Correction of beta-thalassemia major by gene transfer in haematopoietic progenitors of pediatric patients

Beta-thalassemia is a common monogenic disorder due to mutations in the beta-globin gene and gene therapy, based on autologous transplantation of genetically corrected haematopoietic stem cells (HSCs), holds the promise to treat patients lacking a compatible bone marrow (BM) donor. We recently showed correction of murine beta-thalassemia by gene transfer in HSCs with the GLOBE lentiviral vector (LV), expressing a transcriptionally regulated human beta-globin gene. Here, we report successful correction of thalassemia major in human cells, by studying a large cohort of pediatric patients of diverse ethnic origin, carriers of different mutations and all candidates to BM transplantation. Extensive characterization of BM-derived CD34(+) cells before and following gene transfer shows the achievement of high frequency of transduction, restoration of haemoglobin A synthesis, rescue from apoptosis and correction of ineffective erythropoiesis. The procedure does not significantly affect the differentiating potential and the relative proportion of haematopoietic progenitors. Analysis of vector integrations shows preferential targeting of transcriptionally active regions, without bias for cancer-related genes. Overall, these results provide a solid rationale for a future clinical translation

HT-29 and Caco-2 Reporter Cell Lines for Functional Studies of Nuclear Factor Kappa B Activation

The NF-κB is a transcription factor which plays a key role in regulating biological processes. In response to signals, NF-κB activation occurs via phosphorylation of its inhibitor, which dissociates from the NF-κB dimer allowing the translocation to the nucleus, inducing gene expression. NF-κB activation has direct screening applications for drug discovery for several therapeutic indications. Thus, pathway-specific reporter cell systems appear as useful tools to screen and unravel the mode of action of probiotics and natural and synthetic compounds. Here, we describe the generation, characterization, and validation of human epithelial reporter cell lines for functional studies of NF-κB activation by different pro- and anti-inflammatory agents. Caco-2 and HT-29 cells were transfected with a pNF-κB-hrGFP plasmid which contains the GFP gene under the control of NF-κB binding elements. Three proinflammatory cytokines (TNF-α, IL-1β, and LPS) were able to activate the reporter systems in a dose-response manner, which corresponds to the activation of the NF-κB signaling pathway. Finally, the reporter cell lines were validated using lactic acid bacteria and a natural compound. We have established robust Caco-2-NF-κB-hrGFP and HT-29-NF-κB-hrGFP reporter cell lines which represent a valuable tool for primary screening and identification of bacterial strains and compounds with a potential therapeutic interest

Nipple-Sparing Mastectomy After Neoadjuvant Chemotherapy: Definitive Results with a Long-Term Follow-Up Evaluation

Background. Nipple-sparing mastectomy (NSM) is increasingly used for women with breast cancer who are not candidates for conservative surgery. The authors previously reported satisfying results with NSM after neoadjuvant chemotherapy (NACT). Methods. From 2010 to 2020, 1072 women underwent mastectomy at the authors' institution. In this group, 433 NSMs were performed (40%). The only contraindications to NSM were close proximity to the nipple-areola complex (NAC), bloody discharge, and Paget disease. Results. In 112 cases involving 111 women, NSM followed NACT (group 1), whereas it was performed as primary surgery in 321 instances involving 306 women (group 2). At 5 years, local relapse was 7% in group 1 and 2% in group 2, although in the multivariate analysis, locoregional relapses (LRRs) did not differ between the two groups. An increased incidence of local relapse was associated with higher tumor stage (stage III; p = 0.046) and age younger than 51 years (p = 0.038). For 34 (30.3%) of the 111 women in group 1 with a pathologic complete response (pCR), no LRRs were recorded. Only one NAC recurrence was observed. Overall survival with each tumor stage did not differ between the two groups. No differences in complications were observed. Cosmetic results were satisfying in 83.8% of the cases and did not get worse after NACT. Conclusions. The study data definitively confirm that NSM is safe even after NACT, with good cosmetic results and complications comparable with those in the primary surgery setting. Tumor stage and age were the only independent factors for local relapse. Patients with pCR enjoyed optimal locoregional control

Third national surgical consensus conference of the Italian Association of Breast Surgeons (ANISC) on management after neoadjuvant chemotherapy: The difficulty in reaching a consensus

Introduction: Neoadjuvant chemotherapy (NAC) has a profound impact on surgical management of breast cancer. For this reason, the Italian Association of Breast Surgeons (ANISC) promoted the third national Consensus Conference on this subject, open to multidisciplinary specialists. Materials and methods: The Consensus Conference was held on-line in November 2022, and after an introductory session with five core-team experts, participants were asked to vote on eleven controversial issues, while results were collected in real-time with a polling system. Results: A total of 164 dedicated specialists from 74 Breast Centers participated. Consensus was reached for only three of the eleven issues, including: 1) the indication to assess the response with Magnetic Resonance Imaging (79 %); 2) the need to re-assess the biological factors of the residual tumor if present (96 %); 3) the possibility of omitting a formal axillary node dissection for cN1 patients if a pathologic Complete Response (pCR) was confirmed with analysis of one or more sentinel lymph nodes (82 %). The majority voted in favor of mapping both the breast and nodal lesions pre-NAC (59 %), and against the omission of sentinel lymph node biopsy in cN0 patients in the case of pathologic or clinical Complete Response (69 %). In cases of cT3/cN1+ tumors with pCR, only 8 % of participants considered appropriate the omission of Post-Mastectomy Radiation Therapy. Conclusion: There is still a wide variability in surgical approaches after NAC in the "real world". As NAC is increasingly used, multidisciplinary teams should be attuned to conforming their procedures to the rapid advances in this field