Rapid efficacy assessment method for lung cancer therapy

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.

1 . A method for cancer relapse detection which comprises:
 (a) collecting a blood or pleural effusion sample from a human subject;   (b) extracting total RNA from 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 T ) 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 T   (GAPDH)  for the control gene transcript of said total RNA from said sample;   (e) calculating a differential expression ratio Q j  for each gene transcript of j in said panel of gene transcripts of said total RNA according to   
       Q j =2 (ΔC     T     )     j     -(ΔC     T     )     j     ,mean  
 wherein (ΔC T ) j =C T   (GAPDH) −(C T ) j , and
 (ΔC T ) j, mean  is a predetermined mean of (ΔC T ) j  for gene transcript of j over a population of persons not inflicted with lung cancer; 
 
 (f) calculating a normalized expression ratio E j  for each gene transcript of j in said panel of gene transcripts of said total RNA according to
     E   j =( Q   j   −Q   j,mean )/σ j    
 
 wherein Q j, mean  is a predetermined mean of differential expression ratio for gene transcript of j over a population of lung cancer patients and σ 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=ΣE j , where the summation is over all the gene transcripts in said panel of gene transcripts; 
 (h) comparing said Lc from step (g) with a reference load of cancer cells to detect if cancer relapse has occurred in said human subject, wherein said Lc from step (g) higher than said reference load of cancer cells indicates that circulating cancer cells are present in said human subject. 
 
     
     
         2 . A reagent kit for assessment of lung cancer therapy and relapse detection, comprising a panel of PCR primers and probes for the multiple gene transcripts used in real-time quantitative PCR and a scoring method. 
     
     
         3 . A reagent kit of  claim 2 , wherein said multiple gene transcripts are nucleic acid sequences. 
     
     
         4 . A reagent kit of  claim 2 , wherein said nucleic acid sequences are selected from  Homo sapiens  keratin 19 (KRT19), ubiquitin thiolesterase, tripartite motif-containing 28 (TRIM28) and gene highly similar to HSFIB1 for fibronectin.