Assessing the Cost-Effectiveness of Next-Generation Sequencing as a Biomarker Testing Approach in Oncology and Policy Implications: A Literature Review.

OBJECTIVE: A key hurdle in broader next-generation sequencing (NGS) biomarker testing access in oncology is the ongoing debate on NGS's cost-effectiveness. We conducted a systematic review of existing evidence of the costs of NGS as a biomarker testing strategy in oncology and developed policy suggestions. METHODS: We searched multiple databases for studies reporting cost comparisons and cost-effectiveness of NGS across oncology indications and geographies between 2017 and 2022, inclusive. Inclusion criteria were established based on indication and type of cost-effectiveness analysis provided. We validated analyses and policy recommendations with 5 payer/policy maker interviews in the United States, Europe, and United Kingdom. RESULTS: Of the 634 identified studies, 29 met inclusion criteria, spanning 12 countries and 6 indications. Cost comparisons of NGS were evaluated using 3 methodologies: (1) comparison of direct testing costs, (2) comparison of holistic testing costs, and (3) comparison of long-term patient outcomes and costs. Targeted panel testing (2-52 genes) was considered cost-effective when 4+ genes were assessed, and larger panels (hundreds of genes) were generally not cost-effective. Holistic analysis demonstrated that NGS reduces turnaround time, healthcare staff requirements, number of hospital visits, and hospital costs. Finally, studies evaluating NGS testing including the cost of targeted therapies generally found the incremental cost-effectiveness ratio to be above common thresholds but highlighted valuable patient benefits. CONCLUSIONS: Current literature supports NGS's cost-effectiveness as an oncology biomarker testing strategy under specific conditions. These findings underscore the need to develop policies to support holistic assessment of NGS to ensure appropriate reimbursement and access.

Treatment decision for recurrences in non-small cell lung cancer during or after adjuvant osimertinib: an international Delphi consensus report.

Introduction: Osimertinib is recommended by major guidelines for use in the adjuvant setting in patients with EGFR mutation-positive NSCLC following the significant improvement in disease-free survival observed in the Phase III ADAURA trials. Due to limited real-world data in the adjuvant setting, little guidance exists on how to approach potential recurrences either during or after the completion of the treatment. This study aimed to reach a broad consensus on key treatment decision criteria in the events of recurrence. Methods: To reach a broad consensus, a modified Delphi panel study was conducted consisting of two rounds of surveys, followed by two consensus meetings and a final offline review of key statements. An international panel of experts in the field of NSCLC (n=12) was used to provide clinical insights regarding patient management at various stages of NSCLC disease including patient monitoring, diagnostics, and treatment approach for specific recurrence scenarios. This study tested recurrences occurring 1) within or outside the central nervous system (CNS), 2) during or after the adjuvant-osimertinib regimen in NSCLC disease which is 3) amenable or not amenable to local consolidative therapy. Results: Panellists agreed on various aspects of patient monitoring and diagnostics including the use of standard techniques (e.g., CT, MRI) and tumour biomarker assessment using tissue and liquid biopsies. Consensus was reached on 6 statements describing treatment considerations for the specific NSCLC recurrence scenarios. Panellists agreed on the value of osimertinib as a monotherapy or as part of the overall treatment strategy within the probed recurrence scenarios and acknowledged that more clinical evidence is required before precise recommendations for specific patient populations can be made. Discussion: This study provides a qualitative expert opinion framework for clinicians to consider within their treatment decision-making when faced with recurrence during or after adjuvant-osimertinib treatment.

ERG Alteration Due to the rd8 Mutation of the Crb1 Gene in Cln3 +/+ rd8-/rd8- Mice.

Mattapallil et al. described that vendor lines for C57BL/6 N mice may carry the rd8 mutation that leads to an ocular phenotype, which could be mistaken for an induced retinal degeneration. This mouse strain is widely used in ophthalmic research as a background for modeling retinal degeneration. In the process of studying Cln3Δex7/8 knock-in mice on a C57BL/6 N background, we became aware of this issue. The aim of this study thus was to use electroretinography to investigate the age-dependent functional loss in Cln3+/+ rd8-/rd8- mice and compare it to C57BL/6 J mice.The scotopic and photopic amplitudes of the a-wave and b-wave decrease significantly in mutant mice with increasing age, and the implicit time is prolonged. Especially the oscillatory potentials arising from inner retinal interaction seem to be notably affected by the rd8 mutation. Surprisingly, the amplitudes in young C57BL/6 J mice were lower than those measured in C57BL/6 N at any time point.Our results indicate that the rd8 mutation present in C57BL/6 N mice affects the function of the inner and outer retina. This is surprising given that the major retinal morphological alterations due to the rd8 mutation are found in the outer retina.We conclude that the rd8 mutation does affect the retinal function in Cln3+/+ rd8-/rd8- mice in a variable manner. Epigenetic factors and modifying genes lead to a phenotype shift in these mice. Interpreting the results of previous studies in mutant mice on C57BL/6 N background is challenging as comparing results obtained in independent studies or on other mouse backgrounds may be misleading. Using littermates as controls remains the only valid option.

The CLN3 gene and protein: What we know.

BACKGROUND: One of the most important steps taken by Beyond Batten Disease Foundation in our quest to cure juvenile Batten (CLN3) disease is to understand the State of the Science. We believe that a strong understanding of where we are in our experimental understanding of the CLN3 gene, its regulation, gene product, protein structure, tissue distribution, biomarker use, and pathological responses to its deficiency, lays the groundwork for determining therapeutic action plans. OBJECTIVES: To present an unbiased comprehensive reference tool of the experimental understanding of the CLN3 gene and gene product of the same name. METHODS: BBDF compiled all of the available CLN3 gene and protein data from biological databases, repositories of federally and privately funded projects, patent and trademark offices, science and technology journals, industrial drug and pipeline reports as well as clinical trial reports and with painstaking precision, validated the information together with experts in Batten disease, lysosomal storage disease, lysosome/endosome biology. RESULTS: The finished product is an indexed review of the CLN3 gene and protein which is not limited in page size or number of references, references all available primary experiments, and does not draw conclusions for the reader. CONCLUSIONS: Revisiting the experimental history of a target gene and its product ensures that inaccuracies and contradictions come to light, long-held beliefs and assumptions continue to be challenged, and information that was previously deemed inconsequential gets a second look. Compiling the information into one manuscript with all appropriate primary references provides quick clues to which studies have been completed under which conditions and what information has been reported. This compendium does not seek to replace original articles or subtopic reviews but provides an historical roadmap to completed works.

Further Characterization of the Predominant Inner Retinal Degeneration of Aging Cln3 Δex7/8 Knock-In Mice.

Neuronal ceroid lipofuscinosis (NCL) is the most common group of neurogenetic storage diseases typically beginning in childhood. The juvenile form (JNCL), also known as Batten disease, is the most common form. Vision-related problems are often an early sign, appearing prior to motor and mental deficits. We have previously investigated disease progression with age in the Cln3 Δex7/8 KI mouse model for JNCL and showed a decline of visual acuity and a predominant decline of the inner retinal function in mice, similar to human disease. The aim of this study was to further characterize this degeneration by means of flicker ERGs. For the scotopic flicker ERG, we found a significantly lower magnitude for Cln3 Δex7/8 KI mice already at 6 months of age for low stimulus frequencies, while the difference declines with increasing frequency. Under photopic conditions there was no magnitude difference at 6 months, but a cumulative magnitude reduction with further aging. For both conditions the phases were similar for both groups. There was a similar magnitude reduction for the responses of both the slow and fast rod pathway in the 15 Hz experiments, and there were no differences in response phase. Low-frequency flicker responses seem to be sensitive to very early disease manifestations, and while the degeneration is associated with a reduction of predominating inner retinal responses in the scotopic flash ERG, this predominance seems not to be related to a selective involvement of the slow and fast rod pathways.

Retinal function in aging homozygous Cln3 (Δex7/8) knock-in mice.

Neuronal ceroid lipofuscinoses (NCL) are characterized by lysosomal accumulation of autofluorescent material and lead to degeneration of the central nervous system. Patients affected by the juvenile form of NCL (JNCL), the most common form of the disease, develop visual failure prior to mental and motor deficits. It is currently unclear if the corresponding mouse model, Cln3 (Δex7/8) knock-in, develops the same retinal phenotype and electroretinogram (ERG) measurements as affected patients. The aim of our study was to investigate the visual disease progression in the Cln3 (Δex7/8) mice using scotopic and photopic ERGs as well as optokinetic tracking (OKT) at different ages. The results were then compared with age-matched controls.The amplitudes of the a-wave and b-wave (scotopic ERG) decrease significantly in Cln3 (Δex7/8) mice starting at the age of 12 months. A reduction in the b/a-amplitude ratio indicates a degeneration preferentially of the inner retina. An amplitude reduction observed in the Cln3 (+/+) control mice may be attributed to an additional Crb1 (rd8) mutation. Using optokinetic tracking (OKT) the Cln3 (Δex7/8) mice show a progressive decline in visual acuity after 12 months of age.

Progressive retinal degeneration and glial activation in the CLN6 (nclf) mouse model of neuronal ceroid lipofuscinosis: a beneficial effect of DHA and curcumin supplementation.

Neuronal ceroid lipofuscinosis (NCL) is a group of neurodegenerative lysosomal storage disorders characterized by vision loss, mental and motor deficits, and spontaneous seizures. Neuropathological analyses of autopsy material from NCL patients and animal models revealed brain atrophy closely associated with glial activity. Earlier reports also noticed loss of retinal cells and reactive gliosis in some forms of NCL. To study this phenomenon in detail, we analyzed the ocular phenotype of CLN6 (nclf) mice, an established mouse model for variant-late infantile NCL. Retinal morphometry, immunohistochemistry, optokinetic tracking, electroretinography, and mRNA expression were used to characterize retinal morphology and function as well as the responses of Müller cells and microglia. Our histological data showed a severe and progressive degeneration in the CLN6 (nclf) retina co-inciding with reactive Müller glia. Furthermore, a prominent phenotypic transformation of ramified microglia to phagocytic, bloated, and mislocalized microglial cells was identified in CLN6 (nclf) retinas. These events overlapped with a rapid loss of visual perception and retinal function. Based on the strong microglia reactivity we hypothesized that dietary supplementation with immuno-regulatory compounds, curcumin and docosahexaenoic acid (DHA), could ameliorate microgliosis and reduce retinal degeneration. Our analyses showed that treatment of three-week-old CLN6 (nclf) mice with either 5% DHA or 0.6% curcumin for 30 weeks resulted in a reduced number of amoeboid reactive microglia and partially improved retinal function. DHA-treatment also improved the morphology of CLN6 (nclf) retinas with a preserved thickness of the photoreceptor layer in most regions of the retina. Our results suggest that microglial reactivity closely accompanies disease progression in the CLN6 (nclf) retina and both processes can be attenuated with dietary supplemented immuno-modulating compounds.

Immune cells perturb axons and impair neuronal survival in a mouse model of infantile neuronal ceroid lipofuscinosis.

The neuronal ceroid lipofuscinoses are fatal neurodegenerative disorders in which the visual system is affected early in disease progression. A typical accompanying feature is neuroinflammation, the pathogenic impact of which is presently obscure. Here we investigated the role of inflammatory cells in palmitoyl protein thioesterase 1-deficient (Ppt1(-/-)) mice, a model of infantile neuronal ceroid lipofuscinosis (CLN1 disease, infantile), predominantly focusing on the visual system. We detected an early infiltration of CD8+ T-lymphocytes and observed activation of microglia/macrophage-like cells. To analyse the pathogenic impact of lymphocytes, we crossbred Ppt1(-/-) mice with mutants lacking lymphocytes (Rag1(-/-)), and scored axonal transport, axonal perturbation and neuronal survival. This lack of lymphocytes led to a significant amelioration of disease phenotypes, not only in the retino-tectal system, but also in other regions of the central nervous system. Finally, reconstitution experiments revealed a crucial role of CD8+ T-lymphocytes in pathogenesis. Our study provides novel pathomechanistic insights that may be crucial for developing treatment strategies.

Compounds that correct F508del-CFTR trafficking can also correct other protein trafficking diseases: an in vitro study using cell lines.

BACKGROUND: Many genetic diseases are due to defects in protein trafficking where the mutant protein is recognized by the quality control systems, retained in the endoplasmic reticulum (ER), and degraded by the proteasome. In many cases, the mutant protein retains function if it can be trafficked to its proper cellular location. We have identified structurally diverse correctors that restore the trafficking and function of the most common mutation causing cystic fibrosis, F508del-CFTR. Most of these correctors do not act directly as ligands of CFTR, but indirectly on other pathways to promote folding and correction. We hypothesize that these proteostasis regulators may also correct other protein trafficking diseases. METHODS: To test our hypothesis, we used stable cell lines or transient transfection to express 2 well-studied trafficking disease mutations in each of 3 different proteins: the arginine-vasopressin receptor 2 (AVPR2, also known as V2R), the human ether-a-go-go-related gene (KCNH2, also known as hERG), and finally the sulfonylurea receptor 1 (ABCC8, also known as SUR1). We treated cells expressing these mutant proteins with 9 structurally diverse F508del-CFTR correctors that function through different cellular mechanisms and assessed whether correction occurred via immunoblotting and functional assays. Results were deemed significantly different from controls by a one-way ANOVA (p < 0.05). RESULTS: Here we show that F508del-CFTR correctors RDR1, KM60 and KM57 also correct some mutant alleles of other protein trafficking diseases. We also show that one corrector, the cardiac glycoside ouabain, was found to alter the glycosylation of all mutant alleles tested. CONCLUSIONS: Correctors of F508del-CFTR trafficking might have broader applications to other protein trafficking diseases.

Curcumin is a potent modulator of microglial gene expression and migration.

BACKGROUND: Microglial cells are important effectors of the neuronal innate immune system with a major role in chronic neurodegenerative diseases. Curcumin, a major component of tumeric, alleviates pro-inflammatory activities of these cells by inhibiting nuclear factor kappa B (NFkB) signaling. To study the immuno-modulatory effects of curcumin on a transcriptomic level, DNA-microarray analyses were performed with resting and LPS-challenged microglial cells after short-term treatment with curcumin. METHODS: Resting and LPS-activated BV-2 cells were stimulated with curcumin and genome-wide mRNA expression patterns were determined using DNA-microarrays. Selected qRT-PCR analyses were performed to confirm newly identified curcumin-regulated genes. The migration potential of microglial cells was determined with wound healing assays and transwell migration assays. Microglial neurotoxicity was estimated by morphological analyses and quantification of caspase 3/7 levels in 661W photoreceptors cultured in the presence of microglia-conditioned medium. RESULTS: Curcumin treatment markedly changed the microglial transcriptome with 49 differentially expressed transcripts in a combined analysis of resting and activated microglial cells. Curcumin effectively triggered anti-inflammatory signals as shown by induced expression of Interleukin 4 and Peroxisome proliferator activated receptor α. Several novel curcumin-induced genes including Netrin G1, Delta-like 1, Platelet endothelial cell adhesion molecule 1, and Plasma cell endoplasmic reticulum protein 1, have been previously associated with adhesion and cell migration. Consequently, curcumin treatment significantly inhibited basal and activation-induced migration of BV-2 microglia. Curcumin also potently blocked gene expression related to pro-inflammatory activation of resting cells including Toll-like receptor 2 and Prostaglandin-endoperoxide synthase 2. Moreover, transcription of NO synthase 2 and Signal transducer and activator of transcription 1 was reduced in LPS-triggered microglia. These transcriptional changes in curcumin-treated LPS-primed microglia also lead to decreased neurotoxicity with reduced apoptosis of 661W photoreceptor cultures. CONCLUSIONS: Collectively, our results suggest that curcumin is a potent modulator of the microglial transcriptome. Curcumin attenuates microglial migration and triggers a phenotype with anti-inflammatory and neuroprotective properties. Thus, curcumin could be a nutraceutical compound to develop immuno-modulatory and neuroprotective therapies for the treatment of various neurodegenerative disorders.

Rotavirus toxin NSP4 induces diarrhea by activation of TMEM16A and inhibition of Na+ absorption.

Rotavirus infection is the most frequent cause for severe diarrhea in infants, killing more than 600,000 every year. The nonstructural protein NSP4 acts as a rotavirus enterotoxin, inducing secretory diarrhea without any structural organ damage. Electrolyte transport was assessed in the colonic epithelium from pups and adult mice using Ussing chamber recordings. Western blots and immunocytochemistry was performed in intestinal tissues from wild-type and TMEM16A knockout mice. Ion channel currents were recorded using patch clamp techniques. We show that the synthetic NSP4(114-135) peptide uses multiple pro-secretory pathways to induce diarrhea, by activating the recently identified Ca2+ -activated Cl- channel TMEM16A, and by inhibiting Na+ absorption by the epithelial Na+ channel ENaC and the Na+ /glucose cotransporter SGLT1. Activation of secretion and inhibition of Na+ absorption by NSP4(114-135), respectively, could be potently suppressed by wheat germ agglutinin which probably competes with NSP4(114-135) for binding to an unknown glycolipid receptor. The present paper gives a clue as to mechanisms of rotavirus-induced diarrhea and suggests wheat germ agglutinin as a simple and effective therapy.

Expression and function of epithelial anoctamins.

The calcium-activated chloride channel anoctamin1 (ANO1; TMEM16A) is fundamental for the function of epithelial organs. Mice lacking ANO1 expression exhibit transport defects and a pathology similar to cystic fibrosis. They also show a general defect of epithelial electrolyte transport. Here we analyzed expression of all ten members (ANO1-ANO10) in a broad range of murine tissues and detected predominant expression of ANO1, 6, 7, 8, 9, 10 in epithelial tissues, while ANO2, 3, 4, 5 are common in neuronal and muscle tissues. When expressed in Fisher Rat Thyroid (FTR) cells, all ANO proteins localized to the plasma membrane but only ANO1, 2, 6, and 7 produced Ca(2+)-activated Cl(-) conductance, as analyzed by ATP-induced iodide quenching of YFP fluorescence. In contrast ANO9 and ANO10 suppressed baseline Cl(-) conductance and coexpression of ANO9 with ANO1 inhibited ANO1 activity. Patch clamping of ANO-expressing FRT cells indicated that apart from ANO1 also ANO6 and 10 produced chloride currents, albeit with very different Ca(2+) sensitivity and activation time. We conclude that each tissue expresses a set of anoctamins that form cell- and tissue-specific Ca(2+)-dependent Cl(-) channels.

Coiled-coil interactions are required for post-Golgi R-SNARE trafficking.

The sorting of post-Golgi R-SNAREs (vesicle-associated membrane protein (VAMP)1, 2, 3, 4, 7 and 8) is still poorly understood. To address this, we developed a system to investigate their localization, trafficking and cell-surface levels. Here, we show that the distribution and internalization of VAMPs 3 and 8 are determined solely through a new conserved mechanism that uses coiled-coil interactions, and that VAMP4 does not require these interactions for its trafficking. We propose that VAMPs 3 and 8 are trafficked while in a complex with Q-SNAREs. We also show that the dileucine motif of VAMP4 is required for both its internalization and retrieval to the trans-Golgi network. However, when the dileucine motif is mutated, the construct can still be internalized potentially through coiled-coil interactions with Q-SNAREs.