Intrasession Repeatability of OCT Angiography Parameters in Neurodegenerative Disease.

Purpose: To assess the intrasession repeatability of macular OCT angiography (OCTA) parameters in Alzheimer's disease (AD), mild cognitive impairment (MCI), Parkinson's disease (PD), and normal cognition (NC). Design: Cross sectional study. Subjects: Patients with a clinical diagnosis of AD, PD, MCI, or NC were imaged. Images with poor quality and of those with diabetes mellitus, glaucoma, or vitreoretinal disease were excluded from analysis. Methods Intervention or Testing: All participants were imaged using the Zeiss Cirrus HD-5000 with AngioPlex (Carl Zeiss Meditec, Software Version 11.0.0.29946) and repeat OCTA images were obtained for both eyes. Perfusion density (PFD), vessel density (VD), and Foveal avascular zone (FAZ) area were measured from 3 × 3 mm and 6 × 6 mm OCTA images centered on the fovea using an ETDRS grid overlay. Main Outcome Measures: Intraclass correlation coefficients were used to quantify repeatability of PFD, VD, and FAZ area measurements obtained from imaging. Results: 3 × 3 mm scans of 22 AD, 40 MCI, 21 PD, and 26 NC participants and 6 × 6 mm scans of 29 AD, 44 MCI, 29 PD, and 30 NC participants were analyzed. Repeatability values ranged from 0.64 (0.49-0.82) for 6 × 6 mm PFD in AD participants to 0.87 (0.67-0.92) for 3 × 3 mm PFD in AD participants. No significant differences were observed in repeatability between NC participants and those with neurodegenerative disease. Conclusions: Overall, similar OCTA repeatability was observed between NC participants and those with neurodegeneration. Regardless of diagnostic group, macular OCTA metrics demonstrated moderate to good repeatability. Financial Disclosures: The authors have no proprietary or commercial interest in any materials discussed in this article.

PAK4 inhibition improves PD1 blockade immunotherapy in prostate cancer by increasing immune infiltration.

Antitumor immunity requires lymphocytes to localize to the tumor. Prostate cancers (PCs) are immunologically cold and tend to lack T-cell infiltration. Most advanced PCs are insensitive to PD1 blockade therapies. Using syngeneic RM1 prostate tumors, p21-activated kinase-4 (PAK4) knockdown (KD) and pharmacological inhibition was assessed in C57BL/6J mice treated with PD1 antibodies (αPD1). RNASeq was used to characterize the immune response in the tumor. Immunohistochemistry, flow cytometry, and in vivo blocking studies confirmed the role of cell surface proteins in the generation of immune responses. In The Cancer Genome Atlas, PAK4 expression was inversely correlated with immune cell infiltration. PAK4 expression was controlled by the androgen receptor and its pioneering factor, FOXA1. PAK4 KD increased CD8+ T-cell infiltration and expression of IFNγ response genes. PAK4 KD also upregulated angiogenesis and endothelial cell adhesion molecules in the tumor microenvironment, contributing to CD8+ lymphocyte recruitment. Pharmacological inhibition of PAK4 made PC more responsive to immunotherapy with αPD1. A decrease in PAK4 activity increases immune activation and vascularity, which increases CD8+ lymphocyte infiltration into the tumor. Therefore, targeting PAK4 may improve the response of human PC to immunotherapy.

Effect of Nylon Wick Technique on Early Intraocular Pressure Control in Nonvalved Aqueous Shunt Surgery.

PRECIS: The use of nylon wicks with fenestrations in nonvalved aqueous shunt surgery significantly reduces intraocular pressure (IOP) and glaucoma medication usage in the immediate postoperative period compared with the use of fenestrations alone. PURPOSE: To compare early postoperative IOP and medication usage in patients undergoing implantation of a nonvalved aqueous shunt device with fenestrations only or fenestrations with nylon wicks. METHODS: A retrospective review of all nonvalved aqueous shunt insertions completed by one surgeon (L.W.H.) was completed using current procedure terminology. Patients undergoing Baerveldt or ClearPath 350 mm2 aqueous shunt insertion with fenestrations only (n=37) or fenestrations with 2 nylon wicks were identified (n=92). All devices were ligated with 7-0 Vicryl (polyglactin) suture, and either 4 fenestrations or 2 fenestrations and two 9-0 nylon wicks were placed anterior to the ligature. Data regarding visual acuity (VA), IOP, number of glaucoma medications, and complications were collected from the preoperative visit just before surgery, postoperative day 1, week 3 (POW3), week 5, and month 2 (POM2). The main outcome measures were VA, IOP, number of glaucoma medications, and complications at all postoperative time points. RESULTS: There was no difference in logMAR VA between the 2 groups at any time point. At POW3, IOP was significantly lower in the wick group (14.6±7.7 vs. 18.1±8.7 mm Hg, P=0.03). Number of glaucoma medications used was significantly reduced in the wick group at POW3 (0.5±0.9 vs. 1.0±1.2, P=0.02) and POM2 (0.7±1.0 vs. 1.4±1.3, P=0.02). There was no significant increase in the overall rate of complications in the wick group, but there was a higher rate of transient hyphema (28% vs. 8%, P=0.02). CONCLUSIONS: The use of 2 nylon wicks with fenestrations in nonvalved aqueous shunt device implantation can significantly lower IOP and medication burden while awaiting the dissolution of the ligature suture.

Conserved Interferon-γ Signaling Drives Clinical Response to Immune Checkpoint Blockade Therapy in Melanoma.

We analyze the transcriptome of baseline and on-therapy tumor biopsies from 101 patients with advanced melanoma treated with nivolumab (anti-PD-1) alone or combined with ipilimumab (anti-CTLA-4). We find that T cell infiltration and interferon-γ (IFN-γ) signaling signatures correspond most highly with clinical response to therapy, with a reciprocal decrease in cell-cycle and WNT signaling pathways in responding biopsies. We model the interaction in 58 human cell lines, where IFN-γ in vitro exposure leads to a conserved transcriptome response unless cells have IFN-γ receptor alterations. This conserved IFN-γ transcriptome response in melanoma cells serves to amplify the antitumor immune response. Therefore, the magnitude of the antitumor T cell response and the corresponding downstream IFN-γ signaling are the main drivers of clinical response or resistance to immune checkpoint blockade therapy.

Granulomatosis with polyangiitis-associated ischemic optic neuropathy in a previously healthy 50-year-old female.

PURPOSE: To describe a case of anterior ischemic optic neuropathy as a presenting sign of granulomatosis with polyangiitis. OBSERVATIONS: A previously healthy 50-year-old female developed right eye, then left eye, redness and pruritis and was diagnosed with allergic versus viral conjunctivitis. Five days later, she noted an acute decline in vision in the right eye, corresponding with a decrease on Snellen testing from 20/30 to 20/100 with correction. She was noted to have a right relative afferent pupillary defect, 2+ pallid disc edema, and OCT (Spectralis, Heidelberg Engineering, Carlsbad, CA) findings of significant retinal nerve fiber thickening. Review of systems revealed a three-month history fatigue, right-sided headaches, jaw claudication, bronchitis, cough without hemoptysis, and epistaxis, as well as interval development of a petechial rash across her body, migratory polyarthralgias, fevers, and tachycardia. ESR and CRP were markedly elevated, and the patient was admitted to the hospital for a systemic vasculitis workup. She was started on IV methylprednisolone. Her vision improved dramatically with steroids, measuring 20/50 with correction in the right eye after 24 hours and returning to baseline after five days. An extensive workup including imaging, bloodwork, and biopsies led to a diagnosis of granulomatosis with polyangiitis, with PR3-positive ANCA. CONCLUSIONS: Ocular findings, including anterior ischemic optic neuropathy, may be the presenting signs for patients with granulomatosis with polyangiitis. Prompt recognition and treatment with high-dose steroids and immunomodulatory therapy is important for visual recovery. IMPORTANCE: Prompt recognition of potential vasculitis-related vision loss can lead to timely initiation of vision-saving treatment.

Aphakic contact lens use for improved handheld optical coherence tomography imaging in pediatric aphakic patients.

Handheld optical coherence tomography (OCT) makes it possible to acquire intraoperative and clinical imaging in infants and children for whom standard OCT is not possible. In patients with a retinal pathology who are left aphakic after surgery, however, image clarity is affected by insufficient refractive correction with handheld OCT. We describe a technique for supplementing the refractive power of the handheld OCT device with an aphakic contact lens to improve image clarity.

Deep Functional and Molecular Characterization of a High-Risk Undifferentiated Pleomorphic Sarcoma.

Nonrhabdomyosarcoma soft-tissue sarcomas (STSs) are a class of 50+ cancers arising in muscle and soft tissues of children, adolescents, and adults. Rarity of each subtype often precludes subtype-specific preclinical research, leaving many STS patients with limited treatment options should frontline therapy be insufficient. When clinical options are exhausted, personalized therapy assignment approaches may help direct patient care. Here, we report the results of an adult female STS patient with relapsed undifferentiated pleomorphic sarcoma (UPS) who self-drove exploration of a wide array of personalized Clinical Laboratory Improvement Amendments (CLIAs) level and research-level diagnostics, including state of the art genomic, proteomic, ex vivo live cell chemosensitivity testing, a patient-derived xenograft model, and immunoscoring. Her therapeutic choices were also diverse, including neoadjuvant chemotherapy, radiation therapy, and surgeries. Adjuvant and recurrence strategies included off-label and natural medicines, several immunotherapies, and N-of-1 approaches. Identified treatment options, especially those validated during the in vivo study, were not introduced into the course of clinical treatment but did provide plausible treatment regimens based on FDA-approved clinical agents.

Chromosomal instability in untreated primary prostate cancer as an indicator of metastatic potential.

BACKGROUND: Metastatic prostate cancer (PC) is highly lethal. The ability to identify primary tumors capable of dissemination is an unmet need in the quest to understand lethal biology and improve patient outcomes. Previous studies have linked chromosomal instability (CIN), which generates aneuploidy following chromosomal missegregation during mitosis, to PC progression. Evidence of CIN includes broad copy number alterations (CNAs) spanning > 300 base pairs of DNA, which may also be measured via RNA expression signatures associated with CNA frequency. Signatures of CIN in metastatic PC, however, have not been interrogated or well defined. We examined a published 70-gene CIN signature (CIN70) in untreated and castration-resistant prostate cancer (CRPC) cohorts from The Cancer Genome Atlas (TCGA) and previously published reports. We also performed transcriptome and CNA analysis in a unique cohort of untreated primary tumors collected from diagnostic prostate needle biopsies (PNBX) of localized (M0) and metastatic (M1) cases to determine if CIN was linked to clinical stage and outcome. METHODS: PNBX were collected from 99 patients treated in the VA Greater Los Angeles (GLA-VA) Healthcare System between 2000 and 2016. Total RNA was extracted from high-grade cancer areas in PNBX cores, followed by RNA sequencing and/or copy number analysis using OncoScan. Multivariate logistic regression analyses permitted calculation of odds ratios for CIN status (high versus low) in an expanded GLA-VA PNBX cohort (n = 121). RESULTS: The CIN70 signature was significantly enriched in primary tumors and CRPC metastases from M1 PC cases. An intersection of gene signatures comprised of differentially expressed genes (DEGs) generated through comparison of M1 versus M0 PNBX and primary CRPC tumors versus metastases revealed a 157-gene "metastasis" signature that was further distilled to 7-genes (PC-CIN) regulating centrosomes, chromosomal segregation, and mitotic spindle assembly. High PC-CIN scores correlated with CRPC, PC-death and all-cause mortality in the expanded GLA-VA PNBX cohort. Interestingly, approximately 1/3 of M1 PNBX cases exhibited low CIN, illuminating differential pathways of lethal PC progression. CONCLUSIONS: Measuring CIN in PNBX by transcriptome profiling is feasible, and the PC-CIN signature may identify patients with a high risk of lethal progression at the time of diagnosis.

PAK4 inhibition improves PD-1 blockade immunotherapy.

Lack of tumor infiltration by immune cells is the main mechanism of primary resistance to programmed cell death protein 1 (PD-1) blockade therapies for cancer. It has been postulated that cancer cell-intrinsic mechanisms may actively exclude T cells from tumors, suggesting that the finding of actionable molecules that could be inhibited to increase T cell infiltration may synergize with checkpoint inhibitor immunotherapy. Here, we show that p21-activated kinase 4 (PAK4) is enriched in non-responding tumor biopsies with low T cell and dendritic cell infiltration. In mouse models, genetic deletion of PAK4 increased T cell infiltration and reversed resistance to PD-1 blockade in a CD8 T cell-dependent manner. Furthermore, combination of anti-PD-1 with the PAK4 inhibitor KPT-9274 improved anti-tumor response compared with anti-PD-1 alone. Therefore, high PAK4 expression is correlated with low T cell and dendritic cell infiltration and a lack of response to PD-1 blockade, which could be reversed with PAK4 inhibition.

Probabilistic modeling of personalized drug combinations from integrated chemical screen and molecular data in sarcoma.

BACKGROUND: Cancer patients with advanced disease routinely exhaust available clinical regimens and lack actionable genomic medicine results, leaving a large patient population without effective treatments options when their disease inevitably progresses. To address the unmet clinical need for evidence-based therapy assignment when standard clinical approaches have failed, we have developed a probabilistic computational modeling approach which integrates molecular sequencing data with functional assay data to develop patient-specific combination cancer treatments. METHODS: Tissue taken from a murine model of alveolar rhabdomyosarcoma was used to perform single agent drug screening and DNA/RNA sequencing experiments; results integrated via our computational modeling approach identified a synergistic personalized two-drug combination. Cells derived from the primary murine tumor were allografted into mouse models and used to validate the personalized two-drug combination. Computational modeling of single agent drug screening and RNA sequencing of multiple heterogenous sites from a single patient's epithelioid sarcoma identified a personalized two-drug combination effective across all tumor regions. The heterogeneity-consensus combination was validated in a xenograft model derived from the patient's primary tumor. Cell cultures derived from human and canine undifferentiated pleomorphic sarcoma were assayed by drug screen; computational modeling identified a resistance-abrogating two-drug combination common to both cell cultures. This combination was validated in vitro via a cell regrowth assay. RESULTS: Our computational modeling approach addresses three major challenges in personalized cancer therapy: synergistic drug combination predictions (validated in vitro and in vivo in a genetically engineered murine cancer model), identification of unifying therapeutic targets to overcome intra-tumor heterogeneity (validated in vivo in a human cancer xenograft), and mitigation of cancer cell resistance and rewiring mechanisms (validated in vitro in a human and canine cancer model). CONCLUSIONS: These proof-of-concept studies support the use of an integrative functional approach to personalized combination therapy prediction for the population of high-risk cancer patients lacking viable clinical options and without actionable DNA sequencing-based therapy.

Scheduled Postoperative Ripcord Removal in Baerveldt 350 Implants: A Prospective, Randomized Trial.

PURPOSE: Many surgeons remove the ripcord in the Baerveldt glaucoma drainage device to better control tube opening and intraocular pressure (IOP) lowering postoperatively. However, complications following Baerveldt implant surgery with or without ripcord removal are not well-characterized. We performed a prospective, randomized trial to test the hypothesis that scheduled ripcord removal decreases complications and final IOP. METHODS: Eighty-one patients were enrolled and randomized to scheduled ripcord removal at postoperative week 3 or to observation. They were followed for 6 months, and outcomes were compared between the 2 groups. RESULTS: Forty-four patients were randomized to scheduled ripcord removal and 37 to observation. The intervention group had a similar rate of total complications after ripcord removal (36% vs. 24%, P=0.24), a lower rate of tube fibrin obstruction (2.3% vs. 13.5%, P=0.05), and a larger decrease in the number of medications (1.3 vs. 0.49 fewer medications, P=0.01). The removal group's mean IOP decrease was 8.6 mm Hg and success rate was 59%, defined as 5 mm Hg

Identification of copy number alterations in colon cancer from analysis of amplicon-based next generation sequencing data.

The objective of this study was to determine the feasibility to detect copy number alterations in colon cancer samples using Next Generation Sequencing data and to elucidate the association between copy number alterations in specific genes and the development of cancer in different colon segments. We report the successful detection of somatic changes in gene copy number in 37 colon cancer patients by analysis of sequencing data through Amplicon CNA Algorithm. Overall, we have found a total of 748 significant copy number alterations in 230 significant genes, of which 143 showed CN losses and 87 showed CN gains. Validation of results was performed on 20 representative genes by quantitative qPCR and/or immunostaining. By this analysis, we have identified 4 genes that were subjected to copy number alterations in tumors arising in all colon segments (defined "common genes") and the presence of copy number alterations in 14 genes that were significantly associated to one specific site (defined "site-associated genes"). Finally, copy number alterations in ASXL1, TSC1 and IL7R turned out to be clinically relevant since the loss of TSC1 and IL7R was associated with advanced stages and/or reduced survival whereas copy number gain of ASXL1 was associated with good prognosis.

IL-13 receptors as possible therapeutic targets in diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) is a universally fatal childhood cancer of the brain. Despite the introduction of conventional chemotherapy and radiotherapy, improvements in survival have been marginal and long-term survivorship is uncommon. Thus, new targets for therapeutics are critically needed. Early phase clinical trials exploring molecularly-targeted therapies against the epidermal growth factor receptor (EGFR) and novel immunotherapies targeting interleukin receptor-13α2 (IL-13Rα2) have demonstrated activity in this disease. To identify additional therapeutic markers for cell surface receptors, we performed exome sequencing (16 new samples, 22 previously published samples, total 38 with 26 matched normal DNA samples), RNA deep sequencing (17 new samples, 11 previously published samples, total 28 with 18 matched normal RNA samples), and immunohistochemistry (17 DIPG tissue samples) to examine the expression of the interleukin-4 (IL-4) signaling axis components (IL-4, interleukin 13 (IL-13), and their respective receptors IL-4Rα, IL-13Rα1, and IL-13Rα2). In addition, we correlated cytokine and receptor expression with expression of the oncogenes EGFR and c-MET. In DIPG tissues, transcript-level analysis found significant expression of IL-4, IL-13, and IL-13Rα1/2, with strong differential expression of IL-13Rα1/2 in tumor versus normal brain. At the protein level, immunohistochemical studies revealed high content of IL-4 and IL-13Rα1/2 but notably low expression of IL-13. Additionally, a strong positive correlation was observed between c-Met and IL-4Rα. The genomic and transcriptional landscape across all samples was also summarized. These data create a foundation for the design of potential new immunotherapies targeting IL-13 cell surface receptors in DIPG.

Genetic Mechanisms of Immune Evasion in Colorectal Cancer.

To understand the genetic drivers of immune recognition and evasion in colorectal cancer, we analyzed 1,211 colorectal cancer primary tumor samples, including 179 classified as microsatellite instability-high (MSI-high). This set includes The Cancer Genome Atlas colorectal cancer cohort of 592 samples, completed and analyzed here. MSI-high, a hypermutated, immunogenic subtype of colorectal cancer, had a high rate of significantly mutated genes in important immune-modulating pathways and in the antigen presentation machinery, including biallelic losses of B2M and HLA genes due to copy-number alterations and copy-neutral loss of heterozygosity. WNT/ß-catenin signaling genes were significantly mutated in all colorectal cancer subtypes, and activated WNT/ß-catenin signaling was correlated with the absence of T-cell infiltration. This large-scale genomic analysis of colorectal cancer demonstrates that MSI-high cases frequently undergo an immunoediting process that provides them with genetic events allowing immune escape despite high mutational load and frequent lymphocytic infiltration and, furthermore, that colorectal cancer tumors have genetic and methylation events associated with activated WNT signaling and T-cell exclusion.Significance: This multi-omic analysis of 1,211 colorectal cancer primary tumors reveals that it should be possible to better monitor resistance in the 15% of cases that respond to immune blockade therapy and also to use WNT signaling inhibitors to reverse immune exclusion in the 85% of cases that currently do not. Cancer Discov; 8(6); 730-49. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 663.

Integrated molecular analysis of Tamoxifen-resistant invasive lobular breast cancer cells identifies MAPK and GRM/mGluR signaling as therapeutic vulnerabilities.

Invasive lobular breast cancer (ILC) is an understudied malignancy with distinct clinical, pathological, and molecular features that distinguish it from the more common invasive ductal carcinoma (IDC). Mounting evidence suggests that estrogen receptor-alpha positive (ER+) ILC has a poor response to Tamoxifen (TAM), but the mechanistic drivers of this are undefined. In the current work, we comprehensively characterize the SUM44/LCCTam ILC cell model system through integrated analysis of gene expression, copy number, and mutation, with the goal of identifying actionable alterations relevant to clinical ILC that can be co-targeted along with ER to improve treatment outcomes. We show that TAM has several distinct effects on the transcriptome of LCCTam cells, that this resistant cell model has acquired copy number alterations and mutations that impinge on MAPK and metabotropic glutamate receptor (GRM/mGluR) signaling networks, and that pharmacological inhibition of either improves or restores the growth-inhibitory actions of endocrine therapy.

The Short-term Effect of Subtenon Sponge Application Versus Subtenon Irrigation of Mitomycin-C on the Outcomes of Trabeculectomy With Ex-PRESS Glaucoma Filtration Device: A Randomized Trial.

PURPOSE: Traditionally, during trabeculectomy, Mitomycin-C (MMC) is applied to the tissues using surgical sponges. However, alternate modes of application exist. This study assessed the success rates, complication rates, final intraocular pressure (IOP), and bleb characteristics between patients receiving subtenon MMC application by sponge versus irrigation. PATIENTS AND METHODS: A total of 100 patients with glaucoma were enrolled and each was randomized to 1 of the 2 treatment groups. Patients underwent trabeculectomy with Ex-PRESS shunt and MMC placement and were followed for 6 months. Complication rates assessed included bleb failure, bleb leaks, bleb encapsulation, and hypotony, amongst others. Additional factors evaluated included bleb morphology, glaucoma drop usage, Fluorouracil (5-FU) application, bleb revision, and subsequent glaucoma surgery. RESULTS: The irrigation method provided greater IOP lowering effects (P=0.03); correspondingly the irrigation group had higher rates of hypotony (P=0.03) but with no significant consequences. Patients who had trabeculectomy/Ex-PRESS alone had greater IOP reduction than those who had concurrent cataract surgery (P<0.001). The sponge group had higher rates of 5-FU use (P=0.007) and higher reoperation rates (P=0.02) when compared with the irrigation group. Success was defined as achieving 4 mm Hg≤IOP≤15 mm Hg without any anatomical bleb failure or subsequent glaucoma surgery. The overall success rate was 87%. CONCLUSIONS: Application of subtenon MMC by irrigation seems to provide improved short-term outcomes compared with application with sponges. With a similar safety profile, the irrigation method provides better IOP control, and decreases the need for further clinical/surgical intervention in the short-term after trabeculectomy. Longer-term studies will be useful in analyzing if these differences persist with time.

Rapid, ultra low coverage copy number profiling of cell-free DNA as a precision oncology screening strategy.

Current cell-free DNA (cfDNA) next generation sequencing (NGS) precision oncology workflows are typically limited to targeted and/or disease-specific applications. In advanced cancer, disease burden and cfDNA tumor content are often elevated, yielding unique precision oncology opportunities. We sought to demonstrate the utility of a pan-cancer, rapid, inexpensive, whole genome NGS of cfDNA approach (PRINCe) as a precision oncology screening strategy via ultra-low coverage (~0.01x) tumor content determination through genome-wide copy number alteration (CNA) profiling. We applied PRINCe to a retrospective cohort of 124 cfDNA samples from 100 patients with advanced cancers, including 76 men with metastatic castration-resistant prostate cancer (mCRPC), enabling cfDNA tumor content approximation and actionable focal CNA detection, while facilitating concordance analyses between cfDNA and tissue-based NGS profiles and assessment of cfDNA alteration associations with mCRPC treatment outcomes. Therapeutically relevant focal CNAs were present in 42 (34%) cfDNA samples, including 36 of 93 (39%) mCRPC patient samples harboring AR amplification. PRINCe identified pre-treatment cfDNA CNA profiles facilitating disease monitoring. Combining PRINCe with routine targeted NGS of cfDNA enabled mutation and CNA assessment with coverages tuned to cfDNA tumor content. In mCRPC, genome-wide PRINCe cfDNA and matched tissue CNA profiles showed high concordance (median Pearson correlation = 0.87), and PRINCe detectable AR amplifications predicted reduced time on therapy, independent of therapy type (Kaplan-Meier log-rank test, chi-square = 24.9, p < 0.0001). Our screening approach enables robust, broadly applicable cfDNA-based precision oncology for patients with advanced cancer through scalable identification of therapeutically relevant CNAs and pre-/post-treatment genomic profiles, enabling cfDNA- or tissue-based precision oncology workflow optimization.