Nodal staging plays a critical role in the evaluation of disease progression and design of therapeutic strategies in the treatment of most cancer patients. For example, in breast cancer, intradermal (i.d.) or subcutaneous (s.c.) administration of a nonspecific 99mTc-radiocolloid is used to identify the tumor draining or sentinel lymph node for resection. Once the surgical pathologist confirms the presence of cancer cells in the resected sentinel lymph node, additional nodes around the axilla are removed to better assess the extent of lymph node involvement. The accuracy of nodal staging is critical for judicious selection of effective therapy. The inability to surgically resect inaccessible tumor-draining lymph nodes, such as the internal mammary or supraclavicular lymph nodes in breast cancer patients, or the pelvic lymph nodes that drain the prostate in prostate cancer patents, can compromise oncologists’ ability to accurately stage and select proper therapy. Noninvasive, diagnostic imaging of cancer-positive lymph nodes could positively impact the manner in which tumor-node-metastasis (TNM) staging is performed.
Molecular imaging approaches for early-stage diagnosis of metastasis in the lymphatics were first explored by Weinstein et al., who used radio-labeled monoclonal antibodies that were administered (i.d.) through the footpad in preclinical studies.1,2
Since then, several investigators have used immunolymphoscintigraphy as a tool to study lymphatic delivery of agents. Steller et al.3
demonstrated the dose dependence of iodinated monoclonal antibody biodistribution following i.d. administration in a murine animal model, while Wahl et al.4
employed iodinated intact and fragmented antibodies to assess the kinetics of their clearance from the lymphatic system in response to ambulation. Additional reports have used immunolymphoscintigraphy to study lung cancer metastases to pulmonary and mediastinal lymph nodes in dogs.5
The i.d. route of imaging agent delivery may be extremely favorable for nodal staging because antibodies and small molecules drain primarily from the lymph plexus into the lymphatic system. Small molecules pass readily through one or more nodes before exiting the lymphatic circulation via the blood circulatory system, while larger agents may be retained longer within lymph nodes. Some advantages for using a direct lymphatic approach include: (1) high lymph node uptake of antibodies, which enables significantly lower doses of labeled antibodies than that required for intravenous (i.v.) administration, (2) more rapid lymph node uptake, enabling minimal time between i.d. antibody administration and imaging, and (3) increased target-to-background ratios due to reduced nonspecific binding, which would otherwise occur in systemic i.v. administration. A more comprehensive review of lymphatic uptake and transport of proteins following intralymphatic administration can be found in an article by Porter and Charman.6
In addition to animal studies, imaging-based clinical applications of intralymphatic administration have also been explored by several investigators, mostly using radio-labeled monoclonal antibodies in their intact or fragmented form to detect tumor-draining lymph nodes in a spectrum of cancer patients ranging from melanoma, T-cell lymphoma, breast, and prostate cancers. One of the first investigations of lymph node imaging was reported by Order et al. in 1975, in which they demonstrated accumulation of 131
I-labeled antiferrin immunoglobulin in lymph nodes of patients with breast carcinoma and lymphoma.7
Following that, DeLand et al. conjugated 131
I-labeled antibodies to carcinoembryonic antigen (CEA) and showed high sensitivity in detecting nodal metastases after interdigital administration in patients.8
In breast cancer and T-cell lymphoma, immunolymphoscinitigraphy was performed by several investigators with 131
In-, and 99m
Tc-labeled monoclonal antibodies to noninvasively assess regional lymph nodes.9–12
Pelvic nodal metastases in prostate cancer have been investigated13
by bipedal intralymphatic administration of 111
In-PAY 276, while antimelanoma antibodies that have been radio-labeled for lymphoscintigraphy have also been reported in the literature.14–16
Most injections were well tolerated, but some patients have reported pain at the injection site. Most studies have reported a high sensitivity (80 to 100%) using this route of administration, but nonspecific uptake by normal lymph nodes continues to be problematic.
Although lymphoscintigraphy, or the administration of nonspecific 99mTc sulfur colloid, is the “gold standard” for clinically evaluating lymph mapping, some limitations suffered by this imaging technique include the need for long (~20 min) gamma camera integration times, poor temporal and spatial resolution, low signal-to-noise ratios (SNRs), and the finite physical half-life of the tracer [t1/2(99mTc)=6 h]. In contrast, near-infrared (NIR) fluorophores provide an attractive opportunity that outweighs some of these disadvantages inherent in nuclear techniques. Since fluorescent reporters do not have a physical half-life, they can be repeatedly activated using appropriate excitation light. Theoretically, for a fluorophore with a nanosecond fluorescent lifetime and a quantum efficiency of 0.1, 108 photons can be generated per second per molecule. When compared to the one-photon imaging event resulting from the radioactive decay of a radionuclide, the potential advantages of NIR fluorescent optical imaging with NIR fluorophores becomes evident. The increased photon yield can considerably improve signal-to-noise and significantly reduce image acquisition time for molecular imaging, if tissue attenuation does not significantly reduce the number of detected fluorescent photons. While NIR fluorophores also have an absorption cross section, their administration in trace doses does not provide sufficient absorption contrast for in vivo. Hence, NIR contrast may be best provided from the collection of NIR fluorescent photons rather than from the attenuation of excitation photons, which may occur from endogenous chromophores as well as from the exogenous fluorophores.
Herein, we present our work on a dual-labeled conjugate — (111
— which targets the human epidermal growth factor receptor-2 (HER2). Overexpression of HER2 is associated with poor prognosis in about 20 to 30% of breast cancer patients.17
We have previously shown specificity in vivo
in subcutaneous xenograft models.18
In this report, we first review recent results from our group describing lymphatic imaging with nonspecific optical agents. Next, we review our prior results showing tumor targeting of dual-labeled (111
and enhanced performance of optical over nuclear imaging. We additionally show trafficking of the labeled antibody into the lymphatics and its subsequent drainage into lymph nodes using NIR optical imaging.