An absolute approach to using whole exome DNA and RNA workflow for cancer biomarker testing.

Introduction: The concept of personalized medicine in cancer has emerged rapidly with the advancement of genome sequencing and the identification of clinically relevant variants that contribute to disease prognosis and facilitates targeted therapy options. In this study, we propose to validate a whole exome-based tumor molecular profiling for DNA and RNA from formalin-fixed paraffin-embedded (FFPE) tumor tissue. Methods: The study included 166 patients across 17 different cancer types. The scope of this study includes the identification of single-nucleotide variants (SNVs), insertions/deletions (INDELS), copy number alterations (CNAs), gene fusions, tumor mutational burden (TMB), and microsatellite instability (MSI). The assay yielded a mean read depth of 200×, with >80% of on-target reads and a mean uniformity of >90%. Clinical maturation of whole exome sequencing (WES) (DNA and RNA)- based assay was achieved by analytical and clinical validations for all the types of genomic alterations in multiple cancers. We here demonstrate a limit of detection (LOD) of 5% for SNVs and 10% for INDELS with 97.5% specificity, 100% sensitivity, and 100% reproducibility. Results: The results were >98% concordant with other orthogonal techniques and appeared to be more robust and comprehensive in detecting all the clinically relevant alterations. Our study demonstrates the clinical utility of the exome-based approach of comprehensive genomic profiling (CGP) for cancer patients at diagnosis and disease progression. Discussion: The assay provides a consolidated picture of tumor heterogeneity and prognostic and predictive biomarkers, thus helping in precision oncology practice. The primary intended use of WES (DNA+RNA) assay would be for patients with rare cancers as well as for patients with unknown primary tumors, and this category constitutes nearly 20-30% of all cancers. The WES approach may also help us understand the clonal evolution during disease progression to precisely plan the treatment in advanced stage disease.

Human satellite III long noncoding RNA imparts survival benefits to cancer cells.

Long noncoding RNAs (lncRNAs) are heterogeneous group of transcripts that lack coding potential and have essential roles in gene regulations. Recent days have seen an increasing association of noncoding RNAs with human diseases, especially cancers. One interesting group of noncoding RNAs strongly linked to cancers are heterochromatic repetitive Satellite RNAs. Satellite RNAs are transcribed from pericentromeric heterochromatic region of the human chromosomes. Satellite II RNA, most extensively studied, is upregulated in wide variety of epithelial cancer. Similarly, alpha satellite is over expressed in BRCA1-deficient tumors. Though much is known about alpha satellites and SatII repeats, little is known about Satellite III (SatIII) lncRNAs in human cancers. SatIII repeats, though transcriptionally silent in normal conditions is actively transcribed under condition of stress, mainly heat shock. In this study, we show that colon and breast cancer cells aberrantly transcribes SatIII, in a heat shock factor I (HSF1)-independent manner. Our study also reveals that, the overexpression of SatIII RNA favors cancer cell survival by overriding chemo drug-induced cell death. Interestingly, knockdown of SatIII sensitizes cells toward chemotherapeutic drugs. This sensitization is possibly mediated by restoration of p53 protein expression that facilitates cell death. Heat shock however helps SatIII to continue with its pro-cell survival function. Our results, therefore suggest SatIII to be an important regulator of human cancers. Induction of SatIII is not only a response to the oncogenic stress but also facilitates cancer progression by a distinct pathway that is different from heat stress pathway.

Long noncoding RNAs in the regulation of p53-mediated apoptosis in human cancers.

Long noncoding RNAs (lncRNAs) are widely known for their regulatory function in transcriptional and posttranscriptional processes. The involvement of such non-protein-coding RNAs in nuclear organization and chromatin remodeling is often associated with an increased risk of human malignancies. In cancer, lncRNAs either promote cell survival or may act as a growth suppressor, thus conferring a key regulatory function other than their established role in fundamental cellular processes. Interestingly, lncRNAs interfere with the stages of apoptosis and related pathways involving p53. Many of these molecules either regulate or are regulated by p53 while mounting oncogenic events. Consequently, they may confer both prosurvival or proapoptotic functions depending upon the tissue type. Since the mechanism of cell death is bypassed in many human cancers, it has emerged that the lncRNAs are either overexpressed or knocked down to sensitize cells to apoptotic stimuli. Nonetheless, the abundant expression of lncRNAs in tumor cells renders them suitable targets for anticancer therapies. Although the role of lncRNAs in the p53 network and apoptosis has been independently defined, their interplay in activating p53-target genes during cell cycle arrest remains unexplored. Thus, we have specifically reviewed the possible involvement of lncRNAs in the p53-mediated apoptosis of human cancer cells. In particular, we summarize the growing evidence from individual studies and analyze whether lncRNAs are essential to facilitate apoptosis in a p53-dependent manner. This may lead to the identification of p53-associated lncRNAs that are suitable therapeutic targets or diagnostic/prognostic markers.

Nuclear stress bodies: Interaction of its components in oncogenic regulation.

Oncogenesis involves continuous genetic alterations that lead to compromised cellular integrity and immortal cell fate. The cells remain under excessive stress due to endo- and exogenous influences. Human Satellite III long noncoding RNA (SatIII lncRNA) is a key regulator of the global cellular stress response, although its function is poorly explained in cancers. The principal regulator of cancer meshwork is tumor protein p53, which if altered may result in chemoresistance. The heat shock factor 1 (HSF1) being a common molecule between the oncogenic control and global cellular stress acts as an oncogene as well as transcribes SatIII upon heat shock. This prompted us to determine the structure of SatIII RNA and establish the association between SatIII-HSF1-p53. We determined the most stable structure of SatIII RNA with the least energy of - 115.7 kcal/mol. Also, we observed a possible interaction of p53 with SatIII and HSF1 using support vector machine (SVM) algorithm for predicting RNA-protein interaction (RPI). Further, we employ the STRING database to understand if p53 is an interacting component of the nuclear stress bodies (nSBs). A precise inference was drawn from molecular docking which confirmed the interaction of SatIII-HSF1-p53, where a mutated p53 resulted in an altered DNA-binding property with the SatIII molecule. This study being first of its kind infers p53 to be a possible integral component of the nSBs, which may regulate cellular stress response during cancer progression in the presence of HSF1 and SatIII. An extended research on the regulations of SatIII and p53 may open new avenues in the field of apoptosis in cancer and the early approach of molecular targeting.

Emerging roles of long non-coding RNAs in cancer.

Cancer is a physiological condition that has both the endogenous and exogenous influences on its progression. It originates from unusual cell growth, where the cells undergo massive genetic alterations, bypass the signaling machinery and compromise its genetic cohesion. Literature has well narrated the DNA damage studies including driver mutations that interfere with the treatment strategies. However, with evolving medical excellence, recent day studies are trying to unveil the contribution of RNAs in the progression of tumor malignancies. A number of non-coding RNAs have been identified as an active component in cancer genomics. This article aims to review the role of long non-coding RNAs in the spectra of cancers and its prognostic value as the biomarkers in molecular targeting with clinical utility and therapeutic beneficence.