Clinical course

A 49-year-old man presented in February 2017 with chest wall pain and weight loss, leading to the diagnosis of a ccRCC with metastases to the lungs and a rib (Fig. 1). Based on his presentation with anemia, hypercalcemia, and the need for prompt initiation of systemic therapy, his International Metastatic Renal Cell Carcinoma Database Consortium (IMDC) scoring predicted a poor prognosis with a median survival of 7.8 months [8]. Initial management included a right radical cytoreductive nephrectomy, which required a partial hepatectomy. Pathological analyses showed a 9 cm ccRCC invading into the perirenal and renal sinus adipose tissue as well as the ipsilateral adrenal gland of ISUP grade 4 with extensive sarcomatoid differentiation. Eight out of eight lymph nodes were positive for metastatic disease. Staging was consistent with a pT4N1 tumor. IHC studies showed positivity for CK AE1/AE3, and CA-IX. CK7 was negative. PBRM1 and BAP1 were present suggestive of a wild-type state. PD-L1 was expressed in more than 30% of tumor cells. Given the age of the patient, germline testing was pursued using a CancerNext-Expanded genetic panel including genes such as VHL, BAP1, FLCN and PTEN, but did not reveal any mutations.

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Fig. 1

Clinical case. a Coronal contrast-enhanced CT images of a lytic metastasis in the left 10th rib (red arrow) before and after SABR and HD-IL2. b Axial contrast-enhanced CT image of new lytic metastasis in the right distal anterolateral femur (red arrow), which developed after SABR/HD-IL2 therapy. c Coronal proton density fat saturated MR imaging of an osseous metastasis in right glenoid (red arrow) that developed while on pazopanib therapy. d Clinical images illustrating radiation recall dermatitis 11 days after first nivolumab infusion at two prior sites of radiation, the left rib (A, radiated six months prior) and the right knee (B, radiated one month prior). Outlined is an area of subcutaneous edema and discoloration (C) attributed to drainage from lesion A. e Axial contrast-enhanced CT scan of the chest of representative lingular nodule (red arrow) improving with nivolumab therapy. f Hematoxylin and eosin stains of left colon biopsy with increased intraepithelial lymphocytes and cryptitis representative of autoimmune colitis

Within two hours of surgery, a sample of the patient’s tumor was implanted orthotopically into several NOD/SCID immunocompromised mice to generate a tumorgraft (or patient-derived xenograft, PDX) model (Fig. 2). RCC tumorgrafts have shown promise as models in preclinical experimentation preserving the molecular genetics and biology of the corresponding patient tumor [9]. The patient’s tumor was particularly aggressive and a renal mass could be palpated as early as 18 days post-implantation, which is unusual [10]. After 83 days, the tumor had reached 1500 mm³ and was passaged to subsequent cohorts. Histological characterization of the tumorgraft revealed preservation of the morphology of the patient’s tumor, with extensive sarcomatoid differentiation and high levels of PD-L1 expression by IHC (Fig. 2a).

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Fig. 2

Tumorgraft immunoPET studies. a Patient’s tumor (nephrectomy sample) and corresponding tumorgraft demonstrating sarcomatoid differentiation and high PD-L1 expression by IHC. b iPET from representative NOD/SCID mouse with subcutaneous tumorgraft. c-d Images (patient and tumorgraft) from papillary RCC tumor chosen as a control because of low PD-L1 levels. Tumor volumes shown for the individual mice are estimated based on the CT volume quantification of the tumors

One month from initial staging scans, repeat computed tomography (CT) imaging revealed progression of lung and rib metastases. The patient enrolled in a clinical trial combining stereotactic ablative radiotherapy (SABR) and HD-IL2 [11]. He received SABR treatments to his left rib (25 Gy, one fraction) and a left lung metastasis (25 Gy, one fraction) followed by two courses of 600,000 international units/kg IV of HD-IL2 q 8 h. He received ten and nine doses of HD-IL2, two weeks apart. Subsequent imaging studies demonstrated improvement in the radiated lung and rib metastases (Fig. 1a). Otherwise, there was a mixed response with improvement in some non-radiated lung nodules, but also the development of new metastases in the lungs, lymph nodes, and right femur (Fig. 1b).

In June 2017, the patient was switched to pazopanib (800 mg PO qd). He also underwent a right total knee replacement followed by adjuvant radiation (20 Gy over 5 fractions). Repeat scans after three months demonstrated progression of existing lung and nodal metastases, and three new right shoulder metastases, which were painful (Fig. 1c).

One week later, the patient began nivolumab monotherapy (240 mg IV q 2 weeks). A few days after the initial infusion, the patient developed grade 2 dermatitis (by Common Terminology Criteria for Adverse Events, CTCAE) [12] at the sites of prior rib and knee radiation, possibly related to radiation recall (Fig. 1d). Inflammation improved over the following month during which nivolumab was withheld. Upon resolution, the patient resumed treatment with nivolumab and received three additional doses without a dermatologic reaction.

Restaging CT scans three months after the initial nivolumab infusion showed a decrease in size of all lung metastases (Fig. 1e), stable disease at all other sites and no new lesions. The patient continued nivolumab for five additional infusions with interim development of grade 2 hypothyroidism managed with levothyroxine therapy, but then stopped infusions after the development of autoimmune colitis (confirmed by colonoscopy and biopsy; Fig. 1f).

CT and magnetic resonance (MR) scans at this time, eight months from initial nivolumab infusion, confirmed an iRECIST [13] PR with an interval improvement in several sites including the shoulder metastases. Following resolution of the diarrhea, the patient received additional nivolumab but developed grade 3 autoimmune hepatitis requiring intravenous steroids and mycophenolate mofetil, and nivolumab was discontinued.

Seven months after the last infusion of nivolumab and 2 years since diagnosis, the patient remains off immunotherapy without progression and near complete resolution of shoulder metastases (Fig. 1e).

Immuno-PET

We radiolabeled atezolizumab, a monoclonal anti-PD-L1 antibody with a mutant Fc, with zirconium-89 [⁸⁹Zr]. Zirconium-89 is a well-studied positron emitting radioisotope used to label antibodies with a half-life of 78 h, which is compatible with the slower pharmacokinetics of antibodies [14]. This allows imaging to be performed for several days after injection to improve tumor-to-background signal. Accumulation of the isotope in tumor sites over time and clearance from other sites improves contrast.

Methodologically, the antibody was conjugated to the chelator deferoxamine (DFO) at a molar ratio of 1:1.9 and radiolabeled with ⁸⁹Zr (5 mCi per mg of DFO-atezolizumab conjugate) using previously published protocols [15, 16]. Briefly, the DFO-atezolizumab conjugate was incubated with neutralized ⁸⁹Zr for 1 h, and the reaction was quenched with 50 mM diethylenetriamine pentaacetic acid. The radiolabeled antibody fraction was purified using the Zeba™ centrifugal spin columns (40 K MWCO) and eluted in 0.2 M sodium acetate buffer containing 5 mg/mL gentisic acid (pH 5.5–5.6). The conjugate had a specific radioactivity of 2–4 mCi/mg protein, with high radiochemical purity (≥ 99%). The immunoreactivity of the radiolabeled immunoconjugate was confirmed using an in vitro cell-based Lindmo assay [17] and was 86.2 ± 4% (n = 6). In addition, the conjugate was tested for stability in plasma and was found to be quite stable (> 80% of ⁸⁹Zr activity retained with atezolizumab in rat serum at 37 °C after 7 days).

Mice bearing the patient-derived tumorgrafts were injected intravenously (by tail-vein) with ~ 100 μCi of ⁸⁹Zr-DFO-atezolizumab. A second tumorgraft line from a tumor expressing low levels of PD-L1 (< 1%) by IHC was chosen as a negative control (Fig. 2c). PD-L1 IHC procedures and interpretations were standardized (Biocare Medical, Clone ACI3171A,C; 1:300) and results were scored by a pathologist blinded to other results.

Mice were serially imaged on a Siemens Inveon PET/CT system. PET quantification was performed blinded. PET imaging at day 6 post-injection (d.p.i.) showed a statistically significant difference in ⁸⁹Zr signal between the index patient tumorgrafts (4.2 ± 0.6% injected dose/g [%ID/g]; n = 3) and the controls (3.1 ± 0.5% ID/g; n = 3) (p = 0.028) (see Fig. 2). Similar results were observed with a second independent cohort of tumorgrafts (5.2 ± 0.4% ID/g; n = 3) compared to the same control group (p = 0.002).

Differences in tumor uptake could not be accounted for by differences in tumorgraft volumes, which were not significantly different between the index and control groups (831.9 ± 473 mm³ versus 1010.3 ± 492.6 mm³; p = 0.62, respectively). Further, the tumor/muscle contrast in the index tumorgrafts was 4.4 ± 0.4, which is also significantly higher than controls (2.7 ± 0.6) (p < 0.05). (All statistical analyses were performed using GraphPad Prism 7 by un-paired t-tests without correction for multiple comparisons and an alpha value of 0.05.) Following the last PET scan, mice were sacrificed, and tumorgrafts and other vital organs were collected for IHC assays. IHC analyses of the tumorgrafts confirmed expected levels of PD-L1 expression.