Genomic Aberrations of Thymic Epithelial Tumors

Thymic epithelial tumors (TETs) are rare neoplasms classified in thymoma and thymic carcinoma. According to the 2004 WHO classification, thymomas are further subdivided into five subcategories (A, AB, B1, B2, B3), depending on cancer cell shape, degree of atypia and number of intratumoral thymocytes. Surgical resection is recommended for localized tumors, but systemic therapy is the choice for metastatic and not resectable neoplasms. Usually, TETs respond to chemotherapeutic drugs but their effect lasts only for a limited time, thus, new targeted-molecules are currently under evaluation. However, the efficacy of biologic therapies in TET is scant, to date. The main limitation is the lack of a robust rationale to target specific molecules, mainly because the aberrations driving TET growth are obscure. The aim of this project is to clarify the molecular aberration driving the TET growth. Firstly, the frequency of aberrations described in TET case-reports has been evaluated. The presence of BRD4-NUT fusion gene was tested in 148 TETs, but resulted in a rare event, observed in only one thymic carcinoma. Similarly, no KIT mutations were detected in the 13 TETs sequenced. Reviewing literature data, KIT was mutated in only 9% of thymic carcinomas. Therefore, a systematic screening for new genomic aberrations in TET was started using whole genome sequencing to evaluate the presence of mutations, translocation and copy number (CN) aberrations in a B3 thymoma. The translocation t(11;X)(q14.2;q25), the presence of 7 single nucleotide mutations and 1 INDEL were observed together with CN gain of chromosome 1q, 5, 7, X and CN loss of 3p, 6, 13 and part of chromosome 11q. In order to identify which one of these genomic aberrations was recurrent and therefore candidate to drive TET growth, whole exome sequencing and array comparative genomic hybridization (CGH) were adopted. 26 tumors were screened by array CGH and 5 selected to be sequenced using tumor and normal DNA. The identified mutations were confirmed and tested for expression using transcriptome sequencing. The sequencing demonstrated a remarkably heterogeneous pattern of mutations. Recurrent were the amplification of BCL2, observed in 4 thymic carcinomas, and the inactivation of CDKN2A caused by a frame-shift INDEL and 2 CN losses. Because BCL2 and CDKN2A are more likely to be mutated by CN aberrations, 59 formalin fixed paraffin embedded tumors were evaluated using array CGH. Data confirmed the presence of focal CN loss of CDKN2A and amplification of BCL2 as recurrent events in TETs and suggest a possible prognostic role for these CN aberrations. Immunohistochemistry confirmed the lack of P16INK4 expression in tumors with CDKN2A CN loss and showed a poor prognosis for patients with negative staining. P16INK4 is a well-known tumor suppressor gene involved in the control of cell cycle through the binding of CDK4 and 6. The role of anti apoptotic BCL2 family proteins was elucidated using 3 TET cell lines because these molecules could represent targets for therapy. A siRNA approach demonstrated that the expression of BCL2 and MCL1, two anti-apoptotic proteins, was necessary for TET cell line growth. GX15-070, a BH3 mimetic inhibitor of anti-apoptotic BCL2 family members, reduced proliferation of TET cell-lines in vitro, through the induction of autophagy and necroptosis. Gx15-070 inhibited tumor growth in the xenograft model established using TY82 thymic carcinoma cell line; therefore this drug may be tested for patients’ treatment

Editorial: Primary and acquired resistance in lung cancer

No abstract availabl

Hypertermic Intrathoracic Chemotherapy (HITHOC) for thymoma: a narrative review on indications and results

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

ED-B fibronectin expression is a marker of epithelial-mesenchymal transition in translational oncology

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

ED-B-Containing Isoform of Fibronectin in Tumor Microenvironment of Thymomas:A Target for a Theragnostic Approach

Simple Summary The extra-domain B fibronectin (ED-B FN) is highly expressed in thymic epithelial tumors (TETs), as demonstrated by in vivo targeting using 131I-labeled L19 small immunoprotein (131I-L19-SIP) and immunohistochemistry with a predominant expression by stromal cells of a thymoma microenvironment rather than epithelial cells. Such high expression derived from the induction of stromal cells shifts FN production to the ED-B subtype. Our results suggest that Radretumab radioimmunotherapy (R-RIT) inefficacy is not related to low TET ED-B expression but to multifactorial aspects including patients' inherent characteristics, the pattern expression of the target, the biological characteristics of the tumor, and the format of the target agent, which contribute to the resistance of tumor cells to treatment. Aim: to exploit tissue-specific interactions among thymic epithelial tumor (TETs) cells and extra-domain B fibronectin (ED-B FN). Material and methods: The stromal pattern of ED-B FN expression was investigated through tumor specimen collection and molecular profiling in 11 patients with recurrent TETs enrolled in prospective theragnostic phase I/II trials with Radretumab, an ED-B FN specific recombinant human antibody. Radretumab radioimmunotherapy (R-RIT) was offered to patients who exhibited the target expression. Experiments included immunochemical analysis (ICH), cell cultures, immunophenotypic analysis, Western blot, slot-blot assay, and quantitative RT-PCR of two primary thymoma cultures we obtained from patients' samples and in the Ty82 cell line. Results: The in vivo scintigraphic demonstration of ED-B FN expression resulted in R-RIT eligibility in 8/11 patients, of which seven were treated. The best observed response was disease stabilization (n = 5/7) with a duration of 4.3 months (range 3-5 months). IHC data confirmed high ED-B FN expression in the peripherical microenvironment rather than in the center of the tumor, which was more abundant in B3 thymomas. Further, there was a predominant expression of ED-B FN by the stromal cells of the thymoma microenvironment rather than the epithelial cells. Conclusions: Our data support the hypothesis that thymomas induce stromal cells to shift FN production to the ED-B subtype, likely representing a favorable hallmark for tumor progression and metastasis. Collectively, results derived from clinical experience and molecular insights of the in vitro experiments suggested that R-RIT inefficacy is unlikely related to low target expression in TET, being the mechanism of R-RIT resistance eventually related to patients' susceptibility (i.e., inherent characteristics), the pattern expression of the target (i.e., at periphery), the biological characteristics of the tumor (i.e., aggressive and resistant phenotypes), and/or to format of the target agent (i.e., 131I-L19-SIP)

Implications of KRAS mutations in acquired resistance to treatment in NSCLC

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

Nanotopography Induced Human Bone Marrow Mesangiogenic Progenitor Cells (MPCs) to Mesenchymal Stromal Cells (MSCs) Transition

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

Myelodysplastic syndromes: advantages of a combined cytogenetic and molecular diagnostic workup

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)

Contribution of KRAS mutations and c.2369C > T (p.T790M) EGFR to acquired resistance to EGFR-TKIs in EGFR mutant NSCLC: a study on circulating tumor DNA

INTRODUCTION: KRAS oncogene mutations (MUTKRAS) 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 (MUTEGFR). However, inhibition of EGFR may select resistant cells displaying alternative signaling, i.e., KRAS, or restoration of EGFR activity due to additional MUTEGFR, i.e., the c.2369C > T (p.T790MEGFR). AIM: The aim of this study was to investigate the appearance of MUTKRAS 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 MUTKRAS and p.T790MEGFR mutations in 33 advanced NSCLC patients progressing after an EGFR-TKI. RESULTS: p.T790MEGFR was detected in 11 (33.3%) patients, MUTKRAS at codon 12 in 3 (9.1%) while both p.T790MEGFR and MUTKRAS codon 12 were found in 13 (39.4%) patients. Six patients (18.2%) were KRAS wild-type (WTKRAS) and negative for p.T790MEGFR. In 8 subjects paired tumor re-biopsy/plasma samples were available; the percent concordance of tissue/plasma was 62.5% for p.T790MEGFR and 37.5% for MUTKRAS. The analysis of time to progression (TTP) and overall survival (OS) in WTKRAS vs. MUTKRAS were not statistically different, even if there was a better survival with WTKRAS vs. MUTKRAS, 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: MUTKRAS 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

KRAS has a role in acquired resistance to EGFR-TKIs in NSCLC: an analysis on circulating tumor DNA

Abstract not availabl