Timothy M. Illidge

Fusion of Metabolic Function and Morphology: Sequential [18F]Fluorodeoxyglucose Positron-Emission Tomography/Computed Tomography Studies Yield New Insights Into the Natural History of Bone Metastases in Breast Cancer

PURPOSE By monitoring bone metastases with sequential [(18)F]fluorodeoxyglucose positron-emission tomography/computed tomography ([(18)F]FDG-PET/CT) imaging, this study investigates the clinical relevance of [(18)F]FDG uptake features of bone metastases with various radiographic appearances. PATIENTS AND METHODS Bone metastases were found in 67 of 408 consecutive patients with known/suspected recurrent breast cancer on [(18)F]FDG-PET/CT, characterized by CT morphology changes and/or bony [(18)F]FDG uptake. Twenty-five of the patients had sequential [(18)F]FDG-PET/CT examinations (86 studies) over an average follow-up period of 23 months. The temporal changes in [(18)F]FDG uptake and corresponding CT morphology features of 146 bone lesions identified in these 25 patients were followed up and correlated with therapeutic outcome retrospectively. RESULTS The 146 lesions were classified as osteolytic (77), osteoblastic (41), mixed-pattern (11), or no change/negative (17) on CT. The majority of the osteolytic (72; 93.5%) and mixed-pattern lesions (nine; 81.8%), but fewer of the osteoblastic lesions (25; 61%), showed increased [(18)F]FDG uptake. After treatment, 58 osteolytic lesions (80.5%) became [(18)F]FDG negative and osteoblastic on CT and only 14 relatively large lesions (19.5%) remained [(18)F]FDG avid. Of the 25 [(18)F]FDG-avid osteoblastic lesions, 13 (52%) became [(18)F]FDG negative, but 12 (48%) remained [(18)F]FDG avid and increased in size on CT. Five of the mixed-pattern lesions remained [(18)F]FDG avid after treatment. All 17 CT-negative lesions became [(18)F]FDG negative; however, nine of them became osteoblastic. None of the initially [(18)F]FDG-negative lesions showed [(18)F]FDG avidity during follow-up. CONCLUSION [(18)F]FDG uptake reflects the immediate tumor activity of bone metastases, whereas the radiographic morphology changes vary greatly with time among patients.

Endopolyploid cells produced after severe genotoxic damage have the potential to repair DNA double strand breaks

p53 mutant tumour cells respond to genotoxic insults by bypassing G1 arrest and halting in G2. Following release from G2 arrest they undergo mitotic catastrophe, whereby mitotic cycling is suppressed, delayed apoptosis begins and endopolyploid cells are produced. The ability of these endopolyploid cells to participate in the restitution process is controversial. To facilitate recovery, these endopolyploid cells must repair the extensive DNA damage induced. DNA damage and its resolution were studied by observing the kinetics of γ-H2AX foci formation and by comet assay analysis. Subsequently, the kinetics and distribution of Rad51 foci were studied as a measure of homologous recombination. Here we present evidence of the resolution of DNA damage in endopolyploid cells through a decrease of tail moment by comet assay and in the number of cells expressing γ-H2AX foci. Rad51 foci expression reached a maximum in endopolyploid cells on days 5-6 after irradiation, when delayed apoptosis was maximal, indicating that cells were being selected for survival at this time. Furthermore, the proportion of Annexin-V-positive polyploid cells decreased as they continued ongoing rounds of DNA replication, suggesting endoreduplication is involved in selecting cells resistant to apoptosis. Our findings suggest that after severe genotoxic insult endopolyploid cells have a transient survival advantage that may contribute to radioresistance of tumours that undergo mitotic catastrophe.

Segregation of genomes in polyploid tumour cells following mitotic catastrophe

Following irradiation p53‐function‐deficient tumour cells undergo mitotic catastrophe and form endopolyploid cells. A small proportion of these segregates nuclei, and give rise to viable descendants. Here we studied this process in five tumour cell lines. After mitotic failure, tumour cells enter the endocycle and form mono‐nucleated or multi‐nucleated giant cells (MOGC and MNGC). MNGC arise from arrested anaphases, MOGC, from arrested metaphases. In both cases the individual genomes establish a radial pattern by links to a single microtubule organizing centre. Segregation of genomes is also ordered. MNGC present features of mitosis being resumed from late anaphase. In MOGC the sub‐nuclei retain arrangement of stacked metaphase plates and are separated by folds of the nuclear envelope. Mitosis then resumes in sub‐nuclei directly from metaphase. The data presented indicate that endopolyploid tumour cells preserve the integrity of individual genomes and can potentially re‐initiate mitosis from the point at which it was interrupted.

Upregulation of meiosis-specific genes in lymphoma cell lines following genotoxic insult and induction of mitotic catastrophe

BackgroundWe have previously reported that p53 mutated radioresistant lymphoma cell lines undergo mitotic catastrophe after irradiation, resulting in metaphase arrest and the generation of endopolyploid cells. A proportion of these endopolyploid cells then undergo a process of de-polyploidisation, stages of which are partially reminiscent of meiotic prophase. Furthermore, expression of meiosis-specific proteins of the cancer/testis antigens group of genes has previously been reported in tumours. We therefore investigated whether expression of meiosis-specific genes was associated with the polyploidy response in our tumour model.MethodsThree lymphoma cell lines, Namalwa, WI-L2-NS and TK6, of varying p53 status were exposed to a single 10 Gy dose of gamma radiation and their responses assessed over an extended time course. DNA flow cytometry and mitotic counts were used to assess the kinetics and extent of polyploidisation and mitotic progression. Expression of meiotic genes was analysed using RT-PCR and western blotting. In addition, localisation of the meiotic cohesin REC8 and its relation to centromeres was analysed by immunofluorescence.ResultsThe principal meiotic regulator MOS was found to be significantly post-transcriptionally up-regulated after irradiation in p53 mutated but not p53 wild-type lymphoma cells. The maximum expression of MOS coincided with the maximal fraction of metaphase arrested cells and was directly proportional to both the extent of the arrest and the number of endopolyploid cells that subsequently emerged. The meiotic cohesin REC8 was also found to be up-regulated after irradiation, linking sister chromatid centromeres in the metaphase-arrested and subsequent giant cells. Finally, RT-PCR revealed expression of the meiosis-prophase genes, DMC1, STAG3, SYCP3 and SYCP1.ConclusionWe conclude that multiple meiotic genes are aberrantly activated during mitotic catastrophe in p53 mutated lymphoma cells after irradiation. Furthermore, we suggest that the coordinated expression of MOS and REC8 regulate the extent of arrested mitoses and polyploidy.

Recommendations for the use of Yttrium‐90 ibritumomab tiuxetan in malignant lymphoma

Radioimmunotherapy (RIT) with Yttrium‐90 (90Y) ibritumomab tiuxetan (Zevalin®) combines the tumor targeting attributes of a monoclonal antibody against the CD20 antigen and the pure β‐radiation of 90Y. High efficacy and a favorable safety profile have been demonstrated in Phase II and III clinical trials enrolling patients with CD20+ B‐cell non‐Hodgkin lymphoma (B‐NHL). On the basis of these results, 90Y‐ibritumomab tiuxetan was approved in the United States for the treatment of patients with follicular lymphoma (FL) or transformed B‐NHL. In the European Union its use was restricted to FL, refractory to or relapsed after rituximab. There are a number of important clinical trials currently evaluating 90Y‐ibritumomab tiuxetan in other subtypes of lymphoma such as diffuse large‐cell and mantle‐cell lymphoma, as consolidation therapy or as part of myeloablative regimens. In light of the constantly increasing clinical experience with RIT, clinicians face the challenge of how to best integrate this promising new treatment option into existing established treatment algorithms. By incorporating the most recent data in this rapidly developing field, this review article focuses on current recommendations for the use of 90Y‐ibritumomab tiuxetan in patients with malignant lymphoma, outlines future perspectives, and provides practical recommendations for patient management. Cancer 2006.

Cyclophosphamide inhibition of anti-CD40 monoclonal antibody-based therapy of B cell lymphoma is dependent on CD11b+ cells

Monoclonal antibody (mAb)-based immunotherapy is now established as an important option for treating some cancers. The antitumor effects may be further enhanced by combining mAb with conventional chemotherapy. Certain novel immunomodulatory mAbs such as anti-CD40 have shown significant activity in preclinical models. We therefore assessed the efficacy of combining anti-CD40 mAb, known to elicit CTL responses against murine lymphoma models with the commonly used cytotoxic drug, cyclophosphamide. Using the syngeneic tumor model, BCL1, we have shown that timing of cyclophosphamide relative to mAb is critical to therapeutic outcome. Pretreatment with cyclophosphamide 7 to 10 days prior to mAb results in markedly reduced survival levels, similar to that achieved with cyclophosphamide alone. Conversely, when anti-CD40 is given before cyclophosphamide, the level of tumor protection was moderately increased. In vivo tracking experiments reveal that pretreatment with cyclophosphamide leads to diminished CTL expansion, as well as an increased number of CD11b+ cells that display an activated phenotype. These latter cells are able to inhibit T-cell proliferation, at least in part via production of nitric oxide, but do not induce T-cell apoptosis. Furthermore, adoptive transfer of the induced CD11b+ cells is sufficient to inhibit anti-CD40 therapy in tumor-bearing recipients. We have shown that the timing of cyclophosphamide relative to mAb administration is critical to the therapeutic outcome, and although the combination can improve survival, cyclophosphamide given prior to immunotherapy may generate a population of myeloid cells that can interfere with CTL responses and compromise the therapeutic outcome.

Microscopic intratumoral dosimetry of radiolabeled antibodies is a critical determinant of successful radioimmunotherapy in B-cell lymphoma

Radioimmunotherapy is a highly effective treatment for some hematologic malignancies; however, the underlying mechanisms of tumor clearance remain poorly understood. We have previously shown that both targeted radiation using (131)I-labeled anti-MHC class II (MHCII) monoclonal antibody (mAb) plus mAb signaling with unlabeled anti-idiotype are required for the long-term clearance of tumor in syngeneic murine lymphoma models. In this study, we have investigated how the microdistribution of the targeted radiation component of this combination affects the long-term clearance of lymphoma. (131)I-labeled mAb targeting CD45 and MHCII antigens was found to deliver similar doses of radiation to tumor-bearing organ using conventional dosimetry ( approximately 1.0 Gy per MBq when (131)I was labeled to 500 mug mAb and given i.v. per mouse), but when used as radiation vectors in combination therapy only, (131)I-anti-MHCII plus anti-idiotype produced long-term survival. The profound differences in therapy did not seem to be dependent on levels of (131)I-mAb tumor-binding or antibody-dependent cytotoxicity. Instead, the microscopic intratumoral dosimetry seemed to be critical with the (131)I-anti-MHCII, delivering more concentrated and therefore substantially higher radiation dose to tumor cells. When the administered activity of (131)I-anti-CD45 was increased, a radiation dose response was shown in the presence of anti-idiotype and long-term survival was seen. We believe that these new insights should influence the selection of new antigen targets and the design of dosimetric methods in radioimmunotherapy of lymphoma.