Expression of neurotensin receptor-1 (NTS1) in primary breast tumors, cellular distribution, and association with clinical and biological factors.

PURPOSE: Neurotensin receptor-1 (NTS1) is increasingly recognized as a potential target in diverse tumors including breast cancer, but factors associated with NTS1 expression have not been fully clarified. METHODS: We studied NTS1 expression using the Tissue MicroArray (TMA) of primary breast tumors from Institut Bergonié. We also studied association between NTS1 expression and clinical, pathological, and biological parameters, as well as patient outcomes. RESULTS: Out of 1419 primary breast tumors, moderate to strong positivity for NTS1 (≥ 10% of tumoral cells stained) was seen in 459 samples (32.4%). NTS1 staining was cytoplasmic in 304 tumors and nuclear in 155 tumors, a distribution which appeared mutually exclusive. Cytoplasmic overexpression of NTS1 was present in 21.5% of all breast tumors. In multivariate analysis, factors associated with cytoplasmic overexpression of NTS1 in breast cancer samples were higher tumor grade, Ki67 ≥ 20%, and higher pT stage. Cytoplasmic NTS1 was more frequent in tumors other than luminal A (30% versus 17.3%; p < 0.0001). Contrastingly, the main "correlates" of a nuclear location of NTS1 were estrogen receptor (ER) positivity, low E&E (Elston and Ellis) grade, Ki67 < 20%, and lower pT stage. In NTS1-positive samples, cytoplasmic expression of NTS1 was associated with shorter 10-year metastasis-free interval (p = 0.033) compared to NTS1 nuclear staining. Ancillary analysis showed NTS1 expression in 73% of invaded lymph nodes from NTS1-positive primaries. CONCLUSION: NTS1 overexpression was found in about one-third of breast tumors from patients undergoing primary surgery with two distinct patterns of distribution, cytoplasmic distribution being more frequent in aggressive subtypes. These findings encourage the development of NTS1-targeting strategy, including radiopharmaceuticals for imaging and therapy.

Silicon-Containing Neurotensin Analogues as Radiopharmaceuticals for NTS1-Positive Tumors Imaging.

Several independent studies have demonstrated the overexpression of NTS1 in various malignancies, which make this receptor of interest for imaging and therapy. To date, radiolabeled neurotensin analogues suffer from low plasmatic stability and thus insufficient availability for high uptake in tumors. We report the development of 68Ga-radiolabeled neurotensin analogues with improved radiopharmaceutical properties through the introduction of the silicon-containing amino acid trimethylsilylalanine (TMSAla). Among the series of novel radiolabeled neurotensin analogues, [68Ga]Ga-JMV6659 exhibits high hydrophilicity (log D7.4 = -3.41 ± 0.14), affinity in the low nanomolar range toward NTS1 (Kd = 6.29 ± 1.37 nM), good selectivity (Kd NTS1/Kd NTS2 = 35.9), and high NTS1-mediated internalization. It has lower efflux and prolonged plasmatic half-life in human plasma as compared to the reference compound ([68Ga]Ga-JMV6661 bearing the minimum active fragment of neurotensin and the same linker and chelate as other analogues). In nude mice bearing HT-29 xenograft, [68Ga]Ga-JMV6659 uptake reached 7.8 ± 0.54 %ID/g 2 h post injection. Uptake was decreased to 1.38 ± 0.71 %ID/g with injection of excess of non-radioactive neurotensin. Radiation dose as extrapolated to human was estimated as 2.35 ± 0.6 mSv for a standard injected activity of 100MBq. [68Ga]Ga-JMV6659 was identified as a promising lead compound suitable for PET imaging of NTS1-expressing tumors.

Neurotensin Receptor-1 Expression in Human Prostate Cancer: A Pilot Study on Primary Tumors and Lymph Node Metastases.

Neurotensin and its high-affinity receptor, NTR1, are involved in the growth of various tumors. Few data are available regarding NTR1 expression in normal and tumoral human prostate tissue samples. NTR1 expression was assessed using immunohistochemistry in 12 normal prostate tissues, 11 benign prostatic hyperplasia (BPH), 44 prostate cancers, and 15 related metastatic lymph nodes (one per patient, when available). NTR1-staining was negative in normal prostate and BPH samples. NTR1 was overexpressed in four out of 44 (9.1%) primary tumors. There was no clear association between NTR1 overexpression and age, PSA-values, Gleason score, pT-status, nodal-status, or margin. NTR1 was expressed at a high level of five out of 15 (33.3%) metastatic lymph nodes. NTR1 overexpression was thus more frequent in metastatic lymph nodes than in primary tumors (p = 0.038). In this limited series of samples, NTR1 overexpression was observed in few primary prostate cancers. Upregulation was more frequent in related lymph nodes. The presence of this target in metastatic lymph nodes may open new perspectives for imaging and radionuclide therapy of prostate cancer. Factors driving NTR1 expression in primary prostate cancer and in nodal and distant metastases still need to be characterized.

68Ga-Radiolabeling and Pharmacological Characterization of a Kit-Based Formulation of the Gastrin-Releasing Peptide Receptor (GRP-R) Antagonist RM2 for Convenient Preparation of [68Ga]Ga-RM2.

BACKGROUND: [68Ga]Ga-RM2 is a potent Gastrin-Releasing Peptide-receptor (GRP-R) antagonist for imaging prostate cancer and breast cancer, currently under clinical evaluation in several specialized centers around the world. Targeted radionuclide therapy of GRP-R-expressing tumors is also being investigated. We here report the characteristics of a kit-based formulation of RM2 that should ease the development of GRP-R imaging and make it available to more institutions and patients. METHODS: Stability of the investigated kits over one year was determined using LC/MS/MS and UV-HPLC. Direct 68Ga-radiolabeling was optimized with respect to buffer (pH), temperature, reaction time and shaking time. Conventionally prepared [68Ga]Ga-RM2 using an automated synthesizer was used as a comparator. Finally, the [68Ga]Ga-RM2 product was assessed with regards to hydrophilicity, affinity, internalization, membrane bound fraction, calcium mobilization assay and efflux, which is a valuable addition to the in vivo literature. RESULTS: The kit-based formulation, kept between 2 °C and 8 °C, was stable for over one year. Using acetate buffer pH 3.0 in 2.5-5.1 mL total volume, heating at 100 °C during 10 min and cooling down for 5 min, the [68Ga]Ga-RM2 produced by kit complies with the requirements of the European Pharmacopoeia. Compared with the module production route, the [68Ga]Ga-RM2 produced by kit was faster, displayed higher yields, higher volumetric activity and was devoid of ethanol. In in vitro evaluations, the [68Ga]Ga-RM2 displayed sub-nanomolar affinity (Kd = 0.25 ± 0.19 nM), receptor specific and time dependent membrane-bound fraction of 42.0 ± 5.1% at 60 min and GRP-R mediated internalization of 24.4 ± 4.3% at 30 min. The [natGa]Ga-RM2 was ineffective in stimulating intracellular calcium mobilization. Finally, the efflux of the internalized activity was 64.3 ± 6.5% at 5 min. CONCLUSION: The kit-based formulation of RM2 is suitable to disseminate GRP-R imaging and therapy to distant hospitals without complex radiochemistry equipment.

Design, synthesis, and biological evaluation of a multifunctional neuropeptide-Y conjugate for selective nuclear delivery of radiolanthanides.

BACKGROUND: Targeting G protein-coupled receptors on the surface of cancer cells with peptide ligands is a promising concept for the selective tumor delivery of therapeutically active cargos, including radiometals for targeted radionuclide therapy (TRT). Recently, the radiolanthanide terbium-161 (161Tb) gained significant interest for TRT application, since it decays with medium-energy ß-radiation but also emits a significant amount of conversion and Auger electrons with short tissue penetration range. The therapeutic efficiency of radiometals emitting Auger electrons, like 161Tb, can therefore be highly boosted by an additional subcellular delivery into the nucleus, in order to facilitate maximum dose deposition to the DNA. In this study, we describe the design of a multifunctional, radiolabeled neuropeptide-Y (NPY) conjugate, to address radiolanthanides to the nucleus of cells naturally overexpressing the human Y1 receptor (hY1R). By using solid-phase peptide synthesis, the hY1R-preferring [F7,P34]-NPY was modified with a fatty acid, a cathepsin B-cleavable linker, followed by a nuclear localization sequence (NLS), and a DOTA chelator (compound pb12). In this proof-of-concept study, labeling was performed with either native terbium-159 (natTb), as surrogate for 161Tb, or with indium-111 (111In). RESULTS: [natTb]Tb-pb12 showed a preserved high binding affinity to endogenous hY1R on MCF-7 cells and was able to induce receptor activation and internalization similar to the hY1R-preferring [F7,P34]-NPY. Specific internalization of the 111In-labeled conjugate into MCF-7 cells was observed, and importantly, time-dependent nuclear uptake of 111In was demonstrated. Study of metabolic stability showed that the peptide is insufficiently stable in human plasma. This was confirmed by injection of [111In]In-pb12 in nude mice bearing MCF-7 xenograft which showed specific uptake only at very early time point. CONCLUSION: The multifunctional NPY conjugate with a releasable DOTA-NLS unit represents a promising concept for enhanced TRT with Auger electron-emitting radiolanthanides. Our research is now focusing on improving the reported concept with respect to the poor plasmatic stability of this promising radiopeptide.