Search

Remarkable progress has been made in the field of genome engineering after the discovery of CRISPR/Cas9 in 2012 by Jennifer Doudna and Emmanuelle Charpentier. Compared to any other gene-editing tools, CRISPR/Cas9 attracted the attention of the scientific community because of its simplicity, specificity, and multiplex editing possibilities for which the inventors were awarded the Nobel prize for chemistry in 2020. CRISPR/Cas9 allows targeted alteration of the genomic sequence, gene regulation, and epigenetic modifications using an RNA-guided site-specific endonuclease. Though the impact of CRISPR/Cas9 was undisputed, some of its limitations led to key modifications including the use of miniature-Cas proteins, Cas9 Retron precise Parallel Editing via homologY (CRISPEY), Cas-Clover, or development of alternative methods including retron-recombineering, Obligate Mobile Element Guided Activity(OMEGA), Fanzor, and Argonaute proteins. As cancer is caused by genetic and epigenetic alterations, gene-editing was found to be highly useful for knocking out oncogenes, editing mutations to regain the normal functioning of tumor suppressor genes, knock-out immune checkpoint blockade in CAR-T cells, producing ‘off-the-shelf’ CAR-T cells, identify novel tumorigenic genes and functional analysis of multiple pathways in cancer, etc. Advancements in nanoparticle-based delivery of guide-RNA and Cas9 complex to the human body further enhanced the potential of CRISPR/Cas9 for clinical translation. Several studies are reported for developing novel delivery methods to enhance the tumor-specific application of CRISPR/Cas9 for anticancer therapy. In this review, we discuss new developments in novel gene editing techniques and recent progress in nanoparticle-based CRISPR/Cas9 delivery specific to cancer applications.

Autologous dendritic cell (DC)-based immunotherapy is a cell-based advanced therapy medicinal product (ATMP) that was first introduced more than three decades ago. In the current study, our objective was to establish a harmonized protocol using two varied antigenic sources and a good manufacturing practice (GMP)-compliant, manual method for generating clinical-grade DCs at a limited-resource academic setting. After obtaining ethical committee-approved informed consent, the recruited patients underwent leukapheresis, and single-batch DC production was carried out. Using responder-independent flow cytometric assays as quality control (QC) criteria, we propose a differentiation and maturation index (DI and MI, respectively), calculated with the QC cut-off and actual scores of each batch for comparison. Changes during cryopreservation and personnel variation were assessed periodically for up to two to three years. Using our harmonized batch production protocol, the average DI was 1.39 and MI was 1.25. Allogenic responder proliferation was observed in all patients, while IFN-gamma secretion, evaluated using flow cytometry, was detected in 10/36 patients and significantly correlated with CD8+ T cell proliferation (p value-0.0002). Tracking the viability and phenotype of cryopreserved MDCs showed a >90% viability for up to three years, while a mature DC phenotype was retained for up to one year. Our results confirm that the manual/semi-automated protocol was simple, consistent, and cost-effective, without the requirement for expensive equipment and without compromising on the quality of the final product.

Abstract Breast cancer is the most common malignancy among women globally. Development of a reliable plasma biomarker panel might serve as a non-invasive and cost-effective means for population-based screening of the disease. Transcriptomic profiling of breast tumour, paired normal and apparently normal tissues, followed by validation of the shortlisted genes using TaqMan® Low density arrays and Quantitative real-time PCR was performed in South Asian women. Fifteen candidate protein markers and 3 candidate epigenetic markers were validated first in primary breast tumours and then in plasma samples of cases [N = 202 invasive, 16 DCIS] and controls [N = 203 healthy, 37 benign] using antibody array and methylation specific PCR. Diagnostic efficiency of single and combined markers was assessed. Combination of 6 protein markers (Adipsin, Leptin, Syndecan-1, Basic fibroblast growth factor, Interleukin 17B and Dickopff-3) resulted in 65% sensitivity and 80% specificity in detecting breast cancer. Multivariate diagnostic analysis of methylation status of SOSTDC1, DACT2, WIF1 showed 100% sensitivity and up to 91% specificity in discriminating BC from benign and controls. Hence, combination of SOSTDC1, DACT2 and WIF1 was effective in differentiating breast cancer [non-invasive and invasive] from benign diseases of the breast and healthy individuals and could help as a complementary diagnostic tool for breast cancer.

PURPOSEHereditary breast and ovarian cancer (HBOC) syndrome is primarily characterized by mutations in the BRCA1/2 genes. There are several barriers to the implementation of genetic testing and counseling in India that may affect clinical decisions. These consensus recommendations were therefore convened as a collaborative effort to improve testing and management of HBOC in India.DESIGNRecommendations were developed by a multidisciplinary group of experts from the Indian Society of Medical and Pediatric Oncology and some invited experts on the basis of graded evidence from the literature and using a formal Delphi process to help reach consensus. PubMed and Google Scholar databases were searched to source relevant articles.RESULTSThis consensus statement provides practical insight into identifying patients who should undergo genetic counseling and testing on the basis of assessments of family and ancestry and personal history of HBOC. It discusses the need and significance of genetic counselors and medical professionals who have the necessary expertise in genetic counseling and testing. Recommendations elucidate requirements of pretest counseling, including discussions on genetic variants of uncertain significance and risk reduction options. The group of experts recommended single-site mutation testing in families with a known mutation and next-generation sequencing coupled with multiplex ligation probe amplification for the detection of large genomic rearrangements for unknown mutations. Recommendations for surgical and lifestyle-related risk reduction approaches and management using poly (ADP-ribose) polymerase inhibitors are also detailed.CONCLUSIONWith rapid strides being made in the field of genetic testing/counseling in India, more oncologists are expected to include genetic testing/counseling as part of their clinical practice. These consensus recommendations are anticipated to help homogenize genetic testing and management of HBOC in India for improved patient care.

Breast cancer is the most common malignancy among women, with the highest incidence rate worldwide. Dysregulation of long noncoding RNAs during the preliminary stages of breast carcinogenesis is poorly understood. In this study, we performed RNA sequencing to identify long noncoding RNA expression profiles associated with early‐stage breast cancer. RNA sequencing was performed on six invasive ductal carcinoma (IDC) tissues along with paired normal tissue samples, seven ductal carcinoma in situ tissues, and five apparently normal breast tissues. We identified 375 differentially expressed lncRNAs (DElncRNAs) in IDC tissues compared to paired normal tissues. Antisense transcripts (~ 58%) were the largest subtype among DElncRNAs. About 20% of the 375 DElncRNAs were supported by typical split readings leveraging their detection confidence. Validation was performed in n = 52 IDC and paired normal tissue by qRT‐PCR for the identified targets (ADAMTS9‐AS2, EPB41L4A‐AS1, WDFY3‐AS2, RP11‐295M3.4, RP11‐161M6.2, RP11‐490M8.1, CTB‐92J24.3, and FAM83H‐AS1). We evaluated the prognostic significance of DElncRNAs based on TCGA datasets and report that overexpression of FAM83H‐AS1 was associated with patient poor survival. We confirmed that the downregulation of ADAMTS9‐AS2 in breast cancer was due to promoter hypermethylation through in vitro silencing experiments and pyrosequencing.

Cytosine methylation adjacent to adenine, thymine, and cytosine residues but not guanine of the DNA is distinctively known as non-CpG methylation. This CA/CT/CC methylation accounts for 15% of the total cytosine methylation and varies among different cell and tissue types. The abundance of CpG methylation has largely concealed the role of non-CpG methylation. Limitations in the early detection methods could not distinguish CpG methylation from non-CpG methylation. Recent advancements in enrichment strategies and high throughput sequencing technologies have enabled the detection of non-CpG methylation. This review discusses the advanced experimental and computational approaches to detect and describe the genomic distribution and function of non-CpG methylation. We present different approaches such as enzyme-based and antibody-based enrichment, which, when coupled, can also improve the sensitivity and specificity of non-CpG detection. We also describe the current bioinformatics pipelines and their specific application in computing and visualizing the imbalance of CpG and non-CpG methylation. Enrichment modes and the computational suites need to be further developed to ease the challenges of understanding the functional role of non-CpG methylation.

Abstract The EWS-FLI1 chimeric protein uniquely expressed in Ewing’s sarcoma has an obligate role in its aetiology. In our previous report we showed that ectopic expression of the DNA sequences form the junction region (a.a 251–280) can inhibit Ewing’s sarcoma cell growth. In the present report, we introduced a peptide (TAT/NLS/EWS-PEP) comprising of thirty amino acids spanning the junction in conjunction with HIV-1-trans-activating (TAT) and nuclear localization signal sequence (NLS). Peptide uptake and localization studies revealed presence of peptide in ~99% of transduced cells and in the nucleus. Peptide transfection induced cytotoxicity relative to untreated and TAT-NLS peptide treated Ewing’s sarcoma cells. The peptide inhibited clonogenicity, cell cycle, bromo-deoxy uridine (BrdU) uptake and invasion capacity of treated cells. The treatment also affected epithelial to mesenchymal transition (EMT) markers and EWS-FLI1 target gene expression levels. Co-immunoprecipitation experiments involving ectopically expressed full-length EWS-FLI1 protein and the peptide revealed an interaction. Additionally, we found that peptide interaction also occurs with the protein-GGAA microsatellite sequences complex known to contain EWS-FLI1. Further, in the pull-down assay, the peptide was found to interact with proteins known to potentially interact with EWS-FLI1. Based on these results we conclude that peptide could be applied in targeting EWS-FLI1 protein.

EGCG (Epigallocatechin-3-gallate) is the major active principle catechin found in green tea. Skepticism regarding the safety of consuming EGCG is gaining attention, despite the fact that it is widely being touted for its potential health benefits, including anti-cancer properties. The lack of scientific data on safe dose levels of pure EGCG is of concern, while EGCG has been commonly studied as a component of GTE (Green tea extract) and not as a single active constituent. This study has been carried out to estimate the maximum tolerated non-toxic dose of pure EGCG and to identify the treatment related risk factors. In a fourteen day consecutive treatment, two different administration modalities were compared, offering an improved [i.p (intraperitoneal)] and limited [p.o (oral)] bioavailability. A trend of dose and route dependant hepatotoxicity was observed particularly with i.p treatment and EGCG increased serum lipid profile in parallel to hepatotoxicity. Fourteen day tolerable dose of EGCG was established as 21.1 mg/kg for i.p and 67.8 mg/kg for p.o. We also observed that, EGCG induced effects by both treatment routes are reversible, subsequent to an observation period for further fourteen days after cessation of treatment. It was demonstrated that the severity of EGCG induced toxicity appears to be a function of dose, route of administration and period of treatment. Keywords: EGCG, Green tea, Serum lipids, Dose dependant toxicity, Route dependant toxicity, Liver toxicity, Dyslipidemia

Genetic variants located within the 12p13.33/RAD52 locus have been associated with lung squamous cell carcinoma (LUSC). Here, within 5,947 UADT cancers and 7,789 controls from 9 different studies, we found rs10849605, a common intronic variant in RAD52, to be also associated with upper aerodigestive tract (UADT) squamous cell carcinoma cases (OR = 1.09, 95% CI: 1.04-1.15, p = 6x10(-4)). We additionally identified rs10849605 as a RAD52 cis-eQTL inUADT(p = 1x10(-3)) and LUSC (p = 9x10(-4)) tumours, with the UADT/LUSC risk allele correlated with increased RAD52 expression levels. The 12p13.33 locus, encompassing rs10849605/RAD52, was identified as a significant somatic focal copy number amplification in UADT(n = 374, q-value = 0.075) and LUSC (n = 464, q-value = 0.007) tumors and correlated with higher RAD52 tumor expression levels (p = 6x10(-48) and p = 3x10(-29) in UADT and LUSC, respectively). In combination, these results implicate increased RAD52 expression in both genetic susceptibility and tumorigenesis of UADT and LUSC tumors.

Epigenetic modes of gene regulation are important for physiological conditions and its aberrant changes can lead to disease like cancer. 5-hydroxymethylcytosine (5hmC) is an oxidized form of 5-methylcytosine (5mC) catalyzed by Ten Eleven Translocation (TET) enzymes. 5hmC is considered to be a demethylation intermediate and is emerging as a stable and functional base modification. The global loss of 5hmC level is commonly observed in cancers and tumorigenic germline mutations in IDH, SDH and FH are found to be inhibiting TET activity. Although a global loss of 5hmC is characteristic in cancers, locus-specific 5hmC gain implicates selective gene expression control. The definitive role of 5hmC as a tumor suppressing or promoting modification can be deduced by identifying locus-specific 5hmC modification in different types of cancer. Determining the genes carrying 5hmC modifications and its selective variation will open up new therapeutic targets. This review outlines the role of global and locus-specific changes of 5hmC in cancers and the possible mechanisms underlying such changes. We have described major cellular factors that influence 5hmC levels and highlighted the significance of 5hmC in tumor micro environmental condition like hypoxia.

Breast cancer is one of the most common cancers in India as well as worldwide. Among Indian females, the age adjusted incidence rate of breast cancer is as high as 25.8 per 100,000 women and mortality rate of 12.7 per 100,000 women. Accumulating evidence highlights the potential role of long non-coding RNAs (lncRNAs) in breast cancer development. LncRNAs are emerging as important players in tumorigenesis as they are known to participate in various cancerous processes including proliferation, apoptosis, and invasion. LncRNAs are reported to be dysregulated in a number of cancers, demonstrating both oncogenic and tumor suppressive roles, thus suggesting their aberrant expression may be a substantial contributor in cancer development. However, their mechanism of action is poorly understood. Several studies have been published describing lncRNA expression profile of various breast cancer subtypes in different stages of cancer; however, their aberrant expression has not been systematically investigated in early stages. We have carried out expression profiling using deep sequencing of total RNAs to identify lncRNA expression profile in ductal carcinoma in situ and early stage breast cancers. We identified 103 differentially expressed (DE) lncRNAs of which 21 lncRNAs showed progressive pattern based on transcripts per million (TPM) in unmatched normal, adjacent normal, ductal carcinoma in situ, and stage 1 breast cancers. We identified several novel lncRNAs including RP11-295M3.4, LINC01614, RP11-527N22.1, RP11-126H7.4 and RAMP-AS1 that have not been reported in breast cancer before. These findings provide insights into lncRNA expression landscape in early stage breast cancers.

Genetic variants in nicotinic acetylcholine receptor and alcohol metabolism genes have been associated with propensity to smoke tobacco and drink alcohol, respectively, and also implicated in genetic susceptibility to head and neck cancer. In addition to smoking and alcohol, tobacco chewing is an important oral cancer risk factor in India. It is not known if these genetic variants influence propensity or oral cancer susceptibility in the context of this distinct etiology.We examined 639 oral and pharyngeal cancer cases and 791 controls from two case-control studies conducted in India. We investigated six variants known to influence nicotine addiction or alcohol metabolism, including rs16969968 (CHRNA5), rs578776 (CHRNA3), rs1229984 (ADH1B), rs698 (ADH1C), rs1573496 (ADH7), and rs4767364 (ALDH2).The CHRN variants were associated with the number of chewing events per day, including in those who chewed tobacco but never smoked (P = 0.003, P = 0.01 for rs16969968 and rs578776 respectively). Presence of the variant allele contributed to approximately 13% difference in chewing frequency compared to non-carriers. While no association was observed between rs16969968 and oral cancer risk (OR = 1.01, 95% CI = 0.83- 1.22), rs578776 was modestly associated with a 16% decreased risk of oral cancer (OR = 0.84, 95% CI = 0.72- 0.98). There was little evidence for association between polymorphisms in genes encoding alcohol metabolism and oral cancer in this population.The association between rs16969968 and number of chewing events implies that the effect on smoking propensity conferred by this gene variant extends to the use of smokeless tobacco.