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  Pharmaceutical Patents  

 

Title:  Rapid efficacy assessment method for lung cancer therapy
United States Patent: 
7,507,534
Issued: 
March 24, 2009

Inventors:
 Peck; Konan (Taipei, TW), Sher; Yuh-Pyng (Taipei, TW), Shih; Jin-Yuan (Taipei, TW), Yang; Pan-Chyr (Taipei, TW), Wu; Cheng-Wen (Miaoli County, TW)
Assignee: 
National Health Research Institutes (Miaoli County, TW)
Appl. No.: 
11/306,532
Filed: 
December 31, 2005


 

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Abstract

The present invention discloses a method for rapid assessment of lung cancer therapy efficacy in a few days instead of weeks by conventional imaging methods. This method can also be used to detect relapse of the cancer and to improve the current TNM cancer staging method for more accurate prognosis. The rapid assessment of therapy efficacy is based on detecting circulating cancer cells in body fluid with high positive detection rate. The high positive detection rate is achieved by using qPCR amplification of multiple marker genes identified by in silico search of DNA sequence database. This invention also discloses a scoring method to calculate the cancer cell load based on qPCR results to correlate the amount of circulating cancer cells in lung cancer patients and predict the treatment outcomes.

Description of the Invention

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to methods for assessment of carcinoma cancer therapy and relapse detection, and more specifically it relates to an efficacy assessment method for lung cancer therapy to rapidly predict the outcome of lung cancer therapy so treatment with higher likelihood of success can be selected to prevent invalid treatment from wrecking patents, and a routine monitoring method for cancer relapse after the treatment.

2. Description of the Related Art

Lung cancer is the leading cause of cancer-related death and non-small cell lung cancer (NSCLC) accounts for .about.80% of the cases. Attempts to use serum protein markers for the early diagnosis of lung cancer have not yielded satisfactory results for routine screening, and newly developed early diagnostic methods using serum DNA as a diagnostic marker await further validation. Current therapeutic measures remain unable to lower the mortality rate of late-stage lung cancer patients. Surgical resection is still the best cure for the early-stage patients. The tumor, node, metastasis (TNM) classification has been used for cancer staging and prognosis for decades. A large portion of early-stage patients, defined by the current staging system and available imaging modalities, still develop distant metastases although they received surgical removal of the tumor mass. The inability to detect disseminated tumor cells with the current imaging techniques is a major obstacle to accurate cancer staging.

NSCLC is heterogeneous with respect to histology and biological characteristics. Individual NSCLC cells within a tumor and in different patients' tumors express different amounts of marker gene transcripts. The heterogeneity of marker gene expression levels in NSCLC cells limits the reliability of an assay method with a single-marker detection scheme. Several literature reports have described PCR methods for the detection of tumor cells dispersed in the circulation. However, not one tumor marker is consistently and specifically expressed in all of the primary tumors of a particular malignancy. Literature reports have also shown that a panel of marker genes provides a more reliable and informative approach than a single-marker assay for the detection of melanoma and breast cancer cells in blood. Such assays for lung cancer have been limited by the availability of molecular markers.

The presence of epithelial cancer cells in the bone marrow and in the peripheral blood of patients with carcinoma has been reported in literature reports and prior arts. In contrast to bone marrow aspirates, peripheral blood samples can be obtained routinely and more readily. Carcinoma accounts for around 85% of human cancers and the carcinoma cells are of epithelial cell lineage. Techniques such as immunocytology and flow cytometry have been employed in prior arts to detect circulating cancer cells in the peripheral blood. However, both techniques are based on extracting or labeling intact carcinoma cells in circulation by antibodies targeting specifically to the epithelial cell surface antigens such as EpCAM and others. Malignant carcinoma cancer cells often are de-differentiated and lose the characteristic epithelial cell surface antigens. In addition, it is known in cancer research field that EpCAM gene expression is often suppressed to facilitate tumor metastasis. Therefore, the antibody based detection methods have been reported to have low positive detection rates or high false negative rates. Polymerase chain reaction (PCR) has been employed to detect disseminated tumor cells in peripheral blood. Several literature reports have described the use of PCR for detecting circulating cancer cells in the peripheral blood of patients of various cancers. For instance, Peck et al., reported the use of cytokeratin 19 as the maker gene for detecting circulating cancer cells in NSCLC patients with an overall positive detection rate around 40%.

Compared with immunocytology and flow cytometry, PCR has the advantages that it is more readily available, less involved in the operating procedures, less instrument cost, and others. On the other hand, PCR is not able to yield the number of counts of circulating cancer cell in a sample like the other two techniques.

To overcome the current technology difficulties in achieving high positive detection rate and rapid assessment of lung cancer therapy efficacy and relapse detection, a panel of marker genes for achieving high positive detection rate by qPCR and a quantitative analysis method for predicting lung cancer treatment outcome and for prognosis are needed.

SUMMARY OF THE INVENTION

The present invention fulfills the needs in lung cancer treatment by teaching a rapid efficacy assessment method for lung cancer therapy and relapse detection.

The purpose of the present invention is to teach an assessment method for lung cancer therapy. More especially, it teaches a rapid efficacy assessment method for lung cancer therapy by identifying and employing a panel of marker genes for real-time quantitative PCR (qPCR) assay to quantitatively measure the amount of circulating lung cancer cells in body fluids.

Another purpose of the present method is to teach a method for cancer relapse detection by using real time qPCR with a panel of marker genes for detecting circulating lung cancer cells in body fluids.

The present invention identifies a panel of markers for the detection of circulating cancer cells in NSCLC patients by in silico analysis of the National Cancer Institute-Cancer Genome Anatomy Project database. The present invention also teaches a quantitative analysis method to calculate load of cancer cells in the circulation. The quantitative analysis method yields results that are highly correlated with the treatment outcomes of lung cancer patients and serves to predict the treatment outcome in a short time after the treatment is administered.

The method of assessing lung cancer therapy comprises: collecting a body fluid from a subject, extracting total RNA of the body fluid sample, employing qPCR to amplify marker gene transcripts of total RNA for detecting cancer cells in body fluid, and analyzing qPCR threshold cycle number with a set of mathematical formulae.

The present invention further teaches a method to translate expression level of multiple gene transcripts measured by qPCR to the amount of circulating lung cancer cells which is termed cancer cell load (Lc) in this invention.

The present invention further teaches a scoring method and mathematical formulae for calculating cancer cell load, Lc, and predicting lung cancer treatment outcome with the Lc value.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method for lung cancer therapy assessment and a method of cancer relapse detection. Unlike current imaging assessment methods, the present invention provides a rapid assessment that uses multiple marker genes in qPCR assay for detecting circulating lung cancer cells in body fluids.

Compared with immunohistology and flow cytometry assessment methods, the present invention requires no additional antibody antigen interaction process. Extra molecular recognition process reduces the detection rate. Compared with the RT-PCR detection method for circulating cancer cell in prior art, using a panel of marker genes instead of a single marker gene improves the detection rate. On the other hand, it is not trivial to quantitatively analyze and integrate the expression level of multiple transcripts in a qPCR assay and correlating the analysis results to predict treatment outcome. The present invention teaches a set of mathematical formulae which yield results well correlated with lung cancer treatment outcome.

The multiple marker genes that may be selected include, but are not limited to, keratin 19 (KRT19), ubiquitin thiolesterase (UCHL1), Highly similar to HSFIB1 for fibronectin, and tripartite motif-containing 28 (TRIM28).

The method of the present invention for rapid lung cancer therapy efficacy assessment comprises the following steps:

collecting a body fluid sample from a human subject;

extracting total RNA from said body fluid sample;

amplifying a panel of gene transcripts of said total RNA by qPCR;

measuring the expression level of each gene transcript in said panel of gene transcripts of said total RNA from a number of healthy controls and lung cancer patients, wherein a qPCR threshold cycle number is used to represent the expression level of the gene transcripts;

deriving a reference threshold score using the qPCR threshold cycle numbers of the gene transcripts in the panel measured for healthy controls and lung cancer patients;

calculating an indicative score using the qPCR threshold cycle numbers of the gene transcripts in the panel measured for a lung cancer patient both before and after therapy; and

determining therapy efficacy by comparing the indicative scores obtained before and after therapy.

The formulae used in this invention to calculate the values in these steps can be found in the section "Quantitative Analysis (Scoring) of the PCR Results" below.

In the present invention, the body fluids are collected from, but not limit to, peripheral blood or pleural effusion.

In the present invention, the number of multiple gene transcripts selected for amplification is more than two.

In the present invention, the nucleic acid extraction is done without prior antibody/antigen interaction or other molecular recognition processes to isolate cancer cells from normal blood cells.

In the present invention, the quantitative analysis for therapy efficacy is performed as early as one day after the therapeutic regimen is administered.

The method of the present invention for cancer relapse detection comprises the following steps:

collecting a body fluid sample from a human subject;

extracting total RNA from said body fluid sample;

amplifying a panel of gene transcripts of said total RNA by qPCR;

measuring the expression level of each gene transcript in said panel of gene transcripts of said total RNA from a number of healthy controls and lung cancer patients, wherein a qPCR threshold cycle number is used to represent the expression level of the gene transcripts;

deriving a reference threshold score using the qPCR threshold cycle numbers of the gene transcripts in the panel measured for healthy controls and lung cancer patients;

calculating an indicative score using the qPCR threshold cycle numbers of the gene transcripts in the panel measured for a lung cancer patient; and

determining the presence of circulating lung cancer cells by comparing the indicative scores with the reference threshold score.
 

Claim 1 of 2 Claims

1. A method for lung cancer therapy assessment comprising: (a) collecting a blood or pleural effusion sample from a human subject with lung cancer; (b) extracting total RNA of said sample; (c) amplifying a panel of gene transcripts of said total RNA by qPCR, wherein said panel of gene transcripts, named j, comprises keratin 19 (KRT19), ubiquitin thiolesterase (UCHL1), tripartite motif-containing 28 (TRIM28), and Highly similar to HSFIB1 for fibronectin; (d) measuring a qPCR threshold cycle number (C.sub.T).sub.j for each gene transcript of j in said panel of gene transcripts of said total RNA from said sample, and a qPCR threshold cycle number C.sub.T.sup.(GAPDH) for the control gene transcript of said total RNA from said sample; (e) calculating a differential expression ratio Q.sub.j for each gene transcript of j in said panel of gene transcripts of said total RNA according to Q.sub.j=2.sup.(.DELTA.C.sup.T.sup.).sup.j.sup.-(.DELTA.C.sup.T.sup.).sup.- j,mean wherein (.DELTA.C.sub.T).sub.j=C.sub.T.sup.(GAPDH)-(C.sub.T).sub.j, and (.DELTA.C.sub.T).sub.j, mean is a predetermined mean of (.DELTA.C.sub.T).sub.j for gene transcript of j over a population of persons not inflicted with lung cancer; (f) calculating a normalized expression ratio E.sub.j for each gene transcript of j in said panel of gene transcripts of said total RNA according to E.sub.j=(Q.sub.j-Q.sub.j,mean)/.sigma..sub.j wherein Q.sub.j, mean is a predetermined mean of differential expression ratio for gene transcript of j over a population of lung cancer patients and .sigma..sub.j is a predetermined standard deviation of differential expression ratio for gene transcript of j over said population of lung cancer patients; (g) calculating a load of cancer cells (Lc) according to Lc=.SIGMA.E.sub.j, where the summation is over all the gene transcripts in said panel of gene transcripts; (h) administering a therapy for lung cancer to said human subject with lung cancer; (i) performing steps (a) to (g) to yield a load of cancer cells (Lc) for said human subject after said therapy for lung cancer; and (j) comparing said Lc before and after administering said therapy, wherein the therapy is determined to be effective if Lc after the therapy is less than a predetermined value, or is determined to be not effective if Lc after the therapy is not less than the predetermined value.
 

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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