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Purpose: Randomized trials have found that patients with locoregionally advanced p16+ oropharyngeal squamous cell carcinoma (OPSCC) do not benefit from treatment deintensification, even among favorable risk groups. Although various methods have been used to identify candidates for treatment deintensification, the optimal approach is unknown., Methods and Materials: We conducted a multi-institutional cohort study of 444 patients with previously untreated p16+ OPSCC undergoing definitive radiation therapy with or without systemic therapy between 2009 and 2022. We compared the following 2 approaches for identifying candidates for deintensification: (1) favorable versus unfavorable risk, using NRG-HN005 eligibility criteria, and (2) low versus high relative risk of cancer events, using the Head and Neck Cancer Intergroup predictive classifier ("omega score"). We tested differences in outcomes and systemic therapy allocation by risk group using multivariable Cox models, competing event models, and logistic regression, and compared characteristics of hypothetical deintensification trials using the 2 approaches. Progression-free survival events were defined as cancer recurrence (locoregional or distant) or death from any cause., Results: Median follow-up time was 52 months; 120 patients (27.0%) were favorable risk; a different 120 patients had low omega score; 28 patients (6.3%) met both criteria; 184 patients (41.4%) had discordant classification. On ordinal logistic regression, decreasing omega score was associated with a statistically significantly lower odds of receiving intensive therapy (normalized odds ratio, 0.37 per SD; 95% CI, 0.24-0.57), with a greater magnitude than favorable risk group (odds ratio, 0.66; 95% CI, 0.44-0.99). Among patients receiving cisplatin and/or platinum-based induction (n = 374), favorable risk was associated with significantly improved progression-free survival (hazard ratio, 0.59; 95% CI, 0.36-0.99), whereas lower omega score was associated with a significantly decreased relative hazard for cancer events (relative hazard ratio, 0.18; 95% CI, 0.070-0.46). In simulations, selecting patients with low omega scores increased the efficiency of hypothetical noninferiority trials., Conclusions: Considering patients' relative risk of cancer events can help define optimal populations for treatment deintensification in p16+ OPSCC., (Published by Elsevier Inc.)

Adenoid cystic carcinoma is a malignancy that is difficult to treat and often metastasizes to the lung. Systemic chemotherapies are not effective for this tumor type, thus local therapies are frequently used. Here, we report a case demonstrating the use of extensive ablative interventions in controlling the progression of metastatic adenoid cystic carcinoma. A patient with adenoid cystic carcinoma developed numerous metastases to his lungs and liver. Local ablative therapies including interstitial brachytherapy and SBRT were used to treat approximately 80 different metastases over the course of a decade. Over 850 brachytherapy seeds were implanted in this patient, and the tumor control and patient outcome were good. As of the most recent follow-up in March 2024, the patient has survived for approximately 12 years since his diagnosis of adenoid cystic carcinoma. To our knowledge, this case represents the most brachytherapy treatments reported in a single patient. It highlights the utility of interstitial brachytherapy and SBRT in treating extensive lung and liver metastases.

There are over 220 identified genotypes of Human papillomavirus (HPV), and the HPV genome encodes 3 major oncogenes, E5, E6, and E7. Conservation and divergence in protein sequence and function between low-risk versus high-risk oncogenic HPV genotypes has not been fully characterized. Here, we used modern computational and structural folding algorithms to perform a comparative analysis of HPV E5, E6, and E7 between multiple low risk and high risk genotypes. We first identified significantly greater sequence divergence in E5 between low- and high-risk genotypes compared to E6 and E7. Next, we used AlphaFold to model the structure of papillomavirus proteins and complexes with high confidence, including some with no established consensus structure. We observed that HPV E5, but not E6 or E7, had a dramatically different 3D structure between low-risk and high-risk genotypes. To our knowledge, this is the first comparative analysis of HPV proteins using Alphafold artificial intelligence (AI) system. The marked differences in E5 sequence and structure in high-risk HPVs may contribute in important and underappreciated ways to the development of HPV-associated cancers., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: This research was supported by discretionary and startup funds. A.B. Sharabi reports being a paid consultant/advisory board member for AstraZeneca, Primmune, Merck, and Jounce Therapeutics; reports receiving commercial research grants from Varian Medical Systems and Pfizer; reports being the scientific founder and has an equity interest in Toragen Inc. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict-of-interest policies. A. Venuti is a scientific advisory board member for Toragen Inc. J.S. Mayadev reports consulting for Varian Medical Systems, Merck, Primmune, and AstraZeneca., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Neuroblastoma (NB) is considered an immunologically cold tumor and is usually less responsive to immune checkpoint blockade (ICB). Tumor-associated macrophages (TAMs) are highly infiltrated in NB tumors and promote immune escape and resistance to ICB. Hence therapeutic strategies targeting immunosuppressive TAMs can improve responses to ICB in NB. We recently discovered that spleen tyrosine kinase (Syk) reprograms TAMs toward an immunostimulatory phenotype and enhances T-cell responses in the lung adenocarcinoma model. Here we investigated if Syk is an immune-oncology target in NB and tested whether a novel immunotherapeutic approach utilizing Syk inhibitor together with radiation and ICB could provide a durable anti-tumor immune response in an MYCN amplified murine model of NB., Methods: Myeloid Syk KO mice and syngeneic MYCN-amplified cell lines were used to elucidate the effect of myeloid Syk on the NB tumor microenvironment (TME). In addition, the effect of Syk inhibitor, R788, on anti-tumor immunity alone or in combination with anti-PDL1 mAb and radiation was also determined in murine NB models. The underlying mechanism of action of this novel therapeutic combination was also investigated., Results: Herein, we report that Syk is a marker of NB-associated macrophages and plays a crucial role in promoting immunosuppression in the NB TME. We found that the blockade of Syk in NB-bearing mice markedly impairs tumor growth. This effect is facilitated by macrophages that become immunogenic in the absence of Syk, skewing the suppressive TME towards immunostimulation and activating anti-tumor immune responses. Moreover, combining FDA-approved Syk inhibitor, R788 (fostamatinib) along with anti-PDL1 mAb provides a synergistic effect leading to complete tumor regression and durable anti-tumor immunity in mice bearing small tumors (50 mm 3 ) but not larger tumors (250 mm 3 ). However, combining radiation to R788 and anti-PDL1 mAb prolongs the survival of mice bearing large NB9464 tumors., Conclusion: Collectively, our findings demonstrate the central role of macrophage Syk in NB progression and demonstrate that Syk blockade can "reeducate" TAMs towards immunostimulatory phenotype, leading to enhanced T cell responses. These findings further support the clinical evaluation of fostamatinib alone or with radiation and ICB, as a novel therapeutic intervention in neuroblastoma., Competing Interests: AS reports being a paid consultant/advisory board member for AstraZeneca, Primmune, Merck, and Jounce Therapeutics; reports receiving commercial research grants from Varian Medical Systems and Pfizer; holds ownership interest in Toragen Inc. outside of submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Rohila, Park, Pham, Jones, Tapia, Liu, Tamayo, Yu, Sharabi and Joshi.)

The role that human papillomavirus (HPV) oncogenes play in suppressing responses to immunotherapy in cancer deserves further investigation. In particular, the effects of HPV E5 remain poorly understood relative to E6 and E7. Here, we demonstrate that HPV E5 is a negative regulator of anti-viral interferon (IFN) response pathways, antigen processing, and antigen presentation. Using head and neck cancer as a model, we identify that E5 decreases expression and function of the immunoproteasome and that the immunoproteasome, but not the constitutive proteasome, is associated with improved overall survival in patients. Moreover, immunopeptidome analysis reveals that HPV E5 restricts the repertoire of antigens presented on the cell surface, likely contributing to immune escape. Mechanistically, we discover a direct interaction between E5 and stimulator of interferon genes (STING), which suppresses downstream IFN signaling. Taken together, these findings identify a powerful molecular mechanism by which HPV E5 limits immune detection and mediates resistance to immunotherapy., Competing Interests: Declaration of interests E.E.W.C. has received compensation from MSD and Roche for attending advisory boards and is on the scientific advisory board for Toragen Inc. A.B.S. reports research funding and honoraria from Pfizer and Varian Medical Systems, consultant fees from AstraZeneca and Primmune, and other fees from Merck. A.B.S. is the scientific founder and has an equity interest in Toragen Inc., which could potentially benefit from the research results. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict-of-interest policies., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Locally advanced cancers remain therapeutically challenging to eradicate. The most successful treatments continue to combine decades old non-targeted chemotherapies with radiotherapy that unfortunately increase normal tissue damage in the irradiated field and have systemic toxicities precluding further treatment intensification. Therefore, alternative molecularly guided systemic therapies are needed to improve patient outcomes when applied with radiotherapy. In this work, we report a trimodal precision cytotoxic chemo-radio-immunotherapy paradigm using spatially targeted auristatin warheads. Tumor-directed antibodies and peptides conjugated to radiosensitizing monomethyl auristatin E (MMAE) specifically produce CD8 T cell dependent durable tumor control of irradiated tumors and immunologic memory. In combination with ionizing radiation, MMAE sculpts the tumor immune infiltrate to potentiate immune checkpoint inhibition. Here, we report therapeutic synergies of targeted cytotoxic auristatin radiosensitization to stimulate anti-tumor immune responses providing a rationale for clinical translational of auristatin antibody drug conjugates with radio-immunotherapy combinations to improve tumor control., (© 2022. The Author(s).)

Background: To test whether nomograms developed by NRG Oncology for oropharyngeal squamous cell carcinoma (OPSCC) patients could be validated in an independent population-based sample., Methods: The authors tested nomograms for estimating progression-free survival (PFS) and overall survival (OS) in patients from the Veterans Health Administration with previously untreated locoregionally advanced OPSCC, diagnosed between 2008 and 2017, managed with definitive radiotherapy with or without adjuvant systemic therapy. Covariates were age, performance status, p16 status, T/N category, smoking history, education history, weight loss, marital status, and anemia. We used multiple imputation to handle missing data and performed sensitivity analyses on complete cases. Validation was assessed via Cox proportional hazards models, log-rank tests, and c-indexes., Results: A total of 4007 patients met inclusion criteria (658 patients had complete data). Median follow-up time was 3.20 years, with 967 progression events and 471 noncancer deaths. Each risk score was associated with poorer outcomes per unit increase (PFS score, hazard ratio [HR], 1.42 [1.37-1.47]; OS score, HR, 1.40 [1.34-1.45]). By risk score quartile, 2-year PFS estimates were 89.2%, 78.5%, 65.8%, and 48.3%; OS estimates were 92.6%, 83.6%, 73.9%, and 51.3%, respectively (P < .01 for all comparisons). C-indices for models of PFS and OS were 0.65 and 0.67, for all patients, respectively (0.69 and 0.73 for complete cases). The nomograms slightly overestimated PFS and OS in the overall cohort but exhibited high agreement in complete cases., Conclusions: NRG nomograms were effective for predicting PFS and OS for patients with OPSCC, supporting their broader applicability in the OPSCC population undergoing definitive radiotherapy., (© 2022 American Cancer Society.)

Purpose: Cancer treatments can paradoxically appear to reduce the risk of noncancer mortality in observational studies, due to residual confounding. Here we introduce a method, Bias Reduction through Analysis of Competing Events (BRACE), to reduce bias in the presence of residual confounding., Experimental Design: BRACE is a novel method for adjusting for bias from residual confounding in proportional hazards models. Using standard simulation methods, we compared BRACE with Cox proportional hazards regression in the presence of an unmeasured confounder. We examined estimator distributions, bias, mean squared error (MSE), and coverage probability. We then estimated treatment effects of high versus low intensity treatments in 36,630 prostate cancer, 4,069 lung cancer, and 7,117 head/neck cancer patients, using the Veterans Affairs database. We analyzed treatment effects on cancer-specific mortality (CSM), noncancer mortality (NCM), and overall survival (OS), using conventional multivariable Cox and propensity score (adjusted using inverse probability weighting) models, versus BRACE-adjusted estimates., Results: In simulations with residual confounding, BRACE uniformly reduced both bias and MSE. In the absence of bias, BRACE introduced bias toward the null, albeit with lower MSE. BRACE markedly improved coverage probability, but with a tendency toward overcorrection for effective but nontoxic treatments. For each clinical cohort, more intensive treatments were associated with significantly reduced hazards for CSM, NCM, and OS. BRACE attenuated OS estimates, yielding results more consistent with findings from randomized trials and meta-analyses., Conclusions: BRACE reduces bias and MSE when residual confounding is present and represents a novel approach to improve treatment effect estimation in nonrandomized studies., (©2022 American Association for Cancer Research.)

Purpose: B cells play a key role in outcomes of cancer patients and responses to checkpoint blockade immunotherapies. However, the effect of radiation therapy on B cell populations is poorly understood. Here we characterize the effects of radiation on the development, survival, and phenotype of physiological B-cell subsets., Methods and Materials: Naïve and immunized tumor bearing and nontumor bearing mice were treated with large-field or focal stereotactic radiation and distinct B-cell subsets of varying developmental stages were analyzed by flow cytometry and real-time reverse transcription polymerase chain reaction., Results: We first report that focal stereotactic radiation is highly superior to large-field radiation at inducing tumor infiltration of B cells, CD8 +  T cells, and macrophages. We observed that radiation affects B cell development in the bone marrow, increasing frequencies of early pro-B cells and late pro-B cells while inducing upregulation of programmed cell death protein 1. We then demonstrate that class switched B cells and plasma cells are highly resistant to radiation therapy compared with naïve B cells and upregulate activation markers programmed cell death 1 ligand 2 and major histocompatibility complex class II) after radiation. Mechanistically, radiation upregulates Xbp1 and Bcl6 in plasma cells, conferring radioresistance. Furthermore, using an immunization approach, we demonstrate that radiation enhances activation-induced cytidine deaminase mediated class switching and somatic hypermutation in primed B cells., Conclusions: These data demonstrate that stereotactic radiation is superior to large-field radiation at inducing infiltration of immune cells into tumors and that plasma cells and class switched B cells are highly resistant to radiation therapy. These results represent the most comprehensive analysis of the effects of radiation on B cells to date and identify novel mechanisms by which radiation modulates immune cells within the tumor microenvironment., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Durable palliation of advanced lung cancer is a common objective for radiation oncologists. However, there is no consensus on how to deliver the radiation course. Herein we report our experience of using split course radiotherapy and our assessment of outcomes based on planning from three-dimensional (3D) simulation before each treatment course., Methods: All lung cancer patients from 2006-2020 were identified. Of these, 52 patients received a split course treatment of 50-60 Gy in 18-25 fractions intended to provide durable palliation for disease not amenable to curative therapy. Treatment involved 3D planning with repeat computed tomography (CT) simulation prior to the second course. Survival and symptomatic response were analyzed via chart review. We categorized rapid responders versus non-rapid responders from the initial radiation course based on ≥30% gross tumor volume (GTV) reduction at the second CT simulation. We evaluated the impact of response on overall survival and palliative response., Results: Among our cohort treated with split course palliative radiotherapy, 33 (63%) had a rapid response to initial treatment. There was no difference in survival between groups [hazard ratio (HR) =1.30, P=0.47]. There was no significant difference in palliative response rates between rapid and non-rapid responders. On multivariable analysis, only female sex (HR =0.26, P<0.01) and receipt of systemic therapy following radiotherapy (HR =0.19, P<0.01) were associated with improved survival., Conclusions: There is currently significant practice pattern variability for palliative lung radiotherapy. Split course palliative radiation of 50-60 Gy in 18-25 fractions represents an option to consider for patients with advanced lung cancer who do not undergo definitive therapy and may benefit from a higher dose regimen. Our retrospective review suggests that rapid tumor response in a split course model does not predict survival or symptomatic response. Prospective studies are needed to further define which lung cancer patients may benefit from higher dose regimens.

The role of B cells in the tumor microenvironment and B-cell-mediated antitumor immune responses remains relatively understudied. Recent seminal studies have discovered that B cells and associated tertiary lymphoid structures correlate with responses to checkpoint blockade immunotherapy and are prognostic for overall survival of cancer patients. B-cell subsets have remarkable functional diversity and include professional antigen-presenting cells, regulatory cells, memory populations, and antibody-producing plasma cells. Importantly, secreted antibodies can independently activate innate immune responses and induce the cancer immunity cycle. Thus, B cells and B-cell-mediated antibody responses comprise the largely underappreciated second arm of the adaptive immune system and certainly deserve further attention in the field of oncology. Here, we review the known functions of B cells in the tumor microenvironment, the contribution of B cells to the antitumor activity of immunotherapies, and the role of B cells in the overall survival of cancer patients., (©2021 American Association for Cancer Research.)

Background: Image guidance in radiation oncology has resulted in significant improvements in the accuracy and precision of radiation therapy (RT). Recently, the resolution and quality of cone beam computed tomography (CBCT) for image guidance has increased so that tumor masses and lymph nodes are readily detectable and measurable. During treatment of head and neck squamous cell carcinoma (HNSCC), on-board CBCT setup imaging is routinely obtained; however, this CBCT imaging data is not utilized to predict patient outcomes. Here, we analyzed whether changes in CBCT measurements obtained during a course of radiation therapy correlate with responses on routine 3-month follow-up diagnostic imaging and overall survival (OS)., Materials/methods: Patients with oropharyngeal primary tumors who received radiation therapy between 2015 and 2018 were included. Anatomical measurements were collected of largest nodal conglomerate (LNC) at CT simulation, end of radiation treatment (EOT CBCT), and routine 3-month post-RT imaging. At each timepoint anteroposterior (AP), mediolateral (ML) and craniocaudal (CC) measurements were obtained and used to create a 2-dimensional (2D) maximum., Results: CBCT data from 64 node positive patients were analyzed. The largest nodal 2D maximum and CC measurements on EOT CBCT showed a statistically significant correlation with complete response on 3-month post-RT imaging (r = 0.313, p = 0.02 and r = 0.318, p = 0.02, respectively). Furthermore, patients who experienced a 30% or greater reduction in the CC dimension had improved OS (Binary Chi-Square HR 4.85, p = 0.028)., Conclusion: Decreased size of pathologic lymph nodes measured using CBCT setup imaging during a radiation course correlates with long term therapeutic response and overall survival of HNSCC patients. These results indicate that CBCT setup imaging may have utility as an early predictor of treatment response in oropharyngeal HNSCC., (© 2021. The Author(s).)

Radiation therapy exerts a tumoricidal local effect as well as both local and systemic immunomodulation. Immune checkpoint blockade has become a widely used treatment modality across cancer types with a rapidly growing list of agents and US Food and Drug Administration-approved indications. Moreover, there may be synergy between radiation therapy and immune checkpoint blockade. Various strategies have been used, but the optimal sequencing of these therapies is unclear. In this review, the authors discuss the major mechanisms of available immune checkpoint inhibitors and explore the available preclinical and clinical evidence regarding treatment sequencing. They also review safety considerations and conclude with possible future directions., (© 2021 American Cancer Society.)

This patient was found to have a scirrhous carcinoma with extensive perineural invasion and without any evidence of minor salivary gland carcinoma. To our knowledge, this is the first report of isolated scirrhous carcinoma of the oral cavity. Treatment was surgery and adjuvant chemoradiation, and there was complete disease response., Competing Interests: The authors declare no conflicts of interest., (© 2021 The Authors. Clinical Case Reports published by John Wiley & Sons Ltd.)

Background: Radiation recall pneumonitis (RRP) is a poorly understood clinical syndrome in which patients develop radiation pneumonitis triggered by a systemic agent, often years after the completion of radiation therapy. Immune checkpoint blockade agents have only recently been posited as a trigger for RRP. Here, we present three cases of immunotherapy-induced RRP., Case Presentation: Our first patient was diagnosed with primary lung adenocarcinoma, and 4.5 years after completing radiation therapy developed symptomatic RRP immediately following a second dose of nivolumab-containing immunotherapy regimen. Our second patient was diagnosed with primary bladder cancer metastatic to the mediastinum, which was treated twice with radiation therapy. He developed RRP in the days following his second course of ipilimumab-pembrolizumab which was months after his second course of radiation that he received. Our final patient was diagnosed with metastatic small cell lung cancer and received local consolidative radiation therapy in addition to whole-brain radiation. He developed RRP on the 11 th day after concluding his 4 th cycle of nivolumab-ipilimumab, approximately 7 months after having had completed chest radiation therapy., Conclusions: Immunotherapy-induced RRP is a rare diagnosis which can present more focally than traditional immunotherapy pneumonitis and which must be clinically differentiated from other local processes such as pneumonia. Further research should explore the mechanisms underlying these radiation recall reactions as many patients receive radiation and immunotherapy during the course of their cancer treatment., Competing Interests: AS reports research funding and honoraria from Pfizer and Varian Medical Systems, and consultant fees from AstraZeneca and Merck. ABS is the scientific founder and has an equity interest in Toragen Inc. outside the submitted work. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Riviere, Sumner, Cornell, Sandhu, Murphy, Hattangadi-Gluth, Bruggeman, Kim, Randall and Sharabi.)

Novel therapies combined with radiation continue to be of significant interest in the developmental treatment paradigm of gynecologic cancers. Clinical implementation of immunotherapy in oncology has rapidly changed the treatment landscape, options, paradigm, and outcomes through clinical trials. Immunotherapy has emerged as a therapeutic pillar in the treatment of solid tumors with demonstrable synergistic activity when combined with radiation therapy and chemoradiotherapy by an alteration or enhancement of the immune system. In solid tumors, radiation therapy induces migration of dendritic cells, T cell activation, and proliferation, and increases in tumor-infiltrating lymphocytes. These immunomodulatory effects in conjunction with immune checkpoint blockade are currently under active investigation in the adjuvant, definitive, and metastatic settings. Results from early phase trials demonstrate promising efficacy and overall tolerable toxicity profiles of combined modality treatment. There is significant interest in optimizing the treatment for patients with locally advanced cervical cancer beyond the standard of care-chemoradiation-which has been in place for the last 30 years. The majority of cervical cancer emerges after persistent infection with a high-risk subtype of the human papillomavirus, where viral oncoproteins lead to cellular changes and immortalization. As a result, immune tolerance can develop, resulting in cancer. Knowledge of the mechanism of human papillomavirus-related oncogenesis suggests that immune therapy or checkpoint blockade can reinvigorate an antitumor immune response. Current clinical trials are exploring the therapeutic potential of these approaches. Uterine cancers have been grouped into 4 molecular subclasses by their driver mutations, mutational burden, and copy-number alterations. Of these subgroups, the polymerase epsilon-mutated and microsatellite-unstable may represent up to 40% of endometrial cancers, and they have been shown to be immunogenic. Because of the inherent immunogenicity of these MSI-high tumors, combined immune modulation strategies, including chemotherapy, radiation, and immunotherapy and immune checkpoint inhibitor therapy, are being explored to improve treatment outcomes. In this review, we explore current immunomodulatory and multimodality therapeutic approaches in the treatment of cervical and uterine cancer through ongoing clinical trials investigating the combination of immunotherapy and radiation therapy., (Published by Elsevier Inc.)

Higher tumor mutational burden (TMB) has been correlated with response to checkpoint blockade immunotherapy. However, it is unclear whether TMB independently serves as a prognostic biomarker for outcomes in immunotherapy-naïve patients. Here, we evaluated the relationship between TMB and overall survival in 1,415 immunotherapy-naïve patients with diverse advanced malignancies. TMB was studied both as a tiered variable (low ≤5 mutations/Mb, intermediate >5 and <20, high ≥20 and <50, and very high ≥50) and as a continuous variable. Interestingly, we observed a parabolic correlation between TMB and overall survival, in which intermediate-range TMB correlated with decreased survival, whereas low and very high TMB correlated with improved outcomes (median survival: 238, 174, 195, and 350 weeks for low, intermediate, high, and very high TMB, respectively; multivariate P < 0.01). This corresponded to an HR of 1.29 (95% confidence interval, 1.07-1.54; P < 0.01) for intermediate-range TMB on multivariable survival analysis correcting for known confounders, including primary tumor of origin. These results demonstrate that TMB may have utility as a prognostic biomarker in immunotherapy-naïve patients, with a protective effect at higher TMBs, and that studies of survival in immunotherapy-treated patients may need to stratify or randomize by TMB in a nonlinear fashion to account for this confounding., (©2020 American Association for Cancer Research.)

Outcomes for women with node-positive, recurrent, and metastatic cervical cancer remain poor. Persistent infection by the human papilloma virus is related to disordered interactions with the immune system and development of cervical cancer, making the resultant malignancy an attractive target for immunotherapy. Various types of immunomodulatory treatments have been studied, including a bacterial vaccine vector and T cell therapy. Immune checkpoint blockade has shown promise in the recurrent or metastatic settings, and in combination with chemoradiotherapy for definitive treatment with acceptable toxicity profiles. Ongoing trials are investigating timing, dosing, and combinations of immunomodulatory treatments, with potential to improve survival and advance our understanding of the immune system's role in combating cervical cancer., (Published by Elsevier Inc.)

Purpose: To characterize the role of B cells on human papilloma virus (HPV)-associated cancer patient outcomes and determine the effects of radiation and PD-1 blockade on B-cell populations., Experimental Design: Tumor RNA-sequencing data from over 800 patients with head and neck squamous cell carcinoma (HNSCC) and cervical cancer, including a prospective validation cohort, was analyzed to study the impact of B-cell gene expression on overall survival (OS). A novel murine model of HPV + HNSCC was used to study the effects of PD-1 blockade and radiotherapy on B-cell activation, differentiation, and clonality including analysis by single-cell RNA-sequencing and B-cell receptor (BCR)-sequencing. Human protein microarray was then used to quantify B-cell-mediated IgG and IgM antibodies to over 16,000 proteins in the serum of patients treated on a clinical trial with PD-1 blockade., Results: RNA-sequencing identified CD19 and IGJ as novel B-cell prognostic biomarkers for 3-year OS (HR, 0.545; P < 0.001). PD-1 blockade and radiotherapy enhance development of memory B cells, plasma cells, and antigen-specific B cells. BCR-sequencing found that radiotherapy enhances B-cell clonality, decreases CDR3 length, and induces B-cell somatic hypermutation. Single-cell RNA-sequencing identified dramatic increases in B-cell germinal center formation after PD-1 blockade and radiotherapy. Human proteome array revealed enhanced IgG and IgM antibody responses in patients who derived clinical benefit but not those with progressive disease after treatment with PD-1 blockade., Conclusions: These findings establish a key role for B cells in patient outcomes and responses to PD-1 blockade in HPV-associated squamous cell carcinomas and demonstrate the need for additional diagnostics and therapeutics targeting B cells., (©2020 American Association for Cancer Research.)

Purpose: Human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) is associated with daily marijuana use and is also increasing in parallel with increased marijuana use in the United States. Our study is designed to define the interaction between cannabinoids and HPV-positive HNSCC., Experimental Design: The expression of cannabinoid receptors CNR1 and CNR2 was analyzed using The Cancer Genome Atlas (TCGA) HNSCC data. We used agonists, antagonists, siRNAs, or shRNA-based models to explore the roles of CNR1 and CNR2 in HPV-positive HNSCC cell lines and animal models. Cannabinoid downstream pathways involved were determined by Western blotting and analyzed in a primary HPV HNSCC cohort with single-sample gene set enrichment analysis (ssGSEA) and the OncoGenome Positioning System (Onco-GPS)., Results: In TCGA cohort, the expression of CNR1 and CNR2 was elevated in HPV-positive HNSCC compared with HPV-negative HNSCC, and knockdown of CNR1 / CNR2 expression inhibited proliferation in HPV-positive HNSCC cell lines. Specific CNR1 and CNR2 activation as well as nonselective cannabinoid receptor activation in cell lines and animal models promoted cell growth, migration, and inhibited apoptosis through p38 MAPK pathway activation. CNR1/CNR2 antagonists suppressed cell proliferation and migration and induced apoptosis. Using whole-genome expression analysis in a primary HPV HNSCC cohort, we identified specific p38 MAPK pathway activation signature in tumors from HPV HNSCC patients with objective measurement of concurrent cannabinoid exposure., Conclusions: Cannabinoids can promote progression of HPV-positive HNSCC through p38 MAPK pathway activation., (©2020 American Association for Cancer Research.)

Macrophages (MΦ) play a critical role in tumor growth, immunosuppression, and inhibition of adaptive immune responses in cancer. Hence, targeting signaling pathways in MΦs that promote tumor immunosuppression will provide therapeutic benefit. PI3Kγ has been recently established by our group and others as a novel immuno-oncology target. Herein, we report that an MΦ Syk-PI3K axis drives polarization of immunosuppressive MΦs that establish an immunosuppressive tumor microenvironment in in vivo syngeneic tumor models. Genetic or pharmacologic inhibition of Syk and/or PI3Kγ in MΦs promotes a proinflammatory MΦ phenotype, restores CD8 + T-cell activity, destabilizes HIF under hypoxia, and stimulates an antitumor immune response. Assay for transposase-accessible Chromatin using Sequencing (ATAC-seq) analyses on the bone marrow-derived macrophages (BMDM) show that inhibition of Syk kinase promotes activation and binding of NF-κB motif in Syk MC-KO BMDMs, thus stimulating immunostimulatory transcriptional programming in MΦs to suppress tumor growth. Finally, we have developed in silico the "first-in-class" dual Syk/PI3K inhibitor, SRX3207, for the combinatorial inhibition of Syk and PI3K in one small molecule. This chemotype demonstrates efficacy in multiple tumor models and represents a novel combinatorial approach to activate antitumor immunity., (©2020 American Association for Cancer Research.)

There is a critical need to understand mechanisms of resistance and to develop combinatorial strategies to improve responses to checkpoint blockade immunotherapy (CBI). Here, we uncover a novel mechanism by which the human papillomavirus (HPV) inhibits the activity of CBI in head and neck squamous cell carcinoma (HNSCC). Using orthotopic HNSCC models, we show that radiation combined with anti-PD-L1 immunotherapy significantly enhanced local control, CD8 + memory T cells, and induced preferential T-cell homing via modulation of vascular endothelial cells. However, the HPV E5 oncoprotein suppressed immune responses by downregulating expression of major histocompatibility complex and interfering with antigen presentation in murine models and patient tumors. Furthermore, tumors expressing HPV E5 were rendered entirely resistant to anti-PD-L1 immunotherapy, and patients with high expression of HPV16 E5 had worse survival. The antiviral E5 inhibitor rimantadine demonstrated remarkable single-agent antitumor activity. This is the first report that describes HPV E5 as a mediator of resistance to anti-PD-1/PD-L1 immunotherapy and demonstrates the antitumor activity of rimantadine. These results have broad clinical relevance beyond HNSCC to other HPV-associated malignancies and reveal a powerful mechanism of HPV-mediated immunosuppression, which can be exploited to improve response rates to checkpoint blockade. SIGNIFICANCE: This study identifies a novel mechanism of resistance to anti-PD-1/PD-L1 immunotherapy mediated by HPV E5, which can be exploited using the HPV E5 inhibitor rimantadine to improve outcomes for head and neck cancer patients. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/4/732/F1.large.jpg., (©2019 American Association for Cancer Research.)

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Tobacco use is the main risk factor for HNSCC, and tobacco-associated HNSCCs have poor prognosis and response to available treatments. Recently approved anti-PD-1 immune checkpoint inhibitors showed limited activity (≤20%) in HNSCC, highlighting the need to identify new therapeutic options. For this, mouse models that accurately mimic the complexity of the HNSCC mutational landscape and tumor immune environment are urgently needed. Here, we report a mouse HNSCC model system that recapitulates the human tobacco-related HNSCC mutanome, in which tumors grow when implanted in the tongue of immunocompetent mice. These HNSCC lesions have similar immune infiltration and response rates to anti-PD-1 (≤20%) immunotherapy as human HNSCCs. Remarkably, we find that >70% of HNSCC lesions respond to intratumoral anti-CTLA-4. This syngeneic HNSCC mouse model provides a platform to accelerate the development of immunotherapeutic options for HNSCC.

Head and neck squamous cell carcinoma (HNSCC) accounts for more than 600,000 cases and 380,000 deaths annually worldwide. Although human papillomavirus (HPV)-associated HNSCCs have better overall survival compared with HPV-negative HNSCC, loco-regional recurrence remains a significant cause of mortality and additional combinatorial strategies are needed to improve outcomes. The primary conventional therapies to treat HNSCC are surgery, radiation, and chemotherapies; however, multiple other targeted systemic options are used and being tested including cetuximab, bevacizumab, mTOR inhibitors, and metformin. In 2016, the first checkpoint blockade immunotherapy was approved for recurrent or metastatic HNSCC refractory to platinum-based chemotherapy. This immunotherapy approval confirmed the critical importance of the immune system and immunomodulation in HNSCC pathogenesis, response to treatment, and disease control. However, although immuno-oncology agents are rapidly expanding, the role that the immune system plays in the mechanism of action and clinical efficacy of standard conventional therapies is likely underappreciated. In this article, we focus on how conventional and targeted therapies may directly modulate the immune system and the tumor microenvironment to better understand the effects and combinatorial potential of these therapies in the context and era of immunotherapy., (©2019 American Association for Cancer Research.)

Continued discoveries of negative regulators of inflammatory signaling provide detailed molecular insights into peripheral tolerance and anti-tumor immunity. Accumulating evidence indicates that peripheral tolerance is maintained at multiple levels of immune responses by negative regulators of proinflammatory signaling, soluble anti-inflammatory factors, inhibitory surface receptors & ligands, and regulatory cell subsets. This review provides a global overview of these regulatory machineries that work in concert to maintain peripheral tolerance at cellular and host levels, focusing on the direct and indirect regulation of T cells. The recent success of checkpoint blockade immunotherapy (CBI) has initiated a dramatic shift in the paradigm of cancer treatment. Unprecedented responses to CBI have highlighted the central role of T cells in both anti-tumor immunity and peripheral tolerance and underscored the importance of T cell exhaustion in cancer. We discuss the therapeutic implications of modulating the negative regulators of T cell function for tumor immunotherapy with an emphasis on inhibitory surface receptors & ligands-central players in T cell exhaustion and targets of checkpoint blockade immunotherapies. We then introduce a Threshold Model for Immune Activation-the concept that these regulatory mechanisms contribute to defining a set threshold of immunogenic (proinflammatory) signaling required to elicit an anti-tumor or autoimmune response. We demonstrate the value of the Threshold Model in understanding clinical responses and immune related adverse events in the context of peripheral tolerance, tumor immunity, and the era of Checkpoint Blockade Immunotherapy.

Background: Undifferentiated pleomorphic sarcoma (UPS) of the maxillary sinus is an extremely rare malignancy of the head and neck. Surgery is the mainstay of treatment for UPS; however, proximity to vital structures makes it challenging to achieve negative surgical margins. Adjuvant therapy including radiation therapy with or without chemotherapy is generally indicated. Despite advances in multimodality treatment, objective response rates to available therapies and prognosis of metastatic UPS remain dismal. Immunotherapy has become a fourth cornerstone of cancer therapy and checkpoint blockade immunotherapy is a standard of care for recurrent or metastatic cisplatin-refractory head and neck squamous cell carcinoma. Checkpoint blockade immunotherapy is being studied in metastatic sarcoma, including UPS, and while initial results are promising, objective response rates remain below 20%. However, adding radiation therapy to checkpoint blockade immunotherapy has been shown, in both preclinical and retrospective clinical studies, to have combinatorial effects on both local and metastatic disease. Thus, further investigation into the effects of radiation therapy combined with immunotherapy in head and neck sarcomas is warranted. Case Presentation: We present a case of metastatic, chemotherapy-refractory, UPS of the maxillary sinus in a 55-year-old male treated with checkpoint blockade immunotherapy combined with radiation, which resulted in a complete response. Conclusions: This is the first report to our knowledge of metastatic UPS treated with a combination of radiation and dual agent checkpoint blockade immunotherapy. Further investigation is warranted to study the effects of this combination in patients with metastatic UPS that fail to respond to currently available therapies.

Purpose: In the proper context, radiotherapy can promote antitumor immunity. It is unknown if elective nodal irradiation (ENI), a strategy that irradiates tumor-associated draining lymph nodes (DLN), affects adaptive immune responses and combinatorial efficacy of radiotherapy with immune checkpoint blockade (ICB). Experimental Design: We developed a preclinical model to compare stereotactic radiotherapy (Tumor RT) with or without ENI to examine immunologic differences between radiotherapy techniques that spare or irradiate the DLN. Results: Tumor RT was associated with upregulation of an intratumoral T-cell chemoattractant chemokine signature (CXCR3, CCR5-related) that resulted in robust infiltration of antigen-specific CD8 + effector T cells as well as FoxP3 + regulatory T cells (Tregs). The addition of ENI attenuated chemokine expression, restrained immune infiltration, and adversely affected survival when combined with ICB, especially with anti-CLTA4 therapy. The combination of stereotactic radiotherapy and ICB led to long-term survival in a subset of mice and was associated with favorable CD8 effector-to-Treg ratios and increased intratumoral density of antigen-specific CD8 + T cells. Although radiotherapy technique (Tumor RT vs. ENI) affected initial tumor control and survival, the ability to reject tumor upon rechallenge was partially dependent upon the mechanism of action of ICB; as radiotherapy/anti-CTLA4 was superior to radiotherapy/anti-PD-1. Conclusions: Our results highlight that irradiation of the DLN restrains adaptive immune responses through altered chemokine expression and CD8 + T-cell trafficking. These data have implications for combining radiotherapy and ICB, long-term survival, and induction of immunologic memory. Clinically, the immunomodulatory effect of the radiotherapy strategy should be considered when combining stereotactic radiotherapy with immunotherapy. Clin Cancer Res; 24(20); 5058-71. ©2018 AACR ., (©2018 American Association for Cancer Research.)

The cGAS-STING cytosolic DNA sensing pathway may play an integral role in the initiation of antitumor immune responses. Studies evaluating the immunogenicity of various cyclic dinucleotide (CDN) STING agonists administered by intratumoral (i.t.) injection showed potent induction of inflammation, tumor necrosis, and, in some cases, durable tumor-specific adaptive immunity. However, the specific immune mechanisms underlying these responses remain incompletely defined. The majority of these studies have focused on the effect of CDNs on immune cells but have not conclusively interrogated the role of stromal cells in the acute rejection of the CDN-injected tumor. Here, we revealed a mechanism of STING agonist-mediated tumor response that relied on both stromal and immune cells to achieve tumor regression and clearance. Using knockout and bone marrow chimeric mice, we showed that although bone marrow-derived TNFα was necessary for CDN-induced necrosis, STING signaling in radioresistant stromal cells was also essential for CDN-mediated tumor rejection. These results provide evidence for crosstalk between stromal and hematopoietic cells during CDN-mediated tumor collapse after i.t. administration. These mechanistic insights may prove critical in the clinical development of STING agonists. Cancer Immunol Res; 6(4); 422-33. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Background: Stereotactic body radiation therapy (SBRT) has been proposed as a potential alternative to surgery for early lung cancer, although we lack well-powered prospective randomized data comparing these treatments, and existing studies suffer from incomplete information on confounders that can bias results. Here, we evaluated the comparative effectiveness of surgery and SBRT in lung cancer treatment using a large extensively detailed database from the Veteran's Affairs system., Methods: We identified veterans with biopsy-proven clinical stage I non-small cell lung cancer diagnosed between 2006 and 2015 from within the Veteran's Affairs Informatics and Computing Infrastructure. We compared cancer-specific survival among patients receiving lobectomy, sublobar resection, or SBRT using univariable and multivariable competing risk analyses. Multivariable analyses adjusted for confounders including preoperative pulmonary function, smoking status, comorbidity, and staging workup procedures., Results: In all, 4,069 patients were included (449 SBRT, 2,986 lobectomy, 634 sublobar resection). Unadjusted analysis found higher immediate postprocedural mortality in the surgery groups compared with the SBRT group. The multivariable analysis considering long-term survival found higher cancer-specific mortality for SBRT compared with lobectomy (subdistribution hazard ratio 1.45, 95% confidence interval: 1.09 to 1.94, p = 0.01), although no survival difference between SBRT and sublobar resection (subdistribution hazard ratio 1.25, 95% confidence interval: 0.93 to 1.68, p = 0.15)., Conclusions: Among a large cohort of early stage lung cancer patients, we found that lobectomy had improved survival compared with SBRT, although we found no survival difference between sublobar resection and SBRT. Despite these findings, the potential for unmeasured confounding remains and prospective randomized trials are needed to better compare these treatment modalities., (Copyright © 2018 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Purpose: To study the effects of immunosuppression on treatment toxicity, long-term cancer recurrence risk, and survival among human immunodeficiency virus (HIV)-positive anal cancer patients., Methods and Materials: From a nationwide retrospective cohort of veterans with anal cancer we identified 142 HIV-positive patients with stage I-III disease, diagnosed between 2000 and 2015 and treated with definitive-intent chemotherapy and radiation. We used regression models to study the impact of pretreatment CD4 counts and longitudinal posttreatment CD4 counts on outcomes including acute toxicity, long-term ostomy rates, cancer recurrence, cancer-specific survival, and overall survival. All models were adjusted for potential confounders., Results: The median pretreatment CD4 count was 375 cells/mm 3 , which dropped to 157 cells/mm 3 after treatment. Each 100-cell/mm 3 decrease in pretreatment CD4 count was associated with an increased risk of acute hematologic toxicity (odds ratio 1.19, 95% confidence interval [CI] 1.01-1.42, P=.04) and hospitalization for hematologic toxicity (odds ratio 1.24, 95% CI 1.00-1.54, P=.049) but not gastrointestinal toxicity, tumor recurrence, or cancer-specific mortality (P>.05). Each 100-cells/mm 3 decrease in posttreatment CD4 count increased the risk of recurrence by 54% (hazard ratio 1.54, 95% CI 1.09-2.17, P=.01) and cancer mortality by 46% at a trend level (hazard ratio 1.46, 95% CI 0.99-2.14, P=.06). Neither pre- nor posttreatment CD4 count influenced long-term ostomy rates or overall survival (all P>.05)., Conclusions: Lower pretreatment CD4 counts were associated with acute hematologic toxicity, and lower posttreatment CD4 count levels were associated with an increased risk of tumor recurrence. These results suggest that immune surveillance may play an important role in long-term disease control in anal cancer., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Checkpoint blockade immunotherapy has received mainstream attention as a result of striking and durable clinical responses in some patients with metastatic disease and a reasonable response rate in many tumour types. The activity of checkpoint blockade immunotherapy is not restricted to melanoma or lung cancer, and additional indications are expected in the future, with responses already reported in renal cancer, bladder cancer, and Hodgkin's lymphoma among many others. Additionally, the interactions between radiation and the immune system have been investigated, with several studies describing the synergistic effects on local and distant tumour control when radiation therapy is combined with immunotherapy. Clinical enthusiasm for this approach is strengthened by the many ongoing trials combining immunotherapy with definitive and palliative radiation. Herein, we discuss the biological and mechanistic rationale behind combining radiation with checkpoint blockade immunotherapy, with a focus on the preclinical data supporting this potentially synergistic combination. We explore potential hypotheses and important considerations for clinical trial designs. Finally, we reintroduce the notion of radiosensitising immunotherapy, akin to radiosensitising chemotherapy, as a potential definitive therapeutic modality., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Stereotactic radiosurgery and stereotactic body radiation therapy are two contemporary radiation modalities that can treat tumors in any area of the body using highly focused radiation. Recently, immunotherapy has established itself as a viable and powerful anticancer treatment. In this review we detail the rationale supporting a combination of immunotherapy and stereotactic radiation. Additionally, we discuss the evidence for the immune stimulatory effects of focused radiation and the role that radiation may play in enhancing the systemic treatment effects of immunotherapy.

The immune-modulating effects of radiotherapy (XRT) have gained considerable interest recently, and there have been multiple reports of synergy between XRT and immunotherapy. However, additional preclinical studies are needed to demonstrate the antigen-specific nature of radiation-induced immune responses and elucidate potential mechanisms of synergy with immunotherapy. Here, we demonstrate the ability of stereotactic XRT to induce endogenous antigen-specific immune responses when it is combined with anti-PD-1 checkpoint blockade immunotherapy. Using the small animal radiation research platform (SARRP), image-guided stereotactic XRT delivered to B16-OVA melanoma or 4T1-HA breast carcinoma tumors resulted in the development of antigen-specific T cell- and B cell-mediated immune responses. These immune-stimulating effects of XRT were significantly increased when XRT was combined with either anti-PD-1 therapy or regulatory T cell (Treg) depletion, resulting in improved local tumor control. Phenotypic analyses of antigen-specific CD8 T cells revealed that XRT increased the percentage of antigen-experienced T cells and effector memory T cells. Mechanistically, we found that XRT upregulates tumor-associated antigen-MHC complexes, enhances antigen cross-presentation in the draining lymph node, and increases T-cell infiltration into tumors. These findings demonstrate the ability of XRT to prime an endogenous antigen-specific immune response and provide an additional mechanistic rationale for combining radiation with PD-1 blockade in the clinic., (©2014 American Association for Cancer Research.)

Knowledge of the basic mechanisms of the immune system as it relates to cancer has been increasing rapidly. These developments have accelerated the translation of these advancements into medical breakthroughs for many cancer patients. The immune system is designed to discriminate between self and non-self, and through genetic recombination there is virtually no limit to the number of antigens it can recognize. Thus, mutational events, translocations, and other genetic abnormalities within cancer cells may be distinguished as "altered-self" and these differences may play an important role in preventing the development or progression of cancer. However, tumors may utilize a variety of mechanisms to evade the immune system as well. Cancer biologists are aiming to both better understand the relationship between tumors and the normal immune system, and to look for ways to alter the playing field for cancer immunotherapy. Summarized in this review are discussions from the 2013 SITC Primer, which focused on reviewing current knowledge and future directions of research related to tumor immunology and cancer immunotherapy, including sessions on innate immunity, adaptive immunity, therapeutic approaches (dendritic cells, adoptive T cell therapy, anti-tumor antibodies, cancer vaccines, and immune checkpoint blockade), challenges to driving an anti-tumor immune response, monitoring immune responses, and the future of immunotherapy clinical trial design.

Limited treatment options exist for isolated local recurrence of pancreatic ductal adenocarcinoma (PDA) following surgical resection accompanied by neoadjuvant or adjuvant chemoradiation therapy (CRT). While select patients are eligible for re-resection, recurrent lesions are often unresectable. Stereotactic body radiation therapy (SBRT) represents a possible minimally invasive treatment option for these patients, although published data in this setting are currently lacking. This study examines the safety, efficacy, and palliative capacity of re-irradiation with SBRT for isolated local PDA recurrence. All patients undergoing SBRT at two academic centers from 2008-2012 were retrospectively reviewed to identify those who received re-irradiation with SBRT for isolated local recurrence or progression of PDA after previous conventionally fractionated CRT. Information regarding demographics, clinicopathologic characteristics, therapies received, survival, symptom palliation, and toxicity was obtained from patient charts. Kaplan-Meier statistics were used to analyze survival and the log-rank test was used to compare survival among patient subgroups. Eighteen patients were identified. Fifteen had previously undergone resection with neoadjuvant or adjuvant CRT, while 3 received definitive CRT for locally advanced disease. Median CRT dose was 50.4 Gy [interquartile range (IQR), 45.0-50.4 Gy] in 28 fractions. All patients subsequently received gemcitabine-based maintenance chemotherapy, but developed isolated local disease recurrence or progression without evidence of distant metastasis. Locally recurrent or progressive disease was treated with SBRT to a median dose of 25.0 Gy (range, 20.0-27.0 Gy) in 5 fractions. Median survival from SBRT was 8.8 months (95% CI, 1.2-16.4 months). Despite having similar clinicopathologic disease characteristics, patients who experienced local progression greater than vs. less than 9 months after surgery/definitive CRT demonstrated superior median survival (11.3 vs. 3.4 months; P=0.019) and progression-free survival (10.6 vs. 3.2 months; P=0.030) after SBRT. Rates of freedom from local progression at 6 and 12 months after SBRT were 78% (14 of 18 patients) and 62% (5 of 8 patients), respectively. Effective symptom palliation was achieved in 4 of 7 patients (57%) who reported symptoms of abdominal or back pain prior to SBRT. Five patients (28%) experienced grade 2 acute toxicity; none experienced grade ≥3 acute toxicity. One patient (6%) experienced grade 3 late toxicity in the form of small bowel obstruction. In conclusion, re-irradiation with hypofractionated SBRT in this salvage scenario appears to be a safe and reasonable option for palliation of isolated local PDA recurrence or progression following previous conventional CRT. Patients with a progression-free interval of greater than 9 months prior to isolated local recurrence or progression may be most suitable for re-irradiation with SBRT, as they appear to have a better prognosis with survival that is long enough for local control to be of potential benefit.

The self-renewal and multilineage differentiation of embryonic stem cells (ESC) is largely governed by transcription factors or repressors. Extensive efforts have focused on elucidating critical factors that control the differentiation of specific cell lineages, for instance, myeloid lineages in hematopoietic development. In this study, we found that Twist-2, a basic helix-loop-helix (bHLH) transcription factor, plays a critical role in inhibiting the differentiation of ESC. Murine ES cells, in which Twist-2 expression is silenced by lentivirally delivered shRNA, exhibit an enhanced formation of primary embryoid bodies (EB) and enhanced differentiation into mesodermally derived hematopoietic colonies. Furthermore, Twist-2 silenced (LV-siTwist-2) ESC display significantly increased generation of myeloid lineages (Gr-1(+) and F4/80(+) cells) during in vitro hematopoietic differentiation. Treatment with the Toll-like receptor (TLR) 4 ligand synergistically stimulates the generation of primary EB formation as well as of hematopoietic progenitors differentiated from LV-siTwist-2 ES cells. Thus, this study reveals the critical role of the transcriptional repressor Twist-2 in regulating the development of myeloid lineage in hematopoietic differentiation from ESC. This study also suggests a potential strategy for directional differentiation of ESC by inhibiting a transcriptional repressor.

Basic helix-loop-helix (bHLH) transcription factors play critical roles in lymphoid and erythroid development; however, little is known about their role in myeloid lineage development. In this study, we identify the bHLH transcription factor Twist-2 as a key negative regulator of myeloid lineage development, as manifested by marked increases in mature myeloid populations of macrophages, neutrophils, and basophils in Twist-2-deficient mice. Mechanistic studies demonstrate that Twist-2 inhibits the proliferation as well as differentiation of granulocyte macrophage progenitors (GMP) by interacting with and inhibiting the transcription factors Runx1 and C/EBPalpha. Moreover, Twist-2 was found to have a contrasting effect on cytokine production: inhibiting the production of proinflammatory cytokines such as interleukin-12 (IL-12) and interferon-gamma (IFNgamma) while promoting the regulatory cytokine IL-10 by myeloid cells. The data from further analyses suggest that Twist-2 activates the transcription factor c-Maf, leading to IL-10 expression. In addition, Twist-2 was found to be essential for endotoxin tolerance. Thus, this study reveals the critical role of Twist-2 in regulating the development of myeloid lineages, as well as the function and inflammatory responses of mature myeloid cells.