Specific Chromosomal Translocations Research Articles

Extraskeletal Myxoid Chondrosarcoma with Molecularly Confirmed Diagnosis: A Multicenter Retrospective Study Within the Italian Sarcoma Group.

Extraskeletal myxoid chondrosarcoma (EMC) is a rare sarcoma of uncertain origin, marked by specific chromosomal translocations involving the NR4A3 gene, and usually characterized by an indolent course. Surgery (with or without radiotherapy) is the treatment of choice in localized disease. The treatment for advanced disease remains uncertain. In order to better evaluate prognostic factors and outcome, a retrospective pooled analysis of patients with EMC treated at three Italian Sarcoma Group (ISG) referral centers was carried out. All patients with localized EMC surgically treated from 1989 to 2016 were identified. Diagnosis was centrally reviewed according to WHO 2013. Only patients with NR4A3 rearrangement were included. Sixty-seven patients were identified: 13 (20%) female, 54 (80%) male. Median age was 56years (range 18-84). Numbers and type of translocation were: 50 (80%) NR4A3-EWS, 10 (16%) NR4A3-TAF15, 1 (2%) NR4A3-TCF12, and 1 (2%) NR4A3-TFG. Median follow-up was 55months (range 2-312). Five- and ten-year overall survival rates were 94% (86-100 95%CI) and 84% (69-98 95%CI). Thirty-five (52%) patients relapsed: 9 had local recurrence (LR) and 26 had distant metastasis (5 with concomitant LR). The 5- and 10-year disease-free survival rates (DFS) were 51% (38-65 95%CI) and 20% (7-33 95%CI). Size of the primary tumor was significantly related to distant metastasis-free survival (DMFS) (p = 0.004). Patients carrying the NR4A3-EWS translocation had a trend in favor of better DFS (p = 0.08) and DMFS (p = 0.09) compared with the patients with NR4A3-TAF15. Prolonged survival can be expected in patients with EMC, in spite of a high rate of recurrence. Size is significantly associated with distant relapse. The type of NR4A3 translocation could influence outcome.

IgH translocation with undefined partners is associated with superior outcome in multiple myeloma patients.

In multiple myeloma (MM), impact of specific chromosomal translocations involving IgH (14q21 locus, including t(4;14), t(11;14), and t(14;16)) has been explored extensively. However, over 15% MM patients harboring IgH translocation with undefined partners have long been ignored. A prospective non-randomized cohort study with a total of 715 newly-diagnosed MM cases was conducted, 13.6% of whom were t(14;undefined) positive. The whole cohort was divided into four groups: no IgH split (47.7%); t(14;undefined) (13.6%); t(11;14) (17.6%); and t(4;14) or t(14;16) group (21.1%). Median OS for the four groups was 84.2, not reached (NR), 58.7, and 44.2months, respectively, with P values for t(14;undefined) vs no IgH split, t(11;14), and t(4;14)/t(14;16) groups of 0.197, 0.022, and 0.001, respectively. In bortezomib-based group, the survival advantage gained by t(14;undefined) group was much more significant compared to t(11;14) and t(4;14)/t(14;16) groups. Importantly, t(14;undefined) turned out to be an independent predictive factor for longer OS of MM patients in multivariate analysis, especially in the context of bortezomib treatment. Similar results were also observed in the PUMCH external validation cohort. Collectively, our data confirmed and externally validated the favorable prognosis of the t(14;undefined) groups, especially in the era of novel agents.

Jak-Stat Signaling Pathway Related Gene Expressions and Blood Biochemical indicators in Acute Promyelocytic Leukemia

Jak-Stat Signaling Pathway Related Gene Expressions and Blood Biochemical indicators in Acute Promyelocytic Leukemia

Fenretinide induces a new form of dynamin-dependent cell death in pediatric sarcoma

Alveolar rhabdomyosarcoma (aRMS) is a highly malicious childhood malignancy characterized by specific chromosomal translocations mostly encoding the oncogenic transcription factor PAX3-FOXO1 and therefore also referred to as fusion-positive RMS (FP-RMS). Previously, we have identified fenretinide (retinoic acid p-hydroxyanilide) to affect PAX3-FOXO1 expression levels as well as FP-RMS cell viability. Here, we characterize the mode of action of fenretinide in more detail. First, we demonstrate that fenretinide-induced generation of reactive oxygen species (ROS) depends on complex II of the mitochondrial respiratory chain, since ROS scavenging as well as complexing of iron completely abolished cell death. Second, we co-treated cells with a range of pharmacological inhibitors of specific cell death pathways including z-vad (apoptosis), necrostatin-1 (necroptosis), 3-methyladenine (3-MA) (autophagy), and ferrostatin-1 (ferroptosis) together with fenretinide. Surprisingly, none of these inhibitors was able to prevent cell death. Also genetic depletion of key players in the apoptotic and necroptotic pathway (BAK, BAX, and RIPK1) confirmed the pharmacological data. Interestingly however, electron microscopy of fenretinide-treated cells revealed an excessive accumulation of cytoplasmic vacuoles, which were distinct from autophagosomes. Further flow cytometry and fluorescence microscopy experiments suggested a hyperstimulation of macropinocytosis, leading to an accumulation of enlarged early and late endosomes. Surprisingly, pharmacological inhibition as well as genetic depletion of large dynamin GTPases completely abolished fenretinide-induced vesicle formation and subsequent cell death, suggesting a new form of dynamin-dependent programmed cell death. Taken together, our data identify a new form of cell death mediated through the production of ROS by fenretinide treatment, highlighting the value of this compound for treatment of sarcoma patients including FP-RMS.

Pregnancy Complicated with Acute Promyelocytic Leukemia: A Case Report and a Literature Review

Acute promyelocytic leukemia (APL) is a special subgroup of acute myeloid leukemia (AML). About 95% of the patients have specific chromosome translocation t (15 ≤ 17) (q22, q12). APL progresses rapidly and is characterized by extensive and severe bleeding and disseminated intravascular coagulation (DIC). Patients may present with severe clinical manifestation which is often caused by the occurrence of DIC in the early stages of the disease. Pregnancy complicated with APL is rare in the clinical setting. They have many complications, high mortality and are difficult to manage clinically. This case report describes a case of a primary pregnancy complicated with APL, in order to better understand how to manage such complex cases.

Application of CRISPR/Cas9-Mediated Genome Editing in Leishmania.

CRISPR-Cas9 is an RNA guided endonuclease derived from the bacterium Streptococcus pyogenes. Due to its simplicity, versatility, and high efficiency, it has been widely used for genome editing in a variety of organisms including the protozoan parasite Leishmania, the causative agent of human leishmaniasis. Compared to the traditional homologous recombination gene targeting method, CRISPR-Cas9 has been shown to be a more efficient method to delete or disrupt Leishmania genes, generate point mutations, and add tags to endogenous genes. Notably, the stable CRISPR expression systems were shown to delete multicopy family Leishmania genes and genes present in multiploid chromosomes, identify essential Leishmania genes, and create specific chromosome translocations. In this chapter, we describe detailed procedures on using the stable CRISPR expression system for genome editing in Leishmania. These procedures include CRISPR targeting site selection, gRNA design, cloning single and double gRNA coding sequences into the Leishmania CRISPR vector pLdCN, oligonucleotide donor and drug resistance selection donor design, Leishmania cell transfection, screening, and isolation of CRISPR-edited mutants. As the principles of gene editing are generally similar, many of these procedures could also apply to the transient Leishmania CRISPR systems described by other labs.

Differential diagnosis of myxoid soft tissue tumors. Experience in the Clinical University Hospital of Valencia

Soft tissue tumors with myxoid components are often a diagnostic challenge for the pathologist. We retrospectively reviewed 41 cases of soft tissue tumors with myxoid components diagnosed in our center over a five-year period. The most frequent diagnoses were myxofibrosarcoma and myxoid liposarcoma, followed by low-grade fibromyxoid sarcoma, low-grade fibromyxoid tumor and myxoid neurofibroma. Other diagnoses included were extraskeletal myxoid chondrosarcoma, myxoinflammatory fibroblastic sarcoma, low-grade myxoliposarcoma, myofibrosarcoma, fibromatosis, solitary fibrous tumor, non-ossifying variant of ossifying fibromyxoid tumor and ancient neurinoma with myxoid degeneration. Immunohistochemical and molecular biology studies contributed significantly to the diagnosis. We highlight the importance of immunohistochemistry for MUC4 in the diagnosis of low-grade fibromyxoid sarcoma, and underscore the need for molecular studies in selected cases. Furthermore, several myxoid neoplasms present specific chromosomal translocations, therefore molecular biology studies to detect fusion genes are usually essential for the diagnosis. When the characteristics of the sample are not adequate for molecular biology, or no specific alterations are described, an in-depth knowledge of the histology of these lesions is still necessary to decide the most accurate diagnosis.

Detecting MYB and MYBL1 fusion genes in tracheobronchial adenoid cystic carcinoma by targeted RNA-sequencing

Primary tracheobronchial adenoid cystic carcinoma is rare, accounting for less than 1% of all lung tumors. Many adenoid cystic carcinomas have been reported to have a specific chromosome translocation t(6;9)/MYB-NFIB. More recently, t(8;9)/MYBL1-NFIB gene fusion was reported in salivary gland adenoid cystic carcinomas which lacked a t(6;9)/MYB-NFIB. Two prior studies showed t(6;9)/MYB-NFIB in tracheobronchial adenoid cystic carcinoma; however, only rare cases of MYBL1 rearrangement have been reported in this carcinoma. In this study, we used targeted RNA sequencing to investigate fusion genes in tracheobronchial adenoid cystic carcinoma at our institution. Fusions of either MYB or MYBL1 genes were detected in 7 of 7 carcinomas. Three cases had MYB-NFIB, and 3 had MYBL1-NFIB. The remaining case showed a rare MYBL1-RAD51B fusion. These findings suggest that rearrangement involving MYB or MYBL1 is a hallmark of tracheobronchial adenoid cystic carcinoma.

EP1.17-12 Multimodal Aggressive Managment for Askin' Tumor: Results and Prognosis About 3 Cases

Peripheral primitive neuroectodermal tumor (pPNET) is a highly aggressive small round cell tumor belonging to PNET/Ewing sarcoma family. Askin”s tumor is a rare entity of neuroectodermal pathology that starts from the soft parts of the chest wall. It is observed with predilection in the young subject. Herein ; We discuss follow up of three cases after multimodal therapy. We report the case of three patients aged respectively 18; 20; 60 years; whose main symptom in consultation is the appearance of a thoracic parietal mass gradually increasing in size and painful. The general condition was completely preserved in all three patients; and the clinical examination is without abnormalities apart from clubbing in the person aged on a past of smoking. Imaging revealed the presence of a thoracic parietal tumor, the largest of which was 15 X 10X 6 cm without signs of locoregional or distant extension with weak contrast. The histological diagnosis was made on a transparietal mass biopsy with a conclusive immuohistochemical study in favor of a PNET tumor (Askin'tumor) Management was multimodal with 6 aggressive chemotherapy cycles, followed by extensive carcinologic resection surgery and radiation therapy on the tumor bed. We deplore the loss of a patient after distant recurrence in 8th month ; the other two patients remain alive after 25 and 36 months of progression without recurrence. Askin”s tumor is a bone or extra-bone tumor that is histologically similar to Ewing”s sarcoma and PNET, which is characterized by its thoracic location. These three tumors appear to represent different forms of the same entity. They present the same specific chromosomal translocation (11q24-22q12) and, in immunohistochemistry, the same expression of the MIC2 oncogene (CD99) and of the translocation-related FLI-1 protein. The diagnosis is often made at the late stage of locoregional or remote invasion. The treatment is mainly based on surgery and radiotherapy and postoperative chemotherapy to reduce recurrence. The prognosis is unfavorable with a survival at 2 years not exceeding 40% after treatment. Pulmonary metastases are frequently observed even a few years after surgery.

PB1768 PREDICTIVE FACTORS OF SEVERE BLEEDING IN ACUTE PROMYELOCYTIC LEUKEMIA

Background:Acute promyelocytic leukemia (APL) is identified by the predomination of abnormal promyelocytes in the bone marrow (BM) and a specific chromosomal translocation – t(15;17) – resulting in a fusion transcript between the promyelocytic (PML) gene on chromosome 15 and the retinoic acid receptor alpha (RARα) gene on chromosome 17; this transcript is known as PML–RARα. The t(15;17) chromosomal translocation can cause hyperexpression of tissue factor (TF) in cells of patients with APL and render the patient hypercoagulable.Aims:The objective of the current study was to assure more appropriate therapeutic strategies for reducing severe hemorrhaging by assessing the recovery of abnormal coagulation indexes in patients with acute promyelocytic leukemia (APL) during induction therapy.Methods:Retrospective analyses of newly diagnosed with APL were performed during initial treatment. A total of 23 patients newly diagnosed with APL were treated at the Hematology department of Emergency Hospital (Semey, Kazakhstan), between April 2008 and January 2019. This cohort included 9 males and 14 females with a median age of 41 years (range: 12–75). The APL diagnostic criteria were based on the World Health Organization Classification of Tumors–Pathology and Genetics of Tumors of Hematopoietic and Lymphoid Tissues (2008) and French–American–British classification systems (1976) criteria. A molecular diagnosis was confirmed by identification of t(15;17) in a cytogenetic analysis and/or positive detection of PML–RARα using either fluorescence in situ hybridization or reverse transcription‐polymerase chain reaction. The immune phenotype diagnosis of APL was established by positivity for CD33, CD9, CD13, and CD117, and low expression of HLA‐DR and CD34. Early death (ED) was defined as death due to any cause within 30 days of diagnosiResults:Hemorrhage was the principal cause of death during the induction period. The values of white blood cell count, lactate dehydrogenase, prothrombin time (PT), fibrinogen (Fbg), hemoglobin, and bone marrow leukemic promyelocytes were significantly different in the high‐risk group compared to the low/intermediate‐risk groups. There were significant differences in the white blood cell count, bone marrow leukemic promyelocytes, platelet (PLT) count, and the levels of lactate dehydrogenase, d‐dimer, PT, and Fbg, as well as in FLT3‐ITD mutations between patients with major bleeding and those with minor bleeding. Hemostatic variables significantly improved over time during induction therapy. The recovery times of the PLT, PT, and Fbg values were significantly slower in patients with major bleeding than in those with minor bleeding. Particularly, the PLT level in patients with major bleeding was not similar to that in the minor bleeding group until after 4 weeks of treatment. Hemorrhages were the most common cause of induction death in this study. High‐risk patients were more prone to serious clinical bleeding symptoms. Patients with major bleeding had more rapid proliferation characteristics and an increased incidence of FLT3‐ITD mutations compared to patients with minor bleeding. Hemostatic variables recovered significantly more slowly in patients with major bleeding than in those with minor bleeding. Active induction therapy and blood product infusion are efficient in preventing severe bleedingSummary/Conclusion:Our results suggested that low PLT count might be the leading cause of fatal bleeding in patients newly diagnosed with APL.

PS1168 CHROMATIN TOPOLOGY AND THREE‐DIMENSIONAL ORGANIZATION OF CHROMOSOME TERRITORIES 9 AND 22 IN CD34+ BONE MARROW CELLS FROM CHRONIC MYELOID LEUKEMIA PATIENTS

Background:During interphase, chromosomes occupy a limited space within the nucleus, referred to as chromosome territory (CT). The non‐random CT's spatial organization in the nucleus contributes to determining the outcome of chromosomal translocations (t). Comparative analysis of the spatial arrangement of t‐partners and their frequency suggest that this chromosome aberration occurs preferentially among proximally positioned genome regions. The consistence appearance of t(9;22) in hematopoietic stem cells, apart from being etiological factor for chronic myeloid leukemia (CML), is a clear example of what is considered a non‐random chromosome aberration. Besides, the CT's spatial distribution is regulated by epigenetic factors such as post‐translational histone modifications. These modifications are also directly related to the chromatin topology in these CTs, which is relevant since a chromatin misconfiguration has been related to the susceptibility to generate mutations and translocations.Aims:‐ Determine the 3D arrangement of interphase CTs 9 and 22 and BCR‐ABL genes in CD34+ bone marrow (BM) cells from CML patients at the time of diagnosis (CML RxDx) and patients with complete response (CML CR) compared to healthy donors’ CD34+ mobilized bone marrow cells (MBM) and peripheral blood mononuclear cells (PBMC).‐ Map and quantify the distribution of the histone modifications H3K27me3 and H3K9ac across CTs 9 and 22 in our study population.Methods:The study population consisted of 5 BM from CML RxDx and CML CR patients, 5 MBM and PBMC from healthy donors. CD34+ hematopoietic cells were isolated and subsequently processed for 3D‐FISH and 3D immuno‐FISH. Super‐resolution imaging was performed by 3D structured illumination microscopy (SIM) and images reconstructed and analysed using Imaris program (Bitplane).Results:We found that nuclear volumes were significantly larger in CD34 + cells from MBM and CML patients compared to PBMC controls. The absolute CTs volume analysis shows a major volume of CT9 compared to CT22. To determine the distance between BA genes and their CTs we calculated the nearest distance between the mass center of CT9 and CT22. The shorter distance was between de CML RxDx cells since the translocation presence. When we compare the samples without t(9;22), the shorter distances were observed between BA from MBM and CML CR cells compared to PBMC samples. After normalizing the distance between the CTs and BA genes against the radius of the nucleus. We observed that the location of CT22 tended towards the center, whereas that of CT9 towards the nuclear periphery. Meanwhile BA genes were located closer to the nuclear center than the respective CTs’ geometrical center. We have mapped relevant histone modifications like H3K9ac and H3K27me3 across CT9 and 22 using 3D immuno‐FISH. The enrichment of H3K27me3 seems to be significantly decreased in CT22 from samples of CML CR cells.Summary/Conclusion:The consistent appearance of specific chromosomal translocation in leukemias particularly t(9;22) in CML patients has suggested that the positioning and chromatin topology of CTs might play a role in the occurrence of particular chromosomal rearrangements associated with malignant transformation. Using 3D‐FISH and 3D‐SIM we have determined the positions, distances and volumes of CT9 and CT22 and in BCR‐ABL1 genes, in interphase nuclei of CD34+ bone marrow (BM) cells from CML RxDx and in CR patients compared to healthy donors’ CD34+ MBM and PBMC. Likewise, we mapped relevant histone modifications finding a particular difference in TC22 and H3K27me3 histone mark from CML CR CD34+ cells.image

Molecular cytogenetic characterisation of Elytrigia \xd7mucronata, a natural hybrid of E. intermedia and E. repens (Triticeae, Poaceae)

BackgroundInterspecific hybridisation resulting in polyploidy is one of the major driving forces in plant evolution. Here, we present data from the molecular cytogenetic analysis of three cytotypes of Elytrigia ×mucronata using sequential fluorescence (5S rDNA, 18S rDNA and pSc119.2 probes) and genomic in situ hybridisation (four genomic probes of diploid taxa, i.e., Aegilops, Dasypyrum, Hordeum and Pseudoroegneria).ResultsThe concurrent presence of Hordeum (descended from E. repens) and Dasypyrum + Aegilops (descended from E. intermedia) chromosome sets in all cytotypes of E. ×mucronata confirmed the assumed hybrid origin of the analysed plants. The following different genomic constitutions were observed for E. ×mucronata. Hexaploid plants exhibited three chromosome sets from Pseudoroegneria and one chromosome set each from Aegilops, Hordeum and Dasypyrum. Heptaploid plants harboured the six chromosome sets of the hexaploid plants and an additional Pseudoroegneria chromosome set. Nonaploid cytotypes differed in their genomic constitutions, reflecting different origins through the fusion of reduced and unreduced gametes. The hybridisation patterns of repetitive sequences (5S rDNA, 18S rDNA, and pSc119.2) in E. ×mucronata varied between and within cytotypes. Chromosome alterations that were not identified in the parental species were found in both heptaploid and some nonaploid plants.ConclusionsThe results confirmed that both homoploid hybridisation and heteroploid hybridisation that lead to the coexistence of four different haplomes within single hybrid genomes occur in Elytrigia allopolyploids. The chromosomal alterations observed in both heptaploid and some nonaploid plants indicated that genome restructuring occurs during and/or after the hybrids arose. Moreover, a specific chromosomal translocation detected in one of the nonaploids indicated that it was not a primary hybrid. Therefore, at least some of the hybrids are fertile. Hybridisation in Triticeae allopolyploids clearly and significantly contributes to genomic diversity. Different combinations of parental haplomes coupled with chromosomal alterations may result in the establishment of unique lineages, thus providing raw material for selection.

Therapeutic Approaches Targeting PAX3-FOXO1 and Its Regulatory and Transcriptional Pathways in Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a family of soft tissue cancers that are related to the skeletal muscle lineage and predominantly occur in children and young adults. A specific chromosomal translocation t(2;13)(q35;q14) that gives rise to the chimeric oncogenic transcription factor PAX3-FOXO1 has been identified as a hallmark of the aggressive alveolar subtype of RMS. PAX3-FOXO1 cooperates with additional molecular changes to promote oncogenic transformation and tumorigenesis in various human and murine models. Its expression is generally restricted to RMS tumor cells, thus providing a very specific target for therapeutic approaches for these RMS tumors. In this article, we review the recent understanding of PAX3-FOXO1 as a transcription factor in the pathogenesis of this cancer and discuss recent developments to target this oncoprotein for treatment of RMS.

In silico and in vitro screening of small molecule Inhibitors against SYT-SSX1 fusion protein in synovial sarcoma

Synovial sarcoma (SS) is characterized by a tumour specific chromosomal translocation t(X;18) (p11;q11) which results in the formation of SYT-SSX1 fusion protein. This fusion protein represents a clear therapeutic target and molecules specifically targeting SYT-SSX1 fusion protein are currently not available. In this study, SYT-SSX1 fusion protein sequence was retrieved from Uniprot and 3D structure was generated using I-TASSER modeling program. A structure based computational screening approach has been employed using Glide docking software to identify potential SYT-SSX1 small molecule inhibitors that bind to the junction region of the fusion protein. The obtained inhibitors were further filtered based on the docking score and ADME/T properties. Ten best fit compounds were chosen for in vitro studies. The anti-proliferative activities of these 10 compounds were screened in Yamato, ASKA (carries SYT-SSX1 fusion protein) and other sarcoma cell lines such as A673, 143B to understand the specificity of inhibition of the chosen compounds. The in vitro activity was compared against HEK293 cell lines. The compound 5-fluoro-3-(1-phenyl-1H-tetraazol-5-yl)-1H-indole (FPTI) was found to be selectively cytotoxic in synovial sarcoma cell lines (Yamato and ASKA) and this compound also showed insignificant anti proliferative activity on other cell lines. Further, target gene expression study confirmed that FPTI treatment down-regulated SYT-SSX1 and modulated its downstream target genes. Cell cycle analysis revealed the involvement of an apoptotic mechanism of cell death. Further experimental validations may elucidate the therapeutic potentials of FPTI against SYT-SSX1 fusion protein.

Abstract 3378: Variance in the distance between chromosome territories 9 and 22 in lymphocytes from peripheral blood healthy subjects: A proof of concept for a possible risk factor in chronic myeloid leukemia

Abstract The objective of our study was to assess the variations in the 3D organization of Chromosome Territories (CT) of chromosomes 9 and 22 in lymphocytes from healthy subjects. The differences in the distances of CTs 9 and 22 between subjects may provide a new perspective in the predisposition for specific chromosome translocations. We quantified the intra- and inter-subject variation in the distance of CT 9 and 22 as a proof of concept for new questions on the physiological and pathological effects of these differences. Methods: Blood samples from healthy subjects were processed for the isolation of lymphocytes with Ficoll as the gradient media. After the isolation, we fixed the cells directly onto coverslips for a 3D Fish protocol with 9 and 22 whole chromosome probes. Our protocol has been standardized to maintain the integrity of the nucleus with only minor changes in its volume. After the hybridization, we used the Elyra System of Zeiss to obtain super resolution images in Z stack. The images were processed with the Imaris software for its 3D reconstruction, measurement (distances, volumes and colocalization) and analysis. Results: The CTs 9 and 22 behave differently in peripheral blood lymphocytes. The CT 22 has a more internal position (closer to the center of the nucleus) in comparison with the CT 9, which can be seen most of the times in contact with the border of the nucleus. The CT 22 occupies a smaller volume than CT 9, which has almost a twice as larger volume. The CTs 9 have a polarized distribution inside the nucleus, therefore the distance between the CT 9-9 was larger (~7.3 μm ± 0.9) than the distance between CT 22-22. Although the position of the CTs was highly variable in cells from the same individual, we found a statistically significant difference in the inter-individual distance between CT 9-9 and the minimal distance between CT 9-22. Conclusion: Our study suggests that there are differences in the distribution of CT in lymphocytes isolated from peripheral blood between our subjects. These differences could prove to be a new individual characteristic with important implications in the predisposition for specific diseases, mainly to those related with chromosomal rearrangements. Citation Format: David G. Vallejo, Eunice Fabian, Luis A. Herrera. Variance in the distance between chromosome territories 9 and 22 in lymphocytes from peripheral blood healthy subjects: A proof of concept for a possible risk factor in chronic myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3378.

Immunoreactivity of a Monoclonal Antibody to SS18-SSX Fusion Gene Product in Formalin-fixed Paraffin-embedded Synovial Sarcoma Tissue Section.

Synovial sarcoma is an aggressive sarcoma with specific reciprocal chromosomal translocation of SS18 (also known as SYT) and SSX genes. In the present study, we aimed to detect the SS18-SSX fusion gene product in routinely processed pathologic synovial sarcoma tissue section. Monoclonal antibodies to peptide QRPYGYDQ-IMPKKPA, which covered the fusion region of SS18-SSX, were newly established and subsequently characterized by enzyme-linked immunosorbent assay, Western-immunoblotting, and immunohistochemical staining. A monoclonal antibody designated BG35, reacted with boundary region of SS18-SSX chimera protein in a specific manner, when C-terminal or neighboring region of QRPYGYDQ-IMPKKPA is structurally exposed. Immunoreactivity of BG35 was localized in nucleus of synovial sarcoma cells, but not in other sarcoma cells, examined. Interestingly, synovial sarcoma cells with epithelial differentiation exhibited much strong BG35 immunoreactivity than synovial sarcoma cells with sarcomatous differentiation. BG35 may be useful for evaluating molecular kinetics of SSX-SS18 gene product in situ.

256 Monophasic Synovial Sarcoma Presenting as a Parapharyngeal Mass—Report of a Rare Case

Synovial sarcoma (SS) is an aggressive soft tissue sarcoma, most commonly arising in the peri-articular tissue of extremities in young adults, harboring a specific chromosomal translocation t(X;18)(p11.2;q11.2). SS is a misnomer because it does not arise from the synovium; it appears to arise from pluripotent stem cells that are capable of differentiating into mesenchymal and/or epithelial structures and lacks synovial differentiation. SS accounts for ~5%-10% of soft tissue sarcomas. Occasionally, it arises in the head and neck region and carries a poor prognosis. Morphologically, SS can be biphasic (BSS), monophasic (MSS), or poorly differentiated. We report a rare case of MSS presenting as a parapharyngeal mass. The patient is a 27-year-old-man presenting with left-sided facial and mandibular pain. Imaging showed large parapharyngeal soft tissue mass in the region of the left pterygoid musculature measuring 3.6 x 2.9 x 2.5 cm with focal calcifications. He underwent resection of this mass, which showed spindle cell neoplasm with variably prominent hemangiopericytoma-like vascular pattern. Vague nuclear palisading was also noted. Immunohistochemical stains were focally positive for cytokeratins (AE1/AE3, CK7, CK8 (Cam5.2), and CK19), calponin, calretinin, EMA, GFAP, and synaptophysin with Ki67 labeling being 25%-30%. The tumor cells were negative for CD34, CD117, desmin, HMB45, myogenin, P63, S100, and SMA. RT-PCR showed SYT/SSX2 fusion transcript. Combining all, the diagnosis of MSS-spindle cell type was confirmed. Based on AFIP data, ~3% of SS arise in this location. SS is a rare entity to be encountered in the head and neck region and is often a challenge in terms of diagnosis, especially the MSS type. The differential of monophasic tumors is fibrosarcoma, malignant schwannoma, and hemangiopericytoma. We report this case of MSS because of its rare presentation as a parapharyngeal mass. Clinicians and pathologists should be aware of this unusual location and presentation of SS.

SY9-2 - Treatment strategy for advanced soft tissue sarcoma

Soft tissue sarcoma (STS) is a rare tumor with more than 50 histologic subtypes. Many types of STS are not sensitive to chemotherapy, so patients with advanced diseases have poor prognosis. Development of novel anticancer therapy is in urgent need. Doxorubicin monotherapy is currently the standard first line treatment of advanced STS, but there is no standard for second line therapy. Recently, three new drugs: pazopanib, trabectedin, and eribulin have been approved for the second line treatment of patients with advanced STS, but the best treatment sequence of those drugs are not known. Sensitivity of STS to each drug seems to vary by histologic subtypes. For instance, liposarcoma seems be more sensitive to eribulin and less sensitive to pazopanib. Translocation related sarcoma (TRS) such as myxoid liposarcoma seems to be sensitive to trabectedin. Furthermore, these drugs have different safety profiles, so subtypes and patient characteristics must be considered for treatment selection. Development of specific molecular target therapy for each type of STS is necessary. There are specific chromosome translocations in 20-30% of STS, but their products are mostly transcriptional factors, and target therapy for those factors are difficult to develop. Trabectedin inhibits function of transcriptional factors, and shows higher efficacy for TRS. As molecular target therapies for gene mutations, success in molecular target therapy for c-kit and PDGFR mutation in gastrointestinal stromal tumor was followed by efficacy for rare sarcomas such as inflammatory myofibroblastic tumor or dermatofibrosarcoma protuberans, but there are few developments in other sarcomas. STS are associated with angiogenesis to a greater or lesser extent, so angiogenesis inhibitors such as pazopanib show good efficacy in some subtypes. Immune checkpoint inhibitors also have been developed for STS and show some efficacy in subtypes such as undifferentiated pleomorphic sarcoma.

Abstract 700: Characterization of the mode of action of Fenretinide treatment in alveolar rhabdomyosarcoma cells

Abstract Alveolar rhabdomyosarcoma (aRMS) is a highly malicious childhood malignancy characterized by a specific chromosomal translocation encoding the oncogenic transcription factor PAX3-FOXO1. As aRMS cells are addicted to the tumor-specific fusion protein, it may serve as an ideal therapeutic target. Previously, we have identified from a large drug library screen the compound Fenretinide (retinoic acid p-hydroxyanilide), which is already in clinical use, to affect both PAX3-FOXO1 expression as well as aRMS cell viability. The aim of this study was therefore to characterize the mode of action of Fenretinide in more detail. First, we were able to show that Fenretinide induced the generation of reactive oxygen species (ROS) in mitochondria. A more detailed characterization revealed that the Fenretinide-induced ROS derived from an interaction of Fenretinide around complex II of the mitochondrial respiratory chain, leading to the production of superoxides. ROS scavenging as well as complexing of iron ions completely abolished cell death. To identify the mode of cell death involved, we then used a range of pharmacological inhibitors of specific cell death pathways including Z-vad (pan -caspase inhibitor), Necrostatin-1 (necroptosis pathway inhibitor (RIP-1 kinase inhibitor)), 3-Methyadenine (3-MA) (autophagy pathway inhibitor (phosphatidylinositol 3-kinase inhibitor)) and Ferrostatin (ferroptosis pathway inhibitor) during Fenretinide treatment. Surprisingly, none of these inhibitors alone was able to prevent cell death and even different combinations were not sufficient to completely inhibit cell death. CRISPR/Cas9 mediated depletion of key players in the apoptotic and necroptotic pathway (Bak, Bax and RIPK1) confirmed the pharmacological data. We therefore conclude that other, less characterized cell death pathways or a combination of several pathways including apoptosis and necroptosis might be crucial. Interestingly, electron microscopic examination of cells pointed towards an excessive accumulation of vacuoles to be characteristic. Taken together, our data show that Fenretinide shows high potential for the treatment of aRMS, inducing several forms of cell death mediated through the production of ROS. These properties open the search for additional compounds acting in a combinatorial manner. Citation Format: Eva Brack, Marco Wachtel, Beat W. Schaefer. Characterization of the mode of action of Fenretinide treatment in alveolar rhabdomyosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 700. doi:10.1158/1538-7445.AM2017-700

Expression Profiling of Circulating Microvesicles Reveals Intercellular Transmission of Oncogenic Pathways.

Circulating microvesicles have been described as important players in cell-to-cell communication carrying biological information under normal or pathologic condition. Microvesicles released by cancer cells may incorporate diverse biomolecules (e.g., active lipids, proteins, and RNA), which can be delivered and internalized by recipient cells, potentially altering the gene expression of recipient cells and eventually impacting disease progression. Leukemia in vitro model systems were used to investigate microvesicles as vehicles of protein-coding messages. Several leukemic cells (K562, LAMA-87, TOM-1, REH, and SHI-1), each carrying a specific chromosomal translocation, were analyzed. In the leukemic cells, these chromosomal translocations are transcribed into oncogenic fusion transcripts and the transfer of these transcripts was monitored from leukemic cells to microvesicles for each of the cell lines. Microarray gene expression profiling was performed to compare transcriptomes of K562-derived microvesicles and parental K562 cells. The data show that oncogenic BCR-ABL1 transcripts and mRNAs related to basic functions of leukemic cells were included in microvesicles. Further analysis of microvesicles cargo revealed a remarkable enrichment of transcripts related to cell membrane activity, cell surface receptors, and extracellular communication when compared with parental K562 cells. Finally, coculturing of healthy mesenchymal stem cells (MSC) with K562-derived microvesicles displayed the transfer of the oncogenic message, and confirmed the increase of target cell proliferation as a function of microvesicle dosage.Implications: This study provides novel insight into tumor-derived microvesicles as carriers of oncogenic protein-coding messages that can potentially jeopardize cell-directed therapy, and spread to other compartments of the body. Mol Cancer Res; 15(6); 683-95. ©2017 AACR.