Abstract:
The present disclosure relates to a method for assessing if a patient is affected with a lung cancer. The method includes analyzing the presence and amount of cyclohexanone contained in the urine excreted from the patient and determining if the presence and amount of cyclohexanone is indicative of lung cancer.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application claims priority to provisional application Ser. No. 61/302,818, filed Feb. 9, 2010, the contents of which are incorporated by reference herein. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present disclosure relates to a method for determining whether or not a patient is affected with a lung cancer. Patients which are to be the subjects of the method of the present disclosure are preferably mammals including humans. 
     2. Description of the Related Art 
     Substances that are present in large amounts in cancer cells but are either not present, or only present in small amounts in normal cells are known. Such substances are referred to as “tumor markers”. Tumor marker tests, in general, quantitatively determine a tumor marker in blood, and have been used for the purpose of aiding diagnosis of cancer, or confirming the degree of progression of cancer. As such, in order to determine the presence of a tumor marker in a patient, it is typical that a sample of blood is drawn from the patient. 
     Conventional tumor markers are high molecular substances such as hormones, enzymes, isozymes, or fragmented proteins. Since there are great differences among individuals in blood levels of these conventional tumor markers, usability in diagnosing cancer has been unsatisfactory due to the occurrence of false negatives and false positives. 
     SUMMARY OF THE INVENTION 
     The present Applicants investigated utilization of a volatile low molecular compound, which has otherwise has not been investigated as a tumor marker, and consequently discovered that by quantitatively determining cyclohexanone contained in the urine excreted from a mammal, determination is enabled as to whether or not the mammal is affected with a lung cancer. Accordingly, the present disclosure was accomplished. 
     An aspect of the present disclosure relates to a method for determining whether or not a patient is affected with a lung cancer, the method comprising the steps of: 
     obtaining a urine sample from the patient; 
     measuring the concentration of cyclohexanone contained in the urine sample; and 
     determining that the patient is suffering from lung cancer if the concentration is equal to or greater than 1 μM. 
     The patients preferably a primate or a human 
     According to the present disclosure, a method of determination is provided which is useful for diagnosing lung cancer in mammals. 
     The above objects, other objects, features and advantages of the present disclosure will be apparent from the following detailed description of preferred embodiments with reference to attached drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows a scheme for illustrating a procedure of ultrafiltration of a urine sample. 
         FIG. 2  shows a schematic view for illustrating a trapping method of volatile components contained in a urine sample by a SPME method. 
         FIG. 3(   a ) shows a gas chromatogram of a control urine sample. 
         FIG. 3(   b ) shows a gas chromatogram of a urine sample from a human lung cancer patient. 
         FIG. 4  shows graphs demonstrating the peak area of a specified component A in gas chromatograms. 
         FIG. 5(   a ) shows a MS spectrum of the specified component A. 
         FIG. 5(   b ) shows a MS spectrum of cyclohexanone listed in NIST database. 
         FIG. 6(   a ) shows a GC chromatogram of a urine sample from a human lung cancer patient. 
         FIG. 6(   b ) shows a GC chromatogram of cyclohexanone. 
         FIG. 7  shows a standard curve of cyclohexanone. 
         FIG. 8  shows a graph illustrating distribution of cyclohexanone concentration in the urine excreted from the human lung cancer patients and the control urine. 
     
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
     In a general aspect of the disclosure, urine from a patient is collected and the concentration of the cyclohexanone contained in the urine excreted from the patient is measured. Based on the concentration of the cyclohexanone measured, it determined that the patient is suffering from lung cancer if the concentration is equal to or greater than 1 μm. 
     A feature of the above general aspect of the invention may be that the concentration of the cyclohexanone is measured using gas chromatography, liquid chromatography or any other known method or combination of methods of measuring organic compounds. 
     Another feature of the above general aspect of the disclosure may be that the lung cancer is selected from the group consisting of squamous cell, adenocarcinoma, non-small cell lung carcinoma (NSCLC), large cell carcinoma and adenosquamous carcinoma. 
     1. Collection of Urine, and Ultrafiltration of Urine 
     (1) Collecting Method of Urine 
     Urine of a human lung cancer patient was collected from 19 lung cancer patients (Table 1a) corresponding to stages  1 A to  3 B who participated as volunteers. Control urine was collected from 19 human non-lung cancer patients who were from 36 to 86 years old (Table 1b), and participated as volunteers. Each 1 mL of the urine collected from each patient was dispensed into a 1.5 mL tube, and stored at −80° C. until subjecting to an ultrafiltration treatment. 
     Table 1a shows age, sex (represented by “Gender”), race, tissue type, stage, and smoking history (represented by “Tobacco History”) of the human lung cancer patients. Table 1b shows age, sex, race, diagnosis, and smoking history of the human non-lung cancer patients. Blank columns in the smoking history mean that the subject had no history of smoking. The abbreviation of “COPD” in Table 1b means “Chronic Obstructive Pulmonary Disease”. 
     
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 1a 
               
               
                   
               
               
                 Subject  
                   
                   
                   
                   
                 Overall 
                 Tobacco 
               
               
                 No. 
                 Age 
                 Gender 
                 Race 
                 Histology 
                 Stage 
                 History 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 81 
                 F 
                 White 
                 Squamous Cell 
                 1A 
                 Current 
               
               
                 2 
                 84 
                 F 
                 White 
                 Adenocarcinoma 
                 1A 
                   
               
               
                 3 
                 81 
                 F 
                 White 
                 NSCLC-NOS 
                 1A 
                 Former 
               
               
                 4 
                 69 
                 M 
                 White 
                 Squamous Cell 
                 1A 
                 Current 
               
               
                 5 
                 65 
                 F 
                 White 
                 Adenocarcinoma 
                 1A 
                 Former 
               
               
                 6 
                 68 
                 M 
                 White 
                 Adenocarcinoma 
                 1B 
                 Former 
               
               
                 7 
                 73 
                 F 
                 White 
                 Adenosquamous 
                 1B 
                 Former 
               
               
                 8 
                 69 
                 M 
                 White 
                 Squamous Cell 
                 1B 
                 Former 
               
               
                 9 
                 71 
                 M 
                 White 
                 Squamous Cell 
                 2A 
                 Former 
               
               
                 10 
                 54 
                 M 
                 White 
                 Squamous Cell 
                 2B 
                 Former 
               
               
                 11 
                 53 
                 M 
                 White 
                 Large Cell 
                 2B 
                 Former 
               
               
                 12 
                 69 
                 F 
                 White 
                 Adenocarcinoma 
                 3A 
                 Former 
               
               
                 13 
                 64 
                 F 
                 Asian 
                 Adenocarcinoma 
                 3A 
                   
               
               
                 14 
                 64 
                 M 
                 White  
                 Adenocarcinoma 
                 3A 
                 Former 
               
               
                 15 
                 76 
                 F 
                 White  
                 Adenocarcinoma 
                 3A 
                 Current 
               
               
                 16 
                 62 
                 M 
                 White 
                 NSCLC-NOS 
                 3B 
                 Former 
               
               
                 17 
                 81 
                 M 
                 White 
                 Adenocarcinoma 
                 3B 
                 Former 
               
               
                 18 
                 62 
                 M 
                 White 
                 Squamous Cell 
                 3B 
                 Former 
               
               
                 19 
                 55 
                 M 
                 White 
                 Adenosquamous 
                 3B 
                 Former 
               
               
                   
               
               
                 Race (White: Caucasian, Asian: Asian) 
               
               
                 Gender (M = male, F = female) 
               
             
          
         
       
     
     
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1b 
               
               
                   
               
               
                 Subject 
                   
                   
                   
                   
                 Tobacco 
               
               
                 No. 
                 Race 
                 Age 
                 Gender 
                 Diagnosis 
                 History 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 White 
                 72 
                 F 
                 COPD 
                   
               
               
                 2 
                 White 
                 83 
                 M 
                 COPD 
                   
               
               
                 3 
                 Asian 
                 49 
                 M 
                 COPD 
                   
               
               
                 4 
                 Asian 
                 62 
                 M 
                 COPD 
                   
               
               
                 5 
                 White 
                 54 
                 F 
                 Asthma 
                   
               
               
                 6 
                 White 
                 48 
                 F 
                 Healthy 
                 Former 
               
               
                 7 
                 White 
                 49 
                 F 
                 Healthy 
                 Former 
               
               
                 8 
                 Asian 
                 50 
                 F 
                 Healthy 
                 Current 
               
               
                 9 
                 Asian 
                 38 
                 F 
                 Healthy 
                 Current 
               
               
                 10 
                 White 
                 48 
                 F 
                 Healthy 
                 Former 
               
               
                 11 
                 White 
                 38 
                 F 
                 Healthy 
                 Current 
               
               
                 12 
                 White 
                 47 
                 F 
                 Heart disease 
                 Former 
               
               
                 13 
                 White 
                 51 
                 F 
                 Thyroid disease 
                 Current 
               
               
                 14 
                 White 
                 59 
                 M 
                 Healthy 
                 Current 
               
               
                 15 
                 White 
                 55 
                 M 
                 Asthma 
                 Current 
               
               
                 16 
                 Asian 
                 49 
                 F 
                 Healthy 
                 Current 
               
               
                 17 
                 White 
                 61 
                 M 
                 Heart disease 
                 Current 
               
               
                 18 
                 White 
                 62 
                 M 
                 Hypertension 
                 Current 
               
               
                 19 
                 White 
                 36 
                 F 
                 Hypertension 
                 Current 
               
               
                   
               
               
                 Race: White = Caucasian, Asian = Asian 
               
               
                 Gender: M = male, F = female 
               
             
          
         
       
     
     (2) Ultrafiltration of Urine Sample 
     The urine of the human lung cancer patient and the control urine were subjected to ultrafiltration according to the procedure shown in  FIG. 1 . The urine of the human lung cancer patient and the control urine (each 1 mL) which had been frozen and stored at −80° C. were thawed on ice. After thawing, centrifugal separation (13,000 g×10 min) was carried out to remove insoluble matter. The thus obtained supernatant was set on a Microcon centrifugation system filter unit YM-30 (30-kDa cutoff, Millipore, Bedford, Mass.), and subjected to centrifugal separation (12,000 G, 0° C., 90 min). The urine that passed through the filter was set on a Microcon centrifugation system filter unit YM-10 (10-kDa cutoff, Millipore, Bedford, Mass.), and subjected to centrifugal separation in a similar manner as described above. The urine that passed through the filter was set on a Microcon centrifugation system filter unit YM-3 (3-kDa cutoff, Millipore, Bedford, Mass.), and subjected to centrifugal separation in a similar method as described above. 
     When the urine remained on the filter after each centrifugal separation, the remaining urine was transferred on a new filter, and similar centrifugal separation was repeated until no urine remained on the filter. The filtrate that passed through the filter unit YM-3 was dispensed into 1.5 mL tube. The filtrate was stored at 4° C. as a urine sample for analysis until it is analyzed with gas chromatography. 
     2. Analysis of Volatile Components Using Solid Phase Micro Extraction (SPME) Method 
     (1) Extraction of Volatile Components 
     Volatile components in the urine sample for analysis were extracted by a solid phase micro extraction. Each urine sample for analysis in a volume of 50 μL was placed into a 1.5 mL glass vial, and the vial was sealed airtight using a lid with septum. The vial was set into a heating block, and incubated at 40° C. for 10 min. As shown in  FIG. 2 , a Stable Flex DVB/CAR/PDMS fiber (SUPELCO, Inc., 2-layer fiber, film thickness: 50 μm/30 μm) was inserted into the vial for 40 min, to extract the volatile components in the head space to the fiber. 
     Before extracting of the volatile components, the fiber was subjected to a heat treatment at 230° C. for 30 min to eliminate any volatile component from the fiber, and thereafter inserted into the vial. 
     (2) Method for Analyzing Volatile Components 
     After extraction, the volatile components were thermally desorbed and analyzed. The analysis was performed using a gas chromatography (GC) or a gas chromatography-mass spectrometry (GC-MS). The extracted fiber was inserted into the injector of each instrument. 
     As a gas chromatography with a flame ionization detector (FID), GC-4000 (GL Sciences Inc.) was used. Analysis conditions were as shown below. 
     Column: INERTCAP Pure-WAX; internal diameter: 0.25 mm, length: 30 m, film thickness=0.25 μm 
     Column temperature: 40° C. (5 min)→temperature elevation at a rate of 4° C./min→250° C. (5 min) 
     Carrier gas: helium 100 kPa 
     Injection: 230° C., splitless (closed 5 min) 
     Detector: FID (250° C.) 
     As the gas chromatography-mass spectrometry apparatus, GCMS-QP2010 (Shimadzu Corporation) was used. Analysis conditions were as shown below. 
     Column: InertCap® Pure-WAX; internal diameter: 0.25 mm, length: 30 m, film thickness=0.25 μm 
     Column temperature: 40° C. (5 min)→temperature elevation at a rate of 4° C./min→250° C. (5 min) 
     Carrier gas: helium 100 kPa 
     Injection: 230° C., splitless (closed 5 min) 
     Ionization process: Electron Impact method 
     3. Identification and Quantitative Determination of Specified Component that Serves as Marker of Cancer 
     (1) Comparison of Gas Chromatogram 
     A gas chromatogram ( FIG. 3   a ) of the control urine (urine of a human non-lung cancer patient) was compared with a gas chromatogram of the lung cancer urine (urine of a human lung cancer patient) ( FIG. 3   b ). An increased peak present in the urine of the human lung cancer patient was identified by visual inspection. Hereinafter, the peak is referred to as “specified component A”. 
     The peak areas of the specified component A in all samples of the lung cancer urine and the control urine were calculated from each gas chromatogram.  FIG. 4  shows the peak areas of the specified component A. Numerical values presented along the horizontal axis of  FIG. 4  show the patient number listed in Table 1a and Table 1b. As is seen from  FIG. 4 , the peaks of the specified component A for the urine of the human lung cancer patient had significantly greater area than the peaks of the specified component A for the control urine. 
     (2) Searching of Candidate Compound  FIG. 5   a  shows an MS spectrum of the specified component A. Based on the m/z value on the MS spectrum of the specified component A observed, a candidate compound of the specified component A was searched using NIST database (NIST147.LIB) attached to the gas chromatography-mass spectrometry. As a result, cyclohexanone corresponded to the information from the NIST database with a similarity of 98 (perfect match=100).  FIG. 5   b  shows the MS spectrum of cyclohexanone listed in the NIST database. 
     (3) Identification of Specified Component A 
     For the purpose of identifying the specified component A, cyclohexanone (Wako Pure Chemical Industries, Ltd., guaranteed reagent) was purchased. Cyclohexanone was placed into a 1.5 mL vial, and subjected to a gas chromatography analysis on cyclohexanone using the solid phase micro extraction method similarly to the case of the urine samples. As a result, the present Applicants ascertained that the retention time of the specified component A (see,  FIG. 6   a ) corresponded to the retention time of cyclohexanone (see,  FIG. 6   b ). From the agreement on the MS spectrum and of the retention time, the specified component A was identified as being cyclohexanone. 
     (4) Production of Standard Curve of Cyclohexanone 
     Aqueous cyclohexanone solutions of 1 μM, 10 μM, and 100 μM were prepared. The aqueous cyclohexanone solution of each concentration in a volume of 50 μL was placed into a 1.5 mL vial, which was then sealed airtight using a lid with septum. The vial was set into a heating block, and incubated at 40° C. for 10 min. A Stable Flex DVB/CAR/PDMS fiber (SUPELCO, Inc., 2-layer fiber, film thickness: 50 μm/30 μm) was inserted into the head space for 40 min. After the volatile components were extracted to the fiber, the analysis was carried out using the gas chromatography analysis conditions which were similar to those described above. The analysis was repeated three times for the aqueous cyclohexanone solution of each concentration. A standard curve was produced by plotting the peak areas and the concentrations of the aqueous cyclohexanone solutions. 
       FIG. 7  shows a standard curve of cyclohexanone. The longitudinal axis represents the area of the peak corresponding to cyclohexanone, whereas the horizontal axis represents the concentration (μM) of the aqueous cyclohexanone solution. The coefficient of correlation between the concentration of the aqueous cyclohexanone solution and the peak area was 0.9994, indicating high linearity. As shown in Table 2, the coefficient of variation at a concentration of 1 μM was 20.3%. However, the coefficient of variation at each concentration of 10 μM and 100 μM were 1.6% and 0.8%, respectively. 
     
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Peak Area, Standard Deviation, and Coefficient of Variation 
               
               
                 of Cyclohexanone 
               
             
          
           
               
                   
                   
                   
                   
                   
                   
                 Coef- 
               
               
                   
                   
                   
                   
                   
                   
                 ficient of 
               
               
                 Cyclo- 
                 First 
                 Second 
                 Third 
                   
                 Standard 
                 variation 
               
               
                 hexanone 
                 analysis 
                 analysis 
                 analysis 
                 Average 
                 deviation 
                 (%) 
               
               
                   
               
             
          
           
               
                  1 uM 
                 56541 
                 43076 
                 65163 
                 54927 
                 11131.6 
                 20.3 
               
               
                  10 uM 
                 415539 
                 427250 
                 415539 
                 419443  
                 6761.3 
                 1.6 
               
               
                 100 uM 
                 3580449 
                 3602729 
                 3544006  
                 3575728 
                 29644.8 
                 0.8 
               
               
                   
               
             
          
         
       
     
     (5) Measurement of Cyclohexanone Concentration in Urine 
     Using the standard curve shown in  FIG. 7 , cyclohexanone concentrations in the urine of the human lung cancer patient and the control urine were calculated. As shown in Table 3a, the average cyclohexanone concentration in the urine of the human lung cancer patients was 3.4 μM (from minimum concentration of 1.4 μM to maximum concentration of 7.2 μM). On the other hand, as shown in Table 3b, the average cyclohexanone concentration in the control urine was 0.22 μM (from minimum concentration of 0.2 μM to maximum concentration of 0.4 μM). 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3a 
               
             
             
               
                   
               
               
                 Cyclohexanone Concentration in Urine of 
               
               
                 Human Lung Cancer Patients 
               
             
          
           
               
                   
                 Subject 
                   
                   
                 Cyclohexanone 
               
               
                   
                 No. 
                 Histology 
                 Overall Stage 
                 (μM) 
               
               
                   
                   
               
             
          
           
               
                   
                 1 
                 Squamous Cell 
                 1A 
                 7.2 
               
               
                   
                 2 
                 Adenocarcinoma 
                 1A 
                 1.4 
               
               
                   
                 3 
                 NSCLC - NOS 
                 1A 
                 3.8 
               
               
                   
                 4 
                 Squamous Cell 
                 1A 
                 3.5 
               
               
                   
                 5 
                 Adenocarcinoma 
                 1A 
                 2.1 
               
               
                   
                 6 
                 Adenocarcinoma 
                 1B 
                 2.5 
               
               
                   
                 7 
                 Adenosquamous 
                 1B 
                 2.3 
               
               
                   
                 8 
                 Squamous Cell 
                 1B 
                 3.2 
               
               
                   
                 9 
                 Squamous Cell 
                 2A 
                 2.0 
               
               
                   
                 10 
                 Squamous Cell 
                 2B 
                 2.6 
               
               
                   
                 11 
                 Large Cell 
                 2B 
                 3.3 
               
               
                   
                 12 
                 Adenocarcinoma 
                 3A 
                 3.4 
               
               
                   
                 13 
                 Adenocarcinoma 
                 3A 
                 5.8 
               
               
                   
                 14 
                 Adenocarcinoma 
                 3A 
                 3.3 
               
               
                   
                 15 
                 Adenocarcinoma 
                 3A 
                 2.0 
               
               
                   
                 16 
                 NSCLC - NOS 
                 3B 
                 5.2 
               
               
                   
                 17 
                 Adenocarcinoma 
                 3B 
                 2.5 
               
               
                   
                 18 
                 Squamous Cell 
                 3B 
                 5.8 
               
               
                   
                 19 
                 Adenosquamous 
                 3B 
                 3.0 
               
               
                   
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 3b 
               
             
             
               
                   
               
               
                 Cyclohexanone Concentration in Control Urine 
               
             
          
           
               
                 Subject 
                   
                 Cyclohexanone 
               
               
                 No. 
                 Diagnosis 
                 (μM) 
               
               
                   
               
             
          
           
               
                 1 
                 COPD 
                 0.3 
               
               
                 2 
                 COPD 
                 0.4 
               
               
                 3 
                 COPD 
                 0.3 
               
               
                 4 
                 COPD 
                 0.2 
               
               
                 5 
                 Asthma 
                 0.2 
               
               
                 6 
                 Healthy 
                 0.2 
               
               
                 7 
                 Healthy 
                 0.2 
               
               
                 8 
                 Healthy 
                 0.2 
               
               
                 9 
                 Healthy 
                 0.2 
               
               
                 10 
                 Healthy 
                 0.2 
               
               
                 11 
                 Healthy 
                 0.2 
               
               
                 12 
                 Heart 
                 0.2 
               
               
                   
                 disease 
                   
               
               
                 13 
                 Thyroid 
                 0.2 
               
               
                   
                 disease 
                   
               
               
                 14 
                 Healthy 
                 0.2 
               
               
                 15 
                 Asthma 
                 0.2 
               
               
                 16 
                 Healthy 
                 0.2 
               
               
                 17 
                 Heart 
                 0.2 
               
               
                   
                 disease 
                   
               
               
                 18 
                 Hypertension 
                 0.2 
               
               
                 19 
                 Hypertension 
                 0.2 
               
               
                   
               
             
          
         
       
     
       FIG. 8  shows distribution of cyclohexanone concentration in the urine of the human lung cancer patients and control urine. From the aforementioned experimental results, it was identified that the urine excreted from a human contains not less than 1 μM cyclohexanone if he or she is affected with a lung cancer. The dotted line in  FIG. 8  indicates 1 μM that is considered to be a threshold as a marker for lung cancer. 
     Various types of tumor markers have been extensively used in clinical field as markers for diagnosing malignant tumor, determining therapeutic effects, or examining signs of recurrence after surgery. Since almost all conventional tumor markers are proteins or hormones, it has been necessary to collect blood from a subject (mammal), and to measure the concentration in plasma. However, according to the present disclosure, since the tumor marker which is a target of detection is a volatile component contained in urine, an inspection sample can be noninvasively obtained without imposing a burden to the subject. 
     By comparing volatile components contained in the urine excreted from human lung cancer patients and human non-lung cancer patients, cyclohexanone was specified as one component in the urine that increases when affected with a lung cancer, irrespective of the race and the presence/absence of smoking history. Furthermore, Applicants discovered that a patient is affected with a lung cancer when the cyclohexanone concentration in the urine is not less than 1 μM. 
     Thus, the cyclohexanone concentration in urine is useful as a marker for lung cancer. According to the present disclosure, efficient determination as to whether or not a patient is affected with a lung cancer is enabled.