Andrew Sharabi

Stereotactic Radiation Therapy Augments Antigen-Specific PD-1–Mediated Antitumor Immune Responses via Cross-Presentation of Tumor Antigen

Sharabi and colleagues show in two mouse tumor models that radiotherapy combined with PD-1 blockade or Treg depletion improves local tumor control by increasing antigen-experienced and effector-memory T cells, antigen-MHC complexes, and T-cell infiltration into tumors via antigen cross-presentation in the tumor-draining lymph node. 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. Cancer Immunol Res; 3(4); 345–55. ©2014 AACR.

Radiation and checkpoint blockade immunotherapy: radiosensitisation and potential mechanisms of synergy

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 Hodgkins 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.

Hyperprogressors after immunotherapy: Analysis of genomic alterations associated with accelerated growth rate

Purpose: Checkpoint inhibitors demonstrate salutary anticancer effects, including long-term remissions. PD-L1 expression/amplification, high mutational burden, and mismatch repair deficiency correlate with response. We have, however, observed a subset of patients who appear to be “hyperprogressors,” with a greatly accelerated rate of tumor growth and clinical deterioration compared with pretherapy, which was also recently reported by Institut Gustave Roussy. The current study investigated potential genomic markers associated with “hyperprogression” after immunotherapy. Experimental Design: Consecutive stage IV cancer patients who received immunotherapies (CTLA-4, PD-1/PD-L1 inhibitors or other [investigational] agents) and had their tumor evaluated by next-generation sequencing were analyzed (N = 155). We defined hyperprogression as time-to-treatment failure (TTF) <2 months, >50% increase in tumor burden compared with preimmunotherapy imaging, and >2-fold increase in progression pace. Results: Amongst 155 patients, TTF <2 months was seen in all six individuals with MDM2/MDM4 amplification. After anti-PD1/PDL1 monotherapy, four of these patients showed remarkable increases in existing tumor size (55% to 258%), new large masses, and significantly accelerated progression pace (2.3-, 7.1-, 7.2- and 42.3-fold compared with the 2 months before immunotherapy). In multivariate analysis, MDM2/MDM4 and EGFR alterations correlated with TTF <2 months. Two of 10 patients with EGFR alterations were also hyperprogressors (53.6% and 125% increase in tumor size; 35.7- and 41.7-fold increase). Conclusions: Some patients with MDM2 family amplification or EGFR aberrations had poor clinical outcome and significantly increased rate of tumor growth after single-agent checkpoint (PD-1/PD-L1) inhibitors. Genomic profiles may help to identify patients at risk for hyperprogression on immunotherapy. Further investigation is urgently needed. Clin Cancer Res; 23(15); 4242–50. ©2017 AACR.

Tumor immunology and cancer immunotherapy: Summary of the 2013 SITC primer

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.

Proteolytic processing of laminin-5 by MT1-MMP in tissues and its effects on epithelial cell morphology

The extracellular matrix macromolecule laminin‐5 (Ln‐5) is converted by matrix metalloproteinases (MMP) MT1‐MMP and MMP‐2 into a migration‐promoting substrate in vitro. We now report that cleavage of Ln‐5 by MT1‐MMP occurs in vivo and affects epithelial tissue organization and probably Ln‐5 turnover. In MT1‐MMP knockout (KO) mice, the kidneys showed increased levels of total Ln‐5 γ2 subunit, but significantly reduced amounts of γ2′, an amino‐terminal truncated proteolytic form of γ2. The kidney tubular epithelia of KO animals were poorly differentiated, a phenotype reminiscent of human congenital mixed hypoplastic/dysplastic disorders. To establish a better link between Ln‐5 proteolytic cleavage and epithelial morphology, MT1‐MMP expression was reconstituted by transfection of MT1‐MMP into a Ln‐5 positive, MT1‐MMP deficient epithelial cell line. MT1‐MMP transfectants demonstrated increased levels of processed Ln‐5 γ2 chain and enhanced spreading on Ln‐5, but not fibronectin. Recombinant MT1‐MMP cleaved γ2 constructs in vitro at a known in vivo γ2 γ2’ processing site. These results strongly indicate that Ln‐5 is a physiological substrate of MT1‐MMP in vivo. Proteolytic processing of γ2 subunit by MT1‐MMP may influence Ln‐5 turnover in epithelial basement membranes and affect epithelial morphogenesis.

Discrete Proteolysis of Focal Contact and Adherens Junction Components in Porphyromonas gingivalis-Infected Oral Keratinocytes: a Strategy for Cell Adhesion and Migration Disabling

ABSTRACT Adhesive interactions of cells are critical to tissue integrity. We show that infection with Porphyromonas gingivalis, a major pathogen in the periodontal disease periodontitis, interferes with both cell-matrix and cell-cell adhesion in the oral keratinocyte cell line HOK-16. Thus, infected cells showed reduced adhesion to extracellular matrix, changes in morphology from spread to rounded, and impaired motility on purified matrices in Transwell migration assays and scratch assays. Western blot analysis of P. gingivalis-challenged HOK-16 cells revealed proteolysis of focal contact components (e.g., focal adhesion kinase), adherens junction proteins (e.g., catenins), and adhesion signaling molecules (e.g., the tyrosine kinase SRC). Proteolysis was selective, since important components of adherens junctions (E-cadherin) or signaling molecules (extracellular signal-regulated kinases ERK1/2) were not degraded. The virulence factors gingipains, cysteine proteinases expressed by P. gingivalis, are likely responsible for this proteolytic attack, since they directly digested specific proteins in pull-down experiments, and their proteolytic activity was blocked by the cysteine proteinase inhibitor N-α-p-tosyl-l-lysine chloromethyl ketone and also by a caspase inhibitor. Proteolysis was strain dependent, such that ATCC 33277 and 381 had high proteolytic potential, whereas W50 showed almost no proteolytic activity. These findings may help explain the formation of gingival pockets between cementum and periodontal epithelium, a hallmark of periodontitis. Furthermore, they illustrate a new pathogenetic paradigm of infection whereby bacteria may disrupt the integrity of epithelia.

Twist-2 Controls Myeloid Lineage Development and Function

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/EBPα. 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-γ (IFNγ) 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.

Re-irradiation with stereotactic body radiation therapy as a novel treatment option for isolated local recurrence of pancreatic cancer after multimodality therapy: experience from two institutions

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.

Embryonic Stem Cells and Mammary Luminal Progenitors Directly Sense and Respond to Microbial Products

Stem cells are normally maintained in a quiescent state and proliferate only under certain conditions; however, little is known about the biological stimuli that initiate the proliferation and differentiation of stem cells. In this study, we found that functional Toll‐like receptors (TLRs) are expressed on mouse embryonic stem (ES) cells and that TLR ligands stimulate ES cell proliferation and promote their hematopoietic differentiation. TLR ligands activate TLR‐mediated signaling pathways, leading to the altered expression of numerous genes in ES cells. Moreover, TLR ligands efficiently stimulate the proliferation and expansion of adult stem cells and progenitors of nonhematopoietic tissues, such as mammary glands and intestine as well. We further found that mammary luminal progenitor cells (Lin−CD29+CD61+) express TLR4‐MD2 complex and actively proliferate, resulting in the enhanced growth of mammospheres in response to TLR ligands. Thus, mouse ES cells and adult tissue‐specific stem cells/progenitors directly sense and respond to microbial products, which function as a class of foreign, but biological stimuli for stem cell/progenitor proliferation. This finding expands the biological role of TLRs and has implications in understanding stem cell biology, tissue repair/homeostasis, and the role of infection and inflammation in malignant transformation. STEM CELLS 2009;27:1604–1615

Summary and Recommendations from the National Cancer Institute's Clinical Trials Planning Meeting on Novel Therapeutics for Non-Muscle Invasive Bladder Cancer

The NCI Bladder Cancer Task Force convened a Clinical Trials Planning Meeting (CTPM) Workshop focused on Novel Therapeutics for Non-Muscle Invasive Bladder Cancer (NMIBC). Meeting attendees included a broad and multi-disciplinary group of clinical and research stakeholders and included leaders from NCI, FDA, National Clinical Trials Network (NCTN), advocacy and the pharmaceutical and biotech industry. The meeting goals and objectives were to: 1) create a collaborative environment in which the greater bladder research community can pursue future optimally designed novel clinical trials focused on the theme of molecular targeted and immune-based therapies in NMIBC; 2) frame the clinical and translational questions that are of highest priority; and 3) develop two clinical trial designs focusing on immunotherapy and molecular targeted therapy. Despite successful development and implementation of large Phase II and Phase III trials in bladder and upper urinary tract cancers, there are no active and accruing trials in the NMIBC space within the NCTN. Disappointingly, there has been only one new FDA approved drug (Valrubicin) in any bladder cancer disease state since 1998. Although genomic-based data for bladder cancer are increasingly available, translating these discoveries into practice changing treatment is still to come. Recently, major efforts in defining the genomic characteristics of NMIBC have been achieved. Aligned with these data is the growing number of targeted therapy agents approved and/or in development in other organ site cancers and the multiple similarities of bladder cancer with molecular subtypes in these other cancers. Additionally, although bladder cancer is one of the more immunogenic tumors, some tumors have the ability to attenuate or eliminate host immune responses. Two trial concepts emerged from the meeting including a window of opportunity trial (Phase 0) testing an FGFR3 inhibitor and a second multi-arm multi-stage trial testing combinations of BCG or radiotherapy and immunomodulatory agents in patients who recur after induction BCG (BCG failure).