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.

References

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2. 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.

3. 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.

4. 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.

5. 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.