Genetic correction of x-linked chronic granulomatous disease with novel foamy virus vectors

Objective: The X-linked form of chronic granulomatous disease (X-CGD) results from mutations in the CYBB gene encoding gp91phox, the larger subunit of the oxidase flavocytochrome b558. Affected individuals suffer from recurrent life-threatening infections due to impaired superoxide production by the NADPH oxidase in phagocytes. Novel foamy virus (FV) vectors expressing the human codon-optimized gp91phox were evaluated for the genetic correction of the disease in the X-CGD cell line and in X-CGD mouse model.Materials and Methods: The vectors were evaluated in vitro, in the human X-CGD PLB-985 cell line and in the X-CGD BM Lin-cells. Transplantation of transduced Lin-cells was performed in X-CGD mice after busulfan conditioning. Real time PCR was used for chimerism and vector copy number determination. Restoration of NADPH oxidase production was assessed by the NBT and the DHR assays.Results: High and stable gp91phox expression, as well as reconstitution of NADPH activity, was achieved in the human X-CGD PLB-985 cell line and in primary murine X-CGD hematopoietic stem cells (HSCs) ex vivo. Transplantation of transduced bone marrow HSCs in the murine model of X-CGD, even with low MOI, reconstituted the levels of oxidase-producing neutrophils and provided enzymatic activity that reached 70% of normal.Conclusion: FV vectors expressing the human gp91phox transgene constitute potential candidates for the gene therapy of CGD since they combine lack of pathogenicity with efficacy even at low MOI

Olaparib και σχετιζόμενοι μοριακοί βιοδείκτες στον καρκίνο του μαστού - Συστηματική Ανασκόπηση

Η ανάπτυξη νέων τεχνολογιών και η ανεύρεση νέων βιοδεικτών έχουν μεταφέρει την εξατομικευμένη και στοχευμένη θεραπεία στην κλινική πράξη. Η αναζήτηση βιοδεικτών και γονιδίων στόχων, είναι κρίσιμης σημασίας στην επιλογή των ασθενών που θα αποκριθούν καλύτερα και θα έχουν το βέλτιστο κλινικό όφελος. Η συνθετική θνησιμότητα δοκιμάστηκε μέσω των αναστολέων PARP σε συμπαγείς όγκους με ικανοποιητικά και ελπιδοφόρα αποτελέσματα. Ο αναστολέας PARP, Olaparib, εγκρίθηκε πρόσφατα από τον FDA, για τη θεραπεία του καρκίνου των ωοθηκών, του μαστού και του προστάτη υπό συγκεκριμένες προϋποθέσεις με την πιο βασική να είναι η παρουσία γαμετικών μεταλλαγών στα γονίδια BRCA1/2. Στην παρούσα συστηματική ανασκόπηση, ανακτήθηκαν 26 κλινικές μελέτες της περιόδου Δεκ 2011-Δεκ 2021 από τη βάση δεδομένων PubMed, με στόχο την αποτύπωση της αποτελεσματικότητας του Olaparib στον καρκίνο του μαστού αλλά και των σχετιζόμενων βιοδεικτών. Συνολικά, 13 μελέτες πληρούσαν τα κριτήρια και συμπεριλήφθηκαν στην ανασκόπηση. Η πλειοψηφία των μελετών ήταν φάσης Ι ή/και ΙΙ ενώ μόνο τρεις μελέτες ήταν φάσης ΙΙΙ. H αποτελεσματικότητα και η ασφάλεια του Olaparib μελετήθηκε στα πλαίσια: α) της μονοθεραπείας σε μεταστατικό καρκίνο του μαστού β) νέο-επικουρικής και επικουρικής θεραπείας (neoadjuvant/adjuvant) και γ) της συνδυαστικής θεραπείας με χημειοθεραπεία αλλά και με τον αναστολέα PI3K, BKM120. Οι βασικοί μοριακοί βιοδείκτες που εξετάστηκαν ήταν οι γαμετικές/σωματικές παθογόνες μεταλλαγές στα γονίδια BRCA1/2 και σε γονίδια που εμπλέκονται στο μηχανισμό επιδιόρθωσης του ομόλογου ανασυνδυασμού, ενώ εξετάστηκε και η μεθυλίωση του υποκινητή του γονιδίου BRCA1. Γενικότερα οι αποκρίσεις των ασθενών με μεταστατικό καρκίνο του μαστού είχαν μεγάλο εύρος. Συγκεκριμένα στη συνδυαστική θεραπεία το ORR κυμαινόταν από 0-87.5%, στη μονοθεραπεία από 0-60%, σε ασθενείς με γαμετικές μεταλλαγές στο γονίδιο PALB2 και σε άλλα γονίδια του μηχανισμού επιδιόρθωσης η απόκριση κυμαινόταν από 40-82%. Αντιθέτως ασθενείς με μεταλλαγές στα γονίδια ATM ή CHEK2 δεν είχαν απόκριση στη θεραπεία με Olaparib. Υψηλά επίπεδα μεθυλίωσης του υποκινητή του γονιδίου BRCA1 εντοπίστηκαν σε μικρό αριθμό ασθενών με θετική απόκριση χωρίς όμως ισχυρά συμπεράσματα. Οι διαφορετικές προϋπάρχουσες θεραπείες, η ετερογένεια των μοριακών αποτελεσμάτων και το crossing over θεραπειών κατά την πρόοδο νόσου είναι από τις βασικές δυσκολίες για τη σύγκριση των αποτελεσμάτων. Συμπερασματικά υπάρχουν ελπιδοφόρα αποτελέσματα για νέους βιοδείκτες που αφορούν γενετικές αλλά και επιγενετικές τροποποιήσεις των γονιδίων που εμπλέκονται στην επιδιόρθωση του DNA μέσω του ομόλογου ανασυνδυασμού, όμως η περαιτέρω διερεύνηση είναι απαραίτητη για να εξαχθούν ισχυρά συμπεράσματα.The development of new technologies and the discovery of novel biomarkers have made the clinical use of personalized and targeted therapy a reality. Researching for new biomarkers and targets is crucial for the selection of patients who will benefit from such therapies. Synthetic lethality has been put into the test by the use of PARP inhibitors and has provided the scientific community with satisfactory and promising data. Olaparib, a PARP inhibitor has been FDA approved for the therapy of ovarian, breast and prostate cancer under specific requirements, with the most common being the presence of germline BRCA1/2 mutations. Τo identify publications relevant to this review, a systematic literature search of PubMed was conducted for articles published between December 2011 and December 2021, reporting the use of PARP inhibitors in the treatment of breast cancer. A total of 26 publications were identified and 13 of these met the criteria for subsequent review. The majority of the clinical trials were phase I and/or II while only three studies were phase III. The efficacy and safety of Olaparib was determined under different settings: a) monotherapy for metastatic breast cancer b) neoadjuvant or adjuvant therapy and c) combined therapy with chemotherapy or PI3K inhibitor, BKM120. The basic molecular biomarkers tested were germline or somatic mutations in BRCA1/2 and homologous recombination genes, as well as the methylation status of BRCA1 promoter. In general, the responses ranged: a) in the combined therapy setting from 0-87.5%, b) in the monotherapy setting from 0-60%, while patients with germline mutations in PALB2 and in other homologous recombination genes had responses of 40-82%. On the contrary, patients with mutations in ATM or CHEK2 genes, didn’t show any responses to Olaparib. High levels of promoter methylation of BRCA1 gene were detected in a small number of patients with a good response and promising results that need further confirmation. Differences in previous therapeutic regimes, cycles of therapy, the heterogeneity of molecular attributes and the crossing over of therapies upon disease 0progression make these studies hard to compare. Although the results seem to be promising, further examination of the proposed biomarkers of the homologous recombination repair genes is needed in order to have conclusive results and to help identify patients most likely to benefit from PARP inhibitor treatmen

Epigenetic effects following acute and chronic exercise in cardiovascular disease: A systematic review

Introduction: Acute exercise and exercise training may confer epigenetic modifications in healthy subjects. Epigenetic effects after exercise have been showed in patients with cardiovascular disease. The aim of this systematic review was to summarize the evidence from available clinical trials that study epigenetic adaptations after exercise in patients with cardiovascular disease. Methods: The search strategy was performed in PubMed and CENTRAL databases on articles published until September 2020. Studies with titles and abstracts relevant to exercise epigenetic modification applied to cardiovascular patients were fully examined. Inclusion and exclusion criteria were utilized for studies screening. Quality assessment with PEDro scale and evaluation by two independent reviewers was performed. Results: Of the 1714 articles retrieved, 88 articles were assessed for eligibility criteria and 8 articles matched our search criteria and finally included in the systematic analysis. The acute exercise epigenetic (miRNAs) effects were assessed in three studies and the chronic exercise training effects (miRNAs and DNA methylation) in six studies. The results have shown that there is possibly an acute significant exercise effect on epigenetic targets which is more evident after chronic exercise training. Conclusions: By the present systematic review, we provide preliminary evidence of beneficial epigenetic adaptations following acute and chronic exercise in patients with cardiovascular disease. More controlled studies are needed to confirm such evidence

mRNA coexpression patterns of Wnt pathway components and their clinicopathological associations in breast and colorectal cancer

Aberrant Wnt signaling is implicated in carcinogenesis triggering efforts for the development of new therapeutic agents, many of which have entered clinical trials. We extend our previous analysis of WNT3, FZD7, LEFI expression levels in breast and colorectal cancer including WNT2, FZD4 and beta-catenin expression, in an effort to delineate their relative expression levels along with concurrent expression patterns and possible prognostic value. We analyzed 82 breast and 102 colorectal carcinomas for relative mRNA expression levels of the investigated genes by RT-PCR relative quantification with the Delta Delta Ct method. Statistical analysis was performed in order to determine associations of relative mRNA expression and linear correlations. beta-catenin expression was determined by immunochemistry. Regarding breast carcinomas, decreased relative mRNA expression levels of WNT2, FZD4 were found frequently and WNT2 expression was correlated with ER/ PR status (p = 0.045/p = 0.028), whereas beta-catenin with grade (p = 0.026). In colorectal carcinomas, increased relative mRNA expression levels of WNT2 and FZD4 were found in 59% and 32% of cases respectively, whereas beta-catenin showed decreased mRNA expression levels in 57% of cases and a correlation with pN-category (p = 0.037). Linear correlations were observed between WNT2/FZD4 (R=0.542, p < 0.001), WNT2/beta-catenin (R=0.254, p = 0.010), FZD4/beta-catenin (R=0.406, p < 0.001) expression and a correlation between mRNA expression and membranous/cytoplasmic beta-catenin emerged (p = 0.039/0.046). Our results suggest a possible clinical significance for Wnt pathway gene expression levels in both tumour types. The concurrent expression of the investigated genes as well as the different expression profiles, underlines the complexity of this pathway and the necessity of patient selection in order to maximize the efficacy of drugs targeting Wnt pathway

Comprehensive Molecular Analysis of NSCLC; Clinicopathological Associations

Background Selection of NSCLC patients for targeted therapy is currently based upon the presence of sensitizing mutations in EGFR and EML4/ALK translocations. The heterogeneity of molecular alterations in lung cancer has led to the ongoing discovery of potential biomarkers and targets in order to improve survival. Aim This study aimed to detect alterations in EGFR, KRAS, BRAF, PIK3CA, MET-gene copy number and ALK rearrangements in a large cohort of 956 NSCLC patients of Hellenic origin using highly sensitive techniques and correlations with clinicopathological characteristics. Results Mutations were detected in EGFR 10.6%(101 out of 956 samples), KRAS 26.5% (191 out of 720 samples), BRAF 2.5% (12 out of 471 samples), PIK3CA 3.8% (7 out of 184 samples), MET gene amplification was detected in 18% (31 out of 170) and ALK rearrangements in 3.7% (4 out of 107 samples). EGFR mutations were detected in exon 19 (61.4% of mutant cases), exon 21 p.Leu858Arg (19.8%), exon 20 (15.8%), exon 18 (2.9%) and were correlated with gender histology, smoking status and TTF1 staining. p.Thr790Met mutant cases (3.9%) displayed concurrent mutations in exons 19 or 21. Negative TTF-1 staining showed strong negative predictive value for the presence of EGFR mutations. KRAS mutations were associated with histology, the most common mutation being p.Gly12Cys (38%). Discussion In conclusion, only 89 patients were eligible for EGFR-TKIs and ALK inhibitors therapy, whereas 257 patients showed other alterations, highlighting the necessity for a detailed molecular profiling potentially leading to more efficient individualized therapies for NSCLC patients

Comprehensive Molecular Analysis of NSCLC; Clinicopathological Associations.

Selection of NSCLC patients for targeted therapy is currently based upon the presence of sensitizing mutations in EGFR and EML4/ALK translocations. The heterogeneity of molecular alterations in lung cancer has led to the ongoing discovery of potential biomarkers and targets in order to improve survival.This study aimed to detect alterations in EGFR, KRAS, BRAF, PIK3CA, MET-gene copy number and ALK rearrangements in a large cohort of 956 NSCLC patients of Hellenic origin using highly sensitive techniques and correlations with clinicopathological characteristics.Mutations were detected in EGFR 10.6% (101 out of 956 samples), KRAS 26.5% (191 out of 720 samples), BRAF 2.5% (12 out of 471 samples), PIK3CA 3.8% (7 out of 184 samples), MET gene amplification was detected in 18% (31 out of 170) and ALK rearrangements in 3.7% (4 out of 107 samples). EGFR mutations were detected in exon 19 (61.4% of mutant cases), exon 21 p.Leu858Arg (19.8%), exon 20 (15.8%), exon 18 (2.9%) and were correlated with gender histology, smoking status and TTF1 staining. p.Thr790Met mutant cases (3.9%) displayed concurrent mutations in exons 19 or 21. Negative TTF-1 staining showed strong negative predictive value for the presence of EGFR mutations. KRAS mutations were associated with histology, the most common mutation being p.Gly12Cys (38%).In conclusion, only 89 patients were eligible for EGFR -TKIs and ALK inhibitors therapy, whereas 257 patients showed other alterations, highlighting the necessity for a detailed molecular profiling potentially leading to more efficient individualized therapies for NSCLC patients

Raw Data TRAIL.xlsx

Cp raw data from real time RT-PCR experiments are included in this excel file<br

A detailed immunohistochemical analysis of the PI3K/AKT/mTOR pathway in lung cancer: Correlation with PIK3CA, AKT1, K-RAS or PTEN mutational status and clinicopathological features

The phosphoinositide 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homolog (AKT)/mammalian target of rapamycin (mTOR) pathway is upregulated in a number of human cancers, including non-small cell lung cancer (NSCLC). Its potential role in NSCLC progression provides an attractive target for anticancer therapy. The expression of phosphorylated mTOR (p-mTOR), phosphorylated AKT (p-AKT), p85 alpha and p110 gamma subunits of PI3K, phosphorylated p70S6K (p-p70S6K), phosphatase and tensin homolog (PTEN) and phosphorylated 4E-BP1 (p-4E-BP1) was examined by immunohistochemistry in 102 NSCLC specimens. The results were correlated with clinicopathological features. We also examined 61 of our cases for the presence of PIK3CA, AKT1, PTEN and K-RAS mutations. A common PIK3CA mutation was detected at exon 9 in 2 samples (p.E545K), whereas another sample displayed a rare mutation (p.D1018N). Furthermore, 10 out of 54 cases (18.5%) had a K-RAS mutation at codon 12, 5 had a PTEN mutation (exons 7 and 8) and 1 case had an AKT1 mutation (p.E17K). PTEN mutations were associated with nodal metastases. The expression of p-mTOR positively correlated with that of p-AKT and p-p70S6K and was higher in adenocarcinomas along with nuclear p110 gamma PI3K expression, whereas p-4E-BP1 expression was higher in squamous cell carcinomas. We also established a positive association between p85 alpha PI3K or p110 gamma PI3K and cytoplasmic p-AKT and its downstream effectors. An inverse correlation was noted between p-4E-BP1 immunoexpression and tumour status and nuclear p-AKT expression as regards tumour stage. Univariate survival analysis demonstrated that p-4E-BP1 expression, either alone or in combination with cytoplasmic p-AKT expression had an adverse prognostic significance in adenocarcinomas. The combination of p-4E-BP1 and cytoplasmic p-AKT expression remained significant in the multivariate analysis as a function of their interaction with histological type. Our data demonstrate the significance of p-4E-BP1 immunoexpression as a molecular marker of prognostic value in adenocarcinomas, particularly when combined with p-AKT

Histological Classification of 956 NSCLC samples

<p>A. Classification of Resection Samples B. Classification of Small Biopsies/cytology samples and C. Metastasis Samples according to IASLC/ATS/ERS 2011, WHO 2015.</p