Molecular analysis of cellular fluid and liquid cytology specimens for clinical diagnosis, characterization, and integration with microscopic pathology evaluation

Abstract

The application relates to methods, materials, kits, and devices for characterizing fluid specimens of any type or source that may or may not contain cells. The methods, materials, kits, and devices can be used for analyzing fluid obtained from various organs or near a purported tumor site, such as via breast ductal lavage or aspiration of a pancreatic cyst. The methods, materials, kits, and devices generate molecular information that can be used in conjunction with clinical, imaging, and microscopic pathology evaluation to provide a superior pathology diagnosis for clinical and research.

1 . A method for characterizing a biological fluid sample from a patient for evidence of cancer comprising: 
 (a) performing molecular analysis of DNA from the biological fluid sample from a patient comprising: 
 (i) performing an optical density analysis of the biological fluid sample to determine DNA quantity;  
 (ii) performing a quantitative PCR analysis of the biological fluid sample to determine DNA quality;  
 (iii) performing competitive template PCR to determine DNA quality of DNA in the biological fluid sample; and  
   (b) performing mutation analysis of the DNA of the biological fluid sample comprising: 
 (i) determining the presence of mutations in a tumor suppressor gene, and/or the presence of a cancer related genetic marker;  
 (ii) determining tumor suppressor gene loss of heterozygosity (LOH) by analyzing polymorphic microsatellites or other polymorphic markers linked to tumor suppressor genes with respect to their allelic balances, wherein both alleles of each polymorphic microsatellite or other polymorphic marker can be distinguished, thereby distinguishing mutational and/or structural alterations of each allelic copy;  
 (iii) determining point mutations in the K-ras oncogene and/or point mutations in at least one other cancer-associated gene;  
 (iv) determining other structural alterations in DNA;  
 (v) determining the percentage of mutated DNA from steps (b) (ii) and (b) (iii); and  
 (vi) determining the specific temporal sequence of mutation accumulation based on step (v), and  
   (c) determining whether the biological fluid sample has evidence of a cancer, a dysplasia, a pre-cancerous state, or a non-neoplastic condition based on steps (a) to (b).    
     
     
         2 . The method of  claim 1 , further determining whether a focal site of neoplastic progression of a neoplastic lesion is more advanced than other portions of the lesion.  
     
     
         3 . The method of  claim 1 , further comprising confirming the results of steps (a) and (b) by comparing said results with a pathologic analysis of resected tissue obtained from the patient.  
     
     
         4 . The method of  claim 1 , further comprising analyzing the carcinoembryonic antigen (CEA) level of the biological fluid sample.  
     
     
         5 . The method of  claim 1 , wherein the DNA in the biological fluid sample is free-floating, or free and adherent to the surface of cells or tissue constituents of the source of the biological fluid sample.  
     
     
         6 . The method of  claim 5 , wherein the source is a cyst.  
     
     
         7 . The method of  claim 1 , wherein the biological fluid sample is a liquid cytology sample.  
     
     
         8 . The method of  claim 1 , wherein the cycles of quantitative PCR performed in step (b) (ii) is greater than a threshold unique for that specific type of DNA.  
     
     
         9 . The method of  claim 1 , wherein the patient is a mammal.  
     
     
         10 . The method of  claim 9 , wherein the patient is a human.  
     
     
         11 . The method of  claim 1 , wherein the cancer is selected from the group consisting of carcinoma, an epithelial malignancy, a sarcoma, a mesenchymal malignancy, a breast cancer, a pancreatic cancer, an urinary tract cancer, a cervical cancer, a lymphohematopoetic cancer, a neuroepithelial cancer, a central nervous system cancer.  
     
     
         12 . The method of  claim 11 , wherein the central nervous system cancer is a glioma.  
     
     
         13 . The method of  claim 1 , wherein the pre-cancerous state is a mucinous cystadenoma, leukoplakia, a serous cystadenoma, a colon polyp, a mesenchymal precancerous lesions, a neuroglial precancerous lesion, or a lymphohematopoietic precancerous condition.  
     
     
         14 . The method of  claim 1 , wherein the non-neoplastic condition is pancreatitis, a pancreatic pseudocyst, a mesothelial cyst of the pancreas, a lymphoepithelial cyst of the pancreas, an ischemic necrosis of the pancreas, mastitis, a non-neoplastic mesenchymal lesion, a neuroglial non-neoplastic lesion, or a lymphohematopoietic condition.  
     
     
         15 . The method of  claim 1 , wherein the other structural alterations in DNA are selected from the group consisting of: gene amplification, gene translocation, gene rearrangement, and epigenetic modification of DNA by DNA methylation.  
     
     
         16 . A method for diagnosing and/or determining the prognosis of a cancer, a dysplasia, a pre-cancerous state, or a non-neoplastic condition in a patient comprising: 
 (a) performing molecular analysis of DNA from a biological fluid sample of the patient comprising: 
 (i) performing an optical density analysis of the DNA in the biological fluid sample to determine DNA quantity;  
 (ii) performing a quantitative PCR analysis of the DNA in the biological fluid sample to determine DNA quality; and  
 (iii) performing competitive template PCR of the DNA in the biological fluid sample to determine DNA quality; and  
   (b) performing mutation analysis of the DNA in the biological fluid sample comprising: 
 (i) determining the presence of one or more mutations in a tumor suppressor gene, and/or the presence of a cancer related genetic marker;  
 (ii) determining tumor suppressor gene loss of heterozygosity (LOH) by analyzing polymorphic microsatellites or other polymorphic markers linked to tumor suppressor genes with respect to their allelic balances, wherein both alleles of each polymorphic microsatellite or other polymorphic marker can be distinguished, thereby distinguishing mutational and/or structural alterations of each allelic copy;  
 (iii) determining point mutations in the K-ras oncogene and/or point mutations in at least one other cancer-associated gene;  
 (iv) determining other structural alterations in DNA;  
 (v) determining the percentage of mutated DNA from steps (b) (ii) and (b) (iii); and  
 (vi) determining a temporal sequence of mutation accumulation based on step (v); and  
   (c) diagnosing and/or determining the prognosis of the cancer or dysplasia, the pre-cancerous state, or the non-neoplastic condition in the patient in need thereof based on the results of steps (a) and (b).    
     
     
         17 . The method of  claim 16 , further determining whether a focal site of neoplastic progression of a neoplastic lesion is more advanced than other portions of the lesion.  
     
     
         18 . The method of  claim 16 , wherein the cancer or dysplasia is selected from the group consisting of: a carcinoma, an epithelial malignancy, a sarcoma, a mesenchymal malignancy, a breast cancer, a pancreatic cancer, a cervical cancer, an urinary tract cancer, a lymphohematopoetic cancer, a neuroepithelial cancer, and a central nervous system cancer.  
     
     
         19 . The method of  claim 16 , wherein the pre-cancerous state is an epithelial precancerous state selected from the group consisting of a mucinous cystadenoma, leukoplakia, a serous cystadenoma, a colon polyp, a mesenchymal precancerous lesion, a neuroglial precancerous lesion, or a lymphohematopoietic precancerous condition.  
     
     
         20 . The method of  claim 16 , wherein the non-neoplastic condition is pancreatitis, a pancreatic pseudocyst, a mesothelial cyst of the pancreas, a lymphoepithelial cyst of the pancreas, an ischemic necrosis of the pancreas, mastitis, a non-neoplastic mesenchymal lesion, a non-neoplastic neuroglial lesion, or a lymphohematopoietic non-neoplastic condition.  
     
     
         21 . A method for determining a course of treatment for a patient having a cancer or dysplasia, a pre-cancerous state, or a non-neoplastic condition comprising: 
 (a) performing molecular analysis of DNA from a biological fluid sample from the patient comprising: 
 (i) performing an optical density analysis of the DNA in the biological fluid sample to determine DNA quantity;  
 (ii) performing a quantitative PCR analysis of the DNA in the biological fluid sample to determine DNA quality; and  
 (iii) performing competitive template PCR of the DNA in the biological fluid sample to determine DNA quality; and  
   (b) performing mutation analysis of the DNA in the biological fluid sample comprising: 
 (i) determining the presence of mutations in a tumor suppressor gene and/or the presence of a cancer related genetic marker;  
 (ii) determining tumor suppressor gene loss of heterozygosity (LOH) by analyzing polymorphic microsatellites or other polymorphic markers linked to tumor suppressor genes with respect to their allelic balances, wherein both alleles of each polymorphic microsatellite or other polymorphic marker can be distinguished, thereby distinguishing mutational and/or structural alterations of each allelic copy;  
 (iii) determining point mutations in the K-ras oncogene and/or point mutations in at least one other cancer-associated gene;  
 (iv) determining other structural alterations in DNA;  
 (v) determining the percentage of mutated DNA from steps (b) (ii) and (b) (iii); and  
 (vi) determining the specific temporal sequence of mutation accumulation based on step (v); and  
   (c) determining whether the patient has cancer, dysplasia, a pre-cancerous state, or a non-neoplastic condition, and determining a course of treatment for said cancer, dysplasia, precancerous state, or non-neoplastic condition.    
     
     
         22 . The method of  claim 21 , further determining whether a focal site of neoplastic progression of a neoplastic lesion is more advanced than other portions of the lesion.  
     
     
         23 . The method of  claim 21 , wherein the cancer or dysplasia is a carcinoma, an epithelial malignancy, a sarcoma, a mesenchymal malignancy, a breast cancer, a pancreatic cancer, a lymphohematopoetic cancer, a neuroepithelial cancer, or a central nervous system cancer.  
     
     
         24 . The method of  claim 21 , wherein the pre-cancerous state is an epithelial precancerous state selected from the group consisting of: a mucinous cystadenoma, a leukoplakia, a serous cystadenoma, a colon polyp, a mesenchymal precancerous lesion, a neuroglial precancerous lesion, or a lymphohematopoietic condition.  
     
     
         25 . The method of  claim 21 , wherein the non-neoplastic condition is an epithelial non-neoplastic lesion selected from the group consisting of: pancreatitis, a pancreatic pseudocyst, a mesothelial cyst of the pancreas, a lymphoepithelial cyst of the pancreas, an ischemic necrosis of the pancreas, mastitis, a non-neoplastic mesenchymal lesion, a neuroglial and non-neoplastic lesion, and a non-neoplastic lymphohematopoietic condition.  
     
     
         26 . A kit for determining the diagnosis and/or prognosis of a patient comprising: 
 (a) a device for performing molecular analysis of DNA in a biological fluid sample from the patient;    (b) a device for performing mutation analysis of the DNA in the biological fluid sample;    (c) reagents for performing the molecular analysis and the mutation analysis; and    (d) optionally a device for recording data obtained from the devices of (a) and (b).    
     
     
         27 . The kit of  claim 26 , wherein the device for recording data records patient age, patient sex, patient medical history, prior cancer history of patient, patient weight, patient family medical history, diagnosis determined from the mutation analysis of the DNA and molecular analysis of the DNA, temporal sequence of mutation accumulation, and proposed treatment based on determined diagnosis.  
     
     
         28 . A method for determining and characterizing a breast anomaly in a patient comprising: 
 (a) performing a molecular analysis of DNA from a biological sample of patient breast tissue comprising: 
 (i) performing optical density analysis of the biological sample to determine DNA quantity;  
 (ii) performing quantitative PCR analysis of the aspirate to determine DNA quality;  
 (iii) performing competitive template PCR to determine DNA quality; and  
   (b) performing mutation analysis of the DNA of the biological sample comprising: 
 (i) determining the presence of mutations in a tumor suppressor gene and/or the presence of a cancer related genetic marker in the DNA of the biological sample;  
 (ii) determining tumor suppressor gene loss of heterozygosity (LOH) by analyzing polymorphic microsatellites or other polymorphic markers linked to tumor suppressor genes with respect to their allelic balances, wherein both alleles of each polymorphic microsatellite or other polymorphic marker can be distinguished, thereby distinguishing mutational and/or structural alterations of each allelic copy;  
 (iii) determining point mutations in the K-ras oncogene and/or point mutations in at least one other cancer-associated gene;  
 (iv) determining copy number alterations such as homozygous deletion and oncogene amplification;  
 (v) determining other structural alterations in DNA;  
 (vi) determining the percentage of mutated DNA from steps (b)(ii) and (b)(iii); and  
 (vii) determining a temporal sequence of mutation accumulation based on step (v); and  
   (c) assessing the data from steps (a) and (b) to determine the presence of a breast anomaly and characterize the type of breast anomaly.    
     
     
         29 . The method of  claim 28 , further determining whether a focal site of neoplastic progression of a neoplastic lesion is more advanced than other portions of the lesion.  
     
     
         30 . The method of  claim 1 , wherein the biological sample is an aspirate from a breast cyst.  
     
     
         31 . The method of  claim 1 , wherein the breast anomaly is a breast cancer, a breast dysplasia, a pre-cancerous breast condition, or a non-neoplastic condition.  
     
     
         32 . The method of  claim 31 , wherein the breast cancer is selected from the group consisting of invasive ductal carcinoma, invasive lobular carcinoma, breast sarcoma, ductal adenocarcinoma, breast acinar cell carcinoma, metastatic cancer involving the breast, recurrent breast cancer, cystosarcoma phyllodes, Paget's disease, and metastatic breast cancer.  
     
     
         33 . The method of  claim 32 , wherein the metastatic breast cancer is an axillary metastasis with no apparent primary tumor or an axillary adenopathy of a metastatic adenocarcinoma.  
     
     
         34 . The method of  claim 31 , wherein the breast dysplasia is atypical ductal hyperplasia or atypical lobular hyperplasia.  
     
     
         35 . The method of  claim 31 , wherein the pre-cancerous breast condition is selected from the group consisting of mucinous cystadenoma, serous cystadenoma, mucinous duct ectasia, intraductal papillary mucinous neoplasm, breast intraepithelial neoplasia, and a papilloma of the breast.  
     
     
         36 . The method of  claim 31 , wherein the non-neoplastic condition is selected from the group consisting of breast pseudocyst, fibrocystic disease of the breast, fibroadenoma, inflammatory mastitis, soft tissue trauma, lymphedema, and mesothelial cyst.  
     
     
         37 . A method of analyzing a liquid cytology sample for the presence of a mutational change in a patient comprising: 
 (a) performing a molecular analysis of DNA from a liquid cytology sample of the patient comprising: 
 (i) performing optical density analysis of the liquid cytology sample to determine DNA quantity;  
 (ii) performing quantitative PCR analysis of the liquid cytology sample to determine DNA quality;  
 (iii) performing competitive template PCR to determine DNA quality; and  
   (b) performing mutation analysis of the DNA of the liquid cytology sample comprising: 
 (i) determining the presence of mutations in a tumor suppressor gene and/or the presence of a cancer related genetic marker in the DNA of the liquid cytology sample;  
 (ii) determining tumor suppressor gene loss of heterozygosity (LOH) by analyzing polymorphic microsatellites or other polymorphic markers linked to tumor suppressor genes with respect to their allelic balances, wherein both alleles of each polymorphic microsatellite or other polymorphic marker can be distinguished, thereby distinguishing mutational and/or structural alterations of each allelic copy;  
 (iii) determining point mutations in a K-ras oncogene and/or point mutations in at least one other cancer-associated gene;  
 (iv) determining copy number alterations such as homozygous deletion and oncogene amplification;  
 (v) determining other structural alterations in DNA;  
 (vi) determining the percentage of mutated DNA from steps (c)(ii) and (c)(iii); and  
 (vii) determining a temporal sequence of mutation accumulation based on step (v); and  
   (c) assessing the data from steps (a) and (b) to determine the presence of a mutational change in the patient.    
     
     
         38 . The method of  claim 37 , further determining whether a focal site of neoplastic progression of a neoplastic lesion is more advanced than other portions of the lesion.  
     
     
         39 . A method is for analyzing a fluid biological sample, with or without a cell, for detecting focal site of neoplasia progression in a patient with or without a large component of neoplastic or normal tissue comprising: 
 (a) performing a molecular analysis of DNA on a liquid biological sample of the patient comprising: 
 (i) performing optical density analysis of the biological sample to determine DNA quantity;  
 (ii) performing quantitative PCR analysis of the aspirate to determine DNA quality;  
 (iii) performing competitive template PCR to determine DNA quality; and  
   (b) performing mutation analysis of the DNA of the liquid biological sample comprising: 
 (i) using DNA primers to amplify an amplicon of at least 300 nucleotides to 1500 nucleotides, thereby amplifying DNA from more actively replicating tissue sites and excluding degraded DNA from amplification;  
 (ii) determining mutation presence in a tumor suppressor gene and/or mutation presence in a cancer related genetic marker in the DNA of the liquid biological sample;  
 (iii) determining tumor suppressor gene loss of heterozygosity (LOH) by analyzing polymorphic microsatellites or other polymorphic markers linked to tumor suppressor genes with respect to their allelic balances, wherein both alleles of each polymorphic microsatellite or other polymorphic marker can be distinguished, thereby distinguishing mutational and/or structural alterations of each allelic copy;  
 (iv) determining point mutations in the K-ras oncogene and/or point mutations in at least one other cancer-associated gene;  
 (v) determining copy number alterations such as homozygous deletion and oncogene amplification;  
 (vi) determining other structural alterations in the DNA of the liquid biological sample;  
 (vii) determining the percentage of mutated DNA from steps (b)(ii) and (b)(iii); and  
 (viii) determining a temporal sequence of mutation accumulation based on step (v); and  
   (c) determining the focal site of neoplasia progression in the patient by the steps of (a) to (b).    
     
     
         40 . A method of detecting an allelic imbalance in a patient without an internal normal, non-neoplasmic specimen from the patient comprising: 
 (a) performing a molecular analysis of DNA from a biological sample of the patient comprising: 
 (i) performing optical density analysis of the biological sample to determine DNA quantity;  
 (ii) performing quantitative PCR analysis of the DNA quality;  
 (iii) performing competitive template PCR to determine DNA quality; and  
   (b) performing mutation analysis of the DNA of the biological sample comprising: 
 (i) using DNA primers to amplify a polymorphic region of DNA;  
 (ii) defining a combination of specific polymorphic alleles in specimens from the general population;  
 (iii) calculating quantitatively content ratio of polymorphic alleles in specimens from the general population;  
 (iv) defining a statistical average and normal distribution of the content ratio of each combination of polymorphic alleles in specimens from the general population;  
 (v) determining a threshold for designation as within normal limits;  
 (vi) defining content ratios exceeding threshold values as outside the range of content ratio variation in specimens from the general population; and  
   (d) calculating a percentage of cellular DNA subject to allelic imbalance in step (vi) using the average content ratio as a normalizing factor thereby determining the allelic imbalance of the patient without an internal normal, non-neoplasmic specimen.    
     
     
         41 . The method of  claim 39 , wherein the amplicon is at least 500 to 1500 nucleotides long.  
     
     
         42 . The method of  claim 40 , wherein the threshold is at least about 95%.  
     
     
         43 . The method of  claim 40 , wherein the polymorphic region of DNA amplified is a microsatellite, a minisatellite, or a single nucleotide polymorphism.