Letterio S. Politi

Targeting the ANG2/TIE2 Axis Inhibits Tumor Growth and Metastasis by Impairing Angiogenesis and Disabling Rebounds of Proangiogenic Myeloid Cells

Tumor-infiltrating myeloid cells convey proangiogenic programs that counteract the efficacy of antiangiogenic therapy. Here, we show that blocking angiopoietin-2 (ANG2), a TIE2 ligand and angiogenic factor expressed by activated endothelial cells (ECs), regresses the tumor vasculature and inhibits progression of late-stage, metastatic MMTV-PyMT mammary carcinomas and RIP1-Tag2 pancreatic insulinomas. ANG2 blockade did not inhibit recruitment of MRC1(+) TIE2-expressing macrophages (TEMs) but impeded their upregulation of Tie2, association with blood vessels, and ability to restore angiogenesis in tumors. Conditional Tie2 gene knockdown in TEMs was sufficient to decrease tumor angiogenesis. Our findings support a model wherein the ANG2-TIE2 axis mediates cell-to-cell interactions between TEMs and ECs that are important for tumor angiogenesis and can be targeted to induce effective antitumor responses.

Tumor-Targeted Interferon-α Delivery by Tie2-Expressing Monocytes Inhibits Tumor Growth and Metastasis

The use of type I interferons (IFNs) in cancer therapy has been limited by ineffective dosing and significant toxicity. Here, we exploited the tumor-homing ability of proangiogenic Tie2-expressing monocytes (TEMs) to deliver IFN-alpha to tumors. By transplanting hematopoietic progenitors transduced with a Tie2 promoter/enhancer-driven Ifna1 gene, we turned TEMs into IFN-alpha cell vehicles that efficiently targeted the IFN response to orthotopic human gliomas and spontaneous mouse mammary carcinomas and obtained significant antitumor responses and near complete abrogation of metastasis. TEM-mediated IFN-alpha delivery inhibited tumor angiogenesis and activated innate and adaptive immune cells but did not impair myelopoiesis and wound healing detectably. These results illustrate the therapeutic potential of gene- and cell-based IFN-alpha delivery and should allow the development of IFN treatments that more effectively treat cancer.

Epidermal Growth Factor Receptor Expression Identifies Functionally and Molecularly Distinct Tumor-Initiating Cells in Human Glioblastoma Multiforme and Is Required for Gliomagenesis

Epidermal growth factor receptor (EGFR) is a known diagnostic and, although controversial, prognostic marker of human glioblastoma multiforme (GBM). However, its functional role and biological significance in GBM remain elusive. Here, we show that multiple GBM cell subpopulations could be purified from the specimens of patients with GBM and from cancer stem cell (CSC) lines based on the expression of EGFR and of other putative CSC markers. All these subpopulations are molecularly and functionally distinct, are tumorigenic, and need to express EGFR to promote experimental tumorigenesis. Among them, EGFR-expressing tumor-initiating cells (TIC) display the most malignant functional and molecular phenotype. Accordingly, modulation of EGFR expression by gain-of-function and loss-of-function strategies in GBM CSC lines enhances and reduces their tumorigenic ability, respectively, suggesting that EGFR plays a fundamental role in gliomagenesis. These findings open up the possibility of new therapeutically relevant scenarios, as the presence of functionally heterogeneous EGFR(pos) and EGFR(neg) TIC subpopulations within the same tumor might affect clinical response to treatment.

Efficient In Vitro Labeling of Human Neural Precursor Cells with Superparamagnetic Iron Oxide Particles: Relevance for In Vivo Cell Tracking

Recent studies have raised appealing possibilities of replacing damaged or lost neural cells by transplanting in vitro‐expanded neural precursor cells (NPCs) and/or their progeny. Magnetic resonance (MR) tracking of superparamagnetic iron oxide (SPIO)‐labeled cells is a noninvasive technique to track transplanted cells in longitudinal studies on living animals. Murine NPCs and human mesenchymal or hematopoietic stem cells can be efficiently labeled by SPIOs. However, the validation of SPIO‐based protocols to label human neural precursor cells (hNPCs) has not been extensively addressed. Here, we report the development and validation of optimized protocols using two SPIOs (Sinerem and Endorem) to label human hNPCs that display bona fide stem cell features in vitro. A careful titration of both SPIOs was required to set the conditions resulting in efficient cell labeling without impairment of cell survival, proliferation, self‐renewal, and multipotency. In vivo magnetic resonance imaging (MRI) combined with histology and confocal microscopy indicated that low numbers (5 × 103 to 1 × 104) of viable SPIO‐labeled hNPCs could be efficiently detected in the short term after transplantation in the adult murine brain and could be tracked for at least 1 month in longitudinal studies. By using this approach, we also clarified the impact of donor cell death to the MR signal. This study describes a simple protocol to label NPCs of human origin using SPIOs at optimized low dosages and demonstrates the feasibility of noninvasive imaging of labeled cells after transplantation in the brain; it also evidentiates potential limitations of the technique that have to be considered, particularly in the perspective of neural cell‐based clinical applications.

Magnetic Resonance‐Based Tracking and Quantification of Intravenously Injected Neural Stem Cell Accumulation in the Brains of Mice with Experimental Multiple Sclerosis

Eliciting the in situ accumulation and persistence patterns of stem cells following transplantation would provide critical insight toward human translation of stem cell‐based therapies. To this end, we have developed a strategy to track neural stem/precursor cells (NPCs) in vivo using magnetic resonance (MR) imaging. Initially, we evaluated three different human‐grade superparamagnetic iron oxide particles for labeling NPCs and found the optimal labeling to be achieved with Resovist. Next, we carried out in vivo experiments to monitor the accumulation of Resovist‐labeled NPCs following i.v. injection in mice with experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis. With a human MR scanner, we were able to visualize transplanted cells as early as 24 hours post‐transplantation in up to 80% of the brain demyelinating lesions. Interestingly, continued monitoring of transplanted mice indicated that labeled NPCs were still present 20 days postinjection. Neuropathological analysis confirmed the presence of transplanted NPCs exclusively in inflammatory demyelinating lesions and not in normal‐appearing brain areas. Quantification of transplanted cells by means of MR‐based ex vivo relaxometry (R2*) showed significantly higher R2* values in focal inflammatory brain lesions from EAE mice transplanted with labeled NPCs as compared with controls. Indeed, sensitive quantification of low numbers of NPCs accumulating into brain inflammatory lesions (33.3–164.4 cells per lesion; r2 = .998) was also obtained. These studies provide evidence that clinical‐grade human MR can be used for noninvasive monitoring and quantification of NPC accumulation in the central nervous system upon systemic cell injection.

Gene therapy augments the efficacy of hematopoietic cell transplantation and fully corrects mucopolysaccharidosis type I phenotype in the mouse model

Type I mucopolysaccharidosis (MPS I) is a lysosomal storage disorder caused by the deficiency of α-L-iduronidase, which results in glycosaminoglycan accumulation in tissues. Clinical manifestations include skeletal dysplasia, joint stiffness, visual and auditory defects, cardiac insufficiency, hepatosplenomegaly, and mental retardation (the last being present exclusively in the severe Hurler variant). The available treatments, enzyme-replacement therapy and hematopoietic stem cell (HSC) transplantation, can ameliorate most disease manifestations, but their outcome on skeletal and brain disease could be further improved. We demonstrate here that HSC gene therapy, based on lentiviral vectors, completely corrects disease manifestations in the mouse model. Of note, the therapeutic benefit provided by gene therapy on critical MPS I manifestations, such as neurologic and skeletal disease, greatly exceeds that exerted by HSC transplantation, the standard of care treatment for Hurler patients. Interestingly, therapeutic efficacy of HSC gene therapy is strictly dependent on the achievement of supranormal enzyme activity in the hematopoietic system of transplanted mice, which allows enzyme delivery to the brain and skeleton for disease correction. Overall, our data provide evidence of an efficacious treatment for MPS I Hurler patients, warranting future development toward clinical testing.

Chemoimmunotherapy with methotrexate, cytarabine, thiotepa, and rituximab (MATRix regimen) in patients with primary CNS lymphoma: results of the first randomisation of the International Extranodal Lymphoma Study Group-32 (IELSG32) phase 2 trial

BACKGROUND Standard treatment for patients with primary CNS lymphoma remains to be defined. Active therapies are often associated with increased risk of haematological or neurological toxicity. In this trial, we addressed the tolerability and efficacy of adding rituximab with or without thiotepa to methotrexate-cytarabine combination therapy (the MATRix regimen), followed by a second randomisation comparing consolidation with whole-brain radiotherapy or autologous stem cell transplantation in patients with primary CNS lymphoma. We report the results of the first randomisation in this Article. METHODS For the international randomised phase 2 International Extranodal Lymphoma Study Group-32 (IELSG32) trial, HIV-negative patients (aged 18-70 years) with newly diagnosed primary CNS lymphoma and measurable disease were enrolled from 53 cancer centres in five European countries (Denmark, Germany, Italy, Switzerland, and the UK) and randomly assigned (1:1:1) to receive four courses of methotrexate 3·5 g/m(2) on day 1 plus cytarabine 2 g/m(2) twice daily on days 2 and 3 (group A); or the same combination plus two doses of rituximab 375 mg/m(2) on days -5 and 0 (group B); or the same methotrexate-cytarabine-rituximab combination plus thiotepa 30 mg/m(2) on day 4 (group C), with the three groups repeating treatment every 3 weeks. Patients with responsive or stable disease after the first stage were then randomly allocated between whole-brain radiotherapy and autologous stem cell transplantation. A permuted blocks randomised design (block size four) was used for both randomisations, and a computer-generated randomisation list was used within each stratum to preserve allocation concealment. Randomisation was stratified by IELSG risk score (low vs intermediate vs high). No masking after assignment to intervention was used. The primary endpoint of the first randomisation was the complete remission rate, analysed by modified intention to treat. This study is registered with ClinicalTrials.gov, number NCT01011920. FINDINGS Between Feb 19, 2010, and Aug 27, 2014, 227 eligible patients were recruited. 219 of these 227 enrolled patients were assessable. At median follow-up of 30 months (IQR 22-38), patients treated with rituximab and thiotepa had a complete remission rate of 49% (95% CI 38-60), compared with 23% (14-31) of those treated with methotrexate-cytarabine alone (hazard ratio 0·46, 95% CI 0·28-0·74) and 30% (21-42) of those treated with methotrexate-cytarabine plus rituximab (0·61, 0·40-0·94). Grade 4 haematological toxicity was more frequent in patients treated with methotrexate-cytarabine plus rituximab and thiotepa, but infective complications were similar in the three groups. The most common grade 3-4 adverse events in all three groups were neutropenia, thrombocytopenia, anaemia, and febrile neutropenia or infections. 13 (6%) patients died of toxicity. INTERPRETATION With the limitations of a randomised phase 2 study design, the IELSG32 trial provides a high level of evidence supporting the use of MATRix combination as the new standard chemoimmunotherapy for patients aged up to 70 years with newly diagnosed primary CNS lymphoma and as the control group for future randomised trials. FUNDING Associazione Italiana del Farmaco, Cancer Research UK, Oncosuisse, and Swiss National Foundation.

Chlamydophila Psittaci Eradication With Doxycycline As First-Line Targeted Therapy for Ocular Adnexae Lymphoma: Final Results of an International Phase II Trial

PURPOSE The pathogenic association between Chlamydophila psittaci (Cp) and ocular adnexal marginal zone lymphoma (OAMZL) and the efficacy of doxycycline monotherapy have been investigated in retrospective series with variations in stage, management, and follow-up duration. To our knowledge, this is the first international phase II trial aimed at clarifying Cp prevalence and activity of first-line doxycycline in a homogeneous series of consecutive patients with newly diagnosed stage I OAMZL. PATIENTS AND METHODS Forty-seven patients were registered. Tumor tissue, conjunctival swabs, and peripheral blood from 44 patients were assessed for seven Chlamydiaceae infections by three polymerase chain reaction protocols. Thirty-four patients with measurable or parametrable disease were treated with doxycycline and assessed for chlamydial eradication and lymphoma response (primary end point). RESULTS Cp DNA was detected in biopsies of 39 patients (89%); no other Chlamydiaceae were detected. Twenty-nine patients had Cp DNA in baseline swabs and/or blood samples and were evaluable for chlamydial eradication, which was achieved in 14 patients (48%). Lymphoma regression was complete in six patients and partial in 16 (overall response rate, 65%; 95% CI, 49% to 81%); 11 had stable disease, and one had progressive disease. At a median follow-up of 37 months (range, 15 to 62 months), 20 patients remained relapse free (5-year progression-free survival [PFS] ± standard deviation, 55% ± 9%). Cp eradication was associated with improved response rate (86% v 47%; P = .02) and 5-year PFS (68% v 47%; P = .11). CONCLUSION Upfront doxycycline is a rational and active treatment for patients with stage I Cp-positive OAMZL. Lymphoma regression is consequent to Cp eradication, which can easily be monitored on conjunctival and blood samples. Prospective trials aimed at identifying more effective administration schedules for doxycycline are warranted.

Brain conditioning is instrumental for successful microglia reconstitution following hematopoietic stem cell transplantation

The recent hypothesis that postnatal microglia are maintained independently of circulating monocytes by local precursors that colonize the brain before birth has relevant implications for the treatment of various neurological diseases, including lysosomal storage disorders (LSDs), for which hematopoietic cell transplantation (HCT) is applied to repopulate the recipient myeloid compartment, including microglia, with cells expressing the defective functional hydrolase. By studying wild-type and LSD mice at diverse time-points after HCT, we showed the occurrence of a short-term wave of brain infiltration by a fraction of the transplanted hematopoietic progenitors, independently from the administration of a preparatory regimen and from the presence of a disease state in the brain. However, only the use of a conditioning regimen capable of ablating functionally defined brain-resident myeloid precursors allowed turnover of microglia with the donor, mediated by local proliferation of early immigrants rather than entrance of mature cells from the circulation.

Ocular Adnexal Lymphoma: Diffusion-weighted MR Imaging for Differential Diagnosis and Therapeutic Monitoring

PURPOSE To describe the magnetic resonance (MR) imaging and diffusion-weighted (DW) imaging features of ocular adnexal lymphomas (OALs), to determine the diagnostic accuracy of apparent diffusion coefficient (ADC) for discriminating OALs from other orbital mass lesions, and to assess whether variations in ADC constitute a reliable biomarker of OAL response to therapy. MATERIALS AND METHODS Institutional ethical committee approval and informed consent were obtained. In this prospective study, 114 white subjects (65 females and 49 males) were enrolled. Thirty-eight patients with histopathologically proved OAL underwent serial MR and DW imaging examination of the orbits. ADCs of OALs were compared with those of normal orbital structures, obtained in 18 healthy volunteers, and other orbital mass lesions, prospectively acquired in 58 patients (20 primary non-OAL neoplasms, 15 vascular benign lesions, 12 inflammatory lesions, 11 metastases). Interval change in ADC of OALs before and after treatment was analyzed in 29 patients. Analysis of covariance and a paired t test were used for statistical analysis. RESULTS Baseline ADCs in OALs were lower than those in normal structures and other orbital diseases (P < .001). An ADC threshold of 775 x 10(-6) mm(2)/sec resulted in 96% sensitivity, 93% specificity, 88% positive predictive value, 98.2% negative predictive value, and 94.4% accuracy in OAL diagnosis. Following appropriate treatment, 10 (34%) of 29 patients showed OAL volumetric reduction, accompanied (n = 7) or preceded (n = 3) by an increase in ADC (P = .005). Conversely, a further reduction of ADC was observed in the seven patients who experienced disease progression (P < .05). CONCLUSION ADC permits accurate diagnosis of OALs. Interval change in ADC after therapy represents a helpful tool for predicting therapeutic response.