Small Cell Osteosarcoma Research Articles

ENDOBRONCHIAL METASTASIS FROM OSTEOSARCOMA

SESSION TITLE: Disorders of the Pleura SESSION TYPE: Fellow Case Report Posters PRESENTED ON: 10/09/2018 01:15 pm - 02:15 pm INTRODUCTION: Lung is the most frequent site for metastasis from osteosarcoma. Usually, the presentation is with well-defined parenchymal nodules, and unusually with pleural deposits, calcification, or mediastinal nodes. Metastases from osteosarcoma presenting as an isolated endobronchial lesion is discretely rare. CASE PRESENTATION: 36 Y/O man with a diagnosis of osteosarcoma presented with dry cough. The patient CT chest showed a left lower lobe mass with central calcification (figure). The patient underwent bronchoscopy for evaluation left lower lobe mass. The bronchoscopy showed left lower lobe superior segment endobronchial lesion. Hot snare and cryo probe used to debulck the endo bronchial lesion (figure). The lesion was extending deep in the airway and EBUS confirmed the depth of the lesion with the finding for central calcification (figure). Pathology confirmed the diagnosis of metastatic small cell osteosarcoma (figure). DISCUSSION: Airway metastasis of tumors usually rare. The incidence of such metastasis is estimated to be approximately 2%; the most common primary malignancies being renal cell, colorectal, cervical, breast carcinoma, and malignant melanoma.This case presenting an extension of small cell small cell Osteosarcom mass in the left lower lobe into the airway confirmed with EBUS. CONCLUSIONS: Airway inspection is an important test for patient with metastatic tumors to the lung close to a major airways. This will give the chance for a baseline evaluation of the airway for any future progress of disease or future complication of the disease such as hemoptysis. Inexplicable pulmonary manifestations should prompt an early evaluation including bronchoscopy and early institution of local therapy for possible palliation. Reference #1: Mogulkoc N, Goker E, Atasever A, et al. Endobronchial metastasis from osteosarcoma of bone: treatment with intraluminal radiotherapy. Chest. 1999;116:1811–1814 Reference #2: Rubio ER, Chang EE, David O, et al. Pulmonary osteosarcoma: endoscopic management. J Bronchol. 2001;8:277–281 DISCLOSURES: No relevant relationships by Abdul Hamid Alraiyes, source=Web Response No relevant relationships by Mohamed Elrifai, source=Web Response No relevant relationships by Arun Kadamkulam Syriac, source=Web Response

Laryngeal sarcomas: A case series of 5 cases

Primary sarcomas of the larynx are rare and miscellaneous. The most common is chondrosarcoma. Other sarcomas are very rare. Sarcomas can have heterogeneous morphologic features of spindle, small round, epithelioid, pleomorphic and giant cells. Laryngeal sarcomas may mimic carcinomas, lymphomas, small cell carcinoma, mesothelioma and melanoma. This imposes diagnostic challenges for unfamiliar pathologists particularly in small laryngeal biopsies. Our aim was to study the different types of sarcomas that can involve the larynx in our institution, to investigate their diagnostic challenges and potential pitfalls and to find helpful histologic clues to avoid misinterpretation and missed diagnosis. We performed a retrospective review study over 13 years. We retrieved 5 cases of laryngeal sarcomas. They included Kaposi sarcoma, low-grade chondrosarcoma, epithelioid angiosarcoma, polypoid leiomyosarcoma and small cell osteosarcoma. The age range was between 32 and 74 years with an average age of 52 years. The male to female ratio was 3:2. The Kaposi sarcoma and chondrosarcoma were correctly diagnosed. The angiosarcoma was initially missed as recurrent carcinoma. The leiomyosarcoma was initially inferred as polypoid sarcomatoid squamous cell carcinoma. The small cell osteosarcoma initiated the differential diagnosis of high-grade lymphoma, small cell carcinoma, undifferentiated carcinoma, Ewing sarcoma and rhabdomyosarcoma. An implementation of a panel of immunohistochemical markers guided by certain histomorphologic clues was helpful to make the correct diagnosis. Pathologists should be aware of the morphologic spectrum and different growth patterns of laryngeal sarcomas. Immunohistochemistry studies are essential. Correct diagnosis, classification and grading of laryngeal sarcomas are clinically important for the prognosis and appropriate management of patients.

Ewing Sarcoma and the History of Similar and Possibly Related Small Round Cell Tumors: From Whence Have We Come and Where are We Going?

The diagnosis of small round cell tumors always has been extremely difficult, and our current classification systems continue to evolve. Since its initial discovery by Dr James Ewing, the historical context of what is acceptably included under the designation "Ewing sarcoma" has changed. Although Ewing sarcoma and primitive neuroectodermal tumor were both initially described in the early 20th century, these tumors were considered likely distinct entities until the end of that same century, almost 75 years later. With modern immunohistochemistry and more recent advances in molecular techniques, the understanding of Ewing sarcoma and Ewing-like tumors has improved dramatically but also raises new questions and challenges. We now know that this category of tumors is remarkably more heterogenous than initially thought, especially in regards to its cytogenetics and molecular properties, and some of these differences likely have prognostic relevance. Whether we are now expanding the spectrum of Ewing sarcoma or simply recognizing new entities is controversial. Therapeutic approaches to address these new categories and/or entities need further focus and attention. Herein, we provide a comprehensive historical perspective on Ewing sarcoma, Ewing-like tumors (CIC and BCOR-rearranged sarcomas), and related and/or similar small round cell tumors, often included in the differential diagnosis, including mesenchymal chondrosarcoma, desmoplastic small round cell tumor, and small cell osteosarcoma. We also seek to provide updates and insights into the evolving classification and clinical relevance of the Ewing family of tumors.

PAX7 immunohistochemical evaluation of Ewing sarcoma and other small round cell tumours.

Ewing sarcoma is a small round cell tumour that affects bone and soft tissues. Although the detection of the specific fusion gene is a robust method of its diagnosis, immunohistochemistry may serve as a practical surrogate. Recent tissue microarray studies suggested that PAX7 is a novel marker, because it was expressed consistently in Ewing sarcoma, in addition to rhabdomyosarcoma and synovial sarcoma. Here, we evaluated the utility of PAX7 immunohistochemistry in whole-tissue sections of an expanded array of round cell malignancies with adequate molecular characterisation. We stained 30 molecularly confirmed Ewing sarcomas, one EWSR1-NFATC2 sarcoma and 141 non-Ewing round cell tumours by a monoclonal antibody against PAX7. Staining was considered positive if at least 5% of tumour cells were stained. PAX7 was expressed in 27 of 30 Ewing sarcomas (90%), mainly in a diffuse and strong manner. Although NKX2-2 showed similar sensitivity, PAX7 showed more extensive and strong reactivity. One EWSR1-NFATC2 sarcoma co-expressed PAX7 and NKX2-2. PAX7 was also expressed in 24 of 141 non-Ewing tumours, including alveolar rhabdomyosarcomas (seven of 10), poorly differentiated synovial sarcomas (seven of 10), BCOR-CCNB3 sarcomas (eight of 10), small-cell osteosarcoma (one of five) and desmoplastic small round cell tumour (one of 10), one-third of which showed diffuse strong reactivity. Although we confirmed that PAX7 is a sensitive marker for Ewing sarcoma, anti-PAX7 antibody also stained several Ewing sarcoma mimics, whose spectrum was distinct from NKX2-2-positive non-Ewing entities. Further studies are required to determine how PAX7 could be integrated into practice to classify small round cell tumours efficiently.

Array-based DNA-methylation profiling in sarcomas with small blue round cell histology provides valuable diagnostic information

Undifferentiated solid tumors with small blue round cell histology and expression of CD99 mostly resemble Ewing sarcoma. However, they also may include other tumors such as mesenchymal chondrosarcoma, synovial sarcoma, or small cell osteosarcoma. Definitive classification usually requires detection of entity-specific mutations. While this approach identifies the majority of Ewing sarcomas, a subset of lesions remains unclassified and, therefore, has been termed “Ewing-like sarcomas” or small blue round cell tumors not otherwise specified. We developed an approach for further characterization of small blue round cell tumors not otherwise specified using an array-based DNA-methylation profiling approach. Data were analyzed by unsupervised clustering and t-distributed stochastic neighbor embedding analysis and compared with a reference methylation data set of 460 well-characterized prototypical sarcomas encompassing 18 subtypes. Verification was performed by additional FISH analyses, RNA sequencing from formalin-fixed paraffin-embedded material or immunohistochemical marker analyses. In a cohort of more than 1,000 tumors assumed to represent Ewing sarcomas, 30 failed to exhibit the typical EWS translocation. These tumors were subjected to methylation profiling and could be assigned to Ewing sarcoma in 14 (47%), to small blue round cell tumors with CIC alteration in 6 (20%), to small blue round cell tumors with BCOR alteration in 4 (13%), to synovial sarcoma and to malignant rhabdoid tumor in 2 cases each. One single case each was allotted to mesenchymal chondrosarcoma and adamantinoma. 12/14 tumors classified as Ewing sarcoma could be verified by demonstrating either a canonical EWS translocation evading initial testing, by identifying rare breakpoints or fusion partners. The methylation-based assignment of the remaining small blue round cell tumors not otherwise specified also could be verified by entity-specific molecular alterations in 13/16 cases. In conclusion, array-based DNA-methylation analysis of undifferentiated tumors with small blue round cell histology is a powerful tool for precisely classifying this diagnostically challenging tumor group.

Abstract 4835: UD-017, a novel highly selective and orally active CDK7 inhibitor, shows a significant anticancer activity in patient-derived cancers

Abstract Background: Cyclin dependent kinase 7 (CDK7) is an attractive target for anticancer drugs due to its dual roles, cell cycle regulation and gene transcription/RNA processing. We synthesized UD-017, a small-molecule, highly selective, orally active CDK7 inhibitor with a novel chemotype. In this study, we characterized anticancer activities of UD-017 in vitro and in vivo using patient-derived (PD) cancer cells. Methods: We first evaluated an antiproliferative activity of UD-017 broadly in a panel of cancer cell assay including patient-derived 3-dimensional tumor clonogenic assay. To verify the potential of c-Myc expression as a biomarker, we investigated the correlation between c-Myc expression levels and anticancer activity in vitro and in vivo using a colorectal cancer cell. In the main part, anticancer efficacy of UD-017 was investigated in vivo in PD-xenograft (PDX) models using representative PD cancer cells. Results: In antiproliferative panel assays using over 100 cancer cell lines, UD-017 broadly inhibited a wide range of cancer cells from colon, breast, lung, kidney, blood, pancreas, osteosarcoma, sarcoma and urinary bladder cancers. Especially, UD-017 showed high sensitivity in solid tumor such as non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), gastric, osteosarcoma and sarcoma cells with IC50s of 10-200 nM. UD-017 showed a good correlation of c-Myc expression levels with antiproliferative activity using colorectal cancer cells in vitro, and UD-017 reduced the intratumoral MYC mRNA levels by an administration at 100 mg/kg in a HCT-116 xenograft model in mice. In PDX models, UD-017 showed strong inhibition and even regression of tumor growth, and the tumors were almost disappeared on day 14 without body weight loss in non-small cell lung cancer (LXFL1121). In a pleuramesothelioma (PXF541) PDX model, UD-017 also showed regressive effect, and in gastric cancer (GXA3067) and sarcoma (SXFS117) PDX models, UD-017 completely inhibited the tumor growth. Conclusions: We propose UD-017 as a novel type of anticancer drug that shows complete antitumor responses in patient-derived xenograft models of diverse cancer types. c-Myc expression in cancer cells may be a biomarker for the antitumor effect of UD-017. These data support the rationale for further advancing towards clinical development. Citation Format: Takashi Matsushita, Sayaka Ogi, Kazuhiro Onuma, Hidetoshi Sunamoto, Ayumi Ogawa, Toru Hasegawa, Yasunori Tokunaga, Yasuhiro Aga, Shigeru Ushiyama. UD-017, a novel highly selective and orally active CDK7 inhibitor, shows a significant anticancer activity in patient-derived cancers [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 4835.

Abstract B36: A biomarker-guided approach to combining PARP inhibitors with radiotherapy in pediatric sarcomas

Abstract Background: Ewing sarcoma (EWS) expresses high levels of Schlafen-11 (SLFN11). SLFN11 disrupts checkpoint maintenance and may serve as a biomarker to assess sensitivity to Poly (ADP-ribose) polymerase 1 and 2 inhibitors (PARPi). The goal of this study is to evaluate SLFN11 protein expression in a panel of pediatric solid tumors and correlate levels of protein with sensitivity to PARP inhibition combined with ionizing radiation (IR), a component of therapy for many pediatric sarcomas and a potent inducer of DNA damage. Methods: SLFN11 mRNA and protein levels were assessed by quantitative RT-PCR, and immunohistochemistry, Western blot, and immunofluorescence microscopy, respectively. PARPi included talazoparib (TAL), niraparib (NIR), veliparib (VEL), and olaparib (OLA). Approximately 30 minutes after addition of systemic therapy, graded doses of radiation were delivered and viability across a panel of pediatric solid tumor cell lines was measured using the ATP-based Cell TiterGlo assay and confirmed with the colony formation assay. Results: We found that SLFN11 mRNA and protein is expressed at high levels in EWS, and SLFN11 is also variably present in a subset of other pediatric sarcomas, including desmoplastic small round cell tumor, osteosarcoma, and rhabdomyosarcoma. In all tumor cells with detectable SLFN11 expression, viability was reduced by greater than 90% when exposed to 2 Gy IR and 1-10 nM TAL, whereas cells with undetectable levels of SLFN11 were 5-10 times less sensitive. Intriguingly, variation in the potentiation between specific PARPi and IR correlated with the ability to form drug-induced PARP-DNA adducts, with the strong PARP trapper TAL showing ~10-fold higher potency compared to the moderate trapper NIR, and ~300-fold more potency relative to the weak trapper VEL. Consistent with our PARPi findings, the toposiomerase 1 inhibitor irinotecan, which also forms DNA adducts, potentiated with IR similarly to TAL at concentrations <10 nM in tumor cells expressing detectable levels of SLFN11. Conclusion: SLFN11 is present in select pediatric sarcomas and may induce a DNA repair defect that is best exploited by combining low doses of TAL and irinotecan with IR. Citation Format: Anang A. Shelat, Christopher L. Tinkle, Elizabeth Stewart, Sara Michele Federico, Brandon Bianski, Marcia Mellado-Lagarde. A biomarker-guided approach to combining PARP inhibitors with radiotherapy in pediatric sarcomas [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr B36.

SATB2 and TLE1 Expression in BCOR-CCNB3 (Ewing-like) Sarcoma, Mimicking Small Cell Osteosarcoma and Poorly Differentiated Synovial Sarcoma

SATB2 and TLE1 Expression in BCOR-CCNB3 (Ewing-like) Sarcoma, Mimicking Small Cell Osteosarcoma and Poorly Differentiated Synovial Sarcoma

A biomarker-guided approach to combining PARP inhibitors with radiotherapy in pediatric solid tumors.

10556 Background: Ewing sarcoma (EWS) expresses high levels of Schlafen-11 (SLFN11). SLFN11 disrupts checkpoint maintenance and may serve as a biomarker to assess sensitivity to Poly (ADP-ribose) polymerase 1 and 2 inhibitors (PARPi). The goal of this study is to evaluate SLFN11 protein expression in a panel of pediatric solid tumors and correlate levels of protein with sensitivity to PARP inhibition combined with ionizing radiation (IR), a component of therapy for many pediatric solid tumors and a potent inducer of DNA damage. Methods: SLFN11 mRNA and protein levels were assessed by quantitative RT-PCR, and immunohistochemistry, Western blot, and immunofluorescence microscopy, respectively. PARPi included: talazoparib (TAL), niraparib (NIR), veliparib (VEL), and olaparib (OLA). Approximately 30 minutes after addition of systemic therapy, graded doses of radiation were delivered and viability across a panel of pediatric solid tumor cell lines was measured using the ATP-based Cell TiterGlo assay and confirmed with the colony formation assay. Results: We found that SLFN11 mRNA and protein is expressed at high levels in EWS, and SLFN11 is also variably present in a subset of other pediatric solid tumor lines, including desmoplastic small round cell tumor, osteosarcoma, and rhabdomyosarcoma. In all tumor cells with detectable SLFN11 expression, viability was reduced by greater than 90% when exposed to 2Gy IR and 1-10nM TAL, whereas cells with undetectable levels of SLFN11 were 5-10 times less sensitive. Intriguingly, variation in the potentiation between specific PARPi and IR correlated with the ability to form drug-induced PARP-DNA adducts, with the strong PARP trapper TAL showing ~10-fold higher potency compared to the moderate trapper NIR, and ~300-fold more potency relative to the weak trapper VEL. Consistent with our PARPi findings, the toposiomerase 1 inhibitor irinotecan, which also forms DNA adducts, potentiated with IR similarly to TAL at concentrations < 10nM in tumor cells expressing detectable levels of SLFN11. Conclusions: SLFN11 is present in select pediatric solid tumors and may induce a DNA repair defect that is best exploited by combining low-doses of TAL and irinotecan with IR.

Identification of the miRNA-mRNA regulatory network of small cell osteosarcoma based on RNA-seq

Small cell osteosarcoma (SCO) is a rare subtype of osteosarcoma characterized by highly aggressive progression and a poor prognosis. The miRNA and mRNA expression profiles of peripheral blood mononuclear cells (PBMCs) were obtained in 3 patients with SCO and 10 healthy individuals using high-throughput RNA-sequencing. We identified 37 dysregulated miRNAs and 1636 dysregulated mRNAs in patients with SCO compared to the healthy controls. Specifically, the 37 dysregulated miRNAs consisted of 27 up-regulated miRNAs and 10 down-regulated miRNAs; the 1636 dysregulated mRNAs consisted of 555 up-regulated mRNAs and 1081 down-regulated mRNAs. The target-genes of miRNAs were predicted, and 1334 negative correlations between miRNAs and mRNAs were used to construct an miRNA-mRNA regulatory network. Dysregulated genes were significantly enriched in pathways related to cancer, mTOR signaling and cell cycle signaling. Specifically, hsa-miR-26b-5p, hsa-miR-221-3p and hsa-miR-125b-2-3p were significantly dysregulated miRNAs and exhibited a high degree of connectivity with target genes. Overall, the expression of dysregulated genes in tumor tissues and peripheral blood samples of patients with SCO measured by quantitative real-time polymerase chain reaction corroborated with our bioinformatics analyses based on the expression profiles of PBMCs from patients with SCO. Thus, hsa-miR-26b-5p, hsa-miR-221-3p and hsa-miR-125b-2-3p may be involved in SCO tumorigenesis.

Small round blue cell tumors of the sinonasal tract: a differential diagnosis approach

One of the most challenging diagnostic categories within tumors of the sinonasal tract is the small round blue cell tumors. Biopsies are usually small and limited, resulting in considerable diagnostic difficulty for practicing surgical pathologists. These tumors share several overlapping histologic and immunophenotypic findings while also showing considerable variation within and between cases. Specific tumor site of origin, imaging findings, and clinical findings must be combined with the histology and pertinent ancillary studies if the correct diagnosis is to be reached. Discrimination between neoplasms is critical as there are significant differences in therapy and overall outcome. It is important to have a well developed differential diagnosis for this category of tumors, where each of the diagnoses is considered, evaluated, and either confirmed or excluded from further consideration. In an undifferentiated tumor, showing a small round blue cell morphology, using the mnemonic ‘MR SLEEP' helps to highlight tumors to consider: melanoma, mesenchymal chondrosarcoma, rhabdomyosarcoma, sinonasal undifferentiated carcinoma, squamous cell carcinoma (including NUT carcinoma), small cell osteosarcoma, lymphoma, esthesioneuroblastoma (olfactory neuroblastoma), Ewing sarcoma/primitive neuroectodermal tumor, pituitary adenoma, and plasmacytoma. A panel of pertinent immunohistochemistry studies, histochemistries and/or molecular tests should aid in reaching a diagnosis, especially when taking the pattern and intensity of reactions into consideration.

Osteosarcoma of the jaws in Koreans: analysis of 26 cases.

ObjectivesIn order to assess clinical behavior, response to treatment, and factors influencing prognosis of Korean patients with osteosarcoma of the jaws (OSJ).Materials and MethodsA retrospective study of clinical, and pathological records of 26 patients with OSJ treated at the Department of Oral and Maxillofacial Surgery in Yonsei University Dental Hospital from 1990 to March 2017.ResultsOf 26 patients, there were 9 men (34.6%) and 17 women (65.4%). Twenty-one of 26 patients had osteosarcoma of the mandible, and 5 of 26 patients had osteosarcoma of the maxilla. The histopathology of OSJ is highly variable, ranging from chondroblastic type (6 out of 26), osteoblastic type (10 out of 26), fibroblastic type (2 out of 26), to the rare variants like mixed type, small cell osteosarcoma types and more. All patients underwent gross total excision and only a few patients underwent neoadjuvant chemotherapy. Postoperative chemotherapy was given to most of the patients as adjuvant treatment or in combination with radiotherapy. The overall survival rate was 73.1% with an overall 2-year survival rate of 83.3%. The overall 5-,10-,15-year survival rates in this study were 73.5%, 73.5%, 49%, respectively. Using Kaplan-Meier analysis with log rank tests, the size of tumor (T-stage), and resection margins were found to affect the survival rate significantly. The chemotherapy was not significantly associated with improved survival rate.ConclusionSurgical resection with a clear margin is the most important factor in disease survival. The role of chemotherapy and radiotherapy in OSJ remains controversial, and deserves further studies.

The utility of SATB2 immunohistochemical expression in distinguishing between osteosarcomas and their malignant bone tumor mimickers, such as Ewing sarcomas and chondrosarcomas

SATB2 is commonly expressed in osteosarcomas. Although apparently being a valuable diagnostic marker for differentiating between small cell osteosarcoma (SCO) and other small round cell tumors of bone, for instance Ewing sarcoma family of tumors (ESFT), it has not been tested in a large series of ESFT and chondrosarcomas so far. We studied the immunohistochemical expression of SATB2 in 42 osteosarcomas, 31 chondrosarcomas, and 371 genetically confirmed ESFT. SATB2 positivity was detected in 90.4% of osteosarcomas, 87.5% of SCO, 91.3% of osteoblastic osteosarcomas, and in all chondroblastic and parosteal osteosarcomas. The osteoblastic and SCO subtypes expressed SATB2 more intensely than other histological types. SATB2 was expressed in 46.6% of chondrosarcomas, and in 1.3% of ESFT. Sensitivity and specificity of SATB2 immunoexpression were 90.4% and 95.3%, respectively. The positive and negative predictive values in osteosarcoma diagnosis were 66.6% and 98.9%, respectively.In chondrosarcoma, SATB2 immunoexpression was more frequent and intense in high-grade chondrosarcoma (Grade III) and uncommon in chondrosarcoma grade I. SATB2 positivity was detected in 55.6% of chondrosarcomas grade II. SATB2 apparently cannot distinguish between chondroblastic osteosarcoma and high-grade chondrosarcoma. Nevertheless, SATB2 is frequently expressed in osteogenic tumors, but is rarely positive in ESFT, and with the support of CD99 expression and specific molecular studies, it is very useful for distinguishing between these two lesions. Although SATB2 immunoexpression helps to distinguish osteosarcoma from their mimickers, the identification of malignant osteoid matrix formation and the integration of clinical and radiological data remain the corner stone of osteosarcoma diagnosis and as yet no antibody has equalled the diagnostic value of this important morphologic hallmark.

Abstract A12: A retinoblastoma protein phosphorylation code associated with cell adhesion and invasiveness

Abstract Lung cancer remains the leading cause of cancer deaths among men and women worldwide. Its five-year survival rate (17.8%) is by far the lowest among the most common cancers such as colon (65.4%), breast (90.5%), and prostate (99.6%). Early detection and screening still remain challenging, and despite the many recent breakthroughs in lung cancer treatment, improvement in the long-term survival of lung cancer patients is still limited. The inactivation of the retinoblastoma tumor suppressor (Rb) is a major driving force behind tumorigenesis in most cancer types, including lung cancer. Rb is a phosphoprotein traditionally known as a cell cycle repressor. While Rb inactivation in small cell lung carcinomas, osteosarcomas and retinoblastomas occurs by mutational inactivation of the RB1 gene, most other human cancers show Rb inactivation by hyperphosphorylation. The effect of Rb hyperphosphorylation in cell cycle control has been characterized and shown to consist in the abrogation of Rb’s capacity to bind and repress the EF2 transcription factors responsible for triggering S-phase related gene expression. However, our data show that Rb phosphorylation can have novel consequences beyond cell cycle control, and that certain phosphorylations in Rb can impair the cell’s capacity to engage in cell-to-cell interactions. In order to search for phosphorylation patterns that may be associated with cell adhesion defects, we conducted a mass spectroscopy analysis on a panel of E-cadherin-expressing and E-cadherin-deficient cell lines. This analysis identified a four-residues phosphorylation signature (S612, S249, S807 and S821) that is associated to lack of E-cadherin expression. These four residues are strongly phosphorylated in the E-cadherin null cell line H520, relative to the E-cadherin expressing cell line H1666. Consistent with these data, H520 cells overexpress the Cyclin-dependent kinases Cdk2 and Cdk6, both of them known to have Rb as one of its targets. Treatment with CDK inhibitors (Roscovitine, PD00332991, and GW8510) resulted in inhibition of Cdk2, Cdk5, Cdk7, and Cdk9 together with reduced the phosphorylation of Rb S807 and S612, and with the reestablishment of E-cadherin expression. Moreover, shRNA-mediated Rb knockdown caused a marked induction of EMT in our lung cancer cell lines, indicated by decreased E-cadherin expression and elevated N-cadherin levels. Taken together, our data show a Rb phosphorylation signature associated with a non-traditional role for Rb as a regulator of cell adhesion. Given that Rb phosphorylation in specific residues can lead to loss of E-cadherin expression and concomitant loss of cell adhesion, both of them landmarks of the metastatic phenotype, we postulate that this phosphorylation signature may have prognostic value in predicting a proclivity for metastasis of lung cancer cells. Citation Format: Jaileene Perez-Morales, Jonathan Gonzalez-Flores, Alexander De La Rosa, Elvin Estrada, Pedro Santiago-Cardona. A retinoblastoma protein phosphorylation code associated with cell adhesion and invasiveness. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A12.

Establishment and Characterization of a Human Small Cell Osteosarcoma Cancer Stem Cell Line: A New Possible In Vitro Model for Discovering Small Cell Osteosarcoma Biology.

Osteosarcoma (OSA) is the most common primary malignant bone tumor, usually arising in the long bones of children and young adults. There are different subtypes of OSA, among which we find the conventional OS (also called medullary or central osteosarcoma) which has a high grade of malignancy and an incidence of 80%. There are different subtypes of high grade OS like chondroblastic, fibroblastic, osteoblastic, telangiectatic, and the small cell osteosarcoma (SCO). In this study, for the first time, we have isolated, established, and characterized a cell line of cancer stem cells (CSCs) from a human SCO. First of all, we have established a primary finite cell line of SCO, from which we have isolated the CSCs by the sphere formation assay. We have proved their in vitro mesenchymal and embryonic stem phenotype. Additionally, we have showed their neoplastic phenotype, since the original tumor bulk is a high grade osteosarcoma. This research demonstrates the existence of CSCs also in human primary SCO and highlights the establishment of this particular stabilized cancer stem cell line. This will represent a first step into the study of the biology of these cells to discover new molecular targets molecules for new incisive therapeutic strategies against this highly aggressive OSA.

Synchronous multifocal osteosarcoma with small cell histological variant: A double rarity

Multifocal osteosarcoma (MFOS), osteosarcoma involving multiple sites, is a rare variant of osteosarcoma. When the lesions appear within 6 months of initial presentation of the tumor, it is known as synchronous MFOS. Synchronous MFOS has an incidence of 1%–3% only. Moreover, the histological variant of small cell osteosarcoma is even rarer. A case of 14-year-old male with synchronous MFOS of small cell type involving frontal and mandibular bone simultaneously is being reported here. It poses a dilemma to both the clinician and the pathologist to diagnose whether it represents multiple primary tumor or metastatic disease.

A Rare Case of Extraskeletal Small Cell Osteosarcoma: Cytohistologic Correlation and Review of the Literature

Extraskeletal small cell osteosarcoma is a rare soft-tissue tumor with production of osteoid by cytologically malignant small cells. Only six cases have been reported in the literature, two of which have cytologic description. We report a case of a 78-year-old man with a left axillary mass. …

Abstract 1625: Ureidomustine, a novel DNA-crosslinking agent shows activity in sarcoma preclinical models and lacks toxicity in normal tissues

Abstract Ureidomustine (BO-1055) is a water-soluble N- mustard derivative that has antitumor activity against a number of human tumors including prostate, colon cancers and gliomas. In this study, we investigated its pre-clinical activity in sarcoma models. We tested the activity of BO-1055 on early passages from Ewing sarcoma patient samples and patient-derived xenografts, preclinical models of rhabdomyosarcoma, desmoplastic small round cell tumor (DSRCT) and osteosarcoma. We used cell proliferation, viability, clonogenicity and cell death assays, and immunoblotting for differential expression of proteins in cells that are sensitive and resistant to BO-1055. We studied the toxicity of BO-1055 on cardiac myocytes, hematopoietic stem and progenitor cells (HSPCs), epithelial, mesenchymal and endothelial cells, and performed animal toxicity experiments in mice. Efficacy of BO-1055 was tested in NSG mice bearing A673 xenografts (Ewing sarcoma) treated at doses of 10mg/kg, 20mg/kg and 30mg/kg, Q2D×5, via i.v. injection after the tumors grew to 250-500 mm3. We also investigated the efficacy of this agent in NSG mice bearing A204 xenografts and Ewing sarcoma patient-derived xenografts that were resistant to cyclophosphamide. The antitumor effects of BO-1055 in combination with standard chemotherapeutic agents were also evaluated. Ureidomustine exhibited significant cytotoxicity against the in vitro growth of Ewing sarcoma and rhabdomyosarcoma with sub μM IC50 values and was more cytotoxic than conventional DNA damaging agents. BO-1055 had less cytotoxicity against a panel of normal HSPCs. BO-1055 had moderate activity (IC50 = 2-4 μM) on DSRCT cells and had only weak activity (IC50>10μM) against osteosarcoma cell lines. Notably, this agent has no cross-resistance to Taxol and Vinblastine. Growth arrest in G2/M phase was noted at 24h and maximal apoptosis and cell death at 48h and 72h after exposure of A673 cells. Apoptosis was due to the induction of caspase 3 and 7 activity in a dose- and time- dependent manner. Cardiotoxicity was evaluated by binding of BO-1055 to hERG, which was significantly less than that of Astemizole indicating that BO-1055 has less propensity for QTc prolongation. BO-1055 significantly suppressed Ewing sarcoma patient-derived xenografts that were resistant to cyclophosphamide. Remarkably, this agent induced complete suppression of tumors in nude mice bearing A204 xenografts, without significant weight loss. In two-drug combination studies, BO-1055 exhibited synergism with etoposide, SN-38 (active metabolite of irinotecan), doxorubicin and PU-H71 (HSP-90 inhibitor). Because of its broad therapeutic window, lack of cross-resistance, better safety profile and synergistic cytotoxicity with standard drugs, BO-1055 has high potential for clinical application for treatment of Ewing sarcoma and rhabdomyosarcoma patients. Citation Format: Srikanth R. Ambati, Shieh JaeHung, Benet Pera, Elissa W.P. Wong, Eloisi Caldas Lopes, Elizabeth Peguero, Tsann-Long Su, Malcolm A.S. Moore. Ureidomustine, a novel DNA-crosslinking agent shows activity in sarcoma preclinical models and lacks toxicity in normal tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1625. doi:10.1158/1538-7445.AM2015-1625

Small Cell Osteosarcoma

Small round cell osteosarcoma is a very rare type of osteosarcoma, histologically mimicking other small round cell malignancies of bone, most notably Ewing sarcoma. To distinguish small cell osteosarcoma from other primary small cell malignancies of bone, we evaluated the immunohistochemical (IHC) expression of CD99 and SATB2, a marker of osteoblastic differentiation. Second, we analyzed EWSR1 and FUS gene aberrations using fluorescence in situ hybridization and/or reverse transcription-polymerase chain reaction (RT-PCR) techniques to assess whether small cell osteosarcoma and Ewing sarcoma share the same genetic alteration analysis. Thirty-six cases of primitive small cell osteosarcoma of bone were included in this study. All the cases of small cell osteosarcoma showed strong nuclear expression of SATB2 associated with negativity for CD99 antibody or weak, cytoplasmic staining in few neoplastic cells. Reverse transcription-polymerase chain reaction was negative for EWS-FLI1 type 1-2, EWS-ERG type 1, and CIC-DUX4 in the 10 available cases of small cell osteosarcoma analyzed. Fluorescence in situ hybridization analysis was feasible with a readable signal in 13 cases of small cell osteosarcoma, and none of these cases showed any EWSR1 and FUS gene rearrangements. In conclusion, it appears extremely useful to combine IHC analysis of SATB2 and CD99 with molecular analysis of Ewing sarcoma-associated genetic aberrations, to differentiate small cell osteosarcoma from other small round cell malignancies of bone. The strong IHC expression of SATB2 associated with CD99 immunonegativity and the absence of EWSR1 and FUS gene rearrangements in small cell osteosarcoma argues against the existence of a morphologic/genetic continuum with Ewing sarcoma.

Utility of transmission electron microscopy in small round cell tumors.

Small round cell tumors (SRCTs) are a heterogeneous group of neoplasms composed of small, primitive, and undifferentiated cells sharing similar histology under light microscopy. SRCTs include Ewing sarcoma/peripheral neuroectodermal tumor family tumors, neuroblastoma, desmoplastic SRCT, rhabdomyosarcoma, poorly differentiated round cell synovial sarcoma, mesenchymal chondrosarcoma, small cell osteosarcoma, small cell malignant peripheral nerve sheath tumor, and small cell schwannoma. Non-Hodgkin’s malignant lymphoma, myeloid sarcoma, malignant melanoma, and gastrointestinal stromal tumor may also present as SRCT. The current shift towards immunohistochemistry and cytogenetic molecular techniques for SRCT may be inappropriate because of antigenic overlapping or inconclusive molecular results due to the lack of differentiation of primitive cells and unavailable genetic service or limited moleculocytogenetic experience. Although usage has declined, electron microscopy (EM) remains very useful and shows salient features for the diagnosis of SRCTs. Although EM is not always required, it provides reliability and validity in the diagnosis of SRCT. Here, the ultrastructural characteristics of SRCTs are reviewed and we suggest that EM would be utilized as one of the reliable modalities for the diagnosis of undifferentiated and poorly differentiated SRCTs.