RETRACTED ARTICLE: Inter- and intraobserver agreement of the quantitative assessment of [^99mTc]-labelled anti-programmed death-ligand 1 (PD-L1) SPECT/CT in non-small cell lung cancer

Lung cancer is the most commonly diagnosed cancer globally and a leading cause of mortality with over 1.7 million deaths in 2018 alone [1]. Therapeutic molecular-targeting agents have resulted in significant improvements in progression-free and overall survival in advanced non-small cell lung cancer (NSCLC); however, targetable genetic aberrations represent only a small proportion of cases [2]. The introduction of monoclonal antibodies targeting immune checkpoint molecules including programmed cell death protein 1 (PD-1) and its ligand (PD-L1) has revolutionised the treatment paradigm of NSCLC. An important mechanism of immune escape involves the upregulation of co-inhibitory molecule PD-L1 by tumour cells, which on interaction with PD-1, expressed by effector T cells, lead to their dysfunction. Anti-PD-1/PD-L1 therapy improves median overall survival in advanced NSCLC in both first- and second-line settings compared to standard cytotoxic chemotherapy, with durable responses seen in around 20% [3‐6].

PD-L1 expression determined by immunohistochemistry (IHC) is a widely validated biomarker correlating with anti-PD-1/PD-L1 therapeutic response and survival [4‐7]. Despite this correlation, up to 10% of patients deemed ‘non-expressers’ by IHC respond to anti-PD-1/PD-L1 therapy [4]. Heterogeneity of PD-L1 expression both within and between tumours is well reported, as are changes over time particularly following exposure to anti-cancer therapies [8, 9]. Considering that multiple or serial biopsies are impractical and associated with increased risk to individual patients, this temporospatial heterogeneity presents a particular challenge as needle biopsy only samples a small area of the tumour. Additionally, there are multiple PD-L1 assays available which may assess PD-L1 expression on tumour or infiltrative immune cells alone or in combination [10]. Considering a potential for false negative results with IHC and the limitations described, non-invasive imaging techniques present a potential solution and opportunity to improve the predictive value of PD-L1 assessment.

NM-01 is a camelid single-domain antibody against PD-L1 that when radiolabelled with technetium-99 m ([^99mTc]) can be detected by single-photon emission computed tomography (SPECT). Recently, we have reported results from a first-in-human study of [^99mTc]NM-01 that demonstrated both safety and acceptable dosimetry in the first 16 recruited participants with NSCLC [11]. SPECT/computed tomography (CT) scans were obtained 1 and 2 h following [^99mTc]NM-01 injection with primary tumour-to-blood pool ratio (T:BP) assessment correlating with PD-L1 expression determined by IHC. Additionally, uptake was demonstrated in nodal and bone metastases with heterogeneity of expression in 30% of cases. This novel single-domain antibody presents an opportunity for the non-invasive total tumoural assessment of PD-L1 that could help clinicians better stratify patients to receive the most appropriate anti-cancer therapy at the right time in their disease course. Our hypothesis was that quantitative measurement of PD-L1 expression using [^99mTc]NM-01 SPECT/CT is consistent and reproducible between and within observers. The aim of this study was to determine the reproducibility of and agreement between experienced and less experienced observers within a cohort of patients with NSCLC.

Participants aged between 18 and 75 years with histologically confirmed, untreated NSCLC and an Eastern Cooperative Oncology Group (ECOG) performance score of 1 or less were eligible to participate and undergo [^99mTc]NM-01 SPECT/CT. Exclusion criteria included pregnant or lactating females, severe infection and inability to provide biopsy sample for assessment of PD-L1. The study was registered with ClinicalTrials.gov identifier no. NCT02978196. Ethics approval was obtained from Shanghai General Hospital Ethics Committee (approval no. 2016KY220), and all enrolled participants provided written informed consent [11].

Our study demonstrates that the quantitative assessment of [^99mTc]NM-01 using SPECT/CT is both reliable and reproducible within and between independent observers. Interobserver agreement was demonstrated for both T:BP (ICC 0.83) and LN:BP (ICC 0.87). In addition, excellent intraobserver agreement was shown (T:BP ICC 0.95–0.96; LN:BP ICC 0.95). This provides further evidence that [^99mTc]NM-01 has significant potential and clinical utility as a diagnostic agent for the measurement of PD-L1. Non-invasive assessment of PD-L1 is an attractive possibility considering the dynamic nature and heterogeneity of its expression. [^99mTc]NM-01 uptake measured by T:BP on SPECT/CT has already been shown to correlate with PD-L1 expression measured by IHC (r = 0.68, p = 0.014) [11]. This study, which confirms good to excellent inter- and intraobserver agreement of the quantitative assessment of [^99mTc]NM-01 SPECT/CT, supports its potential to provide reliable assessment of PD-L1 expression. It remains unclear whether temporal changes in PD-L1 expression and response assessment using [^99mTc]NM-01 SPECT/CT following anti-PD-1/PD-L1 therapy will be demonstrated and of clinical utility. This will be further explored in a phase II clinical trial, PECan [NCT04436406], which will also compare changes in PD-L1 expression and response to parameters on [¹⁸F]FDG PET/CT in both NSCLC and malignant melanoma.

This study is the first to assess the agreement of SPECT/CT in measuring PD-L1 expression in cancer. Several other radionuclides are currently being developed specifically for imaging the PD-1/PD-L1 axis. ¹⁸F-BMS-986192 (¹⁸Fluor-labelled anti-PD-L1 Adnectin) uptake on positron emission tomography (PET) has been shown to correlate with PD-L1 expression in NSCLC, as has ⁸⁹Zirconium-nivolumab for PD-1 expression, both in early phase clinical trials [13]. In both cases, inter- and intra-tumoural heterogeneity was demonstrated, consistent with the findings described in the early phase trial of [^99mTc]NM-01 SPECT/CT. An important characteristic of [^99mTc]NM-01 is that it is a small (14.3 kDa) antigen-binding fragment radiotracer with rapid blood clearance, with optimal SPECT/CT imaging performed at just 2 h following administration. As [^99mTc]NM-01 does not directly block the PD-L1 binding site, it does not interfere with the PD-1/PD-L1 axis and thus has the potential to assess whole-body PD-L1 status before, during and after anti-PD-L1 therapy. Whilst PET/CT provides a higher degree of spatial resolution, there are some notable benefits to SPECT/CT imaging. [^99mTc] radioisotope and SPECT imaging are both more widely available and relatively inexpensive. Concerns regarding the non-standardised quantification techniques for SPECT/CT may not be fully justified if quantification techniques are reproducible and reliable. Applying simple rules to ROI_max scoring may improve both inter- and intraobserver agreement, as demonstrated in this study where applying a set 3-cm sphere to score the unaffected lung at the level of the aortic arch improved the interobserver ICC. Whilst we did not show any significant improvement in agreement applying a similar rule to the liver, both inter- and intraobserver ICC remained excellent, suggesting that simple rule-based approaches may be used to standardise and simplify image interpretation without significant impact on quantification.

There are some limitations to this study. Firstly, it is limited by its sample size; nevertheless, the relatively narrow confidence intervals suggest a good estimate of the agreement. Despite good to excellent interobserver agreement, the mean sensitivity and specificity were relatively poor with some discrepant cases resulting in a PD-L1 assessment determined by T:BP of [^99mTc]NM-01 discordant with that found on IHC. This is not unexpected considering that heterogeneity of PD-L1 measured by IHC is widely reported in the literature and was demonstrated on [^99mTc]NM-01 assessment in our previous study [11]. In addition, the cut-off value of T:BP ≥ 2.32 correlating with a PD-L1 of ≥ 1% on IHC was determined on a small sample size and requires further validation in larger cohorts [11]. It is also important to note that the patient cohort was relatively heterogenous with regards to tumour staging. Due to the low number of measurable extra-nodal (lung and bone) metastases in the cohort (n = 8), statistical analysis using ICC of the quantitative assessment of [^99mTc]NM-01 in these lesions was not possible. With further understanding of the relationship between PD-L1 expression by IHC and [^99mTc]NM-01 SPECT/CT, it may be possible for both quantitative (as described in this study) and qualitative assessments to be made by observers blind to IHC PD-L1 expression, and their agreement evaluated. SPECT is a highly sensitive imaging modality but has relatively poor resolution; further optimisation with iterative reconstruction methods along with CT attenuation and scatter corrections have the potential to further improve and standardise quantification [14]. Novel SPECT reconstruction techniques that enable standardised quantification will be employed in forthcoming PECan and PELICAN studies [EudraCT 2020-002809-26] to further investigate and validate [^99mTc]NM-01 SPECT/CT clinically. This would also enable quantitative comparison with other PD-L1 PET radionuclides, for example the aforementioned ¹⁸F-BMS-986192 [13].

Overall, good to excellent inter- and intraobserver agreement of the quantitative assessment of [^99mTc]NM-01 SPECT/CT in NSCLC was demonstrated in this study. With correlation between PD-L1 expression determined by [^99mTc]NM-01 SPECT/CT and by immunohistochemistry previously demonstrated, there is considerable potential for [^99mTc]NM-01 SPECT/CT to reliably assess PD-L1 expression, with further analysis in subsequent clinical trials now being conducted.