Establishing a protocol for immunocytochemical staining and chromogenic in situ hybridization of Giemsa and Diff-Quick prestained cytological smears.

BACKGROUND: Protocols for immunocytochemical staining (ICC) and in situ hybridization (ISH) of air-dried Diff-Quick or May-Grünwald Giemsa (MGG)-stained smears have been difficult to establish. An increasing need to be able to use prestained slides for ICC and ISH in specific cases led to this study, aiming at finding a robust protocol for both methods. MATERIALS AND METHODS: The material consisted of MGG- and Diff-Quick-stained smears. After diagnosis, one to two diagnostic smears were stored in the department. Any additional smear(s) containing diagnostic material were used for this study. The majority were fine needle aspirates (FNAC) from the breast, comprising materials from fibroadenomas, fibrocystic disease, and carcinomas. A few were metastatic lesions (carcinomas and malignant melanomas). There were 64 prestained smears. Ten smears were Diff-Quick stained, and 54 were MGG stained. The antibodies used for testing ICC were Ki-67, ER, and PgR, CK MNF116 (pancytokeratin) and E-cadherin. HER-2 Dual SISH was used to test ISH. Citrate, TRS, and TE buffers at pH6 and pH9 were tested, as well as, different heating times, microwave powers and antibody concentrations. The ICC was done on the Dako Autostainer (Dako(®), Glostrup, Denmark), and HER-2 Dual SISH was done on the Ventana XT-machine (Ventana / Roche(®) , Strasbourg, France). RESULTS: Optimal results were obtained with the TE buffer at pH 9, for both ICC and ISH. Antibody concentrations generally had to be higher than in the immunohistochemistry (IHC). The optimal microwave heat treatment included an initial high power boiling followed by low power boiling. No post fixation was necessary for ICC, whereas, 20 minutes post fixation in formalin (4%) was necessary for ISH. CONCLUSIONS: Microwave heat treatment, with initial boiling at high power followed by boiling at low power and TE buffer at pH 9 were the key steps in the procedure. Antibody concentrations has to be adapted for each ICC marker. Post fixation in formalin is necessary for ISH.

Determination of HER-2 status on FNAC material from breast carcinomas using in situ hybridization with dual chromogen visualization with silver enhancement (dual SISH).

During the last years, HER-2 status kits and protocols for chromogen visualization of hybridization signals have come on the market. The first generation using chromogen visualization used single color probes. The second generation, now emerging on the market, uses dual chromogen visualization. The aim of this study has been to test a new dual color chromogen kit (Ventana INFORM HER2 Dual Colour ISH Roche(®)) and compare the results with our in-house method(s). The material consisted primarily of cytological material from invasive breast carcinomas in 49 women. Dual SISH was done on all 49 cytological and histological specimens. The histological specimens were treated according to the manufacturer's recommendations. The procedure was modified in several steps in order to adapt it to the cytological material. Hybridization failed in two cytological specimens. Dual SISH showed concordant results on cytological and histological material as to amplified/not amplified. The included cases had the same HER-2 expression in the invasive and the in situ components on histology. Four IDC showed HER-2 amplification (8.5%). Polysomy was found in two cases. All dual SISH results except for one concurred with the results of the in-house method(s) (1/47=2.1%). The dual SISH is suitable for cytological examination of HER-2 status. The protocol must be optimized for cytological material.

The prognostic impact of occult nodal metastasis in early breast carcinoma.

The clinical relevance of isolated tumor cell (ITC: 0.2-2.0 mm) in axillary lymph nodes (ALNs) remains unknown. The aim of this study was to determine their prognostic significance. A total of 295 patients considered as pN0 after routine histological assessment, were reevaluated with ten-level cytokeratin immunohistochemistry (IHC) and two-level hematoxylin-eosin sections. Survival rates, i.e. disease-free survival (DFS), distant disease-free survival (DDFS) and breast cancer specific survival (BCSS) were compared with those of reevaluated node-negative patients. A total of 84 patients (28%) had ITC/MM identified on IHC sections. ITC had no impact on survival at a median 8.2 years of follow-up, whereas MM showed a trend toward poorer DFS (P = 0.091, log rank) and DDFS (P = 0.066) and significantly reduced BCSS (P = 0.016). In multivariate analyses, detection of MM was an independent prognostic factor for DDFS (P = 0.025) and BCSS (P = 0.01) in adjuvant un-treated patients. Micrometastases (MMs) in axillary lymph nodes have prognostic impact. This was not found for ITC. This finding supports the use of systemic adjuvant therapy in patients with MM.

Breast cancer quantitative proteome and proteogenomic landscape.

In the preceding decades, molecular characterization has revolutionized breast cancer (BC) research and therapeutic approaches. Presented herein, an unbiased analysis of breast tumor proteomes, inclusive of 9995 proteins quantified across all tumors, for the first time recapitulates BC subtypes. Additionally, poor-prognosis basal-like and luminal B tumors are further subdivided by immune component infiltration, suggesting the current classification is incomplete. Proteome-based networks distinguish functional protein modules for breast tumor groups, with co-expression of EGFR and MET marking ductal carcinoma in situ regions of normal-like tumors and lending to a more accurate classification of this poorly defined subtype. Genes included within prognostic mRNA panels have significantly higher than average mRNA-protein correlations, and gene copy number alterations are dampened at the protein-level; underscoring the value of proteome quantification for prognostication and phenotypic classification. Furthermore, protein products mapping to non-coding genomic regions are identified; highlighting a potential new class of tumor-specific immunotherapeutic targets.