Preclinical Evaluation of Podoplanin-Targeted Alpha-Radioimmunotherapy with the Novel Antibody NZ-16 for Malignant Mesothelioma

The prognosis of advanced mesothelioma is poor. Podoplanin (PDPN) is highly expressed in most malignant mesothelioma. This study aimed to evaluate the potential alpha-radioimmunotherapy (RIT) with a newly developed anti-PDPN antibody, NZ-16, compared with a previous antibody, NZ-12. Methods: The in vitro properties of radiolabeled antibodies were evaluated by cell binding and competitive inhibition assays using PDPN-expressing H226 mesothelioma cells. The biodistribution of 111In-labeled antibodies was studied in tumor-bearing mice. The absorbed doses were estimated based on biodistribution data. Tumor volumes and body weights of mice treated with 90Y- and 225Ac-labeled NZ-16 were measured for 56 days. Histologic analysis was conducted. Results: The radiolabeled NZ-16 specifically bound to H226 cells with higher affinity than NZ-12. The biodistribution studies showed higher tumor uptake of radiolabeled NZ-16 compared with NZ-12, providing higher absorbed doses to tumors. RIT with 225Ac- and 90Y-labeled NZ-16 had a significantly higher antitumor effect than RIT with 90Y-labeled NZ-12. 225Ac-labeled NZ-16 induced a larger amount of necrotic change and showed a tendency to suppress tumor volumes and prolonged survival than 90Y-labeled NZ-16. There is no obvious adverse effect. Conclusions: Alpha-RIT with the newly developed NZ-16 is a promising therapeutic option for malignant mesothelioma.

Does primary lesion have to be regarded as a target lesion when evaluating response in metastatic renal cell carcinoma (mRCC) treated with sunitinib?

425 Background: Primary lesion in mRCC often shows necrotic change or only density changes to targeted therapy. As most clinical trials for mRCC have enrolled nephrectomized patients (pts), there is no consensus on including primary lesion in target lesion when evaluating response. We investigated whether best overall response (OR) changes when designate target lesions with or without primary lesion, and such discordance lead to difference in predictive value in terms of time to progression (TTP) and overall survival (OS). Methods: We enrolled mRCC pts with intact primary lesion, at least one measurable metastatic lesion, and proper image data who had received sunitinib in our institution between 2003 and 2011. We documented the variation of the sum of the largest diameters (ΔSLD) with all target lesions and with metastasis-only target lesions separately. Response evaluation was calculated with RECIST v1.1. Results: Of 41 pts, 38 received sunitinib as 1st line treatment and 3 did as 2nd line. The most frequent metastatic site was lung (63.4%) followed by lymph node (46.3%) and bone (42.5%). Median ΔSLD of primary lesion and metastatic target lesion were -6.02% (range: -34.00 to 17.60%), and -18.03% (-100.00 to 120.00%). On response evaluation with metastasis-only target lesions, best OR of 2 pts (4.87%) changed from stable disease to partial response. When categorize pts into responders and non-responders, metastasis-only target lesions resulted in significantly better discrimination of TTP (14.949 vs 4.271 mo, p=0.01) and OS (18.497 vs 9.561 mo, p=0.036) between two groups. Using all target lesions, both TTP (14.949 vs 5.355 mo, p=0.056) and OS (17.971 vs 10.579 mo, p=0.155) were statistically insignificant. Conclusions: Our study showed discordance in OR between using all target lesions and using metastasis-only target lesions, and metastasis-only target lesion could have more accurate predictive value. As response rate is often used as an end point in clinical research of mRCC, further discussion is needed whether primary lesion should be included or not in target lesion. [Table: see text]

Apoptosis precedes necrosis of dystrophin-deficient muscle

The current view that death of dystrophin-deficient muscle fibers is a necrotic process relies primarily upon the histological appearance of the tissue after the degenerative process is well advanced. Here, we tested this view by examining the possibility that apoptosis is a component of dystrophin-deficient muscle cell death. Three assays for apoptosis were employed in analyzing prenecrotic, peak necrotic and regenerated hindlimb muscle of mdx mice: (1) terminal deoxynucleotidyl transferase (TdT) mediated end-labeling of DNA in nuclei in tissue sections; (2) assays for DNA ladders; and (3) electron microscopic assays for the presence of organelles undergoing structural changes characteristic of apoptosis. At all ages sampled, mdx muscle contained apoptotic nuclei, according to TdT-mediated dUTP labeling of tissue sections. Nuclei in regenerated mdx muscle fibers did not display apoptosis. dUTP-labeled nuclei in control C57 muscles were rare or absent at all ages sampled. DNA from 4-week-old mdx mice was found to be cleaved into fragments indicative of preferential cleavage at internucleosomal sites. Electron microscopic analysis showed that organelle structural changes indicating apoptosis appear before pathological changes diagnostic of necrosis. For example, condensed mitochondria, fragmented sarcoplasmic reticulum and nuclei with chromatin condensations resembling apoptosis appear in fibers that otherwise possess normal morphology. Together, the findings show that apoptosis precedes any detectable necrotic change in mdx muscle, and that apoptotic events continue into the stage of dystrophic pathology that is currently viewed as necrosis. Thus, apoptosis characterizes the onset of pathology in dystrophin-deficient muscle which is followed secondarily by necrotic processes.

A fluid percussion model of experimental brain injury in the rat

✓ Fluid percussion models produce brain injury by rapidly injecting fluid volumes into the cranial cavity. The authors have systematically examined the effects of varying magnitudes of fluid percussion injury in the rat on neurological, systemic physiological, and histopathological changes. Acute neurological experiments showed that fluid percussion injury in 53 rats produced either irreversible apnea and death or transient apnea (lasting 54 seconds or less) and reversible suppression of postural and nonpostural function (lasting 60 minutes or less). As the magnitude if injury increased, the mortality rate and the duration of suppression of somatomotor reflexes increased. Unlike other rat models in which concussive brain injury is produced by impact, convulsions were observed in only 13% of survivors. Transient apnea was probably not associated with a significant hypoxic insult to animals that survived. Ten rats that sustained a moderate magnitude of injury (2.9 atm) exhibited chronic locomotor deficits that persisted for 4 to 8 days. Systemic physiological experiments in 20 rats demonstrated that all levels of injury studied produced acute systemic hypertension, bradycardia, and increased plasma glucose levels. Hypertension with subsequent hypotension resulted from higher magnitudes of injury. The durations of hypertension and suppression of amplitude on electroencephalography were related to the magnitudes of injury. While low levels of injury produced no significant histopathological alterations, higher magnitudes produced subarachnoid and intraparenchymal hemorrhage and, with increasing survival, necrotic change and cavitation. These data demonstrate that fluid percussion injury in the rat reproduces many of the features of head injury observed in other models and species. Thus, this animal model could represent a useful experimental approach to studies of pathological changes similar to those seen in human head injury.

STUDIES OF ACUTE PHASE PROTEIN

A method is presented for the immunohistochemical localization of Cx-reactive protein in rabbits, based on the use of a defined antiserum and rigorous fixation techniques requisite for this antigen. In animals in which inflammatory lesions and CxRP response were induced by intramuscular injection of typhoid vaccine, Cx-reactive protein was localized only in the area of local inflammation within muscle fibers showing morphologic evidence of necrotic change. Within such altered fibers, CxRP was observed in peripheral segments of myofiber or in subsarcolemmal sarcoplasm, in scattered deposits in sarcoplasm, and in vacuolar inclusions. No CxRP was found at any time in polymorphonuclear or mononuclear cells in the inflammatory lesion, nor in contralateral muscle, regional or distal lymph nodes, liver, spleen, thymus, heart, or kidney, except as traces in lumens of vessels or interstitium. CxRP was first detected in necrotic myofibers at the inflammatory site after a latent period of 8 to 10 hours following injection of the inflammatory stimulus and could be demonstrated in these sites for the 48 hours of the experiment. It could not be observed at the inflammatory site before appearance in the blood. Identical histologic localization in necrotic myofibers at the site of the local lesions was found following induction of granulocytopenia with nitrogen mustard. These findings are consistent with the hypothesis that CxRP is formed locally at the site of inflammation from tissue elements undergoing necrotic change. Alternatively, secondary deposition from the blood at the inflammatory site cannot be excluded, but is considered less likely in view of the failure to obtain evidence of a cellular localization of CxRP in other organs.