Relationship Between Absorbed Dose and Response in Neuroendocrine Tumors Treated with [177Lu]Lu-DOTATATE.

Peptide receptor radionuclide therapy presents the possibility of tracing and quantifying the uptake of the drug in the body and performing dosimetry, potentially allowing individualization of treatment schemes. However, the details of how neuroendocrine tumors (NETs) respond to different absorbed doses are insufficiently known. Here, we investigated the relationship between tumor-absorbed dose and tumor response in a cohort of patients with NETs treated with [177Lu]Lu-DOTATATE. Methods: This was a retrospective study based on 69 tumors in 32 patients treated within a clinical trial. Dosimetry was performed at each cycle of [177Lu]Lu-DOTATATE, rendering 366 individual absorbed dose assessments. Hybrid planar-SPECT/CT imaging using [177Lu]Lu-DOTATATE was used, including quantitative SPECT reconstruction, voxel-based absorbed dose rate calculation, semiautomatic image segmentation, and partial-volume correction. Changes in tumor volume were used to determine tumor response. The volume for each tumor was manually delineated on consecutive CT scans, giving a total of 712 individual tumor volume assessments. Tumors were stratified according to grade. The relationship between absorbed dose and response was investigated using mixed-effects models and logistic regression. Tumors smaller than 4 cm3 were excluded. Results: In grade 2 NETs, a clear relationship between absorbed dose and volume reduction was observed. Our observations suggest a 90% probability of partial tumor response for an accumulated tumor-absorbed dose of at least 135 Gy. Conclusion: Our findings are in accordance with previous observations regarding the relationship between tumor shrinkage and absorbed dose. Moreover, our data suggest an absorbed dose threshold for partial response in grade 2 NETs. These observations provide valuable insights for the design of dosimetry-guided peptide receptor radionuclide therapy schemes.

Phase II trial demonstrates the efficacy and safety of individualized, dosimetry-based 177Lu-DOTATATE treatment of NET patients.

PURPOSE: Radionuclide therapy with 177Lu-DOTATATE is well established for patients with advanced somatostatin receptor-positive neuroendocrine tumors with a standard schedule of 7.4 GBq at four occasions. However, this approach does not consider individual variability affecting the tumor radiation dose or dose to organs at risk. Therefore, it is important to assess more personalized strategies. The aim of this phase II trial was to evaluate individualized 177Lu-DOTATATE for which the number of cycles varied based on renal dosimetry. METHODS: Patients were eligible if they had a progressive, somatostatin receptor-positive neuroendocrine tumor with a Ki 67 labeling index < 20%. They received cycles of 7.4 GBq of 177Lu-DOTATATE at 10 ± 2-week intervals until a predefined radiation dose to the kidneys was reached. The primary endpoint was objective tumor response (RECIST v 1.1). Secondary endpoints included progression-free survival (PFS), overall survival (OS), and toxicity (CTCAE v. 4.0). RESULTS: Ninety-six patients who had received a median of 5 cycles (range 1-9) were evaluable for efficacy. The objective tumor response was 16% partial response, 66% stable disease, and 19% progressive disease. The median PFS and OS were 29 months and 47 months, respectively, and were significantly associated with kidney dose, performance status, and Ki 67 levels but not with tumor origin. The overall toxicity was mild, and the most common events were grade 1-2 anemia, thrombocytopenia, fatigue, nausea, and diarrhea. Grade 3-4 toxicity occurred in < 10% of patients and was mostly hematological, with no grade 3-4 renal toxicity. CONCLUSION: Individualized treatment with 177Lu-DOTATATE based on renal dosimetry is clearly feasible with low toxicity and promising efficacy, showing the potential to further improve outcome beyond the standard approach, and should be further assessed in randomized trials. TRIAL REGISTRATION: EudraCT 2011-000,240-16. NCT01456078. https://clinicaltrials.gov/ct2/show/NCT01456078.

Selumetinib Plus Adjuvant Radioactive Iodine in Patients With High-Risk Differentiated Thyroid Cancer: A Phase III, Randomized, Placebo-Controlled Trial (ASTRA).

PURPOSE: Selumetinib can increase radioactive iodine (RAI) avidity in RAI-refractory tumors. We investigated whether selumetinib plus adjuvant RAI improves complete remission (CR) rates in patients with differentiated thyroid cancer (DTC) at high risk of primary treatment failure versus RAI alone. METHODS: ASTRA (ClinicalTrials.gov identifier: NCT01843062) is an international, phase III, randomized, placebo-controlled, double-blind trial. Patients with DTC at high risk of primary treatment failure (primary tumor > 4 cm; gross extrathyroidal extension outside the thyroid gland [T4 disease]; or N1a/N1b disease with ≥ 1 metastatic lymph node(s) ≥ 1 cm or ≥ 5 lymph nodes [any size]) were randomly assigned 2:1 to selumetinib 75 mg orally twice daily or placebo for approximately 5 weeks (no stratification). On treatment days 29-31, recombinant human thyroid-stimulating hormone (0.9 mg)-stimulated RAI (131I; 100 mCi/3.7 GBq) was administered, followed by 5 days of selumetinib/placebo. The primary end point (CR rate 18 months after RAI) was assessed in the intention-to-treat population. RESULTS: Four hundred patients were enrolled (August 27, 2013-March 23, 2016) and 233 randomly assigned (selumetinib, n = 155 [67%]; placebo, n = 78 [33%]). No statistically significant difference in CR rate 18 months after RAI was observed (selumetinib n = 62 [40%]; placebo n = 30 [38%]; odds ratio 1.07 [95% CI, 0.61 to 1.87]; P = .8205). Treatment-related grade ≥ 3 adverse events were reported in 25/154 patients (16%) with selumetinib and none with placebo. The most common adverse event with selumetinib was dermatitis acneiform (n = 11 [7%]). No treatment-related deaths were reported. CONCLUSION: Postoperative pathologic risk stratification identified patients with DTC at high risk of primary treatment failure, although the addition of selumetinib to adjuvant RAI failed to improve the CR rate for these patients. Future strategies should focus on tumor genotype-tailored drug selection and maintaining drug dosing to optimize RAI efficacy.

Dosimetric Quantities in Neuroendocrine Tumors over Treatment Cycles with 177Lu-DOTATATE.

Tumor dosimetry was performed for 177Lu-DOTATATE with the aims of better understanding the range and variation of the tumor-absorbed doses (ADs), how different dosimetric quantities evolve over the treatment cycles, and whether this evolution differs depending on the tumor grade. Such information is important for radiobiologic interpretation and may inform the design of alternative administration schemes. Methods: The data came from 41 patients with neuroendocrine tumors (NETs) of grade 1 (n = 23) or 2 (n = 18) who had received between 2 and 9 treatment cycles. Dosimetry was performed for 182 individual lesions, giving a total of 880 individual AD assessments across all cycles. Hybrid planar-SPECT/CT imaging was used, including quantitative SPECT reconstruction, voxel-based absorbed-dose-rate calculation, semiautomatic image segmentation, and partial-volume correction. Linear mixed-effect models were used to analyze changes in tumor ADs over cycles, absorbed-dose rates and activity concentrations on day 1, effective half-times, and tumor volumes. Tumors smaller than 8 cm3 were excluded from analyses. Results: Tumor ADs ranged between 2 and 77 Gy per cycle. On average, the AD decreased over the cycles, with significantly different rates (P < 0.05) of 6% and 14% per cycle for grade 1 and 2 NETs, respectively. The absorbed-dose rates and activity concentrations on day 1 decreased by similar amounts. The effective half-times were less variable but shorter for grade 2 than for grade 1 (P < 0.001). For grade 2 NETs, the tumor volumes decreased, with a similar tendency in grade 1. Conclusion: The tumor AD, absorbed-dose rate, and activity uptake decrease, in parallel with tumor volumes, between 177Lu-DOTATATE treatment cycles, particularly for grade 2 NETs. The effective half-times vary less but are lower for grade 2 than grade 1 NETs. These results may indicate the development of radiation-induced fibrosis and could have implications for the design of future treatment and dosimetry protocols.

Increased risk of Graves´ophthalmopathy in patients with increasing TRAb after radioiodine treatment and the impact of CTLA4 on TRAb titres.

INTRODUCTION: Treatment of Graves´ disease (GD) with radioiodine increases the risk of developing Graves´ ophthalmopathy (GO), and the link between thyroid and orbital tissue may be the presence of TSH-receptors. Radioiodine increases the titers of TRAb and the aim was to investigate the relationship between GO and TRAb titers after treatment with radioiodine and to define the impact of risk genes. METHODS: GD patients without ophthalmopathy or previous treatment with radioiodine were prospectively included at treatment with radioiodine for hyperthyroidism. A follow-up was performed 1 year later for the registration of GO development. The study was performed at a University Hospital Clinic; a referral center of all patients treated with radioiodine in the south of Sweden. The main outcome measures were the development of TRAb, anti-TPO, and anti-TG after 3 months and GO after 12 months and relationship to the genetic background (HLA, CTLA-4, and CYR61). RESULTS: Three months of radioiodine TRAb titers increased in two thirds of patients (p < 0.0005) but not in the other third. Anti-TPO titers were associated with TRAb (R = 0.362, p < 0.0001) but not anti-TG. At follow-up 1 year later (n = 204) 32 patients developed GO with a proportion of 70% in the group increasing in TRAb titers and 30% in the group with unchanged or lower TRAb titers (p-value < 0.0005). Patients with GO had higher titers of TRAb than patients without GO. CTLA-4 (rs231775 SNP) was significantly (p < 0.005) associated with TRAb titers above the median three months after radioiodine. CONCLUSIONS: The increase in TRAb titers after treatment with radioiodine is associated with GO and a genetic variation in CTLA-4 is associated with higher titers of TRAb.

Pituitary Function after High-Dose 177Lu-DOTATATE Therapy and Long-Term Follow-Up.

INTRODUCTION: The pituitary gland has a high expression of somatostatin receptors and is therefore a potential organ at risk for radiation-induced toxicity after 177Lu-DOTATATE treatment. OBJECTIVE: To study changes in pituitary function in patients with neuroendocrine tumors (NETs) treated with dosimetry-based 177Lu-DOTATATE to detect possible late toxicity. METHODS: 68 patients from a phase II clinical trial of dosimetry-based, individualized 177Lu-DOTATATE therapy were included in this analysis. Patients had received a median of 5 (range 3-9) treatment cycles of 7.4 GBq/cycle. Median follow-up was 30 months (range 11-89). The GH/IGF-1 axis, gonadotropins, and adrenal and thyroid axes were analyzed at baseline and on a yearly basis thereafter. Percent changes in hormonal levels over time were analyzed statistically using a linear mixed model and described graphically using box plots. The absorbed radiation dose to the pituitary was estimated based on post-therapeutic imaging, and the results analyzed versus percent change in IGF-1 levels over time. RESULTS: A statistically significant decrease in IGF-1 levels was found (p < 0.005), which correlated with the number of treatment cycles (p = 0.008) and the absorbed radiation dose (p = 0.03). A similar decrease, although non-significant, was seen in gonadotropins in postmenopausal women, while in men there was an increase during the first years after therapy, after which the levels returned to baseline. No change was observed in the adrenal or thyroid axes. CONCLUSIONS: No signs of severe endocrine disorders were detected, although a significant decrease in the GH/IGF-1 axis was found, where dosimetric analyses indicated radiation-induced damage to the pituitary gland as a probable cause.

Substantial intrinsic variability in chemoradiosensitivity of newly established anaplastic thyroid cancer cell-lines.

Background: Well characterized human cell lines are needed for preclinical treatment studies of anaplastic thyroid cancer (ATC).Aims/Objectives: The aim was to establish, verify and characterize a panel of ATC cell lines.Material and methods: Cell lines were established from ATC fine-needle aspiration biopsies and characterized genetically and functionally regarding treatment sensitivities.Results: Eight cell lines were established in vitro and the anaplastic thyroid origin was verified. Seven of the cell lines were also grown as xenografts. The cell lines harboured complex karyotypes with modal numbers in hyperdiploid to near-pentaploid range. Five were TP53 mutated and three carried the BRAFV600E mutation. None had rearrangements of RET. For doxorubicin, IC50 ranged from 0.42 to 46 nmol/L and for paclitaxel from 1.6 to 196 nmol/L. Radiation sensitivity varied between 2.6 and 6.3 Gy. Two of the BRAF mutated cell lines displayed high sensitivity to vemurafenib, while the third was similar to the wild-type ones.Conclusions and significance: We describe a series of new ATC cell lines demonstrating large heterogeneity in the response to cytostatic drugs and the BRAF inhibitor vemurafenib. The observations are relevant to future attempts to optimize treatment combinations for ATC.

The Effect of Radioiodine Treatment on TRAb, Anti-TPO, and Anti-TG in Graves' Disease.

BACKGROUND: In Graves' disease (GD), immunocompetent cells infiltrate thyroid tissue with release of TSH-receptor antibodies (TRAb), and radioiodine treatment is known to elicit an immune response with an increase in TRAb. OBJECTIVES: The aim was to study if all patients treated with radioiodine respond with a release of TRAb, anti-thyroperoxidase (anti-TPO), and anti-thyroglobulin (anti-TG). METHODS: This is a prospective observational study. GD patients (n = 131) were admitted for treatment with radioiodine. Thyroid antibodies were measured before and 3 months after iodine-131 treatment. RESULTS: After 3 months, a fold change > 1.1 was found in 66% of the GD patients, while the remaining 34% did not have a change or decrease in in TRAb. Anti-TPO and anti-TG also increased; the former showed an increase in 73% and the latter of 52%, while 27 and 48% decreased/were unchanged. A significant positive correlation was found between TRAb and anti-TPO, but not between TRAb and anti-TG. In the group with an increase in TRAb, the median fold change was 5.1, but there were no additional effects of tobacco smoking. The proportion of females below the median age (51.5 years) was significantly higher in the group that increased in TRAb compared to the one that decreased/was unchanged (66 vs. 34%). CONCLUSIONS: Treatment with radioiodine elicits an increase in thyroid antibodies, but not in all GD patients. The proportion of responders varied and was affected by age, resulting in a stronger immune response at younger age. However, there were no additional effects of smoking.

Feasibility of simplifying renal dosimetry in 177Lu peptide receptor radionuclide therapy.

BACKGROUND: Recently, 177Lu-dotatate therapy for neuroendocrine tumours has received regulatory approval. Dosimetry can be used to optimize treatment on an individual basis, but there is no international consensus as to how it should be done. The aim of this study is to determine a feasible and accurate dosimetry method to guide individualized peptide receptor radionuclide therapy (PRRT) for patients with neuroendocrine tumours. As part of a clinical trial on 177Lu-dotatate therapy, renal dosimetry was performed for all patients in each treatment cycle, using a hybrid planar-SPECT/CT method. In the present study, we use the image data acquired from 22 patients and 119 cycles and define a set of alternative treatment planning strategies, each representing a simplification in terms of image acquisition and dosimetric calculations. The results from the simplified strategies are compared to the results from the protocol-prescribed hybrid planar-SPECT/CT-based method by analysing differences both in per-cycle and total cumulative absorbed dose (AD) analyses. RESULTS: In general, the SPECT-based methods gave results that were largely consistent with the protocol-specified hybrid method, both in the per-cycle and cumulative AD analyses. Notably, performing one SPECT/CT per cycle at 96 h yielded ADs that were very similar to the protocol method. The methods using planar dosimetry resulted in larger variations, as expected, while giving 4 cycles to all patients resulted in the largest inter-individual differences in cumulative AD. CONCLUSIONS: Performing one SPECT/CT at 96 h in every treatment cycle gives sufficiently reliable dosimetric results to base individualized treatment planning on, with a reasonable demand on resources.

Individualised 177Lu-DOTATATE treatment of neuroendocrine tumours based on kidney dosimetry.

PURPOSE: To present data from an interim analysis of a Phase II trial designed to determine the feasibility, safety, and efficacy of individualising treatment based on renal dosimetry, by giving as many cycles as possible within a maximum renal biologically effective dose (BED). METHOD: Treatment was given with repeated cycles of 7.4 GBq 177Lu-DOTATATE at 8-12-week intervals. Detailed dosimetry was performed in all patients after each cycle using a hybrid method (SPECT + planar imaging). All patients received treatment up to a renal BED of 27 ± 2 Gy (α/ß = 2.6 Gy) (Step 1). Selected patients were offered further treatment up to a renal BED of 40 ± 2 Gy (Step 2). Renal function was followed by estimation and measurement of the glomerular filtration rate (GFR). RESULTS: Fifty-one patients were included in the present analysis. Among the patients who received treatment as planned, the median number of cycles in Step 1 was 5 (range 3-7), and for those who completed Step 2 it was 7 (range 5-8); 73% were able to receive >4 cycles. Although GFR decreased in most patients after the completion of treatment, no grade 3-4 toxicity was observed. Patients with a reduced baseline GFR seemed to have an increased risk of GFR decline. Five patients received treatment in Step 2, none of whom exhibited a significant reduction in renal function. CONCLUSIONS: Individualising PRRT using renal dosimetry seems feasible and safe and leads to an increased number of cycles in the majority of patients. The trial will continue as planned.

Evaluation of immune cell markers in tumor tissue treated with radioimmunotherapy in an immunocompetent rat colon carcinoma model.

BACKGROUND: Immune cells within the tumor can act either to promote growth or rejection of tumor cells. The aim of the present study was to evaluate immune cell markers (number and localization) within the tumor before and during rejection due to radioimmunotherapy, to determine whether there is a change in markers related to rejection and/or tolerance of the tumor cells. METHODS: Thirty immunocompetent rats were inoculated with syngeneic rat colon carcinoma cells and 13-14 days later 21 of these rats were treated with 400 MBq/kg of (177)Lu-DOTA-BR96 monoclonal antibodies. The treated animals were sacrificed and dissected 1, 2, 3, 4, 6, and 8 days post-injection in groups of three animals per day (6 animals on day 8); while the nine untreated animals were sacrificed and dissected on day 0. Paraffin sections were used for immunohistochemical staining of CD2, CD3, CD8α, CD68, and CD163 antigens. Positive cells were counted within: vital tumor cell areas, necrotic areas, granulation tissue surrounding and between the tumor cell areas. The change in the number of positive cells over time in tumors treated with radioimmunotherapy in the same location was evaluated with linear regression models. The number of positive cells in various locations and the number of various antigen-positive cells within the same location were also evaluated over time using box plots. RESULTS: There were a higher number of cells expressing immune cell markers in granulation tissue compared with vital tumor cell areas. Cells expressing markers decreased during radioimmunotherapy, and T-cell markers decreased more than macrophage markers in tumors treated with radioimmunotherapy. The expression of CD8α was higher than that of the other T-cell markers evaluated (CD3 and CD2), which could be explained by the additional expression of CD8α by natural killer (NK) cells and a subset of dendritic cells (DCs). The expression of CD68 (all macrophages, DCs, and neutrophils) tended to be higher than that of CD163 (pro-tumor macrophages). CONCLUSIONS: In this model, we demonstrated a higher number of positive cells for immune cell markers related to augmenting the immune rejection than immune tolerance of tumor cells in tumors and a decrease in markers during radioimmunotherapy.

Long-Term Retention of 177Lu/177mLu-DOTATATE in Patients Investigated by γ-Spectrometry and γ-Camera Imaging.

UNLABELLED: Dosimetry in peptide receptor radionuclide therapy using (177)Lu-DOTATATE is based on patient imaging during the first week after administration and determination of the activity retention as a function of time for different tissues. For calculation of the absorbed dose, it is generally assumed that the long-term activity retention follows the pattern determined from the first week. This work aimed to investigate the validity of this assumption by performing additional patient measurements between 5 and 10 wk after administration. A further aim was to investigate to what extent absorbed dose values were affected when including these measurements, also taking into account the radionuclide impurity of (177m)Lu and build-up of secondary (177)Lu from the (177m)Lu decay. METHODS: A combination of methods was used: planar γ-camera imaging as part of the clinical dosimetry protocol, determination of the whole-body activity between 5 and 9 wk after injection using spectrometric NaI(Tl) and HPGe detectors, and imaging between 5 and 10 wk after injection for assessment of the activity distribution. From these measurements the long-term retention of activity was determined and the relative influence on absorbed doses calculated. RESULTS: The most important finding was a clearly visualized tumor uptake in images from between 5 and 7 wk after injection and in 1 patient also kidney and spleen uptake in images acquired on day 33. As a consequence, the total-body time-activity curve had a tail, which was not completely captured by imaging during the first week. The absorbed doses to total body and tumors obtained when including these late time points were on average 5%-6% higher than those obtained when using data acquired during the first week. The contributions to the absorbed dose from (177m)Lu and secondary (177)Lu were negligible. CONCLUSION: At approximately 5-7 wk after injection, there was a measureable amount of (177)Lu-DOTATATE in patients, which is mainly governed by retention in tumors. For tumor dosimetry, imaging at a later time than the routinely used 7 d may be warranted. The contribution to the absorbed dose from the radionuclide impurity of (177m)Lu was negligible.

Role of CD8-positive cells in radioimmunotherapy utilizing (177)Lu-mAbs in an immunocompetent rat colon carcinoma model.

BACKGROUND: CD8-positive cells might play a crucial role in the therapeutic response to radiation, which has however not been investigated in radioimmunotherapy (RIT). The aim of this study was to evaluate whether cytotoxic T cells affect the response of established tumors and, above all, if they delay or prevent the development of distant metastases after RIT, using an immunocompetent syngeneic rat colon carcinoma model. METHODS: The cytotoxic T cells were depleted in 15 rats by anti-CD8 before the injection of the radioimmunoconjugate (400 MBq/kg body weight (177)Lu-BR96, which binds to the tumor-associated antigen Lewis Y). Fifteen other rats were treated with RIT only. Both groups were followed for 99 days. Blood samples were collected at least once weekly, and tumors were monitored twice weekly. RESULTS: Twenty-nine of the 30 animals exhibited local complete response. The non-responder was treated with anti-CD8 and RIT but succumbed later due to metastases. Five animals in the group given anti-CD8 + RIT were sacrificed due to metastatic disease, and 4 additional animals were found to have metastases at autopsy. In the group given RIT, 4 animals developed metastatic disease, but no metastases were found in the remaining 11 animals at autopsy. Thus, at the end of the study, 6 animals in the anti-CD8 + RIT group were free from metastases, while 11 were free from metastases in the group receiving RIT. CD3(+)CD4(-)CD8(+) lymphocytes were consistently depleted by the anti-CD8 treatment. The myelosuppression was otherwise similar in the two groups. The initial depletion of CD8-positive cells in our syngeneic rat colon carcinoma model resulted in a higher frequency of animals developing metastases. CONCLUSIONS: Depletion of CD8-positive cells during RIT in an immunocompetent rat tumor model might influence the number of animals developing metastases, indicating that the immune system may be important in the long-term outcome of RIT.

Sequential radioimmunotherapy with 177Lu- and 211At-labeled monoclonal antibody BR96 in a syngeneic rat colon carcinoma model.

UNLABELLED: Alpha-particle emitters, such as astatine-211 (211At), are generally considered suitable for the treatment of small cell clusters due to their short path length, while beta-particle emitters, for example, Lutetium-177 (177Lu), have a longer path length and are considered better for small, established tumors. A combination of such radionuclides may be successful in regimens of radioimmunotherapy. In this study, rats were treated by sequential administration of first a 177Lu-labeled antibody, followed by a 211At-labeled antibody 25 days later. METHODS: Rats bearing solid colon carcinoma tumors were treated with 400 MBq/kg body weight 177Lu-BR96. After 25 days, three groups of animals were given either 5 or 10 MBq/kg body weight of 211At-BR96 simultaneously with or without a blocking agent reducing halogen uptake in normal tissues. Control animals were not given any 211At-BR96. Myelotoxicity, body weight, tumor size, and development of metastases were monitored for 120 days. RESULTS: Tumors were undetectable in 90% of the animals on day 25, independent of treatment. Additional treatment with 211At-labeled antibodies did not reduce the proportion of animals developing metastases. The rats suffered from reversible myelotoxicity after treatment. CONCLUSIONS: Sequential administration of 177Lu-BR96 and 211At-BR96 resulted in tolerable toxicity providing halogen blocking but did not enhance the therapeutic effect.

Change in cell death markers during (177)Lu-mAb radioimmunotherapy-induced rejection of syngeneic rat colon carcinoma.

PURPOSE: To monitor cell death in tumors during the rejection process after treatment with an antibody radiolabeled with a ß-emitter. METHODS: Tumors during rejection after treatment with (177)Lu-labeled antibody BR96 and after administration of unlabeled BR96 were compared with untreated tumors from the same immunocompetent syngeneic rat tumor model. Cell death was monitored with the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and immunohistochemical staining of activated caspase-3 and γH2AX. These data were evaluated together with histopathological morphology, BR96-binding antigen expression, and (177)Lu radioactivity distribution imaged by digital autoradiography. RESULTS: The untreated tumors showed staining for all the markers, mainly in and around the necrotic areas. One to 2 days p.i. large areas were stained with anti-γH2AX, followed by a slight decrease. Staining of activated caspase-3 was intense and extensive 1-2 days p.i., while found in and around necrotic areas 3-8 days p.i. TUNEL staining was similar to activated caspase-3 staining 1-2 days p.i. but more extensive than activated caspase-3 staining 3-4 days p.i. Digital autoradiography revealed activity concentration in granulation tissue from 1 day p.i. CONCLUSION: Following radioimmunotherapy in an immunocompetent syngeneic colon carcinoma model, tumor cells did not only die through caspase-3-dependent apoptosis, but also by other mechanisms.

The intratumoral distribution of radiolabeled 177Lu-BR96 monoclonal antibodies changes in relation to tumor histology over time in a syngeneic rat colon carcinoma model.

UNLABELLED: The therapeutic effect of radioimmunotherapy depends on the distribution of the absorbed dose in relation to viable cancer cells within the tumor, which in turn is a function of the activity distribution. The aim of this study was to investigate the distribution of (177)Lu-DOTA-BR96 monoclonal antibodies targeting the Lewis Y antigen over 7 d using a syngeneic rat model of colon carcinoma. METHODS: Thirty-eight tumor-bearing rats were intravenously given 25 or 50 MBq of (177)Lu-DOTA-BR96 per kilogram of body weight and were sacrificed 2, 8, 24, 48, 72, 96, 120, or 168 h after injection, with activity measured in blood and tumor samples. Adjacent cryosections of each tumor were analyzed in 3 ways: imaging using a silicon-strip detector for digital autoradiography, staining for histologic characterization, or staining to determine the distribution of the antigen, vasculature, and proliferating cells using immunohistochemistry. Absorbed-dose rate distribution images at the moment of sacrifice were calculated using the activity distribution and a point-dose kernel. The correlations between antigen expression and both activity uptake and absorbed-dose rate were calculated for several regions of interest in each tumor. Nine additional animals with tumors were given unlabeled antibody to evaluate possible immunologic effects. RESULTS: At 2-8 h after injection, activity was found in the tumor margins; at 24 h, in viable antigen-expressing areas within the tumor; and at 48 h and later, increasingly in antigen-negative areas of granulation tissue. The correlation between antigen expression and both the mean activity and the absorbed-dose rate in regions of interest changed from positive to negative after 24 h after injection. Antigen-negative areas also increased over time in animals injected with unlabeled BR96, compared with untreated tumors. CONCLUSION: The results indicate that viable Lewis Y-expressing tumor cells are most efficiently treated during the initial uptake period. The activity then seems to remain in these initial uptake regions after the elimination of tumor cells and formation of granulation tissue. Further studies using these techniques could aid in determining the effects of the intratumoral activity distribution on overall therapeutic efficacy.

Successful radioimmunotherapy of established syngeneic rat colon carcinoma with 211At-mAb.

BACKGROUND: Most carcinomas are prone to metastasize despite successful treatment of the primary tumor. One way to address this clinical challenge may be targeted therapy with α-emitting radionuclides such as astatine-211 (211At). Radioimmunotherapy utilizing α-particle emitting radionuclides is considered especially suitable for the treatment of small cell clusters and single cells, although lesions of different sizes may also be present in the patient. The aim of this study was primarily to evaluate the toxicity and secondarily in vivo efficacy of a 211At-labeled monoclonal antibody (mAb) directed against colon carcinoma with tumor diameters of approximately 10 mm. METHODS: Eighteen rats with subperitoneal syngeneic colon carcinoma were allocated to three groups of six animals together with three healthy rats in each group. The groups were injected intravenously with either 150 µg of unlabeled mAbs (controls) or 2.5 or 5 MBq 211At-mAbs directed towards the Lewis Y antigen expressed on the cell membrane of several carcinomas. Tumor volume, body weight, and blood cell counts were monitored for 100 days after treatment. RESULTS: Local tumors were non-palpable in five out of six rats after treatment with both activities of 211At-mAbs, compared to one out of six in the control group. At the study end, half of the animals in each group given 211At-BR96 and one animal in the control group were free from disease. Radioimmunotherapy resulted in dose-dependent, transient weight loss and myelotoxicity. Survival was significantly better in the groups receiving targeted alpha therapy than in those receiving unlabeled mAbs. CONCLUSIONS: This study demonstrates the possibility of treating small, solid colon carcinoma tumors with α-emitting radionuclides such as 211At bound to mAbs, with tolerable toxicity.

Two-year survival follow-up of the randomized, double-blind, placebo-controlled phase II study of radium-223 chloride in patients with castration-resistant prostate cancer and bone metastases.

BACKGROUND: This phase II randomized, placebo-controlled study was conducted to evaluate efficacy and safety of radium-223 in patients with castration-resistant prostate cancer (CRPC) and painful bone metastases. Twelve- and 18-month survival results were reported previously. Here we report 24-month overall survival (OS) and safety data from the period 12 to 24 months after the first injection of study medication. METHODS: Patients with CRPC and bone pain were randomized 1:1 to receive 4 injections of radium-223 (50 kBq/kg [n = 33]) or placebo (n = 31) after external-beam radiotherapy; each injection was given every 4 weeks. Endpoints for this report were 24-month OS, long-term safety, and treatment-related adverse events (AEs) occurring in the 12- to 24-month period. RESULTS: After 24 months, 10 (30%) patients were alive in the radium-223 group compared with 4 patients (13%) in the placebo group. Patients who received at least 1 dose of study medication had a median OS of 65 weeks in the radium-223 group vs. 46 weeks in the placebo group (log-rank P = .056). The hazard ratio (HR) for OS, adjusted for baseline covariates, was 0.476 (95% confidence interval [CI], 0.258-0.877; Cox regression P = .017). The most frequent cause of death for both arms was disease progression. There were no reports of treatment-related AEs or long-term hematologic toxicity during the 12- to 24-month follow-up. CONCLUSION: Radium-223 had a highly favorable safety profile, with no evidence of second malignancies at 24-month follow-up. The significant improvement in OS observed in patients receiving radium-223 vs. placebo suggests that treatment of bone disease with radium-223 has survival benefits.

Pattern of antigen expression in metastases after radioimmunotherapy of a syngeneic rat colon carcinoma utilizing the BR96 antibody.

BACKGROUND: Repeated administration of antibody-based therapies such as radioimmunotherapy depends on preserved antigen expression in tumor lesions. The purpose of this study was to evaluate whether the antigen expression in metastases observed after radioimmunotherapy differs from that of untreated primary tumors. FINDINGS: 30 of the 35 Brown Norway rats with syngeneic colon carcinoma treated with 400 MBq/kg 177Lu-DOTA-BR96 exhibited consistent complete response of the primary tumor. 13 animals developed metastases that were detected after treatment. The antigen expression was reduced in 17 of 23 metastases detected after radioimmunotherapy compared with untreated tumors. No tumors completely lacked positively stained tumor cells. CONCLUSIONS: Although it was not possible to demonstrate that the antigen reduction is triggered by the radioimmunotherapy this result stress the importance of considering the risk of reduced antigen expression in metastases after radioimmunotherapy prior to further targeted therapies.