American Journal of Clinical Pathology Advance Access

Cytologic Evaluation of Tumor-Infiltrating Lymphocytes for Adoptive Cell Therapy Sara E Monaco, MD Department of Pathology, University of Pittsburgh Medical Center , Pittsburgh, PA Corresponding author: Sara E. Monaco, MD; Department of Pathology, University of Pittsburgh Medical Center , Pittsburgh, PA Department of Pathology, University of Pittsburgh Medical Center , Pittsburgh, PA Department of Pathology, University of Pittsburgh Medical Center , Pittsburgh, PA Udai S Kammula, MD Solid Tumor Cellular Immunotherapy Program, Hillman Cancer Center, Division of Surgical Oncology, University of Pittsburgh Medical Center , Pittsburgh, PA aqz195, https://doi.org/10.1093/ajcp/aqz195 Sara E Monaco, Liron Pantanowitz, Juan Xing, Jackie Cuda, Udai S Kammula, Cytologic Evaluation of Tumor-Infiltrating Lymphocytes for Adoptive Cell Therapy, aqz195, https://doi.org/10.1093/ajcp/aqz195 Novel immunotherapeutic options for refractory metastatic cancer patients include adoptive cell therapies such as tumor infiltrating lymphocytes (TILs). This study characterizes the clinicopathologic findings in a cohort of TIL specimens. Methods Patients with metastatic malignancy who were eligible had TILs from their metastases grown and expanded and then sent to pathology. Results A total of 11 TIL specimens (10 melanoma, 1 adenocarcinoma) from patients enrolled in an experimental clinical trial were reviewed. All specimens showed more than 200 lymphoid cells, stained positive for lymphoid markers confirming an activated cytotoxic T-cell immunophenotype, and morphologically showed an intermediate-sized population with immature chromatin and frequent mitoses. Six cases (55%) showed large cells with nucleomegaly and prominent nucleoli. Conclusions This report is the first describing cytopathologic findings of autologous TIL therapy including adequacy guidelines and expected cytomorphologic and immunophenotypic findings. To meet this novel clinical demand, a predefined cytology protocol to rapidly process and interpret these specimens needs to be established. In the era of personalized medicine, there has been dramatic emphasis on new biomarker research to discover potential targets for treatment and to find markers associated with critical therapeutic or prognostic information for cancer patients. In addition, research has focused on biomarkers associated with the tumor microenvironment (TME) and inflammatory response to tumors. 1 These new biomarkers and novel therapies, including targeted nonchemotherapeutic therapy and adoptive cell therapy (ACT), are being used to manage a variety of cancer types, either in isolation or as part of combination therapy. 2-11 Accordingly, pathology laboratories are seeing increased demand for the evaluation of novel biomarkers (eg, PD-L1, CD8) so clinicians can make important management decisions and embark on individualized treatment plans for patients. 12-14 Malignant melanoma has been reported to have steadily increasing incidence 15 and has been evaluated in many trials looking at new immunotherapy and ACT with tumor-infiltrating lymphocytes (TILs). 6 , 16-19 The promising results from many of these studies has led to a great variety of treatment options compared with prior limited chemotherapeutic options. 1 , 6 , 16-19 Treatment options for melanoma traditionally consisted of conventional chemotherapy and high-dose interleukin 2 (IL-2) treatment. Around 2010, mutations in the gene encoding the protein kinase B-RAF (BRAF) were identified in the majority of melanoma patients, and this triggered routine testing of pathology specimens from melanoma patients to select patients eligible for BRAF inhibitors. 4 , 5 However, there were failed therapy responses due to acquired resistance to BRAF inhibitors alone or in combination with other therapies. 20 This paved the way for using new immunotherapy with monoclonal antibodies directed at cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed death 1 (PD-1), or programmed death ligand 1 (PD-L1), in addition to ACT options with TILs. 21 , 22 These new immunotherapy options have shown promising results in melanoma patients and provided more treatment options for patients with refractory disease. 23 TIL treatments were initially investigated in patients with metastatic cutaneous melanoma in a series of clinical trials at the National Institutes of Health (NIH) from 1990 to 2015 and showed objective response rates up to 72%, with up to 20% having complete remission and 40% to 50% having a durable clinical response. 16-19 , 23 TIL therapies were initially investigated in the 1970s using in vitro experiments showing that many tumors contained lymphocytes with antitumor activity; TIL therapies were particularly successful when accompanied by IL-2, a T cell growth factor, and more efficacious in attacking autologous tumor cells in a major histocompatibility complex (MHC)–related interaction compared with allogeneic tumor cells. 16 , 18 , 24 , 25 ACT with TIL has several scientific and practical advantages over active immunization and nonspecific immune stimulation. First, by isolating lymphocytes from the TME, immune cells with antitumor specificity can often be preferentially obtained. These T cells often recognize a broad array of tumor antigens including tumor-differentiation antigens, mutated tumor neoantigens, and cancer germ-line antigens. Second, ex vivo expansion of these T cells can be performed in the absence of host suppressive factors such as regulatory T cells, myeloid-derived suppressor cells, and inhibitory macrophages that are present in the TME. Finally, the host patient can be conditioned immediately before adoptive transfer with lymphodepleting regimens that can further eliminate endogenous suppressive influences and enhance the availability of homeostatic cytokines (eg, IL-7 and IL-15) to provide an optimal milieu for the transferred TILs to proliferate and function in vivo. More recently, these in vitro studies led to trials looking at metastasectomies, particularly in uveal melanoma patients, to generate a TIL-enriched specimen for autologous adoptive transfer of TILs in patients with refractory metastatic melanoma. 6 , 17 , 26 In response to these demands for enrolling patients on TIL therapy trials at our cancer center, the cytopathology laboratory worked with surgical oncology to create a workflow process for handling specimens that addressed turnaround and other trial needs. The aim of this study was to review our experience with incorporating autologous TIL therapy evaluation into our cytopathology laboratory and to report the cytomorphologic and immunophenotypic characteristics of TILs used for therapy. Materials and Methods Clinical trial coordinators obtained study materials, protocols, and institutional review board approval before patient enrollment. Informed consent was obtained from all participants. Patients with refractory metastatic cancer were then selected for potential enrollment into the clinical trial (NCT03467516, registered at ClinicalTrials.gov). They underwent metastasectomy for eligibility 26 after being seen and evaluated at the University of Pittsburgh Medical Center and Hillman Cancer Center, University of Pittsburgh, by the Department of Surgical Oncology. Clinical trial coordinators informed the Division of Cytopathology of the date that the TIL specimens would be delivered to the cytopathology laboratory. After surgical procurement of a metastatic lesion, the fresh tumor underwent sterile dissection in the Immune Monitoring and Cell Production Facility at the Hillman Cancer Center. A representative sample of tumor was sent for formal surgical pathology examination, whereas geographically discrete 1- to 2-mm 3 tumor fragments (n = 24) were placed individually in wells of a 24-well culture plate containing complete media with human AB serum and recombinant IL-2 (6,000 IU/mL). Remaining fresh tumor was processed by mechanical and enzymatic digestion to provide a single-cell suspension of autologous tumor targets for TIL reactivity testing. After 2 weeks of growth, individual fragment T-cell cultures were selected for further expansion based on proliferative capacity and evidence of autologous tumor reactivity using a standardized interferon-γ release assay. Final large-scale expansion of selected TIL cultures with the greatest antitumor reactivity was done with anti-CD3 antibody (OKT-3, 30 ng/mL; Miltenyi Biotec) and IL-2 (3,000 IU/mL) in the presence of irradiated (40 Gy) peripheral blood mononuclear feeder cells, as reported previously. 16 , 17 An “in-process” aliquot from each patient’s final expansion autologous TIL specimen (6×10 6 cells received in 50 mL of culture media) was delivered promptly to the cytopathology laboratory and processed on the day of receipt. These in-process specimens were drawn on the same manufacturing day in all specimens, in accordance with US Food and Drug Administration (FDA) requirements to verify the identity of the cell product before infusion. In addition, these cellular products were sent for microbial cultures to prove sterility, and underwent flow cytometry. Flow cytometry was performed to confirm that the cells in the cell product consisted of more than 80% CD3-positive T cells that were enriched in CD8-positive and activated T cells, 16 In the cytology laboratory, specimens were spun down into a cytospin that was stained with Diff-Quik, and the remainder was used to prepare a cell block with the HistoGel (ThermoFisher Scientific) method. The formalin-fixed, paraffin-embedded cell block had immunostains preordered to expedite processing and included a limited panel of stains (leukocyte common antigen [LCA], S100, SOX10, pancytokeratin, and cytokeratin CAM 5.2), with appropriate on-slide tissue controls. Four experimental samples from similarly processed patient expanded samples were used before patient enrollment to verify that processing and reporting were adequate and, in these experimental cases, that additional stains (CD3, CD20, CD4, CD8, TIA1, Epstein-Barr virus [EBV] latent membrane protein [LMP], terminal deoxynucleotidyl transferase [TdT], and CD30) were utilized. Table 1 shows the antibody source and clone information used in our laboratory. Table 1 Immunohistochemistry Methods: Antibody Details and Assay Condition Antibody



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