An Approach to Breast Cancer Diagnosis via PET Imaging of Microcalcifications Using (18)F-NaF

Abstract

Current radiologic methods for diagnosing breast cancer detect specific morphologic features of solid tumors or any associated calcium deposits. These deposits originate from an early molecular microcalcification process of 2 types: type 1 is calcium oxylate and type II is carbonated calcium hydroxyapatite. Type I microcalcifications are associated mainly with benign tumors, whereas type II microcalcifications are produced internally by malignant cells. No current noninvasive in vivo techniques are available for detecting intratumoral microcalcifications. Such a technique would have a significant impact on breast cancer diagnosis and prognosis in preclinical and clinical settings. (18)F-NaF PET has been used solely for bone imaging by targeting the bone hydroxyapatite. In this work, we provide preliminary evidence that (18)F-NaF PET imaging can be used to detect breast cancer by targeting the hydroxyapatite lattice within the tumor microenvironment with high specificity and soft-tissue contrast-to-background ratio while delineating tumors from inflammation.

Methods: Mice were injected with approximately 10(6) MDA-MB-231 cells subcutaneously and imaged with (18)F-NaF PET/CT in a 120-min dynamic sequence when the tumors reached a size of 200-400 mm(3). Regions of interest were drawn around the tumor, muscle, and bone. The concentrations of radiotracer within those regions of interest were compared with one another. For comparison to inflammation, rats with inflamed paws were subjected to (18)F-NaF PET imaging.

Results: Tumor uptake of (18)F(-) was significantly higher (P < 0.05) than muscle uptake, with the tumor-to-muscle ratio being about 3.5. The presence of type II microcalcification in the MDA-MB-231 cell line was confirmed histologically using alizarin red S and von Kossa staining as well as Raman microspectroscopy. No uptake of (18)F(-) was observed in the inflamed tissue of the rats. Lack of hydroxyapatite in the inflamed tissue was verified histologically.

Conclusion: This study provides preliminary evidence suggesting that specific targeting with (18)F(-) of hydroxyapatite within the tumor microenvironment may be able to distinguish between inflammation and cancer.

Keywords: 18F-NaF; 18F−; MDA-MB-231; PET; hydroxyapatite; breast cancer; calcium oxalate; microcalcification.

Figure 1

A three compartment model was used to describe the kinetic distribution of ¹⁸F⁻ ions to tumor and bone. K₁ is the transfer rate of ¹⁸F⁻ from the first compartment 1 (plasma) to compartment 2 (extracellular fluid space) in units of mL/min/g, k₂ is the transfer rate from 2 to 1 (1/min), k₃ is the transfer rate from 2 to compartment 3 (hydroxyapatite lattice) in units of 1/min, and k₄ is the transfer rate. from 3 to 2 (1/min).

Figure 2

A 60–80 min summed PET/CT and PET images of ¹⁸F⁻ uptake in a mouse bearing MDA-MB-231 tumor and imaged for 120 min. With the windowing level set between 0.8 and 12% ID/cc, bone image is saturated but the tumor and brain sand are visible with high contrast-to-background soft-tissue ratio

Figure 3

Time-activity curves (TACs) of different regions in the mouse injected with ¹⁸F-NaF displayed in the previous figure. The percent injected dose per unit volume (%ID/cc) is the ¹⁸F⁻ concentration within each ROI normalized to the total injected dose. The insert is just a zoomed view of the TACs.

Figure 4

Staining of MDA-MB-231 breast cancer samples and normal bone. With von Kossa (red/brown) and alizarin red S (red). The positive von Kossa stains indicate the presence of calcium carbonates suggesting the presence of Type II calcification specifically. A 100 × 60 μm² region of the same tumor section scanned with a Raman microscope revelas the locations within that region where a 960 cm⁻¹ shift was found (green color in the Raman image and Raman spectra) and where no shift near 960 cm⁻¹ (black color). A shift at ~960 cm⁻¹ indicates the presence of HAP.

Figure 5

A coronal view of a PET/CT and CT only images of two mice injected with ¹⁸F-NaF (right) and the corresponding autoradiography/von Kossa staining of a tumor section and normal muscle (left). A localized region, indicated by the thick white arrow, with high ¹⁸F⁻ (about 9 times higher than muscle and 3 times less than bone) was observed in each tumor in the PET images indicating dense HAP. These regions was not observed in the CT images. The autoradiography correlated well with the PET and staining results.

Figure 6

(Left) A rat model of acute inflammation (in right footpad) imaged with 18F- NaF PET at maximal inflammation. Uptake of ¹⁸F⁻ was limited to bone with no accumulation in the inflammatory soft tissue due to lack of HAP. (Right) A section of the inflamed tissue stained with von Kossa. No positive staining (nor red/brown spots) was observed within in the stained tissue indicating no presence of calcium phosphates or type II microcalcification. The lower and upper limits scale bar are 0.8 and 12 %ID/cc.