Immune biomarkers of treatment failure for a patient on a phase I clinical trial of pembrolizumab plus radiotherapy

Pembrolizumab (MK-3475; Pembro) is a humanized IgG4 monoclonal antibody that is directed against the programmed cell death protein 1 (PD-1). PD-1 is expressed by T cells and interacts with its ligands programmed cell death protein ligand 1 (PD-L1) and programmed cell death protein ligand 2 (PD-L2), which are expressed in peripheral tissues and dendritic cells, respectively. PD-L1 expression is variable and involved in the prevention of autoimmunity; however, many tumors upregulate PD-L1 to mediate immune tolerance [1]. By blocking the PD-1 pathway, Pembro can enhance T cell-mediated killing of tumor cells [2]. It has been approved for the treatment of melanoma and non-small cell lung cancer; however, only 20–40 % of patients demonstrate clinical benefit with monotherapy [3‐5]. Radiation therapy (RT) is thought to have immunostimulatory effects by enhancing cancer antigen presentation [6, 7]. Because both modalities work through immune-mediated mechanisms, combination therapy has the potential to augment immune activation by PD-1 checkpoint inhibitors. Preclinical models have demonstrated synergy between RT and PD-1 blockade treatments [8], and several clinical trials combining RT with immunotherapy are underway [9]. A phase 1 clinical trial (NCT02318771) was designed to investigate the immunomodulatory effects of RT/Pembro combination treatment.

We report clinicopathologic and detailed flow cytometry data from a patient with metastatic renal cell carcinoma (RCC) who progressed through combined immunotherapy and radiation.

A 64-year-old Caucasian gentleman presented with flank pain and gross hematuria in February 2010. Initial work-up demonstrated a left renal mass, and a left radical nephrectomy confirmed clear cell RCC, Fuhrman grade 4, pathologically staged as T3 N0. Additionally, at diagnosis, small pulmonary nodules were found that were later confirmed to be RCC by Pax-8 staining (Fig. 3d). He was treated with sunitinib from March 2010 until July 2013, when disease progression occurred. The patient was then treated with axitinib, until progression and the development of additional pulmonary nodules in November 2014.

The patient was thereafter enrolled in a clinical trial (NCT02318771). The trial schema is illustrated in Fig. 1. The trial enrolls patients with metastatic lung cancer, melanoma, head and neck cancer, or RCC. The primary objective is to determine immune activation from single dose 8 Gy vs. fractionated palliative radiotherapy (4 Gy ×5), which is either preceded by one cycle of Pembro or followed by Pembro that continues until disease progression or grade 3 or greater toxicities. Our patient was randomized to group A2, (Fig. 2d) in which he was given a course of palliative RT dose (20 Gy in five fractions to his left chest wall lesion), (Fig. 3a and b) followed by adjuvant Pembro 200 mg once every 3 weeks intravenously. After 5 cycles of treatment, CT scans in May 2015 (Fig. 2b) and a confirmatory scan in June 2015 (Fig. 2c) revealed progression when compared to his baseline scans taken in February 2015 (Fig. 2a). Patient tolerated the combined therapy well without any toxicities and had no complaints of chest wall or sternal discomfort or respiratory symptoms although imaging studies demonstrated an increased size of the left chest wall lesion, multiple bilateral pulmonary nodules, and an anterior mediastinal mass accompanied with erosion of the sternum and the third, fourth, and fifth ribs, as well as new nodules that were not previously identified. As per protocol, the patient was removed from trial following disease progression. A biopsy of the chest wall lesion was performed to rule out possible pseudoprogression.

Peripheral blood draws and tumor biopsies of his target lesion were obtained before, after RT in early March, and at the time of progression in late June. Flow cytometry analysis of his circulating CD3⁺ T lymphocytes revealed a minor change in the proportion of CD8+ lymphocytes from baseline (28 %) compared to post-radiation (24 %) and post-Pembro treatment (24 %). However, due to a decrease in the numbers of circulating CD3⁺ T cells from 1.2 × 10⁶/ml before study to 0.9 × 10⁶/ml at the conclusion of the trial, there was an overall reduction in the number of circulating CD8 T cells by 40 %. This was accompanied by a somewhat lesser 22 % decrease in the numbers of circulating CD4+ T cells post-Pembro. Over the course of treatment, PD1 expression was strongly increased on subsets of circulating CD8⁺ and CD8⁻ CD3⁺ T cells (Fig. 4a, b). Changes in the expression of other immune checkpoint markers including Tim-3 and Lag-3 were also assessed by flow cytometry. Both were detected on circulating CD8+ cells, but there were no significant differences in expression between cells obtained before and after treatment noted (Fig. 4a, b).

There was an absence of tumor-infiltrating lymphocytes (TILs) on tumor biopsy by histological analysis. This finding was confirmed by RT-PCR analyses of tumor biopsies, which failed to detect CD4 or CD8 transcripts (Fig. 4d). RT-PCR also failed to detect TNF-alpha or CD11b transcripts before and after radiotherapy. PD-L1 transcripts were strongly upregulated in biopsy tissue following RT, which was confirmed by immunohistochemistry (Fig. 4c).

In conclusion, we hypothesize that the failure of T lymphocytes to migrate into malignant tissue may have played a role in treatment failure as both RT and Pembro act through T cell-dependent mechanisms. Further optimization of combined RT and immunotherapy requires a more in-depth understanding of the tumor microenvironment. In order to maximize anti-tumor activity, the presence of multiple, non-redundant checkpoint regulators, as well as the dose- and schedule-dependent immunomodulatory effects of RT, must be taken into consideration.