Reducing "atypical squamous cells" overdiagnosis on cervicovaginal smears by diligent cytology screening

The frequency of possible reasons for "atypical squamous cells" (ASC) overdiagnosis on Papanicolaou (Pap) smears was analyzed. Pap smears of 199 women with negative biopsy outcome after an ASC diagnosis were reviewed. Special attention was paid to presence of reproductive tract infections (RTIs), perimenopausal cells (PM cells), immature metaplastic cells, hormone-related alterations, and drying artefacts. Comparisons were made using χ(2) test between the two ASC qualifiers and also between premenopausal and peri/postmenopausal women. Possible reasons for ASC overdiagnosis could be assigned on Pap smear review in 88/199 (44.2%) negative biopsies. Overall, PM cells were the most frequent reason for ASC overdiagnosis, being present in 35/199 (17.6%) smears. RTIs were the next most common cause (14.6%). PM cells were the most significant confounding factors for persistent ASC undetermined significance (ASC-US) over interpretation (20.2%) while in none of the cases these were interpreted as ASC-H (P = 0.004). Of these, 32 smears belonged to peri/postmenopausal women while only three to premenopausal women (P < 0.001). Immature metaplastic cells were significantly more frequent cause of ASC-H rather than ASC-US interpretation (P = 0.007). RTIs and drying artefacts were more frequently overcalled as ASC-US (in premenopausal women) while hormonal changes were interpreted as ASC-H. Hormone related changes, immature metaplastic cells and drying artefacts more commonly resulted in ASC interpretation in peri/ postmenopausal smears. The results of this study suggest that diligent screening can substantially reduce ASC overdiagnosis, thereby reducing the referrals/ follow ups.     

Atypical squamous cells in exfoliative urinary cytology: clinicopathologic correlates

Presence of atypical squamous cells (ASC) in voided urine is an uncommon finding that may be the harbinger of an underlying malignant process. ASCs in urine may precede a de novo histologic diagnosis of malignancy or be the first sign of a recurrence in the lower urinary tract, or the gynecologic tract (in women). This study analyzed all urine cytology specimens with such diagnoses, with reference to their final histologic outcome. All urine cytology cases (n = 17,446) that included ASCs, evaluated at The Johns Hopkins Hospital between 1989 and 2003 (14 yr), were reviewed for diagnoses. ASCs as defined in this study are keratinizing cells with large and hyperchromatic smudgy nuclei, high N/C ratio, abnormal nuclear or cytoplasmic shapes, and densely orangeophilic cytoplasm. These cases lacked the qualitative and quantitative criteria for malignancy. The final reference outcome was determined by subsequent histologic and clinical follow-up. Of these 17,446 urine specimens, 55 cases (0.3%) from 47 patients had ASCs present. Thirty-two of the 47 patients had adequate follow-up. In 8 of these 32 patients (25%), a diagnosis of squamous-cell carcinoma (SCC) of the urinary bladder or urothelial carcinoma (UC) with squamous differentiation was made on subsequent histologic examination. In two cases (6%) a diagnosis of high-grade cervical SCC was established on subsequent follow-up. Twenty two of 32 cases (69%) remained benign on histologic and prolonged clinical follow-up. We conclude that ASCs in urine are rare (0.3% in this series). An interpretation of ASCs in a urine specimen is made when there is insufficient qualitative/quantitative evidence for a carcinoma diagnosis. ASCs in urine are a clinically valid diagnostic category (31% were later diagnosed with SCC). Most patients with urinary ASCs do not develop malignancy and, therefore, these cells may represent a reactive/inflammatory process most commonly due to vaginal contamination (in women) or exfoliation from the distal urethra (in men). Rarely, ASCs may exfoliate from a uterine cervical SCC and, therefore, a pelvic examination should be considered in such patients.     

Atypical squamous cells,cannot exclude high‐grade squamous intraepithelial lesion: Diagnostic features in surepath™ cervical samples

This study was undertaken to identify the situations in which a diagnosis of “Atypical squamous cells, cannot exclude a high‐grade squamous intraepithelial lesion (ASC‐H)” is offered in SurePath? cervical samples and to identify cytological criteria helpful in predicting high‐grade disease. 2,335 (3.4%) SurePath samples reported as atypical squamous cells (ASC) over a period of 2 years, including 1,112 cases with known hrHPV status were retrieved. 105/1,112 cases were categorized into ASC‐H, and slides were available for review in 88/105 cases. These 88 samples were divided into two categories based on follow‐up histological outcome and hrHPV status–category A: cases with CIN2+ lesions on follow‐up (n = 48) and category B: cases with ≤CIN1 lesions or hrHPV negative status (n = 40). 78% (82/105) cases of ASC‐H tested positive for hrHPV. Overall CIN2+ lesions were found in 50.3% (53/105) cases. Of 88 cases reviewed, HCGs were noted in 56.3% (27/48) cases in category A and 75% (30/40) cases in category B. Dispersed metaplastic cells and scattered small atypical cells were seen in 37.5% (18/48) cases in category A and 12.5%(5/40) in category B. The majority of cases with dispersed atypical cells had <20 cells/sample and cases with HCGs had <10 HCGs per sample. The majority of the cases reported as ASC‐H contained HCGs. Of these groups with nuclear crowding, disorganization and those with steep edges (“blocks”) are likely to predict high‐grade disease. The samples with only dispersed atypical cells had <20 cells/sample in majority of cases. In these, a disproportionate andespecially high nuclear: cytoplasmic ratio and irregular chromatin were the most useful features in predicting high‐grade disease. Diagn. Cytopathol. 2013. © 2012 Wiley Periodicals, Inc.     

Atypical parakeratotic spires and HCII HPV results: correlation in liquid-based cervicovaginal cytology specimens interpreted as ASC-US

The 2001 Bethesda System (2nd edition) indicates that parakeratosis with nuclear enlargement, hyperchromasia, irregular contours, and/or three dimensionality should prompt consideration of an interpretation of ASC-US or greater. The authors retrospectively reviewed 812 liquid-based cervicovaginal cytology samples (CVCSs) interpreted as ASC-US during an 11-month period. All slides were studied for the presence or absence of atypical parakeratotic spires (APKSs), a specific form of atypical parakeratosis. Results from corresponding Hybrid Capture II high risk (HR) HPV testing were recorded (blinded). Of 812 women, 28% (230/812) had APKSs and 20% (163/812) had positive HCII assays for HR HPV. Three percent of all reflexed ASC-US vials (26/812) had both APKSs and positive HR HPV assays (sensitivity = 16%, specificity = 69%). APKSs in liquid-based CVCSs interpreted as ASC-US do not independently correlate to a positive reflex test for HR HPV (P < 0.0001). This suggests that APKSs are most often unrelated to underlying HR HPV infection. The finding of APKSs alone in CVCSs does not warrant an interpretation of SIL and should be used only in concert with other findings to label a sample as ASC-US.     

The use of p16INK4A immunocytochemistry in “Atypical squamous cells which cannot exclude HSIL” compared with “Atypical squamous cells of undetermined significance” in liquid‐based cervical smears

Even though p16^INK4a (p16) immunocytochemistry has proven a useful accessory tool verifying the identification of atypical squamous cells of undetermined significance (ASC‐US) categorized smears, the procedure still has limitations. To date few studies examining the usefulness of p16 immunocytochemistry in atypical squamous cells which cannot exclude HSIL (ASC‐H), compared with ASC‐US in liquid‐based cervical smears. Therefore, we examined the correlation of p16 immunocytochemical staining with follow‐up biopsy results on ASC‐H categorized smears and compared the data with those classified as ASC‐US on 105 liquid‐based cytology samples. We found no statistical significance in the p16 expression of ASC‐US smears and the presence of squamous intraepithelial lesions (SIL) in follow‐up biopsies (p = 0.546). However, p16 expression did significantly correlate with the presence of SIL (p = 0.002) in ASC‐H smears. There was a statistically significant relationship between p16 expression and presence of high grade squamous intraepithelial lesions (HSIL) or more on the follow‐up biopsies in both ASC‐US (p = 0.012) and ASC‐H (p < 0.001) categorized smears. In ASC‐US categorized smears, there was no statistical significance between p16 expression and the HR‐HPV viral load (p = 0.091). But there was a statistical significance between p16 expression and the HR‐HPV viral load (p < 0.001) in ASC‐H categorized smears. Our results indicate that p16 immunostaining is a much better useful marker for HR‐HPV infection and detection of SIL in ASC‐H categorized smears compared to those defined as ASC‐US. Diagn. Cytopathol. 2010. © 2009 Wiley‐Liss, Inc.     

Reflex high-risk human papilloma virus DNA test is useful in the triage of women with atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion

This study is aimed to investigate the role of reflex high-risk human papilloma virus (HPV) DNA testing as an alternative triage method to colposcopy for women with atypical squamous cells cannot exclude high-grade squamous intraepithelial lesion (ASC-H) on Papanicolaou (Pap) tests. Reflex HPV DNA testing using Hybrid Capture II method was carried out on 88 women with ASC-H diagnosed by Thin Prep Pap test. Correlation with follow-up biopsies was available on 42 of these patients. The reflex HPV DNA test showed an overall positive rate of 67% and negative rate of 33% in 88 patients with ASC-H. Using age 30 as the cut off point, the positive rate had increased to 83.3% (35/42) in patients 30 yr or younger, while the positive rate for patients older than 30 yr had decreased to 52.2% (24/46). Follow-up colposcopic biopsy results were available in 35 of 59 HPV-positive women, which revealed 15 (43%) high-grade squamous intraepithelial lesions (HSIL), 12 low-grade squamous intraepithelial lesions (LSIL), and 8 negative for dysplasia. In 7 HPV-negative patients, the follow-up biopsies showed no evidence of HSIL or LSIL. Correlation between clinical risk factors and the HPV results demonstrated no significant differences in HPV positivity between the high-risk and low-risk patients. The high sensitivity (100%) and negative predictive rate (100%) in detecting HSIL in our study provide strong evidence that, instead of automatic referral to colposcopy, reflex HPV DNA testing may be used as an alternative triage method for women diagnosed with ASC-H on Thin Prep Pap test, especially for women older than 30 yr of age.     

Triage of cervical cytological diagnoses of atypical squamous cells by DNA methylation of paired boxed gene 1 (PAX1)

Detection of cervical high‐grade squamous intraepithelial lesions (HSIL) in patients with equivocal cytological abnormalities, such as atypical squamous cells (ASC) of undetermined significance (ASCUS) or inability to exclude high‐grade squamous intraepithelial lesions (ASC‐H) is still a challenge. This study tested the efficacy of PAX1 methylation analysis in the triage of cervical ASCUS and ASC‐H and compared its performance with Hybrid Capture 2 (HC2) HPV test. A hospital‐based case–control study was conducted. Cervical scrapings from patients with ASCUS or ASC‐H were used for the quantitative methylation analysis of PAX1 methylation by MethyLight and HPV testing by HC2. Patients with ASC‐H or ASCUS with repeated abnormal smears underwent colposcopic biopsy and subsequent therapies. Diagnoses were made by histopathology at a follow‐up of 2 years. The efficacies of detecting high‐grade lesions were compared. Fifty‐eight cervical scrapings with cytological diagnosis of ASCUS (n = 41) and ASC‐H (n = 17) were analyzed. One of the 41 (2.4%) ASCUS patients and seven of 17 (41.2%) ASC‐H patients were confirmed to have HSIL. After dichotomy of the PMR, PAX1 methylation rates were significantly higher in ASC developing HSIL compared with those developing reactive atypia (87.5% vs. 12.5%, P < 0.001). Testing PAX1 methylation in cervical swabs of patients with ASC confers better sensitivity (87.5% vs. 62.5%) and specificity (98.0% vs. 86.0%) than HC2 HPV testing. We show for the first time that PAX1 hypermethylation analysis may be a better choice than HC2 in the triage of ASCUS and ASC‐H. Diagn. Cytopathol. 2013. © 2011 Wiley Periodicals, Inc.     

Comparison of cervical cytology and the polymerase chain reaction for HPV 16 to identify women with cervical disease in a general practice population.

A comparison of the ability of cervical cytology and the polymerase chain reaction (PCR) for human papilloma virus type 16 (HPV 16) to identify women with cervical disease has been performed in a general practice population of 249 women, none of whom were believed to have current cervical disease prior to examination. Within this population, 29 women were found by colposcopy and subsequent histopathology to have evidence of cervical disease [5 with cervical intraepithelial neoplasia (CIN) 3; 8 with CIN 2; and 16 with CIN 1]. The prevalence of HPV 16 in this population was 18.9% (CIN 3, 80%; CIN 2, 50%, CIN 1, 12.5%, normal, 16.8%). Women with severe disease (CIN 2 and CIN 3) had a significantly higher incidence of HPV 16 DNA than those with mild cervical disease (CIN 1) or no cervical abnormality (P = 0.001). There was no significant difference in the ability of either PCR for HPV 16 or cytology to identify women with cervical disease. The combination of screening by cytology and the presence of HPV 16 DNA resulted in the identification of a higher proportion of the women with disease, but this observation did not reach statistical significance. Although the failure to detect disease by the two screening methods was similar, HPV 16 DNA positivity was associated with a higher false-positive rate for disease detection than cytology (P less than 0.03). The PCR assay for detecting HPV 16 in this investigation was shown to have a false-positive rate of 2.4% and a false-negative rate of 10.4%. The prospect of screening women for cervical disease using PCR for HPV 16 is discussed.     

Microglandular hyperplasia has a cytomorphological spectrum overlapping with atypical squamous cells-cannot exclude high-grade squamous intraepithelial lesion (ASC-H)

We retrospectively evaluated cytological interpretations of conventional cervical smears in 48 cases of biopsy-proven microglandular hyperplasia and compared them with 15 negative controls. A glandular pattern was noted in all 48 cases but was predominant in 73% (35/48) of the cases. Immature metaplastic pattern was present in 71% (34/48) and was predominant in 27% (13/48). These features were not observed in negative control smears. 85% of cases (41/48) were interpreted as negative for epithelial cell abnormality. Two cases with predominantly glandular pattern (6%, 2/35) were interpreted as atypical glandular cells. Five cases with predominantly immature metaplastic pattern (38%, 5/13) showed checkerboard arrangement or rows of single cells with slightly larger atypical nuclei leading to interpretation as "atypical squamous cells-cannot exclude high-grade squamous intraepithelial lesion." The metaplastic cells with normoblast-like karyorrhectic apoptotic debris confined to the nuclear area were scattered among these cells in microglandular hyperplasia with metaplastic pattern.     

Atypical squamous cells in the urine revealing endometrioid adenocarcinoma of the endometrium with squamous cell differentiation: A case report

Urine cytology is mainly used to detect urothelial carcinoma (UC), especially for high‐grade lesions including urothelial carcinoma in situ. Benign squamous cells are often seen in the urine specimens of women, they are either exfoliated from the trigone area of the bladder, the urethra, or the cervicovaginal region. However, abnormal squamous cells in the urine raise concerns of abnormalities of the urinary tract and cervicovaginal area which range from squamous metaplasia of the urothelium, a cervicovaginal squamous intraepithelial lesion, condyloma acuminatum of the bladder, UC with squamous differentiation, and squamous cell carcinoma. We present here a unique case of atypical squamous cells (ASCs) in the urine subsequently leading to the diagnosis of endometrioid adenocarcinoma of the endometrium with squamous differentiation. The presence of ASCs in voided urine is a rare finding that may indicate an underlying malignancy. Careful evaluation of squamous cells in the urine is an important part of our daily cytopathology practice. Diagn. Cytopathol. 2015;43:49–52. © 2014 Wiley Periodicals, Inc.     

Atypical squamous cells, cannot exclude a high-grade squamous intraepithelial lesion: the practice experience of a hospital-based reference laboratory with this new Bethesda system diagnostic category

The 2001 Bethesda System diagnostic category of atypical squamous cells cannot exclude a high grade squamous intraepithelial lesion (ASC-H) was developed and implemented after many studies that proved its clinical relevance. In this study, we describe the practice experience of a hospital-based reference laboratory with this new diagnostic category. The anatomic pathology computer database was searched, and 414 Papanicolaou (Pap) smears signed out as ASC-H were discovered among 60,390 Pap smear accessions over a 17-month period of time. One hundred four cases had corresponding tissue specimens. Slides from all Pap smears and tissue specimens were reviewed, and five Pap smears were reclassified, leaving 99 study cases. In our laboratory, 88.9% of the study cases had either low or high grade dysplasia diagnosed on subsequent tissue specimens. The positive predictive value of ASC-H for histologically proven high-grade squamous intraepithelial lesions (HSIL's) was 40.4%. Of those having human papillomavirus (HPV) hybrid capture II testing, high-risk HPV types were detected in 73.9% of cases. The majority of study cases had less than 25 atypical cells. In two hysterectomy cases and three loop electrosurgical excession procedure (LEEP) conization cases, high-grade dysplasia was present as a single microscopic focus, suggesting that the paucity of atypical cells in ASC-H Pap smears may be secondary to small lesion sampling. Thirteen study patients were postmenopausal and 30.8% had low-grade dysplasia, and of these, 46.2% had high-grade dysplasia on subsequent tissue specimens. In conclusion, our practice experience with ASC-H is similar to that reported in the literature before the 2001 Bethesda System.     

The significance of endocervical cells and metaplastic squamous cells in liquid‐based cervical cytology

We conducted a retrospective study to investigate whether the presence or absence of endocervical cells (EC) and metaplastic squamous cells (MSC) was associated with the detection of squamous intraepithelial lesions in liquid‐based cervical cytology. 90,376 cases of liquid‐based cervical cytology smears received in 2006 were included in the study. Low‐grade (LSIL) and high‐grade squamous intraepithelial lesions (HSIL) were classified according to the Bethesda system (2001). The rates of detecting LSIL and HSIL in smears with and without EC and/or MSC were determined. There were 1,540 LSIL and 396 HSIL. The ratio of HSIL/NILM (no intraepithelial lesion or malignancy) was 0.0022 in smears without EC or MSC, 0.0040 in smears with EC only, 0.0044 in smears with MSC only, and 0.0056 in smears with both EC and MSC present. Compared with smears without EC or MSC, this ratio was significantly higher (P < 0.05) when either EC or MSC was present. Compared with smears with EC only, the ratio was also significantly higher when both EC and MSC were present (P < 0.05). On the other hand, the presence or absence of EC had no effect on the detection rate of LSIL (0.0191 for both groups), while the presence of MSC was actually associated with lower detection rate of LSIL (0.0153, P < 0.05). The presence of endocervical and metaplastic cells was associated with higher detection rates of HSIL. MSC was associated with lower detection or LSIL. Diagn. Cytopathol. 2009. © 2009 Wiley‐Liss, Inc.     

Evaluation of p16 immunostaining to predict high‐grade cervical intraepithelial neoplasia in women with Pap results of atypical squamous cells of undetermined significance

p16 immunostaining has been examined to detect high‐grade cervical intraepithelial neoplasia grade (CIN2+) in Pap cytology specimens. However, the utility of p16 in predicting CIN2+ in Pap specimens with atypical squamous cells of undetermined significance (ASC‐US) or atypical squamous cells, cannot exclude high‐grade squamous intraepithelial neoplasm (ASC‐H), is controversial. In this study, we evaluated the utility of p16 immunostaining for predicting CIN2+ in 78 Pap specimens with ASC‐US/ASC‐H and compared the results in high‐risk HPV DNA and the follow‐up biopsies. p16 immunostaining was positive in 47% (37/78) of the Pap specimens. Of the 13 Pap specimens with follow‐up biopsy results of CIN2+, 7 (54%) were positive for p16. p16 positivity in the Pap specimens was not significantly associated with a CIN2+ biopsy result (P = 0.76). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of p16 immunostaining for predicting CIN2+ were 54%, 52%, 19%, and 85%, respectively. High‐risk HPV DNA was detected in 40% (31/78) of the Pap specimens. The sensitivity, specificity, PPV, and NPV of HPV DNA for predicting CIN2+ were 100%, 72%, 42%, and 100%, respectively. High‐risk HPV genotypes were detected in six p16‐negative specimens with follow‐up biopsy results of CIN2+. Our findings suggest that the utility of p16 immunostaining for predicting CIN2+ in Pap specimens with ASC‐US/ASC‐H is limited. Scant abnormal cells in Pap specimens with ASC‐US/ASC‐H may have contributed to the low p16 sensitivity. Diagn. Cytopathol., 2011. © 2010 Wiley‐Liss, Inc.     

Primary pure squamous cell carcinoma of the breast diagnosed by fine-needle aspiration cytology: a case study using liquid-based cytology

Pure primary squamous cell carcinoma (SCC) is an extremely rare type of breast tumor. We report one of such cases in a 32-year-old woman, diagnosed by fine-needle aspiration cytology (FNAC). Aspiration smears were characterized by squamous cells, both isolated and in aggregates, at various stages of maturation. The tumor was excised, and the histologic sections confirmed the cytologic diagnosis. Pure primary SCC of the breast has a distinctive cytomorphologic appearance, and diagnosis of this tumor by FNAC is possible. For its diagnosis, the exclusion of SCC of local cutaneous structures and metastasis of distant squamous carcinoma are mandatory.     

Limiting factors for cytopathological diagnosis of high-grade squamous intraepithelial lesions: a cytohistological correlation between findings in cervical smears and loop electrical excision procedure.

The present study sought possible factors leading to the cytological diagnosis of atypical squamous cells of uncertain significance (ASCUS) in cases of high-grade squamous intraepithelial lesions (HSIL). Based on retrospective histopathological analysis of loop electrical excision procedure (LEEP) products that diagnosed HSIL, two study groups were randomly selected. The first was consisted of cases with two consecutive Papanicolaou (Pap) smears with the diagnosis of ASCUS. The second (control) group was represented by cases diagnosed as HSIL by cytology. From the Pap smears diagnosed as ASCUS, the sampling limitations was different from control group (P < 0.05). The median size of the largest lesion in each case with ASCUS was 2.66 mm (+/- 1.71 mm). In the control group, the median size of the largest lesion was 5.15 mm (+/-2.58 mm) (P < 0.05). The size of the lesion and sample limitations led patients with cervical intraepithelial neoplasms to be diagnosed as ASCUS for two consecutive times, after a 6-mo period.     

Fine-needle aspiration cytology of metastatic nasopharyngeal carcinoma in cervical lymph nodes: comparison with metastatic squamous-cell carcinoma,and Hodgkin's and non-Hodgkin's lymphoma

Our objective was to study the cytomorphological features of metastatic nasopharyngeal carcinoma (NPC) as compared to squamous-cell carcinoma (SCC) of nonnasopharyngeal origin, Hodgkin's lymphoma (HL), and non-Hodgkin's (NHL) lymphoma in the neck lymph nodes. The aim of this study was to find simple cytological criteria using routine stains that can help in differentiating between these entities. All fine-needle aspiration (FNA) smears of metastatic NPC, SCC, HL, and NHL from neck nodes available in our files were included in this study. Nodal disease was the first clinical manifestation in all cases of NPC, and all the lesions were of the nonkeratinizing type. The cytomorphological features of metastatic NPC, SCC, HL, and NHL have been described. Metastatic NPC and HL had a similar reactive lymphoid background, with eosinophils, plasma cells, and sometimes epithelioid-cell granulomas. The bizarre cells of NPC showed some resemblance to Reed-Sternberg cells of HL. However, the latter can be distinguished from the former on the basis of bulky, pale gray, vacuolated cytoplasm and vesicular nuclei with sharply demarcated prominent macronucleoli. In NPC, the neoplastic cells form distinct aggregates contrasting with the normal lymphoid cells in the background, which was different from the monotonous dispersed population of cells in NHL. Moreover, bizarre cells, prominent nucleoli, and the presence of plasma cells, eosinophils, and granulomas were not common features of NHL. The smears of metastatic SCC revealed keratinized malignant cells in all cases, while eosinophils and plasma cells were absent in the background. Plasma cells were seen intimately related to tumor cells in all cases of NPC. In conclusion, metastatic NPC can be differentiated from metastatic SCC of nonnasopharyngeal origin, HL, and NHL on the basis of simple nuclear and cytoplasmic features of malignant cells and their background. The presence of plasma cells intermingling with tumor-cell clusters is a good indicator of nasopharyngeal origin. On the other hand, a nasopharyngeal origin is unlikely in the presence of keratinized malignant cells.     

Cytology of metastatic cervical squamous cell carcinoma in pleural fluid: Report of a case confirmed by human papillomavirus typing

Cervical squamous cell carcinomas are rarely the cause of malignant effusions. Their identification can be relatively easy when keratinizing atypical squamous cells are present, but may be very difficult when only nonkeratinizing malignant cells are present. We present the case of a 47‐year‐old woman who presented with a large left pleural effusion after having recently completed chemoradiation therapy for stage IIB cervical squamous cell carcinoma. Cytologic examination of the fluid showed a uniform population of single atypical cells with finely vacuolated cytoplasm, ectoendoplasmic demarcation, cell‐in‐cell arrangements, and short rows of cells with intervening “windows,” all features reminiscent of mesothelial cells. No keratinization or three‐dimensional cell clusters were identified. A panel of immunohistochemical stains was performed on the cell block material, and the atypical cells were positive for cytokeratin 5/6, p63, and p16 but not for cytokeratin 7, calretinin, WT1, or Ber‐EP4 or TTF1. These findings were consistent with metastatic squamous cell carcinoma. HPV DNA determination and typing by PCR confirmed the presence of HPV16 in an aliquot of pleural fluid. This is to our knowledge the first reported case of pleural fluid involved by metastatic squamous cell carcinoma where HPV DNA testing was used to confirm the origin of the metastasis. Despite its rarity, metastatic nonkeratinizing squamous cell carcinoma should be considered when a single cell population of large atypical cells is found in effusions. Immunoperoxidase stains and HPV testing can be performed to establish the diagnosis and confirm the origin from a cervical primary. Diagn. Cytopathol. 2009. © 2009 Wiley‐Liss, Inc.