Who, where, what and where to now? A snapshot of publishing patterns in Australian orthopaedic surgery

Background

Development of core research competency is a principle of orthopaedic surgical training in Australia. This paper aims to provide an objective snapshot of publications by Australian orthopaedic trainees and surgeons, to contribute to the discussion on how to identify and build on research capability in the Australian Orthopaedic Association (AOA).

Methods

By analysing journals with a journal impact factor >1 from 2009 to 2015, data were gathered to explore scientific journal publications by Australian orthopaedic surgeons and trainees in relation to who are the authors, what they are reporting and where they are publishing.

Results

One thousand five hundred and thirty-nine articles were identified with 134 orthopaedic trainees and 519 surgeons as authors. The publication rate for both trainees and surgeons was just over two in five. The majority of studies were of level three or four evidence (Oxford's Centre for Evidence-Based Medicine guidelines). Only 5% of trainee papers were published without surgeons' co-authorship. Eighty-six percent of papers published by surgeons did not involve a trainee. The rates of trainees publishing with other trainees were low.

Conclusion

Only 5% of trainee papers were published without surgeons' co-authorship, highlighting the importance of surgeon mentorship in developing trainee research capability. The 86% of papers published by surgeons without trainee co-authorship raises the question of missed mentoring opportunities. Low rates of trainee co-authorship highlight potential for trainees to work together to support each other's research efforts. There is scope for more studies involving higher levels of evidence. This paper raises discussion points and areas for further exploration in relation to AOA trainee research capability.

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Pancreatico-jejunostomy decreases post-operative pancreatic fistula incidence and severity after central pancreatectomy

Backgrounds

Central pancreatectomy (CP) is an alternative to pancreaticoduodenectomy and distal pancreatectomy in benign tumours of pancreatic isthmus management. It is known for a high post-operative pancreatic fistula (POPF) rate. The purpose of this study was to compare POPF incidence between pancreatico-jejunostomy (PJ) and pancreatico-gastrostomy (PG).

Methods

Fifty-eight patients (mean age 53.9 ± 1.9 years) who underwent a CP in four French University Hospitals from 1988 to 2011 were analysed. The distal pancreatic remnant was either anastomosed to the stomach (44.8%, n = 25) or to a Roux-en-Y jejunal loop (55.2%, n = 35) with routine external drainage allowing a systematic search for POPF. POPF severity was classified according to the International Study Group on Pancreatic Fistula (ISGPF) and Clavien-Dindo classifications.

Results

The groups were similar on sex ratio, mean age, ASA score, pancreas texture, operative time and operative blood loss. Mean follow-up was 36.2 ± 3.9 months. POPF were significantly more frequent after PG (76.9 versus 37.5%, P = 0.003). PG was associated with significantly higher grade of POPF both when graded with ISGPF classification (P = 0.012) and Clavien-Dindo classification (P = 0.044). There was no significant difference in post-operative bleeding (0.918) and delayed gastric emptying (0.877) between the two groups. Hospital length of stay was increased after PG (23.6 ± 3.5 days versus 16.5 ± 1.9 days, P = 0.071). There was no significant difference in incidence of long-term exocrine (3.8 versus 19.2%, P = 0.134) and endocrine (7.7 versus 9.4%, P = 0.575) pancreatic insufficiencies.

Conclusion

PG was associated with a significantly higher POPF incidence and severity in CP. We recommend performing PJ especially in older patients to improve CP outcomes.

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Particle Targeting in Complex Biological Media

Over the past few decades, nanoengineered particles have gained increasing interest for applications in the biomedical realm, including diagnosis, imaging, and therapy. When functionalized with targeting ligands, these particles have the potential to interact with specific cells and tissues, and accumulate at desired target sites, reducing side effects and improve overall efficacy in applications such as vaccination and drug delivery. However, when targeted particles enter a complex biological environment, the adsorption of biomolecules and the formation of a surface coating (e.g., a protein corona) changes the properties of the carriers and can render their behavior unpredictable. For this reason, it is of importance to consider the potential challenges imposed by the biological environment at the early stages of particle design. This review describes parameters that affect the targeting ability of particulate drug carriers, with an emphasis on the effect of the protein corona. We highlight strategies for exploiting the protein corona to improve the targeting ability of particles. Finally, we provide suggestions for complementing current in vitro assays used for the evaluation of targeting and carrier efficacy with new and emerging techniques (e.g., 3D models and flow-based technologies) to advance fundamental understanding in bio-nano science and to accelerate the development of targeted particles for biomedical applications.

Nanoengineered particles are gaining increasing interest for applications in the biomedical realm. However, the behavior of targeted particles in complex biological environments is still poorly understood. In this Review, Dai et al. discuss parameters that affect the biological performance of particles and highlight strategies for how the targeting ability of particles can be improved.

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Suppression of AGR2 in a TGF-β-induced Smad regulatory pathway mediates epithelial-mesenchymal transition

Abstract

Background

During cancer progression, epithelial cancer cells can be reprogrammed into mesenchymal-like cells with increased migratory potential through the process of epithelial-mesenchymal transition (EMT), representing an essential step of tumor progression towards metastatic state. AGR2 protein was shown to regulate several cancer-associated processes including cellular proliferation, survival and drug resistance.

Methods

The expression of AGR2 was analyzed in cancer cell lines exposed to TGF-β alone or to combined treatment with TGF-β and the Erk1/2 inhibitor PD98059 or the TGF-β receptor specific inhibitor SB431542. The impact of AGR2 silencing by specific siRNAs or CRISPR/Cas9 technology on EMT was investigated by western blot analysis, quantitative PCR, immunofluorescence analysis, real-time invasion assay and adhesion assay.

Results

Induction of EMT was associated with decreased AGR2 along with changes in cellular morphology, actin reorganization, inhibition of E-cadherin and induction of the mesenchymal markers vimentin and N-cadherin in various cancer cell lines. Conversely, induction of AGR2 caused reversion of the mesenchymal phenotype back to the epithelial phenotype and re-acquisition of epithelial markers. Activated Smad and Erk signaling cascades were identified as mutually complementary pathways responsible for TGF-β-mediated inhibition of AGR2.

Conclusion

Taken together our results highlight a crucial role for AGR2 in maintaining the epithelial phenotype by preventing the activation of key factors involved in the process of EMT.

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PD-L1 Expression in Melanoma: A Quantitative Immunohistochemical Antibody Comparison

Purpose: PD-L1 expression in the pretreatment tumor microenvironment enriches for response to anti-PD-1/PD-L1 therapies. The purpose of this study was to quantitatively compare the performance of five monoclonal anti-PD-L1 antibodies used in recent landmark publications.

Experimental Design: PD-L1 IHC was performed on 34 formalin-fixed paraffin-embedded archival melanoma samples using the 5H1, SP142, 28-8, 22C3, and SP263 clones. The percentage of total cells (including melanocytes and immune cells) demonstrating cell surface PD-L1 staining, as well as intensity measurements/H-scores, were assessed for each melanoma specimen using a computer-assisted platform. Staining properties were compared between antibodies.

Results: Strong correlations were observed between the percentage of PD-L1(+) cells across all clones studied (R² = 0.81–0.96). When present, discordant results were attributable to geographic heterogeneity of the melanoma tissue section rather than differences in PD-L1 antibody staining characteristics. PD-L1 intensity/H-scores strongly correlated with percentage of PD-L1(+) cells (R² > 0.78, all clones).

Conclusions: The 5H1, SP142, 28-8, 22C3, and SP263 clones all demonstrated similar performance characteristics when used in a standardized IHC assay on melanoma specimens. Reported differences in PD-L1 IHC assays using these antibodies are thus most likely due to assay characteristics beyond the antibody itself. Our findings also argue against the inclusion of an intensity/H-score in chromogenic PD-L1 IHC assays. Clin Cancer Res; 23(16); 4938–44. ©2017 AACR.

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A Gene Signature for Selecting Benefit from Hypoxia Modification of Radiotherapy for High-Risk Bladder Cancer Patients

Purpose: Hypoxia modification improves overall survival in muscle-invasive bladder cancer patients who undergo radiotherapy. There is evidence that hypoxic tumors benefit most from hypoxia modification. The study aimed to identify or derive a hypoxia gene signature that predicts benefit from hypoxia-modifying treatment in bladder cancer.

Experimental Design: Published hypoxia signatures were tested and a new one derived by analyzing bladder cancer transcriptomic data from public databases. Tumor samples were available from the BCON phase III randomized trial of radiotherapy alone or with carbogen and nicotinamide (CON). Gene expression data were generated for 151 tumors using Affymetrix Human 1.0 Exon ST arrays and used for independent validation.

Results: A 24-gene signature was derived, which was prognostic in four of six independent surgical cohorts (n = 679; meta HR, 2.32; 95% CI, 1.73–3.12; P < 0.0001). The signature was also prognostic in BCON patients receiving radiotherapy alone (n = 75; HR for local relapse-free survival, 2.37; 95% CI, 1.26–4.47; P = 0.0076). The signature predicted benefit from CON (n = 76; HR, 0.47; 95% CI, 0.26–0.86; P = 0.015). Prognostic significance (P = 0.017) and predictive significance (P = 0.058) remained after adjusting for clinicopathologic variables. A test for interaction between hypoxia status and treatment arms was significant (P = 0.0094).

Conclusions: A 24-gene hypoxia signature has strong and independent prognostic and predictive value for muscle-invasive bladder cancer patients. The signature can aid identification of patients likely to benefit from the addition of carbogen and nicotinamide to radiotherapy. Clin Cancer Res; 23(16); 4761–8. ©2017 AACR.

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Venetoclax in Patients with Previously Treated Chronic Lymphocytic Leukemia

Venetoclax is the first BCL2 inhibitor to enter routine clinical practice. It is an orally bioavailable small molecule that binds BCL2 very specifically. Acting as a pharmacologic mimic of the proteins that initiate apoptosis (a so-called BH3 mimetic), venetoclax rapidly induces apoptosis in chronic lymphocytic leukemia (CLL) cells, which express high levels of BCL2 and rely on it to maintain their survival. As a single agent, daily venetoclax treatment induced durable responses in 79% of patients with relapsed or refractory CLL or small lymphocytic lymphoma in a phase I study, including complete remissions in 20% of patients. Its use was approved by the FDA in April 2016 for patients with previously treated del(17p) CLL on the basis of a single-arm phase II trial demonstrating a 79% response rate and an estimated 1-year progression-free survival of 72% with 400 mg/day continuous therapy. This review focuses on venetoclax, its mechanism of action, pharmacology, and clinical trial data and seeks to place it in the context of rapid advances in therapy for patients with relapsed CLL, especially those with del(17p) CLL. Clin Cancer Res; 23(16); 4527–33. ©2017 AACR.

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Genomic Analysis of Thymic Epithelial Tumors Identifies Novel Subtypes Associated with Distinct Clinical Features

Purpose: To reconcile the heterogeneity of thymic epithelial tumors (TET) and gain deeper understanding of the molecular determinants of TETs, we set out to establish a clinically relevant molecular classification system for these tumors.

Experimental Design: Molecular subgrouping of TETs was performed in 120 patients from The Cancer Genome Atlas using a multidimensional approach incorporating analyses of DNA mutations, mRNA expression, and somatic copy number alterations (SCNA), and validated in two independent cohorts.

Results: Four distinct molecular subtypes of TETs were identified. The most commonly identified gene mutation was a missense mutation in General Transcription Factor II-I (GTF2I group), which was present in 38% of patients. The next group was identified by unsupervised mRNA clustering of GTF2I wild-type tumors and represented TETs enriched in expression of genes associated with T-cell signaling (TS group; 33%). The remaining two groups were distinguished by their degree of chromosomal stability (CS group; 8%) or instability (CIN group; 21%) based upon SCNA analyses. Disease-free survival and overall survival were favorable in the GTF2I group and unfavorable in the CIN group. These molecular subgroups were associated with TET histology and clinical features including disease-free survival. Finally, we demonstrate high expression of PD1 mRNA and correlation of PD1 and CD8A in the TS subgroup.

Conclusions: Molecular subtyping of TETs is associated with disease-free and overall survival. Classification of TETs by a molecular framework could aid in the refinement of staging and in the discovery and development of rational treatment options for patients with TETs. Clin Cancer Res; 23(16); 4855–64. ©2017 AACR.

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U.S. Food and Drug Administration Approval Summary: Atezolizumab for Metastatic Non-Small Cell Lung Cancer

On October 18, 2016, the FDA approved atezolizumab (TECENTRIQ; Genentech, Inc.) for treatment of patients with metastatic non–small cell lung cancer (mNSCLC) whose disease progressed during or following platinum-containing chemotherapy. Approval was based on demonstration of clinically meaningful improvements in overall survival (OS) and an acceptable safety profile in two randomized clinical trials (OAK and POPLAR). Median OS in OAK, a phase III trial, was 13.8 months [95% confidence interval (CI), 11.8–15.7] in the atezolizumab arm compared with 9.6 months (95% CI, 8.6–11.2) in the docetaxel arm [hazard ratio (HR) = 0.74; 95% CI, 0.63–0.87; P = 0.0004]. Median OS in POPLAR, a phase II trial, was 12.6 months (95% CI, 9.7–16.0) and 9.7 months (95% CI, 8.6–12.0; HR = 0.69; 95% CI, 0.52–0.92) for the atezolizumab and docetaxel arms, respectively. In patients treated with atezolizumab, the most common (≥20%) adverse reactions were fatigue, decreased appetite, dyspnea, cough, nausea, musculoskeletal pain, and constipation; the most common (≥2%) grade 3 to 4 adverse events were dyspnea, pneumonia, hypoxia, hyponatremia, fatigue, anemia, musculoskeletal pain, aspartate aminotransferase increase, alanine aminotransferase increase, dysphagia, and arthralgia. Clinically significant immune-related adverse events for patients receiving atezolizumab included 1.4% incidence each of grade 3 to 4 pneumonitis, hepatitis, colitis, and thyroid disease. Clin Cancer Res; 23(16); 4534–9. ©2017 AACR.

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Molecular Pathways: Targeting the Protein Kinase Wee1 in Cancer

Wee1 is a protein kinase that regulates the G₂ checkpoint and prevents entry into mitosis in response to DNA damage. Cyclin-dependent kinases (CDK) are a family of 14 serine/threonine protein kinases that coordinate the progression through the cell cycle. The Cdc2/cyclin B complex controls the progression from G₂ into mitosis. There are two mechanisms by which the G₂ checkpoint is initiated in response to DNA damage: phosphorylation of Cdc25c by CHK1 and of the Wee1 kinase, which phosphorylates Cdc2. Blockade at the G₂ checkpoint is especially important for p53-mutant cells because these tumors mainly rely on DNA repair at the G₂ checkpoint. AZD1775 (formerly MK-1775) is a small-molecule, pyrazol-pyrimidine derivative and potent and ATP-competitive specific inhibitor of the Wee1 kinase. Several preclinical and clinical studies demonstrated encouraging antitumor effects with manageable side effects of the combination of Wee1 inhibition and DNA-damaging agents. Promising combination schedules are being investigated at the moment, for example, combining PARP inhibition and Wee1 inhibition. Also, a weekly schedule with carboplatin and AZD1775 warrants investigation aimed at further improving the antitumor effect. Clin Cancer Res; 23(16); 4540–4. ©2017 AACR.

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Mitochondrial BAX Determines the Predisposition to Apoptosis in Human AML

Purpose: Cell-to-cell variability in apoptosis signaling contributes to heterogenic responses to cytotoxic stress in clinically heterogeneous neoplasia, such as acute myeloid leukemia (AML). The BCL-2 proteins BAX and BAK can commit mammalian cells to apoptosis and are inhibited by retrotranslocation from the mitochondria into the cytosol. The subcellular localization of BAX and BAK could determine the cellular predisposition to apoptotic death.

Experimental Design: The relative localization of BAX and BAK was determined by fractionation of AML cell lines and patient samples of a test cohort and a validation cohort.

Results: This study shows that relative BAX localization determines the predisposition of different AML cell lines to apoptosis. Human AML displays a surprising variety of relative BAX localizations. In a test cohort of 48 patients with AML, mitochondria-shifted BAX correlated with improved patient survival, FLT3-ITD status, and leukocytosis. Analysis of a validation cohort of 80 elderly patients treated with myelosuppressive chemotherapy confirmed that relative BAX localization correlates with probability of disease progression, FLT3-ITD status, and leukocytosis. Relative BAX localization could therefore be helpful to identify elderly or frail patients who may benefit from cytotoxic therapy.

Conclusions: In this retrospective analysis of two independent AML cohorts, our data suggest that Bax localization may predict prognosis of patients with AML and cellular predisposition to apoptosis, combining the actual contribution of known and unknown factors to a final "common path." Clin Cancer Res; 23(16); 4805–16. ©2017 AACR.

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In Vivo Detection of EGFRvIII in Glioblastoma via Perfusion Magnetic Resonance Imaging Signature Consistent with Deep Peritumoral Infiltration: The {varphi}-Index

Purpose: The epidermal growth factor receptor variant III (EGFRvIII) mutation has been considered a driver mutation and therapeutic target in glioblastoma, the most common and aggressive brain cancer. Currently, detecting EGFRvIII requires postoperative tissue analyses, which are ex vivo and unable to capture the tumor's spatial heterogeneity. Considering the increasing evidence of in vivo imaging signatures capturing molecular characteristics of cancer, this study aims to detect EGFRvIII in primary glioblastoma noninvasively, using routine clinically acquired imaging.

Experimental Design: We found peritumoral infiltration and vascularization patterns being related to EGFRvIII status. We therefore constructed a quantitative within-patient peritumoral heterogeneity index (PHI/-index), by contrasting perfusion patterns of immediate and distant peritumoral edema. Application of -index in preoperative perfusion scans of independent discovery (n = 64) and validation (n = 78) cohorts, revealed the generalizability of this EGFRvIII imaging signature.

Results: Analysis in both cohorts demonstrated that the obtained signature is highly accurate (89.92%), specific (92.35%), and sensitive (83.77%), with significantly distinctive ability (P = 4.0033 x 10^–10, AUC = 0.8869). Findings indicated a highly infiltrative-migratory phenotype for EGFRvIII⁺ tumors, which displayed similar perfusion patterns throughout peritumoral edema. Contrarily, EGFRvIII^– tumors displayed perfusion dynamics consistent with peritumorally confined vascularization, suggesting potential benefit from extensive peritumoral resection/radiation.

Conclusions: This EGFRvIII signature is potentially suitable for clinical translation, since obtained from analysis of clinically acquired images. Use of within-patient heterogeneity measures, rather than population-based associations, renders -index potentially resistant to inter-scanner variations. Overall, our findings enable noninvasive evaluation of EGFRvIII for patient selection for targeted therapy, stratification into clinical trials, personalized treatment planning, and potentially treatment-response evaluation. Clin Cancer Res; 23(16); 4724–34. ©2017 AACR.

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Characterization of the T-Cell Receptor Repertoire and Immune Microenvironment in Patients with Locoregionally Advanced Squamous Cell Carcinoma of the Head and Neck

Purpose: Squamous cell carcinoma of the head and neck (SCCHN) is a lethal cancer with a suboptimal 5-year overall survival of approximately 50% with surgery and/or definitive chemoradiotherapy. Novel treatments are thus urgently awaited. Immunotherapy with checkpoint blockade has emerged as a promising option for patients with recurrent/metastatic SCCHN; however, it has not been investigated in the curative-intent setting yet. The purpose of this study was to investigate the T-cell receptor repertoire and the tumor microenvironment in tumor tissues of SCCHN patients with locoregionally advanced disease.

Experimental Design: We performed T-cell receptor sequencing of tumor tissues from 44 patients with locoregionally advanced SCCHN prior to treatment with definitive chemoradiotherapy and correlated the T-cell clonality and the mRNA expression levels of immune-related genes with clinicopathologic parameters.

Results: Clonal expansion of T cells was significantly higher in human papilloma virus (HPV)–negative compared with HPV-positive tumors, signifying more robust antigen presentation in HPV-negative tumors. The latter was supported by the higher percentage of HPV-negative tumors expressing HLA-A protein compared with HPV-positive tumors (P = 0.049). Higher GRZB levels correlated significantly with longer recurrence-free survival (log-rank, P = 0.003) independent of tumor size, nodal stage, and HPV status.

Conclusions: Our findings support clonal expansion of T cells in SCCHN patients with locoregionally advanced disease and imply differences in the antigen presentation capacity between HPV-negative and HPV-positive tumors. Elevated GRZB mRNA levels may also serve as a favorable and independent predictor of outcome in SCCHN patients treated with chemoradiotherapy. These data provide rationale for the introduction of immunotherapeutic approaches in the curative-intent setting. Clin Cancer Res; 23(16); 4897–907. ©2017 AACR.

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Clinical Pathways: Recommendations for Putting Patients at the Center of Value-Based Care

Two major trends that have been affecting the provision of oncology care in the United States are a shift from volume-based to value-based care and a push toward patient-centered healthcare. However, these two trends are not always completely aligned with each other. Value-based payment models, including clinical pathways, are one strategy being implemented by oncology stakeholders to help encourage the uptake of value-based oncology care. If structured with the patient in mind, they can improve quality of care for patients with cancer, decrease inappropriate care while enabling appropriate personalization of care, and constrain rising prices by demanding a stronger link between cost and value. If not structured appropriately, they can limit patient choice, impede access to innovative treatments, and encourage one-size-fits-all oncology care. Clin Cancer Res; 23(16); 4545–9. ©2017 AACR.

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Phase I Study of Fenretinide Delivered Intravenously in Patients with Relapsed or Refractory Hematologic Malignancies: A California Cancer Consortium Trial

Purpose: A phase I study was conducted to determine the MTD, dose-limiting toxicities (DLT), and pharmacokinetics of fenretinide delivered as an intravenous emulsion in relapsed/refractory hematologic malignancies.

Experimental Design: Fenretinide (80–1,810 mg/m²/day) was administered by continuous infusion on days 1 to 5, in 21-day cycles, using an accelerated titration design.

Results: Twenty-nine patients, treated with a median of three prior regimens (range, 1–7), were enrolled and received the test drug. Ninety-seven courses were completed. An MTD was reached at 1,280 mg/m²/day for 5 days. Course 1 DLTs included 6 patients with hypertriglyceridemia, 4 of whom were asymptomatic; 2 patients experienced DLT thrombocytopenia (asymptomatic). Of 11 patients with response-evaluable peripheral T-cell lymphomas, two had complete responses [CR, progression-free survival (PFS) 68+ months; unconfirmed CR, PFS 14+ months], two had unconfirmed partial responses (unconfirmed PR, PFS 5 months; unconfirmed PR, PFS 6 months), and five had stable disease (2–12 cycles). One patient with mature B-cell lymphoma had an unconfirmed PR sustained for two cycles. Steady-state plasma levels were approximately 10 mcg/mL (mid-20s μmol/L) at 640 mg/m²/day, approximately 14 mcg/mL (mid-30s μmol/L) at 905 mg/m²/day, and approximately 22 mcg/mL (mid-50s μmol/L) at 1,280 mg/m²/day.

Conclusions: Intravenous fenretinide obtained significantly higher plasma levels than a previous capsule formulation, had acceptable toxicities, and evidenced antitumor activity in peripheral T-cell lymphomas. A recommended phase II dosing is 600 mg/m² on day 1, followed by 1,200 mg/m² on days 2 to 5, every 21 days. A registration-enabling phase II study in relapsed/refractory PTCL (ClinicalTrials.gov identifier: NCT02495415) is ongoing. Clin Cancer Res; 23(16); 4550–5. ©2017 AACR.

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Sensitivity and Specificity of Cetuximab-IRDye800CW to Identify Regional Metastatic Disease in Head and Neck Cancer

Purpose: Comprehensive cervical lymphadenectomy can be associated with significant morbidity and poor quality of life. This study evaluated the sensitivity and specificity of cetuximab-IRDye800CW to identify metastatic disease in patients with head and neck cancer.

Experimental Design: Consenting patients scheduled for curative resection were enrolled in a clinical trial to evaluate the safety and specificity of cetuximab-IRDye800CW. Patients (n = 12) received escalating doses of the study drug. Where indicated, cervical lymphadenectomy accompanied primary tumor resection, which occurred 3 to 7 days following intravenous infusion of cetuximab-IRDye800CW. All 471 dissected lymph nodes were imaged with a closed-field, near-infrared imaging device during gross processing of the fresh specimens. Intraoperative imaging of exposed neck levels was performed with an open-field fluorescence imaging device. Blinded assessments of the fluorescence data were compared to histopathology to calculate sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV).

Results: Of the 35 nodes diagnosed pathologically positive, 34 were correctly identified with fluorescence imaging, yielding a sensitivity of 97.2%. Of the 435 pathologically negative nodes, 401 were correctly assessed using fluorescence imaging, yielding a specificity of 92.7%. The NPV was determined to be 99.7%, and the PPV was 50.7%. When 37 fluorescently false-positive nodes were sectioned deeper (1 mm) into their respective blocks, metastatic cancer was found in 8.1% of the recut nodal specimens, which altered staging in two of those cases.

Conclusions: Fluorescence imaging of lymph nodes after systemic cetuximab-IRDye800CW administration demonstrated high sensitivity and was capable of identifying additional positive nodes on deep sectioning. Clin Cancer Res; 23(16); 4744–52. ©2017 AACR.

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Phase I Trial of Intratumoral Injection of CCL21 Gene-Modified Dendritic Cells in Lung Cancer Elicits Tumor-Specific Immune Responses and CD8+ T-cell Infiltration

Purpose: A phase I study was conducted to determine safety, clinical efficacy, and antitumor immune responses in patients with advanced non–small cell lung carcinoma (NSCLC) following intratumoral administration of autologous dendritic cells (DC) transduced with an adenoviral (Ad) vector expressing the CCL21 gene (Ad-CCL21-DC). We evaluated safety and tumor antigen–specific immune responses following in situ vaccination (ClinicalTrials.gov: NCT01574222).

Experimental Design: Sixteen stage IIIB/IV NSCLC subjects received two vaccinations (1 x 10⁶, 5 x 10⁶, 1 x 10⁷, or 3 x 10⁷ DCs/injection) by CT- or bronchoscopic-guided intratumoral injections (days 0 and 7). Immune responses were assessed by tumor antigen–specific peripheral blood lymphocyte induction of IFN in ELISPOT assays. Tumor biopsies were evaluated for CD8⁺ T cells by IHC and for PD-L1 expression by IHC and real-time PCR (RT-PCR).

Results: Twenty-five percent (4/16) of patients had stable disease at day 56. Median survival was 3.9 months. ELISPOT assays revealed 6 of 16 patients had systemic responses against tumor-associated antigens (TAA). Tumor CD8⁺ T-cell infiltration was induced in 54% of subjects (7/13; 3.4-fold average increase in the number of CD8⁺ T cells per mm²). Patients with increased CD8⁺ T cells following vaccination showed significantly increased PD-L1 mRNA expression.

Conclusions: Intratumoral vaccination with Ad-CCL21-DC resulted in (i) induction of systemic tumor antigen–specific immune responses; (ii) enhanced tumor CD8⁺ T-cell infiltration; and (iii) increased tumor PD-L1 expression. Future studies will evaluate the role of combination therapies with PD-1/PD-L1 checkpoint inhibition combined with DC-CCL21 in situ vaccination. Clin Cancer Res; 23(16); 4556–68. ©2017 AACR.

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Histone Deacetylase Inhibitor Enhances the Efficacy of MEK Inhibitor through NOXA-Mediated MCL1 Degradation in Triple-Negative and Inflammatory Breast Cancer

Purpose: Inflammatory breast cancer (IBC), diagnosed clinically, and triple-negative breast cancer (TNBC), diagnosed by molecular receptor status, are the two most aggressive forms of breast cancer, and both lack effective targeted therapies. We previously demonstrated involvement of histone deacetylase (HDAC) inhibitor entinostat in regulating apoptosis in IBC and TNBC cells; here, we aimed to identify novel combination therapy candidates.

Experimental Design: Potential therapeutic targets were identified by mRNA expression profiling of TNBC and IBC cells treated with entinostat. Drug action and synergism were assessed by in vitro proliferation assays, tumor growth in vivo, and proteomic analyses. Gain/loss-of-expression studies were utilized to functionally validate the role of identified targets in sensitivity of TNBC and IBC cells to combination therapy.

Results: Entinostat induced activity of the oncogenic ERK pathway and expression of proapoptotic NOXA. These are known to stabilize and degrade, respectively, MCL1, an antiapoptotic Bcl-2 protein. In breast cancer patients, high-MCL1/low-NOXA tumor expression correlated significantly with poor survival outcomes. Combination treatment of entinostat with MEK inhibitor pimasertib reduced the growth of TNBC and IBC cells in vitro and inhibited tumor growth in vivo. The synergistic action of combination therapy was observed in TNBC and IBC cell lines in which NOXA expression was induced following entinostat treatment. The therapeutic activity depended on induction of mitochondrial cell death pathways initiated by NOXA-mediated MCL1 degradation.

Conclusions: Our preclinical findings provide a rationale for the clinical testing of combination HDAC and MEK pathway inhibition for TNBC and IBC that exhibit elevated baseline tumor MCL1 expression. Clin Cancer Res; 23(16); 4780–92. ©2017 AACR.

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Intra- and Interobserver Reproducibility Assessment of PD-L1 Biomarker in Non-Small Cell Lung Cancer

Purpose: Reliable and reproducible methods for identifying PD-L1 expression on tumor cells are necessary to identify responders to anti–PD-1 therapy. We tested the reproducibility of the assessment of PD-L1 expression in non–small cell lung cancer (NSCLC) tissue samples by pathologists.

Experimental Design: NSCLC samples were stained with PD-L1 22C3 pharmDx kit using the Dako Autostainer Link 48 Platform. Two sample sets of 60 samples each were designed to assess inter- and intraobserver reproducibility considering two cut points for positivity: 1% or 50% of PD-L1 stained tumor cells. A randomization process was used to obtain equal distribution of PD-L1 positive and negative samples within each sample set. Ten pathologists were randomly assigned to two subgroups. Subgroup 1 analyzed all samples on two consecutive days. Subgroup 2 performed the same assessments, except they received a 1-hour training session prior to the second assessment.

Results: For intraobserver reproducibility, the overall percent agreement (OPA) was 89.7% [95% confidence interval (CI), 85.7–92.6] for the 1% cut point and 91.3% (95% CI, 87.6–94.0) for the 50% cut point. For interobserver reproducibility, OPA was 84.2% (95% CI, 82.8–85.5) for the 1% cut point and 81.9% (95% CI, 80.4–83.3) for the 50% cut point, and Cohen's coefficients were 0.68 (95% CI, 0.65–0.71) and 0.58 (95% CI, 0.55–0.62), respectively. The training was found to have no or very little impact on intra- or interobserver reproducibility.

Conclusions: Pathologists reported good reproducibility at both 1% and 50% cut points. More adapted training could potentially increase reliability, in particular for samples with PD-L1 proportion, scores around 50%. Clin Cancer Res; 23(16); 4569–77. ©2017 AACR.

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Epigenetic Regulation of KPC1 Ubiquitin Ligase Affects the NF-{kappa}B Pathway in Melanoma

Purpose: Abnormal activation of the NF-B pathway induces a more aggressive phenotype of cutaneous melanoma. Understanding the mechanisms involved in melanoma NF-B activation may identify novel targets for this pathway. KPC1, an E3 ubiquitin ligase, is a regulator of the NF-B pathway. The objective of this study was to investigate the mechanisms regulating KPC1 expression and its clinical impact in melanoma.

Experimental Design: The clinical impact of KPC1 expression and its epigenetic regulation were assessed in large cohorts of clinically well-annotated melanoma tissues (tissue microarrays; n = 137, JWCI cohort; n = 40) and The Cancer Genome Atlas database (TCGA cohort, n = 370). Using melanoma cell lines, we investigated the functional interactions between KPC1 and NF-B, and the epigenetic regulations of KPC1, including DNA methylation and miRNA expression.

Results: We verified that KPC1 suppresses melanoma proliferation by processing NF-B1 p105 into p50, thereby modulating NF-B target gene expression. Concordantly, KPC1 expression was downregulated in American Joint Committee on Cancer stage IV melanoma compared with early stages (stage I/II P = 0.013, stage III P = 0.004), and low KPC1 expression was significantly associated with poor overall survival in stage IV melanoma (n = 137; HR 1.810; P = 0.006). Furthermore, our data showed that high miR-155-5p expression, which is controlled by DNA methylation at its promoter region (TCGA; Pearson's r –0.455; P < 0.001), is significantly associated with KPC1 downregulation (JWCI; P = 0.028, TCGA; P = 0.003).

Conclusions: This study revealed novel epigenetic regulation of KPC1 associated with NF-B pathway activation, promoting metastatic melanoma progression. These findings suggest the potential utility of KPC1 and its epigenetic regulation as theranostic targets. Clin Cancer Res; 23(16); 4831–42. ©2017 AACR.

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Circulating DNA Demonstrates Convergent Evolution and Common Resistance Mechanisms during Treatment of Colorectal Cancer

Purpose: Liquid biopsies allow the tracking of clonal dynamics and detection of mutations during treatment.

Experimental Design: We evaluated under blinded conditions the ability of cell-free DNA (cfDNA) to detect RAS/BRAF mutations in the plasma of 42 metastatic colorectal cancer patients treated on a phase Ib/II trial of FOLFOX and dasatinib, with or without cetuximab.

Results: Prior to treatment, sequencing of archival tissue detected mutations in 25 of 42 patients (60%), while the cfDNA assay detected mutations in 37 of 42 patients (88%). Our cfDNA assay detected mutations with allele frequencies as low as 0.01%. After exposure to treatment, 41 of 42 patients (98%) had a cfDNA-detected RAS/BRAF mutation. Of 21 patients followed with serial measurements who were RAS/BRAF mutant at baseline, 11 (52%) showed additional point mutation following treatment and 3 (14%) no longer had detectable levels of another mutant allele. Of RAS/BRAF wild-type tumors at baseline, 4 of 5 (80%) showed additional point mutations. cfDNA quantitative measurements from this study closely mirrored changes in CEA and CT scan results, highlighting the importance of obtaining quantitative data beyond the mere presence of a mutation.

Conclusions: Our findings demonstrate the development of new RAS/BRAF mutations in patients regardless of whether they had preexisting mutations in the pathway, demonstrating a convergent evolutionary pattern. Clin Cancer Res; 23(16); 4578–91. ©2017 AACR.

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Whole-Exome Sequencing of Metaplastic Breast Carcinoma Indicates Monoclonality with Associated Ductal Carcinoma Component

Purpose: Although most human cancers display a single histology, there are unusual cases where two or more distinct tissue types present within a primary tumor. One such example is metaplastic breast carcinoma, a rare but aggressive cancer with a heterogeneous histology, including squamous, chondroid, and spindle cells. Metaplastic carcinomas often contain an admixed conventional ductal invasive or in situ mammary carcinoma component, and are typically triple-negative for estrogen receptor, progesterone receptor, and HER-2 amplification/overexpression. An unanswered question is the origin of metaplastic breast cancers. While they may arise independently from their ductal components, their close juxtaposition favors a model that postulates a shared origin, either as two derivatives from the same primary cancer or one histology as an outgrowth of the other. Understanding the mechanism of development of these tumors may inform clinical decisions.

Experimental Design: We performed exome sequencing for paired metaplastic and adjacent conventional invasive ductal carcinomas in 8 patients and created a pipeline to identify somatic variants and predict their functional impact, without having normal tissue. We then determined the genetic relationships between the histologically distinct compartments.

Results: In each case, the tumor components have nearly identical landscapes of somatic mutation, implying that the differing histologies do not derive from genetic clonal divergence.

Conclusions: A shared origin for tumors with differing histologies suggests that epigenetic or noncoding changes may mediate the metaplastic phenotype and that alternative therapeutic approaches, including epigenetic therapies, may be required for metaplastic breast cancers. Clin Cancer Res; 23(16); 4875–84. ©2017 AACR.

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Association of Tissue Abiraterone Levels and SLCO Genotype with Intraprostatic Steroids and Pathologic Response in Men with High-Risk Localized Prostate Cancer

Purpose: Germline variation in solute carrier organic anion (SLCO) genes influences cellular steroid uptake and is associated with prostate cancer outcomes. We hypothesized that, due to its steroidal structure, the CYP17A inhibitor abiraterone may undergo transport by SLCO-encoded transporters and that SLCO gene variation may influence intracellular abiraterone levels and outcomes.

Experimental Design: Steroid and abiraterone levels were measured in serum and tissue from 58 men with localized prostate cancer in a clinical trial of LHRH agonist plus abiraterone acetate plus prednisone for 24 weeks prior to prostatectomy. Germline DNA was genotyped for 13 SNPs in six SLCO genes.

Results: Abiraterone levels spanned a broad range (serum median 28 ng/mL, 108 nmol/L; tissue median 77 ng/mL, 271 nmol/L) and were correlated (r = 0.355, P = 0.001). Levels correlated positively with steroids upstream of CYP17A (pregnenolone, progesterone), and inversely with steroids downstream of CYP17A (DHEA, AED, testosterone). Serum PSA and tumor volumes were higher in men with undetectable versus detectable tissue abiraterone at prostatectomy (median 0.10 vs. 0.03 ng/dL, P = 0.02; 1.28 vs. 0.44 cc, P = 0.09, respectively). SNPs in SLCO2B1 associated with significant differences in tissue abiraterone (rs1789693, P = 0.0008; rs12422149, P = 0.03) and higher rates of minimal residual disease (tumor volume < 0.5 cc; rs1789693, 67% vs. 27%, P = 0.009; rs1077858, 46% vs. 0%, P = 0.03). LNCaP cells expressing SLCO2B1 showed two- to fourfold higher abiraterone levels compared with vector controls (P < 0.05).

Conclusions: Intraprostatic abiraterone levels and genetic variation in SLCO genes are associated with pathologic responses in high-risk localized prostate cancer. Variation in SLCO genes may serve as predictors of response to abiraterone treatment. Clin Cancer Res; 23(16); 4592–601. ©2017 AACR.

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Enrichment of PI3K-AKT-mTOR Pathway Activation in Hepatic Metastases from Breast Cancer

Purpose: Little is known about the molecular signatures associated with specific metastatic sites in breast cancer. Using comprehensive multi-omic molecular profiling, we assessed whether alterations or activation of the PI3K–AKT–mTOR pathway is associated with specific sites of breast cancer metastasis.

Experimental Design: Next-generation sequencing–based whole-exome sequencing was coupled with reverse-phase protein microarray (RPPA) functional signaling network analysis to explore the PI3K–AKT–mTOR axis in 32 pretreated breast cancer metastases. RPPA-based signaling data were further validated in an independent cohort of 154 metastatic lesions from breast cancer and 101 unmatched primary breast tumors. The proportion of cases with PI3K–AKT–mTOR genomic alterations or signaling network activation were compared between hepatic and nonhepatic lesions.

Results: PIK3CA mutation and activation of AKT (S473) and p70S6K (T389) were detected more frequently among liver metastases than nonhepatic lesions (P < 0.01, P = 0.056, and P = 0.053, respectively). However, PIK3CA mutations alone were insufficient in predicting protein activation (P = 0.32 and P = 0.19 for activated AKT and p70S6K, respectively). RPPA analysis of an independent cohort of 154 tumors confirmed the relationship between pathway activation and hepatic metastasis [AKT (S473), mTOR (S2448), and 4EBP1 (S65); P < 0.01, P = 0.02, and P = 0.01, respectively]. Similar results were also seen between liver metastases and primary breast tumors [AKT (S473) P < 0.01, mTOR (S2448) P < 0.01, 4EBP1 (S65) P = 0.01]. This signature was lost when primary tumors were compared with all metastatic sites combined.

Conclusions: Breast cancer patients with liver metastasis may represent a molecularly homogenized cohort with increased incidence of PIK3CA mutations and activation of the PI3K–AKT–mTOR signaling network. Clin Cancer Res; 23(16); 4919–28. ©2017 AACR.

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Highlights from Recent Cancer Literature

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GLI1 Blockade Potentiates the Antitumor Activity of PI3K Antagonists in Lung Squamous Cell Carcinoma

Lung squamous cell carcinoma (SCC), strongly associated with smoking, is treated primarily with traditional cytotoxic chemotherapy due to a lack of FDA-approved targeted agents available. Here, we identify the Hedgehog pathway transcription factor GLI1 as a critical driver of lung SCC. Analysis of human lung cancer datasets showed that GLI1 mRNA was highly expressed in human lung SCC and portended a poor prognosis. Inhibition of GLI1 in human lung SCC cell lines suppressed tumor cell clonogenicity and proliferation in culture and in vivo. Addition of SHH ligand, SMO antagonists, or other Hedgehog pathway agonists did not affect GLI1 expression in lung SCC cells. However, GLI1 expression was modulated by either inhibition or activation of the PI3K and MAPK pathways. Furthermore, in vivo growth of SCC harboring amplifications of the PI3K gene PIK3CA was attenuated by antagonizing GLI1 and PI3K. Thus, a combinatorial therapeutic strategy that targets the PI3K–mTOR pathway and GLI1 may lead to effective outcomes for PI3K pathway-dependent cancers, in contrast to recent results of human trials with single-agent PI3K antagonists. Cancer Res; 77(16); 4448–59. ©2017 AACR.

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Micronuclei Frequency in Tumors Is a Predictive Biomarker for Genetic Instability and Sensitivity to the DNA Repair Inhibitor AsiDNA

Therapeutic strategies targeting DNA repair pathway defects have been widely explored, but often only benefit small numbers of patients. Here we characterized potential predictive biomarkers for treatment with AsiDNA, a novel first-in-class DNA repair inhibitor. We evaluated genetic instability and DNA repair defects by direct and indirect assays in 12 breast cancer cell lines to estimate the spontaneous occurrence of single-strand and double-strand breaks (DSB). For each cell line, we monitored constitutive PARP activation, spontaneous DNA damage by alkaline comet assay, basal micronuclei levels, the number of large-scale chromosomal rearrangements (LST), and the status of several DNA repair pathways by transcriptome and genome analysis. Sensitivity to AsiDNA was associated with a high spontaneous frequency of cells with micronuclei and LST and specific alterations in DNA repair pathways that essentially monitor DSB repair defects. A high basal level of micronuclei as a predictive biomarker for AsiDNA treatment was validated in 43 tumor cell lines from various tissues and 15 models of cell- and patient-derived xenografts. Micronuclei quantification was also possible in patient biopsies. Overall, this study identified genetic instability as a predictive biomarker for sensitivity to AsiDNA treatment. That micronuclei frequency can be measured in biopsies and does not reveal the same genetic instability as conventional genome assays opens new perspectives for refining the classification of tumors with genetic instability. Cancer Res; 77(16); 4207–16. ©2017 AACR.

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A Genome-Wide CRISPR Screen Identifies Genes Critical for Resistance to FLT3 Inhibitor AC220

Acute myeloid leukemia (AML) is a malignant hematopoietic disease and the most common type of acute leukemia in adults. The mechanisms underlying drug resistance in AML are poorly understood. Activating mutations in FMS-like tyrosine kinase 3 (FLT3) are the most common molecular abnormality in AML. Quizartinib (AC220) is a potent and selective second-generation inhibitor of FLT3. It is in clinical trials for the treatment of relapsed or refractory FLT3-ITD–positive and –negative AML patients and as maintenance therapy. To understand the mechanisms of drug resistance to AC220, we undertook an unbiased approach with a novel CRISPR-pooled library to screen new genes whose loss of function confers resistance to AC220. We identified SPRY3, an intracellular inhibitor of FGF signaling, and GSK3, a canonical Wnt signaling antagonist, and demonstrated reactivation of downstream FGF/Ras/ERK and Wnt signaling as major mechanisms of resistance to AC220. We confirmed these findings in primary AML patient samples. Expression of SPRY3 and GSK3A was dramatically reduced in AC220-resistant AML samples, and SPRY3-deleted primary AML cells were resistant to AC220. Intriguingly, expression of SPRY3 was greatly reduced in GSK3 knockout AML cells, which positioned SPRY3 downstream of GSK3 in the resistance pathway. Taken together, our study identified novel genes whose loss of function conferred resistance to a selective FLT3 inhibitor, providing new insight into signaling pathways that contribute to acquired resistance in AML. Cancer Res; 77(16); 4402–13. ©2017 AACR.

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Smurf2-Mediated Stabilization of DNA Topoisomerase II{alpha} Controls Genomic Integrity

DNA topoisomerase IIα (Topo IIα) ensures genomic integrity and unaltered chromosome inheritance and serves as a major target of several anticancer drugs. Topo IIα function is well understood, but how its expression is regulated remains unclear. Here, we identify the E3 ubiquitin ligase Smurf2 as a physiologic regulator of Topo IIα levels. Smurf2 physically interacted with Topo IIα and modified its ubiquitination status to protect Topo IIα from the proteasomal degradation in dose- and catalytically dependent manners. Smurf2-depleted cells exhibited a reduced ability to resolve DNA catenanes and pathological chromatin bridges formed during mitosis, a trait of Topo IIα–deficient cells and a hallmark of chromosome instability. Introducing Topo IIα into Smurf2-depleted cells rescued this phenomenon. Smurf2 was a determinant of Topo IIα protein levels in normal and cancer cells and tissues, and its levels affected cell sensitivity to the Topo II–targeting drug etoposide. Our results identified Smurf2 as an essential regulator of Topo IIα, providing novel insights into its control and into the suggested tumor-suppressor functions of Smurf2. Cancer Res; 77(16); 4217–27. ©2017 AACR.

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MET Exon 14 Mutation Encodes an Actionable Therapeutic Target in Lung Adenocarcinoma

Targeting somatically activated oncogenes has revolutionized the treatment of non–small cell lung cancer (NSCLC). Mutations in the gene mesenchymal–epithelial transition (MET) near the exon 14 splice sites are recurrent in lung adenocarcinoma and cause exon skipping (METΔ14). Here, we analyzed 4,422 samples from 12 different malignancies to estimate the rate of said exon skipping. METΔ14 mutation and transcript were most common in lung adenocarcinoma. Endogenously expressed levels of METΔ14 transformed human epithelial lung cells in a hepatocyte growth factor–dependent manner. In addition, overexpression of the orthologous mouse allele induced lung adenocarcinoma in a novel, immunocompetent mouse model. Met inhibition showed clinical benefit in this model. In addition, we observed a clinical response to crizotinib in a patient with METΔ14-driven NSCLC, only to observe new missense mutations in the MET activation loop, critical for binding to crizotinib, upon clinical progression. These findings support genomically selected clinical trials directed toward METΔ14 in a fraction of NSCLC patients, confirm second-site mutations for further therapeutic targeting prior to and beyond acquired resistance, and provide an in vivo system for the study of METΔ14 in an immunocompetent host. Cancer Res; 77(16); 4498–505. ©2017 AACR.

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Acetylation of Mastermind-like 1 by p300 Drives the Recruitment of NACK to Initiate Notch-Dependent Transcription

Although it has long been appreciated that p300 acts as a critical Notch coactivator, the mechanistic details of p300 in Notch-mediated transcription remain unclear. We previously demonstrated that PEAK1-related kinase activating pseudokinase 1 (NACK), also known as SGK223, is a critical coactivator of Notch signaling and binds to the Notch1 ternary complex. Herein we report that p300 and CBP acetylate Mastermind-like 1 (Maml1) on amino acid residues K188 and K189 to recruit NACK to the Notch1 ternary complex, which results in the recruitment of RNA polymerase II to initiate transcription. NACK is recruited to the ternary complexes containing Maml1 and Maml3, but not Maml2. Simultaneous inhibition of p300/CBP and Notch has a synergistic effect in esophageal adenocarcinoma. In summary, this study provides a deeper mechanistic understanding of the assembly of the Notch transcriptional complex and provides rationale and proof of concept for a combinatorial therapeutic attack on Notch-dependent cancers. Cancer Res; 77(16); 4228–37. ©2017 AACR.

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Characterization of MK-4166, a Clinical Agonistic Antibody That Targets Human GITR and Inhibits the Generation and Suppressive Effects of T Regulatory Cells

GITR is a T-cell costimulatory receptor that enhances cellular and humoral immunity. The agonist anti-mouse GITR antibody DTA-1 has demonstrated efficacy in murine models of cancer primarily by attenuation of Treg-mediated immune suppression, but the translatability to human GITR biology has not been fully explored. Here, we report the potential utility of MK-4166, a humanized GITR mAb selected to bind to an epitope analogous to the DTA-1 epitope, which enhances the proliferation of both naïve and tumor-infiltrating T lymphocytes (TIL). We also investigated the role of GITR agonism in human antitumor immune responses and report here the preclinical characterization and toxicity assessment of MK-4166, which is currently being evaluated in a phase I clinical study. Expression of human GITR was comparable with that of mouse GITR in tumor-infiltrating Tregs despite being drastically lower in other human TILs and in many human peripheral blood populations. MK-4166 decreased induction and suppressive effects of Tregs in vitro. In human TIL cultures, MK-4166 induced phosphorylation of NFκB and increased expression of dual specificity phosphatase 6 (DUSP6), indicating that MK-4166 activated downstream NFκB and Erk signaling pathways. Furthermore, MK-4166 downregulated FOXP3 mRNA in human tumor infiltrating Tregs, suggesting that, in addition to enhancing the activation of TILs, MK-4166 may attenuate the Treg-mediated suppressive tumor microenvironment. Cancer Res; 77(16); 4378–88. ©2017 AACR.

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Utility of Genomic Analysis In Circulating Tumor DNA from Patients with Carcinoma of Unknown Primary

Carcinoma of unknown primary (CUP) is a rare and difficult-to-treat malignancy, the management of which might be improved by the identification of actionable driver mutations. We interrogated 54 to 70 genes in 442 patients with CUP using targeted clinical-grade, next-generation sequencing of circulating tumor DNA (ctDNA). Overall, 80% of patients exhibited ctDNA alterations; 66% (290/442) ≥1 characterized alteration(s), excluding variants of unknown significance. TP53-associated genes were most commonly altered [37.8% (167/442)], followed by genes involved in the MAPK pathway [31.2% (138/442)], PI3K signaling [18.1% (80/442)], and the cell-cycle machinery [10.4% (46/442)]. Among 290 patients harboring characterized alterations, distinct genomic profiles were observed in 87.9% (255/290) of CUP cases, with 99.7% (289/290) exhibiting potentially targetable alterations. An illustrative patient with dynamic changes in ctDNA content during therapy and a responder given a checkpoint inhibitor–based regimen because of a mismatch repair gene anomaly are presented. Our results demonstrate that ctDNA evaluation is feasible in CUP and that most patients harbor a unique somatic profile with pharmacologically actionable alterations, justifying the inclusion of noninvasive liquid biopsies in next-generation clinical trials. Cancer Res; 77(16); 4238–46. ©2017 AACR.

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Amlexanox Downregulates S100A6 to Sensitize KMT2A/AFF1-Positive Acute Lymphoblastic Leukemia to TNF{alpha} Treatment

Acute lymphoblastic leukemias (ALL) positive for KMT2A/AFF1 (MLL/AF4) translocation, which constitute 60% of all infant ALL cases, have a poor prognosis even after allogeneic hematopoietic stem cell transplantation (allo-HSCT). This poor prognosis is due to one of two factors, either resistance to TNFα, which mediates a graft-versus-leukemia (GVL) response after allo-HSCT, or immune resistance due to upregulated expression of the immune escape factor S100A6. Here, we report an immune stimulatory effect against KMT2A/AFF1-positive ALL cells by treatment with the anti-allergy drug amlexanox, which we found to inhibit S100A6 expression in the presence of TNF-α. In KMT2A/AFF1-positive transgenic (Tg) mice, amlexanox enhanced tumor immunity and lowered the penetrance of leukemia development. Similarly, in a NOD/SCID mouse model of human KMT2A/AFF1-positive ALL, amlexanox broadened GVL responses and extended survival. Our findings show how amlexanox degrades the resistance of KMT2A/AFF1-positive ALL to TNFα by downregulating S100A6 expression, with immediate potential implications for improving clinical management of KMT2A/AFF1-positive ALL. Cancer Res; 77(16); 4426–33. ©2017 AACR.

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RGS12 Is a Novel Tumor-Suppressor Gene in African American Prostate Cancer That Represses AKT and MNX1 Expression

African American (AA) men exhibit a relatively high incidence and mortality due to prostate cancer even after adjustment for socioeconomic factors, but the biological basis for this disparity is unclear. Here, we identify a novel region on chromosome 4p16.3 that is lost selectively in AA prostate cancer. The negative regulator of G-protein signaling RGS12 was defined as the target of 4p16.3 deletions, although it has not been implicated previously as a tumor-suppressor gene. RGS12 transcript levels were relatively reduced in AA prostate cancer, and prostate cancer cell lines showed decreased RGS12 expression relative to benign prostate epithelial cells. Notably, RGS12 exhibited potent tumor-suppressor activity in prostate cancer and prostate epithelial cell lines in vitro and in vivo. We found that RGS12 expression correlated negatively with the oncogene MNX1 and regulated its expression in vitro and in vivo. Further, MNX1 was regulated by AKT activity, and RGS12 expression decreased total and activated AKT levels. Our findings identify RGS12 as a candidate tumor-suppressor gene in AA prostate cancer, which acts by decreasing expression of AKT and MNX1, establishing a novel oncogenic axis in this disparate disease setting. Cancer Res; 77(16); 4247–57. ©2017 AACR.

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Heme-oxygenase-1 Production by Intestinal CX3CR1+ Macrophages Helps to Resolve Inflammation and Prevents Carcinogenesis

CX3CR1+ macrophages in the intestinal lamina propria contribute to gut homeostasis through the immunomodulatory interleukin IL10, but there is little knowledge on how these cells or the CX3CR1 receptor may affect colorectal carcinogenesis. In this study, we show that CX3CR1-deficient mice fail to resolve gut inflammation despite high production of IL10 and have increased colitis and adenomatous polyps in chemical and genetic models of colon carcinogenesis. Mechanistically, CX3CL1-mediated engagement of the CX3CR1 receptor induced upregulation of heme-oxygenase-1 (HMOX-1), an antioxidant and anti-inflammatory enzyme. CX3CR1-deficient mice exhibited significantly lower expression of HMOX-1 in their adenomatous colon tissues. Combining LPS and CX3CL1 displayed a strong synergistic effect in vitro, but HMOX-1 levels were significantly lower in KO macrophages. Cohousing of wild-type and CX3CR1−/− mice during the AOM/DSS treatment attenuated disease severity in CX3CR1−/− mice, indicating the importance of the microbiome, but did not fully reinstate HMOX-1 levels and did not abolish polyp formation. In contrast, pharmacologic induction of HMOX-1 in vivo by cobalt protoporphyrin-IX treatment eradicated intestinal inflammation and fully protected KO mice from carcinogenesis. Taken together, our results establish an essential role for the receptor CX3CR1 in gut macrophages in resolving inflammation in the intestine, where it helps protects against colitis-associated cancer by regulating HMOX-1 expression. Cancer Res; 77(16); 4472–85. ©2017 AACR.

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Unpaired Extracellular Cysteine Mutations of CSF3R Mediate Gain or Loss of Function

Exclusive of membrane-proximal mutations seen commonly in chronic neutrophilic leukemia (e.g., T618I), functionally defective mutations in the extracellular domain of the G-CSF receptor (CSF3R) have been reported only in severe congenital and idiopathic neutropenia patients. Here, we describe the first activating mutation in the fibronectin-like type III domain of the extracellular region of CSF3R (W341C) in a leukemia patient. This mutation transformed cells via cysteine-mediated intermolecular disulfide bonds, leading to receptor dimerization. Interestingly, a CSF3R cytoplasmic truncation mutation (W791X) found on the same allele as the extracellular mutation and the expansion of the compound mutation was associated with increased leukocytosis and disease progression of the patient. Notably, the primary patient sample and cells transformed by W341C and W341C/W791X exhibited sensitivity to JAK inhibitors. We further showed that disruption of original cysteine pairs in the CSF3R extracellular domain resulted in either gain- or loss-of-function changes, part of which was attributable to cysteine-mediated dimer formation. This, therefore, represents the first characterization of unpaired cysteines that mediate both gain- and loss-of-function phenotypes. Overall, our results show the structural and functional importance of conserved extracellular cysteine pairs in CSF3R and suggest the necessity for broader screening of CSF3R extracellular domain in leukemia patients. Cancer Res; 77(16); 4258–67. ©2017 AACR.

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Functionally Null RAD51D Missense Mutation Associates Strongly with Ovarian Carcinoma

RAD51D is a key player in DNA repair by homologous recombination (HR), and RAD51D truncating variant carriers have an increased risk for ovarian cancer. However, the contribution of nontruncating RAD51D variants to cancer predisposition remains uncertain. Using deep sequencing and case–control genotyping studies, we show that in French Canadians, the missense RAD51D variant c.620C>T;p.S207L is highly prevalent and is associated with a significantly increased risk for ovarian high-grade serous carcinoma (HGSC; 3.8% cases vs. 0.2% controls). The frequency of the p.S207L variant did not significantly differ from that of controls in breast, endometrial, pancreas, or colorectal adenocarcinomas. Functionally, we show that this mutation impairs HR by disrupting the RAD51D–XRCC2 interaction and confers PARP inhibitor sensitivity. These results highlight the importance of a functional RAD51D–XRCC2 interaction to promote HR and prevent the development of HGSC. This study identifies c.620C>T;p.S207L as the first bona fide pathogenic RAD51D missense cancer susceptibility allele and supports the use of targeted PARP-inhibitor therapies in ovarian cancer patients carrying deleterious missense RAD51D variants. Cancer Res; 77(16); 4517–29. ©2017 AACR.

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EIF1AX and NRAS Mutations Co-occur and Cooperate in Low-Grade Serous Ovarian Carcinomas

Low-grade serous ovarian carcinomas (LGSC) are associated with a poor response to chemotherapy and are molecularly characterized by RAS pathway activation. Using exome and whole genome sequencing, we identified recurrent mutations in the protein translational regulator EIF1AX and in NF1, USP9X, KRAS, BRAF, and NRAS. RAS pathway mutations were mutually exclusive; however, we found significant co-occurrence of mutations in NRAS and EIF1AX. Missense EIF1AX mutations were clustered at the N-terminus of the protein in a region associated with its role in ensuring translational initiation fidelity. Coexpression of mutant NRAS and EIF1AX proteins promoted proliferation and clonogenic survival in LGSC cells, providing the first example of co-occurring, growth-promoting mutational events in ovarian cancer. Cancer Res; 77(16); 4268–78. ©2017 AACR.

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Infection Exposure Promotes ETV6-RUNX1 Precursor B-cell Leukemia via Impaired H3K4 Demethylases

ETV6-RUNX1 is associated with the most common subtype of childhood leukemia. As few ETV6-RUNX1 carriers develop precursor B-cell acute lymphocytic leukemia (pB-ALL), the underlying genetic basis for development of full-blown leukemia remains to be identified, but the appearance of leukemia cases in time-space clusters keeps infection as a potential causal factor. Here, we present in vivo genetic evidence mechanistically connecting preleukemic ETV6-RUNX1 expression in hematopoetic stem cells/precursor cells (HSC/PC) and postnatal infections for human-like pB-ALL. In our model, ETV6-RUNX1 conferred a low risk of developing pB-ALL after exposure to common pathogens, corroborating the low incidence observed in humans. Murine preleukemic ETV6-RUNX1 pro/preB cells showed high Rag1/2 expression, known for human ETV6-RUNX1 pB-ALL. Murine and human ETV6-RUNX1 pB-ALL revealed recurrent genomic alterations, with a relevant proportion affecting genes of the lysine demethylase (KDM) family. KDM5C loss of function resulted in increased levels of H3K4me3, which coprecipitated with RAG2 in a human cell line model, laying the molecular basis for recombination activity. We conclude that alterations of KDM family members represent a disease-driving mechanism and an explanation for RAG off-target cleavage observed in humans. Our results explain the genetic basis for clonal evolution of an ETV6-RUNX1 preleukemic clone to pB-ALL after infection exposure and offer the possibility of novel therapeutic approaches. Cancer Res; 77(16); 4365–77. ©2017 AACR.

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Phosphoproteomic Profiling Reveals ALK and MET as Novel Actionable Targets across Synovial Sarcoma Subtypes

Despite intensive multimodal treatment of sarcomas, a heterogeneous group of malignant tumors arising from connective tissue, survival remains poor. Candidate-based targeted treatments have demonstrated limited clinical success, urging an unbiased and comprehensive analysis of oncogenic signaling networks to reveal therapeutic targets and personalized treatment strategies. Here we applied mass spectrometry–based phosphoproteomic profiling to the largest and most heterogeneous set of sarcoma cell lines characterized to date and identified novel tyrosine phosphorylation patterns, enhanced tyrosine kinases in specific subtypes, and potential driver kinases. ALK was identified as a novel driver in the Aska-SS synovial sarcoma (SS) cell line via expression of an ALK variant with a large extracellular domain deletion (ALKΔ2–17). Functional ALK dependency was confirmed in vitro and in vivo with selective inhibitors. Importantly, ALK immunopositivity was detected in 6 of 43 (14%) of SS patient specimens, one of which exhibited an ALK rearrangement. High PDGFRα phosphorylation also characterized SS cell lines, which was accompanied by enhanced MET activation in Yamato-SS cells. Although Yamato-SS cells were sensitive to crizotinib (ALK/MET-inhibitor) but not pazopanib (VEGFR/PDGFR-inhibitor) monotherapy in vitro, synergistic effects were observed upon drug combination. In vivo, both drugs were individually effective, with pazopanib efficacy likely attributable to reduced angiogenesis. MET or PDGFRα expression was detected in 58% and 84% of SS patients, respectively, with coexpression in 56%. Consequently, our integrated approach has led to the identification of ALK and MET as promising therapeutic targets in SS. Cancer Res; 77(16); 4279–92. ©2017 AACR.

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Normal and Malignant Cells Exhibit Differential Responses to Calcium Electroporation

Calcium electroporation may offer a simple general tool for anticancer therapy. Transient permeabilization of cancer cell membranes created by applying short, high-voltage pulses in tumors enables high calcium influxes that trigger cell death. In this study, we compared the relative sensitivity of different human tumor models and normal tissues to calcium electroporation. Plasma membrane Ca2+-ATPase (PMCA) protein expression was confirmed in vitro in all cancer cell lines and normal primary dermal fibroblasts studied. In all tumor types tested in vivo, calcium electroporation effectively induced necrosis, with a range of sensitivities observed (36%–88%) 2 days after treatment. Necrosis was induced using calcium concentrations of 100–500 mmol/L and injection volumes 20%–80% of tumor volume. Notably, only limited effects were seen in normal tissue. Calcium content increased >7-fold in tumor and skin tissue after calcium electroporation but decreased in skin tissue 4 hours after treatment to levels comparable with untreated controls, whereas calcium content endured at high levels in tumor tissue. Mechanistic experiments in vitro indicated that calcium influx was similar in fibroblasts and cancer cells. However, we observed decreased PMCA expression in cancer cells compared with fibroblasts, offering a potential explanation for the different calcium content in tumor cells versus normal tissues. Overall, our results suggest that calcium electroporation can elicit a rapid and selective necrosis of solid tumors, with limited deleterious effects on surrounding normal tissues. Cancer Res; 77(16); 4389–401. ©2017 AACR.

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Targeting SRC Coactivators Blocks the Tumor-Initiating Capacity of Cancer Stem-like Cells

Tumor-initiating cells (TIC) represent cancer stem-like cell (CSC) subpopulations within tumors that are thought to give rise to recurrent cancer after therapy. Identifying key regulators of TIC/CSC maintenance is essential for the development of therapeutics designed to limit recurrence. The steroid receptor coactivator 3 (SRC-3) is overexpressed in a wide range of cancers, driving tumor initiation, cell proliferation, and metastasis. Here we report that SRC-3 supports the TIC/CSC state and induces an epithelial-to-mesenchymal transition (EMT) by driving expression of the master EMT regulators and stem cell markers. We also show that inhibition of SRC-3 and SRC-1 with SI-2, a second-generation SRC-3/SRC-1 small-molecule inhibitor, targets the CSC/TIC population both in vitro and in vivo. Collectively, these results identify SRC coactivators as regulators of stem-like capacity in cancer cells and that these coactivators can serve as potential therapeutic targets to prevent the recurrence of cancer. Cancer Res; 77(16); 4293–304. ©2017 AACR.

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MCAM Mediates Chemoresistance in Small-Cell Lung Cancer via the PI3K/AKT/SOX2 Signaling Pathway

Despite favorable responses to initial therapy, small-cell lung cancer (SCLC) relapse occurs within a year and exhibits resistance to multiple drugs. Because of limited accessibility of patient tissues for research purposes, SCLC patient-derived xenografts (PDX) have provided the best opportunity to address this limitation. Here, we sought to identify novel mechanisms involved in SCLC chemoresistance. Through in-depth proteomic profiling, we identified MCAM as a markedly upregulated surface receptor in chemoresistant SCLC cell lines and in chemoresistant PDX compared with matched treatment-naïve tumors. MCAM depletion in chemoresistant cells reduced cell proliferation and reduced the IC50 inhibitory concentration of chemotherapeutic drugs in vitro. This MCAM-mediated sensitization to chemotherapy occurred via SOX2-dependent upregulation of mitochondrial 37S ribosomal protein 1/ATP-binding cassette subfamily C member 1 (MRP1/ABCC1) and the PI3/AKT pathway. Metabolomic profiling revealed that MCAM modulated lactate production in chemoresistant cells that exhibit a distinct metabolic phenotype characterized by low oxidative phosphorylation. Our results suggest that MCAM may serve as a novel therapeutic target to overcome chemoresistance in SCLC. Cancer Res; 77(16); 4414–25. ©2017 AACR.

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DVH- and NTCP-based dosimetric comparison of different longitudinal margins for VMAT-IMRT of esophageal cancer

Abstract

Purpose

To cover the microscopic tumor spread in squamous cell carcinoma of the esophagus (SCC), longitudinal margins of 3–4 cm are used for radiotherapy (RT) protocols. However, smaller margins of 2–3 cm might be reasonable when advanced diagnostic imaging is integrated into target volume delineation. Purpose of this study was to compare the dose distribution and deposition to the organs at risk (OAR) for different longitudinal margins using a DVH- and NTCP-based approach.

Methods

Ten patients with SCC of the middle or lower third were retrospectively selected. Three planning target volumes (PTV) with longitudinal margins of 4 cm, 3 cm and 2 cm and an axial margin of 1.5 cm to the gross target volume (GTV) were defined for each patient. For each PTV two treatment plans with total doses of 41.4 Gy (neoadjuvant treatment) and 50.4 Gy (definite treatment) were calculated. Dose to the lungs, heart, myelon and liver were then evaluated and compared between different PTVs.

Results

When using a longitudinal margin of 3 cm instead of 4 cm, all dose parameters (Dmin, Dmean, Dmedian and V5-V35), except Dmax could be significantly reduced for the lungs. Regarding the heart, a significant reduction was seen for Dmean and V5, but not for Dmin, Dmax, Dmedian and V10-V35. When comparing a longitudinal margin of 4 cm to a longitudinal margin of 2 cm, a significant difference was calculated for Dmin, Dmean, Dmedian and V5-V35 of the lungs and for Dmax, Dmean and V5-V35 of the heart. Nevertheless, no difference was seen for median heart dose. An additional dose reduction for V10 of the heart was achieved for definite treatment plans when using a longitudinal margin of 3 cm. The NTCP-based risk of pneumonitis was significantly reduced by a margin reduction to 2 cm for neoadjuvant and definite treatment plans.

Conclusion

Reduction of longitudinal margins from 4 cm to 3 cm can significantly reduce the dose to lungs and Dmean of the heart. Despite clinical benefit and oncologic outcome remain unclear, reduction of the longitudinal margins might provide the opportunity to reduce side effects of chemoradiation (CRT) for SCC in upcoming studies.

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Patterns of locoregional failure following post-operative intensity-modulated radiotherapy to oral cavity cancer: quantitative spatial and dosimetric analysis using a deformable image registration workflow

Abstract

Background

We sought to identify spatial/dosimetric patterns of failure for oral cavity cancer patients receiving post-operative IMRT (PO-IMRT).

Methods

Two hundred eighty-nine OCC patients receiving PO-IMRT were retrospectively reviewed from 2000 to 2012. Diagnostic CT documenting recurrence (rCT) was co-registered with planning CT (pCT) using a validated deformable image registration software. Manually segmented recurrent gross disease (rGTV) was deformed to co-registered pCTs. Mapped rGTVs were compared dosimetrically to planned dose and spatially to planning target volumes using centroid-based approaches. Failures types were classified using combined spatial/dosimetric criteria: A (central high-dose), B (peripheral high-dose), C (central intermediate/low-dose), D (peripheral intermediate/low-dose), and E (extraneous-dose).

Results

Fifty-four patients with recurrence were analyzed; 26 local recurrence, 19 regional recurrence, and 9 both local and regional recurrence. Median time to recurrence was 4 months (range 0–71). Median rGTVs volume was 3.7 cm³ (IQR 1.4–10.6). For spatial and dosimetric analysis of the patterns of failure, 30 patients (55.5%) were classified as type A (central high-dose). Non-central high dose failures were distributed as follows: 2 (3.7%) type B, 10 (18.5%) type C, 1 (1.8%) type D, and 9 (16.7%) type E. Non-IMRT failure in the matching low-neck field was seen in two patients. No failures were noted at the IMRT-supraclavicular field match-line.

Conclusions

Approximately half of patients with local/regional failure had non-central high dose recurrence. Peripheral high dose misses were uncommon reflecting adequate delineation and dose delivery. Future strategies are needed to reduce types C and E failures.

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Brentuximab vedotin therapy for CD30-positive cutaneous T-cell lymphoma: a targeted approach to management

Future Oncology, Ahead of Print.


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A review of BF-200 ALA for the photodynamic treatment of mild-to-moderate actinic keratosis

Future Oncology, Ahead of Print.


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Bedaquiline inhibits the ATP synthase in Mycobacterium abscessus and is effective in infected zebrafish [PublishAheadOfPrint]

Pulmonary infections caused by Mycobacterium abscessus are emerging as a global threat, especially in cystic fibrosis patients. Further intensifying the concern of M. abscessus infection is the recent evidence of human-to-human transmission of the infection. M. abscessus is a naturally multidrug resistant, fast-growing pathogen for which pharmacological options are limited. Repurposing antitubercular drugs represents an attractive option for the development of chemotherapeutic alternatives against M. abscessus infections. Bedaquiline (BDQ), an ATP synthase inhibitor, has recently been approved for the treatment of multi-drug resistant tuberculosis. Herein, we show that BDQ has a very low MIC against a vast panel of clinical isolates. Despite being bacteriostatic in vitro, BDQ was highly efficacious in a zebrafish model of M. abscessus infection. Remarkably, a very short period of treatment was sufficient to protect the infected larvae from M. abscessus-induced killing. This was corroborated with reduced numbers of abscesses and cords, considered as major pathophysiological signs in infected zebrafish. Mode of action studies revealed that BDQ triggered a rapid ATP depletion in M. abscessus in vitro, consistent with the drug targeting the F_oF₁ ATP synthase. Importantly, despite failure to select in vitro for spontaneous mutants highly resistant to BDQ, the transfer of single nucleotide polymorphisms leading to D29V or A64P substitutions in atpE conferred high resistance, thus resolving the target of BDQ in M. abscessus. Overall, this study indicates that BDQ is active against M. abscessus in vitro and in vivo and should be considered for clinical use against the difficult-to-manage M. abscessus pulmonary infections.

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Candida albicans Swi/Snf and Mediator Complexes Differentially Regulate Mrr1-induced MDR1 Expression and Fluconazole Resistance. [PublishAheadOfPrint]

Long-term azole treatment of patients with chronic Candida albicans infections can lead to drug resistance. Gain-of-function (GOF) mutations in the transcription factor Mrr1 and the consequent transcriptional activation of MDR1, a drug efflux coding gene, is a common pathway by which this human fungal pathogen acquires fluconazole resistance. This work elucidates the previously unknown downstream transcription mechanisms utilized by hyperactive Mrr1. We've identified the Swi/Snf chromatin remodeling complex as a key co-activator for Mrr1, which is required to maintain basal and induced 'open' chromatin, and Mrr1 occupancy, at the MDR1 promoter. Deletion of snf2, the catalytic subunit of Swi/Snf, largely abrogates the increases in MDR1 expression and fluconazole MIC observed in MRR1^GOF mutant strains. Mediator positively and negatively regulates key Mrr1 target promoters. Deletion of the Mediator tail module med3 subunit reduces, but does not eliminate, the increased MDR1 expression and fluconazole MIC conferred by MRR1^GOF mutations. Eliminating the kinase activity of the Mediator Ssn3 subunit suppresses the decreased MDR1 expression and fluconazole MIC of the snf2 null mutation in MRR1^GOF strains. Ssn3 deletion also suppresses MDR1 promoter histone displacement defects in snf2 null mutants. The combination of this work with studies on other hyperactive zinc cluster transcription factors that confer azole resistance in fungal pathogens reveals a complex picture where the induction of drug efflux pump expression requires the coordination of multiple co-activators. The observed variations in transcription factor and target promoter dependence of this process may make the search for azole sensitivity restoring small molecules more complicated.

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Mediator Tail Module is Required for Tac1 Activated CDR1 Expression and Azole Resistance in Candida albicans. [PublishAheadOfPrint]

The human fungal pathogen Candida albicans develops drug resistance after long-term exposure to azole drugs in the treatment of chronic candidiasis. Gain-of-function (GOF) mutations in the transcription factor Tac1, and the consequent expression of its targets, drug efflux pumps Cdr1 and Cdr2, are a common mechanism by which C. albicans acquires fluconazole resistance. The mechanism by which GOF mutations hyperactivate Tac1 is currently unknown. Here, we define a transcriptional activation domain (TAD) at the C-terminus of Tac1. GOF mutations within the Tac1 TAD, outside the context of full length Tac1, do not generally enhance its absolute potential as a transcriptional activator. Negative regulation of the Tac1 TAD by the Tac1 middle region is necessary for the activating effect of GOF mutations or fluphenazine to be realized. We have found that full-length Tac1, when hyperactivated by xenobiotics or GOF mutations, facilitates the recruitment of the Mediator co-activator complex to the CDR1 promoter. Azole resistance and the activation of Tac1 target genes, such as CDR1, are dependent on the Tac1 TAD and subunits of the Mediator tail module. The dependence of different Tac1 target promoters on the Mediator tail module, however, varies widely. Lastly, we show that hyperactivation of Tac1 is correlated with its Mediator dependent phosphorylation, a potentially useful biomarker for Tac1 hyperactivation. The role of Mediator in events downstream of Tac1 hyperactivation in fluconazole resistant clinical isolates is complex, and provides opportunities and challenges for therapeutic intervention.

http://ift.tt/2w8HAFa

Drug Susceptibility and Replicative Capacity of Multi-Drug Resistant Recombinant Human Cytomegalovirus Harboring Mutations in UL56 and UL54 Genes [PublishAheadOfPrint]

Letermovir is an investigational antiviral agent with a novel mechanism of action involving the viral terminase (pUL56). We evaluated the impact of V236M mutation in UL56 gene alone and combined with E756K mutation in UL54 gene on drug susceptibility and viral replicative capacity of recombinant human cytomegalovirus. The double mutant exhibited at least borderline resistance to all antivirals tested (ganciclovir, foscarnet, cidofovir, brincidofovir and letermovir) and replicated less efficiently than the wild-type virus in vitro.

http://ift.tt/2uJgAZ0

Both 14-day hybrid and bismuth quadruple therapies cure most patients with Helicobacter pylori infection in populations with moderate antibiotic resistance: a randomized controlled trial [PublishAheadOfPrint]

BACKGROUND & AIM: Hybrid therapy is a novel two-step treatment achieving a high eradication rate for H. pylori infection. Currently, whether this new therapy achieves a higher eradication rate than bismuth quadruple therapy remains an unanswered question. The aim of this prospective, randomized, comparive study was to investigate the efficacies of 14-day hybrid therapy and bismuth quadruple therapy in the treatment of H. pylori infection.

METHODS: From July 2013 to June 2015, eligible H. pylori-infected subjects were randomly assigned to receive either 14-day bismuth quadruple therapy (pantoprazole, bismuth subcitrate, tetracycline, and metronidazole for 14 days) or 14-day hybrid therapy (a 7-day dual therapy with pantoprazole plus amoxicillin, followed by a 7-day quadraple therapy with pantoprazole plus amoxicillin, clarithromycin and metronidazole). H. pylori status was examined 6 weeks after the end of treatment.

RESULT: Three hundred and thirty H. pylori-infected participants were randomized to receive 14-day bismuth quadruple therapy (n = 164) or 14-day hybrid therapy (n = 166). The eradication rates by intention-to-treat analysis were similar: 93.9% vs 92.8% (95% confidence interval [CI]: -4.3% - 5.4%; P = 0.68). Per-protocol analysis yielded similar results (96.7% vs 94.9%; P = 0.44). However, bismuth quadruple therapy had a higher frequency of adverse events than hybrid therapy (55.5% vs 15.7%; 95% CI: 30.4% - 49.2%; P < 0.001). The two treatments exhibited comparable drug adherence (93.9% vs 97%, respectively). The resistance rates of antibiotics were: clarithromycin 16.7%; amoxicillin 1.3%; metronidazole 25%, and tetracycline 0% of patients, respectively. In the bismuth quadruple therapy group, the eradication rate of metronidazole-resistant strains was lower than that of metronidazole-susceptible strains (70.0% vs 96.4%; P = 0.04). In the hybrid therapy group, no significant impact of clarithromycin or metronidazole resistance on eradication rates was identified.

CONCULSIONS: Both 14-day hybrid and bismuth quadruple therapies cure most patients with H. pylori infection in populations with moderate antibiotic resistance. However, the former has fewer adverse effects than the latter. ClinicalTrials.gov number: NCT02541864.

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Are standard doses of piperacillin in piperacillin/tazobactam regimens adequate for the management of febrile neutropenia? Answers from population pharmacokinetic modelling and Monte Carlo simulations [PublishAheadOfPrint]

Changes in the pharmacokinetics of piperacillin in febrile neutropenic patients have been reported to result in sub-optimal exposures. This study aimed to develop a population pharmacokinetic model for piperacillin and perform dosing simulation to describe optimal dosing regimens for haematological malignancy patients with febrile neutropenia. Concentration-time data were obtained from previous prospective observational pharmacokinetic and interventional therapeutic drug monitoring studies. Non-parametric population pharmacokinetic analysis and Monte Carlo dosing simulations were performed with Pmetrics® package for R. A two-compartment model with between-subject variability for clearance (CL), adequately described the data from thirty-seven patients (21 males, mean ±SD age 59 ± 12 years and weight 77 ± 16 kg). Parameter estimates were CL, 18.0 ± 4.8 L/h; central compartment V, 14.3 ± 7.3 L; rate constant Kcp, 1.40 ± 1.35 h^-1and Kpc, 4.99 ± 7.81 h^-1. High creatinine clearance (CL_CR) was associated with reduced probability of target attainment (PTA). Extended and continuous infusion regimens achieved a high PTA of > 90% for 50% fT_>MIC. Only continuous regimens achieved > 90% PTA for 100% fT_>MIC when CL_CR was high. The cumulative fraction of response (FTA) was sub-optimal (< 85%) for conventional regimens for both empiric and directed therapy considering 50% and 100% fT_>MIC. FTA was maximized with prolonged infusions. Overall, changes in piperacillin pharmacokinetics and the consequences on to therapeutic dosing requirements appear similar to those observed in intensive care patients. Guidelines should address the altered dosing needs of febrile neutropenic patients exhibiting high CL_CR or with known/presumed infections from high MIC bacteria.

http://ift.tt/2uJxWoO

Relationships of Vancomycin Pharmacokinetics to Body Size and Composition by Computed Tomography Analysis: A novel "Pharmacomorphomic" approach. [PublishAheadOfPrint]

Antibiotics such as vancomycin are empirically dosed on body weight, which may not be optimal across the expanding adult body size distribution. Our aim was to compare the relationships between morphomic parameters generated from computed tomography images to conventional body size metrics as predictors of vancomycin pharmacokinetics (PK). This single center retrospective study included 300 patients with 1622 vancomycin concentration (52% trough) measurements. Bayesian estimation was used to compute individual vancomycin volume of distribution of the central compartment (Vc) and clearance (CL). Approximately 45% of patients were obese with an overall median [5^th, 95^th percentile] weight and body mass index of 87.2 [54.7, 123] kg and 28.8 [18.9, 43.7] kg/m², respectively. Morphomic parameters of body size such as body depth, total body area and T12 to L4 torso volume correlated with Vc. The relationship of vancomycin Vc was poorly predicted by body size but was stronger with T12 to L4 torso volume (R²= 0.11) than weight (R²= 0.04). No relationships between vancomycin CL and traditional body size metrics could be discerned; however, relationships with skeletal muscle volume and total psoas area were found. Vancomycin CL independently correlated with total psoas area and inversely correlated with age. Thus, vancomycin CL was significantly related to total psoas area over age (R²= 0.23, P<0.0001). This proof-of-concept study suggests a potential role for translation of radiographic information into parameters predictive of drug pharmacokinetics. Prediction of individual antimicrobial pharmacokinetic parameters using analytic morphomics has the potential to improve antimicrobial dose selection and outcomes of obese patients.

http://ift.tt/2w8QhiJ

Inhibition of calcineurin or IMPDH exerts moderate to potent antiviral activity against norovirus replication [PublishAheadOfPrint]

Norovirus is a major cause of acute gastroenteritis worldwide and has emerged as an important issue of chronic infection in transplantation patients. Since no approved antiviral is available, we evaluated the effects of different immunosuppressants and ribavirin on norovirus and explored their mechanism-of-actions by using a human norovirus (HuNV) replicon-harboring model and a surrogate murine norovirus (MNV) infectious model. Roles of corresponding drug targets were investigated by gain- or loss-of-function approaches. We found calcineurin inhibitors cyclosporin A (CsA) and tacrolimus (FK506) moderately inhibited HuNV replication. Gene silencing of their cellular targets, cyclophilin A, FKBP12 and calcineurin, significantly inhibited HuNV replication. A therapeutically-speaking low concentration of mycophenolic acid (MPA), an uncompetitive inosine monophosphate dehydrogenase (IMPDH) inhibitor, potently and rapidly inhibited norovirus replication and ultimately cleared HuNV replicons without inducible resistance following long-term drug exposure. Knockdown of MPA cellular targets, IMPDH1 and IMPDH2, suppressed HuNV replication. Consistent with the nucleotide synthesizing function of IMPDH, exogenous guanosine counteracted the anti-norovirus effects of MPA. Furthermore, the competitive IMPDH inhibitor ribavirin efficiently inhibited norovirus and resulted in an additive effect when combined with immunosuppressants. The results from this study demonstrate that calcineurin phosphatase activity and IMPDH guanine synthase activity are crucial in sustaining norovirus infection, thus could be therapeutically targeted. Our results suggest that MPA shall be preferentially considered as immunosuppressive medication for transplantation patients at risk of norovirus infection; whereas ribavirin represents as a potential antiviral for both immunocompromised and immunocompetent patients with norovirus gastroenteritis.

http://ift.tt/2uJ6tDH

PBP4 mediates {beta}-lactam resistance by altered function [PublishAheadOfPrint]

Penicillin Binding Protein 4 (PBP4) can provide high-level β-lactam resistance in S. aureus. A series of missense and promoter mutations associated with pbp4 were detected in strains that displayed high-level resistance. We hereby show that the missense mutations facilitate the β-lactam resistance mediated by PBP4 and the promoter mutations lead to the overexpression of pbp4. Our results also suggest a co-operative interplay between PBPs for β-lactam resistance.

http://ift.tt/2w8taou

No Clinically Relevant Drug-Drug Interactions Between Methadone or Buprenorphine/Naloxone and Anti-Viral Combination Glecaprevir and Pibrentasvir [PublishAheadOfPrint]

The combination of glecaprevir (formerly ABT-493), a nonstructural (NS) protein 3/4A protease inhibitor, and pibrentasvir (formerly ABT-530), a NS5A protein inhibitor, is being developed as treatment for HCV genotype 1-6 infection. The pharmacokinetics, pharmacodynamics, safety, and tolerability of methadone or buprenorphine/naloxone when coadministered with the glecaprevir and pibrentasvir combination in HCV-negative subjects on stable opioid maintenance therapy were investigated in a Phase 1, single-center, two-arm, multiple-dose, open-label sequential study. Subjects received methadone (Arm 1) or buprenorphine/naloxone (Arm 2) once daily (QD) as per their existing individual prescriptions alone (days 1-9) and then in combination with glecaprevir 300 mg QD and pibrentasvir120 mg QD (days 10-16) each morning. Dose-normalized exposures were similar with and without glecaprevir and pibrentasvir for R- and S- methadone (≤ 5% difference) and for buprenorphine and naloxone (≤ 24% difference); norbuprenorphine area under the curve was 30% higher, consistent with maximum and trough plasma concentrations that increased by 21% to 25%. No changes in pupil response, short opiate withdrawal scale, or desire for drugs questionnaire were observed when glecaprevir and pibrentasvir were added to methadone or buprenorphine/naloxone therapy. No dose adjustment is required when glecaprevir and pibrentasvir are coadministered with methadone or buprenorphine/naloxone.

http://ift.tt/2uJgWPq

Safety and Efficacy of MHAA4549A, a Broadly Neutralizing Monoclonal Antibody, in a Human Influenza A Challenge Model: A Phase 2 Randomized Trial [PublishAheadOfPrint]

BACKGROUND: MHAA4549A, a human monoclonal antibody targeting the hemagglutinin stalk region of influenza A virus (IAV), is being developed as a therapeutic for patients hospitalized with severe IAV infection. Safety and efficacy of MHAA4549A were assessed in a randomized, double-blind, placebo-controlled, dose-ranging study in a human IAV-challenge model.

METHODS: One hundred healthy volunteers were inoculated with A/Wisconsin/67/2005 (H3N2) IAV and, 24–36 hours later, administered a single intravenous dose of either placebo, MHAA4549A (400, 1200, or 3600 mg) or a standard oral dose of oseltamivir. Subjects were assessed for safety, pharmacokinetics (PK), and immunogenicity. The intent-to-treat-infected (ITTI) population was assessed for changes in viral load, influenza symptoms, and inflammatory biomarkers.

RESULTS: MHAA4549A was well tolerated in all IAV-challenge subjects. 3600-mg MHAA4549A significantly reduced viral burden relative to placebo as determined by the area under the curve (AUC) of nasopharyngeal virus infection, quantified using quantitative PCR (98%) and 50% tissue culture infective dose (TCID₅₀) assays (100%). Peak viral load, duration of viral shedding, influenza symptoms scores, mucus weight, and inflammatory biomarkers were also reduced. Serum PK was linear with a half-life of ~23 days. No MHAA4549A-treated subjects developed anti-drug antibodies.

CONCLUSIONS: MHAA4549A was well tolerated, and demonstrated statistically significant and substantial antiviral activity in an IAV-challenge model.

REGISTRY: ClinicalTrials.gov, NCT01980966.

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Systematic review and meta-analyses of the effect of chemotherapy on pulmonary Mycobacterium abscessus outcomes and disease recurrence [PublishAheadOfPrint]

Background. In pharmacokinetic/pharmacodynamics models of pulmonary Mycobacterium abscessus complex, the recommended macrolide-containing combination therapy has poor kill rates. However, clinical outcomes are unknown.

Methods. We searched the literature for studies published between 1990-2017 that reported microbial outcomes in patients treated for pulmonary M. abscessus disease. Good outcome was defined as sustained sputum culture conversion (SSCC) without relapse. Random effects models were used to pool studies and estimate proportions of patients with good outcomes. Odds ratio (OR) and 95% confidence intervals [CI] were computed. Sensitivity analyses and meta-regression were used to assess robustness of findings.

Results. In 19 studies of 1533 patients, combination therapy was administered to 508 patients with M. abscessus subspecies abscessus (Maa), 204 with M. abscessus subspecies massiliense (Mam), and 301 with M. abscessus with no species specified. Macrolide-containing regimens achieved SSCC in only 77/233 (34%) new Maa patients versus 117/141 (54%) in Mam patients (OR=0.108 [95% CI, 0.066-0.181]). In refractory disease, SSCC was achieved in 20% (95% CI, 7-36), which was not significantly different across subspecies. The estimated recurrent rates per month were 1.835% (range 1.667%-3.196%) for Maa versus 0.683% (range 0.229%-1.136%) for Mam (OR=6.189 [95%CI, 2.896-13.650]). The proportion of patients with good outcome was 52/223 (23%) with Maa versus 118/141 (84%) with Mam disease (OR=0.059 (95% 0.034-0.101).

Conclusions. Maa pulmonary disease outcomes with the currently recommended regimens are atrocious, with outcomes similar to extensively drug-resistant tuberculosis. Therapeutically, the concept of "nontuberculous mycobacteria" is misguided. There is an urgent need to craft entirely new treatment regimens.

http://ift.tt/2uJfaOe

Activity of the {beta}-Lactamase inhibitor LN-1-255 against carbapenem-hydrolyzing class D {beta}-lactamases from Acinetobacter baumannii [PublishAheadOfPrint]

The number of infections caused by Gram-negative pathogens carrying carbapenemases is increasing, and the group of carbapenem-hydrolyzing class D β-lactamases (CHDLs) is especially problematic. Several clinically important CHDLs have been identified in A. baumannii, including OXA-23, OXA-24/40, OXA-58, OXA-143, OXA-235, and the chromosomally encoded OXA-51. The selection and dissemination of carbapenem-resistant A. baumannii strains constitutes a serious global threat. Carbapenems have been successfully utilized as last resort antibiotics for the treatment of multi-drug-resistant A. baumannii infections. However, the spread of OXA carbapenemases is compromising the continued use of these antimicrobials. In response to this clinical issue, it is necessary and urgent to design and develop new specific inhibitors with efficacy against these enzymes. The aim of this work is to characterize the inhibitory activity of LN-1-255 (a 6-alkylidene-2-substituted penicillin sulfone) and compare it to that of two established inhibitors (avibactam and tazobactam) against the most relevant enzymes of each group of class D carbapenemases in A. baumannii. The β-lactamase inhibitor LN-1-255 demonstrated excellent microbiological synergy and inhibition kinetics parameters against all tested CHDLs, and a significantly higher activity than tazobactam and avibactam. A combination of carbapenems and LN-1-255 was effective against A. baumannii class D carbapenemases. Docking assays confirmed the affinity of LN-1-255 for the active site of these enzymes. LN-1-255 represents a potential new β-lactamase inhibitor, which may have a significant role in eradicating infections caused by A. baumannii isolates carrying CHDLs.

http://ift.tt/2w8J7e9

Development and use of personalized bacteriophage-based therapeutic cocktails to treat a patient with a disseminated resistant Acinetobacter baumannii infection [PublishAheadOfPrint]

Widespread antibiotic use in clinical medicine and the livestock industry has contributed to the global spread of multidrug-resistant (MDR) bacterial pathogens, including Acinetobacter baumannii. We report on a method used to produce a personalized bacteriophage-based therapeutic treatment for a 68-year old diabetic patient with necrotizing pancreatitis complicated by a MDR A. baumannii infection. Despite multiple antibiotic courses and efforts at percutaneous drainage of a pancreatic pseudocyst, the patient deteriorated over a four-month period. In the absence of effective antibiotics, two laboratories identified nine different bacteriophages with lytic activity for an A. baumannii isolate from the patient. Administration of these bacteriophages intravenously and percutaneously into the abscess cavities was associated with reversal of the patient's downward clinical trajectory, clearance of the A. baumannii infection, and a return to health. The outcome of this case suggests that the methods described here for the production of bacteriophage therapeutics could be applied to similar cases and that more concerted efforts to investigate the use of therapeutic bacteriophages for MDR bacterial infections are warranted.

http://ift.tt/2uJauYW

Impact of Type III Secretion Effectors and of Phenoxyacetamide Inhibitors of Type III Secretion on Abscess Formation in a Mouse Model of Pseudomonas aeruginosa Infection [PublishAheadOfPrint]

Pseudomonas aeruginosa is a leading cause of intra-abdominal infections, wound infections, and community-acquired folliculitis, each of which may involve macro- or micro-abscess formation. The rising incidence of multi-drug resistance among P. aeruginosa isolates has increased both the economic burden and the morbidity and mortality associated with P. aeruginosa disease and necessitates a search for novel therapeutics. Previous work from our group detailed novel phenoxyacetamide inhibitors that block type III secretion and injection into host cells in vitro. In this study, we used a murine abscess model of P. aeruginosa infection to test in vivo efficacy of these compounds against the P. aeruginosa type III secretion system (T3SS). Bacteria used the T3SS to intoxicate infiltrating neutrophils to establish abscesses. Despite this antagonism, sufficient numbers of functioning neutrophils remained for proper containment of abscesses, as neutrophil depletion resulted in increased abscess size, formation of dermonecrotic lesions on the skin, and dissemination of P. aeruginosa to internal organs. Consistent with the specificity of the T3SS/neutrophil interaction, P. aeruginosa lacking a functional T3SS was fully capable of causing abscesses in a neutropenic host. Phenoxyacetamide inhibitors attenuated abscess formation and aided in immune clearance of bacteria. Finally, a P. aeruginosa strain resistant to the phenoxyacetamide compound was fully capable of causing abscess formation even in the presence of the T3SS inhibitors. Together our results further define the role of type III secretion in murine abscess formation and demonstrate the in vivo efficacy of phenoxyacetamide inhibitors in P. aeruginosa infection.

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