Background High-risk non-CNS pediatric malignant solid tumors (pMST) have unsatisfactory outcomes, and novel therapies are warranted. Adoptive cellular therapy (ACT) using tumor-infiltrating lymphocytes (TIL) has produced durable responses in melanoma, and improvements in TIL expansion have made ACT-TIL feasible for other solid tumors.1–3 Preclinical mouse models suggest that T-cells from bone marrow (marrow-infiltrating lymphocytes, MIL) have antitumor reactivity offering another source for ACT.4 5 To demonstrate feasibility of ACT in pMST we hypothesized that TIL/MIL can be expanded from these patients.
Methods Patients ≤21 years old undergoing standard-of-care pMST resection were enrolled on an IRB approved protocol. Fresh tumor (≥1 cm3) was collected and bone marrow (10 mL) was obtained when accessible from standard of care procedures. TIL/MIL were cultured in media containing IL-2 (6000 IU/mL). TIL were expanded from tumor fragment cultures (TFC, >1 mm3) or tumor digest. Select TIL samples were further expanded using a rapid expansion protocol (REP). Phenotype of expanded TIL (CD3, CD4, CD8 and CD56) was evaluated using flow cytometry. IFN- γ secretion, measured by ELISA assay, measured tumor-specific reactivity after co-culture with autologous tumor and TIL.
Results Twenty samples were obtained between March 2019-May 2021. Two samples were ineligible (final pathology not pMST), leaving 18 samples for analysis. Five marrow samples were collected. TIL were expanded from 14/18 samples (78%) through TFC with median 5.17 x 10^6 cells (range 1.86 x 10^6–3.21 x 10^8). Average phenotype (%) of TFC-TIL were CD3 (63.17), CD4 (21.46), CD8 (46.19) and CD56 (32.68). 9/10 (90%) of samples successfully underwent REP with median 9.35 x 10^7 cells(range 2.49 x 10^7–5.86 x 10^8) final viable TIL and average fold-change 718.6 (median 458.6). Average phenotype (%) of post-REP TIL were CD3 (96.04), CD4 (75.04), CD8 (19.17) and CD56 (0.43). TIL were expanded from TFC of therapy-naïve (8/10, 80%) and pretreated (chemotherapy and checkpoint immunotherapy) samples (5/8, 63%). Seven samples had sufficient tissue to test tumor-specific reactivity; all were non-reactive. MIL pre-REP was expanded from four samples with median 9.55 x 10^6 cells (range 8.00 x 10^5–1.00 x 10^7). Average phenotype of expanded MIL (%) were CD3 (45.17), CD4 (24.46), CD8 (36.15) and CD56 (28.21) (table 1).
Results of TIL and MIL expansion from 18 pMST samples. Abbreviations: Dx: diagnosis, pre-REP: pre-rapid expansion protocol, post-REP: post-rapid expansion protocol, PBMC: peripheral blood mononuclear cells, GNB: ganglioneuroblastoma, WT: Wilms tumor, OS: osteosarcoma, NB: neuroblastoma, IMT: inflammatory myofibroblastic tumor; ASPS: alveolar soft part sarcoma, SS: synovial sarcoma, ERMS: embryonal rhabdomyosarcoma, N: no systemic therapy, C: chemotherapy, I: immunotherapy, DNG: did not grow, N/A: not applicable, NR: non-reactive
Conclusions This study demonstrates feasibility of pMST TIL expansion ex vivo. Due to tissue volume constraints inherent in pMST sampling, anti-tumor reactivity testing was not feasible for most patients. Determining optimal strategy for TIL-ACT in pMST will require further investigation regarding techniques for expanding tumor-specific TIL.
Acknowledgements The authors would like to thank Swim Across America (www.swimacrossamerica.org) and the Ocala Royal Dames (www.ocalaroyaldames.org) for their generous support of this work.
Rosenberg SA, Restifo NP. Adoptive cell transfer as personalized immunotherapy for human cancer. Science 2015;348(6230):62–68.
Hall M, Mullinax JE, Royster E, et al. Expansion and characterization of tumor-infiltrating lymphocytes from human sarcoma. Journal of Immunotherapy of Cancer 2015;3(Suppl. 2):19.
Mullinax JE, Hall M, Beatty M, et al. Expanded tumor-infiltrating lymphocytes from soft tissue sarcoma have tumor-specific function. J Immunother 2021;44(2):63–70.
Feuerer M, Beckhove P, Bai L, et al. Therapy of human tumors in NOD/SCID mice with patient-derived reactivated memory T cells from bone marrow. Nat Med 2001;7(4):452–458.
Feuerer M, Rocha M, Bai L, et al. Enrichment of memory T cells and other profound immunological changes in the bone marrow from untreated breast cancer patients. Int J Cancer 2001;92(1):96–105.
Ethics Approval This study was approved by the Johns Hopkins All Children’s Hospital IRB (#IRB00193453). Consent was obtained from the patient or parent, as appropriate for age, prior to participating in this study.
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