Combining precision radiotherapy with molecular targeting and immunomodulatory agents: a guideline by the American Society for Radiation Oncology.

The practice of radiation oncology is primarily based on precise technical delivery of highly conformal, image-guided external beam radiotherapy or brachytherapy. However, systematic research efforts are being made to facilitate individualised radiation dose prescriptions on the basis of gene-expressssion profiles that reflect the radiosensitivity of tumour and normal tissue. This advance in precision radiotherapy should complement those benefits made in precision cancer medicine that use molecularly targeted agents and immunotherapies. The personalisation of cancer therapy, predicated largely on genomic interrogation, is facilitating the selection of therapies that are directed against driver mutations, aberrant cell signalling, tumour microenvironments, and genetic susceptibilities. With the increasing technical power of radiotherapy to safely increase local tumour control for many solid tumours, it is an opportune time to rigorously explore the potential benefits of combining radiotherapy with molecular targeted agents and immunotherapies to increase cancer survival outcomes. This theme provides the basis and foundation for this American Society for Radiation Oncology guideline on combining radiotherapy with molecular targeting and immunotherapy agents.

Transcriptional activation of hypoxia-inducible factor-1 (HIF-1) in myeloid cells promotes angiogenesis through VEGF and S100A8.

Emerging evidence indicates that myeloid cells are essential for promoting new blood vessel formation by secreting various angiogenic factors. Given that hypoxia-inducible factor (HIF) is a critical regulator for angiogenesis, we questioned whether HIF in myeloid cells also plays a role in promoting angiogenesis. To address this question, we generated a unique strain of myeloid-specific knockout mice targeting HIF pathways using human S100A8 as a myeloid-specific promoter. We observed that mutant mice where HIF-1 is transcriptionally activated in myeloid cells (by deletion of the von Hippel-Lindau gene) resulted in erythema, enhanced neovascularization in matrigel plugs, and increased production of vascular endothelial growth factor (VEGF) in the bone marrow, all of which were completely abrogated by either genetic or pharmacological inactivation of HIF-1. We further found that monocytes were the major effector producing VEGF and S100A8 proteins driving neovascularization in matrigel. Moreover, by using a mouse model of hindlimb ischemia we observed significantly improved blood flow in mice intramuscularly injected with HIF-1-activated monocytes. This study therefore demonstrates that HIF-1 activation in myeloid cells promotes angiogenesis through VEGF and S100A8 and that this may become an attractive therapeutic strategy to treat diseases with vascular defects.

Association of reactive oxygen species levels and radioresistance in cancer stem cells.

The metabolism of oxygen, although central to life, produces reactive oxygen species (ROS) that have been implicated in processes as diverse as cancer, cardiovascular disease and ageing. It has recently been shown that central nervous system stem cells and haematopoietic stem cells and early progenitors contain lower levels of ROS than their more mature progeny, and that these differences are critical for maintaining stem cell function. We proposed that epithelial tissue stem cells and their cancer stem cell (CSC) counterparts may also share this property. Here we show that normal mammary epithelial stem cells contain lower concentrations of ROS than their more mature progeny cells. Notably, subsets of CSCs in some human and murine breast tumours contain lower ROS levels than corresponding non-tumorigenic cells (NTCs). Consistent with ROS being critical mediators of ionizing-radiation-induced cell killing, CSCs in these tumours develop less DNA damage and are preferentially spared after irradiation compared to NTCs. Lower ROS levels in CSCs are associated with increased expression of free radical scavenging systems. Pharmacological depletion of ROS scavengers in CSCs markedly decreases their clonogenicity and results in radiosensitization. These results indicate that, similar to normal tissue stem cells, subsets of CSCs in some tumours contain lower ROS levels and enhanced ROS defences compared to their non-tumorigenic progeny, which may contribute to tumour radioresistance.

Circulating mouse Flk1+/c-Kit+/CD45- cells function as endothelial progenitors cells (EPCs) and stimulate the growth of human tumor xenografts.

BACKGROUND: Endothelial progenitor cells (EPCs) have been demonstrated to have stem-cell like as well as mature endothelial functions. However, controversy remains as to their origins, immunophenotypic markings, and contribution to the tumor vascular network and tumor survival. METHODS: Flow cytometric analysis and sorting was used to isolate Flk-1+/c-Kit+/CD45- cells. Matrigel and methycellulose assays, flow cytometry, and gene array analyses were performed to characterize several murine EPC cell populations. Human tumor xenografts were used to evaluate the impact of EPCs on tumor growth and vascular development. RESULTS: Flk-1+/c-Kit+/CD45- cells were present at low levels in most murine organs with the highest levels in adipose, aorta/vena cava, and lung tissues. Flk-1+/c-Kit+/CD45- cells demonstrated stem cell qualities through colony forming assays and mature endothelial function by expression of CD31, uptake of acLDL, and vascular structure formation in matrigel. High passage EPCs grown in vitro became more differentiated and lost stem-cell markers. EPCs were found to have hemangioblastic properties as demonstrated by the ability to rescue mice given whole body radiation. Systemic injection of EPCs increased the growth of human xenograft tumors and vessel density. CONCLUSIONS: Flk-1+/C-Kit+/CD45- cells function as endothelial progenitor cells. EPCs are resident in most murine tissue types and localize to human tumor xenografts. Furthermore, the EPC population demonstrates stem-cell and mature endothelial functions and promoted the growth of tumors through enhanced vascular network formation. Given the involvement of EPCs in tumor development, this unique host-derived population may be an additional target to consider for anti-neoplastic therapy.

The role of apoptosis in cancer development and treatment response.

The inactivation of programmed cell death, or apoptosis, is central to the development of cancer. This disabling of apoptotic responses might be a major contributor both to treatment resistance and to the observation that, in many tumours, apoptosis is not the main mechanism for the death of cancer cells in response to common treatment regimens. Importantly, this suggests that other modes of cell death are involved in the response to therapy.

Inhibition of Mac-1 (CD11b/CD18) enhances tumor response to radiation by reducing myeloid cell recruitment.

Despite recent advances in radiotherapy, loco-regional failures are still the leading cause of death in many cancer patients. We have previously reported that bone marrow-derived CD11b(+) myeloid cells are recruited to tumors grown in irradiated tissues, thereby restoring the vasculature and tumor growth. In this study, we examined whether neutralizing CD11b monoclonal antibodies could inhibit the recruitment of myeloid cells into irradiated tumors and inhibit their regrowth. We observed a significant enhancement of antitumor response to radiation in squamous cell carcinoma xenografts in mice when CD11b antibodies are administered systemically. Histological examination of tumors revealed that CD11b antibodies reduced infiltration of myeloid cells expressing S100A8 and matrix metalloproteinase-9. CD11b antibodies further inhibited bone marrow-derived cell adhesion and transmigration to C166 endothelial cell monolayers and chemotactic stimuli, respectively, to levels comparable to those from CD11b knockout or CD18 hypomorphic mice. Given the clinical availability of humanized CD18 antibodies, we tested two murine tumor models in CD18 hypomorphic or CD11b knockout mice and found that tumors were more sensitive to irradiation when grown in CD18 hypomorphic mice but not in CD11b knockout mice. When CD18 hypomorphism was partially rescued by reconstitution with the wild-type bone marrow, the resistance of the tumors to irradiation was restored. Our study thus supports the rationale of using clinically available Mac-1 (CD11b/CD18) antibodies as an adjuvant therapy to radiotherapy.

Elusive identities and overlapping phenotypes of proangiogenic myeloid cells in tumors.

It is now established that bone marrow-derived myeloid cells regulate tumor angiogenesis. This was originally inferred from studies of human tumor biopsies in which a positive correlation was seen between the number of tumor-infiltrating myeloid cells, such as macrophages and neutrophils, and tumor microvessel density. However, unequivocal evidence was only provided once mouse models were used to examine the effects on tumor angiogenesis by genetically or pharmacologically targeting myeloid cells. Since then, identifying the exact myeloid cell types involved in this process has proved challenging because of myeloid cell heterogeneity and the expression of overlapping phenotypic markers in tumors. As a result, investigators often simply refer to them now as "bone marrow-derived myeloid cells." Here we review the findings of various attempts to phenotype the myeloid cells involved and discuss the therapeutic implications of correctly identifying-and thus being able to target-this proangiogenic force in tumors.

Radiation Damage to Tumor Vasculature Initiates a Program That Promotes Tumor Recurrences.

This review, mostly of preclinical data, summarizes the evidence that radiation at doses relevant to radiation therapy initiates a pathway that promotes the reconstitution of the tumor vasculature leading to tumor recurrence. The pathway is not specific to tumors; it promotes repair of damaged and ischemic normal tissues by attracting proangiogenic cells from the bone marrow. For irradiated tumors the pathway comprises: (1) loss of endothelial cells and reduced tumor blood perfusion leading to increased tumor hypoxia and increased levels of hypoxia inducible factor-1 (HIF-1). Alternatively, increased HIF-1 levels may arise by reactive oxygen species (ROS) production caused by tumor reoxygenation. (2) Increased HIF-1 levels lead to increased levels in the tumor of the chemokine stromal cell-derived factor-1 (SDF-1, CXCL12), which captures monocytes/macrophages expressing the CXCR4 receptor of CXCL12. (3) The increased levels of tumor-associated macrophages (TAMs) become highly proangiogenic (M2 polarized) and restore the tumor vasculature, thereby promoting tumor recurrence. The relevance of this pathway for radiation therapy is that it can be blocked in a number of different ways including by inhibitors of monocytes/macrophages, of HIF-1, of CXCL12, of CXCR4, and of CSF-1R, the latter of which is responsible for the M2 polarization of the TAMs. All of these inhibitors produce a robust enhancement of the radiation response of a wide variety of preclinical tumor models. Further, the same inhibitors actually provide protection against radiation damage of several normal tissues. Some of these pathway inhibitors are available clinically, and a first-in-human trial of the CXCR4 inhibitor, plerixafor, with radiation therapy of glioblastoma has yielded promising results, including an impressive increase in local tumor control. Further clinical trials are warranted.

Macrophage exclusion after radiation therapy (MERT): A new and effective way to increase the therapeutic ratio of radiotherapy.

Here we review a variety of preclinical studies and a first-in-human clinical trial of newly diagnosed glioblastoma (GBM) patients that have investigated the significance of the influx of tumor associated macrophages (TAMs) into tumors after irradiation. We summarize the effects on the response of the tumors and normal tissues to radiation of various agents that either reduce the influx of TAMs into tumors after radiation or change their M1/M2 polarization. The studies show that following irradiation there is an accumulation of bone marrow derived TAMs in the irradiated tumors. These TAMs stimulate the resumption of blood flow in the irradiated tumors thereby promoting recurrence of the tumors. A key mechanism for this accumulation of TAMs is driven by the SDF-1/CXCR4 chemokine pathway though other pathways could also be involved for some tumors. Blocking this pathway to prevent the TAM accumulation in the tumors both enhances tumor response to radiation and protects irradiated tissues. A clinical trial in which the CXCR4 antagonist plerixafor was added to standard therapy of glioblastoma validated the preclinical findings by demonstrating i) reduced blood flow in the irradiated site, and ii) significantly improved tumor local control compared to GBM patients not treated with plerixafor. We conclude that macrophage exclusion after radiation therapy (MERT) is an effective way both to enhance the tumor response to radiation and to protect the irradiated normal tissues. Further clinical trials are warranted.

Role of endothelial progenitors and other bone marrow-derived cells in the development of the tumor vasculature.

Increasing evidence suggests the importance of bone marrow-derived cells for blood vessel formation (neovascularization) in tumors, which can occur in two mechanisms: angiogenesis and vasculogenesis. Angiogenesis results from proliferation and sprouting of existing blood vessels close to the tumor, while vasculogenesis is believed to arise from recruitment of circulating cells, largely derived from the bone marrow, and de novo clonal formation of blood vessels from these cells. Although bone marrow-derived cells are crucial for neovascularization, current evidence suggests a promotional role of these cells on the existing blood vessels rather than de novo neovascularization in tumors. This is believed to be due to the highly proangiogenic features of these cells. The bone marrow-derived cells are heterogeneous, consisting of many different cell types including endothelial progenitor cells, myeloid cells, lymphocytes, and mesenchymal cells. These cells are highly orchestrated under the influence of the specific tumor microenvironment, which varies depending on the tumor type, thereby tightly regulating neovascularization in the tumors. In this review, we highlight some of the recent findings on each of these cell types by outlining some of the essential proangiogenic cytokines that these cells secrete to promote tumor angiogenesis and vasculogenesis.

Targeting the Post-Irradiation Tumor Microenvironment in Glioblastoma via Inhibition of CXCL12.

Radiotherapy is a mainstay in glioblastoma therapy as it not only directly targets tumor cells but also depletes the tumor microvasculature. The resulting intra-tumoral hypoxia initiates a chain of events that ultimately leads to re-vascularization, immunosuppression and, ultimately, tumor-regrowth. The key component of this cascade is overexpression of the CXC-motive chemokine ligand 12 (CXCL12), formerly known as stromal-cell derived factor 1 (SDF-1). We here review the role of CXCL12 in recruitment of pro-vasculogenic and immunosuppressive cells and give an overview on future and current drugs that target this axis.

Macrophage Exclusion after Radiation Therapy (MERT): A First in Human Phase I/II Trial using a CXCR4 Inhibitor in Glioblastoma.

PURPOSE: Preclinical studies have demonstrated that postirradiation tumor revascularization is dependent on a stromal cell-derived factor-1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4)-driven process in which myeloid cells are recruited from bone marrow. Blocking this axis results in survival improvement in preclinical models of solid tumors, including glioblastoma (GBM). We conducted a phase I/II study to determine the safety and efficacy of Macrophage Exclusion after Radiation Therapy (MERT) using the reversible CXCR4 inhibitor plerixafor in patients with newly diagnosed glioblastoma. PATIENTS AND METHODS: We enrolled nine patients in the phase I study and an additional 20 patients in phase II using a modified toxicity probability interval (mTPI) design. Plerixafor was continuously infused intravenously via a peripherally inserted central catheter (PICC) line for 4 consecutive weeks beginning at day 35 of conventional treatment with concurrent chemoradiation. Blood serum samples were obtained for pharmacokinetic analysis. Additional studies included relative cerebral blood volume (rCBV) analysis using MRI and histopathology analysis of recurrent tumors. RESULTS: Plerixafor was well tolerated with no drug-attributable grade 3 toxicities observed. At the maximum dose of 400 µg/kg/day, biomarker analysis found suprathreshold plerixafor serum levels and an increase in plasma SDF-1 levels. Median overall survival was 21.3 months [95% confidence interval (CI), 15.9-NA] with a progression-free survival of 14.5 months (95% CI, 11.9-NA). MRI and histopathology support the mechanism of action to inhibit postirradiation tumor revascularization. CONCLUSIONS: Infusion of the CXCR4 inhibitor plerixafor was well tolerated as an adjunct to standard chemoirradiation in patients with newly diagnosed GBM and improves local control of tumor recurrences.

In vivo 1H magnetic resonance spectroscopy of lactate in patients with stage IV head and neck squamous cell carcinoma.

PURPOSE: To investigate in vivo(1)H magnetic resonance spectroscopy imaging of lactate for assessing tumor hypoxia in head and neck cancers and to determine its utility in predicting the response and outcomes. METHODS AND MATERIALS: Volume-localized lactate-edited (1)H magnetic resonance spectroscopy at 1.5 T was performed in vivo on involved neck nodes and control subcutaneous tissues in 36 patients with Stage IV head and neck cancer. The signal intensities (SIs) of lactate, choline, and creatine and the choline/creatine ratio were measured. The tumor partial pressure of oxygen (pO(2)) was obtained in the same lymph node before MRS. Patients were treated with either two cycles of induction chemotherapy (tirapazamine, cisplatin, 5-fluorouracil) followed by simultaneous chemoradiotherapy or the same regimen without tirapazamine. The lactate SI and the choline/creatine ratio correlated with the tumor pO(2), nodal response, and locoregional control. RESULTS: The lactate SI was greater for the involved nodes (median, 0.25) than for the subcutaneous tissue (median, 0.04; p = 0.07). No significant correlation was found between the lactate SI and tumor pO(2) (mean, 0.46 +/- 0.10 for hypoxic nodes [pO(2) < or =10 mm Hg, n = 15] vs. 0.36 +/- 0.07 for nonhypoxic nodes [pO(2) >10 mm Hg, n = 21], p = 0.44). A significant correlation was found between the choline/creatine ratios and tumor pO(2) (mean, 2.74 +/- 0.34 for hypoxic nodes vs. 1.78 +/- 0.31 for nonhypoxic nodes, p = 0.02). No correlation was found between the lactate SI and the complete nodal response (p = 0.52) or locoregional control rates. CONCLUSIONS: The lactate SI did not correlate with tumor pO(2), treatment response, or locoregional control. Additional research is needed to refine this technique.

A role for Lsmlp in response to ultraviolet-radiation damage in Saccharomyces cerevisiae.

A genome-wide screen in Saccharomyces cerevisiae identified LSM1 as a new gene affecting sensitivity to ultraviolet (UV) radiation. Lsmlp is a member of a cytoplasmic complex composed of Lsmlp-7p that interacts with the yeast mRNA degradation machinery. To investigate the potential role of Lsmlp in response to UV radiation, we constructed double mutant strains in which LSM1 was deleted in combination with a representative gene from each of three known yeast DNA repair pathways. Our results show that lsm1delta increases the UV-radiation sensitivity of the rad1delta and rad51delta mutants, but not the radl8delta mutant, placing LSM1 within the post-replication repair/damage tolerance pathway (PRR). When combined with other deletions affecting PRR, lsm1delta increases the UV-radiation sensitivity of the rev3delta, rad30delta and pol30-K164R mutants but not rad5delta. Furthermore, the UV-radiation sensitivity phenotype of lsmldelta is partially rescued by mutations in genes involved in 3' to 5' mRNA degradation, and mutations predicted to function in RNA interactions confer the most UV-radiation sensitivity. Together, these results suggest that Lsmlp may confer protection against UV-radiation damage by protecting the 3' ends of mRNAs from exosome-dependent 3' to 5' degradation as part of a novel RAD5-mediated, PCNA-K164 ubiquitylation-independent subpathway of PRR.

Radiosensitization of yeast cells by inhibition of histone h4 acetylation.

Deletion of genes for proteins involved in histone H4 acetylation produces sensitivity to DNA-damaging agents in both Saccharomyces cerevisiae and mammalian cells. In the present studies, we show that treating wild-type yeast cells with histone acetyl transferase (HAT) inhibitors, which are chemicals that cause a global decrease in histone H4 acetylation, sensitizes the cells to ionizing radiation. Using HAT inhibitors, we have placed histone H4 acetylation into the RAD51-mediated homologous recombination repair pathway. We further show that yeast cells with functionally defective HAT proteins have normal phospho-H2A (gamma-H2A) induction after irradiation but a reduced rate of loss of gamma-H2A. This argues that HAT-defective cells are able to detect DNA double-strand breaks normally but have a defect in the repair of these lesions. We also show that cells treated with HAT inhibitors have intact G1 and G2 checkpoints after exposure to ionizing radiation, suggesting that G1 and G2 checkpoint activation is independent of histone H4 acetylation.

Dynamin impacts homology-directed repair and breast cancer response to chemotherapy.

After the initial responsiveness of triple-negative breast cancers (TNBCs) to chemotherapy, they often recur as chemotherapy-resistant tumors, and this has been associated with upregulated homology-directed repair (HDR). Thus, inhibitors of HDR could be a useful adjunct to chemotherapy treatment of these cancers. We performed a high-throughput chemical screen for inhibitors of HDR from which we obtained a number of hits that disrupted microtubule dynamics. We postulated that high levels of the target molecules of our screen in tumors would correlate with poor chemotherapy response. We found that inhibition or knockdown of dynamin 2 (DNM2), known for its role in endocytic cell trafficking and microtubule dynamics, impaired HDR and improved response to chemotherapy of cells and of tumors in mice. In a retrospective analysis, levels of DNM2 at the time of treatment strongly predicted chemotherapy outcome for estrogen receptor-negative and especially for TNBC patients. We propose that DNM2-associated DNA repair enzyme trafficking is important for HDR efficiency and is a powerful predictor of sensitivity to breast cancer chemotherapy and an important target for therapy.

18F-EF5 PET-based Imageable Hypoxia Predicts Local Recurrence in Tumors Treated With Highly Conformal Radiation Therapy.

PURPOSE: Tumor hypoxia contributes to radiation resistance. A noninvasive assessment of tumor hypoxia would be valuable for prognostication and possibly selection for hypoxia-targeted therapies. 18F-pentafluorinated etanidazole (18F-EF5) is a nitroimidazole derivative that has demonstrated promise as a positron emission tomography (PET) hypoxia imaging agent in preclinical and clinical studies. However, correlation of imageable hypoxia by 18F-EF5 PET with clinical outcomes after radiation therapy remains limited. METHODS AND MATERIALS: Our study prospectively enrolled 28 patients undergoing radiation therapy for localized lung or other tumors to receive pretreatment 18F-EF5 PET imaging. Depending on the level of 18F-EF5 tumor uptake, patients underwent functional manipulation of tumor oxygenation with either carbogen breathing or oral dichloroacetate followed by repeated 18F-EF5 PET. The hypoxic subvolume of tumor was defined as the proportion of tumor voxels exhibiting higher 18F-EF5 uptake than the 95th percentile of 18F-EF5 uptake in the blood pool. Tumors with a hypoxic subvolume ≥ 10% on baseline 18F-EF5 PET imaging were classified as hypoxic by imaging. A Cox model was used to assess the correlation between imageable hypoxia and clinical outcomes after treatment. RESULTS: At baseline, imageable hypoxia was demonstrated in 43% of all patients (12 of 28), including 6 of 16 patients with early-stage non-small cell lung cancer treated with stereotactic ablative radiation therapy and 6 of 12 patients with other cancers. Carbogen breathing was significantly associated with decreased imageable hypoxia, while dichloroacetate did not result in a significant change under our protocol conditions. Tumors with imageable hypoxia had a higher incidence of local recurrence at 12 months (30%) than those without (0%) (P < .01). CONCLUSIONS: Noninvasive hypoxia imaging by 18F-EF5 PET identified imageable hypoxia in about 40% of tumors in our study population. Local tumor recurrence after highly conformal radiation therapy was higher in tumors with imageable hypoxia.

Tumor hypoxia in cancer therapy.

Human solid tumors are invariably less well-oxygenated than the normal tissues from which they arose. This so-called tumor hypoxia leads to resistance to radiotherapy and anticancer chemotherapy as well as predisposing for increased tumor metastases. In this chapter, we examine the resistance of tumors to radiotherapy produced by hypoxia and, in particular, address the question of whether this resistance is the result of the physicochemical free radical mechanism that produces resistance to radiation killing of cells in vitro. We conclude that a major part of the resistance, though perhaps not all, is the result of the physicochemical free radical mechanism of the oxygen effect in sensitizing cells to ionizing radiation. However, in modeling studies used to evaluate the effect of fractionated irradiation on tumor response, it is essential to consider the fact that the tumor cells are at a wide range of oxygen concentrations, not just at the extremes of oxygenated and hypoxic. Prolonged hypoxia of the tumor tissue also leads to necrosis, and necrotic regions are also characteristic of solid tumors. These two characteristics--hypoxia and necrosis--represent clear differences between tumors and normal tissues and are potentially exploitable in cancer treatment. We discuss strategies for exploiting these differences. One such strategy is to use drugs that are toxic only under hypoxic conditions. The second strategy is to take advantage of the selective induction under hypoxia of the hypoxia-inducible factor (HIF)-1. Gene therapy strategies based on this strategy are in development. Finally, tumor hypoxia can be exploited using live obligate anaerobes that have been genetically engineered to express enzymes that can activate nontoxic prodrugs into toxic chemotherapeutic agents.