Cervical FISH Testing for Triage and Support of Challenging Diagnoses: A Case Study of 2 Patients

A 29-year-old Caucasian woman (patient 1) and a 23-year-old Caucasian woman (patient 2).Chief Complaints: Abnormal Pap tests with high risk HPV positivity.

In June 2014, this patient was diagnosed with atypical squamous cells of uncertain significance (ASCUS) after a routine screening cervical Papanicolaou (Pap) test. Reflex high risk human papilloma virus (HPV) testing was performed; the results were positive (genotyping was not performed). Subsequent endocervical curettage showed a small focus of immature, atypical squamous cells with abnormally positive p16 staining and an abnormally increased proliferative index staining pattern (evaluated with Ki-67) (Images 1A-1C). These findings strongly suggested a high-grade lesion; nevertheless, due to the minute and focal nature of these findings, a diagnosis was rendered of squamous dysplasia, cannot exclude high grade dysplasia. Additional follow-up was recommended, and the option of a fluorescent in situ hybridization (FISH) assay (HPV-4C, using reagent manufactured by Cancer Genetics Italia S.r.l.) was also suggested as a method of triage to be performed using the same Thinprep collection media used to create the Pap test. The results of the FISH assay were positive, with 6.6% of cells showing gain of the 3q26 region (Image 1D). With this knowledge, the gynecologist performed a cervical loop electrosurgical excision procedure (LEEP), which revealed moderate squamous dysplasia (cervical intraepithelial neoplasia grade 2 [CIN 2]) supported by strong and abnormal p16/Ki-67 co-expression (Images 1E-1G). PATIENT 2: In June 2013, this patient was diagnosed with atypical glandular cells (AGUS) and was shown to have high-risk human papillomavirus (HPV) positivity. (Again, genotyping was not performed.) The subsequent biopsy showed mild reactive atypia of the glandular cells, which did not completely correlate with the atypical cells revealed by the Papanicolaou (Pap) test. Therefore, further follow-up was recommended. A fluorescent in situ hybridization (FISH) assay was performed on the specimen assayed via the AGUS Pap test; the FISH assay yielded positive results, showing many cells with a gain of 3q26 and 5p15 regions above the established cutoff values. A repeat Pap test was performed, which also was interpreted as indicating AGUS. Results of a second HPV-4C FISH assay showed numerous (14.6%) cells with a gain of 3q26 and 5p15 regions (Image 2A). Repeat cervical and endocervical biopsies showed scant atypical glands in otherwise-generous biopsies (Image 2B). Supported by abnormal p16 and Ki-67 immunohistochemical staining results (Image 2C and 2D) and the knowledge of the abnormal FISH assay results, the pathologist diagnosed the patient with endocervical adenocarcinoma in situ (AIS). The results of a subsequent LEEP confirmed the diagnosis of endocervical adenocarcinoma in situ with negative resection margins.

Abnormal Papanicolaou (Pap) test results with high risk of human papilloma virus (HPV) positivity and scant lesional tissue, as revealed by cervical/endocervical biopsies.