Expansion of tumor-infiltrating and marrow-infiltrating lymphocytes from pediatric malignant solid tumors

Abstract

Introduction: The expansion of tumor-infiltrating lymphocytes (TIL) for adoptive cellular therapy is under investigation in many solid tumors of adulthood. Marrow-infiltrating lymphocytes (MIL) have demonstrated antitumor reactivity preclinically. Successful expansion of TIL/MIL has not been reported across pediatric solid tumor histologies. The objective of this study was to demonstrate successful expansion of TIL from pediatric solid tumors for translation in an adoptive cell therapy (ACT) treatment strategy.

Methods: A prospective study of TIL/MIL expansion was performed on solid tumors of pediatric patients undergoing standard-of-care procedures. TIL/MIL expansions were performed in the presence of high-dose interleukin 2. To demonstrate a full-scale expansion to clinically-relevant cell doses for TIL therapy, initial TIL culture was followed by a rapid expansion protocol for select patients. Expanded specimens were analyzed for phenotype by flow cytometry and for anti-tumor reactivity by the interferon-gamma release assay.

Results: Eighteen tumor samples were obtained. Initial TIL cultures were successfully generated from 14/18 samples (77.7%). A median of 5.52 × 107 (range: 2.5 × 106-3.23 × 108) cells were produced from initial cultures, with 46.9% expressing a CD3 phenotype (46.9%). Eight samples underwent rapid expansion, demonstrating a median 458-fold expansion and a CD3 phenotype of 98%. Initial MIL cultures were successfully generated from five samples, with a predominantly CD3 phenotype (45.2%). Sufficient tumor tissue was only available for seven TIL samples to be tested for reactivity; none demonstrated responsiveness to autologous tumor.

Conclusions: TIL and MIL expansion from pediatric solid tumors was successful, including the full-scale expansion process. This data supports translation to an ACT-TIL treatment strategy in the pediatric population and thus a Phase I trial of ACT-TIL in pediatric high-risk solid tumors is planned.

Keywords: cellular therapy; immunotherapy; pediatrics; solid tumors; tumor-infiltrating lymphocytes.

Fig. 1.

H&E photomicrographs of nongrowing (upper panels) and heavily growing (lower panels) TIL samples from tumor fragment culture. Upper left: GNBL, showing edge of less mature intermixed and stroma rich maturing tumor with no apparent lymphocytes. Upper middle: ASPS CNS metastasis, showing a highly cellular confluent epithelioid cells with no apparent lymphocytes. Upper right: OS metastasis in lung, showing cellular sheets of epithelioid osteogenic sarcoma cells with focal discernible osteoid matrix production and isolated stromal lymphoid cells (center) and adjacent lung (upper right). Lower left: IMT, showing sheets of myofibroblastic cells with vesicular nuclei, prominent nucleoli amid characteristic interstitial plasma cell and other lymphoreticular cell infiltrate (arrows). Lower middle: ERMS, showing embryonal cells with rhabdomyoblastic differentiation in myxoid and alternating cellular areas with isolated intercellular lymphoid cells (arrows). Lower right: OS post-therapy primary tumor, (insert with CD45 IHC) showing postchemotherapy osteogenic sarcoma, residual (10% viable) large neoplastic cells amid dense collagenous tissue and native woven bone spicule right lower corner. H&E, hematoxylin and eosin; IHC, immunohistochemistry; GNBL, ganglioneuroblastoma; ASPS, alveolar soft part sarcoma; CNS, central nervous system; OS, osteosarcoma; IMT, inflammatory myofibroblastic tumor; ERMS, embryonal rhabdomyosarcoma; Pre-REP, prior to rapid expansion.

Fig. 2.

Expansion and phenotypic characterization of lymphocytes from tumors and bone marrow of pediatric malignant solid tumors. (A) TIL and MIL expansion from initial culture (PreREP). (B) PreREP TIL expansion by age, diagnosis, and tumor acquisition site. (C) PreREP TIL expansion comparison among preteenager (12 and under) and teenager (13 and older) patients. (D) PreREP TIL and MIL cell viability. (E) Pre- and post-REP TIL Phenotype comparison. (F) Comparison of TIL and MIL preREP TIL among paired samples. (G) Pre- and post-REP MIL Phenotype comparison. (H) Fold change expansion of TIL and MIL PostREP, (I) cell viability obtained of TIL and MIL PostREP, (J) interferon-gamma concentration in supernatant of three (3) representative PreREP TIL samples when co-cultured with autologous tumor. (K) Co-culture assay positive control (anti-CD3/CD28 stimulation) for interferon-gamma release. PreREP, initial culture before rapid expansion; REP, rapid expansion protocol; PostREP, following rapid expansion; TIL, tumor-infiltrating lymphocytes; MIL, marrow infiltrating lymphocytes; OS, osteosarcoma; WT, Wilms tumor; NB, neuroblastoma/ganglioneuroblastoma; IMT, inflammatory myofibroblastic tumor; ASPS, alveolar soft part sarcoma; SS, synovial sarcoma; ERMS, embryonal rhabdomyosarcoma; ML, myxoid liposarcoma. Statistical significance was determined by two-way ANOVA; **P < .01***P < .001.