Unirradiated Sites Research Articles

Prognostic significance of fludeoxyglucose positron emission tomography delta radiomics following bridging therapy in patients with large B-cell lymphoma undergoing CAR T-cell therapy.

Bridging radiation therapy (bRT) is increasingly being utilized prior to chimeric antigen receptor (CAR) T-cell therapy for large B-cell lymphoma (LBCL). It is unknown how the extent of cytoreduction during bRT impacts outcomes. We retrospectively reviewed patients with LBCL treated with bRT followed by CAR T-cell therapy. Metabolic tumor volume (MTV), maximum standardized uptake value (SUVmax), SUVmean, and total lesion glycolysis (TLG) were extracted from F18-fluorodeoxyglucose positron emission tomography (PET) scans acquired prior to bRT and between completion of bRT and CAR T-cell infusion. Delta radiomics based on changes of these values were then calculated. The association between delta radiomics and oncologic outcomes [progression-free survival (PFS), freedom from distant progression (FFDP), and local control (LC)] were then examined. Thirty-three sites across 23 patients with LBCL were irradiated. All metabolically active disease was treated in 10 patients. Following bRT, median overall decreases (including unirradiated sites) in MTV, SUVmax, SUVmean, and TLG were 22.2 cc (63.1%), 8.9 (36.8%), 3.4 (31.1%), and 297.9 cc (75.8%), respectively. Median decreases in MTV, SUVmax, SUVmean, and TLG in irradiated sites were 15.6 cc (91.1%), 17.0 (74.6%), 6.8 (55.3%), and 157.0 cc (94.6%), respectively. Median follow-up was 15.2 months. A decrease in SUVmax of at least 54% was associated with improved PFS (24-month PFS: 83.3% vs. 28.1%; p = 0.037) and FFDP (24-month FFDP: 100% vs. 62.4%; p < 0.001). A decrease in MTV of at least 90% was associated with improved FFDP (24-month FFDP: 100% vs. 62.4%; p < 0.001). LC was improved in sites with decreases in SUVmax of at least 71% (24-month LC: 100% vs. 72.7%; p < 0.001). Decreases of MTV by at least 90% (100% vs. 53.3%; p = 0.038) and TLG by at least 95% (100% vs. 56.3%; p = 0.067) were associated with an improved complete response rate. bRT led to substantial reductions in MTV, SUVmax, SUVmean, and TLG. The relative extent of these decreases correlated with improved outcomes after CAR T-cell infusion. Prospective cohorts should validate the value of interim PET following bRT for quantifying changes in disease burden and associated prognosis.

Radiotherapy upregulated immune checkpoints contribute to the development of second primary OSCC.

Radiation injury is common after radiotherapy for head and neck cancer. Radiotherapy can reshape the immune microenvironment and cause immunosuppression, including dysregulation of immune checkpoints (ICs). However, the relationship between oral ICs expression after radiation and the development of second primary tumors is unclear. Clinical specimens of second primary oral squamous cell carcinoma after radiotherapy (s-OSCC) and primary OSCC (p-OSCC) were collected. The expression and prognostic value of PD-1, VISTA, and TIM-3 were analyzed using immunohistochemistry. To further clarify the relationship between radiation and ICs alteration, a rat model was constructed to explore the spatiotemporal changes of ICs in the oral mucosa after radiation. In carcinoma tissue, the expression of TIM-3 was higher in s-OSCC than in p-OSCC, while the expression of PD-1 and VISTA was similar between the groups. In para-carcinoma tissue, the expression of PD-1, VISTA, and TIM-3 was higher in s-OSCC. High ICs expression was associated with poor survival. In the rat model, ICs were locally upregulated in the irradiated tongue. Moreover, there was a bystander effect, in which the ICs were also upregulated in the unirradiated site. Radiation may upregulate ICs expression in oral mucosa and contribute to the development of s-OSCC.

Anti-4-1BB immunotherapy enhances systemic immune effects of radiotherapy to induce B and T cell-dependent anti-tumor immune activation and improve tumor control at unirradiated sites.

Radiation therapy (RT) can prime and boost systemic anti-tumor effects via STING activation, resulting in enhanced tumor antigen presentation and antigen recognition by T cells. It is increasingly recognized that optimal anti-tumor immune responses benefit from coordinated cellular (T cell) and humoral (B cell) responses. However, the nature and functional relevance of the RT-induced immune response are controversial, beyond STING signaling, and agonistic interventions are lacking. Here, we show that B and CD4+ T cell accumulation at tumor beds in response to RT precedes the arrival of CD8+ T cells, and both cell types are absolutely required for abrogated tumor growth in non-irradiated tumors. Further, RT induces increased expression of 4-1BB (CD137) in both T and B cells; both in preclinical models and in a cohort of patients with small cell lung cancer treated with thoracic RT. Accordingly, the combination of RT and anti-41BB therapy leads to increased immune cell infiltration in the tumor microenvironment and significant abscopal effects. Thus, 4-1BB therapy enhances radiation-induced tumor-specific immune responses via coordinated B and T cell responses, thereby preventing malignant progression at unirradiated tumor sites. These findings provide a rationale for combining RT and 4-1bb therapy in future clinical trials.

Investigating Metabolic Response after CAR-T Therapy and Bridging Radiotherapy for Relapsed/Refractory B-Cell Lymphoma

Introduction: For patients with aggressive relapsed/refractory B-cell lymphoma (R/R BCL) undergoing CAR-T therapy, metabolic treatment response assessed at the 1-month interval following CAR-T infusion is associated with likelihood of disease progression (Kuhnl A, et al. Blood Adv 2022). In particular, SUV max on 1-month 18F-FDG positron emission tomography (PET) imaging is predictive of disease progression (Zaki A, et al. Blood Adv 2022). However, for patients who undergo bridging radiotherapy (RT) prior to CAR-T infusion, it remains unclear how to interpret metabolic activity for irradiated sites on surveillance PET imaging following CAR-T. We sought to characterize metabolic activity after CAR-T and bridging RT in a cohort of patients with R/R BCL. Methods: This IRB-approved retrospective cohort study included patients with R/R BCL who underwent bridging RT prior to CAR-T infusion at a single institution from January 1, 2019 to April 30, 2022. We measured baseline patient characteristics, treatment factors, and PET metrics prior to bridging RT. We then assessed tumor metabolic activity on surveillance PET imaging following CAR-T infusion until the time of disease progression (defined as Deauville 5 disease) or last follow up. Results: We identified 10 patients who completed bridging RT prior to CAR-T infusion with at least one post-CAR-T disease assessment with PET imaging. Median follow up was 152 days from CAR-T infusion (range: 31-1095). Most patients (90%) had bulky disease (≥7.5 cm) and all had elevated LDH at baseline. Bridging RT dosing ranged from 20-38.5 Gy (median: 20 Gy) over 5-14 fractions (median: 5 fractions). Bridging RT fields were directed at bulky disease sites for 8 of 9 patients with bulky disease. The median metabolic tumor volume (MTV) irradiated with bridging RT was 149 mL (range: 26-803) and the median proportion of total MTV irradiated was 97% (range: 8.7-100). The median time between pre-bridging RT PET imaging and CAR-T infusion was 49 days (range: 37-124) and the median time between the end of bridging RT and CAR-T infusion was 12 days (range: 5-106). On pre-bridging RT PET, the median SUV max in the irradiated field was 23.3 (range: 9.1-36.1) (Figure 1). On 1-month post-CAR-T PET, the median SUV max in the irradiated field decreased to 3.2 (range: 0-6.1) and median delta SUV max was -87% (range: -68% to -100%). At the 1-month post-CAR-T interval, 40% of patients had a complete metabolic response (Deauville 3 or below), 40% had Deauville 4 disease, and 20% had Deauville 5 disease. Among the 4 patients with Deauville 4 disease at 1-month post-CAR-T, 3 patients developed complete metabolic responses and 1 patient had persistent Deauville 4 disease on subsequent PET imaging. No patients in the cohort developed disease progression within the irradiated fields. At last follow up, 3 patients had developed Deauville 5 disease, and for 2 of the 3 patients, areas of disease progression included unirradiated sites of disease that were present on pre-bridging RT PET imaging. Conclusions: In this small retrospective cohort of patients undergoing bridging RT prior to CAR-T therapy for R/R BCL, patients with concern for refractory disease on initial imaging assessment demonstrated complete response or stable disease on subsequent follow up imaging. Caution may be warranted in interpreting metabolic activity levels on initial post-CAR-T surveillance PET imaging, and validation in larger cohorts is needed to better understand metabolic activity dynamics in disease sites treated with bridging RT prior to CAR-T therapy. Figure 1. SUV Max in Disease Sites Treated with Bridging RT* *Each line corresponds to one patient treated with bridging RT prior to CAR-T therapy. SUV max plotted on the vertical axis reflects metabolic activity within the irradiated fields. Time intervals indicated in months on the horizontal axis reflect the approximate time from CAR-T infusion with the exact range in days indicated in parentheses. The magnification box demonstrates metabolic activity at the 1-month post-CAR-T interval with corresponding Deauville score indicated by color. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Irradiation Combined with PD-1 Inhibitor Aggravates Immune-Related Pneumonitis of the Non-Irradiated Lung in a Preclinical Model

<h3>Purpose/Objective(s)</h3> The combination of radiation with PD-1 inhibitors for lung cancer is a trend. However, the combination is limited by immune-related adverse effects, particularly checkpoint inhibitor pneumonitis (CIP). The incidence and the mechanism of CIP of the non-irradiated lung caused by radiation plus PD-1 inhibitors remain unknown. This study was designed to evaluate the incidence of CIP and lung tissue morphology in the preclinical model induced by radiation combined with a PD-1 inhibitor (αPD-1). <h3>Materials/Methods</h3> Fifty-four C57BL/6 mice were divided randomly into the normal group, the irradiation combined with the αPD-1 group (RT + αPD-1 group), and the single irradiation group (RT group). Mice in the RT + αPD-1 group were treated with right lung irradiation (8 Gy once) on day1 and anti-mouse PD-1 monoclonal antibody three times a week. Mice in the RT group received right lung irradiation alone. The mice were euthanized on day10, day15, and day20 after the end RT. Lung tissue morphology and pathological changes were assessed by hematoxylin-eosin staining. Lung fibrosis was evaluated by Masson staining and analysis of hydroxyproline. <h3>Results</h3> Histopathological examination revealed that the inflammation in the irradiated lung is more severe than the unirradiated site. With the extension of time, the lung inflammation of mice in the RT group and RT + αPD-1 group was significantly aggravated, and the whole lung fibrosis appeared on day20. Inflammatory infiltrate scores, alveoli deformation scores, collagen volume fractions, and hydroxyproline contents in lung tissues were all significantly higher in mice administered αPD-1 plus irradiation than in the other groups. In particular, the inflammation is significantly worse in the unirradiated site of the mice administered PD-1 inhibitor plus irradiation than in the RT group on day15 and day20. <h3>Conclusion</h3> Radiotherapy combined with PD-1 inhibitors could aggravate CIP on both sides. Further studies on the mechanism are under investigation. Our findings highlight the potential risk of CIP in non-irradiated lung in patients treated with radiotherapy and PD-1 inhibitors.

Irradiation combined with PD-1 inhibitor aggravates immune-related pneumonitis of the non-irradiated lung in a preclinical model.

e14569 Background: The combination of radiation with PD-1 inhibitors for lung cancer is a trend. However, the combination is limited by immune-related adverse effects, particularly checkpoint inhibitor pneumonitis (CIP). The incidence and the mechanism of CIP of the non-irradiated lung caused by radiation plus PD-1 inhibitors remain unknown. This study was designed to evaluate the incidence of CIP and lung tissue morphology in the preclinical model induced by radiation combined with a PD-1 inhibitor (αPD-1). Methods: Fifty-four C57BL/6 mice were divided randomly into the normal group, the irradiation combined with the αPD-1 group (RT + αPD-1 group), and the single irradiation group (RT group). Mice in the RT + αPD-1 group were treated with right lung irradiation (8 Gy once) on day1 and anti-mouse PD-1 monoclonal antibody three times a week. Mice in the RT group received right lung irradiation alone. The mice were euthanized on day10, day15, and day20 after the end RT. Lung tissue morphology and pathological changes were assessed by hematoxylin-eosin staining. Lung fibrosis was evaluated by Masson staining and analysis of hydroxyproline. Results: Histopathological examination revealed that the inflammation in the irradiated lung is more severe than the unirradiated site. With the extension of time, the lung inflammation of mice in the RT group and RT + αPD-1 group was significantly aggravated, and the whole lung fibrosis appeared on day20. Inflammatory infiltrate scores, alveoli deformation scores, collagen volume fractions, and hydroxyproline contents in lung tissues were all significantly higher in mice administered αPD-1 plus irradiation than in the other groups. In particular, the inflammation is significantly worse in the unirradiated site of the mice administered PD-1 inhibitor plus irradiation than in the RT group on day15 and day20. Conclusions: Radiotherapy combined with PD-1 inhibitors could aggravate CIP on both sides. Further studies on the mechanism are under investigation. Our findings highlight the potential risk of CIP in non-irradiated lung in patients treated with radiotherapy and PD-1 inhibitors. Research Sponsor: This work was supported by the National Natural Science Foundation of China [No. 81972853, No. 81572279] and Clinical Research Startup Program of Southern Medical University by High-level University Construction Funding of Guangdong Provincial Department of Education (LC2019ZD009).

501 Administration of nicotinamide riboside (NR) or pterostilbene (PT) to mice inhibits suppression of contact hypersensitivity (CHS) by mid-range ultraviolet radiation (UVR)

Topical administration of PT inhibits some acute effects of mid-range UVR (UVBR, 280-320 nm) in mouse skin and oral nicotinamide reduces the rate of new non-melanoma skin cancers in high-risk patients. NR+PT inhibits UVBR-induced tissue swelling in mice and does so more effectively than either agent at the dose tested. We have now asked if NR, PT or the combination can inhibit UVBR suppression of CHS in 2 models. In the low-dose model, mice receive a UVBR dose too low to cause gross changes in skin, followed by immunization at the irradiated site, resulting in suppressed CHS. In the high-dose model, mice receive a dose sufficient to cause gross skin changes followed by immunization at an unirradiated site, also with suppressed CHS. C3H mice were placed in groups that were fed standard diet/water or chow containing 0.08% PT, water with 0.4% NR or both for 4 wks. In low-dose experiments, mice in each group had their dorsa shaved and depilated. Half in each group were exposed to 1,700 J/m2 of UVBR daily for 4 d while the other half were mock-irradiated. Mice were immunized on the exposed dorsum with 25 μl of 0.5% dinitrofluorobenzene (DNFB) 4 h after the last irradiation. Mice were challenged 7 d later with 10 μl of 0.2% DNFB to each ear and 24-h ear swelling assessed. CHS in mice not given PT, NR or both was significantly reduced by UVBR exposure compared to mock-irradiated mice. In mice fed NR, PT, or both the UVBR effect was significantly less pronounced. In a preliminary high-dose experiment, groups of mice were treated similarly except that a single dose of 10,000 J/m2 of radiation was administered and immunization was performed on the unirradiated shaved abdomen 3 d later. In this experiment, the UVBR effect was also significantly less pronounced in the NR, PT or NR+PT groups. NR and PT both inhibited UVB-induced immune suppression at the doses administered as did the combination.

Regeneration of Locally Irradiated Bone Marrow I. Dose Dependent, Long-Term Changes in the Rat, with Particular Emphasis upon Vascular and Stromal Reaction

Locally irradiated bone marrow was uniformly depressed during the first week after radiation doses of 2000 to 10,000 rads. The early appearance of hemopoietic regeneration between 7 and 14 days at all dose levels suggested an influx of stem cells from unirradiated sites. Between 2 and 6 months post-irradiation a second wave of hemopoietic depression occurred at all dose levels and was correlated with the disappearance of the sinusoidal microcirculation. Between 6 and 12 months diffuse hemopoietic regeneration occurred only at the 2000 rad level and could be correlated with regeneration of the sinusoidal microcirculation. No significant hemopoiesis or sinusoidal regeneration was observed at the higher dose levels after 6 months or 1 year. Although large vascular channels developed, indicating circulation through the marrow cavity, the characteristic and essential anatomy of the microcirculation was not restored.

Use of a Si/CdTe Compton Camera for In vivo Real-Time Monitoring of Annihilation Gamma Rays Generated by Carbon Ion Beam Irradiation.

The application of annihilation gamma-ray monitoring to the adaptive therapy of carbon ion radiotherapy (C-ion RT) requires identification of the peak intensity position and confirmation of activated elements with annihilation gamma-rays generated at the C-ion-irradiated site from those transported to unirradiated sites. Real-time monitoring of C-ion-induced annihilation gamma-rays was implemented using a Compton camera in a mouse model. An adult C57BL/6 mouse was anesthetized, and C-ion beams were directed into the abdomen at 1 × 109 particles/s for 20 s. The 511 keV annihilation gamma-rays, generated by the interaction between the irradiated C-ion beam and the target mouse, were detected using a silicon/cadmium telluride (Si/CdTe) Compton camera for 20 min immediately after irradiation. The irradiated site and the peak intensity position of 511 keV gamma emissions due to C-ion beam irradiation on a mouse were observed at the abdomen of the mouse by developing Compton images. Moreover, the positron emitter transport was observed by evaluating the range of gamma-ray emission after the C-ion beam irradiation on the mouse. Our data suggest that by confirming the peak intensity and beam range of C-ion RT with Si/CdTe-based Compton camera, it would be possible to reduce the intra-fractional and inter-fractional dose distribution degradation. Therefore, the results of this study would contribute to the future development of adaptive therapy with C-ion RT for humans.

Role of Metastatic Site Irradiation in Pediatric Patients With Metastatic Ewing Sarcoma.

The use of radiation therapy to treat metastases in patients with metastatic Ewing sarcoma (MES) has been controversial and variable. The authors report outcomes and patterns of failure after metastatic site irradiation (MSI). A total of 27 pediatric patients with MES were treated with chemotherapy and received radiation therapy to their primary site. Ten patients additionally received MSI, which consisted of whole-lung irradiation (WLI) in patients with lung metastases. Metastatic sites were followed from diagnosis to the first relapse. Median follow-up was 29 months. Seventy-eight percent of patients relapsed. Two-year progression-free survival (PFS) and overall survival with and without MSI were 30 versus 29% (log rank P=0.38) and 60 versus 70% (log rank P=0.11), respectively. The median time to relapse among patients who relapsed was 19.5 versus 12.3 months for those receiving MSI versus those who did not (P=0.04).Seven of 20 (35%) patients with lung metastases received WLI±other MSI. Two-year PFS with and without MSI was 43% versus 23% (log rank P=0.02). Among patients with a complete response to computed tomography, 5 of 14 (36%) patients received MSI. Two-year PFS with and without MSI was 60% versus 33% (log rank P=0.04).In the cohort of patients who relapsed, among all metastatic sites at diagnosis, the disease recurred at 15% of irradiated sites and 31% of unirradiated sites. On logistic regression, no factors were statistically associated with increased risk of recurrence at initial sites of metastases. Relapses frequently occur at sites of prior unirradiated disease in patients with MES. WLI may improve 2-year PFS, regardless of chemotherapy response. Further investigation of the role of MSI is warranted.

A Prospective, Multicenter Study of Involved-Field Radiation Therapy With Autologous Stem Cell Transplantation for Patients With Hodgkin Lymphoma and Aggressive Non-Hodgkin Lymphoma (ALLG HDNHL04/TROG 03.03)

The role of involved-field radiation therapy (IFRT) with autologous stem cell transplantation (ASCT) for lymphomas remains uncertain. In this prospective, multicenter study, patients undergoing ASCT for relapsed/refractory lymphoma received peritransplant IFRT to disease sites identified at study registration (SR) (before salvage chemotherapy [SC]). Radiation dose was adapted to SC response. Survival, relapse rates/pattern, toxicity, and prognostic factors were evaluated. Forty-five patients were enrolled (23 with Hodgkin lymphoma, 22 with aggressive non-Hodgkin lymphoma). Three-year overall survival and cumulative incidence of posttransplant progression rates were 72% (95% confidence interval [CI], 59%-87%) and 42% (95% CI, 27%-57%), respectively. Stage (P=.03) and elevated lactate dehydrogenase (P=.05) were significant risk factors for disease progression on multivariable analysis. Three-year actuarial in-field, marginal, and distant progression rates were 7% (95% CI, 0%-15%), 9% (95% CI, 0%-18%), and 36% (95% CI, 21%-51%), respectively. Progression occurred in 8 of 30 patients with all sites irradiated and in 13 of 15 patients without all sites irradiated. There were 117 disease sites at SR and 64 post-ASCT progression sites, of which 15 were involved at SR and 12 only at initial diagnosis. Posttransplant relapse occurred in 3 of 83 irradiated and 12 of 34 unirradiated involved sites. Of 28 sites in complete response to SC on computed tomography, there was no relapse in any of the 21 irradiated sites and in 1 of 7 unirradiated sites. Of 72 sites in complete response on positron emission tomography, relapse occurred in 1 of 50 irradiated and 10 of 22 unirradiated sites. No grade 4 nonhematologic radiation therapy toxicities were observed. IFRT was well tolerated and associated with a low rate of in-field progression. Progression rates were lower for patients with all disease sites irradiated. Response to SC on both computed tomography and positron emission tomography warrants further study to select sites for IFRT.

Prognostic factors and patterns of failure in advanced stage Hodgkin lymphoma treated with combined modality therapy

Background and purposeThe role of irradiation to non-bulky and bulky sites of disease in advanced stage Hodgkin lymphoma is controversial. We aimed to review the long-term outcomes of patients treated with combined modality therapy to clarify the role of consolidative radiotherapy. Materials and methodsPatients with stage III or IV Hodgkin lymphoma treated with Stanford V chemotherapy and consolidative radiotherapy to initial sites of disease ≥5 cm were analyzed retrospectively to determine patient outcomes, patterns of failure, and factors associated with treatment failure. ResultsA total of 170 patients were analyzed. Overall survival was 91.2%, freedom from progression was 80.6%, and progression-free survival was 78.9% at 10 years. 5 patients (2.9%) had refractory disease and 27 patients (15.9%) relapsed after treatment. Only an International Prognostic Score (IPS) greater than 2 predicted disease progression. 19 out of 27 relapses occurred exclusively outside of the radiation treatment field, and 17 out of 27 relapses occurred exclusively at original sites of disease. However, only 11 of 170 patients (6.5%) relapsed exclusively at original, non-bulky sites of disease not treated with radiation therapy. The cumulative incidence of local failure at 10 years was 4.6% for unirradiated sites and 2.6% for irradiated sites. ConclusionPatients with advanced stage Hodgkin lymphoma treated with combined modality therapy including consolidative radiotherapy to bulky disease sites had excellent long-term outcomes. Given the low frequency of isolated failures at initial sites, our results suggest that selective radiation therapy to sites at high risk of relapse may be feasible.

The role of granulocyte macrophage colony stimulating factor (GM-CSF) in radiation-induced tumor cell migration.

Recently it has been observed in preclinical models that that radiation enhances the recruitment of circulating tumor cells to primary tumors, and results in tumor regrowth after treatment. This process may have implications for clinical radiotherapy, which improves control of a number of tumor types but which, despite continued dose escalation and aggressive fractionation, is unable to fully prevent local recurrences. By irradiating a single tumor within an animal bearing multiple lesions, we observed an increase in tumor cell migration to irradiated and unirradiated sites, suggesting a systemic component to this process. Previous work has identified the cytokine GM-CSF, produced by tumor cells following irradiation, as a key effector of this process. We evaluated the ability of systemic injections of a PEGylated form of GM-CSF to stimulate tumor cell migration. While increases in invasion and migration were observed for tumor cells in a transwell assay, we found that daily injections of PEG-GM-CSF to tumor-bearing animals did not increase migration of cells to tumors, despite the anticipated changes in circulating levels of granulocytes and monocytes produced by this treatment. Combination of PEG-GM-CSF treatment with radiation also did not increase tumor cell migration. These findings suggest that clinical use of GM-CSF to treat neutropenia in cancer patients will not have negative effects on the aggressiveness of residual cancer cells. However, further work is needed to characterize the mechanism by which GM-CSF facilitates systemic recruitment of trafficking tumor cells to tumors.

Strategies for combining immunotherapy with radiation for anticancer therapy.

Radiation therapy controls local disease but also prompts the release of tumor-associated antigens and stress-related danger signals that primes T cells to promote tumor regression at unirradiated sites known as the abscopal effect. This may be enhanced by blocking inhibitory immune signals that modulate immune activity through a variety of mechanisms. Indeed, abscopal responses have occurred in patients with lung cancer or melanoma when given anti-CTLA4 antibody and radiation. Other approaches involve expanding and reinfusing T or NK cells or engineered T cells to express receptors that target specific tumor peptides. These approaches may be useful for immunocompromised patients receiving radiation. Preclinical and clinical studies are testing both immune checkpoint-based strategies and adoptive immunotherapies with radiation.

Abstract 2941: Local tumor irradiation combined with α-PDL-1 immune checkpoint inhibition results in local and systemic anti-tumor responses: Successful translation of a mouse model to a human case series

Abstract Introduction: Tumor irradiation induces innate and adaptive immune responses which, rarely, lead to tumor regression at distant sites, the abscopal effect. We have previously demonstrated that immunotherapy including Toll-like-receptor agonists (CpG) and checkpoint inhibitors (anti-CTLA4) both preclinically and clinically (NCT00185965 &amp; NCT01769222) can significantly increase the rate of systemic, abscopal responses (Kim, Blood 2012 &amp; Brody, JCO 2010). Here we provide the first report of a preclinical murine model and patient case series following local radiation and systemic anti-PD-L1 (NCT01375842). Methods: Preclinical modeling was performed in a two-tumor, syngenic, A20, lymphoma BALB/c model combining fractionated single tumor radiation and systemic (i.p.) anti-PD-L1. Patients receiving MPDL3280A, a human mAb containing an engineered Fc-domain, as part of the phase 1 clinical trial with mixed responses or asymptomatic progression of disease were eligible for the addition of local radiation therapy. Murine and human immune responses including cell phenotype and function, specifically assessing expression of PD-L1 and production of IFNγ were determined by standard flow cytometry and time of flight mass cytometry (CyTOF). Results: Fractionated radiation delayed tumor growth at the treated site only, and systemic anti-PD-L1 reduced tumor growth rate at both sites, however despite prolonged survival all mice died by day 38 following either monotherapy (radiation or anti-PD-L1). In contrast, combination local fractionated radiation and systemic anti-PD-L1 flattened tumor growth at both the irradiated and un-irradiated site, and prolonged survival with 50% survival at day 48 post-tumor inoculation. Modulation of PD-L1 expression post-radiation and tumor-specific augmentation of IFNγ secretion correlated with the enhanced anti-tumor activity. Five patients including four with solid tumors received fractionated, non-definitive dose radiation with at least stabilization of systemic progression in all patients and a RECIST partial response at systemic sites in 1 patient, notably with a synovial sarcoma. Transient, grade 1-2 inflammatory adverse events (fevers, flu-like symptoms) occurred with no DLTs or serious immune-related toxicities. Modulation of PD-L1 expression, T cell phenotype and IFNγ secretion was observed and updated clinical and immune response will be presented. Conclusion: We provide the first report of combination local radiotherapy with anti-PD-L1 demonstrating synergy in a preclinical model and clinical activity in a limited case series. The magnitude of the immune response and abscopal response rate in mice and humans provides proof-of-concept that anti-PD-L1 may be an equally if not more potent combination immunotherapy with radiation compared to our experience with CpG and/or anti-CTLA4. Citation Format: Idit Sagiv-Barfi, Amanda Rajapaksa, Debra Czerwinski, Serena Chang, Jonathan Hebb, Cariad Chester, Erin Waller, Gregg Fine, Daniel Chen, Marcin Kowanetz, Bryan Irving, Ronald Levy, Holbrook Kohrt. Local tumor irradiation combined with α-PDL-1 immune checkpoint inhibition results in local and systemic anti-tumor responses: Successful translation of a mouse model to a human case series. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2941. doi:10.1158/1538-7445.AM2014-2941

Likelihood of Bone Recurrence in Prior Sites of Metastasis in Patients With High-Risk Neuroblastoma

Despite recent improvements in outcomes, 40% of children with high-risk neuroblastoma will experience relapse, facing a guarded prognosis for long-term cure. Whether recurrences are at new sites or sites of original disease may guide decision making during initial therapy. Eligible patients were retrospectively identified from institutional databases at first metastatic relapse of high-risk neuroblastoma. Included patients had disease involving metaiodobenzylguanidine (MIBG)-avid metastatic sites at diagnosis and first relapse, achieved a complete or partial response with no more than one residual MIBG-avid site before first relapse, and received no total body irradiation or therapy with (131)I-MIBG before first relapse. Anatomically defined metastatic sites were tracked from diagnosis through first relapse to determine tendency of disease to recur at previously involved versus uninvolved sites and to assess whether this pattern was influenced by site irradiation. Of 159 MIBG-avid metastatic sites identified among 43 patients at first relapse, 131 (82.4%) overlapped anatomically with the set of 525 sites present at diagnosis. This distribution was similar for bone sites, but patterns of relapse were more varied for the smaller subset of soft tissue metastases. Among all metastatic sites at diagnosis in our subsequently relapsed patient cohort, only 3 of 19 irradiated sites (15.8%) recurred as compared with 128 of 506 (25.3%) unirradiated sites. Metastatic bone relapse in neuroblastoma usually occurs at anatomic sites of previous disease. Metastatic sites identified at diagnosis that did not receive radiation during frontline therapy appeared to have a higher risk of involvement at first relapse relative to previously irradiated metastatic sites. These observations support the current paradigm of irradiating metastases that persist after induction chemotherapy in high-risk patients. Furthermore, they raise the hypothesis that metastatic sites appearing to clear with induction chemotherapy may also benefit from radiotherapeutic treatment modalities (external beam radiation or (131)I-MIBG).

TH‐A‐BRB‐04: Vascular Response to Microbeam Radiation Therapy in Vivo Using a Murine Window Chamber Tumor Model

Microbeam Radiation Therapy (MRT) has shown a marked tumor‐specific effect. The tissue‐sparing property of this unique treatment is possibly facilitated by efficient normal‐vessel repair mechanisms, contrary to the catastrophic disruption of poorly regulated tumor‐associated vasculature. However, significant controversy exists with regard to the role of endothelial cell death in radiation response. It is possible that the direct killing of tumor vasculature may increase radiation response within the tumor due to either oxygen/nutrient deprivation or through the “bystander effect”. The aim of this study was to characterize the vascular response to MRT in vivo using a murine window chamber model over an extended time course. MRT treatment at various doses (300 micron single beam, 0Gy, 50Gy, 80Gy) was applied to the murine window chamber tumor model. Changes in tumor‐associated vasculature after the MRT treatment utilizing intravital microscopy were profound. A robust angiogenic response was clearly present along the microbeam track in what appeared to be clusters of vessel proliferation following MRT of 50 and 80Gy. This distinctive pattern of angiogenesis was imaged through Day 7, as the irradiated track and regional microenvironment were infiltrated with a dense vascular network. In more recent experiments, we have observed infiltration of tumor cells along pre‐existing vascular networks adjacent to the microbeam, to distant unirradiated sites. These types of responses were not seen in controls or in chambers following wide beam irradiation to the entire window at comparable doses. This behavior strongly suggests that the treatment is promoting epithelial mesenchymal transition, angiogenesis and local invasion. Although these observations are for microbeam treatments, the biology being observed could equally be applied to the issue of marginal miss, which is of concern for high dose conformal radiotherapy. The window chamber model provides a novel approach to direct visualization of the longitudinal vascular changes following treatment with MRT. These findings suggest that radiation induced angiogenesis at sites adjacent to the MRT beam may play an important role in modifying the tumor microenvironment. Such a response may have significant implications with regard to the understood mechanism of radiation‐induced cell death.Learning Objectives:1. Be able to describe concept of microbeam irradiation2. Understand underlying mechanisms and implications of driving angiogenic response of tumor after irradiationThis work was supported by NIH/NCI grant CA40355 and DoD grant BC083195.

Systemic Low-Dose UVB Inhibits CD8 T Cells and Skin Inflammation by Alternative and Novel Mechanisms

Exposure to UVB radiation before antigen delivery at an unirradiated site inhibits functional immunological responses. Mice treated dorsally with suberythemal low-dose UVB and immunized with ova in abdominal skin generated ova-specific CD8 T cells with a significantly decreased activation, expansion, and cytotoxic activity compared with unirradiated mice. UVB also impaired the delayed-type hypersensitivity (DTH) reaction to ova. Transfer of CD4⁺CD25⁺cells from UVB-exposed mice did not suppress the ova-specific CD8 T-cell response or DTH reaction in unexposed mice, confirming that systemic low-dose UVB does not induce long-lived functional regulatory CD4⁺CD25⁺ T cells. Repairing cyclobutane pyrimidine dimer-type DNA damage and blocking aryl hydrocarbon receptor signaling also did not reverse the immunosuppressive effect of UVB on ova-specific CD8 T cells and DTH, suggesting that cyclobutane pyrimidine dimers and the aryl hydrocarbon receptor are not required in systemic low-dose UVB-induced immunosuppression. The known UVB chromophore, cis-urocanic acid, and reactive oxygen species triggered the inhibition of DTH caused by UVB, but they were not involved in the modulation of CD8 T cells. These findings indicate that systemic low-dose UVB impedes the primary response of antigen-specific CD8 T cells by a novel mechanism that is independent of pathways known to be involved in systemic suppression of DTH.

Efficacy of Abbreviated Stanford V Chemotherapy and Involved Field Radiotherapy in Early Stage Hodgkin's Disease: Mature Results of the G4 Trial.

Abstract 1670Poster Board I-696The standard management for early stage Hodgkin's disease (HD) is combined modality therapy. In the G4 protocol, patients (pts) with non-bulky, supra-diaphragmatic stage I-IIA disease received 8 weeks of Stanford V chemotherapy + 30 Gy involved field radiotherapy (IFRT).87 pts were enrolled and treated between 4/1996 and 4/2001. Median age was 30 years (16-59) and stages were IA (n=23), IIA (n=64). Unfavorable risk factors were present in 47 patients (54%) according to German Hodgkin Study Group (GHSG) criteria (> 2 AA sites, ESR > 50 or EN involvement) and 38 (44%) according to EORTC criteria (> 3 AA sites, ESR > 50). Therapy was well tolerated with grade 3-4 non-hematologic toxic events in 7 % of pts. These included constipation (n=3), peripheral neuropathy (n=1), generalized weakness (n=2), chest pain (n=1), mylagias (n=1), abdominal pain (n=1) and an allergic reaction to etoposide (n=1). 42 patients received cytokine support for grade 3-4 neutropenia however only 2 pts developed fever with neutropenia. No cases of clinical bleomycin toxicity or radiation pneumonitis were observed. At a median follow-up of 9 (2-12) years, freedom from progression (FFP) and survival (OS) are 94% and 96% respectively. FFP was 100% for favorable and 89% for unfavorable patients by GHSG criteria (p=0.04) with no differences in OS (96.9% versus 95.7%). All relapses (n=5) occurred in the RT field: limited in 2 patients and combined with distant disease in 3 pts. All relapses were in “unfavorable” risk patients: 5 per GHSG and 4 per EORTC criteria. Secondary therapy included chemotherapy followed by high dose therapy and stem cell support (n=3) and ABVD (n=2). Three pts died, 2 due to disease progression after second-line therapy and one due to metastatic colon cancer. 5 patients developed a second cancer (2 breast, 2 melanomas at unirradiated sites and 1 colon cancer). The cases of breast cancer were considered unrelated to RT as both occurred within 5-years of therapy in women who were > 30 yrs at time of treatment. No secondary AML and no late cardiac or pulmonary toxicities have been observed.In conclusion, for pts with non-bulky stage I/II A HD, abbreviated Stanford V with 8 weeks of chemotherapy and 30 Gy IFRT is a safe and highly effective regimen. It is still too early to assess potential RT-related complications. Our outcomes in “unfavorable” stage I-IIA patients without bulky or symptomatic disease compare favorably with more intensive or prolonged regimens employed by the GHSG and EORTC. DisclosuresNo relevant conflicts of interest to declare.

Ultraviolet radiation reduces resistance to Mycobacterium tuberculosis infection in BCG-vaccinated guinea pigs

Ultraviolet radiation (UVR) in sunlight suppresses anti-microbial immunity and may impair vaccine inefficacy. To investigate whether UVR reduces the protective efficacy of BCG vaccine, guinea pigs were exposed to a single dose of 5.6, 11.2, or 22.5 kJ/m(2) of UVR either 3 days before or 3 or 28 days after BCG vaccination and then challenged by the aerosol route with virulent Mycobacterium tuberculosis 6 weeks later. Pre-challenge skin test responses to purified protein derivative (PPD) were significantly reduced in the UV-irradiated animals. Similarly, exposure to UVR reduced the proliferation of spleen cells to PPD, altered cytokine mRNA expression in spleen and lung digest cells, and increased the colony forming units (CFU) in the lungs of M. tuberculosis-infected guinea pigs at 5 weeks post-infection. Spleen IFN-gamma, TNF-alpha, IL-10 and IL-12p40 mRNA levels were higher when exposed to 5.6 kJ/m(2) UVR, while the levels were significantly reduced at 11.2 and 22.5 kJ/m(2). Lung cell IL-10 mRNA expression increased at all UVR doses. Thus, UVR exposure a few days before or after BCG vaccination at a distant unirradiated site impaired vaccine-induced resistance against virulent M. tuberculosis in guinea pigs possibly by altering cytokine responses in a UVR dose-dependent manner.