Duchenne and Becker Muscular Dystrophies: A Review of Animal Models, Clinical End Points, and Biomarker Quantification.

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are neuromuscular disorders that primarily affect boys due to an X-linked mutation in the DMD gene, resulting in reduced to near absence of dystrophin or expression of truncated forms of dystrophin. Some newer therapeutic interventions aim to increase sarcolemmal dystrophin expression, and accurate dystrophin quantification is critical for demonstrating pharmacodynamic relationships in preclinical studies and clinical trials. Current challenges with measuring dystrophin include the variation in protein expression within individual muscle fibers and across whole muscle samples, the presence of preexisting dystrophin-positive revertant fibers, and trace amounts of residual dystrophin. Immunofluorescence quantification of dystrophin can overcome many of these challenges, but manual quantification of protein expression may be complicated by variations in the collection of images, reproducible scoring of fluorescent intensity, and bias introduced by manual scoring of typically only a few high-power fields. This review highlights the pathology of DMD and BMD, discusses animal models of DMD and BMD, and describes dystrophin biomarker quantitation in DMD and BMD, with several image analysis approaches, including a new automated method that evaluates protein expression of individual muscle fibers.

Quantitative assessment of pancreatic cancer precursor lesions in IHC-stained tissue with a tissue image analysis platform.

Tissue image analysis (tIA) is emerging as a powerful tool for quantifying biomarker expression and distribution in complex diseases and tissues. Pancreatic ductal adenocarcinoma (PDAC) develops in a highly complex and heterogeneous tissue environment and, generally, has a very poor prognosis. Early detection of PDAC is confounded by limited knowledge of the pre-neoplastic disease stages and limited methods to quantitatively assess disease heterogeneity. We sought to develop a tIA approach to assess the most common PDAC precursor lesions, pancreatic intraepithelial neoplasia (PanIN), in tissues from KrasLSL-G12D/+; Trp53LSL-R172H/+; Pdx-Cre (KPC) mice, a validated model of PDAC development. tIA profiling of training regions of PanIN and tumor microenvironment (TME) cells was utilized to guide identification of PanIN/TME tissue compartment stratification criteria. A custom CellMap algorithm implementing these criteria was applied to whole-slide images of KPC mice pancreata sections to quantify p53 and Ki-67 biomarker staining in each tissue compartment as a proof-of-concept for the algorithm platform. The algorithm robustly identified a higher percentage of p53-positive cells in PanIN lesions relative to the TME, whereas no difference was observed for Ki-67. Ki-67 expression was also quantified in a human pancreatic tissue sample available to demonstrate the translatability of the CellMap algorithm to human samples. Together, our data demonstrated the utility of CellMap to enable objective and quantitative assessments, across entire tissue sections, of PDAC precursor lesions in preclinical and clinical models of this disease to support efforts leading to novel insights into disease progression, diagnostic markers, and potential therapeutic targets.

Commentary: Roles for Pathologists in a High-throughput Image Analysis Team.

Historically, pathologists perform manual evaluation of H&E- or immunohistochemically-stained slides, which can be subjective, inconsistent, and, at best, semiquantitative. As the complexity of staining and demand for increased precision of manual evaluation increase, the pathologist's assessment will include automated analyses (i.e., "digital pathology") to increase the accuracy, efficiency, and speed of diagnosis and hypothesis testing and as an important biomedical research and diagnostic tool. This commentary introduces the many roles for pathologists in designing and conducting high-throughput digital image analysis. Pathology review is central to the entire course of a digital pathology study, including experimental design, sample quality verification, specimen annotation, analytical algorithm development, and report preparation. The pathologist performs these roles by reviewing work undertaken by technicians and scientists with training and expertise in image analysis instruments and software. These roles require regular, face-to-face interactions between team members and the lead pathologist. Traditional pathology training is suitable preparation for entry-level participation on image analysis teams. The future of pathology is very exciting, with the expanding utilization of digital image analysis set to expand pathology roles in research and drug development with increasing and new career opportunities for pathologists.

Evaluating tumor heterogeneity in immunohistochemistry-stained breast cancer tissue.

Quantitative clinical measurement of heterogeneity in immunohistochemistry staining would be useful in evaluating patient therapeutic response and in identifying underlying issues in histopathology laboratory quality control. A heterogeneity scoring approach (HetMap) was designed to visualize a individual patient's immunohistochemistry heterogeneity in the context of a patient population. HER2 semiquantitative analysis was combined with ecology diversity statistics to evaluate cell-level heterogeneity (consistency of protein expression within neighboring cells in a tumor nest) and tumor-level heterogeneity (differences of protein expression across a tumor as represented by a tissue section). This approach was evaluated on HER2 immunohistochemistry-stained breast cancer samples using 200 specimens across two different laboratories with three pathologists per laboratory, each outlining regions of tumor for scoring by automatic cell-based image analysis. HetMap was evaluated using three different scoring schemes: HER2 scoring according to American Society of Clinical Oncology and College of American Pathologists (ASCO/CAP) guidelines, H-score, and a new continuous HER2 score (HER2(cont)). Two definitions of heterogeneity, cell-level and tumor-level, provided useful independent measures of heterogeneity. Cases where pathologists had disagreement over reads in the area of clinical importance (+1 and +2) had statistically significantly higher levels of tumor-level heterogeneity. Cell-level heterogeneity, reported either as an average or the maximum area of heterogeneity across a slide, had low levels of dependency on the pathologist choice of region, while tumor-level heterogeneity measurements had more dependence on the pathologist choice of regions. HetMap is a measure of heterogeneity, by which pathologists, oncologists, and drug development organizations can view cell-level and tumor-level heterogeneity for a patient for a given marker in the context of an entire patient cohort. Heterogeneity analysis can be used to identify tumors with differing degrees of heterogeneity, or to highlight slides that should be rechecked for QC issues. Tumor heterogeneity plays a significant role in disconcordant reads between pathologists.

Validation of a Muscle-Specific Tissue Image Analysis Tool for Quantitative Assessment of Dystrophin Staining in Frozen Muscle Biopsies.

CONTEXT.­: Duchenne muscular dystrophy is a rare, progressive, and fatal neuromuscular disease caused by dystrophin protein loss. Common investigational treatment approaches aim at increasing dystrophin expression in diseased muscle. Some clinical trials include assessments of novel dystrophin production as a surrogate biomarker of efficacy, which may predict a clinical benefit from treatment. OBJECTIVES.­: To establish an immunofluorescent scanning and digital image analysis workflow that provides an objective approach for staining intensity assessment of the immunofluorescence dystrophin labeling and determination of the percentage of biomarker-positive fibers in muscle cryosections. DESIGN.­: Optimal and repeatable digital image capture was achieved by a rigorously qualified fluorescent scanning process. After scanning qualification, the MuscleMap (Flagship Biosciences, Westminster, Colorado) algorithm was validated by comparing high-power microscopic field total and dystrophin-positive fiber counts obtained by trained pathologists to data derived by MuscleMap. Next, the algorithm was tested on whole-slide images of immunofluorescent-labeled muscle sections from Duchenne muscular dystrophy, Becker muscular dystrophy, and control patients. RESULTS.­: When used under the guidance of a trained pathologist, the digital image analysis tool met predefined validation criteria and demonstrated functional and statistical equivalence with manual assessment. This work is the first, to our knowledge, to qualify and validate immunofluorescent scanning and digital tissue image-analysis workflow, respectively, with the rigor required to support the clinical trial environments. CONCLUSIONS.­: MuscleMap enables analysis of all fibers within an entire muscle biopsy section and provides data on a fiber-by-fiber basis. This will allow future clinical trials to objectively investigate myofibers' dystrophin expression at a greater level of consistency and detail.

Long-term treatment with eteplirsen in nonambulatory patients with Duchenne muscular dystrophy.

This analysis aims to describe the outcomes of two nonambulatory patients with Duchenne muscular dystrophy (DMD) who participated in two clinical studies. The two consecutive trials of eteplirsen (studies 201 and 202) were conducted in patients with DMD (N = 12) and confirmed genetic mutations amenable to exon 51 skipping.In study 201, 12 patients were randomized to receive once-weekly, double-blind intravenous infusions of eteplirsen 30 or 50 mg/kg or placebo for 24 weeks; patients then received open-label eteplirsen during weeks 25 through 28. All 12 patients continued onto open-label extension study 202 and received long-term treatment with eteplirsen. We compared cardiac, pulmonary, and upper limb function and dystrophin production in the nonambulatory twin patients versus the 10 ambulatory patients through 240 combined treatment weeks.Ten study patients remained ambulatory through both studies, while the identical twin patients both experienced early, rapid loss of ambulation. The twin patients had greater disease severity at baseline (6-minute walk test [6MWT], 330 and 256 m) versus the other patients (n = 10; 6MWT range, 341-418 m). They maintained cardiac and upper limb function through combined week 240, with outcomes similar to those of the patients who remained ambulatory. Dystrophin production was confirmed following eteplirsen treatment.Despite the loss of ambulation, other markers of disease progression remained relatively stable in the eteplirsen-treated twin patients and were similar to those of the ambulatory patients.

Eteplirsen treatment for Duchenne muscular dystrophy: Exon skipping and dystrophin production.

OBJECTIVE: To describe the quantification of novel dystrophin production in patients with Duchenne muscular dystrophy (DMD) after long-term treatment with eteplirsen. METHODS: Clinical study 202 was an observational, open-label extension of the randomized, controlled study 201 assessing the safety and efficacy of eteplirsen in patients with DMD with a confirmed mutation in the DMD gene amenable to correction by skipping of exon 51. Patients received once-weekly IV doses of eteplirsen 30 or 50 mg/kg. Upper extremity muscle biopsy samples were collected at combined study week 180, blinded, and assessed for dystrophin-related content by Western blot, Bioquant software measurement of dystrophin-associated immunofluorescence intensity, and percent dystrophin-positive fibers (PDPF). Results were contrasted with matched untreated biopsies from patients with DMD. Reverse transcription PCR followed by Sanger sequencing of newly formed slice junctions was used to confirm the mechanism of action of eteplirsen. RESULTS: Reverse transcription PCR analysis and sequencing of the newly formed splice junction confirmed that 100% of treated patients displayed the expected skipped exon 51 sequence. In treated patients vs untreated controls, Western blot analysis of dystrophin content demonstrated an 11.6-fold increase (p = 0.007), and PDPF analysis demonstrated a 7.4-fold increase (p < 0.001). The PDPF findings were confirmed in a re-examination of the sample (15.5-fold increase, p < 0.001). Dystrophin immunofluorescence intensity was 2.4-fold greater in treated patients than in untreated controls (p < 0.001). CONCLUSION: Taken together, the 4 assays, each based on unique evaluation mechanisms, provided evidence of eteplirsen muscle cell penetration, exon skipping, and induction of novel dystrophin expression. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence of the muscle cell penetration, exon skipping, and induction of novel dystrophin expression by eteplirsen, as confirmed by 4 assays.

The evaluation of E-Cadherin and vimentin as biomarkers of clinical outcomes among patients with non-small cell lung cancer treated with erlotinib as second- or third-line therapy.

E-Cadherin and vimentin protein expression was assessed in late stage non-small cell lung cancer tumors from the placebo controlled clinical trial, NCIC-CTG BR.21, to determine if these markers had the potential to predict outcome of erlotinib therapy. E-Cadherin and vimentin protein expression levels were assessed in tumors from 95 patients, who were representative of the overall population, using semi-quantitative immunohistochemistry. The percentage of tumor cells with grades 0, 1, 2, or 3 membrane staining of E-cadherin and cytoplasmic staining of vimentin was measured. Three scoring methods and multiple cut-offs were explored to determine if these markers were able to divide patients into groups with different overall survival (OS). A cut-off point for E-cadherin of ≥40% tumor cells with staining of +2 and +3 and a cut-off for vimentin of ≥10% of tumors cell with any staining provided the optimal stratification. The OS hazard ratio (HR) for E-cadherin(+) versus E-cadherin(-) in the erlotinib-treated patients was 0.68 (0.35-1.33) compared with 1.48 (0.69-3.15) in the placebo patients and the OS (HR) for erlotinib versus placebo was 0.47 (0.26-0.88) in E-cadherin(+) patients compared with 1.12 (0.52-2.44) in the E-cadherin(-) patients. The OS (HR) for vimentin(+) versus vimentin(-) in the erlotinib-treated patients was 0.65 (0.31-1.38) compared to 2.32 (1.09-4.94) in the placebo-treated patients and the OS (HR) for erlotinib versus placebo was 0.26 (0.11-0.63) in vimentin(+) compared to 0.99 (0.55-1.76) in the vimentin(-) patients. Similar trends were observed for progression-free survival and response rate. E-Cadherin and vimentin are biomarkers worthy of additional study as predictive markers of outcome of erlotinib therapy.

Multiplexed measurement of proteins in tissue in a clinical environment.

There is an emerging need for more effective approaches to accurately quantitate protein expression in tissue samples. In many clinical studies and particularly in pharmaceutical clinical trials, access to adequate tissue samples is a major bottleneck, and thus techniques to measure protein expression in these valuable tissue specimens is important. This study will review current approaches in multiplexing of protein expression in tissue, and discusses new approaches using a novel image registration technique across multiple tissue sections.

Inducible expression of TGFß, snail and Zeb1 recapitulates EMT in vitro and in vivo in a NSCLC model.

The progression of cancer from non-metastatic to metastatic is the critical transition in the course of the disease. The epithelial to mesenchymal transition (EMT) is a mechanism by which tumor cells acquire characteristics that improve metastatic efficiency. Targeting EMT processes in patients is therefore a potential strategy to block the transition to metastatic cancer and improve patient outcome. To develop models of EMT applicable to in vitro and in vivo settings, we engineered NCI-H358 non-small cell lung carcinoma cells to inducibly express three well-established drivers of EMT: activated transforming growth factor ß (aTGFß), Snail or Zeb1. We characterized the morphological, molecular and phenotypic changes induced by each of the drivers and compared the different end-states of EMT between the models. Both in vitro and in vivo, induction of the transgenes Snail and Zeb1 resulted in downregulation of epithelial markers and upregulation of mesenchymal markers, and reduced the ability of the cells to proliferate. Induced autocrine expression of aTGFß caused marker and phenotypic changes consistent with EMT, a modest effect on growth rate, and a shift to a more invasive phenotype. In vivo, this manifested as tumor cell infiltration of the surrounding mouse stromal tissue. Overall, Snail and Zeb1 were sufficient to induce EMT in the cells, but aTGFß induced a more complex EMT, in which changes in extracellular matrix remodeling components were pronounced.

The role and impact of quantitative discovery pathology.

The decision to advance an early-stage compound into formal preclinical testing depends on confidence in mechanism, efficacy and toxicity profiles. A substantial percentage of this confidence comes from histopathology interpretation, as the local tissue environment contains strong signals of both efficacy and toxicity. Accessing this tissue information is made difficult by biological variability across organs and tissues, an insufficient pool of pathology experts working in discovery, and the high subjectivity and individual isolation of microscope-based observations. This article describes how whole-slide imaging and quantitative analysis by trained pathologists are improving early-stage decision-making.

Intracranial drug-delivery scaffolds: biocompatibility evaluation of sucrose acetate isobutyrate gels.

INTRODUCTION: Sucrose acetate isobutyrate (SAIB) is a water insoluble, biodegradable gel used for controlled-release oral and subcutaneous drug delivery. We investigated SAIB compatibility in the rat central nervous system (CNS) by implanting solutions of SAIB in adult and in neonatal brains. METHODS: 10-15 microL solutions of SAIB gels in 0-30% ethanol were injected into the cerebral cortex of adult Fischer 344 rats. Control animals were implanted with a 10 mg biodegradable poly anhydride copolymer of poly [bis (p-carboxyphenoxy) propane] anhydride and sebacic acid (PCPP:SA). Adult rats were evaluated for signs of pain and distress, including changes in posture, facial signs, and grooming behavior. 1-2 microL solutions of SAIB gels in 15% ethanol were injected into brains of 12-24 h-old rats. Neonatal rats were evaluated for survival. Adult and neonatal brains were examined by histopathology 3-48 days after implant. RESULTS: Gel implants produced elliptical compression of cortical tissue, cell loss, and inflammation. Cell loss appeared to be confined to the implantation wound and associated neuronal fields. In adult rats, neurophil compression, inflammation, and cell loss appeared similar with the 10-mg PCPP:SA implants and the 10-mg SAIB implants. There was no clinical evidence of pain or distress from SAIB implants. 1-2 microL implants of SAIB-15% ethanol had no effect on survival of neonatal animals. CONCLUSION: Brain implants of SAIB induce a mild to moderate inflammatory response and associated neuronal cell damage. The implants appeared to be biocompatible in adult and neonatal animals. These results suggest that further studies of SAIB as an injectable drug-delivery scaffold for CNS therapeutic agents are warranted.