Distribution of exogenous complement factor H in mice in vivo.

Factor H is an important regulator of complement activation in plasma and on cell surfaces in both humans and mice. If FH function is compromised, inappropriate complement activation on self-surfaces can have disastrous effects as seen in the kidney diseases atypical haemolytic uremic syndrome (aHUS) and C3 glomerulopathy. As FH constructs have been proposed to be used in treatment for these diseases, we studied the distribution of exogenous FH fragments in mice. Full-length mFH, mFH1-5 and mFH18-20 fragments were radiolabelled, and their distribution was examined in WT, FH-/- and FH-/- C3-/- mice in vivo. Whole body scintigraphy revealed accumulation of radioactivity in the abdominal part of the mice, but also to the thyroid gland and urinary bladder. At organ level in WT mice, some full-length FH accumulated in internal organs, but most of it remained in the circulation. Both of the mFH fragments accumulated in the kidneys and were excreted in urine. For mFH1-5, urinary secretion is the likely cause for the accumulation. Concentration of mFH18-20 to kidneys was slower, and at tissue level, mFH18-20 was localized at the proximal tubuli in WT and FH-/- C3-/- mice. No C3-independent binding to glomeruli was detected. In conclusion, these results show that glomerular glycosaminoglycans and sialic acids alone do not collect FH in kidneys. Deposition of C3 fragments is also needed, which implies that in aHUS, the problem is in simultaneous recognition of C3 fragments and glycosaminoglycans or sialic acids by FH, not just the inability of FH to recognize glomerular endothelium as such.

Sodium fluoride (Na18F) PET Response Criteria in Solid Tumors (NAFCIST): a framework for response assessment in bone tumors.

The current Response Evaluation Criteria in Solid Tumors for measuring tumor response in osteosarcoma may be sub-optimal, as even responsive bone tumors may show limited change in tumor diameters. This limits the use of traditional imaging assessment tools. Therefore, discerning osteosarcoma response to therapy on magnetic resonance imaging before surgery is often difficult, and it is typically evaluated after surgery by assessing the amount of necrosis in resected surgical specimens. To address these challenges, sodium fluoride (Na18F) positron emission tomography/computed tomography (PET/CT) scans can be utilized to better image bone response to therapy, as, fluoride is avidly taken up by bone. Na18F Response Criteria in Solid Tumors (NAFCIST) has been developed as a novel method to evaluate treatment response using Na18F PET/CT. Current evidence supporting NAFCIST comes from a pilot study that evaluated alpha particle radium-223 in patients with osteosarcoma. In this review, practical guidance for utilizing NAFCIST in the context of bone tumors is illustrated to aid future studies.

Immunolymphoscintigraphy with 99mTc-labeled monoclonal antibody (BW 431/26) reacting with carcinoembryonic antigen in breast cancer.

In breast cancer, the most interesting sites showing the regional spread of the disease are the axillary and internal mammary lymph nodes. Monoclonal antibodies are specific in detecting tumor metastases. The aim of this study is to present a simple method whereby the immunolymphoscintigraphic approach is introduced whether bimanually or parasternally. Twenty consecutive female breast cancer patients were imaged with 99mTc-labeled monoclonal intact IgG1 antibody (BW 431/26, Marburg, Federal Republic of Germany), which reacts with carcinoembryonic antigen. The labeling yield was in 10 cases over 98%. The patients had a suspicion of scar recurrency, skin metastases, or palpable lymph node affixions. The patients were imaged twice after bimanual s.c. injections (at 2-3 h and 20-22 h); the bimanual injections were given into first and fourth interdigital interstitial spaces and in one case parasternally following Sappey's lines. A total of 105 lesions in 18 patients were detected. Twenty-five lesions in 18 patients were verified cytologically or histologically. Sensitivity of morphological data was 84%. In supraclavicular and axillary lymph node regions the sensitivity was 90% and the specificity 88% compared to other findings. Most of the undetected lesions were skeletal. The carcinoembryonic antigen concentration in serum had no correlation with the findings. The human anti-murine antibodies showed in two patients of 15 elevated response. This immunolymphoscintigraphy method could be of clinical importance because it enables detection of both regional and systemic lesions in a common type of cancer. The method is sensitive, except for bone lesions, and might be applied for screening purposes in selected patients.

Immunolymphoscintigraphy and immunoscintigraphy of ovarian and fallopian tube cancer using F(ab')2 fragments of monoclonal antibody OC 125.

We have used immunolymphoscintigraphy (ILS) alone or in combination with immunoscintigraphy with 131I-labeled F(ab')2 fragments of monoclonal OC 125 antibodies to improve detection of retroperitoneal lymph node metastases of ovarian and fallopian tube cancer. ILS was carried out with bilateral dorsopedal s.c. injections on nine patients and with bilateral iliopelvic injections into the ischiorectal fossa on two other patients. Radioimaging was performed 2-4 times between 0 and 5 days. An additional dose of labeled antibody fragments was given i.v., and imaging was done 2-3 days later. Conventional immunoscintigraphy without preceding ILS was carried out on another nine patients. Dorsopedal ILS improved detection of pelvic and paraaortic lymph node metastases. Malignant lymph nodes were detectable as early as 3 h after s.c. injection of the tracer. Combined results of ILS and immunoscintigraphy in 16 surgically verified cases indicated a true positive finding in 9 patients, true negative finding in 5, false positive in one, and false negative in 1. Calculated from these figures the sensitivity, specificity, and accuracy of the method were 90, 83, and 88%, respectively. Involved lymph nodes were found more frequently in those patients whose serum CA 125 concentration was elevated demonstrating that an elevated serum CA 125 level does not preclude successful radioimmunodetection.

Endoglin is a suitable target for efficient imaging of solid tumors: in vivo evidence in a canine mammary carcinoma model.

Increasing evidence suggests that endoglin (CD105) is a new powerful marker of neovascularization in solid malignancies; thus, using breast cancer as a model, we investigated whether targeting of CD105 by monoclonal antibody (mAb) MAEND3 can be used for in vivo imaging of solid tumors. Immunohistochemistry and flow cytometry identified differential expression of CD105 on breast cancer and endothelial cells; in fact, neoplastic cells were weakly and rarely stained by mAb MAEND3, which in contrast, strongly and invariably stained blood vessel endothelia within the breast adenocarcinomas investigated and cultured endothelial cells. Moreover, in contrast to CD31, which currently represents the reference marker to assess angiogenetic activity, CD105 expression was highest in semiconfluent and actively proliferating endothelial cells, and it progressively decreased as cells reached tight confluency and low [3H]thymidine uptake. i.v. administration of 18 MBq of 125I-labeled mAb MAEND3 efficiently imaged spontaneous mammary adenocarcinomas in two dogs; the uptake of radiolabeled mAb was rapid and intense because tumor: background ratios of 8.2:1 and 9.3:1 were reached 8 h after mAb administration, in the absence of immediate and/or long-term clinical side effects. Altogether, our present data suggest that targeting of CD105 on tumor-associated blood vessels may represent a new strategy for in vivo imaging of solid malignancies, regardless of their histological origin.

Repeated intratumoral administration of ONCOS-102 leads to systemic antitumor CD8+ T-cell response and robust cellular and transcriptional immune activation at tumor site in a patient with ovarian cancer.

Adenoviruses are excellent immunotherapeutic agents with a unique ability to prime and boost immune responses. Recombinant adenoviruses cause immunogenic cancer cell death and subsequent release of tumor antigens for antigen presenting cells, resulting in the priming of potent tumor-specific immunity. This effect may be further enhanced by immune-stimulating transgenes expressed by the virus. We report a case of a 38-year-old female with Stage 3 metastatic micropapillary serous carcinoma of the ovary. She was treated in a Phase I study with a granulocyte-macrophage colony stimulating factor (GMCSF)-expressing oncolytic adenovirus, Ad5/3-D24-GMCSF (ONCOS-102). The treatment resulted in progressive infiltration of CD8+ lymphocytes into the tumor and concomitant systemic induction of several tumor-specific CD8+ T-cell populations. The patient was alive at the latest follow up more than 20 months after initiation of the study.

Gene therapy using antisense oligodeoxynucleotides labeled with Auger-emitting radionuclides.

Antisense oligomers may be used as a vehicle for carrying a radiation source into a specific location inside a tumor cell. The effects of radioactive-labeled oligodeoxynucleotides (ODNs) may have both direct antisense inhibition and radiation. Thus far, the use of radioactive ODNs has been limited mostly to clinical biokinetic studies. Therapeutic possibilities remain unknown if the basic question of the optimal source of radiation is unanswered. We have shown previously that oligonucleotide therapy can be effective theoretically with the internally labeled ODN phosphorothioates 32P, 33P, and 35S. Here, we expand the selection of radionuclides; we calculated in vivo subcellular tissue distribution for ODN phosphorothioates using the decay characteristics of several beta- and Auger-emitting radionuclides: 32P, 35S, 51Cr, 67Ga, 111In, (1114m)In, 123I, 125I, 131I, and 201Tl. The absorbed nuclear doses of these radiolabeled oligonucleotides were estimated in different cellular dimensions using the subcellular biodistribution data for two oligonucleotides (ISIS 2105 and ISIS 2922). Our results indicate that Auger-emitter isotopes do not give higher absorbed cell nuclear doses than the isotopes suitable for internal labeling of ODN phosphorothioates. However, the biological difference is difficult to estimate. The best isotope for subcellular targeting was 35S, which gives the smallest variation of nuclear dose in the different cell dimensions we studied (nuclear diameter, 6-16 microm; cellular diameter, 12-20 microm). Therefore, we conclude that in oligonucleotide radiotherapy, nuclear targets should be treated with short-range beta-emitters (35S or 33P) that are suitable for the internal labeling of oligonucleotides unless the relative biological effectiveness of Auger-emitters could be remarkably improved. Dual labeling with 32P and 35S may provide therapeutic benefits when treating smaller and larger targets simultaneously. Further in vivo development, especially with 33P and 35S labels for ODNs, is strongly indicated.

Imaging of soft-tissue sarcomas with indium-111-labeled monoclonal antimyosin Fab fragments.

Some soft-tissue sarcomas contain intracellular myosin. We therefore studied the possibility of localizing various soft-tissue sarcomas with 111In-labeled monoclonal antibody Fab fragments binding specifically to myosin, assuming that damage to the cell membrane could expose intracellular myosin. Nineteen patients with different types of soft-tissue sarcomas were studied. Eighteen patients were found to have abnormal antibody uptakes. Antibody uptake was not observed in an additional patient operated for a benign tumor (gastric leiomyoma). The immunoscintigraphy results were generally in good agreement with those of other radiologic findings (computed tomography, ultrasound, magnetic resonance imaging). Surprisingly, the immunohistochemistry results showed that tumors not stainable for myosin can also be imaged with antimyosin. Thus, the mechanism of antibody uptake does not seem to be related entirely to specific antigen recognition. Irrespective of the exact mechanism for the uptake of labeled antibody this method appears to be useful for localizing soft-tissue sarcomas.

In vivo detection of intervertebral disk injury using a radiolabeled monoclonal antibody against keratan sulfate.

UNLABELLED: In the intervertebral disk, proteoglycans form the major part of the extracellular matrix, surrounding chondrocytelike disk cells. Keratan sulfate is a major constituent of proteoglycans. METHODS: We have radioiodinated a monoclonal antibody raised against keratan sulfate. This antibody was injected into rats (n = 6), and the biodistribution was studied. A model of intervertebral disk injury was developed, and two tail disks in each animal with both acute (2 wk old) and subacute (7 wk old) injuries were studied for in vivo antibody uptake. RESULTS: The biodistribution at 72 h was as follows: blood, 0.0018 percentage injected dose per gram of tissue (%ID/g); lung, 0.0106 %ID/g; esophagus, 0.0078 %ID/g; kidney, 0.0063 %ID/g; liver, 0.0047 %ID/g; spleen, 0.0046 %ID/g; heart, 0.0036 %ID/g; thyroid, 0.0034 %ID/g; muscle, 0.0017 %ID/g; and bone, 0.0016 %ID/g. In the subacute stage, a significant difference (P < 0.006) was found in antibody uptake between injured disks (n = 12) and adjacent healthy disks (n = 12). In vivo gamma imaging showed increased uptake in other animals having lumbar disk injuries (2, 7, and 17 d after injury). Cartilage tissue, such as the trachea, was studied separately and showed extremely high antibody uptake, 0.10 %ID/g. Rat trachea was also visualized on gamma images. CONCLUSION: Our data suggest that antibodies against nucleus pulposus components, such as proteoglycans, can be used for in vivo detection of intervertebral disk injury. This finding is in spite of the minimal circulation present in intervertebral disks.

Modulation of antibody kinetics by the cell membrane active agent tween 80 in vivo.

The modulation of antibody uptake by the tumour has been crucial in many radioantibody applications for delivering optimal dose for therapy. Our approach was to modulate the monoclonal antibody (MAb) uptake by using a surface detergent (Tween 80). Our Mab was raised against the tyrosine kinase receptor recombinant protein tie. Mice bearing Lewis lung carcinoma xenografts were studied after injecting I-125 labeled IgG1 subclass monoclonal antibody 3c4c7g6 tie protein. The biodistribution was studied at 4, 24, 48, 72, 96 and 120 hours after intravenous injection. Tween 80 was administered intratumourally, 0.04% of tumour volume. Without Tween 80 the antibody half-lives in tumour were 90 hours, in blood 39 hours, in liver 22 hours, and in kidney 52 hours, whereas using intratumoural Tween 80 half-lives were in tumour 66 hours, in blood 26 hours, in liver 27.5 hours and in kidney 27.5 hours. Although the Tween manipulation did not increase uptakes by organs, it did enhance clearance rate from the blood. This data indicates that antibody dose can be optimized by surface detergent, enhancing clearance without any burden to critical organs. This might be crucial in adjusting delivered radioimmunotherapy dose by changing the mean residence time of an antibody.

Effects of lithium gammalinolenate on the perfusion of liver and pancreatic tissues in pancreatic cancer.

Because of its poor prognosis, new modalities to treat pancreatic cancer are highly welcome. Gammalinolenate (GLA) has been shown to possess antitumor activity on various human cancer cell lines in vitro and some evidence has been found of its modulative activity on tubulin active agents, such as vinca alkaloids. GLA treatment is thought to change the penetration and distribution of chemotherapeutic agents in pancreatic tumor tissue. The in vivo effects of GLA are widely unknown. This is the first study on the modulation effects of both oral or intravenous GLA on blood perfusion in vivo. We analysed tissue perfusion prior to treatment and on the 10th day of GLA treatment in patients with pancreatic cancer. Dynamic gamma imaging was performed for 20 minutes after Tc-99m-MIBI injection, and the whole body was scanned after the dynamic study and at 4 hours. Half-lives in liver, left kidney, spleen, pancreas and tumor were recorded using a developed macro program for background corrected geometric mean data from irregular region of interests. Half-lives in the liver did not change due to oral GLA treatment, but they decreased dramatically in two of three patients after i.v. GLA treatment. Additionally, individual changes were observed in pancreatic half-lives, as in four out of five cases the half-life increased and in one case it decreased. No major changes were observed in kidney and spleen half-lives. GLA treatment had no effects on the blood brain barrier. This technique demonstrates perfusion in salivary glands, thyroid, lungs, heart, spleen, kidneys, muscles, spine and bladder, but no changes in perfusion could be detected due to GLA treatment. However, qualitatively enhanced blood flow through the pancreatic tumor was observed. In all patients irrespective of the route of administration of GLA, the organ-to-background ratios in liver decreased. The effect is, however, smallest after oral dosing. The pancreas-to-background ratio was increased in 3/5 patients, these patients exhibited stabilized disease. In a patient with large liver metastases the pancreas-to-background ratio decreased, and she showed a rapid disease progression during GLA therapy. The change in the pancreatic uptake was inversely proportional to the change in CA 19-9 concentration. Our results indicate the that GLA treatment dramatically changes tissue perfusion, especially in liver and pancreatic tumors, even at low doses, and these changes may predict response to GLA therapy.

Labelling of bleomycin with Auger-emitter increases cytotoxicity in squamous-cell cancer cell lines.

PURPOSE: To investigate the cytotoxicity of bleomycin (BLM), two Auger-emitting bleomycin complexes (indium-111 ((111)In)-BLMC) and (111)InCl3 in three squamous cell cancer (SCC) cell lines. MATERIAL AND METHODS: Three recently established SCC cell lines were investigated using the 96-well clonogenic assay. Concentrations causing 50% inhibition in cell survival (IC50) were calculated for BLM and two specific activities of (111)In-BLMC (40 MBq/mg BLM (low) and 195 MBq/mg BLM (high)). RESULTS: (111)In-BLMC (low) was the most toxic to the SCC cell lines. (111)In-BLMC containing 4.9-fold more activity of (111)In (195 MBq/mg BLM) was more effective than BLM (p=0.0029), but not as toxic as (111)In-BLMC (low) (p=0.0023). UT-SCC-19A had a IC50 value for BLM as low as 4.1 nM, whereas IC50 values for (111)In-BLMC (low) and (111)In-BLMC (high) were 2.0 nM and 2.6 nM, respectively. The most chemoresistant cell line UT-SCC-12A had a IC50 value for BLM of 18.8 nM, for (111)In-BLMC (low) 10.7 nM and for (111)In-BLMC (high) 12.7 nM. (111)InCl3 had no cell killing effect. CONCLUSIONS: This study shows that (111)In-BLMC is superior in SCC cell killing compared with BLM. These data provide the basis for further clinical investigations of (111)In-BLMC.

Imaging of pharyngeal and laryngeal carcinomas with indium-111-labeled monoclonal anti-CEA antibodies.

Localization of primary tumors, metastases, or recurrences in 13 consecutive patients with histological verification of squamous cell or adenocarcinoma was made with radioimmunodetection using monoclonal radiolabeled anti-CEA antibody. All surgical specimens stained immunohistochemically, except one, were positive for CEA. Of the known 19 tumor sites 17 were visualized in antibody scans. There were two positive findings that did not prove to be positive during 12 month follow-up. The scintigram findings did not correlate with CEA serum concentrations that, with one exception, were normal in all patients.

Treatment of olfactory neuroblastoma. A report of 11 cases.

Eleven olfactory neuroblastomas treated at Helsinki University Central Hospital between 1970 and 1991 were reviewed retrospectively. The distribution of the patients was according to Morita's staging (modified Kadish's classification) as follows: one stage A, one stage B and nine stage C. Tumor resections were performed in all cases, and five were considered radical. All patients received radiotherapy: total doses ranged from 42 to 70 Gy. At least a short treatment response was achievable in all cases. Chemotherapy was given to two patients: one remission was obtained by methotrexate with leucovorin rescue and doxorubicin for residual disease after radiotherapy. Distant metastases were observed in three cases, two in the lungs and one intraperitoneally. After a median follow-up of 63 (range, 6-140) months, five patients are alive and well, two patients have died with no evidence of disease; two patients who received only 42 and 50 Gy in 5 and 6 weeks have died of local recurrences and two of distant metastases. None of the patients with advanced (stage B or C) disease who received radical radiotherapy ( > or = 60 Gy given in 6 to 9 weeks) developed local recurrence. The increasing incidence of distant metastasis justifies an intensification of initial treatment, especially in state C disease.

Detection of pseudomyxoma peritonei by radioimmunohistochemistry and radioimmunoscintigraphy.

Pseudomyxoma peritonei (PP) is a local slowly progressing disease with typical abdominal swelling. Treatment is uneffective and the long-term prognosis is poor. Conventional radiology provides usually only a delineation of low density area relating gelatinous masses accumulating in the peritoneal cavity. In this study, immunohistochemistry based on digital quantitative autoradiography utilizing radiolabelled monoclonal antibody B72.3 (MoAb) recognizing TAG-72 antigen on epithelial carcinomas was used for diagnosis of pseudomyxoma (7 patients). The PP patients were studied with radioiodinated I-131-labeled MoAb after intravenous (2 patients) and intraperitoneal (7 patients) injections. Radioactivities of MoAbs varied considerably in the tumors. Both intra- and extracellular staining pattern was observed by immunohistochemistry. Gamma imaging at 1, 3, 7 and 14 days after i.v. injection (2 patients) revealed targeting of all known lesions. The intraperitoneally injected MoAb (7 patients) retained long time in the peritoneal cavity, specific tumour targeting was seen up to 16 days by an antibody-SPECT, while maximum blood radioactivity was measured between 8-12 hrs. Radiolabelled B72.3 MoAb recognizing TAG-72 antigen is also present within pseudomyxoma cells. It can be used for radioimmunohistochemistry of PP. Accurate imaging of PP is possible by MoAb suggesting earlier diagnosis and more accurate location of residual disease after operations, and evaluating treatment response. Estimated tumour dose for intraperitoneal tumour (MIRD formalism) was 13 mGy/MBq. This indicates that the radioiodinated B72.3 antibody can be used for in vivo targeting and therapeutic applications of intraperitoneal pseudomyxoma.

[191mIr: distribution and retention in animal experiments]. / 191mIr: Verteilung und Retention im Tierversuch.

Ultrashort-lived tracer 191mIr (T1/2 = 4.92 s) can be obtained with a high yield from an 191Os/191mIr generator with a low 191Os breakthrough. It was eluated directly into the tail veins of Wistar rats. These animals were imaged dynamically (five frames/s) up to 40 s. The measurement was repeated five times on each animal. The whole-body retention and biodistribution of 191Os was studied by sacrificing the rats at one and four days, respectively, after injection. The activity retained was highest in the kidneys and the spleen, followed by the muscles and the liver. These values indicate that the breakthrough is by no means dangerous and that investigations can be repeated immediately with a radiation exposure of no significance. Furthermore, all investigations in the same animal were reproducible, suggesting that 191mIr might be a good tracer for nuclear angiograms.

Absorbed dose estimates for 131I-labelled monoclonal antibody therapy in patients with intraperitoneal pseudomyxoma.

Seven patients with intraperitoneal pseudomyxoma originating from the appendix (4 cases) and from the ovary (3 cases) were treated with radioimmunotherapy. During the therapy, nine infusions of 3.0-4.2 GBq of 131I-labelled B72.3 monoclonal antibody were administered. We developed three-dimensional dose calculation software that can utilize activity maps based on SPET images to calculate the absorbed dose distribution using point source kernels. The dose calculation program was employed to calculate absorbed doses to various organs. The calculated dose distributions enable us to evaluate the variation in dose within the organs, which is normally not available using approaches based on geometric models. The patient-specific absorbed dose calculations were compared with doses based on a model that uses photon S-factors derived from a standard phantom. The compared doses agreed well on average, but in some organs showed large discrepancies.

A dual-tracer method for studying intragastric distribution and gastric emptying of solids and liquids in functional dyspepsia.

Twenty-five patients with functional dyspepsia and 11 healthy controls matched for age and sex were examined. The patients were divided into two groups: patients with dysmotility-like symptoms and those with ulcer-like symptoms. In a dual-tracer gastric emptying study, dysmotility-like and ulcer-like symptoms could not be distinguished from each other on the basis of gastric emptying times. The intragastric distribution and the solid lag time in dysmotility-like dyspepsia differed significantly from those of the controls.

A novel method for monitoring liver ethanol injections: activity tissue distribution in rat liver using digital quantitative autoradiography and CH3(14)CH2OH.

Accurate ethanol microdistribution during percutaneous ethanol injections (PEI) have not previously been reported by in vivo monitoring methods (e.g. by using ultrasound or other flow methods). Any real-time imaging method is insufficient to show the gradual diffusion of ethanol and its ultimate spread. Therefore a novel method to study the microdistribution and radiopharmacokinetics of labeled ethanol in rat liver was developed. Rats were injected with C-14-labeled ethanol into the right liver lobe. Liver slices were investigated at 1, 5, 15, and 30 min using a novel digital quantitative autoradiographic (DQAR) method. Tissue slices at 1 and 5 min demonstrated increased activity of C-14-labeled ethanol around the injection site, resulting in a uniform distribution at 15 min. At 30 min, a weak elimination was observed. Our results indicate that in PEI treatment, toxic effects may be found outside the primary injection site. Our DQAR method shows the dynamic spread of ethanol with great anatomical detail. It may therefore be used in future studies on ethanol kinetics in the liver and tumor tissues to optimize the antitumor effect of PEI while minimizing the potential for adverse spread and complications.