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Biodistribution analysis of 89Zr-labelled anti-Gal3 mAb in three different animal models.

The high prevalence of thyroid nodules in the adult population and the relatively low incidence of thyroid cancer make the preoperative identification of malignant lesions challenging. The β-galactoside–binding protein galectin-3 is widely expressed in well-differentiated thyroid carcinomas, but not in normal thyrocytes and benign thyroid nodules. This molecule offers a candidate biomarker to improve thyroid cancer diagnosis. Here we report the development of an immunoPET approach for noninvasive imaging of thyroid cancer. The method employs a 89Zr-labeled mAb to galectin-3, which shows high specificity and binding affinity in vitro. Reliable and specific immunoPET imaging was obtained of thyroid cancer in vivo in murine xenograft models of human thyroid cancer. Our findings provide a method to improve the clinical management of patients with thyroid nodules while reducing unnecessary surgery and social costs. Cancer Res; 76(12); 3583–92. ©2016 AACR.

Description of additional methods and procedures used in the study.

Immunohistochemical analysis of galectin-3 expression in normal tissues ex vivo.

Legends for Supplementary Figures S1-S2.

Background Recent developments in abscopal effect strongly support the use of radiotherapy for the treatment of metastatic disease. However, deeper understanding of the molecular mechanisms underlying the abscopal effect are required to best benefit a larger proportion of patients with metastasis. Several groups including ours, reported the involvement of wild-type (wt) p53 in radiation-induced abscopal effects, however very little is known on the role of wtp53 dependent molecular mechanisms. Methods We investigated through in vivo and in vitro approaches how wtp53 orchestrates radiation-induced abscopal effects. Wtp53 bearing (A549) and p53-null (H1299) NSCLC lines were xenotransplanted in nude mice, and cultured in 2D monolayers and 3D tumor spheroids. Extracellular vesicles (EVs) were isolated from medium cell culture by ultracentrifugation protocol followed by Nanoparticle Tracking Analysis. Gene expression was evaluated by RT-Real Time, digital qRT-PCR, and dot blot technique. Protein levels were determined by immunohistochemistry, confocal anlysis, western blot techniques, and immunoassay. Results We demonstrated that single high-dose irradiation (20 Gy) induces significant tumor growth inhibition in contralateral non-irradiated (NIR) A549 xenograft tumors but not in NIR p53-null H1299 or p53-silenced A549 (A549sh/p53) xenografts. We further demonstrates that irradiation of A549 cells in vitro induces a senescence-associated secretory phenotype (SASP) producing extracellular vesicles (EVs) expressing CD63 and carrying DNA:RNA hybrids and LINE-1 retrotransposon. IR-A549 EVs also hamper the colony-forming capability of recipient NIR A549 cells, induce senescent phenotype, nuclear expression of DNA:RNA hybrids, and M1 macrophage polarization. Conclusions In our models, we demonstrate that high radiation dose in wtp53 tumors induce the onset of SASP and secretion of CD63+ EVs loaded with DNA:RNA hybrids and LINE-1 retrotransposons that convey senescence messages out of the irradiation field triggering abscopal effect in NIR tumors.

Mammalian development, adult tissue homeostasis and the avoidance of severe diseases including cancer require a properly orchestrated cell cycle, as well as error-free genome maintenance. The key cell-fate decision to replicate the genome is controlled by two major signalling pathways that act in parallel—the MYC pathway and the cyclin D–cyclin-dependent kinase (CDK)–retinoblastoma protein (RB) pathway1,2. Both MYC and the cyclin D–CDK–RB axis are commonly deregulated in cancer, and this is associated with increased genomic instability. The autophagic tumour-suppressor protein AMBRA1 has been linked to the control of cell proliferation, but the underlying molecular mechanisms remain poorly understood. Here we show that AMBRA1 is an upstream master regulator of the transition from G1 to S phase and thereby prevents replication stress. Using a combination of cell and molecular approaches and in vivo models, we reveal that AMBRA1 regulates the abundance of D-type cyclins by mediating their degradation. Furthermore, by controlling the transition from G1 to S phase, AMBRA1 helps to maintain genomic integrity during DNA replication, which counteracts developmental abnormalities and tumour growth. Finally, we identify the CHK1 kinase as a potential therapeutic target in AMBRA1-deficient tumours. These results advance our understanding of the control of replication-phase entry and genomic integrity, and identify the AMBRA1–cyclin D pathway as a crucial cell-cycle-regulatory mechanism that is deeply interconnected with genomic stability in embryonic development and tumorigenesis. AMBRA1-mediated degradation of cyclin D through CRL4–DDB1 regulates cell proliferation and prevents replication stress in neurodevelopment and cancer.

Differentiated thyroid cancer (DTC) cells may lose NIS expression and iodine uptake, but usually express TSH receptors (TSHR). Therefore, the aim of our study was to compare two radiolabeled superagonist TSH analogues for DTC imaging. These analogues (namely TR1401 and TR1402) have a higher TSHR binding affinity than recombinant human TSH (Thyrogen®). Radiolabeling was performed with technetium-99m using an indirect method via HYNIC conjugation and was followed by in vitro quality controls and binding assay on TSHR-positive cell lines (ML-1). An in vitro binding assay was also performed and compared with radiolabeled human recombinant TSH. In vivo imaging was performed in four dogs with spontaneous follicular thyroid carcinoma with solid poorly differentiated areas with 99mTc-TR1401 SPECT/CT, 99mTc-TR1402 SPECT/CT, and [18F]FDG PET/CT on different days within 2 weeks. TR1401 and TR1402 were labeled with high specific activity (8.3 ± 1.2 MBq/µg) and retention of their biological activity and structural integrity. Both agonists were able to efficiently bind TSHR receptors expressed by cell lines with dissociation constants (Kd) of 2.7 nM for 99mTc-TR1401 and 0.5 nM for 99mTc-TR1402 compared with 99mTc-Thyrogen (Kd = 8.4 nM). In tumor-targeting experiments, a focal uptake was observed in dogs with spontaneous intraglandular thyroid carcinoma, in which TSHR expression was confirmed by immunohistochemistry. 99mTc-TR1402 provided higher T/B than 99mTc-TR1401 and [18F]FDG (12.9 ± 1.3, 10.2 ± 0.7, and 3.8 ± 0.6, respectively, all p &lt, 0.001). Given these results, 99mTc-TR1402 appears to be a useful tool for in vivo imaging of thyroid cancer.

MYCN drives aggressive behavior and refractoriness to chemotherapy, in several tumors. Since MYCN inactivation in clinical settings is not achievable, alternative vulnerabilities of MYCN-driven tumors need to be explored to identify more effective and less toxic therapies. We previously demonstrated that PARP inhibitors enhance MYCN-induced replication stress and promote mitotic catastrophe, counteracted by CHK1. Here, we showed that PARP and CHK1 inhibitors synergized to induce death in neuroblastoma cells and in primary cultures of SHH-dependent medulloblastoma, their combination being more effective in MYCN amplified and MYCN overexpressing cells compared to MYCN non-amplified cells. Although the MYCN amplified IMR-32 cell line carrying the p.Val2716Ala ATM mutation showed the highest sensitivity to the drug combination, this was not related to ATM status, as indicated by CRISPR/Cas9-based correction of the mutation. Suboptimal doses of the CHK1 inhibitor MK-8776 plus the PARP inhibitor olaparib led to a MYCN-dependent accumulation of DNA damage and cell death in vitro and significantly reduced the growth of four in vivo models of MYCN-driven tumors, without major toxicities. Our data highlight the combination of PARP and CHK1 inhibitors as a new potential chemo-free strategy to treat MYCN-driven tumors, which might be promptly translated into clinical trials.

The lack of a non-invasive test for malignant thyroid nodules makes the diagnosis of thyroid cancer (TC) challenging. Human galectin-3 (hGal3) has emerged as a promising target for medical TC imaging and diagnosis because of its exclusive overexpression in malignant thyroid tissues. We previously developed a human-chimeric αhGal3 Fab fragment derived from the rat monoclonal antibody (mAb) M3/38 with optimized clearance characteristics using PASylation technology. Here, we describe the elucidation of the hGal3 epitope recognized by mAb M3/38, X-ray crystallographic analysis of its complex with the chimeric Fab and, based on the three-dimensional structure, the rational humanization of the Fab by CDR grafting. Four CDR-grafted versions were designed using structurally most closely related fully human immunoglobulin VH/VL regions of which one—employing the acceptor framework regions of the HIV-1 neutralizing human antibody m66—showed the highest antigen affinity. By introducing two additional back-mutations to the rodent donor sequence, an affinity toward hGal3 indistinguishable from the chimeric Fab was achieved (KD = 0.34 ± 0.02 nM in SPR). The PASylated humanized Fab was site-specifically labelled with the fluorescent dye Cy7 and applied for the immuno-histochemical staining of human tissue sections representative for different TCs. The same protein was conjugated with the metal chelator Dfo, followed by radiolabelling with 89Zr(IV). The resulting protein tracer allowed the highly sensitive and specific PET/CT imaging of orthotopic tumors in mice, which was confirmed by quantitative analysis of radiotracer accumulation. Thus, the PASylated humanized αhGal3 Fab offers clinical potential for the diagnostic imaging of TC.

Rationale: The high expression of Galectin-3 (Gal3) in macrophages of atherosclerotic plaques suggests its participation in atherosclerosis pathogenesis, and raises the possibility to use it as a target to image disease severity in vivo. Here, we explored the feasibility of tracking atherosclerosis by targeting Gal3 expression in plaques of apolipoprotein E knockout (ApoE-KO) mice via PET imaging. Methods: Targeting of Gal3 in M0-, M1- and M2 (M2a/M2c)-polarized macrophages was assessed in vitro using a Gal3-F(ab')2 mAb labeled with AlexaFluor®488 and 89Zr- desferrioxamine-thioureyl-phenyl-isothiocyanate (DFO). To visualize plaques in vivo, ApoE-KO mice were injected i.v. with 89Zr-DFO-Gal3-F(ab')2 mAb and imaged via PET/CT 48 h post injection. Whole length aortas harvested from euthanized mice were processed for Sudan-IV staining, autoradiography, and immunostaining for Gal3, CD68 and α-SMA expression. To confirm accumulation of the tracer in plaques, ApoE-KO mice were injected i.v. with Cy5.5-Gal3-F(ab')2 mAb, euthanized 48 h post injection, followed by cryosections of the body and acquisition of fluorescent images. To explore the clinical potential of this imaging modality, immunostaining for Gal3, CD68 and α-SMA expression were carried out in human plaques. Single cell RNA sequencing (scRNA-Seq) analyses were performed to measure LGALS3 (i.e. a synonym for Gal3) gene expression in each macrophage of several subtypes present in murine or human plaques. Results: Preferential binding to M2 macrophages was observed with both AlexaFluor®488-Gal3-F(ab')2 and 89Zr-DFO-Gal3-F(ab')2 mAbs. Focal and specific 89Zr-DFO-Gal3-F(ab')2 mAb uptake was detected in plaques of ApoE-KO mice by PET/CT. Autoradiography and immunohistochemical analyses of aortas confirmed the expression of Gal3 within plaques mainly in macrophages. Moreover, a specific fluorescent signal was visualized within the lesions of vascular structures burdened by plaques in mice. Gal3 expression in human plaques showed similar Gal3 expression patterns when compared to their murine counterparts. Conclusions: Our data reveal that 89Zr-DFO-Gal3-F(ab')2 mAb PET/CT is a potentially novel tool to image atherosclerotic plaques at different stages of development, allowing knowledge-based tailored individual intervention in clinically significant disease.

Preoperative characterization of thyroid nodules is challenging since thyroid scintigraphy fails to distinguish between benign and malignant lesions. Galectin-3 (gal-3) is expressed in well-differentiated and in undifferentiated thyroid cancer types but not in normal thyrocytes and benign thyroid lesions. Herein, we aimed to validate gal-3 targeting as a specific method to detect non–radioiodine-avid thyroid cancer in thyroid orthotopic tumor models. Methods: Papillary (BcPAP) and anaplastic (CAL62 and FRO82-1) thyroid carcinoma cell lines were characterized via Western blot and polymerase chain reaction for gal-3 and sodium-iodide symporter (NIS) expression. An (89)Zr-labeled F(ab′)(2) antigal-3 was generated and characterized for binding versus (125)I on 2- and 3-dimensional cell cultures. The thyroid carcinoma cells were inoculated into the left thyroid lobe of athymic nude mice, and the orthotopic tumor growth was monitored via ultrasound and fluorescence molecular tomography. Head-to-head PET/CT comparison of (124)I versus (89)Zr-deferoxamine (DFO)-F(ab′)(2) antigal-3 was performed, followed by biodistribution studies and immunohistochemical analysis for gal-3 and NIS expression. Results: The thyroid carcinoma cells investigated were invariably gal-3–positive while presenting low or lost NIS expression. (89)Zr-DFO-F(ab′)(2) antigal-3 tracer showed high affinity to gal-3 (dissociation constant, ∼3.9 nM) and retained immunoreactivity (>75%) on 2-dimensional cell cultures and on tumor spheroids. (125)I internalization in FRO82-1, BcPAP, and CAL62 was directly dependent on NIS expression, both in 2-dimensional and tumor spheroids. PET/CT imaging showed (89)Zr-DFO-F(ab′)(2) antigal-3 signal associated with the orthotopically implanted tumors only; no signal was detected in the tumor-free thyroid lobe. Conversely, PET imaging using (124)I showed background accumulation in tumor-infiltrated lobe, a condition simulating the presence of non–radioiodine-avid thyroid cancer nodules, and high accumulation in normal thyroid lobe. Imaging data were confirmed by tracer biodistribution studies and immunohistochemistry. Conclusion: A specific and selective visualization of thyroid tumor by targeting gal-3 was demonstrated in the absence of radioiodine uptake. Translation of this method into the clinical setting promises to improve the management of patients by avoiding the use of unspecific imaging methodologies and reducing unnecessary thyroid surgery.

Colorectal cancer (CRC) is one of the most common malignant tumors worldwide and understanding its underlying molecular mechanisms is crucial for the development of therapeutic strategies. The mitogen-activated protein kinase-kinase 3 (MKK3) is a specific activator of p38 MAP kinases (p38 MAPKs), which contributes to the regulation of several cellular functions, such as proliferation, differentiation, apoptosis as well as response to drugs. At present, the exact MKK3/p38 MAPK pathway contribution in cancer is heavily debated because of its pleiotropic function. In this work, we retrospectively explored the prognostic and pathobiologic relevance of MKK3 in a cohort of CRC patients and assessed MKK3 molecular functions in a panel of CRC lines and colonocytes primary cultures. We found increased MKK3 levels in late-stage CRC patients which correlated with shorter overall survival. Herein, we report that the MKK3 targeting by inducible RNA interference univocally exerts antitumor effects in CRC lines but not in primary colonocytes. While MKK3 depletion per se affects growth and survival by induction of sustained autophagy and death in some CRC lines, it potentiates response to chemotherapeutic drug 5-fluorouracil (5-FU) in all of the tested CRC lines in vitro. Here, we demonstrate for the first time that in CRC the MKK3 specifically activates p38delta MAPK isoform to sustain prosurvival signaling and that such effect is exacerbated upon 5-FU challenge. Indeed, p38delta MAPK silencing recapitulates MKK3 depletion effects in CRC cells in vitro and in vivo. Overall, our data identified a molecular mechanism through which MKK3 supports proliferation and survival signaling in CRC, further supporting MKK3 as a novel and extremely attractive therapeutic target for the development of promising strategies for the management of CRC patients.

Checkpoint inhibitor-based immunotherapy is opening a promising scenario in oncology, with objective responses registered in multiple cancer types. However, reliable predictive markers of tumor responsiveness are still lacking. These markers need to be urgently identified for a better selection of patients that can be candidates for immunotherapy. In this pilot study, a cohort of 34 consecutive patients bearing programmed death-ligand 1 (PD-L1)-positive non-small cell lung carcinoma (NSCLC), treated with pembrolizumab, was considered. The retrospective immuno-phenotypic analysis performed on the original tumor biopsies allowed for the identification of a specific “galectin signature”, which strongly correlated with tumor responsiveness to anti PD-1 immunotherapy. We observed that the large majority of patients (about 90%) with high galectin-3 tumor expression (score 3+) showed an early and dramatic progression of the disease after three cycles of treatments. In contrast, all patients with negative or low/intermediate expression of galectin-3 in tumor cells showed an early and durable objective response to pembrolizumab, indicating galectin-3 as an interesting predictive marker of tumor responsiveness. The galectin-3 signature, at least in NSCLCs, promises a better selection of patient candidates for immunotherapy, reducing unnecessary treatment exposures and social costs. A large multicenter study is ongoing to validate this finding.

Sinonasal melanomas are rare neoplasms with poor prognosis that may harbor KIT and NRAS genes mutations. Molecular alterations represent possible targets of tailored therapeutic approaches. We describe the case of a 74-year-old patient with primary melanoma of the nasal cavity. Mutational analysis of KIT demonstrated a point missense mutation D820G in exon 17. This represents, to our knowledge, the first case of sinonasal melanoma harboring this specific KIT mutation. Although KIT mutations confer sensibility to thyrosine-kinase inhibitor, it has been proved that this is strongly dependent on the region in which this alteration occurs. Thus it seems very important to perform an accurate gene mutational analysis to provide a biological rationale to the tailored therapy.

Background Combination therapy with BRAF and MEK inhibitors significantly improves survival in BRAF mutated melanoma patients but is unable to prevent disease recurrence due to the emergence of drug resistance. Cancer stem cells (CSCs) have been involved in these long-term treatment failures. We previously reported in lung cancer that CSCs maintenance is due to altered lipid metabolism and dependent upon Stearoyl-CoA-desaturase (SCD1)-mediated upregulation of YAP and TAZ. On this ground, we investigated the role of SCD1 in melanoma CSCs. Methods SCD1 gene expression data of melanoma patients were downloaded from TCGA and correlated with disease progression by bioinformatics analysis and confirmed on patient’s tissues by qRT-PCR and IHC analyses. The effects of combination of BRAF/MEKi and the SCD1 inhibitor MF-438 were monitored by spheroid-forming and proliferation assays on a panel of BRAF-mutated melanoma cell lines grown in 3D and 2D conditions, respectively. SCD1, YAP/TAZ and stemness markers were evaluated in melanoma cells and tissues by qRT-PCR, WB and Immunofluorescence. Results We first observed that SCD1 expression increases during melanoma progression. BRAF-mutated melanoma 3D cultures enriched for CSCs overexpressed SCD1 and were more resistant than 2D differentiated cultures to BRAF and MEK inhibitors. We next showed that exposure of BRAF-mutated melanoma cells to MAPK pathway inhibitors enhanced stemness features by upregulating the expression of YAP/TAZ and downstream genes but surprisingly not SCD1. However, SCD1 pharmacological inhibition was able to downregulate YAP/TAZ and to revert at the same time CSC enrichment and resistance to MAPK inhibitors. Conclusions Our data underscore the role of SCD1 as prognostic marker in melanoma and promote the use of SCD1 inhibitors in combination with MAPK inhibitors for the control of drug resistance. Electronic supplementary material The online version of this article (10.1186/s13046-018-0989-7) contains supplementary material, which is available to authorized users.

International audience; Human AKTIP and mouse Ft1 are orthologous ubiquitin E2 variant proteins involved in telomere maintenance and DNA replication. AKTIP also interacts with A- and B-type lamins. These features suggest that Ft1 may be implicated in aging regulatory pathways. Here, we show that cells derived from hypomorph Ft1 mutant (Ft1kof/kof ) mice exhibit telomeric defects and that Ft1kof/kof animals develop progeroid traits, including impaired growth, skeletal and skin defects, abnormal heart tissue, and sterility. We also demonstrate a genetic interaction between Ft1 and p53. The analysis of mice carrying mutations in both Ft1 and p53 (Ft1kof/kof ; p53ko/ko and Ft1kof/kof ; p53+/ko ) showed that reduction in p53 rescues the progeroid traits of Ft1 mutants, suggesting that they are at least in part caused by a p53-dependent DNA damage response. Conversely, Ft1 reduction alters lymphomagenesis in p53 mutant mice. These results identify Ft1 as a new player in the aging process and open the way to the analysis of its interactions with other progeria genes using the mouse model.

Cutaneous melanoma (CM) is a malignancy with increasing occurrence. Its microRNA repertoire has been defined in a number studies, leading to candidates for biological and clinical relevance: miR-200a/b/c, miR-203, miR-205, miR-204, miR-211, miR-23b and miR-26a/b. Our work was aimed to validate the role of these candidate miRNAs in melanoma, using additional patients cohorts and in vitro cultures. miR-26a, miR-204 and miR-211 were more expressed in normal melanocytes, while miR-23b, miR-200b/c, miR-203 and miR-205 in epidermis and keratinocytes. None of the keratinocyte-related miRNAs was associated with any known mutation or with clinical covariates in melanoma. On the other hand, the loss of miR-204 was enriched in melanomas with NRAS sole mutation (Fisher exact test, P = 0.001, Log Odds = 1.67), and less frequent than expected in those harbouring CDKN2A mutations (Fisher exact test, P = 0.001, Log Odds − 1.09). Additionally, miR-204 was associated with better prognosis in two independent melanoma cohorts and its exogenous expression led to growth impairment in melanoma cell lines. Thus, miR-204 represents a relevant mechanism in melanoma, with potential prognostic value and its loss seems to act in the CDKN2A pathway, in cooperation with NRAS. Electronic supplementary material The online version of this article (10.1186/s12943-018-0819-8) contains supplementary material, which is available to authorized users.

Pseudomyogenic hemangioendothelioma (PMH) is a rare, mostly indolent, endothelial neoplasm of low-grade malignancy, often mimicking myoid and epithelioid tumors histologically. It is more frequent in young adult males and it usually presents with multiple cutaneous nodules, mostly localized at the extremities. It traverses several tissue planes simultaneously and can involve dermis, subcutis, skeletal muscle, and bone. Histologically, it is characterized by plump spindle cells with eosinophilic cytoplasm, often arranged in fascicles and epithelioid cells with "pseudomyogenic" morphology. Immunohistochemically, PMH is positive for Factor VIII, FLI-1, INI-1, vimentin, MDM2, CDK4, CD31, AE1/AE3, EMA, and P63. The efficacy of treatments is only partially known. Because of the frequent multifocal aspect of PMH, which contraindicates surgery, systemic treatments, such as gemcitabine, sirolimus, and everolimus are used. Based on our observation of multifocal PMH of the foot in a 17-year-old male patient, treated with gemcitabine with complete cutaneous response in a 2-year follow-up, we decided to discuss this rare tumor and underline its progression and therapeutic approaches. Thanks to a correct diagnosis, it is possible to avoid aggressive therapeutic approaches, which would be necessary for nonindolent diseases, such as sarcoma, which often needs amputation.

There is accumulating evidence that the vitamin D pathway may play a role in melanoma. The aim of this study was to investigate the association between 25-hydroxyvitamin D [25(OH)D] serum levels and the risk of cutaneous melanoma. A case-control study with 137 incident cases of melanoma (serum samples collected at the time of diagnosis) and 99 healthy controls (serum samples collected between October and April) was carried out and evaluated in the framework of an evidence synthesis of clinical epidemiological studies on the topic to facilitate comparisons and summarize the scientific evidence produced so far. There was a statistically significant difference in the median levels of serum vitamin D between melanoma patients and healthy controls (18.0 vs. 27.8 ng/ml, P0.001). Among melanoma patients, 66.2%, compared with 15.2% of healthy controls, had vitamin D deficiency (≤20 ng/ml), whereas vitamin D sufficiency (≥30 ng/ml) was observed in only 7.4% of melanoma patients and in 37.4% of the healthy controls (P0.001). A multivariate model including age, sex, and BMI showed a statistically significant inverse association between melanoma and vitamin D sufficiency versus deficiency (odds ratio=0.04; 95% confidence interval: 0.02-0.10, P0.001). Also, vitamin D insufficiency versus deficiency was significantly inversely associated with melanoma (odds ratio=0.13; 95% confidence interval: 0.06-0.27, P0.001). These results suggest that both deficient and insufficient serum levels of vitamin D are associated with melanoma and that a trend seems to be present with a reduced risk of melanoma when vitamin D approaches normal values.

Objective Galectin-3 is constitutively expressed in bone cells and was recently shown to modulate osteogenic transdifferentiation of vascular smooth muscle cells and atherosclerotic calcification. However, the role of galectin-3 in bone physiology is largely undefined. To address this issue, we analyzed (1) the skeletal features of 1-, 3- and 6-month-old galectin-3 null (Lgals3−/−) and wild type (WT) mice and (2) the differentiation and function of osteoblasts and osteoclasts derived from these animals. Methods Long bone phenotype, gene expression profile, and remodeling were investigated by micro-computed tomography, real time-PCR, static and dynamic histomorphometry, and assessment of biochemical markers of bone resorption and formation. Bone competence was also evaluated by biomechanical testing at 3 months. In vitro, the effects of galectin-3 deficiency on bone cell differentiation and function were investigated by assessing (a) gene expression of osteoblast markers, alkaline phosphatase activity, mineralization assay, and WNT/β-catenin signaling (of which galectin-3 is a known regulator) in osteoblasts; and (b) tartrate-resistant acid phosphatase activity and bone resorption activity in osteoclasts. Results Lgals3−/− mice revealed a wide range of age-dependent alterations including lower bone formation and higher bone resorption, accelerated age-dependent trabecular bone loss (p Conclusions These data provide evidence for a contribution of galectin-3 to bone cell maturation and function, bone remodeling, and biomechanical competence, thus identifying galectin-3 as a promising therapeutic target for age-related disorders of bone remodeling.

Indeterminate cytology arises from the limit inherent in traditional cyto-morphological analysis. Numerous benign processes can cause subtle cellular or nuclear changes, sometimes indistinguishable from findings of well-differentiated carcinoma. Moreover, follicular carcinoma can be diagnosed only if the tumor’s ability to penetrate capsular or vascular structures is documented, but such a property can only be assessed at final histology.

MRE11 is a component of the MRE11/RAD50/NBS1 (MRN) complex, whose activity is essential to control faithful DNA replication and to prevent accumulation of deleterious DNA double-strand breaks. In humans, hypomorphic mutations in these genes lead to DNA damage response (DDR)-defective and cancer-prone syndromes. Moreover, MRN complex dysfunction dramatically affects the nervous system, where MRE11 is required to restrain MYCN-dependent replication stress, during the rapid expansion of progenitor cells. MYCN activation, often due to genetic amplification, represents the driving oncogenic event for a number of human tumors, conferring bad prognosis and predicting very poor responses even to the most aggressive therapeutic protocols. This is prototypically exemplified by neuroblastoma, where MYCN amplification occurs in about 25% of the cases. Intriguingly, MRE11 is highly expressed and predicts bad prognosis in MYCN-amplified neuroblastoma. Due to the lack of direct means to target MYCN, we explored the possibility to trigger intolerable levels of replication stress-dependent DNA damage, by inhibiting MRE11 in MYCN-amplified preclinical models. Indeed, either MRE11 knockdown or its pharmacological inhibitor mirin induce accumulation of replication stress and DNA damage biomarkers in MYCN-amplified cells. The consequent DDR recruits p53 and promotes a p53-dependent cell death, as indicated by p53 loss- and gain-of-function experiments. Encapsulation of mirin in nanoparticles allowed its use on MYCN-amplified neuroblastoma xenografts in vivo, which resulted in a sharp impairment of tumor growth, associated with DDR activation, p53 accumulation, and cell death. Therefore, we propose that MRE11 inhibition might be an effective strategy to treat MYCN-amplified and p53 wild-type neuroblastoma, and suggest that targeting replication stress with appropriate tools should be further exploited to tackle MYCN-driven tumors.

Hodgkin lymphoma (HL) associated with Warthin tumor (WT) is extremely rare, accounting for only 3 cases of classical HLs. Here, we report for the first time the occurrence of a nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) involving the lymphoid stroma of a WT of the parotid gland. Pathogenesis of WT is controversial, with both a nodal and a parenchymal possible origin. On the other hand, extranodal involvement by HLs is uncommon. In our case, the coexistence of a WT and of a NLPHL within its stroma and in cervical lymph node emphasizes the importance of a careful evaluation of the lymphoid tissue in WT in order to exclude the possibility of an associated lymphoid malignancy.

Galectins (S-type lectins) are an evolutionarily-conserved family of lectin molecules, which can be expressed intracellularly and in the extracellular matrix, as well. Galectins bind β-galactose-containing glycoconjugates and are functionally active in converting glycan-related information into cell biological programs. Altered glycosylation notably occurring in cancer cells and expression of specific galectins provide, indeed, a fashionable mechanism of molecular interactions able to regulate several tumor relevant functions, among which are cell adhesion and migration, cell differentiation, gene transcription and RNA splicing, cell cycle and apoptosis. Furthermore, several galectin molecules also play a role in regulating the immune response. These functions are strongly dependent on the cell context, in which specific galectins and related glyco-ligands are expressed. Thyroid cancer likely represents the paradigmatic tumor model in which experimental studies on galectins’ glycobiology, in particular on galectin-3 expression and function, contributed greatly to the improvement of cancer diagnosis. The discovery of a restricted expression of galectin-3 in well-differentiated thyroid carcinomas (WDTC), compared to normal and benign thyroid conditions, contributed also to promoting preclinical studies aimed at exploring new strategies for imaging thyroid cancer in vivo based on galectin-3 immuno-targeting. Results derived from these recent experimental studies promise a further improvement of both thyroid cancer diagnosis and therapy in the near future. In this review, the biological role of galectin-3 expression in thyroid cancer, the validation and translation to a clinical setting of a galectin-3 test method for the preoperative characterization of thyroid nodules and a galectin-3-based immuno-positron emission tomography (immuno-PET) imaging of thyroid cancer in vivo are presented and discussed.

The introduction of multigene panel testing for hereditary breast/ovarian cancer screening has greatly improved efficiency, speed, and costs. However, its clinical utility is still debated, mostly due to the lack of conclusive evidences on the impact of newly discovered genetic variants on cancer risk and lack of evidence‐based guidelines for the clinical management of their carriers. In this pilot study, we aimed to test whether a systematic and multiparametric characterization of newly discovered mutations could enhance the clinical utility of multigene panel sequencing. Out of a pool of 367 breast/ovarian cancer families Sanger‐sequenced for BRCA1 and BRCA2 gene mutations, we selected a cohort of 20 BRCA1/2‐negative families to be subjected to the BROCA‐Cancer Risk Panel massive parallel sequencing. As a strategy for the systematic characterization of newly discovered genetic variants, we collected blood and cancer tissue samples and established lymphoblastoid cell lines from all available individuals in these families, to perform segregation analysis, loss‐of‐heterozygosity and further molecular studies. We identified loss‐of‐function mutations in 6 out 20 high‐risk families, 5 of which occurred on BRCA1,CHEK2 and ATM and are esteemed to be risk‐relevant. In contrast, a novel RAD50 truncating mutation is most likely unrelated to breast cancer. Our data suggest that integrating multigene panel testing with a pre‐organized, multiparametric characterization of newly discovered genetic variants improves the identification of risk‐relevant alleles impacting on the clinical management of their carriers.

// Salvatore Sciacchitano 1, 2 , Luca Lavra 2 , Alessandra Ulivieri 2 , Fiorenza Magi 2 , Gian Paolo De Francesco 3 , Carlo Bellotti 4 , Leila B. Salehi 2, 5 , Maria Trovato 6 , Carlo Drago 7 and Armando Bartolazzi 8, 9 1 Department of Clinical and Molecular Medicine, Sapienza University, 00161 Rome, Italy 2 Laboratory of Biomedical Research, Niccolo Cusano University Foundation, 00166 Rome, Italy 3 Department of Oncological Science, Breast Unit, St Andrea University Hospital, 00189 Rome, Italy 4 Operative Unit Surgery of Thyroid and Parathyroid, Sapienza University of Rome, St Andrea Hospital, 00189 Rome, Italy 5 Department of Biopathology and Diagnostic Imaging, Tor Vergata University, 00133 Rome, Italy 6 Department of Clinical and Experimental Medicine, Pad D 2° piano-AOU Policlinico “G. Martino”, 98125 Messina, Italy 7 Department of Economics, Niccolo Cusano University, 00166 Rome, Italy 8 Laboratory of Surgical and Experimental Pathology, St Andrea University Hospital, 00189 Rome, Italy 9 Department of Oncology-Pathology, Cancer Center Karolinska Universitetssjukhuset Solna, S-17176 Stockholm, Sweden Correspondence to: Salvatore Sciacchitano, email: salvatore.sciacchitano@fondazioneniccolocusano.it Keywords: thyroid FNA cytology, indeterminate thyroid nodules, systematic review, meta-analysis, diagnostic performance Received: January 27, 2017 Accepted: March 22, 2017 Published: April 19, 2017 ABSTRACT Since it is impossible to recognize malignancy at fine needle aspiration (FNA) cytology in indeterminate thyroid nodules, surgery is recommended for all of them. However, cancer rate at final histology is

111 Background: Checkpoint inhibitor-based immunotherapy is opening a promising scenario in oncology with objective responses registered in several tumors. However reliable predictive markers of tumor responsiveness are still lacking. These markers need to be urgently identified for a better selection of patients candidate to immunotherapy. In NSCLC setting, the pleiotropic molecules Galectin 1 and 3 regulate apoptosis and tumor immune-escape and are in the right position to play as predictive marker in anti-PD1 based immunotherapy. Methods: 27 consecutive patients with non small cell lung cancer (NSCLC) stage IV, treatment-naive, selected on the basis of PD-L1 tumor proportion score (TPS) ≥ 50 % were considered for immunotherapy with anti PD-1 antibodies as a first-line treatment. Patients assessments were conducted at baseline and after 3 cycles of therapy (week 12) and classified according to immune-related response criteria (iRECIST). Immunohistochemical analysis was performed on primary tumor biopsies before treatment in order to evaluate Gal-1 and Gal-3 expression both in cancer cells and tumor interstitium. Tumors were grouped in 2 different classes depending on the expression level of these molecules. Results: In our preliminary data there seems to be a strong correlation between the responses to anti-PD-1 treatment and a low-intermediate expression of intratumoral Gal-3 and interstitial Gal-1. In fact, all patients in the intratumoral Gal-3 low-intermediate expression group had an excellent response to immunotherapic treatment (10/10 patients), while 15/17 patients with high intracellular Gal-3 expression had clinical and radiographic progression of the disease after only three cycles of treatment, despite the high expression of PD-L1. In contrast, 8/11 patients with a low-intermediate interstitial expression of Gal-1 had an excellent response to treatment, whereas in the group with high interstitial expression of Gal-1, the response rate was drastically lower (5/15 patients). Conclusions: The expression of interstitial Gal-1 protein and intratumoral Gal-3 protein appear to be a good candidates as predictive markers of response to immunotherapy.