Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism.

Treatment of tuberculosis, a complex granulomatous disease, requires long-term multidrug therapy to overcome tolerance, an epigenetic drug resistance that is widely attributed to nonreplicating bacterial subpopulations. Here, we deploy Mycobacterium marinum-infected zebrafish larvae for in vivo characterization of antitubercular drug activity and tolerance. We describe the existence of multidrug-tolerant organisms that arise within days of infection, are enriched in the replicating intracellular population, and are amplified and disseminated by the tuberculous granuloma. Bacterial efflux pumps that are required for intracellular growth mediate this macrophage-induced tolerance. This tolerant population also develops when Mycobacterium tuberculosis infects cultured macrophages, suggesting that it contributes to the burden of drug tolerance in human tuberculosis. Efflux pump inhibitors like verapamil reduce this tolerance. Thus, the addition of this currently approved drug or more specific efflux pump inhibitors to standard antitubercular therapy should shorten the duration of curative treatment.

[18F]FDG-PET/CT atypical response patterns to immunotherapy in non-small cell lung cancer patients: long term prognosis assessment and clinical management proposal.

AIM: To determine the long-term prognosis of immune-related response profiles (pseudoprogression and dissociated response), not covered by conventional PERCIST criteria, in patients with non-small-cell lung cancer (NSCLC) treated with immune checkpoint inhibitors (ICPIs). METHODS: 109 patients were prospectively included and underwent [18F]FDG-PET/CT at baseline, after 7 weeks (PETinterim1), and 3 months (PETinterim2) of treatment. On PETinterim1, tumor response was assessed using standard PERCIST criteria. In the event of PERCIST progression at this time-point, the study design provided for continued immunotherapy for 6 more weeks. Additional response patterns were then considered on PETinterim2: pseudo-progression (PsPD, subsequent metabolic response); dissociated response (DR, coexistence of responding and non-responding lesions), and confirmed progressive metabolic disease (cPMD, subsequent homogeneous progression of lesions). Patients were followed up for at least 12 months. RESULTS: Median follow-up was 21 months. At PETinterim1, PERCIST progression was observed in 60% (66/109) of patients and ICPI was continued in 59/66. At the subsequent PETinterim2, 14% of patients showed PsPD, 11% DR, 35% cPMD, and 28% had a sustained metabolic response. Median overall survival (OS) and progression-free-survival (PFS) did not differ between PsPD and DR (27 vs 29 months, p = 1.0; 17 vs 12 months, p = 0.2, respectively). The OS and PFS of PsPD/DR patients were significantly better than those with cPMD (29 vs 9 months, p < 0.02; 16 vs 2 months, p < 0.001), but worse than those with sustained metabolic response (p < 0.001). This 3-group prognostic stratification enabled better identification of true progressors, outperforming the prognostic value of standard PERCIST criteria (p = 0.03). CONCLUSION: [18F]FDG-PET/CT enables early assessment of response to immunotherapy. The new wsPERCIST ("wait and see") PET criteria proposed, comprising immune-related atypical response patterns, can refine conventional prognostic stratification based on PERCIST criteria. TRIAL REGISTRATION: HDH F20230309081206. Registered 20 April 2023. Retrospectively registered.

Efficient polymer nanoparticle-mediated delivery of gene editing reagents into human hematopoietic stem and progenitor cells.

Clinical applications of hematopoietic stem cell (HSC) gene editing are limited due to their complex and expensive logistics. HSC editing is commonly performed ex vivo using electroporation and requires good manufacturing practice (GMP) facilities, similar to bone marrow transplant centers. In vivo gene editing could overcome this limitation; however, electroporation is unsuitable for systemic in vivo applications to HSCs. Here we evaluated polymer-based nanoparticles (NPs), which could also be used for in vivo administration, for the delivery of mRNA and nucleases to human granulocyte colony-stimulating factor (GCSF)-mobilized CD34+ cells. NP-mediated ex vivo delivery showed no toxicity, and the efficiency was directly correlated with the charge of the NPs. In a side-by-side comparison with electroporation, NP-mediated gene editing allowed for a 3-fold reduction in the amount of reagents, with similar efficiency. Furthermore, we observed enhanced engraftment potential of human HSCs in the NSG mouse xenograft model using NPs. Finally, mRNA- and nuclease-loaded NPs were successfully lyophilized for storage, maintaining their transfection potential after rehydration. In conclusion, we show that polymer-based NP delivery of mRNA and nucleases has the potential to overcome current limitations of HSC gene editing. The predictable transfection efficiency, low toxicity, and ability to lyophilize NPs will greatly enhance the portability and provide a highly promising platform for HSC gene therapy.

Learning a confidence score and the latent space of a new supervised autoencoder for diagnosis and prognosis in clinical metabolomic studies.

BACKGROUND: Presently, there is a wide variety of classification methods and deep neural network approaches in bioinformatics. Deep neural networks have proven their effectiveness for classification tasks, and have outperformed classical methods, but they suffer from a lack of interpretability. Therefore, these innovative methods are not appropriate for decision support systems in healthcare. Indeed, to allow clinicians to make informed and well thought out decisions, the algorithm should provide the main pieces of information used to compute the predicted diagnosis and/or prognosis, as well as a confidence score for this prediction. METHODS: Herein, we used a new supervised autoencoder (SAE) approach for classification of clinical metabolomic data. This new method has the advantage of providing a confidence score for each prediction thanks to a softmax classifier and a meaningful latent space visualization and to include a new efficient feature selection method, with a structured constraint, which allows for biologically interpretable results. RESULTS: Experimental results on three metabolomics datasets of clinical samples illustrate the effectiveness of our SAE and its confidence score. The supervised autoencoder provides an accurate localization of the patients in the latent space, and an efficient confidence score. Experiments show that the SAE outperforms classical methods (PLS-DA, Random Forests, SVM, and neural networks (NN)). Furthermore, the metabolites selected by the SAE were found to be biologically relevant. CONCLUSION: In this paper, we describe a new efficient SAE method to support diagnostic or prognostic evaluation based on metabolomics analyses.

Development and validation of a radiomic model for the diagnosis of dopaminergic denervation on [18F]FDOPA PET/CT.

PURPOSE: FDOPA PET shows good performance for the diagnosis of striatal dopaminergic denervation, making it a valuable tool for the differential diagnosis of Parkinsonism. Textural features are image biomarkers that could potentially improve the early diagnosis and monitoring of neurodegenerative parkinsonian syndromes. We explored the performances of textural features for binary classification of FDOPA scans. METHODS: We used two FDOPA PET datasets: 443 scans for feature selection, and 100 scans from a different PET/CT system for model testing. Scans were labelled according to expert interpretation (dopaminergic denervation versus no dopaminergic denervation). We built LASSO logistic regression models using 43 biomarkers including 32 textural features. Clinical data were also collected using a shortened UPDRS scale. RESULTS: The model built from the clinical data alone had a mean area under the receiver operating characteristics (AUROC) of 63.91. Conventional imaging features reached a maximum score of 93.47 but the addition of textural features significantly improved the AUROC to 95.73 (p < 0.001), and 96.10 (p < 0.001) when limiting the model to the top three features: GLCM_Correlation, Skewness and Compacity. Testing the model on the external dataset yielded an AUROC of 96.00, with 95% sensitivity and 97% specificity. GLCM_Correlation was one of the most independent features on correlation analysis, and systematically had the heaviest weight in the classification model. CONCLUSION: A simple model with three radiomic features can identify pathologic FDOPA PET scans with excellent sensitivity and specificity. Textural features show promise for the diagnosis of parkinsonian syndromes.

Prognostic value of immunotherapy-induced organ inflammation assessed on 18FDG PET in patients with metastatic non-small cell lung cancer.

PURPOSE: We evaluated the prognostic value of immunotherapy-induced organ inflammation observed on 18FDG PET in patients with non-small cell lung cancer (NSCLC) treated with immune checkpoint inhibitors (ICPIs). METHODS: Data from patients with IIIB/IV NSCLC included in two different prospective trials were analyzed. 18FDG PET/CT exams were performed at baseline (PETBaseline) and repeated after 7-8 weeks (PETInterim1) and 12-16 weeks (PETInterim2) of treatment, using iPERCIST for tumor response evaluation. The occurrence of abnormal organ 18FDG uptake, deemed to be due to ICPI-related organ inflammation, was collected. RESULTS: Exploratory cohort (Nice, France): PETInterim1 and PETInterim2 revealed the occurrence of at least one ICPI-induced organ inflammation in 72.8% of patients, including midgut/hindgut inflammation (33.7%), gastritis (21.7%), thyroiditis (18.5%), pneumonitis (17.4%), and other organ inflammations (9.8%). iPERCIST tumor response was associated with improved progression-free survival (p < 0.001). iPERCIST tumor response and immuno-induced gastritis assessed on PET were both associated with improved overall survival (OS) (p < 0.001 and p = 0.032). Combining these two independent variables, we built a model predicting patients' 2-year OS with a sensitivity of 80.3% and a specificity of 69.2% (AUC = 72.7). Validation cohort (Genova, Italy): Immuno-induced gastritis (19.6% of patients) was associated with improved OS (p = 0.04). The model built previously predicted 2-year OS with a sensitivity and specificity of 72.0% and 63.6% (AUC = 70.7) and 3-year OS with a sensitivity and specificity of 69.2% and 80.0% (AUC = 78.2). CONCLUSION: Immuno-induced gastritis revealed by early interim 18FDG PET in around 20% of patients with NSCLC treated with ICPI is a novel and reproducible imaging biomarker of improved OS.

Genome editing in large animal models.

Although genome editing technologies have the potential to revolutionize the way we treat human diseases, barriers to successful clinical implementation remain. Increasingly, preclinical large animal models are being used to overcome these barriers. In particular, the immunogenicity and long-term safety of novel gene editing therapeutics must be evaluated rigorously. However, short-lived small animal models, such as mice and rats, cannot address secondary pathologies that may arise years after a gene editing treatment. Likewise, immunodeficient mouse models by definition lack the ability to quantify the host immune response to a novel transgene or gene-edited locus. Large animal models, including dogs, pigs, and non-human primates (NHPs), bear greater resemblance to human anatomy, immunology, and lifespan and can be studied over longer timescales with clinical dosing regimens that are more relevant to humans. These models allow for larger scale and repeated blood and tissue sampling, enabling greater depth of study and focus on rare cellular subsets. Here, we review current progress in the development and evaluation of novel genome editing therapies in large animal models, focusing on applications in human immunodeficiency virus 1 (HIV-1) infection, cancer, and genetic diseases including hemoglobinopathies, Duchenne muscular dystrophy (DMD), hypercholesterolemia, and inherited retinal diseases.

Primal-dual for classification with rejection (PD-CR): a novel method for classification and feature selection-an application in metabolomics studies.

BACKGROUND: Supervised classification methods have been used for many years for feature selection in metabolomics and other omics studies. We developed a novel primal-dual based classification method (PD-CR) that can perform classification with rejection and feature selection on high dimensional datasets. PD-CR projects data onto a low dimension space and performs classification by minimizing an appropriate quadratic cost. It simultaneously optimizes the selected features and the prediction accuracy with a new tailored, constrained primal-dual method. The primal-dual framework is general enough to encompass various robust losses and to allow for convergence analysis. Here, we compare PD-CR to three commonly used methods: partial least squares discriminant analysis (PLS-DA), random forests and support vector machines (SVM). We analyzed two metabolomics datasets: one urinary metabolomics dataset concerning lung cancer patients and healthy controls; and a metabolomics dataset obtained from frozen glial tumor samples with mutated isocitrate dehydrogenase (IDH) or wild-type IDH. RESULTS: PD-CR was more accurate than PLS-DA, Random Forests and SVM for classification using the 2 metabolomics datasets. It also selected biologically relevant metabolites. PD-CR has the advantage of providing a confidence score for each prediction, which can be used to perform classification with rejection. This substantially reduces the False Discovery Rate. CONCLUSION: PD-CR is an accurate method for classification of metabolomics datasets which can outperform PLS-DA, Random Forests and SVM while selecting biologically relevant features. Furthermore the confidence score provided with PD-CR can be used to perform classification with rejection and reduce the false discovery rate.

Impact of Systematic Whole-body 18F-Fluorodeoxyglucose PET/CT on the Management of Patients Suspected of Infective Endocarditis: The Prospective Multicenter TEPvENDO Study.

BACKGROUND: Diagnostic and patients' management modifications induced by whole-body 18F-FDG-PET/CT had not been evaluated so far in prosthetic valve (PV) or native valve (NV) infective endocarditis (IE)-suspected patients. METHODS: In sum, 140 consecutive patients in 8 tertiary care hospitals underwent 18F-FDG-PET/CT. ESC-2015-modified Duke criteria and patients' management plan were established jointly by 2 experts before 18F-FDG-PET/CT. The same experts reestablished Duke classification and patients' management plan immediately after qualitative interpretation of 18F-FDG-PET/CT. A 6-month final Duke classification was established. RESULTS: Among the 70 PV and 70 NV patients, 34 and 46 were classified as definite IE before 18F-FDG-PET/CT. Abnormal perivalvular 18F-FDG uptake was recorded in 67.2% PV and 24.3% NV patients respectively (P < .001) and extracardiac uptake in 44.3% PV and 51.4% NV patients. IE classification was modified in 24.3% and 5.7% patients (P = .005) (net reclassification index 20% and 4.3%). Patients' managements were modified in 21.4% PV and 31.4% NV patients (P = .25). It was mainly due to perivalvular uptake in PV patients and to extra-cardiac uptake in NV patients and consisted in surgery plan modifications in 7 patients, antibiotic plan modifications in 22 patients and both in 5 patients. Altogether, 18F-FDG-PET/CT modified classification and/or care in 40% of the patients (95% confidence interval: 32-48), which was most likely to occur in those with a noncontributing echocardiography (P < .001) or IE classified as possible at baseline (P = .04), while there was no difference between NV and PV. CONCLUSIONS: Systematic 18F-FDG-PET/CT did significantly and appropriately impact diagnostic classification and/or IE management in PV and NV-IE suspected patients. CLINICAL TRIALS REGISTRATION: NCT02287792.

CRISPR/Cas9 for the treatment of haematological diseases: a journey from bacteria to the bedside.

Genome editing therapies represent a significant advancement in next-generation, precision medicine for the management of haematological diseases, and CRISPR/Cas9 has to date been the most successful implementation platform. From discovery in bacteria and archaea over three decades ago, through intensive basic research and pre-clinical development phases involving the modification of therapeutically relevant cell types, CRISPR/Cas9 genome editing is now being investigated in ongoing clinic trials. Despite the widespread enthusiasm brought by this new technology, significant challenges remain before genome editing can be routinely recommended and implemented in the clinic. These include risks of genotoxicity resulting from off-target DNA cleavage or chromosomal rearrangement, and suboptimal efficacy of homology-directed repair editing strategies, which thus limit therapeutic options. Practical hurdles such as high costs and inaccessibility to patients outside specialised centres must also be addressed. Future improvements in this rapidly developing field should circumvent current limitations with novel editing platforms and with the simplification of clinical protocols using in vivo delivery of editing reagents.

Targeted homology-directed repair in blood stem and progenitor cells with CRISPR nanoformulations.

Ex vivo CRISPR gene editing in haematopoietic stem and progenitor cells has opened potential treatment modalities for numerous diseases. The current process uses electroporation, sometimes followed by virus transduction. While this complex manipulation has resulted in high levels of gene editing at some genetic loci, cellular toxicity was observed. We have developed a CRISPR nanoformulation based on colloidal gold nanoparticles with a unique loading design capable of cellular entry without the need for electroporation or viruses. This highly monodispersed nanoformulation avoids lysosomal entrapment and localizes to the nucleus in primary human blood progenitors without toxicity. Nanoformulation-mediated gene editing is efficient and sustained with different CRISPR nucleases at multiple loci of therapeutic interest. The engraftment kinetics of nanoformulation-treated primary cells in humanized mice are better relative to those of non-treated cells, with no differences in differentiation. Here we demonstrate non-toxic delivery of the entire CRISPR payload into primary human blood progenitors.

RGD-functionalized magnetosomes are efficient tumor radioenhancers for X-rays and protons.

Although chemically synthesized ferro/ferrimagnetic nanoparticles have attracted great attention in cancer theranostics, they lack radio-enhancement efficacy due to low targeting and internalization ability. Herein, we investigated the potential of RGD-tagged magnetosomes, bacterial biogenic magnetic nanoparticles naturally coated with a biological membrane and genetically engineered to express an RGD peptide, as tumor radioenhancers for conventional radiotherapy and proton therapy. Although native and RGD-magnetosomes similarly enhanced radiation-induced damage to plasmid DNA, RGD-magnetoprobes were able to boost the efficacy of radiotherapy to a much larger extent than native magnetosomes both on cancer cells and in tumors. Combined to magnetosomes@RGD, proton therapy exceeded the efficacy of X-rays at equivalent doses. Also, increased secondary emissions were measured after irradiation of magnetosomes with protons versus photons. Our results indicate the therapeutic advantage of using functionalized magnetoparticles to sensitize tumors to both X-rays and protons and strengthen the case for developing biogenic magnetoparticles for multimodal nanomedicine in cancer therapy.

Correction to: 18F-DOPA PET/CT in brain tumors: impact on multidisciplinary brain tumor board decisions.

Jérôme Barriere was inadvertently missing in the original version of this article. He has participated to the study design, protocol writing and inclusion of a significant number of patients.

18F-DOPA PET/CT in brain tumors: impact on multidisciplinary brain tumor board decisions.

PURPOSE: This study aimed to assess the therapeutic impact and diagnostic accuracy of 18F-DOPA PET/CT in patients with glioblastoma or brain metastases. METHODS: Patients with histologically proven glioblastoma or brain metastases were prospectively included in this monocentric clinical trial (IMOTEP). Patients were included either due to a clinical suspicion of relapse or to assess residual tumor infiltration after treatment. Multimodality brain MRI and 18F-DOPA PET were performed. Patients' data were discussed during a Multidisciplinary Neuro-oncology Tumor Board (MNTB) meeting. The discussion was first based on clinical and MRI data, and an initial diagnosis and treatment plan were proposed. Secondly, a new discussion was conducted based on the overall imaging results, including 18F-DOPA PET. A second diagnosis and therapeutic plan were proposed. A retrospective and definitive diagnosis was obtained after a 3-month follow-up and considered as the reference standard. RESULTS: One hundred six cases were prospectively investigated by the MNTB. All patients with brain metastases (N = 41) had a clinical suspicion of recurrence. The addition of 18F-DOPA PET data changed the diagnosis and treatment plan in 39.0% and 17.1% of patients' cases, respectively. Concerning patients with a suspicion of recurrent glioblastoma (N = 12), the implementation of 18F-DOPA PET changed the diagnosis and treatment plan in 33.3% of cases. In patients evaluated to assess residual glioblastoma infiltration after treatment (N = 53), 18F-DOPA PET data had a lower impact with only 5.7% (3/53) of diagnostic changes and 3.8% (2/53) of therapeutic plan changes. The definitive reference diagnosis was available in 98/106 patients. For patients with tumor recurrence suspicion, the adjunction of 18F-DOPA PET increased the Younden's index from 0.44 to 0.53 in brain metastases and from 0.2 to 1.0 in glioblastoma, reflecting an increase in diagnostic accuracy. CONCLUSION: 18F-DOPA PET has a significant impact on the management of patients with a suspicion of brain tumor recurrence, either glioblastoma or brain metastases, but a low impact when used to evaluate the residual glioblastoma infiltration after a first-line radio-chemotherapy or second-line bevacizumab.

SUV calculation in breast cancer: which normalization should be applied when using 18F-FDG PET?

BACKGROUND: When using 18F-FDG PET, glucose metabolism quantification is affected by various factors. We aimed to investigate the benefit of different standardized uptake value (SUV) normalizations to improve the accuracy of 18F-FDG uptake to predict breast cancer aggressiveness and response to treatment. METHODS: Two hundred fifty-two women with locally advanced breast cancer treated with neoadjuvant chemotherapy (NAC) were included. Women underwent 18F-FDG PET before and after the first course of NAC. Glucose serum levels, patient heights and weights were recorded at the time of each PET exam. Four different procedures for SUV normalization of the primary tumor were used: by body weight (SUVBW) by blood glucose level (SUVG), by lean body mass (SUL) and then corrected for both lean body mass and blood glucose level (SULG). RESULTS: At baseline, SUL was significantly lower than SUVBW (5.9±4.0 and 9.5±6.5, respectively; P<0.0001), whereas SUVG and SUVBW were not significantly different (9.7±6.4 and 9.5±6.5, respectively; P=0.67). Concerning SUV changes (ΔSUV), the different normalizations methods did not induce significant quantitative differences. The correlation coefficients were high between the four normalizations methods of SUV1, SUV2 and ΔSUVB (R>0.95; P<0.0001). High baseline SUVBW measures were positively correlated with the biological tumor characteristics of aggressiveness and proliferation (P<0.001): ductal carcinoma, high tumor grading, high mitotic activity, negative estrogen receptor status and the TNBC subtype. ΔSUVBW was highly predictive of pCR (AUC=0.76 on ROC curve analysis; P<0.0001). The different SUV normalizations yields identical statistical results and AUC to predict tumor biological aggressiveness and response to therapy. CONCLUSIONS: In the present setting, SUVBW and SUL can be considered as robust measures and be used in future multicenter trials. The additional normalization of SUV by glycemia involves stringent methodologic procedures to avoid biased risk measurements and offers no statistical advantages.

Long-term multilineage engraftment of autologous genome-edited hematopoietic stem cells in nonhuman primates.

Genome editing in hematopoietic stem and progenitor cells (HSPCs) is a promising novel technology for the treatment of many human diseases. Here, we evaluated whether the disruption of the C-C chemokine receptor 5 (CCR5) locus in pigtailed macaque HSPCs by zinc finger nucleases (ZFNs) was feasible. We show that macaque-specific CCR5 ZFNs efficiently induce CCR5 disruption at levels of up to 64% ex vivo, 40% in vivo early posttransplant, and 3% to 5% in long-term repopulating cells over 6 months following HSPC transplant. These genome-edited HSPCs support multilineage engraftment and generate progeny capable of trafficking to secondary tissues including the gut. Using deep sequencing technology, we show that these ZFNs are highly specific for the CCR5 locus in primary cells. Further, we have adapted our clonal tracking methodology to follow individual CCR5 mutant cells over time in vivo, reinforcing that CCR5 gene-edited HSPCs are capable of long-term engraftment. Together, these data demonstrate that genome-edited HSPCs engraft, and contribute to multilineage repopulation after autologous transplantation in a clinically relevant large animal model, an important step toward the development of stem cell-based genome-editing therapies for HIV and potentially other diseases as well.

Development of Third-generation Cocal Envelope Producer Cell Lines for Robust Lentiviral Gene Transfer into Hematopoietic Stem Cells and T-cells.

Lentiviral vectors (LVs) pseudotyped with vesicular stomatitis virus envelope glycoprotein (VSV-G) have demonstrated great promise in gene therapy trials employing hematopoietic stem cell and T-cells. The VSV-G envelope confers broad tropism and stability to the vector but is toxic when constitutively expressed, which has impeded efforts to generate stable producer cell lines. We previously showed that cocal pseudotyped LVs offer an excellent alternative to VSV-G vectors because of their broad tropism and resistance to human serum inactivation. In this study, we demonstrate that cocal LVs transduce CD34(+) and CD4(+) T-cells more efficiently than VSV-G LVs and share the same receptor(s) for cell entry. 293T-cells stably expressing the cocal envelope produced significantly higher LV titers than VSV-G expressing cells. We developed cocal pseudotyped, third-generation, self-inactivating LV producer cell lines for a GFP reporter and for a WT1 tumor-specific T-cell receptor, which achieved concentrated titers above 10(8) IU/ml and were successfully adapted for growth in suspension, serum-free culture. The resulting LVs were at least as effective as standard LVs in transducing CD34(+) and CD4(+) T-cells. Our stable cocal LV producer cell lines should facilitate the production of large-scale, high titer clinical grade vectors.

Novel fluorescent genome editing reporters for monitoring DNA repair pathway utilization at endonuclease-induced breaks.

The creation of a DNA break at a specific locus by a designer endonuclease can be harnessed to edit a genome. However, DNA breaks may engage one of several competing repair pathways that lead to distinct types of genomic alterations. Therefore, understanding the contribution of different repair pathways following the introduction of a targeted DNA break is essential to further advance the safety and efficiency of nuclease-induced genome modification. To gain insight into the role of different DNA repair pathways in resolving nuclease-induced DNA breaks into genome editing outcomes, we previously developed a fluorescent-based reporter system, designated the Traffic Light Reporter, which provides a readout of gene targeting and gene disruption downstream of a targeted DNA double-strand break. Here we describe two related but novel reporters that extend this technology: one that allows monitoring of the transcriptional activity at the reporter locus, and thus can be applied to interrogate break resolution at active and repressed loci; and a second that reads out single-strand annealing in addition to gene targeting and gene disruption. Application of these reporters to assess repair pathway usage in several common gene editing contexts confirms the importance that chromatin status and initiation of end resection have on the resolution of nuclease-induced breaks.

Role of positron emission tomography for the monitoring of response to therapy in breast cancer.

This review considers the potential utility of positron emission tomography (PET) tracers in the setting of response monitoring in breast cancer, with a special emphasis on glucose metabolic changes assessed with (18)F-fluorodeoxyglucose (FDG). In the neoadjuvant setting of breast cancer, the metabolic response can predict the final complete pathologic response after the first cycles of chemotherapy. Because tumor metabolic behavior highly depends on cancer subtype, studies are ongoing to define the optimal metabolic criteria of tumor response in each subtype. The recent multicentric randomized AVATAXHER trial has suggested, in the human epidermal growth factor 2-positive subtype, a clinical benefit of early tailoring the neoadjuvant treatment in women with poor metabolic response after the first course of treatment. In the bone-dominant metastatic setting, there is increasing clinical evidence that FDG-PET/computed tomography (CT) is the most accurate imaging modality for assessment of the tumor response to treatment when both metabolic information and morphologic information are considered. Nevertheless, there is a need to define standardized metabolic criteria of response, including the heterogeneity of response among metastases, and to evaluate the costs and health outcome of FDG-PET/CT compared with conventional imaging. New non-FDG radiotracers highlighting specific molecular hallmarks of breast cancer cells have recently emerged in preclinical and clinical studies. These biomarkers can take into account the heterogeneity of tumor biology in metastatic lesions. They may provide valuable clinical information for physicians to select and monitor the effectiveness of novel therapeutics targeting the same molecular pathways of breast tumor.

Targeted gene therapies: tools, applications, optimization.

Many devastating human diseases are caused by mutations in a single gene that prevent a somatic cell from carrying out its essential functions, or by genetic changes acquired as a result of infectious disease or in the course of cell transformation. Targeted gene therapies have emerged as potential strategies for treatment of such diseases. These therapies depend upon rare-cutting endonucleases to cleave at specific sites in or near disease genes. Targeted gene correction provides a template for homology-directed repair, enabling the cell's own repair pathways to erase the mutation and replace it with the correct sequence. Targeted gene disruption ablates the disease gene, disabling its function. Gene targeting can also promote other kinds of genome engineering, including mutation, insertion, or gene deletion. Targeted gene therapies present significant advantages compared to approaches to gene therapy that depend upon delivery of stably expressing transgenes. Recent progress has been fueled by advances in nuclease discovery and design, and by new strategies that maximize efficiency of targeting and minimize off-target damage. Future progress will build on deeper mechanistic understanding of critical factors and pathways.