Development of99m Tc-EC-tyrosine for Early Detection of Breast Cancer Tumor Response to the Anticancer Drug Melphalan

Rationale and Objectives

Radiolabeled tyrosine analogues that have been successfully used in tumor imaging accumulate in tumor cells via an upregulated L-type amino acid transporter system. The anticancer drug melphalan is an L-type amino acid transporter substrate. Therefore, radiolabeled tyrosine analogues may have great potential in evaluating treatment responses to melphalan. In this study, a 99m Tc-labeled tyrosine analogue, 99m Tc tyrosine using N,N′-ethylene-di-L-cysteine (EC) as a chelator, was developed and its potential for noninvasively assessing tumors’ early response to melphalan determined.

Materials and Methods

EC-tyrosine was synthesized in a three-step procedure and labeled with 99m Tc. To assess cellular uptake kinetics, the percentage uptake of 99m Tc-EC-tyrosine in the rat breast cancer cell line 13762 was measured. Planar imaging was performed in rats with 13762 cell-derived tumors. To determine the transport mechanisms of 99m Tc-EC-tyrosine, a competitive inhibition study using L-tyrosine as an inhibitor was performed in vitro and in vivo. To assess tumors’ response to melphalan, tumor-bearing rats were treated with different doses of melphalan, and planar imaging was performed 0 and 3 days after treatment. Immunohistochemical analyses were conducted to determine expressions of L-type amino acid transporter 1 and cellular proliferation marker Ki-67.

Results

L-tyrosine significantly inhibited 99m Tc-EC-tyrosine uptake in vitro and in vivo. Tumor volume decreased in a dose-dependent manner with melphalan, and tumor/muscle ratios of 99m Tc-EC-tyrosine were significantly reduced in treated groups. Immunohistochemical data indicated that about 70% of tumor cells in the melphalan-treated groups underwent apoptosis, and the changes in tumor/muscle ratios reflected the decreased percentage of viable cells in treated tumors.

Conclusions

These findings suggest that 99m Tc-EC-tyrosine has great potential for monitoring tumor response to melphalan in breast tumor–bearing rats.

Currently, 18 F-2-fluoro-2-deoxy-glucose ( 18 F-FDG) is the most commonly used radiotracer in positron emission tomography (PET) for tumor detection and staging. However, it has several limitations that can give rise to false-positive and false-negative diagnoses. For instance, it has poor contrast in brain tumors because of the high glucose uptake in normal and neoplastic brain tissues . For breast cancer, whole-body 18 F-FDG PET is not quite suitable for primary breast cancer diagnosis, especially for low-grade tumors and those <1 cm in diameter . In addition, 18 F-FDG PET is not sensitive enough in detecting early-stage micrometastases and small tumor–infiltrated axillary lymph nodes .

Therefore, radiolabeled amino acids have been developed as alternatives to 18 F-FDG. Their application in tumor detection is based on the increased uptake of amino acids in tumor cells, which is presumed to reflect enhanced amino acid transporter expression, metabolism, and protein synthesis. The uptake of amino acids in macrophages and other inflammatory cells is lower than that of glucose analogue 18 F-FDG; thus, amino acid–based radiotracers are considered more specific than 18 F-FDG . Aromatic radiolabeled amino acids such as tyrosine and its derivatives are more suitable given their more readily chemical modifications and more favorable biologic characterizations. To date, tyrosine and its α-methyl-substituted analogue α-methyl tyrosine (AMT) have been successfully labeled with 11 C, 18 F, and 124/125 I for PET and with 123/131 I for single-photon emission computed tomography (SPECT) . Most of these radiolabeled tyrosine analogues have shown promising preclinical and clinical results, particularly in brain tumor staging. They have also been successfully applied to breast cancer imaging. For instance, 11 C-labeled tyrosine appears to be better than 18 F-FDG because of its lower uptake in noncancerous fibrocystic disease . Radiolabeled tyrosine analogues have been found to accumulate in tumor cells, predominantly via the L-type amino acid transporter (LAT) system, which is the only system that can transport large neutral amino acids with aromatic rings . LAT, especially its subtype LAT1, is an ideal target for tumor imaging because it is upregulated and highly expressed in many cancer cell lines, and its expression is positively correlated with tumor growth . In fact, LAT1 has been determined as a prognostic factor in patients with breast cancer, even in those with triple-negative breast cancer .

Get Radiology Tree app to read full this article<

Get Radiology Tree app to read full this article<

Get Radiology Tree app to read full this article<

Materials and methods

Chemicals and Analysis

Get Radiology Tree app to read full this article<

Synthesis of Precursor EC-tyrosine

N-t-butoxycarbonyl-O-[3-Br-propyl]-L-tyrosine methyl ester (compound 1)

Get Radiology Tree app to read full this article<

N-t-butoxycarbonyl-O-[3-(N-ethylenedicysteine) propyl]-L-tyrosine (compound 2)

Get Radiology Tree app to read full this article<

O-[3-(N-ethylenedicysteine) propyl]-L-tyrosine (EC-tyrosine) (compound 3)

Get Radiology Tree app to read full this article<

Radiolabeling EC-tyrosine and EC with 99m Tc

Get Radiology Tree app to read full this article<

Determination of the Partition Coefficient

Get Radiology Tree app to read full this article<

In Vitro Cellular Uptake Studies

Get Radiology Tree app to read full this article<

In Vitro Competitive Inhibition Study

Get Radiology Tree app to read full this article<

Blood Clearance

Get Radiology Tree app to read full this article<

In Vivo Tissue Distribution Study of 99m Tc-EC-tyrosine

Get Radiology Tree app to read full this article<

Planar Scintigraphic Imaging Study

Get Radiology Tree app to read full this article<

In Vivo Uptake Blocking Study

Get Radiology Tree app to read full this article<

Melphalan Treatment Response Assessment

Get Radiology Tree app to read full this article<

Statistical Analysis

Get Radiology Tree app to read full this article<

Results

Chemical and Radiochemical Characteristics

Get Radiology Tree app to read full this article<

Get Radiology Tree app to read full this article<

Get Radiology Tree app to read full this article<

In Vitro Cellular Uptake Studies

Get Radiology Tree app to read full this article<

Get Radiology Tree app to read full this article<

Competitive Inhibition Study of 99m Tc-EC-tyrosine

Get Radiology Tree app to read full this article<

Get Radiology Tree app to read full this article<

Blood Clearance

Get Radiology Tree app to read full this article<

Get Radiology Tree app to read full this article<

In Vivo Tissue Distribution of 99m Tc-EC-tyrosine

Get Radiology Tree app to read full this article<

Table 1

Tissue Distribution Results for 99m Tc-EC-tyrosine in Normal Fischer 344 Rats

Organ Time Point (minutes) 30 120 240 Blood 0.96 ± 0.076 0.41 ± 0.039 0.23 ± 0.006 Heart 0.24 ± 0.007 0.12 ± 0.011 0.08 ± 0.009 Lungs 0.47 ± 0.041 0.33 ± 0.001 0.20 ± 0.025 Thyroid 0.43 ± 0.036 0.24 ± 0.001 0.11 ± 0.002 Pancreas 0.21 ± 0.018 0.17 ± 0.016 0.10 ± 0.007 Liver 1.47 ± 0.045 2.42 ± 0.075 0.97 ± 0.007 Spleen 0.47 ± 0.054 1.41 ± 0.117 0.62 ± 0.036 Kidneys 11.12 ± 0.545 16.50 ± 0.395 11.13 ± 0.234 Stomach 0.27 ± 0.006 0.19 ± 0.002 0.08 ± 0.000 Intestine 1.13 ± 0.009 0.33 ± 0.023 0.17 ± 0.017 Muscles 0.10 ± 0.009 0.05 ± 0.006 0.03 ± 0.000 Bones and joints 0.28 ± 0.021 0.10 ± 0.003 0.07 ± 0.009 Brain 0.04 ± 0.003 0.01 ± 0.001 0.01 ± 0.001

EC, EC, N,N′-ethylene-di-L-cysteine.

Values are the percentage of injected dose per gram weight ( n = 3) per time interval. Data represent the mean of three measurements ± standard deviation.

Get Radiology Tree app to read full this article<

Planar Scintigraphic Imaging Study

Get Radiology Tree app to read full this article<

Get Radiology Tree app to read full this article<

In Vivo Uptake Blocking Study

Get Radiology Tree app to read full this article<

Get Radiology Tree app to read full this article<

Melphalan Treatment Response Assessment

Get Radiology Tree app to read full this article<

Get Radiology Tree app to read full this article<

Discussion

Get Radiology Tree app to read full this article<

Get Radiology Tree app to read full this article<

Get Radiology Tree app to read full this article<

Get Radiology Tree app to read full this article<

Get Radiology Tree app to read full this article<

Get Radiology Tree app to read full this article<

Get Radiology Tree app to read full this article<

Get Radiology Tree app to read full this article<

Conclusions

Get Radiology Tree app to read full this article<

Get Radiology Tree app to read full this article<

References

• 1. Rosenbaum S.J., Lind T., Antoch G., et. al.: False-positive FDG PET uptake—the role of PET/CT. Eur Radiol 2006; 16: pp. 1054-1065.

• 2. Lim H.S., Yoon W., Chung T.W., et. al.: FDG PET/CT for the detection and evaluation of breast diseases: usefulness and limitations. Radiographics 2007; 27: pp. S197-S213.

• 3. Aukema T.S., Rutgers E.J., Vogel W.V., et. al.: The role of FDG PET/CT in patients with locoregional breast cancer recurrence: a comparison to conventional imaging techniques. Eur J Surg Oncol 2010; 36: pp. 387-392.

• 4. Jager P.L., Vaalburg W., Pruim J., et. al.: Radiolabeled amino acids: basic aspects and clinical applications in oncology. J Nucl Med 2001; 42: pp. 432-445.

• 5. McConathy J., Goodman M.M.: Non-natural amino acids for tumor imaging using positron emission tomography and single photon emission computed tomography. Cancer Metastasis Rev 2008; 27: pp. 555-573.

• 6. Kole A.C., Nieweg O.E., Pruim J., et. al.: Standardized uptake value and quantification of metabolism for breast cancer imaging with FDG and L-[1-11C]tyrosine PET. J Nucl Med 1997; 38: pp. 692-696.

• 7. Kaira K., Oriuchi N., Imai H., et. al.: Prognostic significance of L-type amino acid transporter 1 (LAT1) and 4F2 heavy chain (CD98) expression in early stage squamous cell carcinoma of the lung. Cancer Sci 2009; 100: pp. 248-254.

• 8. Furuya M., Horiguchi J., Nakajima H., et. al.: Correlation of L-type amino acid transporter 1 and CD98 expression with triple negative breast cancer prognosis. Cancer Sci 2012; 103: pp. 382-389.

• 9. del Amo E.M., Urtti A., Yliperttula M.: Pharmacokinetic role of L-type amino acid transporters LAT1 and LAT2. Eur J Pharm Sci 2008; 35: pp. 161-174.

• 10. Samuels B.L., Bitran J.D.: High-dose intravenous melphalan: a review. J Clin Oncol 1995; 13: pp. 1786-1799.

• 11. Harada N., Nagasaki A., Hata H., et. al.: Down-regulation of CD98 in melphalan-resistant myeloma cells with reduced drug uptake. Acta Haematol 2000; 103: pp. 144-151.

• 12. Moscow J.A., Swanson C.A., Cowan K.H.: Decreased melphalan accumulation in a human breast cancer cell line selected for resistance to melphalan. Br J Cancer 1993; 68: pp. 732-737.

• 13. Jurisson S., Cutler C., Smith S.V.: Radiometal complexes: characterization and relevant in vitro studies. Q J Nucl Med Mol Imaging 2008; 52: pp. 222-234.

• 14. Marzilli L.G.: Linking deprotonation and denticity of chelate ligands. Rhenium(V) oxo analogs of technetium-99m radiopharmaceuticals containing N2S2 chelate ligands. Inorg Chem 1994; 33: pp. 4850-4860.

• 15. Anderson C.J., John C.S., Li Y.J., et. al.: N, N′-ethylene-di-L-cysteine (EC) complexes of Ga(III) and In(III): molecular modeling, thermodynamic stability and in vivo studies. Nucl Med Biol 1995; 22: pp. 165-173.

• 16. Feldman J.G.R., Mark S., Schwartz J., et. al.: A methematical model for tumor volume evaluation using two-dimensions. J Appl Quan Meth 2009; 4: pp. 8.

• 17. Kong F.L., Zhang Y., Ali M.S., et. al.: Synthesis of (99m)Tc-EC-AMT as an imaging probe for amino acid transporter systems in breast cancer. Nucl Med Commun 2010; 31: pp. 699-707.

• 18. Yanagida O., Kanai Y., Chairoungdua A., et. al.: Human L-type amino acid transporter 1 (LAT1): characterization of function and expression in tumor cell lines. Biochim Biophys Acta 2001; 1514: pp. 291-302.

• 19. Shikano N., Kawai K., Nakajima S., et. al.: Renal accumulation and excretion of radioiodinated 3-iodo-alpha-methyl-L-tyrosine. Ann Nucl Med 2004; 18: pp. 263-270.

• 20. Amano S., Inoue T., Tomiyoshi K., et. al.: In vivo comparison of PET and SPECT radiopharmaceuticals in detecting breast cancer. J Nucl Med 1998; 39: pp. 1424-1427.

• 21. Bergstrom J., Furst P., Noree L.O., et. al.: Intracellular free amino acid concentration in human muscle tissue. J Appl Physiol 1974; 36: pp. 693-697.

• 22. Lin J., Raoof D.A., Thomas D.G., et. al.: L-type amino acid transporter-1 overexpression and melphalan sensitivity in Barrett’s adenocarcinoma. Neoplasia 2004; 6: pp. 74-84.

• 23. He B., Frey E.C.: Comparison of conventional, model-based quantitative planar, and quantitative SPECT image processing methods for organ activity estimation using In-111 agents. Phys Med Biol 2006; 51: pp. 3967-3981.

• 24. Pereira J.M., Stabin M.G., Lima F.R., et. al.: Image quantification for radiation dose calculations—limitations and uncertainties. Health Phys 2010; 99: pp. 688-701.