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Abstract Soft tissue sarcomas represent an heterogeneous group of rare mesenchymal tumors comprising 1% of all solid malignancies. Among them, liposarcoma is one of the most common histotypes with atypical lipomatous tumor/well differentiated liposarcoma and dedifferentiated liposarcoma (ALT/WDLPS and DDLPS) as the major sub-entities. The unavailability of predictive, prognostic and druggable biomarkers makes the management of these lesions challenging. In recent years CDK4 and its inhibitors have emerged as potential agents for these lesions especially for ALT/WDLPS and DDLPS but the results are not conclusive and need to be elucidated. This study involved 21 ALT/WDLPS and DDLPS patients. Histological analyses of MDM2 and CDK4 were carried out. Moreover, a DDLPS patient-derived cancer model was established in vitro and in vivo assessing the efficacy of palbociclib in combination and sequential treatment. Finally, in silico analyses on CDK4 expression were carried out. The results showed a higher expression of CDK4 and MDM2 in DDLPS compared to ALT/WDLPS. Moreover, no correlation between MDM2 expression and CDK4 was observed. Next, in vitro analysis of CDK4 inhibitor palbociclib showed an antagonistic effect when combined to other chemotherapeutics, while it exhibited a significant synergy when administered in sequential schedule with lenvatinib. Next, in vivo analysis on DDLPS xenotransplanted embryos assessing the efficacy and safety profile of the in vitro tested schedules confirmed the observed data. This proof-of-concept study sheds light on the natural history of ALT/WDLPS and DDLPS and provides the rationale for the clinical applicability of sequential treatment with palbociclib in the management of DDLPS.

Immunotherapy has emerged as promising treatment in sarcomas, but the high variability in terms of histology, clinical behavior and response to treatments determines a particular challenge for its role in these neoplasms. Tumor immune microenvironment (TiME) of sarcomas reflects the heterogeneity of these tumors originating from mesenchymal cells and encompassing more than 100 histologies. Advances in the understanding of the complexity of TiME have led to an improvement of the immunotherapeutic responsiveness in sarcomas, that at first showed disappointing results. The proposed immune-classification of sarcomas based on the interaction between immune cell populations and tumor cells showed to have a prognostic and potential predictive role for immunotherapies. Several studies have explored the clinical impact of immune therapies in the management of these histotypes leading to controversial results. The presence of Tumor Infiltrating Lymphocytes (TIL) seems to correlate with an improvement in the survival of patients and with a higher responsiveness to immunotherapy. In this context, it is important to consider that also immune-related genes (IRGs) have been demonstrated to have a key role in tumorigenesis and in the building of tumor immune microenvironment. The IRGs landscape in soft tissue and bone sarcomas is characterized by the connection between several tumor-related genes that can assume a potential prognostic and predictive therapeutic role. In this paper, we reviewed the state of art of the principal immune strategies in the management of sarcomas including their clinical and translational relevance.

Abstract Background Soft tissue sarcomas (STS) are a rare group of solid neoplasm including among others liposarcoma, leiomyosarcoma (L-sarcoma) and undifferentiated pleomorphic sarcoma (UPS) entities. The current first-line treatment is represented by anthracycline based- regimens, second-line may include trabectedin. Currently the activity of trabectedin and its mechanism of action is not completely elucidated. Methods Taking the advantages of our 3D patient-derived primary culture translational model we performed genomic-, chemobiogram, proteomic- and in vivo analysis in a UPS culture (S1). Furthermore pharmacological profiling of a UPS and L-sarcoma patient-derived case series and in silico analysis were carried out. Results Trabectedin exhibited an increased activity in 3D respect to 2D cultures suggesting an extracellular matrix (ECM) and timp1 involvement in its mechanism of action. Moreover 3D S1 xenotranspanted zebrafish model showed an increased sensitivity to trabectedin. Finally the results were further validated in a UPS and L-sarcoma case series. Conclusions Taken together these results confirmed the activity of trabectedin in these STS histotypes. Moreover the data underline the ECM involvement in the cytotoxic effect mediated by trabectedin and could open the door for researches aimed to focus on the patient setting that could benefit from this agent.

Abstract Bone metastases (BM) are still the main cause of morbidity in cancer patients because of skeletal-related events (SREs) that reduce quality of life. They have also led to increased social and healthcare costs. At present, data available on BM are insufficient. This was a multicentre prospective observational study of patients with BM from breast cancer (BC) with at least 6 months’ follow-up. Information on patients at the first diagnosis of BM, including demographics and characteristics of the primary tumor and BM. Data were periodically updated by participating centres and reviewed by the coordinator centre. From October 2014 to July 2019, 618 patients with BM from solid tumors were enrolled and 220 were eligible for the present study. Median age was 62 years (range 26–86). Median follow-up was 34 months (range 6–149). At the time of enrolment, 109 (50%) had only BM (BOM) and 109 (50%) had concomitant visceral lesions and BM (BVM). Median time-to-first BM was 47 months (range 0–312) in BOM and 78.6 months in BVM patients. Disease-free interval differed on the basis of BC molecular subtype and stage. Ninety-eight BM patients had at least on SRE. Zoledronate was used in 69.1% of cases and denosumab in 28.3%. First-line treatment was hormone-based (50.7%), chemotherapy-based (38.7%) or chemotherapy- + hormone therapy-based (9.7%). Median progression-free and overall survival were 15.1 months (95% CI 12.6–18.4) and 66.8 months (95% CI 52.1–79.2), respectively. Our prospective study could substantially help to better understand the natural history of BM from BC.

BackgroundNTRK (neurotrophic tyrosine receptor kinase)-rearranged spindle cell neoplasms are a new group of tumors included in the new 5th edition of the World Health Organization (WHO) classification of soft Tissue and Bone Sarcomas. These tumors are characterized by NTRK gene fusions and show a wide spectrum of histologies and clinical behavior. Several targeted therapies have recently been approved for tumors harboring NTRK fusions, including STS.Case PresentationA 26-year-old male with advanced, pretreated NTRK rearranged spindle cell neoplasm and liver, lung and bone metastases was treated with larotrectinib on a continuous 28-day schedule, at a dose of 100 mg twice daily. An 18FDG-PET/CT scan performed after 7 days of treatment showed tumor shrinkage in both visceral and bone lesions. There was no drug-related toxicity. Subsequent evaluations confirmed continued tumor regression in disease sites. The patient is well and continues treatment.ConclusionThe clinical and radiological response of our patient with an uncommon TPM4 (exon 7)-NTRK1 (exon 12) gene fusion tumor treated with a first-generation TRK inhibitor could contribute to a better understanding of the biology of this new STS entity and help to improve patient management.

IntroductionBone metastases (BMs) are a negative prognostic factor in patients with non-small cell lung cancer (NSCLC). Although immune-checkpoint inhibitors (ICIs) have dramatically changed the therapeutic landscape of NSCLC, little information is available on BMs from NSCLC treated with ICIs alone or in association with bone-targeted therapy (BTT) such as zoledronate or denosumab.MethodsFrom 2014 to 2020, 111 of the 142 patients with BMs secondary to NSCLC extrapolated from the prospective multicenter Italian BM Database were eligible for analysis. Information on blood count, comorbidities, and toxicity was retrospectively collected. The neutrophil-to-lymphocyte ratio (NLR) pre- and post-treatment was calculated. Survival was analyzed using the Kaplan–Meier method, with statistical significance of survival differences assessed using the log-rank test.ResultsMedian age was 66 (range, 42–84) years. Performance status (PS) Eastern Cooperative Oncology Group (ECOG) was 0–1 in 79/111 patients. The majority of patients (89.2%) had adenocarcinoma histology. At a median follow-up of 47.4 months, median progression-free (mPFS) and overall survival (mOS) was 4.9 (95%CI, 2.8–10.0) and 11.9 (95%CI, 8.2–14.4) months, respectively. Forty-six (43.4%) patients with BM NSCLC underwent first- or further-line therapy with ICIs: 28 (60.8%) received nivolumab, 9 (19.6%) pembrolizumab, and 9 (19.6%) atezolizumab. Of the 46 patients treated with ICIs, 30 (65.2%) underwent BTT: 24 (80.0%) with zoledronate and 6 (20.0%) with denosumab. The ICI-alone group had an mOS of 15.8 months [95%CI, 8.2–not evaluable (NE)] vs. 21.8 months (95%CI, 14.5–not evaluable) for the ICI plus BTT group and 7.5 (95%CI, 6.1–10.9) months for the group receiving other treatments (p < 0.001). NLR ≤5 had a positive impact on OS.ConclusionBTT appears to have a synergistic effect when used in combination with ICIs, improving patient survival.

Background: Several phase-II trials have been designed to evaluate tyrosine kinase inhibitors (TKIs), in particular, pazopanib in neuroendocrine neoplasia (NEN), but its efficacy has not yet been demonstrated in a randomised-controlled Phase III trial. A systematic review of the published clinical trials of metastatic NEN patients could reduce the possible bias of single phase II studies. The present systematic review focuses on the efficacy and safety of pazopanib in patients with metastatic and locally advanced NEN.Methods: A systematic search in the major databases Medline/PubMed, Cochrane and Embase and in supplementary material from important international Meetings was performed to identify publications on pazopanib for the treatment of neuroendocrine neoplasia. English language was defined as a restriction. Four authors of the present review independently performed the study selection, assessed the risk of bias and extracted study data. Four published clinical trials and 2 abstracts were identified. One trial was excluded because the topic was Von-Hippel Landau disease and one abstract was eliminated because of the lack of information on meeting proceedings.Results: In all of the trials pazopanib was orally administered at a dose of 800 mg daily continuously with a 28-day cycle. The intention-to-treat population for efficacy was composed of 230 patients with a median age of 62 years. The partial response rate was 10.7% (95% confidence interval 2.6–20.5). The rate for stable disease was 79.6% (range: 61.7–92.1%) with a disease control rate (DCR) of 90.3%. Progressive disease was reported in 9.7% (range 5.2–17.6) of patients. No complete responses were observed. Median progression-free survival was 11.6 months (95% CI: 9.2–13.9). Overall survival from all the trials was 24.6 (95% CI: 18.7–40.8) months. Severe adverse events (grade III–IV) included hypertension 31%, 16% increase in AST/ALT, diarrhoea 10% and fatigue 10%.Conclusions: Pazopanib monotherapy achieved a DCR of 90.3% in patients with locally advanced and/or metastatic neuroendocrine neoplasia, with an overall response rate comparable to other TKIs and mTOR inhibitors and a safety profile similar to that of drugs of the same class.

The incidence of neuroendocrine neoplasia (NEN) is higher in individuals ≥70 years of age (elderly) who are underrepresented in clinical trials because of comorbidities and low performance status. We retrospectively analyzed the outcome of elderly patients with metastatic NEN (mNEN). Comorbidities were summarized by Charlson Comorbidity Index (CCI), Kaplan–Meier method was applied to estimate overall survival (OS) and Cox’s proportional hazard model was used to assess the impact of known prognostic factors. We retrieved data on 145 mNEN patients aged ≥70 years seen at our center from June 2007 to March 2016. Fifty-six (38.6%) were aged ≥75 years. ECOG PS was 0 in 45.7% of cases and CCI was 0 in 41.0% and 1 in 37.4%. A total of 75.4% of patients had grade (G)1/G2 NEN and 24.6%, G3. Octreoscan/Gallium PET/CT and FDG-PET/CT were positive in 94.2% and 70.3% of cases, respectively. Median follow-up was 72.3 (53.2–85.1) months. Seventy-nine patients received first-line somatostatin analogs (SSA), 23 peptide receptor radionuclide therapy (PRRT) and 36 chemotherapy (CHT). Seven did not undergo first-line therapy and 102 received more than one line. Median overall survival (mOS) was 5.1 years (95% CI: 3.4–6.6). No differences in mOS were seen according to CCI. First-line PRRT patients had a mOS of 6.5 years (95% CI: 3.3–not reached (NR)), SSA 5.7 years (95% CI: 4.2–7) and CHT 5.9 years (95% CI: 0.4–NR). mOS in CHT-treated G3 patients was 1.5 years (1.0–2.5). ECOG PS and FDG PET/CT were identified as independent prognostic factors. Results suggest that the above treatments positively impacted OS in elderly mNEN patients, including those aged ≥75 years.

Giant cell tumor of bone (GCTB) and desmoplastic fibroma (DF) are bone sarcomas with intermediate malignant behavior and unpredictable prognosis. These locally aggressive neoplasms exhibit a predilection for the long bone or mandible of young adults, causing a severe bone resorption. In particular, the tumor stromal cells of these lesions are responsible for the recruiting of multinucleated giant cells which ultimately lead to bone disruption. In this regard, the underlying pathological mechanism of osteoclastogenesis processes in GCTB and DF is still poorly understood. Although current therapeutic strategy involves surgery, radiotherapy and chemotherapy, the benefit of the latter is still debated. Thus, in order to shed light on these poorly investigated diseases, we focused on the molecular biology of GCTB and DF. The expression of bone-vicious-cycle- and neoangiogenesis-related genes was investigated. Moreover, combining patient-derived primary cultures with 2D and 3D culture platforms, we investigated the role of denosumab and levantinib in these diseases. The results showed the upregulation of RANK-L, RANK, OPN, CXCR4, RUNX2 and FLT1 and the downregulation of OPG and CXCL12 genes, underlining their involvement and promising role in these neoplasms. Furthermore, in vitro analyses provided evidence for suggesting the combination of denosumab and lenvatinib as a promising therapeutic strategy in GCTB and DF compared to monoregimen chemotherapy. Furthermore, in vivo zebrafish analyses corroborated the obtained data. Finally, the clinical observation of retrospectively enrolled patients confirmed the usefulness of the reported results. In conclusion, here we report for the first time a molecular and pharmacological investigation of GCTB and DF combining the use of translational and clinical data. Taken together, these results represent a starting point for further analyses aimed at improving GCTB and DF management.

Abstract The use of patient-derived primary cell cultures in cancer preclinical assays has increased in recent years. The management of resected tumor tissue remains complex and a number of parameters must be respected to obtain complete sample digestion and optimal vitality yield. We provide an overview of the benefits of correct primary cell culture management using different preclinical methodologies, and describe the pros and cons of this model with respect to other kinds of samples. One important advantage is that the heterogeneity of the cell populations composing a primary culture partially reproduces the tumor microenvironment and crosstalk between malignant and healthy cells, neither of which is possible with cell lines. Moreover, the use of patient-derived specimens in innovative preclinical technologies, such as 3D systems or bioreactors, represents an important opportunity to improve the translational value of the results obtained. In vivo models could further our understanding of the crosstalk between tumor and other tissues as they enable us to observe the systemic and biological interactions of a complete organism. Although engineered mice are the most common model used in this setting, the zebrafish (Danio rerio) species has recently been recognized as an innovative experimental system. In fact, the transparent body and incomplete immune system of zebrafish embryos are especially useful for evaluating patient-derived tumor tissue interactions in healthy hosts. In conclusion, ex vivo systems represent an important tool for cancer research, but samples require correct manipulation to maximize their translational value.

The skeleton is one of the most common sites of metastatic spread from advanced clear-cell renal carcinoma (ccRCC). Most of the bone lesions observed in RCC patients are classified as osteolytic, causing severe pain and morbidity due to pathological bone destruction. Nowadays, it is well known that cancer induced bone loss in lytic metastasis is caused by the triggering of a vicious cycle between cancer and bone resident cells that leads to an imbalance between bone formation and degradation. Targeting the mammalian target of rapamycin (mTOR) is an efficient treatment option for metastatic renal carcinoma patients. Moreover, bone targeted therapy could benefit bone metastatic cancer patients caused by advanced RCC. However, more data is needed to support the hypothesis of the beneficial effect of a combined therapy. The aim of this work is to investigate the effect of targeting mTOR and the sequential combination with bone targeted therapy as a strategy to break the vicious cycle between ccRCC cells and osteoclasts. A previously optimized fully human co-culture model is used to mimic the crosstalk between Caki-2 cells (ccRCC) and osteoclasts. Cells are treated at fixed timing with everolimus, zoledronic acid and denosumab as single or sequential combined treatment. We show that Caki-2 cells can induce osteoclast cells differentiation from isolated human monocytes, as demonstrated by specific tartrate-resistant acid phosphatase (TRAP) staining and f-actin ring formation, in a statistically significant manner. Moreover, differentiated osteoclasts proved to be functionally active by pit formation assay. Caki-2 cells co-cultured with osteoclasts acquire a more aggressive phenotype based on gene expression analysis. Interestingly, the sequential combined treatment of everolimus and zoledronic acid is the most effective in the inhibition of both Caki-2 cells survival and osteoclastogenic potential, making it an effective strategy to inhibit the vicious cycle of bone metastasis. At preclinical level, this observation confirms the value of our co-culture model as a useful tool to mimic the bone microenvironment and to assess drug sensitivity in vitro. A better understanding of the molecular mechanisms involved in tumor-bone cells crosstalk will be investigated next. Keywords: Bone metastasis, Renal carcinoma, Osteoclasts, Vicious cycle, Co-culture, Targeted therapy

Patient-derived specimens are an invaluable resource to investigate tumor biology. However, in vivo studies on primary cultures are often limited by the small amount of material available, while conventional in vitro systems might alter the features and behavior that characterize cancer cells. We present our data obtained on primary dedifferentiated liposarcoma cells cultured in a 3D scaffold-based system and injected into a zebrafish model. Primary cells were characterized in vitro for their morphological features, sensitivity to drugs and biomarker expression, and in vivo for their engraftment and invasiveness abilities. The 3D culture showed a higher enrichment in cancer cells than the standard monolayer culture and a better preservation of liposarcoma-associated markers. We also successfully grafted primary cells into zebrafish, showing their local migratory and invasive abilities. Our work provides proof of concept of the ability of 3D cultures to maintain the original phenotype of ex vivo cells, and highlights the potential of the zebrafish model to provide a versatile in vivo system for studies with limited biological material. Such models could be used in translational research studies for biomolecular analyses, drug screenings and tumor aggressiveness assays.

Background: Myxofibrosarcoma (MFS), formerly considered as a myxoid variant of malignant fibrous histiocytoma, is the most common sarcoma of the extremities in adults and is characterized by a high frequency of local recurrence. The clinical behavior of MFS is unpredictable and the efficacy of chemotherapy is still not well documented. Furthermore, given the relatively recent recognition of MFS as a distinct pathologic entity its cellular and molecular biology has still not been extensively studied in patient-derived preclinical models. We examined the molecular biology and treatment outcomes of high-grade, patient-derived MFS primary cultures. Methods: A total of three patient-derived MFS primary cultures were analyzed. We evaluated the role of CD109 expression and also looked for a correlation between transforming growth factor-beta (TGF-β) expression and sensitivity of the primary cultures to different drugs. Results: CD109 was a promising marker for the identification of more aggressive high-grade MFS and a potential therapeutic target. The results also highlighted the potential role of TGF-β in chemoresistance. Pharmacological analysis confirmed the sensitivity of the cultures to chemotherapy. The most active treatments were epirubicin alone and epirubicin in combination with ifosfamide, the latter representing the current standard of care for soft tissue sarcomas (STSs), including MFS. Conclusions: Our results provide a starting point for further research aimed at improving the management of MFS patients undergoing chemotherapy.

Myxofibrosarcoma (MFS) belongs to the group of sarcoma tumors, which represent only 1% of the totality of adult tumors worldwide. Thus, given the rare nature of this cancer, this makes the availability of MFS cell lines difficult. In an attempt to partially fill this gap, we immortalized a primary culture of MFS (IM-MFS-1) and compared the cell morphology with patient’s tumor tissue. IM-MFS-1 was genetically characterized through a Comparative Genomic Hybridization (CGH) array and the mesenchymal phenotype was evaluated using Polymerase chain reaction (PCR) and immunofluorescence staining. Drug sensitivity for MFS therapies was monitored over time in cultures. We confirmed the conservation of the patient’s tumor cell morphology and of the mesenchymal phenotype. Conversely, the synthesis and expression of CD109, a TGFβ co-receptor used to facilitate the diagnosis of high-grade MFS diagnosis, was maintained constant until high cancer cell line passages. The CGH array revealed a complex karyotype with cytogenetic alterations that include chromosome regions associated with genes involved in tumor processes. Cytotoxicity assays show drug sensitivity constantly increased during the culture passages until a plateau was reached. In conclusion, we established and characterized a new MFS cell line that can be used for future preclinical and molecular studies on soft tissue sarcomas.

Eribulin mesylate is a novel, non-taxane, synthetic microtubule inhibitor showing antitumor activity in a wide range of tumors including soft tissue sarcomas (STS). Eribulin has been recently approved for the treatment of metastatic liposarcoma (LPS) patients previously treated with anthracyclines. This work investigated the mechanism of action of this innovative antitubulin agent in well-differentiated/dedifferentiated LPS (ALT/DDLPS) which represents one of the most common adipocytic sarcoma histotypes. A primary culture of ALT/DDLPS from a 54-year-old patient was established. The anticancer activity of eribulin on the patient-derived primary culture was assessed by MTT and tunel assays. Eribulin efficacy was compared to other drugs approved for the treatment of STS. Cell migration and morphology were examined after exposure to eribulin to better understand the drug mechanism of action. Finally, Western blot analysis of apoptosis and migration proteins was performed. The results showed that eribulin exerts its antiproliferative effect by the arrest of cell motility and induction of apoptosis. Our results highlighted the activity of eribulin in the treatment of ALT/DDLPS patients.