Discovery of a septin-4 covalent binder with antimetastatic activity in a mouse model of melanoma.

Cancer spreading through metastatic processes is one of the major causes of tumour-related mortality. Metastasis is a complex phenomenon which involves multiple pathways ranging from cell metabolic alterations to changes in the biophysical phenotype of cells and tissues. In the search for new effective anti-metastatic agents, we modulated the chemical structure of the lead compound AA6, in order to find the structural determinants of activity, and to identify the cellular target responsible of the downstream anti-metastatic effects observed. New compounds synthesized were able to inhibit in vitro B16-F10 melanoma cell invasiveness, and one selected compound, CM365, showed in vivo anti-metastatic effects in a lung metastasis mouse model of melanoma. Septin-4 was identified as the most likely molecular target responsible for these effects. This study showed that CM365 is a promising molecule for metastasis prevention, remarkably effective alone or co-administered with drugs normally used in cancer therapy, such as paclitaxel.

Predictors of Recurrence and Progression in Poorly Differentiated Cutaneous Squamous Cell Carcinomas: Insights from a Real-Life Experience.

INTRODUCTION: Surgery represents the primary treatment option for cutaneous squamous cell carcinoma (cSCC) aiming for complete tumor resection (R0). Recurrence and metastasis significantly affect survival and outcomes, and poorly differentiated (G3) cSCC is associated with a higher risk of recurrence. However, the specific clinical and histopathological features that predict recurrence and progression in G3-cSCC remain unclear. METHODS: A retrospective analysis was conducted on a series of patients with primary G3-cSCC diagnosed at the Turin University Hospital between January 2016 and January 2021. After independent histological revision, logistic regression models were used to identify clinico-pathological predictors of cutaneous recurrence, lymphnode/metastatic progression, and both types of progression. RESULTS: Among the 161 G3-cSCC patients, 80.1% (129/161) showed no signs of local recurrence or metastatic progression, while 19.9% (32 patients) had progressed. In the univariate logistic regression, tumor clinical diameter, depth of infiltration (DOI), and lymphovascular invasion (LVI) were identified as significant predictors across the various types of progression (p < 0.05). In the context of multivariate logistic regression, distinct models proved to be significant. For skin recurrence, a 3-variable model incorporating DOI (OR 1.16, 95% CI, 1.01-1.35, p = 0.050), LVI (OR 3.61, 95% CI, 1.11-11.8, p = 0.034), and desmoplasia (OR 3.45, 95% CI, 1.25-9.5, p = 0.017) was selected. Regarding lymphnode/metastatic progression, a 3-variable model combining pT2 (OR 6.10, 95% CI, 1.15-32.35, p = 0.034), pT3 (OR 14.33, 95% CI, 2.79-73.63, p = 0.001), and LVI (OR 3.86, 95% CI, 1.10-13.62, p = 0.036) was identified. Lastly, a 2-variable model for both types of progression consisted of vertical tumor thickness (OR 5.45, 95% CI, 1.11-27.32, p = 0.039) and LVI (OR 1.15, 95% CI, 1.04-1.26, p = 0.006). CONCLUSION: Tumor size, DOI, and LVI were significant predictors of recurrence and metastatic progression. Notably, the size of histologically defined tumor-free margins did not affect the risk of recurrence, whilst LVI emerged as a key predictor of all forms of progression. These findings provide insights into risk stratification and suggest that close monitoring and potential adjuvant therapies, such as radiation therapy, may be necessary especially for patients with lymphovascular involvement.

First pilot case-control interventional study using autologous extracellular vesicles to treat chronic venous ulcers unresponsive to conventional treatments.

Current therapeutic approaches for chronic venous ulcers (CVUs) still require evidence of effectiveness. Diverse sources of extracellular vesicles (EVs) have been proposed for tissue regeneration, however the lack of potency tests, to predict in-vivo effectiveness, and a reliable scalability have delayed their clinical application. This study aimed to investigate whether autologous serum-derived EVs (s-EVs), recovered from patients with CVUs, may be a proper therapeutic approach to improve the healing process. A pilot case-control interventional study (CS2/1095/0090491) has been designed and s-EVs recovered from patients. Patient eligibility included two or more distinct chronic lesions in the same limb with 11 months as median persistence of active ulcer before enrollment. Patients were treated three times a week, for 2 weeks. Qualitative CVU analysis demonstrated that s-EVs-treated lesions displayed a higher percentage of granulation tissue compared to the control group (Sham) (s-EVs 3 out of 5: 75-100 % vs Sham: none), further confirmed at day 30. s-EVs-treated lesions also displayed higher sloughy tissue reduction at the end of treatment even increased at day 30. Additionally, s-EV treatment led to a median surface reduction of 151 mm2 compared to 84 mm2 in the Sham group, difference even more evident at day 30 (s-EVs 385 mm2vs Sham 106 mm2p = 0.004). Consistent with the enrichment of transforming growth factor-ß1 in s-EVs, histological analyses showed a regenerative tissue with an increase in microvascular proliferation areas. This study first demonstrates the clinical effectiveness of autologous s-EVs in promoting the healing process of CVUs unresponsive to conventional treatments.

BRAFV600 variant allele frequency predicts outcome in metastatic melanoma patients treated with BRAF and MEK inhibitors.

BACKGROUND: The prognostic impact of variant allele frequency (VAF) on clinical outcome in BRAFV600 mutated metastatic melanoma patients (MMPs) receiving BRAF (BRAFi) and MEK inhibitors (MEKi) is unclear. MATERIALS AND METHODS: A cohort of MMPs receiving first line BRAFi and MEKi was identified by inspecting dedicated databases of three Italian Melanoma Intergroup centres. VAF was determined by next generation sequencing in pre-treatment baseline tissue samples. Correlation between VAF and BRAF copy number variation was analysed in an ancillary study by using a training and a validation cohort of melanoma tissue samples and cell lines. RESULTS: Overall, 107 MMPs were included in the study. The VAF cut-off determined by ROC curve was 41.3%. At multivariate analysis, progression-free survival (PFS) was significantly shorter in patients with M1c/M1d [HR 2.25 (95% CI 1.41-3.6, p < 0.01)], in those with VAF >41.3% [HR 1.62 (95% CI 1.04-2.54, p < 0.05)] and in those with ECOG PS ≥1 [HR 1.82 (95% CI 1.15-2.88, p < 0.05)]. Overall survival (OS) was significantly shorter in patients with M1c/M1d [HR 2.01 (95% CI 1.25-3.25, p < 0.01)]. Furthermore, OS was shorter in patients with VAF >41.3% [HR 1.46 (95% CI 0.93-2.29, p = 0.06)] and in patients with ECOG PS ≥1 [HR 1.52 (95% CI 0.94-2.87, p = 0.14)]. BRAF gene amplification was found in 11% and 7% of samples in the training and validation cohort, respectively. CONCLUSIONS: High VAF is an independent poor prognostic factor in MMP receiving BRAFi and MEKi. High VAF and BRAF amplification coexist in 7%-11% of patients.

The lobular neoplasia enigma: management and prognosis in a long follow-up case series.

BACKGROUND: Many oncologists debate if lobular neoplasia (LN) is a risk factor or an obligatory precursor of more aggressive disease. This study has three aims: (i) describe the different treatment options (surgical resection vs observation), (ii) investigate the upgrade rate in surgically treated patients, and (iii) evaluate the long-term occurrences of aggressive disease in both operated and unoperated patients. METHODS: A series of 122 patients with LN bioptic diagnosis and follow-up information were selected. Clinical, radiological, and pathological data were collected from medical charts. At definitive histology, either invasive or ductal carcinoma in situ was considered upgraded lesions. RESULTS: Atypical lobular hyperplasia (ALH), lobular carcinoma in situ (LCIS), and high-grade LN (HG-LN) were diagnosed in 44, 63, and 15 patients, respectively. The median follow-up was 9.5 years. Ninety-nine patients were surgically treated, while 23 underwent clinical-radiological follow-up. An upgrade was observed in 28/99 (28.3%). Age ≥ 54 years (OR 4.01, CI 1.42-11.29, p = 0.009), Breast Imaging-Reporting and Data System (BI-RADS) categories 4-5 (OR 3.76, CI 1.37-10.1, p = 0.010), and preoperatory HG-LN diagnosis (OR 8.76, 1.82-42.27, p = 0.007) were related to upgraded/aggressive disease. During follow-up, 8 patients developed an ipsilateral malignant lesion, four of whom were not initially operated (4/23, 17%). CONCLUSIONS: BI-RADS categories 4-5, HG-LN diagnosis, and age ≥ 54 years were features associated with an upgrade at definitive surgery. Moreover, 17% of unoperated cases developed an aggressive disease, emphasizing that LN patients need close surveillance due to the long-term risk of breast cancer.

The Molecular Landscape of Primary Acral Melanoma: A Multicenter Study of the Italian Melanoma Intergroup (IMI).

Acral melanoma (AM) is a rare and aggressive subtype of melanoma affecting the palms, soles, and nail apparatus with similar incidence among different ethnicities. AM is unrelated to ultraviolet radiation and has a low mutation burden but frequent chromosomal rearrangements and gene amplifications. Next generation sequencing of 33 genes and somatic copy number variation (CNV) analysis with genome-wide single nucleotide polymorphism arrays were performed in order to molecularly characterize 48 primary AMs of Italian patients in association with clinicopathological and prognostic features. BRAF was the most commonly mutated gene, followed by NRAS and TP53, whereas TERT promoter, KIT, and ARID1A were less frequently mutated. Gains and losses were recurrently found in the 1q, 6p, 7, 8q, 20 and 22 chromosomes involving PREX2, RAC1, KMT2C, BRAF, CCND1, TERT, and AKT3 genes, and in the 6q, 9, 10, 11q and 16q chromosomes including CDKN2A, PTEN, and ADAMTS18 genes, respectively. This study confirmed the variety of gene mutations and the high load of CNV in primary AM. Some genomic alterations were associated with histologic prognostic features. BRAF mutations, found with a higher rate than previously reported, correlated with a low Breslow thickness, low mitotic count, low CNV of the AMs, and with early-stage of disease.

Prognostic and Predictive Biomarkers in Stage III Melanoma: Current Insights and Clinical Implications.

Melanoma is one of the most aggressive skin cancers. The 5-year survival rate of stage III melanoma patients ranges from 93% (IIIA) to 32% (IIID) with a high risk of recurrence after complete surgery. The introduction of target and immune therapies has dramatically improved the overall survival, but the identification of patients with a high risk of relapse who will benefit from adjuvant therapy and the determination of the best treatment choice remain crucial. Currently, patient prognosis is based on clinico-pathological features, highlighting the urgent need of predictive and prognostic markers to improve patient management. In recent years, many groups have focused their attention on identifying molecular biomarkers with prognostic and predictive potential. In this review, we examined the main candidate biomarkers reported in the literature.

NTRK Gene Fusion Detection in Atypical Spitz Tumors.

Atypical Spitz tumors (AST) deviate from stereotypical Spitz nevi for one or more atypical features and are now regarded as an intermediate category of melanocytic tumors with uncertain malignant potential. Activating NTRK1/NTRK3 fusions elicit oncogenic events in Spitz lesions and are targetable with kinase inhibitors. However, their prevalence among ASTs and the optimal approach for their detection is yet to be determined. A series of 180 ASTs were screened with pan-TRK immunohistochemistry and the presence of NTRK fusions was confirmed using FISH, two different RNA-based NGS panels for solid tumors, and a specific real time RT-PCR panel. Overall, 26 ASTs showed pan-TRK immunostaining. NTRK1 fusions were detected in 15 of these cases showing cytoplasmic immunoreaction, whereas NTRK3 was detected in one case showing nuclear immunoreaction. Molecular tests resulted all positive in only two ASTs (included the NTRK3 translocated), RNA-based NGS and real time RT-PCR were both positive in three cases, and FISH and real time RT-PCR in another two cases. In seven ASTs NTRK1 fusions were detected only by FISH and in two cases only by real time RT-PCR. The frequency of NTRK fusions in ASTs is 9%, with a clear prevalence of NTRK1 compared to NTRK3 alterations. Pan-TRK immunohistochemistry is an excellent screening test. Confirmation of NTRK fusions may require the use of different molecular techniques.

NTRK Fusions in Central Nervous System Tumors: A Rare, but Worthy Target.

The neurotrophic tropomyosin receptor kinase (NTRK) genes (NTRK1, NTRK2, and NTRK3) code for three transmembrane high-affinity tyrosine-kinase receptors for nerve growth factors (TRK-A, TRK-B, and TRK-C) which are mainly involved in nervous system development. Loss of function alterations in these genes can lead to nervous system development problems; conversely, activating alterations harbor oncogenic potential, promoting cell proliferation/survival and tumorigenesis. Chromosomal rearrangements are the most clinically relevant alterations of pathological NTRK activation, leading to constitutionally active chimeric receptors. NTRK fusions have been detected with extremely variable frequencies in many pediatric and adult cancer types, including central nervous system (CNS) tumors. These alterations can be detected by different laboratory assays (e.g., immunohistochemistry, FISH, sequencing), but each of these approaches has specific advantages and limitations which must be taken into account for an appropriate use in diagnostics or research. Moreover, therapeutic targeting of this molecular marker recently showed extreme efficacy. Considering the overall lack of effective treatments for brain neoplasms, it is expected that detection of NTRK fusions will soon become a mainstay in the diagnostic assessment of CNS tumors, and thus in-depth knowledge regarding this topic is warranted.

Dp71 Expression in Human Glioblastoma.

BACKGROUND: Dp71 is the most abundant dystrophin (DMD) gene product in the nervous system. Mutation in the Dp71 coding region is associated with cognitive disturbances in Duchenne muscular dystrophy (DMD) patients, but the function of dystrophin Dp71 in tumor progression remains to be established. This study investigated Dp71 expression in glioblastoma, the most common and aggressive primary tumor of the central nervous system (CNS). METHODS: Dp71 expression was analyzed by immunofluorescence, immunohistochemistry, RT-PCR, and immunoblotting in glioblastoma cell lines and cells isolated from human glioblastoma multiforme (GBM) bioptic specimens. RESULTS: Dp71 isoform was expressed in normal human astrocytes (NHA) cell lines and decreased in glioblastoma cell lines and cells isolated from human glioblastoma multiforme bioptic specimens. Moreover, Dp71 was localized in the nucleus in normal cells, while it was localized into the cytoplasm of glioblastoma cells organized in clusters. We have shown, by double labeling, that Dp71 colocalizes with lamin B in normal astrocytes cells, confirming the roles of Dp71 and lamin B in maintaining nuclear architecture. Finally, we demonstrated that decreased Dp71 protein in cells isolated from human bioptic specimens was inversely correlated with the Ki-67 tumor proliferative index. CONCLUSION: A decreased Dp71 expression is associated with cancer proliferation and poor prognosis in glioblastoma.

Caveolin 1 expression favors tumor growth and is associated with poor survival in primary lung adenocarcinomas.

Despite the consolidated clinico-pathological correlates of Caveolin 1 expression in non-small cell lung cancer, the available data on the role of Caveolin 1 in relation to proliferation, migration, and metastasis in lung adenocarcinoma cells is still scant. Here, we aimed to confirm whether Caveolin 1 may act as a promoter of cell growth in human lung adenocarcinoma using in vitro and in vivo models, supported by a survival analysis of Caveolin 1 expression in a series of 116 primary lung adenocarcinomas. The silencing of endogenous Caveolin 1 expression in H522 lung adenocarcinoma cells through stable shRNA transfection significantly inhibited cellular proliferation in vitro and in vivo, in a lung adenocarcinoma xenograft mouse model. The bioluminescence imaging analysis revealed that tumors derived from Caveolin 1 shRNA-transfected cells grew slower than control xenografts. However, this difference progressively diminished over time and was definitively lost after 21 days. This was consistent with a progressive Caveolin 1 re-expression, which started at day 7. The association between the restored expression of Caveolin 1 and the restart of tumor growth in vivo supports the booster role of Caveolin 1 in lung adenocarcinoma progression. To further confirm this role, Caveolin 1 expression was assessed by immunohistochemistry in a series of 116 human lung adenocarcinomas. Positive Caveolin 1 tumors accounted for 20% of cases and were associated with a significantly worse overall survival compared to Caveolin 1-negative cancers. Taken together, these data highlight that Caveolin 1 expression confers a proliferative advantage in lung adenocarcinoma cells, thus fostering increased tumor aggressiveness.

Diagnostic revision of 206 adult gliomas (including 40 oligoastrocytomas) based on ATRX, IDH1/2 and 1p/19q status.

The diagnosis of 206 low and high grade adult gliomas, including 40 oligoastrocytomas, was revised based on the immunohistochemical reactivity for the ATRX protein, IDH1/2 mutation status and 1p/19q chromosomal status. All oligodendrogliomas kept the initial diagnosis. Astrocytomas did not change diagnosis in 30 of 36 cases (83.3 %); four of 36 (11.1 %) cases were reclassified as oligodendroglioma, one (2.8 %) as DNT and the other (2.8 %) as reactive gliosis. Oligoastrocytomas changed diagnosis in 35 of 40 (87.5 %) cases, being reclassified 22 of 40 (55 %) as astrocytoma, 11 of 40 (27.5 %) as oligodendroglioma and two of 40 (5 %) as reactive gliosis. Four (10 %) remained unclassifiable. In one case only (2.5 %), the diagnosis of oligoastrocytoma could not be excluded since tumor astrocytes and tumor oligodendrocytes coexisted in mixed tumor areas. In the GBM tumor subgroup, GBMO disappeared because they were not substantiated by molecular genetics. Pilocytic astrocytomas retained ATRX expression. Loss of nuclear ATRX protein expression was strongly associated to IDH1/2 mutations (p = 0.0001) and mutually exclusive with total 1p/19q co-deletion (p = 0.0001). In astrocytic tumors, loss of immunoreactivity for the ATRX protein was significantly associated to the ALT phenotype (p = 0.0003). The constitutive ATRX expression in microglia/macrophages may be misleading, especially in the identification of an oligodendroglial tumor infiltration. Of paramount importance in the recognition of oligodendroglial and astrocytic tumor cells were the double immunostainings for ATRX/GFAP, ATRX/IDH1R132H, ATRX/Iba-1 and ATRX/CD68.

Mesenchymal/radioresistant traits in granular astrocytomas: evidence from a combined clinical and molecular approach.

AIMS: Granular-cell astrocytomas (GCAs) are morphologically characterized by a prominent component of granular periodic acid-Schiff-positive cells, and show increased aggressiveness as compared with 'ordinary' astrocytomas. The aim of this study was to investigate, in a small series of three GCAs, the expression of mesenchymal/radioresistance-associated biomarkers [such as chitinase-3-like protein 1 (YKL-40), hepatocyte growth factor receptor (c-Met), and caveolin 1 (Cav1)] that could contribute to the poor outcome associated with this glioma subgroup. METHODS AND RESULTS: Our results show that GCAs, according to the new molecular glioma classifications, consistently show a prognostically negative molecular trait (IDH1wt-ATRX noloss-1p/19q nocodeletion). Furthermore, GCAs significantly differed from a control series of 33 'conventional' astrocytomas, because of diffuse and strong immunohistochemical coexpression of YKL-40, c-Met, and Cav1. CONCLUSIONS: Our findings show that specific morphological traits, such as a granular-cell component, could represent useful features in guiding the search for prognostic and predictive biomarkers that could eventually be therapy-targetable (e.g. Met inhibitors aimed at reducing radioresistance).

Squamocellular Carcinoma of the Skin: Clinicopathological Features Predicting the Involvement of the Surgical Margins and Review of the Literature.

BACKGROUND: The new AJCC classification has highlighted some particular risk factors for squamous cell carcinoma (SCC) relevant for prognosis. Incomplete excision is not infrequent in SCC. The aim of this study is to examine features that can predict an incomplete excision on the basis of the new AJCC classification and to review the literature on this topic. MATERIALS AND METHODS: 81 SCC patients were included. All patients were submitted to excisional biopsy with a margin of at least 4 mm from the clinical edges as recommended. Histological characteristics of the lesions analysed were maximum diameter, grading, site, Breslow thickness, Clark level, deep tissue invasion (neural, bone, muscle), presence of ulceration and positivity of the margins. RESULTS: The average Breslow thickness was 3.93 mm. Out of the 81 patients included, 14 showed involved margins. The 2 parameters that were implicated in predicting involvement of the margins in the multivariable model were Breslow thickness and location of the lesion on the ear or lip. Grading was not associated with involvement of margins. CONCLUSION: According to the new AJCC classification, this study could be useful to plan the most suitable surgical technique in order to avoid the risk of incomplete surgery.

Meningeal carcinomatosis underdiagnosis and overestimation: incidence in a large consecutive and unselected population of breast cancer patients.

BACKGROUND: The incidence of meningeal carcinomatosis appears to be higher than in the past due to advances in neuro-imaging diagnostic techniques and improvements in cancer survival. Among solid tumors, breast cancer is the cancer most commonly associated with meningeal carcinomatosis, with an incidence rate of between 0.8 and 16%. Aim of this study has been i) to evaluate the incidence of meningeal carcinomatosis in a continuous breast cancer unselected series treated in a dedicated Breast Unit and ii) to define the clinico-pathological and molecular parameters associated with meningeal carcinomatosis development. METHODS: A retrospective series of 1915 consecutive patients surgically treated for breast cancer between 1998 and 2010 was collected. Clinico-pathological data were recorded from medical charts and pathological reports, including the date of development of symptomatic meningeal carcinomatosis. Meningeal carcinomatosis incidence was determined at both 5- and 10-year follow-ups. RESULTS: Three patients in the first 5 years of follow-up and six patients in 10 years of follow-up developed meningeal carcinomatosis. An incidence rate of 5.44 per 10,000 patients (95% CI: 1.75-16.9) was observed, with a 5-year risk of 0.3%. At 10-year follow up, the rate increased to 7.55 per 10,000 patients (95% CI: 3.39-16.8). In a univariate analysis, young age, tumor size larger than 15 mm, histological grade 3, more than three metastatic lymph nodes, negative estrogen receptor, positive HER2 and high proliferative index were significantly associated with meningeal carcinomatosis development. CONCLUSIONS: In an unselected breast cancer population, meningeal carcinomatosis is a rare event that is associated with adverse prognostic factors. Meningeal carcinomatosis incidence is overestimated when recorded in biased/high-risk selected breast cancer patients and should not be considered to accurately reflect the overall breast cancer population.