Comparison of anti-CD20 and anti-CD45 antibodies for conventional and pretargeted radioimmunotherapy of B-cell lymphomas

Radiolabeled anti-CD20 antibodies produce responses in 60% to 95% of patients with relapsed non-Hodgkin lymphoma (NHL); however, absorbed radiation ratios between tumors and normal organs are relatively low, and many patients have relapses. In this study we compared the abilities of anti-CD45 (BC8) and anti-CD20 (1F5) antibodies to target human Ramos lymphoma xenografts in athymic mice. When direct radioiodination was performed with conventional methods, BC8 delivered 2- to 4-fold more radioiodine to tumors than 1F5, with tumor-to-normal organ ratios as high as 20:1 using radiolabeled BC8 compared with a maximal ratio of 9.8:1 using radioiodinated 1F5. To optimize the biodistribution of radioactivity, we performed studies following a pretargeting method using streptavidin (SA)-conjugated BC8 and 1F5. Injection of a synthetic clearing agent decreased the circulating level of conjugates by 80% to 90% within 1 hour. Pretargeting with BC8-SA resulted in a 2- to 4-fold greater tumor uptake of radiolabeled biotin than with 1F5-SA, with maximal tumor-to-normal organ ratios of more than 80:1 and approximately 16:1, respectively. Therapy experiments demonstrated that 400 microCi (14.8 MBq) or more of yttrium-90-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-biotin cured 100% of mice treated with BC8-SA and more than 90% of mice pretargeted with 1F5-SA, with complete remission occurring 8 to 10 days sooner in mice receiving BC8-SA. After treatment with 200 microCi (7.4 MBq) (90)Y-DOTA-biotin, 70% of the mice treated with BC8-SA were cured, but no mice were cured using 1F5-SA. Doses up to 800 microCi (29.6 MBq) (90)Y-DOTA-biotin were delivered with minor toxicity using either antibody conjugate. These lymphoma xenograft data suggest that pretargeted radioimmunotherapy using either anti-CD20 or anti-CD45 conjugates is highly effective and minimally toxic.     

BRAF mutation testing with a rapid,fully integrated molecular diagnostics system

Fast and accurate diagnostic systems are needed for further implementation of precision therapy of BRAF-mutant and other cancers. The novel Idylla^TM BRAF Mutation Test has high sensitivity and shorter turnaround times compared to other methods. We used Idylla to detect BRAF V600 mutations in archived formalin-fixed paraffin-embedded (FFPE) tumor samples and compared these results with those obtained using the cobas 4800 BRAF V600 Mutation Test or MiSeq deep sequencing system and with those obtained by a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory employing polymerase chain reaction–based sequencing, mass spectrometric detection, or next-generation sequencing. In one set of 60 FFPE tumor samples (15 with BRAF mutations per Idylla), the Idylla and cobas results had an agreement of 97%. Idylla detected BRAF V600 mutations in two additional samples. The Idylla and MiSeq results had 100% concordance. In a separate set of 100 FFPE tumor samples (64 with BRAF mutation per Idylla), the Idylla and CLIA-certified laboratory results demonstrated an agreement of 96% even though the tests were not performed simultaneously and different FFPE blocks had to be used for 9 cases. The Idylla^TM BRAF Mutation Test produced results quickly (sample to results time was about 90 minutes with about 2 minutes of hands on time) and the closed nature of the cartridge eliminates the risk of PCR contamination. In conclusion, our observations demonstrate that the Idylla test is rapid and has high concordance with other routinely used but more complex BRAF mutation–detecting tests.     

Studies on aorta during development. II. Differences in ontogeny of the key enzymes involved in cholesteryl ester synthesis and hydrolysis in rabbit aorta

It is well known that cholesteryl ester accumulation is dramatically increased in the atherosclerotic artery. The enzymes acyl-CoA: cholesterol acyltransferase (ACAT), acid cholesteryl esterase (ACE) and neutral cholesteryl esterase (NCE) may play key roles in the accumulation of cholesteryl esters in the arterial wall. However, very little is known regarding the developmental pattern of the key enzymes involved in cholesteryl ester synthesis and hydrolysis. The total activities of ACAT, ACE and NCE were measured by radioassay using liposomal substrates in rabbit aortic homogenates. Our results indicate that ACAT activity decreases as a quadratic function with age (P less than 0.05). ACAT activity (pmol/100 mg protein/min) decreased from a high value in the fetus at term (63.3 +/- 7.4) to gradually lower values with increasing age. On the other hand, ACE activity (pmol/mg protein/min) was low in the fetus at term, and changed as a quadratic function with age (P less than 0.05) increasing gradually to higher activities with age up to a maximum at 12 weeks then decreased at 21 weeks. NCE activity (pmol/mg protein/min) increased dramatically from a low value in the fetus at term (3.34 +/- 0.48) to a maximum value at 1.5 weeks (14.65 +/- 2.73) then decreased as a linear function with increasing age up to 21 weeks (P less than 0.05). Plasma total cholesterol (mg/dl) also increased sharply from the fetal value at term of 98.5 +/- 5.2 to a maximum value at 1.5 weeks of 666.4 +/- 33.4, then decreased as a quadratic function with increasing age up to 21 weeks (40.8 +/- 6.7) (P less than 0.05). The free cholesterol content (microgram/mg protein) of the aortic tissue was initially high in the fetus (24.8 +/- 5.9) then increased with age. Examination of the ratio of synthesis to hydrolysis of cholesteryl esters as an index of enzyme activity units demonstrated a very high index in the fetus of 6.1 that rapidly decreased with increasing age in the young adult rabbit down to a value of 0.4 by 21 weeks of age. Correlation coefficients between enzyme activities, plasma cholesterol levels and aortic cholesterol levels indicated (a) a positive correlation of NCE activity with plasma cholesterol, (b) a negative correlation of NCE and ACE with aortic-cholesteryl ester content, and (c) no significant correlation of ACAT activity with either plasma cholesterol or aortic cholesterol content, indicating other factors are involved.(ABSTRACT TRUNCATED AT 400 WORDS)     

Factors related to the recurrence of thyroid nodules after surgery for benign radiation-related nodules.

OBJECTIVE: Benign thyroid nodules are increased by radiation exposure and recurrences are common. The goal of this study was to determine the factors related to recurrence of nodular disease and the efficacy of thyroid hormone therapy in reducing them. DESIGN, SETTING, AND SUBJECTS: The study population was drawn from a cohort of 4296 people treated before the age of 16 with conventional external radiation for benign conditions of the head and neck between 1939 and the early 1960s. The study group consisted of 632 subjects who had benign nodules removed surgically. At the discretion of their physicians, 426 subjects were treated with thyroid hormone after surgery, 198 were not, and in 8 subjects, thyroid hormone treatment status was unknown. MAIN OUTCOMES: There were 129 (20.4%) subjects who developed new nodules during follow-up. Women had a greater risk of recurrent nodules than men (27.5% vs. 13.5%) and the rate of recurrence correlated inversely with the extent of surgery. The group who took thyroid hormone had a lower recurrence rate than the untreated group (14.2% vs. 34.2%). The risk of recurrence was reduced to 0.69 (0.47-1.01) in thyroid hormone-treated subjects, regardless of extent of surgery, and to 0.66 (0.46-0.97) when only the subjects with less than 75% of their thyroid removed were considered. CONCLUSION: Thyroid hormone therapy reduced recurrences in this irradiated cohort. It should be recommended to all patients with irradiated thyroids and previous thyroid surgery.     

Comparison of immunoscintigraphy, efficacy, and toxicity of conventional and pretargeted radioimmunotherapy in CD20-expressing human lymphoma xenografts.

Pretargeted radioimmunotherapy (RIT) using streptavidin (sAv)-conjugated antibodies before radiolabeled-biotin is a promising approach to improve absorbed dose ratios and achieve high durable remission rates with diminished systemic toxicity. This study compared the immunoscintigraphy, toxicity, and therapeutic efficacy of pretargeted RIT with conventional RIT using an anti-CD20 antibody. METHODS: Athymic mice bearing Ramos human Burkitt's lymphoma xenografts were injected intraperitoneally with a 1F5-sAv conjugate followed 24 h later by a galactosylated, biotinylated clearing agent (CA) and, finally, 3 h later by (111)In- or (90)Y-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-biotin. The comparison groups consisted of mice injected with conventional, directly labeled (111)In- or (90)Y-1F5. RESULTS: Rapid tumor uptake of radioactivity within 2 h was observed with the pretargeting approach, resulting in high-contrast tumor images at 24 h with minimal blood-pool radioactivity. Although conventional radiolabeled antibodies produced clear tumor images at 24 h, a large amount of radioactivity was present in the blood pool. The tumor-to-blood ratio was 3.5:1 with pretargeting compared with 0.4:1 with conventional (111)In-1F5. Pretargeted RIT with 29.6 MBq (800 micro Ci) (90)Y-DOTA-biotin cured 100% of mice with tolerable toxicity, whereas conventional RIT with (90)Y-1F5 at a dose of 14.8 MBq (400 micro Ci) produced no cures, induced profound pancytopenia, and was lethal to all mice. CONCLUSION: These results suggest that anti-CD20 pretargeted RIT may be superior to conventional radiolabeled antibodies in terms of radioimmunoscintigraphy, toxicity, and therapeutic efficacy for treatment of B-cell lymphomas.     

Age-related interrelationships of blood pressure and arterial sterol accumulation in spontaneously atherosclerosis-susceptible and atherosclerosis-resistant pigeons.

Interrelationships of arterial sterol accumulation and blood pressures were examined in age-matched White Carneau and Show Racer pigeons. It was found that the systolic blood pressure of White Carneaux changed from 155.0 mm Hg at nine months of age to 168.8 mm Hg at 17 months of age (P less than 0.005) while that of the Show Racers did not show any age-related increases. In White Carneaux, the total sterol content in the aorta increased by 43.6% from 9 months to 17 months of age (P less than 0.001) with major changes in the steryl ester fraction while no such changes were evident in the Show Racers. This indicates interrelationships of the arterial sterol content and blood pressure in the White Carneau pigeon.     

Human breast-milk factors influencing lipid metabolism by fetal rabbit aorta in organ culture

Effects of human or rabbit milk on cholesterol content, incorporation of [14C]oleate, and DNA synthesis were investigated in organ cultures of aorta from fetal and suckling rabbits. Human skim milk (50 mL/L) in organ culture decreased content (mumol/g protein) of aortic total cholesterol (control: 162.9 +/- 24.6 milk: 117.6 +/- 4.9) with significant decrease in cholesteryl esters (control: 16.5 +/- 5.1, milk: 2.3 +/- 0.5). The effect was observed in lipoprotein (total cholesterol: 120.2 +/- 8.8) and lipoprotein-free (total cholesterol: 85.4 +/- 5.1) fractions with molecular weight greater than 100,000. The human milk fraction with molecular weight greater than 100,000 was also the most active in promoting [3H]thymidine incorporation into DNA in fetal aorta. Although milk stimulated the incorporation of [14C] oleate into triglycerides (control: 38.8 +/- 2.5%, milk: 82.1 +/- 4.2%), decreased incorporation to phospholipids (control: 55.0 +/- 3.0%, milk: 11.3 +/- 1.8%) was observed. These studies suggest that milk contains factors influencing aortic lipid metabolism during development.     

Regional differences in cholesterol content of aorta in response to experimental coarctation in spontaneously atherosclerosis-susceptible pigeons

Summary The effect of experimental coarctation of the aorta at a site above the coeliac bifurcation (site of occurance of spontaneous atherosclerotic lesions) in White Carneau pigeons was examined. Cholesterol content of the aorta in the site where spontaneous atherosclerosis usually occurs (coeliac bifurcation=lesion site) was decreased in pigeons with aortic coarctation. On the other hand, in the region proximal to the site of coarctation which is usually free of atherosclerotic events cholesterol accumulation was increased. A decrease in percent composition of oleic acid (the major fatty acid which increases during atherosclerosis) was noted in the lesion site. These studies have shown regional differences in response of aorta to experimental coarctation in spontaneously atherosclerotic-susceptible pigeons and suggest that lowering of blood pressure at lesion site might decrease cholesterol accumulation and perhaps might retard subsequent atherogenic process.

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Örtliche Unterschiede des Cholesterin-Gehalts der Aortenwand nach experimenteller Aorteneinengung bei Atherosklerose-anfälligen Tauben

Zusammenfassung Die Auswirkungen einer experimentellen Einengung der Aorta oberhalb der Bifurkation (Prädilektionsstelle spontaner atheroskleroser Läsionen) wurde bei weißen Carneau-Tauben untersucht. An der Stelle, an der üblicherweise eine spontane Atherosklerose auftritt, war der Cholesteringehalt der Aorta bei Tauben mit Aortenkonstriktion vermindert. Weiterhin wurde eine Abnahme des prozentualen Gehalts von Ölsäure im Bereich der Läsionsstelle verzeichnet. Andererseits war die Cholesterineinlagerung im Arterienbereich proximal von der Einengung gesteigert, d. h. an einer Stelle, die normalerweise frei von Atherosklerose ist.Die Untersuchungen zeigen regionale Differenzen der Gefäßwandreaktion auf experimentelle Konstriktion bei Atherosklerose-anfälligen Tauben und lassen vermuten, daß mit vermindertem Blutdruck die Cholesterin-Einlagerung an Prädilektionsstellen abnimmt und möglicherweise ein nachfolgender atherosklerotischer Prozeß verzögert wird.

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With 1 figure and 3 tables

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This study was supported in part by grants HL-24162 (To M.T.R.S.) and HL-21592 (To B.A.K.) from National Heart, Lung and Blood Institute.