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To the Editor: We previously demonstrated in vivo imaging of cell death in the myocardia of patients with acute myocardial infarction using technetium Tc 99m–labeled annexin-V (^99mTc-p-annexin-V [Apomate], Theseus Imaging, Cambridge, Mass) and nuclear imaging.¹ Because high proliferation and apoptotic indices have been reported in rapidly growing malignant tumors,² information about the extent of apoptosis in the tumor may also provide insight into the tumor's biology and prognosis.

Report of a Case
A 65-year-old man was referred to our hospital for treatment of a tumor of the left ventricle (Figure 1). Prior to surgery, we attempted to visualize apoptosis in the tumor using ^99mTc-p-annexin-V. To localize possible ^99mTc-p-annexin-V uptake, a thallous chloride Tl 201 perfusion scintigram was performed simultaneously using a dual-isotope imaging technique.

View larger version (73K): [in this window] [in a new window] Figure 1. Imaging Studies of the Heart A, Echocardiogram of the heart in short-axis orientation shows a large tumor in the left ventricle originating from the lateral area (white arrow). B, Thallium perfusion scintigraphy shows the perfused myocardium of the left ventricle in a short-axis orientation. C, Increased uptake of technetium Tc 99m-p-annexin-V can be seen (black arrows) within the contour of the left ventricle on short-axis single-photon emission computed tomographic imaging, using a similar orientation as the perfusion scintigram.

This imaging protocol showed a large area of enhanced ^99mTc-p-annexin-V, which localized to the cardiac region, as indicated by ²⁰¹Tl perfusion scintigraphy, and resembled the echocardiographic image of the tumor (Figure 1). The tumor could not be completely excised, and scintigraphic imaging of the tumor after surgery showed ^99mTc-p-annexin-V but not ²⁰¹Tl activity. Immunostaining of the excised tumor tissue using a polyclonal antibody against annexin-V (Hyphen Biomed, Villejuif, France) showed specific binding of annexin-V to the plasma membrane of cells with apoptotic morphology (Figure 2). This colocalized with the presence of activated caspase-3 in corresponding sections (CM1 antibody, IDUN Pharmaceuticals, La Jolla, Calif), suggesting that annexin-V was bound to cells with apoptosis, which was confirmed by double staining with both antibodies (Figure 2). Histologic analysis of the tumor revealed high-grade undifferentiated sarcoma. The patient refused further treatment and died 2.5 months after surgery. Postmortem analysis showed a large intracardiac tumor mass and multiple metastases.

View larger version (137K): [in this window] [in a new window] Figure 2. Immunohistochemical Analysis of the Tumor Specimen A, Immunostaining for annexin-V shows binding of annexin-V to the plasma membrane of tumor cells (brown staining, arrows). B, Corresponding section stained with CM1 antibody specific for activated caspase-3 shows extensive binding to the cytosol of cells, which colocalize with annexin-V–positive cells in A (brown staining, arrows). C, Double staining with annexin-V antibody (brown staining) and CM1 antibody (red staining) shows specific binding of annexin-V of the plasma membrane of cells with activated caspase-3 in the cytosol.

Comment
Our data indicate that ^99mTc-p-annexin-V accumulated in the tumor because it bound to a large number of cells with phosphatidylserine on the surfaces, which results from apoptosis.³ Because a high apoptotic index is found mainly in malignant tumors and may be related to poor prognosis, ^99mTc-p-annexin-V imaging may be helpful to obtain prognostic information about tumors in a noninvasive way.^{1, 4} This could help select the best therapeutic approach. Another promising application for ^99mTc-p-annexin-V imaging may be to monitor the effect of antitumor treatment.⁵ We observed a large number of apoptotic cells within the tumor, indicating that these cells still contain essential parts of the cell death program and suggesting that they may still be susceptible to chemotherapeutic agents. Therefore, this technique may aid diagnosis and therapy selection in localized malignant tumors.

AUTHOR INFORMATION
Funding/Support: This study was supported by grant 98.195 from the Dutch Heart Foundation and grant D96.025 from the NHS. The CM1 antibody used was a gift from IDUN Pharmaceuticals, La Jolla, Calif.

Acknowledgment: We thank M. T. Pakbiers and J. Habets for technical support.

L. Hofstra, MD,PhD; E. A. Dumont, MD
Department of Cardiology

P. W. L. Thimister, MD,PhD; G. A. K. Heidendal, MD,PhD
Department of Nuclear Medicine

A. P. DeBruine, MD,PhD
Department of Pathology

T. W. O. Elenbaas, MD
Department of Cardiac Surgery

H. H. Boersma, PharmD
Department of Clinical Pharmacy and Toxicology
University Hospital Maastricht
Maastricht, the Netherlands

W. L. van Heerde, PhD; C. P. M. Reutelingsperger, PhD
Department of Biochemistry
University of Maastricht
Maastricht

1. Hofstra L, Liem IH, Dumont E, et al. Visualisation of cell death in vivo in patients with acute myocardial infarction. Lancet. 2000;356:209-212. FULL TEXT | ISI | PUBMED 2. Gisbertz IA, Schouten HC, Bot FJ, Arends JW. Cell turnover parameters in small cell and large cell varieties of primary intestinal non-Hodgkin's lymphoma. Cancer. 1998;83:158-165. PUBMED 3. Martin SJ, Reutelingsperger CP, McGahon AJ, et al. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus. J Exp Med. 1995;182:552-556. 4. Naresh KN, Lakshminarayanan K, Pai SA, Borges AM. Apoptosis index is a predictor of metastatic phenotype in patients with early stage squamous carcinoma of the tongue. Cancer. 2001;91:578-584. PUBMED 5. Belhocine TZ, Hustinnx R, Jerusalem G, et al. 99m-Tc RH Annexin-V (Apomate) as a marker resulting from chemotherapy: preliminary results. Abstract presented at: 47th annual meeting of the Society of Nuclear Medicine; 2000; St Louis, Mo.

Letters Section Editors: Stephen J. Lurie, MD, PhD, Senior Editor; Jody W. Zylke, MD, Contributing Editor.

JAMA. 2001;285:1841-1842.

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