Genome Engineering as a Therapeutic Approach in Cancer Therapy: A Comprehensive Review.

Cancer is one of the foremost causes of mortality. The human genome remains stable over time. However, human activities and environmental factors have the power to influence the prevalence of certain types of mutations. This goes to the excessive progress of xenobiotics and industrial development that is expanding the territory for cancers to develop. The mechanisms involved in immune responses against cancer are widely studied. Genome editing has changed the genome-based immunotherapy process in the human body and has opened a new era for cancer treatment. In this review, recent cancer immunotherapies and the use of genome engineering technology are largely focused on.

T-cell lymphocytes' aging clock: telomeres, telomerase and aging.

Aging is the decline of physiological capabilities required for life maintenance and reproduction over time. The human immune cells, including T-cells lymphocytes, undergo dramatic aging-related changes, including those related to telomeres and telomerase. It was demonstrated that telomeres and telomerase play crucial roles in T-cell differentiation, aging, and diseases, including a well-documented link between short telomeres and telomerase activation demonstrated in several T-cells malignancies. Herein, we provide a comprehensive review of the literature regarding T-cells' telomeres and telomerase in health and age related-diseases.

Genetic markers of chronic lymphocytic leukemia: a retrospective study of 312 patients from a reference center in Lebanon.

Aim: Chronic lymphocytic leukemia (CLL) is a highly heterogenous hemopathy. Genetic stratification of CLL patients has important prognostic and therapeutic values - mainly immunoglobulin heavy chain variable region gene (IGHV) mutational status and the presence of cytogenetic abnormalities. The genetics of CLL in Lebanon is scarcely described in the literature. Patients & methods: In this work, we studied the genetic biomarkers of 312 Lebanese CLL patients. Results: Prominent IGHV genes were IGHV4-34, IGHV1-69 and IGHV3-30; and CLL #1 and #5 presented major subsets. Some similarities as well as major differences were highlighted when comparing our data with previously published data. Conclusion: The distribution of IGHV alleles in our series differed from previously described distributions, suggesting involvement of antigenic selection and regional variables in CLL pathogenesis.

A homozygous missense variant in PTPN2 with early-onset Crohn's disease, growth failure and dysmorphic features in an infant: a case report.

Crohn's disease (CD) is a chronic idiopathic inflammatory bowel condition that can affect any part of the gastrointestinal tract. Several hundred candidate loci or genes including PTPN2 have been reportedly associated with CD. A whole-exome sequencing (WES) was conducted in a 9-year-old Lebanese girl with a CD onset at 13 months and in both her asymptomatic parents. The analysis detected an extremely rare homozygous variant in PTPN2: c.359C>T, p.(Ser120Leu) in the patient, while both her parents were heterozygous. This variant, located in the protein tyrosine phosphatase (PTP) domain within a highly conserved amino acid, is classified as VUS according to the American College of Medical Genetics (ACMG) criteria. To evaluate the hypothetical functional consequences of the identified variant, a quantitative expression analysis of PTPN2 was performed in blood tissues of the patient, her parents, and two healthy controls. PTPN2 expression was not noted in the patient compared to her parents and the normal controls, suggesting a functional PTPN2 impairment caused by c.359C>T. This variant c.359C>T, p.(Ser120Leu) in PTPN2 has never been previously described in the literature. Our report suggests an association of PTPN2: c.359C>T with early-onset CD.

Mesenchymal stem cells-derived secretome and extracellular vesicles: perspective and challenges in cancer therapy and clinical applications.

Stem cell-based therapies have been foreshowed as a promising therapeutic approach for the treatment of several diseases. However, in the cancer context, results obtained from clinical studies were found to be quite limited. Deeply implicated in inflammatory cues, Mesenchymal, Neural, and Embryonic Stem Cells have mainly been used in clinical trials as a vehicle to deliver and stimulate signals in tumors niche. Although these stem cells have shown some therapeutical promises, they still face several challenges, including their isolation, immunosuppression potential, and tumorigenicity. In addition, regulatory and ethical concerns limit their use in several countries. Mesenchymal stem cells (MSC) have emerged as a gold standard adult stem cell medicine tool due to their distinctive characteristics, such as self-renewal and potency to differentiate into numerous cell types with lower ethical restrictions. Secreted extracellular vesicles (EVs), secretomes, and exosomes play a crucial role in mediating cell-to-cell communication to maintain physiological homeostasis and influence pathogenesis. Due to their low immunogenicity, biodegradability, low toxicity, and ability to transfer bioactive cargoes across biological barriers, EVs and exosomes were considered an alternative to stem cell therapy through their immunological features. MSCs-derived EVs, exosomes, and secretomes showed regenerative, anti-inflammatory, and immunomodulation properties while treating human diseases. In this review, we provide an overview of the paradigm of MSCs derived exosomes, secretome, and EVs cell-free-based therapies, we will focus on MSCs-derived components in anti-cancer treatment with decreased risk of immunogenicity and toxicity. Astute exploration of MSCs may lead to a new opportunity for efficient therapy for patients with cancer.

Spotlight on hTERT Complex Regulation in Cutaneous T-Cell Lymphomas.

As a major cancer hallmark, there is a sustained interest in understanding the telomerase contribution to carcinogenesis in order to therapeutically target this enzyme. This is particularly relevant in primary cutaneous T-cell lymphomas (CTCL), a malignancy showing telomerase dysregulation with few investigative data available. In CTCL, we examined the mechanisms involved in telomerase transcriptional activation and activity regulation. We analyzed 94 CTCL patients from a Franco-Portuguese cohort, as well as 8 cell lines, in comparison to 101 healthy controls. Our results showed that not only polymorphisms (SNPs) located at the promoter of human telomerase reverse transcriptase (hTERT) gene (rs2735940 and rs2853672) but also an SNP located within the coding region (rs2853676) could influence CTCL occurrence. Furthermore, our results sustained that the post-transcriptional regulation of hTERT contributes to CTCL lymphomagenesis. Indeed, CTCL cells present a different pattern of hTERT spliced transcripts distribution from the controls, mostly marked by an increase in the hTERT ß+ variants proportion. This increase seems to be associated with CTCL development and progression. Through hTERT splicing transcriptome modulation with shRNAs, we observed that the decrease in the α-ß+ transcript induced a decrease in the cell proliferation and tumorigenic capacities of T-MF cells in vitro. Taken together, our data highlight the major role of post-transcriptional mechanisms regulating telomerase non canonical functions in CTCL and suggest a new potential role for the α-ß+ hTERT transcript variant.

Telomeric Repeat-Containing RNA (TERRA): A Review of the Literature and First Assessment in Cutaneous T-Cell Lymphomas.

Telomeric Repeat-containing RNA (TERRA) are long non-coding RNAs transcribed from telomeric DNA sequences from multiple chromosome ends. Major research efforts have been made to understand TERRA roles and functions in several physiological and pathological processes. We summarize herein available data regarding TERRA's roles in human cells and we report the first investigation in cutaneous T-cells lymphomas (CTCL) using real-time PCR. Among the TERRA analysed, our data suggest a particular role for TERRA 16p downregulation and TERRA 11q upregulation in CTCL lymphomagenesis.

Exploring hTERT promoter methylation in cutaneous T-cell lymphomas.

Cutaneous T-cell lymphomas (CTCLs) are telomerase-positive tumors expressing hTERT, although neither gene rearrangement/amplification nor promoter hotspot mutations could explain the hTERT re-expression. As the hTERT promoter is rich in CpG, we investigated the contribution of epigenetic mechanisms in its re-expression. We analyzed hTERT promoter methylation status in CTCL cells compared with healthy cells. Gene-specific methylation analyses revealed a common methylation pattern exclusively in tumor cells. This methylation pattern encompassed a hypermethylated distal region from -650 to -150 bp and a hypomethylated proximal region from -150 to +150 bp. Interestingly, the hypermethylated region matches with the recently named TERT hypermethylated oncogenic region (THOR). THOR has been associated with telomerase reactivation in many cancers, but it has so far not been reported in cutaneous lymphomas. Additionally, we assessed the effect of THOR on two histone deacetylase inhibitors (HDACi), romidepsin and vorinostat, both approved for CTCL treatment and a DNA methyltransferase inhibitor (DNMTi) 5-azacytidine, unapproved for CTCL. Contrary to our expectations, the findings reported herein revealed that THOR methylation is relatively stable under these epigenetic drugs' pressure, whereas these drugs reduced the hTERT gene expression.

Pediatric M5 acute myeloid leukemia with MLL-SEPT6 fusion and a favorable outcome.

Acute myeloid leukemia (AML) patients with MLL-SEPT6 fusion represent a small subset of AML. The uncommon MLL-SEPT6 rearrangement results from t(X;11) or other variants like ins(X;11), and it is usually associated with complex cytogenetic abnormalities. We herein report a case of AML-M5-infant with ins(X;11)(q24;q23q13) and MLL-SEPT6. The one-year-old boy presented with leukocytosis, anemia and thrombocytopenia. He had a favorable response to chemotherapy according to ELAM02protocol and is currently in complete remission. We here, highlight the occurrence of MLL-SEPT6 as the sole abnormality in a pediatric-AML-M5 case, discuss the prognostic implication of this genetic variant, while reviewing previously reported AML-MLL-SEPT6 cases.

Targeting Epigenetic Modifiers Can Reduce the Clonogenic Capacities of Sézary Cells.

Sézary syndrome (SS) is an aggressive leukemic variant of cutaneous T-cell lymphomas (CTCL) in which the human Telomerase Reverse Transcriptase (hTERT) gene is re-expressed. Current available treatments do not provide long-term response. We previously reported that Histone deacetylase inhibitors (HDACi, romidespin and vorinostat) and a DNA methyltransferase inhibitor (DNMTi, 5-azacytidine) can reduce hTERT expression without altering the methylation level of hTERT promoter. Romidepsin and vorinostat are approved for CTCL treatment, while 5-azacytidine is approved for the treatment of several hematological disorders, but not for CTCL. Here, using the soft agar assay, we analyzed the functional effect of the aforementioned epidrugs on the clonogenic capacities of Sézary cells. Our data revealed that, besides hTERT downregulation, epidrugs' pressure reduced the proliferative and the tumor formation capacities in Sézary cells in vitro.

An unusual case of chronic lymphocytic leukemia with trisomy 12 and t(14;18) and a favorable response to ibrutinib.

Chronic lymphocytic leukemia (CLL) is the most common type of adult leukemia. Chromosomal abnormalities are reported to play important roles in CLL pathogenesis and evolution, including deletions of 11q, 13q, 17p, and trisomy12, that are frequently observed and have a known prognostic value. Furthermore, the mutational status of the IGHV gene was reported as an independent prognostic marker in CLL impacting the choice of therapy. We herein, report an unusual presentation of a Lebanese CLL patient with two cytogenetic abnormalities: trisomy 12 and t(14;18)(q32;q21), along with an unmutated IGHV, displaying a favorable response to ibrutinib with a maintained complete remission.

Diagnosis and treatment of lymphomas in the era of epigenetics.

Lymphomas represent a heterogeneous group of cancers characterized by clonal lymphoproliferation. Over the past decades, frequent epigenetic dysregulations have been identified in hematologic malignancies including lymphomas. Many of these impairments occur in genes with established roles and well-known functions in the regulation and maintenance of the epigenome. In hematopoietic cells, these dysfunctions can result in abnormal DNA methylation, erroneous chromatin state and/or altered miRNA expression, affecting many different cellular functions. Nowadays, it is evident that epigenetic dysregulations in lymphoid neoplasms are mainly caused by genetic alterations in genes encoding for enzymes responsible for histone or chromatin modifications. We summarize herein the recent epigenetic modifiers findings in lymphomas. We focus also on the most commonly mutated epigenetic regulators and emphasize on actual epigenetic therapies.

Whole-exome and whole-genome sequencing in chronic lymphocytic leukemia: new biomarkers to target.

Many biomarkers indicate prognosis in chronic lymphocytic leukemia; such as fluorescence in situ hybridization testing: 17p or 11q deletions have a worse prognosis than trisomy 12, 13q deletion or normal result, or the mutational status of the immunoglobulin heavy chain (IGHV): unmutated IGHV have a worse prognosis than mutated IGHV. Recently, many gene mutations (TP53, NOTCH1 etc.,) have been linked to a worse prognosis. With the new era of high-throughput sequencing, it has become easier to study gene mutations and their implication in predicting prognosis. In this review, we aim to review all the studies that performed whole-exome sequencing or whole-genome sequencing on chronic lymphocytic leukemia cells and explore the implication of various genes in disease prognosis.

A rare case of acute myeloid leukemia with t(12;19)(q13;q13).

Acute myeloid leukemia (AML) is characterized by chromosomal abnormalities affecting both prognosis and course of treatment. While most AML patients have well described chromosomal aberrations, around 10% present with rare chromosomal abnormalities. We herein, report a rare balanced translocation t(12;19)(q13;q13) in a 66-year old M5-AML patient identified by Conventional cytogenetic analysis and confirmed by SNP array. We suggest that t(12;19) as a sole chromosomal abnormality could be associated with a poor prognosis. Further studies are needed to understand the molecular basis of this translocation in AML.

Reliable blood cancer cells' telomere length evaluation by qPCR.

BACKGROUND: Telomere shortening is linked to a range of different human diseases, hence reliable measurement methods are needed to uncover such associations. Among the plethora of telomere length measurement methods, qPCR is reported as easy to conduct and a cost-effective approach to study samples with low DNA amounts. METHODS: Cancer cells' telomere length was evaluated by relative and absolute qPCR methods. RESULTS: Robust and reproducible telomere length measurements were optimized taking into account a careful reference gene selection and by knowing the cancer cells ploidy. qPCR data were compared to "gold standard" measurement from terminal restriction fragment (TRF). CONCLUSIONS: Our study provides guidance and recommendations for accurate telomere length measurement by qPCR in cancer cells, taking advantage of our expertise in telomere homeostasis investigation in primary cutaneous T-cell lymphomas. Furthermore, our data emphasize the requirement of samples with both, high DNA quality and high tumor cells representation.

First molecular study in Lebanese patients with Cockayne syndrome and report of a novel mutation in ERCC8 gene.

BACKGROUND: Cockayne Syndrome (CS) is a rare autosomal recessive disorder characterized by neurological and sensorial impairment, dwarfism, microcephaly and photosensitivity. CS is caused by mutations in ERCC6 (CSB) or ERCC8 (CSA) genes. METHODS: Three patients with CS were referred to the Medical Genetics Unit of Saint Joseph University. Sanger sequencing of both ERCC8 and ERCC6 genes was performed: ERCC8 was tested in all patients while ERCC6 in one of them. RESULTS: Sequencing led to the identification of three homozygous mutations, two in ERCC8 (p.Y322* and c.843 + 1G > C) and one in ERCC6 (p.R670W). All mutations were previously reported as pathogenic except for the c.843 + 1G > C splice site mutation in ERCC8 which is novel. CONCLUSIONS: Molecular diagnosis was established in all patients included in our study. A genotype-phenotype correlation is discussed and a link, between mutations and some specific religious communities in Lebanon, is suggested.

Untreated chronic lymphocytic leukemia in Lebanese patients: an observational study using standard karyotyping and FISH.

AIM: We aimed to understand the biology of chronic lymphocytic leukemia (CLL) patients in Lebanon. MATERIALS & METHODS: We applied conventional cytogenetic and FISH studies on Lebanese patients diagnosed with CLL and undergoing a watch and wait approach. RESULTS: Our study disclosed 53.6% of patients with aberrant karyotypes among which 26.7% were complex karyotypes. Genetic aberrations included del(13q14) 46.4%, 14q32 translocation in 25%, trisomy 12 in 14.3%, del(17p13) and del(11q22) in 7.1% each. The deletion of 6q21/6q23 was not found in any of our patients. CONCLUSION: The higher prevalence of del(13q14) as a sole abnormality could be the primary event in inducing CLL. The del(17p13) and del(11q22) followed as potential drivers for progression in CLL patients with a watch and wait approach.