CUTOFF POINT DELTA CT METHOD FOR GENETIC PCR TESTING IN HUMAN CANCER

Abstract

The present invention is related to an improved method for HER2 gene test by using quantitative real-time PCR (Polymerase Chain Reaction) technique. Our invention streamlines test process, and incorporates quality control for each major step, including sample, reagent, operation, and data report. We eliminate the need for reference genes which is hard to standardize in HER2 PCR test. We develop a cutoff reference point by using the statistical mean of tumor tissue population, and adopt a simplified scoring scheme for evaluation of HER2 status. Our invention produces consistent result across machines and labs, and has proven to be clinically successful in HER2 test.

Claims

1. A streamlined and standardized method for determining the status of a particular tumor-associated gene whose mRNA expression fits a normal distribution measured by real-time PCR technique, a simplified scoring system without use of reference genes and comprising the steps of: (a) preparing a specimen sample with suspected invasive cancer content; (b) extracting total RNA from the specimen sample and measuring its concentration; (c) running reverse transcription and PCR with a constant amount of total RNA for cDNA input along with synthetic oligos (sDNA) as a reference material having at least one embedded cutoff reference point (CRP) and without using a reference gene and wherein the CRP is defined in terms of a specify copy number of gene's mRNA; and (d) analyzing the mRNA output of the PCR analysis by applying the dynamic and normalized cutoff reference point reading to score the result as positive or negative.

2. The method of claim 1, wherein the specimen sample can be flexible, depending on its availability and wherein the specimen sample can be freshly-frozen tissue (FF), freshly frozen optimal-cutting-temperature-compound tissue (FF OCT), or formalin-fixed paraffin-embedded tissue (FFPE) and wherein the specimen sample is cut at 10 to 25 mg for FF tissue and ten 10-μm in 5 to 10 sections for FF OCT or FFPE tissue.

3. The method of claim 1, wherein different sources of specimen samples can be tested on the same PCR plate.

4. The method of claim 1, wherein sample RNA input to each reaction is constant in volume (i.e., 4 μl) and wherein the assays are equivalent if the sample RNA concentration is maintained (i.e., 2 ng/μl of reaction) after changing reaction volume.

5. The method of claim 1, wherein sDNA is prepared at various concentrations to cover the full range of mRNA expression for different sources of tumor samples and wherein the assays are equivalent if sDNA concentration is maintained in each standard after changing reaction volume.

6. The method of claim 1, wherein the cutoff reference point is the statistical mean and standard deviations of the normal specimen tissue mRNA expression.

7. The method of claim 1, wherein a dual RNA control system is implemented for scoring the result as positive or negative, to provide quality check for potential false reading arising from technical defects in reagents and testing procedures and wherein control RNAs are prepared from breast cancer cell lines.

8. The method of claim 1, wherein the PCR test results of step (d) will be scored in terms of Ct, threshold either automatically set by the PCR instrument at which first detects fluorescence above background noise or manually set at a value that renders the reading of a sDNA standard (i.e., 0.002 pg/μl) equivalent to CRP.

9. The method of claim 1, wherein the Ct. of test result of step (d) is adjusted for experimental variance and for cutoff reference check with the following equations:
cΔCt=Ct(CRP)−Ct(Unknown Sample); if cΔCt>1, then the test result is positive; else, the test result is negative and wherein CRP can be established by using different pairs of primers and probes that are able to specifically amplify and detect the relevant gene mRNA molecules.

10. The method of claim 1, wherein the gene status by PCR test of step (d) is validated with FISH companion test and wherein the valid test results are ranged between 85% and 95% in agreement, which will depend on sample forms and tumor biological heterogeneity tested and wherein, if adjacent tumor slices from the same tumor block are used for RT-PCR and for FISH, the agreement between two test results will be the highest.

11. The method of claim 1, wherein the cancer tumor is a breast cancer and the gene can be any human cancer-associated marker whose mRNA expression fits a normal distribution, such as EGFR, ERa, BRAC1 and BRAC2, and the status of the gene, as either over-expressed or under-expressed, can be determined by CRP and cΔCt scoring.

12. The method of claim 1, wherein the cancer tumor can be any cancers where gene expression needs to be tested for treatment decision making and wherein the gene's mRNA expression is a normal distribution statistically; whereby the CRP concept is applicable to gene test in other cancers.

13. The method of claim 1, wherein the status of HER2 or other cancer-associated genes can be tested by using the circulating tumor cell mRNA obtained from blood sample and wherein the Cutoff Reference Point for mRNA expression can be set at a value corresponding to the level determined by a previously established method, such as Enzyme-linked Immunosorbent Assay.

14. A streamlined and standardized method for determining the HER2 status of a tumor sample by measuring HER2 mRNA expression level using quantitative real-time PCR technique without use of reference genes, comprising the steps of: (a) preparing a specimen sample with invasive cancer content for HER2 test; (b) extracting total RNA from the specimen sample and measuring its concentration; (c) running reverse transcription and PCR with a constant amount of total RNA for cDNA input along with synthetic oligos (sDNA) as a reference material having at least one embedded cutoff reference point (CRP) and without using a reference gene and wherein the CRP is defined in terms of a specify copy number of HER2 mRNA and wherein sDNA is prepared at various concentrations to cover the full range of HER2 mRNA expression levels for multiple types of tumor tissues and wherein the volume of sDNA added to reaction can be changed accordingly if a different reaction volume is used and provided assays remain equivalent; and (d) analyzing the mRNA output of the PCR analysis by applying the dynamic and normalized cutoff reference point reading to score the result as HER2 positive or HER2 negative.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 is an example plate setup format.

[0017] FIG. 2 is a flowchart of an example HER2 PCR testing procedure illustrating standardization of assay process and data report.

DETAILED DESCRIPTION

[0018] Method

[0019] Sample Collection—

[0020] Immediately after biopsy or surgery, the tumor specimen can be preserved in formalin-fixed paraffin embedded (FFPE), freshly frozen in liquid nitrogen or in optimal-cutting temperature compound (OCT).

[0021] RNA Extraction—

[0022] The total RNA is extracted from 25 mg of frozen sample or from ten 10-μm sections by using a commercial RNA Isolation Kit. The total RNA concentration was determined by a spectrophotometer or fluorometer.

[0023] Reagent Content—

[0024] Reagents are stored at −20° C. in two groups. HER2 sDNA is a synthetic single-stranded oligo nucleotide, and has a molecular weight of 22570.6. Control RNA is prepared from breast cancer cell lines.

TABLE-US-00001 Pack A: Pack B: RT Mix (650 μl) Her-2 sDNA (0.2 pg/μl, 50 μl) PCR Mix (4400 μl) Her-2 sDNA (0.02 pg/μl, 50 μl) RT Enzyme (200 U/μl, 12 μl) Her-2 sDNA (0.002 pg/μl, 50 μl) PCR Enzyme (5U/μ, 25 μl) Her-2 sDNA (0.0002 pg/μl, 50 μl) RNase/DNase-free water (500 μl) Her-2 sDNA (0.00002 pg/μl, 50 μl) tRNA solution (0.1 mg/ml, 50 μl) Control RNA (80 μl, at 25 ng/μl)

[0025] Assay and Plate Design—

[0026] A HER2 test contains the reactions: i) sDNA solutions and one NTC in duplicate for generation of Standard Curve; ii) Unknown sample RNA in triplicate, plus one No RT well for each sample; iii) Control RNA in triplicate plus one No RT well. Refer to the User Manual of real-time PCR machine for the procedures of assigning plate wells for the above reactions. Choose ‘FAM’ for Collect fluorescence data and ‘ROX’ as Reference dye. See FIG. 1 for an example of plate setup.

[0027] Running RT and PCR—

[0028] Real-time PCR assay consists of two consecutive steps, i.e., reverse transcription (RT) and polymerase chain reaction (PCR). In RT process, mRNA is converted to a single stranded cDNA which is then amplified with a specific pair of primers in PCR process. The quantity of amplified DNA fragments is monitored with a fluorescence-labeled oligo probe in each PCR cycle. The C.sub.t value is recorded for each amplification curve, and is used for calculation of gene expression level in sample. Following are detailed steps. [0029] 1. Take out RT Mix, RT enzyme, tRNA, and water from Pack A, and thaw them on ice; meanwhile, take out sDNA solutions from Pack B and put them on a separate ice bath. Invert each vial several times after thawing reagent. [0030] 2. Determine the number of reactions for your assay and calculate the quantity of RT Mix and RT Enzyme. For each reaction, including Standard, Unknown, NTC and No RT, you need 5.9 μl RT Mix and 0.1 μl RT Enzyme, but in No RT control, add 0.1 μl water to replace RT Enzyme. Mix solution several times with pipet. Do not vortex. [0031] 3. Load 6 μl of the above solution to each well uniformly. It is not necessary to change tip after each loading. [0032] 4. Determine RNA concentration for each sample and adjust to 25 ng/μ1. Add 4 μl of RNA sample to each Unknown or No RT well, the total RNA in each reaction will be 100 ng; meanwhile, add 4 μl of Control RNA to the control well. [0033] 5. Add 4 μl of sDNA solution to each corresponding Standard well. In NTC well, add 4 μl of tRNA to replace sDNA. The total volume of each RT reaction is 10 μl. [0034] 6. Seal PCR plate tightly with membrane across plate edges and around each well. Put the plate into real-time PCR machine and perform RT reaction using a thermal profile: 50° C. for 30 min, and 72° C. for 5 min, then cool down to 20° C. or room temperature. [0035] 7. When running RT, take out PCR Mix and PCR Enzyme from Pack A. Thaw the reagents on ice, and re-suspend the reagents by inverting vials several times. [0036] 8. Calculate the quantity of PCR Mix and PCR Enzyme needed for your assay. For each PCR reaction, you need 39.8 μl of PCR Mix and 0.2 μl of PCR Enzyme. Add appropriate amount of PCR enzyme to PCR Mix, and pipet solution up and down several times. Do not vertex solution. [0037] 9. Load 40 μl of above solution to each well. Seal PCR plate again with membrane and ensure that the edges and wells are tightly sealed. The total volume of solution in each well will be 50 μl. [0038] 10. Put the plate back to a real-time PCR machine and set up thermal profile to: 95° C. for 1 min/40 cycles of 95° C. for 12 s/55° C. for 1 min/72° C. for 30 s. [0039] 11. Collect data after running, and trash the plate in a different location to avoid potential contamination to the future assays.

[0040] Data Analysis and Test Requirements—

[0041] Our method offers a tool for accurate measurement of HER2 expression level in breast cancer cells, as well as a tool for identification of cancer patients who might benefit from Herceptin treatment. Tests of breast tumor samples lead to establishment of a cutoff reference point for HER2 positive tumors. Following is detailed discussion on obtaining high quality test results. [0042] 1. Threshold setup. Threshold is defined as a fluorescence intensity level, above which the fluorescence signal is considered to be significantly higher than the background. Software provided with real-time machine setup a threshold following each PCR run for calculation of sample C.sub.t; however, the threshold may vary in different assays. Our tests indicate that sDNA at 0.002 ng/μl has an average C.sub.t of 21.65 (Table 1); accordingly, setup of a threshold to yield a C.sub.t value of 21.65 for 0.002 sDNA will produce consistent results across assays. [0043] 2. Validation of standard curve. Standard Curve is a plot of the initial template quantity in the standard wells (X-axis) versus the C.sub.t (threshold cycle). Usually, a least mean square curve fitting algorithm is adopted to generate a standard curve. R.sup.2 is a key parameter used to evaluate the quality of a standard curve. The highest value of R.sup.2 is 1. Standard curve that has a R.sup.2 value higher than 0.95 is considered valid. [0044] 3. Amplification Efficiency. Amplification Efficiency measures the percentage of the template molecules which are doubled every cycle, and is a comprehensive parameter that reflects the overall quality of assay reagents and conditions. In a real-time PCR, amplification efficiency is estimated from standard curve using the equation: Efficiency=10.sup.(−1/slope)−1. A valid assay should have an Amplification Efficiency above 95%. [0045] 4. Replicate Variation. Replicates are wells which contain the same RNA sample, and, theoretically, should have the same C.sub.t value. Therefore, variation among the replicates would represent the errors of assay operations. A valid test should have a replicate variation of less than 0.5 C.sub.t units. [0046] 5. False Positive. If a sample contains normal level of HER2 mRNA but displays a significantly low C.sub.t, this phenomenon is termed as ‘False Positive’. A major cause of false positive sample is DNA contamination, which may come from many different sources, such as reagents and amplified products from neighboring wells or previous assay plates. A “No Template Control” (NTC) is included in the test for each sample, and should have no amplification signal or no C.sub.t under normal conditions. [0047] 6. False Negative. If a sample contains an over-expression level of HER2 mRNA but displays a normal or even higher C.sub.t, this phenomenon is termed as ‘False Negative’. The sample quality is a major cause of false negative results. For example, the presence of PCR inhibitors or RNA degradation can lead to high shift of C.sub.t value. Our tests indicate that effects of both PCR inhibitors and RNA degradation are highly sensitive to sample dilution. If a sample at two different concentrations shows consistent result, then, it is considered having acceptable quality. [0048] 7. Control RNA. Test also incorporates control RNAs which are isolated from breast cancer cell lines. These RNAs are used as true controls for HER2 positive and HER2 negative. Inclusion of these RNAs also monitors reagent quality and assay operation. Under standard test conditions, control RNAs show constant delta C.sub.t value (ΔC.sub.t) against cutoff reference point. [0049] 8. Cutoff Reference Point. Prior to routine HER2 PCR testing, a HER2 cutoff reference point(s) (CRP) must be established in order to compute the cΔC.sub.t which is equal to CRP−C.sub.t (Unknown Sample); if cΔC.sub.t>1, the test result is HER2 positive; else, negative. CRP is derived from the statistical mean of a breast tumor tissue population, and is monitored by a synthetic DNA (sDNA) in each test.

[0050] Validation with FISH Test—

[0051] HER2 gene amplification was determined by the FDA-approved Pathway Her2 DNA Probe Kit (Vysis/Abbott, USA). According to the manufacturer's instruction, HER2 gene was considered non-amplified if an average Her2 (red) to CEP17 (green) ratio was <2.0, and low amplification between 2.0 and 4.0, and highly amplified if the ratio was >4.0. For comparison purpose, the samples with a ratio of >2.0 were classified as HER2 positive; otherwise, as HER2 negative. FISH tests were conducted either at NCKU Pathology Lab or by an outsourced reference lab. FISH test were done selectively (mostly for IHC 2+ samples), not for all the samples.

Examples

[0052] Testing Procedure Flow Chart—FIG. 2 is included to demonstrate the essence of the quality assurance and result validation before and after the PCR run with the invented cutoff point method.

[0053] Test Data and Result Analysis—Table 1 shows establishment of HER2 standard curve baseline and calculation of cΔC.sub.t, for the determination of HER2 expression status in breast tissue samples. As detailed in ‘Claim 10’, if cΔC.sub.t is greater than 1, then HER2 test is positive; otherwise, the HER2 test will be negative. The cutoff reference point (CRP) for the tissues preserved in optimal-cutting temperature compound (OCT) is 21.65, for the tissues in formalin-fixed paraffin embedded (FFPE) is 25.13. Tissue samples from sources, such as OCT vs. FFPR, can be tested in the same PCR run.

[0054] Distribution of HER2 mRNA Expression in Breast Tissues—Table 2 shows that HER2 mRNA gene expression in both tumor and normal tissues exhibits a normal distribution, regardless the tissue processing methods. Analysis also shows that HER2 expression in the tumor in normal tissues belongs to different populations. Showing a normal distribution of HER2 mRNA expression offers a statistical basis for establishment of cutoff reference point (CRP) for HER2 PCR testing.

[0055] Establishment of Cutoff Reference Point (CRP) by using The Statistical Mean of Breast Tumor HER2 mRNA expression level. Table 3 shows adoption of a C.sub.t value as cutoff reference point that is around the statistical mean of HER2 mRNA expression level in the tumor samples and is equivalent the statistical mean of normal tissue minus 3.5 standard deviations. Given that the statistical mean varies by sample sources, such as OCT vs. FFPE, different CRP is used for different tissue sources accordingly. Table 3 also shows that CRP may be monitored by a defined quantity of sDNA (a synthetic single-stranded DNA) in each test for easy calculation of cΔCt in a real-time manner, which is further described in ‘Claim 10’.

[0056] Test Result Validation, PCR vs. FISH—Table 4 shows the detail test results of the invented method and FISH method on different types and sources of breast tumor samples. Table 5 is included to summarize the agreement score between the invented method and the existing FISH method. Given that the sample acquisition in this study is random and non-consecutive slicing between the PCR test and the FISH test for the same patient, the agreement score of 90% reported here is conservative.

[0057] With Reference to FIG. 1, it will be noted that ‘Standard’ is the well containing different amount of Her-2 sDNA for generation of Standard Curve; ‘Unknown’ is the well that contains sample RNA, and each sample is run in triplicate; ‘NTC’ is a control which contains all the reaction components except sDNA; ‘No RT’ is a control which contains RNA and all other reaction components, but no RT enzyme; ‘Control RNA’ is included as control for the quality of both RT and PCR reactions.

[0058] It is also established that the foregoing disclosed methods and protocols, which omit the use of a reference gene, may be utilized to determine the expression of genes other than HER2 that also possess a normal mRNA distribution to provide a simplified scoring system to confirm the status of genes in human cancer.