^99mTc-anti-TNF-α antibody for the imaging of disease activity in pulmonary sarcoidosis

Discussion

In this study we observed selective accumulation of ^99mTc-infliximab in the target tissue of pulmonary sarcoidosis patients. The accumulation was highest and most robust in the hilar lymph nodes. While some patients showed significant accumulation, others did not, which suggests varying levels of TNF-α expression between subjects. The most pronounced accumulation was observed in patients with an indication for systemic treatment. The accumulation of ^99mTc-infliximab as measured by the ratio of T/B values (increase between 6 h and 20 h) correlated positively with all four laboratory parameters (sIL-2R concentration, serum ACE, ACE Z-score and the BAL CD4/CD8 ratio), while accumulation correlated negatively with all four clinical parameters (VC, FEV₁, Tiffeneau index and D_LCO).

The finding that TNF-α expression differs between sarcoidosis patients is in agreement with previous research. Asano et al. [7] measured TNF-α levels in lymph node samples of sarcoidosis patients and observed highest levels in lymph nodes in the active stage as opposed to the early and healing stages. Fehrenbach et al. [5] showed that the level of bioactive TNF-α spontaneously released by isolated BAL immune cells differed between patients. Highest levels were observed in the presence of aggregates of alveolar macrophages, which represent predecessors to granulomas. Well-developed granulomas were associated with lower levels of TNF-α release, confirming that TNF-α is important during the initial phase of granuloma formation [5].

A variable TNF-α expression as measured by ^99mTc-infliximab scintigraphy was also observed by Galli et al. [22] in a similar group of newly diagnosed pulmonary sarcoidosis patients. They observed that the pulmonary accumulation of ^99mTc-infliximab was very low and variable and showed no correlation with the pulmonary ¹⁸F-FDG PET/CT SUV_max values. A moderate correlation was observed with the CD4/CD8 ratio in peripheral blood lymphocytes, but not with the CD4/CD8 ratio or cellularity of BAL, possibly because of the small sample size. The authors also explained the differences between ¹⁸F-FDG and ^99mTc-infliximab by the different mechanism of uptake of the two radiopharmaceuticals and by the individual variability in the production of TNF-α.

In the present study we expanded the scope with several clinical and laboratory parameters. Results confirmed a limited correlation between ^99mTc-infliximab accumulation in the lung parenchyma and all laboratory and clinical parameters, with the highest radiopharmaceutical accumulation observed in the lymph nodes. An explanation could be a lower TNF-α concentration in the lung tissue, potentially caused by a washout of soluble TNF-α due to high perfusion. An additional explanation for the lower overall uptake in the lung parenchyma could be the normal or only mildly altered lung function observed in our population. This is confirmed by ¹⁸F-FDG PET/CT, demonstrating limited or no metabolic activity in the lung parenchyma in six out of 10 patients. The mild pulmonary involvement suggests a limited parenchymal TNF-α expression and subsequent low ^99mTc-infliximab accumulation.

We observed no correlation between the accumulation of ^99mTc-infliximab and ¹⁸F-FDG PET/CT SUV_max values.

Our findings of ^99mTc-infliximab accumulation correlating with clinical parameters are in line with literature on rheumatoid arthritis. In a study using ^99mTc-adalimumab, another TNF-α inhibitor, Barrera et al. [24] found that joint uptake of ^99mTc-adalimumab in five rheumatoid arthritis patients decreased after treatment with systemic corticosteroids and was indicative of a decrease in the rheumatoid arthritis disease activity score. In a similar study, Roimicher et al. [25] found good correlation between ^99mTc-adalimumab uptake and magnetic resonance imaging of hands and wrists in eight rheumatoid arthritis patients. ^99mTc-adalimumab scintigraphy showed a higher sensitivity than clinical examination. The value of ^99mTc-infliximab scintigraphy in the prediction of the response of intra-articular infliximab in 12 rheumatoid arthritis and spondylarthropathy patients was illustrated by Conti et al. [20]. Affected joints showed higher tracer uptake than nonaffected joints and the degree of tracer accumulation prior to therapy allowed to distinguish responders, partial responders and nonresponders. In contrast, D’Alessandria et al. [21] found that ^99mTc-infliximab scintigraphy could not predict clinical outcome in 10 patients with Crohn's disease. Possible explanations discussed by the authors were either low TNF-α production in the gut of Crohn's disease patients, low ^99mTc-infliximab extravasation at the radiopharmaceutical dose of 200 μg or the pre-treatment with oral prednisone (1 mg·kg⁻¹) which could have resulted in downregulation of bowel TNF-α expression.

Serum TNF-α levels in serum in our patients were in the range 13.9–111 pg·mL⁻¹, which is in agreement with reported values [26]. Although a statistically significant correlation was found with the sIL-2R and serum ACE concentrations, no significant correlation was found with the ACE Z-score, BAL CD4/CD8 ratio or any of the clinical parameters. This finding illustrates that the measurement of circulating (soluble) TNF-α is not equivalent to measurement of local TNF-α expression in tissue that includes transmembrane TNF-α, which plays an important role in granulomatous diseases such as sarcoidosis [27].

A correlation between accumulation of ^99mTc-infliximab and serum sIL-2R and ACE concentrations is plausible from the perspective of existing knowledge. Keicho et al. [28] and Ina et al. [29] observed higher levels of sIL-2R in serum of patients with active sarcoidosis compared with inactive disease and found a significant correlation with serum ACE levels. Alveolar macrophages are a source of sIL-2R, ACE and TNF-α [4, 5, 28, 29]. It therefore seems that the initial phase of granuloma formation is associated with higher levels of sIL-2R, ACE and TNF-α, and based on the latter, a higher accumulation of ^99mTc-infliximab. Despite these associations, sIL-2R, ACE and TNF-α are not interchangeable. While alveolar macrophages are the main source of TNF-α in sarcoidosis, T-lymphocytes and monocytes also release sIL-2R, and epitheloid cells and monocytes contribute to the production of ACE [5, 29]. In our study group, the accumulation of ^99mTc-infliximab showed better correlation with clinical parameters than sIL-2R, serum ACE/ACE Z-score and serum TNF-α.

Correlation analysis with ¹⁸F-FDG PET/CT SUV_max values in our population showed limited correlation with laboratory parameters, clinical parameters or ^99mTc-infliximab accumulation. These findings support that ¹⁸F-FDG PET/CT and ^99mTc-infliximab scintigraphy are not equivalent and confirms the notion that high activity on ¹⁸F-FDG PET/CT is not necessarily indicative of high TNF-α expression.

A limitation of our study is the small group size (10 patients) and subsequent limited statistical power. Since it involved a pilot study we chose newly diagnosed patients without prior immunosuppressive therapy to avoid the risk of suppression of TNF-α expression. Future research should focus on a population with an indication for anti-TNF-α therapy to evaluate the correlation between ^99mTc-infliximab pre-therapy accumulation and treatment response.

Compared with the SUV_max values obtained with ¹⁸F-FDG PET/CT the ratios of T/B values in the SPECT/CT images obtained with ^99mTc-infliximab are less pronounced. A major contribution in this respect is the high activity in the blood pool, which is due to the long biological half-life of infliximab (9 days). The short physical half-life of ^99mTc (6 h) necessitates scanning within 24 h. An interesting possible way of addressing this issue is the use of a radioactive isotope with a longer physical half-life, e.g. zirconium-89 (physical half-life 78 h), which has been used successfully in the radiolabelling and clinical application of other monoclonal antibodies [30]. Finally, a tool to quantify the total activity in one organ (e.g. the lungs), as opposed to the focal activity measurements used in our study, might have great clinical value [31]. If such tools become available and are properly validated, they should be used in future studies.

Our population consisted of pulmonary sarcoidosis patients. Wijnen et al. [19] recently discussed that the higher infliximab response rate observed for sarcoidosis patients suffering from uveitis is probably determined by the shorter time between diagnosis and initiation of anti-TNF-α treatment compared with small-fibre neuropathy and respiratory functional impairment. At least a partial explanation may be that TNF-α expression is higher during early granuloma formation phases. As our results illustrate that serum TNF-α levels correlate poorly with clinical parameters, it will be important to quantify TNF-α expression in tissue, including transmembrane TNF-α, e.g. by means of scintigraphic measurement. Early intervention with infliximab as part of an aggressive treatment has already been investigated for early rheumatoid arthritis and has been proven to be beneficial, although the increased effects were sometimes limited [32, 33]. An aggressive anti-TNF-α intervention strategy for clinically threatened sarcoidosis patients with the highest TNF-α expression, even as second- or first-line therapy, has the potential of increased efficacy, prevention of disease progression and higher cost-effectiveness.

In conclusion, our results show that a variable, selective accumulation of ^99mTc-infliximab occurs in newly diagnosed sarcoidosis patients, mainly in the hilar lymph nodes. Thus, ^99mTc-infliximab accumulation reflects the TNF-α expression in each individual patient and therefore provides valuable information on the presence of the biological target for anti-TNF-α therapy. Further research is warranted to evaluate the role of ^99mTc-infliximab scintigraphy in predicting anti-TNF-α therapy response in sarcoidosis patients.