Optimal Fixation Conditions and DNA Extraction Methods for MLPA Analysis on FFPE Tissue-Derived DNA.

OBJECTIVES: Molecular genetic analysis of formalin-fixed, paraffin-embedded (FFPE) tissues is of great importance both for research and diagnostics. Multiplex ligation-dependent probe amplification (MLPA) is a widely used technique for gene copy number determination, and it has been successfully used for FFPE tissue-extracted DNA analysis. However, there have been no studies addressing the effect of tissue fixation procedures and DNA extraction methods on MLPA. This study therefore focuses on selecting optimal preanalytic conditions such as FFPE tissue preparation conditions and DNA extraction methods. METHODS: Healthy tissues were fixed in buffered or nonbuffered formalin for 1 hour, 12 to 24 hours, or 48 to 60 hours at 4 °C or at room temperature. DNA extracted from differently fixed and subsequently paraffin-embedded tissues was used for MLPA. Four commercial DNA extraction kits and one in-house method were compared. RESULTS: Tissues fixed for 12 to 24 hours in buffered formalin at room temperature produced DNA with the most optimal quality for MLPA. The in-house FFPE DNA extraction method was shown to perform as efficient as or even superior to other methods in terms of suitability for MLPA, time and cost-efficiency, and ease of performance. CONCLUSIONS: FFPE-extracted DNA is well suitable for MLPA analysis, given that optimal tissue fixation and DNA extraction methods are chosen.

Validity of whole slide images for scoring HER2 chromogenic in situ hybridisation in breast cancer.

AIM: Whole slide images (WSIs) have stimulated a paradigm shift from conventional to digital pathology in several applications within pathology. Due to the fact that WSIs have not yet been approved for primary diagnostics, validating their use for different diagnostic purposes is still mandatory. The aim of this study was to test the validity of WSI in assessing human epidermal growth factor receptor 2 (HER2) status in breast cancer specimens using chromogenic in situ hybridisation (CISH). MATERIALS AND METHODS: Ninety-six HER2 CISH slides were scored by two observers on a light microscope (400× viewing magnification) and on WSI (40× scanning magnification, one focus plane) with a minimum of 6 weeks washout period. The concordance between digital and microscopic HER2 scores was assessed. RESULTS: Digitally, 93/96 cases could be assessed (96.8%). Microscopic and digital evaluation of HER2 amplification status were concordant in 68/93 cases ((73.1%, 95% CI: 0.639 -0.823), κ 0.588). CISH underscoring was most noticeable in the amplified and equivocal categories while the highest level concordance was seen in cases with a normal copy number. Additionally there was a noticeable tendency to underestimate the average HER2 scores on WSI: lower in 59 and higher in 11 cases. There was no major difference in time spent for microscopic scoring (86.9 s) and digital scoring (81.7 s). CONCLUSIONS: There was a reasonable concordance between microscopic scoring and WSI-based scoring of HER2 copy number of CISH slides. Nevertheless, WSIs scanned on a single focal plane are insufficient to assess HER2 gene amplification status by scoring CISH due to the noticeable tendency towards digitally underestimating the number of HER2 spots. Scanning at multiple focus planes may offer better resolution for improved digital CISH spot counting.

Added value of HER-2 amplification testing by multiplex ligation-dependent probe amplification in invasive breast cancer.

BACKGROUND: HER-2 is a prognostic and predictive marker, but as yet no technique is perfectly able to identify patients likely to benefit from HER-2 targeted therapies. We aimed to prospectively assess the added value of first-line co-testing by IHC, and multiplex ligation-dependent probe amplification (MLPA) and chromogenic in situ hybridization (CISH). METHODS: As local validation, HER-2 MLPA and CISH were compared in 99 breast cancers. Next, we reviewed 937 invasive breast cancers, from 4 Dutch pathology laboratories, that were prospectively assessed for HER-2 by IHC and MLPA (and CISH in selected cases). RESULTS: The validation study demonstrated 100% concordance between CISH and MLPA, if both methods were assessable and conclusive (81.8% of cases). Significant variation regarding percentages IHC 0/1+ and 2+ cases was observed between the laboratories (p<0.0001). Overall concordance between IHC and MLPA/CISH was 98.1% (575/586) (Kappa = 0.94). Of the IHC 3+ cases, 6.7% failed to reveal gene amplification, whereas 0.8% of the IHC 0/1+ cases demonstrated gene amplification. Results remained discordant after retrospective review in 3/11 discordant cases. In the remaining 8 cases the original IHC score was incorrect or adapted after repeated IHC staining. CONCLUSIONS: MLPA is a low-cost and quantitative high-throughput technique with near perfect concordance with CISH. The use of MLPA in routinely co-testing all breast cancers may reduce HER-2 testing variation between laboratories, may serve as quality control for IHC, will reveal IHC 0/1+ patients with gene amplification, likely responsive to trastuzumab, and identify IHC 3+ cases without gene amplification that may respond less well.

Intra-operative rapid diagnostic method based on CK19 mRNA expression for the detection of lymph node metastases in breast cancer.

Staging by sentinel node (SN) biopsy is the standard procedure for clinically node-negative breast cancer patients. Intra-operative analysis of the SN allows immediate axillary lymph node (ALN) dissection in SN positive patients, but a quick, reliable and reproducible method is lacking. We tested the suitability of a quantitative cytokeratin 19 (CK19) mRNA one step nucleic acid amplification (OSNA#) technique (OSNA-CK19) for intra-operative SN analysis. OSNA-CK19 involves a short manual sample preparation step and subsequent fully automated amplification of CK19 mRNA based on reverse transcription loop-mediated isothermal amplification, with results available within 30-40 min. OSNA-CK19 was compared to histological staining (Hematoxylin&Eosin and CAM5.2 and CK19 immunostaining) of 346 frozen ALNs from 32 breast cancer patients, using half of the lymph node for each method. 267 samples were negative and 61 positive by both methods. Three samples were histology positive and OSNA-CK19 negative. Fifteen samples were histology negative and OSNA-CK19 positive, 11 of which had copy numbers close to the cut-off level of OSNA-CK19. Seven of these 15 samples were RT-PCR positive for epithelial markers and/or showed CK19 protein expression by Western blot suggesting the presence of tumor deposits in the lymph node part investigated by OSNA-CK19. Concordance with histology was 94.8%, and 96.8% after exclusion of the latter 7 discordant cases. Sensitivity was 95.3% and specificity was 94.7% before and 97.1% after discordant case investigation. Our results indicate that OSNA-CK19 can potentially be useful in an intra-operative clinical setting to detect SN tumor involvement in breast cancer patients.