Evolution and Impact of Subclonal Mutations in Papillary Thyroid Cancer.

Unlike many cancers, the pattern of tumor evolution in papillary thyroid cancer (PTC) and its potential role in relapse have not been elucidated. In this study, multi-region whole-exome sequencing (WES) was performed on early-stage PTC tumors (n = 257 tumor regions) from 79 individuals, including 17 who had developed relapse, to understand the temporal and spatial framework within which subclonal mutations catalyze tumor evolution and its potential clinical relevance. Paired primary-relapse tumor tissues were also available for a subset of individuals. The resulting catalog of variants was analyzed to explore evolutionary histories, define clonal and subclonal events, and assess the relationship between intra-tumor heterogeneity and relapse-free survival. The multi-region WES approach was key in correctly classifying subclonal mutations, 40% of which would have otherwise been erroneously considered clonal. We observed both linear and branching evolution patterns in our PTC cohort. A higher burden of subclonal mutations was significantly associated with increased risk of relapse. We conclude that relapse in PTC, while generally rare, does not follow a predictable evolutionary path and that subclonal mutation burden may serve as a prognostic factor. Larger studies utilizing multi-region sequencing in relapsed PTC case subjects with matching primary tissues are needed to confirm these observations.

Telomerase reverse transcriptase promoter mutations in cancers derived from multiple organ sites among middle eastern population.

Sanger Sequencing and immunohistochemistry was employed to investigate the TERT promoter mutations and TERT protein expression with their association to clinicopathological characteristics in over 2200 samples of Middle Eastern origin from 13 different types of cancers. The TERT promoter mutations were most frequently present in bladder cancer (68.6%), followed by central nervous system tumors (28.7%), thyroid cancer (15.4%), prostate cancer (9.3%), endometrial carcinoma (3.7%), rhabdomyosarcoma (1.4%), colorectal cancer (1%), epithelial ovarian carcinoma (0.7%) and breast cancer (0.7%). No mutations were observed in other types of cancers. In bladder cancer, we found significant inverse association with metastasis and a trend to good survival in patients with TERT mutations. In gliomas, TERT promoter mutations predicted poor prognosis. In thyroid cancer, high frequency of TERT mutation was observed in poorly differentiated carcinoma. In addition, TERT promoter mutations were associated with aggressive markers and poor outcome in follicular thyroid carcinomas.

Genetic heterogeneity and evolutionary history of high-grade ovarian carcinoma and matched distant metastases.

BACKGROUND: High-grade serous ovarian carcinoma (HGSOC) is the most frequent type of ovarian carcinoma, associated with poor clinical outcome and metastatic disease. Although metastatic processes are becoming more understandable, the genomic landscape and metastatic progression in HGSOC has not been elucidated. METHODS: Multi-region whole-exome sequencing was performed on HGSOC primary tumours and their metastases (n = 33 tumour regions) from six patients. The resulting somatic variants were analysed to delineate tumour evolution and metastatic dissemination, and to compare the repertoire of events between primary HGSOC and metastasis. RESULTS: All cases presented branching evolution patterns in primary HGSOC, with three cases further showing parallel evolution in which different mutations on separate branches of a phylogenetic tree converge on the same gene. Furthermore, linear metastatic progression was observed in 67% of cases with late dissemination, in which the metastatic tumour mostly acquires the same mutational process active in primary tumour, and parallel metastatic progression, with early dissemination in the remaining 33.3% of cases. Metastatic-specific SNVs were further confirmed as late dissemination events. We also found the involvement of metastatic-specific driver events in the Wnt/ß-catenin pathway, and identified potential clinically actionable events in individual patients of the metastatic HGSOC cohort. CONCLUSIONS: This study provides deeper insights into clonal evolution and mutational processes that can pave the way to new therapeutic targets.

Germline POLE and POLD1 proofreading domain mutations in endometrial carcinoma from Middle Eastern region.

BACKGROUND: Endometrial carcinoma (EC) accounts for 5.8% of all cancers in Saudi females. Although most ECs are sporadic, 2-5% tend to be familial, being associated with Lynch syndrome and Cowden syndrome. In this study, we attempted to uncover the frequency, spectrum and phenotype of germline mutations in the proofreading domain of POLE and POLD1 genes in a large cohort of ECs from Middle Eastern region. METHODS: We performed Capture sequencing and Sanger sequencing to screen for proofreading domains of POLE and POLD1 genes in 432 EC cases, followed by evaluation of protein expression using immunohistochemistry. Variant interpretation was performed using PolyPhen-2, MutationAssessor, SIFT, CADD and Mutation Taster. RESULTS: In our cohort, four mutations (0.93%) were identified in 432 EC cases, two each in POLE and POLD1 proofreading domains. Furthermore, low expression of POLE and POLD1 was noted in 41.1% (170/1414) and 59.9% (251/419) of cases, respectively. Both the cases harboring POLE mutation showed high nuclear expression of POLE protein, whereas, of the two POLD1 mutant cases, one case showed high expression and another case showed low expression of POLD1 protein. CONCLUSIONS: Our study shows that germline mutations in POLE and POLD1 proofreading region are a rare cause of EC in Middle Eastern population. However, it is still feasible to screen multiple cancer related genes in EC patients from Middle Eastern region using multigene panels including POLE and POLD1.

Multivapor-Responsive Controlled Actuation of Starch-Based Soft Actuators.

Multivapor-responsive biocompatible soft actuators have immense potential for applications in soft robotics and medical technology. We report fast, fully reversible, and multivapor-responsive controlled actuation of a pure cassava-starch-based film. Notably, this starch-based actuator sustains its actuated state for over 60 min with a continuous supply of water vapor. The durability of the film and repeatability of the actuation performance have been established upon subjecting the film to more than 1400 actuation cycles in the presence of water vapor. The starch-based actuators exhibit intriguing antagonistic actuation characteristics when exposed to different solvent vapors. In particular, they bend upward in response to water vapor and downward when exposed to ethanol vapor. This fascinating behavior opens up new possibilities for controlling the magnitude and direction of actuation by manipulating the ratio of water to ethanol in the binary solution. Additionally, the control of the bending axis of the starch-based actuator, when exposed to water vapor, is achieved by imprinting-orientated patterns on the surface of the starch film. The effect of microstructure, postsynthesis annealing, and pH of the starch solution on the actuation performance of the starch film is studied in detail. Our starch-based actuator can lift 10 times its own weight upon exposure to ethanol vapor. It can generate force ∼4.2 mN upon exposure to water vapor. To illustrate the vast potential of our cassava-starch-based actuators, we have showcased various proof-of-concept applications, ranging from biomimicry to crawling robots, locomotion near perspiring human skin, bidirectional electric switches, ventilation in the presence of toxic vapors, and smart lifting systems. These applications significantly broaden the practical uses of these starch-based actuators in the field of soft robotics.

APOBEC SBS13 Mutational Signature-A Novel Predictor of Radioactive Iodine Refractory Papillary Thyroid Carcinoma.

Standard surgery followed by radioactive iodine (131I, RAI) therapy are not curative for 5−20% of papillary thyroid carcinoma (PTC) patients with RAI refractory disease. Early predictors indicating therapeutic response to RAI therapy in PTC are yet to be elucidated. Whole-exome sequencing was performed (at median depth 198x) on 66 RAI-refractory and 92 RAI-avid PTCs with patient-matched germline. RAI-refractory tumors were significantly associated with distinct aggressive clinicopathological features, including positive surgical margins (p = 0.016) and the presence of lymph node metastases at primary diagnosis (p = 0.012); higher nonsilent tumor mutation burden (p = 0.011); TERT promoter (TERTp) mutation (p < 0.0001); and the enrichment of the APOBEC-related single-base substitution (SBS) COSMIC mutational signatures 2 (p = 0.030) and 13 (p < 0.001). Notably, SBS13 (odds ratio [OR] 30.4, 95% confidence intervals [CI] 1.43−647.22) and TERTp mutation (OR 41.3, 95% CI 4.35−391.60) were revealed to be independent predictors of RAI refractoriness in PTC (p = 0.029 and 0.001, respectively). Although SBS13 and TERTp mutations alone highly predicted RAI refractoriness, when combined, they significantly increased the likelihood of predicting RAI refractoriness in PTC. This study highlights the APOBEC SBS13 mutational signature as a novel independent predictor of RAI refractoriness in a distinct subgroup of PTC.

Prognostic Value and Function of KLF5 in Papillary Thyroid Cancer.

The Krüppel-like factor 5 (KLF5), a zinc-finger transcriptional factor, is highly expressed in several solid tumors, but its role in PTC remains unclear. We investigated the expression of KLF5 protein in a large cohort of PTC patient samples and explored its functional role and mechanism in PTC cell lines in vitro and in vivo. KLF5 overexpression was observed in 65.1% of all PTC cases and it was significantly associated with aggressive clinico-pathological parameters and poor outcome. Given the significant association between KLF5 and HIF-1α overexpression in PTC patients, we investigated the functional correlation between KLF5 and HIF-1α in PTC cells. Indeed, the analysis revealed the co-immunoprecipitation of KLF5 with HIF-1α in PTC cells. We also identified KLF5-binding sites in the HIF-1α promoter that specifically bound to KLF5 protein. Mechanistically, KLF5 promoted PTC cell growth, invasion, migration, and angiogenesis, while KLF5 downregulation via specific inhibitor or siRNA reverses its action in vitro. Importantly, the silencing of KLF5 decreases the self-renewal ability of spheroids generated from PTC cells. In addition, the depletion of KLF5 reduces PTC xenograft growth in vivo. These findings suggest KLF5 can be a possible new molecular therapeutic target for a subset of PTC.

Krupple-Like Factor 5 is a Potential Therapeutic Target and Prognostic Marker in Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy. Despite current therapeutic and surgical options, advanced EOC shows poor prognosis. Identifying novel molecular therapeutic targets is highly needed in the management of EOC. Krupple-like factor 5 (KLF5), a zinc-finger transcriptional factor, is highly expressed in a variety of cancer types. However, its role and expression in EOC is not fully illustrated. Immunohistochemical analysis was performed to assess KLF5 protein expression in 425 primary EOC samples using tissue microarray. We also addressed the function of KLF5 in EOC and its interaction with signal transducer and activator of transcription 3 (STAT3) signaling pathway. We found that KLF5 overexpressed in 53% (229/425) of EOC samples, and is associated with aggressive markers. Forced expression of KLF5 enhanced cell growth in low expressing EOC cell line, MDAH2774. Conversely, knockdown of KLF5 reduced cell growth, migration, invasion and progression of epithelial to mesenchymal transition in KLF5 expressing cell lines, OVISE and OVSAHO. Importantly, silencing of KLF5 decreased the self-renewal ability of spheroids generated from OVISE and OVSAHO cell lines. In addition, downregulation of KLF5 potentiated the effect of cisplatin to induce apoptosis in these cell lines. These data reveals the pro-tumorigenic role of KLF5 in EOC and uncover its role in activation of STAT3 signaling pathway, suggesting the importance of KLF5 as a potential therapeutic target for EOC therapy.

Whole-Exome Sequencing of Matched Primary and Metastatic Papillary Thyroid Cancer.

Background: Distant metastasis is a rare occurrence in thyroid cancer, and it can be associated with poor prognosis. The genomic repertoires of various solid malignancies have previously been reported but remain underexplored in metastatic papillary thyroid cancer (PTC). Furthermore, whether distant metastases harbor distinct genetic alterations beyond those observed in primary tumors is unknown. Methods: We performed whole-exome sequencing on 14 matched distant metastases, primary PTC tumors, and normal tissues. Point mutations, copy number alterations, cancer cell fractions, and mutational signatures were defined using the state-of-the-art bioinformatics methods. All likely deleterious variants were validated by orthogonal methods. Results: Genomic differences were observed between primary and distant metastatic deposits, with a median of 62% (range 21-92%) of somatic mutations detected in metastatic tissues, but absent from the corresponding primary tumor sample. Mutations in known driver genes including BRAF, NRAS, and HRAS were shared and preferentially clonal in both sites. However, likely deleterious variants affecting DNA methylation and transcriptional repression signaling genes including SIN3A, RBBP1, and CHD4 were found to be restricted in the metastatic lesions. Moreover, a mutational signature shift was observed between the mutations that are specific or enriched in the metastatic and primary lesions. Conclusions: Primary PTC and distant metastases differ in their range of somatic alterations. Genomic analysis of distant metastases provides an opportunity to identify potentially clinically informative alterations not detected in primary tumors, which might influence decisions for personalized therapy in PTC patients with distant metastasis.

Clonal Evolution and Timing of Metastatic Colorectal Cancer.

Colorectal cancer (CRC) is the third most frequently diagnosed cancer worldwide, where ~50% of patients develop metastasis, despite current improved management. Genomic characterisation of metastatic CRC, and elucidating the effects of therapy on the metastatic process, are essential to help guide precision medicine. Multi-region whole-exome sequencing was performed on 191 sampled tumour regions of patient-matched therapy-naïve and treated CRC primary tumours (n = 92 tumour regions) and metastases (n = 99 tumour regions), in 30 patients. Somatic variants were analysed to define the origin, composition, and timing of seeding in the metastatic progression of therapy-naïve and treated metastatic CRC. High concordance, with few genomic differences, was observed between primary CRC and metastases. Most cases supported a late dissemination model, via either monoclonal or polyclonal seeding. Polyclonal seeding appeared more common in therapy-naïve metastases than in treated metastases. Whereby, treatment prompted for the selection of distinct resistant clones, through monoclonal seeding to distant metastatic sites. Overall, this study reinforces the importance of early clinical detection and surgical excision of the CRC tumour, whilst further highlighting the clinical challenges for metastatic CRC with increased intratumour heterogeneity (either due to early dissemination or polyclonal metastatic spread) and the underlying risk of future therapeutic resistance in treated patients.