Final Report Summary - IMAGING LYMPHOMA (New imaging methods for detecting treatment response in lymphoma)
A targeted imaging agent for detecting cell death
We have developed a targeted imaging agent, based on the C2A domain of Synaptotagmin-I, which is capable of binding, with nanomolar affinity, to the phosphatidylserine exposed during tumor cell death. Our novel probe (C2Am) includes a unique cysteine residue, which was introduced by site-directed mutagenesis and can be used for controlled attachment of metal ion chelates for subsequent incorporation of radiometals for nuclear imaging.
We labeled the cysteine residue with maleimide-DOTA and demonstrated that the 111In labeled agent was capable of detecting cell death in a murine lymphoma model. As part of our objective of developing C2Am as a clinical tool for detecting lymphoma cell death post-treatment, we undertook a systematic comparison of the performance of C2Am and Annexin V. 99mTc labeled Annexin V reached early clinical trials for detection of cell death in a variety of diseases, including lymphoma, however there were problems with its biodistribution, which limited the success of this agent in the clinic.
Previously, the laboratory compared fluorescently labeled C2Am and Annexin V as agents for detecting cell death using flow cytometry and demonstrated that C2Am showed up to four-fold more specific binding to dead cells than Annexin V due to lower binding to viable cells. In order to make a comparison of these agents using radionuclide imaging in vivo we obtained, during this grant period, Annexin-V modified with the HYNIC chelator, for labeling with 99mTc, and have optimized modification of C2Am with the same chelator. The success of modification was assessed using ESI mass spectrometry.
With the successful implementation of FDG-PET imaging in the laboratory, the full range of radionuclide imaging techniques are now established. This allowed their systematic comparison with hyperpolarized 13C MRI techniques for detecting treatment response in lymphoma(both in EL4 xenograft mice and in Emu-myc mice) .
Genetically engineered mouse models of lymphoma
We initially employed a human NPM-ALK transgenic mouse model that used the haematopoietic cell-specific Vav promoter. However we had problems with the long latency period for development of lymphoid malignancies (12-24 months) and high rates of mortality. Then we started to use the well-characterized Emu-myc transgenic mouse model, which almost invariably develops pre-B or B-cell lymphomas with associated leukemia (90% succumbing in the first 5 months of life) due to constitutive expression of the c-myc oncogene in the B cell lineage under control of the Emu immunoglobulin enhancer. This model has already been proven to be useful for identifying new therapies for aggressive Myc-driven lymphomas that have failed standard therapies. The model shares histological and genetic similarities with Burkitt's lymphoma. A remarkable observation from the studies in the Vav-driven NPM-ALK tumor model was the very high levels of labeled alanine observed in these tumors when compared to the implanted EL4 model. Interestingly these Emu-myc tumors have very low levels of alanine and are much more similar to the implanted EL4 tumor model.
A targeted imaging agent for detecting lymphoma cell death post-treatment
Quality assessment of 99mTc-labelled C2Am and Annexin V were performed using HPLC followed by cell binding assays using the EL4 lymphoma cell line. Biodistribution studies were also performed using these optimized agents, which were compared for their facility to image cell death in the implanted EL4 lymphoma model and in the new genetically engineered mouse tumor models that we have established.
Imaging of in genetically engineered mouse (GEM) models of lymphoma
With the establishment of two new GEM lines, which give tumors with shorter latency periods and greater penetrance than the model used initially, with the optimization of the C2Am and Annexin V radiolabeling and with the establishment of FDG-PET in the laboratory we were ideally placed to conduct a systematic comparison of these established methods of detecting treatment response with the novel hyperpolarized 13C MRS methods of detecting treatment response that we have developed. The use of a transgenic mouse model allowed a more realistic analysis of treatment response in spontaneous lymphomas. These tumours present genetically defined lesions that are treated at their natural site of origin.
The treatment response detected with FGD-PET imaging approach was much smaller at 24 h after cyclophosphamide treatment than the 13C MRS methods (hyperpolarized pyruvate). Cyclophosphamide was chosen as the cytotoxic agent because this drug is used in clinical settings; however, recent treatments with this drug can provoke an increase of activated inflammatory cells, leading to an overestimation of the viable tumour fraction because the inflammatory cells also show high FDG uptake. In the clinic, a decrease in the SUV from baseline of at least 30% after one cycle is necessary to obtain a partial response. On these grounds, a transient influx in inflammatory cells and its contribution to FDG uptake can be of importance. With clinical trials set to commence, utilization of hyperpolarized pyruvate may be preferentially used in pathologies and/or for drugs where the traditional approaches to assess treatment response tend to fail and an increased understanding of the underlying biological nature of the response seen with hyperpolarized pyruvate is required to help interpret the human data. We demonstrated the feasibility of using DNP hyperpolarized 13C-pyruvate to detect early response to cyclophosphamide treatment in a transgenic mouse of lymphoma which may complement FDG-PET as a clinical tool.
We have developed a targeted imaging agent, based on the C2A domain of Synaptotagmin-I, which is capable of binding, with nanomolar affinity, to the phosphatidylserine exposed during tumor cell death. Our novel probe (C2Am) includes a unique cysteine residue, which was introduced by site-directed mutagenesis and can be used for controlled attachment of metal ion chelates for subsequent incorporation of radiometals for nuclear imaging.
We labeled the cysteine residue with maleimide-DOTA and demonstrated that the 111In labeled agent was capable of detecting cell death in a murine lymphoma model. As part of our objective of developing C2Am as a clinical tool for detecting lymphoma cell death post-treatment, we undertook a systematic comparison of the performance of C2Am and Annexin V. 99mTc labeled Annexin V reached early clinical trials for detection of cell death in a variety of diseases, including lymphoma, however there were problems with its biodistribution, which limited the success of this agent in the clinic.
Previously, the laboratory compared fluorescently labeled C2Am and Annexin V as agents for detecting cell death using flow cytometry and demonstrated that C2Am showed up to four-fold more specific binding to dead cells than Annexin V due to lower binding to viable cells. In order to make a comparison of these agents using radionuclide imaging in vivo we obtained, during this grant period, Annexin-V modified with the HYNIC chelator, for labeling with 99mTc, and have optimized modification of C2Am with the same chelator. The success of modification was assessed using ESI mass spectrometry.
With the successful implementation of FDG-PET imaging in the laboratory, the full range of radionuclide imaging techniques are now established. This allowed their systematic comparison with hyperpolarized 13C MRI techniques for detecting treatment response in lymphoma(both in EL4 xenograft mice and in Emu-myc mice) .
Genetically engineered mouse models of lymphoma
We initially employed a human NPM-ALK transgenic mouse model that used the haematopoietic cell-specific Vav promoter. However we had problems with the long latency period for development of lymphoid malignancies (12-24 months) and high rates of mortality. Then we started to use the well-characterized Emu-myc transgenic mouse model, which almost invariably develops pre-B or B-cell lymphomas with associated leukemia (90% succumbing in the first 5 months of life) due to constitutive expression of the c-myc oncogene in the B cell lineage under control of the Emu immunoglobulin enhancer. This model has already been proven to be useful for identifying new therapies for aggressive Myc-driven lymphomas that have failed standard therapies. The model shares histological and genetic similarities with Burkitt's lymphoma. A remarkable observation from the studies in the Vav-driven NPM-ALK tumor model was the very high levels of labeled alanine observed in these tumors when compared to the implanted EL4 model. Interestingly these Emu-myc tumors have very low levels of alanine and are much more similar to the implanted EL4 tumor model.
A targeted imaging agent for detecting lymphoma cell death post-treatment
Quality assessment of 99mTc-labelled C2Am and Annexin V were performed using HPLC followed by cell binding assays using the EL4 lymphoma cell line. Biodistribution studies were also performed using these optimized agents, which were compared for their facility to image cell death in the implanted EL4 lymphoma model and in the new genetically engineered mouse tumor models that we have established.
Imaging of in genetically engineered mouse (GEM) models of lymphoma
With the establishment of two new GEM lines, which give tumors with shorter latency periods and greater penetrance than the model used initially, with the optimization of the C2Am and Annexin V radiolabeling and with the establishment of FDG-PET in the laboratory we were ideally placed to conduct a systematic comparison of these established methods of detecting treatment response with the novel hyperpolarized 13C MRS methods of detecting treatment response that we have developed. The use of a transgenic mouse model allowed a more realistic analysis of treatment response in spontaneous lymphomas. These tumours present genetically defined lesions that are treated at their natural site of origin.
The treatment response detected with FGD-PET imaging approach was much smaller at 24 h after cyclophosphamide treatment than the 13C MRS methods (hyperpolarized pyruvate). Cyclophosphamide was chosen as the cytotoxic agent because this drug is used in clinical settings; however, recent treatments with this drug can provoke an increase of activated inflammatory cells, leading to an overestimation of the viable tumour fraction because the inflammatory cells also show high FDG uptake. In the clinic, a decrease in the SUV from baseline of at least 30% after one cycle is necessary to obtain a partial response. On these grounds, a transient influx in inflammatory cells and its contribution to FDG uptake can be of importance. With clinical trials set to commence, utilization of hyperpolarized pyruvate may be preferentially used in pathologies and/or for drugs where the traditional approaches to assess treatment response tend to fail and an increased understanding of the underlying biological nature of the response seen with hyperpolarized pyruvate is required to help interpret the human data. We demonstrated the feasibility of using DNP hyperpolarized 13C-pyruvate to detect early response to cyclophosphamide treatment in a transgenic mouse of lymphoma which may complement FDG-PET as a clinical tool.