WO2015017125A2 - Procédé de traitement du syndrome de barrett et de l'adénocarcinome œsophagien - Google Patents

Procédé de traitement du syndrome de barrett et de l'adénocarcinome œsophagien Download PDF

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WO2015017125A2
WO2015017125A2 PCT/US2014/046702 US2014046702W WO2015017125A2 WO 2015017125 A2 WO2015017125 A2 WO 2015017125A2 US 2014046702 W US2014046702 W US 2014046702W WO 2015017125 A2 WO2015017125 A2 WO 2015017125A2
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mutational load
subject
clonality
dna
mutations
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PCT/US2014/046702
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WO2015017125A3 (fr
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Eric Matthew Gayle Ellsworth
Sydney David Finkelstein
Sara Ann Jackson
Brendan Corcoran
Dennis Morgan SMITH
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Redpath Integrated Pathology Inc.
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Priority claimed from US13/954,247 external-priority patent/US10131942B2/en
Application filed by Redpath Integrated Pathology Inc. filed Critical Redpath Integrated Pathology Inc.
Publication of WO2015017125A2 publication Critical patent/WO2015017125A2/fr
Publication of WO2015017125A3 publication Critical patent/WO2015017125A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • 61/640,527 entitled “Methods for diagnosing low and high grade dysplasia in Barrett's esophagus” filed April 30, 2012 and U.S. Provisional Application Serial No. 61/661,256 entitled “Methods for diagnosing low and high grade dysplasia in Barrett's esophagus” filed June 18, 2012, each of which are hereby incorporated herein by reference in their entirety.
  • determining mutational load as a predictor of the risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma in a subject, the method comprising: amplifying DNA sequences from a biological specimen from the subject; detecting mutations in microsatellite regions of the amplified DNA sequences; categorizing clonality of each mutation; calculating a mutational load based on the sum of low and high clonality mutations; wherein calculating the mutational load further comprises assigning a score to each mutation based on detection of DNA microsatellite instability at a particular locus, calculating a mutational load based on the sum of low and high clonality mutations; wherein DNA microsatellite instability at a single locus is defined as 0.75zi, and wherein DNA microsatellite instability at multiple loci is defined as 0.75zi + ATTORNEY REF. NO.130341.01212 PATENT
  • 0.5z 2 wherein zi represent a single locus displaying DNA micros atellite instability and Z2 is the number of loci displaying DNA microsatellite instability greater than 1 locus; wherein the score for low clonality is 0.5x, wherein x is the number of low clonality mutations and the score for high clonality is y, wherein y is the number of high clonality mutations; and wherein the overall mutational load is y + 0.5x + 0.75zi + 0.5z 2 ; comparing the mutational load with a series of pre-determined mutational load cut-offs defining risk categories; and assigning the subject to a risk category corresponding to the subject's mutational load, wherein each risk category is indicative of the risk of disease progression.
  • the pre-determined mutational load cut-offs defining risk categories are derived from a pre-determined patient population distribution with known mutational loads corresponding to a known disease state diagnosis.
  • the known disease state diagnosis is selected from normal squamous, columnar epithelium without Barrett's metaplasia, Barrett's metaplasia, Barrett's metaplasia intermediate for dysplasia, low-grade dysplasia and high-grade dysplasia.
  • the risk categories are selected from no mutational load, low mutational load, and high mutational load.
  • the subject is assigned to the no mutational load risk category when the subject has mutational load of 0.0.
  • no mutational load is indicative of no risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
  • no mutational load is indicative of the absence of actionable disease.
  • the absence of actionable disease is categorized as Barrett's metaplasia with a lower risk of progression than the baseline risk for Barrett's metaplasia, wherein surveillance of the patient can be safely discontinued.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less 1.75.
  • a low mutational load is indicative of a low risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
  • a low mutational load is indicative of suitability of the subject for monitoring.
  • the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than or equal to 1.75.
  • a high mutational load is indicative of high risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
  • a high mutational load is indicative of suitability of the subject for at least one treatment modality selected from endoscopic mucosal resection, radiofrequency ablation, cryoablation, endoscopic submucosal dissection, photodynamic therapy and combinations thereof.
  • the subject is a human. In some embodiments, the subject is a human diagnosed with Barrett's esophagus.
  • the biological specimen is a mucosal lining of the esophagus. In some embodiments, the biological specimen is representative of a disease region.
  • amplifying DNA sequences comprises selecting a primer pair corresponding to a specific microsatellite region; adding the primer pair to the DNA sequences; and performing quantitative polymerase chain reaction on the DNA sequences with the primer.
  • detecting mutations comprises determining the sequence of the amplified DNA and comparing the amplified DNA to a known wild type control sequence for the specific microsatellite region and identifying differences between the sequence of the amplified DNA and the known wild type control sequence.
  • the specific microsatellite regions are selected from lp (CMM1 , Lmyc), 3p (VHL, OGG1), 5q (MCC, APC), 9p (CDKN2A, CDKN2B), lOq (PTEN, MXI1), 17p (TP53), 17q (NME1), 18q (DCC), 21q, 22q (NF2) and combinations thereof.
  • categorizing clonality of each mutation comprises assigning one of three categories selected from the group consisting of no clonality, low clonality and high clonality.
  • high clonality is assigned where loss of heterozygosity is present in greater than about 75% of DNA analyzed.
  • low clonality is assigned where loss of heterozygosity is present in about 50% to about 75% of DNA analyzed.
  • no clonality is assigned where loss of heterozygosity is present in less than about 50% of DNA analyzed.
  • DNA microsatellite instability is determined by the presence of at least one of the shortening and lengthening of a DNA microsatellite region.
  • calculating a mutational load further comprises summing the clonality weighting for each specific microsatellite region showing a mutation or DNA microsatellite instability.
  • determining mutational load as a predictor of disease progression is independent of a histological standard.
  • calculating the mutational load further comprises assigning a score to each mutation based on detection of DNA microsatellite instability at a particular locus, calculating a mutational load based on the sum of low and high clonality mutations; wherein DNA microsatellite instability at a single locus is defined as 0.75z l 5 and wherein DNA microsatellite instability at multiple loci is defined as 0.75zi + 0.5z 2 , wherein zi represent a single locus displaying DNA microsatellite instability and Z2 is
  • the pre-determined mutational load cut-offs defining risk categories are derived from a pre-determined patient population distribution with known ATTORNEY REF. NO.130341.01212 PATENT mutational loads corresponding to a known disease state diagnosis.
  • the known disease state diagnosis is selected from normal squamous, columnar epithelium without Barrett's metaplasia, Barrett's metaplasia, Barrett's metaplasia intermediate for dysplasia, low-grade dysplasia and high-grade dysplasia.
  • the risk categories are selected from no mutational load, low mutational load, and high mutational load.
  • the subject is assigned to the no mutational load risk category when the subject has mutational load of 0.0.
  • no mutational load is indicative of no risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
  • no mutational load is indicative of the absence of actionable disease.
  • the absence of actionable disease is categorized as Barrett's metaplasia with a lower risk of progression than the baseline risk for Barrett's metaplasia, wherein surveillance of the patient can be safely discontinued.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than 1.75.
  • a low mutational load is indicative of a low risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
  • a low mutational load is indicative of suitability of the subject for monitoring.
  • the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than 1.75.
  • a high mutational load is indicative of high risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
  • a high mutational load is indicative of suitability of the subject for at least one treatment modality selected from endoscopic mucosal resection, radiofrequency ablation, cryoablation, endoscopic submucosal dissection, photodynamic therapy and combinations thereof.
  • the subject is a human. In some embodiments, the subject is a human diagnosed with Barrett's esophagus.
  • the biological specimen is a mucosal lining of the esophagus. In some embodiments, the biological specimen is representative of a disease region.
  • amplifying DNA sequences comprises selecting a primer pair corresponding to a specific microsatellite region; adding the primer pair to the DNA sequences; and performing quantitative polymerase chain reaction on the DNA sequences with the primer.
  • detecting mutations comprises determining the sequence of the amplified DNA and comparing the amplified DNA to a known wild type control sequence for the specific microsatellite region and identifying differences between the sequence of the amplified DNA and the known wild type control sequence.
  • the specific microsatellite regions are selected from lp (CMM1, Lmyc), 3p (VHL, OGG1), 5q (MCC, APC), 9p (CDKN2A, CDKN2B), lOq (PTEN, MXI1), 17p (TP53), 17q (NME1), 18q (DCC), 21q, 22q (NF2) and combinations thereof.
  • categorizing clonality of each mutation comprises assigning one of three categories selected from the group consisting of no clonality, low clonality and high clonality.
  • high clonality is assigned where loss of heterozygosity is present in greater than about 75% of DNA analyzed.
  • low clonality is assigned where loss of heterozygosity is present in about 50% to about 75% of DNA analyzed.
  • no clonality is assigned where loss of heterozygosity is present in less than about 50% of DNA analyzed.
  • DNA microsatellite instability is determined by the presence of at least one of the shortening and lengthening of a DNA microsatellite region.
  • calculating a mutational load further comprises summing the clonality weighting for each specific microsatellite region showing a mutation or DNA microsatellite instability.
  • determining mutational load as a predictor of disease progression is independent of a histological standard.
  • Figure 1 depicts representative formalin-fixed, paraffin embedded (FFPE), hematoxylin and eosin (H&E) stained slide of Barrett's epithelium. Multiple re-cuts of several biopsies from the same patient are present on the slide. Multiple histological targets ATTORNEY REF. NO.130341.01212 PATENT were microdissected from such slides for molecular analyses. IN indicates a normal, non- Barrett's epithelial target used as a baseline control. 2x, 3x, and 4x indicate individual microdissected targets containing Barrett's epithelium.
  • FFPE formalin-fixed, paraffin embedded
  • H&E hematoxylin and eosin
  • Figure 2 depicts mutational load (ML) in microdissected targets by histological classification.
  • B. Levels of mutational load (no ML, low ML, and high ML) were established within targets histologically diagnosed as intestinal metaplasia and then applied to other histological classifications.
  • NS normal squamous epithelium
  • Col columnar, non-Barrett's epithelium
  • IM intestinal metaplasia
  • IND "indefinite for dysplasia”
  • LGD low-grade dysplasia
  • HGD high-grade dysplasia.
  • Figure 3 depicts the correlation of ML with histological classifications of 60 microdissected targets in 6 progressor (38 targets) and 5 non-progressor (22 targets) patients.
  • NORM normal squamous epithelium
  • IM intestinal metaplasia
  • IND "indefinite for dysplasia”
  • LGD low-grade dysplasia
  • HGD high-grade dysplasia.
  • Figure 4 depicts the number of targets with 1, 2, 3, or 4 micros atellite markers affected at particular loci.
  • COL columnar mucosa
  • IM intestinal metaplasia
  • IND "indefinite for dysplasia”
  • LGD low-grade dysplasia
  • HGD high-grade dysplasia
  • EAC esophageal adenocarcinoma.
  • patient and “subject” are meant to include any mammal including, but not limited to, humans, bovines, primates, equines, porcines, caprines, ovines, felines, canines, and any rodent (e.g., rats, mice, hamsters, and guinea pigs).
  • mammals include agricultural animals, domesticated animals, and primates, especially humans.
  • anomaly is meant a broad, encompassing term to indicate a disease related change in a cell or tissue of an organ.
  • anomaly includes cancer or dysplasia, a pre-cancerous neoplastic state, or a non-neoplastic condition.
  • Pre-cancerous states include proliferative lesions that can span a spectrum from low-grade to high-grade neoplasia.
  • non-neoplastic condition and “non-neoplastic abnormality” are meant to indicate specimens from sites known not to contain neoplasia.
  • the non-neoplastic condition may be inflammatory or any adaptive state that may include features of cell proliferation but needs to be clearly discriminated from neoplasia.
  • Bio specimen is meant to include, but is not limited to, any sample containing DNA or cells from a subject.
  • biological specimens include, but are not limited to, biopsies, fine needle aspirates, a cytology sample, a blood sample, a spinal tap, resected tissue, frozen tissue, blood sample, fixed tissue, a urine sample, a tissue swab (e.g., buccal swab or pap smear), and the like.
  • a biological specimen may include a fine needle aspiration; a biliary brushing; a core needle biopsy, an incisional biopsy, ATTORNEY REF.
  • the biological specimen may be a breast lavage sample, an ascites fluid sample, fine needle aspirates from a cyst or other region of the subject's body, urine, blood, cerebrospinal fluid, a liquid cytology sample obtained by any medically available method, and/or saliva.
  • the sample can contain cells or may contain only free-floating DNA (non-nuclear DNA) in the fluid sample.
  • the biological specimen is cellular, paucicellular, or cell-free which are meant to include the abundant presence of cells, the sparse presence of cells or the complete absence of cells respectively.
  • the biological samples can be any sample containing DNA or cells from a patient.
  • Such samples include but are not limited to fine needle aspirates, a cytology sample, a blood sample, a spinal tap, resected tissue, frozen tissue, blood sample, fixed tissue, a urine sample, a tissue swab (e.g., buccal swab or pap smear), and the like.
  • the biological specimen is a solid tissue section obtained from a subject.
  • a biological specimen may include "tissue” and "cells" as well as “fluid samples”.
  • the subject is a human with Barrett's metaplasia (BM).
  • Biological specimens may be routinely fixed in standard fixative chemical agents, of any size including minute needle biopsy specimens and cell blocks of cytology material, and of any age including those stored in paraffin for over thirty years. Solid tissue specimens, removed at surgery or through biopsy procedures, may be exposed to fixative agents designed to prevent tissue breakdown and preserve morphologic integrity for microscopic analysis and archival storage.
  • tissue and “cells,” is meant to include resected tissue (fixed, stained, or treated), cytology specimens, blood and blood fractions from a patient or from a tissue bank.
  • tissue aggressiveness or “biological aggressiveness” are meant to include the phenotypic expression of a malignancy that is associated with increased adverse biological behavior. This includes phenomenon such as capacity for early metastatic seeding, capacity for wide visceral organ dissemination, rapid growth and invasion, lack of treatment responsiveness, short treated disease free interval and short overall patient survival.
  • clonal expansion is meant a unidirectional process replacing precursor neoplastic cells with a dominant tumor cell population of cells with progressively more mutations.
  • tumor is meant to include any malignant or non-malignant tissue or cellular containing material or cells.
  • non-malignant tissue is meant to include any abnormal tissue or cell phenotype and/or genotype associated with metaplasia, hyperplasia, a ATTORNEY REF. NO.130341.01212 PATENT polyp, or pre-cancerous conditions (e.g., leukoplakia, colon polyps), regenerative change, physiologic adaption to stress or injury and cellular change in response to stress of injury.
  • Tumor is meant to include solid tumors as well as leukemia's and lymphomas.
  • Neoplasm "malignancy”, and “cancer” are used interchangeably.
  • loss of heterozygosity is meant to include the loss of normal function of one allele of a gene in which the other allele was already inactivated.
  • a common occurrence in cancer, loss of heterozygosity may indicate the absence of a functional tumor suppressor gene in a particular gene.
  • clonality or loss of heterozygosity is determined by detecting mutations in pre-determined regions of DNA.
  • the microsatellites are chosen to survey genomic instability by examining clonally expanded mutations in regions adjacent to, and in linkage disequilibrium with tumor suppressor genes commonly involved in many types of cancers.
  • the type of cancer is a carcinoma.
  • clonality is meant to include but not limited to the state of a cell or cellular DNA.
  • clonality may represent the proportion of cells from a sample or sub-sample which is affected by a particular mutation or genetic alteration.
  • clonality may represent the proportion of cellular DNA from a sample or sub-sample which is affected by a particular mutation or genetic alteration.
  • a tumor may derive from one mutated cell, wherein the progeny of this cell may be clones of the mutated cell and carry the same mutations such that they are technically a single clone of that cell. However, during course of cell division, it is possible for the progeny to acquire new mutations and acquire new characteristics to diverge as a new clone.
  • microsatellite region is meant to include, but is not limited to, repeating sequences of about 2-6 base pairs of DNA.
  • the repeating sequence is often simple, consisting of two, three or four nucleotides (di-, tri-, and tetranucleotide repeats respectively), and can be repeated 3 to 100 times.
  • BM Barrett's metaplasia
  • BM also known as Barrett's esophagus
  • Microscopic classification of dysplasia in can be subjective and, importantly, there are no histologic features that signal progression risk in early BM.
  • BM is ATTORNEY REF. NO.130341.01212 PATENT believed to be multifocal, and changes to the genome may precede morphological alteration and herald progression risk.
  • determining mutational load as a predictor of the risk of disease progression from BM to esophageal adenocarcinoma (EA) in a subject comprising: amplifying DNA sequences from a biological specimen from the subject; detecting mutations in micros atellite regions of the amplified DNA sequences; categorizing clonality of each mutation; calculating a mutational load based on the sum of low and high clonality mutations; comparing the mutational load with a series of predetermined mutational load cut-offs defining risk categories; and assigning the subject to a risk category corresponding to the subjects mutational load, wherein each risk category is indicative of the risk of disease progression.
  • EA esophageal adenocarcinoma
  • the pre-determined mutational load cut-offs defining risk categories are derived from a pre-determined patient population distribution with known mutational loads corresponding to a known disease state diagnosis.
  • the known disease state diagnosis is selected from normal squamous, columnar epithelium without BM, BM, BM intermediate for dysplasia, low grade dysplasia (LGD) and high grade dysplasia (HGD).
  • the risk categories are selected from no mutational load, low mutational load, and high mutational load.
  • the subject is assigned to the no mutational load risk category when the subject has mutational load of 0.0.
  • no mutational load is indicative of no risk of disease progression from BM to EA.
  • no mutational load is indicative of the absence of actionable disease.
  • actionable disease is categorized as.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than or equal to 2.0.
  • a low mutational load is indicative of a low risk of disease progression from BM to EA. In some embodiments, a low mutational load is indicative of suitability of the subject for monitoring.
  • the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than 2.0.
  • a high mutational load is indicative of high risk of disease progression from BM to EA.
  • a high mutational load is indicative of suitability of the subject for at least one treatment modality selected from endoscopic mucosal resection, radiofrequency ablation, cryoablation, endoscopic submucosal dissection, photodynamic therapy and combinations thereof.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than or equal to 1.0. In some embodiments, the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than 1.0.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than or equal to 1.5. In some embodiments, the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than 1.5.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than or equal to 2.5. In some embodiments, the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than 2.5.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than or equal to 3.0. In some embodiments, the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than 3.0.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than or equal to 3.5. In some embodiments, the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than 3.5.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than or equal to 4.0. In some embodiments, the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than 4.0.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than or equal to a mutational load of about 0.1 to about 5.0. In some embodiments, the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than between 0.1 and 5.0.
  • the risk categories are selected from no mutational load, low mutational load, and high mutational load.
  • the subject is assigned to the no mutational load risk category when the subject has mutational load of 0.0.
  • no mutational load is indicative of no risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
  • no mutational load is indicative of the absence of actionable disease.
  • the absence of actionable disease is categorized as Barrett's metaplasia with a lower risk of progression than the baseline risk for Barrett's metaplasia, wherein surveillance of the patient can be safely discontinued.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than 1.75.
  • a low mutational load is indicative of a low risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
  • a low mutational load is indicative of suitability of the subject for monitoring.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than 2.0.
  • a low mutational load is indicative of a low risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
  • a low mutational load is indicative of suitability of the subject for monitoring.
  • the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than 2.0. In some embodiments, the subject is assigned to the high mutational load risk category when the subject has a ATTORNEY REF. NO.130341.01212 PATENT mutational load greater than or equal to 2.0. In some embodiments, the wherein a high mutational load is indicative of high risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
  • a high mutational load is indicative of suitability of the subject for at least one treatment modality selected from endoscopic mucosal resection, radiofrequency ablation, cryoablation, endoscopic submucosal dissection, photodynamic therapy and combinations thereof.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than or equal to 1.75.
  • a low mutational load is indicative of a low risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
  • a low mutational load is indicative of suitability of the subject for monitoring.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than 1.75.
  • a low mutational load is indicative of a low risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
  • a low mutational load is indicative of suitability of the subject for monitoring.
  • the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than 1.75. In some embodiments, the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than or equal to 1.75. In some embodiments, the wherein a high mutational load is indicative of high risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma. In some embodiments, a high mutational load is indicative of suitability of the subject for at least one treatment modality selected from endoscopic mucosal resection, radiofrequency ablation, cryoablation, endoscopic submucosal dissection, photodynamic therapy and combinations thereof.
  • the pre-determined mutational load cut-offs are variable.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than or equal to a mutation load ranging from about 1.75 to about 2.0.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than a mutation load ranging from about 1.75 to about 2.0.
  • the subject is assigned to the high mutational load risk category when the subject has a mutational load greater from about 1.75 to about 2.0.
  • the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than or equal from about 1.75 to about 2.0.
  • the subject is a human. In some embodiments, the subject is a human diagnosed with BM. In some embodiments, the biological specimen is a mucosal lining of the esophagus. In some embodiments, the biological specimen comprises the mucosal lining of the esophagus. In some embodiments, the biological specimen comprises a deeper tissue type. These deeper tissue types are capable of forming sarcomas. In other embodiments, the specimen is a tissue section. In some embodiments, the biological specimen is representative of a disease region.
  • the biological specimen is a formalin-fixed, paraffin embedded tissue section.
  • the tissue section is from the esophagus of the subject.
  • the biological specimen is a fresh section of esophagus.
  • biological specimens are selected for the presence of BM. In other embodiments, selection is accomplished by identification of histological features characteristic of BM. In some embodiments, biological specimens are selected independently of a histological standard.
  • the subject is a human with squamous dysplasia.
  • the majority of EA cases arise from BM were the squamous mucosa transforms first to a mucinous lining and then to a mucinous cancer. This is defined for about 80% of EA cases. The remainder is squamous cancers where the squamous lining directly transforms to squamous cancer.
  • the subject is a human diagnosed with esophageal squamous cancer.
  • amplifying DNA sequences comprises selecting a primer pair corresponding to a specific microsatellite region; adding the primer pair to the DNA sequences; and performing quantitative polymerase chain reaction on the DNA sequences with the primer.
  • the first step in the qPCR process is to adjust the DNA concentration to a value of about 5 ng/ ⁇ so that the absolute amount of DNA present in each reaction is the same, but so that the integrity may vary which is the purpose of the analysis.
  • 5 ATTORNEY REF. NO.130341.01212 PATENT ng/ ⁇ is preferred as it has been found to be a minimal value for robust amplification.
  • other amounts may be used. For example, from about 10 ng/ ⁇ to about 10 ⁇ g/ ⁇ L and more preferably from about 5 ng/ ⁇ to about 5 ⁇ g/ ⁇ L may be used.
  • the number of qPCR cycles may be used as a marker of DNA quality.
  • the lower the number cycles required to reach a desired threshold is indicative of higher quality DNA.
  • the DNA quality is considered suboptimal due to, for example, allelic imbalance resulting from inadequate amounts of template DNA.
  • Ct values i.e., threshold values for quantitative PCR product detection
  • Ct values of 29.0-30.0 are considered borderline. Values of 29.0 or less are indicative of good quality DNA.
  • DNA quality may be further assessed by performing competitive template polymerase chain reaction (PCR) amplification for a unique pair of genes, (e.g., Glucocerebrosidase Gene and its pseudogene) at a particular point where the two genes have virtually identical sequences, with the exception of a 55 base pair deletion in the pseudogene. This is not the only gene that can serve this purpose. In some embodiments, any pairing of gene or genomic segments of similar sequence but differences in length can be substituted.
  • PCR template polymerase chain reaction
  • this PCR reaction creates two amplicons that are identical in sequence except for the deletional region. During the reaction, a competition exists between the two similar templates (but having different lengths). In some embodiments, the degree of DNA degradation in the sample will be reflected by less effective amplification of the longer template as compared to the shorter template. In some embodiments, this serves as a measure of DNA integrity. In some embodiments, the amount of each product, short and long, may be quantitatively measured by capillary electrophoresis. Methods of performing the PCR reaction and electrophoresis are well known in the art.
  • reagents are added to the final sample the purpose of which is to enhance DNA availability, to enhance the ability to amplify the DNA, and DNA quantity.
  • any system that utilizes similar primers to amplify products of different lengths can be substituted.
  • the procedure for qPCR and PCR amplification has been well described and variations on its performance will not impact the various embodiments of the invention.
  • the recommended procedures of the manufacturers for the PCR reagents are closely followed (Gene Amp kit, Applied Biosystems).
  • other commercial and non-commercial systems for qPCR and PCR amplification can be readily substituted.
  • the qPCR or PCR reaction is performed in a manner that is highly robust, especially when using minute DNA samples.
  • robust in this context is meant a qPCR or PCR reaction that reliably generates abundant amplified DNA that accurately reflects the starting composition mixture of normal and mutated DNA derived from a particular specimen.
  • Reagents such as dimethylsulfoxide or dextran sulfate may be added to the amplification reaction to enhance amplification. In some embodiments, other similar reagents can be substituted. In some embodiments, manipulations, such as nested PCR, may be performed to further enhance amplification.
  • DNA is amplified from fixative treated biological specimens. Once DNA amplification has been carried out from a fixative treated biological specimen it is vital that rigorous separation of primers and most importantly small sized nonspecific amplification products may be performed in such a way as to isolate the desired amplification product as purely as possible. Due to the factors listed above, fixative treated biological specimens tend to produce a relatively greater amount of such nonspecific products, which can be seen as a smear effect on horizontal gel electrophoresis. If these products are carried into the subsequent genotyping steps such as DNA sequencing, they may result in artificial bands or weak ineffective sequencing reactions.
  • One approach to isolating specific amplification product is to carry out agarose gel electrophoresis at relatively high agarose concentrations such as 3% to free the desired appropriate amplification product from nonspecific contaminants.
  • a rate limiting step when handling fixative treated biological specimens for genetic analysis may be effective and specific DNA amplification.
  • DNA that has been exposed to chemical fixative such as formaldehyde are often unsuccessfully or only poorly amplified.
  • chemical fixative such as formaldehyde
  • the reason for the inability to efficiently amplify fixed tissues is known to be related to chemical action of the fixative agent upon nucleic acids, the precise mechanism most directly related to poor amplification is only poorly understood.
  • the most suitable measures to overcome this ATTORNEY REF. NO.130341.01212 PATENT detrimental effect are therefore not fully appreciated. Instead, recourse is usually taken to sacrificing large amounts of fixative treated tissue or abandoning the use of fixative treated tissues altogether.
  • any region of a gene can be amplified provided sufficient sequence information is available upon which to formulate amplifying and sequencing primers; short DNA sequences, such as 18-30 base pair long, most easily created by means of an oligonucleotide synthesizer apparatus.
  • these primers direct the amplification and sequencing of DNA.
  • Oligonucleotide primer pairs are usually designed to amplify a genomic region approximately 200 base pairs in length, although longer lengths can be effectively amplified from fixative treated tissues. Either amplifying primer can serve as a sequencing primer, but design and use of an internal primer may in some case be worthwhile to achieve a clean sequencing band pattern.
  • sequencing will be performed by means of dideoxy chain termination with 35 S radionucleotide incorporation, it is important to select a radionucleotide that will be incorporated as close to the 30 end of the ultimate sequencing primer, ideally within three bases and several times within the first 10 bases.
  • DNA sequences from a biological specimen or amplified DNA can be analyzed for quantity, quality or a combination thereof.
  • optical density (OD) analysis is performed to quantify the DNA.
  • One approach uses the nanodrop technique, because it requires only one microliter to be sacrificed for the purpose of obtaining the DNA concentration. Other techniques for quantifying DNA will serve quite adequately for this purpose.
  • the higher optical density (OD) value the larger amount of DNA is present.
  • the quantity of DNA extracted can vary.
  • the DNA can be quantified by measurement of the optical density to fluorescent light at wavelengths of 230, 260, and 280 nm.
  • the 260/280 and 260/230 ratios may be 1.7-2.0, in keeping with extraction of purified DNA and for the purpose to exclude protein and other contaminants.
  • any technique that defines the amount of DNA in the sample can be a suitable substitute.
  • DNA quality, or degree of degradation is also determined.
  • quantitative PCR qPCR
  • ATTORNEY REF. NO.130341.01212 PATENT competitive template PCR as described above.
  • a competitive duplex PCR reaction of highly similar DNA sequence but differing in length is needed. In some embodiments, this can be accomplished by simply carrying out a short and long product PCR reaction in one container (e.g., test tube) on one source of DNA.
  • qPCR quantitative PCR
  • the qPCR reaction can be performed using sybr green as the indicator in a suitable thermo cycler capable of measuring fluorescence during the amplification process as this is the simplest and least costly technique.
  • Other techniques for qPCR determination using fluorescent labeled primers can substitute just as well.
  • Known quantitative controls and replicate analysis of samples may be used to standardize amplification reactions and is recommended; the exact use and configuration of controls and replicate analysis may be varied as determined by the user. Standardization of quantitative PCR amplification of the first exon of the K-ras-2 gene may be used.
  • any PCR product from any gene or genomic segment that amplifies reliably may be effectively used.
  • detecting mutations comprises determining the sequence of the amplified DNA and comparing the amplified DNA to a known wild type control sequence for the specific microsatellite region and identifying differences between the sequence of the amplified DNA and the known wild type control sequence.
  • detecting mutations comprises determining the sequence of the amplified DNA by capillary gel electrophoresis and comparing the amplified DNA to a known wild type control sequence for the specific microsatellite regions. Wild type sequences can be obtained from databases known to those skilled in the art.
  • the NCBI database may be used to design primers and obtain current sequence information.
  • the Ensembl database may be used to cross reference a sequence and determine the intron-exon boundaries for a particular gene.
  • sequences may be obtained from the UCSC Genome Bowser, the NCBI databases or combinations thereof. ATTORNEY REF. NO.130341.01212 PATENT
  • the specific microsatellite regions are selected from lp (CMM1, Lmyc), 3p (VHL, OGG1), 5q (MCC, APC), 9p (CDKN2A, CDKN2B), lOq (PTEN, MXI1), 17p (TP53), 17q (NME1), 18q (DCC), 21q, 22q (NF2) and combinations thereof.
  • mutations are in detected in specific chromosomal regions.
  • the regions including but not limited to those in the table below or combinations thereof.
  • the microsatellite panel is comprised of the chromosomal regions in the table 1 below.
  • primers flanking a specific chromosomal region can be designed based on currently known primer design principles of DNA sequencing.
  • primer sets flanking one or more of the chromosomal regions described in the table below can be designed based on currently known primer design principles of DNA sequencing.
  • primers flanking a specific chromosomal region can be designed based on currently known primer design principles of PCR.
  • primer sets flanking one or more of the chromosomal regions described in the table below can be designed based on currently known primer design principles of PCR.
  • PCR amplification may be used to generate amplicons of less than 200 nucleotides using synthetic oligonucleotide primers flanking each microsatellite.
  • Allele peak heights and lengths may be used to define the presence or absence of allelic imbalance and clonality (i.e., LOH) for a given sample.
  • Allelic imbalance is reported when the ratio of polymorphic allelic bands for a particular marker is beyond about 95% confidence limits for the variation in peak heights for individual allele pairings derived from analysis using nonneoplastic specimen samples. In general, this is below about 0.5 or above about 2.0.
  • the allele ratio is two standard deviations beyond the average for the ratio of the specific pairing of polymorphic alleles.
  • allelic imbalance mutations are treated as genomic deletions associated with tumor suppressor genes.
  • the ratio of allele peak heights is a measure of an admixture of mutated and non-mutated cells or DNA, and varies according to the individual pairing of specific microsatellite marker alleles. Allele ratios of 2.0 or 0.5 is ATTORNEY REF. NO.130341.01212 PATENT said to be present when 50% of the total DNA is derived from cells possessing the loss. The deviation from ideal normal ratio of 1.0 indicates which specific allele was affected. Allele ratios below about 0.5 or above about 2.0 are mathematically correlated with the proportion of cells affected by genomic loss.
  • allelic imbalance determination can be carried out as follows. Post-amplification products are electrophoresed and relative fluorescence determined for individual allele peak height. The ratio of peaks is calculated by dividing the value for the shorter-sized allele by that of the longer-sized allele. Thresholds for significant allelic imbalance have been determined beforehand in studies using normal (i.e., nonneoplastic) specimens representing each unique pairing of individual alleles for every marker used in the panel. In Some embodiments, the normal range of variability is established for a particular marker. In further embodiments, the range may be characterized by two standard deviations, or quantiles of the distribution of peak height ratios, or similar techniques aimed at characterizing normal variation of the marker in non-mutated DNA.
  • Peak height ratios falling outside of the normal range of variability are assessed as showing significant allelic imbalance.
  • the non-neoplastic tissue targets are used to establish informativeness status and then to determine the individual pattern of polymorphic marker alleles. Having established significant allelic imbalance, it is then possible to calculate the proportion of cellular DNA that was subject to hemizygous loss. For example, a polymorphic marker pairing whose peak height ratio was ideally 1.00 in normal tissue with a standard deviation in non-neoplastic tissue of 0.23, could be inferred to have 50% of its cellular content affected by hemizygous loss if the peak height ratio was 0.5 or 2.0.
  • allelic imbalance requires that a minimum of 50% of the DNA in a given sample be derived from cells possessing deletion of the specific microsatellite marker. The deviation from ideal normal ratio of 1.0 indicated which specific allele was affected. In a similar fashion, allele ratios below 0.5 or above 2.0 could be mathematically correlated with the proportion of cells affected by genomic loss. Other algorithms for quantitative determination of allelic imbalance can be used with equal effectiveness.
  • categorizing clonality of each mutation comprises assigning one of three categories selected from the group consisting of no clonality, low clonality and high clonality.
  • high clonality is assigned where loss of heterozygosity is present in greater than about 75% of DNA analyzed.
  • low clonality is assigned where loss of heterozygosity is present in about 50% to about 75% of DNA analyzed.
  • no clonality is assigned where loss of heterozygosity is present in less than about 50% of DNA analyzed.
  • microsatellite mutation detection, and allelic imbalance indicates copy number change.
  • clonality is interpreted as the percentage of cells affected by a copy number change, nominally a deletion that confers loss of heterozygosity in a linked tumor suppressor gene.
  • clonality is interpreted as the percentage of DNA affected by a copy number change, nominally a deletion that confers loss of heterozygosity in a linked tumor suppressor gene.
  • allelic imbalance mutations are treated as genomic deletions associated with tumor suppressor genes.
  • the ratio of allele peak heights is a measure of an admixture of mutated and non-mutated cells or DNA, and varies according to the individual pairing of specific microsatellite marker alleles. Allele ratios of 2.0 or 0.5 is said to be present when 50% of the total DNA is derived from cells possessing the loss. The deviation from ideal normal ratio of 1.0 indicates which specific allele was affected. Allele ratios below about 0.5 or above about 2.0 are mathematically correlated with the proportion of cells affected by genomic loss.
  • determining clonality can be carried out as follows. Post-amplification products are electrophoresed and relative fluorescence determined for individual allele peak height. The ratio of peaks is calculated by dividing the value for the shorter-sized allele by that of the longer-sized allele. Thresholds for significant allelic imbalance have been determined beforehand in extensive studies using normal (i.e., nonneoplastic) specimens representing each unique pairing of individual alleles for every marker being used. Peak height ratios falling outside of two standard deviations beyond the mean for each polymorphic allele pairing were assessed as showing significant allelic imbalance. In each case, the non-neoplastic tissue targets are used to establish informativeness status and then to determine the individual pattern of polymorphic marker alleles.
  • allelic imbalance it is then possible to calculate the proportion of cellular DNA that was subject to hemizygous loss.
  • a polymorphic marker pairing whose peak height ratio was ideally 1.00 in normal tissue with a standard deviation in non-neoplastic tissue of 0.23, could be inferred to have 50% of its cellular content affected by hemizygous loss if the peak height ratio was 0.5 or 2.0.
  • the deviation from ideal normal ratio of 1.0 indicated which specific allele was affected.
  • allele ratios below 0.5 or above 2.0 could be mathematically correlated with the proportion of cells affected by genomic loss.
  • Other algorithms for quantitative determination of allelic imbalance can be used with equal effectiveness.
  • calculating the mutational load comprises assigning a score to each mutation based on a categorization of low or high clonality of each mutation, wherein the score for low clonality is 0.5x, wherein x is the number of low clonality mutations and the score for high clonality is y, wherein y is the number of high clonality mutations; and wherein the overall mutational load is y + 0.5x.
  • calculating the mutational load further comprises assigning a score to each mutation based on detection of DNA microsatellite instability at a particular locus, wherein DNA microsatellite instability at a single locus is defined as 0.75z, wherein z is the number of loci displaying DNA microsatellite instability; wherein the score for low clonality is 0.5x, wherein x is the number of low clonality mutations and the score for high clonality is y, wherein y is the number of high clonality mutations; and wherein the overall mutational load is y + 0.5x + 0.75z.
  • DNA microsatellite instability is determined by the presence of at least one of the shortening and lengthening of a DNA microsatellite region. In some embodiments, the emergence of new alleles, manifested as DNA microsatellite instability mutations" as this is the first MSI that has been seen in these markers.
  • calculating the mutational load further comprises assigning a score to each mutation based on detection of DNA microsatellite instability at multiple loci, wherein DNA microsatellite instability at multiple loci is defined as 2z, wherein z is the number of loci displaying DNA microsatellite instability; wherein the score for low clonality is 0.5x, wherein x is the number of low clonality mutations and the score for high clonality is y, wherein y is the number of high clonality mutations; and wherein the overall mutational load is y + 0.5x + 2z.
  • DNA microsatellite instability is determined by the presence of at least one of the shortening and lengthening of a DNA microsatellite region. In some embodiments, the emergence of new alleles, manifested ATTORNEY REF. NO.130341.01212 PATENT as DNA micros atellite instability mutations" as this is the first MSI that has been seen in these markers.
  • calculating the mutational load further comprises assigning a score to each mutation based on detection of DNA microsatellite instability at multiple loci, wherein DNA microsatellite instability at multiple loci is defined as lz, wherein z is the number of loci displaying DNA microsatellite instability; wherein the score for low clonality is 0.5x, wherein x is the number of low clonality mutations and the score for high clonality is y, wherein y is the number of high clonality mutations; and wherein the overall mutational load is y + 0.5x + lz.
  • DNA microsatellite instability is determined by the presence of at least one of the shortening and lengthening of a DNA microsatellite region. In some embodiments, the emergence of new alleles, manifested as DNA microsatellite instability mutations" as this is the first MSI that has been seen in these markers.
  • calculating the mutational load further comprises assigning a score to each mutation based on detection of DNA microsatellite instability at multiple loci, wherein DNA microsatellite instability at multiple loci is defined as 0.5z, wherein z is the number of loci displaying DNA microsatellite instability; wherein the score for low clonality is 0.5x, wherein x is the number of low clonality mutations and the score for high clonality is y, wherein y is the number of high clonality mutations; and wherein the overall mutational load is y + 0.5x + 0.5z.
  • DNA microsatellite instability is determined by the presence of at least one of the shortening and lengthening of a DNA microsatellite region. In some embodiments, the emergence of new alleles, manifested as DNA microsatellite instability mutations" as this is the first MSI that has been seen in these markers.
  • calculating the mutational load further comprises assigning a score to each mutation based on detection of DNA microsatellite instability at a particular locus, wherein DNA microsatellite instability at a single locus is defined as 0.75zi, and wherein DNA microsatellite instability at multiple loci is defined as 0.5z 2 , wherein zi is the number of loci displaying DNA microsatellite instability and Z2 is the number of loci displaying DNA microsatellite instability greater than 1 ; wherein the score for low clonality is 0.5x, wherein x is the number of low clonality mutations and the score for high clonality is ATTORNEY REF. NO.130341.01212 PATENT y, wherein y is the number of high clonality mutations; and wherein the overall mutational load is y + 0.5x + 0.75z ! + 0.5z 2 .
  • calculating the mutational load further comprises assigning a score to each mutation based on detection of DNA microsatellite instability at a particular locus, wherein DNA microsatellite instability at a single locus is defined as 0.75zi, and wherein DNA microsatellite instability at multiple loci is defined as 0.75zi + 0.5z 2 , wherein zi represent a single locus displaying DNA microsatellite instability and Z2 is the number of loci displaying DNA microsatellite instability greater than 1 locus; wherein the score for low clonality is 0.5x, wherein x is the number of low clonality mutations and the score for high clonality is y, wherein y is the number of high clonality mutations; and wherein the overall mutational load is y + 0.5x + 0.75zi + 0.5(z 2 ).
  • zi 1.
  • mutational load y + 0.5x + 0.75(1) + 0.5(2).
  • mutational load y + 0.5x + 0.75(1).
  • calculating a mutational load further comprises summing the clonality weighting for each specific microsatellite region showing a mutation or DNA microsatellite instability.
  • weightings for high clonality mutations range from 2.0 to 2.5, and those for low clonality mutations range from 1 to 1.4. Since the absolute overall score can be arbitrarily scaled (i.e. 1-10 or 1-100 can be equivalent scores), any weighting maintaining these proportions will produce equivalent cutoffs scaled by the chosen scaling factor.
  • the overall score for a case is the maximum score of any target. This is consistent with standard practice in microscopic evaluation of Barrett's esophagus and many other neoplastic conditions.
  • particular weightings may be assigned to a particular mutation, DNA microsatellite region or a combination thereof. In some embodiments, weightings are equal for all mutations in particular DNA microsatellite regions analyzed. In some embodiments, mutations in particular DNA microsatellite regions may be given higher weightings than other mutations in particular DNA microsatellite regions. In some ATTORNEY REF. NO.130341.01212 PATENT embodiments, certain mutations in particular DNA microsatellite regions may have a greater influence on the ML for a particular subject. In some embodiments, mutations in particular DNA microsatellite regions may include, but are not limited to mutations in 17p (TP53) and 9p (CDKN2A, CDKN2B).
  • calculating a mutational load further comprises assigning a distinct weighting for a mutation depending on the genomic locus of the mutations.
  • a low clonality mutation at 17p (TP53) or 9p (pi 6) may be weighted as 0.55 up to 0.75 rather than 0.5, and a high clonality mutation may be weighted as 1.05 up to 1.5.
  • multiple microsatellite markers may flank a particular tumor suppressor gene.
  • the count of mutations may count the presence of any or all mutations for a given locus (i.e. all markers in proximity to a particular tumor suppressor, or if only one tumor suppressor gene per chromosome arm is interrogated, all markers per chromosome arm) as a single mutation.
  • weighting of mutation is defined and clonality is determined by statistical techniques such as logistic regression, discriminant analysis or machine learning techniques such as neural networks or support vector machines. Many such techniques will also produce estimated cut-offs between distinct disease states in the progression of BM (i.e., between disease states of non-dysplastic BM (i.e., intestinalized columnar mucosal metaplasia).
  • cut-offs may be calculated without regression by use of receiver-operator characteristics to define desired performance characteristics of a particular cut-off depending the desired balance of test performance characteristics (e.g. sensitivity, specificity and overall accuracy).
  • analysis of clonal diversity can supplement the embodiments described herein.
  • clonal diversity can in addition to the embodiments described herein serve as a factor in the prediction of disease progression.
  • the presence of a mutation spanning multiple markers near a particular tumor suppressor gene can serve as additional weighting for a mutation.
  • this weighting is 0.25 per additional microsatellite marker affected (i.e., 1.25 for a high clonality mutation where 2 markers are affected, 1.5 where 3 are affected, ATTORNEY REF. NO.130341.01212 PATENT etc.).
  • mutations with lengths of mutated segment of DNA greater than 5 million base pairs, measured by the number of base pairs between distinct microsatellite markers at the same genomic locus are given an extra weighting above that assigned for clonality.
  • mutations affecting more than 1 million bases are given an extra weighting of 0.25.
  • mutations affecting more than 5 million base pairs are given an extra weighting of 0.5
  • determining mutational load is independent of a histological standard.
  • microscopic slides associated with a biological specimen, if present, are first reviewed, as well as any clinical information pertinent to the individual patient.
  • analysis of DNA from a biological specimen can be performed without preliminary review of microscopic features.
  • Some embodiments are directed to methods of identifying the presence of dysplasia in a subject comprising: amplifying DNA sequences from a biological specimen from a subject with Barrett's esophagus; detecting mutations in specific microsatellite regions of amplified DNA sequences; categorizing clonality to each mutation; calculating a clonality weighting for each mutation; calculating a weighted mutation count; and comparing the weighted mutation count with a cut-off weighted mutation count, wherein a weighted mutation count above the cut-off weighted mutation count indicates the presence of dysplasia.
  • the weighted mutation count may indicate a greater degree of neoplastic change than is evident from morphologic change on histology examination. For example, regions of intestinal metaplasia taken from a patient with dysplasia can show a greater degree of change than intestinal metaplasia from a non- dysplastic patient. This "progressor metaplasia" may indicate more advanced disease in the patient than is indicated by morphologic inspection. This may explain why a substantial proportion of esophageal adenocarcinoma patients present without a previous diagnosis of dysplasia.
  • the detection of progressor metaplasia may be used to determine risk of disease progression in a non-dysplastic patient, which in turn may be used to determine the appropriateness of therapeutic interventions such as radiofrequency ablation of the esophagus.
  • techniques to relate independent variables to class- based outcomes/dependent variables can used to predict non-dysplastic or dysplastic disease from number and clonality of mutations, including linear discriminant analysis to discriminate non-dysplastic from dysplastic disease, and including regression technique such as proportional odds logistic regression to predict disease state based on number and clonality of mutations.
  • the cut-off weighted mutation count cut-off separating low grade dysplasia from high grade dysplasia is 5.3. In some embodiments, the cut-off weighted mutation count cutoff separating low grade dysplasia from high grade dysplasia is 7.9.
  • Some embodiments are directed to methods of treating a subject with a high risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma, the method comprising: amplifying DNA sequences from a biological specimen from the subject; detecting mutations in micros atellite regions of the amplified DNA sequences; categorizing clonality of each mutation; calculating a mutational load based on the sum of low and high clonality mutations; comparing the mutational load with a series of pre-determined mutational load cut-offs defining risk categories; assigning the subject to a risk category corresponding to the subjects mutational load, wherein each risk category is indicative of the risk of disease progression; determining if the subject is in a risk category where treatment is indicated; and administering to the subject a at least one treatment modality selected from endoscopic mucosal resection, endoscopic submucosal dissection, a therapeutically effective amount of radiofrequency ablation, a therapeutically effective amount of cry
  • the pre-determined mutational load cut-offs defining risk categories are derived from a pre-determined patient population distribution with known mutational loads corresponding to a known disease state diagnosis.
  • the known disease state diagnosis is selected from normal squamous, columnar epithelium without Barrett's metaplasia, Barrett's metaplasia, Barrett's metaplasia intermediate for dysplasia, low grade dysplasia and high grade dysplasia.
  • the risk categories are selected from no mutational load, low mutational load, and high mutational load.
  • the subject is assigned to the no mutational load risk category when the subject has mutational load of 0.0.
  • no mutational load is indicative of no risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
  • no mutational load is indicative of the absence of actionable disease.
  • no actionable disease is categorized as Barrett's metaplasia with a lower risk of progression than the baseline risk for Barrett's metaplasia, such that surveillance of the patient can be safely discontinued.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than or equal to 2.0.
  • a low mutational load is indicative of a low risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
  • a low mutational load is indicative of suitability of the subject for monitoring.
  • the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than 2.0.
  • a high mutational load is indicative of high risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
  • a high mutational load is indicative of suitability of the subject for at least one treatment modality selected from endoscopic mucosal resection, endoscopic submucosal dissection, a therapeutically effective amount of radiofrequency ablation, a therapeutically effective amount of cryoablation, a therapeutically effective amount of photodynamic therapy and combinations thereof.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than or equal to 1.0. In some embodiments, the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than 1.0.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than or equal to 1.5. In some embodiments, the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than 1.5.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than or equal to 2.5.
  • the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than 2.5.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than or equal to 3.0. In some embodiments, the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than 3.0.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than or equal to 3.5. In some embodiments, the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than 3.5.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than or equal to 4.0. In some embodiments, the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than 4.0.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than or equal to a mutational load of about 0.1 to about 5.0. In some embodiments, the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than between 0.1 and 5.0.
  • the risk categories are selected from no mutational load, low mutational load, and high mutational load.
  • the subject is assigned to the no mutational load risk category when the subject has mutational load of 0.0.
  • no mutational load is indicative of no risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
  • no mutational load is indicative of the absence of actionable disease.
  • the absence of actionable disease is categorized as Barrett's metaplasia with a lower risk of progression than the baseline risk for Barrett's metaplasia, wherein surveillance of the patient can be safely discontinued.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than 1.75.
  • a low mutational load is indicative of a low risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
  • a low mutational load is indicative of suitability of the subject for monitoring.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than 2.0.
  • a low mutational load is indicative of a low risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
  • a low mutational load is indicative of suitability of the subject for monitoring.
  • the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than 2.0. In some embodiments, the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than or equal to 2.0. In some embodiments, the wherein a high mutational load is indicative of high risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma. In some embodiments, a high mutational load is indicative of suitability of the subject for at least one treatment modality selected from endoscopic mucosal resection, radiofrequency ablation, cryoablation, endoscopic submucosal dissection, photodynamic therapy and combinations thereof.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than or equal to 1.75.
  • a low mutational load is indicative of a low risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
  • a low mutational load is indicative of suitability of the subject for monitoring.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than 1.75.
  • a low mutational load is indicative of a low risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
  • a low mutational load is indicative of suitability of the subject for monitoring.
  • the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than 1.75. In some embodiments, the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than or equal to 1.75. In some embodiments, the ATTORNEY REF. NO.130341.01212 PATENT wherein a high mutational load is indicative of high risk of disease progression from Barrett's metaplasia to esophageal adenocarcinoma.
  • a high mutational load is indicative of suitability of the subject for at least one treatment modality selected from endoscopic mucosal resection, radiofrequency ablation, cryoablation, endoscopic submucosal dissection, photodynamic therapy and combinations thereof.
  • the pre-determined mutational load cut-offs are variable.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than or equal to a mutation load ranging from about 1.75 to about 2.0.
  • the subject is assigned to the low mutational load risk category when the subject has a mutational load greater than 0.0 but less than a mutation load ranging from about 1.75 to about 2.0.
  • the subject is assigned to the high mutational load risk category when the subject has a mutational load greater from about 1.75 to about 2.0.
  • the subject is assigned to the high mutational load risk category when the subject has a mutational load greater than or equal from about 1.75 to about 2.0.
  • the indication for a particular clinical management of a patient to prevent or eradicate cancer depends on the cost of the management, the risk of disease progression toward cancer, and the tolerability and side effects associated with that management choice.
  • ML stratifies the risk of disease progression, and the cost of the intervention does not depend on the ML observed in the patient, the utility of ML in improving risk assessment for disease progression is the ability to intervene earlier in those patients that need it while sparing other patients intervention and allowing a reduction of surveillance in others.
  • different ML levels would be associated with distinct clinical management strategies. In some embodiments, improves the estimation of risk of disease progression.
  • disease eradications include ablation radiofrequency, cryotherapy, plasma/photodynamic therapy), esophageal mucosal resection, esophageal submucosal enucleation, and combinations thereof.
  • the subject is a human. In some embodiments, the subject is a human diagnosed with Barrett's esophagus. In some embodiments, the biological specimen is a mucosal lining of the esophagus. In some embodiments, the biological specimen is representative of a disease region.
  • amplifying DNA sequences comprises: selecting a primer pair corresponding to a specific microsatellite region; adding the primer pair to the DNA sequences; and performing quantitative polymerase chain reaction on the DNA sequences with the primer.
  • detecting mutations comprises determining the sequence of the amplified DNA and comparing the amplified DNA to a known wild type control sequence for the specific microsatellite region and identifying differences between the sequence of the amplified DNA and the known wild type control sequence.
  • the specific microsatellite regions are selected from lp (CMM1, Lmyc), 3p (VHL, OGG1), 5q (MCC, APC), 9p (CDKN2A, CDKN2B), lOq (PTEN, MXI1), 17p (TP53), 17q (NME1), 18q (DCC), 21q, 22q (NF2) and combinations thereof.
  • categorizing clonality of each mutation comprises assigning one of three categories selected from the group consisting of no clonality, low clonality and high clonality.
  • high clonality is assigned where loss of heterozygosity is present in greater than about 75% of DNA analyzed.
  • low clonality is assigned where loss of heterozygosity is present in about 50% to about 75% of DNA analyzed.
  • no clonality is assigned where loss of heterozygosity is present in less than about 50% of DNA analyzed.
  • calculating the mutational load comprises assigning a score to each mutation based on a categorization of low or high clonality of each mutation, wherein the score for low clonality is 0.5x, wherein x is the number of low clonality mutations and the score for high clonality is y, wherein y is the number of high clonality mutations; and wherein the overall mutational load is y + 0.5x.
  • mutation load can be expressed generally as X*W Low + y*Wmg h , where W L OW and Wmg h , are weightings for low and high clonality respectively.
  • calculating the mutational load further comprises assigning a score to each mutation based on detection of DNA microsatellite instability at a particular locus, wherein DNA microsatellite instability at a single locus is defined as 0.75z, wherein z is the number of loci displaying DNA microsatellite instability; wherein the score for low clonality is 0.5x, wherein x is the number of low clonality mutations and the score for high clonality is y, wherein y is the number of high clonality mutations; and wherein the overall mutational load is y + 0.5x + 0.75z.
  • DNA microsatellite instability is determined by the presence of at least one of the shortening and lengthening of a DNA microsatellite region. In some embodiments, the emergence of new alleles, manifested as DNA microsatellite instability mutations" as this is the first MSI that has been seen in these markers.
  • calculating the mutational load further comprises assigning a score to each mutation based on detection of DNA microsatellite instability at ATTORNEY REF. NO.130341.01212 PATENT multiple loci, wherein DNA micros atellite instability at multiple loci is defined as 2z, wherein z is the number of loci displaying DNA microsatellite instability; wherein the score for low clonality is 0.5x, wherein x is the number of low clonality mutations and the score for high clonality is y, wherein y is the number of high clonality mutations; and wherein the overall mutational load is y + 0.5x + 2z.
  • DNA microsatellite instability is determined by the presence of at least one of the shortening and lengthening of a DNA microsatellite region. In some embodiments, the emergence of new alleles, manifested as DNA microsatellite instability mutations" as this is the first MSI that has been seen in these markers.
  • calculating the mutational load further comprises assigning a score to each mutation based on detection of DNA microsatellite instability at multiple loci, wherein DNA microsatellite instability at multiple loci is defined as lz, wherein z is the number of loci displaying DNA microsatellite instability; wherein the score for low clonality is 0.5x, wherein x is the number of low clonality mutations and the score for high clonality is y, wherein y is the number of high clonality mutations; and wherein the overall mutational load is y + 0.5x + lz.
  • DNA microsatellite instability is determined by the presence of at least one of the shortening and lengthening of a DNA microsatellite region. In some embodiments, the emergence of new alleles, manifested as DNA microsatellite instability mutations" as this is the first MSI that has been seen in these markers.
  • calculating the mutational load further comprises assigning a score to each mutation based on detection of DNA microsatellite instability at multiple loci, wherein DNA microsatellite instability at multiple loci is defined as 0.5z, wherein z is the number of loci displaying DNA microsatellite instability; wherein the score for low clonality is 0.5x, wherein x is the number of low clonality mutations and the score for high clonality is y, wherein y is the number of high clonality mutations; and wherein the overall mutational load is y + 0.5x + 0.5z.
  • DNA microsatellite instability is determined by the presence of at least one of the shortening and lengthening of a DNA microsatellite region. In some embodiments, the emergence of new alleles, manifested as DNA microsatellite instability mutations" as this is the first MSI that has been seen in these markers.
  • ATTORNEY REF. NO.130341.01212 PATENT is the first MSI that has been seen in these markers.
  • calculating the mutational load further comprises assigning a score to each mutation based on detection of DNA microsatellite instability at a particular locus, wherein DNA microsatellite instability at a single locus is defined as 0.75zi, and wherein DNA microsatellite instability at multiple loci is defined as 0.5z 2 , wherein zi is the number of loci displaying DNA microsatellite instability and Z2 is the number of loci displaying DNA microsatellite instability greater than 1 ; wherein the score for low clonality is 0.5x, wherein x is the number of low clonality mutations and the score for high clonality is y, wherein y is the number of high clonality mutations; and wherein the overall mutational load is y + 0.5x + 0.75z ! + 0.5z 2 .
  • calculating the mutational load further comprises assigning a score to each mutation based on detection of DNA microsatellite instability at a particular locus, wherein DNA microsatellite instability at a single locus is defined as 0.75zi, and wherein DNA microsatellite instability at multiple loci is defined as 0.75z !
  • zi 1.
  • mutational load y + 0.5x + 0.75(1) + 0.5(2).
  • mutational load y + 0.5x + 0.75(1).
  • calculating a mutational load further comprises summing the clonality weighting for each specific microsatellite region showing a mutation or DNA microsatellite instability.
  • particular weightings may be assigned to a particular mutation, DNA microsatellite region or a combination thereof. In some embodiments, weightings are equal for all mutations in particular DNA microsatellite regions analyzed. In some embodiments, mutations in particular DNA microsatellite regions may be given higher weightings than other mutations in particular DNA microsatellite regions. In some embodiments, certain mutations in particular DNA microsatellite regions may have a greater influence on the ML for a particular subject. In some embodiments, mutations in particular ATTORNEY REF. NO.130341.01212 PATENT
  • DNA microsatellite regions may include, but are not limited to mutations in 17p (TP53) and 9p (CDKN2A, CDKN2B).
  • calculating a mutational load further comprises assigning a distinct weighting for a mutation depending on the genomic locus of the mutations.
  • a low clonality mutation at 17p (TP53) or 9p (pi 6) may be weighted as 0.55 up to 0.75 rather than 0.5, and a high clonality mutation may be weighted as 1.05 up to 1.5.
  • analysis of clonal diversity can supplement the embodiments described herein.
  • clonal diversity can in addition to the embodiments described herein serve as a factor in the prediction of disease progression.
  • determining mutational load as a predictor of disease progression is independent of a histological standard.
  • RMA is shown to induce regression of mutation bearing and cause reversion of intestinalized to normal squamocolumnar cells. Regression is time dependent and can occur at 6-12 months following treatment. Intestinalized mucosal cells bearing highly clonally expanded mutations are more resistant to regression but can be eliminated by repeat treatment. Integrated microscopic/molecular analysis provides sensitive parameters with which to classify, plan RMA and monitor patient with Barrett's metaplasia on a more personalized basis.
  • Barrett's specimens from 215 patients were microdissected into 420 distinct targets comprising disease states of columnar (non-Barrett's) metaplasia, intestinal (Barrett's) metaplasia, low-grade dysplasia, and high-grade dysplasia.
  • Microdissection targets were tested for loss of heterozygosity in a panel of 16 microsatellite markers using PCR/capillary electrophoresis.
  • the presence or absence of mutation and the proportion of cells affected by mutation were quantitatively determined, with high clonality mutations representing >75% of ATTORNEY REF. NO.130341.01212 PATENT cells, and low clonality representing 50%-75%. Numbers of high and low clonality mutations were fit to disease state using proportional odds logistic regression.
  • a panel of molecular markers was evaluated to analyze the overall degree of molecular change in conjunction with histologic diagnosis.
  • Microdissection targets with histologic HGD (range 2.0-6.0, average 3.3) had higher mutation load than LGD (range 1.50-4.5, average 2.4).
  • Targets with carcinoma in situ (CIS) showed significantly higher mutational load (range 3.0-7.0, average 5.3) than targets showing only high-grade dysplasia.
  • 30% of HGD targets had mutation loads in the range associated with LGD (i.e., below average for HGD).
  • 33% of LGD targets had mutation loads associated with IM.
  • Standard histological sections (4 ⁇ thick) of archival, formalin-fixed, paraffin-embedded (FFPE) from 271 patients histologically known to have BM tissue were microscopically reviewed. Patients in the study cohort had previously undergone upper GI endoscopy and pathology review of biopsy specimens. Patients with a histological classification of intestinal metaplasia, "indefinite for dysplasia", and various grades of dysplasia were selected for inclusion in the study. Patients without evidence of BM were excluded, as were patients with intramucosal and/or invasive carcinoma.
  • Hematoxylin and eosin (H&E) stained, formalin-fixed, paraffin embedded (FFPE) slides were used to guide microdissections of histologically classified targets from 6- 8 unstained, serial FFPE slides. Multiple distinct regions were microdissected from each slide to obtain cells corresponding to distinct foci of histologically classified disease. Multiple targets were taken according to the availability of topographically separate tissue ATTORNEY REF. NO.130341.01212 PATENT fragments, even when these fragments were from the same histological classification of BM ( Figure 1). Microdissections typically resulted in 3-8 targets for a given slide ( Figure 1).
  • microdissected targets of histologically classified normal squamous epithelium and epithelium containing columnar cells that were not intestinalized were examined as a baseline control for mutational markers.
  • These targets were microdissected from the same FFPE slides as targets with various histological classifications of BM. Accuracy of microdissection was confirmed by microscopic review of post-microdissection stained slides.
  • a histological classification for each target was assigned based on review of the specimens and accompanying histopathology reports from the original microscopic review. When consensus between pathologists was available, it was used as the histological classification for the target. When consensus was not available, we relied on the histological classification of the expert pathologist in our group.
  • Targets were classified as non-Barrett's epithelium and Barrett's epithelium using the following histological classifications in order of increasing severity: normal squamous and columnar mucosa for non-Barrett's epithelial targets, and intestinal metaplasia, "indefinite for dysplasia", LGD, and HGD for Barrett's epithelial targets.
  • the panel contained markers at the following chromosomal loci (associated genes in parenthesis): lp (CMM1, L-myc), 3p (VHL, HoGGl), 5q (MCC, APQ, 9p (CDKN2A), lOq (PTEN, MXI1), 17p (TPS 3), 17q (NME1), 18q (DCQ, 21q (TFF1 and PSEN2) and 22q (NF2).
  • Marker qualification studies were performed using surgically resected EA specimen's microdissected at sites of intestinal metaplasia, dysplasia, and EA, with histological classification at each site representing consensus of four GI pathologists. In that study, numerous genomic loci adjacent to tumor suppressor genes were analyzed for LOH. The results of such studies were used to select a smaller, more relevant panel of genomic loci to examine. The smaller panel used here was limited to LOH ATTORNEY REF. NO.130341.01212 PATENT mutational markers next to tumor suppressor genes with a mutation in at least 20% of the surgical EA specimens.
  • Normal variability was characterized in preliminary studies for each pairing of allele lengths examined for LOH in order to account for differing nucleic acid amplifications related to differences in allele length.
  • Normal squamous, non-Barrett's epithelial targets were used to characterize this variability. By analyzing this variability, it was possible to quantitatively estimate the proportion (clonality) of cells mutated at a particular genomic locus. Each microdissected target was tested for each molecular marker in duplicate or triplicate in order to ensure reproducibility. Mutational profiles in cell populations were defined as high clonality when >75% of the DNA was mutated and low clonality when 50-75% of the DNA was mutated. When ⁇ 50% of the DNA was mutated, no mutations were reported due to the analytical limit of detection of the assay, which was 50% for each LOH.
  • POLR Proportional odds logistic regression
  • Weightings were also evaluated using fractional allelic loss (FAL), an analysis for the proportions of low and high clonality mutations to the number of informative markers. All results from various analyses consistently determined a weight of 0.5 for low clonality mutations and 1 for high clonality mutations.
  • FAL fractional allelic loss
  • the low ML level included all intestinal metaplasia targets that had mutations but had an ML below the high ML cutoff. We used these levels of mutational load to evaluate the mutational load in other histological classifications. The frequency of mutations in various genomic loci of each target was also determined for each histological class.
  • Esophageal biopsies were examined for LOH mutational profiles adjacent to tumor suppressor genes.
  • Each FFPE biopsy slide was microdissected at multiple target sites as guided by histologically observed cellular morphology ( Figure 1). Microdissections of distinct targets were performed on patient samples with various demographics (Table 5). There were 199 males and 72 females from which 568 distinct microdissection targets were analyzable.
  • the number of LOH mutations was determined in targets with various histological classifications. Table 6 summarizes the number of mutated LOH loci per microdissected target averaged for all targets across the range of histological classes ATTORNEY REF. NO.130341.01212 PATENT examined. The number of mutated LOH loci increased with increasing severity of histological classification. Most LOH mutations were detected in HGD targets, and in those HGD targets a relatively high proportion of cells were found with these mutations (high clonality). While most mutations found in HGD targets were high clonality, mutations found in non-dysplastic histological classifications (intestinal metaplasia, "indefinite for dysplasia”) were typically low clonality.
  • Mutational load (Table 6, Figure 2) represents tumor suppressor gene LOH mutations within clonally expanded cell populations in microdissected targets. Higher clonality mutations were indicative of a greater number of cells with the same mutations within a microdissected target. Semi-quantitative analysis was used that incorporated the number of mutations and the proportion of cell populations that had mutations to assess mutational load. In this system, low clonality mutations were weighted with a numerical value of 0.5 and high clonality mutations with a value of 1 in order to determine the overall LOH mutational load for each target.
  • the mutational load for each microdissected target was correlated to the histological class of the target ( Figure 2).
  • mutational load was positively correlated to histological classification, with the overall number and clonality of mutations increasing with increasingly severe histological classification (Figure 2A).
  • ML Mutational Load
  • Figure 2B Three levels of Mutational Load (ML) with respect to each histological classification.
  • the first level contained targets that lacked mutations and, as such, had no detectable ML.
  • the second level contained targets with 1 low clonality mutation to 2 high clonality mutations with a mutational load greater than 0 but less than or equal to 2 and was defined as having low ML.
  • the third level contained targets with greater than two high clonality mutations with a mutational load greater than 2 and was defined as having high ML.
  • Table 5 summarizes the proportion of microdissected targets for each level of mutational load in each histological class.
  • the majority of histological targets with normal squamous epithelium and epithelium containing columnar cells that were not intestinalized (90% of normal squamous epithelial targets and 61% of columnar, non-Barrett's epithelial targets) had no detectable ML (Table 8).
  • Table 8 Of the proportion of squamous and columnar targets that had mutations, all were low clonality mutations falling into levels of low ML (Table 6).
  • genomic instability was surveyed by assessing LOH mutations adjacent to tumor suppressor genes across cell populations with a range of histological classifications. The presence and extent of LOH mutations in cell populations was correlated to each histological class. Microdissected targets, guided by morphological features, were taken at multiple sites, as available, in biopsy specimens. The overall number of mutations and clonality of cells with mutations were formulated into a mutational load that increased in correlation with increasingly severe histological classification. This correlation is consistent with the known fact that increasing genomic instability drives clonal expansion of cells and disease progression in BM.
  • BM targets with a histological classification of intestinal metaplasia were used to define three levels of Mutational Load (ML) with respect to each histological class: ATTORNEY REF. NO.130341.01212 PATENT no ML, low ML and high ML ( Figure 2B, Table 7).
  • ML Mutational Load
  • Levels of mutational load in tumor suppressor genes were established with respect to specimens histologically classified with intestinal metaplasia because i) the presence of intestinal metaplasia can be relatively reliably diagnosed; and, ii) intestinal metaplasia is more prevalent in the clinical population than more severe histological classes of BM.
  • levels of mutational load with respect to intestinal metaplasia makes the levels most relevant to the most frequent and reliable histology found in patients with BM.
  • Targets with no ML were found in non-dysplastic histological classifications.
  • Targets with low ML had relatively low levels of LOH mutational accumulation without evidence of clonal expansion of mutated cells.
  • Targets with high ML had relatively high levels of LOH mutational load and clonal expansion of cells with these mutations.
  • High ML was consistently found in higher levels of histological dysplasia; however, high ML was also seen in some cases with less severe histological classifications, such as intestinal metaplasia.
  • “Indefinite for dysplasia” targets tended to have higher mutation load than those with intestinal metaplasia, with mutations detected in 86% of targets (Table 8). Since these targets were histologically indefinite, some may, in fact, have more advanced mutational damage than others. As with intestinal metaplasia, some "indefinite for dysplasia” targets may have mutations that precede morphological changes consistent with dysplasia. Therefore, mutational analysis may provide additional information to aid in clinical diagnosis and management when such microscopic changes have yet to occur or are indefinite.
  • Targets that displayed low ML and were histologically classified as normal squamous epithelium and epithelium containing non-intestinalized columnar cells (10% of normal squamous epithelial targets; 39% of columnar, non-Barrett's epithelial targets) could represent actual mutations within histologically normal appearing mucosa or detection of mutated DNA from adjacent cells or intercellular fluids.
  • the squamous and columnar mucosal targets were microdissected from the same FFPE biopsy slides as those histologically diagnosed with BE, making it possible that mutations from the adjacent Barrett's epithelium or intercellular fluids were detected.
  • the mutational load in these squamous and columnar epithelial targets could also represent chromosomal aberrations that have yet to become morphologically visible by histology.
  • HGD is considered a severe premalignant event that requires clinical intervention, because it is associated with greater risk of progression to EA.
  • cutoffs derived from intestinal metaplasia targets classified all but one HGD target as having high ML. This supports the association of high levels of genomic instability with more severe histological classifications of BM and is in line with the concept that patients with high ML may also be at greater risk of progression to EA. Consistently, the presence of three or more DNA abnormalities in patients has been associated with a greater risk of progression towards cancer.
  • High ML may, therefore, provide support for associated interventions, even when histological classification of BM may be less than severe dysplasia (intestinal metaplasia, "indefinite for dysplasia", LGD). High ML in less severe histological classifications of BM may be indicative of impending morphological changes that have yet to become histologically visible.
  • this type of mutational profiling may also facilitate monitoring using sequential biopsies taken over varying periods of time prior to determining if ablation is needed. Furthermore, it can provide objective molecular information with respect to the success of ablation. As with other forms of neoplasia, distinct clones of disease acquire distinct mutations, and a new clone is unlikely to have the same mutational profile as an existing clone. Incomplete elimination of the original clonal cell populations would be reflected in the same mutations persisting after ablation. In contrast, when the mutational profile in follow up biopsies differs from that of the initial biopsy, new clones of cells, as identified by different mutations, have likely evolved. When there is no evidence of mutations in follow up biopsies, complete eradication of atypical clones has likely been achieved.
  • a chief limitation in this study and all studies of BM is the variability associated with histological classification and the resulting lack of standardized histological classes for comparison to molecular results. Another limitation of this study concerns specimen type. Histology slides from biopsies are valuable specimens for studies such as this ATTORNEY REF. NO.130341.01212 PATENT one, as they represent "real-world" specimens. However, biopsies are subject to sampling variation because, although current guidelines call for four-quadrant biopsies every 1cm across the region of dysplastic BE, in clinical practice, more limited sampling often occurs.
  • Example 6 Evaluation of mutational load in microdissection target of dysplastic and non-dysplastic Barrett's esophagus
  • FFPE biopsies of 59 patients yielded 146 targets that were classified as intestinal metaplasia (IM), indefinite for dysplasia (IND), low grade dysplasia (LGD) or high grade dysplasia (HGD) by 3 pathologists blinded to molecular results. Consensus diagnosis was defined as agreement between at least 2 pathologists. Targets were microdissected and analyzed for loss of heterozygosity (LOH) mutations using a panel of 16 DNA markers.
  • LHO heterozygosity
  • Table 9 shows the frequency of pathologist agreement on histological classification. At least one pathologist disagreed the majority of the time for IND (95%) and LGD (88%) classifications.
  • Biopsy specimens from 370 patients with BE were microdissected into 787 distinct targets based on histological features from 4 independent study cohorts. Each target was histologically classified as intestinal metaplasia (IM), indefinite for dysplasia (IND), low grade dysplasia (LGD), high grade dysplasia (HGD), or carcinoma in situ (CIS). Each microdissection target was tested for loss of heterozygosity (LOH) and/or microsatellite instability (MSI) in a panel of 22 markers targeting known tumor suppressor genes using PCR/capillary electrophoresis. The presence or absence of LOH and/or MSI and the proportion of DNA affected by LOH mutation (clonality) were quantitatively determined.
  • IM intestinal metaplasia
  • IND indefinite for dysplasia
  • LGD low grade dysplasia
  • HFD high grade dysplasia
  • CIS carcinoma in situ
  • High clonality LOH mutations contained >75% of mutated DNA and low clonality LOH ATTORNEY REF.
  • NO.130341.01212 PATENT contained 50%-75%.
  • Mutational load (ML) was estimated using the number of low and high clonality mutations and the number of loci affected by MSI.
  • the no ML level consisted only of intestinal metaplasia microdissected targets that lacked detectable mutations.
  • the high ML level was defined as the level that captured 5% of intestinal metaplasia microdissected targets that had the highest level of ML.
  • the low ML level included all intestinal metaplasia microdissected targets that had mutations but had an ML below the high ML cutoff.
  • Table 11 describes the percentage of microdissected targets with the indicated histology that had each level of ML. HGD and CIS frequently had high ML, while other less severe histological classifications had a heterogeneous range of ML.
  • Example 8 A Preliminary longitudinal assessment of mutational load in patients with Barrett's Esophagus ATTORNEY REF. NO.130341.01212 PATENT
  • results ML increased with the degree of neoplasia (Figure 3).
  • the mean ML for non-BE (Norm), intestinal metaplasia (IM), indefinite for dysplasia (IND), low grade dysplasia (LGD), and HGD targets in the progressor and non-progressor groups is listed in Table 11.
  • the progressor group had a higher ML compared to the non-progressor group (average ML 1.6 versus 0.2, respectively, p ⁇ 0.001).
  • patients from the non-progressors never had an ML greater than 1.
  • ML consistently provides an objective measure of the presence and extent of genomic instability in BE.
  • Subjects with advanced neoplasia have a higher ML than those with less advanced neoplasia.
  • Patients with progressive neoplasia have a greater ML than those without progressive disease.
  • Histology-guided assessment of ML in patients with less advanced BE may provide a more sensitive marker for progressive neoplasia than histology alone.
  • Specimens were from biopsies of 25 patients with confirmed dysplasia. These specimens were microdissected into 93 distinct microdissection targets, each encompassing a specific area of disease on a specific biopsy slide. Each microdissection target was reviewed by a pathologist blinded to the molecular results and assigned a microscopic diagnosis of normal, intestinal metaplasia, low grade dysplasia, high grade dysplasia or carcinoma.
  • CCM1 genomic loci
  • Lmyc 3p
  • VHL 3p
  • MCC 5q
  • MCC CDKN2B
  • lOq PTEN, MXI1
  • 17p TP53
  • NME1 17q
  • the presence and absence of allelic imbalance mutations, and clonality of mutation were determined for each microsatellite.
  • an overall clonality was determined as the maximum clonality observed at any microsatellite marker located near that locus (e.g. 4 markers on 17p).
  • Mutational load was calculated for each microdissection target as a weighted count of mutations using weightings as previous described (e.g. 1 for high clonality, 0.5 for ATTORNEY REF. NO.130341.01212 PATENT low clonality, 0.75 for microsatellite instability (MSI) when it was the only mutation and 1 for MSI together mutations at any other loci).
  • weightings as previous described (e.g. 1 for high clonality, 0.5 for ATTORNEY REF. NO.130341.01212 PATENT low clonality, 0.75 for microsatellite instability (MSI) when it was the only mutation and 1 for MSI together mutations at any other loci).
  • Table 12 and Figure 4 shows the number of targets with 1,2, 3, or 4 microsatellite markers affected at a particular locus. Mutations encompassing multiple markers at a given locus were only found in targets diagnosed as high grade dysplasia and carcinoma, and were particularly common at 9p and 17p.
  • Table 13 depicts the calculation of mutational load in a series of 20 patients. In some cases, several specimens from a single patient were tested (see Table 13, "Target" column). Mutational load was calculated based on the number of loci with high clonality mutations, the number of low clonality mutations and the number of loci displaying microsatellite instability.
  • Table 14 shows the calculation of mutational load based on differing weightings being assigned to particular loci in a group of 20 patients. In some cases, several specimens from a single patient were tested (see Table 14, "Target" column). Certain loci were assigned higher weightings based on known associations with disease progression. The mutational loads calculated this was correlated with disease diagnosis based on histology.
  • Example 12 Assessment of mutational load in biopsy tissue provides additional information about genomic instability to histological classifications of Barrett's Esophagus
  • ML summarized the presence and clonality of loss of heterozygosity (LOH) mutations and the emergence of new alleles, manifested as microsatellite instability (MSI) mutations, in 10 genomic loci around tumor suppressor genes associated with EAC.
  • LH loss of heterozygosity
  • MSI microsatellite instability
  • Esophageal adenocarcinoma exhibits the highest rate of increasing incidence of any solid cancer in the U.S. today, and Barrett's esophagus (BE) represents a precursor of and largest risk factor for EAC.
  • the carcinogenesis of BE has been associated with morphologic changes in esophageal tissue as well as activation of oncogenes and inactivation of tumor suppressor genes. Studies have shown that variable degrees of mutational change take place in the microsatellite regions of tumor suppressor genes at the histological onset of BE. The cumulative buildup of various mutations has been closely associated with the different histological grades of BE and EAC.
  • Histological progression to EAC is associated with a relatively poor prognosis, with a 5-year survival rate for regional cancer below 18%. Consequently, emphasis has been placed upon understanding the risk for progression to EAC of each histological stage of BE such that patients can be appropriately managed with intervention or surveillance.
  • IM intestinal metaplasia
  • IND indefinite for dysplasia
  • LGD low-grade dysplasia
  • HGD high-grade dysplasia
  • Both BE and EAC can be readily identified by microscopic examination, but the presence and different grades of dysplasia can be difficult to diagnose due to challenges in discriminating reactive epithelial atypia from true dysplasia.
  • the classification of indefinite for dysplasia is sometimes provided when cellular atypia is observed, but the criteria for the histological diagnosis of dysplasia are not fully met. Poor orientation of the ATTORNEY REF. NO.130341.01212 PATENT histological sections and presence of inflammatory infiltrate are among the most common factors interfering with the pathologist's ability to differentiate between the presence or absence of dysplasia.
  • Inter-observer variability in the histological classification of BE has been reported by various studies. Most variability is linked to LGD, yet some variability is also seen in cases of HGD, complicating the decision as to how to clinically manage patients.
  • Non-dysplastic histological features are limited when it comes to determining whether or not a case of BE is likely to progress to cancer or remain stable, as there are no observable non-dysplastic histological microscopic features that can reveal a patient's likelihood of cancer progression. Because of this uncertainty, many choose ablation interventions for low grade dysplastic and non-dysplastic BE, which has provoked concerns about unnecessary healthcare expenditures. Supplementary diagnostic information that enables better characterization of the risk for less advanced stages of BE disease would be valuable in improving patient care and controlling healthcare costs.
  • Genomic instability was assessed by mutational load in histological targets microdissected from BE patient biopsy tissue. Mutational load summarized the presence and extent of LOH next to TP53 and CDKN2A as well as LOH in 8 additional genomic loci next to tumor suppressor genes. Microsatellite instability (MSI) around these tumor suppressor genes was also included in the assessment of mutational load. Our results demonstrate that histology-guided assessment for mutational load provides an objective measure of the presence and extent of genomic instability. Assessment for ATTORNEY REF. NO.130341.01212 PATENT mutational load provides an added dimension to less advanced BE histology that could help to better manage BE patients in early or uncertain histological stages of disease.
  • Study Cohort Standard histological sections (4 ⁇ thick) of formalin-fixed, paraffin-embedded (FFPE) tissue were examined from 415 patients histologically known to have BE. Microdissection of 661 biopsy slides yielded 877 targets in total from three study sites, each with IRB approval of their corresponding study protocol (IRB# 26163, IRB #11- 29, IRB # 5658 and IRB# 5629). All patients in the study had previously undergone upper GI endoscopy. Patients without evidence of BE were excluded.
  • FFPE formalin-fixed, paraffin-embedded
  • Histological Classification Hematoxylin and eosin (H&E) stained, FFPE histology slides underwent microscopic evaluation for the selection of targets for subsequent microdissection. Each target was histologically classified as follows: normal squamous epithelium, columnar mucosa (COL), and, in order of increasing severity: intestinal metaplasia (IM), indefinite for dysplasia (IND), low grade dysplasia (LGD), high grade dysplasia (HGD), and esophageal adenocarcinoma (EAC).
  • BE histological classification began with intestinal metaplasia histology. A histological classification was assigned based on pathologist review of tissue. All pathologists were blinded to molecular results. In one study cohort from Norwalk Hospital, three pathologists classified the same microdissected targets. Consensus diagnosis was defined as agreement between at least two pathologists.
  • Microdissection Hematoxylin and eosin (H&E) stained, FFPE slides were used as guides to microdissect tissue (targets) with BE histology from 1-3 unstained, serial FFPE slides of each patient.
  • Microdissected targets corresponded to distinct foci of tissue with EAC histology, BE histology, columnar mucosa histology, or normal epithelial histology, as identified by a pathologist.
  • Normal epithelial and columnar mucosa (COL) targets were microdissected from the same FFPE slides as targets with various histological classifications of BE or EAC histology. Microscopic review confirmed the accuracy of all microdissections.
  • Detection of LOH and MSI Detection of LOH and new alleles consistent with microsatellite instability (MSI) were investigated at 10 individual genomic loci, using a panel of 22 DNA markers associated with common tumor suppressor genes relevant to BE. The presence of MSI at BAT25 and BAT26 loci were also examined in a subset of ATTORNEY REF. NO.130341.01212 PATENT microdissected targets. LOH and MSI were assessed using PCR and quantitative capillary electrophoresis of DNA extracted from each microdissected target, as previous described.
  • DNA markers for the following chromosomal loci comprised the panel (associated genes in parentheses): lp (CMM1 , L-myc), 3p (VHL, HoGGl), 5q (MCC, APC), 9p (CDKN2A), lOq (PTEN, MXI1), 17p (TP53), 17q (NME1), 18q (DCC), 21q (TFF1 and PSEN2) and 22q (NF2).
  • Quantitative PCR for housekeeping genes was used to ensure there was sufficient, high quality DNA available for analysis prior to LOH and MSI analysis.
  • the analyses were performed on both BE microdissection samples as well as internal controls (normal appearing squamous and columnar mucosa), which were all subject to equivalent formalin fixation and histological processing.
  • PCR amplification and subsequent mutational analysis using quantitative capillary electrophoresis methods were then performed on all microdissected samples with adequate qPCR results.
  • each LOH analysis was assessable in each patient, the informativeness (heterozygosity) of each LOH marker in normal epithelial from each patient was first examined by quantitative capillary electrophoresis methods. Normal epithelial targets were also used to account for minor differences in the amplification rates of the two allele lengths during PCR. PCR amplification and subsequent quantitative capillary electrophoresis of DNA from each microdissected target was then performed to assess LOH and MSI.
  • LOH was called "present" in microdissected targets when there was a degree of allelic imbalance that was equal to or beyond two standard deviations above the average difference in allele peak heights for DNA in normal epithelial microdissection targets.
  • the extent (clonality) of LOH was determined using the ratio of allele peak heights in DNA from microdissected targets, which is proportional to the amount of LOH mutated DNA present in the sample. All DNA specimens with LOH were tested in duplicate or triplicate to ensure reproducibility. LOH mutations were considered high clonality when >75% of the DNA had LOH mutation and low clonality when 50-75% of the DNA had LOH mutation.
  • MSI was defined by the presence of additional minor, but reproducible, peaks in the electropherograms after PCR amplification and subsequent quantitative capillary electrophoresis of DNA from each target.
  • the minor peaks did not correspond to either of ATTORNEY REF. NO.130341.01212 PATENT the two major allele lengths present in normal epithelial control DNA and were not accountable by the presence of shadow band formation during capillary gel electrophoresis.
  • the minor peaks were reproducible through replicate confirmatory PCR amplification and further quantitative capillary electrophoresis testing.
  • Mutational Load Mutational load measured the presence and clonality of LOH mutations and the presence of MSI at each genomic locus examined. The presence and clonality of LOH mutation at each genomic locus was determined for each microdissected target. Low clonality LOH was defined as 50-75% of the DNA containing LOH, and high clonality LOH was defined as >75% of the DNA containing LOH. All LOH mutations at a given genomic loci were assigned a numerical value based on their low or high clonality. A proportional value of 0.5 was assigned for low clonality mutations and 1 for high clonality mutations using proportional odds logistic regression (POLR), as previously described.
  • POLR proportional odds logistic regression
  • MSI The presence of MSI was also assigned a proportional value using POLR.
  • the proportional value of MSI present at a single genomic locus was 0.75.
  • the proportional value of each additional MSI present, beyond one locus, was 0.5.
  • These numerical values for low clonality and high clonality LOH mutations ( Figure 5A) as well as MSI mutations ( Figure 5B) were added together for all loci containing LOH and/or MSI in a microdissected target. The resulting cumulative value was defined as the mutation load (ML) for that microdissected target.
  • Polyserial correlation coefficient was used to examine the correlation between histological class and mutational load of microdissected targets when mutational load was assessed using only LOH mutations ( Figure 5A) or both LOH and MSI mutations ( Figure 5B).
  • An analysis of variance (ANOVA) model was used to examine the difference in mutational load between advanced (HGD, EAC) and less advanced (IM, IND, LGD) histological classifications when mutational load included only LOH analysis as compared to when it included both LOH and MSI analysis.
  • ANOVA was performed with an interaction term between the two methods of mutational load assessment (LOH only, LOH and MSI) and the two categories of histological classifications of BE (advanced histology, less advanced histology).
  • Table 17 Frequency of pathologist agreement on histological classification in one study cohort.
  • IM intestinal metaplasia
  • IND indefinite for dysplasia
  • LGD low- grade dysplasia
  • HGD high-grade dysplasia
  • Genomic Instability The presence and extent (clonality) of genomic instability in each microdissected target was assessed by mutational load. Mutational load of a target was calculated based on the presence and clonality of LOH mutations as well as the presence of MSI in DNA from each histological target. In this system, numerical values were determined by POLR as follows: 0.5 for low clonality LOH mutations (50-75% of DNA had LOH), 0.75 for the first MSI, 0.5 for each additional MSI, and 1 for high clonality LOH mutations (>75% of DNA had LOH).
  • ML mutational load
  • the first level contained microdissected targets that lacked mutations and, as such, had no ML.
  • the second level (low ML) contained targets with a mutational load found in the majority of intestinal metaplasia microdissected targets. Low ML targets had mutations but were below the top fifth percentile of intestinal metaplasia targets with the highest ML.
  • the third level contained microdissected targets with a mutational load similar to those targets in the top fifth percentile of intestinal metaplasia targets with the highest mutational load.
  • Table 18 summarizes the proportion of microdissected targets for each level of mutational load in each histological class.
  • COL columnar mucosa
  • non-BE targets COL
  • indefinite for dysplasia microdissected targets 18% had no ML; while the remaining proportion of indefinite for dysplasia targets had mutations, including 66% that had low ML and 16% that had high ML.
  • Most microdissected targets histologically classified as LGD had ATTORNEY REF.
  • genomic loci had LOH mutations in non-dysplastic targets (IM, indefinite for dysplasia) in comparable frequency to CDKN2A and TP53 associated mutations, suggesting that other genomic loci beyond those related to TP53 and CDKN2A are present at even early stages of BE.
  • MSI mutations at these 10 genomic loci occurred more often in dysplastic microdissected targets but were also present in non-dysplastic targets across nearly all loci.
  • a system having at least one of A, B, and C would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.).
  • a convention analogous to "at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g. , " a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.).
  • a range includes each individual member.
  • a group having 1-3 substituents refers to groups having 1 , 2, or 3 substituents.
  • a group having 1-5 substituents refers to groups having 1 , 2, 3, 4, or 5 substituents, and so forth.

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Abstract

L'invention concerne des procédés de traitement du syndrome de Barrett et de l'adénocarcinome œsophagien et des procédés de détermination d'une charge de mutation en tant que facteur de prévision du risque d'évolution pathologique du syndrome de Barrett en adénocarcinome œsophagien.
PCT/US2014/046702 2013-07-30 2014-07-15 Procédé de traitement du syndrome de barrett et de l'adénocarcinome œsophagien WO2015017125A2 (fr)

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