Characterization of EpCAM in thyroid cancer biology by three-dimensional spheroids in vitro model.

BACKGROUND: Thyroid cancer (TC) is the most common endocrine malignancy. Nowadays, undifferentiated thyroid cancers (UTCs) are still lethal, mostly due to the insurgence of therapy resistance and disease relapse. These events are believed to be caused by a subpopulation of cancer cells with stem-like phenotype and specific tumor-initiating abilities, known as tumor-initiating cells (TICs). A comprehensive understanding of how to isolate and target these cells is necessary. Here we provide insights into the role that the protein Epithelial Cell Adhesion Molecule (EpCAM), a known TICs marker for other solid tumors, may have in TC biology, thus considering EpCAM a potential marker of thyroid TICs in UTCs. METHODS: The characterization of EpCAM was accomplished through Western Blot and Immunofluorescence on patient-derived tissue samples, adherent cell cultures, and 3D sphere cultures of poorly differentiated thyroid cancer (PDTC) and anaplastic thyroid cancer (ATC) cell lines. The frequency of tumor cells with putative tumor-initiating ability within the 3D cultures was assessed through extreme limiting dilution analysis (ELDA). EpCAM proteolytic cleavages were studied through treatments with different cleavages' inhibitors. To evaluate the involvement of EpCAM in inducing drug resistance, Vemurafenib (PLX-4032) treatments were assessed through MTT assay. RESULTS: Variable EpCAM expression pattern was observed in TC tissue samples, with increased cleavage in the more UTC. We demonstrated that EpCAM is subjected to an intense cleavage process in ATC-derived 3D tumor spheres and that the 3D model faithfully mimics what was observed in patient's samples. We also proved that the integrity of the protein appears to be crucial for the generation of 3D spheres, and its expression and cleavage in a 3D system could contribute to drug resistance in thyroid TICs. CONCLUSIONS: Our data provide novel information on the role of EpCAM expression and cleavage in the biology of thyroid TICs, and our 3D model reflects the variability of EpCAM cleavage observed in tissue samples. EpCAM evaluation could play a role in clinical decisions regarding patient therapy since its expression and cleavage may have a fundamental role in the switch to a drug-resistant phenotype of UTC cells.

Extra-nuclear TERT counteracts oxidative stress and promotes progression in papillary thyroid carcinoma.

The reactivation of TERT is associated with poor outcome in papillary thyroid cancer (PTC). Extra-telomeric functions of TERT were reported, with a protective role against oxidative stress (OS). The aim of the present study was to explore the extra-nuclear TERT localization in PTC and its role in cancer progression. TERT nuclear export under OS were analyzed in K1 PTC cell line. We investigated the role of different TERT localizations using specific TERT constructs that limit its localization to the nucleus or to the mitochondria. The effect of SRC kinase inhibitor PP2, which reduces TERT nuclear export, was investigated as well. Moreover, TERT localization was analyzed in 39 PTC tissues and correlated with the genetic profile and the level of OS, DNA damage and apoptosis in the tumors and with the clinical characteristics of the patients. We demonstrated that TERT is exported from the nucleus in response to OS induced either from H2O2 or the BRAF inhibitor PLX4720. We proved that extra-nuclear TERT reduces mitochondrial OS and induces mitochondrial fragmentation. Moreover, limiting mitochondrial TERT localization reduced proliferation, migration, AKT phosphorylation and glycolysis and increased DNA damage and p21 expression. Finally, in PTC tissues the fraction of mitochondrial/nuclear TERT resulted inversely correlated with OS and p21 expression and associated with tumor persistence. In conclusion, our data indicate that extra-nuclear TERT is involved in reducing the effect of excessive OS, thus promoting cancer cell survival. Extra-nuclear TERT may thus represent a marker of cancer progression and a possible therapeutic target in PTC.

Time-Activity data fitting in molecular Radiotherapy: Methodology and pitfalls.

Absorbed radiation doses are essential in assessing the effects, e.g. safety and efficacy, of radiopharmaceutical therapy (RPT). Patient-specific absorbed dose calculations in the target or the organ at risk require multiple inputs. These include the number of disintegrations in the organ, i.e. the time-integrated activities (TIAs) of the organs, as well as other parameters describing the process of radiation energy deposition in the target tissue (i.e. mean energy per disintegration, radiation dose constants, etc). TIAs are then estimated by incorporating the area under the radiopharmaceutical's time-activity curve (TAC), which can be obtained by quantitative measurements of the biokinetics in the patient (typically based on imaging data such as planar scintigraphy, SPECT/CT, PET/CT, or blood and urine samples). The process of TAC determination/calculation for RPT generally depends on the user, e.g., the chosen number and schedule of measured time points, the selection of the fit function, the error model for the data and the fit algorithm. These decisions can strongly affect the final TIA values and thus the accuracy of calculated absorbed doses. Despite the high clinical importance of the TIA values, there is currently no consensus on processing time-activity data or even a clear understanding of the influence of uncertainties and variations in personalised RPT dosimetry related to user-dependent TAC calculation. As a first step towards minimising site-dependent variability in RPT dosimetry, this work provides an overview of quality assurance and uncertainty management considerations of the TIA estimation.

On the use of solid 133Ba sources as surrogate for liquid 131I in SPECT/CT calibration: a European multi-centre evaluation.

INTRODUCTION: Commissioning, calibration, and quality control procedures for nuclear medicine imaging systems are typically performed using hollow containers filled with radionuclide solutions. This leads to multiple sources of uncertainty, many of which can be overcome by using traceable, sealed, long-lived surrogate sources containing a radionuclide of comparable energies and emission probabilities. This study presents the results of a quantitative SPECT/CT imaging comparison exercise performed within the MRTDosimetry consortium to assess the feasibility of using 133Ba as a surrogate for 131I imaging. MATERIALS AND METHODS: Two sets of four traceable 133Ba sources were produced at two National Metrology Institutes and encapsulated in 3D-printed cylinders (volume range 1.68-107.4 mL). Corresponding hollow cylinders to be filled with liquid 131I and a mounting baseplate for repeatable positioning within a Jaszczak phantom were also produced. A quantitative SPECT/CT imaging comparison exercise was conducted between seven members of the consortium (eight SPECT/CT systems from two major vendors) based on a standardised protocol. Each site had to perform three measurements with the two sets of 133Ba sources and liquid 131I. RESULTS: As anticipated, the 131I pseudo-image calibration factors (cps/MBq) were higher than those for 133Ba for all reconstructions and systems. A site-specific cross-calibration reduced the performance differences between both radionuclides with respect to a cross-calibration based on the ratio of emission probabilities from a median of 12-1.5%. The site-specific cross-calibration method also showed agreement between 133Ba and 131I for all cylinder volumes, which highlights the potential use of 133Ba sources to calculate recovery coefficients for partial volume correction. CONCLUSION: This comparison exercise demonstrated that traceable solid 133Ba sources can be used as surrogate for liquid 131I imaging. The use of solid surrogate sources could solve the radiation protection problem inherent in the preparation of phantoms with 131I liquid activity solutions as well as reduce the measurement uncertainties in the activity. This is particularly relevant for stability measurements, which have to be carried out at regular intervals.

Safety injections of nuclear medicine radiotracers: towards a new modality for a real-time detection of extravasation events and 18F-FDG SUV data correction.

BACKGROUND: 18F-FDG PET/CT imaging allows to study oncological patients and their relative diagnosis through the standardised uptake value (SUV) evaluation. During radiopharmaceutical injection, an extravasation event may occur, making the SUV value less accurate and possibly leading to severe tissue damage. The study aimed to propose a new technique to monitor and manage these events, to provide an early evaluation and correction to the estimated SUV value through a SUV correction coefficient. METHODS: A cohort of 70 patients undergoing 18F- FDG PET/CT examinations was enrolled. Two portable detectors were secured on the patients' arms. The dose-rate (DR) time curves on the injected DRin and contralateral DRcon arm were acquired during the first 10 min of injection. Such data were processed to calculate the parameters ΔpinNOR = (DRinmax- DRinmean)/DRinmax and ΔRt = (DRin(t) - DRcon(t)), where DRinmax is the maximum DR value, DRinmean is the average DR value in the injected arm. OLINDA software allowed dosimetric estimation of the dose in the extravasation region. The estimated residual activity in the extravasation site allowed the evaluation of the SUV's correction value and to define an SUV correction coefficient. RESULTS: Four cases of extravasations were identified for which ΔRt [(390 ± 26) µSv/h], while ΔRt [(150 ± 22) µSv/h] for abnormal and ΔRt [(24 ± 11) µSv/h] for normal cases. The ΔpinNOR showed an average value of (0.44 ± 0.05) for extravasation cases and an average value of (0.91 ± 0.06) and (0.77 ± 0.23) in normal and abnormal classes, respectively. The percentage of SUV reduction (SUV%CR) ranges between 0.3% and 6%. The calculated self-tissue dose values range from 0.027 to 0.573 Gy, according to the segmentation modality. A similar correlation between the inverse of ΔpinNOR and the normalised ΔRt with the SUV correction coefficient was found. CONCLUSIONS: The proposed metrics allowed to characterised the extravasation events in the first few minutes after the injection, providing an early SUV correction when necessary. We also assume that the characterisation of the DR-time curve of the injection arm is sufficient for the detection of extravasation events. Further validation of these hypotheses and key metrics is recommended in larger cohorts.

The length of FOXE1 polyalanine tract in congenital hypothyroidism: Evidence for a pathogenic role from familial, molecular and cohort studies.

Introduction: FOXE1 is required for thyroid function and its homozygous mutations cause a rare syndromic form of congenital hypothyroidism (CH). FOXE1 has a polymorphic polyalanine tract whose involvement in thyroid pathology is controversial. Starting from genetic studies in a CH family, we explored the functional role and involvement of FOXE1 variations in a large CH population. Methods: We applied NGS screening to a large CH family and a cohort of 1752 individuals and validated these results by in silico modeling and in vitro experiments. Results: A new heterozygous FOXE1 variant segregated with 14-Alanine tract homozygosity in 5 CH siblings with athyreosis. The p.L107V variant demonstrated to significantly reduce the FOXE1 transcriptional activity. The 14-Alanine-FOXE1 displayed altered subcellular localization and significantly impaired synergy with other transcription factors, when compared with the more common 16-Alanine-FOXE1. The CH group with thyroid dysgenesis was largely and significantly enriched with the 14-Alanine-FOXE1 homozygosity. Discussion: We provide new evidence that disentangle the pathophysiological role of FOXE1 polyalanine tract, thereby significantly broadening the perspective on the role of FOXE1 in the complex pathogenesis of CH. FOXE1 should be therefore added to the group of polyalanine disease-associated transcription factors.

FAM83B is involved in thyroid cancer cell differentiation and migration.

FAM83B has been recently identified as an oncogene, but its role in thyroid cancers (TC) is still unclear. We examined the expression of FAM83B and its possible involvement in cell migration and differentiation, in neoplastic/normal thyroid tissues and in TC human cell lines. FAM83B expression in TC varies according to the tumor histotype, being significantly downregulated in more aggressive and metastatic tissues. FAM83B levels in cell lines recapitulate patients' samples variations, and its total and cytoplasmic levels decrease upon the induction of migration, together with an increase in its nuclear localization. Similar variations were detected in the primary tumor and in the metastatic tissues from a follicular TC. FAM83B knock down experiments confirmed its role in thyroid differentiation and cell migration, as demonstrated by the reduction of markers of thyroid differentiation and the increase of the mesenchymal marker vimentin. Moreover, the silencing of FAM83B significantly increased cells migration abilities, while not affecting the oncogenic RAS/MAPK/PI3K pathways. Our data indicate for the first time a role for FAM83B in TC cell differentiation and migration. Its expression is reduced in dedifferentiated tumors and its nuclear re-localization could favour distant migration, suggesting that FAM83B should be considered a possible diagnostic and prognostic biomarker.

Emerging Roles of DLK1 in the Stem Cell Niche and Cancer Stemness.

DLK1 is a maternally imprinted, paternally expressed gene coding for the transmembrane protein Delta-like homologue 1 (DLK1), a non-canonical NOTCH ligand with well-described roles during development, and tumor-supportive functions in several aggressive cancer forms. Here, we review the many functions of DLK1 as a regulator of stem cell pools and tissue differentiation in tissues such as brain, muscle, and liver. Furthermore, we review recent evidence supporting roles for DLK1 in the maintenance of aggressive stem cell characteristics of tumor cells, specifically focusing on central nervous system tumors, neuroblastoma, and hepatocellular carcinoma. We discuss NOTCH -dependent as well as NOTCH-independent functions of DLK1, and focus particularly on the complex pattern of DLK1 expression and cleavage that is finely regulated from a spatial and temporal perspective. Progress in recent years suggest differential functions of extracellular, soluble DLK1 as a paracrine stem cell niche-secreted factor, and has revealed a role for the intracellular domain of DLK1 in cell signaling and tumor stemness. A better understanding of DLK1 regulation and signaling may enable therapeutic targeting of cancer stemness by interfering with DLK1 release and/or intracellular signaling.

Role of Epigenetic Therapy in the Modulation of Tumor Growth and Migration in Human Castration-Resistant Prostate Cancer Cells with Neuroendocrine Differentiation.

INTRODUCTION: Neuroendocrine transdifferentiation (NED) of prostate cancer (PC) cells is associated with the development of resistance to antiandrogen therapy and poor prognosis in patients with castration-resistant PC (CRPC). Many of the molecular events, involved in NED, appear to be mediated by epigenetic mechanisms. In this study, we evaluated the antitumor activity and epigenetic modulation of 2 epigenetic drugs, such as the demethylating agent 5-aza-2'-deoxycytidine (AZA) and the methyl donor S-adenosylmethionine (SAM), in 2 human CRPC cell lines with NED (DU-145 and PC-3). METHODS: The effects of AZA and SAM on cell viability, cell cycle, apoptosis, migration, and genome-wide DNA methylation profiling have been evaluated. RESULTS: Both drugs showed a prominent antitumor activity in DU-145 and PC-3 cells, through perturbation of cell cycle progression, induction of apoptosis, and inhibition of cell migration. AZA and SAM reversed NED in DU-145 and PC-3, respectively. Moreover, AZA treatment modified DNA methylation pattern in DU-145 cells, sustaining a pervasive hypomethylation of the genome, with a relevant effect on several pathways involved in the regulation of cell proliferation, apoptosis, and cell migration, in particular Wnt/ß-catenin. CONCLUSIONS: A relevant antitumor activity of these epigenetic drugs on CRPC cell lines with NED opens a new scenario in the therapy of this lethal variant of PC.

Biomechanical, operative and biological aspects of the cervical margin relocation: a case report / Aspectos biomecânicos, operatórios e biológicos da recolocação da margem cervical com resina composta em cavidades classe II com margens profundas: um relato de caso

The clinical success of tooth-colored indirect restorations has been confirmed in several studies. However, inlays and onlays restorations in Class II cavities with deep gingival margins can still be considered a clinical challenge. With the purpose of facilitating the execution of the operative procedures in intrasulcular margins and reducing the risk of restorative failures, the technique of cervical margin relocation has been explored as a noninvasive alternative to surgical crown lengthening. This work aims at discussing through a case report the biomechanical, operative and biological aspects in the treatment of teeth with deep gingival margins. Therefore, given the therapy applied in the clinical case presented, it is concluded that the cervical margin relocation with composite resin is advantageous since it eliminates the need for surgery, allowing the implementation of indirect restorations in fewer clinical sessions, not causing damage to periodontal tissues once it provided good finishing and polishing with the establishment of a correct emergence profile, allowing flawless maintenance of gingival health after one year. (AU)

Sucesso clínico das restaurações indiretas livres de metal tem sido confirmado em diversos estudos. No entanto, restaurações parciais indiretas em cavidades do tipo classe II com margens profundas ainda podem ser consideradas um desafio clínico. Com a proposta de facilitar a execução dos procedimentos operatórios em margens intra-sulculares e reduzir a ocorrência de falhas, a técnica de elevação da margem gengival em resina composta tem sido explorada como alternativa não invasiva à cirurgia de aumento de coroa clínica. Este trabalho tem a intenção de discutir através de um relato de caso clínico os aspectos biomecânicos, operatórios e biológicos no tratamento de dentes com margens cervicais profundas. Sendo assim, conclui-se que a técnica de elevação da margem gengival com resina composta é vantajosa, pois elimina a necessidade de cirurgia permitindo a execução de restaurações indiretas em menos sessões clínicas, não gerando danos aos tecidos periodontais, desde que haja um bom acabamento e polimento, com estabelecimento de um correto perfil de emergência. (AU)

Current Therapeutic Strategies in BRAF-Mutant Metastatic Colorectal Cancer.

Around 8-12% of patients with advanced colon rectal cancer (CRC) present with BRAF alterations, in particular V600E mutation, which is associated with right-side, poorly differentiated and mucinous type tumors. The presence of BRAF mutation (BRAF-mt) has been identified as a hallmark of poor prognosis and treatment optimization in this patient subgroup is an important goal. Currently, the standard of care is an aggressive strategy involving triplet chemotherapy and anti-VEGF agents, but new therapeutic approaches are emerging. Very promising results have been obtained with targeted therapy combinations, such as anti-BRAF agents plus anti-EGFR agents. Furthermore, around 60% of BRAF-mt patients show a strong association with high microsatellite instability (MSI-H) and immune checkpoint inhibitors could represent the new standard of care for this subgroup. The focus of this review is to summarize current strategies for BRAF-mt CRC treatment and highlight new therapeutic options.

A multicentre and multi-national evaluation of the accuracy of quantitative Lu-177 SPECT/CT imaging performed within the MRTDosimetry project.

PURPOSE: Patient-specific dosimetry is required to ensure the safety of molecular radiotherapy and to predict response. Dosimetry involves several steps, the first of which is the determination of the activity of the radiopharmaceutical taken up by an organ/lesion over time. As uncertainties propagate along each of the subsequent steps (integration of the time-activity curve, absorbed dose calculation), establishing a reliable activity quantification is essential. The MRTDosimetry project was a European initiative to bring together expertise in metrology and nuclear medicine research, with one main goal of standardizing quantitative 177Lu SPECT/CT imaging based on a calibration protocol developed and tested in a multicentre inter-comparison. This study presents the setup and results of this comparison exercise. METHODS: The inter-comparison included nine SPECT/CT systems. Each site performed a set of three measurements with the same setup (system, acquisition and reconstruction): (1) Determination of an image calibration for conversion from counts to activity concentration (large cylinder phantom), (2) determination of recovery coefficients for partial volume correction (IEC NEMA PET body phantom with sphere inserts), (3) validation of the established quantitative imaging setup using a 3D printed two-organ phantom (ICRP110-based kidney and spleen). In contrast to previous efforts, traceability of the activity measurement was required for each participant, and all participants were asked to calculate uncertainties for their SPECT-based activities. RESULTS: Similar combinations of imaging system and reconstruction lead to similar image calibration factors. The activity ratio results of the anthropomorphic phantom validation demonstrate significant harmonization of quantitative imaging performance between the sites with all sites falling within one standard deviation of the mean values for all inserts. Activity recovery was underestimated for total kidney, spleen, and kidney cortex, while it was overestimated for the medulla. CONCLUSION: This international comparison exercise demonstrates that harmonization of quantitative SPECT/CT is feasible when following very specific instructions of a dedicated calibration protocol, as developed within the MRTDosimetry project. While quantitative imaging performance demonstrates significant harmonization, an over- and underestimation of the activity recovery highlights the limitations of any partial volume correction in the presence of spill-in and spill-out between two adjacent volumes of interests.

Thyroid Cancer Stem-Like Cells: From Microenvironmental Niches to Therapeutic Strategies.

Thyroid cancer (TC) is the most common endocrine malignancy. Recent progress in thyroid cancer biology revealed a certain degree of intratumoral heterogeneity, highlighting the coexistence of cellular subpopulations with distinct proliferative capacities and differentiation abilities. Among those subpopulations, cancer stem-like cells (CSCs) are hypothesized to drive TC heterogeneity, contributing to its metastatic potential and therapy resistance. CSCs principally exist in tumor areas with specific microenvironmental conditions, the so-called stem cell niches. In particular, in thyroid cancer, CSCs' survival is enhanced in the hypoxic niche, the immune niche, and some areas with specific extracellular matrix composition. In this review, we summarize the current knowledge about thyroid CSCs, the tumoral niches that allow their survival, and the implications for TC therapy.

Pancreatic mucinous cystadenocarcinoma in a patient harbouring BRCA1 germline mutation effectively treated with olaparib: A case report.

BACKGROUND: Pancreatic mucinous cystadenocarcinoma (MCAC) is a rare malignancy with a poor prognosis when it presents metastases at diagnosis. Due to its very low incidence, there are no clear recommendations for the treatment of advanced disease. Olaparib (an oral PARP inhibitor) has been approved for the maintenance treatment of patients with metastatic pancreatic adenocarcinoma harbouring germline BRCA1/2 mutations. Herein, we report the first case of a germline BRCA1 mutated unresectable MCAC which was effectively treated with olaparib. CASE SUMMARY: A 41-year-old woman, without personal or family history of cancer, was diagnosed with ovarian and peritoneal metastases of MCAC. She underwent 12 cycles of gemcitabine plus oxaliplatin (GEMOX) obtaining a partial response and allowing radical surgery. One year later, local recurrence was documented, and other 12 cycles of GEMOX were administered obtaining a complete response. Seven years later, another local recurrence, not amenable to surgical resection, was diagnosed. She started FOLFIRINOX (oxaliplatin, irinotecan, leucovorin and fluorouracil), obtaining a partial response after 8 cycles. Given the excellent response to platinum-based chemotherapy, BRCA testing was performed, and a BRCA1 germline mutation was detected. She was switched to maintenance olaparib due to chemotherapy-related toxicities and achieved an almost complete metabolic response, with a reduction in the diameter of the lesion, after three months of therapy. CONCLUSION: The current case suggests the beneficial effect of olaparib in BRCA mutated MCAC. However, further studies are required.

Niche-derived soluble DLK1 promotes glioma growth.

Tumor cell behaviors associated with aggressive tumor growth such as proliferation, therapeutic resistance, and stem cell characteristics are regulated in part by soluble factors derived from the tumor microenvironment. Tumor-associated astrocytes represent a major component of the glioma tumor microenvironment, and astrocytes have an active role in maintenance of normal neural stem cells in the stem cell niche, in part via secretion of soluble delta-like noncanonical Notch ligand 1 (DLK1). We found that astrocytes, when exposed to stresses of the tumor microenvironment such as hypoxia or ionizing radiation, increased secretion of soluble DLK1. Tumor-associated astrocytes in a glioma mouse model expressed DLK1 in perinecrotic and perivascular tumor areas. Glioma cells exposed to recombinant DLK1 displayed increased proliferation, enhanced self-renewal and colony formation abilities, and increased levels of stem cell marker genes. Mechanistically, DLK1-mediated effects on glioma cells involved increased and prolonged stabilization of hypoxia-inducible factor 2alpha, and inhibition of hypoxia-inducible factor 2alpha activity abolished effects of DLK1 in hypoxia. Forced expression of soluble DLK1 resulted in more aggressive tumor growth and shortened survival in a genetically engineered mouse model of glioma. Together, our data support DLK1 as a soluble mediator of glioma aggressiveness derived from the tumor microenvironment.

Uncertainty analysis of tumour absorbed dose calculations in molecular radiotherapy.

BACKGROUND: Internal dosimetry evaluation consists of a multi-step process ranging from imaging acquisition to absorbed dose calculations. Assessment of uncertainty is complicated and, for that reason, it is commonly ignored in clinical routine. However, it is essential for adequate interpretation of the results. Recently, the EANM published a practical guidance on uncertainty analysis for molecular radiotherapy based on the application of the law of propagation of uncertainty. In this study, we investigated the overall uncertainty on a sample of a patient following the EANM guidelines. The aim of this study was to provide an indication of the typical uncertainties that may be expected from performing dosimetry, to determine parameters that have the greatest effect on the accuracy of calculations and to consider the potential improvements that could be made if these effects were reduced. RESULTS: Absorbed doses and the relative uncertainties were calculated for a sample of 49 patients and a total of 154 tumours. A wide range of relative absorbed dose uncertainty values was observed (14-102%). Uncertainties associated with each quantity along the absorbed dose calculation chain (i.e. volume, recovery coefficient, calibration factor, activity, time-activity curve fitting, time-integrated activity and absorbed dose) were estimated. An equation was derived to describe the relationship between the uncertainty in the absorbed dose and the volume. The largest source of error was the VOI delineation. By postulating different values of FWHM, the impact of the imaging system spatial resolution on the uncertainties was investigated. DISCUSSION: To the best of our knowledge, this is the first analysis of uncertainty in molecular radiotherapy based on a cohort of clinical cases. Wide inter-lesion variability of absorbed dose uncertainty was observed. Hence, a proper assessment of the uncertainties associated with the calculations should be considered as a basic scientific standard. A model for a quick estimate of uncertainty without implementing the entire error propagation schema, which may be useful in clinical practice, was presented. Ameliorating spatial resolution may be in future the key factor for accurate absorbed dose assessment.

Comparison of different calculation techniques for absorbed dose assessment in patient specific peptide receptor radionuclide therapy.

AIM: The present work concerns the comparison of the performances of three systems for dosimetry in RPT that use different techniques for absorbed dose calculation (organ-level dosimetry, voxel-level dose kernel convolution and Monte Carlo simulations). The aim was to assess the importance of the choice of the most adequate calculation modality, providing recommendations about the choice of the computation tool. METHODS: The performances were evaluated both on phantoms and patients in a multi-level approach. Different phantoms filled with a 177Lu-radioactive solution were used: a homogeneous cylindrical phantom, a phantom with organ-shaped inserts and two cylindrical phantoms with inserts different for shape and volume. A total of 70 patients with NETs treated by PRRT with 177Lu-DOTATOC were retrospectively analysed. RESULTS: The comparisons were performed mainly between the mean values of the absorbed dose in the regions of interest. A general better agreement was obtained between Dose kernel convolution and Monte Carlo simulations results rather than between either of these two and organ-level dosimetry, both for phantoms and patients. Phantoms measurements also showed the discrepancies mainly depend on the geometry of the inserts (e.g. shape and volume). For patients, differences were more pronounced than phantoms and higher inter/intra patient variability was observed. CONCLUSION: This study suggests that voxel-level techniques for dosimetry calculation are potentially more accurate and personalized than organ-level methods. In particular, a voxel-convolution method provides good results in a short time of calculation, while Monte Carlo based computation should be conducted with very fast calculation systems for a possible use in clinics, despite its intrinsic higher accuracy. Attention to the calculation modality is recommended in case of clinical regions of interest with irregular shape and far from spherical geometry, in which Monte Carlo seems to be more accurate than voxel-convolution methods.

AtezoTRIBE: a randomised phase II study of FOLFOXIRI plus bevacizumab alone or in combination with atezolizumab as initial therapy for patients with unresectable metastatic colorectal cancer.

BACKGROUND: Immune checkpoint inhibitors (ICIs) reported remarkable achievements in several solid tumours. However, in metastatic colorectal cancer (mCRC) promising results are limited to patients with deficient mismatch repair/microsatellite instability-high (dMMR/MSI-high) tumours due to their immune-enriched microenvironment. Combining cytotoxic agents and bevacizumab in mCRC with proficient mismatch repair/microsatellite stability (pMMR/MSS) could make ICIs efficacious by increasing the exposure of neoantigens, especially with highly active chemotherapy regimens, inducing immunogenic cell death, increasing the tumoral infiltration of CD8+ T-cells and reducing tumour-associated myeloid-derived suppressor cells. VEGF-blockade also plays an immunomodulatory role by inhibiting the expansion of T regulatory lymphocytes. Consistently with this rationale, a phase Ib study combined the anti-PDL-1 atezolizumab with FOLFOX/bevacizumab as first-line treatment of mCRC, irrespective of microsatellite status, and reported interesting activity and efficacy results, without safety concerns. Phase III trials led to identify FOLFOXIRI plus bevacizumab as an upfront therapeutic option in selected mCRC patients. Drawing from these considerations, the combination of atezolizumab with an intensified upfront treatment (FOLFOXIRI) and bevacizumab could be worthy of investigation. METHODS: AtezoTRIBE is a prospective, open label, phase II, comparative trial in which initially unresectable and previously untreated mCRC patients, irrespective of microsatellite status, are randomized in a 1:2 ratio to receive up to 8 cycles of FOLFOXIRI/bevacizumab alone or in combination with atezolizumab, followed by maintenance with bevacizumab plus 5-fluoruracil/leucovorin with or without atezolizumab according to treatment arm until disease progression. The primary endpoint is PFS. Assuming a median PFS of 12 months for standard arm, 201 patients should be randomized in a 1:2 ratio to detect a hazard ratio of 0.66 in favour of the experimental arm. A safety run-in phase including the first 6 patients enrolled in the FOLFOXIRI/bevacizumab/atezolizumab arm was planned, and no unexpected adverse events or severe toxicities were highlighted by the Safety Monitoring Committee. DISCUSSION: The AtezoTRIBE study aims at assessing whether the addition of atezolizumab to an intensified chemotherapy plus bevacizumab might be an efficacious upfront strategy for the treatment of mCRC, irrespective of the microsatellite status. TRIAL REGISTRATION: AtezoTRIBE is registered at Clinicaltrials.gov ( NCT03721653 ), October 26th, 2018 and at EUDRACT (2017-000977-35), Februray 28th, 2017.

Long-term survival of two patients with recurrent pancreatic acinar cell carcinoma treated with radiofrequency ablation: A case report.

BACKGROUND: Pancreatic acinar cell carcinoma (PACC) is a rare type of malignant pancreatic cancer that represents approximately 1% of all pancreatic neoplasms. Due to its very low incidence, only a few retrospective studies are available. Although surgery is the first choice for treatment, most patients experience recurrence (mainly in the liver) and there are no clear recommendations for patients with advanced disease. CASE SUMMARY: We report two patients with PACC treated with surgery who experienced tumour recurrence in the liver. Patient 1 carried a germline mutation in the APC gene. Both patients were treated with gemcitabine plus oxaliplatin and gemcitabine plus capecitabine as first- and second-line therapies, respectively. After a favourable response to chemotherapy, the patients underwent radiofrequency ablation of the remaining liver metastases. For patient 1, we documented a relapse in the liver after a disease-free period of 9 mo, and treatment with gemcitabine plus capecitabine was restarted. The patient achieved a complete response, and he remains alive without evidence of disease recurrence after six years. After radiofrequency ablation, patient 2 experienced disease-free survival for 21 mo, when peritoneal relapse was diagnosed and treated with chemotherapy. The patient achieved a stable disease state for nearly two years; nevertheless, further progressive disease was documented, and he died seven years after the first relapse. CONCLUSION: PACC presents different biological behaviours than pancreatic adenocarcinoma. Multidisciplinary treatment involving local ablative therapies may be considered for PACC.

Hypoxia-induced release, nuclear translocation, and signaling activity of a DLK1 intracellular fragment in glioma.

Glioblastoma multiforme is characterized in part by severe hypoxia associated with tumor necrosis. The cellular response to hypoxia can influence several properties of tumor cells associated with aggressive tumor growth, including metabolic adaptations and tumor cell migration and invasion. Here, we found that Delta Like Non-Canonical Notch Ligand 1 (DLK1) expression was elevated as compared with normal brain in a genetically engineered mouse model of glioma, and that DLK1 expression increased with tumor grade in human glioma samples. DLK1 expression was highest in hypoxic and perivascular tumor areas, and we found that hypoxia induced the release and nuclear translocation of an intracellular fragment of DLK1 in murine glioma as well as in human glioma cultures. Release of the intracellular fragment was dependent on ADAM17 and Hypoxia-inducible Factor 1alpha and 2alpha (HIF-1alpha/HIF-2alpha), as ADAM17 inhibitors and HIF1A/HIF2A siRNA blocked DLK1 cleavage. Expression of a cleavable form of DLK1 amplified several hypoxia-induced traits of glioma cells such as colony formation, stem cell marker gene expression, a PI3K-pathway-mediated metabolic shift, and enhanced invasiveness. Effects of DLK1 were dependent on DLK1-cleavage by ADAM17, as expression of non-cleavable DLK1 could not replicate the DLK1-induced hypoxic phenotype. Finally, forced expression of DLK1 resulted in more invasive tumor growth in a PDGFB-induced glioma mouse model without affecting overall survival. Together, our findings suggest a previously undescribed role for DLK1 as an intracellular signaling molecule.