HLA typing of patient samples was performed at the Puget Sound Blood Center (PSBC). The interventional and pilot studies using either NY-ESO-1-specific T cells or IFN-γ were registered with clinicaltrials.gov.

Leukapheresis was performed at either PSBC or at the University of Washington (UW) General Clinical Research Center if the patient expressed HLA A*0201, had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1, and if tumor biopsies stained positive for NY-ESO-1 in >25% of cells by immunohistochemistry (IHC). Subsequent processing of leukapheresed product was performed at FHCRC, including the cell processing facility (CPF), under current good manufacturing practice (GMP) guidelines. This form of ACT using peripheral blood as a source of antigen-specific T cells is known as endogenous T-cell (ETC) therapy and yields a T-cell product of near uniform specificity with central memory properties.10 ETC has been used in first-in-human studies targeting tumor-associated antigens with evidence of clinical efficacy.11 12

To facilitate detailed functional analysis, an aliquot of T-cell product was removed from the CPF and then expanded in the research laboratory. In vitro assays were used to assess recognition of target cells pulsed with various peptide concentrations and to assess cytokine production secondary to antigen stimulation. Cell surface phenotype and T cell receptor beta chain variable region (TCR Vβ) sequencing were performed on aliquots from the second expansion.13 Specificity of expanded T-cell products was confirmed using chromium release assays performed on day 12 of the rapid expansion protocol. T2 target cells were cultured alone for 2 hours or in media with target peptide; when relevant, the HLA A*0201+, NY-ESO-1+tumor line, Mel A375 (A375), was also used as target cell.

Cell surface phenotype of persisting cells was determined by costaining with NY-ESO-1 tetramer (FHCRC, Seattle, Washington, USA) and fluorochrome-conjugated monoclonal antibodies (mAbs). Multicolor flow cytometry data were collected using a LSRII (Becton Dickinson, Franklin Lakes, New Jersey, USA) and FlowJo software. For staining, cells were fixed and stained for CD8 (BD Bioscience, Franklin Lakes, New Jersey, USA).

TCR Vβ sequencing and normalization was performed on suspensions of tumor-infiltrating lymphocytes, as well as paraffin-mbedded specimens by Adaptive Biotechnologies (Seattle, Washington, USA). The number of total cells, T cells, T-cell fraction, the number of unique rearrangements, and clonality were calculated for each sample as previously described.14

Immunohistochemical analyses of myocardium for CD3, CD4, CD8, CD20, and CD68 expression were performed using an automated immunostainer (Bond III) and the following mAbs: CD3 (clone LN10, dilution 1:100; Novacastra, Newcastle, UK), CD4 (clone SP35, dilution 1:25; Cell Marque, Rocklin, California, USA), CD8 (clone 4B11, dilution 1:200; Novacastra), CD20 (clone L26, dilution 1:800; Dako, Glostrup, Denmark), and CD68 (clone Kp-1, dilution 1:1000; Cell Marque). Tonsils were used as positive controls.

Immunohistochemical expression to confirm eligibility of patients for treatment with NY-ESO-1-specific T cells was performed at the UW Pathology Lab with mAb specific for NY-ESO-1 (1:100, E978 clone; Life Technologies, Grand Island, New York, USA), CD3 (clone LN10, dilution 1:200; Novacastra), and MHC class I (1:8000, EMR8-5 clone; Abcam, Cambridge, Massachusetts, USA).

IFN#1 was a man in his mid-40s with intractable pain in his left shoulder. He visited a local emergency department, and an investigative chest X-ray showed a hilar mass. Subsequent CT of the chest, abdomen, and pelvis again demonstrated the hilar mass, along with a 2.5×3.6 cm pelvic mass. Biopsy showed diffuse expression of Bcl-2, vimentin, CD99, and neoplastic cells expressing TLE-1, indicative of SS. He received two cycles of doxorubicin and ifosfamide, with disease progression. Next, he received two cycles of trabectedin, again with disease progression (table 1).

Multiple brain metastases were identified, and the patient underwent gamma knife radiation. Given that his tumor demonstrated strong homogenous NY-ESO-1 expression and that he was HLA-A0201+, he was enrolled in the ACT trial.

Despite the aggressive metastatic chemoresistant disease, 4 weeks after T-cell infusion, all six lung tumors showed a measurable decrease in size. A hilar lymph node decreased from 3.0×3.0 cm to 1.9×3.0 cm, and the small pleural-based lung nodules also decreased in size. The most dramatic response was in a large right upper lobe (RUL) tumor that previously received 3600 cGy of radiation 3 months prior to ACT. This mass was 10.8×8.1 cm prior to radiation and remained stable at 10.4×8.0 cm 3 months after radiation therapy. However, 4 weeks after T-cell therapy, the tumor decreased markedly in size to 6.9×5.3 cm, and by 10 weeks to 6.9×4.5 cm (figure 2B). Importantly, biopsy of lung lesion pretreatment and forehead lesion post-treatment were stained for NY-ESO-1 and MHC class I. The post-treatment sample showed both increased NY-ESO-1 staining (figure 2D) and increased MHC class I expression (figure 2E). No lung tumors (including subcentimeter nodules) had progressed 4 weeks after T-cell therapy. Subjectively, he reported a marked improvement in fatigue and respiratory status following ACT and even returned to work after missing weeks because of fatigue. He experienced common self-limited toxicities of HD Cy (leukopenia, anemia, malaise, and mild mucositis), IFN-γ and IL-2 (fatigue, malaise, and myalgias), but no unexpected toxicities.

Prior to T-cell infusion, NY-ESO-1 tet⁺ T cells were undetectable in the blood (<0.01% of CD8+ cells). Four weeks after infusion, following complete reconstitution of the absolute lymphocyte and white blood cell counts and cyclophosphamide conditioning, NY-ESO-1 tet⁺ T cells represented 5% of all peripheral CD8+ T cells (figure 2A). At 10 weeks postinfusion, this cell population remained detectable.

Despite the marked response of the pulmonary metastases, the overall response was mixed as a soft tissue lesion on his forehead progressed 10 weeks after ACT (figure 2C). To investigate the lack of response at some tumor sites, his forehead lesion was excised. The pathology demonstrated homogenous staining of NY-ESO-1 by IHC, but few infiltrating T cells were found and the tissue lacked MHC class I expression (data not shown), which was thought to be responsible for the lack of response and may indicate that the impact of IFN-γ in the tumor microenvironment is not durable.

The IFN#2 was a man in his mid-40s who first noticed lower back pain. His pain progressed, and a small lump eventually developed. MRI at an outside hospital showed a right flank mass measuring 7.8×8.7×12.9 cm. Subsequent biopsy demonstrated poorly differentiated SS. He underwent six cycles of neoadjuvant doxorubicin with ifosfamide and vincristine, followed by surgical resection. He also received radiation and concurrent ifosfamide for two cycles after the surgical resection. Unfortunately, 2 years later during routine surveillance, lung metastases were discovered in both lungs. After discussing multiple treatment options, the patient elected for ACT.

Before trial enrollment, his poorly controlled type 2 diabetes was noted with a hemoglobin A1c of 10.1%, complicated by diabetic nephropathy and a baseline serum creatinine of 1.4 mg/dL (1.5 mg/dL was the trial max). Prior to ACT, a cardiac stress echocardiogram was performed and deemed normal. His ECOG performance status was 1.

On trial initiation, he experienced no symptoms from the single-agent IFN-γ and tolerated Cy well. He had no immediate complications during T-cell infusion. Two days following T-cell infusion, he reported fatigue and was found to have an elevated creatinine of 1.9, attributed to Cy and an underlying renal dysfunction. He received intravenous hydration with normal saline in the outpatient infusion center, and his creatinine returned to baseline. On the evening between days 3 and 4 post-T cell infusion, he developed malaise, a cough with pink-tinged sputum, and a fever to 101°F. CBC performed earlier that day confirmed an appropriate neutrophil count. He took acetaminophen at home and elected not to go to the emergency room, with planned follow-up at the clinic in 1–2 days. On day 4 after T-cell infusion, he suddenly lost consciousness and was found in cardiac arrest. Resuscitation was attempted but was unsuccessful.

Autopsy revealed disease that was more extensive than originally appreciated on CT with innumerable tiny (<0.5 cm) bilateral lung metastases. Autopsy also showed a myocarditis with an immune infiltrate primarily composed of CD68+ cells (figure 3D) and CD3+ cells (figure 3B). Almost no CD4+ or CD8+ cells were detected (data not shown). Since the infusion product was greater than 98% CD8+, his myocarditis was attributed to the conditioning regimen, further complicated by his pre-existing comorbidities. T-cell receptor (TCR) sequencing from myocardial tissue was attempted, but an insufficient number of reads were observed. This was consistent with the IHC that showed only a small fraction of infiltrating T cells, again suggesting that T cell-induced myocardial toxicity was not the cause of death. We also confirmed that the heart tissue did not express NY-ESO-1 and concluded that the addition of IFN-γ to HD Cy and low-dose IL-2 as administered in this regimen was not safe, and this cohort was terminated.