Genomic characterization of cervical lymph node metastases in papillary thyroid carcinoma following the Chornobyl accident.

Childhood radioactive iodine exposure from the Chornobyl accident increased papillary thyroid carcinoma (PTC) risk. While cervical lymph node metastases (cLNM) are well-recognized in pediatric PTC, the PTC metastatic process and potential radiation association are poorly understood. Here, we analyze cLNM occurrence among 428 PTC with genomic landscape analyses and known drivers (131I-exposed = 349, unexposed = 79; mean age = 27.9 years). We show that cLNM are more frequent in PTC with fusion (55%) versus mutation (30%) drivers, although the proportion varies by specific driver gene (RET-fusion = 71%, BRAF-mutation = 38%, RAS-mutation = 5%). cLNM frequency is not associated with other characteristics, including radiation dose. cLNM molecular profiling (N = 47) demonstrates 100% driver concordance with matched primary PTCs and highly concordant mutational spectra. Transcriptome analysis reveals 17 differentially expressed genes, particularly in the HOXC cluster and BRINP3; the strongest differentially expressed microRNA also is near HOXC10. Our findings underscore the critical role of driver alterations and provide promising candidates for elucidating the biological underpinnings of PTC cLNM.

Radiation-related genomic profile of papillary thyroid carcinoma after the Chernobyl accident.

The 1986 Chernobyl nuclear power plant accident increased papillary thyroid carcinoma (PTC) incidence in surrounding regions, particularly for radioactive iodine (131I)-exposed children. We analyzed genomic, transcriptomic, and epigenomic characteristics of 440 PTCs from Ukraine (from 359 individuals with estimated childhood 131I exposure and 81 unexposed children born after 1986). PTCs displayed radiation dose-dependent enrichment of fusion drivers, nearly all in the mitogen-activated protein kinase pathway, and increases in small deletions and simple/balanced structural variants that were clonal and bore hallmarks of nonhomologous end-joining repair. Radiation-related genomic alterations were more pronounced for individuals who were younger at exposure. Transcriptomic and epigenomic features were strongly associated with driver events but not radiation dose. Our results point to DNA double-strand breaks as early carcinogenic events that subsequently enable PTC growth after environmental radiation exposure.

Extracting DNA from FFPE Tissue Biospecimens Using User-Friendly Automated Technology: Is There an Impact on Yield or Quality?

DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue blocks is amenable to analytical techniques, including sequencing. DNA extraction protocols are typically long and complex, often involving an overnight proteinase K digest. Automated platforms that shorten and simplify the process are therefore an attractive proposition for users wanting a faster turn-around or to process large numbers of biospecimens. It is, however, unclear whether automated extraction systems return poorer DNA yields or quality than manual extractions performed by experienced technicians. We extracted DNA from 42 FFPE clinical tissue biospecimens using the QiaCube (Qiagen) and ExScale (ExScale Biospecimen Solutions) automated platforms, comparing DNA yields and integrities with those from manual extractions. The QIAamp DNA FFPE Spin Column Kit was used for manual and QiaCube DNA extractions and the ExScale extractions were performed using two of the manufacturer's magnetic bead kits: one extracting DNA only and the other simultaneously extracting DNA and RNA. In all automated extraction methods, DNA yields and integrities (assayed using DNA Integrity Numbers from a 4200 TapeStation and the qPCR-based Illumina FFPE QC Assay) were poorer than in the manual method, with the QiaCube system performing better than the ExScale system. However, ExScale was fastest, offered the highest reproducibility when extracting DNA only, and required the least intervention or technician experience. Thus, the extraction methods have different strengths and weaknesses, would appeal to different users with different requirements, and therefore, we cannot recommend one method over another.

The effect of long-term -80°C storage of thyroid biospecimens on RNA quality and ensuring fitness for purpose.

AIMS: To establish whether RNA degrades in long-term storage at -80°C and whether RNA integrity numbers (RINs) determine 'fitness for purpose' in severely degraded RNA. METHODS: RNA was extracted from 549 thyroid biospecimens stored at -80°C for 0.1-10.9 years then their RINs correlated with storage time. RT-PCR for 65, 265, 534 and 942 base pair amplicons of hydroxymethylbilane synthase was used to measure amplicon length in RNA from cryopreserved and FFPE biospecimens that were equally degraded according to RIN. RESULTS: Storage time did not correlate with RIN. Longer amplicons were obtained from cryopreserved samples than FFPE samples with equal RINs. CONCLUSIONS: RNA does not degrade in thyroid biospecimens stored for long periods of time at -80°C. Although RINs are known to predict amenability to analytical platforms in good quality samples, this prediction is unreliable in severely degraded samples.

DNA fingerprinting: a quality control case study for human biospecimen authentication.

This case study illustrates the usefulness of the DNA fingerprinting method in biobank quality control (QC) procedures and emphasizes the need for detailed and accurate record keeping during processing of biological samples. It also underlines the value of independent third-party assessment to identify points at which errors are most likely to have occurred when unexpected results are obtained from biospecimens.

Simultaneously extracting DNA, RNA, and protein using kits: is sample quantity or quality prejudiced?

Interdisciplinary "omics" research and the stringent quality requirements of array-based technologies require the simultaneous yet efficient extraction of DNA, RNA, and protein from the same tissue block. However, the few commercially available simultaneous extraction kits have not been evaluated. We compare the TriplePrep (GE Healthcare) and AllPrep (Qiagen) kits using good, intermediate, and poor quality tissue with specialist single-extract methods: Puregene (DNA), RNeasy (RNA), and homogenizations into buffer (protein). The following parameters were evaluated: DNA-yield (total DNA and double-stranded), purity (260:280 and 260:230), and integrity (gel electrophoresis); RNA-yield, purity, and integrity (RNA integrity numbers [RINs] and quantitative reverse transcription polymerase chain reaction [Q-RT-PCR]); protein-yield and quality (two-dimensional difference gel electrophoresis [2D-DIGE]). Puregene DNA yields were 183% and 506% those of TriplePrep and AllPrep, respectively. For RNA, AllPrep and RNeasy were indistinguishable, but their yields were 412% to 588% those of TriplePrep (depending on block condition) and their between-sample variability was better. TriplePrep protein yields were 57% those of the control, and 6.9% of the gel spots were more than 2-fold altered. However, AllPrep yields were 20% of the control, with 11% of the gel spots being more than 2-fold altered. Therefore, TriplePrep outperformed AllPrep in DNA and protein extractions, the reverse was true for RNA, but neither kit achieved optimal efficiency because both yield and quality were compromised.

Antimicrobials and in vitro systems: antibiotics and antimycotics alter the proteome of MCF-7 cells in culture.

Cell culture is widely used to study gene or protein changes in response to experimental conditions. The value of such experiments depends on stringent control and understanding of the in vitro environment. Despite well-documented evidence describing toxic effects in the clinical setting, antibiotics and antimycotics are routinely used in cell culture without regard for their potential toxicity. We cultured MCF-7 breast cancer cells in the presence/absence of antibiotics (penicillin/streptomycin) and/or the antimycotic amphotericin B. Differential protein expression was assessed using 2D-DIGE and MALDI-MS/MS. Antibiotics caused 8/488 spots (1.3% of the protein) to be generally down-regulated. The affected proteins were principally chaperones and cytoskeletal. In marked contrast, amphotericin B induced a more dramatic response, with 33/488 spots (9.5% of the total protein) generally up-regulated. The proteins were mostly involved in chaperoning and protein turnover. Combining antibiotics and amphotericin B had little overall effect, with only one (unidentified) protein being up-regulated. As this study identifies differential protein expression attributable to antibiotics/antimycotics, we urge caution when comparing and interpreting proteomic results from different laboratories where antibiotics/antimycotics have been used. We conclude that as antibiotics and antimycotics alter the proteome of cultured cells in markedly different ways their use should be avoided where possible.

The in vitro anti-tumour activity of zoledronic acid and docetaxel at clinically achievable concentrations in prostate cancer.

Bisphosphonates and chemotherapy have increasingly gained favour in the treatment of metastatic hormone resistant prostate cancer. We investigated whether zoledronic acid, at a concentration found at the bone, would enhance the anti-tumour activity of docetaxel in the hormone resistant prostate cancer cell line PC-3. Cells were exposed to zoledronic acid (1 mM) in combination or in sequence with docetaxel (3 nM). Cell viability, apoptosis and markers for inhibition of the mevalonate pathway were analyzed 48 or 72 hours after drug treatment. Reduction in cell viability and increased apoptosis levels were most pronounced with single agent zoledronic acid. Western blot analysis showed an overall reduction in the proliferation marker Mini chromosome maintenance protein 2 (MCM2) and reduction in caspase-3 precursor for all drug treatments and a marked reduction in Rho A levels with single agent zoledronic acid and zoledronic acid-docetaxel sequence. This study highlights the potency of zoledronic acid, when used at concentrations similar to those found at the bone, in reducing cell viability and causing apoptosis. Clinically, these findings suggest that in patients with bone metastases due to hormone resistance prostate cancer, who are not fit enough for systemic chemotherapy, single agent zoledronic acid may have a direct effect on viability of prostate cancer epithelial cells.

TP53 codon 72 polymorphism in radiation-associated human papillary thyroid cancer.

The study investigated an association between the germline polymorphism at TP53 codon 72 and the development of papillary thyroid cancer (PTC) following exposure to radiation from the Chernobyl accident. TP53 genotype was examined in 48 pediatric/adolescent (age at diagnosis <18 years) and 68 adult post-Chernobyl patient with PTC, 53 adult patients with sporadic PTC and 313 healthy individuals from Russian-Ukrainian population. In addition, we evaluated loss of heterozygosity for TP53 and the allele expression ratio. The genotype of the patients was correlated with clinicopathological data. Arg TP53 homozygotes were found to be significantly underrepresented among adults with post-Chernobyl PTC, but not in children and adolescents when compared with sporadic PTC cases and the general population. In the tumors, cell transformation did not lead to allelic loss or biased TP53 allele expression in heterozygous individuals. None of TP53 genotypes specifically associated with tumor stage and morphology, however there were particular correlations with lymph node status in certain age groups of radiation-associated cases not seen in sporadic PTCs. The findings suggest TP53 allele combinations other than Arg/Arg may contribute to the risk of development of PTC in individuals exposed to radiation during their late childhood, adolescence or in young adulthood.

Low prevalence of BRAF mutations in radiation-induced thyroid tumors in contrast to sporadic papillary carcinomas.

Point mutations of the BRAF gene have been recently described with high prevalence in papillary thyroid carcinomas. However, this molecular alteration has not been studied in radiation-induced thyroid tumors. We analyzed the prevalence of BRAF point mutations and RET/PTC rearrangements in 55 post-Chernobyl papillary carcinomas, compared with 82 sporadic papillary carcinomas. Radiation-induced tumors demonstrated a low prevalence (4%) of BRAF point mutations and high prevalence (58%) of RET/PTC rearrangements. Sporadic papillary carcinomas revealed a clearly distinct pattern, with 37% of tumors harboring BRAF mutations and 20% RET/PTC rearrangements. These results demonstrate a significant difference in the molecular genetic profile of sporadic and radiation-induced thyroid tumors.