Tryptophan metabolism in breast cancers: molecular imaging and immunohistochemistry studies

Abstract

Introduction: Tryptophan oxidation via the kynurenine pathway is an important mechanism of tumoral immunoresistance. Increased tryptophan metabolism via the serotonin pathway has been linked to malignant progression in breast cancer. In this study, we combined quantitative positron emission tomography (PET) with tumor immunohistochemistry to analyze tryptophan transport and metabolism in breast cancer.

Methods: Dynamic α-[(11)C]methyl-l-tryptophan (AMT) PET was performed in nine women with stage II-IV breast cancer. PET tracer kinetic modeling was performed in all tumors. Expression of L-type amino acid transporter 1 (LAT1), indoleamine 2,3-dioxygenase (IDO; the initial and rate-limiting enzyme of the kynurenine pathway) and tryptophan hydroxylase 1 (TPH1; the initial enzyme of the serotonin pathway) was assessed by immunostaining of resected tumor specimens.

Results: Tumor AMT uptake peaked at 5-20 min postinjection in seven tumors; the other two cases showed protracted tracer accumulation. Tumor standardized uptake values (SUVs) varied widely (2.6-9.8) and showed a strong positive correlation with volume of distribution values derived from kinetic analysis (P<.01). Invasive ductal carcinomas (n=6) showed particularly high AMT SUVs (range, 4.7-9.8). Moderate to strong immunostaining for LAT1, IDO and TPH1 was detected in most tumor cells.

Conclusions: Breast cancers show differential tryptophan kinetics on dynamic PET. SUVs measured 5-20 min postinjection reflect reasonably the tracer's volume of distribution. Further studies are warranted to determine if in vivo AMT accumulation in these tumors is related to tryptophan metabolism via the kynurenine and serotonin pathways.

Figure 1

Three-compartment model for alpha-[¹¹C]methyl-L-tryptophan (AMT) kinetics in breast tumor tissue using first-order rate constants. The rate constant K₁ (mL/g/min) represents the forward and k₂ (min⁻¹) represents the reverse combined transport of AMT across the blood vessel, interstitial space, and cell membrane into the cytoplasm, in which it comprises the free compartment (C_f). Irreversible enzymatic conversion of AMT to its metabolites and accumulation in the metabolic compartment (C_m) is characterized by the metabolic rate constant k₃ (min⁻¹). Finally, the rate constant k₄ (min⁻¹) characterizes the reverse transport of AMT metabolites across the interstitial space and blood vessel back into the blood pool. C_P: plasma compartment; C_T: tissue compartment.

Figure 2

(A) AMT tracer accumulation in breast tumor between 5 and 20 minutes post injection. There is a good contrast between AMT tracer accumulation in tumor tissue and surrounding breast tissue. (B). Corresponding time-activity curve (black circles) shows high initial uptake, which peaks within the first 20 minutes with subsequent steady decline, in the same tumor. This time course was seen in most patients with breast cancer. (C). In two subjects a more prolonged accumulation was seen with a peak at 20–30 minutes post injection. The curves with open circles indicate the very low uptake in normal breast tissue (contralateral to the tumor).

Figure 3

Correlations between VD’ values and SUV. Left panel: 7 patients (Group 1; G₁) with an early plateau and then decline of tumoral radioactivity 20 min after injection; right panel: whole patient group (n=9).

Figure 4

IDO and LAT1 staining in benign breast disease (A, C) vs. breast cancer (carcinosarcoma, patient #1 [B, D]). A. Ductal cells in benign breast disease demonstrate IDO immunoreactivity. B. Section from the breast cancer shows extensive IDO immunoreactivity in both ductal cells and infiltrating tumor cells. C. LAT1 staining in duct cells from benign breast disease. D. Strong LAT1 staining in both duct and infiltrating tumor cells in breast cancer. Original magnification 20×.

Figure 5

Strong IDO and LAT1 staining in an invasive ductal carcinoma (patient #8). A. No-primary control staining. B. IDO staining at 3+ intensity in 100% of tumor cells. C. LAT1 staining at 3+ intensity in 100% of cells. Original magnification 20×.

Figure 6

TPH1 immunostaining in benign breast disease vs. invasive ductal breast carcinoma (patient #9; original resected breast cancer specimen). A. No-primary control staining. B. Ductal epithelial cells from benign breast disease demonstrate TPH1 immunoreactivity. C. Both ductal epithelial and infiltrating tumor cells show extensive, strong TPH1 immunoreactivity. Original magnification 20×.