US20020058251A1 - Diagnostic method using expression of mn/ca9 protein in agus pap smears - Google Patents
Diagnostic method using expression of mn/ca9 protein in agus pap smears Download PDFInfo
- Publication number
- US20020058251A1 US20020058251A1 US09/461,938 US46193899A US2002058251A1 US 20020058251 A1 US20020058251 A1 US 20020058251A1 US 46193899 A US46193899 A US 46193899A US 2002058251 A1 US2002058251 A1 US 2002058251A1
- Authority
- US
- United States
- Prior art keywords
- cells
- atypical
- agus
- antigen
- pap smear
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000009595 pap smear Methods 0.000 title claims abstract description 102
- 108090000623 proteins and genes Proteins 0.000 title description 12
- 102000004169 proteins and genes Human genes 0.000 title description 10
- 238000002405 diagnostic procedure Methods 0.000 title description 3
- 102100024423 Carbonic anhydrase 9 Human genes 0.000 claims abstract description 152
- 206010008263 Cervical dysplasia Diseases 0.000 claims abstract description 130
- 230000003902 lesion Effects 0.000 claims abstract description 77
- 239000000427 antigen Substances 0.000 claims abstract description 72
- 102000036639 antigens Human genes 0.000 claims abstract description 72
- 108091007433 antigens Proteins 0.000 claims abstract description 72
- 238000009826 distribution Methods 0.000 claims abstract description 17
- 208000032124 Squamous Intraepithelial Lesions Diseases 0.000 claims description 96
- 238000000034 method Methods 0.000 claims description 55
- 201000007490 Adenocarcinoma in Situ Diseases 0.000 claims description 47
- 208000009956 adenocarcinoma Diseases 0.000 claims description 25
- 201000009030 Carcinoma Diseases 0.000 claims description 24
- 208000024312 invasive carcinoma Diseases 0.000 claims description 5
- 238000009396 hybridization Methods 0.000 claims description 4
- 238000003364 immunohistochemistry Methods 0.000 claims description 3
- 108020004999 messenger RNA Proteins 0.000 claims description 3
- 230000003321 amplification Effects 0.000 claims description 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 description 159
- 238000003745 diagnosis Methods 0.000 description 54
- 238000012744 immunostaining Methods 0.000 description 41
- 230000000762 glandular Effects 0.000 description 35
- 238000010186 staining Methods 0.000 description 27
- 230000002962 histologic effect Effects 0.000 description 25
- 230000001613 neoplastic effect Effects 0.000 description 24
- 208000020077 squamous cell intraepithelial neoplasia Diseases 0.000 description 20
- 206010028980 Neoplasm Diseases 0.000 description 18
- 210000003679 cervix uteri Anatomy 0.000 description 18
- 238000001574 biopsy Methods 0.000 description 14
- 230000027455 binding Effects 0.000 description 13
- 230000000875 corresponding effect Effects 0.000 description 13
- 108090000765 processed proteins & peptides Proteins 0.000 description 12
- 206010058314 Dysplasia Diseases 0.000 description 11
- 230000001413 cellular effect Effects 0.000 description 11
- 229920001184 polypeptide Polymers 0.000 description 11
- 102000004196 processed proteins & peptides Human genes 0.000 description 11
- 210000001519 tissue Anatomy 0.000 description 11
- 241000264877 Hippospongia communis Species 0.000 description 10
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 10
- 206010008342 Cervix carcinoma Diseases 0.000 description 9
- 230000005856 abnormality Effects 0.000 description 9
- 201000010881 cervical cancer Diseases 0.000 description 9
- 208000007951 cervical intraepithelial neoplasia Diseases 0.000 description 8
- 206010050808 Hyperchromasia Diseases 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 230000002380 cytological effect Effects 0.000 description 6
- 230000009826 neoplastic cell growth Effects 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 5
- 230000007170 pathology Effects 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 208000007879 Atypical Squamous Cells of the Cervix Diseases 0.000 description 4
- 108060003951 Immunoglobulin Proteins 0.000 description 4
- 206010054949 Metaplasia Diseases 0.000 description 4
- 102000003992 Peroxidases Human genes 0.000 description 4
- 229960002685 biotin Drugs 0.000 description 4
- 239000011616 biotin Substances 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 230000002596 correlated effect Effects 0.000 description 4
- 230000001086 cytosolic effect Effects 0.000 description 4
- 102000018358 immunoglobulin Human genes 0.000 description 4
- 239000003446 ligand Substances 0.000 description 4
- 230000003211 malignant effect Effects 0.000 description 4
- 230000015689 metaplastic ossification Effects 0.000 description 4
- 108040007629 peroxidase activity proteins Proteins 0.000 description 4
- 239000002243 precursor Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 3
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 241001529936 Murinae Species 0.000 description 3
- 230000002159 abnormal effect Effects 0.000 description 3
- 125000003275 alpha amino acid group Chemical group 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 201000006662 cervical adenocarcinoma Diseases 0.000 description 3
- 208000019065 cervical carcinoma Diseases 0.000 description 3
- 210000002777 columnar cell Anatomy 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000000104 diagnostic biomarker Substances 0.000 description 3
- 210000005168 endometrial cell Anatomy 0.000 description 3
- 230000002357 endometrial effect Effects 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 230000000877 morphologic effect Effects 0.000 description 3
- 206010001197 Adenocarcinoma of the cervix Diseases 0.000 description 2
- 238000006418 Brown reaction Methods 0.000 description 2
- 102000003846 Carbonic anhydrases Human genes 0.000 description 2
- 108090000209 Carbonic anhydrases Proteins 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 241000701806 Human papillomavirus Species 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 241001490312 Lithops pseudotruncatella Species 0.000 description 2
- 208000034179 Neoplasms, Glandular and Epithelial Diseases 0.000 description 2
- 241000042032 Petrocephalus catostoma Species 0.000 description 2
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 210000000254 ciliated cell Anatomy 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 206010020718 hyperplasia Diseases 0.000 description 2
- 230000002055 immunohistochemical effect Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 230000036210 malignancy Effects 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000002356 single layer Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 208000034246 Adenocarcinoma of the cervix uteri Diseases 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 206010059313 Anogenital warts Diseases 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 1
- 208000004746 Atrophic Vaginitis Diseases 0.000 description 1
- 206010003693 Atrophic vulvovaginitis Diseases 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 208000009458 Carcinoma in Situ Diseases 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 101100492693 Danio rerio atp5if1a gene Proteins 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 201000009273 Endometriosis Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010017533 Fungal infection Diseases 0.000 description 1
- 108010015776 Glucose oxidase Proteins 0.000 description 1
- 239000004366 Glucose oxidase Substances 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 208000031888 Mycoses Diseases 0.000 description 1
- BKAYIFDRRZZKNF-VIFPVBQESA-N N-acetylcarnosine Chemical compound CC(=O)NCCC(=O)N[C@H](C(O)=O)CC1=CN=CN1 BKAYIFDRRZZKNF-VIFPVBQESA-N 0.000 description 1
- 208000008636 Neoplastic Processes Diseases 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 208000006994 Precancerous Conditions Diseases 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 206010041848 Squamous cell carcinoma of the cervix Diseases 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- GPKUGWDQUVWHIC-UHFFFAOYSA-N [4-(4-hydrazinylphenyl)phenyl]hydrazine tetrahydrochloride Chemical compound Cl.Cl.Cl.Cl.NNC1=CC=C(C=C1)C1=CC=C(NN)C=C1 GPKUGWDQUVWHIC-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000001745 anti-biotin effect Effects 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 102000023732 binding proteins Human genes 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 239000008366 buffered solution Substances 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000002621 cervical conization Methods 0.000 description 1
- 201000006612 cervical squamous cell carcinoma Diseases 0.000 description 1
- 201000003565 cervix uteri carcinoma in situ Diseases 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 239000003433 contraceptive agent Substances 0.000 description 1
- 230000002254 contraceptive effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000002498 deadly effect Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003413 degradative effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 201000003683 endocervical adenocarcinoma Diseases 0.000 description 1
- 201000003908 endometrial adenocarcinoma Diseases 0.000 description 1
- 208000029382 endometrium adenocarcinoma Diseases 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 229940116332 glucose oxidase Drugs 0.000 description 1
- 235000019420 glucose oxidase Nutrition 0.000 description 1
- 230000003118 histopathologic effect Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 238000009802 hysterectomy Methods 0.000 description 1
- 229940027941 immunoglobulin g Drugs 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 238000002991 immunohistochemical analysis Methods 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 201000004933 in situ carcinoma Diseases 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 210000005170 neoplastic cell Anatomy 0.000 description 1
- 230000010309 neoplastic transformation Effects 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 201000010808 postmenopausal atrophic vaginitis Diseases 0.000 description 1
- 230000001855 preneoplastic effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 208000028172 protozoa infectious disease Diseases 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 208000022159 squamous carcinoma in situ Diseases 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000013517 stratification Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 231100000588 tumorigenic Toxicity 0.000 description 1
- 230000000381 tumorigenic effect Effects 0.000 description 1
- 208000022625 uterine cervix carcinoma in situ Diseases 0.000 description 1
- 210000001215 vagina Anatomy 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 210000003905 vulva Anatomy 0.000 description 1
- -1 without limitation Chemical group 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
Definitions
- Carcinoma of the cervix is one of the most common malignancies in women, and worldwide it is second only to breast cancer in both incidence and mortality (NIH Consensus Statement (1) Cervical Cancer. 1996.14:1-18).
- NIH Consensus Statement (1) Cervical Cancer. 1996.14:1-18 In developed countries in general, and the United States in particular, the wide acceptance of the Pap smear screening program has resulted in dramatic decreases in the incidence of, and mortality from, cervical cancer.
- a recent survey conducted in United States still showed that an estimated 15,700 women were diagnosed with invasive cervical carcinoma, and an estimated 4,900 women died of cervical cancer per annum (NIH Consensus Statement (1) Cervical Cancer. 1996.14:1-18).
- dysplastic glandular cells as exfoliative endometrial cells and/or reactive endocervical cells (Kos et al., 1993; and Lee K R, Manna E A, St. John T. Atypical endocervical glandular cells: accuracy of cytologic diagnosis. Diagn. Cytopathol. 1995. 13:202-208).
- a broad morphologic spectrum of benign and neoplastic lesions are included in the category of AGUS. These include atypical endocervical repair, endometriosis, microglandular hyperplasia, tubal metaplasia, squamous intraepithelial lesion (SIL) with glandular involvement, and glandular dysplasia, and adenocarcinoma (Lee K R. Atypical glandular cells in cervical smears from women who have undergone cone biopsy. A potential diagnostic pitfall. Acta. Cytol., 1993. 37:705-709; Pacey F, Ayer B, Greenberg M.
- Endocervical glandular atypia and adenocarcinoma A correlation of cytology and histology., Int. J. Gynecol. Pathol., 1993. 12:208-218; Taylor R, Guerriere J, Nash J D, Henry M R, O'Conner D M. Atypical cervical cytology: Colposcopic follow-up using The Bethesda System, J. Reprod. Med., 1993. 38:443-447; Kennedy A W, Salmieri S S, Wirth S L, Biscotti C V, Tuason L J, Travarca M J. Results of the clinical evaluation of atypical glandular cells of undetermined significance (AGUS) detected on cervical cytology screening. Gynecol. Oncol., 1996. 63:14-18; and Bose S, Kannan V, Kline T S. Abnormal endocervical cells. Really abnormal? Really endocervical? Am. J. Clin. Pathol., 1994. 101:708-713).
- Atypical glandular cells of undetermined significance cytologic criteria to separate clinically significant from benign lesions.
- MN/CA9 a novel antigen, termed MN/CA9. It is a transmembrane glycoprotein that was discovered in the cervical adenocarcinoma cell line, Hela (Zavada J, Zavadova Z, Pastorekova S, Ciampor F, Pastorek J, Zelnik V. Expression of MaTu-MN protein in human tumor cultures and in clinical specimens. Int. J. Cancer 1993. 54:268-274).
- the gene encoding the MN/CA9 product is unusual—the only homologous functional domain identified to date being a carbonic anhydrase domain.
- MN/CA9 protein in neoplastic progression includes its association with the tumorigenic phenotype in human cell hybrids and neoplastic transformation of mouse 3T3 cells following transfection with MN cDNA (Zavada et al., 1993; and Pastorek J, Pastorekova S, Callebaut I, Morrion J P, Zelnik V, Opavsky R, Zatovicova M, Liao S, Portelle D, Stanbridge E J, Zavada J, Burny A, Kettmann R. Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment.
- MN/CA9 is expressed in all cases of AIS and in more than 90% of cervical squamous neoplasms. High levels of MN/CA9 protein expression were frequently observed in the normal-looking endocervical cells in regions adjacent to dysplastic tissues but the normal cervix does not express MN/CA9 protein.
- a study of 305 Pap smears has also indicated that the MN/CA9 expression seen in exfoliative cells in Pap smears recapitulates MN/CA9 expression in the corresponding tissue sections of the cervix.
- the present invention fulfills the need for a more accurate diagnosis of AGUS than the current routine conventional Pap smear screening.
- the invention provides new insight into the distribution of MN/CA9 antigen on atypical and/or normal cells on AGUS-diagnosed Pap smear specimens and serves as a clinical roadmap enabling MN/CA9 antigen to at last serve as a powerful diagnostic biomarker for cancerous or pre-cancerous conditions.
- the present invention for the first time provides a reliable comprehensive method to determine the presence of cancerous or pre-cancerous cervical lesions from Pap smear cells that have been cytologically diagnosed as atypical glandular cells of undetermined significance (AGUS) under the Bethesda System of terminology.
- AGUS-diagnosed Pap smear cells are subjected to a procedure that detects expression of MN/CA9 antigen, such as immunohistochemistry or in situ cytohybridization.
- AGUS-diagnosed Pap smears typically lack readily ascertainable dysplastic cells, but do include atypical and normal endocervical cells.
- MN/CA9 antigen it is the distribution of MN/CA9 antigen observed on atypical or normal cells that is used to diagnose the presence of significant or low grade lesions. More specifically, significant lesions, including adenocarcinoma, invasive carcinoma (CA), or high grade squamous intraepithelial lesions (HSIL) are diagnosed when MN/CA9 antigen is observed on atypical cells. Low grade lesions, including low grade squamous intraepithelial lesions (LSIL) or atypia, are diagnosed when MN/CA9 antigen is absent from atypical cells but is present on normal endocervical cells. A benign condition is diagnosed when MN/CA9 is absent from atypical and normal endocervical cells.
- LSIL low grade squamous intraepithelial lesions
- a benign condition is diagnosed when MN/CA9 is absent from atypical and normal endocervical cells.
- a significant lesion is diagnosed whenever MN/CA9 antigen is observed on atypical cells.
- the presence of adenocarcinoma including adenocarcinoma in situ (AIS) and invasive adenocarcinoma, is diagnosed when MN/CA9 antigen is detected on atypical cells in a columnar or honeycomb configuration.
- the presence of HSIL is diagnosed when MN/CA9 antigen is detected on atypical cells in a tight cluster.
- the methods of the present invention overcome problems of uncertainty and false negatives associated with the AGUS diagnosis for Pap smear specimens.
- the method discriminates among atypical Pap smears that are associated with significant lesions, low grade lesions or a benign condition.
- FIG. 1 shows a spectrum of squamous and glandular alterations to illustrate the criteria for diagnosis of atypia, preneoplastic and neoplastic lesions:
- Panel A is a normal cervix showing orderly arrangement of squamous and glandular epithelium
- Panel B is an example of atypical squamous metaplasia (left) and reserve cell proliferation (right)
- Panel C is an example of glandular atypia with epithelial stratification
- Panels D and E illustrate LSIL and HSIL, respectively
- Panel F is an example of AIS (Magnification: A 100 ⁇ , B-F 200 ⁇ );
- FIG. 2 shows staining patterns of MN/CA9 immunoreactivity in AGUS Pap smears: Panel A is a smear that shows no immunostaining; Panel B shows positive immunostaining in both atypical cells (arrow) and normal ECs (arrowhead), Panel C shows MN/CA9 positive atypical cells only, Panel D is an example of immunoreactivity of normal ECs only, with the corresponding Pap stain in panel E, moreover the pattern of immunoreactivity may be focal (panel F) or diffuse (panel G), and the morphology of the immunostained cells may be tight clusters (panel H) or honeycomb configuration (panel I) (Magnification: A, F and G 100 ⁇ ; H and I 200 ⁇ ; B, C, D and E, 400 ⁇ );
- FIG. 3 shows distributions of MN/CA9 immunostaining of AGUS Pap smears and their correlation with histologic diagnosis
- FIG. 4 shows the distribution of MN/CA9 immunostaining of AGUS Pap smears in the three categories: favor reactive, not otherwise specified (NOS), and favor neoplastic, and their correlation with histologic diagnosis;
- FIG. 5 shows cytologic features of atypical cell clusters in AGUS-favor reactive Pap smears, such as enlarged nuclear size with minimal cellular overlap and mild degree of nuclear pleomorphism (panels A, B and C), and the same cell clusters, destained and then immunostained to detect MN/CA9 expression provide examples where no immunoreactivity correlated with no histological lesion (panel D), and strong (panel E) and weak immunoreactivity (panel F) was associated with histologic diagnoses of HSIL: note that the ciliated cells pointed to in panel E are MN/CA9 negative (Magnification: 400 ⁇ );
- FIG. 6 shows cytologic features of atypical cell clusters in AGUS-NOS Pap smears (panels A, B and C), such as nuclei 3 ⁇ larger than the nuclei of the adjacent intermediate cells (arrow), and increased cellular overlap and nuclear hyperchromasia, however corresponding MN/CA9 immunostains of the same specimens demonstrate a negative immunostain in Panel D that corresponds with a follow-up biopsy that was negative, a positive immunostain in panel E corresponding with a histologic diagnosis of HSIL, and a characteristic honeycomb pattern of immunostaining in panel F that corresponds with a follow-up biopsy diagnosed as adenocarcinoma. (Magnification: 400 ⁇ ); and
- FIG. 7 shows cytologic features of atypical cell clusters in AGUS-favor neoplastic Pap smears, such as significant cellular overlap, nuclear pleomorphism and hyperchromasia (panels A, B and C) and the corresponding MN/CA9 immunostains show examples where a negative immunostain (panel D) corresponds with a follow-up biopsy that is benign, a positive immunostain (panel E) corresponds with a histologic diagnosis of HSIL, and stained cells, which exhibit honeycomb and columnar features (panel F), corresponds with a follow-up biopsy diagnosed as AIS (Magnification: 400 ⁇ ).
- the method of the present invention entails examining of the levels and distribution of expression of MN/CA9 antigen to confirm diagnoses obtained by cytological examinations, e.g., Pap smears, when observing atypical glandular cells of undetermined significance.
- a positive result serves as an early marker of dysplasia even in the absence of its clinical manifestations.
- the first step in any cytological diagnostic method is obtaining suitable Pap smear cells for review.
- a cytologist examines an exfoliative cell specimen, obtained by scraping some cells from the lining of the cervix, smearing the cells onto a slide and staining with Papanicolaou stain.
- the cytologist examines the stained smears for the presence of abnormal-looking cells that indicate the presence of a malignant condition.
- the term “malignant condition” refers to the presence of dysplasia including adenocarcinoma in situ (AIS), invasive carcinoma (CA), neoplastic, malignant or tumor cells or the like.
- an exfoliative cell specimen is obtained from a patient, who may or may not harbor a malignant condition.
- the specimen may be obtained by rotating a cervical sampling device, such as a swab, spatula, or cytobrush along a portion of cervix or vaginal mucosa to obtain a cell sample.
- a suitable specimen will contain endocervical cells with squamous and/or glandular cells.
- the exfoliative cell specimen is generally smeared on the slide to provide a thin layer of the specimen on the surface of the slide.
- a “monolayer” is defined as substantially a two-dimensional layer of uniformly distributed cellular material, predominantly made up of single cells and small clusters of cells.
- Previous methods for disaggregating the exfoliated cells and using such disaggregated cells to produce a “monolayer” of cells on a specimen slide have included ultrasonic vibration, shearing with a rotor, syringing, forced filtration, centrifugation, sedimentation, and filter transfer.
- one smear can be stained according to the conventional Papanicolaou technique and the other smear can be used to detect expression of MN/CA9 antigen.
- a conventional Pap smear sample can be destained to remove Pap stain and then re-stained, for example using immunohistochemical techniques, to detect expression of MN/CA9 antigen.
- TBS Bethesda System
- the Bethesda System first reports the adequacy of the sample, e.g., if endocervical cells are present, and uses descriptive terms for abnormal results.
- the System may also describe any benign cellular changes detected on the Pap smear due to fungal, bacterial, protozoal, or viral infection.
- the results may report if the Pap smear detected reactive cellular changes associated with inflammation, atrophic vaginitis, radiation, or an intrauterine contraceptive device. (IUD).
- Pap smears containing atypical cells or cell clusters that do not correspond to benign cellular changes are given a descriptive diagnosis of epithelial cell abnormalities.
- the descriptive diagnosis of epithelial cell abnormalities is further categorized as squamous or glandular cell abnormalities.
- the descriptive diagnosis of squamous cell abnormalities can include: (1) atypical squamous cells of undetermined significance (ASCUS); (2) low-grade squamous intraepithelial lesions (LSIL); or (3) high-grade squamous intraepithelial lesions (HSIL).
- ASCUS indicates abnormalities that do not fit the criteria for a squamous intraepithelial lesion (SIL), but are nevertheless noteworthy.
- the ASCUS category may be further qualified with Favor Reactive Process, Changes Associated with Atrophy, or Favor Neoplasia.
- LSIL includes mild dysplasia and may include changes suggestive of human papilloma virus (HPV).
- HSIL includes moderate to severe dysplasia, carcinoma in situ, and squamous cell carcinoma.
- glandular cell abnormalities can include: (1) glandular endometrial cells, which are cytologically benign, in a post-menopausal woman; (2) atypical glandular cells of undetermined significance (AGUS); and (3) pre-invasive or malignant neoplasms such as endocervical, endometrial, extrauterine, or other adenocarcinoma.
- AGUS atypical glandular cells of undetermined significance
- pre-invasive or malignant neoplasms such as endocervical, endometrial, extrauterine, or other adenocarcinoma.
- the AGUS category may be further qualified with Favor Reactive Process, Favor Neoplastic Process and Not Otherwise Specified (NOS).
- Pap smear cells that have been cytologically diagnosed as atypical glandular cells of undetermined significance (AGUS) under the Bethesda System of terminology are subjected to a procedure whereby expression of MN/CA9 antigen is detected.
- MN/CA9 antigen is herein defined to mean proteins and/or polypeptides encoded by an MN/CA9 gene or fragments thereof.
- a “polypeptide” is a chain of amino acids covalently bound by peptide linkages and is herein considered to be composed of 50 or less amino acids.
- a “protein” is herein defined to be a polypeptide composed of more than 50 amino acids.
- the MN/CA9 gene encodes an MN/CA9 protein of about 48 kDa.
- MN/CA9 protein is a transmembrane glycoprotein, which may be localized on the cell surface and in the nucleus of HeLa cells and in some human carcinomas.
- the MN/CA9 protein is expressed in HeLa cells as twin proteins of 54 and 58 kDa, which can form disulfide-linked oligomers.
- the gene for the MN/CA9 protein includes the nucleotide sequence of SEQ ID NO:1, which encodes the amino acid sequence of SEQ ID NO:2., as described in U.S. Pat. No. 5,387,676, incorporated herein by reference.
- MN/CA9 antigens include proteins and/or polypeptides encoded by MN/CA9 alleles, which have varying amino acid sequences, including without limitation, amino acid substitutions, extensions, deletions, truncations and combinations thereof, that fall within the scope of this invention.
- a protein is subject to post-translational modifications, such as proteolytic or degradative processes in vivo; thus, truncated MN/CA9 polypeptides or MN/CA9 proteins that are significantly modified, e.g., by the presence or absence of glycosylated, phosphorylated, adenylated, or myristoylated residues, may also be found in clinical specimens.
- MN/CA9 antigen is used herein to encompass modified MN/CA9 proteins and/or polypeptides that retain a characterizing fraction of an MN/CA9 protein and/or polypeptide, such as an antigenic determinant or immunoreactive epitope, which binds detectably to an anti-MN/CA9 antibody.
- MN/CA9 antigen may be reacted with a binding moiety capable of specifically binding the MN/CA9 protein/polypeptide, thereby producing a binding moiety/MN/CA9 antigen complex.
- the specimen is contacted with an antibody that is specific for MN/CA9 antigen under conditions for binding of the antibody to the antigenic site. After contact, the presence or absence of an antibody/antigen complex is determined.
- the binding moiety is typically an antibody capable of forming an antibody/MN/CA9 antigen complex, i.e., an anti-MN/CA9 antibody.
- antibody is understood to mean a binding protein, for example, an immunoglobulin or other protein comprising an immunoglobulin variable region-like binding domain, having the appropriate binding affinity and specificity for MN/CA9 antigen. Methods for generating polyclonal or monoclonal antibodies are well known in the art.
- the antibody may be from a mammalian source, including human, murine, or a combination thereof.
- the antibody is preferably IgG, but may be an IgM, IgE, IgA, or the like.
- antibody fragments such as Fab, F(ab′) 2 , Fv, and so forth.
- the antibody fragments may be prepared by conventional techniques, for example, by peptidase digestion of the antibody using papain or pepsin.
- antibody fragments may be genetically engineered, preferably from the variable regions of the light and/or heavy chains (V H and V L ), including the hyper variable regions, and still more preferably from the V H and V L region.
- V H and V L variable regions of the light and/or heavy chains
- hybrid antibodies capable of binding more than one antigen, constant-variable region chimeras, “composite” immunoglobulins with heavy and light chains from different origins, and “altered” antibodies with improved specificity and other characteristics can be prepared by recombinant techniques for use in the present invention.
- Monoclonal antibodies are preferred.
- the means for producing a detectable signal is incorporated into the assay system.
- a primary antibody employed in the assay e.g., an anti-MN/CA9 antibody
- the label may be a chromophore, including fluorescent dyes such as fluorescein, rhodamine, Cy5, and the like; or an enzyme, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase or the like.
- the primary antibody can be conjugated to a ligand that specifically binds a second binding moiety, wherein the ligand can include haptens, such as DNP or biotin.
- An indirect labeling procedure may also be used wherein one may contact the washed slide with a second binding moiety, that binds specifically to the primary antibody or to a ligand conjugated to the primary antibody.
- a monoclonal antibody derived from a murine source is the primary antibody
- a labeled anti-mouse immunoglobulin may be used as a secondary antibody.
- an avidin containing molecule or an anti-biotin antibody can specifically bind and form a complex with a primary or secondary antibody conjugated to biotin.
- the primary or secondary binding moieties or their ligands may be linked with a detectable label, such as an enzymatic, fluorescent, radioactive, phosphorescent or colored particle label.
- a detectable label such as an enzymatic, fluorescent, radioactive, phosphorescent or colored particle label.
- the labeled complex may be detected, e.g., by microscopic examination or with the aid of an image detector.
- MN/CA9 expression can be determined using in situ cytohybridization to selectively detect a target RNA molecule encoding an MN/CA9 protein/polypeptide.
- the target mRNA molecule may be amplified and detected, for example, with primers and hybridization probes capable of hybridizing specifically with at least a portion of the mRNA molecule encoding the MN/CA9 protein/polypeptide or its complementary sequences.
- the primers and/or hybridization probes are oligonucleotides substantially complementary or identical to portions of SEQ ID NO:1.
- the probes are capable of hybridizing with sequences complementary to SEQ ID NO:1 under moderate to stringent hybridization conditions.
- the next step of the method is observing the distribution of MN/CA9 antigen expressed on atypical and/or normal cells of the AGUS cytologically diagnosed Pap smear.
- Morphologically normal reserve and columnar cells constitute the cell population that we define as normal endocervical cells (ECs). Any cell clusters that morphologically deviate from normal squamous cells, endocervical reserve cells, or columnar cells are considered atypical cell clusters.
- the nuclei of atypical cells are, in general, 3 to 5 times larger than normal ECs and exhibit significant hyperchromasia with an increased nuclear/cytoplasmic ratio.
- Atypical cells may be derived from atypical squamous metaplasia, atypical reserve cell proliferation, and endocervical glandular atypia.
- the distribution of MN/CA9 antigen expression may fall within one or more general patterns.
- a negative MN/CA9 expression pattern exists either when no label is observed or two or less normal endocervical cells are detectably labeled.
- a positive MN/CA9 expression pattern is present when detectable label is observed in any atypical cell cluster or in more than two normal endocervical cell clusters.
- a diffuse staining pattern is when the majority of the atypical cells/cell clusters or more than 100 normal endocervical cells/cell clusters in each smear exhibit strong MN/CA9 expression.
- a focal pattern of staining is observed when only isolated endocervical cell clusters and /or atypical cells are positive.
- the method of the present invention lowers the level of false negative results in Pap smear cells previously diagnosed as AGUS.
- the precursor to cervical cancer is dysplasia, also known in the art as cervical intraepithelial neoplasia (CIN) or squamous intraepithelial lesions (SIL).
- CIN cervical intraepithelial neoplasia
- SIL squamous intraepithelial lesions
- Cells are diagnosed as AGUS when atypical cells are observed, putatively of glandular origin, which the cytologist does not consider to be dysplasia.
- false negative results are avoided because significant or serious cancerous or pre-cancerous lesions are associated with MN/CA9 antigen detection on atypical cells of AGUS-diagnosed Pap smear specimens.
- the significant lesions can include adenocarcinoma, invasive carcinoma (CA), and high grade SIL (HSIL).
- CA invasive carcinoma
- HSIL high grade SIL
- LSIL low grade SIL
- Preferred embodiments of the present invention include two particular MN/CA9 antigen staining patterns, typically found on atypical cells, that are always diagnostic of Pap smears obtained from patients having a significant or serious lesions associated with cervical cancer or its precursor.
- the Pap smear cells exhibit a pattern of MN/CA9 expression described herein as focally columnar or as a honeycomb configuration.
- Examples of the honeycomb pattern can be found in FIG. 2 (panel I), FIG. 6 (panel F) and FIG. 7 (panel F).
- the honeycomb configuration on atypical cells and clusters in a diffuse pattern is a characteristic of Pap smears obtained from patients with adenocarcinoma (AIS) or invasive adenocarcinoma.
- AIS adenocarcinoma
- AIS adenocarcinoma
- the Pap smear cells exhibit a pattern of MN/CA9 expression on atypical cells that are arranged in tight clusters (for reference see FIG. 2, panels B, C, F, G, and H; FIG. 5, panels E and F; FIG. 6, panel E; and FIG. 7, panel E).
- the pattern of staining of atypical cells in tight clusters can be diffuse or focal (see, e.g., FIG. 2, panels F and G) and is usually a characteristic of Pap smear cells from patients with high grade SIL (HSIL).
- HSIL high grade SIL
- low grade lesions are distinguishable from more serious lesions.
- Such low grade lesions are associated with another staining pattern, wherein expression of MN/CA9 antigen is not detected on atypical cells, but is restricted to normal cells.
- Staining that detects the presence of MN/CA9 antigen only on normal ECs is diagnostic of Pap smears obtained from patients having less serious low grade lesions, including low grade SIL (LSIL) and atypia.
- LSIL low grade SIL
- preferred versions of the present invention include a step wherein the absence of any pattern of MN/CA9 expression described above generally correlates with a benign condition. Consequently, a negative staining pattern, where neither normal nor atypical cells exhibit staining for MN/CA9 antigen, is diagnostic of a benign condition.
- the present invention provides an improved method for diagnosing the presence of significant and low grade lesions associated with cervical cancer and its precursors.
- the method overcomes the problems of uncertainty and/or false negative results in Pap smear specimens, previously categorized as atypical glandular cells of undetermined significance (AGUS).
- AGUS atypical glandular cells of undetermined significance
- the method can distinguish less serious lesions from significant cancerous or pre-cancerous lesions in the cervix.
- the absence of MN/CA9 expression is a reliable indicator of a benign condition.
- Table 1 compares the present study with follow-up studies conducted by several other laboratories in those cases diagnosed as endocervical atypia. The cumulative results, as shown in Table 1, indicate that approximately 40% of AGUS cases represent high grade SIL, adenocarcinoma in situ (AIS), or carcinoma, and correspondingly 60% represent benign or insignificant lesions.
- SIL high grade SIL
- AIS adenocarcinoma in situ
- carcinoma adenocarcinoma in situ
- the median age of the patients included in the study was 39 (range was 20-81 years). All of the Pap smears were collected by a cytobrush and a wooden spatula. The sources of tissue sections of the cervices were from colposcopic-directed cervical biopsies, endocervical curettage, cervical conization or hysterectomy.
- Cytology The Pap smears were screened by 6 cytotechnicians and reviewed by 8 pathologists, including three board certified cytopathologists. The TBS classification was used in the study and cytologic diagnosis of AGUS was further classified as AGUS-favor reactive, AGUS-favor neoplastic, and AGUS-not otherwise specified (AGUS-NOS). The Pap smears were not reviewed for diagnostic stringency; thus the results are concordant with the actual cytologic screening in a private practice situation. The cytologic and histologic data were correlated. The MN/CA9 immunoreactivity was interpreted in a blinded fashion and the results were then correlated with the histologic data. Only in those cases where there was a discrepancy between the original histologic diagnosis and MN/CA9 immunoreactivity were the histologic sections reviewed with knowledge of the results of MN/CA9 immunostaining.
- ABSC avidin-biotin peroxidase complex
- the slides were then incubated with appropriate blocking serum (5% normal horse serum in PBS), followed by incubation with purified ascites fluid-derived primary antibody MN75 (1: 10,000 dilution in PBS containing 0.1% BSA) for 60 minutes.
- the secondary biotinylated horse antimouse immunoglobulin G antibody (1:200 dilution in PBS) was then added for 30 min., followed by incubation with avidin-biotin peroxidase complex (ABC Elite) for 30 min. (Vector Laboratories, Burlingame, Calif.).
- Diaminobenzidine tetrahydrochloride was used as chromagen (Sigma Chemical Co., St. Louis, Mo.). After treatment, the sections were washed with distilled H 2 O, counterstained with hematoxylin, and mounted with Permount.
- any MN/CA9 immunoreactive cells in cell clusters that morphologically deviated from normal squamous cell, endocervical reserve cell or columnar cell were considered as atypical cell clusters.
- the nuclei of those atypical cells were, in general, 3 to 5 times larger than normal endocervical cells (ECs) and exhibited significant hyperchromasia with an increased nuclear/cytoplasmic ratio.
- the numbers of positive cells or cell clusters in each smear were counted under 4 ⁇ scanning power.
- Diffuse MN/CA9 immunoreactivity was defined as when more than 50% of the atypical cells/cell clusters or more than 100 normal endocervical cells/cell clusters in each smear exhibited strong (++/+++) MN/CA9 immunoreactivity.
- the smear was scored as positive when brown staining was identified in any atypical cell cluster or in more than two normal endocervical cell clusters, and negative when no brown reaction was seen or staining was limited to two or less normal endocervical cell Clusters in each smear.
- Histology The tissue sections of the cervix from each case were reviewed and interpreted as benign, atypia, cervical intraepithelial neoplasia (CIN), or AIS/Carcinoma (AIS/CA).
- CIN cervical intraepithelial neoplasia
- AIS/CA AIS/Carcinoma
- the criteria used for diagnoses of glandular atypia/neoplasia and CIN are those defined by Crum & Kurman (Crum C P, Cibas E S, Lee K R. Criteria for grading squamous intraepithelial lesions. In: Pathology of Early Cervical Neoplasia, New York Churchill Living stone, 1997. 47-91; and Kurman R J, Norris H J, Wilkinson E.
- FIG. 1 The benign category included normal cervix with or without inflammation (panel A).
- the diagnosis of atypia included atypical squamous metaplasia/atypical reserve cell proliferation and endocervical glandular atypia (panels B and C).
- a two scale system was used for the diagnosis of CIN, namely low grade (Condyloma/CIN I) (panel D) and high grade (CIN II and CIN III) (panel E).
- An example of AIS is shown in panel F.
- Biopsy follow-up showed thirty four (14%) of the cervices had no obvious abnormalities.
- the corresponding Pap smears were 13 specimens diagnosed as AGUS-favor reactive, 20 as AGUS NOS and one specimen diagnosed as AGUS-favor neoplastic.
- cytologic diagnoses were: 3 AGUS-favor reactive, 8 AGUS-NOS, and 1 AGUS-favor neoplastic.
- MN/CA9 Immunoreactivity in Pap Smears In normal Pap smears the exfoliative endocervical cells/cell clusters and endometrial cells from the lower uterine segment are consistently MN/CA9 negative (data not shown). In the AGUS Pap smears we found a relatively complex pattern of cytologic morphology /immunoreactivity combinations. These are illustrated in FIG. 2 and serve as a framework for succeeding interpretations in the study. Cytologic criteria for atypical glandular cells, delineated in the TBS classification, were used in the interpretation (National Cancer Institute Workshop. The revised Bethesda System for reporting cervical/vaginal cytologic diagnosis: report of the 1991 Bethesda Workshop.
- Panel A shows an AGUS Pap smear that exhibits no MN/CA9 immunoreactivity.
- positive immunostaining In smears where positive immunostaining is seen there are two broad categories: those where atypical cells, with or without normal ECs, are positive and those where normal ECs only stain (panels B-D). In those smears that do exhibit positive immunostaining, variations in intensity of stain are encountered.
- Panel B illustrates clusters of atypical cells that are strongly positive (arrow) and normal ECs that are weakly positive (arrowhead). The pattern of immunostaining may also vary.
- Panel F shows an example of focal staining and panel G shows an example of diffuse immunoreactivity.
- FIG. 4 shows how predictive AGUS diagnoses (i.e. AGUS-favor reactive, AGUS-NOS and AGUS-favor neoplastic) were when compared to their respective patterns of MN/CA9 immunostaining.
- FIG. 5 Examples of the Pap staining and immunostaining are shown in FIG. 5. All of the atypical cell clusters in panels A, B, and C show minimal cellular overlap and nuclear pleomorphism, with a mild degree of hyperchromasia. In panel B the atypical cells are associated with ciliated metaplastic cells (arrow). However, when there was MN/CA9 immunostaining of the cells, including ciliated cell clusters (panels E and F), high grade lesions (HSIL) were found in the cervices. Conversely, no dysplastic tissue was identified when no immunostaining was seen (panel D).
- HSIL high grade lesions
- FIG. 6 Examples of the Pap staining and immunostaining are shown in FIG. 6. All of the atypical cells in panels A, B and C show cellular overlap, moderate nuclear pleomorphism and slightly increased nuclear/cytoplasmic ratio. Again, HSIL (panel E) and AIS (panel F) were histologically identified only in those cases where atypical cells stained positive. No dysplastic lesion was observed in those cases in which no MN/CA9 immunostaining was detected (panel D).
- FIG. 7 Examples of the Pap staining and immunostaining are shown in FIG. 7. All of the atypical cell clusters in panels A, B and C show cellular overlap, marked nuclear pleomorphism, hyperchromasia and increased nuclear/cytoplasmic ratio. HSIL and AIS were observed in the corresponding cervices of those Pap smears where MN/CA9 immunostaining was seen in the atypical cell clusters (panels E and F). Benign histology was seen in the MN/CA9 negative Pap smear (panel D).
- LSIL cases showed staining of atypical ⁇ normal ECs.
- the immunostaining pattern seen in AIS cases is always diffuse and the stained cell clusters exhibit columnar or honeycomb configurations. This is in contrast to cases of HSIL, where the cells are arranged in tight clusters, and in the majority of cases they exhibit a focal pattern of immunostaining. Therefore, on this basis of the difference in morphologies of the stained cell clusters and their immunostaining patterns one is able to discriminate between AIS and HSIL in the AGUS Pap smear diagnoses. This has clear and significant clinical implications.
- MN/CA9 immunostaining of atypical cells predicted all of the cases where significant lesions were found.
- MN/CA9 antigen expression in atypical cells is an excellent biomarker for use in AGUS diagnoses, and can be used by a clinician with great advantage as an adjunct in determining which AGUS diagnoses are likely yield significant lesions. This has a distinct cost-benefit potential since it has been shown that only approximately 40% of AGUS diagnoses are correspondingly associated with significant lesions (see Table 1).
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Pregnancy & Childbirth (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Reproductive Health (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gynecology & Obstetrics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
Description
- This application claims the benefit of Provisional Patent Application No. 60/147,556, filed Aug. 5, 1999.
- Carcinoma of the cervix is one of the most common malignancies in women, and worldwide it is second only to breast cancer in both incidence and mortality (NIH Consensus Statement (1) Cervical Cancer. 1996.14:1-18). In developed countries in general, and the United States in particular, the wide acceptance of the Pap smear screening program has resulted in dramatic decreases in the incidence of, and mortality from, cervical cancer. However, a recent survey conducted in United States still showed that an estimated 15,700 women were diagnosed with invasive cervical carcinoma, and an estimated 4,900 women died of cervical cancer per annum (NIH Consensus Statement (1) Cervical Cancer. 1996.14:1-18). Moreover, the incidence of adenocarcinorna has tripled in the past two decades (McGonigle K F, and Berek J S. Early stage squamous cell and adenocarcinoma of the cervix. Curr. Opin. Obstet. Gynecol., 1992. 4:109-119), and currently represents up to 25% of all cervical cancers diagnosed (Miller B E, Flax S D, Arheart K, Photopulos G. The presentation of adenocarcinoma of the uterine cervix. Cancer 1993. 72:1281-1285; and Crum C P, Cibas E S, Lee K R. Glandular precursors, adenocarcinomas, and their mimics. In: Pathology of early cervical neoplasia. New York: Churchill Living stone, 1997. 177-240).
- Many factors have been implicated in the relatively high incidence of cervical cancer but the inherently high rate of false negatives appears to be one of the major concerns in the current system of screening. The errors have been attributed, in part, to technical problems, but also may well be based primarily on human factors that are not remedied readily by rules and regulations (Kos L G. Cervical (Pap) smear. New directions. Cancer (Phial.), 71 (suppl.); 1993. 1406-1412).
- In recent years, the use of new cervical sampling devices (e.g. cytobrushes) have improved the detection of both glandular and squamous neoplasms involving the endocervical canal. However, the increased endocervical cell yields have also created many new patterns of benign and neoplastic endocervical cytology with significant overlapping features. These patterns are unfamiliar to cytologists in routine daily practice. This uncertainty has been reflected in an increase in the cytologic diagnosis of endocervical glandular atypia.
- The morphological criteria for a Pap smear diagnosis of AIS and adenocarcinoma have been well described (Ayer B, Pacey F, Greenberg M, Bousfield L. The cytologic diagnosis of adenocarcinoma in situ of the cervix uteri and related lesions. I. Adenocarcinoma in situ. Acta Cytol., 1987. 31:397-411; and Pacey N F. Glandular neoplasms of the uterine cervix. In: Bibbo M, ed. Comprehensive Cytopatholog. Philadelphia: W B Saunders, 1991. 243-255). However, false-negative rates of up to 40 percent in Pap smears from women later found to have AIS and/or invasive endocervical adenocarcinoma have been reported (Crum et al., 1997, 177-240; and Kim H S, Underwood D, Frable W J. Adenocarcinoma in the cervicovaginal Papanicolaou smear: analysis of a 12-year experience. Diagn. Cytopathol., 1991. 7:119-124). The earlier misdiagnosing of AIS/CA was attributed to the presence of low numbers of endocervical glandular cells in the Pap smears, and/or technical artifacts. However, studies have also indicated that human error may play an important role. This includes the incorrect diagnosis of dysplastic glandular cells as exfoliative endometrial cells and/or reactive endocervical cells (Kos et al., 1993; and Lee K R, Manna E A, St. John T. Atypical endocervical glandular cells: accuracy of cytologic diagnosis. Diagn. Cytopathol. 1995. 13:202-208).
- In an attempt to clarify the situation, the Bethesda System committee in 1988 established cytologic criteria for atypical glandular cells, and proposed a new diagnostic terminology with a subclassification scheme, in order to aid in patient triage. It was recommended that endocervical glandular abnormalities that exceed reactive change but fall short of invasive adenocarcinoma be reported as atypical glandular cells of undetermined significance (AGUS) (National Cancer Institute Workshop. The revised Bethesda System for reporting cervical/vaginal cytologic diagnosis: report of the 1991 Bethesda Workshop. JAMA, 1992. 267:1892; and Kurman R J, Solomon D. In The Bethesda System for Reporting Cervical/Vaginal Cytologic Diagnoses. New York: Springer-Verlag, 1994. 64-76). Up to 1.25 million American women receive a diagnosis of AGUS annually (Raab S S, Geisinger K R, Silverman J F, Thomas P A, Stanley M W. Interobserver variability of a Papanicolaou smear diagnosis of atypical glandular cells of undetermined significance. Am. J. Clin. Pathol., 1998. 110:653-659). However, the results of this subclassification have yet to be proven clinically effective.
- A broad morphologic spectrum of benign and neoplastic lesions are included in the category of AGUS. These include atypical endocervical repair, endometriosis, microglandular hyperplasia, tubal metaplasia, squamous intraepithelial lesion (SIL) with glandular involvement, and glandular dysplasia, and adenocarcinoma (Lee K R. Atypical glandular cells in cervical smears from women who have undergone cone biopsy. A potential diagnostic pitfall. Acta. Cytol., 1993. 37:705-709; Pacey F, Ayer B, Greenberg M. The cytologic diagnosis of adenocarcinoma in situ of the cervix uteri and related lesions. III. Pitfalls in diagnosis. Acta. Cytol., 1988. 32:325-330; Yahr U, Lee K R. Cytologic findings in microglandular hyperplasia of the cervix. Diagn. Cytopathol. 1991. 7:248-251; Novotny D B, Maygarden S J, Johnson D E, Frable W J. Tubal metaplasia. A frequent potential pitfall in the cytologic diagnosis of endocervical glandular dysplasia on cervical smears. Acta. Cytol., 1992. 36:1-10; Pacey et al., 1991; and Kos L G. Diagnostic cytology and its histopathologic bases. Vol. 1. 4th ed. Philadelphia; J B Lippincott: 1992. 387:452-454).
- Several laboratories have conducted follow-up studies in those cases diagnosed as endocervical atypia. The results indicate that approximately 40% of AGUS cases represent high grade SIL, adenocarcinoma in-situ (AIS), or carcinoma, and correspondingly 60% represent benign or insignificant lesions. A surprising range (15-58%) of patients had significant lesions (HSIL, AIS/CA) in follow-up biopsies, after receiving a Pap smear diagnosis of AGUS. Furthermore, only a small fraction of the significant lesions were glandular neoplasms (AIS/CA). Thus, the AGUS diagnosis appears to be a misnomer inasmuch that many AGUS lesions are not glandular at all (Wilbur D C, Mulford D M, Sickel J Z, Atkinson K M. The problem of endocervical atypia: new cytologic presentations of normal endocervical cells. Mod. Pathol., 1994. 38:808; Goff B A., Atanasoff P, Brown E, Muntz H G, Bell D A, Rice L W. Endocervical glandular atypia in Papanicolaou smears. Obstet. Gynecol., 1992. 79:101-104; Lee et al., 1995; Nasu I, Meurer W, Fu Y S. Endocervical glandular atypia and adenocarcinoma: A correlation of cytology and histology., Int. J. Gynecol. Pathol., 1993. 12:208-218; Taylor R, Guerriere J, Nash J D, Henry M R, O'Conner D M. Atypical cervical cytology: Colposcopic follow-up using The Bethesda System, J. Reprod. Med., 1993. 38:443-447; Kennedy A W, Salmieri S S, Wirth S L, Biscotti C V, Tuason L J, Travarca M J. Results of the clinical evaluation of atypical glandular cells of undetermined significance (AGUS) detected on cervical cytology screening. Gynecol. Oncol., 1996. 63:14-18; and Bose S, Kannan V, Kline T S. Abnormal endocervical cells. Really abnormal? Really endocervical? Am. J. Clin. Pathol., 1994. 101:708-713).
- There have been many attempts to define cytologic criteria that aid in the diagnosis of glandular lesions of the cervix, with the goal of separating neoplastic glandular cells from reactive changes and squamous neoplasia. Yet, there remains poor agreement among cytopathologists in reclassifying lesions originally diagnosed as AGUS, and in separating clinically significant from benign AGUS lesions (Raab et al., 1998; Novotny et al., 1992; Nasu et al.,1993; Raab S S, Isacson C, Layfield L J, Lenel, J C, Slagel, D D, Thomas P A. Atypical glandular cells of undetermined significance: cytologic criteria to separate clinically significant from benign lesions. Am. J. Clin. Pathol., 1995. 104:574-582; Raab S S, Snider T E, Potts S A, McDaniel H L, Robinson R A, Nelson D L, Sigman J D, Thomas P A. Atypical glandular cells of undetermined significance. Diagnostic accuracy and interobserver variability using select cytologic criteria. Am. J. Clin. Pathol., 1997. 107:299-307; and DiTomasso J P, Ramzy I, Mody D R. Glandular lesions of the cervix.: Validity of cytologic criteria used to differentiate reactive changes, glandular intraepithelial lesions and adenocarcinoma. Acta. Cytol., 1996. 40:1127-1135).
- This diagnostic uncertainty has posed a particular dilemma in clinical management decisions, both from a cost-benefit standpoint and a desire not to subject patients to unnecessary invasive procedures. Therefore, there is a need for a useful discriminator of AGUS diagnoses that can separate glandular cells that are atypical due to reactive-reparative changes from cells that are atypical due to dysplasia and carcinoma.
- Recently, a novel antigen, termed MN/CA9, has been described. It is a transmembrane glycoprotein that was discovered in the cervical adenocarcinoma cell line, Hela (Zavada J, Zavadova Z, Pastorekova S, Ciampor F, Pastorek J, Zelnik V. Expression of MaTu-MN protein in human tumor cultures and in clinical specimens. Int. J. Cancer 1993. 54:268-274). The gene encoding the MN/CA9 product is unusual—the only homologous functional domain identified to date being a carbonic anhydrase domain. Evidence supporting the role of the MN/CA9 protein in neoplastic progression includes its association with the tumorigenic phenotype in human cell hybrids and neoplastic transformation of mouse 3T3 cells following transfection with MN cDNA (Zavada et al., 1993; and Pastorek J, Pastorekova S, Callebaut I, Morrion J P, Zelnik V, Opavsky R, Zatovicova M, Liao S, Portelle D, Stanbridge E J, Zavada J, Burny A, Kettmann R. Cloning and characterization of MN, a human tumor-associated protein with a domain homologous to carbonic anhydrase and a putative helix-loop-helix DNA binding segment. Oncogene 1994. 9:2877-2888). A preliminary screen of clinical specimens indicated that expression of MN/CA9 protein is restricted to very few normal tissues, but significant levels of expression were noted in certain malignancies, including cervical neoplasms (Pastorek et al., 1994).
- A study of several hundred benign and neoplastic cervical specimens has shown that MN/CA9 is expressed in all cases of AIS and in more than 90% of cervical squamous neoplasms. High levels of MN/CA9 protein expression were frequently observed in the normal-looking endocervical cells in regions adjacent to dysplastic tissues but the normal cervix does not express MN/CA9 protein. In addition, a study of 305 Pap smears has also indicated that the MN/CA9 expression seen in exfoliative cells in Pap smears recapitulates MN/CA9 expression in the corresponding tissue sections of the cervix. Virtually all atypical glandular cells derived from AIS and adenocarcinoma expressed high levels of MN antigen, whereas endocervical cells obtained from benign cervices were negative (Liao S Y, Brewer C, Zavada J, Pastorek J, Pastorekova S, Marietta A, Berman M L, DiSaia P J, Stanbridge E J. Identification of the MN antigen as a diagnostic biomarker of cervical intraepithelial squamous and glandular neoplasia and cervical carcinomas. Am. J. Pathol., 1994. 145: 598-609; (Liao S Y, Stanbridge E J. Expression of the MN antigen in cervical Papanicolaou smears is an early diagnostic biomarker of cervical dysplasia. Canc. Epid. Biom. Prev., 1996. 5:549-557).
- While these results have shown that expression of the MN/CA9 antigen can indicate AIS and adencarcinoma, they have not enabled reliable distinction between high grade and low grade lesions and benign conditions. Enabling such a distinction is critical to meaningful clinical diagnosis. The deadly harm caused by false negatives, and the anguish and expense caused by false positives demands a more reliable clinical procedure.
- The present invention fulfills the need for a more accurate diagnosis of AGUS than the current routine conventional Pap smear screening. The invention provides new insight into the distribution of MN/CA9 antigen on atypical and/or normal cells on AGUS-diagnosed Pap smear specimens and serves as a clinical roadmap enabling MN/CA9 antigen to at last serve as a powerful diagnostic biomarker for cancerous or pre-cancerous conditions.
- Specifically, the present invention for the first time provides a reliable comprehensive method to determine the presence of cancerous or pre-cancerous cervical lesions from Pap smear cells that have been cytologically diagnosed as atypical glandular cells of undetermined significance (AGUS) under the Bethesda System of terminology. The AGUS-diagnosed Pap smear cells are subjected to a procedure that detects expression of MN/CA9 antigen, such as immunohistochemistry or in situ cytohybridization. AGUS-diagnosed Pap smears typically lack readily ascertainable dysplastic cells, but do include atypical and normal endocervical cells. It is the distribution of MN/CA9 antigen observed on atypical or normal cells that is used to diagnose the presence of significant or low grade lesions. More specifically, significant lesions, including adenocarcinoma, invasive carcinoma (CA), or high grade squamous intraepithelial lesions (HSIL) are diagnosed when MN/CA9 antigen is observed on atypical cells. Low grade lesions, including low grade squamous intraepithelial lesions (LSIL) or atypia, are diagnosed when MN/CA9 antigen is absent from atypical cells but is present on normal endocervical cells. A benign condition is diagnosed when MN/CA9 is absent from atypical and normal endocervical cells.
- In preferred versions of the present invention, not only the presence, but the nature of, a significant lesion is diagnosed whenever MN/CA9 antigen is observed on atypical cells. In one embodiment, the presence of adenocarcinoma, including adenocarcinoma in situ (AIS) and invasive adenocarcinoma, is diagnosed when MN/CA9 antigen is detected on atypical cells in a columnar or honeycomb configuration. In another embodiment, the presence of HSIL is diagnosed when MN/CA9 antigen is detected on atypical cells in a tight cluster.
- Accordingly, the methods of the present invention overcome problems of uncertainty and false negatives associated with the AGUS diagnosis for Pap smear specimens. In addition, the method discriminates among atypical Pap smears that are associated with significant lesions, low grade lesions or a benign condition.
- These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, appended claims, and accompanying drawings, where:
- FIG. 1 shows a spectrum of squamous and glandular alterations to illustrate the criteria for diagnosis of atypia, preneoplastic and neoplastic lesions: Panel A is a normal cervix showing orderly arrangement of squamous and glandular epithelium, Panel B is an example of atypical squamous metaplasia (left) and reserve cell proliferation (right), Panel C is an example of glandular atypia with epithelial stratification, Panels D and E illustrate LSIL and HSIL, respectively, and Panel F is an example of AIS (Magnification: A 100×, B-F 200×);
- FIG. 2 shows staining patterns of MN/CA9 immunoreactivity in AGUS Pap smears: Panel A is a smear that shows no immunostaining; Panel B shows positive immunostaining in both atypical cells (arrow) and normal ECs (arrowhead), Panel C shows MN/CA9 positive atypical cells only, Panel D is an example of immunoreactivity of normal ECs only, with the corresponding Pap stain in panel E, moreover the pattern of immunoreactivity may be focal (panel F) or diffuse (panel G), and the morphology of the immunostained cells may be tight clusters (panel H) or honeycomb configuration (panel I) (Magnification: A, F and
G 100×; H and I 200×; B, C, D and E, 400×); - FIG. 3 shows distributions of MN/CA9 immunostaining of AGUS Pap smears and their correlation with histologic diagnosis;
- FIG. 4 shows the distribution of MN/CA9 immunostaining of AGUS Pap smears in the three categories: favor reactive, not otherwise specified (NOS), and favor neoplastic, and their correlation with histologic diagnosis;
- FIG. 5 shows cytologic features of atypical cell clusters in AGUS-favor reactive Pap smears, such as enlarged nuclear size with minimal cellular overlap and mild degree of nuclear pleomorphism (panels A, B and C), and the same cell clusters, destained and then immunostained to detect MN/CA9 expression provide examples where no immunoreactivity correlated with no histological lesion (panel D), and strong (panel E) and weak immunoreactivity (panel F) was associated with histologic diagnoses of HSIL: note that the ciliated cells pointed to in panel E are MN/CA9 negative (Magnification: 400×);
- FIG. 6 shows cytologic features of atypical cell clusters in AGUS-NOS Pap smears (panels A, B and C), such as
nuclei 3× larger than the nuclei of the adjacent intermediate cells (arrow), and increased cellular overlap and nuclear hyperchromasia, however corresponding MN/CA9 immunostains of the same specimens demonstrate a negative immunostain in Panel D that corresponds with a follow-up biopsy that was negative, a positive immunostain in panel E corresponding with a histologic diagnosis of HSIL, and a characteristic honeycomb pattern of immunostaining in panel F that corresponds with a follow-up biopsy diagnosed as adenocarcinoma. (Magnification: 400×); and - FIG. 7 shows cytologic features of atypical cell clusters in AGUS-favor neoplastic Pap smears, such as significant cellular overlap, nuclear pleomorphism and hyperchromasia (panels A, B and C) and the corresponding MN/CA9 immunostains show examples where a negative immunostain (panel D) corresponds with a follow-up biopsy that is benign, a positive immunostain (panel E) corresponds with a histologic diagnosis of HSIL, and stained cells, which exhibit honeycomb and columnar features (panel F), corresponds with a follow-up biopsy diagnosed as AIS (Magnification: 400×).
- The method of the present invention entails examining of the levels and distribution of expression of MN/CA9 antigen to confirm diagnoses obtained by cytological examinations, e.g., Pap smears, when observing atypical glandular cells of undetermined significance. A positive result serves as an early marker of dysplasia even in the absence of its clinical manifestations.
- I. Obtain Pap Smear
- The first step in any cytological diagnostic method is obtaining suitable Pap smear cells for review. In a conventional Pap smear test, a cytologist examines an exfoliative cell specimen, obtained by scraping some cells from the lining of the cervix, smearing the cells onto a slide and staining with Papanicolaou stain. The cytologist examines the stained smears for the presence of abnormal-looking cells that indicate the presence of a malignant condition. The term “malignant condition” refers to the presence of dysplasia including adenocarcinoma in situ (AIS), invasive carcinoma (CA), neoplastic, malignant or tumor cells or the like.
- In the method of the invention an exfoliative cell specimen is obtained from a patient, who may or may not harbor a malignant condition. The specimen may be obtained by rotating a cervical sampling device, such as a swab, spatula, or cytobrush along a portion of cervix or vaginal mucosa to obtain a cell sample. A suitable specimen will contain endocervical cells with squamous and/or glandular cells.
- The exfoliative cell specimen is generally smeared on the slide to provide a thin layer of the specimen on the surface of the slide. However, the manual observation of cellular abnormalities or the automated analysis of cytological material can be optimized by preparing “monolayers” of cells on the specimen slides. A “monolayer” is defined as substantially a two-dimensional layer of uniformly distributed cellular material, predominantly made up of single cells and small clusters of cells. Previous methods for disaggregating the exfoliated cells and using such disaggregated cells to produce a “monolayer” of cells on a specimen slide have included ultrasonic vibration, shearing with a rotor, syringing, forced filtration, centrifugation, sedimentation, and filter transfer. Examples of such techniques include the automated ThinPrep® System (Cytyc Corporation, Marlborough, Mass.), which was described by Hutchinson, M. L., et al., Anatomic Pathology, Vol. 96, No. 3, pp. 300-305(1991), and the CytoRich process system (Hoffman-La Roche Inc., Nutley, N.J.) described in U.S. Pat. No. 5,346,831.
- When duplicate smears are available, one smear can be stained according to the conventional Papanicolaou technique and the other smear can be used to detect expression of MN/CA9 antigen. Alternatively, after the cytological examination, a conventional Pap smear sample can be destained to remove Pap stain and then re-stained, for example using immunohistochemical techniques, to detect expression of MN/CA9 antigen.
- II. Cytological Dx: The Bethesda System (TBS)
- The TBS classification system is used for the preliminary cytological diagnosis of Pap smear cells. The Clinical Laboratory Improvement Act of 1988 mandated a uniform system for reporting Pap smear screening results. The system that was developed pursuant to that Act is known as The Bethesda System (TBS), and has been in effect since 1991, replacing the earlier classification system. There is no accurate way to correlate the two systems, except in the broadest of terms.
- The Bethesda System first reports the adequacy of the sample, e.g., if endocervical cells are present, and uses descriptive terms for abnormal results. The System may also describe any benign cellular changes detected on the Pap smear due to fungal, bacterial, protozoal, or viral infection. In addition the results may report if the Pap smear detected reactive cellular changes associated with inflammation, atrophic vaginitis, radiation, or an intrauterine contraceptive device. (IUD).
- Pap smears containing atypical cells or cell clusters that do not correspond to benign cellular changes are given a descriptive diagnosis of epithelial cell abnormalities. The descriptive diagnosis of epithelial cell abnormalities is further categorized as squamous or glandular cell abnormalities.
- The descriptive diagnosis of squamous cell abnormalities can include: (1) atypical squamous cells of undetermined significance (ASCUS); (2) low-grade squamous intraepithelial lesions (LSIL); or (3) high-grade squamous intraepithelial lesions (HSIL). ASCUS indicates abnormalities that do not fit the criteria for a squamous intraepithelial lesion (SIL), but are nevertheless noteworthy. The ASCUS category may be further qualified with Favor Reactive Process, Changes Associated with Atrophy, or Favor Neoplasia. LSIL includes mild dysplasia and may include changes suggestive of human papilloma virus (HPV). HSIL includes moderate to severe dysplasia, carcinoma in situ, and squamous cell carcinoma.
- The descriptive diagnosis of glandular cell abnormalities can include: (1) glandular endometrial cells, which are cytologically benign, in a post-menopausal woman; (2) atypical glandular cells of undetermined significance (AGUS); and (3) pre-invasive or malignant neoplasms such as endocervical, endometrial, extrauterine, or other adenocarcinoma. The AGUS category may be further qualified with Favor Reactive Process, Favor Neoplastic Process and Not Otherwise Specified (NOS).
- III. Detection Procedure
- Pap smear cells that have been cytologically diagnosed as atypical glandular cells of undetermined significance (AGUS) under the Bethesda System of terminology are subjected to a procedure whereby expression of MN/CA9 antigen is detected.
- A. MN/CA9 antigen
- The phrase “MN/CA9 antigen” is herein defined to mean proteins and/or polypeptides encoded by an MN/CA9 gene or fragments thereof. A “polypeptide” is a chain of amino acids covalently bound by peptide linkages and is herein considered to be composed of 50 or less amino acids. A “protein” is herein defined to be a polypeptide composed of more than 50 amino acids. The MN/CA9 gene encodes an MN/CA9 protein of about 48 kDa. MN/CA9 protein is a transmembrane glycoprotein, which may be localized on the cell surface and in the nucleus of HeLa cells and in some human carcinomas. The MN/CA9 protein is expressed in HeLa cells as twin proteins of 54 and 58 kDa, which can form disulfide-linked oligomers. The gene for the MN/CA9 protein includes the nucleotide sequence of SEQ ID NO:1, which encodes the amino acid sequence of SEQ ID NO:2., as described in U.S. Pat. No. 5,387,676, incorporated herein by reference.
- It can be appreciated that a protein or polypeptide produced by a neoplastic cell in vivo could be altered in sequence from that produced by a tumor cell in cell culture. Thus, MN/CA9 antigens include proteins and/or polypeptides encoded by MN/CA9 alleles, which have varying amino acid sequences, including without limitation, amino acid substitutions, extensions, deletions, truncations and combinations thereof, that fall within the scope of this invention.
- It can also be appreciated that a protein is subject to post-translational modifications, such as proteolytic or degradative processes in vivo; thus, truncated MN/CA9 polypeptides or MN/CA9 proteins that are significantly modified, e.g., by the presence or absence of glycosylated, phosphorylated, adenylated, or myristoylated residues, may also be found in clinical specimens. Accordingly, the phrase “MN/CA9 antigen” is used herein to encompass modified MN/CA9 proteins and/or polypeptides that retain a characterizing fraction of an MN/CA9 protein and/or polypeptide, such as an antigenic determinant or immunoreactive epitope, which binds detectably to an anti-MN/CA9 antibody.
- B. Immunohistochemistry
- In one embodiment of the present invention, MN/CA9 antigen may be reacted with a binding moiety capable of specifically binding the MN/CA9 protein/polypeptide, thereby producing a binding moiety/MN/CA9 antigen complex. Typically, the specimen is contacted with an antibody that is specific for MN/CA9 antigen under conditions for binding of the antibody to the antigenic site. After contact, the presence or absence of an antibody/antigen complex is determined.
- The binding moiety is typically an antibody capable of forming an antibody/MN/CA9 antigen complex, i.e., an anti-MN/CA9 antibody. As used herein, the term “antibody” is understood to mean a binding protein, for example, an immunoglobulin or other protein comprising an immunoglobulin variable region-like binding domain, having the appropriate binding affinity and specificity for MN/CA9 antigen. Methods for generating polyclonal or monoclonal antibodies are well known in the art. The antibody may be from a mammalian source, including human, murine, or a combination thereof. The antibody is preferably IgG, but may be an IgM, IgE, IgA, or the like. Moreover, other useful antibodies having suitable binding domains include antibody fragments, such as Fab, F(ab′)2, Fv, and so forth. The antibody fragments may be prepared by conventional techniques, for example, by peptidase digestion of the antibody using papain or pepsin.
- Alternatively, antibody fragments may be genetically engineered, preferably from the variable regions of the light and/or heavy chains (VH and VL), including the hyper variable regions, and still more preferably from the VH and VL region. Accordingly, “hybrid” antibodies capable of binding more than one antigen, constant-variable region chimeras, “composite” immunoglobulins with heavy and light chains from different origins, and “altered” antibodies with improved specificity and other characteristics can be prepared by recombinant techniques for use in the present invention.
- Monoclonal antibodies are preferred. A murine hybridoma that produces a representative anti-MN/CA9 antibody, the monoclonal antibody M75, was deposited at the American Type Culture Collection [ATCC, Rockville, Md. (USA)] on Sep. 17, 1992, under ATCC Number HB 11128 as disclosed in U.S. Pat. No. 5,387,676 (incorporated herein by reference).
- Contact between the antibody and the specimen is generally carried out in an aqueous buffered system. After a period of contact between the specimen and the antibody, the slide is washed with an aqueous buffered solution to remove the unreacted antibody. Next, the binding of antibody to antigen is detected via the use of a labeling system.
- To make the determination of the presence of an antigen/antibody(immune) complex, the means for producing a detectable signal is incorporated into the assay system. For example, in direct labeling procedures one may conjugate a primary antibody employed in the assay, e.g., an anti-MN/CA9 antibody, to a label, which is capable of producing a detectable signal. The label may be a chromophore, including fluorescent dyes such as fluorescein, rhodamine, Cy5, and the like; or an enzyme, such as alkaline phosphatase, horseradish peroxidase, glucose oxidase or the like. Alternatively, the primary antibody can be conjugated to a ligand that specifically binds a second binding moiety, wherein the ligand can include haptens, such as DNP or biotin. An indirect labeling procedure may also be used wherein one may contact the washed slide with a second binding moiety, that binds specifically to the primary antibody or to a ligand conjugated to the primary antibody. For example, where a monoclonal antibody derived from a murine source is the primary antibody, a labeled anti-mouse immunoglobulin may be used as a secondary antibody. Alternatively, an avidin containing molecule or an anti-biotin antibody can specifically bind and form a complex with a primary or secondary antibody conjugated to biotin.
- Moreover, the primary or secondary binding moieties or their ligands may be linked with a detectable label, such as an enzymatic, fluorescent, radioactive, phosphorescent or colored particle label. The labeled complex may be detected, e.g., by microscopic examination or with the aid of an image detector.
- C. Amplification and In Situ Hybridization
- In another embodiment of the present invention, MN/CA9 expression can be determined using in situ cytohybridization to selectively detect a target RNA molecule encoding an MN/CA9 protein/polypeptide. The target mRNA molecule may be amplified and detected, for example, with primers and hybridization probes capable of hybridizing specifically with at least a portion of the mRNA molecule encoding the MN/CA9 protein/polypeptide or its complementary sequences. Preferably, the primers and/or hybridization probes are oligonucleotides substantially complementary or identical to portions of SEQ ID NO:1. Most preferably, the probes are capable of hybridizing with sequences complementary to SEQ ID NO:1 under moderate to stringent hybridization conditions.
- IV. Distribution of MN/CA9 Antigen and Dx
- The next step of the method is observing the distribution of MN/CA9 antigen expressed on atypical and/or normal cells of the AGUS cytologically diagnosed Pap smear. Morphologically normal reserve and columnar cells, including metaplastic squamous cells, constitute the cell population that we define as normal endocervical cells (ECs). Any cell clusters that morphologically deviate from normal squamous cells, endocervical reserve cells, or columnar cells are considered atypical cell clusters. The nuclei of atypical cells are, in general, 3 to 5 times larger than normal ECs and exhibit significant hyperchromasia with an increased nuclear/cytoplasmic ratio. Atypical cells may be derived from atypical squamous metaplasia, atypical reserve cell proliferation, and endocervical glandular atypia.
- The distribution of MN/CA9 antigen expression may fall within one or more general patterns. A negative MN/CA9 expression pattern exists either when no label is observed or two or less normal endocervical cells are detectably labeled. On the other hand, a positive MN/CA9 expression pattern is present when detectable label is observed in any atypical cell cluster or in more than two normal endocervical cell clusters. A diffuse staining pattern is when the majority of the atypical cells/cell clusters or more than 100 normal endocervical cells/cell clusters in each smear exhibit strong MN/CA9 expression. In addition, a focal pattern of staining is observed when only isolated endocervical cell clusters and /or atypical cells are positive.
- The method of the present invention lowers the level of false negative results in Pap smear cells previously diagnosed as AGUS. The precursor to cervical cancer is dysplasia, also known in the art as cervical intraepithelial neoplasia (CIN) or squamous intraepithelial lesions (SIL). Cells are diagnosed as AGUS when atypical cells are observed, putatively of glandular origin, which the cytologist does not consider to be dysplasia. In the method of the present invention, false negative results are avoided because significant or serious cancerous or pre-cancerous lesions are associated with MN/CA9 antigen detection on atypical cells of AGUS-diagnosed Pap smear specimens. The significant lesions can include adenocarcinoma, invasive carcinoma (CA), and high grade SIL (HSIL). Expression of MN/CA9 antigen on atypical cells is occasionally associated with low grade SIL (LSIL), however this “false positive” result may forewarn of lesions that will progress to HSIL.
- Preferred embodiments of the present invention include two particular MN/CA9 antigen staining patterns, typically found on atypical cells, that are always diagnostic of Pap smears obtained from patients having a significant or serious lesions associated with cervical cancer or its precursor.
- In one preferred embodiment of the present invention, the Pap smear cells exhibit a pattern of MN/CA9 expression described herein as focally columnar or as a honeycomb configuration. Examples of the honeycomb pattern can be found in FIG. 2 (panel I), FIG. 6 (panel F) and FIG. 7 (panel F). The honeycomb configuration on atypical cells and clusters in a diffuse pattern, is a characteristic of Pap smears obtained from patients with adenocarcinoma (AIS) or invasive adenocarcinoma.
- In another preferred embodiment of the present invention, the Pap smear cells exhibit a pattern of MN/CA9 expression on atypical cells that are arranged in tight clusters (for reference see FIG. 2, panels B, C, F, G, and H; FIG. 5, panels E and F; FIG. 6, panel E; and FIG. 7, panel E). The pattern of staining of atypical cells in tight clusters can be diffuse or focal (see, e.g., FIG. 2, panels F and G) and is usually a characteristic of Pap smear cells from patients with high grade SIL (HSIL).
- In yet another embodiment of the present invention low grade lesions are distinguishable from more serious lesions. Such low grade lesions are associated with another staining pattern, wherein expression of MN/CA9 antigen is not detected on atypical cells, but is restricted to normal cells. Staining that detects the presence of MN/CA9 antigen only on normal ECs is diagnostic of Pap smears obtained from patients having less serious low grade lesions, including low grade SIL (LSIL) and atypia.
- Finally, preferred versions of the present invention include a step wherein the absence of any pattern of MN/CA9 expression described above generally correlates with a benign condition. Consequently, a negative staining pattern, where neither normal nor atypical cells exhibit staining for MN/CA9 antigen, is diagnostic of a benign condition.
- The present invention provides an improved method for diagnosing the presence of significant and low grade lesions associated with cervical cancer and its precursors. The method overcomes the problems of uncertainty and/or false negative results in Pap smear specimens, previously categorized as atypical glandular cells of undetermined significance (AGUS). Despite the absence of readily ascertainable dysplasia on the Pap smear specimen, the method can distinguish less serious lesions from significant cancerous or pre-cancerous lesions in the cervix. Moreover, the absence of MN/CA9 expression is a reliable indicator of a benign condition.
- Although the present invention has been described in considerable detail with reference to certain preferred versions thereof, other versions are possible. For example, it is contemplated that the screening assays of the present invention may be automated, thereby facilitating the screening of a large number of specimens at the same time. Therefore, the spirit and scope of the appended claims should not be limited to the description of the preferred versions described herein.
- A total of 245 Pap smears with individual cytologic diagnoses of AGUS, with or without SIL, were studied. Corresponding histologic examination of biopsy material was conducted in each case. We found that MN/CA9 immunoreactivity was seen in all cases where atypical and neoplastic cervical tissues were diagnosed. In contrast, Pap smears of normal cervices with a cytologic diagnosis of AGUS were MN/CA9 negative. Importantly, high levels of the MN/CA9 protein expression were seen in all cases of AIS and invasive adenocarcinoma.
- Table 1 compares the present study with follow-up studies conducted by several other laboratories in those cases diagnosed as endocervical atypia. The cumulative results, as shown in Table 1, indicate that approximately 40% of AGUS cases represent high grade SIL, adenocarcinoma in situ (AIS), or carcinoma, and correspondingly 60% represent benign or insignificant lesions.
TABLE 1 Follow-up Histologic Diagnosis of Cases with Cytologic Diagnosis of Atypical Glandular Cells (AGUS) in Routine Pap Smears Reference Follow-up Wilbur Gaff Lee Bose Taylor Kenney This diagnosis et al et al et al et al et al et al study Average Benign/atypia 46* 40 42 20 64 81 19 44 LSIL 13 24 0 37 17 4 31 18 HSIL 32 30 44 41 20 5 39 30 AIS/Adenoca. 9 13 14 0 0 10 11 8 Significant lesions** 41 43 58 41 20 15 50 38 Total No. of cases 225 56 74 44 77 30 245 - Materials and Methods
- Tissue Specimens: Individual Pap smears from 557 patients with a cytologic diagnosis of AGUS with or without SIL (incidence: 0.3%) were retrieved from 185,414 Pap smears examined by a private cytologic laboratory in Orange County, Southern California, between July, 1994 to September, 1998. The follow-up histologic confirmation was identified in 251 Pap smears (follow-up rate: 43%). Among these, six cases were excluded due to insufficient material in the endocervical curettage (n=3), endometrial curettage only (n=2), and air-dried smear (n=1). Thus, the total number of Pap smears included in the study was 245. The median age of the patients included in the study was 39 (range was 20-81 years). All of the Pap smears were collected by a cytobrush and a wooden spatula. The sources of tissue sections of the cervices were from colposcopic-directed cervical biopsies, endocervical curettage, cervical conization or hysterectomy.
- Cytology: The Pap smears were screened by 6 cytotechnicians and reviewed by 8 pathologists, including three board certified cytopathologists. The TBS classification was used in the study and cytologic diagnosis of AGUS was further classified as AGUS-favor reactive, AGUS-favor neoplastic, and AGUS-not otherwise specified (AGUS-NOS). The Pap smears were not reviewed for diagnostic stringency; thus the results are concordant with the actual cytologic screening in a private practice situation. The cytologic and histologic data were correlated. The MN/CA9 immunoreactivity was interpreted in a blinded fashion and the results were then correlated with the histologic data. Only in those cases where there was a discrepancy between the original histologic diagnosis and MN/CA9 immunoreactivity were the histologic sections reviewed with knowledge of the results of MN/CA9 immunostaining.
- Immunohistochemical Studies: The mouse monoclonal antibody used to detect the MN/CA9 protein has been described previously (Pastorek et al., 1994). The antibody recognizes the antigen in formalin fixed, paraffin embedded sections and archived Pap smears (Liao et al., 1994; and Liao et al., 1996). Immunohistochemical staining of tissue sections and decolorized Pap smears with the anti-MN/CA9 Mab (MN75) was done using a peroxidase technique described previously (Hsu S M, Raine L, Fanger H. Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem., 1981. 29:577-580). Known positive and negative tissue specimens were included in each run. Briefly, the smears were decolorized with 1% acid alcohol and rinsed with distilled water. Five micron sections of paraffin-embedded tissues were deparaffinized. The endogenous peroxidase was blocked by incubating the slides in a solution of 3.0% hydrogen peroxide in methanol for 10 min. The slides were then incubated with appropriate blocking serum (5% normal horse serum in PBS), followed by incubation with purified ascites fluid-derived primary antibody MN75 (1: 10,000 dilution in PBS containing 0.1% BSA) for 60 minutes. The secondary biotinylated horse antimouse immunoglobulin G antibody (1:200 dilution in PBS) was then added for 30 min., followed by incubation with avidin-biotin peroxidase complex (ABC Elite) for 30 min. (Vector Laboratories, Burlingame, Calif.). Diaminobenzidine tetrahydrochloride was used as chromagen (Sigma Chemical Co., St. Louis, Mo.). After treatment, the sections were washed with distilled H2O, counterstained with hematoxylin, and mounted with Permount.
- Immunohistochemical analysis: The specificity of staining was defined by the presence of a brown reaction product predominantly on the plasma membrane. The intensity of the staining was subjectively defined by using a two scale system; namely weak (+) and strong (++ to +++). Very weak staining of the cytoplasm, detectable only at a high-power magnification, was considered as negative because of the lack of specificity (Liao et al., 1996). All of the cells and/or cell clusters that stained, either weakly or strongly, were evaluated under the high-power magnification. Any MN/CA9 immunoreactive cells in cell clusters that morphologically deviated from normal squamous cell, endocervical reserve cell or columnar cell were considered as atypical cell clusters. The nuclei of those atypical cells were, in general, 3 to 5 times larger than normal endocervical cells (ECs) and exhibited significant hyperchromasia with an increased nuclear/cytoplasmic ratio. The numbers of positive cells or cell clusters in each smear were counted under 4× scanning power. Diffuse MN/CA9 immunoreactivity was defined as when more than 50% of the atypical cells/cell clusters or more than 100 normal endocervical cells/cell clusters in each smear exhibited strong (++/+++) MN/CA9 immunoreactivity. The smear was scored as positive when brown staining was identified in any atypical cell cluster or in more than two normal endocervical cell clusters, and negative when no brown reaction was seen or staining was limited to two or less normal endocervical cell Clusters in each smear.
- Histology: The tissue sections of the cervix from each case were reviewed and interpreted as benign, atypia, cervical intraepithelial neoplasia (CIN), or AIS/Carcinoma (AIS/CA). The criteria used for diagnoses of glandular atypia/neoplasia and CIN are those defined by Crum & Kurman (Crum C P, Cibas E S, Lee K R. Criteria for grading squamous intraepithelial lesions. In: Pathology of Early Cervical Neoplasia, New York Churchill Living stone, 1997. 47-91; and Kurman R J, Norris H J, Wilkinson E. Tumors of the cervix, vagina, and vulva. In: Atlas of Tumor Pathology, 3rd series, fasc. 4, Armed Forces Institute of Pathology, 1992. 37-139). Representative examples are shown in FIG. 1. The benign category included normal cervix with or without inflammation (panel A). The diagnosis of atypia included atypical squamous metaplasia/atypical reserve cell proliferation and endocervical glandular atypia (panels B and C). A two scale system was used for the diagnosis of CIN, namely low grade (Condyloma/CIN I) (panel D) and high grade (CIN II and CIN III) (panel E). An example of AIS is shown in panel F.
- Correlation between cytology and histology: The comparative analysis between the various categories of AGUS Pap smears and the histologic diagnoses of the cervical specimens is outlined in table 2.
TABLE 2 Biopsy Follow-Up of Patients with Cytologic Diagnosis of AGUS Cytologic Diagnosis AGUS-ALL AGUS-FAVOR AGUS-FAVOR CATEGORIES REACTIVE AGUS-NOS NEOPLASTIC N = 245 N − 29 N = 57 N = 59 Without With Without With Without With Histologic SIL SIL SIL SIL SIL SIL Diagnosis No. (%) n = 28 n = 1 n = 137 n = 20 N = 51 n = 8 Benign 34 (14) 13 0 20 0 1 0 Atypia* 12 (5) 3 0 8 0 1 0 LSIL 76 (31) 9 1 55 2 7 2 HSIL 95 (39) 1 0 49 18 22 5 Endometrial 3 (1) 0 0 0 0 3 0 Adenoca. AIS** 25 (10) 2 0 5 0 17 1 - Of the 245 cases studied, 29 (12%) of the Pap smears were diagnosed as AGUS-favor reactive, 157 (64%) as AGUS-NOS, and 59 (24%) as AGUS-favor neoplastic. Coexisting cytologic diagnosis of SIL was found in Pap smears of one AGUS-favor reactive, 20 AGUS-NOS and 8 AGUS-favor neoplastic.
- Biopsy follow-up showed thirty four (14%) of the cervices had no obvious abnormalities. The corresponding Pap smears were 13 specimens diagnosed as AGUS-favor reactive, 20 as AGUS NOS and one specimen diagnosed as AGUS-favor neoplastic.
- Twelve (5%) of the cervical specimens received a histologic diagnosis of atypia. The cytologic diagnoses were: 3 AGUS-favor reactive, 8 AGUS-NOS, and 1 AGUS-favor neoplastic.
- A significant fraction of the histologic diagnoses were LSIL (76 cases) or HSIL (95 cases). In the cases of LSIL diagnosis the corresponding cytologic diagnoses were: 10 AGUS-favor reactive, 57 AGUS-NOS, and 9 AGUS-favor neoplastic. A similar broad distribution of cytologic diagnoses was seen in the HSIL cohort. There were 1 AGUS-favor reactive, 67 AGUS-NOS, and 27 AGUS-favor neoplastic.
- In the most serious lesion category, that of AIS/CA, there were 28 cases (11%), of which three were endometrial adenocarcinomas. The corresponding cytologic diagnoses were: 2 AGUS-favor reactive, 5 AGUS-NOS, and 21 AGUS-favor neoplastic.
- Thus, our study indicated that 50% of the AGUS Pap smears had significant lesions (HSIL and AIS/CA) in the follow-up biopsies. Although the majority (81%) of AGUS-favor neoplastic Pap smears were histologically confirmed to be HSIL or AIS/CA, only 46% of the AGUS-NOS Pap smears had significant lesions found in the cervices. Thus, these diagnoses clearly reflect the quandary facing the clinician. The diagnosis of AGUS-NOS was easily the most frequently used of the cytologic diagnostic categories of AGUS and was not very helpful in predicting clinical outcome.
- MN/CA9 Immunoreactivity in Pap Smears: In normal Pap smears the exfoliative endocervical cells/cell clusters and endometrial cells from the lower uterine segment are consistently MN/CA9 negative (data not shown). In the AGUS Pap smears we found a relatively complex pattern of cytologic morphology /immunoreactivity combinations. These are illustrated in FIG. 2 and serve as a framework for succeeding interpretations in the study. Cytologic criteria for atypical glandular cells, delineated in the TBS classification, were used in the interpretation (National Cancer Institute Workshop. The revised Bethesda System for reporting cervical/vaginal cytologic diagnosis: report of the 1991 Bethesda Workshop. JAMA, 1992. 267:1892; and Kurman et al., 1994). Panel A shows an AGUS Pap smear that exhibits no MN/CA9 immunoreactivity. In smears where positive immunostaining is seen there are two broad categories: those where atypical cells, with or without normal ECs, are positive and those where normal ECs only stain (panels B-D). In those smears that do exhibit positive immunostaining, variations in intensity of stain are encountered. Panel B illustrates clusters of atypical cells that are strongly positive (arrow) and normal ECs that are weakly positive (arrowhead). The pattern of immunostaining may also vary. Panel F shows an example of focal staining and panel G shows an example of diffuse immunoreactivity. In those Pap smears where atypical cells were stained, there are morphological variations. Some of the stained atypical cells are arranged in tight cell clusters (panel H), where in other smears focally columnar or honey-comb configurations were observed (panel 1).
- Correlation between MN/CA9 immunostaining and histologic diagnosis: The distribution of immunostaining patterns described above, and their correlation with histologic diagnosis, is illustrated in FIG. 3. It is immediately apparent that all specimens with histologic diagnoses ranging from atypia to AIS/CA are positive for MN/CA9 expression. Conversely, all cases of benign cervices had MN/CA9 negative Pap smears.
- There was an interesting correlation between the degree of dysplasia diagnosed histologically and the morphology of the MN/CA9 positive cells in the corresponding Pap smears. Those specimens with a histologic diagnosis of AIS/CA or HSIL exhibited, in all cases, immunostaining of atypical cells±normal ECs in the corresponding Pap smears. Moreover, immunoreactive atypical cells in cases of AIS/CA always exhibited focally columnar or honeycomb features, with a diffuse staining pattern. Conversely, in cases of HSIL, the MN/CA9 positive atypical cells usually were arranged in tight clusters (for reference see FIG. 2, panels B, C, H and I), and only a fraction of the cases (28%) exhibited a diffuse immunostaining pattern. In the cases of diagnoses of LSIL, 14/76 showed a similar pattern, whereas the remainder exhibited staining of normal ECs only (for reference see FIG. 2, panel D). The number of cases with a histologic diagnosis of atypia is small (n=12) but all of them exhibited immunostaining of normal ECs only. Finally, all of the benign cases were MN/CA9 negative.
- What is immediately apparent from these data is that in the vast majority of cases where atypical cells exhibit MN/CA9, the presence of a significant lesion (HSIL and AIS/CA) is indicated. The picture is less clear for low grade lesions. Whereas, a fraction of LSIL cases (14/76) do, indeed, show immunostaining of atypical cells, the remainder showed only normal ECs expressing the antigen. This latter feature is also seen in all cases of atypia (n=12). Thus, from these criteria it is not possible to distinguish with confidence these latter two categories of histologic diagnosis on the basis of MN/CA9 immunoreactivity.
- Statistical analysis: The immunopositive smears were subjected to a true diagnosis analysis (Dunn G, Everitt B. Clinical problems and statistical solutions. In Clinical Biostatistics, New York Halstead Press 1995; 1-32). In this analysis the association of immunostained atypical cells with a significant lesion in the corresponding biopsy is considered to have positive predictive value. Correspondingly, the correlation of immunostained normal ECs only with a significant lesion is considered to have a negative predictive value. The analysis is presented in Table 3. Clearly, sensitivity, specificity, and positive predictive values are excellent.
TABLE 3 True Diagnosis Analysis of Immunopositive Smears True diagnosis Test result HSIL/AIS/CA LSIL/Atpia Atypical cell 123 14 Positive Normal cell only 0 74 Positive - The categories of AGUS diagnosis and their patterns of immunostaining: It is clear from the preceding data that high grade lesions are predicted on the basis of MN/CA9 immunostaining of atypical cells in the relevant Pap smears that have received a general diagnosis of AGUS. FIG. 4 shows how predictive AGUS diagnoses ( i.e. AGUS-favor reactive, AGUS-NOS and AGUS-favor neoplastic) were when compared to their respective patterns of MN/CA9 immunostaining.
- AGUS-FAVOR REACTIVE: A total of 29 cases were diagnosed in this category. In keeping with the cytologic diagnoses, follow up biopsies were benign in 45% (n=13). All of these were MN/CA9 negative. Three cases of atypia and 10 cases of LSIL were diagnosed. All of these showed MN/CA9 immunostaining of normal ECs only. There were also 1 case of HSIL and 2 cases of AIS. All were MN/CA9 positive, with atypical cells staining. Importantly, the few cases where significant lesions were seen were predicted by MN/CA9 immunostaining of atypical cells.
- Examples of the Pap staining and immunostaining are shown in FIG. 5. All of the atypical cell clusters in panels A, B, and C show minimal cellular overlap and nuclear pleomorphism, with a mild degree of hyperchromasia. In panel B the atypical cells are associated with ciliated metaplastic cells (arrow). However, when there was MN/CA9 immunostaining of the cells, including ciliated cell clusters (panels E and F), high grade lesions (HSIL) were found in the cervices. Conversely, no dysplastic tissue was identified when no immunostaining was seen (panel D).
- AGUS-NOS (not otherwise specified): This was the largest category of the AGUS diagnoses (n=157). The histologic diagnoses clearly indicate the clinical problem with this area. The full range of benign, low grade and high grade lesions were found, with significant numbers in each category (Table 2). Once again, all high grade lesions showed MN/CA9 immunostaining of atypical cells in Pap smears. In the cases of LSIL, only 14/76 showed this pattern; immunostaining, of normal ECs only was seen in the remainder, as was seen in the cases of the histologic diagnoses of atypia. Thus, in those cases where significant lesions (HSIL and AIS) were present, they were readily diagnosed on the basis of MN/CA9 positivity. Importantly, they represented 46% of the total in this category of AGUS-NOS.
- Examples of the Pap staining and immunostaining are shown in FIG. 6. All of the atypical cells in panels A, B and C show cellular overlap, moderate nuclear pleomorphism and slightly increased nuclear/cytoplasmic ratio. Again, HSIL (panel E) and AIS (panel F) were histologically identified only in those cases where atypical cells stained positive. No dysplastic lesion was observed in those cases in which no MN/CA9 immunostaining was detected (panel D).
- AGUS-FAVOR NEOPLASTIC: The majority of histologic diagnoses in this category confirmed the presence of significant lesions (HSIL, AIS/CA). As illustrated in Table 2, there were 27 (46%) HSIL and 21 (36%) AIS/CA cases. All showed MN/CA9 immunostaining of atypical cells. Those cases diagnosed as LSIL and atypia (n=9 and n=1, respectively) showed positive staining of normal ECs only. Once again the MN/CA9 immunostaining pattern accurately predicted the presence of significant lesions.
- Examples of the Pap staining and immunostaining are shown in FIG. 7. All of the atypical cell clusters in panels A, B and C show cellular overlap, marked nuclear pleomorphism, hyperchromasia and increased nuclear/cytoplasmic ratio. HSIL and AIS were observed in the corresponding cervices of those Pap smears where MN/CA9 immunostaining was seen in the atypical cell clusters (panels E and F). Benign histology was seen in the MN/CA9 negative Pap smear (panel D).
- Discussion
- In the present invention, we have uncovered a clear association between MN/CA9 immunostaining of atypical cells±normal ECs in AGUS Pap smears and the presence of significant lesions in the cervix, whether they be HSIL or AIS/CA (FIG. 3). Similarly, we have uncovered a clear correlation between the lack of MN/CA9 immunostaining and the absence of lesions in the cervix. However, the MN/CA9 immunostaining pattern does not discriminate between LSIL and atypia. In all cases of atypia and 82% (n=62) of LSIL cases, immunostaining of normal ECs only was observed. The remainder of the LSIL cases showed staining of atypical±normal ECs. This small number (n=14) of LSIL cases may represent a small percentage of false positives (in the sense that staining of atypical cells is diagnostic of the presence of significant lesions in the cervix) or may be a prognostic indication that these lesions will progress to HSIL. In addition, the immunostaining pattern seen in AIS cases is always diffuse and the stained cell clusters exhibit columnar or honeycomb configurations. This is in contrast to cases of HSIL, where the cells are arranged in tight clusters, and in the majority of cases they exhibit a focal pattern of immunostaining. Therefore, on this basis of the difference in morphologies of the stained cell clusters and their immunostaining patterns one is able to discriminate between AIS and HSIL in the AGUS Pap smear diagnoses. This has clear and significant clinical implications.
- The value of the diagnostic procedures of this invention as an adjunct to cytologic diagnosis is particularly apparent when the AGUS smears are clustered into the three subcategories of AGUS-favor reactive, AGUS-NOS and AGUS-favor neoplastic (FIG. 4). As expected, the majority of AGUS-favor neoplastic diagnoses correlated with the presence of significant lesions. All of these also showed MN/CA9 immunostaining of atypical cells. The cases of LSIL and atypia (total n=12) showed positive staining of normal ECs only. The category of AGUS-NOS showed the broadest range of lesions in follow-up biopsies. Again, MN/CA9 immunostaining of atypical cells predicted all of the cases where significant lesions were found. A small fraction (n=14) of “false positives” were found here, which are LSIL cases where atypical cells are MN/CA9 positive. It is interesting that this “false positive” staining pattern is found only in the AGUS NOS category and may, as discussed above, be prognostic of a lesion that is destined to progress. In the AGUS-favor reactive category relatively few (n=3) had significant lesions. All were correctly diagnosed by MN/CA9 immunostaining pattern and no false positives were noted.
- These data demonstrate that in the context of the present invention, MN/CA9 antigen expression in atypical cells is an excellent biomarker for use in AGUS diagnoses, and can be used by a clinician with great advantage as an adjunct in determining which AGUS diagnoses are likely yield significant lesions. This has a distinct cost-benefit potential since it has been shown that only approximately 40% of AGUS diagnoses are correspondingly associated with significant lesions (see Table 1).
- Although the present invention has been described in considerable detail with reference to certain preferred versions thereof, other versions are possible. Therefore, the spirit and scope of the appended claims should not be limited to the description of the preferred versions described herein.
Claims (12)
Priority Applications (12)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US09/461,938 US6379907B1 (en) | 1999-08-05 | 1999-12-15 | Diagnostic method using expression of MN/CA9 protein in AGUS Pap smears |
US09/572,756 US6403327B1 (en) | 1999-08-05 | 2000-05-16 | Diagnostic method using expression of MN/CA9 protein in ASCUS Pap smears |
CA002380646A CA2380646A1 (en) | 1999-08-05 | 2000-08-02 | Diagnostic method using expression of mn/ca9 protein in ascus pap smears |
EP00953790A EP1200475A4 (en) | 1999-08-05 | 2000-08-02 | Diagnostic method using expression of mn/ca9 protein in ascus pap smears |
PCT/US2000/021027 WO2001010910A1 (en) | 1999-08-05 | 2000-08-02 | Diagnostic method using expression of mn/ca9 protein in ascus pap smears |
EP00952388A EP1261641B1 (en) | 1999-08-05 | 2000-08-02 | Diagnostic method using expression of mn/ca9 protein in agus pap smears |
JP2001515716A JP2003526777A (en) | 1999-08-05 | 2000-08-02 | Diagnostic method using expression of MN / CA9 protein in AGUS / Pap smear |
PCT/US2000/021017 WO2001010909A1 (en) | 1999-08-05 | 2000-08-02 | Diagnostic method using expression of mn/ca9 protein in agus pap smears |
JP2001515717A JP2003514217A (en) | 1999-08-05 | 2000-08-02 | Diagnostic method using MN / CA9 protein expression in ASCUS PAP smear |
AT00952388T ATE331957T1 (en) | 1999-08-05 | 2000-08-02 | DIAGNOSTIC METHOD USING EXPRESSION OF MN/CA9 PROTEIN IN AGUS-PAP SWABES |
CA002384744A CA2384744A1 (en) | 1999-08-05 | 2000-08-02 | Diagnostic method using expression of mn/ca9 protein in agus pap smears |
DE60029140T DE60029140D1 (en) | 1999-08-05 | 2000-08-02 | DIAGNOSTIC PROCEDURE USING EXPRESSION OF THE MN / CA9 PROTEIN IN AGUS-PAP SLAKES |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14755699P | 1999-08-05 | 1999-08-05 | |
US09/461,938 US6379907B1 (en) | 1999-08-05 | 1999-12-15 | Diagnostic method using expression of MN/CA9 protein in AGUS Pap smears |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/572,756 Continuation-In-Part US6403327B1 (en) | 1999-08-05 | 2000-05-16 | Diagnostic method using expression of MN/CA9 protein in ASCUS Pap smears |
Publications (2)
Publication Number | Publication Date |
---|---|
US6379907B1 US6379907B1 (en) | 2002-04-30 |
US20020058251A1 true US20020058251A1 (en) | 2002-05-16 |
Family
ID=26845025
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US09/461,938 Expired - Fee Related US6379907B1 (en) | 1999-08-05 | 1999-12-15 | Diagnostic method using expression of MN/CA9 protein in AGUS Pap smears |
Country Status (7)
Country | Link |
---|---|
US (1) | US6379907B1 (en) |
EP (1) | EP1261641B1 (en) |
JP (1) | JP2003526777A (en) |
AT (1) | ATE331957T1 (en) |
CA (1) | CA2384744A1 (en) |
DE (1) | DE60029140D1 (en) |
WO (1) | WO2001010909A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050020937A1 (en) * | 2003-03-31 | 2005-01-27 | West Virginia University | Method for detecting pathogenic agents |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040137551A1 (en) * | 2003-01-13 | 2004-07-15 | Markovic Nenad S. | Cervical acid phosphatase - papanicolaou (CAP-PAP) test kit, method and accesories, processes for producing and using the same |
US20050136405A1 (en) * | 2003-12-17 | 2005-06-23 | James Linder | Novel method for the detection of cancer biomarkers in cervical specimens |
US20060160151A1 (en) * | 2005-01-10 | 2006-07-20 | Baylor College Of Medicine | Method of quantitative immunohistochemistry and in situ hybridization |
MX346624B (en) * | 2011-09-13 | 2017-03-27 | Koninklijke Philips Nv | System and method for the detection of abnormalities in a biological sample. |
CN112528852A (en) * | 2020-12-10 | 2021-03-19 | 深思考人工智能机器人科技(北京)有限公司 | Recognition method and system of glandular cells |
Family Cites Families (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6297051B1 (en) | 1997-01-24 | 2001-10-02 | Institute Of Virology, Slovak Academy Of Sciences | MN gene and protein |
US5989838A (en) | 1992-03-11 | 1999-11-23 | Institute Of Virology, Slovak Academy Of Sciences | Immunological methods of detecting MN proteins and MN polypeptides |
US5972353A (en) | 1992-03-11 | 1999-10-26 | Institute Of Virology, Slovak Academy Of Sciences | MN proteins, polypeptides, fusion proteins and fusion polypeptides |
US6204370B1 (en) | 1992-03-11 | 2001-03-20 | Institute Of Virology, Slovak Academy Of Sciences | MN gene and protein |
US6297041B1 (en) | 1992-03-11 | 2001-10-02 | Institute Of Virology, Slovak Academy Of Sciences | MN gene and protein |
US5387676A (en) | 1992-03-11 | 1995-02-07 | Ciba Corning Diagnostics Corp. | MN gene and protein |
US6027887A (en) | 1992-03-11 | 2000-02-22 | Institute Of Virology, Solvak Academy Of Sciences | MN gene and protein |
US5981711A (en) | 1992-03-11 | 1999-11-09 | Institute Of Virology, Slovak Academy Of Sciences | MN-specific antibodies and hybridomas |
US5955075A (en) | 1992-03-11 | 1999-09-21 | Institute Of Virology, Slovak Academy Of Sciences | Method of inhibiting tumor growth using antibodies to MN protein |
US6093548A (en) | 1992-03-11 | 2000-07-25 | Institute Of Virology, Slovak Academy Of Sciences | Detection and quantitation of MN-specific antibodies. |
US6069242A (en) | 1992-03-11 | 2000-05-30 | Institute Of Virology, Slovak Academy Of Sciences | MN gene and protein |
US5346831A (en) | 1992-09-29 | 1994-09-13 | Hoffman-La Roche Inc. | Cytorich process system |
-
1999
- 1999-12-15 US US09/461,938 patent/US6379907B1/en not_active Expired - Fee Related
-
2000
- 2000-08-02 WO PCT/US2000/021017 patent/WO2001010909A1/en active IP Right Grant
- 2000-08-02 JP JP2001515716A patent/JP2003526777A/en active Pending
- 2000-08-02 AT AT00952388T patent/ATE331957T1/en not_active IP Right Cessation
- 2000-08-02 DE DE60029140T patent/DE60029140D1/en not_active Expired - Lifetime
- 2000-08-02 CA CA002384744A patent/CA2384744A1/en not_active Abandoned
- 2000-08-02 EP EP00952388A patent/EP1261641B1/en not_active Revoked
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20050020937A1 (en) * | 2003-03-31 | 2005-01-27 | West Virginia University | Method for detecting pathogenic agents |
Also Published As
Publication number | Publication date |
---|---|
US6379907B1 (en) | 2002-04-30 |
ATE331957T1 (en) | 2006-07-15 |
CA2384744A1 (en) | 2001-02-15 |
EP1261641A4 (en) | 2005-06-15 |
EP1261641A1 (en) | 2002-12-04 |
WO2001010909A1 (en) | 2001-02-15 |
DE60029140D1 (en) | 2006-08-10 |
EP1261641B1 (en) | 2006-06-28 |
JP2003526777A (en) | 2003-09-09 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8609351B2 (en) | Detection of dysplastic or neoplastic cells using anti-MCM4 antibodies | |
US7157233B2 (en) | Methods and compositions for the detection of cervical disease | |
EP1025444B1 (en) | Determination of cellular growth abnormality | |
US8076093B2 (en) | Monoclonal antibodies and methods for their use in the detection of cervical disease | |
US6379907B1 (en) | Diagnostic method using expression of MN/CA9 protein in AGUS Pap smears | |
US6403327B1 (en) | Diagnostic method using expression of MN/CA9 protein in ASCUS Pap smears | |
AU2011202593A1 (en) | Methods and compositions for the detection of cervical disease |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: REGENTS OF THE UNIVERSITY OF CALIFORNIA, THE, CALI Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:LIAO, SHU-YUAN M.D.;STANBRIDGE, ERIC J., PH.D.;REEL/FRAME:010484/0416 Effective date: 19991119 |
|
RR | Request for reexamination filed |
Effective date: 20020703 |
|
RR | Request for reexamination filed |
Effective date: 20030612 |
|
FEPP | Fee payment procedure |
Free format text: PAYOR NUMBER ASSIGNED (ORIGINAL EVENT CODE: ASPN); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY |
|
FPAY | Fee payment |
Year of fee payment: 4 |
|
B1 | Reexamination certificate first reexamination |
Free format text: THE PATENTABILITY OF CLAIMS 3, 4 AND 5 IS CONFIRMED. CLAIMS 1, 2, 6, 7, 8 AND 9 WERE PREVIOUSLY DISCLAIMED. |
|
AS | Assignment |
Owner name: BAYER HEALTHCARE LLC, NEW YORK Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:BAYER PHARMACEUTICALS CORPORATION;REEL/FRAME:023027/0804 Effective date: 20071219 Owner name: BAYER HEALTHCARE LLC,NEW YORK Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:BAYER PHARMACEUTICALS CORPORATION;REEL/FRAME:023027/0804 Effective date: 20071219 |
|
REMI | Maintenance fee reminder mailed | ||
FPAY | Fee payment |
Year of fee payment: 8 |
|
SULP | Surcharge for late payment |
Year of fee payment: 7 |
|
REMI | Maintenance fee reminder mailed | ||
LAPS | Lapse for failure to pay maintenance fees | ||
STCH | Information on status: patent discontinuation |
Free format text: PATENT EXPIRED DUE TO NONPAYMENT OF MAINTENANCE FEES UNDER 37 CFR 1.362 |
|
FP | Lapsed due to failure to pay maintenance fee |
Effective date: 20140430 |