validated biomarkers as predictors (reference for cluster analysis).
- Variance in duplicate measures is obtained by squaring the difference in duplicates and dividing by 2. The mean value of these duplicate variances (S A 2 ) can be determined. Then, using the formula to determine coefficient of variation, the square root of the mean variance divided by the overall mean ' marker level multiplied by 100 will give CVA .
- Si 2 + S A 2 /2 mean measured within subject variance
- Mean measured within subject variance is determined from the variance between samples collected two weeks apart.
- CVi is then determined using the formula for CV as above.
- S ⁇ 2 is calculated using the following formula. Then CVo is calculated as previously discussed.
- index of heterogeneity is significant then a distribution of true within subject variances can be developed and the upper percentile points used to calculate the critical difference.
- an individual marker level is determined as positive or negative based on a population based reference level. If the within-subject variation is small compared to the between-subject variation, a significant change in an individual marker level may not be perceived as significant when using population-based cut-off point.
- Harris has shown that if index levels are less that 0.6 the population-based reference value is usually insensitive to significant fluctuations in an individual subject. In this case the probability that an observed level will fall within the conventional normal range is greater than the specified probability that is derived from the distribution of the population as a whole (Harris E. K. Clin Chem 1974;20: 1535-42).
- the reference values is insensitive to individual changes, following marker levels over time in an individual is usually more useful than comparing one set of marker levels to a reference level.
- Cochran's test can be used to test the ratio of the maximum variance to the sum of the variances.
- Reed's criterion can be used (Reed AH, Henry RJ and Mason Va. Clin Chem 1971 ;17:275-84). This criterion analyzes the difference between the extreme value and the next highest (or lowest value). The value is rejected if the difference exceeds one-third the range of all values. This criterion assumes that the true distribution of values for a given parameter are normal.
- Example 2 The Salivary Soluble CD44 Test [0092] Subject Characteristics: Seventy-three HNSCC patients, 54 patients with benign diseases of the upper aerodigestive tract (UADT) and history of tobacco and/or alcohol use, and 10 normal nonsmoking controls were studied according to the protocol approved by the Institutional Review Board. All HNSCC patients had biopsy proven newly diagnosed or recurrent squamous cell carcinoma. Included were all stages and sites except nasopharynx, since nasopharyngeal carcinoma tends to behave differently than squamous cell carcinoma in other sites.
- Patients with benign diseases of the upper aerodigestive tract (UADT) (from here on referred to as benign disease) were obtained from a general otolaryngology clinic. Most of these patients also had a history of tobacco or alcohol use.
- Nonsmoking, normal subjects were volunteers from healthcare and research fields. All subjects completed a written consent prior to enrollment [0093] Saliva Collection: Five milliliters of normal saline was placed in the subject's mouths. Patients were asked to swish for five seconds, gargle for five seconds and then spit into a specimen cup.
- SolCD44 ELlSA Levels of solCD44s using an ELISA assay (Bender MedSystems, Vienna, Austria) that recognizes all CD44 normal and variant isoforms were measured. This assay has been used extensively in serum and other body fluids and correlates with cancer progression in many tumors. Specificity of the CD44 antibody is described in detail in the Bender MedSystems Manual. They detected no cross reactivity between this test and TNF- ⁇ , TNF- ⁇ , TNF-R, IFN- ⁇ 2c, INF- ⁇ , IL-8 annexin, sELAM-l 5 sl- selectin, slCAMl, or HER-2.
- the specificity of the antibody was confirmed by Western blot.
- the test involves a sandwich-type ELISA where a monoclonal anti-solCD44 antibody, adsorbed onto microwells, binds CD44 in the sample.
- Horseradish peroxidase- conjugated monoclonal anti-solCD44 antibody binds the CD44-antibody complex and reacts with a substrate solution to produce a colored product with an absorbance measured quantitatively at 450nm. Sample concentrations are determined by a standard curve.
- this ELISA plate is designed for use with plasma, serum and urine samples. Any matrix, i.e., serum, urine, saliva, may contain factors that affect ELISA test results; a matrix effect.
- the standards are prepared in a synthetic saliva matrix (Salimetrics) diluted 1 :5 in normal saline (since patients swish and gargle with 5cc saline) and use a sample diluents (Sal metrics) developed for saliva samples. Samples were vortexed, centrifuged at 3,000 G and the supernatant was used for study. The test was performed at full, 1 :2 and 1 :4 dilutions. To adjust for hydration status, the solCD44 levels are normalized to protein using a protein assay (Bio-Rad). All sample assays were performed in triplicate. The protein and solCD44 concentrations for each sample were averaged and divided by the average protein concentration for that sample.
- solCD44 ELISA Quality Control The standard curve was generated using cubic spline curve fit. Standard curves were run in duplicate on each plate. Coefficient of determination ranged from 0.98- 0.99 for all of the standard curves. A representative curve is shown in Figure 6. [0097] The precision of an assay is defined by the agreement between replicate measures. Samples (73 HNSCC and 54 control specimens) were repeated in triplicate at full concentration, 1 :2 and 1 :4 dilutions. The average coefficient of variation for the resulting 381 duplicate measurements was 4.5 %. [0098] Analytical sensitivity is defined as the lowest concentration detected that is significantly different than zero. Mean absorbance of our blanks run on 17 different plates was 0.02 ⁇ .015.
- a good screening test for HNSCC must have high sensitivity and specificity. Using results from 73 HNSCC patients and 64 subjects without HNSCC the sensitivity and specificity of the solCD44 test was calculated at several cut-off points, thereby deriving its receiver-operator characteristic (ROC) curve.
- ROC receiver-operator characteristic
- SolCD44 Levels Seventy-three HNSCC patients had disease of the oral cavity, oropharynx, larynx or hypoparynx. The mean solCD44 level was 27.3 ( ⁇ ) 36.2 ng/ml for HNSCC patients, 7.4 ⁇ 6.0 ng/ml for the patients with benign disease (p ⁇ .0001) 5 and 4.8 ⁇ 2.8 ng/ml (p ⁇ 0.0001) for normal nonsmokers. Results are shown in Figure 7. Four of the HNSCC patients with cervical lymph node disease had no identified mucosal primary. One of the four had newly diagnosed disease and the other three had recurrences to neck lymph nodes.
- Salivary protein levels and normalized solCD44 Since salivary protein levels correlate with hydration status, protein assays were performed as previously described to determine if correcting for hydration status would improve results. Mean protein levels were significantly higher in the 73 cancer patients (1.1 ⁇ 0.9 mg/ml, p ⁇ 0.0001) compared to patients with benign disease (0.5 ⁇ 0.3 mg/ml) and normal nonsmokers (0.6 ⁇ 0.4 mg/ml). It is possible that HNSCC patients were more dehydrated than normals due to swallowing difficulty. However, this is unlikely since small tumors do not usually cause swallowing difficulty and protein levels did not increase with increasing tumor size (Table 1).
- Sensitivity and specificity of the solCD44 test for HNSCC was calculated. A cut-off point set at 8.0 ng/ml resulted in a sensitivity of 78% and specificity of 70%, while a cut-off point of 12.0 ng/ml resulted in a sensitivity of 67% and specificity of 91 %.
- solCD44 levels were significantly elevated in patients with primary tumors in the oral cavity and pharynx compared to the larynx and hypopharynx we also calculated sensitivity and specificity for this subgroup compared to the 64 patients without HNSCC. For these 42 patients, a cutoff point of 8.0 ng/ml resulted in a sensitivity of 81% and a specificity of 70% while a cut-off point of 12.0 ng/ml resulted in a sensitivity of 74% and a specificity of 91%.
- the solCD44 test detected 75% of oral cavity, 50% of laryngeal, 72% of oropharyngeal and 57% of hypopharyngeal primaries. [00112]
- the level of expression of solCD44 was also statistically significantly elevated in this cancer group compared to the benign disease group (23.9 ⁇ 31.3 ng/ml vs 7.0 ⁇ 5.9 ng/ml, p ⁇ .05).
- the distribution of potentially important covariates was compared between the two groups by Chi-square analysis. The groups differed significantly with respect to several factors.
- Soluble CD44 test In our pilot study we showed that salivary solCD44 levels were elevated in HNSCC patients compared to normal controls. In this subsequent work, tobacco and alcohol use, gender, race, and SES are controlled for and evaluate the association of salivary solCD44 levels with common benign diseases of the UADT. One hundred and two HNSCC patients and 69 controls were enrolled from otolaryngology clinics at the
- Results are summarized in Table 4 for 97 HNSCC with mucosal disease and 84 control patients.
- the mean salivary solCD44 level was 24.7 ng/ml for subjects with HNSCC and 9.4 ng/ml for the controls (pO.0001).
- Levels tended to be higher in patients with oral cavity and oropharynx tumors compared to larynx and hypopharynx tumors. There was a tendency toward higher levels in patients with spread to the local lymph nodes compared to patients without nodal spread though this difference did not reach statistical significance. Levels did not correlate significantly with tumor size or stage, suggesting that the solCD44 test can detect HNSCC regardless of tumor size.
- We also evaluated whether oral health impacted solCD44 levels. From the questionnaire we determined that HNSCC patients had more teeth removed due to periodontal disease or decay than controls. Adjusting for this, the difference in SO1CD44 level between groups was still significant (p 0.0559). We also assessed HNSCC subjects and control subjects for ability to gargle. The two groups did not differ with respect to gargling ability.
- biomarkers are not normalized to protein levels when comparing tumor and control patients. Dehydration in the cancer patients (secondary to swallowing disturbance by the tumor) is one explanation for the difference in protein level between tumor and normal subjects. However, protein levels did not increase with increasing tumor size as would be expected if this occurred.
- the subgroup labeled "other disease” included 4 patients with tonsillitis or pharyngitis with mean solCD44 level of 6.85ng/ml. There were a total of 80 active diagnoses among 63 patients who completed questionnaires. Each subgroup was compared to the remaining control patients using a t-test.
- solCD44 level None of the subgroups had statistically significant elevations in solCD44 level.
- solCD44 levels were elevated in 50% of cases. The most significant finding is illustrated by patient 6 where solCD44 levels were high and the subject underwent disease progression. Patient 1 is interesting because this patient had low solCD44 levels and never progressed after 4 years follow-up, supporting a conclusion that solCD44 level distinguishes individuals who are likely to progress from those unlikely to progress.
- Example 3
- Western blots Western blot analysis was performed on HNSCC cell lines UMSCC- 1 IB, UMSCC-9, SCC-25 wild-type and SCC-25 CD44s transfectants. We also examined 2 solCD44 false positive control salivas, 2 HNSCC salivas, and 2 true negative control saliva samples. Proteinase inhibitors were added prior to saliva storage to prevent post-collection degradation. All blots were performed using a standard western blot protocol.
- CD44s CD44 standard form
- DNA from HNSCC cell lines was amplified by PCR using primers in CD44 exon 1 and exon 19.
- the PCR amplification product was cloned into the TOPO Vector (Invitrogen, pcDNA3.1/V5-His TOPO TA expression Kit) pcDNA3.1/V5-His. Following transformation, colonies were screened by PCR and grown. The construct was then purified by Miniprep (Qiagen) and confirmed by sequencing.
- the purified CD44s construct was transfected into SCC-25 cells following the lipofectamine protocol provided by the manufacturer (Invitrogen). For transient transfection, cells were collected at 24 hour and analyzed for CD44 expression. For stable transfection, cells were passaged into selective medium 1 day after the start of transfection. At 2 days the G 148. antibiotic was added to select for expression of the transfected antibiotic-resistance gene. For this preliminary experiment, stably transfected cells were grown as a pool as described by Recillas-Targa rather than isolating individual clones. At the present time, we are isolating individual clones to confirm these results.
- Results of the stable transfection are shown for the cell lysate ( Figure 2A) and conditioned media ( Figure 2B).
- Overexpression of CD44s results in increased expression of a doublet at 75kDa in the cell lysate as well as a higher band around 130 kDa. Stronger expression of the 13OkDa band in the transfectant cell lysate raises the possibility that this is a variably glycosylated form of CD44s rather than an alternatively spliced isoform.
- the doublet at 75 kDa may be from contamination by cell lysate rather than the soluble form of CD44, while the bands at 65-70 kDa and 5OkDa correspond to published solCD44 products (205).
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Also Published As
Publication number | Publication date |
---|---|
AU2007249805A1 (en) | 2007-11-22 |
US8088591B2 (en) | 2012-01-03 |
EP2032716A1 (en) | 2009-03-11 |
US20090325201A1 (en) | 2009-12-31 |
CA2652043A1 (en) | 2007-11-22 |
US20120115165A1 (en) | 2012-05-10 |
US20140024042A1 (en) | 2014-01-23 |
AU2007249805B2 (en) | 2013-01-10 |
US20160341728A1 (en) | 2016-11-24 |
EP2032716A4 (en) | 2010-03-17 |
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