US20060068418A1 - Identification of markers in lung and breast cancer - Google Patents

Identification of markers in lung and breast cancer Download PDF

Info

Publication number
US20060068418A1
US20060068418A1 US11/178,109 US17810905A US2006068418A1 US 20060068418 A1 US20060068418 A1 US 20060068418A1 US 17810905 A US17810905 A US 17810905A US 2006068418 A1 US2006068418 A1 US 2006068418A1
Authority
US
United States
Prior art keywords
mrna species
lymph node
specific
overabundant
pcr
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US11/178,109
Other languages
English (en)
Inventor
Tony Godfrey
Liqiang Xi
Siva Raja
William Gooding
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Pittsburgh
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Priority to US11/178,109 priority Critical patent/US20060068418A1/en
Assigned to UNIVERSITY OF PITTSBURGH-OF THE COMMONWEALTH SYSTEM OF HIGHER EDUCATION reassignment UNIVERSITY OF PITTSBURGH-OF THE COMMONWEALTH SYSTEM OF HIGHER EDUCATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: RAJA, SIVA, GODFREY, TONY E., XI, LIQIANG, GOODING, WILLIAM E.
Publication of US20060068418A1 publication Critical patent/US20060068418A1/en
Priority to US13/030,003 priority patent/US20110312515A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • Metastatic lesions commonly are detected by histological techniques, including immunohistochemical techniques. Metastasized cells typically infiltrate the lymph nodes, and, thus in most instances, certain sentinel lymph nodes (lymph nodes where metastasized cells typically first infiltrate), or staging lymph nodes (lymph nodes typically analyzed for presence of certain types of cancers), are recognized for each cancer type and are analyzed for the presence of lesions, including micrometastases. Trained histologists often can detect metastatic lesions visually after tissue from a sentinel or staging lymph node is sectioned and stained. Highly trained histologists often can visualize micrometasteses, but the ability to visualize such lesions varies from histologist-to-histologist.
  • the present invention relates to a diagnostic method for detecting the presence of cancer cells in a patient by identifying the expression of certain markers indicative of the presence of cancer cells.
  • the present invention relates to a method of identifying the expression of markers indicative of the presence of breast cancer cells in a lymph node of a patient.
  • the method comprises determining if an mRNA species specific to one or more of TACSTD1, CK19, MGB1, MGB2, PIP and CK7 is overabundant in an RNA sample prepared from the lymph node.
  • the overabundance of the mRNA species is indicative of the presence of displaced breast cells in the lymph node.
  • the present invention relates to a method for identifying the expression of markers indicative of the presence of lung cancer cells in a lymph node of a patient.
  • the method comprises determining if an mRNA species specific to one or more of CEA, CK7, CK19, PVA, SCCA1.2 (SCCA1+SCCA2), SFTPB and TACSTD1 is overabundant in an RNA sample prepared from the lymph node.
  • the overabundance of the mRNA species is indicative of the presence of displaced lung cells in the lymph node.
  • the present invention relates to an article of manufacture comprising packaging material and one or more nucleic acids specific to one or more of CK7, CK19, MGB1, MGB2, PIP and TACSTD1.
  • the packaging material comprises an indicia, for example and without limitation, a writing, illustration, label, tag, book, booklet and/or package insert, indicating that the one or more nucleic acids can be used in a method of identifying expression of markers indicative of the presence of breast cancer cells in a lymph node of a patient.
  • the present invention relates to an article of manufacture comprising packaging material and one or more nucleic acids specific to one or more of CEA, CK7, CK19, PVA, SCCA1.2, SFTPB and TACSTD1.
  • the packaging material comprises an indicia indicating that the one or more nucleic acids can be used in a method of identifying expression of markers indicative of the presence of lung cancer cells in a lymph node of a patient.
  • the present invention relates to a composition
  • a composition comprising one or more primers or probes specific to one or more of CK7, CK19, MGB1, MGB2, PIP and TACSTD1 and RNA extracted from the lymph node of a patient diagnosed with or suspected of having breast cancer, or a nucleic acid, or analog thereof, derived from the RNA.
  • the present invention relates to a composition
  • a composition comprising one or more primers or probes specific to one or more of CEA, CK7, CK19, PVA, SCCA1.2, SFTPB and TACSTD1 and RNA extracted from the lymph node of a patient diagnosed with or suspected of having lung cancer, or a nucleic acid, or analog thereof, derived from the RNA.
  • FIG. 1 is a listing of a cDNA sequence of the cytokeratin 7 (CK7) marker (SEQ ID NO: 1).
  • FIG. 2 is a listing of a cDNA sequence of the cytokeratin 19 (CK19) marker (SEQ ID NO: 2).
  • FIG. 3 is a listing of a cDNA sequence of the mammaglobin 1 (MGB1) marker (SEQ ID NO: 3).
  • FIG. 4 is a listing of a cDNA sequence of the mammaglobin 2 (MGB2) marker (SEQ ID NO: 4).
  • FIG. 5 is a listing of a cDNA sequence of the prolactin-inducible protein (PIP) marker (SEQ ID NO: 5).
  • FIG. 6 is a listing of a cDNA sequence of the pemphigus vulgaris (PVA) marker (SEQ ID NO: 6).
  • FIG. 7 is a listing of a cDNA sequence of the squamous cell carcinoma antigen 1 (SCCA1) marker (SEQ ID NO: 7).
  • FIG. 8 is a listing of a cDNA sequence of the squamous cell carcinoma antigen 2 (SCCA2) marker (SEQ ID NO: 8).
  • FIG. 9 is a listing of a cDNA sequence of the surfactant, pulmonary-associated protein b (SFTPB) marker (SEQ ID NO: 9).
  • FIG. 10 is a listing of a cDNA sequence of the tumor-associated calcium signal transducer 1 (TACSTD1) marker (SEQ ID NO: 10).
  • FIG. 11 is a listing of a cDNA sequence of the carcinoembryonic antigen-related cell adhesion molecule 5 (CEA) marker (SEQ ID NO: 11).
  • FIG. 12 is a scatter plot showing the expression levels of CK7, CK19, MGB1, MGB2, PIP and TACSTD1 in primary tumor, tumor-positive lymph nodes and benign lymph nodes of a breast cancer patient.
  • FIGS. 13 A-O provide scatter plots illustrating the ability of two-marker systems to distinguish between benign and malignant cells in a lymph node of a breast cancer patient (negative—gray circle; positive—black circle).
  • FIG. 15 A-BB provide scatter plots illustrating the ability of two-marker systems to distinguish between benign and malignant cells in a lymph node of a lung cancer patient (negative—circle; positive—“+”).
  • FIG. 16 is a plot of the best combination of three markers for detecting lung cancer in different histological types, plotting the Fold Change-Positive Lymph Nodes PLN vs. Highest Benign Nodes BN (PVA—asterix; SFTPB—circle; and TACSTD1—triangle).
  • FIG. 17A provides data obtained from the secondary screening set of lymph nodes on individual gene expression observed in primary tumors, benign and positive nodes.
  • the horizontal line indicates the most accurate cutoff value calculated by a receiver-operator characteristic curve analysis. Classification characteristics of the individual markers are reported in Table I, below.
  • FIGS. 17 B-E provide secondary screening set data on gene expression for potential two-marker combinations using a linear discriminator decision rule. As with the individual markers, the black line indicates the decision rule generated from the secondary screening set data that produces the most accurate characterization. Classification characteristics of the marker combinations are reported in Table I.
  • FIGS. 17 F-I provide secondary screening set data on gene expression for potential two-marker combinations applying equal probability contour statistical analysis. Equal probability curves were generated around the mean expression value observed for the 2 markers in benign lymph nodes. This demonstrates that while the marker combination of CK19 and MGB1 accurately characterizes the lymph nodes (see Table I), the wide distribution of expression observed in benign nodes for these markers increases optimism in applying the decisicion rule. By this method of analysis, the marker combination of TACSTD1 and PIP more confidently characterizes the lymph nodes.
  • FIG. 18A provides data obtained from the validation set of SLN on individual gene expression observed in negative and positive nodes.
  • the horizontal line indicates the decision rule calculated from data obtained from the secondary screening set. Classification characteristics of the individual markers are reported in Table J.
  • FIGS. 18 B-G provide validation set data on gene expression for two-marker combinations using linear discriminator decision rule for all potential marker pairs. As with the individual markers, the black line indicates the decision rule generated from the secondary screening set data that produces the most accurate characterization. Classification characteristics of the marker combinations are reported in Table J.
  • FIGS. 18 H-M provide validation set data analyzed using the equal probability contours generated from the secondary screening set data.
  • the relative levels of expression observed for all but one of the positive lymph nodes were well outside the 0.999 confidence contour.
  • Some of the positive nodes are positive for only one marker (crosses located in the left upper or right lower quadrants), demonstrating that a 2 marker assay improves sensitivity while maintaining high specificity.
  • FIGS. 19 A-B provides results of a fully automated, 2-marker QRT-PCR analysis of lymph nodes.
  • the assay accurately characterized all 18 lymph nodes (9 negative, 9 positive) evaluated.
  • nucleic acid detection assays described herein are much more discriminating than are histological studies in most instances (a few, excellent histologists are capable of identifying micrometastases in lymph node sections), and are robust and repeatable in the hands of any minimally-trained technician.
  • markers that are expressed in certain cancer types, including breast cancer and lung cancer. These markers are markers specific to the tissue from which the particular cancer type arises and typically are not expressed, at least to the same levels, in lymphoid tissue. The presence and/or elevated expression of one or more of these markers in sentinel lymph node tissue is indicative of displaced cells in the lymphoid tissue, which correlates strongly with a cancer diagnosis.
  • the terms “expression” and “expressed” mean production of a gene-specific mRNA by a cell.
  • a “marker” is a gene that is expressed abnormally in a lymphatic biopsy.
  • the markers described herein are mRNA species that are expressed in cells of a specific tumor source at a significantly higher level as compared to expression in lymphoid cells.
  • Expression levels of mRNA can be quantifyd by a number of methods. Traditional methods include Northern blot analysis. More recently, nucleic acid detection methods have been devised that facilitate quantification of transcripts. Examples of PCR methods are described in U.S. patent application Ser. No. 10/090,326 (U.S. Ser. No. 10/090,326), incorporated herein by reference in its entirety. Other methods for determining expression levels of a given mRNA include isothermic amplification or detection assays and array technologies, as are known in the art, such as, without limitation, those described below.
  • a typical PCR reaction includes multiple amplification steps, or cycles that selectively amplify a target nucleic acid species. Because detection of transcripts is necessary, the PCR reaction is coupled with a reverse transcription step (reverse transcription PCR, or RT-PCR).
  • a typical PCR reaction includes three steps: a denaturing step in which a target nucleic acid is denatured; an annealing step in which a set of PCR primers (forward and backward primers) anneal to complementary DNA strands; and an elongation step in which a thermostable DNA polymerase elongates the primers. By repeating this step multiple times, a DNA fragment is amplified to produce an amplicon, corresponding to the target DNA sequence.
  • Typical PCR reactions include 30 or more cycles of denaturation, annealing and elongation. In many cases, the annealing and elongation steps can be performed concurrently, that is at the same temperature, in which case the cycle contains only two steps.
  • the lengths of the denaturation, annealing and elongation stages may be any desirable length of time. However, in attempting to shorten the PCR amplification reaction to a time suitable for intraoperative diagnosis, the lengths of these steps can be in the seconds range, rather than the minutes range.
  • the denaturation step may be conducted for times of one second or less.
  • the annealing and elongation steps optimally are less than 10 seconds each, and when conducted at the same temperature, the combination annealing/elongation step may be less than 10 seconds.
  • each cycle may be shortened considerably without substantial deterioration of production of amplicons.
  • Use of high concentrations of primers is helpful in shortening the PCR cycle time.
  • High concentrations typically are greater than about 400 nM, and often greater than about 800 nM, though the optimal concentration of primers will vary somewhat from assay-to-assay.
  • Sensitivity of RT-PCR assays may be enhanced by the use of a sensitive reverse transcriptase enzyme (described below) and/or high concentrations of reverse transcriptase primer to produce the initial target PCR template.
  • PCR primer sets are pairs of forward and reverse oligonucleotide primers that anneal to a target DNA sequence to permit amplification of the target sequence, thereby producing a target sequence-specific amplicon.
  • PCR primer sets can include two primers internal to the target sequence, or one primer internal to the target sequence and one specific to a target sequence that is ligated to the DNA or cDNA target, using a technique known as “ligation-anchored PCR” (Troutt, A. B., et al. (1992), “Ligation-anchored PCR: A Simple Amplification Technique with Single-sided Specificity,” Proc. Natl. Acad. Sci. USA, 89:9823-9825).
  • a “derivative” of a specified oligonucleotide is an oligonucleotide that binds to the same target sequence as the specified oligonucleotide and amplifies the same target sequence to produce essentially the same amplicon as the specified oligonucleotide but for differences between the specified oligonucleotide and its derivative.
  • the derivative may differ from the specified oligonucleotide by insertion, deletion and/or substitution of any residue of the specified sequence so long as the derivative substantially retains the characteristics of the specified sequence in its use for the same purpose as the specified sequence.
  • reagents for any assay or reaction, such as a reverse transcription and PCR, are any compound or composition that is added to the reaction mixture including, without limitation, enzyme(s), nucleotides or analogs thereof, primers and primer sets, probes, antibodies or other binding reagents, detectable labels or tags, buffers, salts and co-factors.
  • a “reaction mixture” for a given assay or reaction includes all necessary compounds and/or compositions necessary to perform that assay or reaction, even if those compounds or compositions are not expressly indicated.
  • Reagents for many common assays or reactions, such as enzymatic reaction are known in the art and typically are provided and/or suggested when the assay or reaction kit is sold.
  • multiplexed PCR assays may be optimized, or balanced, by time-shifting the production of amplicons, rather than by manipulating primer concentrations. This may be achieved by using two primer sets, each primer set having a different Tm so that a two-stage PCR assay can be performed, with different annealing and/or elongation temperatures for each stage to favor the production of one amplicon over another.
  • This time and temperature shifting method permits optimal balancing of the multiplex reaction without the difficulties faced when manipulation of primer concentrations is used to balance the reaction. This technique is especially useful in a multiplex reaction where it is desirable to amplify a rare cDNA along with a control cDNA.
  • a quantitative reverse transcriptase polymerase chain reaction for rapidly and accurately detecting low abundance RNA species in a population of RNA molecules (for example, and without limitation, total RNA or mRNA), includes the steps of: a) incubating an RNA sample with a reverse transcriptase and a high concentration of a target sequence-specific reverse transcriptase primer under conditions suitable to generate cDNA; b) subsequently adding suitable polymerase chain reaction (PCR) reagents to the reverse transcriptase reaction, including a high concentration of a PCR primer set specific to the cDNA and a thermostable DNA polymerase to the reverse transcriptase reaction, and c) cycling the PCR reaction for a desired number of cycles and under suitable conditions to generate PCR product (“amplicons”) specific to the cDNA.
  • PCR polymerase chain reaction
  • reaction vessel all tubes, containers, vials, cells and the like in which a reaction is performed may be referred to herein, from time to time, generically, as a “reaction vessel”), removing a source of contamination typically found in two-tube reactions.
  • the reaction c) may be performed in the same tube as the reverse transcriptase reaction by adding sufficient reagents to the reverse transcriptase (RT) reaction to create good, or even optimal conditions for the PCR reaction to proceed.
  • a single tube may be loaded, prior to the running of the reverse transcriptase reaction, with: 1) the reverse transcriptase reaction mixture, and 2) the PCR reaction mixture to be mixed with the cDNA mixture after the reverse transcriptase reaction is completed.
  • the reverse transcriptase reaction mixture and the PCR reaction mixture may be physically separated by a solid, or semi-solid (including amorphous, glassy substances and waxy) barrier of a composition that melts at a temperature greater than the incubation temperature of the reverse transcriptase reaction, but below the denaturing temperature of the PCR reaction.
  • the barrier composition may be hydrophobic in nature and forms a second phase with the RT and PCR reaction mixtures when in liquid form.
  • a barrier composition is wax beads, commonly used in PCR reactions, such as the AMPLIWAX PCR GEM products commercially available from Applied Biosystems of Foster City, Calif.
  • the separation of the reverse transcriptase and the PCR reactions may be achieved by adding the PCR reagents, including the PCR primer set and thermostable DNA polymerase, after the reverse transcriptase reaction is completed.
  • the PCR reagents are added mechanically by a robotic or fluidic means to make sample contamination less likely and to remove human error.
  • the products of the QRT-PCR process may be compared after a fixed number of PCR cycles to determine the relative quantity of the RNA species as compared to a given reporter gene.
  • One method of comparing the relative quantities of the products of the QRT-PCR process is by gel electrophoresis, for instance, by running the samples on a gel and detecting those samples by one of a number of known methods including, without limitation, Southern blotting and subsequent detection with a labeled probe, staining with ethidium bromide and incorporating fluorescent or radioactive tags in the amplicons.
  • the progress of the quantitative PCR reactions typically is monitored by determining the relative rates of amplicon production for each PCR primer set.
  • Monitoring amplicon production may be achieved by a number of processes, including without limitation, fluorescent primers, fluorogenic probes and fluorescent dyes that bind double-stranded DNA.
  • a common method is the fluorescent 5′ nuclease assay. This method exploits the 5′ nuclease activity of certain thermostable DNA polymerases (such as Taq or Tfl DNA polymerases) to cleave an oligomeric probe during the PCR process. The oligomer is selected to anneal to the amplified target sequence under elongation conditions.
  • the probe typically has a fluorescent reporter on its 5′ end and a fluorescent quencher of the reporter at the 3′ end. So long as the oligomer is intact, the fluorescent signal from the reporter is quenched. However, when the oligomer is digested during the elongation process, the fluorescent reporter no longer is in proximity to the quencher.
  • the relative accumulation of free fluorescent reporter for a given amplicon may be compared to the accumulation of the same amplicons for a control sample and/or to that of a control gene, such as ⁇ -actin or 18S rRNA to determine the relative abundance of a given cDNA product of a given RNA in a RNA population.
  • Products and reagents for the fluorescent 5′ nuclease assay are readily available commercially, for instance from Applied Biosystems.
  • Equipment and software also are readily available for monitoring amplicon accumulation in PCR and QRT-PCR according to the fluorescent 5′ nuclease assay and other QPCR/QRT-PCR procedures, including the Smart Cycler, commercially available from Cepheid of Sunnyvale, Calif., the ABI Prism 7700 Sequence Detection Systemt (TaqMan), commercially available from Applied Biosystems.
  • a cartridge-based sample preparation system (GenXpert) combines a thermal cycler and fluorescent detection device having the capabiliti es of the Smart Cycler product with fluid circuits and processing elements capable of automatically extracting specific nucleic acids from a tissue sample and performing QPCR or QRT-PCR on the nucleic acid.
  • the system uses disposable cartridges that can be configured and pre-loaded with a broad variety of reagents. Such a system can be configured to disrupt tissue and extract total RNA or mRNA from the sample.
  • the reverse transcriptase reaction components can be added automatically to the RNA and the QPCR reaction components can be added automatically upon completion of the reverse transcriptase reaction.
  • the PCR reaction may be monitored of production (or loss) of a particular fluorochrome from the reaction.
  • the automated system will automatically alter the PCR conditions. In one example, this is particularly useful in the multiplexed embodiment described above, where a more-abundant (control) target species is amplified by the first, lower Tm, primer set at a lower temperature than the less abundant species amplified by the second, higher Tm, primer set.
  • the annealing temperature is lower than the effective Tm of the first primer set.
  • the annealing temperature then is automatically raised above the effective Tm of the first primer set when production of the first amplicon by the first primer set is detected.
  • a first PCR reaction may be conducted at the first Tm and, when the first PCR reaction proceeds past a threshold level, a second primer with a different Tm is added, resulting in a sequential multiplexed reaction.
  • the amounts of certain reverse transcriptase and the PCR reaction components typically are atypical in order to take advantage of the faster ramp times of some thermal cyclers.
  • the primer concentrations are very high. Typical gene-specific primer concentrations for reverse transcriptase reactions are less than about 20 nM.
  • the reverse transcriptase primer concentration was raised to greater than 20 nM, preferably at least about 50 nM, and typically about 100 nM.
  • Standard PCR primer concentrations range from 100 nM to 300 nM. Higher concentrations may be used in standard PCR reactions to compensate for Tm variations. However, the referenced primer concentrations are for circumstances where no Tm compensation is needed.
  • Proportionately higher concentrations of primers may be empirically determined and used if Tm compensation is necessary or desired.
  • the PCR primer concentrations typically are greater than 200 nM, preferably greater than about 500 nM and typically about 800 nM.
  • the ratio of reverse transcriptase primer to PCR primer is about 1 to 8 or more. The increase in primer concentrations permitted PCR experiments of 40 cycles to be conducted in less than 20 minutes.
  • a sensitive reverse transcriptase may be preferred in certain circumstances where either low amounts of RNA are present or a target RNA is a low abundance RNA.
  • sensitive reverse transcriptase it is meant a reverse transcriptase capable of producing suitable PCR templates from low copy number transcripts for use as PCR templates.
  • the sensitivity of the sensitive reverse transcriptase may derive from the physical nature of the enzyme, or from specific reaction conditions of the reverse transcriptase reaction mixture that produces the enhanced sensitivity.
  • One example of a sensitive reverse transcriptase is SensiScript RT reverse transcriptase, commercially available from Qiagen, Inc. of Valencia, Calif.
  • This reverse transcriptase is optimized for the production of cDNA from RNA samples of ⁇ 50 ng, but also has the ability to produce PCR templates from low copy number transcripts. In practice, in the assays described herein, adequate results were obtained for samples of up to, and even in excess of, about 400 ng RNA. Other sensitive reverse transcriptases having substantially similar ability to reverse transcribe low copy number transcripts would be equivalent sensitive reverse transcriptase for the purposes described herein. Notwithstanding the above, the ability of the sensitive reverse transcriptase to produce cDNA from low quantities of RNA is secondary to the ability of the enzyme, or enzyme reaction system to produce PCR templates from low copy number sequences.
  • a multiplex PCR process involves production of two or more amplicons in the same reaction vessel. Multiplex amplicons may be analyzed by gel electrophoresis and detection of the amplicons by one of a variety of methods, such as, without limitation ethidium bromide staining, Southern blotting and hybridization to probes, or by incorporating fluorescent or radioactive moieties into the amplicons and subsequently viewing the product on a gel. However, real-time monitoring of the production of two or more amplicons is preferred. The fluorescent 5′ nuclease assay is the most common monitoring method.
  • Equipment for example, the above-described Smart Cycler and TaqMan products that permits the real-time monitoring of accumulation of two or more fluorescent reporters in the same tube.
  • oligomers are provided corresponding to each amplicon species to be detected.
  • the oligomer probe for each amplicon species has a fluorescent reporter with a different peak emission wavelength than the oligomer probe(s) for each other amplicons species.
  • the accumulation of each unquenched fluorescent reporter can be monitored to determine the relative amounts of the target sequence corresponding to each amplicon.
  • PCR primer sets having similar annealing and elongation kinetics and similar sized amplicons are desirable.
  • the design and selection of appropriate PCR primer sets is a process that is well known to a person skilled in the art.
  • the process for identifying optimal PCR primer sets, and respective ratios thereof to achieve a balanced multiplex reaction also is known.
  • balanced it is meant that certain amplicon(s) do not out-compete the other amplicon(s) for resources, such as dNTPs or enzyme. For instance, by limiting the abundance of the PCR primers for the more abundant RNA species in an RT-PCR experiment will allow the detection of less abundant species.
  • Equalization of the Tm (melting temperature) for all PCR primer sets also is encouraged. See, for instance, ABI PRISM 7700 Sequence Detection System User Bulletin #5, “Multiplex PCR with TaqMan VIC Probes”, Applied Biosystems (1998/2001).
  • a method has been described in WO 02/070751 for performing a multiplex PCR process, including QRT-PCR and QPCR, capable of detecting low copy number nucleic acid species along with one or more higher copy number species.
  • the difference between low copy number and high copy number nucleic acid species is relative, but is referred to herein as a difference in the prevalence of a low (lower) copy number species and a high (higher) copy number species of at least about 30-fold, but more typically at least about 100-fold.
  • the relative prevalence of two nucleic acid species to be amplified is more salient than the relative prevalence of the two nucleic acid species in relation to other nucleic acid species in a given nucleic acid sample because other nucleic acid species in the nucleic acid sample do not directly compete with the species to be amplified for PCR resources.
  • the prevalence of any given nucleic acid species in a given nucleic acid sample, prior to testing, is unknown.
  • the “expected” number of copies of a given nucleic acid species in an nucleic acid sample often is used herein and is based on historical data on the prevalence of that species in nucleic acid samples. For any given pair of nucleic acid species, one would expect, based on previous determinations of the relative prevalence of the two species in a sample, the prevalence of each species to fall within a range. By determining these ranges one would determine the difference in the expected number of target sequences for each species.
  • mRNA species is identified as “overabundant” if it is present in statistically significant amounts over normal prevalence of the mRNA species in a sample from a normal patient or lymph node. As is abundantly illustrated in the examples and plots provided herein, a person of skill in the art would be able to ascertain statistically significant ranges or cutoffs for determining the precise definition of “overabundance” for any one or more mRNA species.
  • the multiplex method involves performing a two- (or more) stage PCR amplification, permitting modulation of the relative rate of production of a first amplicon by a first primer set and a second amplicon by a second primer set during the respective amplification stages.
  • PCR amplifications to produce amplicons directed to a lower abundance nucleic acid species are effectively “balanced” with PCR amplifications to produce amplicons directed to a higher abundance nucleic acid species.
  • Separating the reaction into two or more temporal stages may be achieved by omitting the PCR primer set for any amplicons that are not to be produced in the first amplification stage. This is best achieved through use of automated processes, such as the GenXpert prototype system described above.
  • Two or more separate amplification stages may be used to tailor and balance multiplex assays, along with, or to the exclusion of tailoring the concentration of the respective primer sets.
  • a second method for temporally separating the PCR amplification process into two or more stages is to select PCR primer sets with variation in their respective Tm.
  • primers for a lower copy number nucleic acid species would have a higher Tm (Tm 1 ) than primers for a higher abundance species (Tm 2 ).
  • Tm 1 primers for a lower copy number nucleic acid species
  • Tm 2 primers for a higher abundance species
  • the first stage of PCR amplification is conducted for a predetermined number of cycles at a temperature sufficiently higher than Tm 2 so that there is substantially no amplification of the higher abundance species.
  • the annealing and elongation steps of the PCR reaction are conducted at a lower temperature, typically about Tm 2 , so that both the lower abundance and the higher abundance amplimers are amplified.
  • Tm refers to “effective Tm,” which is the Tm for any given primer in a given reaction mix, which depends on factors, including, without limitation, the nucleic acid sequence of the primer and the primer concentration in the reaction mixture.
  • PCR amplification is a dynamic process.
  • the higher temperature annealing stage may be carried out at any temperature typically ranging from just above the lower Tm to just below the higher Tm, so long as the reaction favors production of the amplicon by the higher Tm primer set.
  • the annealing for the lower temperature reaction typically is at any temperature below the Tm of the low temperature primer set.
  • the amplicon for the low abundance RNA is amplified at a rate faster than that the amplicon for the higher abundance RNA (and preferably to the substantial exclusion of production of the second amplicon), so that, prior to the second amplification stage, where it is desirable that amplification of all amplicons proceeds in a substantially balanced manner, the amplicon for the lower abundance RNA is of sufficient abundance that the amplification of the higher abundance RNA does not interfere with the amplification of the amplicon for the lower abundance RNA.
  • the annealing and elongation steps may be performed above Tm 1 to gain specificity over efficiency (during the second stage of the amplification, since there is a relatively large number of low abundance nucleic acid amplicons, selectivity no longer is a significant issue, and efficiency of amplicon production is preferred). It, therefore, should be noted that although favorable in many instances, the temperature variations may not necessarily result in the complete shutdown of one amplification reaction over another.
  • a first primer set with a first Tm may target a more-abundant template sequence (for instance, the control template sequence) and a second primer set with a higher Tm may target a less-abundant template sequence.
  • the more-abundant template and the less-abundant template may both be amplified in a first stage at a temperature below the (lower) Tm of the first primer set.
  • the annealing and/or elongation temperature of the reaction is raised above the Tm of the first primer set, but below the higher Tm of the second primer set to effectively shut down amplification of the more abundant template.
  • Selection of three or more sets of PCR primer sets having three or more different Tms can be used to amplify sequences of varying abundance in a stepwise manner, so long as the differences in the Tms are sufficiently large to permit preferential amplification of desired sequences to the substantial exclusion of undesired sequences for a desired number of cycles.
  • the lowest abundance sequences are amplified in a first stage for a predetermined number of cycles.
  • the lowest abundance and the lesser abundance sequences are amplified in a second stage for a predetermined number of cycles.
  • all sequences are amplified in a third stage.
  • the minimum temperature for each stage may vary, depending on the relative efficiencies of each single amplification reaction of the multiplex reaction. It should be recognized that two or more amplimers may have substantially the same Tm, to permit amplification of more than one species of similar abundance at any stage of the amplification process.
  • the three-stage reaction may also proceed stepwise from amplification of the most abundant nucleic acid species at the lowest annealing temperature to amplification of the least abundant species at the highest annealing temperature.
  • an additional tool is provided for the “balancing” of multiplex PCR reactions besides the matching of Tms and using limiting amounts of one or more PCR primer sets.
  • the exploitation of PCR primer sets with different Tms as a method for sequentially amplifying different amplicons may be preferred in certain circumstances to the sequential addition of additional primer sets.
  • the use of temperature-dependent sequencing of multiplex PCR reactions may be coupled with the sequential physical addition of primer sets to a single reaction mixture.
  • the internal positive controls are DNA oligonucleotides that have the same primer sequences as the target gene (CEA or tyrosinase) but have a different internal probe sequence. Selected sites in the IPC's optionally may be synthesized with uracil instead of thymine so that contamination with the highly concentrated mimic could be controlled using uracil DNA glycosylase, if required.
  • the IPCs may be added to any PCR reaction mastermix in amounts that are determined empirically to give Ct values typically greater than the Ct values of the endogenous target of the primer set.
  • the PCR assays are then performed according to standard protocols, and even when there is no endogenous target for the primer set, the IPC would be amplified, thereby verifying that the failure to amplify the target endogenous DNA is not a failure of the PCR reagents in the mastermix.
  • the IPC probe fluoresces differently than the probe for the endogenous sequences.
  • a variation of this for use in RT-PCR reactions is where the IPC is an RNA and the RNA includes an RT primer sequence.
  • the IPC verifies function of both the RT and PCR reactions. Both RNA and DNA IPCs (with different corresponding probes) may also be employed to differentiate difficulties in the RT and PCR reactions.
  • the rapid QRT-PCR protocols described herein may be run in about 20 minutes. This short time period permits the assay to be run intraoperatively so that a surgeon can decide on a surgical course during a single operation (typically the patient will remain anesthetized and/or otherwise sedated in a single “operation”, though there may be a waiting period between when the sample to be tested is obtained and the time the interoperative assay is complete), rather than requiring a second operation, or requiring the surgeon to perform unneeded or overly broad prophylactic procedures. For instance, in the surgical evaluation of certain cancers, including breast cancer, melanoma, lung cancer, esophageal cancer and colon cancer, tumors and sentinel lymph nodes are removed iii a first operation.
  • the sentinel nodes are later evaluated for micrometastases, and, when micrometastases are detected in a patient's sentinel lymph node, the patient will need a second operation, thereby increasing the patient's surgical risks and patient discomfort associated with multiple operations.
  • a physician can make an immediate decision on how to proceed without requiring the patient to leave the operating room or associated facilities.
  • the rapid test also is applicable to needle biopsies taken in a physician's office. A patient need not wait for days to get the results of a biopsy (such as a needle biopsy of a tumor or lymph node), but can now get more accurate results in a very short time.
  • a probe in the context of gene expression analysis, is “specific to” a gene or transcript if under reaction conditions it can hybrizide specifically to transcripts of that gene within a sample, or sequences complementary thereto, and not to other transcripts.
  • a probe in a diagnostic assay, is specific to a gene if it can bind to a specific transcript or desired family of transcripts in mRNA extracted from a specimen, to the practical exclusion (does not interfere substantially with the detection assay) of other transcripts.
  • primers are specific to a gene if they specifically amplify a sequence of that gene, to the practical exclusion of other sequences in a sample.
  • Table B provides primer and probe sequences for the mRNA quantification assays described and depicted in the Examples and Figures.
  • FIGS. 1-11 provide non-limiting examples of cDNA sequences of the various mRNA species detected in the Examples. Although the sequences provided in Table B were found effective in the assays described in the examples, other primers and probes would likely be equally suited for use in the QRT-PCR and other mRNA detection and quantification assays, either described herein or as are known in the art. Design of alternate primer and probe sets for PCR assays, as well as for other mRNA detection assays is well within the abilities of one of average skill in the art.
  • a number of computer software programs will generate primers and primer sets for PCR assays from cDNA sequences according to specified parameters.
  • Non limiting examples of such software include, NetPrimer and Primer Premier 5, commercially available from PREMIER Biosoft International of Palo Alto, Calif., which also provides primer and probe design software for molecular beacon and array assays.
  • Primers and/or probes for two or more different mRNAs can be identified, for example and without limitation, by aligning the two or more target sequences according to standard methods, determining common sequences between the two or more mRNAs and entering the common sequences into a suitable primer design computer program.
  • a “primer or probe” for detecting a specific mRNA species is any primer, primer set and/or probe that can be utilized to detect and/or quantify the specific mRNA species.
  • An “mRNA species” can be a single mRNA species, corresponding to a single mRNA expression product of a single gene, or can be multiple mRNAs that are detected by a single common primer and/or probe combination, such as the SCCA1.2 and MAGEA136-plex pecies described below.
  • a test typically comprises one or more reagents, such as, without limitation, nucleic acid primers or probes, packaged in a container, such as, without limitation, a vial, tube or bottle, in a package suitable for commercial distribution, such as, without limitation, a box, a sealed pouch, a blister pack and a carton.
  • the package typically contains an indicia, for example and without limitation, a writing, illustration, label, book, booklet, tag and/or packaging insert, indicating that the packaged reagents can be used in a method for identifying expression or markers indicative of the presence of cancer cells in a lymph node of a patient.
  • packaging materials includes any article used in the packaging for distribution of reagents in a kit, including without limitation cointainers, vials, tubes, bottles, pouches, blister packaging, labels, tags, instruction sheets and package inserts.
  • a kit would include reagents necessary for the one-tube QRT-PCR process described above.
  • the kit would include the above-described reagents, including reverse transcriptase, a reverse transcriptase primer, a corresponding PCR primer set, a thermostable DNA polymerase, such as Taq polymerase, and a suitable fluorescent reporter, such as, without limitation, a probe for a fluorescent 5′ nuclease assay, a molecular beacon probe, a single dye primer or a fluorescent dye specific to double-stranded DNA, such as ethidium bromide.
  • the primers may be present in quantities that would yield the high concentrations described above.
  • Thermostable DNA polymerases are commonly and commercially available from a variety of manufacturers.
  • kits may include: suitable reaction tubes or vials, a barrier composition, typically a wax bead, optionally including magnesium; reaction mixtures (typically 10 ⁇ ) for the reverse transcriptase and the PCR stages, including necessary buffers and reagents such as dNTPs; nuclease- or RNase-free water; RNase inhibitor; control nucleic acid(s) and/or any additional buffers, compounds, co-factors, ionic constituents, proteins and enzymes, polymers, and the like that may be used in reverse transcriptase and/or PCR stages of QRT-PCR reactions.
  • suitable reaction tubes or vials typically a wax bead, optionally including magnesium
  • reaction mixtures typically 10 ⁇
  • reagents such as dNTPs
  • nuclease- or RNase-free water such as RNase inhibitor
  • PCR primers and reverse transcriptase may be packaged individually to facilitate flexibility in configuring the assay, or together to increase ease of use and to reduce contamination.
  • buffers, salts and co-factors can be packaged separately or together.
  • kits also may include reagents and mechanical components suitable for the manual or automated extraction of nucleic acid from a tissue sample.
  • reagents are known to those skilled in the art and typically are a matter of design choice.
  • tissue is disrupted ultrasonically in a suitable lysis solution provided in the kit.
  • the resultant lysate solution is then filtered and RNA is bound to RNA-binding magnetic beads also provided in the kit or cartridge.
  • the bead-bound RNA is washed, and the RNA is eluted from the beads and placed into a suitable reverse transcriptase reaction mixture prior to the reverse transcriptase reaction.
  • reagents and their mode of packaging typically are dictated by the physical configuration of the robotics and fluidics of the specific RNA extraction system, for example and without limitation, the GenXpert system.
  • International Patent Publication Nos. WO 04/48931, WO 03/77055, WO 03/72253, WO 03/55973, WO 02/52030, WO 02/18902, WO 01/84463, WO 01/57253, WO 01/45845, WO 00/73413, WO 00/73412 and WO 00/72970 provide non-limiting examples of cartridge-based systems and related technology useful in the methods described herein.
  • kits may be packaged together or separately, and each constituent may be presented in one or more tubes or vials, or in cartridge form, as is appropriate.
  • the constituents, independently or together, may be packaged in any useful state, including without limitation, in a dehydrated, lyophilized, a glassified or an aqueous state.
  • the kits may take the physical form of a cartridge for use in automated processes, having two or more compartments including the above-described reagents. Suitable cartridges are disclosed for example in U.S. Pat. Nos. 6,440,725, 6,431,476, 6,403,037 and 6,374,684.
  • Array technologies also can facilitate determining the expression level of two or more genes by facilitating performance of the desired reactions and their analysis by running multiple parallel reactions at the same time.
  • One example of an array is the GeneChip® gene expression array, commercially available from Affymetrix, Inc. of Santa Clara, Calif.
  • Patents illustrating array technology and uses therefor include, without limitation, U.S. Pat. Nos. 6,040,138, 6,245,517, 6,251,601, 6,261,776, 6,306,643, 6,309,823, 6,346,413, 6,406,844 and 6,416,952.
  • An “array”, such as a “microarray” can be a substrate containing one or more binding reagents, typically in discrete physical locations, permitting high throughput analysis of the binding of a sample to the array.
  • an array contains probes specific to transcripts of one or more of the genes described herein affixed to a substrate.
  • the probes can be nucleic acids or analogs thereof, as are known in the art.
  • An array also can refer to a plurality of discrete reaction chambers, permitting multiple parallel reactions and detection events on a miniaturized scale.
  • the nucleic acid quantification method is a rolling circle amplification method.
  • rolling circle amplification methods are described in U.S. Pat. Nos. 5,854,003; 6,183,960; 6,344,329; and 6,210,884, each of which are incorporated herein by reference to the extent they teach methods for detecting and quantifying RNA species.
  • a padlock probe is employed to facilitate the rolling circle amplification process.
  • a padlock probe is a linear oligonucleotide or polynucleotide designed to include one target-complementary sequence at each end, and which is designed such that the two ends are brought immediately next to each other upon hybridization to the target sequence.
  • the probe also includes a spacer between the target-complementary sequences that includes a polymerase primer site and a site for binding to a probe, such as a molecular beacon probe, for detecting the padlock probe spacer sequence.
  • the probe ends can then be joined by enzymatic DNA ligation to form a circular template that can be amplified by polymerase extension of a complementary primer.
  • Thousands of concatemerized copies of the template can be generated by each primer, permitting detection and quantification of the original RNA template.
  • Quantification can be automated by use, for example and without limitation, of a molecular beacon probe or other probe capable of detecting accumulation of a target sequence.
  • Padlock probe sequences target unique portions of the target RNA in order to ensure specific binding with limited or no cross-reactivity.
  • RCA is an isothermic method in that the amplification is performed at one temperature.
  • NASBA nucleic acid sequence-based amplification
  • a typical NASBA reaction is initiated by the annealing of a first oligonucleotide primer to an RNA target in an RNA sample.
  • the 3′ end of the first primer is complementary to the target analyte; the 5′ end encodes the T7 RNA polymerase promoter.
  • AMV-RT reverse transcription
  • RNA is digested with RNase H, permitting a second primer (sense) to anneal to the cDNA strand, permitting the DNA polymerase activity of the reverse transcriptase to be engaged, producing a double-stranded cDNA copy of the original RNA template, with a functional T7 RNA polymerase promoter at one end. T7 polymerase is then used to produce an additional RNA template, which is further amplified, though in reverse order, according to the same procedure.
  • T7 polymerase is then used to produce an additional RNA template, which is further amplified, though in reverse order, according to the same procedure.
  • a variety of other nucleic acid detection and/or amplification methods are known to those of skill in the art, including variations on the isothermic strand displacement, PCR and RCA methods described herein.
  • Tissues and Pathological Evaluation Tissue specimens were obtained from tissue banks at the University of Pittsburgh Medical Center through IRB approved protocols. All specimens were snap frozen in liquid nitrogen and later embedded in OCT for frozen sectioning. Twenty 5-micron sections were cut from each tissue for RNA isolation. In addition, sections were cut and placed on slides for H&E and IHC analysis at the beginning, middle (between the tenth and eleventh sections for RNA), and end of the sections for RNA isolation. All three H&E slides from each specimen underwent pathological review to confirm presence of tumor, percentage of tumor, and to identify the presence of any contaminating tissues. All of the unstained slides were stored at ⁇ 20° C.
  • Immunohistochemistry evaluation was performed using the AE1/AE3 antibody cocktail (DAKO, Carpinteria, Calif.), and Vector Elite ABC kit and Vector AEC Chromagen (Vecta Laboratories, Burlingame, Calif.). IHC was used as needed as needed to confirm the H&E histology.
  • the screening was conducted in two phases. All potential markers entered the primary screening phase and expression was analyzed in 6 primary tumors and 10 benign lymph nodes obtained from patients without cancer (5 RNA pools with 2 lymph node RNA's per pool). Markers that showed good characteristics for lymph node metastasis detection passed into the secondary screening phase.
  • the secondary screen consisted of expression analysis on 20-25 primary tumors, 20-25 histologically positive lymph nodes and 21 benign lymph nodes without cancer.
  • RNA Isolation and cDNA Synthesis RNA was isolated using the RNeasy minikit (Qiagen, Valencia, Calif.) essentially as described by the manufacturer. The only modification was that we doubled the volume of lysis reagent and loaded the column in two steps. This was found to provide better RNA yield and purity, probably as a result of diluting out the OCT in the tissue sections. Reverse transcription was performed in 100- ⁇ l reaction volumes either with random hexamer priming or sequence-specific priming using a probe indicated in Table C, and Superscript II (Invitrogen, Carlsbad, Calif.) reverse transcriptase. For the primary screen, three reverse transcription reactions were performed, each with 500 ng of RNA.
  • RNA input for primary tumors and positive nodes was also 500 ng.
  • the RNA input was 2000 ng resulting in the equivalent of 80 ng RNA per QPCR reaction.
  • Quantitative PCR All quantitative PCR was performed on the ABI Prism 7700 Sequence Detection Instrument (Applied Biosystems, Foster City, Calif.). Relative expression of the marker genes was calculated using the delta-C T methods previously described and with ⁇ -glucuronidase as the endogenous control gene. All assays were designed for use with 5′ nuclease hybridization probes although the primary screening was performed using SYBER Green quantification in order to save cost. Assays were designed using the ABI Primer Express Version 2.0 software and where possible, amplicons spanned exon junctions in order to provide cDNA specificity. All primer pairs were tested for amplification specificity (generation of a single band on gels) at 60, 62 and 64° C. annealing temperature. In addition, PCR efficiency was estimated using SYBER green quantification prior to use in the primary screen. Further optimization and more precise estimates of efficiency were performed with 5′nuclease probes for all assays used in the secondary screen.
  • RNAs from human placenta, thyroid, heart, colon, PCI13 cell line and SKBR3 cell line served as a universal positive expression control for all the genes in the marker screening process.
  • each 50 ⁇ l reaction contained 1 ⁇ TaqMan buffer A (Applied Biosystems), 300 nM each dNTP, 3.5 mM MgCl 2 , 0.06 units/ ⁇ l Amplitaq Gold (Applied Biosystems), 0.25 ⁇ SYBR Green I (Molecular Probes, Eugene, Oreg.) and 200 nM each primer.
  • the amplification program comprised 2-stages with an initial 95° C. Taq activation stage for 12 min followed by 40 cycles of 95° C. denaturation for 15 s, 60 or 62 or 64° C. anneal/extend for 60 s and a 10 second data collection step at a temperature 2-4° C.
  • Probe-based QPCR was performed as described previously (Godfrey, et al., Clin Cancer Res. 2001 December, 7(12):4041-8). Briefly, reactions were performed with a probe concentration of 200 nM and a 60 second anneal/extend phase at 60° C., or 62° C., or 64° C.
  • the sequences of primers and probes purchased from IDT, Coralville, Iowa) for genes evaluated in the secondary screen are listed in Table B, below.
  • Table A identifies genes used in the following studies.
  • Table B provides PCR primer and TAQMAN probe sequences used in the quantitative PCR and RT-PCR amplifications described herein.
  • Table C provides RT primer sequences as used instead of random hexamer primers. All PCR and RT-PCR reactions were conducted using standard methods.
  • T primary tumor
  • FIG. 12 is a scatter plot showing the expression levels of CK7, CK19, MGB1, MGB2, PIP, and TACSTD1 in primary tumor, tumor-positive lymph nodes and benign lymph nodes.
  • FIGS. 13 A-O provide scatter plots illustrating the ability of two-marker systems to distinguish between benign and malignant cells in a lymph node. Tables D and E provide the raw data from which the graphs of FIGS. 12 and 13 A-O were generated.
  • FIG. 14 is a scatter plot showing the expression levels of CEA, CK7, CK19, LUNX, PVA, SCCA1.2, SFTPB, and TACSTD1 in primary tumor, tumor-positive lymph nodes and benign lymph nodes.
  • FIGS. 15 A-BB provide scatter plots illustrating the ability of two-marker systems to distinguish between benign and malignant cells in a lymph node.
  • FIG. 16 is a plot of the best combination of three markers for detecting lung cancer in different histological types. Tables F and G provide the raw data from which the graphs of FIGS. 14 and 15 A-BB were generated.
  • This data illustrates the strong correlation of expression of CEA, CK7, CK19, PVA, SCCA1.2, SFTPB, and TACSTD1 markers, alone or in combination, in sentinel lymph nodes with the presence of malignant cells arising from a lung cancer in the sentinel lymph node.
  • Example 2 An extensive literature and database survey identified potential mRNA markers for detection of lymph node metastases in breast cancer.
  • a primary screen analyzed the relative expression of 43 potential markers in 6 primary breast tumors and 10 benign lymph nodes obtained from patients without cancer.
  • Six markers showed good characteristics for lymph node metastasis detection and entered a secondary screening phase where expression was analyzed in 25 primary tumors, 27 histologically positive lymph nodes and 21 benign lymph nodes from patients without cancer (73 independent patients). Based on the classification characteristics, 4 markers were selected for an external validation study of 90 SLN from independent patients with breast cancer using a rapid, multiplex real-time PCR assay.
  • 9 histologically negative and 9 histologically positive lymph nodes were analyzed using a completely automated and rapid RNA isolation and real-time PCR assay on the GeneXpert®.
  • Tissues for the marker screening and the GeneXpert® study were obtained from tissue banks at the University of Pittsburgh Medical Center and SLN for the marker validation study were obtained from the Minimally Invasive Molecular Staging of Breast Cancer Trial (MIMS) initiated at the Medical University of South Carolina.
  • MIMS Minimally Invasive Molecular Staging of Breast Cancer Trial
  • SLN specimens For the validation study, 115 chronologically-obtained SLN specimens from individual patients were identified. Five-micron serial sections were cut from each tissue, and the initial and final two tissue sections were mounted on slides for histological analysis with H&E staining and pancytokeratin IHC. The intervening sections were distributed 4:1:4:1:4 etc., such that four sections were immediately placed in chaotropic lysis buffer for RNA isolation and every fifth section was mounted on a slide for histology review. The total number of sections cut was dependent on size of the SLN (range 50-60). All specimens were reviewed to confirm adequate preservation of histology for pathologic analysis resulting in the exclusion of 25 specimens. For the remaining 90 SLN, sections from three levels (beginning, middle and end) underwent pathologic review with both H&E and IHC staining, and remaining slides were reviewed as needed.
  • RNA Isolation For the screening and validation studies, RNA was isolated using the RNeasy minikit (Qiagen, Valencia, Calif.) as described by the manufacturer. The only modification was that the volume of lysis reagent was doubled and loaded on the column in two steps. All RNA's were DNAse treated using the DNA-free Kit from Ambion.
  • Quantitative RT-PCR Analysis For the marker screening study, cDNA was synthesized using random hexamers. Quantitative real-time PCR was performed on the ABI Prism 7700 Sequence Detection instrument and expression of each marker gene was measured relative to the endogenous control gene ⁇ -glucuronidase using ⁇ Ct calculations. To save cost, the primary screen was performed using quantification with SYBR green. In the secondary screen, 5′ nuclease hybridization probes were used to increase assay specificity. All assays were designed using the ABI Primer Express Version 2.0 software and where possible, amplicons spanned exon junctions to provide cDNA specificity. Negative controls were included in each PCR plate.
  • RNAs from human placenta, thyroid, heart, colon, PCI13 cell line and SKBR3 cell line served as a universal positive expression control for all the genes in the marker screening process.
  • RNA input for each lymph node sample was 50-200 ng per QRT-PCR reaction and all reactions were performed in duplicate.
  • Each reaction incorporated an internal positive control (IPC) oligonucleotide to demonstrate adequate assay sensitivity in the case of negative results.
  • IPC internal positive control
  • GeneXpert Analysis Twenty-four, 5- ⁇ m sections of OCT embedded tissue were sectioned into 800 ⁇ l of GeneXpert® lysis buffer (Cepheid, Sunnyvale, Calif.). The lysis buffer was filtered through a 0.22- ⁇ m syringe filter (Osmonics Inc, West borough, Mass.), and loaded into a GeneXpert® cartridge. The automated processes of RNA isolation, reverse transcription, and QRT-PCR on the GeneXpert®.
  • the characteristics used to evaluate markers were sensitivity, specificity, classification accuracy and negative and positive predictive values.
  • the evaluation included characterizing the distributions of the markers and testing the fit of the data to the log-normal distribution. For individual markers, a cutoff value was determined that maximized the classification accuracy. In cases where classification accuracy was 100%, the cutoff was set at the midpoint between the highest expressing benign node and the lowest expressing histologically positive node. Markers were also evaluated in paired combinations and a linear prediction rule was generated for each pair. The rule was equivalent to the linear predictor that equalized the fitted probabilities above and below the linear boundary. That is, points on the boundary line had a predicted probability midway between the numeric scores assigned to positive and negative nodes.
  • Prediction characteristics of marker combinations were also determined by generating equal probability contours.
  • the joint distributions of marker pairs were assumed to follow a bivariate log-normal distribution. From estimates of the means, variances and covariances of benign nodes, equal probability contours were constructed around the estimated mean values obtained for relative level of expression in benign lymph nodes. Observed values were then plotted against these equal probability ellipsoids and compared to contours for the more extreme quantiles, including the 95 th , 99 th and 99.9 th percentiles. This method of analyzing the data attaches a value to each point that is the approximate probability that the plotted node is benign.
  • the ratio was calculated between the median expression in tumors and the highest expressing benign node and between the lowest expressing tumor and the highest expressing benign node.
  • TACSTD1 cytokeratin 7
  • CK19 cytokeratin 19
  • MGB1 mammoglobin 1
  • MGB2 Mammoglobin 2
  • PIP prolactin inducible protein
  • TACSTD1/PIP CK19/TACSTD1
  • TACSTD1/MGB1 TACSTD1/MGB2
  • TACSTD1/MGB2 TACSTD1/MGB2
  • FIGS. 19A and 19B To externally validate the classification accuracy of selected markers tested in the secondary screen, an independent, validation set of 90 breast cancer sentinel lymph nodes was prospectively analyzed ( FIGS. 19A and 19B ). Furthermore, to demonstrate the potential for intraoperative analysis, this study was performed on the SmartCycler® instrument (Cepheid) using rapid, multiplex QRT-PCR. Subtle differences in calculated relative expression values were observed (data not shown) from this change in thermocycler platform, but in an effort to indirectly evaluate the robustness of the QRT-PCR analysis, the classification algorithms from the secondary screen were applied to the validation set data without any correction factors.
  • Previous studies have aimed to determine if RT-PCR analysis of lymph nodes is more sensitive than IHC and thus capable of further improving the clinical staging of breast cancer patients.
  • the present study differs from those studies in that the present aim was not to determine if QRT-PCR identified metastatic disease in definitively analyzed, histologically negative SLN, but rather to surpass current methods of analysis with regards to timeliness, reproducible objectivity, and automation.
  • this assay may prove to be capable of surpassing current techniques in this regard.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Hospice & Palliative Care (AREA)
  • Biophysics (AREA)
  • Oncology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
US11/178,109 2004-07-09 2005-07-08 Identification of markers in lung and breast cancer Abandoned US20060068418A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US11/178,109 US20060068418A1 (en) 2004-07-09 2005-07-08 Identification of markers in lung and breast cancer
US13/030,003 US20110312515A1 (en) 2004-07-09 2011-02-17 Identification of markers in lung and breast cancer

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US58659904P 2004-07-09 2004-07-09
US58701904P 2004-07-09 2004-07-09
US11/178,109 US20060068418A1 (en) 2004-07-09 2005-07-08 Identification of markers in lung and breast cancer

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US13/030,003 Continuation US20110312515A1 (en) 2004-07-09 2011-02-17 Identification of markers in lung and breast cancer

Publications (1)

Publication Number Publication Date
US20060068418A1 true US20060068418A1 (en) 2006-03-30

Family

ID=35839738

Family Applications (4)

Application Number Title Priority Date Filing Date
US11/178,134 Expired - Fee Related US7662561B2 (en) 2004-07-09 2005-07-08 Identification of markers in esophageal cancer, colon cancer, head and neck cancer, and melanoma
US11/178,109 Abandoned US20060068418A1 (en) 2004-07-09 2005-07-08 Identification of markers in lung and breast cancer
US12/637,862 Abandoned US20100240037A1 (en) 2004-07-09 2009-12-15 Identification of markers in esophageal cancer, colon cancer, head and neck cancer, and melanoma
US13/030,003 Abandoned US20110312515A1 (en) 2004-07-09 2011-02-17 Identification of markers in lung and breast cancer

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US11/178,134 Expired - Fee Related US7662561B2 (en) 2004-07-09 2005-07-08 Identification of markers in esophageal cancer, colon cancer, head and neck cancer, and melanoma

Family Applications After (2)

Application Number Title Priority Date Filing Date
US12/637,862 Abandoned US20100240037A1 (en) 2004-07-09 2009-12-15 Identification of markers in esophageal cancer, colon cancer, head and neck cancer, and melanoma
US13/030,003 Abandoned US20110312515A1 (en) 2004-07-09 2011-02-17 Identification of markers in lung and breast cancer

Country Status (6)

Country Link
US (4) US7662561B2 (fr)
EP (6) EP2468889A3 (fr)
JP (3) JP2008510454A (fr)
AU (2) AU2005271961B8 (fr)
CA (2) CA2571642A1 (fr)
WO (2) WO2006017151A2 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050112622A1 (en) * 2003-08-11 2005-05-26 Ring Brian Z. Reagents and methods for use in cancer diagnosis, classification and therapy
US20060003391A1 (en) * 2003-08-11 2006-01-05 Ring Brian Z Reagents and methods for use in cancer diagnosis, classification and therapy
US20060019290A1 (en) * 2004-07-09 2006-01-26 Godfrey Tony E Identification of markers in esophageal cancer, colon cancer, head and neck cancer, and melanoma
US20060068433A1 (en) * 2004-09-20 2006-03-30 Godfrey Tony E Multiple mode multiplex reaction quenching method
US20070218496A1 (en) * 2006-03-11 2007-09-20 Roche Molecular Systems, Inc. Method for quick determination of cytokeratin 19 (ck19) and primers and probes therefore
EP1876245A1 (fr) * 2006-07-03 2008-01-09 Sysmex Corporation Procédé et appareil pour déterminer la taille des métastases
US20080102495A1 (en) * 2006-06-20 2008-05-01 Lynn Kozma Multi-stage amplification reactions by control of sequence replication times
US20080131916A1 (en) * 2004-08-10 2008-06-05 Ring Brian Z Reagents and Methods For Use In Cancer Diagnosis, Classification and Therapy
WO2008063413A3 (fr) * 2006-11-13 2008-09-04 Source Precision Medicine Inc Détermination du profil de l'expression génique dans l'identification, la surveillance et le traitement du cancer du poumon
US20130231578A1 (en) * 2012-03-02 2013-09-05 Nihon Kohden Corporation Electrocardiogram analysis report, electrocardiogram analysis apparatus, and electrocardiogram analysis program
US9151762B2 (en) 2009-06-12 2015-10-06 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Identification of DSG-3 as a biomarker for the detection of metastasis in lymph nodes
WO2023081988A1 (fr) * 2021-11-09 2023-05-19 Coelho Guilherme Portela Méthode de prédiction d'efficacité d'un traitement contre le cancer du sein

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7622456B2 (en) * 2006-11-06 2009-11-24 Non-Profit Organization Chang Gung Memorial Hospital Method on clinical applications in head neck cancer by using DSG3 molecule for predicting malignant degree of cancer, serving as a molecular target and using RNA jamming sequence on inhibition-specific of DSG3 expression
MX368365B (es) 2009-12-22 2019-09-30 Avon Prod Inc Composiciones estimuladoras de paxilina y usos cosmeticos de las mismas.
CN102686745B (zh) * 2009-12-24 2014-01-29 国立大学法人爱媛大学 颈部淋巴结转移分析方法及头颈癌的肿瘤标记物
PL2659004T3 (pl) 2010-12-30 2017-12-29 Avon Products, Inc. Modulacja dyneiny w skórze
US9771617B2 (en) 2011-06-27 2017-09-26 Eisai R&D Management Co., Ltd. Microrna biomarkers indicative of alzheimer's disease
US10195136B2 (en) 2011-08-24 2019-02-05 Avon Products, Inc. Collagen and elastin stimulating compositions and uses thereof
US8632827B2 (en) 2011-12-13 2014-01-21 Avon Products, Inc Modulation of thymosin beta-4 in skin
WO2013090228A2 (fr) 2011-12-13 2013-06-20 Avon Products, Inc. Extraits de maesa japonica et procédés d'utilisation
CN108949992B (zh) * 2018-08-14 2021-11-05 高鑫 一种与食管鳞癌及其分级相关的生物标志物
CN109913552B (zh) * 2019-03-27 2020-08-04 河北医科大学第四医院 一种食管鳞癌诊疗靶点及应用
EP4116435A1 (fr) * 2021-07-08 2023-01-11 Universidad de Granada Procédé in vitro pour le diagnostic et/ou le pronostic de mélanome malin

Citations (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5314809A (en) * 1991-06-20 1994-05-24 Hoffman-La Roche Inc. Methods for nucleic acid amplification
US5814491A (en) * 1995-06-06 1998-09-29 Vijg; Jan Method of and apparatus for diagnostic DNA testing
US5837442A (en) * 1995-11-29 1998-11-17 Roche Molecular Systems, Inc. Oligonucleotide primers for amplifying HCV nucleic acid
US5843761A (en) * 1986-08-13 1998-12-01 Bayer Corporation (Formerly Molecular Diagnostics, Inc.) CDNA coding for carcinoembryonic antigen
US5854003A (en) * 1997-07-03 1998-12-29 Tularik Inc. Screening method for agents that modulate human NIK activity
US5882856A (en) * 1995-06-07 1999-03-16 Genzyme Corporation Universal primer sequence for multiplex DNA amplification
US5958349A (en) * 1997-02-28 1999-09-28 Cepheid Reaction vessel for heat-exchanging chemical processes
US6040138A (en) * 1995-09-15 2000-03-21 Affymetrix, Inc. Expression monitoring by hybridization to high density oligonucleotide arrays
US6057105A (en) * 1995-03-17 2000-05-02 Ngi/Cancer Tech Company, Llc Detection of melanoma or breast metastasis with a multiple marker assay
US6183960B1 (en) * 1995-11-21 2001-02-06 Yale University Rolling circle replication reporter systems
US6245517B1 (en) * 1998-09-29 2001-06-12 The United States Of America As Represented By The Department Of Health And Human Services Ratio-based decisions and the quantitative analysis of cDNA micro-array images
US6251601B1 (en) * 1999-02-02 2001-06-26 Vysis, Inc. Simultaneous measurement of gene expression and genomic abnormalities using nucleic acid microarrays
US6261776B1 (en) * 1989-06-07 2001-07-17 Affymetrix, Inc. Nucleic acid arrays
US6306643B1 (en) * 1998-08-24 2001-10-23 Affymetrix, Inc. Methods of using an array of pooled probes in genetic analysis
US6309823B1 (en) * 1993-10-26 2001-10-30 Affymetrix, Inc. Arrays of nucleic acid probes for analyzing biotransformation genes and methods of using the same
US6346413B1 (en) * 1989-06-07 2002-02-12 Affymetrix, Inc. Polymer arrays
US6374684B1 (en) * 2000-08-25 2002-04-23 Cepheid Fluid control and processing system
US6403037B1 (en) * 2000-02-04 2002-06-11 Cepheid Reaction vessel and temperature control system
US6406844B1 (en) * 1989-06-07 2002-06-18 Affymetrix, Inc. Very large scale immobilized polymer synthesis
US6416952B1 (en) * 1989-06-07 2002-07-09 Affymetrix, Inc. Photolithographic and other means for manufacturing arrays
US6431476B1 (en) * 1999-12-21 2002-08-13 Cepheid Apparatus and method for rapid ultrasonic disruption of cells or viruses
US6440725B1 (en) * 1997-12-24 2002-08-27 Cepheid Integrated fluid manipulation cartridge
US20050123976A1 (en) * 2003-12-02 2005-06-09 Michael Mitas Methods and compositions for diagnosing epithelial cell cancer
US20060068433A1 (en) * 2004-09-20 2006-03-30 Godfrey Tony E Multiple mode multiplex reaction quenching method
US7101663B2 (en) * 2001-03-02 2006-09-05 University of Pittsburgh—of the Commonwealth System of Higher Education PCR method

Family Cites Families (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0732929B1 (fr) 1993-10-29 2008-05-14 The Brigham And Women's Hospital, Inc. Utilisation therapeutique de leurres d'elements cis in vivo
US7625697B2 (en) * 1994-06-17 2009-12-01 The Board Of Trustees Of The Leland Stanford Junior University Methods for constructing subarrays and subarrays made thereby
US6031129A (en) * 1995-10-03 2000-02-29 Quantum Chemical Corporation Use of pentavalent group VA oxides in acetic acid processing
AUPO188396A0 (en) 1996-08-26 1996-09-19 Insearch Limited Detection of protozoan parasites
GB9719044D0 (en) 1997-09-08 1997-11-12 Inst Of Ophthalmology Assay
US6660228B1 (en) 1998-03-02 2003-12-09 Cepheid Apparatus for performing heat-exchanging, chemical reactions
US6037129A (en) * 1998-05-28 2000-03-14 Medical University Of South Carolina Multi-marker RT-PCR panel for detecting metastatic breast cancer
AU2736400A (en) 1999-01-27 2000-08-18 University Of South Florida Inhibition of stat3 signal transduction for human cancer therapy
US6300069B1 (en) 1999-05-03 2001-10-09 Qiagen Gmbh Generation and amplification of nucleic acids from ribonucleic acids
ES2272289T5 (es) 1999-05-28 2011-10-21 Cepheid Cartucho para realizar una reacción química.
DE60022025T2 (de) 1999-05-28 2006-06-29 Cepheid, Sunnyvale Anlage zum brechen von zellen
SE9902410D0 (sv) 1999-06-24 1999-06-24 Cavidi Tech Ab Reverse transcriptase assay kit, use thereof and method for analysis of RT activity in biological samples
AU5830400A (en) * 1999-06-30 2001-01-22 Arto Orpana Diagnostic method for squamous cell carcinoma
ATE469242T1 (de) * 2000-03-27 2010-06-15 Univ Jefferson Zusammensetzungen und methoden zur identifizierung und zum targeting von krebszellen aus dem verdauungskanal
US6783934B1 (en) 2000-05-01 2004-08-31 Cepheid, Inc. Methods for quantitative analysis of nucleic acid amplification reaction
AU2001280987A1 (en) * 2000-08-03 2002-02-18 Corixa Corporation Compositions and methods for the therapy and diagnosis of head and neck cancer
US8048386B2 (en) 2002-02-25 2011-11-01 Cepheid Fluid processing and control
WO2002037113A2 (fr) * 2000-11-03 2002-05-10 Michael Giesing Validation clinique et fonctionnelle de cibles a partir de cellules cancereuses disseminees
US6312929B1 (en) 2000-12-22 2001-11-06 Cepheid Compositions and methods enabling a totally internally controlled amplification reaction
AU2002359915A1 (en) 2001-12-26 2003-07-15 Olympus Corporation Reaction container and reaction container holding mechanism
US6819027B2 (en) 2002-03-04 2004-11-16 Cepheid Method and apparatus for controlling ultrasonic transducer
JP2005519624A (ja) * 2002-03-13 2005-07-07 ジェノミック ヘルス， インコーポレイテッド 生検腫瘍組織での遺伝子発現プロファイリング
WO2003087766A2 (fr) * 2002-04-05 2003-10-23 The Government Of The United States Of America, As Represented By The Secretary Of The Department Of Health And Human Services Procedes de diagnostic de potentiel de metastase ou de developpement de carcinome hepatocellulaire et d'identification de cibles therapeutiques
AU2003272367A1 (en) * 2002-09-12 2004-04-30 Yissum Research Development Company Of The Hebrew University Of Jerusalem Method for detection of micro-metastasis
EP1572105B1 (fr) * 2002-11-14 2011-08-31 John Wayne Cancer Institute Detection de micro metastases de melanomes et de cancer du sein dans des ganglions lymphatiques drainant des tumeurs incorporees dans de la paraffine par rt-pcr quantitative a l'aide de marqueurs multiples
US20040101859A1 (en) 2002-11-25 2004-05-27 Cepheid Compositions, methods and kits for polynucleotide amplification reactions and microfluidic devices
JP2008510454A (ja) * 2004-07-09 2008-04-10 ユニバーシティ オブ ピッツバーグ オブ ザ コモンウェルス システム オブ ハイヤー エデュケイション 肺癌および乳癌におけるマーカーの同定
US9032602B2 (en) 2011-07-15 2015-05-19 The Boeing Company Methods and systems for in-process quality control during drill-fill assembly

Patent Citations (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5843761A (en) * 1986-08-13 1998-12-01 Bayer Corporation (Formerly Molecular Diagnostics, Inc.) CDNA coding for carcinoembryonic antigen
US6416952B1 (en) * 1989-06-07 2002-07-09 Affymetrix, Inc. Photolithographic and other means for manufacturing arrays
US6346413B1 (en) * 1989-06-07 2002-02-12 Affymetrix, Inc. Polymer arrays
US6406844B1 (en) * 1989-06-07 2002-06-18 Affymetrix, Inc. Very large scale immobilized polymer synthesis
US6261776B1 (en) * 1989-06-07 2001-07-17 Affymetrix, Inc. Nucleic acid arrays
US5314809A (en) * 1991-06-20 1994-05-24 Hoffman-La Roche Inc. Methods for nucleic acid amplification
US6309823B1 (en) * 1993-10-26 2001-10-30 Affymetrix, Inc. Arrays of nucleic acid probes for analyzing biotransformation genes and methods of using the same
US6057105A (en) * 1995-03-17 2000-05-02 Ngi/Cancer Tech Company, Llc Detection of melanoma or breast metastasis with a multiple marker assay
US5814491A (en) * 1995-06-06 1998-09-29 Vijg; Jan Method of and apparatus for diagnostic DNA testing
US5882856A (en) * 1995-06-07 1999-03-16 Genzyme Corporation Universal primer sequence for multiplex DNA amplification
US6040138A (en) * 1995-09-15 2000-03-21 Affymetrix, Inc. Expression monitoring by hybridization to high density oligonucleotide arrays
US6210884B1 (en) * 1995-11-21 2001-04-03 Yale University Rolling circle replication reporter systems
US6183960B1 (en) * 1995-11-21 2001-02-06 Yale University Rolling circle replication reporter systems
US6344329B1 (en) * 1995-11-21 2002-02-05 Yale University Rolling circle replication reporter systems
US5837442A (en) * 1995-11-29 1998-11-17 Roche Molecular Systems, Inc. Oligonucleotide primers for amplifying HCV nucleic acid
US5958349A (en) * 1997-02-28 1999-09-28 Cepheid Reaction vessel for heat-exchanging chemical processes
US5854003A (en) * 1997-07-03 1998-12-29 Tularik Inc. Screening method for agents that modulate human NIK activity
US6440725B1 (en) * 1997-12-24 2002-08-27 Cepheid Integrated fluid manipulation cartridge
US6306643B1 (en) * 1998-08-24 2001-10-23 Affymetrix, Inc. Methods of using an array of pooled probes in genetic analysis
US6245517B1 (en) * 1998-09-29 2001-06-12 The United States Of America As Represented By The Department Of Health And Human Services Ratio-based decisions and the quantitative analysis of cDNA micro-array images
US6251601B1 (en) * 1999-02-02 2001-06-26 Vysis, Inc. Simultaneous measurement of gene expression and genomic abnormalities using nucleic acid microarrays
US6431476B1 (en) * 1999-12-21 2002-08-13 Cepheid Apparatus and method for rapid ultrasonic disruption of cells or viruses
US6403037B1 (en) * 2000-02-04 2002-06-11 Cepheid Reaction vessel and temperature control system
US6374684B1 (en) * 2000-08-25 2002-04-23 Cepheid Fluid control and processing system
US7101663B2 (en) * 2001-03-02 2006-09-05 University of Pittsburgh—of the Commonwealth System of Higher Education PCR method
US20050123976A1 (en) * 2003-12-02 2005-06-09 Michael Mitas Methods and compositions for diagnosing epithelial cell cancer
US20060068433A1 (en) * 2004-09-20 2006-03-30 Godfrey Tony E Multiple mode multiplex reaction quenching method

Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080199867A1 (en) * 2001-03-02 2008-08-21 Godfrey Tony E Multiple mode multiplex reaction quenching method
US20080199891A1 (en) * 2003-08-11 2008-08-21 Ring Brian Z Reagents and Methods For Use In Cancer Diagnosis, Classification and Therapy
US20060003391A1 (en) * 2003-08-11 2006-01-05 Ring Brian Z Reagents and methods for use in cancer diagnosis, classification and therapy
US8399622B2 (en) 2003-08-11 2013-03-19 Clarient Diagnostic Services, Inc. Reagents and methods for use in cancer diagnosis, classification and therapy
US20110003709A1 (en) * 2003-08-11 2011-01-06 Ring Brian Z Reagents and methods for use in cancer diagnosis, classification and therapy
US7811774B2 (en) 2003-08-11 2010-10-12 Applied Genomics, Inc. Reagents and methods for use in cancer diagnosis, classification and therapy
US20050112622A1 (en) * 2003-08-11 2005-05-26 Ring Brian Z. Reagents and methods for use in cancer diagnosis, classification and therapy
US8440410B2 (en) 2003-08-11 2013-05-14 Clarient Diagnostic Services, Inc. Reagents and methods for use in cancer diagnosis, classification and therapy
US20060019290A1 (en) * 2004-07-09 2006-01-26 Godfrey Tony E Identification of markers in esophageal cancer, colon cancer, head and neck cancer, and melanoma
US20100240037A1 (en) * 2004-07-09 2010-09-23 Godfrey Tony E Identification of markers in esophageal cancer, colon cancer, head and neck cancer, and melanoma
US7662561B2 (en) * 2004-07-09 2010-02-16 University Of Pittsburgh-Of The Commonwealth System Of Higher Education Identification of markers in esophageal cancer, colon cancer, head and neck cancer, and melanoma
US20080131916A1 (en) * 2004-08-10 2008-06-05 Ring Brian Z Reagents and Methods For Use In Cancer Diagnosis, Classification and Therapy
US20060068433A1 (en) * 2004-09-20 2006-03-30 Godfrey Tony E Multiple mode multiplex reaction quenching method
US20070218496A1 (en) * 2006-03-11 2007-09-20 Roche Molecular Systems, Inc. Method for quick determination of cytokeratin 19 (ck19) and primers and probes therefore
CN101054603B (zh) * 2006-04-11 2011-03-30 霍夫曼-拉罗奇有限公司 快速测定细胞角蛋白19(ck19)的方法和其引物及探针
EP1845167A1 (fr) * 2006-04-11 2007-10-17 Roche Diagnostics GmbH Procédé de détermination rapide de cytokératine 19 (CK19) et les amorces et les sondes correspondantes
US8119352B2 (en) 2006-06-20 2012-02-21 Cepheld Multi-stage amplification reactions by control of sequence replication times
US20080102495A1 (en) * 2006-06-20 2008-05-01 Lynn Kozma Multi-stage amplification reactions by control of sequence replication times
EP1876245A1 (fr) * 2006-07-03 2008-01-09 Sysmex Corporation Procédé et appareil pour déterminer la taille des métastases
US20080096204A1 (en) * 2006-07-03 2008-04-24 Sysmex Corporation Method and apparatus for determining cancer metastasis
KR101403803B1 (ko) * 2006-07-03 2014-06-03 시스멕스 가부시키가이샤 암 전이의 판정 방법 및 장치
US20100184034A1 (en) * 2006-11-13 2010-07-22 SOURCE PRECISION MEDICINE, INC d/b/a SOURCE MDX Gene Expression Profiling for Identification, Monitoring and Treatment of Lung Cancer
EP2087140A2 (fr) * 2006-11-13 2009-08-12 Source Precision Medicine, Inc. Détermination du profil de l'expression génique dans l'identification, la surveillance et le traitement du cancer du poumon
WO2008063413A3 (fr) * 2006-11-13 2008-09-04 Source Precision Medicine Inc Détermination du profil de l'expression génique dans l'identification, la surveillance et le traitement du cancer du poumon
US9151762B2 (en) 2009-06-12 2015-10-06 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Identification of DSG-3 as a biomarker for the detection of metastasis in lymph nodes
US20130231578A1 (en) * 2012-03-02 2013-09-05 Nihon Kohden Corporation Electrocardiogram analysis report, electrocardiogram analysis apparatus, and electrocardiogram analysis program
WO2023081988A1 (fr) * 2021-11-09 2023-05-19 Coelho Guilherme Portela Méthode de prédiction d'efficacité d'un traitement contre le cancer du sein

Also Published As

Publication number Publication date
US20110312515A1 (en) 2011-12-22
JP2008510454A (ja) 2008-04-10
AU2005271960B2 (en) 2011-12-08
EP1774030A4 (fr) 2008-07-09
US20060019290A1 (en) 2006-01-26
EP2275578A3 (fr) 2011-11-30
EP1774030A2 (fr) 2007-04-18
AU2005271960A1 (en) 2006-02-16
AU2005271961A1 (en) 2006-02-16
EP2287329A2 (fr) 2011-02-23
EP2272987A3 (fr) 2012-04-11
CA2571642A1 (fr) 2006-02-16
WO2006017151A2 (fr) 2006-02-16
EP1774025A4 (fr) 2009-09-16
EP2468889A3 (fr) 2012-07-11
US7662561B2 (en) 2010-02-16
EP2287329A3 (fr) 2011-08-03
US20100240037A1 (en) 2010-09-23
JP2012005500A (ja) 2012-01-12
CA2571650A1 (fr) 2006-02-16
AU2005271961B2 (en) 2011-10-13
EP2468889A2 (fr) 2012-06-27
EP2275578A2 (fr) 2011-01-19
EP2272987A2 (fr) 2011-01-12
WO2006017151A3 (fr) 2007-01-11
JP2008505644A (ja) 2008-02-28
WO2006017150A3 (fr) 2009-04-09
EP1774025A2 (fr) 2007-04-18
WO2006017150A2 (fr) 2006-02-16
AU2005271961B8 (en) 2011-11-24

Similar Documents

Publication Publication Date Title
AU2005271960B2 (en) Identification of markers in lung and breast cancer
US20090226881A1 (en) Multiple mode multiplex reaction quenching method
AU2005250479B2 (en) Diagnosing or predicting the course of breast cancer
WO2002070751A1 (fr) Procede de reaction en chaine de la polymerase
AU2002247248A1 (en) PCR method
CN111394457B (zh) 生物标志物的应用、检测肾透明细胞癌的试剂及其试剂盒
KR20140125647A (ko) 조기 유방암 예후 예측 진단용 자동화 시스템
EP2496710B1 (fr) Détermination du gain 17q dans le neuroblastome de patients par l'analyse de l'adn circulant
CA2709106C (fr) Procedes et reactifs pour la detection hative de melanomes
AU2011253566A1 (en) Identification of markers in esophageal cancer, colon cancer, head and neck cancer and melanoma

Legal Events

Date Code Title Description
AS Assignment

Owner name: UNIVERSITY OF PITTSBURGH-OF THE COMMONWEALTH SYSTE

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:GODFREY, TONY E.;XI, LIQIANG;RAJA, SIVA;AND OTHERS;REEL/FRAME:017031/0844;SIGNING DATES FROM 20050815 TO 20050830

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION