Contralateral breast dose with electronic compensators and conventional tangential fields - A clinical dosimetric study.

Dose to the contralateral breast (CLB) from radiotherapy treatment has the potential to induce secondary breast cancer. Electronic tissue compensation (eComp) for breast cancer patients is one of the alternative methods to conventional 3D-conformal radiotherapy that eliminates the use of wedges. Several studies have investigated dose to the CLB using tangential fields involving wedges, intensity-modulated radiation therapy and volumetric modulated arc radiation therapy and various other techniques via treatment planning system calculations, Monte Carlo methods and phantoms. However, there are limited data published in assessing the actual dose received by the CLB from treatment with eComp-based tangential fields. In this study, the CLB dose for patients undergoing tangential field radiotherapy with eComp and enhanced dynamic wedged (standard) tangential fields was measured and compared to assess the CLB dose between the two methods. Measurements were conducted on a randomised trial of 40 patients, 20 of them had undergone standard planning, and the remaining 20 were treated with eComp. The mean surface dose measured with TLDs at 3cm from the medial tangential border for eComp and standard techniques was 10.04±1.37% and 10.14±2.05%, respectively for a prescription dose of 2.65Gy/fraction. The estimated dose at 1cm depth in tissue, measured with the use of perspex domes placed over the TLD at the same location, was 5.12±0.87% and 6.29±2.01% for eComp and standard, respectively. The CLB dose is dependent on the proximity of the medial tangential field edge to the contralateral breast and is patient-specific. The results of this study show that at 1cm depth, eComp technique delivers significantly less dose (p<0.05) to the CLB as compared to standard tangential fields. Furthermore, the surface dose measured for both eComp and standard are comparable indicating that the eComp-based tangential field technique does not contribute any excess dose to CLB when compared to standard tangential fields. The excess relative risk (ERR) for radiation-induced cancers for eComp was found to be 0.08, compared to 0.11 for standard tangential fields.

Regulatory T Cells Shape the Differential Impact of Radiation Dose-Fractionation Schedules on Host Innate and Adaptive Antitumor Immune Defenses.

PURPOSE: We examined how radiation dose per fraction (DPF) and total dose, as represented by biological effective dose (BED), can independently and differentially affect the immunomodulatory capacity of radiation therapy (RT). METHODS AND MATERIALS: AT3-OVA mammary and MC38 colorectal tumors in C57BL/6 mice were irradiated with rationally selected dose-fractionation schedules, alone or with immune-modulating or -depleting agents. Tumor growth was monitored as a readout of therapeutic response. Flow cytometry and RNA sequencing of mouse tumors and analysis of transcriptomic data sets from irradiated human cancers were used to examine the immunomodulatory effects of the different radiation schedules. RESULTS: In AT3-OVA tumors, radiation DPF rather than BED determined the ability of RT to evoke local antitumor CD8+ T cell responses and synergize with anti-PD-1 therapy. Natural killer cell-mediated control of irradiated tumors was more sensitive to radiation BED. Radiation-induced regulatory T cell (Treg) responses, which were detected in both mouse and human tumors, were a major factor underlying the differential activation of adaptive immunity by radiation DPF and the activity of natural killer cells during the early phase of response to RT. Targeted inhibition of Treg responses within irradiated tumors rescued and enhanced local tumor control by RT and permitted the generation of abscopal and immunologic memory responses, irrespective of radiation schedule. MC38 tumors did not support the induction of an amplified Treg response to RT and were highly vulnerable to its immunoadjuvant effects. CONCLUSIONS: Local radiation-induced Treg responses are influenced by radiation schedule and tumor type and are a critical determinant of the immunoadjuvant potential of RT and its ability to synergize with T cell-targeted immunotherapy.

Follistatin attenuates radiation-induced fibrosis in a murine model.

PURPOSE: Fibrosis can be a disabling, severe side effect of radiotherapy that can occur in patients, and for which there is currently no effective treatment. The activins, proteins which are members of the TGFß superfamily, have a major role in stimulating the inflammatory response and subsequent fibrosis. Follistatin is an endogenous protein that binds the activins virtually irreversibly and inhibits their actions. These studies test if follistatin can attenuate the fibrotic response using a murine model of radiation-induced fibrosis. EXPERIMENTAL DESIGN: C57BL/6 mice were subcutaneously injected with follistatin 24 hours prior to irradiation. Mice were irradiated in a 10 x 10 mm square area of the right hind leg with 35 Gy and were given follistatin 24 hours before radiation and three times a week for six months following. Leg extension was measured, and tissue was collected for histological and molecular analysis to evaluate the progression of the radiation-induced fibrosis. RESULTS: Leg extension was improved in follistatin treated mice compared to vehicle treated mice at six months after irradiation. Also, epidermal thickness and cell nucleus area of keratinocytes were decreased by the follistatin treatment compared to the cells in irradiated skin of control mice. Finally, the gene expression of transforming growth factor ß1 (Tgfb1), and smooth muscle actin (Acta2) were decreased in the irradiated skin and Acta2 and inhibin ßA subunit (Inhba) were decreased in the irradiated muscle of the follistatin treated mice. CONCLUSIONS: Follistatin attenuated the radiation-induced fibrotic response in irradiated mice. These studies provide the data to support further investigation of the use of follistatin to reduce radiation-induced fibrosis in patients undergoing radiotherapy for cancer.

Building immunity to cancer with radiation therapy.

Over the last decade there has been a dramatic shift in the focus of cancer research toward understanding how the body's immune defenses can be harnessed to promote the effectiveness of cytotoxic anti-cancer therapies. The ability of ionizing radiation to elicit anti-cancer immune responses capable of controlling tumor growth has led to the emergence of promising combination-based radio-immunotherapeutic strategies for the treatment of cancer. Herein we review the immunoadjuvant properties of localized radiation therapy and discuss how technological advances in radio-oncology and developments in the field of tumor-immunotherapy have started to revolutionize the therapeutic application of radiotherapy.

The curative outcome of radioimmunotherapy in a mouse breast cancer model relies on mTOR signaling.

Radiotherapy is a successful treatment modality for localized cancer. Our group has been exploring radiotherapy in combination with immunotherapy (radioimmunotherapy) to enhance systemic antitumor responses. Previously, we have shown that when local radiotherapy was combined with monoclonal antibodies (mAbs) (that enable T-cell responses by engaging costimulation [anti (α)-CD137] and blocking coinhibition [α-PD-1] [corrected], up to 100% of mice bearing established syngeneic AT-3 mammary tumors were cured, but single modality treatments were not curative. Here, we investigated the molecular mechanisms underlying responses to this radioimmunotherapy approach. We observed that inhibition of signaling through the mammalian target of rapamycin (mTOR) pathway during the first 10 days of treatment severely impaired the curative effect of radioimmunotherapy, at least in part by reducing MHC class I expression on tumor cells, reducing dendritic cell (DC) activation status and CD8+ T-cell function. This data indicates that the efficacy of this type of radioimmunotherapy approach involves mTOR signaling and therefore, mTOR inhibitory drugs may impede the efficacy of similar radioimmunotherapy approaches in humans.

Enhanced lithium-induced brain recovery following cranial irradiation is not impeded by inflammation.

Radiation-induced brain injury occurs in many patients receiving cranial radiation therapy, and these deleterious effects are most profound in younger patients. Impaired neurocognitive functions in both humans and rodents are associated with inflammation, demyelination, and neural stem cell dysfunction. Here we evaluated the utility of lithium and a synthetic retinoid receptor agonist in reducing damage in a model of brain-focused irradiation in juvenile mice. We found that lithium stimulated brain progenitor cell proliferation and differentiation following cranial irradiation while also preventing oligodendrocyte loss in the dentate gyrus of juvenile mice. In response to inflammation induced by radiation, which may have encumbered the optimal reparative action of lithium, we used the anti-inflammatory synthetic retinoid Am80 that is in clinical use in the treatment of acute promyelocytic leukemia. Although Am80 reduced the number of cyclooxygenase-2-positive microglial cells following radiation treatment, it did not enhance lithium-induced neurogenesis recovery, and this alone was not significantly different from the effect of lithium on this proinflammatory response. Similarly, lithium was superior to Am80 in supporting the restoration of new doublecortin-positive neurons following irradiation. These data suggest that lithium is superior in its restorative effects to blocking inflammation alone, at least in the case of Am80. Because lithium has been in routine clinical practice for 60 years, these preclinical studies indicate that this drug might be beneficial in reducing post-therapy late effects in patients receiving cranial radiotherapy and that blocking inflammation in this context may not be as advantageous as previously suggested.

Radiotherapy increases the permissiveness of established mammary tumors to rejection by immunomodulatory antibodies.

It is becoming increasingly evident that radiotherapy may benefit from coincident or subsequent immunotherapy. In this study, we examined whether the antitumor effects of radiotherapy, in established triple-negative breast tumors could be enhanced with combinations of clinically relevant monoclonal antibodies (mAb), designed to stimulate immunity [anti-(α)-CD137, α-CD40] or relieve immunosuppression [α-programmed death (PD)-1]. While the concomitant targeting of the costimulatory molecules CD137 and CD40 enhanced the antitumor effects of radiotherapy and promoted the rejection of subcutaneous BALB/c-derived 4T1.2 tumors, this novel combination was noncurative in mice bearing established C57BL/6-derived AT-3 tumors. We identified PD-1 signaling within the AT-3 tumors as a critical limiting factor to the therapeutic efficacy of α-CD137 therapy, alone and in combination with radiotherapy. Strikingly, all mice bearing established orthotopic AT-3 mammary tumors were cured when α-CD137 and α-PD-1 mAbs were combined with single- or low-dose fractionated radiotherapy. CD8+ T cells were essential for curative responses to this combinatorial regime. Interestingly, CD137 expression on tumor-associated CD8+ T cells was largely restricted to a subset that highly expressed PD-1. These CD137+PD-1High CD8+ T cells, persisted in irradiated AT-3 tumors, expressed Tim-3, granzyme B and Ki67 and produced IFN-γ ex vivo in response to phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation. Notably, radiotherapy did not deplete, but enriched tumors of functionally active, tumor-specific effector cells. Collectively, these data show that concomitant targeting of immunostimulatory and inhibitory checkpoints with immunomodulatory mAbs can enhance the curative capacity of radiotherapy in established breast malignancy.

EGFR blockade with ZD1839 ("Iressa") potentiates the antitumor effects of single and multiple fractions of ionizing radiation in human A431 squamous cell carcinoma. Epidermal growth factor receptor.

PURPOSE: Signaling pathways initiated by the epidermal growth factor receptor (EGFR) play important roles in the response to ionizing radiation. In this study the consequences of inhibiting the EGFR on the response of A431 cells (human vulvar squamous cell carcinoma cells that overexpress EGFR) to radiation, were investigated in vitro and in vivo, using the selective EGFR-tyrosine kinase inhibitor, ZD1839 ("Iressa"). METHODS AND MATERIALS: The effect of ZD1839 on proliferation, apoptosis, and clonogenic survival after radiation was determined in vitro. For in vivo studies, athymic nude mice with established subcutaneous A431 xenografts (approximately 100 mm(3)) were treated with either a single 10 Gy fraction or 4 daily 2.5 Gy fractions of radiation with or without ZD1839 (75 mg/kg/day intraperitoneally for 10 days) to determine effects on tumor growth delay. RESULTS: Treatment of A431 cells with ZD1839 in vitro reduced proliferation, increased apoptosis, and reduced clonogenic survival after radiation. Strikingly greater than additive effects of ZD1839 in combination with radiation on tumor growth delay were observed in vivo after either a single 10 Gy fraction (enhancement ratio: 1.5) or multiple 4 x 2.5 Gy fractions (enhancement ratio: 4). ZD1839 reduced tumor vascularity, as well as levels of vascular endothelial growth factor (VEGF) protein and mRNA induced by stimulation with epidermal growth factor (EGF), suggesting a possible role of inhibition of angiogenesis in the effect. CONCLUSIONS: Inhibiting EGFR-mediated signal transduction cascades with ZD1839 potentiates the antitumor effect of single and multiple fractions of radiation. These data provide preclinical rationale for clinical trials of EGFR inhibitors including ZD1839 in combination with radiation.