Elisabeth Ouwerkerk-Noordam

Exploiting the residual of cervical thin layer brush samples through cytohistology in cases with invasive carcinoma with application of antibodies.

OBJECTIVE To exploit cervical thin layer brush samples through cytohistology in cases with invasive carcinoma with application of antibodies. STUDY DESIGN Fourteen cases from women with carcinoma diagnosed in 2006 were selected out of 29 invasive carcinomas. From these 14 cases liquid-based cervical cytology material was available to prepare cytohistology. Eight women had squamous cell carcinoma, 4 endocervical adenocarcinoma, 1 endometrial adenocarcinoma and 1 ovarian adenocarcinoma. The residual material from the thin layer sample, collected by brushes by general practitioners, was used to prepare paraffin sections. These were stained with the Papanicolaou method and for the biomarkers Ki-67 and p16 and, if desired, for differentiation markers, including carcinoembryonic antigen, vimentin, cytokeratin 7 and cytokeratin 20 to establish the immunoprofile of the carcinoma. RESULTS The morphologic details in the cancer nuclei in the paraffin sections were excellent, while in all cases the thin layer cytology slide contained thick epithelial fragments with blurred nuclei. In 5 of the 6 adenocarcinomas, the glandular architecture diagnostic of adenocarcinoma was visible in the cytohistology, which was highlighted in the biomarker stainings, particularly so in the Ki-67 sections. With the exception of endometrial adenocarcinoma, all p16(INK4a) stainings were positive, as they were in the ovarian adenocarcinoma case. CONCLUSION Cytohistology is an adjunct to routine cervical cytologic examination of thin layer samples, allowing an unequivocal and refined diagnosis.

Endometrial Cells in Liquid-Based Cervical Cytology: A Diagnostic Pitfall Solved by Preparing Cytohistology from the Residual Thin Layer Sample

Objective: It was our aim to assess the usefulness of cytohistology in cervical thin layer brush samples with problems in the differential diagnosis of endometrial cells. Study Design: This study reveals the cytological, cytohistological and immunohistochemistry findings of 8 cases suspicious of adenocarcinoma in situ (AIS)/adenocarcinoma (AC) in cervical liquid-based cytology (LBC) preparations that turned out to be normal endometrial cells. Results: All 8 cervical LBCs featured endometrial and atypical endocervical-like columnar cells with frequent ragged ‘feathered’ edge appearance and rosette formations. Overlapping atypical glandular cell groups were present on 2 ThinPrep slides as well. In cytohistology of 7 cases, the recognition of endometrial stroma with endometrial glands easily allowed the diagnosis of normal endometrium. In 1 case with very small loose tissue fragments without glands, the diagnosis could be established by positivity for CD10 marker (endometrial stroma) and without proliferative activity in the Ki-67 immunostaining. Conclusion: In cervical LBC preparations, nuclear hyperchromasia, pleomorphism and nucleoli in normal endometrial cells are more obvious than in conventional smears, and their arrangement is sometimes suggestive of AIS or AC. In the 8 cases presented, we could avoid a false-positive diagnosis of AIS or AC through cytohistology/immunohistochemistry, and in consequence, unnecessary colposcopical/histological examination.

Biomarker p16INK4a on Paraffin Sections Prepared from Cervical Brush Samples Highlights Nuclear Changes Resulting in Unquestionable Cytohistodiagnosis

OBJECTIVE To optimize the morphology of pl6-positive atypical and (pre)neoplastic cells in paraffin cytoblock sections with the aim to minimize equivocal diagnoses on cytology samples. STUDY DESIGN Patients with negative cytology results or results within normal limits (WNL) (n=38), atypical squamous cells of undetermined significance (ASCUS) (n=25) and high grade squamous intraepithelial lesion (HSIL) (n=16) were selected. The residual material of the cervical brush samples was processed to cytoblock paraffin sections and stained for pl1. The cytohistodiagnosis of the pl1-stained paraffin sections was based on the cytologic and histologic morphology. RESULTS Of the 38 cytologically negative cases, only 4 contained afe w faintly positive pl61cells. Of the 25AS CUS cases, I 1as cytohistologically upgraded to low grade squamous intraepithelial lesion (LSIL). All 16 HSIL cases contained cells with outspoken diffuse positive immunostaining, highlighting chromatin clumping in the (pre)malignant cells. In the paraffin sections the tissue fragments showed architecture consistent with that of HSIL. The nuclear to cytoplasmic ratio in the HSIL was severely disturbed. CONCLUSION Cervical brush samples allow an unequivocal cytohistodiagnosis based on the (pre)malignant nuclear changes highlighted by the p16 staining of the paraffin sections.

Switching from Neural Networks (PAPNET) to the Imager (Hologic) for Computer-Assisted Screening

Objective: The large set of ThinPrep slides prepared in the Leiden Cytology and Pathology Laboratory is exploited for calculating the impact of the transition from PAPNET neural network scanning to the Imager technology. Study Design: All cervical samples were suspended and fixed in the coagulant fixative BoonFix. We compared 57,541 ThinPrep slides which were scanned by PAPNET and 64,273 ThinPrep slides processed with the Imager: 99,157 cases originated from the Dutch population screening program of asymptomatic women (screenees) and the remaining 22,657 samples were of symptomatic women. In the PAPNET series, 23% were diagnosed by additional light microscopy; in the Imager method, all slides were studied light microscopically. The cytoscores (positive cytology per 1,000 samples) were calculated for normal, atypical squamous cells of undetermined significance (ASC-US), cervical intraepithelial neoplasia (CIN) grades I–II, and for CIN III+. The odds ratios (ORs) for the positive cytoscores were assessed for both the screenees and the symptomatic women. Results: The cytoscores, per 1,000 cases, for ASC-US varied from 17.77 to 40.59, for CIN I–II from 7.17 to 33.35, and for CIN III+ from 2.81 to 8.8. These 6 cytoscores were higher for symptomatic women than for screenees. We observe significantly elevated ORs for the Imager for ASC-US (1.26 and 1.23), CIN I–II (1.45) and for CIN III+ (1.58 and 1.45). These 3 ORs are higher for screenees than for symptomatic women. Conclusion: The Imager technology is more efficacious, particularly for handling screenee slides.

Biomarker p16INK4a on Paraffin Sections from Residual Material: Cancerous Tissue Fragments Overlooked in False Negative Papanicolaou-Stained Thin-Layer Slide

The cytoblock technique, as used in our Leiden laboratory on a large scale, processes a liquid cytologic sample into a histologic paraffin slide.1 This ensures thin cell sections in which the chromatin changes are visualized in each nucleus, in contrast to cytologic specimens in which the tissue fragments show extensive nuclear blurring. Most important, however, the cytoblock technique allows the interpreter to examine the immunohistologically positive cells within the context of the tissue. We present an exemplary false negative case on which we could use our cellblock p16 technique described in this issue of Acta Cytologica.2 The cell sample, as well as brush head, was placed immediately into germand DNA-free 20 mL-volumes of BoonFix and sent by overnight mail to the testing laboratory (Leiden Cytology and Pathology Laboratory, Leiden, the Netherlands). A thin-layer slide was prepared according to liquid-based cytology methods using automated processing of cell suspensions (ThinPrep 3000 Processor, Cytyc Corporation, Marlborough, Massachusetts, U.S.A.). The original diagnosis of the ThinPrep slide in the false negative case we describe was within normal limits in December 2006. In February 2007 the patient was referred to the Leiden University Hospital for complaint of postcoital bleeding, and a cervical polyp was removed in which (histologically) high grade squamous intraepithelial lesion and adenocarcinoma were detected. A hysterectomy was performed. The general practitioner informed us that we had missed the diagnosis of cancer in the thin-layer slide of December 2006. We keep the vials with the rest of the material after the ThinPrep slide is made, and thus we could retrieve it from the archive. We could observe that some reddish material was still in the endocervical part of the brush. We decided to place the vial with the brush head in the paint shaker again to remove all material left behind in the bristles of the brush and enrich the sample with possibly valuable diagnostic material. This enriched sample was histoprocessed in the Pathos (Milestone, Sorisole [BG], Italy), applying microwave technology. From each block, several 4-mm sections were cut and stained for p16 as described in this issue of Acta Cytologica.2 In the sections we encountered, numerous small tissue fragments from cancerous epithelium with p16-positive nuclei in which the chromatin changes were highlighted (Figure 1A and B). Also, the cytoplasm of these cells stained strongly positive. Accordingly, it was easy to identify the malignant nature of these minibiopsy specimens. In our present screening practice of 70,000 cases per year, when we encounter such undiagnosable clusters LETTERS TO THE EDITORS