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Abstract We previously reported the results of a randomized phase II trial (NCT02904954) in patients with early-stage non-small cell lung cancer (NSCLC) who were treated with either two preoperative cycles of the anti-PD-L1 antibody durvalumab alone or combined with immunomodulatory doses of stereotactic radiation (DRT). The trial met its primary endpoint of major pathological response, which was significantly higher following DRT with no new safety signals. Here, we report on the prespecified secondary endpoint of disease-free survival (DFS) regardless of treatment assignment and the prespecified exploratory analysis of DFS in each arm of the trial. DFS at 2 and 3 years across patients in both arms of the trial were 73% (95% CI: 62.1–84.5) and 65% (95% CI: 52.5–76.9) respectively. For the exploratory endpoint of DFS in each arm of the trial, three-year DFS was 63% (95% CI: 46.0–80.4) in the durvalumab monotherapy arm compared to 67% (95% CI: 49.6–83.4) in the dual therapy arm. In addition, we report post hoc exploratory analysis of progression-free survival as well as molecular correlates of response and recurrence through high-plex immunophenotyping of sequentially collected peripheral blood and gene expression profiles from resected tumors in both treatment arms. Together, our results contribute to the evolving landscape of neoadjuvant treatment regimens for NSCLC and identify easily measurable potential biomarkers of response and recurrence.

The IRE1α-XBP1 arm of the unfolded protein response (UPR) has been associated with immunosuppression and cancer progression. Here the authors show that IRE1α-XBP1 activation is associated with poor overall survival in patients with non-small cell lung cancer and that IRE1α loss in cancer cells promotes anti-tumor immune responses in lung cancer preclinical models.

Tsp-1 in the tumor microenvironment is known to suppress tumor growth and progression. Here the authors show that PRSS2 represses Tsp-1 by binding to lipoprotein receptor-related protein 1 and suggest targeting PRSS2 mediated Tsp-1 repression as a potential therapeutic strategy.

Copper depletion has been reported to improve survival in patients with triple negative breast cancer (TNBC) but the underlying mechanisms are not completely understood. Here, the authors show that copper chelation reduces mitochondrial oxidative phosphorylation leading to decreased TNBC metastasis.

Abstract Tetrathiomolybdate (TM) is a novel, copper-depleting compound associated with promising survival in a phase II study of patients with high-risk and triple-negative breast cancer. We sought to elucidate the mechanism of TM by exploring its effects on collagen processing and immune function in the tumor microenvironment (TME). Using an exploratory cohort, we identified markers of collagen processing (LOXL2, PRO-C3, C6M, and C1M) that differed between those with breast cancer versus controls. We measured these collagen biomarkers in TM-treated patients on the phase II study and detected evidence of decreased collagen cross-linking and increased degradation over formation in those without disease compared to those who experienced disease progression. Preclinical studies revealed decreased collagen deposition, lower levels of myeloid-derived suppressor cells, and higher CD4+ T-cell infiltration in TM-treated mice compared with controls. This study reveals novel mechanisms of TM targeting the TME and immune response with potential applications across cancer types.

Summary: To investigate changes in the tumor microenvironment (TME) during lung cancer progression, we interrogate tumors from two chest computed tomography (CT)-defined groups. Pure non-solid (pNS) CT density nodules contain preinvasive/minimally invasive cancers, and solid density nodules contain invasive cancers. Profiling data reveal a dynamic interaction between the tumor and its TME throughout progression. Alterations in genes regulating the extracellular matrix and genes regulating fibroblasts are central at the preinvasive state. T cell-mediated immune suppression is initiated in preinvasive nodules and sustained with rising intensity through progression to invasive tumors. Reduced T cell infiltration of the cancer cell nests is more frequently associated with preinvasive cancers, possibly until tumor evolution leads to a durable, viable invasive phenotype accompanied by more varied and robust immune suppression. Upregulation of immune checkpoints occurs only in the invasive nodules. Throughout progression, an effector immune response is present but is effectively thwarted by the immune-suppressive elements.

Breast cancer is the most common type of cancer in women and notwithstanding important therapeutic advances, remains the second leading cause of cancer-related death. Despite extensive research relating to the hormone ghrelin, responsible for the stimulation of growth hormone release and appetite, little is known of the effects of its unacylated form, especially in cancer. The present study aimed to characterize effects of unacylated ghrelin on breast cancer cells, define its mechanism of action, and explore the therapeutic potential of unacylated ghrelin or analog AZP-531. We report potent anti-tumor effects of unacylated ghrelin, dependent on cells being cultured in 3D in a biologically-relevant extracellular matrix. The mechanism of unacylated ghrelin-mediated growth inhibition involves activation of Gαi and suppression of MAPK signaling. AZP-531 also suppresses the growth of breast cancer cells in vitro and in xenografts, and may be a novel approach for the safe and effective treatment of breast cancer.

Summary: Epigenetic changes are increasingly being appreciated as key events in breast cancer progression. However, breast cancer subtype-specific epigenetic regulation remains poorly investigated. Here we report that EZH2 is a leading candidate of epigenetic modulators associated with the TNBC subtype and that it predicts poor overall survival in TNBC patients. We demonstrate that specific pharmacological or genetic inhibition of EZH2 catalytic activity impairs distant metastasis. We further define a specific EZH2high population with enhanced invasion, mammosphere formation, and metastatic potential that exhibits marked sensitivity to EZH2 inhibition. Mechanistically, EZH2 inhibition differentiates EZH2high basal cells to a luminal-like phenotype by derepressing GATA3 and renders them sensitive to endocrine therapy. Furthermore, dissection of human TNBC heterogeneity shows that EZH2high basal-like 1 and mesenchymal subtypes have exquisite sensitivity to EZH2 inhibition compared with the EZH2low luminal androgen receptor subtype. These preclinical findings provide a rationale for clinical development of EZH2 as a targeted therapy against TNBC metastasis. : TNBC presents a clinical challenge because it exhibits a high incidence of metastatic recurrence. Yomtoubian et al. identify a highly metastatic basal-like subpopulation in the primary tumor that expresses elevated EZH2 levels. EZH2 inhibition induces luminal differentiation by derepressing GATA3, rendering tumors sensitive to endocrine therapy, and inhibiting metastasis. Keywords: breast cancer, metastasis, circulating tumor cells, epigenetics, EZH2, GATA3, differentiation

Molecularly targeted therapies benefit approximately 15–20% of non-small cell lung cancer (NSCLC) patients carrying specific drug-sensitive mutations. Thus, there is a clinically unmet need for the identification of novel targets for drug development. Here, we performed RNA-deep sequencing to identify altered gene expression between malignant and non-malignant lung tissue. Matrix Metalloproteinase 14 (MMP14), a membrane-bound proteinase, was significantly up-regulated in the tumor epithelial cells and intratumoral myeloid compartments in both mouse and human NSCLC. Overexpression of a soluble dominant negative MMP14 (DN-MMP14) or pharmacological inhibition of MMP14 blocked invasion of lung cancer cells through a collagen I matrix in vitro and reduced tumor incidence in an orthotopic K-RasG12D/+p53−/− mouse model of lung cancer. Additionally, MMP14 activity mediated proteolytic processing and activation of Heparin-Binding EGF-like Growth Factor (HB-EGF), stimulating the EGFR signaling pathway to increase proliferation and tumor growth. This study highlights the potential for development of therapeutic strategies that target MMP14 in NSCLC with particular focus on MMP14-HB-EGF axis.

ABSTRACT IMPACT: T cell dysfunction is a dominant suppressor of anti-tumor immunity, reducing immunotherapeutic efficacy and benefit to patients; our work will identify novel mediators of this process for both therapeutic potential and underlying mechanism, allowing for both potential immediate clinical utility and identification of future targets based on new mechanistic insights. OBJECTIVES/GOALS: T cell dysfunction is a dominant suppressor of anti-tumor immunity, reducing immunotherapeutic efficacy and clinical benefit to the majority of patients. We aim to interrogate a novel mediator of dysfunction identified from transcriptome analyses, pyruvate kinase muscle isozyme isoform 2 (PKM2), for therapeutic utility and underlying mechanism. METHODS/STUDY POPULATION: Transcriptome analyses of CD8+ lymphocytes from tumor-bearing lungs from both murine KrasG12D p53-/- and human non-small cell lung cancer (NSCLC) patients were performed, and differentially expressed genes identified. Flow cytometric analyses for PKM isoform expression and effects of target knockdown on accumulation of dysfunctional characteristics, including checkpoint and transcription factor expression, proliferation, and cytokine production, were performed using an in vitro co-culture of murine antigen-specific T (OT-I) cells and antigen-expressing NSCLC (HKP1-ova) cells. In vivo examination of the same was performed using adoptive transfer of OT-I cells into immunocompetent recipient mice with engraftment of HKP1-ova cells, and subsequent evaluation of mouse survival and T cell phenotypes. RESULTS/ANTICIPATED RESULTS: Transcriptome analyses demonstrated that PKM expression was upregulated in dysfunctional T cells from both murine and human samples. This was confirmed both in vitro with co-culture and in vivo with adoptive transfer approaches, with both activated and dysfunctional OT-I cells expressing higher levels of isoform 2 of PKM than naive OT-I cells. Expression of PKM2 mimicked the kinetics of the transcription factor Tox, a known driver of dysfunction, and knockdown of PKM2 resulted in reduced granzyme B expression, and increased proportions of progenitors with fewer terminally differentiated dysfunctional cells. Knockdown of PKM2 in adoptively-transferred OT-I cells led to enhanced tumor control; results are being extended to other tumor models, and T cells metabolically profiled with PKM2 manipulation. DISCUSSION/SIGNIFICANCE OF FINDINGS: This work identified a novel mediator of dysfunction whose targeting has the potential to enhance anti-tumor immunity. Mechanistically, targeting PKM2 led to altered T cell differentiation to a dysfunctional state, linking metabolic phenotypes to these traits and underlining the importance and therapeutic potential of T cell metabolic pathways.

Emerging studies have begun to demonstrate that reprogrammed stromal cells play pivotal roles in tumor growth, metastasis, and resistance to therapy. However, the contribution of stromal cells to non-small-cell lung cancer (NSCLC) has remained underexplored. We used an orthotopic model of Kras-driven NSCLC to systematically dissect the contribution of specific hematopoietic stromal cells in lung cancer. RNA deep-sequencing analysis of individually sorted myeloid lineage and tumor epithelial cells revealed cell-type-specific differentially regulated genes, indicative of activated stroma. We developed a computational model for crosstalk signaling discovery based on ligand-receptor interactions and downstream signaling networks and identified known and novel tumor-stroma paracrine and tumor autocrine crosstalk-signaling pathways in NSCLC. We provide cellular and molecular insights into components of the lung cancer microenvironment that contribute to carcinogenesis. This study has the potential for development of therapeutic strategies that target tumor-stroma interactions and may complement conventional anti-cancer treatments.

Lung cancer is the leading cause of cancer related mortality worldwide, with non-small cell lung cancer (NSCLC) as the most prevalent form. Despite advances in treatment options including minimally invasive surgery, CT-guided radiation, novel chemotherapeutic regimens, and targeted therapeutics, prognosis remains dismal. Therefore, further molecular analysis of NSCLC is necessary to identify novel molecular targets that impact prognosis and the design of new-targeted therapies. In recent years, tumor "activated/reprogrammed" stromal cells that promote carcinogenesis have emerged as potential therapeutic targets. However, the contribution of stromal cells to NSCLC is poorly understood. Here, we show increased numbers of bone marrow (BM)-derived hematopoietic cells in the tumor parenchyma of NSCLC patients compared with matched adjacent non-neoplastic lung tissue. By sorting specific cellular fractions from lung cancer patients, we compared the transcriptomes of intratumoral myeloid compartments within the tumor bed with their counterparts within adjacent non-neoplastic tissue from NSCLC patients. The RNA sequencing of specific myeloid compartments (immature monocytic myeloid cells and polymorphonuclear neutrophils) identified differentially regulated genes and mRNA isoforms, which were inconspicuous in whole tumor analysis. Genes encoding secreted factors, including osteopontin (OPN), chemokine (C-C motif) ligand 7 (CCL7) and thrombospondin 1 (TSP1) were identified, which enhanced tumorigenic properties of lung cancer cells indicative of their potential as targets for therapy. This study demonstrates that analysis of homogeneous stromal populations isolated directly from fresh clinical specimens can detect important stromal genes of therapeutic value.

Purpose. To assess outcomes of minimally invasive plate osteosynthesis (MIPO) using a locking compression plate (LCP) for 42 humeral shaft fractures. Methods. 28 men and 14 women aged 18 to 68 (mean, 34; median, 29) years underwent closed reduction and MIPO using a LCP for type 12-A (n=26) and type 12-B (n=16) humeral shaft fractures. Eight of the patients were aged ≥50 years. Patients were followed up monthly until radiological union in at least 3 of the 4 cortices. Functional assessment was based on the Disabilities of Arm, Shoulder and Hand (DASH) score. Results. The mean follow-up period was 25 (range, 14–35) months. The mean DASH score was 35.1 at month 3 and improved to 8.9 at month 6 and 5.2 at year 1. The mean angulation was 4° in the coronal plane and 7° in the sagittal plane. All fractures united after a mean of 14 weeks. Two patients with transverse fractures had delayed union and received bone marrow injections at 12 or 13 weeks; they achieved union at week 20. One patient developed a radial nerve palsy immediately after surgery and underwent surgical exploration through the anterolateral approach. The plate was re-applied, and the nerve recovered in 48 hours with full power in all the muscle groups. Conclusion. MIPO with LCP is a safe and effective technique for fixation of diaphyseal humeral fractures, and results in faster bone union, better cosmesis, and minimal complications.

Purpose. To compare outcomes in Indian women who underwent total knee arthroplasty (TKA) using the standard Legacy Posterior Stabilised (LPS) versus the gender-specific LPS high-flexion knee prosthesis. Methods. 100 women (200 knees) aged 60 to 80 years with an arc of flexion of ≥90° underwent simultaneous sequential TKA for primary osteoarthritis of both knees. They were randomised to receive the standard NexGen LPS prosthesis (n=50) or the gender-specific NexGen LPS High-Flex prosthesis (n=50). Both knees in each patient received the same prosthesis. The resected bone from the posterior femoral condyle was 2 mm greater when the gender-specific prosthesis was used. Patients were followed up at 3, 6, 12, and 24 months. Range of motion, Knee Society score, and Hospital for Special Surgery score were evaluated pre- and post-operatively by a single assessor. Results. The mean follow-up duration was 2.1 (range, 1.6–2.5) years. Respectively for the standard and gender-specific groups, the mean range of motion was 111° and 112° preoperatively and 120° and 123° at the latest follow-up. The gender-specific group gained approximately 3° more in range of motion (p=0.007). The Knee Society score and the Hospital for Special Surgery score between groups were not significantly different pre- and post-operatively. Conclusion. The perceived advantage of a gender-specific prosthesis over a standard prosthesis did not translate into better clinical and functional outcome scores.

MicroRNAs (miRNAs) are key regulators of gene expression and contribute to a variety of biological processes. Abnormal miRNA expression has been reported in various diseases including pathophysiology of breast cancer, where they regulate protumorigenic processes including vascular invasiveness, estrogen receptor status, chemotherapy resistance, invasion and metastasis. The miRBase sequence database, a public repository for newly discovered miRNAs, has grown rapidly with approximately >10,000 entries to date. Despite this rapid growth, many miRNAs have not yet been validated, and several others are yet to be identified. A lack of a full complement of miRNAs has imposed limitations on recognizing their important roles in cancer, including breast cancer. Using deep sequencing technology, we have identified 189 candidate novel microRNAs in human breast cancer cell lines with diverse tumorigenic potential. We further show that analysis of 500-nucleotide pri-microRNA secondary structure constitutes a reliable method to predict bona fide miRNAs as judged by experimental validation. Candidate novel breast cancer miRNAs with stem lengths of greater than 30 bp resulted in the generation of precursor and mature sequences in vivo. On the other hand, candidates with stem length less than 30 bp were less efficient in producing mature miRNA. This approach may be used to predict which candidate novel miRNA would qualify as bona fide miRNAs from deep sequencing data with approximately 90% accuracy.

Abnormal regulation of angiogenesis in tumors results in the formation of vessels that are necessary for tumor growth, but compromised in structure and function. Abnormal tumor vasculature impairs oxygen and drug delivery and results in radiotherapy and chemotherapy resistance, respectively. Alpha particles are extraordinarily potent, short-ranged radiations with geometry uniquely suitable for selectively killing neovasculature.Actinium-225 ((225)Ac)-E4G10, an alpha-emitting antibody construct reactive with the unengaged form of vascular endothelial cadherin, is capable of potent, selective killing of tumor neovascular endothelium and late endothelial progenitors in bone-marrow and blood. No specific normal-tissue uptake of E4G10 was seen by imaging or post-mortem biodistribution studies in mice. In a mouse-model of prostatic carcinoma, (225)Ac-E4G10 treatment resulted in inhibition of tumor growth, lower serum prostate specific antigen level and markedly prolonged survival, which was further enhanced by subsequent administration of paclitaxel. Immunohistochemistry revealed lower vessel density and enhanced tumor cell apoptosis in (225)Ac-E4G10 treated tumors. Additionally, the residual tumor vasculature appeared normalized as evident by enhanced pericyte coverage following (225)Ac-E4G10 therapy. However, no toxicity was observed in vascularized normal organs following (225)Ac-E4G10 therapy.The data suggest that alpha-particle immunotherapy to neovasculature, alone or in combination with sequential chemotherapy, is an effective approach to cancer therapy.

Loss of Id1 in the bone marrow (BM) severely impairs tumor angiogenesis resulting in significant inhibition of tumor growth. This phenotype has been associated with the absence of circulating endothelial progenitor cells (EPCs) in the peripheral blood of Id1 mutant mice. However, the manner in which Id1 loss in the BM controls EPC generation or mobilization is largely unknown. Using genetically modified mouse models we demonstrate here that the generation of EPCs in the BM depends on the ability of Id1 to restrain the expression of its target gene p21. Through a series of cellular and functional studies we show that the increased myeloid commitment of BM stem cells and the absence of EPCs in Id1 knockout mice are associated with elevated p21 expression. Genetic ablation of p21 rescues the EPC population in the Id1 null animals, re-establishing functional BM-derived angiogenesis and restoring normal tumor growth. These results demonstrate that the restraint of p21 expression by Id1 is one key element of its activity in facilitating the generation of EPCs in the BM and highlight the critical role these cells play in tumor angiogenesis.