Molecular diagnostic and computerized decision support system for selecting the optimum treatment for human cancer

• US 20030143572A1
• Filed: 08/09/2002
• Published: 07/31/2003
• Est. Priority Date: 08/13/2001
• Status: Abandoned Application

• Current Assignee: Genetics Development Corporation
• Original Assignee: Genetics Development Corporation
• Inventors: Yu, Rong, Lu, Mou-Ying Fu
• Application Number:
  US10/216,509
• Publication Number:
  US 20030143572A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/20 Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation
• CPC Class Codes: G16B 50/00 ICT programming tools or database systems specially adapted for bioinformatics
• CPC Class Codes: G16H 20/10 relating to drugs or medications, e.g. for ensuring correct administration to patients
• CPC Class Codes: G16H 50/20 for computer-aided diagnosis, e.g. based on medical expert systems

Abstract

A computerized decision support system and method for predicting which of one or more drugs suitable to treat a cancerous condition in a patient are the optimum drug(s), where such selection is based upon the particular patient'"'"'s genotype. A PCR kit and/or a gene chip detects multiple genes, expressions and/or mutations associated with a particular cancer using a sample of the patient'"'"'s tissue or blood. A detector accepts the gene chip and analyzes the patient'"'"'s genotype; and a computerized system using a database which associates patient genotypes and the efficacy and toxicity of various anti-cancer drugs used in treating patients with a particular cancerous condition connected to the detector correlates the output of the detector to the database to provide a recommendation as to which drugs are optimum for treating the patient'"'"'s cancer.

Claims

1. A computerized decision support system and apparatus for selecting the optimum treatment for a cancerous condition in a human patient, the apparatus comprising:

a PCR kit and/or a gene chip designed to detect, expressions and/or mutations of multiple genes associated with a particular cancer, using patient'"'"'s tissue or blood samples;

an integrated detector for analyzing both PCR and gene chip results.

a detector for accepting receipt of the gene chip toward analyzing the patient'"'"'s genotype;

a database describing the correlation of patient genotypes and the efficacy and toxicity of various anti-cancer drugs used in treating patients with a particular cancerous condition; and

a computerized decision support system operably connected to the detector for correlating the output of the detector to the database;

whereby the operator is provided with a definitive recommendation as to which drug or drugs are deemed optimum for treating the patient'"'"'s cancer;

2. A method for selecting the optimum treatment for a cancerous condition in a human patient, the method comprising:

preparing PCR kit and gene chip;

isolating mRNA from a patient'"'"'s tumor or blood sample with an extraction buffer;

synthesizing and amplifying cDNA in a patient'"'"'s tumor or blood sample with primers highly specific for targeted cancer genes;

detecting cancer genes, mutations using a gene chip;

analyzing and interpreting PCR and/or gene chip results using a detector linked to a computerized decisions support system running a diagnostic software program with accompanying database for providing an indication of the drug which is optimum for treating the patient'"'"'s cancer with the least likely chance for a drug side effect.

3. The method according to claim 2, wherein the step of isolating mRNA from a patient'"'"'s tumor or blood sample comprises the substeps of:

homogenizing a sample of the patient'"'"'s tumor, blood or serum in 1 ml of denaturing solution containing 4M guanidine thiocyanate, 25 mM sodium citrate, and 0.1 mM 2-mercaptoethanol;

mixing the resultant and homogenizing sequentially with 0.1 ml of 49;

1 chloroform/isoamyl alcohol;

incubating the resulting mixture for 15 minutes on ice and centrifuging at 10,000×

g for 20 minutes at 4 degrees C.;

transferring the upper aqueous phase into a new container and mixing with 1 ml of 100% isopropanol;

incubating the resulting mixture at −

20 degrees C. for thirty minutes at 10,000×

g for 10 minutes;

washing the resulting pellet with 1 ml of 75% ethanol and redissolving in RNase-free water; and

quantifying the resulting RNA sample on a spectophotometer at 260 nm and stored at −

70 degrees C.

4. The method according to claim 2, wherein the step of synthesizing and amplifying cDNA in a patient'"'"'s tumor or blood sample with specific primers for breast cancer genes further comprises the substeps of:

adding the RNA sample (1 μ

g) into 25 μ

g of 2×

reaction mix containing 0.4 mM of each dNTP, 2.4 mM MgSO4, 16 U reverse transcriptase, and 2.5 U Tag DNA polymerase, and 10 μ

M cDNA amplification primers for breast cancer genes;

adjusting the final solution volume to 50 μ

l with autoclaved distilled water;

performing cDNA synthesis and amplification using a DNA Thermal Cycler with the following programs,

cDNA synthesis performed at 1 cycle of 45-55 degrees C. for 20-30 minutes, followed by an incubation at 94 degrees C. for two minutes;

cDNA amplification performed at 35-40 cycles of 94 degrees C. for 15 s (Denature)/55-60 degrees C. for 30 s (Anneal)/68-72 degrees C. for 1 minute (Extend); and

Final extension performed at 1 cycle of 72 degrees C. for 5-10 minutes.

5. The method according to claim 4 comprises the specific primers for breast cancer genes, ER Alpha, Her2, ErbB1, BRCA1 and BRCA2.

6. The method according to claim 2 for detecting and analyzing the PCR product further comprises the substeps of:

resolving the PCR product by electrophoresis in 1.5% agarose gel;

visualizing by electrofluores,ence; and

analyzing the number of PCR fragments using a detector device linked to a computerized decision support system.

7. A method using a clinical computerized decision support system for selecting the optimum treatment for a patient suffering from breast cancer, the method comprising:

combining a gene chip with PCR primers for the detection of particular breast cancer genes in a sample of the patient'"'"'s tissue or blood;

optically inspecting the resulting chemical and biological reaction using a automated detector;

correlating using the software output from the integrated detector with a disease analytical models database and/or a database comprising the results of clinical studies testing the efficacy and toxicity of various drugs in treating patients with particular genotypes having breast cancer; and

providing an indication of one or more drugs which is optimum for treating the patient'"'"'s breast cancer with the most effective outcome and the least amount of side effect.

8. The method according to claim 7, wherein the PCR primers comprise:

ERα

5′

-gctactgtgcagtgtgcaat (F), 5′

-tcgtatcccacctttcatca (B);

Her2 5′

-aggatatccaggaggtgcag (F), 5′

-actgctcatggcagcagtca (B);

ErbB1 5′

-gtggagaactctgagtgcat (F), 5′

-cgaggatttccttgttggct (B);

BRCA2 5′

-ctgtccaggtatcagatgct (F), 5′

-atgtgtggcatgacttggca (B); and

BRCA1 5′

-tagctgatgtattggacgtt (F), 5′

-gagatctttggggtcltcag (B).