Molecular and Immunohistochemical Characterization of Historical Long-Term Preserved Fixed Tissues from Different Human Organs

University and museum collections are very important sources of biological samples that can be used to asses the past and present genetic diversity of many species. Modern genetic and immunohistochemical techniques can be used on long-term preserved fixed tissues from museum specimens to answer epidemiological questions. A proof of principle was established to apply modern molecular genetics and immunohistochemical methods to these old specimens and to verify the original diagnosis. We analysed 19 specimens from our university collection including human organs that had been in fixative for more than 80 years. The tissues originated from lung, colon, brain, heart, adrenal gland, uterus and skin. We isolated amplifiable DNA from these wet preparations and performed mutational analysis of BRAF, KRAS and EGFR. The tissues were also embedded in paraffin and used for modern histology and immunohistochemistry. Our data show that amplifiable DNA is extractable and ranged from 0.25 to 22.77 μg of total DNA. In three specimens BRAFV600E or KRASG12D mutations were found. Additionally, expression of different proteins like vimentin and GFAP was detected immunohistochemical in six investigated specimens. On the basis of our results the original diagnosis was altered in three specimens. Our work showed that it is possible to extract amplifiable DNA suitable for sequence analysis from long-term fixed tissue. Furthermore, histology and immunohistochemistry is feasible in specimens fixed long time ago. We conclude that these old preparations are suitable for further epidemiological research and that our methods open up new opportunities for future studies.


Introduction
The Pathologic-Anatomical Collection of the University of Rostock contains objects mostly being between 50 and 100 years old. Most objects are wet preparations of common infectious and neoplastic diseases of the first half of the last century such as tuberculosis, syphilis or

Ethics statement
All specimens investigated had been collected in an anonymous manner a long time ago, so that an informed consent of the patients could not be obtained. As a matter of fact, any personal impairment of persons-be they alive or dead-can be excluded by the design of the study. The use of human tissue samples for this study was endorsed by the local ethics committee (ethics commission / university medicine of Rostock, Rostock, Germany: project number: A2015-0061).

Origin of tissue
Sampling was carried out from preparations from the University Pathological Collection Rostock. To be included in our study, the wet preparations needed to meet the following criteria: (1) the original diagnosis was one of the targeted neoplastic diseases (2) the tumour was macroscopically visible (3) there were no signs of mould or autolysis. Accordingly, 19 wet preparations of skin, lung, adrenal gland, colon, heart, brain and uterus were selected from our collection with fixation dates between 1923 to 1964 ( Table 1, Fig 1). Neither original fixation solution nor long-term storage conditions were known for any case. It was assumed that the tissues were fixed and preserved in formalin and alcohol (glycerine or ethanol) since these were the most common fixation procedures in Germany in the last century. In every case, the original glass container was carefully opened and the original fixation solution was discarded. A biopsy of approximately 5 mm 3 was taken from an area that was considered to be representative for the particular illness without destroying the overall structure of the preparation. Afterwards, the wet preparation was put back into its original glass container and covered with

DNA extraction
Tumour DNA was extracted from cryo preserved biopsies of 19 wet preparations. For each specimen, sixty 10 μmslices were collected into 1.5 ml micro tubes containing 400 μl digestion solution (10 mM Tris, 0.1 mM EDTA, pH 8.0, and 0.5% Tween-20) and 40 μl proteinase K solution (20 mg/ml). The samples were incubated at 56°C for up to 72 h and 20 μl fresh proteinase K solution (20 mg/ml) was added after 36 h to achieve a final concentration of 1.2 mg proteinase K solution. After centrifugation, the supernatant was transferred into a new micro tube, mixed with 5 M NaCl and incubated at 60°C for 4 h in order to reverse cross-links from formalin-fixation. Then, DNA was isolated using one volume phenol/chloroform/isoamyl alcohol. After ethanol precipitation, the DNA pellet was dissolved in 30 μl of H 2 O dd . DNA quantity and quality were evaluated using the full absorption spectrum (220-750 nm) obtained by the NanoDrop ND-1000 spectrophotometer. From 2 μl samples DNA concentration in ng/μl and absorbance ratio at 260/280 nm were calculated.

Molecular characterization
The mutational status of KRAS exon 2-4, BRAF exon 15 and EGFR exon 18-21 was analysed by PCR and sequenced in selected specimens. PCR primers (MWG-Biotech, Ebersberg, Germany) and amplification conditions are shown in Table 2. Twenty-five microliter of reaction mixture contained 0.2 μl MyTaq polymerase (Bioline, Luckenwalde, Germany) with 5 μl 5x PCR buffer, 1 μM of each primer set and 75 ng of template DNA. PCR started at 95°C for 10 min. This was followed by 35 cycles at 94°C for 30 s, annealing for 30 s (temperature see Table 2) and 72°C for 45 s, followed by a 10 min extension at 72°C. The obtained PCR-product (1 μl) was used for reamplification under the same PCR reaction conditions, because of no or only weak signals in gel electrophoresis. PCR products were purified with alkaline phosphatase (Thermo Scientific, Darmstadt, Germany) and Exonuclease I (Thermo Scientific, Darmstadt, Germany). Subsequently, sequencing reaction was performed using BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems, Darmstadt, Germany) with each pair of forward and reverse primers, followed by analysis on a 3500 genetic analyzer (Applied Biosystems). The sequence data was compared with their corresponding sequence, KRAS (Genbank GI:
Previous to the experiments, immunostaining protocols were optimized by dilution series and positive and negative controls. Positive controls consisted of specimens with well known expression of the antigen. Negative controls were performed by substituting the primary antibody with IgG1 (mouse, code: X0931, DAKO), or IgG (rabbit, code: X0936, DAKO). During the experiments, positive controls were included with each run. Negative controls were checked by an experienced pathologist "on slide" by evaluating the negative staining of stromal cells or leukocytes

Extraction of DNA from wet preparations
We used 19 wet preparations, with ages ranging from 50-91 years. Tumour DNA was successfully extracted from all these 19 wet preparations by using proteinase K and Phenol protocol [21]. The total amount of DNA ranged from 0.25 to 22.77 μg (mean: 6.28 ±5.53 μg) ( Table 3). In specimens with known fixation date, no correlation between DNA amount and age of the specimen was seen. The purity of DNA, assessed by the ratio of absorbance at 260/280 nm, was low in all samples (mean: 1.46) ( Table 3). The median DNA amount with regard to the different organs ranged from 2.15 ng in heart to 9.0±0.4 ng in brain ( Table 4). The A 260 /A 280 ratio ranged from 1.37±0.06 in brain to 1.52 ±0.09 in skin (Table 4).

Amplification and molecular characterization
PCR amplification was performed targeting 158 bp to 381 bp gene fragments of KRAS, BRAF or EGFR (Table 5). In one case (specimen Nn23) the genes for both EGFR and BRAF were analysed. KRAS was successfully amplified in 2/2, BRAF in 9/10 and EGFR in 4/5 specimens, altogether a positive amplification was detected in 14/16 of all investigated specimens ( Table 5). Sequencing of KRAS (exon 2-4), BRAF (exon 15) and EGFR (exon 18-21) of the corresponding tissue was possible with all amplifiable PCR products (Table 5). Interestingly, in specimen Da236 the KRAS G12D mutation and in two specimens (Ht13 and Ht9b) the BRAF V600E mutation was found (Fig 2).

Methylation analysis
We attempted to determine the MGMT methylation status of three specimens (ZNS33, ZNS212 and ZNS270). However, neither MGMT nor COL2A1 amplification was detected in the investigated specimens. In order to exclude the possibility that the absence of methylation might be attributed to technical errors, we included a known fully methylated DNA as reference, where DNA methylation of both MGMT and COL2A1 was detectable, so that any technical errors in the Methy-Light analyses could be excluded.

Morphological evaluation (haematoxylin-eosin stains)
In order to validate the original diagnoses, we performed H&E staining in each specimen. Histology was assessed by a professionally trained pathologist and evaluated according to today's standards. The quality of the histological sections varied according to a bundle of factors like age of the specimen, tissue type, type of disease, and manipulations prior to histological embedding, but was considered to be sufficient to make an accurate diagnosis in every case. Most of the specimens showed a histological picture that matched the originally labelled diagnosis. However, two specimens (Nn28 and Lu176) originally designated as "lung cancer" showed spindle-shaped tumour cells in a herringbone pattern (Fig 3B and 3C). Therefore, the rather unspecific original diagnosis of "lung cancer" could be further restricted to lung sarcoma. Additionally, the specimen Ht32 showed typical squamous-cell morphology and was therefore considered as squamous-cell carcinoma, rather than melanoma, as originally indicated.

Analysis of tissues by immunohistochemistry
Six specimens were chosen to be analysed by immunohistochemistry (Lu170, Lu176, He146, Nn23, Nn28, and ZNS212) and were classified according to the immunoreactive score (IRS) (Table 6, Fig 3). As we did not know whether formalin-or alcohol-based fixation reagents or a mixture of both were used for original fixation, we performed heat-induced epitope retrieval (HIER) before every staining procedure to enhance the antigenicity as far as possible. Immunohistochemical staining with antibodies against vimentin was performed and showed positivity in 2/2 specimens (100%) with IRS of 2 (mild) and 6 (moderate). TTF-1 staining was performed in four bronchial carcinomas but no expression was found. Four specimens were stained with antibodies against cytokeratin AE1/AE3 but no convincing signal could be detected. The most convincing immunohistochemical staining was found with GFAP in the specimen ZNS212, labelled as pilocytic astrocytoma (Fig 3J). The percentage of positive cells was over 80% with a mild staining reaction leading to an IRS of 8 which displays a mild expression of GFAP. All immunohistologically stained sections showed some background labeling, even when no tumour cells were marked by the antibodies.

Discussion
In this study we investigated 19 specimens from our university collection labelled as different neoplastic diseases using molecular, histological and immunohistochemical methods. The goal was to show feasibility of the application of some of today's standard laboratory methods to study old preparations. We were able to isolate amplifiable DNA from long-term stored tissue and to perform mutational analyses of defined genes. Furthermore, it was possible to conduct histological and immunohistochemical investigations on up to 91 year old specimens and to use the results in order to confirm the original diagnosis.
The mutational analysis of DNA from old tissue was limited by different factors. In a variety of tissues, Karlsen et al. showed that formalin inhibits amplification [22]. Therefore, several protocols indicate that formalin-fixed tissue yields only low quantities and sometimes fragmented DNA [12,[23][24][25]. So the bottleneck is on the one hand the extraction of the DNA and on the other hand the amplifiability of the isolated DNA. We solved the first problem by prolonging the digestion period up to 3 days as it was also proposed by Paireder et al. [19]. In preliminary tests, we had also tried different column-based extraction methods; however only a low amount of DNA (approximately 1ng/μl) was isolated, which was not amplifiable. The extraction with phenol/chloroform was found to be suitable for DNA isolation from our specimens. In general, most extraction attempts yielded DNA concentration above 100 ng/μl, but with a A 260 /A 280 ratio below the optimal range of 1.8 to 2.0, indicating that all DNA extracts were contaminated with proteins ( Table 4). The formalin fixation leads to a network of covalent protein-protein and protein-nucleic acid cross-links which poses significant challenge to the recovery of DNA for downstream applications [14,[26][27][28]. Possibly, these cross-links were not fully destroyed by digestion and as a consequence downstream applications like amplification of DNA were affected. However, it is important to get amplifiable DNA, which was only possible in 16 of 19 specimens. We used a variant of nested PCR to increase the yield of the PCR product. In contrast, the DNA from three glioma specimens was not amplifiable most likely because the DNA was too severely degraded. This might be due to the fact that brain tissue consists to a great extent of fat and therefore is not very stable against outer influences like prolonged formalin fixation. Ferrer et al. demonstrated that 4% buffered formalin is better suited as a fixating agent for brain tissue than 10% non-buffered formalin to preserve DNA [29]. Thus, the suspected formalin fixation might have hampered the isolation of amplifiable DNA from the fatty brain tissue, although no definite statement about the fixation agent can be made.
Despite the low purity, the remaining 16 extracts allowed the amplification of sequences ranging between 211 bp to 381 bp as well as sequence analyses. Interestingly, in two melanomas (Ht9b and Ht13) we detected the BRAF V600E mutation, which is a predictor for clinical IRS represents the product of score resulting from percentage of positive cells and score resulting from intensity of staining. a efficacy of RAF inhibitors such as vemurafenib today. Additionally, a KRAS G12D mutation was found in the colon carcinoma of Da236, which is a negative predictor of response to anti-epidermal growth factor receptor antibodies in today's practice. The quality of all H&E stained slices was considered to be sufficient to histologically validate the original diagnosis. However, we did not obtain satisfactory results by performing immunohistochemistry on long-term fixed tissue. Encouraging IRS scores were reached in two specimens for vimentin and in one preparation for GFAP, with IRS scores of 2, 6 and 8 respectively. This corresponds well with observations from Lyck et al. who showed that, among other markers, GFAP was relatively robust against formalin fixation [30]. Nevertheless, no signal at all could be detected for TTF-1, cytokeratin AE1/AE3 and CD56. This could either be due to the modification of antigenic sites by the fixating agent or because the tumour do not express the particular antigens at all. The latter assumption is likely in two of four specimens tested for AE1/AE3 since they were histologically labelled as sarcomas and therefore do not express AE1/ AE3. Several authors showed that formalin fixation over a few weeks already decreases immunoreactivity of various antibodies [31][32][33] due to cross-linking as well as formation of coordinative bonds for calcium ions. Less experience is available in tissue fixed with ethanol which is suspected to irreversibly destroy the original protein structure by coagulation [33]. Furthermore, several authors suggest that not only the fixating agent but also its delayed penetration into the organ causes frustrating immunohistochemical results, as this may lead to autolytical destruction of antigenic sides [31][32]34]. In summary, immunohistochemistry on long-term preserved tissue is a challenging task which could not be satisfactorily solved so far.
Nevertheless, due to the good quality of H&E staining results, the original diagnosis could be confirmed in most cases as shown in Table 1. However, in one specimen that was originally categorized as melanoma (Ht32) the diagnosis had to be changed into squamous cell carcinoma of the skin because of the presence of keratin pearls and proliferating atypical keratinocytes. The corresponding wet preparation, created in 1924, showed a black colour and it could be speculated that the original diagnosis was made without performing any microscopy. Surprisingly, two specimens of lung cancer, one primary and one adrenal metastasis, (Nn28 and Lu176) showed a roughly identical microscopic pattern like it occurs in a sarcoma. In addition, positive staining for vimentin and negative staining for cytokeratin AE1/AE3 was found in both specimens which supports the diagnosis of sarcoma. Due to the very similar histological characteristics and as lung sarcoma is a rather rare neoplastic entity [35]; it could well be assumed that both specimens were obtained from the same patient. No documentation about the fixation time or other circumstances were found so that unfortunately a final statement can not be given.
Our results show that the assessment of the microscopic structure of old long-term stored specimens is firstly successfully realizable and secondly necessary when museum specimens are intended to be used for educational and research purposes. Even in our small number of cases there were three newly made diagnoses that either mismatched or specified the original description of the entity.

Conclusion
We used conventional histology, immunohistochemistry and molecular analyses in preserved specimens up to 91 years old. For DNA extraction, the method had to be modified, because conventional column-based methods were not suitable. The molecular methods needed adjustments, like a higher amount of starting material (60 slices of 10μm), a longer digestion period (up to 72h) and a more sensitive, nested PCR protocol. It could be shown that mutation analyses are possible in those specimens. Thus, we assume that these old preparations are suitable for further epidemiological research. One limitation of our study is that only specimens from one centre were analyzed, but the experiences and methods presented here should also be applicable in other university or museum collections. Valuable findings about pathogenesis and epidemiology of cancer and infectious diseases could be acquired by investigations of specimen collections from different centres.