Abstract: Deletions of the spinal muscular atrophy (SMA)-determining gene, SMN1, NAIP, and a third multicopy gene, BTF2p44tel were investigated in 60 unrelated Turkish SMA patients. SMN1 was deleted for at least exons 7 and 8 in 85% of the Turkish SMA patients. The NAIP gene was deleted in 75 and 33% of type I and type II SMA patients, respectively. Analysis of the 5′end of the BTF2p44tel gene indicated the extension of deletion in 13.3% of the cases, mainly in type I patients. Deletions of the NAIP and BTF2p44tel genes were detected in 1.3 and 3.9% of carrriers, respectively, in Turkish SMA families. Two patients were detected to harbor the hybrid SMN gene, one type II with deletion of the NAIP gene, and one type III without deletion of the NAIP gene.

Abstract: Mutations of the TP53 gene have been associated with resistance to chemotherapy as well as poor prognosis in many different malignancies. This is the first prospective study of the prognostic value of somatic TP53 mutations in patients with newly diagnosed extremity osteosarcoma. Patients and Methods One hundred ninety-six patients with high-grade, nonmetastatic osteosarcoma of the extremities were enrolled from seven tertiary care institutions and observed prospectively for tumor recurrence (median follow-up duration, 44 months). All patients received neoadjuvant or adjuvant chemotherapy and surgery. Tumors were analyzed for the presence of TP53 mutations by polymerase chain reaction single-strand conformation polymorphism analysis and direct DNA sequencing. The association of the status of the TP53 gene with the risk of systemic recurrence was examined using survival analyses with traditional and histologic markers as prognostic factors. Results Patient age was the only factor that varied with TP53 gene status (P = .05). No relationship was identified between TP53 status and systemic relapse (relative risk, 1.24; P = .41). Analyses based on missense or nonsense mutations gave similar results (P 〉 .10). In multivariate analysis, large ( 〉 9 cm) tumor size (relative risk, 1.9; P = .006) and poor histologic response (≤ 90% necrosis) to chemotherapy (relative risk, 2.14; P = .02) were the only significant independent predictors of systemic outcome. Conclusion We found no evidence that TP53 mutations predict for development of metastases in patients with high-grade osteosarcoma. Identification of other genes that influence chemotherapy response and clinical outcome in osteosarcoma is needed to facilitate further improvements in patient outcomes.

Abstract: Sarcoma is a group of rare bone and soft tissue tumors with over 50 distinct subtypes. Survival rate ranges widely due to the lack of efficacious treatments. Immunotherapy, such as adoptive cell therapy (ACT), has drawn significant interest due to its minimal toxicity. In ACT, tumor infiltrating lymphocytes (TILs) are isolated from patients, expanded, and autologously infused back. We recently observed TILs’ presence in Undifferentiated Pleomorphic Sarcoma (UPS) and Myxofibrosarcoma (MFS) tumors and found that tumor’s PD-L1 overexpression is correlated with better clinical outcome in UPS but not MFS. 1 The Th1 anti-tumoral inflammatory pathway was highly activated in the former cohort, which may explain the favorable outcome. We hypothesize that there are phenotypic and functional differences between TILs of UPS with differential PD-L1 expression, which may be related to clinical outcomes. However, sarcoma TILs are rare and challenging to culture, which significantly impedes their studies. We first aim to robustly expand sarcoma TILs to sufficient numbers. Methods Tumors’ PD-L1 expression was determined by RT-qPCR (table 1). To initiate the tumor-fragment (TF) method of TIL culturing, primary tumors were fragmented and cultured in IMDM, IL-2, and 10% HSA. We further optimized the TF protocol to expand rare sarcoma TILs. Rapid expansion protocol (REP) with anti-CD3/anti-CD28 co-stimulating beads was employed for additional expansion. During REP, TILs were co-treated with gamma-chain cytokines (IL-2, 7, 15, 21). Results Of the 15 MFS TIL populations expanded, only 40% achieved sufficient growth (1x10 6 ) for analysis (figure 1A). Our optimized TF protocol expanded TILs from 8 UPS cases with a 62.5% success rate (figure 1B). UPS TILs were further stimulated with REP and various gamma-chain cytokine treatments. In ACT, prolonged culturing with IL-2 is known to cause activation-induced cell death, problematic in clinical treatments. We demonstrated that treatments with a Trio-cocktail (IL-7, 15, and 21) or IL-15 alone can achieve TIL proliferation comparable to that of IL-2 (figure 2). Abstract 168 Figure 1 Initial TIL Culturing with Tumor Fragment Method. Figure A.. The traditional tumor fragment protocol was used to expand TILs of four MFS cases. TILs were cultured and expanded from primary tumor fragments in IL-2 (6000IU/mL) supplemented complete media (CM) over four weeks in duration. Fifteen TIL populations were derived from the four MFS cases. Populations were categorized based on their growth rates and labeled as ‘1’ or ‘2’ representing ‘fast’ or ‘slow’ growing TILs, respectively. Additional populations ‘A’ and ‘B’ represent biological replicates. Population TIL164 ‘2’ had no replicates. At Week 4, populations’ cell counts were determined via hemocytometer. As shown, only 6 out of 15 populations achieved 〉 1x10^6 cells (40% success rate). Figure B. An optimized tumor fragment protocol was used to expand TILs of eight UPS cases. Optimization includes shortening the culturing duration from four weeks to two weeks, reducing frequency of cell culture disruption, and adjusting cell culture environments. TILs were expanded from primary tumor fragments in CM over two weeks in duration. At Week 2, populations’ cell counts were determined via hemocytometer. As shown, 5 out of 8 cases achieved 〉 1x10^6 cells (62.5% success rate). Abstract 168 Figure 2 Gamma-chain cytokine treatments of UPS TILs. Gamma-chain cytokine treatments of UPS TILs with CD3/CD28 stimulation. Magnetic Dynabeads coated with CD3 and CD28 monoclonal antibodies were used to stimulate cells at a bead to cell ratio of 1:3. In ACT, IL-2 is a gold-standard cytokine that facilitates potent T-cell growth. However, it is known to cause activation-induced cell death. Resulting TIL population also possesses an exhausted-effector phenotype with low durability. UPS TIL Case 52 and Case 166 were treated with various interleukins during two weeks of REP, including gamma-chain IL-7, 15, 21 and inflammatory IL-12. IL-7, IL-12, and IL-21 individually did not elicit significant T-cell growth. IL-15 alone and in combination with IL-7 and IL-21 yield growth comparable to IL-2. IL-2 was obtained from Novartis (50ng/mL). All other cytokines were obtained from PeproTech (25ng/mL). Abstract 168 Table 1 Tumor PD-L1 RNA Expression. Four MFS and eight UPS cases were processed. Tumors’ PD-L1 RNA expression was determined via RT-qPCR and evaluated as a ratio with the housekeeping gene STAM2. TIL359’s PD-L1 status has yet to be evaluated. Conclusions Sarcoma infiltrates are difficult to culture, and their roles remain largely unstudied. By optimizing the TF protocol in conjunction with anti-CD3/CD28 treatments, we developed a robust in vitro pipeline to expand rare sarcoma TILs, enabling downstream characterization. We also demonstrated the potential for alternate gamma-chain cytokines to favorably replace IL-2 during TIL expansion. Future phenotypic and functional evaluation of UPS TILs would elucidate the impact of tumors’ differential PD-L1 expression on UPS patients‘ prognoses. These findings would inform the implementation of ACT in sarcoma treatments. References Wunder J, Lee M, Nam J, Lau B, Dickson B, Pinnaduwage D, Bull S, Ferguson P, Seto A, Gokgoz N, Andrulis I. Osteosarcoma and soft-tissue sarcomas with an immune infiltrate express PD-L1: relation to clinical outcome and Th1 pathway activation. Onco Immunology 2020; 9 : e1737385-1- e1737385-13. Ethics Approval Patients provided signed consent before study entry, as approved by Mount Sinai Hospital’s Ethics Board (REB#01-0138-U).

Abstract: Objectives: Sarcomas are mesodermal cancers of bone and soft tissue of which there are & gt;60 malignant varieties, many of which can be difficult to diagnose or subtype using traditional histopathology. A universal molecular definition of sarcoma types would therefore be an invaluable tool to the diagnostic pathologist. RNA has the potential to offer a complimentary perspective to cytogenetic- and methylation-based diagnostics, as it represents the active state of the disease at sampling and better reveals its phenotype. Recognizing the potential for RNA-based classification, we set out to create a first-generation transcriptional atlas of sarcoma. Methods: To develop transcriptional definitions of cancers with the potential to further subclassify tumor types, we designed a self-optimizing and scale-adaptive unsupervised method (RACCOON), which groups samples into hierarchically organized clusters. We used this approach on the UCSC Treehouse Childhood Cancer Compendium, a set of 2,178 pediatric and 9,400 adult tumors, 1,130 of which are sarcomas, as well as 1,735 non-neoplastic samples. We then trained an ensemble of convolutional neural networks to classify tumors to these transcriptional clusters. We have now added 624 more sarcoma samples from Toronto centers and international collaborators to better represent the breadth of sarcoma. We are actively sequencing 500 additional samples in partnership with the Gabriella Miller Kids First Research Program to yield an expanded cohort of & gt;2,200 uniformly processed and analyzed sarcomas. Results: Sarcomas organize into two clusters at the highest hierarchical level: one characterized by entities which occur primarily in adults and resemble mature tissue, the other by primarily pediatric entities which exhibit high stemness and resemble embryonic tissue. Several included entities are not bona fide sarcomas but originate from the mesoderm (e.g., Wilms Tumor) signifying a common transcriptional identity for mesodermal neoplasms. Additionally, we demonstrate the first transcriptional subtypes of central osteosarcoma reflecting its major histotypes and representing divergent clinical courses. We also determine Ewing Sarcoma (ES) to be a distinct entity which clusters separately from all other cancers, raising questions of its origin and affinity to sarcoma. When classifying ongoing patients to the atlas, we correctly classified & gt;85% of tumors and corrected the diagnosis of 7%. We find 14% of ES in our dataset were likely misdiagnosed CIC- or BCOR-driven sarcomas. Critically, assigned subtypes are consistent between primary and relapse pairs. Conclusion: RNA-seq is a promising tool for both subtype discovery and classifying sarcoma in ongoing patients. We have already included this tool in tumor boards to help inform patient care. Our method reveals the overarching organization of sarcoma for the first time and specifies its underlying biology. This atlas is ever-growing and is open to the community to contribute. Citation Format: Joshua O. Nash, Federico Comitani, Rose Chami, Sarah Cohen-Gogo, Astra Chang-Schwertschkow, Yael Babichev, Jodi Lees, Noa Alon, Nalan Gokgoz, Stephen Man Yu, Kyoko Yuki, Miranda Lorenti, Zhanqin Liu, Alaina McGoey, Famida Spatare, Bernarld Castro, Kim Tsoi, Hagit Peretz Soroka, Jack Brzezinski, Anita Villani, Albiruni Razak, Abha Gupta, Elizabeth Demicco, Gino Somers, Brendan C. Dickson, Jay S. Wunder, Irene L. Andrulis, David Malkin, Rebecca A. Gladdy, Adam Shlien. The development of a multiscale transcriptional atlas of sarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B027.