Lyophilized brain tumor specimens can be used for histologic, nucleic acid, and protein analyses after 1 year of room temperature storage

Abstract

Frozen tissue, a gold standard biospecimen, can yield well preserved nucleic acids and proteins after over a decade but is vulnerable to thawing and has substantial fiscal, spatial, and environmental costs. A long-term room temperature biospecimen storage alternative that preserves broad analytical utility can potentially empower tissue-based research. As there is scant data on the analytical utility of lyophilized brain tumor biospecimens, we evaluated lyophilized (freeze-dried) samples stored for 1 year at room temperature. Lyophilized tumor tissue processed into paraffin sections produced good histology. Yields of extracted DNA, RNA, and protein approximated those of frozen tissue. After 1 year, lyophilized samples yielded high molecular weight DNA that permitted copy number variation analysis, IDH 1 mutation detection, and MGMT promoter methylation PCR. A 27 % decrease in RIN scores over the 1 year suggests that RNA degradation was inhibited though incompletely. Nevertheless, RT-PCR studies on lyophilized tissue performed similarly to frozen tissue. In contrast to FFPE tissues where protein bands were absent or shifted to a lower molecular weight, lyophilized samples showed similar protein bands as frozen tissue on SDS-PAGE analysis. Lyophilized tissue performed similarly to frozen tissue for Western blots and enzyme activity assays. Immunohistochemistry of lyophilized tissue that were processed into FFPE blocks often required longer incubation times for staining than standard FFPE samples but generally provided robust antigen detection. This preliminary study suggests that lyophilization has promise for long-term room temperature storage while permitting varied tests; however, further work is required to better stabilize nucleic acids particularly RNA.

Fig. 1

Genomic DNA extracted from frozen, lyophilized, and FFPE brain tumor tissue M–DNA marker, MEN(F) frozen meningioma, MEN(L) lyophilized meningioma MEN(P) FFPE meningioma, LGG(F) frozen low grade glioma, LGG(L) lyophilized low grade glioma, LGG(P) FFPE low grade glioma, GBM(F) frozen glioblastoma, GBM(L) lyophilized glioblastoma, GBM(P) FFPE glioblastoma

Fig. 2

SNP analysis of genomic DNA, lyophilized (L) vs. frozen (F) tissue of three types of brain tumor: meningioma (MEN), low grade glioma (LGG), glioblastoma (GBM). On the left half of the figure, copy number variation is shown for chromosome 7 and 19, where the colored squares represent gain at specific chromosome loci (yellow–1 copy, blue–3 copies, red–4 copies). On the right half of the figure, loss of heterozygosity (LOH) on chromosome 10 and 22 q are shown in a B Allele frequency plot

Fig. 3

Assessment of RNA quality. A–RNA integrity number (RIN) of total RNA extracted from lyophilized (open bars) and frozen (grey bars) brain tumor specimens B–RT-PCR products amplified from mRNA of following specimens: MEN(F) frozen meningioma, MEN(L) lyophilized meningioma, LGG(F) frozen low grade glioma, LGG(L) lyophilized low grade glioma, GBM(F) frozen glioblastoma, GBM(L) lyophilized glioblastoma

Fig. 4

Analysis of protein samples. A Coomassie staining of total proteins on 4–20 % gradient tris–glycine gel, where M protein marker, MEN(F) frozen meningioma, MEN(L) lyophilized meningioma, MEN(P) FFPE meningioma, LGG(F) frozen low grade glioma, LGG(L) lyophilized low grade glioma, LGG(P) FFPE low grade glioma, GBM(F) frozen glioblastoma, GBM(L) lyophilized glioblastoma, GBM(P) FFPE glioblastoma. B–Western blot for GFAP and GAPDH

Fig. 5

Immunohistochemical staining of frozen–A, D, G, J, M, P,lyophilized–B, E, H, K, N, Q and FFPE–C, F, I, L, O, R brain tumor tissue. (Aperio digital image 200× magnification)