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Myxofibrosarcoma (MFS) and undifferentiated pleomorphic sarcoma (UPS) can be considered as a spectrum of the same disease entity, representing one of the most common adult soft tissue sarcoma (STS) of the extremities. While MFS is rarely metastasizing, it shows an extremely high rate of multiple frequent local recurrences (50–60% of cases). On the other hand, UPS is an aggressive sarcoma prone to distant recurrence, which is correlated to a poor prognosis. Differential diagnosis is challenging due to their heterogeneous morphology, with UPS remaining a diagnosis of exclusion for sarcomas with unknown differentiation lineage. Moreover, both lesions suffer from the unavailability of diagnostic and prognostic biomarkers. In this context, a genomic approach combined with pharmacological profiling could allow the identification of new predictive biomarkers that may be exploited for differential diagnosis, prognosis and targeted therapy, with the aim to improve the management of STS patients. RNA-Seq analysis identified the up-regulation of MMP13 and WNT7B in UPS and the up-regulation of AKR1C2, AKR1C3, BMP7, and SGCG in MFS, which were confirmed by in silico analyses. Moreover, we identified the down-regulation of immunoglobulin genes in patient-derived primary cultures that responded to anthracycline treatment compared to non-responder cultures. Globally, the obtained data corroborated the clinical observation of UPS as an histotype refractory to chemotherapy and the key role of the immune system in determining chemosensitivity of these lesions. Moreover, our results confirmed the validity of genomic approaches for the identification of predictive biomarkers in poorly characterized neoplasms as well as the robustness of our patient-derived primary culture models in recapitulating the chemosensitivity features of STS. Taken as a whole, this body of evidence may pave the way toward an improvement of the prognosis of these rare diseases through a treatment modulation driven by a biomarker-based patient stratification.

Adult rhabdomyosarcoma (RMS) represents an uncommon entity with an incidence of less than 3% of all soft tissue sarcomas (STS). Consequently, the natural history and the clinical management of this disease are infrequently reported. In order to fill this gap, we investigated the molecular biology of an adult RMS case series. The expression of epithelial mesenchymal transition-related gene and chemoresistance-related gene panels were evaluated. Moreover, taking advantage of our STS translational model combining patient-derived primary culture and 3D-scaffold, the pharmacological profile of an adult head and neck sclerosing RMS was assessed. Furthermore, NGS, microsatellite instability, and in silico analyses were carried out. RT-PCR identified the upregulation of CDH1, SLUG, MMP9, RAB22a, S100P, and LAPTM4b, representing promising biomarkers for this disease. Pharmacological profiling showed the highest sensitivity with anthracycline-based regimen in both 2D and 3D culture systems. NGS analysis detected RAB3IP-HMGA2 in frame gene rearrangement and FGFR4 mutation, microsatellite instability analysis did not detect any alteration. In silico analysis confirmed the mutation of FGFR4 as a promising marker for poor prognosis and a potential therapeutic target. We report for the first time the molecular and pharmacological characterization of rare entities of adult head and neck and posterior trunk RMS. These preliminary data could shed light on this poorly understood disease.

Bone is one of the most preferred sites of metastatic spread from different cancer types, including breast cancer. However, different breast cancer subtypes exhibit distinct metastatic behavior in terms of kinetics and anatomic sites of relapse. Despite advances in the diagnosis, the identification of patients at high-risk of bone recurrence is still an unmet clinical need. We conducted a retrospective analysis, by gene expression and immunohistochemical assays, on 90 surgically resected breast cancer samples collected from patients who experienced no evidence of distant metastasis, bone or visceral metastasis in order to identify a primary tumor-derived marker of bone recurrence. We identified trefoil factor-1 (pS2 or TFF1) as strictly correlated to bone metastasis from ER+ breast cancer. In silico analysis was carried out to confirm this observation, linking gene expression data with clinical characteristics available from public clinical datasets. Then, we investigated TFF1 function in ER+ breast cancer tumorigenesis and bone metastasis through xenograft in vivo models of MCF 7 breast cancer with gain and loss of function of TFF1. As a response to microenvironmental features in primary tumors, TFF1 expression could modulate ER+ breast cancer growth, leading to a less proliferative phenotype. Our results showed it may not play a role in late stages of bone metastasis, however further studies are warranted to understand whether it could contribute in the early-stages of the metastatic cascade. In conclusion, TFF1 upregulation in primary ER+ breast cancer could be useful to identify patients at high-risk of bone metastasis. This could help clinicians in the identification of patients who likely can develop bone metastasis and who could benefit from personalized treatments and follow-up strategies to prevent metastatic disease.

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.

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.

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.

Introduction Pulmonary carcinoids (PCs) are classed according to the World Health Organization 2004 classification as typical or atypical carcinoids. Owing to their rarity, no dedicated clinical trials with somatostatin analogs (SSAs) have been carried out on primary PCs. Patients and Methods From January 2007 to December 2015, 30 patients with metastatic PCs underwent first-line SSA treatment (20 with octreotide long-acting repeatable 30 mg and 10 with lanreotide 120 mg every 28 days). Eight (23.3%) patients had typical carcinoids and 23 (76.7%) had atypical carcinoids. Results The median age was 65.5 years (range, 47-82 years). All patients (23 males and 7 females) were Gallium-68-DOTA-TOC-positron emission tomography/computed tomography (PET/CT)-positive (29 patients) or octreoscan-positive (1 patient). Of the 20 patients who performed fluorine-18-fluorodeoxyglucose positron emission tomography/computed tomography ( 18 FDG-PET/CT), 14 (70.0%) were positive and 6 negative (30.0%). The median treatment duration was 10 months (range, 2-59 months). One patient achieved a partial response (3.3%), and 26 (86.6%) showed stable disease. One patient interrupted SSA treatment owing to symptomatic cholelithiasis. Five-year survival was 53.0% (95% confidence interval [CI], 15.0%-80.0%). The median progression-free survival (mPFS) was 11.1 months (95% CI, 7.0-15.0 months). Negative 18 FDG-PET/CT patients had an mPFS of 15.2 months (95% CI, 7.6 months to not reached) compared with 7.0 months (95% CI, 4.0-10.1 months) for 18 FDG-PET/CT-positive patients. No differences in mPFS were found in relation to TTF1-value, histologic subtype, and presence of extrahepatic metastases. Conclusion SSAs showed antitumor activity in terms of disease control rate and PFS and proved safe, even in patients with poor Eastern Cooperative Oncology Group status. 18 FDG-PET/CT would appear to be a prognostic factor.

Cancer metastasis is the leading cause of morbidity and cancer-related death. Bone is one of the most favored sites of metastatic spread, particularly from breast cancer. However, our understanding on how the host tissue can impact tumor growth is weak. This is hampered by the lack of a reliable in vitro model ables to replicate the complex microenvironment. Indeed, bone microenvironment provides physical support to cells but it can also affect cell behaviour and phenotype. We developed a hybrid 3D collagen-based scaffold functionalized with hydroxyapatite material (HA) in order to mimic natural bone features. Scaffolds were synthesized in our laboratory through collagen solution mixed and cross-linked with hydroxyapatite. Then, we cultured MDA-MB-231, a triple negative breast cancer cell line and MCF-7, an ER+ breast cancer cell line in collagen or hybrid scaffold, in order to evaluate how extracellular matrix affects cell behavior. Cell growth was evaluated by MTT cell-proliferation assay in a time course analysis. Moreover, we evaluated cell morphology and cell disposition within the scaffold by confocal microscopy. Finally, gene profiling was evaluated by qRT-PCR. MCF-7 cells acquire an organized and linear structure, whereas MDA-MB-231 an epithelial-like morphology, when cultured in hybrid scaffold. MDA-MB-231 cells proliferate faster than MCF-7 in both models. However, this is not affected by hydroxyapatite since the proliferation rate is similar when cells are cultured in collagen or hybrid scaffold. MDA-MB-231 grown in hybrid scaffold show a significant increase in RANKL/OPG ratio, pathway strictly involved in bone homeostasis. Moreover, they show a significant increased expression in JAG1, MMP2 and SNAIL1. For MCF-7, we observe a decrease in CDH1/VIM expression ratio and a significant decrease in OPG, osteoclastogenesis inhibitory factor. Overall, these results suggest a shift induced by hydroxyapatite scaffold towards an osteolytic phenotype. Whereas collagen-scaffold could better simulate in vivo primary tumor (Liverani C. et al, Sci Rep 9, 12263, 2019), we highlight that hybrid hydroxyapatite/collagen scaffold is more suitable to study breast cancer cells behaviour in bone metastasis. Then, we will implement it with a direct co-culture of osteoclast and osteoblast cells, in order to better investigate the contribution of the host microenvironment in bone metastasis. This model could provide a reliable 3D in vitro model for the study of bone metastasis mechanisms and for drug screening assays. Citation Format: Chiara Spadazzi, Laura Mercatali, Alessandro De Vita, Chiara Liverani, Claudia Cocchi, Giacomo Miserocchi, Alberto Bongiovanni, Federica Recine, Sebastiano Calpona, Nada Riva, Giandomenico Di Menna, Lorena Gurrieri, Valentina Fausti, Silvia Angela De Bonis, Giovanni Martinelli, Nicola Baldini, Toni Ibrahim. Development of a biomimetic 3D scaffold to study breast cancer bone metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 333.

Soft tissue sarcomas (STS) represent a variety of tumors of mesenchymal origin, accounting for about 1% of all adult cancers. This group of tumors comprises over 60 different histotypes with different biology showing different sensitivity to therapeutic agents. For decades, the standard first-line systemic treatment of metastatic STS has comprised anthracycline based-chemotherapy. Second-line therapy options include agents such as ifosfamide, gemcitabine, and pazopanib, but the optimal sequential therapy for the management of metastatic disease has yet to be defined. Trabectedin is one of the new molecules approved for patients in progression after first-line chemotherapy with anthracyclines or for those unfit for these agents. The compound is characterized by multiple potential mechanisms of action combining cytotoxic, targeted, and immunological effects. This article takes an in-depth look at the role of trabectedin in the management of metastatic STS, including L-sarcoma and non-L-sarcoma.

e13072 Background: Bone Metastases (BM) are still the main cause of morbidity and morbility in cancer patients because of their complications defined as skeletal-related events (SREs).SREs reduce pts quality of life and are associated with an increasing in social and health costs. At present, data concerning BM are mainly obtained retrospectively from monocentric experiences. Methods: We performed a multicentre prospective observational study of patients with BM from breast cancer (BC) with at least 6 month (m)'s follow-up who were registered in a prospective BM database (BMDB). Detailed information on patients at first diagnosis of BM was recorded in a custom-built software system, updated every 6 m by participating centres and reviewed by the coordinator centre.All pts have signed an informed consent. Results: Since October 2014,618 pts with BM from solid tumors were enrolled of whom 220 have BC as primitive site with a 6 m follow-up. Median age was 62 y (range 26-86). Median Follow up was 34 m (6-149). At enrolment 109 (50%) had only BM and 109 (50%) pts had concomitant visceral and BM. Median time to first BM was 47 m (range 0-312) in Bone only disease and 78.6 m in pts with visceral bone metastases. Disease Free interval (DFI) was different according to BC molecular subtypes and stage. The univariate hazard ratio (HR) for visceral or bone metastasis was higher in luminal B tumors (1.56, 95% confidence interval [CI]:1.1-2.3) (p = 0.002), basal-like (2.50, 95% CI:1.1-6.0) (p = 0.043), and HER2-enriched tumors (1.37, 95% CI:0.78-2.4) (p = 0.273). DFI for pts with stage I disease at diagnosis of primary BC was longer than that for stage III pts (median 67.2 m, 95%CI:53.1-96.1, vs. 58.1 m, 95%CI:41.9-73.4), with a HR of 1.84 (95% CI:1.1-3.0) (p = 0.015) for the stage III group, and 0.98 (95% C.I.:0.6-1.5) (p = 0.930) for the stage II group. During BM disease, 98 pts had at least 1 SREs . Zoledronate was used in 69.1% and Denosumab in 28.3% of cases. First line treatment was hormone-based (n = 105, 50.7%) chemo-based therapy (n = 80, 38.7%) and chemo+ormono based (n = 20, 9.7%). During follow up progression disease occurred in 167 pts. Median progression-free (mPFS) and overall survival calculated from metastatic disease diagnosis (mOS) were 15.1 m (95%CI 12.6-18.4) and 66.8 m (95%CI 52.1-79.2),respectively. Conclusions: This study presents prospective data about a cohort of BC pts enrolled at the first BM occurrence and followed over the time, extrapolated by the multicentric observational BMDB in order to better understed the clinical history of breast cancer and bone metastases.

e17030 Background: Urothelial carcinoma (UC) accounts for 90% of cases of bladder cancer. In advanced UC diagnosis, skeletal involvement is frequent. Here we report the establishment of a primary culture of bone metastasis (BM) from High Grade UC derived from 81 years-old patient with a previous diagnosis of papillary urothelial cancer in 2016. After 2 years, patient relapsed to bone and visceral sites and because of poor clinical condition, patient underwent palliative best supportive care. Methods: Patient-derived bone metastatic cell culture was obtained from a surgical specimen. Tumor cells were isolated by collagenase digestion. Cells were seeded on in vitro 2D plates or into 3D collagen scaffolds composed of bovine collagen type I. The establishment of tumor cell isolation was confirmed through the evaluation of cytomorphologic features and positive pan-citokeratin staining. Then, drug treatments was performed and cell survival was evaluated. The study was approved by Ethical Committee and patient signed an informed consent. Results: After a long-term culture, we were able to isolate from a 3D scaffold a tumor clone that successfully growth until passage 20. Stabilized cells generate spheroid-like aggregates, recreating acinar-structures typical of the primary papillary urothelial tumor. Next, we treated cells with: Gemcitabine (Gem), Carboplatin (Carbo), Docetaxel, Carbo+Gem and Bone targeted drugs (Zoledronic Acid and Denosumab). The most effective treatment was Gem+Carbo, in particular 24% of survival in 2D platform and 87% on 3D collagen scaffold. For all treatments, cells cultured on 3D scaffold were more resistant to drugs, mimicking more closely the in-vivo condition. Conclusions: We were able to isolate and establish a BM primary culture from UC using a 3D in-vitro collagen scaffold. This system can recreate microenvironmental conditions more similar to in-vivo ones and it promoted the isolation of tumor cell clones from stromal components of the heterogeneous primary culture. This cell line could be useful to investigate the molecular and genetic profile in order to identify promising molecular targets and to better understard the natural history of BM from UC.

e23542 Background: Soft tissue sarcomas (sts) represent a rare group of solid neoplasm of mesenchymal origin with a 1% incidence of all adult cancers. Liposarcoma and leiomyosarcoma (L-sarcoma) are the most common histotypes. The current first-line treatment for advanced/metastatic L-sarcomas is represented by anthracycline based-regimens. Second-line therapy options include trabectedin, eribulin, ifosfamide, gemcitabine and dacarbazine. In particular, the activity of trabectedin, a tetrahydroisoquinoline molecule, as well as its mechanism of action is not completely elucidated. Methods: The study involved six patient affected L-sarcoma. Patient-derived primary cultures were established after patient surgical treatment. Primary cells were cultured in standard monolayer culture and 3D collagen-based scaffolds. In order to gain inside into the mechanism of action of the drugs, genomic-, chemobiogram, proteomic-, cytometer and in silico analysis were performed. Results: The results confirmed the preservation of tumor gene expression in 3D culture model compared to standard monolayer culture. In particular 3D culture system was able to resemble the patient gene expression of TGFb, SLUG, SNAIL, MMP9 compared to 2D. In silico analysis showed an upregulation of COL1A1 gene, the extracellular matrix component of the present 3D model, in sts compared to other tumors. Pharmacological analysis displayed an increased sensitivity to trabectedin in all the 3D L-sarcoma primary culture compared to 2D culture system, while this was not observed with the other drugs. As previous preclinical evidences suggested that trabectedin mechanism of action include the impairing of ECM proteolytic degradation mediated by tumor cells, we think that its high observed sensitivity depend on the presence of collagen in our 3D- model. Conclusions: These results were suggestive of the losing credibility of monolayer standard culture for pharmacology studies and highlight the promising role of 3D patient-derived culture model for the study of sts biology. Moreover these results shed the light on the potential role of ECM in the mechanism of action of trabectedin in L-sarcomas. The work points out the role of this tumor microenvironment component in predicting response to trabectedin and provide the rationale for better stratifying patients which will be candidate for this drug. Further researches are needed to confirm these evidences.

4611 Background: Neuroendocrine neoplasms (NENs) are a rare subgroup of tumors with challenging management due to their unpredictable and heterogeneous behaviour. The identification of clinically useful biomarkers is a top priority need in this disease. The negative notch regulator DLL3 has gained increasing attention in small cell lung carcinoma, large cell neuroendocrine carcinoma and neuroendocrine prostate cancer, confirming the tumor suppressor function of Notch-1 signaling in neuroendocrine cells. Methods: A retrospective immunohistochemical analysis of DLL3, PD-L1 and RB1 was performed on FFPE samples from 43 patients with gastroenteropancreatic (GEP)-NENs and correlated with clinical characteristics. Results: DLL3 was expressed in high-grade (G3) GEP-NENs. The presence of DLL3 was significantly associated with poorly differentiated NEC (77.8% positive tumors), while none of the patients with well-differentiated NET expressed this marker. Expression of DLL3 was correlated with loss of RB1 and negative 68Ga-PET/CT scan. The 85.7% of DLL3- positive tumors showed loss of RB1 expression, while only 1 out of 35 DLL3- negative tumors had RB1 loss. DLL3 was expressed in 75% of patients with negative 68Ga-PET/CT, while only in 25% of patients with positive 68Ga-PET/CT scan. The presence of DLL3 was negatively associated with PFS and OS. Median PFS was 41.9 months in DLL3-negative patients versus 7.9 months in DLL3-positive patients; median OS was 72.9 months in DLL3-negative patients versus 11.7 months in DLL3-positive patients. No correlation was found with DLL3 and PD-L1 expression. The presence of PD-L1 was not associated with any clinical characteristics. Conclusions: DLL3 is expressed in high grade GEP-NENs and is associated with loss of RB1, negative 68Ga-PET/CT scan and unfavourable clinical outcome. The presence of DLL3 discriminates poorly differentiated NEC from well-differentiated NET. DLL3 could represent the ideal prognostic factor to stratify patients with GEP-NENs and a candidate therapeutic target in NEC patients.

Background: Limited data is available on the role, and extent of, postchemotherapy lymphadenectomy (PC-LND) in patients with clinical evidence of pelvic (cN1-3) or retroperitoneal (RP) lymph node spread from urothelial bladder carcinoma. Objective: To compare the outcomes of operated versus nonoperated patients after first-line chemotherapy. Design, setting, and participants: Data from 34 centers was collected, totaling 522 patients, treated between January 2000 and June 2015. Criteria for patient selection were the following: bladder primary tumor, lymph node metastases (pelvic. ±. RP) only, first-line platinum-based chemotherapy given. Intervention: LND (with cystectomy) versus observation after first-line chemotherapy for metastatic urothelial bladder carcinoma. Outcome measures and statistical analysis: Overall survival (OS) was the primary endpoint. Multiple propensity score techniques were adopted, including 1:1 propensity score matching and inverse probability of treatment weighting. Additionally, the inverse probability of treatment weighting analysis was performed with the inclusion of the covariates, that is, with doubly robust estimation. Results and limitations: Overall, 242 (46.4%) patients received PC-LND and 280 (53.6%) observation after chemotherapy. There were 177 (33.9%) and 345 (66.1%) patients with either RP or pelvic LND only, respectively. Doubly robust estimation-adjusted comparison was not significant for improved OS for PC-LND (hazard ratio [HR]: 0.86, 95% confidence interval [CI]: 0.56-1.31, p = 0.479), confirmed by matched analysis (HR: 0.91, 95% CI: 0.60-1.36, p = 0.628). This was also observed in the RP subgroup (HR: 1.12, 95% CI: 0.68-1.84). The retrospective nature of the data and the heterogeneous patient population were the major limitations. Conclusions: Although there were substantial differences between the two groups, after accounting for major confounders we report a nonsignificant OS difference with PC-LND compared with observation only. These findings may be hypothesis-generating for future prospective trials. Patient summary: We found no differences in survival by adding postchemotherapy lymphadenectomy in patients with pelvic or retroperitoneal lymph node metastatic bladder cancer. The indication to perform postchemotherapy lymphadenectomy in the most suitable patients requires additional studies. In contemporary cohorts of patients with metastatic pelvic or retroperitoneal lymph nodes from bladder cancer, we found no survival benefit from postchemotherapy surgery versus observation in a retrospective study. Performing postchemotherapy lymphadenectomy remains investigational in patients with metastatic bladder cancer.

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

Improvement in the understanding of bone disease biology has led to the development of bone-targeted agents (BTAs). The most widely used BTAs are bisphosphonates, which are inhibitors of osteoclastogenesis and osteoclast activation, and the new bone-targeted therapy, which is denosumab, an inhibitor of receptor activator of nuclear factor kappa-B ligand (RANKL).

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the starting point to achieve reproducible and reliable results. All samples should be treated in the same way from blood collection to storage. Depending on the analyses to be performed, whole blood and serum samples should be stored at -20 °C or -80 °C until use. Blood/serum samples should also be aliquoted to avoid multiple freeze-thawing. Another important issue is the sample conditions during shipment from one laboratory to another. If dry ice is not available or the shipment takes longer than a few days, alternative approaches are needed. One option is to use filter paper for blood collection. Here, we propose a method for blood and serum sample collection that takes advantage of dried blood spots (DBS) and dried serum spots (DSS). We developed the procedure to extract DNA from DBS for the downstream evaluation of some single nucleotide polymorphisms (SNPs) by real time PCR. We also optimized an ELISA assay starting from proteins eluted from DSS. This method can be used with other ELISA assays or procedures evaluating proteins.

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

Bone metastases (BMs) are responsible for high morbidity in patients. A multidisciplinary approach involving a team of specialists offers an effective therapeutic strategy based on disease characteristics, medical history, and performance status. We evaluated the impact of our 10-year multidisciplinary experience on the management of patients with BM. We retrospectively analyzed 2194 medical reports of 1628 patients referred to our Osteoncology Center from 2005 to 2015. Cases were discussed weekly by a multidisciplinary team. Eight hundred thirty-eight (38.2%) of the 2194 visits were requested because of a risk of complications from BM. Antiblastic treatment and bone-targeted therapy were modified in 709 (66.3%) and 309 (31%) of cases, respectively. Radiotherapy was scheduled in 220 (20%) of the 1099 patients for whom information was recorded. Patients completed the Brief Pain Inventory (BPI) during their first visit, 1296 (59.1%) reporting pain (median intensity 4), and 537 (41.4%) experiencing a level that interfered substantially with daily activities. New ortheses and/or antalgic therapy was prescribed accordingly. After 7 days, 208 (16%) patients were re-evaluated and a new BPI administered. A significant improvement in the worst (p

A 70-year-old man affected by bone metastases from castration resistant prostate cancer underwent Alpharadin ((223)Ra-dichloride) therapy (6 administrations of 50 kBq per kg i.v., once every 4 weeks). The inter-fraction variability of the absorbed dose to lesions was evaluated for four injections. Dosimetric assessments were performed following the MIRD approach and a recently published methodology. The mean absorbed dose and standard deviation for 4 lesions [mean (σ %)] were: 434 mGy (15%) and 516 mGy (21%) for the right and left humeral head, 1205 mGy (14%) and 781 mGy (8%) for the right and left glenoid. The estimated total absorbed dose after the whole treatment, considering also the relative-biological effectiveness of alpha particles (RBE = 5), yielded a D RBE range of 13-36 Gy. A good correlation between (99m)Tc and (223)Ra uptake was obtained (R (2) = 0.7613). The tumour-non-tumour (TNT) ratio of 8 lesions (those above, plus 4 additional), monitored by six (99m)Tc-MDP bone scans over a period of about 10 months, evidenced a TNT reduction in two lesions (-42 and -48 %), but in most lesions the TNT remained fairly constant, evidencing that (223)Ra-dichloride therapy tends to prevent further progression of osseous disease, leading to chronicity of the metastatic status.

There is a growing need for more accurate biomarkers to facilitate the diagnosis and prognosis of patients with grade (G) 3 neuroendocrine carcinomas (NECs). In particular, the discrimination between well-differentiated neuroendocrine carcinomas (WD-NECs) and poorly differentiated neuroendocrine carcinomas (PD-NECs) is still an unmet need. We previously showed that 68Gallium-(68Ga-) PET/CT positivity is a prognostic factor in patients with gastroenteropancreatic (GEP) G3 NECs, correlating with a better outcome in terms of overall survival. Here, we hypothesize that 68Ga-PET/CT could help to discriminate between WD-NECs and PD-NECs, adding complementary information to that obtained from morphologic and biologic factors. A retrospective, single-institution study was performed on 11 patients with histologically confirmed, measurable G3 large- or small-cell GEP-NECs according to the 2017 WHO classification. The staging procedures included a 68Ga-PET/CT scan. Results of 68Ga-PET/CT were correlated in univariate analysis with loss of tissue immunohistochemical expression of DAXX/ATRX or RB1 frequently associated with WD-NECs or PD-NECs, respectively. None of the patients with positive 68Ga-PET/CT showed loss of RB1 expression, whereas among those (n=6) with negative 68Ga-PET/CT, 4 showed loss of expression. A trend towards a correlation between loss of RB1 expression and negative 68Ga-PET/CT was observed. Our preliminary data support the hypothesis that PD-NECs carrying RB1 mutation and loss of its expression may be associated with negative 68Ga-PET/CT. If confirmed in a larger clinical trial, 68Ga-PET/CT would help in the stratification of G3 NECs.

Undifferentiated pleomorphic sarcoma (UPS) is an aggressive mesenchymal neoplasm with no specific line of differentiation. Eribulin, a novel synthetic microtubule inhibitor, has shown anticancer activity in several tumors, including soft tissue sarcomas (STS). We investigated the molecular biology of UPS, and the mechanisms of action of this innovative microtubule-depolymerizing drug. A primary culture from a patient with UPS was established and characterized in terms of gene expression. The activity of eribulin was also compared with that of other drugs currently used for STS treatment, including trabectedin. Finally, Western blot analysis was performed to better elucidate the activity of eribulin. Our results showed an upregulation of epithelial mesenchymal transition-related genes, and a downregulation of epithelial markers. Furthermore, genes involved in chemoresistance were upregulated. Pharmacological analysis confirmed limited sensitivity to chemotherapy. Interestingly, eribulin exhibited a similar activity to that of standard treatments. Molecular analysis revealed the expression of cell cycle arrest-related and pro-apoptotic-related proteins. These findings are suggestive of aggressive behavior in UPS. Furthermore, the identification of chemoresistance-related genes could facilitate the development of innovative drugs to improve patient outcome. Overall, the results from the present study furnish a rationale for elucidating the role of eribulin for the treatment of UPS.

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

IF 3.156; International audience; Background: Radical cystectomy (RC) and radiochemotherapy (RCT) are curative options for muscle-invasive bladder cancer (MIBC). Optimal treatment strategy remains unclear in elderly patients.Material and methods: Patients aged 80 years old and above with T2-T4aN0-2M0-Mx MIBC were identified in the Retrospective International Study of Cancers of the Urothelial Tract (RISC) database. Patients treated with RC were compared with those treated with RCT. The impact of surgery on overall survival (OS) was assessed using a Cox proportional hazard model. Progression included locoregional and metastatic relapse and was considered a time-dependent variable.Results: Between 1988 and 2015, 92 patients underwent RC and 72 patients had RCT. Median age was 82.5 years (range 80–100) and median follow-up was 2.90 years (range 0.04–11.10). Median OS was 1.99 years (95%CI 1.17–2.76) after RC and 1.97 years (95%CI 1.35–2.64) after RCT (p = .73). Median progression-free survival (PFS) after RC and RCT were 1.25 years (95%CI 0.80–1.75) and 1.52 years (95%CI 1.01–2.04), respectively (p = .54). In multivariate analyses, only disease progression was significantly associated with worse OS (HR = 10.27 (95%CI 6.63–15.91), p

Although bone metastases represent a major challenge in the natural history of breast cancer (BC), the complex interactions involved have hindered the development of robust in vitro models. The aim of this work is the development of a preclinical model of cancer and bone stromal cells to mimic the bone microenvironment. We studied the effects on osteoclastogenesis of BC cells and Mesenchymal stem cells (MSC) cultured alone or in combination. We also analyzed: (a) whether the blockade of the Epithelial Growth Factor Receptor (EGFR) pathway modified their influence on monocytes towards differentiation, and (b) the efficacy of bone-targeted therapy on osteoclasts. We evaluated the osteoclastogenesis modulation of human peripheral blood monocytes (PBMC) indirectly induced by the conditioned medium (CM) of the human BC cell line SCP2, cultured singly or with MSC. Osteoclastogenesis was evaluated by TRAP analysis. The effect of the EGFR blockade was assessed by treating the cells with gefitinib, and analyzed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Western Blot (WB). We observed that SCP2 co-cultured with MSC increased the differentiation of PBMC. This effect was underpinned upon pre-treatment of the co-culture with gefitinib. Co-culture of SCP2 with MSC increased the expression of both the bone-related marker Receptor Activator of Nuclear Factor κB (RANK) and EGFR in BC cells. These upregulations were not affected by the EGFR blockade. The effects of the CM obtained by the cells treated with gefitinib in combination with the treatment of the preosteoclasts with the bone-targeted agents and everolimus enhanced the inhibition of the osteoclastogenesis. Finally, we developed a fully human co-culture system of BC cells and bone progenitor cells. We observed that the interaction of MSC with cancer cells induced in the latter molecular changes and a higher power of inducing osteoclastogenesis. We found that blocking EGFR signaling could be an efficacious strategy for breaking the interactions between cancer and bone cells in order to inhibit bone metastasis.

Introduction: Pharmacotherapy for metastatic renal cell carcinoma (mRCC) has been revolutionized during the last years due to improved understanding of the molecular pathways that underlie tumor development and progression.Areas covered: This manuscript reviews all the approved drugs for the treatment of mRCC and clinical trials that led to registration and the safety profiles of each molecule. An update of the newer targeted drugs that are currently under investigation, such as cabozantinib and immunologic therapies, is also presented.Expert opinion: At present, medical oncologists have at least seven agents to treat mRCC; however, progression-free survival and overall survival are still limited and other more personalized therapeutic approaches are needed. In the first-line setting, the COMPARZ and PISCES trials showed that pazopanib presents noninferior efficacy but a better safety profile compared to sunitinib. This could help identify the most suitable drug on the basis of the clinical status of pati...