Search

BACKGROUND: Thymomas can benefit of cytoreductive surgery even if a complete resection is not feasible. The pleural cavity is the most common site of progression and the resection of pleural metastases can be performed in selected patients. We evaluated the results of stereotactic body radiation therapy for the treatment of pleural metastases in patients not eligible for surgery. METHODS: We retrospectively selected 22 patients treated with stereotactic body radiation therapy for pleural metastases between 2013 and 2019. According to RECIST criteria 1.1 modified for thymic epithelial tumors, time to local failure and progression free survival were calculated using Kaplan-Meier method. RESULTS: The median age was 40 years (range, 29–73 years). There were 1 A, 3 AB, 3 B1, 3 B2, 3 B2/B3 and 9 B3 thymomas. Pleural metastases and primary tumor were synchronous in 8 patients. Five patients had a single pleural metastatic site and 17 presented multiple localizations. Sixteen patients received stereotactic body radiation therapy on multiple sites of pleural metastases. The median dose of radiation was 30 Gy (range, 24–40 Gy). With a median follow-up of 33.2 months (95% CI: 13.1–53.3 months), ten patients experienced disease progression with a median progression free survival was 20.4 months (95% CI: 10.7–30.0 months). The disease control rate was 79% and 41% after 1 and 2 years, respectively. Local disease control rate was 92% and 78% after 1 and 2 years, respectively. There were not significant differences in progression free survival between patients diagnosed with synchronous and metachronous metastases (P=0.477), across those treated or not with chemotherapy (P=0.189) and between those who received or not a previous surgical resection of the pleural metastases (P=0.871). There were not grade 3–4 toxicities related to the treatment. CONCLUSIONS: Stereotactic body radiation therapy of pleural metastases is feasible and offers a promising local control of diseases. The impact of this treatment on patients’ survival is hardly predictable because of the heterogeneous clinical behavior of thymomas.

Background Insights into the mechanism of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) could provide important information for further patient management, including the choice of second-line treatment. The EGFR T790M mutation is the most common mechanism of resistance to first- and second-generation EGFR TKIs. Owing to its biologic relevance in the response of non–small-cell lung cancer (NSCLC) to the selective pressure of treatment, the present study investigated whether the occurrence of T790M at progression differed among patients receiving gefitinib, erlotinib, or afatinib. Patients and Methods The present retrospective study included patients with NSCLC with an EGFR activating mutation, who had received gefitinib, erlotinib, or afatinib as first-line treatment. Plasma samples for the analysis of cell-free DNA were taken at disease progression and analyzed using a digital droplet polymerase chain reaction EGFR mutation assay. Results A total of 83 patients were enrolled; 42 had received gefitinib or erlotinib and 41afatinib. The patient characteristics were comparable across the 2 groups. The median time to progression (TTP) was 14.4 months for the gefitinib and erlotinib group and 10.2 months for the afatinib group (P = .09). Of the 83 patients, 47 (56.6%) were positive for the T790M in plasma. A greater incidence of T790M was observed in patients with progression during gefitinib or erlotinib therapy compared with patients treated with afatinib (33 [79%] vs. 14 [34%], respectively; odds ratio, 7.1; 95% confidence interval, 2.7-18.5; P = .0001). Conclusions Although gefitinib, erlotinib, and afatinib showed a comparable TTP in patients receiving first-line therapy, the incidence of T790M differed among them, as demonstrated by the present study, which could have implications for the choice of second-line treatment.

Lung cancer has become a paradigm for precision medicine in oncology, and liquid biopsy (LB) together with radiomics may have a great potential in this scenario. They are both minimally invasive, easy to perform, and can be repeated during patient’s follow-up. Also, increasing evidence suggest that LB and radiomics may provide an efficient way to screen and diagnose tumors at an early stage, including the monitoring of any change in the tumor molecular profile. This could allow treatment optimization, improvement of patients' quality of life, and healthcare-related costs reduction. Latest reports on lung cancer patients suggest a combination of these two strategies, along with cutting-edge data analysis, to decode valuable information regarding tumor type, aggressiveness, progression, and response to treatment. The approach seems more compatible with clinical practice than the current standard, and provides new diagnostic companions being able to suggest the best treatment strategy compared to conventional methods. To implement radiomics and liquid biopsy directly into clinical practice, an artificial intelligence (AI)-based system could help to link patients' clinical data together with tumor molecular profiles and imaging characteristics. AI could also solve problems and limitations related to LB and radiomics methodologies. Further work is needed, including new health policies and the access to large amounts of high-quality and well-organized data, allowing a complementary and synergistic combination of LB and imaging, to provide an attractive choice e in the personalized treatment of lung cancer.

BackgroundMaintenance treatment after autologous bone marrow transplantation in multiple myeloma improves the outcome of patients. We designed a phase II clinical trial to evaluate the treatment with IL2 and zoledronate after autologous bone marrow transplantation in myeloma patients.MethodsPatients with a histologically proven diagnosis of multiple myeloma become eligible if achieved a very good partial remission in bone marrow samples after 3 months from autologous bone marrow transplantation. IL2 was administered from day 1 to 7. In the first cycle, the daily dose was 2 × 106 IU, whereas, in subsequent ones the IL2 dose was progressively escalated, with +25% increases at each cycle, until evidence of toxicity or up to 8 × 106 IU. Four mg of zoledronic acid were infused on day 2. Flow cytometry analysis of γδ-lymphocytes was performed at days 1 and 8 of treatment cycles.ResultsForty-four patients have been enrolled between 2013 and 2016. The median time to progression was 22.5 months (95% CI 9.7–35.2). A complete remission with a negative immunofixation was obtained in 18% of patients and correlated with a significantly longer time to progression (p = 0.015). Treatment was well tolerated without G3 or 4 toxicities. After a week of treatment with IL2 and zoledronate, γδ lymphocytes, Vγ9δ2, CD57+, effector, late effector, and memory γδ increased but in subsequent cycles, there was a progressive reduction of this expansion.ConclusionsThe maintenance treatment with IL2 and Zoledronate has a modest activity in myeloma patients after autologous bone marrow transplantation.EudraCT Number2013-001188-22.

Resistance to EGFR tyrosin kinase inhibitors (TKI) inevitably occurs. Here it is reported the case of a young patient affected by lung adenocarcinoma harboring the L858R EGFR sensitive mutation. The patient developed multiple TKI resistance mechanisms: T790M EGFR resistance mutation, detected only on tumor cell-free DNA, squamous cell transformation and MET amplification, both detected on a tumor re-biopsy. The co-occurrence of squamous cell transformation and de novo MET amplification is an extremely rare event, and this case confirms how dynamic and heterogeneous can be the temporal and spatial tumor evolution under treatment pressure.

OBJECTIVE: With this narrative review, we retraced the history of hypertermic intrathoracic chemotherapy (HITHOC) since the beginning, analyzing literature on operative technique, feasibility and efficacy of this treatment. Moreover, we report the fifteen-year experience of our center in this relatively new technique, for what concerns both early postoperative results and long-term oncological outcomes. BACKGROUND: Thymomas are frequently misdiagnosed and recognized in advanced stage, often with pleural dissemination, especially when not associated to Myasthenia Gravis that allows an early diagnosis during the initial assessment. Moreover, the natural history of locally advanced thymoma is characterized by a high rate of pleural or pericardial relapses. Surgery has always been considered a milestone in thymoma’s treatment, even in case of serous dissemination or relapses, although his role as exclusive therapy does not guarantee an acceptable local disease control. In case of disseminated disease, different multidisciplinary protocols have been experimented, from chemotherapy to radiation therapy, alone or associated to surgery, in order to increase overall and disease-free survival, but the breakthrough happened in the early 90s with the introduction of HITHOC following surgery. Combination of surgery and HITHOC resulted in less toxic than systemic chemotherapy and providing a good local disease control in patients with stage IVa thymomas or thymoma’s pleural recurrences. METHODS: We searched PubMed for relevant literature, up to January 2020, on hypertermic intrapleural chemotherapy for thymomas (TPR or DNT), selecting only those reporting information about HITHOC protocol used, postoperative course and oncological outcomes. CONCLUSIONS: HITHOC is a safe and feasible procedure, with a very low complication rate and negligible systemic effects of chemotherapeutic agents, effective in controlling both TPR and DNT, in particular as regards local disease-free survival. KEYWORDS: Hypertermic intrathoracic chemotherapy (HITHOC); thymoma; intracavitary chemotherapy; hyperthermia; redo-surgery

The Italian Association of Medical Oncology recommendations on thymic epithelial tumors, which have been drawn up for the first time in 2020 through an evidence-based approach, report indications on all the main aspects of clinical management of this group of rare diseases, from diagnosis and staging, to new available systemic treatments, such as targeted therapies and immunotherapies. A summary of key recommendations is presented here and complete recommendations are reported as Supplementary Materials, available at https://doi.org/10.1016/j.esmoop.2021.100188., Highlights • Thymic epithelial tumors (TETs). • Multidisciplinary management of patients with TETs. • Rare cancers. • Updated recommendations.

Alterations in the genetic content, such as Copy Number Variations (CNVs) is one of the hallmarks of cancer and their detection is used to recognize tumoral DNA. Analysis of cell-free DNA from plasma is a powerful tool for non-invasive disease monitoring in cancer patients. Here we exploit third generation sequencing (Nanopore) to obtain a CNVs profile of tumoral DNA from plasma, where cancer-related chromosomal alterations are readily identifiable.Compared to Illumina sequencing -the only available alternative- Nanopore sequencing represents a viable approach to characterize the molecular phenotype, both for its ease of use, costs and rapid turnaround (6 hours).

BackgroundEGFR-positive Non-small Cell Lung Cancer (NSCLC) is a dynamic entity and tumor progression and resistance to tyrosine kinase inhibitors (TKIs) arise from the accumulation, over time and across different disease sites, of subclonal genetic mutations. For instance, the occurrence of EGFR T790M is associated with resistance to gefitinib, erlotinib, and afatinib, while EGFR C797S causes osimertinib to lose activity. Sensitive technologies as radiomics and liquid biopsy have great potential to monitor tumor heterogeneity since they are both minimally invasive, easy to perform, and can be repeated over patient’s follow-up, enabling the extraction of valuable information. Yet, to date, there are no reported cases associating liquid biopsy and radiomics during treatment.Case presentationIn this case series, seven patients with metastatic EGFR-positive NSCLC have been monitored during target therapy. Plasma-derived cell free DNA (cfDNA) was analyzed by a digital droplet PCR (ddPCR), while radiomic analyses were performed using the validated LifeX® software on computed tomography (CT)-images. The dynamics of EGFR mutations in cfDNA was compared with that of radiomic features. Then, for each EGFR mutation, a radiomic signature was defines as the sum of the most predictive features, weighted by their corresponding regression coefficients for the least absolute shrinkage and selection operator (LASSO) model. The receiver operating characteristic (ROC) curves were computed to estimate their diagnostic performance. The signatures achieved promising performance on predicting the presence of EGFR mutations (R2 = 0.447, p 2 = 0.301, p = 0.003 for T790M; and R2 = 0.354, p = 0.001 for activating plus resistance mutations), confirmed by ROC analysis.ConclusionTo our knowledge, these are the first cases to highlight a potentially promising strategy to detect clonal heterogeneity and ultimately identify patients at risk of progression during treatment. Together, radiomics and liquid biopsy could detect the appearance of new mutations and therefore suggest new therapeutic management.

A number of treatments have been developed for HER1, 2 and 3-driven non-small cell lung cancer (NSCLC), of which the most successful have been the epidermal growth factor receptor-tyrosine kinase inhibitors in HER1-mutant tumours resulting in highly improved progression-free survival. Human epidermal growth factor (HER)2 and 3-driven tumours represent the minority of NSCLC, and effective therapies in these patients still represent an unmet medical need. The encouraging results seen with anti-HER2 and anti-HER3 monoclonal antibodies need to be validated in larger studies, even if the greatest obstacle is represented by the exiguous number of patients bearing deregulated HER2/3 system and abnormalities of signal transduction pathway. Considering NSCLC tumour heterogeneity, which affects response and resistance to treatment, combined multiparametric approaches, such as liquid biopsy together with radiomics, may provide a better understanding of the tumour dynamics and clonal selection during the treatments.

Background It is still unclear how to combine biomarkers to identify patients who will truly benefit from anti-PD-1 agents in NSCLC. This study investigates exosomal mRNA expression of PD-L1 and IFN-γ, PD-L1 polymorphisms, tumor mutational load (TML) in circulating cell-free DNA (cfDNA) and radiomic features as possible predictive markers of response to nivolumab and pembrolizumab in metastatic NSCLC patients. Methods Patients were enrolled and blood (12 ml) was collected at baseline before receiving anti-PD-1 therapy. Exosome-derived mRNA and cfDNA were extracted to analyse PD-L1 and IFN-γ expression and tumor mutational load (TML) by digital droplet PCR (ddPCR) and next-generation sequencing (NGS), respectively. The PD-L1 single nucleotide polymorphisms (SNPs) c.-14-368 T > C and c.*395G > C, were analysed on genomic DNA by Real-Time PCR. A radiomic analysis was performed on the QUIBIM Precision® V3.0 platform. Results Thirty-eight patients were enrolled. High baseline IFN-γ was independently associated with shorter median PFS (5.6 months vs. not reached p = 0.0057), and levels of PD-L1 showed an increase at 3 months vs. baseline in patients who progressed (p = 0.01). PD-L1 baseline levels showed significant direct and inverse relationships with radiomic features. Radiomic features also inversely correlated with PD-L1 expression in tumor tissue. In subjects receiving nivolumab, median PFS was shorter in carriers of c.*395GG vs. c.*395GC/CC genotype (2.3 months vs. not reached, p = 0.041). Lastly, responders had higher non-synonymous mutations and more links between co-occurring genetic somatic mutations and ARID1A alterations as well. Conclusions A combined multiparametric approach may provide a better understanding of the molecular determinants of response to immunotherapy.

Background Thymoma and myasthenia gravis share several pathogenetic aspects including the role of surgery as a therapeutic option. Extended thymectomy is associated with excellent survival and good local control, especially in early stages, and its role for the neurologic disease has been recently validated. The aim of this study is evaluating oncologic and neurologic outcomes of myasthenic patients with thymoma who underwent extended thymectomy. Methods We retrospectively collected surgical, oncologic, and neurologic data of all myasthenic patients with thymoma who underwent extended thymectomy at our department from January 1994 to December 2016. Clinical and pathologic data, neurologic remission rate, and overall survival and disease-free interval were analyzed. Results In all, 219 patients underwent extended thymectomy. The B2 histotype was the most represented thymoma (24.2%), and the most prevalent pathologic Masaoka stage was IIB (37.9%). The overall survival and disease-free survival were statistically different between early stage and advanced stage. During the surveillance, 33 patients (15.1%) had recurrences, treated in 21 cases with iterative surgery. Regarding neurologic outcomes, 75 patients (34.2%) reached a complete stable remission, 84 (38.4%) a pharmacologic remission, 51 (23.3%) had an improvement of their symptoms, and in 9 patients (4.1%) myasthenia was unchanged or worsened. Conclusions Surgery is a cornerstone in the treatment of patients with both thymoma and myasthenia gravis. Extended thymectomy, as proposed by Masaoka, offers considerable oncologic outcomes with an excellent survival and low recurrence rate of thymoma; moreover, surgery leads to remarkable neurologic results.

Background Circulating cell-free DNA (cfDNA) may help understand the molecular response to pharmacologic treatment and provide information on dynamics of clonal heterogeneity. Therefore, this study evaluated the correlation between treatment outcome and activating EGFR mutations (act-EGFR) and T790M in cfDNA in patients with advanced NSCLC given osimertinib. Methods Thirty-four NSCLC patients resistant to first/second-generation EGFR-TKIs, positive for both act-EGFR and T790M in cfDNA at the time of progression were enrolled in this study. Plasma samples were obtained at osimertinib baseline and after 3 months of therapy; cfDNA was analyzed by droplet digital PCR and results were expressed as mutant allele frequency (MAF). Results At baseline, act-EGFR MAF was significantly higher than T790M (p 2.6% and 0.22 (6 months vs. not reached, respectively, p = 0.01). Conclusion act-EGFR MAF and T790M/act-EGFR MAF ratio are potential markers of outcome in patients treated with osimertinib.

Background In patients with anaplastic lymphoma kinase (ALK)-positive non–small-cell lung cancer (NSCLC), disease progression occurs after a median of 9 to 10 months of crizotinib treatment. Several mechanisms of resistance have been identified and include ALK mutations and amplification or the activation of bypassing signaling pathways. Rebiopsy in NSCLC patients represents a critical issue and the analysis of circulating cell-free DNA (cfDNA) has a promising role for the identification of resistance mechanisms. Patients and Methods Twenty patients with advanced ALK-positive NSCLC were enrolled after disease progression during crizotinib treatment; cfDNA was analyzed using digital droplet polymerase chain reaction (BioRad, Hercules, CA) for ALK (p.L1196M, p.G1269A, and p.F1174L) and Kirsten rat sarcoma (KRAS) (codons 12 and 13) mutations. Results ALK secondary mutations (p.L1196M, p.G1269A, and p.F1174L) were identified in 5 patients; 1 patient had 2 ALK mutations (p.L1196M and p.G1269A). Overall, 10 patients presented KRAS mutations (7 p.G12D, 2 p.G12V, and 1 p.G12C mutations, respectively). In 3 patients KRAS mutations were associated with ALK mutations. cfDNA was monitored during the treatment with second-generation ALK inhibitors and the amount of ALK as well as KRAS mutations decreased along with tumor regression. Conclusion ALK and KRAS mutations are associated with acquired resistance to crizotinib in ALK-positive NSCLC. In particular, ALK acquired mutations can be detected in plasma and could represent a promising tumor marker for response monitoring.

Background Mesangiogenic progenitor cells (MPCs) have shown the ability to differentiate in-vitro toward mesenchymal stromal cells (MSCs) as well as angiogenic potential. MPCs have so far been described in detail as progenitors of the mesodermal lineage and appear to be of great significance in tissue regeneration and in hemopoietic niche regulation. On the contrary, information regarding the MPC angiogenic process is still incomplete and requires further clarification. In particular, genuine MPC angiogenic potential should be confirmed in-vivo. Methods In the present article, markers and functions associated with angiogenic cells have been dissected. MPCs freshly isolated from human bone marrow have been induced to differentiate into exponentially growing MSCs (P2-MSCs). Cells have been characterized and angiogenesis-related gene expression was evaluated before and after mesengenic differentiation. Moreover, angiogenic potential has been tested by in-vitro and in-vivo functional assays. Results MPCs showed a distinctive gene expression profile, acetylated-low density lipoprotein uptake, and transendothelial migration capacity. However, mature endothelial markers and functions of endothelial cells, including the ability to form new capillaries, were absent, thus suggesting MPCs to be very immature endothelial progenitors. MPCs showed marked 3D spheroid sprouting activating the related molecular machinery, a clear in-vitro indication of early angiogenesis. Indeed, MPCs applied to chicken chorioallantoic membrane induced and participated in neovessel formation. All of these features were lost in mesengenic terminally differentiated P2-MSCs, showing definite separation of the two differentiation lineages. Conclusion Our results confirm the bona-fide angiogenic potential of MPCs and suggest that the high variability reported for MSC cultures, responsible for the controversies regarding MSC angiogenic potential, could be correlated to variable percentages of co-isolated MPCs in the different culture conditions so far used. Electronic supplementary material The online version of this article (doi:10.1186/s13287-017-0562-x) contains supplementary material, which is available to authorized users.

// Elena Ciabatti 1,3 , Angelo Valetto 2 , Veronica Bertini 2 , Maria Immacolata Ferreri 2 , Alice Guazzelli 2 , Susanna Grassi 1,3 , Francesca Guerrini 1 , Iacopo Petrini 4 , Maria Rita Metelli 1 , Maria Adelaide Caligo 2 , Simona Rossi 2 and Sara Galimberti 1 1 Department of Clinical and Experimental Medicine, Section of Hematology, University of Pisa, Pisa, Italy 2 Laboratory of Medical Genetics, Azienda Ospedaliero-Universitaria Pisana, S. Chiara Hospital, Pisa, Italy 3 GenOMec, University of Siena, Siena, Italy 4 Department of Translational Research and New Technologies in Medicine, University of Pisa, Pisa, Italy Correspondence to: Elena Ciabatti, email: // Keywords : myelodysplastic syndromes, aCGH, cytogenetics, TP53, SF3B1 Received : August 23, 2016 Accepted : March 14, 2017 Published : March 25, 2017 Abstract In this study we present a new diagnostic workup for the myelodysplastic syndromes (MDS) including FISH, aCGH, and somatic mutation assays in addition to the conventional cytogenetics (CC). We analyzed 61 patients by CC, FISH for chromosome 5, 7, 8 and PDGFR rearrangements, aCGH, and PCR for ASXL1, EZH2, TP53, TET2, RUNX1, DNMT3A, SF3B1 somatic mutations. Moreover, we quantified WT1 and RPS14 gene expression levels, in order to find their possible adjunctive value and their possible clinical impact. CC analysis showed 32% of patients with at least one aberration. FISH analysis detected chromosomal aberrations in 24% of patients and recovered 5 cases (13.5%) at normal karyotype (two 5q- syndromes, one del(7) case, two cases with PDGFR rearrangement). The aGCH detected 10 “new” unbalanced cases in respect of the CC, including one with alteration of the ETV6 gene. After mutational analysis, 33 patients (54%) presented at least one mutation and represented the only marker of clonality in 36% of all patients. The statistical analysis confirmed the prognostic role of CC either on overall or on progression-free-survival. In addition, deletions detected by aCGH and WT1 over-expression negatively conditioned survival. In conclusion, our work showed that 1) the addition of FISH (at least for chr. 5 and 7) can improve the definition of the risk score; 2) mutational analysis, especially for the TP53 and SF3B1, could better define the type of MDS and represent a “clinical warning”; 3) the aCGH use could be probably applied to selected cases (with suboptimal response or failure).

// Marzia Del Re 1, * , Paola Bordi 2, * , Iacopo Petrini 3, * , Eleonora Rofi 1 , Francesca Mazzoni 4 , Lorenzo Belluomini 5 , Enrico Vasile 3 , Giuliana Restante 1 , Francesco Di Costanzo 4 , Alfredo Falcone 3 , Antonio Frassoldati 5 , Ron H.N. van Schaik 6 , Christi M.J. Steendam 7 , Antonio Chella 8 , Marcello Tiseo 2 , Riccardo Morganti 9 and Romano Danesi 1 1 Clinical Pharmacology and Pharmacogenetics Unit, Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy 2 Medical Oncology Unit, University Hospital of Parma, Parma, Italy 3 Medical Oncology Unit, Department of Translational Research and New Technologies in Medicine and Surgery, University Hospital of Pisa, Pisa, Italy 4 Medical Oncology Unit, University Hospital Careggi, Firenze, Italy 5 Medical Oncology Unit, Civil Hospital Arcispedale S. Anna, Ferrara, Italy 6 Department of Clinical Chemistry, Erasmus University Medical Center, Rotterdam, The Netherlands 7 Department of Pulmonology, Erasmus University Medical Center, Rotterdam, and Amphia Hospital, Breda, The Netherlands 8 Lung Diseases Unit, Azienda Ospedaliero-Universitaria, Pisa, Italy 9 Section of Statistics, Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy * These authors have contributed equally to this work Correspondence to: Iacopo Petrini, email: iacopo.petrini@unipi.it Keywords: circulating tumor DNA, EGFR, personalized medicine, predictive biomarkers Received: July 12, 2017 Accepted: July 26, 2017 Published: September 15, 2017 ABSTRACT Introduction: NSCLC harboring activating mutations of EGFR is highly sensitive to first-line EGFR-tyrosine kinase inhibitors (TKIs), but drug resistance depending on the EGFR mutation p.T790M will occur in about 50-60% of patients. Detailed information on the amount of p.T790M plasmatic level associated with resistance to EGFR-TKIs and guidance to treatment with p.T790M-effective TKI depending on these levels, is lacking. Methods: This study enrolled p.T790M-positive patients (n=49) affected by EGFR-mutated NSCLC at progression to first-line EGFR-TKIs and, in selected cases (n=5), after second-line treatment with osimertinib. Cell-free circulating tumor DNA (cftDNA) was extracted from plasma and the quantitative analysis of EGFR ex19del, p.L858R and p.T790M was performed by digital droplet PCR. Results: The mean amount of mutated alleles at progression to first-line EGFR-TKIs was 108,492 copies/ml for ex19del, 97,336 copies/ml for p.L858R, but only 8,754 copies/ml for p.T790M. There was no significant correlation between progression-free survival and the ratio of p.T790M over EGFR activating mutations. The analysis of cftDNA in 5 patients treated with osimertinib revealed a marked decrease of all EGFR mutant alleles. Conclusions: The amount of p.T790M in plasma can be much lower than activating EGFR mutations. Despite this finding, osimertinib is effective in p.T790M-positive patients. These results indicate that clones driving resistance to EGFR-TKIs represent a minority among cells bearing activating EGFR-mutations. In addition, the identification of a threshold level of p.T790M is not a strict requirement for the selection of patients to be treated with osimertinib, since treatment showed a decrease in all EGFR mutated cells.

Background Systemic treatment of advanced non-small cell lung cancer (NSCLC) has changed dramatically since the introduction of targeted therapies. The analysis of circulating tumor DNA (ctDNA) is a valuable approach to monitor the clonal evolution of tumors during treatment with EGFR-tyrosine kinase inhibitors (TKIs) and to detect resistance mutations. Case presentation A NSCLC patient with exon 19 deletion (ex19del) of EGFR was treated with osimertinib after multiple lines of treatment and obtained a partial response that lasted over 26 months. Blood was collected at each visit and ctDNA was extracted to monitor ex19del by digital droplet PCR. Within a few weeks from the beginning of osimertinib, ex19del disappeared from plasma but appeared again and steadily increased a few months later anticipating tumor progression. Interestingly, the change in ex19del was much more pronounced than other mutations, since T790M appeared 3 months after the increase of ex19del, and C797S was detectable a few weeks before clinical disease progression. Then the patient received cytotoxic chemotherapy, which was associated with a decrease in ex19del and disappearance of T790M and C797S; however, at disease progression, all EGFR mutations increased again in plasma together with MET amplification which was detected by NGS. Conclusions The measurement of ex19del changes in ctDNA is a simple and sensitive approach to monitor clinical outcome to osimertinib and, potentially, to other therapeutic interventions.

Analysis of circulating tumor DNA (ctDNA) for the identification of T790M mutation in advanced EGFR-mutated NSCLC patients can replace tissue re-biopsy for resistance characterization and, being non-invasive, may be applied for disease monitoring. We analysed ctDNA during osimertinib treatment to correlate mutational levels with clinical outcome and to predict pattern of resistance.Forty patients with advanced NSCLC receiving osimertinib for T790M + disease after previous EGFR-TKI were enrolled in a pilot study to collect plasma at baseline and every 12 weeks until progression. Molecular analysis of ctDNA was performed by ddPCR and Therascreen®. When feasible at progression, tissue re-biopsy and NGS analysis were performed.Thirty-eight patients had baseline plasma samples suitable for molecular analysis. Patients with low levels of the EGFR activating mutation in ctDNA [2200 copies/mL or allele frequency (AF)6.1%] showed better progression-free survival (17.8 or 17.8 months vs. 4.3 or 2.7, p = 0.022 or p = 0.018, respectively) and overall survival (23.6 or 23.6 vs. 7.7 or 7.3, p = 0.016 or p = 0.013, respectively) than patients with high levels (≥ 2200 copies/mL or AF ≥ 6.1%). Patients with detectable EGFR mutations in plasma (shedders) presented worse outcome than negative subjects (non-shedders). Low levels of T790M, higher T790M/activating mutation ratio and complete clearance after 2 months were associated with a trend towards better outcome. Tissue re-biopsy at resistance showed 3 patients with EGFR C797S, 1 with MET amplification, 1 with MYC amplification, 1 with PTEN loss, 3 with SCLC transformation.The mutational analysis performed on plasma plays a significant role in prognostic stratification, especially for the EGFR activating mutation, since patients with absence or low levels of mutations presented a better outcome to osimertinib. At progression, tissue re-biopsy remains a crucial issue for the identification of resistance mechanisms.

Thymic epithelial tumors are rare malignancies. Thymic carcinoma represents about 20% of all thymic epithelial tumors and has aggressive behavior, with a greater tendency to metastatic spread. Thymic carcinoma is often diagnosed in advanced stages for which systemic treatment is the main therapeutic option. The association of chemotherapy and antiangiogenic agents in the first-line setting has never been investigated in this very rare cancer. However, preclinical and clinical evidence has suggested that inhibition of angiogenesis could be beneficial. The RELEVENT trial is a multicenter, open-label, single-arm, phase II study aimed at investigating the activity and safety of ramucirumab combined with paclitaxel and carboplatin in chemotherapy-naive patients affected by thymic carcinoma or B3 thymoma with area of carcinoma. The primary endpoint of the trial is the overall response rate. Progression-free survival, overall survival, and safety are secondary endpoints. Patient-reported outcomes will be collected at each visit. The mutational status of a subset of genes, polymorphisms, and selected micro-RNA expression will be evaluated.

Therapy resistance represents a clinical challenge for advanced non-small cell lung cancer (NSCLC), which still remains an incurable disease. There is growing evidence that cancer-initiating or cancer stem cells (CSCs) provide a reservoir of slow-growing dormant populations of cells with tumor-initiating and unlimited self-renewal ability that are left behind by conventional therapies reigniting post-therapy relapse and metastatic dissemination. The metabolic pathways required for the expansion of CSCs are incompletely defined, but their understanding will likely open new therapeutic opportunities. We show here that lung CSCs rely upon oxidative phosphorylation for energy production and survival through the activity of the mitochondrial citrate transporter, SLC25A1. We demonstrate that SLC25A1 plays a key role in maintaining the mitochondrial pool of citrate and redox balance in CSCs, whereas its inhibition leads to reactive oxygen species build-up thereby inhibiting the self-renewal capability of CSCs. Moreover, in different patient-derived tumors, resistance to cisplatin or to epidermal growth factor receptor (EGFR) inhibitor treatment is acquired through SLC25A1-mediated implementation of mitochondrial activity and induction of a stemness phenotype. Hence, a newly identified specific SLC25A1 inhibitor is synthetic lethal with cisplatin or with EGFR inhibitor co-treatment and restores antitumor responses to these agents in vitro and in animal models. These data have potential clinical implications in that they unravel a metabolic vulnerability of drug-resistant lung CSCs, identify a novel SLC25A1 inhibitor and, lastly, provide the first line of evidence that drugs, which block SLC25A1 activity, when employed in combination with selected conventional antitumor agents, lead to a therapeutic benefit.

Thymic epithelial tumors (TETs) are a heterogenous group of rare tumors, with a complex histopatological classification. Furthermore, the recent introduction of the first TNM staging system, that is scheduled to replace the Masaoka-Koga system, may create further difficulties in TET management, that remains challenging. Several guidelines for treatment of TETs are available and provide recommendations based mainly on non randomized trials and retrospective or limited series. Often the lack of evidence leads to formulation of indications based on expert opinions. As for other rare cancers it is crucial to create networks to coordinate the work among centres involved in treatment of these diseases in order to offer the best diagnostic and therapeutic tools. For this purpose, in 2014 a network named TYME (ThYmic MalignanciEs), was founded in Italy with the aim of improving care and research in TETs. In September 2017 a panel of multidisciplinary experts from TYME network and from other Italian centres strongly involved in TET diagnosis and treatment convened a first Italian Expert meeting together with representatives of association for patients affected by rare thoracic cancers Tu.To.R, to explore how these tumors are managed in the different centres of Italy compared to ESMO guidelines. In this paper we summarize the issues discussed during that meeting and we propose recommandations based on Masaoka Koga and the new TNM staging system.

There are not established treatments for patients with advanced malignant pleural mesothelioma that progressed after first-line chemotherapy with cisplatin and pemetrexed. Retrospective analyses suggest a possible role for rechallenge with pemetrexed for selected patients. Phase II trials demonstrate a modest efficacy of vinorelbine monotherapy with a response rate ranging between 0% and 18% and a tolerable toxicity profile. Combination schedules, despite an increased toxicity, fail to demonstrate an improved efficacy. To date, genome wide analyses did not show molecular targets suitable for therapy and biological drugs did not exert a significant efficacy in clinical trials. Immunotherapy has given a hint of efficacy in early clinical trials but definitive evaluations are still ongoing.

Background: PD-L1 expression in tumour tissues is widely used to select patients to receive anti-PD-1/PD-L1 antibodies, but data are lacking on the correlation of plasma PD-L1 levels with the effect of treatments. Methods: To investigate the association between PD-L1 mRNA in plasma-derived exosomes and response to nivolumab and pembrolizumab in patients with melanoma (n=18) and NSCLC (n=8), blood was obtained at time point 0 and after 2 months. Exosomal PD-L1 mRNA was measured by digital droplet PCR. Results: The mean±s.e.m. PD-L1 levels in patients with complete and partial responses were 830.4±231.3 and 242.5±82.5 copies per ml at time 0 vs 2 months, respectively (P=0.016). In patients with stable disease the mean±s.e.m. values were 298.8±97.2 vs 247.5±29.8 copies per ml (P=0.586), while in progressive disease, PD-L1 mRNA levels were 204.0±68.8 vs 416.0±87.8 copies per ml at time 0 vs 2 months, respectively (P=0.001). Conclusions: This study demonstrates that exosomal PD-L1 is significantly associated with response to treatment.

Purpose The evaluation of molecular targets in gastric cancer has demonstrated the predictive role of HER2 amplification for trastuzumab treatment in metastatic gastric cancer. Besides HER2, other molecular targets are under evaluation in metastatic gastric tumors. However, very little is known about their role in resected tumors. We evaluated the expression of HER2, EGFR, MET, AKT1 and phospho-mTOR in resected stage II-III adenocarcinomas. Methods Ninety-two patients with resected stomach (63%) or gastro-esophageal adenocarcinomas (27%) were evaluated. Antibodies anti-HER2, EGFR, MET, AKT1 and phospho-mTOR were used for immunostaining of formalin-fixed paraffin-embedded slides. Using FISH, HER2 amplification was evaluated in cases with an intermediate (+2) staining. Results EGFR overexpression (11%) was a poor prognostic factor for overall survival (3-year OS: 47% vs 77%; Log-Rank p= 0.033). MET overexpression (36%) was associated with a trend for a worse survival (3-year OS: 65% vs 77%; Log-Rank p= 0.084). HER2 amplification/overexpression and mTOR hyper-phosphorylation were observed in 13% and 48% of tumors, respectively. AKT1 overexpression (8%) was not a prognostic factor by itself (p= 0.234). AKT1 and EGFR overexpression was mutually exclusive and patients with EGFR or AKT1 overexpression experienced a poor prognosis (3-year OS: 52% vs. 79%, Log-Rank p= 0.005). Conclusions EGFR is confirmed a poor prognostic factor in resected gastric cancers. We firstly describe a mutually exclusive overexpression of EGFR and AKT1 with potential prognostic implications, suggesting the relevance of this pathway for the growth of gastric cancers.

Background Hypereosinophilia-associated syndromes are a heterogeneous group of diseases characterized by sustained and elevated blood eosinophilia with evidence of eosinophil-induced organ damage. Classically, Eosinophilic granulomatosis with polyangiitis (EGPA) and Hypereosinophilic syndrome (HES) present several overlapping clinical and laboratory features, making it challenging to correctly insert patients in restricted and well-defined categories with specific and more effective therapeutic approaches. Therefore, great efforts are ongoing searching for novel biomarkers able to differentiate these two disorders in daily practice. Objectives To detect T cell receptor gamma (TCRG) clonal rearrangements in EGPA and HES, comparing the frequency distribution of V region and J region segment utilization in the study population. Methods In this single center study, we included consecutive patients with a diagnosis of EGPA and HES. Inclusion criteria were: documentation of a persistent peripheral eosinophilic count of ≥1.5 x109/L and signs or symptoms of organ involvement. Clinical and laboratory data of the patients were collected. Sequence-based determination of the frequency distribution of TCRG Gene Rearrangements was performed using next-generation sequencing with the Illumina MiSeq (LymphoTrack TRG assay, Invivoscribe). Results We included 21 patients (9 with EGPA and 12 with HES). Four EGPA patients were MPO-ANCA positive. We detected TCRG clonal rearrangements in 44% (4/9) patients with EGPA and in 42% (5/12) patients with HES (p-value = n.s). No association was observed between TCRG clonal rearrangements and ANCA status in EGPA patients. Recurrent TCRG gene rearrangements were observed; in particular, Vg10JgP1 (5 cases) and Vg4Jg1/2 (4 cases) were detected in both EGPA and HES, whereas Vg9Jg1/2 (2 cases) and Vg10Jg1/2 (2 cases) were found only in patients with HES. Conclusions Even if preliminary, this study reveals a similar T cell receptor gamma repertoire in EGPA and HES, thus suggesting a possible antigen-driven inflammatory response underlying hypereosinophilia in both EGPA and HES. Moreover, our results would suggest that the TCR clonality cannot be used as a tool for the differential diagnosis between EGPA and HES. Disclosure of Interest None declared

// Marzia Del Re 1 , Marcello Tiseo 2 , Paola Bordi 2 , Armida D’Incecco 3 , Andrea Camerini 4 , Iacopo Petrini 5 , Maurizio Lucchesi 6 , Alessandro Inno 7 , Daniele Spada 8 , Enrico Vasile 6 , Valentina Citi 1 , Giorgio Malpeli 9 , Enrica Testa 8 , Stefania Gori 7 , Alfredo Falcone 6 , Domenico Amoroso 4 , Antonio Chella 10 , Federico Cappuzzo 3 , Andrea Ardizzoni 2 , Aldo Scarpa 9 , Romano Danesi 1 1 Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy 2 Medical Oncology Unit, Azienda Ospedaliero-Universitaria, Parma, Italy 3 Medical Oncology Unit, AUSL6, Istituto Toscano Tumori, Livorno, Italy 4 Medical Oncology Unit, AUSL12, Istituto Toscano Tumori, Lido di Camaiore, Italy 5 Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy 6 Medical Oncology Unit 2, Azienda Ospedaliero-Universitaria, Pisa, Italy 7 Medical Oncology Unit, Ospedale Sacro Cuore, Negrar, Italy 8 Medical Oncolgy Unit, Ospedale Santa Maria della Misericordia, Urbino, Italy 9 ARC-NET Research Centre and Department of Pathology and Diagnostics, Azienda Ospedaliero-Universitaria, Verona, Italy 10 Lung Diseases Unit, Azienda Ospedaliero-Universitaria, Pisa, Italy Correspondence to: Romano Danesi, email: romano.danesi@unipi.it Keywords: cell-free circulating tumor DNA, KRAS, EGFR, activating mutation, tyrosine kinase inhibitors Received: June 24, 2015 Accepted: December 29, 2015 Published: January 20, 2016 ABSTRACT Introduction: KRAS oncogene mutations ( MUT KRAS ) drive resistance to EGFR inhibition by providing alternative signaling as demonstrated in colo-rectal cancer. In non-small cell lung cancer (NSCLC), the efficacy of treatment with EGFR tyrosine kinase inhibitors (EGFR-TKIs) depends on activating EGFR mutations ( MUT EGFR ). However, inhibition of EGFR may select resistant cells displaying alternative signaling, i.e., KRAS , or restoration of EGFR activity due to additional MUT EGFR , i.e., the c.2369C > T ( p.T790M EGFR ). Aim: The aim of this study was to investigate the appearance of MUT KRAS during EGFR-TKI treatment and their contribution to drug resistance. Methods: This study used cell-free circulating tumor DNA (cftDNA) to evaluate the appearance of codon 12 MUT KRAS and p.T790M EGFR mutations in 33 advanced NSCLC patients progressing after an EGFR-TKI. Results: p.T790M EGFR was detected in 11 (33.3%) patients, MUT KRAS at codon 12 in 3 (9.1%) while both p.T790M EGFR and MUT KRAS codon 12 were found in 13 (39.4%) patients. Six patients (18.2%) were KRAS wild-type ( WT KRAS ) and negative for p.T790M EGFR . In 8 subjects paired tumor re-biopsy/plasma samples were available; the percent concordance of tissue/plasma was 62.5% for p.T790M EGFR and 37.5% for MUT KRAS . The analysis of time to progression (TTP) and overall survival (OS) in WT KRAS vs. MUT KRAS were not statistically different, even if there was a better survival with WT KRAS vs. MUT KRAS , i.e., TTP 14.4 vs. 11.4 months ( p = 0.97) and OS 40.2 vs. 35.0 months ( p = 0.56), respectively. Conclusions: MUT KRAS could be an additional mechanism of escape from EGFR-TKI inhibition and cftDNA is a feasible approach to monitor the molecular development of drug resistance.

Background Mutation of general transcription factor IIi (GTF2I) (chromosome 7 c.74146970T>A) is common in thymic epithelial tumors and is a candidate driver aberration for cancer growth. To our knowledge, this mutation has not been described in other diseases. We evaluated the presence of GTF2I mutation in hematological malignancies. Materials and methods We sequenced samples from 31 patients with acute leukemia, 29 with chronic leukemia and 12 with myelodysplastic syndrome. The genomic fragment of exon 15 containing the hotspot of mutation was amplified using polymerase chain reaction (PCR) and sequenced. Results We did not identify any GTF2I mutation in patients with hematological malignancies. Conclusion Even though our sample size was limited, our data and reports from the literature suggest that GTF2I mutation is not present or is uncommon in these diseases.

We present on a new case of myelodysplastic syndrome characterized by array Comparative Genomic Hybridization. This technique confirmed the monosomy 7, detected by conventional cytogenetics, and revealed also a deletion on the short arm of chromosome 12. This deletion extends for about 14.8 Mb and breaks ETV6 gene. 12p deletion extents in hematological malignancies may vary, but the minimally deleted region almost invariably contains ETV6, that is considered the main candidate tumor suppressor genes within the region for tumor progression. It has been shown that levels of ETV6 were significantly decreased in cases with 12p13 deletions, whereas expression of other genes in the deleted region, like BCL2L14, LRP6, DUSP16 and GPRC5D, did not show any variation, independently of their copy number status. This observation strengthens the fact that ETV6 may play a potential role in the tumorigenesis process. The role of ETV6 in our patient myelodysplastic syndrome is showed by his clinical history and his poor prognosis.

// Marzia Del Re 1, * , Eleonora Rofi 1, * , Giuliana Restante 1 , Stefania Crucitta 1 , Elena Arrigoni 1 , Stefano Fogli 1 , Massimo Di Maio 2 , Iacopo Petrini 3 and Romano Danesi 1 1 Clinical Pharmacology and Pharmacogenetics Unit, Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy 2 Department of Oncology, University of Turin, Ordine Mauriziano Hospital, Turin, Italy 3 General Pathology, Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy * These authors contributed equally to this work Correspondence to: Romano Danesi, email: romano.danesi@unipi.it Keywords: KRAS; NSCLC; treatment acquired resistance; TKI; pharmacogenetics Received: September 06, 2017 Accepted: October 27, 2017 Published: December 21, 2017 ABSTRACT Rationale: KRAS is the most common and, simultaneously, the most ambiguous oncogene implicated in human cancer. Despite KRAS mutations were identified in Non Small Cell Lung Cancers (NSCLCs) more than 20 years ago, selective and specific inhibitors aimed at directly abrogating KRAS activity are not yet available. Nevertheless, many therapeutic approaches have been developed potentially useful to treat NSCLC patients mutated for KRAS and refractory to both standard chemotherapy and targeted therapies. The focus of this review will be to provide an overview of the network related to the intricate molecular KRAS pathways, stressing on preclinical and clinical studies that investigate the predictive value of KRAS mutations in NSCLC patients. Materials and Methods: A bibliographic search of the Medline database was conducted for articles published in English, with the keywords KRAS, KRAS mutations in non-small cell lung cancer, KRAS and tumorigenesis, KRAS and TKIs, KRAS and chemotherapy, KRAS and monoclonal antibody, KRAS and immunotherapy, KRAS and drugs, KRAS and drug resistance.

Background Insights into the mechanism of resistance to first generation EGFR-TKIs may provide important information for further patient management, including the choice of second-line treatments. T790M is a gatekeeper mutation of the ATP binding pocket of the EGFR kinase domain and is the most common mechanism of resistance to first-generation EGFR-TKIs. Owing to its biologic relevance in the response of NSCLC to the selective pressure of treatments, the present study investigated in circulating cell-free DNA (cfDNA) if the occurrence of T790M at progression differed among gefitinib, afatinib, and erlotinib. Methods This study included patients with NSCLC bearing EGFR activating mutation, and given gefitinib (G), erlotinib (E) or afatinib (A) as first-line treatment. Plasma samples for the analysis of cfDNA were taken at disease progression (PD) and analyzed by a ddPCR using the ddPCR EGFR Mutation Assay. In selected cases, a rebiopsy was performed to confirm the absence of the T790M in negative plasma. Results A total of 83 patients were enrolled; 42 patients received G/E and 41 received A. Patients’ characteristics were comparable across the two groups. Median time to progression (TTP) was 14.4 in G/E vs 10.2 months in A group (p = 0.09). Forty-seven out of 83 patients (56.6%) were positive for the T790M in plasma. There was a higher incidence of the T790M in patients who progressed to G/E than in patients treated with A: 33 (79%) vs 14 (34%), respectively (p = 0.0001). To confirm the absence of the T790M, a rebiopsy was feasible in 7 patients of the G/E group and in 23 of the A group. The analysis of the cytological sample confirmed the absence of the T790M, and PI3K mutation was found in both groups in 1 patient (2%). Three patients (7%) had MET amplification in the A group. Afatinib dosage was reduced in 15 patients to 30 mg; T790M was not correlated with the dose reduction, being detectable in 6 patients who needed the reduction and in 8 who received the full standard dose (p = 0.54). Conclusions In conclusion, even though gefitinib, erlotinib, and afatinib belong to the same class of EGFR-TKIs, differences in the appearance of resistance mutation are demonstrated in the present study and this finding may have implications in the choice of 2nd-line treatment. Legal entity responsible for the study The authors. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Liquid biopsy has emerged as an alternative source of nucleic acids for the management of Epidermal Growth Factor Receptor (EGFR)-mutant non-Small Cell Lung Cancer (NSCLC). The use of circulating cell-free DNA (cfDNA) has been recently introduced in clinical practice, resulting in the improvement of the identification of druggable EGFR mutations for the diagnosis and monitoring of response to targeted therapy. EGFR-dependent (T790M and C797S mutations) and independent (Mesenchymal Epithelial Transition [MET] gene amplification, Kirsten Rat Sarcoma [KRAS], Phosphatidyl-Inositol 4,5-bisphosphate 3-Kinase Catalytic subunit Alpha isoform [PI3KCA], and RAF murine sarcoma viral oncogene homolog B1 [BRAF] gene mutations) mechanisms of resistance to EGFR tyrosine kinase inhibitors (TKIs) have been evaluated in plasma samples from NSCLC patients using highly sensitive methods (i.e., digital droplet PCR, Next Generation Sequencing), allowing for the switch to other therapies. Therefore, liquid biopsy is a non-invasive method able to detect the molecular dynamic changes that occur under the pressure of treatment, and to capture tumor heterogeneity more efficiently than is allowed by tissue biopsy. This review addresses how liquid biopsy may be used to guide the choice of treatment strategy in EGFR-mutant NSCLC.

e14054 Background: Immunotherapy has revolutionized the treatment of NSCLC. However, response rate is variable, with a substantial failure rate. Thus, the identification of predictive biomarkers of response to immunotherapy is an area of great interest. Methods: Patients with locally advanced or metastatic NSCLC treated with nivolumab or pembrolizumab were enrolled. Disease response was defined following RECIST criteria (v. 1.1). Four ml of plasma were collected for the analysis of exosomal mRNA levels of PD-L1 (e-PD-L1) and IFN-γ (e-IFN-γ) at baseline and at the time of first radiological assessment. Exosome isolation and mRNA extraction was obtained by the exoRNeasy kit (Qiagen, Valencia, CA). e-PD-L1 and e-IFN-γ were evaluated by the QX100 ddPCR (Bio-Rad, Hercules, CA) and expressed as allele frequency (%). Chest computed tomography (CT) scan at baseline was used for the radiomic analysis. Tumor segmentation was performed on DICOM-formed images taken from the picture archiving, and the regions of interest were delineated manually and analyzed using the QUIBIM SL software. Survival was calculated stratifying patients based on e-PD-L1 and e-IFN-γ median values. Results: Nivolumab was given to 17 patients as 2nd line and to 8 subjects as further line of treatment, while 13 patients received 1st line pembrolizumab. Median PFS was 11 vs 16.2 months (mos) in patients with baseline e-PD-L1 of < 0.3% vs ≥0.3%, respectively (p = 0.16). e-PD-L1 significantly increased in disease progression (PD) vs partial response (PR) and disease stabilization (SD) (p = 0.01) after 2 mos of treatment. In patients with e-IFN-γ ≥4.1% vs < 4.1% at baseline, median PFS was 5.6 mos vs not reached, respectively (p = 0.003). The multiparametric radiomic analysis identified the Cluster Prominence Value (CPV, p = 0.012) and the Cluster Shade Value (CSV, p = 0.034) as significantly correlated with treatment outcome. Moreover, the D2d parameter and e-IFN-γ were inversely correlated (p < 0,0001). CPV and D2d reflect the intra-tumor architecture, including necrosis, cell proliferation, and angiogenesis. Conclusions: Liquid biopsy data correlate with radiomic parameters and predict response to immunotherapy.

8565 Background: Thymomas but not thymic carcinomas can benefit of cytoreductive surgery even if a complete resection is not achievable. Surgical resection of pleural metastases, the most common site of progression, can be performed in selected patients. We evaluated the outcome of stereotactic body radiation therapy (SBRT) for treatment of pleural metastases in patients’ not eligible for surgery. Methods: We retrospectively identified 22 patients treated with SBRT for pleural metastases between 2004 and 2019. According to RECIST criteria, time to local failure and progression free survival (PFS) were calculated using Kaplan-Meier estimation. Results: Twelve of the 22 patients were male. The median age was 40 years (range 29-73). There were 1 A, 3 AB, 3 B1, 3 B2, 3 B2/B3 and 9 B3 thymomas. The Masaoka stage at diagnosis was IIA in 2, IIB in 7, III in 5, IVA in 7 and IVB in 1 patient. Pleural metastases and primary tumor were synchronous in 8 patients. Thymectomy was performed in 21 patients. Seven patients received pre-operative chemotherapy and 12 post-operative radiotherapy. One patient received chemotherapy and radiotherapy after a macroscopically incomplete thymectomy. Five patients had a single pleural metastatic site and 17 presented multiple localizations. Sixteen patients received SBRT on multiple sites of pleural metastases. At the time of the analysis a patient received SBRT exclusively on one of 3 pleural metastases. The median dose of radiation was 30Gy (range 25-40) given in 3 fractions. Ten patients experienced a progression of treated lesions with a median time to local failure of 25.5 months (95%CI 20.9-30.1). The median PFS was 20.4 months (95%CI 10.7-30). There were not significant differences in PFS between patients diagnosed with synchronous and metachronous metastases (p=0.477), across those treated with chemotherapy or naive (p=0.189) and between those who received or not a previous surgical resection of the pleural metastases (p=0.871). Conclusions: SBRT of pleural metastases is feasible and offer an interesting local control of diseases. The impact of this treatment on patients’ survival is hardly predictable because of the heterogenous clinical behavior of thymomas.

The WHO-classification was shown to be an independent prognostic marker in some but not all retrospective studies possibly due to lack of reproducibility. We investigated the reproducibility of the WHO-classification and its prognostic implication using a large series of resected thymomas.Four independent pathologists histologically classified a surgical series of 129 thymic tumors in a blinded fashion. Fleiss' kappa-coefficient was used to assess the pathologists' overall agreement, and Cohen-Kappa to assess the agreement between two observers. Disease-related-survival (DRS) and progression-free-survival (PFS) curves were generated by Kaplan-Meier method and compared by log-rank test.In 63/129 (48.8%) cases there was a complete agreement; in 43/129 (33.3%) cases 3/4 pathological diagnoses were identical; in 15/129 (11.6%) cases the diagnoses were identical by pair; in 8/129 (6.2%) cases three different pathological diagnoses were on record. The Kappa-correlation coefficient was only moderate (0.53). A following web review carried out on the 23 cases with at least two different diagnoses reached a complete consensus. The histotype showed a statistically significant impact on PFS and DRS in the classification provided by only two pathologists.In this study, the agreement on WHO classification of thymomas was only moderate and this impacted on patients management. Web consensus conference on the diagnosis, more stringent diagnostic criteria or the adoption of referral diagnostic centres may substantially reduce discrepancies.

// Iacopo Petrini 1 , Serena Barachini 2 , Vittoria Carnicelli 3 , Sara Galimberti 2 , Letizia Modeo 4 , Roberto Boni 4 , Martina Sollini 5 , Paola Anna Erba 4 1 General Pathology, Department of Translational Research and New Technology in Medicine, University of Pisa, Pisa, Italy 2 Laboratory of Hematology, Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy 3 Biochemistry, Department of Translational Research and New Technology in Medicine, University of Pisa, Pisa, Italy 4 Nuclear Medicine, Department of Translational Research and New Technology in Medicine, University of Pisa, Pisa, Italy 5 Department of Biomedical Sciences, Humanitas University, Rozzano, Milan, Italy Correspondence to: Iacopo Petrini, email: iacopo.petrini@unipi.it Keywords: fibronectin, endothelial-to-mesenchymal transition, ED-B, prostate cancer, TGF-β Received: July 25, 2016 Accepted: November 09, 2016 Published: November 25, 2016 ABSTRACT Fibronectin is a component of the extracellular matrix that links collagen fibers to integrins on the cell’s surface. The splicing isoforms, containing the ED-B domain, are not expressed in adult tissues but only in tumor stroma or during embryonic development. Fibroblasts and endothelial cells express ED-B fibronectin during angiogenesis. Also cancer cells can synthetize ED-B fibronectin, but its function in tumor growth needs to be further elucidated. We evaluated the expression of ED-B fibronectin in prostate cancer cell lines: PC3 and DU145. Using TGF-β, we induced epithelial to mesenchymal transition in culture and observed an increase of ED-B fibronectin expression. Thereafter, we evaluated the expression of ED-B fibronectin in multipotent mesangiogenic progenitor cells, and in mesenchymal stromal cells. The expression of ED-B fibronectin was much higher in mesenchymal than prostate cancer cells even after the epithelial to mesenchymal transition. Epithelial to mesenchymal transition is a key step for tumor progression contributing to the metastatic spread. Therefore, circulating cancer cells could seed into the metastatic niche taking advantage from the ED-B fibronectin that secrete their own.

Mesangiogenic progenitor cells (MPCs) are a very peculiar population of cells present in the human adult bone marrow, only recently discovered and characterized. Owing to their differentiation potential, MPCs can be considered progenitors for mesenchymal stromal cells (MSCs), and for this reason they potentially represent a promising cell population to apply for skeletal tissue regeneration applications. Here, we evaluate the effects of surface nanotopography on MPCs, considering the possibility that this specific physical stimulus alone can trigger MPC differentiation toward the mesenchymal lineage. In particular, we exploit nanogratings to deliver a mechanical, directional stimulus by contact interaction to promote cell morphological polarization and stretching. Following this interaction, we study the MPC-MSC transition by i. analyzing the change in cell morphotype by immunostaining of the key cell-adhesion structures and confocal fluorescence microscopy, and ii. quantifying the expression of cell-phenotype characterizing markers by flow cytometry. We demonstrate that the MPC mesengenic differentiation can be induced by the solely interaction with the NGs, in absence of any other external, chemical stimulus. This aspect is of particular interest in the case of multipotent progenitors as MPCs that, retaining both mesengenic and angiogenic potential, possess a high clinical appeal.

Mesangiogenic Progenitor Cells (MPCs) are human bone marrow-derived multipotent cells, isolated in vitro under selective culture conditions and shown to retain both mesengenic and angiogenic potential. MPCs also co-isolated with multipotent stromal cells (MSCs) when bone marrow primary cultures were set up for clinical applications, using human serum (HS) in place of fetal bovine serum (FBS). MPC culture purity (over 95%) is strictly dependent on HS supplementation with significant batch-to-batch variability. In the present paper we screened different sources of commercially available pooled human AB type serum (PhABS) for their ability to promote MPC production under selective culture conditions. As the majority of “contaminating” cells in MPC cultures were represented by MSC-like cells, we hypothesized a role by differentiating agents present in the sera. Therefore, we tested a number of growth factors (hGF) and found that higher concentrations of FGF-2, EGF, PDGF-AB, and VEGF-A as well as lower concentration of IGF-1 give sub-optimal MPC recovery. Gene expression analysis of hGF receptors was also carried out both in MSCs and MPCs, suggesting that FGF-2, EGF, and PDGF-AB could act promoting MSC proliferation, while VEGF-A contribute to MSC-like cell contamination, triggering MPC differentiation. Here we demonstrated that managing hGF contents, together with applying specific receptors inhibitors (Erlotinib-HCl and Nintedanib), could significantly mitigate the batch-to-batch variability related to serum supplementation. These data represent a fundamental milestone in view of manufacturing MPC-based medicinal products.

e20526Background: Crizotinib is an effective treatment for patients with ALK rearranged NSCLC but, unfortunately, patients progress with a median of 9-10 months. Resistance could be acquired throug...

Purpose: The aim of this study was to evaluate the activity of the combination of 5-fluorouracil/folinic acid and irinotecan (FOLFIRI) as third-line chemotherapy (CT) in metastatic gastric cancer (mGC) patients pretreated with platinum derivatives, fluoropyrimidines, and taxanes. Methods: We prospectively collected data of mGC patients treated with third-line FOLFIRI at our institution from 2009 to 2014. Eligible patients should be treated with a fluoropyrimidine-platinum first-line CT and a subsequent taxane-based second-line CT. FOLFIRI consisted of irinotecan 180 mg/m2 and leucovorin 200 mg/m2, followed by 5-fluorouracil 2,800 mg/m2 (administered as 48-hour i.v. continuous infusion from day 1 to 3), with cycles repeated every 2 weeks. Response rate (RR) was evaluated according to RECIST version 1.0, while progression-free (PFS) and overall survival (OS) were estimated using the Kaplan-Meier method. Results: A total of 33 patients were included. The majority (97%) had good performance status (0-1 according to ECOG), while median PFS after first-line and second-line CT was 5.2 and 4.4 months, respectively. Two patients experienced an objective response (RR: 6%), while 14 patients achieved disease stabilization (disease control rate: 42%). Median PFS and OS from the start of third-line CT were 3.3 and 7.5 months, respectively. Hematological and nonhematological grade 3-4 toxicities were uncommon and included neutropenia (6.1%), diarrhea (9.1%), vomiting (3%), and asthenia (3%). Febrile neutropenia was not reported. Conclusions: Third-line CT with FOLFIRI may be an option in heavily pretreated mGC patients with preserved performance status and organ function. This regimen has a favorable safety profile, and signs of activity have been observed after standard first- and second-line CT.

Introduction Thymic carcinomas represent the most aggressive histotype of thymic epithelial tumors (TETs). The 2004 World Health Organization classification has assigned a subgroup of thymic carcinomas as t(15;19) carcinomas based on the presence of t(15;19), a translocation found in poorly differentiated and highly aggressive NUT midline carcinomas. These tumors are characterized byrearrangement of the NUT (nuclear protein in testis) gene on chromosome 15q14, which in most cases fuses to the bromodomain containing 4 ( BRD 4) gene on chromosome 19 p13.1 through reciprocal t(15;19) translocation, resulting in constitutive BRD4-NUT fusion protein expression. To our knowledge, NUT translocation has been reported only in four thymic carcinomas. Due to the rarity of TETs, the prevalence of NUT rearrangement in TETs has however never been systematically explored. Methods Formalin-fixed paraffin-embedded samples of histologically confirmed TETs were evaluated for NUT expression and rearrangement by immunohistochemistry and fluorescence in situ hybridization, respectively. Results A series of 148 TETs (37 carcinomas and 111 thymomas) were examined for NUT expression and rearrangement. Only one thymic carcinoma (2.7% of thymic carcinomas or 0.68% of TETs) was found positive for NUT expression and rearrangement. Conclusions Rearrangement of NUT is infrequent in TETs. We propose that caution should be taken to distinguish t(15;19) thymic carcinoma from other mediastinal carcinomas, as NUT midline carcinomas are often associated with dreadful prognosis or overt lethality.

Purposes: Pancreatic cancer is the fourth leading cause of cancer-related death, and studies on the clinical relevance of its genomic imbalances are warranted. Experimental Design: Recurrent copy number alterations of cytobands and genes were analyzed by array comparative genomic hybridization (aCGH) in 44 resected pancreatic cancer specimens. Prognostic markers identified by aCGH were validated by PCR gene copy number assay in an independent validation cohort of 61 resected pancreatic cancers. The functions of gene identified were evaluated by proliferation, cell cycle, and migration assays in pancreatic cancer cells. Results: We showed recurrent copy number gains and losses in the first cohort. Loss of 18q22.3 was significantly associated with short-term overall survival in the first cohort (P = 0.019). This cytoband includes the carboxypeptidase of glutamate-like (CPGL) gene. CPGL gene deletion was associated with shorter overall survival in the validation cohort (P = 0.003). CPGL deletion and mutations of TP53 or Kras seem to be independent events. A Cox model analysis of the two cohorts combined showed that loss of 18q22.3/deletion of the CPGL gene was an independent poor prognostic factor for overall survival (HR = 2.72, P = 0.0007). Reconstitution of CPGL or its splicing variant CPGL-B into CPGL-negative pancreatic cancer cells attenuated cell growth, migration, and induced G1 accumulation. Conclusion: Loss of 18q22.3/deletion of the CPGL gene is a poor prognostic marker in resected pancreatic cancer, and functional studies suggest the CPGL gene as growth suppressor gene in pancreatic cancer. Clin Cancer Res; 18(2); 524–33. ©2011 AACR.

Eur J Clin Invest 2011; 41 (11): 1186–1194 Abstract Background Melanoma is an immunogenic tumour but, despite the wide range of immunotherapies tested, only few promising results have been reported to date. Both in vitro and in xenograft models, γδ lymphocyte-mediated cytotoxicity against melanoma cells has been reported. IL-2/zoledronate treatment can expand γδ cells in vitro and in animal models. This could represent an immunotherapeutic strategy against melanoma. To evaluate the feasibility of this approach, we studied γδ lymphocyte phenotype from patients with melanoma, their ability to be expanded by IL-2/zoledronate and their cytotoxic activity against SK-MEL-30 cell line. Materials and methods Peripheral blood samples were collected from 30 patients with melanoma and 10 healthy donors. Percentage of γδ lymphocytes and CD45RO+CD27+, CD45RA+CD27−, CD57+, Vγ9Vδ2 subpopulations were evaluated by flow cytometry. IL-2/zoledronate γδ cell expansion rate and their cytotoxicity against SK-MEL-30 cell line were studied. Results A percentage decrease in circulating Vγ9Vδ2 and an increase in CD45RA+CD27− and CD57+ γδ lymphocytes were observed in melanoma. IL-2/zoledronate expansion rate did not differ between controls and patients with melanoma but cytotoxicity against SK-MEL-30 appeared reduced. Conclusions Our results show that γδ cell function is impaired in patients with advanced melanoma and suggest a possible role in tumour progression.

Background Overexpression of c-kit, a tyrosine kinase receptor protein encoded by the protooncogene kit, has been previously reported in thymic epithelial tumors and in other neoplasms such as gastrointestinal stromal tumors, myeloproliferative disorders, melanoma, and seminoma. Mutations in the kit gene have been related to response to imatinib in gastrointestinal stromal tumor and one case report of thymic carcinoma. We studied expression of c-kit in a large retrospective series of thymic epithelial malignancies and sequenced the whole gene in a subset of patients. Methods Thymic epithelial tumors from 120 patients (13 thymic carcinomas and 107 thymomas) were examined. Immunohistochemical staining with an antic-kit polyclonal antibody was performed on a tissue microarray. Mutation analyses of exons 1 to 20 were conducted by direct DNA sequencing of polymerase chain reaction products in eight thymic carcinomas, five thymomas, and one thymic carcinoma cell line. Results The percentage of c-kit positive cells was significantly higher in thymic carcinoma (46%) than in thymoma (4%). Decreased disease-related survival and progression-free survival were observed in c-kit positive tumors. No mutations were detected. Conclusion c-kit expression is strongly but not exclusively related to thymic carcinoma histotype, and it is of prognostic value. Mutations are very rare.

210 Background: The anti-PD-1 monoclonal antibodies (moAb) nivolumab and pembrolizumab have improved the survival of melanoma and non-small cell lung cancer (NSCLC) patients. However, treatment selection is based on tumor PD-L1 expression by immuno-histochemistry and no specific approaches are available to monitor treatment response. The aim of this study was to investigate the association between PD-L1 mRNA levels in plasma-derived exosomes and response to nivolumab and pembrolizumab in patients affected by melanoma (n = 18) and NSCLC (n = 8). Methods: Blood (6 ml) was obtained at 1) baseline (before initiation of anti-PD1 moAb or at the time of last available radiological evaluation of disease response) and 2) after two months of treatment (at the time of first response evaluation or disease re-assessment). Exosomes were extracted from plasma and PD-L1 mRNA expression was measured by digital PCR and expressed as copies/ml. Results: Overall, the number of copies of mRNA PD-L1/ml plasma varied according to tumor response; in particular, an increase was found in patients with PD and a decrease was observed in patients who achieved a CR/PR. The mean±SEM values of PD-L1 in patients responding to treatment (CR+PR) were 830.4±231.3 and 242.5±82.5 copies/ml (baseline vs. 2 months, p = 0.016), respectively. In patients with stable disease the mean±SEM values were as expected 298.8±97.2 vs. 247.5±29.8 copies/ml (p = 0.586), while in progressive disease PD-L1 expression levels were 204.0±68.8 vs. 416.0±87.8 copies/ml (p = 0.001), respectively. Conclusions: In the present study, we demonstrate for the first time that changes in exosomal PD-L1 expression occur in melanoma and NSCLC patients treated with nivolumab or pembrolizumab and may correlate with the radiological tumor response. This proof-of-concept study demonstrates the feasibility of detecting PD-L1 in plasma and its relationship with response to treatment.