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Summary: Background: Tertiary Lymphoid Structures (TLS) correlate with positive outcomes in patients with NSCLC and the efficacy of immune checkpoint blockade (ICB) in cancer. The actin regulatory protein hMENA undergoes tissue-specific splicing, producing the epithelial hMENA11a linked to favorable prognosis in early NSCLC, and the mesenchymal hMENAΔv6 found in invasive cancer cells and pro-tumoral cancer-associated fibroblasts (CAFs). This study investigates how hMENA isoforms in tumor cells and CAFs relate to TLS presence, localization and impact on patient outcomes and ICB response. Methods: Methods involved RNA-SEQ on NSCLC cells with depleted hMENA isoforms. A retrospective observational study assessed tissues from surgically treated N0 patients with NSCLC, using immunohistochemistry for tumoral and stromal hMENA isoforms, fibronectin, and TLS presence. ICB-treated patient tumors were analyzed using Nanostring nCounter and GeoMx spatial transcriptomics. Multiparametric flow cytometry characterized B cells and tissue-resident memory T cells (TRM). Survival and ICB response were estimated in the cohort and validated using bioinformatics pipelines in different datasets. Findings: Findings indicate that hMENA11a in NSCLC cells upregulates the TLS regulator LTβR, decreases fibronectin, and favors CXCL13 production by TRM. Conversely, hMENAΔv6 in CAFs inhibits LTβR-related NF-kB pathway, reduces CXCL13 secretion, and promotes fibronectin production. These patterns are validated in N0 NSCLC tumors, where hMENA11ahigh expression, CAF hMENAΔv6low, and stromal fibronectinlow are associated with intratumoral TLS, linked to memory B cells and predictive of longer survival. The hMENA isoform pattern, fibronectin, and LTβR expression broadly predict ICB response in tumors where TLS indicates an anti-tumor immune response. Interpretation: This study uncovers hMENA alternative splicing as an unexplored contributor to TLS-related Tumor Immune Microenvironment (TIME) and a promising biomarker for clinical outcomes and likely ICB responsiveness in N0 patients with NSCLC. Funding: This work is supported by AIRC (IG 19822), ACC (RCR-2019-23669120), CAL.HUB.RIA Ministero Salute PNRR-POS T4, “Ricerca Corrente” granted by the Italian Ministry of Health.

Abstract Background Immune checkpoint blockade (ICB) has significantly prolonged survival of non-small cell lung cancer (NSCLC) patients, although most patients develop mechanisms of resistance. Recently single-cell RNA-sequencing (scRNA-Seq) revealed a huge T-cell phenotypic and (dys)functional state variability. Accordingly, T-cell exhaustion is recognized as a functional adaptation, with a dynamic progression from a long-lived “pre-exhausted stem-like progenitor” to a “terminally exhausted” state. In this scenario it is crucial to understand the complex interplay between co-stimulatory and inhibitory molecules in CD8+ T-cell functionality. Methods To gain a baseline landscape of the composition, functional states, and transcriptomic signatures predictive of prognosis, we analyzed CD8+ T-cell subsets characterized by the presence/absence of PD1 and CD28 from periphery, adjacent non-tumor tissue and tumor site of a cohort of treatment-naïve NSCLC patients, by integrated multiparametric flow cytometry, targeted multi-omic scRNA-seq analyses, and computational pipelines. Results Despite the increased PD1 levels, an improved PD1+CD28+ T-cell polyfunctionality was observed with the transition from periphery to tumor site, associated with lack of TIGIT, TIM-3 and LAG-3, but not with Ag-experienced-marker CD11a. Differently from CD28+ T cells, the increased PD1 levels in the tumor were associated with reduced functionality in PD1+CD28− T cells. CD11ahigh, although expressed only in a small fraction of this subset, still sustained its functionality. Absence of TIGIT, TIM-3 and CTLA-4, alone or combined, was beneficial to CD28− T cells. Notably, we observed distinct TRM phenotypes in the different districts, with CD28+ T cells more capable of producing TGFβ in the periphery, potentially contributing to elevated CD103 levels. In contrast CD28− TRM mainly produced CXCL13 within the tumor. ScRNA-seq revealed 5 different clusters for each of the two subsets, with distinctive transcriptional profiles in the three districts. By interrogating the TCGA dataset of patients with lung adenocarcinoma (LUAD) and metastatic NSCLC treated with atezolizumab, we found signatures of heterogeneous TRM and "pre-exhausted" long-lived effector memory CD8+ T cells associated with improved response to ICB only in the presence of CD28. Conclusions Our findings identify signatures able to stratify survival of LUAD patients and predict ICB response in advanced NSCLC. CD28 is advocated as a key determinant in the signatures identified, in both periphery and tumor site, thus likely providing feasible biomarkers of ICB response. Graphical Abstract

IntroductionEpithelioid hemangioendothelioma (EHE) is an ultra-rare sarcoma, marked by distinctive molecular and pathological features and with a variable clinical behavior. Its natural history is still partially understood, reliable prognostic and predictive factors are lacking and many questions are still open on the optimal management. In the context of EURACAN, a prospective registry specifically dedicated to EHE was developed and launched with the aim of providing, through high-quality prospective data collection, a better understanding of this disease.Study designRegistry-based cohort study including only new cases of patients with a pathological and molecularly confirmed diagnosis of EHE.ObjectivesTo improve the understanding of EHE natural history, validate and identify new prognostic and predictive factors, clarify the activity and efficacy of currently available treatment options, describe treatment pattern.MethodsSettings and participantsIt is an hospital-based registry established in centers with expertise in EHE including adult patients with a new pathological and molecularly confirmed diagnosis of EHE starting from the 1st December 2023. The characteristics of each patient in the facility who meets the above-mentioned inclusion criteria will be collected prospectively and longitudinally with follow-up at cancer progression and / or cancer relapse or patient death. It is a secondary use of data which will be collected from the clinical records. The data collected for the registry will not entail further examinations or admissions to the facility and/or additional appointments to those normally provided for routine patient follow-up. VariablesFull details on patients and disease features, treatment and outcome will be collected, according to common clinical practice guidelines developed and shared with all the contributing centers. In addition, data on potential confounders (e.g. comorbidity; functional status etc.) will also be collected. Statistical methodsThe data analyses will include descriptive statistics and analytical analyses. Multivariable Cox's proportional hazards model and Hazard ratios (HR) for all-cause or cause-specific mortality will be used to determine independent predictors of overall survival, recurrence and progression.ResultsThe registry has been joined by 21 sarcoma reference centers across EU and UK, covering 10 countries. Patients' recruitment started in December 2023. The estimated completion date is December 2033 upon agreement on the achievement of all the registry objectives. The already established collaboration and participation of EHE patient's associations involved in the project will help in promoting the registry and fostering accrual.

Objectives: To develop a mutation-based radiomics signature to predict response to imatinib in Gastrointestinal Stromal Tumors (GISTs). Methods: Eighty-two patients with GIST were enrolled in this retrospective study, including 52 patients from one center that were used to develop the model, and 30 patients from a second center to validate it. Reference standard was the mutational status of tyrosine-protein kinase (KIT) and platelet-derived growth factor α (PDGFRA). Patients were dichotomized in imatinib sensitive (group 0 - mutation in KIT or PDGFRA, different from exon 18-D842V), and imatinib non-responsive (group 1 - PDGFRA exon 18-D842V mutation or absence of mutation in KIT/PDGFRA). Initially, 107 texture features were extracted from the tumor masks of baseline computed tomography scans. Different machine learning methods were then implemented to select the best combination of features for the development of the radiomics signature. Results: The best performance was obtained with the 5 features selected by the ANOVA model and the Bayes classifier, using a threshold of 0.36. With this setting the radiomics signature had an accuracy and precision for sensitive patients of 82 % (95 % CI:60–95) and 90 % (95 % CI:73–97), respectively. Conversely, a precision of 80 % (95 % CI:34–97) was obtained in non-responsive patients using a threshold of 0.9. Indeed, with the latter setting 4 patients out of 5 were correctly predicted as non-responders. Conclusions: The results are a first step towards using radiomics to improve the management of patients with GIST, especially when tumor tissue is unavailable for molecular analysis or when molecular profiling is inconclusive.

The therapeutic scenario of Human Epidermal Growth Factor Receptor 2 positive advanced breast cancer (ABC) has been recently enriched by a number of innovative agents, which are reshaping treatment sequence. While randomized trials have documented an advantage in terms of efficacy, for the newly available agents we lack effectiveness and tolerability evidence from the real-world setting. Similarly, the identification of predictive biomarkers might improve clinical decision. We herein describe the outline of a prospective/retrospective study which aims to explore the optimal sequence of treatment in HER2+, pertuzumab pre-treated ABC patients treated in II line with anti-HER2 agents in clinical practice. As part of the pre-clinical tasks envisioned by the STEP study, in vitro cell models of resistance were exploited to investigate molecular features associated with reduced efficacy of HER2 targeting agents at the transcript level. The aggressive behavior of resistant cell populations was measured by growth assessment in mouse models. This approach led to the identification of DARPP-32 and t-DARPP proteins as possible predictive biomarkers of efficacy of anti-HER2 agents. Biomarkers validation and the clinical goals will be reached through patients’ inclusion into two independent cohorts, i.e., the prospective and retrospective cohorts, whose setup is currently ongoing.

Background Understanding how cancer signaling pathways promote an immunosuppressive program which sustains acquired or primary resistance to immune checkpoint blockade (ICB) is a crucial step in improving immunotherapy efficacy. Among the pathways that can affect ICB response is the interferon (IFN) pathway that may be both detrimental and beneficial. The immune sensor retinoic acid-inducible gene I (RIG-I) induces IFN activation and secretion and is activated by actin cytoskeleton disturbance. The actin cytoskeleton regulatory protein hMENA, along with its isoforms, is a key signaling hub in different solid tumors, and recently its role as a regulator of transcription of genes encoding immunomodulatory secretory proteins has been proposed. When hMENA is expressed in tumor cells with low levels of the epithelial specific hMENA11a isoform, identifies non-small cell lung cancer (NSCLC) patients with poor prognosis. Aim was to identify cancer intrinsic and extrinsic pathways regulated by hMENA11a downregulation as determinants of ICB response in NSCLC. Here, we present a potential novel mechanism of ICB resistance driven by hMENA11a downregulation.Methods Effects of hMENA11a downregulation were tested by RNA-Seq, ATAC-Seq, flow cytometry and biochemical assays. ICB-treated patient tumor tissues were profiled by Nanostring IO 360 Panel enriched with hMENA custom probes. OAK and POPLAR datasets were used to validate our discovery cohort.Results Transcriptomic and biochemical analyses demonstrated that the depletion of hMENA11a induces IFN pathway activation, the production of different inflammatory mediators including IFNβ via RIG-I, sustains the increase of tumor PD-L1 levels and activates a paracrine loop between tumor cells and a unique macrophage subset favoring an epithelial-mesenchymal transition (EMT). Notably, when we translated our results in a clinical setting of NSCLC ICB-treated patients, transcriptomic analysis revealed that low expression of hMENA11a, high expression of IFN target genes and high macrophage score identify patients resistant to ICB therapy.Conclusions Collectively, these data establish a new function for the actin cytoskeleton regulator hMENA11a in modulating cancer cell intrinsic type I IFN signaling and extrinsic mechanisms that promote protumoral macrophages and favor EMT. These data highlight the role of actin cytoskeleton disturbance in activating immune suppressive pathways that may be involved in resistance to ICB in NSCLC.

Abstract Profiling the T-Cell Receptor (TCR) repertoire is establishing as a potent approach to investigate autologous and treatment-induced antitumor immune response. Technical and computational breakthroughs, including high throughput next-generation sequencing (NGS) approaches and spatial transcriptomics, are providing unprecedented insight into the mechanisms underlying antitumor immunity. A precise spatiotemporal variation of T-cell repertoire, which dynamically mirrors the functional state of the evolving host-cancer interaction, allows the tracking of the T-cell populations at play, and may identify the key cells responsible for tumor eradication, the evaluation of minimal residual disease and the identification of biomarkers of response to immunotherapy. In this review we will discuss the relationship between global metrics characterizing the TCR repertoire such as T-cell clonality and diversity and the resultant functional responses. In particular, we will explore how specific TCR repertoires in cancer patients can be predictive of prognosis or response to therapy and in particular how a given TCR re-arrangement, following immunotherapy, can predict a specific clinical outcome. Finally, we will examine current improvements in terms of T-cell sequencing, discussing advantages and challenges of current methodologies.

Effective treatment of advanced/metastatic bone and soft tissue sarcomas still represents an unmet medical need. Recent advances in targeted therapies have highlighted the potential of cyclin-dependent kinases (CDK) inhibitors in several cancer types, including sarcomas. CDKs are master regulators of the cell cycle; their dysregulation is listed among the “hallmarks of cancer” and sarcomas are no exception to the rule. In this review, we report both the molecular basis, and the potential therapeutic implications for the use of CDK inhibitors in sarcoma treatment. What is more, we describe and discuss the possibility and biological rationale for combination therapies with conventional treatments, target therapy and immunotherapy, highlighting potential avenues for future research to integrate CDK inhibition in sarcoma treatment.

BackgroundAdvanced and unresectable bone and soft tissue sarcomas (BSTS) still represent an unmet medical need. We demonstrated that the alkylating agent trabectedin and the PARP1-inhibitor olaparib display antitumor activity in BSTS preclinical models. Moreover, in a phase Ib clinical trial (NCT02398058), feasibility, tolerability and encouraging results have been observed and the treatment combination is currently under study in a phase II trial (NCT03838744).MethodsDifferential expression of genes involved in DNA Damage Response and Repair was evaluated by Nanostring® technology, extracting RNA from pre-treatment tumor samples of 16 responder (≥6-month progression free survival) and 16 non-responder patients. Data validation was performed by quantitative real-time PCR, RNA in situ hybridization, and immunohistochemistry. The correlation between the identified candidate genes and both progression-free survival and overall survival was investigated in the publicly available dataset “Sarcoma (TCGA, The Cancer Genome Atlas)”.ResultsDifferential RNA expression analysis revealed an 8-gene signature (CDKN2A, PIK3R1, SLFN11, ATM, APEX2, BLM, XRCC2, MAD2L2) defining patients with better outcome upon trabectedin+olaparib treatment. In responder vs. non-responder patients, a significant differential expression of these genes was further confirmed by RNA in situ hybridization and by qRT-PCR and immunohistochemistry in selected experiments. Correlation between survival outcomes and genetic alterations in the identified genes was shown in the TCGA sarcoma dataset.ConclusionsThis work identified an 8-gene expression signature to improve prediction of response to trabectedin+olaparib combination in BSTS. The predictive role of these potential biomarkers warrants further investigation.

In this work we consider differential equations of the type $$\pm\, u^{(k)}=f(u),$$ and study the extinction profile of their solutions. Emphasis is placed on the special case $-u^{(4)}=\sign(u)$, which is related to the Kuramoto-Sivashinsky equation. In this case we describe in more detail the extinction phenomenon and prove a conjecture by Galaktionov and Svirshchevskii.

Abstract Background Tracking the genetic variability of Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) is a crucial challenge. Mainly to identify target sequences in order to generate robust vaccines and neutralizing monoclonal antibodies, but also to track viral genetic temporal and geographic evolution and to mine for variants associated with reduced or increased disease severity. Several online tools and bioinformatic phylogenetic analyses have been released, but the main interest lies in the Spike protein, which is the pivotal element of current vaccine design, and in the Receptor Binding Domain, that accounts for most of the neutralizing the antibody activity. Methods Here, we present an open-source bioinformatic protocol, and a web portal focused on SARS-CoV-2 single mutations and minimal consensus sequence building as a companion vaccine design tool. Furthermore, we provide immunogenomic analyses to understand the impact of the most frequent RBD variations. Results Results on the whole GISAID sequence dataset at the time of the writing (October 2020) reveals an emerging mutation, S477N, located on the central part of the Spike protein Receptor Binding Domain, the Receptor Binding Motif. Immunogenomic analyses revealed some variation in mutated epitope MHC compatibility, T-cell recognition, and B-cell epitope probability for most frequent human HLAs. Conclusions This work provides a framework able to track down SARS-CoV-2 genomic variability.

Gastrointestinal stromal tumors (GISTs) are rare, mesenchymal tumors of the gastrointestinal tract, characterized by either KIT or PDGFRA mutation in about 85% of cases. KIT/PDGFRA wild type gastrointestinal stromal tumors (wtGIST) account for the remaining 15% of GIST and represent an unmet medical need: their prevalence and potential medical vulnerabilities are not completely defined, and effective therapeutic strategies are still lacking. In this study we set a patient-derived preclinical model of wtGIST to investigate their phenotypic features, along with their susceptibility to cellular immunotherapy with cytokine-induced killer lymphocytes (CIK) and interferons (IFN). We generated 11 wtGIST primary cell lines (wtGISTc). The main CIK ligands (MIC A/B; ULBPs), along with PD-L1/2, were expressed by wtGISTc and the expression of HLA-I molecules was preserved. Patient-derived CIK were capable of intense killing in vitro against wtGISTc resistant to both imatinib and sunitinib. We found that CIK produce a high level of granzyme B, IFNα and IFNγ. CIK-conditioned supernatant was responsible for part of the observed tumoricidal effect, along with positive bystander modulatory activities enhancing the expression of PD-L1/2 and HLA-I molecules. IFNα, but not In, had direct antitumor effects on 50% (4/8) of TKI-resistant wtGISTc, positively correlated with the tumor expression of IFN receptors. wtGIST cells that survived IFNα were still sensitive to CIK immunotherapy. Our data support the exploration of CIK immunotherapy in clinical studies for TKI-resistant wtGIST, proposing reevaluation for IFNα within this challenging setting.

Background Sarcomas exhibit low expression of factors related to immune response, which could explain the modest activity of PD-1 inhibitors. A potential strategy to convert a cold into an inflamed microenvironment lies on a combination therapy. As tumor angiogenesis promotes immunosuppression, we designed a phase Ib/II trial to test the double inhibition of angiogenesis (sunitinib) and PD-1/PD-L1 axis (nivolumab).Methods This single-arm, phase Ib/II trial enrolled adult patients with selected subtypes of sarcoma. Phase Ib established two dose levels: level 0 with sunitinib 37.5 mg daily from day 1, plus nivolumab 3 mg/kg intravenously on day 15, and then every 2 weeks; and level −1 with sunitinib 37.5 mg on the first 14 days (induction) and then 25 mg per day plus nivolumab on the same schedule. The primary endpoint was to determine the recommended dose for phase II (phase I) and the 6-month progression-free survival rate, according to Response Evaluation Criteria in Solid Tumors 1.1 (phase II).Results From May 2017 to April 2019, 68 patients were enrolled: 16 in phase Ib and 52 in phase II. The recommended dose of sunitinib for phase II was 37.5 mg as induction and then 25 mg in combination with nivolumab. After a median follow-up of 17 months (4–26), the 6-month progression-free survival rate was 48% (95% CI 41% to 55%). The most common grade 3–4 adverse events included transaminitis (17.3%) and neutropenia (11.5%).Conclusions Sunitinib plus nivolumab is an active scheme with manageable toxicity in the treatment of selected patients with advanced soft tissue sarcoma, with almost half of patients free of progression at 6 months.Trial registration number NCT03277924.

Bone sarcomas are a group of heterogeneous malignant mesenchymal tumors. Complete surgical resection is still the cornerstone of treatment, but, in the advanced/unresectable setting, their management remains challenging and not significantly improved by target- and immuno-therapies. We focused on the tyrosine kinase Eph type-A receptor-2 (EphA2), a key oncoprotein implicated in self-renewal, angiogenesis, and metastasis, in several solid tumors and thus representing a novel potential therapeutic target. Aiming at better characterizing its expression throughout the main bone sarcoma histotypes, we investigated EPHA2 expression in the Cancer Cell Lines Encyclopedia and in public datasets with clinical annotations. looking for correlations with molecular, histopathological and patients’ features and clinical outcomes in a total of 232 osteosarcomas, 197 Ewing’s sarcomas, and 102 chondrosarcomas. We observed EPHA2 expression in bone sarcoma cell lines. We demonstrated higher EPHA2 expression in tumor tissues when compared to normal counterparts. A significant correlation was found between EPHA2 expression and Huvos grade (osteosarcoma) and with worse overall survival (dedifferentiated chondrosarcoma). Next, we characterized EPHA2 expression and activation in bone sarcoma primary tissues and in patient-derived xenografts generated in our laboratory to verify their reliability as in vivo models of osteosarcoma, Ewing’s sarcoma and chondrosarcoma. Furthermore, for the first time, we demonstrated EPHA2 expression in chondrosarcoma, suggesting its potential key role in this histotype. Indeed, we observed a significant dose-dependent antitumor effect of the EphA2-inhibitor ALW-II-41-27 in patient-derived in vitro models. In conclusion, EphA2 targeting represents a promising novel therapeutic strategy against bone sarcomas.

Abstract Background Nuclear protein in testis (NUT) midline carcinoma is a rarely diagnosed and potentially under-recognized type of squamous carcinoma that is considered one of the most aggressive human solid tumors. Alpha-fetoprotein elevation has been associated with chronic liver diseases and a limited number of cancers. In particular, in presence of a mediastinal mass in a young man, alpha-fetoprotein elevation is considered nearly pathognomonic of a non-seminoma germ-cell tumor. Case presentation A 22-year old man without any comorbidity was diagnosed with a large mediastinal mass with skeletal and lymph node metastases. The clinical picture was dominated by a life-threatening superior vena cava syndrome with elevated alpha-fetoprotein and lactate dehydrogenase that supported the diagnostic suspicion of mediastinal germ-cell tumor. However, a biopsy showed a poorly-differentiated and diffusely necrotic carcinoma. We eventually reached the diagnosis of the peculiar entity of NUT midline carcinoma, but the differential diagnosis was quite challenging also because alpha-fetoprotein is not reported as a marker of NUT midline carcinoma. Notably, alpha-fetoprotein levels correlated with disease course. Conclusions The life-threatening aggressiveness of NUT midline carcinoma mandates to reach the right diagnosis in the shortest possible time. In this regard, poorly differentiated carcinomas lacking glandular differentiation mandate testing for NUT expression by immunohistochemistry. Awareness of a potentially misleading tumor marker elevation can help to broaden the differential diagnosis and establish the most appropriate treatment.

Abstract Background Enhancing the antitumor activity of the DNA-damaging drugs is an attractive strategy to improve current treatment options. Trabectedin is an isoquinoline alkylating agent with a peculiar mechanism of action. It binds to minor groove of DNA inducing single- and double-strand-breaks. These kinds of damage lead to the activation of PARP1, a first-line enzyme in DNA-damage response pathways. We hypothesized that PARP1 targeting could perpetuate trabectedin-induced DNA damage in tumor cells leading finally to cell death. Methods We investigated trabectedin and PARP1 inhibitor synergism in several tumor histotypes both in vitro and in vivo (subcutaneous and orthotopic tumor xenografts in mice). We searched for key determinants of drug synergism by comparative genomic hybridization (aCGH) and gene expression profiling (GEP) and validated their functional role. Results Trabectedin activated PARP1 enzyme and the combination with PARP1 inhibitors potentiated DNA damage, cell cycle arrest at G2/M checkpoint and apoptosis, if compared to single agents. Olaparib was the most active PARP1 inhibitor to combine with trabectedin and we confirmed the antitumor and antimetastatic activity of trabectedin/olaparib combination in mice models. However, we observed different degree of trabectedin/olaparib synergism among different cell lines. Namely, in DMR leiomyosarcoma models the combination was significantly more active than single agents, while in SJSA-1 osteosarcoma models no further advantage was obtained if compared to trabectedin alone. aCGH and GEP revealed that key components of DNA-repair pathways were involved in trabectedin/olaparib synergism. In particular, PARP1 expression dictated the degree of the synergism. Indeed, trabectedin/olaparib synergism was increased after PARP1 overexpression and reduced after PARP1 silencing. Conclusions PARP1 inhibition potentiated trabectedin activity in a PARP1-dependent manner and PARP1 expression in tumor cells might be a useful predictive biomarker that deserves clinical evaluation.

Metastatic bone and soft tissue sarcomas often relapse after chemotherapy (CHT) and molecular targeted therapy (mTT), maintaining a severe prognosis. A subset of sarcoma cancer stem cells (sCSC) is hypothesized to resist conventional drugs and sustain disease relapses. We investigated the immunotherapy activity of cytokine induced killer cells (CIK) against autologous sCSC that survived CHT and mTT. The experimental platform included two aggressive bone and soft tissue sarcoma models: osteosarcoma (OS) and undifferentiated-pleomorphic sarcoma (UPS). To visualize putative sCSC we engineered patient-derived sarcoma cultures (2 OS and 3 UPS) with a lentiviral sCSC-detector wherein the promoter of stem-gene Oct4 controls the expression of eGFP. We visualized a fraction of sCSC (mean 24.2 ± 5.2%) and confirmed their tumorigenicity in vivo. sCSC resulted relatively resistant to both CHT and mTT in vitro. Therapeutic doses of doxorubicin significantly enriched viable eGFP+sCSC in both OS (2.6 fold, n = 16) and UPS (2.3 fold, n = 29) compared to untreated controls. Treatment with sorafenib (for OS) and pazopanib (for UPS) also determined enrichment (1.3 fold) of viable eGFP+sCSC, even if less intense than what observed after CHT. Sarcoma cells surviving CHT and mTT were efficiently killed in vitro by autologous CIK even at minimal effector/target ratios (40:1 = 82%, 1:4 = 29%, n = 13). CIK immunotherapy did not spare sCSC that were killed as efficiently as whole sarcoma cell population. The relative chemo-resistance of sCSC and sensitivity to CIK immunotherapy was confirmed in vivo. Our findings support CIK as an innovative, clinically explorable, approach to eradicate chemo-resistant sCSC implicated in tumor relapse.

Background: The evidence on high-dose ifosfamide (HD-IFO) use in patients with relapsed osteosarcoma is limited. We performed a retrospective study to analyze HD-IFO activity. Methods: Patients with osteosarcoma relapsed after standard treatment [methotrexate, doxorubicin, cisplatin +/− ifosfamide (MAP+/−I)] with measurable disease according to RECIST1.1 were eligible to ifosfamide (3 g/m2/day) continuous infusion (c.i.) days 1–5 q21d. RECIST1.1 overall response rate (ORR) (complete response (CR) + partial response (PR)), progression-free survival at 6-month (6m-PFS), duration of response (DOR), and 2-year overall survival (2y-OS) were assessed. PARP1 expression and gene mutations were tested by immunohistochemistry and next-generation sequencing. Results: 51 patients were included. ORR was 20% (1 CR + 9 PR). Median DOR was 5 months (95%CI 2–7). Median PFS, 6m-PFS, OS, and 2y-OS were 6 months (95%CI 4–9), 51%, 15 months (10–19), and 30%, respectively. A second surgical complete remission (CR2) was achieved in 26 (51%) patients. After multivariate analysis, previous use of ifosfamide (HR 2.007, p = 0.034) and CR2 (HR 0.126, p < 0.001) showed a significant correlation with PFS and OS, respectively. No significant correlation was found between outcomes and PARP1 or gene mutations. Conclusions: HD-IFO should be considered as the standard first-line treatment option in relapsed osteosarcoma and control arm of future trial in this setting.

Background: Rechallenge with imatinib is an option in advanced gastrointestinal stromal tumor (GIST) patients following progression with standard tyrosine-kinase inhibitors (TKIs), imatinib, sunitinib and regorafenib. We retrospectively collected data from metastatic Italian GIST patients treated with imatinib resumption after progression to conventional TKIs. Methods: A total of 104 eligible advanced GIST patients, previously treated with imatinib, sunitinib and regorafenib, were collected from six referral Italian institutions. Mutational analysis was recorded and correlated with survival and response according to RECIST 1.1 or CHOI criteria. Results: Overall, 71 patients treated with imatinib 400 mg as rechallenge were included. Mutational status was available in all patients. The median follow up was 13 months. In patients who received a rechallenge therapy, the median time to progression (TTP) was 5.4 months [95% confidence interval (CI) 1.9–13.5] and overall survival (OS) was 10.6 months (95% CI 2.8–26.9). A correlation between mutational status, response rate, TTP and OS was not found but comparing deleted versus nondeleted KIT exon 11 patients, a significant difference was identified in terms of TTP and OS ( p = 0.04 and p = 0.02, respectively). Conclusions: Our retrospective data confirm that imatinib rechallenge is a reasonable option in advanced GIST. The prognostic value of the specific KIT mutations was confirmed in our series.

Background: Regorafenib (REG) has now been approved as the standard third-line therapy in metastatic gastrointestinal stromal tumour (GIST) patients at the recommended dose and schedule of 160 mg once daily for the first 3 weeks of each 4-week cycle. However, it has a relevant toxicity profile that mainly occurs within the first cycles of therapy, and dose and schedule adjustments are often required to reduce the frequency or severity of adverse events and to avoid early treatment discontinuation. To date, large amounts of data on the use of REG in metastatic GIST patients in daily clinical practice are not available, and we lack information about how this treatment personalization really affects the quality of life (QoL) of patients. The aim of the present retrospective study is to build a comprehensive picture of all alternative REG strategies adopted in daily clinical practice for use in metastatic GIST patients. Methods: Metastatic GIST patients treated with dose adjustment or alternative schedules of REG at seven reference Italian centres were retrospectively included. Results: For a total of 62 metastatic GIST patients, we confirmed that REG treatment adjustment is common in clinical practice and that it is very heterogeneous, with approximately 20 different strategies being adopted. Independent of which strategy is chosen, treatment personalization has led to a clinical benefit defined as complete or partial resolution of side effects in almost all patients, affecting the duration of REG treatment. Conclusions: The personalization of REG, even if it is heterogeneous, seems to be crucial to maximize the overall treatment duration.