Widening the spectrum of players affected by genetic changes in Wilms tumor relapse.

Few studies investigated the genetics of relapsed Wilms tumor (WT), suggesting the SIX1 gene, the microRNA processing genes, and the MYCN network as possibly involved in a relevant percentage of relapses. We investigated 28 relapsing WT patients (10 new cases and 18 cases in which the involvement of SIX and miRNAPG had been excluded) with a panel of ∼5000 genes. We identified variants affecting genes involved in DNA damage prevention and repair in 12/28 relapsing patients (42.9%), and affecting genes involved in chromatin modification and regulation in 6/28 relapsing patients (21.4%), widening the spectrum of anomalies detected in relapsed tumors. The disclosure of molecular pathways possibly underlying tumor progression might allow to use molecularly targeted therapies at relapse. Surprisingly, germline anomalies, mostly affecting DNA damage prevention and repair genes, were identified in 13/28 patients (46.4%), raising the issue of performing a genetic testing to all children presenting with a WT.

Timeline of surgery in localized angiosarcoma of the breast: Improving outcome following multidisciplinary treatment optimization.

INTRODUCTION: Primary (PAS) and radiation-associated angiosarcomas (RAAS) of the breast are rare tumors of vascular origin with poor survival. In this retrospective cohort study, we aimed to assess the impact of multidisciplinary treatment optimization on the prognosis of patients who underwent surgery at a national referral center. MATERIALS AND METHODS: Cases of operable angiosarcoma of the breast evaluated by a multidisciplinary team including surgeons, medical oncologists and radiation oncologists expert in the field and treated from January 2012 to January 2023 were retrieved from a prospectively maintained database. The outcomes of three treatment groups, defined by the timing of surgery in relation to adjuvant and neoadjuvant therapies, were compared. RESULTS: Fifty-nine patients with operable angiosarcomas of the breast (49 RAAS and 10 PAS) were retrospectively identified. The five-year overall survival was 85.2 % (95 % CI 73.9-98.2) and event-free survival was significantly better in patients with grade 1 than those with grade 2 or 3 tumors. Patients receiving neoadjuvant chemotherapy had significantly better outcomes than those treated with primary surgery. Pathological complete response was significantly higher in patients receiving neoadjuvant radiotherapy after neoadjuvant chemotherapy, and a trend towards better distant-disease-free survival was found for patients with complete response at time of surgery. CONCLUSIONS: Optimization of angiosarcoma treatment based on specialized, multidisciplinary assessment regarding the type and timing of surgery and the use of neoadjuvant chemoradiotherapy can improve outcomes. The findings of this study support the use of neoadjuvant chemotherapy as well as adjuvant and neoadjuvant radiotherapy in clinical practice.

Pleomorphic Liposarcoma of the Extremity and Trunk: Multimodality Therapy for Some but Not All?

BACKGROUND: Pleomorphic liposarcoma (PLPS) is an ultra-rare malignancy distinct from well-differentiated/dedifferentiated and myxoid liposarcoma. In this study, we sought to (1) assess outcomes after surgery for primary, non-metastatic PLPS and (2) explore potential indications for multimodality therapy. METHODS: Clinicopathologic data were retrospectively collected for patients treated from 2002 to 2019 at our sarcoma referral center. Descriptive data were summarized and Kaplan-Meier plots were constructed for overall survival (OS) and crude cumulative incidences (CCI) of disease-specific death (DSD), local recurrence (LR), and distant metastasis (DM). Univariable models were performed to assess the association of specific variables of interest on outcome. RESULTS: Forty-four pathology-verified PLPS cases were included in this study. Median tumor size was 8.5 cm; 75% were FNCLCC Grade 3. All patients underwent complete resection, including 15 patients (34%) who required re-excision to secure microscopic negative margins. Radiation therapy was given to 75% of patients, chemotherapy in 36%. At 5 years, OS was 75.3%; CCI of DSD, LR, and DM were 17.5%, 2.3%, and 32.5%. Larger tumor size was strongly associated with worse OS (p = 0.028) and DSD (p ≤ 0.001). A subgroup of patients (n = 10, 23%) with smaller, predominantly Grade 2 tumors underwent surgery alone without any LR or DM event at a median follow-up of 7.9 years. CONCLUSIONS: In PLPS, aggressive surgery and when appropriate, radiation therapy, results in excellent local control. Chemotherapy can be considered for larger tumors. Patients with smaller, Grade 2 tumors may be potentially cured with surgery alone.

GDF-15 predicts epithelioid hemangioendothelioma aggressiveness and is down-regulated by sirolimus through ATF4/ATF5 suppression.

PURPOSE: Epithelioid hemangioendothelioma (EHE) poses a therapeutic challenge due to limited efficacy of conventional chemotherapy in advanced cases, necessitating exploration of new treatment avenues and identification of novel aggressiveness biomarkers. This study aimed at i) utilizing an EHE patient-derived xenograft (PDX) model and its associated cell line to assess the efficacy of sirolimus and ii) analyzing two distinct patient cohorts to pinpoint circulating biomarkers of EHE aggressiveness. EXPERIMENTAL DESIGN: A PDX model and corresponding cell line were established from an advanced EHE patient, demonstrating consistency with the original tumor in terms of histomorphology, WWTR1::CAMTA1 fusion presence, and genomic and transcriptomic profiles. Two independent patient series were employed to investigate the association between Growth/Differentiation Factor 15 (GDF-15) serum levels and EHE aggressiveness. RESULTS: ELISA analyses on EHE cell culture medium and blood from EHE-carrying mice revealed the release of GDF-15 by EHE cells. Sirolimus exhibited markedly higher anti-tumor activity compared to doxorubicin, concurrently reducing GDF-15 expression/release both in vivo and in vitro. This reduction was attributed to the drug-induced inhibition of phosphorylation/activation of 4E-BP1 and subsequent downregulation of the GDF-15 transcription factors ATF4 and ATF5. Blood sample analyses from two independent patient series showed a significant correlation between GDF-15 and EHE aggressiveness. CONCLUSION: This study identifies GDF-15 as a novel biomarker of EHE aggressiveness and underscores the superior efficacy of sirolimus compared to doxorubicin in our experimental models. The observed inhibition of GDF-15 release by sirolimus suggests its potential as a biomarker for monitoring the drug's activity in patients.

Extent of macroscopic vascular invasion predicts distant metastasis in primary leiomyosarcoma of the inferior vena cava.

BACKGROUND: In retroperitoneal leiomyosarcoma (RP LMS), the predominant issue is distant metastasis (DM). We sought to determine variables associated with this outcome and disease-specific death (DSD). METHODS: Data were retrospectively collected on patients with primary RP LMS treated at a high-volume center from 2002 to 2023. For inferior vena cava (IVC)-origin tumors, the extent of macroscopic vascular invasion was re-assessed on each resection specimen and correlated with preoperative cross-sectional imaging. Crude cumulative incidences were estimated for DM and DSD and univariable and multivariable models were performed. RESULTS: Among 157 study patients, median tumor size was 11.0 cm and 96.2% of cases were intermediate or high grade. All patients underwent complete resection, 56.7% received chemotherapy (43.9% neoadjuvant) and 14.6% received radiation therapy. Only tumor size and grade and not site of tumor origin (e.g., IVC vs. other) were associated with DM and DSD (p < 0.05). Among 64 patients with IVC-origin tumors, a novel 3-tier classification was devised based on the level of intimal disruption, which was associated with both DM (p = 0.007) and DSD (0.002). CONCLUSION: In primary RP LMS, only tumor size and grade are predictive of DM and DSD. In IVC-origin tumors, the extent of macroscopic vascular invasion is also strongly predictive of these outcomes.

Spatial distribution of tumour immune infiltrate predicts outcomes of patients with high-risk soft tissue sarcomas after neoadjuvant chemotherapy.

BACKGROUND: Anthracycline-based neoadjuvant chemotherapy (NAC) may modify tumour immune infiltrate. This study characterized immune infiltrate spatial distribution after NAC in primary high-risk soft tissue sarcomas (STS) and investigate association with prognosis. METHODS: The ISG-STS 1001 trial randomized STS patients to anthracycline plus ifosfamide (AI) or a histology-tailored (HT) NAC. Four areas of tumour specimens were sampled: the area showing the highest lymphocyte infiltrate (HI) at H&E; the area with lack of post-treatment changes (highest grade, HG); the area with post-treatment changes (lowest grade, LG); and the tumour edge (TE). CD3, CD8, PD-1, CD20, FOXP3, and CD163 were analyzed at immunohistochemistry and digital pathology. A machine learning method was used to generate sarcoma immune index scores (SIS) that predict patient disease-free and overall survival (DFS and OS). FINDINGS: Tumour infiltrating lymphocytes and PD-1+ cells together with CD163+ cells were more represented in STS histologies with complex compared to simple karyotype, while CD20+ B-cells were detected in both these histology groups. PD-1+ cells exerted a negative prognostic value irrespectively of their spatial distribution. Enrichment in CD20+ B-cells at HI and TE areas was associated with better patient outcomes. We generated a prognostic SIS for each tumour area, having the HI-SIS the best performance. Such prognostic value was driven by treatment with AI. INTERPRETATION: The different spatial distribution of immune populations and their different association with prognosis support NAC as a modifier of tumour immune infiltrate in STS. FUNDING: Pharmamar; Italian Ministry of Health [RF-2019-12370923; GR-2016-02362609]; 5 × 1000 Funds-2016, Italian Ministry of Health; AIRC Grant [ID#28546].

Clinical recommendations for treatment of localized angiosarcoma: A consensus paper by the Italian Sarcoma Group.

Angiosarcoma (AS) represents a rare and aggressive vascular sarcoma, posing distinct challenges in clinical management compared to other sarcomas. While the current European Society of Medical Oncology (ESMO) clinical practice guidelines for sarcoma treatment are applicable to AS, its unique aggressiveness and diverse tumor presentations necessitate dedicated and detailed clinical recommendations, which are currently lacking. Notably, considerations regarding surgical extent, radiation therapy (RT), and neoadjuvant/adjuvant chemotherapy vary significantly in localized disease, depending on each different site of onset. Indeed, AS are one of the sarcoma types most sensitive to cytotoxic chemotherapy. Despite this, uncertainties persist regarding optimal management across different clinical presentations, highlighting the need for further investigation through clinical trials. The Italian Sarcoma Group (ISG) organized a consensus meeting on April 1st, 2023, in Castel San Pietro, Italy, bringing together Italian sarcoma experts from several disciplines and patient representatives from "Sofia nel Cuore Onlus" and the ISG patient advocacy working group. The objective was to develop specific clinical recommendations for managing localized AS within the existing framework of sarcoma clinical practice guidelines, accounting for potential practice variations among ISG institutions. The aim was to try to standardize and harmonize clinical practices, or at least highlight the open questions in the local management of the disease, to define the best evidence-based practice for the optimal approach of localized AS and generate the recommendations presented herein.

A Gradual Transition Toward Anaplasia in Wilms Tumor Through Tolerance to Genetic Damage.

Patients with Wilms tumor (WT) in general have excellent survival, but the prognosis of patients belonging to the subgroup of WT with diffuse anaplasia (DA) is poor due to frequent resistance to chemotherapy. We hypothesized that DA WT cells might undergo changes, such as acquiring a persistent tolerance to DNA damage and copy number aberrations (CNAs), which could eventually lead to their resistance to chemotherapy treatment. Tissue sections from chemotherapy-treated DA WTs (n = 12) were compared with chemotherapy-treated nonanaplastic WTs (n = 15) in a tissue microarray system, enabling analysis of 769 tumor regions. All regions were scored for anaplastic features and immunohistochemistry was used to quantify p53 expression, proliferation index (Ki67), and DNA double-strand breaks (γH2AX). CNAs were assessed by array-based genotyping and TP53 mutations using targeted sequencing. Proliferation index and the frequency of DNA double-strand breaks (γH2AX dot expression) increased with higher anaplasia scores. Almost all (95.6%) areas with full-scale anaplasia had TP53 mutations or loss of heterozygosity, along with an increased amount of CNAs. Interestingly, areas with wild-type TP53 with loss of heterozygosity and only one feature of anaplasia (anaplasia score 1) also had significantly higher proliferation indices, more DNA double-strand breaks, and more CNAs than regions without any anaplastic features (score 0); such areas may be preanaplastic cell populations under selective pressure for TP53 mutations. In conclusion, we suggest that chemoresistance of DA WTs may be partly explained by a high proliferative capability of anaplastic cells, which also have a high burden of double-stranded DNA breaks and CNAs, and that there is a gradual emergence of anaplasia in WT.

Regorafenib in advanced solitary fibrous tumour: Results from an exploratory phase II clinical study.

BACKGROUND: To investigate the activity of regorafenib in advanced solitary fibrous tumour (SFT). METHODS: An Italian monocentric investigator-initiated exploratory single-arm Phase II trial was conducted of regorafenib in adult patients with advanced and progressive SFT, until progression or limiting toxicity. Prior treatment with antiangiogenics was allowed. Primary and secondary end-points were: overall response rate (ORR) by Choi criteria, and ORR by RECIST, progression-free survival (PFS), overall survival (OS). RESULTS: From January 2016 to February 2021, 18 patients were enroled [malignant-SFT = 13; dedifferentiated-SFT (D-SFT) = 4; typical-SFT (T-SFT) = 1]. Fourteen patients were pre-treated, in 12 cases with antiangiogenics (median [m-] lines of treatment = 3). Sixteen patients were evaluable for response (one screening failure; one early discontinuation). Six/16 (35.2%) required a definitive dose reduction. ORR by Choi was 37.5% (95% CI: 15.2-64.6), with 6/16 (37.5%) partial responses (PR), 6/16 (37.5%) stable disease (SD) and 4/16 (25%) progressions; 5/6 responses occurred in patients pre-treated with antiangiogenics. No responses were detected in D-SFT. Best RECIST responses were: 1/16 (6.2%) PR, 12/16 (75%) SD, 3/16 (18.8%) progressions. At 48.4 month m-FU, m-PFS by Choi was 4.7 (inter-quartile range: 2.4-13.1) months, with 31.2% patients progression-free at 1 year. CONCLUSION: Regorafenib showed activity in SFT, with 30% patients free-from-progression at one year. Responses were observed also in patients pretreated and refractory to another antiangiogenic agents. However, ORR and m-PFS were lower than reported with other antiangiogenics, and this was possibly due to discrepancies in the patient population and the high-rate of dose reductions.

Pathological and radiological response following neoadjuvant treatments in primary localized resectable myxofibrosarcoma and undifferentiated pleomorphic sarcoma of the extremities and trunk wall.

BACKGROUND: To explore the correlation between pathological and radiological response to preoperative treatments and outcome in surgically treated patients with myxofibrosarcoma (MFS) and undifferentiated pleomorphic sarcoma (UPS). METHODS: All consecutive patients with primary localized MFS and UPS of the extremities and trunk wall surgically treated with curative intent at our center (2005-2021) were included. Clinical data including residual visible tumor (VT%) on surgical specimen and Response Evaluation Criteria in Solid Tumor (RECIST) were retrieved. Kaplan-Meier curves for overall survival and disease-free survival, and cumulative incidence of local relapse and distant metastasis were estimated in a competing risk framework according to RECIST and VT%, overall and by treatment group. Cox and Fine and Gray multivariable models were performed. RESULTS: Of 693 patients affected by primary MFS and UPS, 233 (66 MFS and 167 UPS) were treated by neoadjuvant chemotherapy (naChT), radiotherapy (naRT), or both (naChT-RT). VT% was ≤5% in 13/46 (28.2%), 24/99 (24.2%), and 40/88 (45.4%) patients, respectively. There were 11/46 (29.7%), 22/99 (22.7%), and 23/88 (26.1%) RECIST partial responses and 18/46 (48.6%), 59/99 (60.8%), and 60/88 (68.2%) RECIST stable disease, respectively. In naChT, a trend for a better survival was observed when VT% ≤5% (p = .09), whereas RECIST partial responses and stable disease had the same outcome. VT% was not associated with outcome in naRT or naChT-RT, whereas RECIST response was. CONCLUSION: In primary localized MFS and UPS treated with neoadjuvant therapies, VT% seems more relevant than size reduction after naChT, whereas the opposite is true when naRT is administered alone or concurrent to ChT.

Effectiveness of irinotecan plus trabectedin on a desmoplastic small round cell tumor patient-derived xenograft.

This study exploited a novel patient-derived xenograft (PDX) of desmoplastic small round cell tumor (DSRCT), which reproduces histomorphological and molecular characteristics of the clinical tumor, to assess the activity of cytotoxic and targeted anticancer agents. Antitumor effect was moderate for doxorubicin, pazopanib and larotrectenib [maximum tumor volume inhibition (max TVI), 55-66%], while trabectedin had higher activity (max TVI, 82%). Vinorelbine, irinotecan and eribulin achieved nearly complete tumor growth inhibition (max TVI, 96-98%), although tumors regrew after the end of treatment. The combination of irinotecan with either eribulin or trabectedin resulted in complete responses, which were maintained until the end of the experiment for irinotecan plus trabectedin. Irinotecan-based combinations nearly abrogated the expression of proteins of the G2/M checkpoint, preventing cell entrance in mitosis, and induced apoptotic and necroptotic cell death. Consistently, irinotecan plus trabectedin resulted in reprogramming of DSCRT transcriptome, with downregulation of E2F targets, G2/M checkpoint and mitotic spindle gene sets. This study emphasizes the importance of patient-derived preclinical models to explore new treatments for DSRCT and fosters clinical investigation into the activity of irinotecan plus trabectedin.

Treating secondary malignant neoplasms: A burden of childhood cancer survivors.

Each year approximately 35,000 children and adolescents are diagnosed with cancer in Europe. Five-year survival rates have improved and now reach 80% in most European countries, thanks to a combination of chemotherapy, radiotherapy, and surgery. To date, there are more than 44,000 Italians still living several years after being diagnosed with cancer in developmental age. The risk of premature morbidity and mortality for cancer survivors is well known and documented. Approximately 60% of survivors of cancer in childhood and adolescence have at least one chronic health condition in later life, and more than one in four develop severe or life-threatening disorders. Among the various long-term iatrogenic sequelae of cancer treatments, the most worrisome are second malignant neoplasms. We reported on our mono-institutional experiences of screening and treating secondary breast cancer, secondary thyroid cancer and secondary osteosarcoma. Recommendations on the surveillance needed for cancer survivors because of the risk of late effects of their disease or its treatment suggest that discussing the potential problems early on can be crucial to a patient's future health. These considerations and our consolidated experience strengthen our conviction that survivors of cancer in childhood and adolescence who develop second malignant neoplasms should be treated at highly-specialized centers. Multidisciplinary care requires close communications and high levels of up-to-date professional expertise. This challenging area of health care is also changing rapidly because cancer survivorship is a work in progress, but we cannot wait for definitive conclusions on many aspects because this will take decades, especially for pediatric patients.

Long-term survivors with desmoplastic small round cell tumor (DSRCT): Results from a retrospective single-institution case series analysis.

OBJECTIVE: To report on a retrospective study of primary DSRCT aiming at characterizing long-term survivors (LTS). METHODS: All consecutive patients treated at our institution for a primary DSRCT between 2000 and 2021 were retrospectively identified. Patients received multiagent chemotherapy ± surgery ± hyperthermic intraperitoneal chemotherapy (HIPEC) ± whole abdomino-pelvic radiotherapy (WAP-RT) ± high-dose chemotherapy ± maintenance chemotherapy (MC). Event-free survival (EFS) and overall survival (OS) were estimated by Kaplan-Meier method. Patients alive, without evidence of disease at ≥36 months from diagnosis, were defined as LTS. RESULTS: Thirty-eight patients were identified. All received multiagent chemotherapy; 27/38 (71%) surgery (7/27 [26%] plus HIPEC), 9/38 (24%) WAP-RT, 12/38 (32%) MC. At a median-follow-up of 37 months (IQR 18-63), overall median-EFS and median-OS were 15 and 37 months, respectively. All events occurred within 35 months. In patients who underwent surgery, median-EFS and median-OS were 19 and 37 months (23 and 43 months after R0/R1, and 10 and 19 months after R2 resection), respectively. LTS were 5/38 (13%), alive at 37, 39, 53, 64, 209 months. None had liver or extra-abdominal metastasis at diagnosis, they all received R0/R1 resection, 3/5 had WAP-RT, 2/5 MC, 1/5 received high-dose chemotherapy, none HIPEC. CONCLUSIONS: In our series cure was likely achieved in 13% of DSRCT. LTS had no liver/extra-abdominal disease, were treated with complete surgery, and possibly WAP-RT/MC.

Molecular Signature of Biological Aggressiveness in Clear Cell Sarcoma of the Kidney (CCSK).

Clear cell sarcoma of the kidney (CCSK) is a rare pediatric renal tumor with a worse prognosis than Wilms' tumor. Although recently, BCOR internal tandem duplication (ITD) has been found as a driver mutation in more than 80% of cases, a deep molecular characterization of this tumor is still lacking, as well as its correlation with the clinical course. The aim of this study was to investigate the differential molecular signature between metastatic and localized BCOR-ITD-positive CCSK at diagnosis. Whole-exome sequencing (WES) and whole-transcriptome sequencing (WTS) were performed on six localized and three metastatic BCOR-ITD-positive CCSKs, confirming that this tumor carries a low mutational burden. No significant recurrences of somatic or germline mutations other than BCOR-ITD were identified among the evaluated samples. Supervised analysis of gene expression data showed enrichment of hundreds of genes, with a significant overrepresentation of the MAPK signaling pathway in metastatic cases (p < 0.0001). Within the molecular signature of metastatic CCSK, five genes were highly and significantly over-expressed: FGF3, VEGFA, SPP1, ADM, and JUND. The role of FGF3 in the acquisition of a more aggressive phenotype was investigated in a cell model system obtained by introducing the ITD into the last exon of BCOR by Crispr/Cas9 gene editing of the HEK-293 cell line. Treatment with FGF3 of BCOR-ITD HEK-293 cell line induced a significant increase in cell migration versus both untreated and scramble cell clone. The identification of over-expressed genes in metastatic CCSKs, with a particular focus on FGF3, could offer new prognostic and therapeutic targets in more aggressive cases.

Secondary osteosarcoma: a challenge indeed.

BACKGROUND: The risk of survivors developing a secondary bone sarcoma after being treated for pediatric cancers is well established. The aim of this study was to examine the clinical characteristics and outcomes of patients with secondary osteosarcoma (SOS). METHODS: The study concerns survivors of childhood and adolescence primary neoplasms (PN) treated with chemotherapy, with or without radiotherapy and surgery, subsequently diagnosed with SOS. RESULTS: We identified 26 patients (13 females, 13 males) who developed SOS a median 7.3 years after being diagnosed with a PN (5/7 of these patients tested for Li-Fraumeni and found positive for the syndrome). The sample's median age was 8.0 and 15.0 years when their PN and SOS were diagnosed, respectively. To treat their PN, 24 out of 26 patients had been given radiotherapy, and 19 had received chemotherapy including doxorubicin. A considerable number of SOS occurred at unfavorable sites (nine hip bone, six skull). All but one patient received chemotherapy with tailored schedules, omitting doxorubicin in 19 cases. Eighteen of the 26 patients underwent surgery. The 5- and 10-year overall survival and probabilities after the diagnosis of SOS (95% confidence interval) were 50% (32.7-76.5%) and 38.9% (22.4-67.4%); 5- and 10-year progression-free survival was 47% (29.9-73.7%) and 35.2% (19.3-64.4%), respectively. CONCLUSIONS: The survival rates after SOS are lower than in patients with primary osteosarcoma, but not negligible. It is therefore mandatory to discuss the best choice of treatment for such patients at a referral center, in terms of their chances of cure and quality of life.

Establishment of 6 pediatric rhabdomyosarcoma patient's derived xenograft models closely recapitulating patients' tumor characteristics.

INTRODUCTION: The prognosis for patients with metastatic and recurrent pediatric rhabdomyosarcoma (RMS) remains poor. The availability of preclinical models is essential to identify promising treatments We established a series of pediatric RMS patient derived xenografts (PDXs), all faithfully mirroring primary tumor characteristics and representing a unique tool for clarifying the biological processes underlying RMS progression and relapse. METHODS: Fresh tumor samples from 12 RMS patients were implanted subcutaneously in both flanks of immunocompromised mice. PDXs were considered as grafted after accomplishing three passages in mice. Characterization of tumor tissues and models was performed by comparing both morphology and immunoistochemical and fluorescence in situ hybridization (FISH) characteristics. RESULTS: Six PDXs were established, with a successful take rate of 50%. All models closely mirrored parental tumor characteristics. An increased grafting rate for tumors derived from patients with worse outcome (p = 0.006) was detected. For 50% PDXs grafting occurred when the corresponding patient was still alive. CONCLUSION: Our findings increase the number of available RMS PDX models and strengthen the role of PDXs as useful preclinical tools for patients with unmet medical needs and to develop personalized therapies.

Targeted RNA-sequencing analysis for fusion transcripts detection in tumor diagnostics: assessment of bioinformatic tools reliability in FFPE samples.

Aim: Diagnostic laboratories are progressively introducing next-generation sequencing (NGS) technologies in the routine workflow to meet the increasing clinical need for comprehensive molecular characterization in cancer patients for diagnosis and precision medicine, including fusion-transcripts detection. Nevertheless, the low quality of messenger RNA (mRNA) extracted from formalin-fixed paraffin-embedded (FFPE) samples may affect the transition from traditional single-gene testing approaches [like fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), or polymerase chain reaction (PCR)] to NGS. The present study is aimed at assessing the overall accuracy of RNA fusion transcripts detection by NGS analysis in FFPE samples in real-world diagnostics. Methods: Herein, NGS data from 190 soft tissue tumors (STTs) and carcinoma cases, discussed in the context of the institutional Molecular Tumor Board, are reported and analyzed by FusionPlex© Solid tumor kit through the manufacturer's pipeline and by two well-known fast and accurate open-source tools [Arriba (ARR) and spliced transcripts alignment to reference (STAR)-fusion (SFU)]. Results: The combination of FusionPlex© Solid tumor with ArcherDX® Analysis suite (ADx) analysis package has been proven to be sensitive and specific in STT samples, while partial loss of sensitivity has been found in carcinoma specimens. Conclusions: Albeit ARR and SFU showed lower sensitivity, the use of additional fusion-detection tools can contribute to reinforcing or extending the output obtained by ADx, particularly in the case of low-quality input data. Overall, our results sustain the clinical use of NGS for the detection of fusion transcripts in FFPE material.

Gene expression-based dissection of inter-histotypes, intra-histotype and intra-tumor heterogeneity in pediatric tumors.

Intra-tumor heterogeneity (ITH) fosters tumor evolution, resistance to therapy, and relapse. Recently, many evidence have been accumulated on the occurrence of genetic ITH in pediatric cancers. With this study we aimed to address the downstream effects that genetic and epigenetic ITH, and tumor-microenvironment interactions may produce within a tumor mass. To this aim, we investigated by high-throughput gene expression multiple samples of 5 hepatoblastomas, 5 neuroblastomas, 5 rhabdomyosarcomas, and 5 Wilms tumors. Principal component analysis, single sample hallmark gene sets analysis, and weighted gene co-expression network analysis were performed on gene expression data. We observed that the different tumors clustered by histotype, and then by case, and in addition, a variable degree of ITH was visible in all the investigated cases. The ITH highlighted in this study can represent a challenge in tumor treatment since we demonstrated that different druggable hallmarks and targets may be heterogeneously present within the same tumor mass, and this can potentially lead to therapeutic failure. Despite this heterogeneity, we could highlight some commonalities among the different histotypes investigated, supporting the feasibility to move in the clinic from a histotype-driven to a target-driven, sometimes agnostic, approach at least in some cases.

ISG15 as a prognostic biomarker in solitary fibrous tumour.

BACKGROUND: Solitary fibrous tumour (SFT) is a rare mesenchymal malignancy that lacks robust prognostic and predictive biomarkers. Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like modifier, associated with tumour progression, and with poor survival of SFT patients, as previous published by our group. Here, we describe the role of ISG15 in the biology of this rare tumour. METHODS: ISG15 expression was assessed by immunohistochemistry in tissue microarrays from SFT patients and tested for correlation with progression-free survival and overall survival (OS). The effects of ISG15 knockdown or induction were investigated for cancer stem cell (CSC) characteristics and for drug sensitivity in unique in vitro models of SFT. RESULTS: The prognostic value of ISG15 for OS was validated at protein level in malignant SFT patients, prospectively treated with pazopanib and enrolled in GEIS-32 trial. In SFT in vitro models, ISG15 knockdown lead to a decrease in the expression of CSC-related genes, including SOX2, NANOG, ALDH1A1, ABCB1 and ABCC1. Likewise, ISG15 downregulation decreased the clonogenic/ tumoursphere-forming ability of SFT cells, while enhancing apoptotic cell death after doxorubicin, pazopanib or trabectedin treatment in 3D cell cultures. The regulation of CSC-related genes by ISG15 was confirmed after inducing its expression with interferon-ß1; ISG15 induction upregulated 1.28- to 451-fold the expression of CSC-associated genes. CONCLUSIONS: ISG15 is a prognostic factor in malignant SFT, regulating the expression of CSC-related genes and CSCs maintenance. Our results suggest that ISG15 could be a novel therapeutic target in SFT, which could improve the efficacy of the currently available treatments.

Renal cell carcinoma in children and adolescents: a retrospective study of a French-Italian series of 93 cases.

AIMS: Renal cell carcinomas (RCCs) represent 2-5% of kidney malignancies in children and adolescents. Appropriate diagnostic and classification are crucial for the correct management of the patients and in order to avoid inappropriate pre-operative chemotherapy, which is usually recommended if a Wilms' tumour is suspected. METHODS AND RESULTS: A French-Italian series of 93 renal cell carcinomas collected from 1990 to 2019 in patients aged less than 18 years was reclassified according to the 2016 World Health Organization (WHO) classification and the latest literature. TFE3 and TFEB fluorescence in-situ hybridisation (FISH) analyses and a panel of immunohistochemical stains were applied. The median age at diagnosis was 11 years (range = 9 months-17 years). MiT family (MiTF) translocation RCCs accounted for 52% of the tumours, followed by papillary (20%) and unclassified RCCs (13%). Other subtypes, such as SDHB-deficient and fumarate hydratase-deficient RCCs, represented 1-3% of the cases. We also described a case of ALK-rearranged RCC with a metanephric adenoma-like morphology. CONCLUSION: A precise histological diagnosis is mandatory, as targeted therapy could be applied for some RCC subtypes, i.e. MiTF-translocation and ALK-translocation RCC. Moreover, some RCC subtypes may be associated with a predisposition syndrome that will impact patients' and family's management and genetic counselling. A precise RCC subtype is also mandatory for the clinical management of the patients and inclusion in new prospective clinical trials.