Radioimmunotherapy with alpha-particle-emitting immunoconjugates.

Alpha particles are energetic short-range ions whose higher linear energy transfer produces extreme cytotoxicity. An alpha-particle-emitting radioimmunoconjugate consisting of a bismuth-212-labeled monoclonal immunoglobulin M specific for the murine T cell/neuroectodermal surface antigen Thy 1.2 was prepared. Analysis in vitro showed that the radioimmunoconjugate was selectively cytotoxic to a Thy 1.2+ EL-4 murine tumor cell line. Approximately three bismuth-212-labeled immunoconjugates per target cell reduced the uptake of [3H]thymidine by the EL-4 target cells to background levels. Mice inoculated intraperitoneally with EL-4 cells were cured of their ascites after intraperitoneal injection of 150 microcuries of the antigen-specific radioimmunoconjugate, suggesting a possible role for such conjugates in intracavitary cancer therapy.

E2F4 regulates a stable G2 arrest response to genotoxic stress in prostate carcinoma.

The retinoblastoma (pRB) family proteins regulate the E2F transcription factors; their complexes regulate critical transitions through the cell cycle. The function of these pRB family/E2F complexes, which includes p130/E2F4, in response to genotoxic agents, is not well understood. We investigated the role of E2F4 in the genotoxic stress response. Following radiation treatment, E2F4 colocalized with p130 in the nucleus during a radiation-induced stable G(2)-phase arrest. Arrested cells had significantly decreased expression of Cyclins A2 and B1 and decreased phosphorylation of mitotic protein monoclonal-2 (MPM-2) mitotic proteins. Small interference RNA (siRNA)-mediated knockdown of E2F4 sensitized cells to subsequent irradiation, resulting in enhanced cellular DNA damage and cell death, as determined by caspase activation and decreased clonogenic cell survival. Downstream E2F4 targets potentially involved in the progression from G(2) into M phase were identified by oligonucleotide microarray expression profiling. Chromatin immunoprecipitation localized E2F4 at promoter regions of the Bub3 and Pttg1 mitotic genes following irradiation, which were among the downregulated genes identified by the microarray. These data suggest that in response to radiation, E2F4 becomes active in the nucleus, enforces a stable G(2) arrest by target gene repression, and thus provides increased cell survival ability by minimizing propagation of cells that have irreparable DNA damage.

Influence of killer immunoglobulin-like receptor/HLA ligand matching on achievement of T-cell complete donor chimerism in related donor nonmyeloablative allogeneic hematopoietic stem cell transplantation.

Achievement of complete donor chimerism (CDC) after allogeneic nonmyeloablative hematopoietic stem cell transplantation (NMHSCT) is important for preventing graft rejection and for generating a graft-vs-malignancy effect. The alloreactivity of NK cells and some T-cell subsets is mediated through the interaction of their killer immunoglobulin-like receptors (KIRs) with target cell HLA/KIR ligands. The influence of KIR matching on the achievement of T-cell CDC after NMHSCT has not been previously described. We analyzed 31 patients undergoing T-cell replete related donor NMHSCT following fludarabine and 200 cGy TBI. Recipient inhibitory KIR genotype and donor HLA/KIR ligand matches were used to generate an inhibitory KIR score from 1 to 4 based upon the potential number of recipient inhibitory KIRs that could be engaged with donor HLA/KIR ligands. Patients with a score of 1 were less likely to achieve T-cell CDC (P=0.016) and more likely to develop graft rejection (P=0.011) than those with scores greater than 1. Thus, patients with lower inhibitory KIR scores may have more active anti-donor immune effector cells that may reduce donor chimerism. Conversely, patients with greater inhibitory KIR scores may have less active NK cell and T-cell populations, which may make them more likely to achieve CDC.

Synthesis of hemoglobin F in adult simian erythroid progenitor-derived colonies.

The simian hematopoietic system is known to respond to anemic stress with the production of erythrocytes containing large amounts of fetal hemoglobin. To determine the regulatory mechanism responsible for hemoglobin F (HbF) production in stress erythropoiesis, adult simian bone marrow cells were cultured in plasma clots in the presence or absence of erythropoietin and burst-promoting activities, and the HbF content of progenitor-derived colonies was determined by radioimmunoligand assay. Three classes of erythroid progenitors were detected: BFU-E, CFU-E, and a very mature cohort of dense highly erythropoietin-responsive cells (HERC). These classes varied in inverse proportion to their maturity with respect to their potential for HbF accumulation in the colonies to which they give rise. Both erythropoietin and burst-promoting activity stimulated HbF production, particularly in colonies derived from immature progenitors. For example, under conditions of high erythropoietin stimulation, BFU-E colonies contained 13.7-37.7% HbF, CFU-E colonies contained 2.8-13.5% HbF, and HERC colonies 0-1% HbF. These results suggest that under nonstress conditions simian erythrocytes are derived almost entirely from HERC progenitors and proerythroblasts in which gamma chain synthesis is suppressed. During stress erythropoiesis, augmented HbF accumulation could be explained by the rapid entrance into the marrow of proerythroblasts directly derived from immature progenitors. Gamma chain production in these proerythroblasts is additionally regulated by the changes in environmental erythropoietin and burst-promoting activities.

The late increase in intracellular free radical oxygen species during apoptosis is associated with cytochrome c release, caspase activation, and mitochondrial dysfunction.

Mitochondria play central roles in cellular metabolism and apoptosis and are a major source of reactive oxygen species (ROS). We investigated the role of ROS and mitochondria in radiation-induced apoptosis in multiple myeloma cells. Two distinct levels of ROS were generated following irradiation: a small increase observed early, and a pronounced late increase, associated with depletion of reduced glutathione (GSH) and collapse of mitochondrial membrane potential (deltapsi(m)). Exogenous ROS and caspase-3 induced deltapsi(m) drop and cytochrome c release from mitochondria, which could be prevented by molecular (dominant-negative caspase-9) and pharmacologic (zVAD-fmk) caspase inhibitors and overexpression of Bcl-2. Exogenous ROS also induced mitochondrial permeability transition (PT) pore opening and cytochrome c release in isolated mitochondria, which could be blocked by inhibition of PT with cyclosporin A. These results indicate that the late ROS production is associated with increased PT pore opening and decreased deltapsi(m), and GSH, events associated with caspase activation and cytochrome c release.

Error rates in clinical radiotherapy.

PURPOSE: Error rates in clinical oncology are undergoing increasing scrutiny. The purpose of this study was to understand error frequency, error patterns, underlying causal links, consequences, and possible prevention strategies in clinical radiotherapy. PATIENTS AND METHODS: Treatment information, self-reported error documentation, and retrospective analyses of electronic treatment verification transcripts for 1,925 consecutive patients treated with a total of 93,332 individual radiotherapy fields were reviewed and analyzed. RESULTS: A total of 59 separate errors that affected 168 individual treatment fields were detected, which yielded a crude radiation delivery error rate of 0.18%. All 59 errors were judged to be level I (negligible chance of adverse medical outcome) with the most common error category being a minor treatment field block misplacement. A comprehensive quality assurance program and an electronic record-and-verify linear accelerator interlock system seem to have prevented the occurrence of many additional errors. However, nine of the 59 errors were directly related to the use of this system and generally involved the transposition of similar numbers within series of treatment coordinate data-sets. Overall, radiotherapy error rates favorably compare with reported error rates for pharmaceutical administration in large tertiary care hospitals. CONCLUSION: When modern automated error-minimization methods are used along with nonpunitive error reporting systems, clinical radiotherapy seems to be highly safe. Formal error analysis studies may allow the rational design of prevention strategies that are attuned to the frequency, seriousness, and antecedent causes of many classes of potential radiotherapy errors.

Family history of prostate cancer in patients with localized prostate cancer: an independent predictor of treatment outcome.

PURPOSE: To determine if familial prostate cancer patients have a less favorable prognosis than patients with sporadic prostate cancer after treatment for localized disease with either radiotherapy (RT) or radical prostatectomy (RP). PATIENTS AND METHODS: One thousand thirty-eight patients treated with either RT (n = 583) or RP (n = 455) were included in this analysis. These patients were noted as having a positive family history if they confirmed the diagnosis of prostate cancer in a first-degree relative. The outcome of interest was biochemical relapse-free survival (bRFS). We used proportional hazards to analyze the effect of the presence of family history and other potential confounding variables (ie, age, treatment modality, stage, biopsy Gleason sum [GS], and initial prostate-specific antigen [iPSA] levels) on treatment outcome. RESULTS: Eleven percent of all patients had a positive family history. The 5-year bRFS rates for patients with negative and positive family histories were 52% and 29%, respectively (P < .001). The potential confounders with bRFS rates were iPSA levels, biopsy GS, and clinical tumor stage; treatment modality and age did not appear to be associated with outcome. After adjusting for potential confounders, family history of prostate cancer remained strongly associated with biochemical failure. CONCLUSION: This is the first study to demonstrate that the presence of a family history of prostate cancer correlates with treatment outcome in a large unselected series of patients. Our findings suggest that familial prostate cancer may have a more aggressive course than nonfamilial prostate cancer, and that clinical and/or pathologic parameters may not adequately predict this course.

Stage III neuroblastoma over 1 year of age at diagnosis: improved survival with intensive multimodality therapy including multiple alkylating agents.

PURPOSE: A nonrandomized, single-arm trial was conducted to assess the efficacy of multimodality therapy including intensive chemotherapy with multiple alkylating agents in the treatment of children with Evans stage III neuroblastoma older than 1 year at diagnosis. PATIENTS AND METHODS: Twenty-five patients with a median age of 18 months at diagnosis were treated with multimodality therapy including surgery and chemotherapy using either nitrogen mustard (mechlorethamine), doxorubicin, cisplatin, dacarbazine (DTIC), vincristine, and cyclophosphamide (MADDOC) or cisplatin and cyclophosphamide induction followed by maintenance MADDOC (induction MADDOC) protocols. Sixteen of 25 patients also received radiotherapy to the tumor bed and primary lymph nodes. Event-free survival (EFS) was compared with that reported previously in the literature. N-myc amplification was evaluated prospectively and the Shimada classification was evaluated retrospectively as potential prognostic factors. RESULTS: We report a 72% EFS (95% confidence interval +/- 18%) with a median follow-up of 85 months. EFS was significantly worse for patients with tumors demonstrating N-myc amplification (P = .018). Patients classified as favorable according to the Shimada system experienced a significantly better EFS (P = .04), but unfavorable patients still maintained a 60% EFS. CONCLUSION: Intensive multimodality treatment including MADDOC and induction MADDOC chemotherapy provides a very good EFS for children older than 1 year who have stage III neuroblastoma. Children classified as favorable according to the Shimada system have a better prognosis. Patients whose tumors demonstrate N-myc amplification have a poor prognosis despite therapy.

Treatment with nephrectomy only for small, stage I/favorable histology Wilms' tumor: a report from the National Wilms' Tumor Study Group.

PURPOSE: Children younger than 24 months with small (< 550 g), favorable histology (FH) Wilms tumors (WTs) were shown in a pilot study to have an excellent prognosis when treated with nephrectomy only. PATIENTS AND METHODS: A study of nephrectomy only for the treatment of selected children with FH WT was undertaken. Stringent stopping rules were designed to insure closure of the study if the true 2-year relapse-free survival rate was 90% or lower. RESULTS: Seventy-five previously untreated children younger than 24 months with stage I/FH WTs for which the surgical specimen weighed less than 550 g were treated with nephrectomy only. Three patients developed metachronous, contralateral WT 1.1, 1.4, and 2.3 years after nephrectomy, and eight patients relapsed 0.3 to 1.05 years after diagnosis (median, 0.4 years; mean, 0.51 years). The sites of relapse were lung (n = 5) and operative bed (n = 3). The 2-year disease-free (relapse and metachronous contralateral WT) survival rate was 86.5%. The 2-year survival rate is 100% with a median follow-up of 2.84 years. The 2-year disease-free survival rate (excluding metachronous contralateral WT) was 89.2%, and the 2-year cumulative risk of metachronous contralateral WT was 3.1%. CONCLUSION: Children younger than 24 months treated with nephrectomy only for a stage I/FH WT that weighed less than 550 g had a risk of relapse, including the development of metachronous contralateral WT, of 13.5% 2 years after diagnosis. All patients who experienced relapse on this trial are alive at this time. This approach will be re-evaluated in a clinical trial using a less conservative stopping rule.

Radiation therapy in pregnancy: risk calculation and risk minimization.

The benefits of radiation therapy (RT) as part of a treatment regimen for cancer must be weighed against the potential risk of harm to the patient and in the pregnant patient, the risk to the developing fetus. Information necessary for determining the potential effects of RT on the developing fetus include the gestational age, absorbed fetal dose-equivalent, and dose-rate. The risk periods in humans for RT-induced prenatal or neonatal death, congenital anomalies, severe mental retardation (SMR), temporary (TGR) or permanent growth retardation (PGR), carcinogenesis, sterility, and germ cell mutations have been elicited directly from the study of Japanese victims of the atomic bombs and unintentional medical exposures, and indirectly from animal experiments. The wide range of congenital anomalies elicited from animal studies have not occurred in the Japanese atomic bomb victims exposed in utero. The major congenital anomaly observed in the Japanese cohort has been microcephaly. The highest risk period for SMR correlates with the proliferation, differentiation, and, most importantly, migration of neurons from their proliferative zones. PGR was apparent 17 years after ionizing radiation (IR) exposure at Hiroshima in children who were within 1,500 meters of the hypocenter. Children were on average 2.25 cm shorter, 3 kg lighter, and had head diameters 1.1 cm smaller than age-matched children. The projected lifetime risk of cancer mortality in the Japanese cohort is 14% per gray. The risk of a radiation-induced hereditary disorder is reported to be approximately 1% per gray. RT plays a major role in the definitive treatment of cervical and breast carcinomas, Hodgkin's disease, and non-Hodgkin's lymphoma. With appropriate abdominal shielding in place, the estimated fetal dose can be reduced by 50% or greater in most cases. In certain clinical situations, RT may be administered during pregnancy.

Lymphoma and pregnancy.

Coincident lymphoma and pregnancy is rare, occurring in only one of 1,000 or fewer deliveries. Patients with Hodgkin's disease usually present with typical manifestations, and most studies suggest that the disease probably has little or no effect on the pregnancy and vise versa. In contrast, patients with non-Hodgkin's lymphoma often have unusual manifestations and their diagnosis is frequently delayed. These patients usually have aggressive, advanced-stage disease and a poor outcome. The optimal management of these women requires special considerations. Treatment during the first trimester is associated with significant risk to the developing fetus and should be avoided. Women requiring treatment at this time should have a therapeutic abortion. Beyond the first trimester, the management options for women with Hodgkin's disease include observation until disease progression or delivery, single-agent vinblastine, modified or standard combination chemotherapy, and radiation therapy (with appropriate shielding). Because of their poor prognosis, women with non-Hodgkin's lymphoma should receive standard chemotherapy (despite the potential risk to the developing fetus).

The palliative role of radiotherapy in the management of the cancer patient.

Radiotherapy (RT) plays a major role in the palliation of cancer symptoms. Cancer-related bone pain, bronchial obstruction, superior vena cava syndrome, spinal cord compression, and central neurologic dysfunction due to brain metastases are all treated effectively and safely by RT. Although numerous techniques and radiation dose fractionation schemes are used in practice, substantial palliation often may be accomplished by just two to 10 appropriately targeted treatments. Thus, palliative RT regimens need not be highly protracted to be effective. Several new techniques, including strontium 89 radiopharmaceutical therapy, high-dose-rate endobronchial brachytherapy, and stereotactic radiosurgery, are now being evaluated in clinical trials to determine their efficacy in the relief of various oncologic symptoms. Palliative RT continues to be a mainstay of treatment in the management of cancer patients with advanced disease.

Palliative radiotherapy.

Radiotherapy is often used for the palliative treatment of advanced cancer. Although each cancer is unique in its histology and natural history, there are several scenarios that repeatedly challenge physicians. Specifically, skeletal metastases, spinal cord compression, brain metastasis, bronchial obstruction, and vena cava obstruction are regularly encountered. This review will discuss the diagnosis and treatment of these common important situations.

Wilms' tumor patients with pulmonary metastases.

We have evaluated the clinicopathologic characteristics, survival probabilities, patterns of treatment failure, and late toxicities in a group of 51 Wilms' tumor patients with pulmonary metastases evaluated at our center between 1968 and 1988 and treated intensively with surgery, multiagent chemotherapy, and radiotherapy. Twenty-one patients had pulmonary metastases at diagnosis (Stage IV-p) and 30 had pulmonary relapse after initial treatment for nonmetastatic disease. With a median follow-up time of 83 months, actuarial 5- and 10-year survival probability for the group as a whole is 59%. For the subset of patients with favorable histology Stage IV-p disease, 10-year survival probability is 77%, whereas for the subset with relapsed anaplastic or sarcomatous disease, 10-year survival probability is 22%. The lung was the predominant site of failure even in patients who had received relatively high doses of pulmonary radiotherapy. Late effects seen in patients with over 10 years of follow-up included musculoskeletal and soft tissue growth abnormalities (93% incidence) as well as breast hypoplasia and endocrinologic abnormalities. Second malignant neoplasms of the breast, thyroid, and pancreas were observed at post-treatment intervals of 17, 3.5 and 12 years, respectively.

A mobile shield to reduce scatter radiation to the contralateral breast during radiotherapy for breast cancer: preclinical results.

PURPOSE: To design a practical breast shield and to investigate its efficacy in reducing scattered radiation to the contralateral breast of patients undergoing radiation therapy for breast cancer. METHODS AND MATERIALS: We constructed a mobile shield consisting of (a) a mobile base and a counterweight; (b) a vertical column adjustable in height and a diagonal arm adjustable in angle; (c) a curved, 2.5-cm thick lead sheet with a 1-cm thick polystyrene liner for blocking scattered radiation; and (d) diode detectors to verify that the edge of the lead sheet is not in the useful beam in addition to the use of the field light. Measurements were performed with thermoluminescent dosimeters on 10 patients without the shield and on an anthropomorphic phantom with a pair of wax breasts with and without the shield. All of the patients were treated with 6-MV photons (Varian 6/100). The scattered radiation from the medial and lateral fields was measured separately. RESULTS: The contribution of the medial field to the total scattered dose was 70% to 75%, whether a medial wedge was used or not. However, without a medial wedge, the scattered dose was reduced by nearly 33% at 3 to 9 cm away from the medial border. In the anthropomorphic phantom study with wax breast, the mobile shield reduced the medial field contribution to the total scatter dose to less than the contribution from the lateral field without a shield. With a prescribed dose of 50 Gy and a medial wedge, the median scatter dose to the contralateral breast from 6 patients was 5.3 Gy; without a medial wedge, it was 3.8 Gy from 4 patients at 6 cm from the medial border. In the phantom study, with the shield the total dose to the contralateral breast was 1.0 Gy at 6 cm from the medial border with a same prescribed dose. CONCLUSION: The mobile shield reduced the scatter dose to the contralateral breast from the linear accelerator (Varian 6/100, 6-MV photons) by a factor of 3 to 4. The shield greatly reduced the scattered dose in the wax phantom. Equivalent reductions in patients may be clinically significant by reducing the risk of radiation-induced breast cancer in the contralateral breast of woman undergoing radiation therapy for breast cancer. The shield is safe and easy to adjust to each patient.

Cell cycle alterations, apoptosis, and response to low-dose-rate radioimmunotherapy in lymphoma cells.

PURPOSE: In an attempt to elucidate some aspects of the radiobiological basis of radioimmunotherapy, we have evaluated the in vitro cellular response patterns for malignant lymphoma cell lines exposed to high- and low-dose-rate radiation administered within the physiological context of antibody cell-surface binding. METHODS AND MATERIALS: We used two different malignant lymphoma cell lines, a Thy1.2+ murine T-lymphoma line called EL-4 and a CD20+ human B-lymphoma line called Raji. Cells were grown in suspension cultures and exposed to high-dose-rate gamma radiation from an external 137Cs source or low-dose-rate beta radiation from DTPA-solubilized 90Y in solution. In some experiments, cells were pre-incubated with an excess of nonradioactive antibody in order to assess the effects of immunoglobulin surface binding during radiation exposure. Irradiated cells were evaluated for viability, cell-cycle changes, patterns of post-radiation morphologic changes, and biochemical hallmarks of radiation-associated necrosis and programmed cell death. RESULTS: The EL-4 line was sensitive to both high-dose-rate and low-dose-rate irradiation, while the Raji showed efficient cell kill only after high-dose-rate irradiation. Studies of radiation-induced cell cycle changes demonstrated that both cell lines were efficiently blocked at the G2/M interface by high-dose-rate irradiation, with the Raji cells appearing somewhat more susceptible than the EL-4 cells to low-dose-rate radiation-induced G2/M block. Electron microscopy and DNA gel electrophoresis studies showed that a significant proportion of the EL-4 cells appeared to be dying by radiation-induced programmed cell death (apoptosis) while the Raji cells appeared to be dying primarily by classical radiation-induced cellular necrosis. CONCLUSION: We propose that the unusual clinical responsiveness of some high and low grade lymphomas to modest doses of low-dose-rate radioimmunotherapy may be explained in part by the induction of apoptosis. The unusual dose-response characteristics observed in some experimental models of radiation-induced apoptosis may require a reappraisal of standard linear quadratic and alpha/beta algorithms used to predict target tissue cytoreduction after radioimmunotherapy.

Alpha particle radio-immunotherapy: animal models and clinical prospects.

Short-lived isotopes that emit alpha particles have a number of physical characteristics which make them attractive candidates for radioimmunotherapy. Among these characteristics are high linear energy transfer and correspondingly high cytotoxicity; particle range limited to several cell diameters from the parent atom; low potential for repair of alpha-induced DNA damage; and low dependence on dose rate and oxygen enhancement effects. This report reviews the synthesis, testing and use in animal models of an alpha particle emitting radioimmunoconjugate constructed via the noncovalent chelation of Bismuth-212 to a monoclonal IgM antibody specific for the murine T cells/neuroectodermal surface antigen, Thy 1.2. These 212Bi-anti-Thy 1.2 immunoconjugates are capable of extraordinary cytotoxicity in vitro, requiring approximately three 212Bi-labeled conjugates per target cell to suppress 3H-thymidine incorporation to background levels. The antigen specificity afforded by the monoclonal antibody contributes a factor of approximately 40 to the radiotoxicity of the immunoconjugate. Animals inoculated with a Thy 1.2+ malignant ascites were cured of their tumor in an antigen-specific fashion by intraperitoneal doses of approximately 200 microCi per mouse. Alpha particle emitting radioimmunoconjugates show great potential for regional and intracavitary molecular radiotherapy.

Biodistribution studies of anti-Thy 1.2 IgM immunoconjugates: implications for radioimmunotherapy.

We have prepared 111In radioimmunoconjugates (RICs) of the IgM isotype with specificity for the murine T cell/neuroectodermal surface antigen, Thy 1.2. Using gamma camera immunoscintigraphy, we have analyzed the biodistribution patterns of the RICs after intravenous and intraperitoneal injection into normal Thy 1.2+ and Thy 1.2- mice. Both routes of administration show antigen-specific uptake by the splenic T lymphocyte population. A high degree of nonspecific uptake by the reticuloendothelial system is also observed. Analysis of the specific activity of various segments of spleens from RIC-injected animals shows inhomogeneous uptake of the RIC not readily apparent by immunoscintigraphy. Animals injected with the RIC and then given high dose total body irradiation showed rapid shifts in radionuclide distribution away from the target cell population and into the general reticuloendothelial system, suggesting that death of the target cell can alter RIC biodistribution. Analyses of RIC biodistribution patterns will contribute to optimization of treatment by radioimmunotherapy.

Radiation hormesis.

"Radiation hormesis" is the name given to the putative stimulatory effects of low level ionizing radiation (generally in the range of 1-50 cGy of low-LET radiation). Based on historical and pharmacologic principles reminiscent of some of the major tenets of homeopathy, most of these effects are now generally ascribed to protective feedback systems that, upon exposure to low concentrations of toxins, proceed to stimulate metabolic detoxification and repair networks. The activation of these networks may then result in net beneficial effects on the cell, organism or species. Discussions of possible stimulatory effects of low levels of ionizing radiation have recently become entangled with the separate but related question of whether a threshold dose level exists on the radiotoxicologic dose-response curve. This review summarizes some of the relevant historical and scientific data bearing on the question of radiation hormesis. We find the data in support of most of the hormesis postulates intriguing but inconclusive.

Image analysis for the study of radionuclide distribution in tissue sections.

UNLABELLED: Tissue section autoradiographs are often prepared to review the precise spatial locations of a radiolabeled molecule relative to cells, such as in the study of radiolabeled antibody distribution. The objective of this work was to develop and evaluate a method to automatically detect both grains and cell nuclei from stained tissue autoradiographs using a microscope and an image analyzer. METHOD: Using a sequence of morphological image operations, the densely stained regions of the section, representing the cell nuclei are identified first, and then subtracted from the original image. This enables the identification of autoradiographic grains under conditions of variable contrast, by separation of the grains overlapping the cell nuclei from the extracellular spaces, permitting a more accurate and robust automatic segmentation routine. RESULTS: The accuracy of the method to detect grains has been evaluated at different threshold levels. The highest accuracy obtained was approximately 90%. The accuracy in the detection of cell nuclei was histology-dependent. As examples, we have estimated accuracies of approximately: 86%, 81% and 77% for kidney, EL-4 lymphoma and pneumonocyte sections, respectively. CONCLUSION: This method was tested using specimens designed to study radiolabeled antibody distribution, but it should be applicable with comparable accuracy to other radiolabeled compounds for which quantitative information on the heterogeneity of distribution is required.