Intra-image referencing for simplified assessment of HER2-expression in breast cancer metastases using the Affibody molecule ABY-025 with PET and SPECT

Dan Sandberg, Vladimir Tolmachev, Irina Velikyan, Helena Olofsson, Anders Wennborg, Joachim Feldwisch, Jörgen Carlsson, Henrik Lindman, Jens Sörensen, Dan Sandberg, Vladimir Tolmachev, Irina Velikyan, Helena Olofsson, Anders Wennborg, Joachim Feldwisch, Jörgen Carlsson, Henrik Lindman, Jens Sörensen

Abstract

Purpose: In phase I/II-studies radiolabelled ABY-025 Affibody molecules identified human epidermal growth factor receptor 2 (HER2) expression in breast cancer metastases using PET and SPECT imaging. Here, we wanted to investigate the utility of a simple intra-image normalization using tumour-to-reference tissue-ratio (T/R) as a HER2 status discrimination strategy to overcome potential issues related to cross-calibration of scanning devices.

Methods: Twenty-three women with pre-diagnosed HER2-positive/negative metastasized breast cancer were scanned with [111In]-ABY-025 SPECT/CT (n = 7) or [68Ga]-ABY-025 PET/CT (n = 16). Uptake was measured in all metastases and in normal spleen, lung, liver, muscle, and blood pool. Normal tissue uptake variation and T/R-ratios were established for various time points and for two different doses of injected peptide from a total of 94 whole-body image acquisitions. Immunohistochemistry (IHC) was used to verify HER2 expression in 28 biopsied metastases. T/R-ratios were compared to IHC findings to establish the best reference tissue for each modality and each imaging time-point. The impact of shed HER2 in serum was investigated.

Results: Spleen was the best reference tissue across modalities, followed by blood pool and lung. Spleen-T/R was highly correlated to PET SUV in metastases after 2 h (r = 0.96, P < 0.001) and reached an accuracy of 100% for discriminating IHC HER2-positive and negative metastases at 4 h (PET) and 24 h (SPECT) after injection. In a single case, shed HER2 resulted in intense tracer retention in blood. In the remaining patients shed HER2 was elevated, but without significant impact on ABY-025 biodistribution.

Conclusion: T/R-ratios using spleen as reference tissue accurately quantify HER2 expression with radiolabelled ABY-025 imaging in breast cancer metastases with SPECT and PET. Tracer binding to shed HER2 in serum might affect quantification in the extreme case.

Keywords: Affibody; HER2-receptor; PET; SPECT; Shedding; T/R.

Conflict of interest statement

Funding

This work was sponsored by The Swedish Cancer Society (Cancerfonden) and The Swedish Breast Cancer Foundation (Bröstcancerfonden). ABY-025 was donated by Affibody AB.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study was approved by the regional ethics committee and all patients signed informed consent.

Figures

Fig. 1
Fig. 1
Examples of abdominal PET/CT and SPECT/CT images including normal liver, liver metastases and spleen for reference. Left column: low peptide dose [68Ga]-ABY-025-PET/CT at 1, 2, and 4 hours after injection. Middle column: high peptide dose [68Ga]-ABY-025-PET/CT at corresponding time points in the same patient. Right column (other patient): [111In]-ABY-025--SPECT/CT at 4, 24, and 48 hours after injection. The PET colour scale is thresholded at SUV 32. The SPECT images are corrected for the decay of 111In to represent uptake and the colour scale arbitrarily normalised. Arrows indicate a liver metastasis of breast cancer in each of the two patients. Examples of dual spleen VOI:s (spherical) and single slice circular ROI are shown in yellow (first row PET images). 68Ga LD and 68Ga HD stand respectively for low and high peptide content [68Ga]-ABY-025. 111In stands for [111In]-ABY-025
Fig. 2
Fig. 2
(a): Maximum intensity projection (MIP) example of extraordinary shedding after start of HER2-targeted therapy (PET#12) at 2 h after injection (the only available time point in this case). (b): Bar graph of the SUV for this patient’s tissues relative the average of all 16 patients at the corresponding time point. Highest value (370%) was found for the aortic blood pool, with 260% for cardiac blood pool (partly myocardium). Notably, a tilted distribution of activity towards blood and blood rich organs and away from kidney, muscle and liver was seen. (c): For comparison, a MIP of a patient with HER2-positive metastases in liver and bone marrow, but no visible activity in the blood at 2 h. This pattern was seen in 22/23 patients in both ABY-025 studies
Fig. 3
Fig. 3
(a) HD [68Ga]-ABY-025 PET at 4 h after injection: Scatter plot of spleen T/R against SUV. Suggested cut off for T/R-based HER2-diagnostics using these criteria is 6.5. At 2 h after injection the distribution was similar, but with a suggested cut off of 2.75 with one false positive (HercepTest 2+/ISH negative) metastasis, correctly classified here at 4 h. (b) [111In]-ABY-025 SPECT at 24 h after injection. Scatter plot of spleen T/R against the previously described 24/4 h uptake ratio (dual time point) method. Suggested cutoff of 2 for HER2-diagnostics using these criteria. All data points represent metastases with HER2-status verified by immunohistochemistry. Red circles: HER2 negative, blue squares: HER2-positive. Blue arrow: actual T/R-value of 27.3

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