Multimodal molecular landscape of response to Y90-resin microsphere radioembolization followed by nivolumab for advanced hepatocellular carcinoma.

BACKGROUND: Combination therapy with radioembolization (yttrium-90)-resin microspheres) followed by nivolumab has shown a promising response rate of 30.6% in a Phase II trial (CA209-678) for advanced hepatocellular carcinoma (HCC); however, the response mechanisms and relevant biomarkers remain unknown. METHODS: By collecting both pretreatment and on-treatment samples, we performed multimodal profiling of tissue and blood samples and investigated molecular changes associated with favorable responses in 33 patients from the trial. RESULTS: We found that higher tumor mutation burden, NCOR1 mutations and higher expression of interferon gamma pathways occurred more frequently in responders. Meanwhile, non-responders tended to be enriched for a novel Asian-specific transcriptomic subtype (Kaya_P2) with a high frequency of chromosome 16 deletions and upregulated cell cycle pathways. Strikingly, unlike other cancer types, we did not observe any association between T-cell populations and treatment response, but tumors from responders had a higher proportion of CXCL9+/CXCR3+ macrophages. Moreover, biomarkers discovered in previous immunotherapy trials were not predictive in the current cohort, suggesting a distinctive molecular landscape associated with differential responses to the combination therapy. CONCLUSIONS: This study unraveled extensive molecular changes underlying distinctive responses to the novel treatment and pinpointed new directions for harnessing combination therapy in patients with advanced HCC.

Therapeutic efficacy and safety of a human fusion construct targeting the TWEAK receptor Fn14 and containing a modified granzyme B.

BACKGROUND: Antibody-drug conjugates are an exceptional and useful therapeutic tool for multiple diseases, particularly for cancer treatment. We previously showed that the fusion of the serine protease granzyme B (GrB), the effector molecule or T and B cells, to a binding domain allows the controlled and effective delivery of the cytotoxic payload into the target cell. The production of these constructs induced the formation of high molecular aggregates with a potential impact on the efficacy and safety of the protein. METHODS: Our laboratory designed a new Fn14 targeted fusion construct designated GrB(C210A)-Fc-IT4 which contains a modified GrB payload for improved protein production and preserved biological activity. We assessed the construct's enzymatic activity, as well as in vitro cytotoxicity and internalization into target cells. We also assessed pharmacokinetics, efficacy and toxicology parameters in vivo. RESULTS: GrB(C210A)-Fc-IT4 protein exhibited high affinity and selective cytotoxicity within the nanomolar range when tested against a panel of Fn14-positive human cancer cell lines. The construct rapidly internalized into target cells, activating the caspase cascade and causing mitochondrial membrane depolarization. Pharmacokinetic studies in mice revealed that GrB(C210A)-Fc-IT4 displayed a bi-exponential clearance from plasma with a fast initial clearance (t1/2α=0.36 hour) followed by a prolonged terminal-phase plasma half-life (t1/2ß=35 hours). Mice bearing MDA-MB-231 orthotopic tumor xenografts treated with vehicle or GrB(C210A)-Fc-IT4 construct (QODx5) demonstrated tumor regression and long-term (>80 days) suppression of tumor growth. Treatment of mice bearing established, subcutaneous A549 lung tumors showed impressive, long-term tumor suppression compared with a control group treated with vehicle alone. Administration of GrB(C210A)-Fc-IT4 (100 mg/kg total dose) was well-tolerated by mice and resulted in significant reduction of tumor burden in a lung cancer patient-derived xenograft model. Toxicity studies revealed no statistically significant changes in aspartate transferase, alanine transferase or lactate dehydrogenase in treated mice. Histopathological analysis of tissues from treated mice did not demonstrate any specific drug-related changes. CONCLUSION: GrB(C210A)-Fc-IT4 demonstrated excellent, specific cytotoxicity in vitro and impressive in vivo efficacy with no significant toxicity in normal murine models. These studies show GrB(C210A)-Fc-IT4 is an excellent candidate for further preclinical development.

Production of Recombinant Gelonin Using an Automated Liquid Chromatography System.

Advances in recombinant DNA technology have opened up new possibilities of exploiting toxic proteins for therapeutic purposes. Bringing forth these protein toxins from the bench to the bedside strongly depends on the availability of production methods that are reproducible, scalable and comply with good manufacturing practice (GMP). The type I ribosome-inhibiting protein, gelonin, has great potential as an anticancer drug, but is sequestrated in endosomes and lysosomes. This can be overcome by combination with photochemical internalization (PCI), a method for endosomal drug release. The combination of gelonin-based drugs and PCI represents a tumor-targeted therapy with high precision and efficiency. The aim of this study was to produce recombinant gelonin (rGel) at high purity and quantity using an automated liquid chromatography system. The expression and purification process was documented as highly efficient (4.4 mg gelonin per litre induced culture) and reproducible with minimal loss of target protein (~50% overall yield compared to after initial immobilized metal affinity chromatography (IMAC)). The endotoxin level of 0.05-0.09 EU/mg was compatible with current standards for parenteral drug administration. The automated system provided a consistent output with minimal human intervention and close monitoring of each purification step enabled optimization of both yield and purity of the product. rGel was shown to have equivalent biological activity and cytotoxicity, both with and without PCI-mediated delivery, as rGelref produced without an automated system. This study presents a highly refined and automated manufacturing procedure for recombinant gelonin at a quantity and quality sufficient for preclinical evaluation. The methods established in this report are in compliance with high quality standards and compose a solid platform for preclinical development of gelonin-based drugs.

A novel in vivo model using immunotoxin in the absence of p-glycoprotein to achieve ultra selective depletion of target cells: Applications in trogocytosis and beyond.

A commonly employed method to determine the function of a particular cell population and to assess its contribution to the overall system in vivo is to selectively deplete that population and observe the effects. Using monoclonal antibodies to deliver toxins to target cells can achieve this with a high degree of efficiency. Here, we describe an in vivo model combining the use of immunotoxins and multidrug resistant (MDR) gene deficient mice so that only MDR deficient cells expressing the target molecule would be depleted while target molecule expressing, but MDR sufficient, cells are spared. This allows targeted depletion at a higher degree of specificity than has been previously achieved. We have applied this technique to study trogocytosis, the intercellular transfer of cell surface molecules, but this principle could also be adapted using technology already available for use in other fields of study.

Identification of the cis­molecular neighbours of the immune checkpoint protein B7­H4 in the breast cancer cell­line SK­BR­3 by proteomic proximity labelling.

The immune checkpoint protein B7­H4 plays an important role in the positive as well as the negative regulation of immune T­cell responses. When expressed on cancer cells, B7­H4 inhibits T­cell activity, and numerous types of cancer cells use upregulation of B7­H4 as a survival strategy. Thus, B7­H4 is a potential target for anticancer drug therapy. Unfortunately, the cell biology of this molecule has yet to be fully elucidated. Even basic properties, such as the nature of B7­H4 interactors, are controversial. In particular, the cis­interactors of B7­H4 on cancer cell plasma membranes have not been investigated to date. The present study used a proteomic proximity­labelling assay to investigate the molecular neighbours of B7­H4 on the surface of the human breast cancer cells SK­BR­3. By comparison to a comprehensive proteome analysis of SK­BR­3 cells, the proximity method detected a relatively small number of low abundance plasma membrane proteins highly enriched for proteins known to modulate cell adhesion and immune recognition. It may be inferred that these molecules contribute to the immunosuppressive behaviour that is characteristic of B7­H4 on cancer cells.

Development of a human immuno-oncology therapeutic agent targeting HER2: targeted delivery of granzyme B.

BACKGROUND: Immunotherapeutic approaches designed to augment T and B cell mediated killing of tumor cells has met with clinical success in recent years suggesting tremendous potential for treatment in a broad spectrum of tumor types. After complex recognition of target cells by T and B cells, delivery of the serine protease granzyme B (GrB) to tumor cells comprises the cytotoxic insult resulting in a well-characterized, multimodal apoptotic cascade. METHODS: We designed a recombinant fusion construct, GrB-Fc-4D5, composed of a humanized anti-HER2 scFv fused to active GrB for recognition of tumor cells and internal delivery of GrB, simulating T and B cell therapy. We assessed the construct's antigen-binding specificity and GrB enzymatic activity, as well as in vitro cytotoxicity and internalization into target and control cells. We also assessed pharmacokinetic and toxicology parameters in vivo. RESULTS: GrB-Fc-4D5 was highly cytotoxic to Her2 positive cells such as SKBR3, MCF7 and MDA-MB-231 with IC50 values of 56, 99 and 27 nM, respectively, and against a panel of HER2+ cell lines regardless of endogenous expression levels of the PI-9 inhibitor. Contemporaneous studies with Kadcyla demonstrated similar levels of in vitro activity against virtually all cells tested. GrB-Fc-4D5 internalized rapidly into target SKOV3 cells within 1 h of exposure rapidly delivering GrB to the cytoplasmic compartment. In keeping with its relatively high molecular weight (160 kDa), the construct demonstrated a terminal-phase serum half-life in mice of 39.2 h. Toxicity studies conducted on BALB/c mice demonstrated no statistically significant changes in SGPT, SGOT or serum LDH. Histopathologic analysis of tissues from treated mice demonstrated no drug-related changes in any tissues examined. CONCLUSION: GrB-Fc-4D5 shows excellent, specific cytotoxicity and demonstrates no significant toxicity in normal, antigen-negative murine models. This construct constitutes a novel approach against HER2-expressing tumors and is an excellent candidate for further development.

Design, Characterization, and Evaluation of scFvCD133/rGelonin: A CD133-Targeting Recombinant Immunotoxin for Use in Combination with Photochemical Internalization.

The objective of this study was to develop and explore a novel CD133-targeting immunotoxin (IT) for use in combination with the endosomal escape method photochemical internalization (PCI). scFvCD133/rGelonin was recombinantly constructed by fusing a gene (scFvCD133) encoding the scFv that targets both non-glycosylated and glycosylated forms of both human and murine CD133/prominin-1 to a gene encoding the ribosome-inactivating protein (RIP) gelonin (rGelonin). RIP-activity was assessed in a cell-free translation assay. Selective binding and intracellular accumulation of scFvCD133/rGelonin was evaluated by flow cytometry and fluorescence microscopy. PCI of scFvCD133/rGelonin was explored in CD133high and CD133low cell lines and a CD133neg cell line, where cytotoxicity was evaluated by the MTT assay. scFvCD133/rGelonin exhibited superior binding to and a higher accumulation in CD133high cells compared to CD133low cells. No cytotoxic responses were detected in either CD133high or CD133low cells after 72 h incubation with <100 nM scFvCD133/rGelonin. Despite a severe loss in RIP-activity of scFvCD133/rGelonin compared to free rGelonin, PCI of scFvCD133/rGelonin induced log-fold reduction of viability compared to PCI of rGelonin. Strikingly, PCI of scFvCD133/rGelonin exceeded the cytotoxicity of PCI of rGelonin also in CD133low cells. In conclusion, PCI promotes strong cytotoxic activity of the per se non-toxic scFvCD133/rGelonin in both CD133high and CD133low cancer cells.

Advancing scholarship by publishing curriculum as an e-book.

Background: Medical educators provide service by developing curricula and writing learning material. In addition, academic institutions expect medical educators to publish scholarship to be considered for promotion and academic advancement. Unfortunately, educators may receive limited time to execute these duties and expectations. One way medical educators can streamline their workload is by publishing educational coursework they have previously written into an e-book through an online publisher. This allows them to transform educational service they have already completed into scholarship required for academic recognition, thus maximizing the efficient use of their time. Intervention: Publishing educational material as an e-book requires four steps. First, medical educators must determine which of their educational materials is best suited for publishing as an e-book. Second, educators must rank the features of each e-book publisher and choose the one that best meets their needs. Third, the educational material must be adapted as a manuscript and submitted for publication. Finally, the e-book must be advertised, promoted, and distributed to its intended audience. In addition, the success of the project should be evaluated. To illustrate this process, we describe the steps we took to publish the learning material we created for our internal medicine residents into an e-book. Lessons Learned: The overall process took approximately 3 months and went smoothly. For future publications, we would determine better ways to track the number of downloads of the e-book, ensure all of our images are adequately large, and consider the use of academic, rather than commercial e-book publishers.

Light-enhanced VEGF121/rGel: A tumor targeted modality with vascular and immune-mediated efficacy.

Interactions between stromal cells and tumor cells pay a major role in cancer growth and progression. This is reflected in the composition of anticancer drugs which includes compounds directed towards the immune system and tumor-vasculature in addition to drugs aimed at the cancer cells themselves. Drug-based treatment regimens are currently designed to include compounds targeting the tumor stroma in addition to the cancer cells. Treatment limiting adverse effects remains, however, one of the major challenges for drug-based therapy and novel tolerable treatment modalities with diverse high efficacy on both tumor cells and stroma is therefore of high interest. It was hypothesized that the vascular targeted fusion toxin VEGF121/rGel in combination with the intracellular drug delivery technology photochemical internalization (PCI) stimulate direct cancer parenchymal cell death in addition to inhibition of tumor perfusion, and that an immune mediated response is relevant for treatment outcome. The aim of the present study was therefore to elucidate the anticancer mechanisms of VEGF121/rGel-PCI. In contrast to VEGF121/rGel monotherapy, VEGF121/rGel-PCI was found to mediate its effect through VEGFR1 and VEGFR2, and a targeted treatment effect was shown on two VEGFR1 expressing cancer cell lines. A cancer parenchymal treatment effect was further indicated on H&E stains of CT26-CL25 and 4 T1 tumors. VEGF121/rGel-PCI was shown, by dynamic contrast enhanced MRI, to induce a sustained inhibition of tumor perfusion in both tumor models. A 50% complete remission (CR) of CT26.CL25 colon carcinoma allografts was found in immunocompetent mice while no CR was detected in CT26.CL25 bearing athymic mice. In conclusion, the present report indicate VEGF121/rGel -PCI as a treatment modality with multimodal tumor targeted efficacy that should be further developed towards clinical utilization.

CONTeMPLATE-a mnemonic to help medical educators infuse reflection into their residency curriculum.

Reflection, where clinical experiences are analyzed to gain greater understanding and meaning, is an important step in workplace learning. Residency programs must teach their residents the skills needed for deep reflection. Medical educators may find it difficult to construct a curriculum which includes the key elements needed to enable learners to attain these skills. When we first implemented reflection into our residency curriculum, we soon realized that our curriculum only taught residents how to engage in superficial reflection. Our curriculum lacked some key elements. To help guide the transformation of our curriculum, we combed the literature for best practices. The CONTeMPLATE mnemonic was born out of this process. It is a tool to help medical educators consider and implement key elements required to enable deep reflection. The purpose of this article is to show medical educators how they can use the CONTeMPLATE mnemonic to incorporate reflective practice into their own curriculum.

SAFE QI - a framework to overcome the challenges of implementing a quality improvement curriculum into a residency program.

Quality improvement (QI) is an essential component of medical practice. Medical students and residents must learn the skills to conduct clinical QI during their educational programs. Medical educators must create and implement a curriculum in QI to empower their students to develop this skill and knowledge. However, developing and implementing a QI curriculum may be challenging for some residency programs. Residency programs with a relatively short duration of training - for example, only 2 years - may be unable to implement an extensive QI curriculum without siphoning away time for other learning objectives. Small residency programs may lack faculty with expertise to teach this topic. Residency programs with only a few residents may find it difficult to evaluate the success of a QI curriculum using robust statistical analysis. These residency programs need a QI curriculum with several features. The curriculum must be deliverable in a short period of time. There must be tools to assess the residents' attainment of the curricular objectives. The curriculum must give the residents practical skills to develop their own QI initiatives. Finally, there must be simple methods to evaluate the curriculum's effectiveness. To address these goals, we developed the SAFE QI (QI curriculum which is short, assessed, functional, and effective) framework for the 2-year subspecialty respirology residency program at the University of Alberta. There are 2-3 entrants per year for a total of 4-6 residents. This framework helps medical educators overcome the challenges of implementing a QI curriculum into their educational programs. This article illustrates how this framework was used to develop and deliver an institution's own QI curriculum.

Applying a reflexive framework to evaluate a communication skills curriculum.

After creating and delivering an educational curriculum, medical educators must ultimately evaluate the effectiveness of the implemented curriculum. Seasoned educators can benefit from using an established framework to help them structure a thorough, complete curricular evaluation; however, novice educators may have difficulty in transforming the concept of evaluation into a concrete process. The RUFDATA (Reasons and purpose, Uses, Focus, Data and evidence, Audience, Timing, and Agency) framework is one such paradigm. It is a well-recognized tool consisting of a reflexive framework that can guide medical educators to evaluate their own medical education curriculum. Just as important, it enables medical educators to reflect on the reasons behind the evaluation. This insight, in turn, can foster a spirit of evaluation, thus helping to ingrain it into the local educational culture. By using the evaluation of our communication skills curriculum as an example, this article describes how educators can apply the RUFDATA framework to evaluate their own curriculum.

Early Bronchus-Associated Lymphoid Tissue Lymphoma Diagnosed with Immunoglobulin Heavy Chain Molecular Testing.

When extranodal marginal zone B-cell lymphoma of mucosa associated lymphoid tissue (MALT), a low grade B-cell lymphoma, arises in the lung it is referred to as bronchus-associated lymphoid tissue (BALT) lymphoma. We describe a patient with a history of Sjögren's syndrome and rheumatoid arthritis with dyspnea and imaging consistent with lymphoid interstitial pneumonia (LIP). However, while histology and immunohistochemistry lacked definitive features of a lymphoma, immunoglobulin heavy chain (IgH) polymerase chain reaction testing demonstrated B-cell monoclonality, consistent with an early BALT lymphoma.

Using an Instructional Design Model to Teach Medical Procedures.

Educators are often tasked with developing courses and curricula that teach learners how to perform medical procedures. This instruction must provide an optimal, uniform learning experience for all learners. If not well designed, this instruction risks being unstructured, informal, variable amongst learners, or incomplete. This article shows how an instructional design model can help craft courses and curricula to optimize instruction in performing medical procedures. Educators can use this as a guide to developing their own course instruction.

Allele-Specific Reprogramming of Cancer Metabolism by the Long Non-coding RNA CCAT2.

Altered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the major nutrients that fuel cellular metabolism, and the pathways utilizing these nutrients are often altered in cancer. Here, we show that the long ncRNA CCAT2, located at the 8q24 amplicon on cancer risk-associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo in an allele-specific manner by binding the Cleavage Factor I (CFIm) complex with distinct affinities for the two subunits (CFIm25 and CFIm68). The CCAT2 interaction with the CFIm complex fine-tunes the alternative splicing of Glutaminase (GLS) by selecting the poly(A) site in intron 14 of the precursor mRNA. These findings uncover a complex, allele-specific regulatory mechanism of cancer metabolism orchestrated by the two alleles of a long ncRNA.

Coaching patients during pulmonary function testing: A practical guide.

Pulmonary function tests are an important tool to assist in the diagnosis and management of patients with respiratory disease. Ensuring that the tests are of acceptable quality is vital. Acceptable pulmonary function test quality requires, among others, optimal patient performance. Optimal patient performance, in turn, requires adequate coaching from registered respiratory therapists (RRTs) and other pulmonary function laboratory personnel. The present article provides techniques and tips to help RRTs coach patients during testing. The authors briefly review the components of pulmonary function testing, then describe factors that may hinder a patient's performance, list common mistakes that patients make during testing, and provide tips that RRTs can use to help patients optimize their performance.

L'exploration fonctionnelle pulmonaire est un outil important pour contribuer au diagnostic et à la prise en charge des patients atteints d'une maladie respiratoire. Il est essentiel de s'assurer que les tests sont d'une qualité acceptable. Pour parvenir à une analyse des explorations fonctionnelles respiratoires de qualité acceptable, il faut, entre autres, obtenir le rendement optimal du patient. Pour ce faire, l'inhalothérapeute et le reste du personnel du laboratoire de fonction pulmonaire doivent donner des conseils pertinents. Le présent article présente des techniques et des trucs pour aider les inhalothérapeutes à conseiller les patients pendant les tests. Les auteurs analysent brièvement les éléments de l'exploration fonctionnelle pulmonaire, décrivent les facteurs qui nuisent au rendement du patient, énumèrent les erreurs courantes que font les patients pendant les tests et donnent des conseils que les inhalothérapeutes peuvent utiliser pour aider les patients à optimiser leur rendement.