Antibodies against a variety of tumor-associated antigens have been studied, as well as a number of modifications to the antibodies themselves, including Fab' fragments and chimeric, humanized, and human
Antibodies against a variety of tumor-associated antigens have been studied, as well as a number of modifications to the antibodies themselves, including Fab' fragments and chimeric, humanized, and human antibodies. The appropriate use of radioimmunoconjugates in the evaluation of cancer patients has not yet been clearly defined. Only a few studies have assessed their use, primarily through the intravenous route, in initial disease staging. To date, immunolymphoscintigraphy has not proven promising in the staging of cancers. More emphasis has been given to the use of IV radioimmunoconjugates to detect residual and recurrent disease, with generally favorable results. In addition, radioimmunoguided surgery, using small, handheld probes to detect foci of antibody accumulation, appears to be a valuable tool. As better production techniques become available and large clinical trials provide more clearly defined indications for radioimmunoconjugate use, these agents should enter the arena for routine diagnostic use.
In 1993, the FDA approved the first radiolabeled monoclonal antibody for diagnostic use in patients with cancer, CYT-103 (OncoScint OV/CR). This indium-111-labeled whole murine antibody to a tumor-associated glycoprotein, TAG-72, is found on a variety of mucin-producing adenocarcinomas. Many other antibodies recognizing other tumor-associated antigens are being investigated currently for tumor imaging. An understanding of the underlying principles of this unique modality is necessary for its appropriate and optimal use in the management of oncology patients.
The purpose of this paper is twofold: (1) to explore some of the technical issues surrounding the selection and optimal use of radiolabeled antibodies for diagnostic use and (2) to present a sampling of clinical trial results using some of the more promising agents for radioimmunodetection.
In order to image tumors with antibodies, one must target those antigens on the tumor cell that are different, either qualitatively or quantitatively, from antigens on surrounding normal cells. Ideally, the targeted antigens would be unique to tumor cells (ie, not found in any normal tissue in any amount). However, in the real world, most "tumor-associated antigens" are also found in some normal tissues, although sometimes at lower density or in less accessible sites (eg, intracellular) than in tumors. The sensitivity and specificity of radiolabeled antibody imaging depends, in part, on the degree of expression of accessible antigen in the tumor site vs normal tissue.
Among the first antigens targeted for immunoscintigraphy were the oncofetal antigens (eg, carcinoembryonic antigen [CEA] and alpha-fetoprotein [AFP]). Later, monoclonal antibody development led to the discovery of other, previously unidentified tumor-associated antigens, (eg, tumor-associated glycoprotein 72 [TAG-72], novel CEA epitopes, and epithelial membrane antigen [EMA]), which has expanded the repertoire of available agents.
In selecting radioisotopes for imaging, one must consider a variety of factors, many of which are mutually exclusive (the "no free lunch" phenomenon). For example, one would like to have high count rates while limiting the radiation dose to the patient. Therefore, isotopes that have a high efficiency of interaction with the gamma camera crystal (eg, technetium-99m) are preferred over those with a low efficiency (eg, iodine-131). Technetium-99m also has a relatively short half-life (approximately 6 hours), which, coupled with its relatively low-energy gamma photon, means that a larger administered activity (which translates to more counts per second) can be given for the same patient radiation dose. Iodine-131, on the other hand, emits not only a higher-energy gamma radiation but also beta particulate radiation, which increases patient radiation dose without contributing to imaging. Both technetium-99m and iodine-131 are relatively inexpensive--an advantage in this era of cost consciousness.
For optimal lesion detection, however, a high target-background ratio is needed, which means that blood and normal tissue levels have decreased while tumor levels remain high. With whole antibodies, this frequently requires waiting 24 hours or more for clearance from normal tissues to occur. The "ideal" isotope from the standpoints of efficiency and radiation dose, technetium-99m, has a short, 6-hour half-life. This means that by 24 hours, the count rate will have dropped to a point that would require prolonged imaging time to acquire suitable images, which, in turn, increases the probability that patient movement will degrade those images. Iodine-131, with its 8-day half-life, presents no such problem. The chemistry of technetium-99m is also less favorable for antibody labeling than is that of iodine.
Iodine-123 is chemically identical to iodine-131 but has no beta emission. Its efficiency of interaction with the gamma camera crystal is almost as good as that of technetium-99m. The half-life of iodine-123 is 13 hours, which is acceptable for 24-hour imaging but marginal past that point. Unfortunately, iodine-123 is relatively expensive and is less readily available than iodine-131 or technetium-99m.
Indium-111 has a favorable half-life (approximately 3 days) for delayed imaging. However, it tends to accumulate in normal liver tissue, which decreases its usefulness for the detection of liver metastases. It is also expensive, but is currently the only isotope used with an FDA-approved monoclonal antibody imaging agent.
The first antibodies used for imaging were produced by immunizing an animal (usually a mouse) with human tumor cells or cell extracts and then harvesting and purifying antibody from the animal's serum or ascitic fluid. This method yielded a variety of antibodies against a wide spectrum of antigens, some of which were "tumor specific" and others of which were more ubiquitous in tissues. Since these antibodies were derived from many B-lymphocyte clones, the term "polyclonal" is used to describe them. As might be expected, substantial lot-to-lot variation occurred when this production method was used.
In 1975, Kohler and Milstein  developed a method for selecting specific clones of cells that produced pure antibody against a single antigen; hence these antibodies were termed "monoclonal." As initially described, animals were immunized with a target substance (eg, whole cells, membrane extract, or a purified antigen source), as is the case for the production of polyclonal antibodies. Subsequently, however, the splenic B lymphocytes were harvested and fused with mouse myeloma cells to form immortal "hybridomas," which could be grown in cell culture. Each hybridoma clone produced a single antibody, which could be screened for immunoreactivity with the desired antigen. Cells from desirable clones could then be grown in animals (ascitic fluid) or in artificial cell culture systems to produce large amounts of pure antibody. This technology permitted the mass production of antibodies for clinical use.
Modifications of Monoclonal and Polyclonal Antibodies
Various modifications of these antibodies have been explored. Whole antibody has a rather slow clearance half-time from blood and normal tissues, necessitating prolonged delays between antibody injection and imaging. Not only does this inconvenience the patient and prolong the diagnostic process, but longer delays necessitate the use of isotopes that are suboptimal with respect to imaging characteristics and patient radiation dose.
Fab' Fragments--The fact that smaller molecules tend to clear more rapidly has led to the development of a technique whereby whole antibodies are digested into antibody fragments in such a way as to maintain immunoreactivity. Since the specificity of antibody binding depends on the variable region of the IgG molecule, elimination of the constant region should produce faster clearance without compro- mising affinity. Fab' fragments consist of variable regions of one heavy chain (VH) and one light chain (VL), while F(ab')2 fragments consist of two Fab(v) fragments connected at the hinge region. They are approximately one third and two thirds, respectively, the molecular weight of whole antibody .
Even smaller fragments consisting of only the hypervariable regions of the heavy and light chains have been produced, and are sometimes referred to as Fv fragments. The smallest unit that would retain immunologic specificity is the hypervariable region peptide or molecular recognition unit, the sites that confer the specific antigen-binding properties to the antibody molecule . Not only are these fragments cleared from nontumor sites more quickly due to their smaller size, they should also be better able to migrate through the extravascular space into the interior of the tumor, thus expanding the available antigen-binding sites .
Chimeric Antibodies--Another method of limiting the amount of mouse protein in an antibody is to fuse the variable region of the murine monoclonal antibody of interest to the constant region of human antibody, either chemically or using genetic engineering techniques . The resulting antibodies are known as "chimeric" antibodies (named for the chimera of Greek mythology, creatures that were composed of parts of several different animals). The antigenicity of chimeric antibodies is similar to that of antibody fragments.
"Humanized" antibodies have been developed using genetic engineering techniques to graft the complementarity-determining regions of mouse antibodies into human molecules . These antibodies would be expected to have antigenicity (incidence of human antimouse antibody formation) similar to FV fragments while retaining other characteristics of whole antibody.
Human Antibodies--A few fully human antitumor antibodies have been studied. Antigen-stimulated human B lymphocytes from cancer patients are virus transformed and grown in nutrient media within a device that allows harvesting of high-purity antibody. As there is no foreign (nonhuman) protein content, antigenicity of these human antibodies would be expected to be quite low. This has been found to be the case in clinical trials, in which no antigenic responses were detected in patients receiving multiple injections of human antibodies 
Bifunctional Antibodies--An additional modification is the development of bifunctional antibodies that bind to both tumor-associated antigens and to a radiolabeled ligand. This allows for a two-step process of radiolabeling in which the unlabeled antibody is injected first, followed by injection of the labeled ligand after clearance of unbound antibody from the blood pool . This technique significantly decreases background activity, allowing detection of smaller lesions and/or enhanced detection of liver lesions using indium-111 for labeling.
Human Antimouse Antibodies
The tendency of mammalian immune systems to generate antibodies against foreign proteins, the very basis of monoclonal antibody production, also creates difficulties in the use of these substances in humans. The incidence of human antimouse antibodies after injection of whole murine antibody depends, in part, on the impact of the primary malignancy on the patient's immune response, as well as on the amount of protein administered. Therefore, patients with malignancies such as the lymphomas have a lower incidence of human antimouse antibodies (0 of 12 patients receiving up to four injections, with 1 patient developing antimouse antibodies after a fifth injection in one study)  than do patients with solid tumors (32% to 100%) [11-14].
The production of human antimouse antibodies in patients is worrisome for several reasons. The obvious concern when dealing with the injection of a foreign protein into patients is that of allergic reactions. While a variety of such reactions have been noted in the many trials using murine antibodies in human subjects, serious allergic responses, such as anaphylaxis, have fortunately been extremely rare. Minor reactions in patients who have developed human antimouse antibodies, although more common, are not as frequent as one might expect.
Another problem is that once a patient has developed his or her own antibodies against the murine antibodies, any subsequently injected murine antibody will form complexes with the patient's antimouse antibody. These antibody complexes are quickly cleared by the reticuloendothelial system, which prevents the murine antibody from reaching its intended target. Therefore, imaging these patients would produce the equivalent of a liver/spleen scan rather than a "tumor scan."
The presence of human antimouse antigens in a patient's serum can also result in erroneous results in a variety of laboratory assays that utilize murine antibodies (eg, radio-immunoassay and enzyme-linked immunosorbent assay). Perhaps most important for the oncology patient is the fact that many assays for tumor-associated serum markers used to detect early recurrence may become difficult to interpret in the presence of significant levels of human antimouse antibodies.
Murine antibody fragments and chimeric antibodies are an improvement over whole antibodies from the standpoint of having a lower incidence of human antimouse antibody formation (2% to 15%) [12,15-16], while complementarity-determining region-grafted antibodies and fully human antibodies appears to have almost no immunogenicity . In general, the amount of mouse-derived protein injected into the patient is directly related to the incidence of human antimouse antibodies.
Although nuclear imaging can provide physiologic information about living systems that cannot be obtained with other imaging modalities, it has inherent limitations on the spatial resolution that can be achieved. This pertains to radiolabeled antibody imaging as well as more familiar nuclear imaging studies. One means of improving the lower limit of detection for lesions is to employ a technique called single-photon emission computed tomography (SPECT). This technique results in the equivalent of a three-dimensional map of isotope distribution in the body and enhances contrast resolution, making possible the detection of smaller and/or less intense concentrations of isotope. Lesions as small as 0.5 cm have been detected using technetium-99m SPECT . Single-photon emission computed tomography is also helpful in anatomic localization of lesions, compared to planar imaging alone.
Despite the improvements provided by SPECT imaging, radiolabeled antibody imaging alone is rarely sufficient to pinpoint the location of malignant lesions. This is due partly to the spatial resolution problem mentioned above and partly to the fact that normal structures (ideally) are not imaged to any great extent, therefore removing the usual reference points upon which one depends for exact anatomic localization. A potential solution to this problem is the fusion, by means of computer applications, of SPECT images of the distribution of labeled antibodies with CT or MR images . This fusion process is being studied at several centers, and eventually commercial software packages should become available for routine clinical use.
Initial Disease Staging
After a primary malignancy has been detected, accurate disease staging is important for treatment planning in order to avoid unnecessary morbidity from surgical procedures or other attempts at curative therapy in patients who cannot benefit from these procedures. Radiolabeled antibodies may have some role in the staging of certain malignancies. Since antibody imaging does not depend on gross anatomic alteration of structures, metastases may be detectable earlier with this modality than with CT or MRI, although lesions smaller than approximately 1 cm are not reliably detected using current antibody imaging technology [19-20].
Fewer studies have been performed to assess the utility of radioimmunodetection in staging prior to initial therapy than for management of known or suspected recurrent disease. However, inferences can be made with regard to this application from a number of studies that involved patients with primary and recurrent tumors and reported the results for these two groups separately.
Colorectal Cancer--Results of studies using a variety of antibodies to colorectal carcinoma have yielded mixed results in presurgical evaluation trials. In a study of mixed primary and recurrent colon cancer, indium-111-CYT-103 had a sensitivity of 92% and a specificity of 67% and changed patient management in 33% of patients. Of 11 patients with primary disease, three were found by immunoscintigraphy to have extrahepatic intra-abdominal sites of involvement undetected by other imaging modalities . A separate study of the same antibody in 155 patients, which compared immunoscintigraphy to CT, found that the overall sensitivity and specificity of the two modalities were similar. However, CT was more sensitive for liver metastases and immunoscintigraphy was more sensitive for pelvic tumors and extrahepatic abdominal sites .
Indium-111-ICR2, which recognizes EMA, was found in a study of 22 patients to have a sensitivity of 80% and specificity of 20% for primary tumor on unblinded reading (due to localization in dysplastic benign tumors and inflammatory lesions), and a poor sensitivity for liver or lymph node metastases .
Head and Neck Cancers--Technetium-99m-labeled-174H.64, an antibody against a cytokeratin-associated antigen, was used in the preoperative imaging of 21 patients with histologically proven squamous cell carcinomas of the head and neck, 18 of whom had remaining primary tumor. All 18 primary tumors were detected, as well as 15 of 18 lymph node metastases, the smallest of which was less than 1 cm. One of these nodes was detected by immunoscintigraphy only. Two of the three false-negative nodes contained only microscopic disease by histology. Two of three distant metastases were identified by immunoimaging .
Breast Cancer--In a study of eight patents with nine known primary breast carcinomas, preoperative technetium-99m-IMMU-4 Fab' imaging was compared to surgical pathology reports of breast and axillary nodes. All nine primary tumors were detected (100% sensitivity, 100% specificity). Adequate imaging of the ipsilateral axillae was possible in seven of the nine involved breasts. Four axillae were judged positive, and of these, two were found to have metastases. The three negative axillae were all found to be free of disease, yielding a sensitivity of 100% and a specificity of 72%. The smallest primary tumor measured 1.4 cm .
Assessment of Lymph Node Involvement--In addition to being administered intravenously, radioimmuno- conjugates have also been injected subcutaneously for imaging of the lymphatic system in the tumor region to assess nodal involvement prior to surgery. Lymphoscintigraphy has been performed routinely for many years, using radiopharmaceuticals such as ultrafine sulfur colloid. However, these studies depend on tumor-induced alterations of the lymph node architecture. Thus, a nidus of cells that has not yet grown to the extent of causing these changes will not be detected. Intuitively, immunolymphoscintigraphy should enhance sensitivity due to the active targeting of smaller foci of cancer cells. Unfortunately, clinical trials to date have yielded mixed results.
In an early study by Epenetos, whole HMFG2 was used as a specific antibody and UJ13A, an antibody against neuroblastoma, was used as a nonspecific antibody in six patients with cervical carcinoma. All six had significant uptake of both antibodies in inguinal nodes and lymphatic channels after subcutaneous pedal injection. All patients also underwent standard lymphoscintigraphy, which was abnormal in one of six. This patient had additional uptake of HMFG2 in the pelvic wall bilaterally and was found to have evidence of disease on physical examination .
In a study of 11 patients with malignant melanoma for whom node resection was planned, patients received iodine-131-labeled Fab 96.5, an anti-p97 antibody, or Fab 48.7, an anti-high-molecular-weight antigen of melanoma, as a specific antibody, and iodine-125- labeled Fab 1.4, an anti-leukemia antibody, as a nonspecific antibody. Of six patients with surgically positive nodes, three had localization of specific antibody by imaging, as well as one of five surgically negative patients. (Imaging was not performed with nonspecific antibody due to the characteristics of iodine-125.) Tissue counting of nodes yielded similar levels of both specific and nonspecific antibody, and autoradiography showed similar distribution of both within tumor-bearing nodes . Both this study and that by Epenetos seem to indicate that, for at least some tumor-antibody combinations, the uptake of antibody in tumor-bearing nodes is a nonspecific occurrence.
A prospective study of 40 patients with suspected breast cancer using two different specific monoclonal antibodies (3E1.2 and RCC-1) found that the sensitivity and specificity of RCC-1, the better performing of the two antibodies, was comparable to clinical assessment in detecting axillary node involvement, determined histologically in those patients who were found at surgery to have cancer (36 patients). The sensitivity was 50% for both imaging and clinical examination, and the specificity was 60% and 50%, respectively. Although imaging was not found to be superior to physical examination, the investigators noted that in some women, positive nodes that were not suspected by clinical examination were detected by immunoscintigraphy .
Detection of Residual or Recurrent Disease
In patients who have already received treatment, it is often difficult to differentiate residual or recurrent disease from a nonmalignant pathologic process. For example, after radiation therapy to the pelvic region, tissue planes are frequently indistinct or distorted on subsequent CT studies; this makes it hard to arrive at definitive statements concerning residual or recurrent disease. Helping clinicians make these distinctions may prove to be one of the best diagnostic uses of radiolabeled antibodies; this application has been the subject of most of the clinical trials to date. If adequate specificity can be achieved for a given antibody-tumor system, positive antibody imaging could result in lower costs by avoiding the need for multiple other imaging studies while shortening the time to therapy and/or avoiding unhelpful surgical procedures.
Colorectal Cancer--Again citing colorectal carcinoma trials, indium-111-CYT-103 imaging changed management decisions in 10 (55%) of 18 patients with known or suspected recurrence (based on elevated serum CEA levels): Antibody imaging detected occult local recurrence in six patients, identified an isolated liver metastasis in a patient with fatty liver and an equivocal CT, avoided liver resection in two patients who were found to have additional abdominal lesions, and led to the cancellation of surgery in one patient who was thought to have an isolated liver metastasis but was found to have widespread metastases .
A comparison of iodine-131-FO23C5, an anti-CEA antibody, with CT, MRI, ultrasound, endoscopy, or other diagnostic procedures in evaluating local relapse of colorectal carcinoma showed antibody imaging to have a higher sensitivity (89%) than any other modality except MRI (93%) and a higher specificity (78%) than any other modality . Similarly, a comparison of techetium-99m-IMMU-4 Fab' to conventional diagnostic procedures demonstrated that immunoscintigraphy was more accurate than conventional procedures and would have changed management in 58% of patients .
The use of radioimmunoconjugates for the detection of occult recurrence in patients with a rising CEA but negative standard imaging studies and colonoscopy has been the focus of a number of studies. Indium-111-ZCE-025 imaging was positive in 19 of 20 patients with CEA-producing tumors and occult recurrence by these criteria. Of these, 2 had positive biopsies, and 13 underwent exploratory surgery. Although only 10 of the 13 were found to have malignant lesions at the time of surgery, the remaining 3 patients, as well as 4 who did not undergo surgery, were found on follow-up to have recurrence at sites that had been positive on radioimmunoscintigraphy .
In a similar study, 15 patients underwent imaging with technetium-99m-IMMU-4 prior to exploratory laparot- omy, and positive foci were correlated with surgical and pathologic findings. There were 12 true-positive studies, and one false-positive study. The remaining two studies were true negatives by surgical correlation. A total of 26 malignant lesions were found surgically, of which 21 were positive by imaging; 30 biopsies were negative, 25 of which were negative by imaging and 5 of which were false positive. Therefore, the per lesion sensitivity was 81% and specificity was 83%. Four of the five false-positive lesions were inflammatory lesions of the peritoneum, and one was a hemorrhagic ovarian cyst. Of the 15 patients, 5 underwent complete disease resection .
Other Cancers--A trial evaluating the ability of intravenous indium-111-CYT-103 to detect recurrent ovarian carcinoma found a sensitivity of 68% and specificity of 58% . In another trial, intraperitoneal iodine-131-C0C183B2 had a sensitivity and specificity of 95% and 89%, respectively, in the detection of recurrent ovarian cancer . Intravenous CYT-103 has been shown to be able to detect peritoneal carcinomatosis-an advantage over other imaging modalities.
In a study of 27 patients with known metastatic disease, technetium-99m-labeled F(ab')2 fragments of antibody 9.2.27, which recognize melanoma-associated antigen, were able to image metastatic melanoma in 43 (81%) of 53 known sites and 36 previously unknown sites. However, relatively poor detection rates were noted for the lungs (60% of known lesions) .
Despite these generally encouraging results, it has yet to be proven whether the use of these and other immunoconjugates will result in improved patient survival or well-being. Nor is it known whether this modality will be cost effective in the routine management of cancer patients. This information may be forthcoming in the next few years, as more large-scale trials address these key issues.
Although it does not involve imaging, another potential use of radiolabeled antibodies for diagnostic purposes involves the use of iodine-125-labeled antibodies and a small handheld gamma detector for locating nodes or other tissues containing tumor during surgery. The long half-life of iodine-125 allows clearance of background activity prior to surgery, while its low gamma energy is effectively blocked by surrounding tissue, making accurate localization possible. Indium-111-labeled antibodies have also been used for this application, but the characteristics of this isotope are less desirable.
Trials of this technique prior to second-look surgery for ovarian carcinoma , radical prostatectomy , surgery for primary breast  or primary or recurrent colorectal cancer [39-45] have been performed or are underway. The technique could be applied in the surgical management of other cancers as well. In colorectal carcinoma trials, for which the most data have been accumulated, radioimmunoguided surgery (RIGS) has been found to have a high lesion detection rate (67% to 97%), with alteration of planned management in 18% to 46% of patients (most commonly, avoidance of unnecessary liver resections in patients found to have additional abdominal metastases or identification of resectable local-regional recurrence in patients with suspected disease not localized by conventional methods).
Of particular interest is one study  for which long-term follow-up data are available. In this study, 86 patients with recurrent colorectal carcinoma were evaluated first by conventional surgical exploration and then using RIGS. Patients were classified into RIGS resectable (those resectable by both techniques), RIGS nonresectable (resectable by traditional exploration but with additional RIGS-positive sites), and traditional nonresectable. Follow-up data at 2, 3, and 4 years showed dramatically increased survival rates for the RIGS-resectable group compared to the two nonresectable groups, and for the RIGS-nonresectable group compared to the traditional nonresectable group. No patients from either of the nonresectable groups survived 5 years, while 60% of the RIGS-resectable group were alive at that time point. These data would seem to indicate that RIGS can be used to improve the selection of resectable patients during second-look surgery for recurrent colorectal cancer.
Dosimetry Prior to Radiolabeled Antibody Therapy
One last use of radiolabeled antibody imaging should be mentioned briefly. Although a discussion of the therapeutic use of radiolabeled antibodies is beyond the scope of this paper, one problem that arises with use of this form of systemic radiation therapy is the estimation of radiation dose to be delivered to the tumor. Estimates cannot be based solely on body weight or surface area, since antigen expression varies between individuals and even between different sites in the same individual. By quantifying the accumulation of antibody labeled with an "imaging" isotope, one can predict how much antibody labeled with a "therapy" isotope will reach the tumor, and thus how much radiation will be delivered.
Some isotopes used for therapy (eg, iodine-131, rhenium-186, copper-67) produce gamma photons that can be imaged in addition to beta particles useful for therapy, so that the "imaging" isotope is the same as the "therapy" isotope. Other beta emitters (eg, yttrium-90) emit no gamma photons, and thus, cannot be used to produce images of sufficient quality for dosimetric purposes. Another isotope with similar chemical properties, but with favorable gamma emissions, is necessary to act as a surrogate for these isotopes in imaging.
Unfortunately, there is currently no consensus on the best way to calculate dosimetry estimates. An additional problem posed by this use of isotopes is the risk of inducing a human antimouse antibody response in the patient. This, in itself, will change the kinetics of the system, making estimates based on the imaging data unreliable, as well as diminishing therapeutic efficacy.
It seems clear that although the clinical use of radioimmunoconjugates is still in its infancy, this modality will prove to be a useful addition to the evaluation of oncology patients. As advanced techniques for generating less immunogenic proteins are perfected and become available on a commercial scale, and as large-scale clinical trials continue to define their most effective use, additional radioimmunoconjugates should become available for diagnostic use. Although these agents will not completely replace more conventional diagnostic testing, it is to be hoped that they will allow for earlier and/or more efficacious therapy while avoiding unnecessary morbidity.
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