Factors affecting chemistry of reduction-mediated 99mTc-labelling of monoclonal antibodies and polyclonal human immunoglobulins.

In developing a new method for preparing a radiopharmaceutical for clinical investigation, a thorough understanding of reaction stoichiometry is crucial in optimizing the labelling chemistry. Factors determining labelling efficiency of the 2-mercaptoethanol (2-ME)-mediated 99mTc-labelling of antibody molecules were elucidated using anti-tumor monoclonal antibodies of different IgG subclasses (i.e. IOR-CEA(IgG1), M170(IgG1), 3F8(IgG3) and EMD (IgG2a)) and polyclonal human immunoglobulins (Sandoglobulin). Antibodies which were sensitive to 2-ME reduction (i.e. required 500-1000 molar excess of 2-ME) could tag 99mTc with high efficiency since they possessed abundant reactive sites (i.e. sulfydryl groups) for 99mTc binding. Reduction sensitivity of antibodies was unlikely to be affected by IgG subclass and could be rated as follows: Sandoglobulin > IOR-CEA > 3F8 > M170 > EMD. Concentrations of the reduced antibodies for effective labelling appeared to be related to the reduction sensitivity, i.e. 0.2, 0.4 and 0.6 mg/ml were required for labelling of IOR-CEA, 3F8 and M170 respectively. In addition, susceptibility to 2-ME reduction seemed to reflect the rate of antibody labelling. For 2-ME resistant molecules, i.e. M170 and EMD, successful labelling could be achieved by using a slow 99mTc reducing agent such as SnCl2 instead of SnF2 which reacted more rapidly. Since 2-ME generates reactive sulfhydryl groups that are distal to antigen binding sites, the immunoreactivity of the modified antibody was not affected by the effect of reduction.

Performance characteristics of fluorescence endoscope in detection of head and neck cancers.

Laser-induced fluorescence endoscopy (LIFE) is a noninvasive method for detecting early cancers in hollow organs. A laser-induced fluorescence endoscope that was designed for lung imaging was investigated for its effectiveness in comparison to white light endoscopy (WLE) in localizing head and neck cancers. A total of 196 images from 98 sites in 56 patients were graded into 5 levels from normal to definitely abnormal. A cutoff level to differentiate normal from abnormal images was chosen from a receiver operating characteristic curve. On this basis, LIFE (sensitivity, 92.86%; specificity, 78.57%) was observed to be more effective and reliable than WLE (sensitivity, 67.86%; specificity, 70%) in locating neoplastic foci and precancerous lesions. Nevertheless, WLE was helpful in presenting anatomic details. The reliability of LIFE varied from site to site. It excelled in examination of the oropharynx (sensitivity, 100%; specificity, 96.20%), but was less effective for lesions of the nasopharynx (sensitivity, 66.70%; specificity, 75.00%). In standardizing the test, interobserver variation was assessed, and good agreement in image interpretation was confirmed by statistical analysis. In conclusion, LIFE was found to be an effective and reliable tool for detecting head and neck cancers.

Detection of colorectal carcinoma by anti-CEA monoclonal antibody (IOR-CEA1) labeled with 99mTc scintigraphy.

BACKGROUND/AIMS: This study shows that a new monoclonal antibody (IOR-CEA1) labeled with technetium-99m has high diagnostic efficacy for colorectal adenocarcinoma. This immunoscintigraphy is helpful in clinical management especially for patients whose serum carcinoembryonic antigen and computed tomography are questionable for recurrent diseases. The study aims to evaluate the efficiency of a new monoclonal antibody (IOR-CEA1) labeled with technetium-99m in the detection of colorectal carcinoma. METHODOLOGY: Forty colorectal carcinoma patients were examined. They were divided into 2 groups: Group I (9 patients) with untreated primary tumor; and Group II (31 patients) who were suspected of recurrent or residual diseases from 1) equivocal computed tomography or magnetic resonance imaging, or 2) rising serum carcinoembryonic antigen but normal imaging or clinical findings. One milligram of the antibody labeled with 25mCi of technetium-99m was slowly infused intravenously and images were obtained by nuclear medicine techniques. Sensitivity, specificity, accuracy, positive and negative predictive values were determined. RESULTS: 99mTc-IOR-CEA1 had 86% sensitivity, 71% specificity, 83% accuracy, 94% positive predictive value and 50% negative predictive value for the detection of colorectal cancer in 42 studies (2 patients had repeated studies). Serum carcinoembryonic antigen had only 33% sensitivity for detection of the primary cancer and 58% sensitivity in detection of recurrent diseases. Carcinoembryonic antigen had 100% positive predictive value but only 31.3% negative predictive value for diagnosis of the recurrence of tumor. Fifty-two percent of the antibody scans provided more information than computed tomography scans with clinical impact on further management in group II patients. CONCLUSIONS: The 99mTc-IOR-CEA1 scintigraphy is a promising investigative method which is safe and has high accuracy in the detection of recurrent colorectal carcinoma, especially in the patients whose serum carcinoembryonic antigen and computed tomography findings are equivocal for recurrent diseases.

Photodynamic therapy in management of head and neck cancers and precancerous lesions.

Photodynamic therapy (PDT) is a new form of cancer treatment with low morbidity. In this study, PDT was evaluated for its effectiveness in management of recurrent or widespread precancerous lesions, primary cancers in inoperable sites, recurrent or residual cancers which were refractory to radiotherapy and chemotherapy, and advanced tumors in the head and neck. Fifty-one patients were treated over a period of 5 years. A 91.67 per cent complete response rate was observed for T1 tumors (primary and recurrence) with a recurrent rate of 27.27 per cent. Nasopharyngeal carcinoma was highly responsive to PDT since all T1 and T2 tumors responded completely. This was in contrast to cancers in the soft palate which failed in most cases possibly due to inadequate light dose distribution. PDT was remarkably effective in curing premalignant diseases (100% complete response rate). Postoperative PDT was equally effective in treating the microscopic residual malignancy. For advanced tumors, PDT in adjunct to conventional modalities could induce complete response in 5 out of the 10 patients and resolve symptoms in 4 cases. The mean follow-up time for this series was 28.3 months (range 3-66 months). In conclusion, PDT is a useful modality for the treatment of head and neck tumors and precancerous lesions presenting in forms or under conditions that posed considerable difficulties in management by conventional approaches.

Fluorescence-guided biopsy in the diagnosis of an unknown primary cancer in patients with metastatic cervical lymph nodes.

This study was conducted to determine the effectiveness of fluorescence-guided biopsy in diagnosis of unknown primary cancers in the head and neck. Thirteen patients with biopsy-proven cervical node metastases were evaluated prospectively with laser-induced fluorescence (LIF) endoscopy in parallel to panendoscopy. Among the 13 positive sites suggested by LIF imaging, 5 were confirmed by histopathology as squamous cell carcinoma, 4 as dysplasia, 2 as inflammation, and 2 as normal. Panendoscopy with random biopsies located 2. The 2 cases of squamous cell carcinomas discovered conventionally were identified by histologic sections from ipsilateral tonsillectomy. In this series, the unknown primary cancers were localized by LIF imaging and conventional means at rates of 38.5% and 15.4%, respectively. The LIF imaging not only aided in revealing a greater proportion of occult primary cancers, but also helped in reducing the number of unnecessary biopsies. Fluorescence-guided biopsy diagnosis provides useful information for therapeutic planning with curative aim or for adequate palliation.

Photodynamic therapy for residual or recurrent cancer of the nasopharynx.

Adequate or effective treatments are not always available for most recurrent or residual nasopharyngeal cancers (NPC). Photodynamic therapy (PDT) using hematoporphyrin derivative (HpD) was evaluated for its effectiveness in treating patients, who conventionally failed, with curative or palliative intent. Thirteen patients were treated from March 1994 to November 1998. PDT was given to eradicate tumor cells, debulk tumor mass for other treatment options, and to resolve obstruction. Long-term tumor control could be achieved in 6 patients with localized lesions at T1-T2 stages. The mean disease free survival time was 25.8 months (range 5-61 months). For tumors beyond T2 stage (7 cases), PDT in combination with chemotherapy, laser surgery or radiotherapy induced complete response in 1 out of 5 patients (survival time = 40 months) and partial response in the rest (survival time = 16-37 months). In two patients who refused or were in tolerable to further treatment, PDT yielded useful palliative results (i.e. resolve nasal obstruction and epistaxis). On an overall basis, the average survival time for these patients with relatively advanced diseases was 24.7 months (range 9-40 months). Our study demonstrated that HpD-PDT could effectively control locally recurrent or residual NPC at T1-T2 stages and offered good palliation for more advances. Combined PDT and chemotherapy seemed to prolong survival time for a period longer than 2 years in T3-T4 tumors.

Reduction-mediated 99mTc-labeling of antitumor monoclonal antibodies: effect of increasing specific activity on antibody binding kinetics.

Reduction-mediated 99mTc-labeling of antibodies has gained widespread acceptance in preparation of tumor imaging agents. Increased specific activity to enhance detection signals has raised the question of whether such an attempt would cause change in antibody binding kinetics. To answer this question, two antitumor monoclonal antibodies, i.e. IOR-CEA (IgG1) and EMD (IgG2a) were labeled with 99mTc to yield specific activities ranging from 549-4414 MBq/mg. Regression analysis of the binding data revealed that the binding kinetics of IOR-CEA were shifted from monovalent to bivalent binding upon increasing the specific activities. This phenomenon of affinity enhancement was confirmed by the dissociation study where we found soluble CEA had greater difficulty in extracting the cell-bound IOR-CEA labeled at higher specific activity. The bivalent bindings was further supported by the finding that IOR-CEA with higher specific activities delivered less than expected radioactivity to tumor targets despite their immunoreactivities being well preserved. For EMD, the kinetics seemed to be shifted from bivalent to monovalent interaction. At higher specific activities, adverse changes in immunoreactivity were recognized. Breakage of EMD into 99mTc-Fab fragments was likely to occur and was supported by the observation that EMD delivered more than expected radioactivities to target cells upon increasing specific activity. Precaution should be taken when one deals with high specific activity labeling since this might alter the antibody binding kinetics either favorably or unfavorably.

Laser-induced fluorescence imaging in localization of head and neck cancers.

Laser-induced fluorescence tumor imaging exploits the difference in tissue autofluorescence properties between normal and cancerous tissues. The effectiveness and reliability of fluorescence imaging with a lung imaging fluorescence endoscopy (LIFE) system for cancers in the head and neck were compared to those of white light endoscopy (WLE). Examinations by WLE and LIFE were conducted on 25 patients suspected for malignancy. Histologic diagnosis was confirmed by biopsy. Posttreatment evaluations were performed on 6 cancer patients identified by this study. By LIFE, all 16 cancerous lesions, including 2 occult cancers, were identified (100%), while WLE achieved only an 87.5% detection rate. LIFE (specificity 87.5%) was greatly helpful to WLE (specificity 50%) in differentiating inflammation from malignancy, though it failed to exclude granuloma. The results of this study suggest potential roles of LIFE in early detection, correct staging, and treatment evaluation of cancers in the head and neck.

Differences in in vitro photodynamic sensitivity among head and neck cancers.

This study was conducted to address the question of how cancers of different histologies of the head and neck region responded to photodynamic therapy (PDT). Five human cancer cell lines were investigated: two squamous cell carcinoma lines (pharynx and tongue), mucoepidermoid carcinoma (submaxillary gland), rhabdomyosarcoma (embryonic) and adenocarcinoma (colon). The cell lines were treated with haematoporphyrin derivative (HpD) at doses of 0.78-25Mug ml(-1), with excitation of the absorbed drug by a 'black light' source (340-380 nm). An MTT assay demonstrated different PDT responses among the various cell types. On the basis of LD(50), the sensitivity of the different cell lines was ranked as follows: adenocarcinoma> squamous cell carcinoma> rhabdomyosarcoma> mucoepidermoid carcinoma. The magnitude of the LD(50) was suggested by a drug uptake study to be governed in part by cellular levels of sensitizer and in part by intrinsic cell sensitivity. This study provided information that may help to identify the histological types of head and neck cancers that would respond favourably to PDT.

Photodynamic therapy in the treatment of head and neck cancers: a two-year experience.

This report describes the effectiveness of photodynamic therapy (PDT) in treating patients with widespread superficial premalignant and malignant lesions, primary and second primary, nonresponsive residual or recurrent cancers in head and neck. Twelve patients whose disease was at T1 stage responded completely. Severe dysplasia and field cancerous lesions involving a large area of oral mucosa (3 cases) also yielded excellent results. Average disease-free period was 13.6 months. The longest survival period was 2 years. All patients as mentioned are still alive without any relapse or recurrence. Combined PDT with other treatment was required to control T2-3 carcinomas. PDT adjunct, however, permitted the prescription of conventional treatment, i.e. radiotherapy or surgery, in a less morbid manner. No unacceptable treatment complications and skin photosensitivity were observed in this study. This indicated the potential role of PDT in the management of long-standing problems in head and neck cancers.

Differential radiosensitization of radioresistant human cancer cells by caffeine.

The effect of caffeine, the methylated xanthine, in sensitizing the lethal action of ionizing radiation in vitro was investigated in human cancer cells which were clinically known to be radioincurable. The tumor lines were hepatocellular carcinoma and colon adenocarcinoma. Plateau phase cultures, after absorbing doses of 2 Gy, survived at a rate of 56.30 per cent for colon cancer and at 66.05 per cent for liver cancer. Both lines were radiosensitized by caffeine but at different potencies. Noteworthily, hepatocellular carcinoma whilst less radiosensitive than colon adenocarcinoma was 4 times more susceptible to caffeine. The lowest effective caffeine concentration for liver cancer was 2 mM which slightly exceeded the anticipated lethal concentration in humans. Research on radiosensitizing effect of methylated xanthines on hepatoma system still remains intriguing. Future work should be pursued with the use of less toxic compounds, such as theobromine.

Variables influencing cell binding assays for antibody binding kinetics.

Owing to growing interest in the use of cell binding assays for quality testing of radiolabelled antibodies in radioimmunoscintigraphy, factors in the assay which might affect final outcome were evaluated in order to accumulate information for effective and reliable assay design. For unmodified antibody, magnitude of binding at certain antibody concentrations changed with alteration in number of target cells attached to the microtitre plate. The level of signal detection could either be enhanced or suppressed by changing the dilution of the enzyme-linked tester antibody as in the case of the enzyme-linked immunosorbent assay (ELISA). Accordingly, changes in either assay factor resulted in a shift in the slope of the binding curve even when the immunoreactivity of the antibody remained unchanged. A conventional technique of single cell preparation by trypsin-EDTA was investigated for the trypsin action on tumour cell antigenicity. No adverse effects could be observed if dissociated cells were incubated for 24 hours before the assay. Variation in number of target cells seemed to be a key factor in determining the power of the assay to reveal changes in the quality of the radiolabelled antibody. Our findings suggested the need to standardize factors such as the size of the seed, determination of plating efficiency and the time for culture in order to assure interassay reproducibility.

Analysis of immunoradiological properties of radiolabelled monoclonal antibody for tumour localization.

This investigation demonstrates the effect of 131I incorporation on antibody function and radionuclide delivery to target antigens by radiolabelled antibody. For our antihepatoma monoclonal antibody with affinity constant K = 1.12 x 10(8) M-1 and maximum binding capacity of 1.66 x 10(6) sites on each hepatoma cell, a linear decrease in K was observed with the increase in specific activity from 1.90 to 97.12 microCi microgram-1. Radioiodination also inactivated nearly half of the immunoreactive molecules. Most interestingly, when magnitudes of antibody mass and radioactivity which bound target cells were compared for preparations of different specific activities, each contained similar nanomolar of antibody molecules. We recognized the slight gain in nanogram bound opposing to the great loss in bound counts per minute (cpm) as consequences of decreasing levels of 131I incorporation from 97.12 and 26.64 to 1.90 microCi microgram-1. Our findings suggest that the quality of radiolabelled antibody should be assessed in both its immunological function and radioactive transfer capability.

Quantification of antibiotic drug potency by a two-compartment radioassay of bacterial growth.

The two-compartment radioassay for microbial kinetics based on continuous measurement of the 14CO2 released by bacterial metabolism of 14C-labeled substrate offers a valuable approach to testing the potency of antimicrobial drugs. By using a previously validated radioassay with gram-positive and gram-negative bacteria, a group of protein synthesis inhibitors was evaluated for their effect on microbial growth kinetics. All tested drugs induced changes in both the slopes and intercepts of the growth curves. An exponential growth model was applied to quantify the drug effect on the processes of bacterial 14CO2 liberation and cell generation. The response was measured in terms of a generation rate constant. A linear dependence of the generation rate constant on the dose of spectinomycin was observed with Escherichia coli. Sigmoidal-shaped curves were found in the assays of chloramphenicol and tetracycline. The implications of dose-response curves are discussed on the basis of the receptor site concept for drug action. The assay sensitivities for chloramphenicol and tetracycline were similar to those obtained by the cell counting method, but the sensitivity of the radioassay was at least 10 times greater for spectinomycin.

Assessing performances of Iodogen-coated surfaces used for radioiodination of proteins.

Performances of Iodogen-coated surfaces were assessed by measuring kinetics of iodide oxidation with a spectrophotometer. The iodide was effectively oxidized by a surface of high Iodogen concentration. The oxidation rate tended to reach a plateau at 8.45 micrograms cm-2. Increasing the total mass of coating without changing the surface concentration did not increase the rate of oxidation. At each level of concentration, the magnitude of iodide oxidized was proportional to the amount applied in a log-linear pattern providing that the quantity of Iodogen was in constant excess. Oxidation on polypropylene, borosilicate and soda-lime glasses were also investigated. Nonpolar polypropylene surfaces yielded the lowest rate of oxidation while polar soda-lime glass yielded the highest. Borosilicate surface was rated at an intermediate level, but it did not provide good matrix for coating due to its poor reproducibility. It appeared that both polypropylene and borosilicate surfaces might trap a certain fraction of the Iodogen and reduce the effective surface concentration. Findings in this study supply fundamental guidelines for surface selection and preparation.

Bacterial growth kinetics: modelling and evaluation of two-compartment radioassay.

Quantitative measurements of bacterial growth may be made using a radioassay technique. This method measures, by scintillation counting, the 14CO2 derived from the bacterial metabolism of a 14C-labeled substrate. Mathematical growth models may serve as reliable tools for estimation of the generation rate constant (or slope of the growth curve) and provide a basis for evaluating assay performance. Two models, i.e., exponential and logistic, are proposed. Both models yielded an accurate fit to the data from radioactive measurement of bacterial growth. The exponential model yielded high precision values of the generation rate constant, with an average relative standard deviation of 1.2%. Under most conditions the assay demonstrated no changes in the slopes of growth curves when the number of bacteria per inoculation was changed. However, the radiometric assay by scintillation method had a growth-inhibiting effect on a few strains of bacteria. The source of this problem was thought to be hypersensitivity to trace amounts of toluene remaining on the detector.

Radiometric assay of bacterial growth: analysis of factors determining system performance and optimization of assay technique.

A quantitative technique for the measurement of 14CO2 released from a bacterial culture was evaluated. The technique uses liquid scintillation counting to record 14CO2 accumulation on a fluor-impregnated filter paper within a double-chambered scintillation vial that also houses the bacterial growth medium. We have successfully identified and corrected the major causes for a variably low detection efficiency, and also established the optimum mixture of reagents for the detection system. Incorporation of Triton X-100 into the scintillation fluid used for the detector reduced the variability between identical assays in a single batch from 50% to 5%, and, in conjunction with an increase in the scintillator concentration, raised the counting efficiency from 30% to 70-88%. The response of the improved detector is linear over a wide range of count-rates. Another significant modification was the interchange of growth and detector chambers. Overall, a 40-fold increase in count-rate during the exponential phase of bacterial growth was obtained by improving 14CO2 detection efficiency, increasing the rate of 14CO2 transfer from liquid to gas phases and enlarging the growth supporting capacity of the detector system. The minimum detection time for bacterial growth was shortened and the exponential phase of bacterial proliferation was lengthened by at least 2 hr. High counting efficiency, precision, and linearity make the improved detector a sensitive and reliable tool for radiometry of bacterial growth and metabolism.