1. HISTOLOGICAL QUALITY CONTROL OF FROZEN SAMPLES USING MATCHING FFPE TISSUE – PRELIMINARY RESULTS OF OUR INSTITUTIONAL BIOBANK E. Mattioli, E. Foglia Manzillo, V. Rubini, F. Palma, A. Paradiso, G. Simone

2. QUALITY CONTROL IN BIOBANKING  Definition: Quality Control (QC) is a complex of procedures aimed at verifying the suitability of the collected samples for research  International publications provide guidelines and technical details: ISBER, 2012 Best Practices for Repositories, www.isber.org ISBER, 2012 Best Practices for Repositories, www.isber.orgwww.isber.org IARC, Technical Standards and Protocols for Biological Resource Centres, 2009 IARC, Technical Standards and Protocols for Biological Resource Centres, 2009 Mager SR et al, Standard operating procedure for the collection of fresh frozen tissue samples, Eur J Cancer 2007 Mager SR et al, Standard operating procedure for the collection of fresh frozen tissue samples, Eur J Cancer 2007 Morente MM et al, TuBaFrost 2: Standardising tissue collection and quality control procedures for a European virtual frozen tissue bank network, Eur J Cancer 2006 Morente MM et al, TuBaFrost 2: Standardising tissue collection and quality control procedures for a European virtual frozen tissue bank network, Eur J Cancer 2006

3.  histological  molecular 2 levels of QC TYPES OF QUALITY CONTROL

4. AIMS: AIMS: verification that the Biobank sample is representative of the whole lesion verification that the Biobank sample is representative of the whole lesion verification that the Biobank sample possesses adequate morphological and immunohistochemical features verification that the Biobank sample possesses adequate morphological and immunohistochemical features HISTOLOGICAL QC MEANS: observation of frozen sections of the banked sample and/or sections from matching paraffin blocks MEANS: observation of frozen sections of the banked sample and/or sections from matching paraffin blocks

5. AIM: verification that the banked samples are suitable for research use (preservation of relevant molecules) MOLECULAR QC MEANS: assay of the degree of fragmentation of nucleic acids (DNA/RNA) via extraction, amplification (PCR) and gel electrophoresis

6. QC PROCEDURE: evaluation of critical immuno- morphological features on frozen biobank aliquots and on matching FFPE material representative of the whole lesion tumor cellularity tumor cellularity MIB-1 staining MIB-1 staining AIM: testing the concordance (i.e. reproducibility) of these features on sections from frozen tissue and from the corresponding paraffin tissue blocks. DESIGN OF THE STUDY

7. 60 cases from our Institutional Biobank were enrolled, including both primary and metastatic tumors. MATERIALS & METHODS Site/typen. Primary tumors breast29 colorectal9 gastric5 endometrial5 Metastatic tumors nodal metastases from breast 6 liver metastases from colorectal 6 Total60 1/2

8.  At our Pathology Department, as a routine procedure, each biobank-dedicated tissue sample is cut in two mirror-matching halves: one half is reduced into aliquots, which are weighed and frozen → @Institutional Biobank one half is reduced into aliquots, which are weighed and frozen → @Institutional Biobank the other half is sent to routine histological processing → “specular” paraffin block (@Pathology Unit) the other half is sent to routine histological processing → “specular” paraffin block (@Pathology Unit) MATERIALS & METHODS For each case tumor cellularity and MIB-1 staining were evaluated on sections from both frozen and FFPE tissue. 2/2

9. The comparison of frozen aliquots with specular FFPE tissue showed a variable degree of discrepancy, due to the intrinsic heterogeneity of tumor tissue. RESULTS 1/5

10. The fraction of concordant cases significantly varied according to the amount of deviation tolerated in the definition of “concordance”. Tumor cellularity MIB-1 staining Low tolerance 55% (33/60) 52.1% (25/48) Intermediate tolerance 63.3% (38/60) 68.7% (33/48) High tolerance 71.7% (43/60) 81.2% (39/48) Tolerated deviation Tumor cellularity MIB-1 staining Low ±10% ± 5% Intermediate ± 15% ± 10% High ± 20% ± 15% RESULTS 2/5

11. Concordance appeared to be influenced by anatomic site, histotype and cytoarchitectural features RESULTS – TUMOR CELLULARITY 3/5  Endometrial carcinomas and liver metastases showed the highest concordance fractions in tumor cellularity. Tumor groupLow tolerance Intermediate tolerance High tolerance Discordant Endometrial cr80%100% 0% Breast cr51.7%58.6%65.5%34.5% Colorectal cr44.4%55.5%77.7%22.3% Gastric cr60% 80%20% Node metastases (breast) 50%66.7% 33.3% Liver metastases (colon) 66.6% 33.4% Tumor cellularity - % concordance

12.  Metastatic tumors showed the highest concordance fractions in MIB-1 staining RESULTS – MIB-1 STAINING 4/5  gastric carcinomas were discordant in 50% of cases. Tumor groupLow tolerance Intermediate tolerance High tolerance Discordant Endometrial cr20%40%60%40% Breast cr55.2%75.9%86.2%13.8% Colorectal cr40%60%80%20% Gastric cr50% Node metastases (breast)66.6%66.6%100%0% Liver metastases (colon)100%100%100%0% MIB-1 staining - % concordance

13. MIB-1 was non-assessable in 20% of cases (12/60) because of technical artefacts on the sections from frozen aliquots. RESULTS 5/5  this finding may indicate a higher lability of the molecular components compared to morphological attributes.

14.  immunomorphological features were not as reproducible between matching frozen and FFPE tissue as expected, because of the inherent heterogeneity of tumor tissue.  mirror-matching FFPE blocks can still play a role in biobanking QC procedures  however, sample collection and storage procedures need to be optimized with the intent to minimize the effects of tumor heterogeneity and to improve the preservation of molecular attributes. CONCLUSIONS

15. THANKS!

17. WHY MATCHING PARAFFIN SECTIONS??? WAIT! easier to cut better morphological detail/image quality better morphological detail/image quality no need to thaw samples! no need to thaw samples!