Comprehensive Molecular Profiling of NPM1-Mutated Acute Myeloid Leukemia Using RNAseq Approach.

Acute myeloid leukemia (AML) is a complex hematologic malignancy with high morbidity and mortality. Nucleophosmin 1 (NPM1) mutations occur in approximately 30% of AML cases, and NPM1-mutated AML is classified as a distinct entity. NPM1-mutated AML patients without additional genetic abnormalities have a favorable prognosis. Despite this, 30-50% of them experience relapse. This study aimed to investigate the potential of total RNAseq in improving the characterization of NPM1-mutated AML patients. We explored genetic variations independently of myeloid stratification, revealing a complex molecular scenario. We showed that total RNAseq enables the uncovering of different genetic alterations and clonal subtypes, allowing for a comprehensive evaluation of the real expression of exome transcripts in leukemic clones and the identification of aberrant fusion transcripts. This characterization may enhance understanding and guide improved treatment strategies for NPM1mut AML patients, contributing to better outcomes. Our findings underscore the complexity of NPM1-mutated AML, supporting the incorporation of advanced technologies for precise risk stratification and personalized therapeutic strategies. The study provides a foundation for future investigations into the clinical implications of identified genetic variations and highlights the importance of evolving diagnostic approaches in leukemia management.

CD99 Modulates the Proteomic Landscape of Ewing Sarcoma Cells and Related Extracellular Vesicles.

Ewing sarcoma (EWS) is an aggressive pediatric bone tumor characterized by unmet clinical needs and an incompletely understood epigenetic heterogeneity. Here, we considered CD99, a major surface molecule hallmark of EWS malignancy. Fluctuations in CD99 expression strongly impair cell dissemination, differentiation, and death. CD99 is also loaded within extracellular vesicles (EVs), and the delivery of CD99-positive or CD99-negative EVs dynamically exerts oncogenic or oncosuppressive functions to recipient cells, respectively. We undertook mass spectrometry and functional annotation analysis to investigate the consequences of CD99 silencing on the proteomic landscape of EWS cells and related EVs. Our data demonstrate that (i) the decrease in CD99 leads to major changes in the proteomic profile of EWS cells and EVs; (ii) intracellular and extracellular compartments display two distinct signatures of differentially expressed proteins; (iii) proteomic changes converge to the modulation of cell migration and immune-modulation biological processes; and (iv) CD99-silenced cells and related EVs are characterized by a migration-suppressive, pro-immunostimulatory proteomic profile. Overall, our data provide a novel source of CD99-associated protein biomarkers to be considered for further validation as mediators of EWS malignancy and as EWS disease liquid biopsy markers.

Exosome-Based Liquid Biopsy Approaches in Bone and Soft Tissue Sarcomas: Review of the Literature, Prospectives, and Hopes for Clinical Application.

The knowledge of exosome impact on sarcoma development and progression has been implemented in preclinical studies thanks to technological advances in exosome isolation. Moreover, the clinical relevance of liquid biopsy is well established in early diagnosis, prognosis prediction, tumor burden assessment, therapeutic responsiveness, and recurrence monitoring of tumors. In this review, we aimed to comprehensively summarize the existing literature pointing out the clinical relevance of detecting exosomes in liquid biopsy from sarcoma patients. Presently, the clinical utility of liquid biopsy based on exosomes in patients affected by sarcoma is under debate. The present manuscript collects evidence on the clinical impact of exosome detection in circulation of sarcoma patients. The majority of these data are not conclusive and the relevance of liquid biopsy-based approaches in some types of sarcoma is still insufficient. Nevertheless, the utility of circulating exosomes in precision medicine clearly emerged and further validation in larger and homogeneous cohorts of sarcoma patients is clearly needed, requiring collaborative projects between clinicians and translational researchers for these rare cancers.

Integrated Antitumor Activities of Cellular Immunotherapy with CIK Lymphocytes and Interferons against KIT/PDGFRA Wild Type GIST.

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.

Phage Display-Based Nanotechnology Applications in Cancer Immunotherapy.

Phage display is a nanotechnology with limitless potential, first developed in 1985 and still awaiting to reach its peak. Awarded in 2018 with the Nobel Prize for Chemistry, the method allows the isolation of high-affinity ligands for diverse substrates, ranging from recombinant proteins to cells, organs, even whole organisms. Personalized therapeutic approaches, particularly in oncology, depend on the identification of new, unique, and functional targets that phage display, through its various declinations, can certainly provide. A fast-evolving branch in cancer research, immunotherapy is now experiencing a second youth after being overlooked for years; indeed, many reports support the concept of immunotherapy as the only non-surgical cure for cancer, at least in some settings. In this review, we describe literature reports on the application of peptide phage display to cancer immunotherapy. In particular, we discuss three main outcomes of this procedure: (i) phage display-derived peptides that mimic cancer antigens (mimotopes) and (ii) antigen-carrying phage particles, both as prophylactic and/or therapeutic vaccines, and (iii) phage display-derived peptides as small-molecule effectors of immune cell functions. Preclinical studies demonstrate the efficacy and vast potential of these nanosized tools, and their clinical application is on the way.

Trabectedin and olaparib in patients with advanced and non-resectable bone and soft-tissue sarcomas (TOMAS): an open-label, phase 1b study from the Italian Sarcoma Group.

BACKGROUND: Trabectedin is an alkylating drug with a unique mechanism of action causing single-strand and double-strand DNA breaks that activate DNA damage-response pathways. Based on our preclinical data, we hypothesised that poly(ADP-ribose) polymerase 1 (PARP1) inhibitors might be an ideal partner of trabectedin and aimed to assess the safety, identify the recommended phase 2 dose, and explore preliminary signs of activity of trabectedin and olaparib combination treatment in patients with bone and soft-tissue sarcoma. METHODS: We did an open-label, multicentre, phase 1b study, recruiting patients from the national Italian sarcoma network aged 18 years and older with histologically confirmed bone and soft-tissue sarcoma progressing after standard treatments with Eastern Cooperative Oncology Group performance status of 1 or less. In a classic 3 + 3 design, patients received a 24 h infusion of trabectedin on day 1 and olaparib orally twice a day in 21-day cycles across six dose levels (trabectedin 0·675-1·3 mg/m2 every 3 weeks; olaparib 100-300 mg twice a day from day 1 to 21). Intermediate dose levels were permitted to improve safety and tolerability. The primary endpoint was determination of the recommended phase 2 dose (the maximum tolerated dose). Safety and antitumour activity were assessed in all patients who received at least one dose of the study drugs. We report the results of the dose-escalation and dose-expansion cohorts. The trial is still active but closed to enrolment, and follow-up for patients who completed treatment is ongoing. This trial is registered with ClinicalTrials.gov, number NCT02398058. FINDINGS: Between Nov 17, 2014, and Jan 30, 2017, of 54 patients assessed for eligibility, we enrolled 50 patients: 28 patients in the dose-escalation cohort and 22 patients in the dose-expansion cohort. Patients received a median of four cycles of treatment (IQR 2-6; range 1-17 [the patients who received the highest number of cycles are still on treatment]) with a median follow-up of 10 months (IQR 5-23). Considering all dose levels, the most common grade 3-4 adverse events were lymphopenia (32 [64%] of 50 patients), neutropenia (31 [62%]), thrombocytopenia (14 [28%]), anaemia (13 [26%]), hypophosphataemia (20 [40%]), and alanine aminotransferase concentration increase (9 [18%]). No treatment-related life-threatening adverse events or deaths occurred. One (2%) patient interrupted treatment without progression without reporting any specific toxicity. Observed dose-limiting toxicities were thrombocytopenia, neutropenia for more than 7 days, and febrile neutropenia. We selected intermediate dose level 4b (trabectedin 1·1 mg/m2 every 3 weeks plus olaparib 150 mg twice a day) as the recommended phase 2 dose. Seven (14%; 95% CI 6-27) of 50 patients achieved a partial response according to Response Evaluation Criteria In Solid Tumors 1.1. INTERPRETATION: Trabectedin and olaparib in combination showed manageable toxicities at active dose levels for both drugs. Preliminary data on antitumour activity are encouraging. Two dedicated phase 2 studies are planned to assess activity of this combination in both ovarian cancer (EudraCT2018-000230-35) and soft-tissue sarcomas. FUNDING: Italian Association for Cancer Research, Italian Sarcoma Group, Foundation for Research on Musculoskeletal and Rare Tumors, and Italian Ministry of Health.

PARP1 expression drives the synergistic antitumor activity of trabectedin and PARP1 inhibitors in sarcoma preclinical models.

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.

Sorafenib and everolimus for patients with unresectable high-grade osteosarcoma progressing after standard treatment: a non-randomised phase 2 clinical trial.

BACKGROUND: Results of previous study showed promising but short-lived activity of sorafenib in the treatment of patients with unresectable advanced and metastatic osteosarcoma. This treatment failure has been attributed to the mTOR pathway and might therefore be overcome with the addition of mTOR inhibitors. We aimed to investigate the activity of sorafenib in combination with everolimus in patients with inoperable high-grade osteosarcoma progressing after standard treatment. METHODS: We did this non-randomised phase 2 trial in three Italian Sarcoma Group centres. We enrolled adults (≥18 years) with relapsed or unresectable osteosarcoma progressing after standard treatment (methotrexate, cisplatin, and doxorubicin, with or without ifosfamide). Patients received 800 mg sorafenib plus 5 mg everolimus once a day until disease progression or unacceptable toxic effects. The primary endpoint was 6 month progression-free survival (PFS). All analyses were intention-to-treat. This trial is registered with ClinicalTrials.gov, number NCT01804374. FINDINGS: We enrolled 38 patients between June 16, 2011, and June 4, 2013. 17 (45%; 95% CI 28-61) of 38 patients were progression free at 6 months. Toxic effects led to dose reductions, or short interruptions, or both in 25 (66%) of 38 patients and permanent discontinuation for two (5%) patients. The most common grade 3-4 adverse events were lymphopenia and hypophosphataemia each in six (16%) patients, hand and foot syndrome in five (13%), thrombocytopenia in four (11%), and fatigue, oral mucositis, diarrhoea, and anaemia each in two (5%). One patient (3%) had a grade 3 pneumothorax that required trans-thoracic drainage, and that recurred at the time of disease progression. This was reported as a serious adverse event related to the study drugs in both instances. No other serious adverse events were reported during the trial. There were no treatment-related deaths. INTERPRETATION: Although the combination of sorafenib and everolimus showed activity as a further-line treatment for patients with advanced or unresectable osteosarcoma, it did not attain the prespecified target of 6 month PFS of 50% or greater. FUNDING: Italian Sarcoma Group.

The combination of sorafenib and everolimus shows antitumor activity in preclinical models of malignant pleural mesothelioma.

BACKGROUND: Malignant Pleural Mesothelioma (MPM) is an aggressive tumor arising from mesothelial cells lining the pleural cavities characterized by resistance to standard therapies. Most of the molecular steps responsible for pleural transformation remain unclear; however, several growth factor signaling cascades are known to be altered during MPM onset and progression. Transducers of these pathways, such as PIK3CA-mTOR-AKT, MAPK, and ezrin/radixin/moesin (ERM) could therefore be exploited as possible targets for pharmacological intervention. This study aimed to identify 'druggable' pathways in MPM and to formulate a targeted approach based on the use of commercially available molecules, such as the multikinase inhibitor sorafenib and the mTOR inhibitor everolimus. METHODS: We planned a triple approach based on: i) analysis of immunophenotypes and mutational profiles in a cohort of thoracoscopic MPM samples, ii) in vitro pharmacological assays, ii) in vivo therapeutic approaches on MPM xenografts. No mutations were found in 'hot spot' regions of the mTOR upstream genes (e.g. EGFR, KRAS and PIK3CA). RESULTS: Phosphorylated mTOR and ERM were specifically overexpressed in the analyzed MPM samples. Sorafenib and everolimus combination was effective in mTOR and ERM blockade; exerted synergistic effects on the inhibition of MPM cell proliferation; triggered ROS production and consequent AMPK-p38 mediated-apoptosis. The antitumor activity was displayed when orally administered to MPM-bearing NOD/SCID mice. CONCLUSIONS: ERM and mTOR pathways are activated in MPM and 'druggable' by a combination of sorafenib and everolimus. Combination therapy is a promising therapeutic strategy against MPM.

AI Based Discovery of a New AKR1C3 Inhibitor for Anticancer Applications.

AKR1C3 is an upregulated enzyme in prostate and other cancers; in addition to regulating hormone synthesis, this enzyme is thought to play a role in the aggressiveness of tumors and in the defense against drugs. We here used an unbiased method to discover new potent AKR1C3 inhibitors: through an AI-based virtual drug screen, compound 4 was identified as a potent and selective enzymatic inhibitor able to translate this activity into a pronounced antiproliferative effect in the 22RV1 prostate cancer cell model. As other known AKR1C3 inhibitors, compound 4 determined a significantly increased activity of abiraterone, a drug approved for advanced prostate cancer. Compound 4 also showed a synergic effect with doxorubicin in osteosarcoma cell lines; specifically, the effect is correlated with AKR1C3 expression. In this research work, therefore, the use of AI allowed the identification of a new structure as an AKR1C3 inhibitor and its potential to enhance the effect of chemotherapeutics.

Prolonged disease stability with trabectedin in a heavily pretreated elderly patient with metastatic leiomyosarcoma of the thigh and renal failure: a case report and review of the literature.

Leiomyosarcoma represents about 24% of all soft tissue sarcomas and can originate from retroperitoneum, uterus, or extremities. Adequate local control may be achieved with surgery and radiotherapy. In the presence of unresectable metastases either doxorubicin- or gemcitabine-based chemotherapy is the standard of treatment. Nevertheless, prognosis remains poor regardless of the selected chemotherapy regimen, and new effective therapeutic agents for patients with advanced leiomyosarcoma are needed. Trabectedin, a promising new DNA-damaging agent with a mechanism of action that is different from that of traditional alkylating agents, is approved in Europe for the treatment of patients with advanced soft tissue sarcoma, after failure of anthracyclines and ifosfamide, or who are unsuited to receive these agents and in combination with pegylated liposomal doxorubicin (PLD) for the treatment of patients with relapsed platinum-sensitive ovarian cancer. We present a case of a 76-year-old patient with progressive metastatic lung lesions from a previously resected primary leiomyosarcoma of the thigh and moderate renal failure, who achieved 17 months of disease stability during third-line treatment with trabectedin. Trabectedin was not associated with any cumulative toxicity and was consistently well tolerated for a total of 22 treatment cycles. Current evidence on trabectedin is also presented.

DNA damage response and repair genes in advanced bone and soft tissue sarcomas: An 8-gene signature as a candidate predictive biomarker of response to trabectedin and olaparib combination.

Background: Advanced 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). Methods: Differential 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)". Results: Differential 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. Conclusions: This 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.

Epidermal Growth Factor Receptor (EGFR) mutation analysis, gene expression profiling and EGFR protein expression in primary prostate cancer.

BACKGROUND: Activating mutations of the epidermal growth factor receptor (EGFR) confer sensitivity to the tyrosine kinase inhibitors (TKi), gefitinib and erlotinib. We analysed EGFR expression, EGFR mutation status and gene expression profiles of prostate cancer (PC) to supply a rationale for EGFR targeted therapies in this disease. METHODS: Mutational analysis of EGFR TK domain (exons from 18 to 21) and immunohistochemistry for EGFR were performed on tumour tissues derived from radical prostatectomy from 100 PC patients. Gene expression profiling using oligo-microarrays was also carried out in 51 of the PC samples. RESULTS: EGFR protein overexpression (EGFRhigh) was found in 36% of the tumour samples, and mutations were found in 13% of samples. Patients with EGFRhigh tumours experienced a significantly increased risk of biochemical relapse (hazard ratio-HR 2.52, p=0.02) compared with patients with tumours expressing low levels of EGFR (EGFRlow). Microarray analysis did not reveal any differences in gene expression between EGFRhigh and EGFRlow tumours. Conversely, in EGFRhigh tumours, we were able to identify a 79 gene signature distinguishing mutated from non-mutated tumours. Additionally, 29 genes were found to be differentially expressed between mutated/EGFRhigh (n=3) and mutated/EGFRlow tumours (n=5). Four of the down-regulated genes, U19/EAF2, ABCC4, KLK3 and ANXA3 and one of the up-regulated genes, FOXC1, are involved in PC progression. CONCLUSIONS: Based on our findings, we hypothesize that accurate definition of the EGFR status could improve prognostic stratification and we suggest a possible role for EGFR-directed therapies in PC patients. Having been generated in a relatively small sample of patients, our results warrant confirmation in larger series.

EphA2 Expression in Bone Sarcomas: Bioinformatic Analyses and Preclinical Characterization in Patient-Derived Models of Osteosarcoma, Ewing's Sarcoma and Chondrosarcoma.

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.

Enhancing Proteotoxic Stress in Leiomyosarcoma Cells Triggers Mitochondrial Dysfunctions, Cell Death, and Antitumor Activity in vivo.

Leiomyosarcomas are rare and aggressive tumors characterized by a complex karyotype. Surgical resection with or without radiotherapy and chemotherapy is the standard curative treatment. Unfortunately, a high percentage of leiomyosarcomas recurs and metastasizes. In these cases, doxorubicin and ifosfamide represent the standard treatment but with low response rates. Here, we evaluated the induction of proteotoxic stress as a possible strategy to kill leiomyosarcoma cells in a therapeutic perspective. We show that aggressive leiomyosarcomas coexist with high levels of proteotoxic stress. As a consequence, we hypothesized that leiomyosarcoma cells are vulnerable to further increases of proteotoxic stress. The small compound 2c is a strong inducer of proteotoxic stress. In leiomyosarcoma cells, it triggers cell death coupled to a profound reorganization of the mitochondrial network. By using stimulated emission depletion microscopy, we have unveiled the existence of DIABLO/SMAC clusters that are modulated by 2c. Finally, we have engineered a new version of 2c linked to polyethylene glycol though a short peptide, named 2cPP. This new prodrug is specifically activated by proteases present in the tumor microenvironment. 2cPP shows a strong antitumor activity in vivo against leiomyosarcomas and no toxicity against normal cells.

Crosstalk between Macrophages and Myxoid Liposarcoma Cells Increases Spreading and Invasiveness of Tumor Cells.

Myxoid liposarcoma (MLPS) is the second most common subtype of liposarcoma and has tendency to metastasize to soft tissues. To date, the mechanisms of invasion and metastasis of MLPS remain unclear, and new therapeutic strategies that improve patients' outcomes are expected. In this study, we analyzed by immunohistochemistry the immune cellular components and microvessel density in tumor tissues from patients affected by MLPS. In order to evaluate the effects of primary human MLPS cells on macrophage polarization and, in turn, the ability of macrophages to influence invasiveness of MLPS cells, non-contact and 3D organotypic co-cultures were set up. High grade MLPS tissues were found heavily vascularized, exhibited a CD3, CD4, and CD8 positive T lymphocyte-poor phenotype and were massively infiltrated by CD163 positive M2-like macrophages. Conversely, low grade MLPS tissues were infiltrated by a discrete amount of CD3, CD4, and CD8 positive T lymphocytes and a scarce amount of CD163 positive macrophages. Kaplan-Meier analysis revealed a shorter Progression Free Survival in MLPS patients whose tumor tissues were highly vascularized and heavily infiltrated by CD163 positive macrophages, indicating a clear-cut link between M2-like macrophage abundance and poor prognosis in patients. Moreover, we documented that, in co-culture, soluble factors produced by primary human MLPS cells induce macrophage polarization toward an M2-like phenotype which, in turn, increases MLPS cell capability to spread into extracellular matrix and to cross endothelial monolayers. The identification of M2-like polarization factors secreted by MLPS cells may allow to develop novel targeted therapies counteracting MLPS progression.

PARP1 Inhibitor and Trabectedin Combination Does Not Increase Tumor Mutational Burden in Advanced Sarcomas-A Preclinical and Translational Study.

Drug-induced tumor mutational burden (TMB) may contribute to unleashing the immune response in relatively "immune-cold" tumors, such as sarcomas. We previously showed that PARP1 inhibition perpetuates the DNA damage induced by the chemotherapeutic agent trabectedin in both preclinical models and sarcoma patients. In the present work, we explored acquired genetic changes in DNA repair genes, mutational signatures, and TMB in a translational platform composed of cell lines, xenografts, and tumor samples from patients treated with trabectedin and olaparib combination, compared to cells treated with temozolomide, an alkylating agent that induces hypermutation. Whole-exome and targeted panel sequencing data analyses revealed that three cycles of trabectedin and olaparib combination neither affected the mutational profiles, DNA repair gene status, or copy number alterations, nor increased TMB both in homologous recombinant-defective and proficient cells or in xenografts. Moreover, TMB was not increased in tumor specimens derived from trabectedin- and olaparib-treated patients (5-6 cycles) when compared to pre-treatment biopsies. Conversely, repeated treatments with temozolomide induced a massive TMB increase in the SJSA-1 osteosarcoma model. In conclusion, a trabectedin and olaparib combination did not show mutagenic effects and is unlikely to prime subsequent immune-therapeutic interventions based on TMB increase. On the other hand, these findings are reassuring in the increasing warning of treatment-induced hematologic malignancies correlated to PARP1 inhibitor use.

Targeting EGFR/HER2 pathways enhances the antiproliferative effect of gemcitabine in biliary tract and gallbladder carcinomas.

BACKGROUND: Advanced biliary tract carcinomas (BTCs) have poor prognosis and limited therapeutic options. Therefore, it is crucial to combine standard therapies with molecular targeting. In this study EGFR, HER2, and their molecular transducers were analysed in terms of mutations, amplifications and over-expression in a BTC case series. Furthermore, we tested the efficacy of drugs targeting these molecules, as single agents or in combination with gemcitabine, the standard therapeutic agent against BTC. METHODS: Immunohistochemistry, FISH and mutational analysis were performed on 49 BTC samples of intrahepatic (ICCs), extrahepatic (ECCs), and gallbladder (GBCs) origin. The effect on cell proliferation of different EGFR/HER2 pathway inhibitors as single agents or in combination with gemcitabine was investigated on BTC cell lines. Western blot analyses were performed to investigate molecular mechanisms of targeted drugs. RESULTS: EGFR is expressed in 100% of ICCs, 52.6% of ECCs, and in 38.5% of GBCs. P-MAPK and p-Akt are highly expressed in ICCs (>58% of samples), and to a lower extent in ECCs and GBCs (<46%), indicating EGFR pathway activation. HER2 is overexpressed in 10% of GBCs (with genomic amplification), and 26.3% of ECCs (half of which has genomic amplification). EGFR or its signal transducers are mutated in 26.5% of cases: 4 samples bear mutations of PI3K (8.2%), 3 cases (6.1%) in K-RAS, 4 (8.2%) in B-RAF, and 2 cases (4.1%) in PTEN, but no loss of PTEN expression is detected. EGI-1 cell line is highly sensitive to gemcitabine, TFK1 and TGBC1-TKB cell lines are responsive and HuH28 cell line is resistant. In EGI-1 cells, combination with gefitinib further increases the antiproliferative effect of gemcitabine. In TFK1 and TGBC1-TKB cells, the efficacy of gemcitabine is increased with addiction of sorafenib and everolimus. In TGBC1-TKB cells, lapatinib also has a synergic effect with gemcitabine. HuH28 becomes responsive if treated in combination with erlotinib. Moreover, HuH28 cells are sensitive to lapatinib as a single agent. Molecular mechanisms were confirmed by western blot analysis. CONCLUSION: These data demonstrate that EGFR and HER2 pathways are suitable therapeutic targets for BTCs. The combination of gemcitabine with drugs targeting these pathways gives encouraging results and further clinical studies could be warranted.

Delving into PARP inhibition from bench to bedside and back.

With the ever-expanding therapeutic indications and ongoing clinical trials with Poly(adenosine diphosphate-ribose) Polymerase (PARP) inhibitors, it is of outmost importance to stop and rethink what we know and still do not know concerning one of the major revolutions in target therapies in the last decades. Indeed, many PARP inhibitors (PARPi) are able to bind multiple targets, with a plethora of potential interactions with cancer cell signaling, metabolism and the tumor microenvironment (TME). These interactions can mediate both response and resistance to PARPi, but also represent an opportunity for sequential and/or combinatorial therapies. Here we advocate a "look before you leap" approach in reviewing available clinical and preclinical evidence concerning PARPi, delving into this complex entanglement, trying to unravel the potential for innovative therapeutic strategies revolving on PARP inhibition.

High Dose Ifosfamide in Relapsed and Unresectable High-Grade Osteosarcoma Patients: A Retrospective Series.

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.