Patent Application: US-57275600-A

Abstract:
determining the presence of cancerous or pre - cancerous cervical lesions from ascus - diagnosed pap smear cells by observing the distribution of mn / ca9 antigen expressed on atypical or normal cells and diagnosing significant lesions when mn / ca9 antigen is observed on atypical cells , low grade lesions when mn / ca9 antigen is absent from atypical cells but is present on normal endocervical cells , and a benign condition when mn / ca9 antigen is absent from both atypical cells and normal endocervical cells .

Description:
the method of the present invention entails examination 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 squamous cells of undetermined significance ( ascus ). a positive result serves as an early marker of dysplasia even in the absence of its clinical manifestations . the mn / ca9 antigen has shown to be useful in identifying cervical dysplasia / carcinorna and , as shown in our previous patent application ser . no . 09 / 461 , 930 , provides an effective method for dealing with the atypical glandular cells of undetermined significance ( agus ) diagnostic category . our studies now indicate that mn / ca9 expression will provide a similar discriminator of associated significant lesions for the ascus category . we have examined 82 specimens with a pap smear diagnosis of ascus that have a corresponding tissue biopsy with a histologic diagnosis . as illustrated in table 1 , there were 44 specimens that stained positively for mn / ca9 expression . of these , the majority ( n = 35 ) had an lsil - diagnosed tissue biopsy . only one mn / ca9 positive smear had a benign tissue biopsy . in recent years , it has become relatively common practice among cytopathologists to subcategorize ascus diagnoses into ascus - favor reactive , ascus - not otherwise specified ( nos ), and ascus favor sil . a similar subcategorization is also used for agus - diagnosed pap smears . in a limited pilot study or such subcategorized ascus diagnoses we , again , found that mn / ca9 expression is significantly associated with the presence of sil ( hsil and lsil ) in the tissue biopsies ( table 2 ). our data clearly indicates that ascus diagnoses may be aided - in the sense of predicting the presence of an sil - by the identification of mn / ca9 immunopositivity in the pap smear . to the best of our knowledge , we are the first to evaluate mn / ca9 expression as a useful biomarker for determining whether a significant cervical lesion is present in women who receive a pap test diagnosis of ascus . other investigators have independently confirmed our earlier observations of the association of mn / ca9 expression and the presence of cervical dysplasia and / or carcinoma in tissue specimens ; see costa , m . j . ( 1996 ), infra ; and costa , m . j ., et al ( 1995 ), infra . clinical specimens are accessed from archival material , derived from routine clinical practice , a method of resource acquisition we have used successfully for all of our previous studies ; see liao , s . y ., brewer , c ., et al , supra ; and liao , s . y , and stanbridge , e . j ., supra ). the pap smear specimens are a combination of conventional smears that have been spray fixed and thin prep specimens . all of the immunostained pap smears are destained and restained with papanicolaou stain , and kept in a permanent file . examples of this process are shown in fig1 which provides comparative illustrations of original pap stained smears ( a and d ), mn / ca9 immunostained smears ( b and e ) and re - stained pap smears after immunostaining ® and f ). the re - stained pap smears still retain the cytologic property as the original smears . of a total of 726 ascus cases ( all with histologic confirmation ) available to us , 149 were ascus - favor reactive , 416 ascus - nos , and 161 ascus - favor lsil . those patients with a diagnosis of ascus - favor lsil receive an immediate colposcopy and histologic diagnosis . patients in the other two categories receive a repeat pap test . if the repeat diagnosis is ascus or lsil , the patient is referred to colposcopy . mn / ca9 : a mouse monoclonal antibody ( mab ), designated m75 , is used ( liao , s . y ., brewer , c ., et al , supra ). it recognizes the proteoglycan domain of the mn / ca9 peptide . the mn / ca9 antigen is remarkably robust and survives papanicolaou staining of pap smears ( thereby allowing for destaining of routine pap smears followed by immunostaining with the m75 mab ), as well as fixation and paraffin embedding of tissues ( liao , s . y ., brewer , c ., et al , supra ; and liao , s . y , and stanbridge , e . j ., supra ). for tissue sections , five micron sections of paraffin - embedded tissues are deparaffinized . the endogenous peroxidase is blocked by incubating the slides in a solution of 2 . 5 % hydrogen peroxide in methanol for 45 minutes . the slides are then incubated with the appropriate blocking serum ( 5 % normal horse serum in pbs ) for 20 minutes . all incubations are performed at room temperature in humidified chambers . the slides are then incubated with ascites fluid - derived primary antibody m75 ( 1 : 10 , 000 dilution in pbs - containing 0 . 1 % bovine serum albumin ) for 60 minutes , then a 30 minute incubation with secondary biotinylated horse antimouse immunoglobulin g antibody ( 1 : 200 dilution in pbs ), followed by incubation with avidinbiotin peroxidase complexes ( abc elite ) for 30 minutes ( vector laboratories , burlingame , calif . ). diaminobenzidine tetrahydrochloride ( dab ) is used as chromagen ( sigma chemical co ., st . louis , mo .). after treatment , the sections are washed with distilled h 2 o , counterstained with hematoxylin , and mounted with permount . duplicate deparaffinized sections of selected samples are also pretreated with microwaves . in this regard , the sections are rehydrated in 10 mmol / l citric acid monohydrate solution ( ph 6 . 0 ), placed in the microwave oven and subjected to 2 × 5 minute exposures at the high setting . the slides are allowed to cool and then are rinsed with distilled h 2 o , and pbs . the slides are then processed for staining as described above . known positive and negative specimens are included in each staining run . for cervical smears , the routine pap - stained smears are first decolorized with 1 % acid alcohol and rinsed with distilled water . the smears are then immunostained following the procedure described above . the specificity of immunostaining is identified by the presence of a brown reaction product , predominantly on the plasma membrane ( see fig1 ). the patterns of immunostaining — focal versus diffuse and normal cells only versus atypical cells — are identified and recorded . the metaplastic squamous cell , reserve cell , and columnar cell constitute the cell population that we define as normal endocervical cells ( ecs ). any mn / ca9 immunoreactive parabasal dyskeratotic cells and / or cells that morphologically deviate from normal ecs are considered as atypical cells or cell clusters . the nuclei of those atypical cells are , in general , 2 - 3 times larger than normal ecs and exhibit significant hyperchromasia with an increased nuclear / cytoplasmic ratio . referring to fig2 in panels a and b , no immunoreactivity is seen . the cervix has benign histology ( panel c ). in panels d and e , diffuse positivity is seen in the majority of normal endocervical cells , and low - grade dysplasia ( lsil / cin i ) is identified in the cervical tissue ( panel f ). in panels g and h , mn / ca9 immunoreactivity is seen in atypical cells , characterized by increased nuclear / cytoplasmic ratio and nuclear hyperchromasia . the cervical tissue section contains a high - grade lesion ( cin iii , panel 1 ). ( magnification 400 ×) shows a spectrum of squamous and glandular alterations , illustrating 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 ×); the ki67 mouse monoclonal antibody is obtained from commercial sources ( e . g . immunotech corp ., maine ), and is used according to the manufacturer &# 39 ; s protocol . ki67 immunostaining is performed on tissue sections . the immunostaining pattern is used as an index of proliferation , to confirm the histologic diagnosis ( bulten , j ., supra ) and to correlate with mn / ca9 expression . we have found that immunostaining of tissue sections works well , using the mn / ca9 protocol . examples of ki67 immunostaining are shown in fig3 where , in benign cervix ( a ), the ki 67 positivity is restricted to the basal cell layer ( b ) and no mn / ca9 immunoreactivity is seen ( c ). in contrast , the dysplastic cervix ( d , cin iii ) shows ki 67 nuclear staining in all layers of dysplastic cells ( e ), and there is diffuse mn / ca9 immunoreactivity in the corresponding section ( f ). ( magnification 200 ×). fig3 shows staining patterns of mn / ca9 immunoreactivity in agus pap smears . the pattern of immunostaining ( focal or diffuse staining of atypical cells versus normal cells only ) discriminates between biopsies that contain low grade squamous intraepithelial lesion ( lsil ) ( immunostaining of normal cells only ) versus a high grade squamous intraepithelial lesion ( hsil ) and / or ca ( focal or diffuse staining of atypical cells ). our study population is derived from women who have received a pap smear diagnosis of ascus . all cases studied are accompanied by a histologic diagnosis , based upon a tissue biopsy taken at the time of the initial ascus diagnosis or following a repeat pap smear diagnosis that is abnormal or ascus , specifically . archival material is used for all of the study specimens . both pap smears and corresponding cervical tissue specimens are derived from routine diagnostic material . smears and tissue sections are obtained from the same patient . in those cases where a routine conventional . pap smear specimen is used , the immunostained smear is destained and restained with papanicolaou stain . basically , there are two categories of patients with an ascus diagnosis : 1 ) those patients who receive a pap smear diagnosis of ascus - favor sil . these patients receive an immediate colposcopic examination and cervical biopsy and / or curettage . in this group we are able to directly ascertain the correlation between mn / ca9 expression and the presence of lesions ( sil and / or ca ). 2 ) those patients who receive a pap smear diagnosis of ascus - favor reactive or ascus - nos . these patients are followed - up with repeat pap smear ( s ). if the patient receives an sil or persistent ascus diagnosis then a colposcopic examination and cervical biopsy and / or curettage are performed , in 3 - 12 months . several results are possible , namely : in a small study we have found examples of possibilities 1 and 2 , but not possibility 3 . this is consistent with the notion that the presence of a significant lesion or lsil would be accompanied by positive mn / ca9 expression . furthermore , the likelihood that one would encounter the situation where the progression from benign → sil would occur within the 3 - 12 month follow - 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