Identification of HPV Types and Mycobacterium Tuberculosis Complex in Historical Long-Term Preserved Formalin Fixed Tissues in Different Human Organs.

University anatomical-pathological collections represent huge sources of human tissues and preparations from a variety of different diseases. With the help of modern genetic and histological methods, preserved fixed tissues from pathological collections can be used to re-evaluate former diagnoses. We analysed 25 specimens from our pathological collection with ages ranging from 78 to 112 years. The tissues originated from the oral cavity, lip, tongue, lung, bone, kidney, spleen, thymus, larynx, lymph node, penis and uterine cervix with an original diagnosis of epithelial cancers or tuberculosis. Amplifiable DNA was extracted and in epithelial cancers, potential HPV infection was investigated. Specimens with an original diagnosis of tuberculosis were examined for mycobacterial infection. The tissues were also examined using modern histological methods. Our data showed that in 24/25 specimens the histological structure was preserved and in 10/11 specimens the diagnosis of squamous cell carcinoma could be confirmed. Additionally, HPV type 16 was detected in 8 specimens. The histological pattern of tuberculosis was found in 11/14 specimens and the Mycobacterium tuberculosis complex was ascertained in four specimens. Our study showed that pathogens such as HPV or Mycobacterium tuberculosis can be detected in historical pathological preparations, and that these collections are suitable for further epidemiological research.

The dark matter of the cancer genome: aberrations in regulatory elements, untranslated regions, splice sites, non-coding RNA and synonymous mutations.

Cancer is a disease of the genome caused by oncogene activation and tumor suppressor gene inhibition. Deep sequencing studies including large consortia such as TCGA and ICGC identified numerous tumor-specific mutations not only in protein-coding sequences but also in non-coding sequences. Although 98% of the genome is not translated into proteins, most studies have neglected the information hidden in this "dark matter" of the genome. Malignancy-driving mutations can occur in all genetic elements outside the coding region, namely in enhancer, silencer, insulator, and promoter as well as in 5'-UTR and 3'-UTR Intron or splice site mutations can alter the splicing pattern. Moreover, cancer genomes contain mutations within non-coding RNA, such as microRNA, lncRNA, and lincRNA A synonymous mutation changes the coding region in the DNA and RNA but not the protein sequence. Importantly, oncogenes such as TERT or miR-21 as well as tumor suppressor genes such as TP53/p53, APC, BRCA1, or RB1 can be affected by these alterations. In summary, coding-independent mutations can affect gene regulation from transcription, splicing, mRNA stability to translation, and hence, this largely neglected area needs functional studies to elucidate the mechanisms underlying tumorigenesis. This review will focus on the important role and novel mechanisms of these non-coding or allegedly silent mutations in tumorigenesis.

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.