Molecular Profiling in Thyroid cancer- Using next generation sequencing to differentiate rare cases of papillary thyroid hyperplasia from papillary thyroid carcinoma

Introduction/Objective Differentiating papillary thyroid hyperplasia from papillary thyroid carcinoma is made primarily on differences in key histologic and cytomorphologic features. These include architectural features such as invasion and nuclear changes such as chromatin pallor, nuclear grooves, and intranuclear pseudoinclusions. Distinguishing between these two diagnoses is not always straightforward in a minority of cases, as papillary thyroid hyperplasia nodules can be misinterpreted as papillary thyroid carcinoma and vice versa. This is because the nuclear changes can be subtle and be induced artificially by poor or inadequate formalin fixation. Immunostains such as cytokeratin 19 (CK19), galectin-3, and HBME1 may be used to distinguish between papillary thyroid hyperplastic nodules and papillary thyroid carcinoma; however, as immunostaining results are not always definitive, other more definitive ancillary tests are sometimes utilized such as molecular testing. Methods/Case Report Thyroid cases with indeterminate diagnoses (n=6) were obtained from the archives. Such cases included papillary thyroid hyperplasia and cases with descriptive diagnoses such as well-differentiated encapsulated and low-grade papillary carcinoma. Five cases of definitive papillary thyroid carcinoma and one case of follicular thyroid carcinoma (n=6) were used as a comparison group. Next generation sequencing (NGS) assays were performed using a custom SLIMamp targeted DNA solid tumor panel (Pillar Biosciences) and a targeted multi gene RNA fusion panel (Invitae). Results (if a Case Study enter NA) All of the definitive cases of papillary thyroid carcinoma showed characteristic mutations such as BRAF V600E as well as RNA fusions such as RET/PTC while all of the indeterminate cases did not show any detectable DNA mutations or RNA fusions, confirming their benign nature. A separate case that was signed out as non-invasive follicular thyroid neoplasm with papillary-like nuclear features showed an NRAS Q61R mutation and a novel ETV1-ERG fusion. Conclusion This study demonstrates the utility of molecular testing as an adjunct to histopathological evaluation in definitive diagnosis and differentiation of papillary thyroid hyperplasia from papillary carcinoma. Next generation sequencing with targeted DNA and RNA panels will be beneficial for both known and novel variant or fusion detection in suspect cases.

Quantitative spectrofluorometric assay detecting nuclear condensation and fragmentation in intact cells

At present, nuclear condensation and fragmentation have been estimated also using Hoechst probes in fluorescence microscopy and flow cytometry. However, none of the methods used the Hoechst probes for quantitative spectrofluorometric assessment. Therefore, the aim of the present study was to develop a spectrofluorometric assay for detection of nuclear condensation and fragmentation in the intact cells. We used human hepatoma HepG2 and renal HK-2 cells cultured in 96-well plates treated with potent apoptotic inducers (i.e. cisplatin, staurosporine, camptothecin) for 6–48 h. Afterwards, the cells were incubated with Hoechst 33258 (2 µg/mL) and the increase of fluorescence after binding of the dye to DNA was measured. The developed spectrofluorometric assay was capable to detect nuclear changes caused by all tested apoptotic inducers. Then, we compared the outcomes of the spectrofluorometric assay with other methods detecting cell impairment and apoptosis (i.e. WST-1 and glutathione tests, TUNEL, DNA ladder, caspase activity, PARP-1 and JNKs expressions). We found that our developed spectrofluorometric assay provided results of the same sensitivity as the TUNEL assay but with the advantages of being fast processing, low-cost and a high throughput. Because nuclear condensation and fragmentation can be typical markers of cell death, especially in apoptosis, we suppose that the spectrofluorometric assay could become a routinely used method for characterizing cell death processes.

Noninvasive Encapsulated Papillary RAS-Like Thyroid Tumor (NEPRAS)

Introduction: The diagnosis of thyroid tumors arising from follicular cells that are encapsulated/well delimited and noninvasive is a challenge. When unequivocal nuclear alterations are present, the final diagnosis can range from noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) and classical/encapsulated papillary thyroid cancer (PTC), including aggressive subtypes, to poorly differentiated carcinoma. As proposed recently, the presence of papillae in the absence of exuberant nuclear alterations (nuclear score 2), given that the other criteria for NIFTP are met, may not be sufficient for the diagnosis of PTC. This condition is called “noninvasive encapsulated papillary RAS-like thyroid tumor” (NEPRAS), whose nature would be borderline and not malignant. Revising our cases of tumors > 1 cm that were diagnosed previously as PTC and that were encapsulated/well delimited and noninvasive, we found three cases of NEPRAS. We now revised our cases of tumors > 1 cm diagnosed previously as well-differentiated tumor of uncertain malignant potential (WDT-UMP) because the nuclear alterations were not considered to be sufficient for the diagnosis of PTC on that occasion. Case: In a 29-year-old euthyroid male patient with a single thyroid nodule whose fine-needle aspiration had revealed indeterminate cytology, a single tumor measuring 3.2 cm was reclassified from WDT-UMP to NEPRAS. For this diagnosis, the tumor met the following criteria: encapsulation or clear demarcation, no vascular or capsular invasion, presence of papillae, < 30% solid/trabecular/insular growth pattern, no tumor necrosis or high mitotic activity, and nuclear score 2. The BRAFV600E mutation was absent. The patient continues to show no signs of recurrence 7 years after lobectomy. Conclusion: Despite the presence of papillae, some tumors may be reclassified from malignant (encapsulated PTC) to borderline (NEPRAS). This proposal would result in a change of management, with the same implications as those seen for the change from noninvasive encapsulated follicular variant of PTC to NIFTP. References: Ohba K et al. Encapsulated Papillary Thyroid Tumor with Delicate Nuclear Changes and a KRAS Mutation as a Possible Novel Subtype of Borderline Tumor. J Pathol Transl Med. 2019;53:136-41 AND Rosario PW. Noninvasive encapsulated papillary RAS-like thyroid tumor (NEPRAS) or encapsulated papillary thyroid carcinoma (PTC). J Pathol Transl Med. 2020;54:263-4.

Quantitative Spectrofluorometric Assay Detecting Nuclear Condensation and Fragmentation in Intact Cells

At present, nuclear condensation and fragmentation have been estimated also using Hoechst probes in fluorescence microscopy and flow cytometry. However, none of the methods has used the Hoechst probes for quantitative spectrofluorometric assessment. Therefore, the aim of present study was to develop a spectrofluorometric assay for detection of nuclear condensation and fragmentation in intact cells. We used HepG2 and HK‑2 cells cultured in 96-well plates which were treated with potent apoptotic inducers (i.e. cisplatin, staurosporine, camptothecin) for 6-48 h. Then, the cells were incubated with Hoechst 33258 (2 µg/mL) and the increase of fluorescence after binding of the dye to DNA was measured. The developed spectrofluorometric assay was capable to detect nuclear changes caused by all tested apoptotic inducers. Then, we compared the outcomes of the spectrofluorometric assay with other methods detecting apoptosis (i.e. TUNEL, DNA ladder, caspase activity). We found that the developed assay provides results of same sensitivity as TUNEL assay but the advantages of the spectrofluorometric assay are fast processing, low-cost and high throughput. Because nuclear condensation and fragmentation can be typical markers of cell death, especially in apoptosis, we suppose that the spectrofluorometric assay could become a routinely used method for characterizing cell death processes.

NUCLEAR DEGENERATION AS A BY-PRODUCT OF AGING AND ENVIRONMENT

Background: The process of aging in the immediate toxic environment occurring in this Industrialized world is detrimental for human body. This is accelerating aging process and at the cellular level, the toxins present in environment are inducing chromosomal(micronuclei) and nuclear degenerative changes (karyorrhexis, karyolysis, pyknosis, condensed chromatin). The given study evaluates the effects of aging and environment on the chromosomal/nuclear degenerative changes, thus affecting the exfoliated cells collected from buccal mucosa. Methods: The sample included 86 healthy subjects divided into two groups according to age: 46 women aged above 60 years and 40 women of 20- 25 years of age. A questionnaire was prepared to retrieve all the data related to health and drug related history. Buccal smears were prepared and stained with both Papanicolaou and H&E stain. Hundred cells were counted from each slide to determine the number of micronuclei and other nuclear degenerative changes. Results: The number of micronuclei and other nuclear degenerative changes were signicantly higher among the elderly women (p<0.05) when compared with young women volunteers. Conclusion: Aging along with environmental factors appear to be detrimental in inducing mutagenic/ genotoxic effects at the cellular level. Cytological evaluation is clearly indicative of nuclear changes evident with aging.

Complete chloroplast genome sequencing and comparative analysis reveals changes to the chloroplast genome after allopolyploidization in Cucumis

Allopolyploids undergo ‘genomic shock’ leading to large genetic and epigenetic modifications. Previous studies mainly focused on nuclear changes, while little is known about the inheritance and changes of organelle genome in allopolyploidization. The synthetic allotetraploid Cucumis ×hytivus, which is generated via hybridization between C. hystrix and C. sativus, is a useful model system to study cytonuclear variation. Here, we report the chloroplast genome of allotetraploid C. ×hytivus and its diploid parents via sequencing and comparative analysis. The obtained chloroplast genomes ranged in length from 154,673 to 155,760 bp, and their gene contents, gene orders, and GC contents were similar to each other. Comparative genome analysis support chloroplast maternally inheritance. However, we identified 51 Indels and 292 SNPs genetic variation of chloroplast genome in allopolyploid C. ×hytivus relative to its female parent C. hystrix. Nine intergenic regions with rich variation were identified through comparative chloroplast genome analysis in the Cucumis subgenus. The phylogenetic network based on the chloroplast genome sequences clarified the evolution and taxonomic position of the synthetic allotetraploid C. ×hytivus. The results of this study provide us with an insight into the changes of organelle genome after allopolyploidization, and a new understanding of the cytonuclear evolution.

The Role of Histone Acetylation-/Methylation-Mediated Apoptotic Gene Regulation in Hepatocellular Carcinoma

Epigenetics, an inheritable phenomenon, which influences the expression of gene without altering the DNA sequence, offers a new perspective on the pathogenesis of hepatocellular carcinoma (HCC). Nonalcoholic steatohepatitis (NASH) is projected to account for a significant share of HCC incidence due to the growing prevalence of various metabolic disorders. One of the major molecular mechanisms involved in epigenetic regulation, post-translational histone modification seems to coordinate various aspects of NASH which will further progress to HCC. Mounting evidence suggests that the orchestrated events of cellular and nuclear changes during apoptosis can be regulated by histone modifications. This review focuses on the current advances in the study of acetylation-/methylation-mediated histone modification in apoptosis and the implication of these epigenetic regulations in HCC. The reversibility of epigenetic alterations and the agents that can target these alterations offers novel therapeutic approaches and strategies for drug development. Further molecular mechanistic studies are required to enhance information governing these epigenetic modulators, which will facilitate the design of more effective diagnosis and treatment options.

CaMKIIδC Drives Early Adaptive Ca 2+ Change and Late Eccentric Cardiac Hypertrophy

Rationale: CaMKII (Ca 2+ -Calmodulin dependent protein kinase) δC activation is implicated in pathological progression of heart failure (HF) and CaMKIIδC transgenic mice rapidly develop HF and arrhythmias. However, little is known about early spatio-temporal Ca 2+ handling and CaMKII activation in hypertrophy and HF. Objective: To measure time- and location-dependent activation of CaMKIIδC signaling in adult ventricular cardiomyocytes, during transaortic constriction (TAC) and in CaMKIIδC transgenic mice. Methods and Results: We used human tissue from nonfailing and HF hearts, 4 mouse lines: wild-type, KO (CaMKIIδ-knockout), CaMKIIδC transgenic in wild-type (TG), or KO background, and wild-type mice exposed to TAC. Confocal imaging and biochemistry revealed disproportional CaMKIIδC activation and accumulation in nuclear and perinuclear versus cytosolic regions at 5 days post-TAC. This CaMKIIδ activation caused a compensatory increase in sarcoplasmic reticulum Ca 2+ content, Ca 2+ transient amplitude, and [Ca 2+ ] decline rates, with reduced phospholamban expression, all of which were most prominent near and in the nucleus. These early adaptive effects in TAC were entirely mimicked in young CaMKIIδ TG mice (6–8 weeks) where no overt cardiac dysfunction was present. The (peri)nuclear CaMKII accumulation also correlated with enhanced HDAC4 (histone deacetylase) nuclear export, creating a microdomain for transcriptional regulation. At longer times both TAC and TG mice progressed to overt HF (at 45 days and 11–13 weeks, respectively), during which time the compensatory Ca 2+ transient effects reversed, but further increases in nuclear and time-averaged [Ca 2+ ] and CaMKII activation occurred. CaMKIIδ TG mice lacking δB exhibited more severe HF, eccentric myocyte growth, and nuclear changes. Patient HF samples also showed greatly increased CaMKIIδ expression, especially for CaMKIIδC in nuclear fractions. Conclusions: We conclude that in early TAC perinuclear CaMKIIδC activation promotes adaptive increases in myocyte Ca 2+ transients and nuclear transcriptional responses but that chronic progression of this nuclear Ca 2+ -CaMKIIδC axis contributes to eccentric hypertrophy and HF.

Daily Grape Juice Consumption Promotes Weight Loss, Improved Stability and Reduced the DNA Damage in the Elderly

The aim of this study was to determine the influence of chronic supplementation with grape juice (400ml), in modulating the anthropometric and biochemical parameters. Thirty-nine seniors participated and were evaluated at baseline after 30 days. The chronic consumption of grape juice reduced weight, BMI and waist circumference. Indeed, the consumption modulated biochemical parameters, decreased the total cholesterol levels, Low-density lipoprotein (LDL) cholesterol, Urea and GGT. In addition, the intake of juice improved the performance in the TUG test, and caused more stability in the elderly. The levels of protein oxidation declined and the antioxidant potential significantly increased as well as SOD and the ratio SOD / CAT. In contrast, levels reduced sulfhydryl groups to consumption. In nuclear changes there are a decrease in the frequency of MN and picnosis. In conclusion, grape juice could be an excellent option to improve quality of life in elderly.