Utilization of fluorescence in situ hybridization with cytokeratin discriminators in TOP2A assessment of chemotherapy-treated patients with breast cancer.

Tumor biomarkers increasingly provide information for predicting outcomes with chemotherapeutic regimens (personalized medicine). Topo2A is a DNA helicase targeted by anthracyclines, cytotoxic therapeutics used in both adjuvant and palliative treatments of breast cancer. TOP2A gene amplification/deletion is implicated in response to anthracycline-based chemotherapy. We describe an approach for analyzing formalin-fixed, paraffin-embedded breast tumors on tissue microarrays with TOP2A fluorescence in situ hybridization coupled with cytokeratin immunofluorescence to target tumor cells. Stained tissue from patient specimens was imaged and analyzed using Metafer/Metacyte (Metasystems, Waltham, MA, USA), including customized image classifiers. TOP2A/CEN17 ratios of 2.0 or greater (amplified) and 0.8 or less (deleted) were observed for 10.0% and 6.1% of the patients, respectively. Patient outcomes for adjuvant chemotherapy (cyclophosphamide-epirubicin-fluorouracil, cyclophosphamide-methotrexate-fluorouracil, no chemotherapy) were evaluated. No statistical significance was achieved for clinical end points regarding TOP2A status in anthracycline-treated patients. However, patients with TOP2A aberrations receiving methotrexate-based therapy exhibited a significant decrease in 5-year distant disease-free survival and breast cancer-specific overall survival, especially for patients with TOP2A deletions (disease-free survival: hazard ratio, 5.31 [P = .001], and breast cancer-specific overall survival: hazard ratio, 6.45 [P ≤ .001]). No significant differences were seen in patients included in the no-chemotherapy group. Topo2A protein levels were assessed by immunohistochemistry with no correlative statistical relevance to immunofluorescence/fluorescence in situ hybridization-based prognosis for cyclophosphamide-epirubicin-fluorouracil or cyclophosphamide-methotrexate-fluorouracil groups. Interestingly, aberrant (under)expressing patients in the no-chemotherapy group exhibited better 5-year distant disease-free survival (hazard ratio, 0.39; P = .004), trending toward more favorable breast cancer-specific overall survival (hazard ratio, 0.61; P = .11). Our results indicate a strategy by which fluorescence in situ hybridization scoring targeted to cytokeratin-positive tumor cells may provide a tool for added precision and efficiency in TOP2A evaluation from tumor tissue.

Proteomic analysis of high-grade dysplastic cervical cells obtained from ThinPrep slides using laser capture microdissection and mass spectrometry.

The purpose of this discovery phase study was to identify candidate protein biomarkers for high-grade dysplastic cervical cells using mass spectrometry. Laser Capture Microdissection (LCM) was utilized to isolate high-grade dysplastic and normal cells from ThinPrep slides prepared from cervical cytological specimens. Following cell capture, samples were solubilized and proteins separated by gel electrophoresis in preparation for enzymatic digestion and liquid chromatography mass spectrometry analysis (LC-MS). Processed samples were subsequently analyzed using a linear ion trap coupled with a Fourier transform mass spectrometer (LTQ-FT MS). It was determined that both PreservCyt Solution and ThinPrep Pap Stain (Cytyc Corporation) were compatible with the sample processing and LC-MS analysis. In total, from 9 normal and 9 abnormal cervical cytological specimens, more than 1000 unique proteins were identified with high confidence, based on approximately 12,000 captured cells per specimen. Quantitative protein differences between HSIL (High-Grade Squamous Intraepithelial Lesion) and NILM (Negative for Intraepithelial Lesions or Malignancy) samples were determined by comparing the intensities of the representative (label-free) peptide ions. More than 200 proteins were found to exhibit a 3-fold difference in protein level. Interestingly, significant up-regulation of nuclear and mitochondrial proteins in HSIL specimens was noted. In several cases, the increased protein abundance observed in high-grade cells, as determined by quantitative LC-MS, was validated by immunocytochemical methods using ThinPrep cervical specimens. With the study of additional clinical specimens, the differential abundance of proteins in high-grade dysplastic cells versus morphologically normal cervical cells may lead to validated novel biomarkers for cervical disease.

Evaluation of p16INK4a expression in ThinPrep cervical specimens with the CINtec p16INK4a assay: correlation with biopsy follow-up results.

BACKGROUND: The aim of this study was to examine p16(INK4a) protein expression in ThinPrep (Cytyc Corporation, Marlborough, Mass) cervical specimens by using the CINtec p16(INK4a) Cytology Kit (Dako, Glostrup, Denmark). The ability of this assay to accurately identify underlying high-grade lesions was assessed by using follow-up biopsies and comparing these results with Hybrid Capture 2 (Digene, Gaithersburg, Md) high-risk HPV (hc(2)) results. METHODS: Three hundred ninety-eight residual ThinPrep samples were collected, and histological follow-up data were retrieved for abnormal cytology specimens. After preparation of a Papanicolaou-stained slide, a second slide was processed in preparation for p16(INK4a) immunostaining. High-risk human papillomavirus testing (hc(2)) was also performed. RESULTS: Of the 163 cytologically abnormal samples, 6-month biopsy follow-up data were available for 45% of the specimens. At initial blinded evaluation, 21 of the 26 cases with cervical intraepithelial neoplasia (CIN) II/III follow-up were positive for p16(INK4a), yielding an overall diagnostic sensitivity of 81%; 29 of the 47 cases diagnosed as CIN I or less were p16(INK4a) negative, yielding a diagnostic specificity of 62%. In comparison, the hc(2) test results indicated a diagnostic sensitivity of 100% with a diagnostic specificity of 15%. After review of selected cases with CIN II/III follow-up, 25 of 26 slides were deemed to be positive for p16(INK4a), increasing the diagnostic sensitivity to 96%. CONCLUSIONS: The CINtec p16(INK4a) Cytology Kit, in combination with ThinPrep cervical samples, allowed clear evaluation of p16(INK4a) protein overexpression. Diagnostic specificity of the CINtec p16(INK4a) assay was significantly improved relative to hc(2). To increase p16(INK4a) immunostaining in abnormal cells, a modified kit version with improved staining performance has been developed and is currently being evaluated.