Ki-67 immunostaining in Pap smears of cervix: assessment of proliferation in preinvasive and invasive squamous epithelial lesions

Ki-67 is a novel proliferation marker. Its use has been extensively studied in biopsy sections of preinvasive and invasive squamous epithelial lesions of cervix. However, only limited work has been done on its application on Pap smears of cervix. We applied Ki-67 immunostaining on cytology smears of various grades of squamous epithelial lesions of cervix. Sixty cases were chosen for immunostaining by Peroxidase antiperoxidase method using DAB as a chromogen. High level of Ki-67 labelled proliferation was measured in squamous cell carcinomas of cervix. Statistically significant difference was observed between preinvasive and invasive squamous epithelial lesions of cervix. Ki-67 could prove as a useful adjunctive test to routine Pap smear in triage of patients harboring lesions of cervix.     

Fine-needle aspiration of neurilemoma (schwannoma). A clinicocytopathologic study of 116 patients

The preoperative fine-needle aspiration cytology (FNAC) diagnoses in 116 surgically excised neurilemomas were reviewed and compared with the corresponding histopathologic diagnoses made on surgical specimens and with clinical data. In addition, the utility of adjunctive techniques was analyzed and other spindle-cell lesions in the differential diagnoses were discussed. An unequivocal, benign diagnosis was rendered by FNAC in 80 cases, 67 of which were correctly labelled as neurilemoma in a review of the original cytology reports. There were 6 false-positive malignant diagnoses while 23 smears were considered insufficient and 7 inconclusive as to whether benign or malignant. On reevaluation, the diagnostic smears in most cases contained spindle cells with wavy nuclei embedded in a fibrillar, occasionally collagenous, and/or myxoid matrix and Antoni A/Antoni B tissue fragments. A moderate to abundant admixture of round to oval cells was also frequent. Nuclear palisading was seen in 41 smears with distinctive Verocay bodies in 10. Markedly pleomorphic nuclei were seen in smears from 8 ancient and 6 conventional neurilemomas, and slight to moderate nuclear pleomorphism was observed in 38 additional cases. Thus most neurilemomas have distinct cytomorphologic features that allow correct diagnosis. The major problem in FNAC of neurilemoma is to obtain sufficient material. Furthermore aspirates showing predominantly Antoni A features, nuclear pleomorphism, and/or myxoid changes can easily be confused with other types of benign or malignant soft-tissue tumors.     

Cytopathologic features of secondary peripheral ameloblastic carcinoma: A case report

Peripheral ameloblastic carcinoma is an extremely rare odontogenic tumor derived from the remnants of dental lamina and/or mucosal epithelium of the oral mucosa. We present a case of secondary peripheral ameloblastic carcinoma of the mandibular gingiva. The patient was a 71‐year‐old man with gingival swelling and persistent bleeding. Exfoliative cytology revealed cohesive clusters composed of basaloid cells with nuclear atypia and various forms of keratinized cells of dysplastic squamous appearance. Some cell groups had a peripheral palisade. Histology of the biopsy and surgically removed specimens revealed characteristic features resembling squamous cell carcinoma, basal cell carcinoma, and benign follicles of ameloblastoma. These neoplastic structures, as well as proliferation and elongation of the mucosal epithelium, comprised an extensive network. The varied cytopathologic findings may be related to proliferation and transformation of basal cells of the mucosal epithelium toward ameloblastic carcinoma and variable squamous differentiation. Diagn. Cytopathol. 2011;39:354–358. © 2010 Wiley‐Liss, Inc.     

Chromosome in situ hybridisation, Ki-67, and telomerase immunocytochemistry in liquid based cervical cytology

AIMS: To assess the potential value of chromosome in situ hybridisation (CISH), Ki-67, and telomerase immunocytochemistry in liquid based cervical cytology to help detect carcinoma cells and precursors. METHOD: Sixty ThinPrep processed cervical cytology samples were studied: 23 cases within the normal limit, 13 low grade squamous intraepithelial lesions (LSILs), 10 high grade squamous intraepithelial lesions (HSILs), six squamous cell carcinomas, three endocervical adenocarcinomas, two cervical adenosquamous cell carcinomas, and three endometrial adenocarcinomas. CISH was performed with DNA probes specific for the pericentromeric regions of chromosome 11 and 16. Hybridisation signals were visualised with the streptavidin-biotin peroxidase technique. The monoclonal MIB1 and polyclonal TRT-H231 antibodies were used to detect Ki-67 and telomerase immunoreactivity, respectively. RESULTS: Non-specific background staining was almost absent in CISH slides. Normal squamous and glandular cells showed a diploid chromosomal pattern. A relative gain in chromosomes 11 and 16 (aneusomy) was seen in HSIL and the carcinomas (p<0.0001). In MIB1 stained smears, normal cells and koilocytes showed inconspicuous immunoreactivity, whereas strongly immunoreactive nuclei were found in cancer cells and HSIL (p<0.0001). Not only carcinoma and HSIL cells, but also some normal cells, showed cytoplasmic staining for telomerase. CONCLUSIONS: These preliminary results indicate that ThinPrep processed cervical smears are suitable for CISH and immunocytochemical studies. The neoplastic squamous and glandular cells were easily identified based on nuclear aneusomy and strong Ki-67 immuoreactivity in the context of abnormal nuclear morphology. This is the first study to apply CISH in cervical cytology using an immunoenzymatic approach.     

Selected problems in fine needle aspiration of head and neck masses.

A wide variety of masses in the head and neck, including those in the major salivary glands, can be approached by fine needle aspiration. In many instances, a correct definitive diagnosis con be rendered after examination of smears or cell block material. However, several significant but uncommon areas can lead to diagnostic difficulties, with the potential for clinically important diagnostic errors. Many of these occur in salivary gland lesions. The most frequent problems involve variations in the expected cytology of pleomorphic adenoma. Then, there are several benign-malignant "look-alike" pairs of lesions. The first of these is related to small-cell epithelial neoplasms of low nuclear grade; the most frequent problem is between basal cell adenomas and adenoid cystic carcinoma, particularly the solid (anaplastic) type. The next area contrasts mucoepidermoid carcinoma with its cytologic mimic, benign salivary gland duct obstruction. The final difficulty in salivary gland aspiration contrasts large-cell epithelial lesions of low nuclear grade: oncocytic proliferations and acinic cell carcinoma. The clinical implications of cytologically benign squamous cell-containing cyst aspirates from the lateral neck will be discussed. Finally, a brief consideration of methodological optimization for thyroid aspirations will be offered.     

AgNOR counts are useful in cervical smears.

Because they present marked cellular changes, the cytological appearances of epithelial repair may be confused with those of cancer. To see whether the Ag-NOR staining technique for nucleolar organizer regions would be useful to distinguish benign proliferative reactions (squamous metaplasia and repair) from cervical intra-epithelial neoplasia (CIN I, CIN II, and CIN III), we studied a series of cervical smears. The smears, previously stained with the Papanicolaou technique, were destained and restained with Ag-NOR silver. Significantly different (P less than 0.05) Ag-NOR counts were found in squamous metaplasia, epithelial repair, and various degrees of CIN. The Ag-NOR technique appears useful in the diagnosis of cervical cytology and particularly in cases with marked cellular change, which could be confused with neoplasia.     

Application of modified ultrafast Papanicolaou stain in cytology of various organs

This prospective study was conducted to assess the use of modified ultrafast Papanicolaou (MUFP) stain for fine-needle aspiration cytology (FNAC) of various organs. In India, many reagents are not easily available. In this modification, Gill's haematoxylin, EA-36, and isopropanol substitute Richard-Allan haematoxylin, cytostain, and ethanol, respectively. Group I included 40 FNAC smears of various organs. In each case, three smears were prepared and stained by MUFP, Papanicolaou, and May-Grunwald Giemsa (MGG) stains. In group II, 10 intraoperative cytology smears of different organs were stained with MUFP and rapid haematoxylin-eosin (H&E). For assessment of MUFP stain, scores were given on four parameters: background of smears, overall staining pattern, cell morphology, and nuclear staining. Quality index was calculated from ratio of score achieved to maximum possible score. Diagnosis made by MUFP stain was compared with standard stains. It was observed that MUFP-stained smears had clear RBC free background, crisp nuclear chromatin, well-stained nucleoli, and transparent cytoplasm. The diagnosis made was correct except in three cases of metastatic squamous-cell carcinoma. Hence, it was concluded that MUFP stain is useful for rapid diagnosis by FNAC, but is not useful for squamous-cell lesions.     

Fine-needle aspiration cytopathology of soft tissue: Chondromyxoid and myxoid lesions

Soft-tissue lesions pose an intimidating challenge to diagnostic interpretation by fine-needle aspiration cytology. to better characterize such aspirates, we investigated a subgroup of lesions which yielded a large amount of chondromyxoid and myxoid stroma (CMS). Thirty-three cases (22 malignant lesions, 11 benign lesions) in which CMS was a prominent feature of the smears were studied. the most common malignant neoplasms were extraskeletal chondrosarcoma (6 cases), myxoid liposarcoma (6), and malignant fibrous histiocytoma (5). the benign entities consisted of ganglion cyst (4), myxoma (3), and neurofibroma (2). of the aspirates from 22 malignant neoplasms, 15 (68%) contained opaque stroma, and in 18 (81%) the stroma was distributed as discrete clumps or fragments; all extraskeletal chondrosarcomas and chordomas contained these features. Additionally, in five of six chondrosarcomas the matrix exhibited cells within lacunae. Except for a tracheal chondroma which extended into the adjacent soft tissue, no benign aspirates had all three of these cytologic features. Twenty-three aspirates (70%) were moderately or highly cellular; all but two of these were malignant. Moderate to marked nuclear atypia occurred in 13/22 (59%) malignant and 1/11 (9.0%) benign aspirates. With a few exceptions, aspiration smears of benign lesions with abundant CMS were characterized by low cellularity, semitransparent amorphous stroma, and lack of nuclear irregularity. Recognition of variations in the character of the background material in addition to traditional cytologic criteria can aid in the diagnosis of benign and malignant chondromyxoid and myxoid aspirates of soft tissue. © 1995 Wiley- Liss, Inc.     

The proliferative cell fraction in cytology specimens. A study of human esophageal carcinoma.

Immunostaining of cell cycle-related antigens, especially Ki-67, DNA polymerase alpha, and proliferating cell nuclear antigen, has become an important method to assess the proliferative activity of tumors. These three nuclear antigens were studied by immunohistochemical analysis of cytologic smears. These smears were obtained by scraping the cut surface of 10 cases of esophageal squamous cell carcinoma and were fixed and prepared by different methods. The results were compared with those of tissue sections to apply the immunocytochemical findings of these antigens to cytology specimens. Smears that were placed on Denhardt- or Neoprene-coated slides and subsequently fixed in 4% paraformaldehyde and methanol exhibited the best cell adherence to the slides, had minimal loss of antigenicity, and had good preservation of cell morphologic features for all three antigens examined. The percentage of positive tumor cells in the cytology smear was generally in good agreement with that in the tissue section. For these three antigens, proliferating cell nuclear antigen demonstrated a much higher percentage of positive cells than either Ki-67 or DNA polymerase alpha, in both the smears and the tissue sections. In summary, Ki-67, DNA polymerase alpha, and proliferating cell nuclear antigen can be immunolocalized successfully in cytology smears and may become another parameter to assess the proliferative activity of tumors in the field of diagnostic pathology.     

Bone lesions: Role of sediment cytology

This study was conducted to evaluate the role of sediment cytology of biopsy specimen fixatives, which is usually discarded, in early diagnosis of bone lesions. Cytological smears prepared from sediments of biopsy specimen fixatives (sediment cytology) were used to study 65 bone specimens biopsied with suspicion of malignancy. The cytological diagnosis was then compared with histological diagnosis, taking the latter as gold standard. Smears were adequately cellular and showed good preservation of cellular morphology. Some of the smears showed microfragments of tissue. Cytology labeled 29 lesions as malignant, 26 lesions as benign, 3 as inflammatory, and 7 smears as inconclusive because of low cell yield. Sediment cytology was able to correctly diagnose 58 of 65 lesions. There was no false‐positive or false‐negative case. The sediment cytology could be considered as an easy and effective diagnostic tool that can provide early diagnosis for the lesion of bone. Diagn. Cytopathol. 2009. © 2009 Wiley‐Liss, Inc.     

Evaluation of a cervical cytology device (cell-sweep) based on comparison to colposcopic findings.

A new cytology sampling device, the CellSweep, identifies squamous intraepithelial lesions with a sensitivity of 75%. The purpose of this study was to evaluate the accuracy of cervical cytology using a new sampling device combining an endocervical brush and ectocervical spatula into one unit (CellSweep, patented by R. Mohajer, Troy, Michigan). From April 1995 to July 1995, 71 patients referred to the Allegheny University Hospitals Colposcopy Clinic had cervical cytology obtained with the CellSweep and underwent colposcopic evaluation of the cervix. The ability of the CellSweep to detect an abnormality confirmed by colposcopic evaluation was studied. Colposcopically directed ectocervical biopsies were obtained only in patients with identifiable lesions (n = 32). No random biopsies were obtained. The cytology smear was unsatisfactory for interpretation in one case. The remaining 70 Papanicolaou smears were read as normal in 17 (24%) cases and atypical squamous cells in 19 (27%). A squamous intraepithelial lesion (SIL) was detected in 34 (48%) smears. The colposcopic evaluation was normal in 50 patients who had satisfactory Papanicolaou smears, whereas SIL was detected in 20 cases. In 31 patients, SIL was not present in either colposcopy or cytology. In this preliminary study, the CellSweep identified SIL with a sensitivity of 75% and a specificity of 62%. The CellSweep, which combines an endocervical brush and an ectocervical spatula into a single unit, seems to be an acceptable device for obtaining cervical cells for cytologic screening.     

Positive correlation between the frequency of micronucleated cells and dysplasia in Papanicolaou smears

Most cancers arise in epithelial tissues and these tissues are typically the targets of the carcinogens responsible for the tumors. Exfoliated epithelial cells have traditionally been used for cancer screening by cytopathologists and these cells also can be used for biomonitoring of genotoxic effects in humans. Cervical cancer results from the progression of preinvasive precursor lesions, called low-grade squamous intraepithelial lesions (LGSILs), to high-grade squamous intraepithelial lesions (HGSILs). The gradient from low to high-grade lesions is characterized by increasing nuclear atypia and the failure of cellular differentiation in progressively more superficial levels of the epithelium. These phenotypic changes are hypothesized to be accompanied by increased genetic instability that can be documented using the micronucleus (MN) assay in exfoliated cervical cells. A retrospective study was performed to investigate the frequency of micronucleated cells in cervical smears from women at high risk for developing cervical cancer. Papanicolaou (Pap) smears from 275 women previously studied at a cancer clinic were coded and analyzed for the frequency of micronucleated cells. LGSIL, HGSIL, and invasive carcinoma smears had significantly higher frequencies than normal and ASCUS (abnormal squamous cells of undetermined origin) smears. HGSIL or severely dysplastic smears had the highest frequency of micronucleated cells (although not significantly higher than LGSIL smears), an observation that that could be useful in confirming these types of lesions. The results indicate that the MN frequency in exfoliated cervical cells may be an additional criterion for establishing cervical cancer risk.     

Cytologic features of high-grade squamous intraepithelial lesions involving endocervical glands on ThinPrep cytology

Usage of liquid-based cytology has resulted in better cellular preservation with enhancement of nuclear features. The purpose of this retrospective 2-yr study (January 1999 through December 2000) was to evaluate the cellular features of endocervical gland involvement by a high-grade squamous intraepithelial lesion (HSIL) vs. endocervical adenocarcinoma in situ (AIS) on cell samples processed by the ThinPrep method as compared to conventional smears. Of the 97 cases of CIN III diagnosed on cytology, 52 (54%) showed surface endocervical gland involvement by CIN III and form the basis of this study. There were also six cases of endocervical AIS diagnosed on histology with prior cytology. The architectural features of HSIL involving endocervical glands and AIS were similar to those previously reported on conventional smears. A consistent finding of HSIL involving endocervical glands was the loss of central cell polarity and piling within cell groups, a finding not present in AIS. Central cell polarity was maintained in cellular groupings of AIS. In addition to the cellular feature present on conventional smears, micronucleoli were clearly visualized in cells of HSIL involving endocervical glands and prominent nucleoli were present in AIS. Apoptosis and mitoses were clearly visualized in both entities. Endocervical gland involvement by HSIL has characteristic cell patterns and features on liquid-based/thin-layer cytology that permit their distinction from AIS.     

Effects of tamoxifen on the cytology of the uterine cervix in breast cancer patients

Tamoxifen, a nonsteroidal antiestrogen, is the endocrine therapy of choice for all stages of breast cancer. Because tamoxifen is well tolerated and has minimal side effects, it is currently being evaluated in large scale trials as a chemopreventive agent for women at risk for developing breast cancer. The potential adverse effects of tamoxifen, specifically the development of proliferative lesions of the endometrium, coupled with the prospect of its wider use, places new emphasis on recognizing tamoxifen-associated histologic and cytologic changes in the female genital tract. The current study evaluated cervical smears from 52 breast cancer patients treated with tamoxifen compared with 21 smears from breast cancer patients who had not received tamoxifen. Cytologic diagnoses were classified according to the Bethesda system. The presence of blood, inflammation, and hormonal effect were also assessed. No squamous intraepithelial lesions were identified. A total of 21 of 38 smears (55%) from patients receiving tamoxifen alone and 11 of 14 smears (78%) from women who received tamoxifen in combination with adjuvant cytotoxic chemotherapy showed atypias compared with only 6 of the 21 breast cancer patients (28%) who did not have hormonal therapy. The number of smears showing atypia was equally divided into changes interpreted as benign reactive and atypical squamous cells of undetermined significance (ASCUS). Of the 19 patients whose smears were classified as ASCUS, 13 patients had a subsequent cervical biopsy and none showed dysplasia or diagnostic human papilloma virus changes. Tamoxifen therapy was not associated with an increase in the presence of blood or inflammation, and no discernible alteration in the hormonal state was seen in the cervical smears. We conclude that the use of tamoxifen may be associated with benign squamous atypia in cervical smears and that the atypia is not associated with intraepithelial lesions. Diagn. Cytopathol. 1998;19:417–422. © 1998 Wiley-Liss, Inc.     

Small cell carcinoma of the cervix in liquid-based Pap test: utilization of split-sample immunocytochemical and molecular analysis

Small cell (neuroendocrine) carcinoma of the uterine cervix (SMCC) is a rare, highly aggressive malignant neoplasm. Both conventional and liquid-based cytology (LBC) cervical smears have low sensitivity in diagnosing SMCC, requiring immunocytochemical (ICH) confirmation. We present the first series of SMCC primarily diagnosed in cytology specimens, and ICH studies performed on the residual LBC specimens with subsequent confirmation of the diagnosis on surgical pathology specimens. Immunocytochemical stains for keratin, p16INK4, and neuroendocrine markers (synaptophysin, chromogranin, CD56) were performed on additional ThinPrep slides. HPV test used chromogenic in situ hybridization high risk HPV DNA probe. The Pap smears in all three specimens were highly cellular with a mixture of squamous cells and numerous well-preserved single or small cohesive clusters of malignant epithelial cells. Tumor cells were small, monomorphic with minimal cytoplasm and high nuclear/cytoplasmic ratio. There was significant nuclear overlap, but no nuclear molding, or smudging of nuclear chromatin. The chromatin pattern was stippled. A background tumor diathesis was prominent. Atypical squamous cells of undetermined significance (ASCUS) were noted in one case, and markedly abnormal squamous cells were seen in another case. The main cytology differential diagnoses included high-grade squamous intraepithelial lesion and an endometrial adenocarcinoma. Immunocytochemical positivity for the neuroendocrine markers supported the diagnoses of SMCC in all three cases. The morphologic features of the concurrent surgical pathology specimens were typical of SMCC. The tissue diagnoses were also confirmed by immunohistochemistry. Our study allows us to conclude that SMCC can be primarily diagnosed in LBC specimens using a panel of immunocytochemical stains.     

Cytologic features of “dry-type” cutaneous leishmaniasis

Exfoliative cytology smears from the lesions of 179 patients with cutaneous leishmaniasis due to Leishmania tropica were studied with specific reference to cellular reactions and their effect on the parasite. Aggregates of the parasite (so-called Leishman Donovan bodies) were present within macrophages and in some fibroblasts. The nature of the inflammatory reaction to the disease was studied by performing differential counts of the inflammatory cells present in the smears. These were correlated with the number of Leishman Donovan bodies. There was an inverse relationship between the number of Leishman Donovan bodies and the percentage of small lymphocytes, neutrophils, and type I macrophages. It is postulated that aggregates of activated macrophages (designated types II and III) and the Leishmanian milieu (sticky matrix) protect the amastigote Leishmania parasites from being eradicated by the inflammatory and immune reaction. The cytoplasmic blebbing of the parasitophorous vacuoles and cell to cell connection of the activated histiocytes could be shown by the CD-68 immunostaining of the tissue biopsy. Diagn. Cytopathol. 1998;19:182–185. © 1998 Wiley-Liss, Inc.     

The estimation of mean nuclear volume in the diagnosis of breast carcinoma.

Sixty-two cases of breast pathology were randomly selected from the files of the Dunedin Public Hospital for evaluation of mean epithelial nuclear volume. The cases were comprised of both benign and malignant ductal epithelial disease, diagnosed in cytological smears or in histological sections. Nuclear volume in histological preparations was estimated by the stereological technique of point-line intercept measurements to derive volume-weighted mean volumes (vV). An index of the nuclear volume (Vi) of cytology smears was calculated from measurements of nuclear areas by either image analysis Vi(e) or point-line intercepts Vi(p). By all methods of analysis a clear distinction of nuclear volume was found between the benign [means for the cytology were 148 microns 3 (Vi(e)) or 246 microns 3 (Vi(p)), and 203 microns 3 (Vv) for the histology specimens] and malignant diseased cases [means for cytology: 524 microns 3 (Vi(e)) or 886 microns 3 (Vi(p)), and 587 microns 3 (vV) for the histology specimens].     

Outcome of "Atypical squamous cells" in a cervical cytology screening program: implications for follow up in resource limited settings

(1) To study the incidence and outcome of "Atypical squamous cells (ASC)" diagnosis in a hospital based cytology screening programme. (2) To work out a feasible strategy for follow up of Atypical squamous cells-undetermined significance (ASC-US) and Atypical squamous cells-cannot exclude high grade squamous intraepithelial lesion (ASC-H) in resource limited settings. A total of 29,475 women were screened cytologically through Pap tests. The epithelial cell abnormalities (ECA) detected on screening were reviewed by the cytopathologists and classified according to Bethesda 2001 system. The women with ASC-US reports were followed up by two repeat cytology tests at 3 and 6 months of the initial visit. The persistent ASC-US cases or the cases which revealed squamous intraepithelial lesion (SIL) on follow up smears; as well as all ASC-H and above cases were referred for coloposcopic evaluation. ASC diagnosis comprised 3.6% of all reports. These were qualified as ASC-US (3.36%) and ASC-H (0.22%). On follow up, only 7 CIN 2 or worse (3.2%) lesions were detected on 218 biopsies in ASC-US category while in ASC-H qualifier 16 CIN 2 and above lesions (30.8%) were picked up on 52 colposcopic biopsies. The difference between ASC-US and ASC-H categories for a CIN 2 or worse outcome was highly significant (P < 0.001). ASC-H qualifier has a high likelihood for an ominous histological outcome and warrants an immediate colposcopic evaluation. On the other hand, ASC-US cases can be managed conservatively by repeat cytology tests at regular intervals without a significant risk of missing a high grade lesion. Diligent screening of cervical smears can judiciously downgrade some cases overcalled as ASC because of inflammatory atypia and thereby reduce referrals in geographic settings with high prevalence of reproductive tract infections. High risk HPV (HR HPV) testing may be a useful adjunct to further reduce referrals by selecting the women who require colposcopic evaluation.