Dermal collagen matrices for ventral hernia repair: comparative analysis in a rat model.

PURPOSE: The purpose of this study was to compare inflammatory responses, tissue integration, and strength of the acellular dermal collagen matrices AlloDerm(®)* Regenerative Tissue Matrix, Permacol™**Surgical Implant (Permacol), and CollaMend™*** Implant in a rat model for ventral hernia repair. METHODS: Rats were randomized into four groups and abdominal wall defects repaired with an inlay graft of AlloDerm, Permacol, or CollaMend. Rats were sacrificed at six time points and the defect area was removed and analyzed for tissue integration and physical strength. RESULTS: Variable cell infiltration was seen for the three implant groups. At of the all time points examined, cellular infiltration was most rapid in the AlloDerm implants and slowest for CollaMend. At 14 days, significant cell infiltration along with putative blood vessel formation was observed for AlloDerm, while Permacol implants exhibited a moderate level of infiltration. Very few cells penetrated CollaMend implants at 2 weeks. Cells had reached the center of the Permacol implants by 1 month, whereas CollaMend implants were encapsulated with a loose coat of disconnected cells, with very few cells infiltrating past the surface. At 6 months, AlloDerm and Permacol had evidence of cell penetration throughout the implants, while the CollaMend samples exhibited limited infiltration. Animals for each implant developed seromas: AlloDerm 40%, Permacol 33%, and CollaMend 83%. Mechanical testing revealed that AlloDerm at 6 months showed the lowest tensile strength, CollaMend the highest, and Permacol an intermediate level. CONCLUSIONS: The three biologics exhibited different patterns and rates of cellular and vascular permeation in our rat model. AlloDerm implants exhibited the most rapid and extensive cellular infiltration, followed by Permacol. However, on gross examination, the AlloDerm implants thinned significantly by 6 months. In contrast, the Permacol and CollaMend implants appeared to be largely intact.

Long-term histologic and mechanical results of a Permacol™ abdominal wall explant.

PURPOSE: We hypothesize that Permacol™ may allow controlled integration over time while providing long-term mechanical stability and native tissue remodeling. The purpose of this report is to investigate these properties in an explanted piece of Permacol™ after 2 years in vivo. METHODS: A 62-year-old female presented with a complex abdominal wall history having undergone a transverse rectus abdominis musculocutaneous (TRAM) flap breast reconstruction 10 years ago, followed by an abdominal wall repair with Marlex™ mesh for weakness 3 years later. Two years ago, she developed an abdominal bulge repaired with a Permacol™ overlay. Twenty-three months postoperatively, she presented with abdominal distension. Computed tomography (CT) scanning demonstrated a fluid collection behind the Permacol™. She underwent incision and drainage of the hematoma/bursa and quilting repair of the abdominal wall. A 1 × 6-cm Permacol™ section was resected as part of closure. Histology, immunohistochemistry, and mechanical testing of the Permacol™ explant were performed. RESULTS: Histology showed fibroblast and blood vessel ingrowth with no cellular infiltrates reflective of inflammation. Immunohistochemistry for human-specific collagen types I and III and elastin detected staining throughout. Sections stained with non-specific control antibody exhibited no discernable staining. Elastin highlighted blood vessels. Native Permacol™ had a breaking strength of ~20 N, while for explanted Permacol™, it was ~33 N. CONCLUSIONS: Permacol™ maintained durability while allowing vascular ingrowth without residual inflammation. Explant demonstrated integration with human collagen and elastin remodeling throughout. Increase in mechanical strength may reflect newly synthesized collagen and elastin. These histologic findings and clinical result support the use of Permacol™ in complex abdominal wall reconstruction.

IL-10 and GM-CSF expression and the presence of antigen-presenting cells in chronic venous ulcers.

BACKGROUND: White cell trapping and activation occurs in the legs of patients having chronic venous insufficiency (CVI), and it is thought that this process may be important in the development of CVI ulcers. This study has compared the tissue distribution of proinflammatory (GM-CSF) and anti-inflammatory cytokines (IL-10) and inflammatory cell markers (CD68, HLA-DR) between CVI ulcers, other chronic and acute wounds, and autologous nonwounded skin to determine whether cell-mediated immunity (CMI) is impaired in CVI ulcers. METHODS: Wound and donor site tissue was obtained from 10 patients with CVI ulcers and 10 patients with other chronic and acute wounds. Serial Formalin-fixed sections were processed by standard hematoxylin and eosin staining or by indirect immunoperoxidase histochemical staining. RESULTS: HLA-DR-positive antigen-presenting cells (APC), including CD68-positive macrophages and dermal dendritic cells, were found with greater frequency in CVI ulcers than in other chronic or acute wounds (P = 0.0015) or in the autologous CVI donor site tissue (P = 0.006). CVI ulcers also demonstrated increased IL-10 staining of the entire epidermis compared to non-CVI wounds (P = 0.0019) or autologous donor site tissue (P = 0.004), whereas there was no significant change in the presence of the counteracting cytokine, GM-CSF. CONCLUSIONS: These findings suggest that although the cellular components of CMI are present in CVI ulcers, their function may be impaired by the increased level of IL-10. Future studies will examine whether IL-10-mediated suppression of CMI and/or inhibition of GM-CSF-stimulated keratinocyte proliferation may contribute to the chronic nature of CVI ulcers.

Experimental studies on the role of antibody fragments in cancer radio-immunotherapy: Influence of radiation dose and dose rate on toxicity and anti-tumor efficacy.

Whereas bivalent fragments have been widely used for radio-immunotherapy, no systematic study has been published on the therapeutic performance of monovalent conjugates in vivo. The aim of our study was, therefore, to determine the therapeutic performance of (131)I-labeled Fab as compared to bivalent conjugates and to analyze factors that influence dose-limiting organ toxicity and anti-tumor efficacy. The maximum tolerated doses (MTDs) and dose-limiting organ toxicities of the (131)I-labeled anti-CEA antibody MN-14 [IgG, F(ab')2 and Fab] were determined in nude mice bearing s.c. human colon cancer xenografts. Mice were treated with or without bone marrow transplantation (BMT) or inhibition of the renal accretion of antibody fragments by D-lysine or combinations thereof. Toxicity and tumor growth were monitored. Radiation dosimetry was calculated from biodistribution data. With all 3 (131)I-labeled immunoconjugates [IgG, F(ab')2 and Fab], the red marrow was the only dose-limiting organ; MTDs were 260 microCi for IgG, 1,200 microCi for F(ab')2 and 3 mCi for Fab, corresponding to blood doses of 17 Gy, 9 Gy and 4 Gy, respectively. However, initial dose rates were 10 times higher with Fab as compared to IgG and 3 times higher as compared to F(ab')2. The MTD of all 3 immunoconjugates was increased by BMT by approximately 30%. In accordance with renal doses below 10 Gy, no signs of nephrotoxicity were observed. Despite lower absorbed tumor doses, at equitoxic dosing, Fab fragments were more effective at controlling tumor growth than the respective bivalent fragment or IgG, probably due to higher intratumoral dose rates. Our data indicate that the improved anti-tumor effectiveness of antibody fragments as compared to IgG and the higher myelotoxicity at comparably lower red marrow doses are most likely due to the higher initial dose rates observed with antibody fragments.

Morphological and clinical aspects of scapular fasciocutaneous free flap transfer for treatment of venous insufficiency in the lower extremity.

We have recently shown that free scapular fasciocutaneous flaps transferred to the lower extremities of patients with chronic venous insufficiency and cutaneous ulcers have resulted in improvement in venous refilling times measured by photoplethysmography in the flap areas and that recurrent ulceration does not recur for up to 7 years. We hypothesized that the transferred flaps contained valves in their microvascular bed, which facilitated venous return, and using scanning electron microscopy of vascular corrosion casts and light and transmission electron microscopy of tissue sections prepared from human dorsal thoracic fascia, we showed that valves were most abundant in veins with a luminal diameter of 30-120 microm (59.3% of 905 valves). The depth of these valves increased with venous diameter, but the size of valve sinuses was not different for individual valves. Except for veins > 1,000 microm in diameter, there was no significant difference in the number of valves in different parts of an individual flap or between different flaps. Most valves were bicuspid; only in the vein Category 30-120 microm were unicuspid valves encountered. Valves were sometimes located in series in a short segment of a vein; occasionally they were found at the merging of two veins. Transmission electron microscopy showed that valve leaflets had collagen fibers that ascended toward the tip of the leaflet and were occasionally accompanied by elastic fibers. Myofibroblasts were regularly present in the valve leaflets. The present report reviews and updates these anatomic data about the human scapular region, focusing on venous valvular microstructure, and suggests that the high number of smaller-size valves contributes to improved hemodynamic of the leg and thus the clinical success of free scapular flaps used to treat cutaneous ulcerations in the lower extremity.

Comparison of IL-10 levels in chronic venous insufficiency ulcers and autologous donor tissue.

In previous immunohistochemical studies, chronic venous insufficiency (CVI) ulcers have been shown to display positive staining for interleukin-10 (IL-10), while other wounds (including autologous donor wound tissue) show a reduced staining level. IL-10 inhibits the synthesis of many proinflammatory cytokines, while also inhibiting antigen presentation by antigen-presenting cells. It is possible that abnormally high amounts of IL-10 in chronic wounds may be related to the failure of these wounds to progress to final wound healing. The purpose of this study was to quantify the levels of IL-10 in CVI ulcers and autologous donor tissue using Western blotting. Extracts were prepared from frozen wound tissue samples and equal amounts of protein were concentrated by immune-precipitation for Western blot analysis. Densitometric analysis was performed on nonsaturated chemilumigraphs and normalized to an IL-10 standard run on each gel. The quantity of IL-10 in CVI ulcers was found to be 490% of the quantity in autologous donor tissue. This study provides confirmatory quantitative data which supports previous immunohistochemical findings showing elevated levels of IL-10 in CVI ulcers.

Improved treatment of medullary thyroid cancer in a nude mouse model by combined radioimmunochemotherapy: doxorubicin potentiates the therapeutic efficacy of radiolabeled antibodies in a radioresistant tumor type.

Whereas in advanced metastatic medullary thyroid cancer (MTC), a variety of chemotherapeutic regimens have achieved only limited success clinically, more recently, radioimmunotherapy (RIT) with 131I-labeled anti-carcinoembryonic antigen (CEA) monoclonal antibodies (MAbs) has shown promising results. The aims of this study were to compare, in an animal model, the therapeutic efficacy of RIT to clinically used "standard" chemotherapeutic regimens and to evaluate whether combination strategies of both modalities may be feasible and may help to improve therapeutic results in this rather radioresistant tumor type. Nude mice, bearing s.c. xenografts of the human MTC cell line, TT, were treated either with the 131I-labeled anti-CEA MAb, F023C5 IgG, or were administered chemotherapeutic regimens that had shown promising results in patients with metastatic MTC (doxorubicin and cisplatinum monotherapy, combinations of both agents, and a 5-fluorouracil/dacarbazine/streptozotocin scheme). Control groups were left untreated or were injected with an irrelevant radiolabeled antibody at equitoxic dose levels. The maximum tolerated dose (MTD) of each agent was determined. Combinations of chemotherapy and RIT were evaluated as well. Toxicity and tumor growth were monitored at weekly intervals. From the chemotherapeutic agents and schemes tested, doxorubicin monotherapy was the most effective; combination therapies did not result in an increased antitumor efficacy, but they did result in more severe toxicity. At equitoxic doses, no significant difference was found between the therapeutic efficacy of doxorubicin and that of RIT. Myelotoxicity was dose limiting with radiolabeled MAbs (MTD, 600 microCi), as well as with chemotherapeutic regimens containing alkylating agents (cisplatinum, dacarbazine, or streptozotocin). At its MTD (200 microg), doxorubicin caused only mild myelotoxicity, and despite signs of cardiac toxicity, gastrointestinal side effects were dose limiting. Accordingly, bone marrow transplantation (BMT) enabled dose intensification with RIT (MTD with BMT, 1100 microCi), which led to further increased antitumor efficacy, whereas BMT was unable to increase the MTD of doxorubicin. Due to the complementarity of toxic side effects but an anticipated synergism of antitumor efficacy, combinations of RIT with doxorubicin were tested. Administrations of 500 microCi of 131I-labeled anti-CEA and, 48 h later, 200 microg of doxorubicin (i.e., 83 and 100% of the respective single-agent MTDs), were the highest doses that did not result in an increased lethality; with bone marrow support, 1000 microCi of 131I-labeled anti-CEA could be combined with 200 microg of doxorubicin (i.e., 90 and 100% of the individual MTDs). Therapeutic results of this combined radioimmunochemotherapy were superior to equitoxic monotherapy with either agent, and indication for synergistic antitumor effects is given. At its respective MTD, radioimmunochemotherapy led to a 36% cure rate if it was given without bone marrow support and to a 85% permanent cure rate if it was given with bone marrow support. The animal model, as presented in this study, seems to be useful for the preclinical testing of therapeutic agents for the systemic treatment of MTC. At equitoxic doses, RIT with radiolabeled anti-CEA antibodies seems to be equally as effective as chemotherapy with doxorubicin. Combination of RIT and doxorubicin chemotherapy seems to have synergistic therapeutic efficacy, which may be due to a radiosensitizing effect of doxorubicin.

Anti-carcinoembryonic antigen antibodies versus somatostatin analogs in the detection of metastatic medullary thyroid carcinoma: are carcinoembryonic antigen and somatostatin receptor expression prognostic factors?

BACKGROUND: Surgery is currently the only potentially curative approach in the treatment of medullary thyroid carcinoma (MTC). In many instances however, postsurgically elevated or rising plasma calcitonin and/or carcinoembryonic antigen (CEA) levels indicate persistent metastatic disease, although conventional diagnostic procedures (computed tomography (CT), magnetic resonance imaging (MRI), and invasive venous catheterization) fail to localize the responsible lesions. Recently, anti-CEA antibodies and somatostatin analogs have shown promising results in the staging of MTC. The aim of this study was to compare the sensitivity of both methodologies, especially for the detection of occult MTC, and to assess whether there may be correlations between the scintigraphic behavior and the patients' prognosis. METHODS: A total of 26 patients with medullary thyroid carcinoma were examined at our institution between 1977 and 1996. Ten of them had known disease, 14 had occult metastatic MTC, and 2 were free of disease at the time of presentation. Fourteen patients were investigated with anti-CEA monoclonal antibodies (MAbs) (receiving a total of 35 injections: clones BW431/26, BW431/31, IMACIS, or F023C5, labeled with 99mTc, (111)In or (131)I), and 8 patients were studied with (111)In-labeled octreotide. Two patients received potentially therapeutic doses of (131)I-labeled anti-CEA antibodies. All patients underwent conventional radiologic evaluation (ultrasonography, CT, and MRI) and/or biopsy within 4 weeks. Additional imaging was performed with 99mTc-(V)-DMSA, (131)I-metaiodobenzylguanidine, 201thallium chloride, 99mTc-methylene diphosphate, and/or 18F-fluorodeoxyglucose-positron emission tomography. Clinical follow-up was obtained. RESULTS: All patients with established disease had elevated plasma CEA (range, 6.8-345 ng/mL; calcitonin levels between 92 and 11,497 pg/mL), whereas in 9 of 14 occult cases, levels were < or = 5 ng/mL (range, 0.6-829 ng/mL; calcitonin, 72-2920 pg/mL). In patients with known disease, the overall lesion-based sensitivity was 86% for the anti-CEA MAbs, whereas octreotide was unable to target any tumor in patients with rapidly progressing disease or distant metastases (overall sensitivity, 47%). In all patients with occult MTC, anti-CEA MAbs and octreotide were able to localize at least one lesion (patient-based sensitivity, virtually 100%). In patients with postsurgically persistent hypercalcitoninemia, cervical lymph nodes were identified as the most frequent site of metastases, whereas in patients with occult and slowly progressing disease several years after primary surgery, anti-CEA MAbs and octreotide showed bilateral involvement of mediastinal lymph nodes; however, tumor to nontumor ratios were usually higher with octreotide in these cases. With anti-CEA Mabs, the highest tumor to nontumor ratios were observed in clinically aggressive, rapidly progressing disease. The sensitivity of all other diagnostic modalities was, at < or = 50%, significantly lower. Indication for antitumor effects was observed in a patient receiving 65 mCi of (111)I-labeled F(ab')2 fragments of the clone F023C5. CONCLUSIONS: For the detection of occult MTC, anti-CEA MAbs and octreotide seem to have a sensitivity that is superior to conventional diagnostic modalities, especially also when used in combination. Better detectability with anti-CEA antibodies (which may result in higher CEA expression) seems to be associated with more aggressively growing forms of MTC, whereas somatostatin receptor expression at normal CEA plasma levels and weaker MAb targeting may be associated with a more benign clinical course. This is in accordance with the study of Busnardo et al. (Cancer 1984; 53:278-85), who showed higher CEA serum levels to be associated with a worse prognosis, as well as with the in vitro findings of Reubi et al. (Lab Invest 1991;64:567-73), who demonstrated lower somatostatin receptor expression in less differentiated MTC. Fu

Overcoming the nephrotoxicity of radiometal-labeled immunoconjugates: improved cancer therapy administered to a nude mouse model in relation to the internal radiation dosimetry.

BACKGROUND: Elevated renal uptake and extended retention of radiolabeled antibody fragments and peptides is a problem in the therapeutic application of such agents. However, cationic amino acids have been shown to reduce renal accretion. The aims of the current study were to evaluate whether this methodology would benefit therapy with yttrium 90 (90Y)-labeled antibody fragments (Fab, F(ab)2), to establish the relationship between radiation dosimetry and observed biologic effects, and to compare the antitumor efficacy of antibody fragments with that of whole immunoglobulin (Ig)G. METHODS: The maximum tolerated dose (MTD) and the dose-limiting organ toxicity of 90Y-labeled anti-carcinoembryonic antigen (CEA) MN-14 monoclonal antibodies (Fab, F(ab)2, and IgG) were determined in nude mice bearing GW-39 human colon carcinoma xenografts. The mice were treated with or without kidney protection by administration of D-lysine, with or without bone marrow transplantation (BMT), or with combinations of each. Toxicity and tumor growth were monitored at weekly intervals after radioimmunotherapy. Dosimetry was calculated from biodistribution studies using 88Y-labeled antibody. Three different dosimetric models were examined: 1) taking solely self-to-self doses into account, using S factors for 90Y in spheroids from 0.1 to 1 g; 2) correcting for cross-organ radiation; and 3) using actual mouse anatomy as represented by nuclear magnetic resonance imaging with a three-dimensional internal dosimetry package (3D-ID). RESULTS: The kidney was the first dose-limiting organ with the use of Fab fragments. Acute radiation nephritis occurred at injected activities > or = 325 microCi, and chronic nephrosis at doses > or = 250 microCi. Activities of 200 microCi were tolerated by 100% of the animals (i.e., the MTD). Application of lysine decreased the renal dose by approximately fivefold, facilitating a 25% increase in the MTD (to 250 microCi), because myelotoxicity became dose-limiting despite red marrow doses of less than 5 gray (Gy). By using BMT and lysine, the MTD could be doubled from 200 to 400 microCi, where no biochemical or histologic evidence of renal damage was observed (kidney dose, < or = 40 Gy). With injected activities of > or = 325 microCi without kidney protection, and with a hepatic self-to-self dose of only 4 Gy, rising liver enzymes were observed, which could be explained only by cross-organ radiation from radioactivity in the kidneys (in the immediate neighborhood of the right kidney up to > or = 150 Gy). The MTD of F(ab)2 fragments could be elevated only by a combination of BMT and lysine. With IgG, the bone marrow alone was dose-limiting. Tumor dosimetry correlated well with antitumor effects; Fab was more effective than F(ab)2, which was consistent with its more favorable dosimetry, and it may also be more effective than IgG due to its higher dose rate and more homogenous distribution. Dosimetry Model 1 was insufficient for predicting biologic effects. Model 2 seemed to be more accurate, accounting for interorgan crossfire. However, Model 3 showed an additional substantial contribution to the red bone marrow dose due to crossfire from the abdominal organs. CONCLUSIONS: These data show that radiation nephrotoxicity is an important effect of cancer therapy with radiometal-conjugated antibody fragments or peptides. However, this effect can be overcome successfully with the application of cationic amino acids, which substantially increase the anti-tumor efficacy of radiometal-labeled immunoconjugates. For understanding the biologic effects (e.g., liver toxicity) of 90Y in a mouse model, accounting for cross-organ radiation is essential. Further studies with radiometal-conjugated monoclonal antibody fragments and peptides are necessary to determine the MTD, dose-limiting organs, antitumor effectiveness, and nephroprotective effects of cationic amino acids in humans.

Can occult metastases be treated by radioimmunotherapy?

BACKGROUND: Tumor lesions in the millimeter (mm) range may escape detection with nuclear medicine imaging methods (including single photon emission computed tomography [SPECT]) using radiolabeled monoclonal antibodies (MoAbs). We hypothesized that these lesions still could receive a potentially therapeutic radiation absorbed dose, and therefore should be treated, despite the lack of detection. METHODS: To simulate this situation, 2-mm beads (0.004 mL) containing approximately 1.15 microCi of iodine-131 (131I) were used. The beads were placed centrally in a 1200-mL liver phantom containing approximately 3 mCi of 131I. The resultant activity concentration on the beads was approximately 288 microCi/mL compared with approximately 2.5 microCi/mL in the phantom, corresponding to a maximum tumor uptake of approximately 0.3% injected dose per gram (%ID/g) if 100 mCi of 131I-labeled immunoglobulin G were administered. The phantom, containing the beads, was imaged by both planar and SPECT techniques at hypothetical Day 1 (time of maximum tumor uptake) and at hypothetical Day 7 to examine the improved target-to-nontarget ratio over time. In addition to imaging the beads, the radiation absorbed dose to the simulated lesions from the beta component emissions of 131I was calculated using absorbed fractions based on Berger's point kernels. RESULTS: Regardless of the conditions used, the beads could not be observed by either planar or SPECT imaging. However, the radiation-absorbed dose to the simulated lesion was calculated to be as high as approximately 6200 centigray (cGy), with an average dose rate of approximately 89.5 cGy/hour. CONCLUSIONS: This simulation demonstrates that a relatively high absorbed dose and dose rate can be delivered to mm-sized lesions not observed by conventional nuclear imaging methods, and that these lesions should be considered for radioimmunotherapy with 1311 MoAbs. However, for micrometastases of <1 mm, other radionuclides with shorter path length beta particles than 131I, Auger electrons, or alpha particles should be considered.

Phase I/II clinical radioimmunotherapy with an iodine-131-labeled anti-carcinoembryonic antigen murine monoclonal antibody IgG.

UNLABELLED: The aim of this study was to determine, in a Phase I/II clinical trial, the pharmacokinetics, dosimetry and toxicity, as well as antitumor activity, of the 131I-labeled murine anti-carcinoembryonic antigen (CEA) monoclonal antibody, NP-4 (IgG1 subtype). METHODS: A total of 57 patients with CEA-expressing tumors (29 colorectal, 9 lung, 7 pancreas, 6 breast and 4 medullary thyroid cancer patients), mostly in very advanced stages, were treated. The patients underwent a diagnostic study (1-3 mg of IgG and 8-30 mCi of 131I) to assess tumor targeting and to estimate dosimetry, followed by the therapeutic dose (4-23 mg and 44-268 mCi), based on the radiation dose to the red marrow. Imaging was performed from 4-240 hr postinjection (planar and SPECT). Blood and whole-body clearance were determined; radiation doses were calculated by the Medical Internal Radiation Dose scheme. RESULTS: Red marrow doses ranged from 45 to 706 cGy, and whole-body doses ranged from 31 to 344 cGy. Differences in pharmacokinetics were found between different types of CEA-producing tumors: blood T 1/2 was significantly lower in colorectal cancer when compared to all other tumor types (21.4 +/- 11.1 hr versus 35.8 +/- 13.2 hr, p < 0.01), as was also whole-body t 1/2. Myelotoxicity was dose-limiting, and its severity was related to the types of prior therapy and extent of bone marrow involvement. In patients without prior radiation or chemotherapy, marrow doses as high as 600 cGy were tolerated without evidence of dose-limiting toxicity. No major toxicity to other organs was observed. Tumor doses were inversely related to the tumor mass and ranged between 2 and 218 cGy/mCi. Modest antitumor effects were seen in 12 of 35 assessable patients (1 partial remission, 4 minor/mixed responses and 7 with stabilization of previously rapidly progressing disease). CONCLUSION: These results suggest that prior chemotherapy or external beam radiation is an important risk factor for the development of hematological toxicity in radioimmunotherapy and that higher radiation doses may be delivered to tumors of patients without prior therapy compromising the bone marrow reserve. The different and, in the individual cases, unpredictable clearance rates suggest the necessity of dosimetry-based treatment planning rather than mCi/m2 dosing. Small tumors seem to be more suitable for radioimmunotherapy because of their favorable dosimetry, but to achieve better therapeutic results in patients with bulky disease, the application of higher, potentially myeloablative doses is indicated.

Advantage of residualizing radiolabels for an internalizing antibody against the B-cell lymphoma antigen, CD22.

LL2 is an anti-CD22 pan-B-cell monoclonal antibody which, when radiolabeled, has a high sensitivity for detecting B-cell, non-Hodgkin's lymphoma (NHL), as well as an antitumor efficacy in therapeutic applications. The aim of this study was to determine whether intracellularly retained radiolabels have an advantage in the diagnosis and therapy of lymphoma with LL2. In vitro studies showed that iodinated LL2 is intracellularly catabolized, with a rapid release of the radioiodine from the cell. In contrast, residualizing radiolabels, such as radioactive metals, are retained intracellularly for substantially longer. In vivo studies were performed using LL2-labeled with radioiodine by a non-residualizing (chloramine-T) or a residualizing method (dilactitol-tyramine, DLT), or with a radioactive metal (111In). The biodistribution of a mixture of 125I (non-residualizing chloramine-T compared to residualizing DLT), 111In-labeled LL2 murine IgG2a or its fragments [F(ab')2, Fab'], as well as its humanized, CDR-grafted form, was studied in nude mice bearing the RL human B-cell NHL cell line. Radiation doses were calculated from the biodistribution data according to the Medical International Radiation Dose scheme to assess the potential advantage for therapeutic applications. At all assay times, tumor uptake was higher with the residualizing labels (i.e., 111In and DLT-125I) than with the non-residualizing iodine label. For example, tumor/blood ratios of 111In-labeled IgG were 3.2-, 3.5- and 2.8-fold higher than for non-residualizing iodinated IgG on days 3, 7 and 14, respectively. Similar results were obtained for DLT-labeled IgG and fragments with residualized radiolabels. Tumor/organ ratios also were higher with residualizing labels. No significant differences in tumor, blood and organ uptake were observed between murine and humanized LL2. The conventionally iodinated anti-CD20 antibody, 1F5, had tumor uptake values comparable to those of iodinated LL2, the uptake of both antibodies being strongly dependent on tumor size. These data suggest that, with internalizing antibodies such as LL2, labeling with intracellularly retained isotopes has an advantage over released ones, which justifies further clinical trials with residualizing 111In-labeled LL2 for diagnosis, and residualizing 131I and 90Y labels for therapy.

Cancer imaging with radiolabeled antibodies: new advances with technetium-99m-labeled monoclonal antibody Fab' fragments, especially CEA-Scan and prospects for therapy.

The use of radiolabeled anticancer antibodies to detect cancer sites by external scintigraphy has had a relatively long history. With the advent of monoclonal antibodies (MAbs), which precluded the need for purifying the antibodies by laborious purification steps, there was a surge of interest and efforts to develop these reagents for both imaging and therapy applications (1). Today, many thousands of patients have received different forms and doses of MAbs for various purposes, and four MAb-based products have been licensed for manufacture and sale in the U.S. (2,3). This article describes the most recent MAb product to be approved in the U.S. for colorectal cancer imaging, including discussions of using this agent and its therapeutic counterpart in several cancer types.

Variables influencing tumor dosimetry in radioimmunotherapy of CEA-expressing cancers with anti-CEA and antimucin monoclonal antibodies.

UNLABELLED: In this study, we examined the factors that may influence tumor dosimetry in the radioimmunotherapy of solid, CEA-expressing cancers. METHODS: Data from 119 tumors in 93 patients with CEA-expressing cancers were analyzed. The patients underwent radioimmunotherapy with the 131I-labeled IgG1 anti-CEA antibodies NP-4 (Ka = 10(8) M-1) or MN-14 (Ka = 10(9) M-1), its humanized form hMN-14, as well as the anticolon-specific antigen-p (CSAp) antibody, Mu-9. For dosimetry, the biodistribution, targeting kinetics and cumulated activity of tumors and organs were determined from planar and SPECT imaging. RESULTS: An inverse logarithmic relationship between tumor size and antibody uptake was found for both anti-CEA antibodies, whereas no such relationship was found for Mu-9. The absolute tumor uptake was identified as the most important factor determining the radiation dose to the tumor (r = 0.9), with the biological half-life of the antibody in the tumor being of secondary importance (r = 0.5). No significant difference in tumor uptake was found between both anti-CEA antibodies, despite their tenfold difference in affinity. At comparable masses, colorectal and medullary thyroid cancers had significantly higher tumor uptakes (p = 0.02), as well as tumor-to-red marrow dose ratios, than other cancer types. The tumor half-lives of the anti-CEA antibodies were significantly lower in colorectal than in all other tumor types (p = 0.01). CONCLUSION: In radioimmunotherapy, tumor uptake appears to be the most important dose-determining factor. Differences in antibody affinity are reflected by differences in the biological half-life, not the absolute uptake. Especially favorable conditions for anti-CEA antibodies seem to prevail in colorectal cancer patients having minimal disease, as well as in medullary thyroid cancer, where cytotoxic tumor doses might be expected. Antimucin antibodies may have a particular advantage in the treatment of patients with larger colorectal tumors.

99Tcm-LL1: a potential new bone marrow imaging agent.

LL1, a monoclonal antibody (MAb) to HLA Class-II-like antigen (li determinant) on the surface of B-lymphocytes, monocytes and histiocytes, was evaluated as an agent for bone marrow imaging. Six patients with diverse diseases (non-Hodgkin's lymphoma, n = 2; multiple myeloma, n = 1; polycythaemia vera, n = 1; lung cancer, n = 1; breast cancer, n = 1) were given low protein doses (< 1 mg) of 99Tcm (30 mCi) labelled Fab' of LL1. 99Tcm-sulphur colloid (SC) imaging was performed in three patients for comparison. Both planar and single photon emission tomographic images were acquired using Sopha gamma cameras. As early as 2 h post-MAb injection, excellent bone marrow images were achieved in all patients, demonstrating both normal or hyperproliferative marrow, as well as 'cold' bone marrow abnormalities such as radiation defects or cancer metastases. Similar to SC, relatively high uptake of LL1 was found in the liver and spleen. However, the bone marrow-to-liver and -spleen uptake ratios were approximately 19-fold higher (0.75 vs 0.04) and 6-fold higher (1.23 vs 0.22), respectively, with LL1 than with SC. The higher bone marrow uptake allowed clearly superior visualization of the thoracic spine when compared to SC. The mean T1/2 of blood and whole-body clearance were 0.4 and 66 h, respectively. The highest radiation absorbed doses (in cGy mCi-1) were observed in the spleen (0.47 +/- 0.24), kidneys (0.25 +/- 0.09) and liver (0.14 +/- 0.04). The bone marrow dose was only 0.05 +/- 0.02 cGy mCi-1. These results indicate that bone marrow imaging with 99Tcm-LL1 is feasible, and that LL1 may be a suitable alternative to SC because of better visualization of the lower thoracic spine. Potential applications include the improved detection of bone marrow metastases of solid tumours and the assessment of haematological disorders.

Enhanced bilateral somatostatin receptor expression in mediastinal lymph nodes ("chimney sign") in occult metastatic medullary thyroid cancer: a typical site of tumour manifestation?

In medullary thyroid cancer (MTC), post-surgically elevated plasma calcitonin and/or carcinoembryonic antigen levels frequently indicate persisting metastatic disease, although conventional diagnostic procedures fail to localize the responsible lesions (occult disease). Somatostatin analogues have been used successfully in disease localization, but recently concerns have been raised that increased thoracic uptake of indium-111 pentetreotide in patients with previous external beam irradiation may represent a false-positive finding, caused by post-irradiation pulmonary fibrosis. We recently examined seven patients with metastatic MTC by somatostatin receptor scintigraphy (six with occult and one with established disease). In four patients, all of whom had stable or slowly rising tumour marker levels over several years, a chimney-like bilateral mediastinal uptake of indium-111 pentetreotide was found. In two patients with persisting hypercalcitonaemia immediately after primary surgery, supraclavicular lymph node metastases were identified as the responsible lesions. None of these seven patients had prior external beam radiation therapy. In two cases, histological confirmation was obtained. In one patient, disease progression could be shown during follow-up. These data suggest that bilateral mediastinal lymph node involvement is a typical site of disease in slowly progressing occult metastatic MTC; the "chimney sign" may represent a typical finding with somatostatin analogues in such cases. Therefore, we believe that even in the case of prior external beam irradiation, mediastinal uptake of octreotide might represent metastatic MTC rather than radiation fibrosis.

The microvenous valvular anatomy of the human dorsal thoracic fascia.

The use of free scapular fasciocutaneous flaps for reconstruction of recalcitrant grade 6 venous stasis ulcers has shown excellent early success rates. Venous refilling times measured postoperatively over the flaps by photoplethysmography have noted improvements to normal levels. Preliminary anatomic studies have demonstrated valves in the circumflex scapular veins of flaps used in reconstruction. The purpose of this study was to investigate and document the number, morphology, size, and location of valves in the human dorsal thoracic fascia. Ten scapular flaps were obtained from unembalmed cadavers and injected with methyl methacrylate. Each flap cast was divided into four parts: proximal, right and left, and distal, right and left. We reduced the size of specimens (the largest being 24 x 11 mm) and studied them in a scanning electron microscope. We identified all valves, estimated the diameter of the corresponding vein, calculated the depth of the valvular sinus, and related it to the corresponding venous size. Light microscopy and transmission electron microscopy were used as assisting tools applied to glutaraldehyde-fixed specimens. Analysis of injected specimens showed that valves were most abundant in veins with a luminal diameter of 30 to 120 microns (59.3 percent of 905 valves). The depth of valves became larger with increasing venous diameter. The sizes of valve sinuses were not different for individual valves. Except for veins larger than 1000 microns in diameter, there was no significant difference between the number of valves in different parts of an individual flap, nor were there significant differences between the valve numbers in different flaps. Most valves were bicuspid; only in the vein category of 30 to 120 microns were unicuspid valves encountered. Valves sometimes were located in series in a short segment of a vein; occasionally, they were found at the merging site of two veins. Transmission electron microscopy showed that valve leaflets had collagen fibers that ascended toward the tip of the leaflet and occasionally were accompanied by elastic fibers. Myofibroblasts were regularly present in the valve leaflets. These data show that fasciocutaneous flaps from the scapular region have numerous valves (90 valves on average in each flap) in the venous microcirculation. The microvenous valves in the dorsal thoracic fascia appear to be structurally similar to valves in larger veins. These valves may play a role in the improved hemodynamics and promising clinical outcome of patients with chronic venous insufficiency who have undergone free scapular flap reconstruction.

Thyroid radiation doses during radioimmunotherapy of CEA-expressing tumours with 131I-labelled monoclonal antibodies.

A number of radioimmunotherapy (RAIT) trials with iodinated antibodies have shown a high variability in the radiation doses to the thyroid. Therefore, the aim of this study was to evaluate which factors influence these thyroid doses during RAIT with 131iodinated monoclonal anti-carcinoembryonic antigen (CEA) antibodies. Data from 36 patients with CEA-expressing tumours were analysed. The patients underwent RAIT with the 131I-labelled IgG1 anti-CEA antibody, MN-14 (Ka = 10(9) l mol-1) or its F(ab')2 fragment (activity range 45.8-220.0 mCi). The thyroid was blocked with 120 mg iodine (lugol's orSSKI solution) and 400 mg perchlorate per day, starting 1 day prior to the first study. Blood clearance and molecular composition of labelled plasma compounds were determined by blood sampling and size-exclusion high-performance liquid chromatography analysis. The cumulated activities of tissues were determined from daily imaging and blood clearance data. Doses were derived from the MIRD scheme. Thyroid radiation doses showed a high variability, between 1.2 and 37.7 cGy mCi-1 (mean +/- S.D.: 11.1 +/- 8.3 cGy mCi-1), corresponding to absolute doses between 2.5 and 43.6 Gy. However, the maximal iodine uptake in the thyroid was 2.4 +/- 1.9 microCi mCi-1 (range 0.2-10.0 microCi mCi-1), which was less than 1% of the injected activity, indicating that more than 99% of the thyroid was blocked in all cases. No correlation was found between these thyroid doses and conditions leading to an enhanced exposure to free radioiodine, such as unbound I- in the mAb preparation, rapid metabolic breakdown of the labelled antibody due to human anti-mouse antibodies (HAMA), or immune complex formation with circulating antigen. However, a relationship between the thyroid doses and the patients' compliance in taking their Lugol's and perchlorate blocking medications, as well as to a relatively high variability in the biological half-life of the iodine in the thyroid (range from 31.1 h to virtual infinity), is indicated. No rising TSH titres or other signs of (latent) hypothyroidism were seen in these patients during a 2 year follow-up period. Longer follow-up was not possible because of the terminal condition of most of the patients. These data show that thyroid doses in an appropriately blocked individual given a standard, non-myeloablative dose of RAIT, are generally lower than those assumed to be required to cause late hypothyroidism. Even if higher activities are used, potential hypothyroidism may be overcome easily by hormone replacement. Thyroid doses are independent of parameters leading to an enhanced exposure of the thyroid to free radioiodine, suggesting that patient compliance in taking their blocking medication may be the most crucial factor for reducing thyroid doses in RAIT with 131I-labelled antibodies.

Reduction of renal uptake of monoclonal antibody fragments by amino acid infusion.

UNLABELLED: The renal uptake of radiolabeled antibody fragments and peptides presents a problem in radioimmunodetection and therapy, compromising lesion sensitivity, especially with intracellularly-retained isotopes. Previously, we showed that cationic amino acids and their derivatives are capable of significantly reducing kidney uptake in animals. We report our initial clinical results of successful renal uptake reduction in five patients who underwent cancer radioimmunodetection with 99mTc-anti-CEA Fab' fragments. METHODS: The patients were infused with two liters of a commercially-available nutritive amino acid solution (containing approximately 2.25 g/liter lysine-glutamate and 2.50 g/liter arginine), whereas 75 control patients received the same volume of saline (quantification of organ and tumor kinetics from conjugate whole-body views by ROI technique). RESULTS: The renal uptake in the amino acid group was significantly lower (p<0.05) than in the control group (11.1 +/- 2.0% injected dose versus 17.7 +/- 7.0% injected dose at 24 hr postinjection), whereas the uptake of all other organs remained unaffected. Gel filtration chromatography of the urine taken from amino-acid-treated patients showed that a significantly higher amount of excreted activity was bound to intact Fab' (53% of excreted activity) in contrast to only less than 10% in the control group. CONCLUSION: The renal uptake of monoclonal antibody fragments in patients can be reduced significantly by amino acid infusion, even at considerably lower doses than those that were safe and effective in animals. As was found in animals, the mechanism seems to rely on an inhibition of the re-absorption of tubularly-filtered proteins by the proximal tubule cells. These results encourage further clinical trials to lower the renal uptake experienced in radioimmunodetection, as well as in therapeutic trials with antibody fragments and peptides.

Factors influencing the pharmacokinetics, dosimetry, and diagnostic accuracy of radioimmunodetection and radioimmunotherapy of carcinoembryonic antigen-expressing tumors.

The aim of this study was to examine factors that may influence the pharmacokinetics, diagnostic accuracy, and dosimetry in radioimmunodetection and radioimmunotherapy with anti-carcinoembryonic antigen (CEA) monoclonal antibodies (mAbs). Data from 275 patients with CEA expressing tumors were analyzed retrospectively. Of these, 69 patients devoid of human antimouse antibody (i.e., 31 colorectal, 9 lung, 7 breast, 4 ovarian, 6 pancreatic, 9 medullary thyroid, 1 gallbladder, and 1 salivary gland cancer, and 1 primary tumor of unknown origin) underwent a low-protein-dose diagnostic study (0.3-2.6 mg of protein; 6.8-28.8 mCi 131I-labeled IgG or fragments), followed within 4 weeks by a high-protein-dose therapy injection (4.0-27.5 mg of protein; 29.8-238.9 mCi). The anti-CEA antibodies NP-4 (Ka=10(8)M-1) and MN-14 (ka=10(9)M-1) were used. Plasma clearance, the molecular composition of radioactivity in the plasma, and the cumulated activity in organs and tumors were determined. Radiation doses were derived from the Medical Internal Radiation Dose scheme. At a low-protein dose and over a similar range of plasma CEA, a significantly higher percentage of MN-14 than of NP-4 was complexed with circulating CEA, consistent with its higher affinity. Complexation was reduced with increasing protein doses. However, the targeting sensitivity was not affected. Profound differences were found in the clearance of the antibody between different types of cancer. Colorectal cancer patients cleared the antibody significantly faster from blood (T1/2=17.6+/-12.6 versus 44.2 +/- 23.7 h) and whole body (t1/2= 53.2 +/- 30.1 versus 114.6+/-59.7 h) than all other tumor types (P <0.001). Consequently, significantly lower red marrow (2.1 +/- 1.0 cGy/mCi versus 4.3 +/- 1.6 cGy/mCi) and whole-body doses (0.5 +/- 0.3 cGy/mCi versus 1.0 +/- 0.4 cGy/mCi) were seen in colorectal cancer patients as compared with other tumor types (P < 0.001). This clearance is probably due to hepatic metabolism of the immune complexes. Clearance rates were especially high in patients with colorectal cancer having large liver metastases and elevated liver enzymes (rapid hepatic clearance with liberation of free I-). In contrast, a disease-stage and plasma CEA-matched cohort of colorectal cancer patients, examined with the 131 I-labeled anti-colon-specific antigen p mAb Mu-9, showed normal murine IgG pharmacokinetics (n=22;3 of them compared intraindividually to MN-14). Only in colorectal cancer patients did complexes between mAb and CEA tend to clear rapidly, whereas Mu-9 had normal kinetics in these patients. This suggests that different CEA-expressing cancer types may produce heterogeneous CEA molecules and that the variability in mAb clearance is due to varying clearance rates of these different circulating CEA subspecies. Disease-related alterations in antibody metabolism are unlikely, given that only anti-CEA antibodies exhibit this phenomenon.