Alternative Tissue Fixation Protocols Dramatically Reduce the Impact of DNA Artifacts, Unraveling the Interpretation of Clinical Comprehensive Genomic Profiling.

Formalin-fixed paraffin-embedded (FFPE) samples represent the cornerstone of tissue-based analysis in precision medicine. Targeted next-generation sequencing panels are routinely used to analyze a limited number of genes to guide treatment decision-making for advanced-stage patients. The number and complexity of genetic alterations to be investigated are rapidly growing; in several instances, a comprehensive genomic profiling analysis is needed. The poor quality of genetic material extracted from FFPE samples may impact the feasibility/reliability of sequencing data. We sampled 9 colorectal cancers to allow 4 parallel fixations: (1) neutral buffered formalin (NBF), (2) acid-deprived formalin fixation (ADF), (3) precooled ADF (coldADF), and (4) glyoxal acid free (GAF). DNA extraction, fragmentation analysis, and sequencing by 2 large next-generation sequencing panels (OCAv3 and TSO500) followed. We comprehensively analyzed library and sequencing quality controls and the quality of sequencing results. Libraries from coldADF samples showed significantly longer reads than the others with both panels. ADF-derived and coldADF-derived libraries showed the lowest level of noise and the highest levels of uniformity with the OCAv3 panel, followed by GAF and NBF samples. The data uniformity was confirmed by the TSO500 results, which also highlighted the best performance in terms of the total region sequenced for the ADF and coldADF samples. NBF samples had a significantly smaller region sequenced and displayed a significantly lower number of evaluable microsatellite loci and a significant increase in single-nucleotide variations compared with other protocols. Mutational signature 1 (aging and FFPE artifact related) showed the highest (37%) and lowest (17%) values in the NBF and coldADF samples, respectively. Most of the identified genetic alterations were shared by all samples in each lesion. Five genes showed a different mutational status across samples and/or panels: 4 discordant results involved NBF samples. In conclusion, acid-deprived fixatives (GAF and ADF) guarantee the highest DNA preservation/sequencing performance, thus allowing more complex molecular profiling of tissue samples.

Case report: Surgical management of vitreous obstruction following PreserFlo ® MicroShunt implantation.

Purpose: To describe a clinical case of lumen obstruction a few days after implantation of the PreserFlo® Microshunt which has been resolved by anterior vitrectomy. Observation: A 76-year-old patient with advanced and progressing primary open-angle glaucoma (POAG) presented ten days after PreserFlo® Microshunt implantation in his left eye with an intraocular pressure (IOP) of 24 mmHg because of vitreous obstruction. Anterior vitrectomy with 25 Gauge vitrector was performed to remove the vitreous using a bimanual technique with two corneal accesses. The surgery was successful in lowering uncontrolled IOP without device repositioning. A free lumen and a IOP in the low range of tens was observed during follow-up. Conclusions and importance: PreserFlo ® MicroShunt obstruction by vitreous in pseudophakic patient is a possible complication. Anterior vitrectomy without the need of tube repositioning was successful in lowering uncontrolled IOP.

Apoptosis in mesenchymal stromal cells activates an immunosuppressive secretome predicting clinical response in Crohn's disease.

In vivo apoptosis of human mesenchymal stromal cells (MSCs) plays a critical role in delivering immunomodulation. Yet, caspase activity not only mediates the dying process but also death-independent functions that may shape the immunogenicity of apoptotic cells. Therefore, a better characterization of the immunological profile of apoptotic MSCs (ApoMSCs) could shed light on their mechanistic action and therapeutic applications. We analyzed the transcriptomes of MSCs undergoing apoptosis and identified several immunomodulatory factors and chemokines dependent on caspase activation following Fas stimulation. The ApoMSC secretome inhibited human T cell proliferation and activation, and chemoattracted monocytes in vitro. Both immunomodulatory activities were dependent on the cyclooxygenase2 (COX2)/prostaglandin E2 (PGE2) axis. To assess the clinical relevance of ApoMSC signature, we used the peripheral blood mononuclear cells (PBMCs) from a cohort of fistulizing Crohn's disease (CD) patients who had undergone MSC treatment (ADMIRE-CD). Compared with healthy donors, MSCs exposed to patients' PBMCs underwent apoptosis and released PGE2 in a caspase-dependent manner. Both PGE2 and apoptosis were significantly associated with clinical responses to MSCs. Our findings identify a new mechanism whereby caspase activation delivers ApoMSC immunosuppression. Remarkably, such molecular signatures could implicate translational tools for predicting patients' clinical responses to MSC therapy in CD.

Functional and anatomic changes between early postoperative recovery and long-term follow-up after combined epiretinal and internal limiting membrane peeling.

OBJECTIVE: To evaluate short- and long-term changes in best-corrected visual acuity (BCVA) and retinal layer thicknesses after combined epiretinal membrane (ERM) and internal limiting membrane (ILM) peeling for macular holes and symptomatic ERMs. DESIGN: Retrospective observational case series. PARTICIPANTS: Patients with ERMs or with macular holes and ERMs treated with combined ERM and ILM peeling. METHODS: Study eyes (n = 36) and healthy fellow eyes (n = 17) were evaluated using the automated segmentation of retinal layers performed by SPECTRALIS software that automatically calculated the average central retinal thickness and the average thickness in each of the individual retinal layers. The analysis was performed at 6-18 months after surgery and after 60 months. MAIN OUTCOME MEASURES: Changes in BCVA and retinal layer thicknesses determined by automated segmentation at the first and last follow-up visits. RESULTS: BCVA improved from a baseline 0.48 ± 0.25 logMAR (20/60 Snellen) to 0.18 ± 0.18 logMAR (20/30 Snellen) at the short-term postoperative examination (p < 0.0001). Between first and last follow-up visit, 5 eyes (14%) were classified as better, 28 (78%) as stable, and 3 (8%) as worse. BCVA of the control fellow eyes remained stable during the follow-up. The thicknesses of retinal layers decreased significantly (p < 0.009). At the last follow-up, the ganglion cell layer was thinner and the inner nuclear layer was thicker in the operated eyes compared with the healthy fellow eyes. CONCLUSION: Combined ERM and ILM peeling may improve BCVA in some patients. However, over a long follow-up period, it can be associated with progressive ganglion cell layer thinning that could affect BCVA stability.

Full-Thickness Macular Hole: Are Supra-RPE Granular Deposits Remnants of Photoreceptors Outer Segments? Clinical Implications.

PURPOSE: To describe the presence of specific morphological characteristics of idiopathic, full-thickness macular hole (MH) potentially influencing postoperative best corrected visual acuity (BCVA) and surgical outcomes. DESIGN: Retrospective, multicenter and interventional case series. METHODS: Clinical charts and multimodal imaging pictures of 149 eyes of 143 consecutive patients diagnosed with MH, treated surgically and with a minimum follow-up of 12 months, were reviewed. RESULTS: Supra-retinal pigment epithelium (RPE) granular deposits were diagnosed in 121 of 149 eyes (81.2%). A smooth morphology was identified in 58 of 149 eyes (38.9%), whereas a bumpy border was present 91 of 149 eyes (61.1%). Photoreceptor disruption was mainly located close to the MH aperture. In 8% of the included cases, preoperative anatomical progression from smooth to bumpy morphology was noted. The presence of supra-RPE granular deposits was a significant predictor of lower postoperative BCVA only in univariate analysis (P < .001). The presence of a bumpy border was significantly correlated with lower postoperative BCVA in both univariate and multivariate analysis (P < .001). BCVA gain was significantly lower in MH with bumpy borders (P < .001). A bumpy border was also significantly associated with poor postoperative anatomical restoration (P < .001). CONCLUSIONS: Supra RPE-granular deposits and a bumpy morphology may be indicators of photoreceptor disruption in MH. A bumpy morphology may suggest deeper and potentially irreversible photoreceptor damage, and may negatively influence both functional and anatomical recovery.

Novel Lymphocyte-Independent Antitumor Activity by PD-1 Blocking Antibody against PD-1+ Chemoresistant Lung Cancer Cells.

PURPOSE: Antibodies against the lymphocyte PD-1 (aPD-1) receptor are cornerstone agents for advanced non-small cell lung cancer (NSCLC), based on their ability to restore the exhausted antitumor immune response. Our study reports a novel, lymphocyte-independent, therapeutic activity of aPD-1 against NSCLC, blocking the tumor-intrinsic PD-1 receptors on chemoresistant cells. EXPERIMENTAL DESIGN: PD-1 in NSCLC cells was explored in vitro at baseline, including stem-like pneumospheres, and following treatment with cisplatin both at transcriptional and protein levels. PD-1 signaling and RNA sequencing were assessed. The lymphocyte-independent antitumor activity of aPD-1 was explored in vitro, by PD-1 blockade and stimulation with soluble ligand (PD-L1s), and in vivo within NSCLC xenograft models. RESULTS: We showed the existence of PD-1+ NSCLC cell subsets in cell lines and large in silico datasets (Cancer Cell Line Encyclopedia and The Cancer Genome Atlas). Cisplatin significantly increased PD-1 expression on chemo-surviving NSCLC cells (2.5-fold P = 0.0014), while the sequential treatment with anti-PD-1 Ab impaired their recovery after chemotherapy. PD-1 was found to be associated with tumor stemness features. PD-1 expression was enhanced in NSCLC stem-like pneumospheres (P < 0.0001), significantly promoted by stimulation with soluble PD-L1 (+27% ± 4, P < 0.0001) and inhibited by PD-1 blockade (-30% ± 3, P < 0.0001). The intravenous monotherapy with anti-PD-1 significantly inhibited tumor growth of NSCLC xenografts in immunodeficient mice, without the contribution of the immune system, and delayed the occurrence of chemoresistance when combined with cisplatin. CONCLUSIONS: We report first evidence of a novel lymphocyte-independent activity of anti-PD-1 antibodies in NSCLC, capable of inhibiting chemo-surviving NSCLC cells and exploitable to contrast disease relapses following chemotherapy. See related commentary by Augustin et al., p. 505.

FOXA1 regulates alternative splicing in prostate cancer.

Dysregulation of alternative splicing in prostate cancer is linked to transcriptional programs activated by AR, ERG, FOXA1, and MYC. Here, we show that FOXA1 functions as the primary orchestrator of alternative splicing dysregulation across 500 primary and metastatic prostate cancer transcriptomes. We demonstrate that FOXA1 binds to the regulatory regions of splicing-related genes, including HNRNPK and SRSF1. By controlling trans-acting factor expression, FOXA1 exploits an "exon definition" mechanism calibrating alternative splicing toward dominant isoform production. This regulation especially impacts splicing factors themselves and leads to a reduction of nonsense-mediated decay (NMD)-targeted isoforms. Inclusion of the NMD-determinant FLNA exon 30 by FOXA1-controlled oncogene SRSF1 promotes cell growth in vitro and predicts disease recurrence. Overall, we report a role for FOXA1 in rewiring the alternative splicing landscape in prostate cancer through a cascade of events from chromatin access, to splicing factor regulation, and, finally, to alternative splicing of exons influencing patient survival.

Integrative genomic and transcriptomic analyses illuminate the ontology of HER2-low breast carcinomas.

BACKGROUND: The "HER2-low" nomenclature identifies breast carcinomas (BCs) displaying a HER2 score of 1+/2+ in immunohistochemistry and lacking ERBB2 amplification. Whether HER2-low BCs (HLBCs) constitute a distinct entity is debated. METHODS: We performed DNA and RNA high-throughput analysis on 99 HLBC samples (n = 34 cases with HER2 score 1+/HLBC-1, n = 15 cases with HER2 score 2+ and ERBB2 not amplified/HLBC-2N, and n = 50 cases with score 2+ and ERBB2 copy number in the equivocal range/HLBC-2E). We compared the mutation rates with data from 1317 samples in the Memorial Sloan-Kettering Cancer Center (MSKCC) BC cohort and gene expression data with those from an internal cohort of HER2-negative and HER2-positive BCs. RESULTS: The most represented mutations affected PIK3CA (31/99, 31%), GATA3 (18/99, 18%), TP53 (17/99, 17%), and ERBB2 (8/99, 8%, private to HLBC-2E). Tumor mutational burden was significantly higher in HLBC-1 compared to HLBC-2E/N (P = 0.04). Comparison of mutation spectra revealed that HLBCs were different from both HER2-negative and HER2-positive BCs, with HLBC-1 resembling more HER2-negative tumors and HLBC-2 mutationally related to HER2-addicted tumors. Potentially actionable alterations (annotated by using OncoKB/ESCAT classes) affected 52 patients. Intra-group gene expression revealed overlapping features between HLBC-1 and control HER2-negative BCs, whereas the HLBC-2E tumors showed the highest diversity overall. The RNA-based class discovery analysis unveiled four subsets of tumors with (i) lymphocyte activation, (ii) unique enrichment in HER2-related features, (iii) stromal remodeling alterations, and (iv) actionability of PIK3CA mutations (LAURA classification). CONCLUSIONS: HLBCs harbor distinct genomic features when compared with HER2-positive and HER2-negative BCs; however, differences across IHC classes were also unveiled thus dissecting the full picture of heterogeneity across HER2-low disease. The HLBC-2E category harbors most distinctive features, whereas HLBC-1 seems superimposable to HER2-negative disease. Further studies are needed to ascertain whether the four genomic-driver classes of the LAURA classification hold prognostic and/or predictive implications.

MULTIMODAL IMAGING CHARACTERISTICS AND FUNCTIONAL CORRELATES IN RIP HEALING.

PURPOSE: To report the imaging and functional features of the repair tissue following retinal pigment epithelium (RPE) tears. METHODS: This cross-sectional observational study included patients with RPE tears secondary to neovascular age-related macular degeneration and at least 12 months of follow-up. The following variables were analyzed: best-corrected visual acuity; retinal sensitivity using microperimetry; outer retinal layers status and RPE resurfacing on optical coherence tomography; fibrosis; autofluorescence signal recovery using blue-light and near-infrared autofluorescence. RESULTS: Overall, 48 eyes were included (age: 82 ± 5 years) and 34 of them showed signs of healing. Retinal pigment epithelium resurfacing was noticed in 22 cases, whereas fibrosis appeared in 21 eyes. Autofluorescence improved in 17 cases using blue-light infrared autofluorescence and 7 eyes on near-infrared autofluorescence. Outer retinal layers were more frequently preserved when RPE resurfacing and autofluorescence improvement occurred ( P < 0.05). Although best-corrected visual acuity was higher for smaller RPE tears ( P = 0.01), retinal sensitivity of the healing tissue was positively affected by autofluorescence improvement ( P < 0.001) and by absence of fibrosis ( P = 0.03). CONCLUSION: Autofluorescence signal recovery after rip occurrence possibly reflects the underlying status of the RPE and is associated with better functional outcomes. Our findings highlight the importance of blue-light infrared autofluorescence and especially near-infrared autofluorescence assessment in the setting of rip healing.

Six Drivers to Face the XXI Century Challenges and Build the New Healthcare System: "La Salute in Movimento" Manifesto.

The aging of the population, the burden of chronic diseases, possible new pandemics are among the challenges for healthcare in the XXI century. To face them, technological innovations and the national recovery and resilience plan within the European Union can represent opportunities to implement changes and renovate the current healthcare system in Italy, in an effort to guarantee equal access to health services. Considering such scenario, a panel of Italian experts gathered in a multidisciplinary Think Tank to discuss possible design of concepts at the basis of a new healthcare system. These ideas were summarized in a manifesto with six drivers for change: vision, governance, competence, intelligence, humanity and relationship. Each driver was linked to an action to actively move toward a new healthcare system based on trust between science, citizens and institutions.

Collision of germline POLE and PMS2 variants in a young patient treated with immune checkpoint inhibitors.

The onset of multiple and metachronous tumors in young patients induces to suspect the presence of genetic variants in genes associated with tumorigenesis. We describe here the unusual case of a 16-year-old patient who developed a synchronous bifocal colorectal adenocarcinoma with distant metastases. We provide high throughput molecular characterization with whole-exome sequencing (WES) and DNA targeted sequencing of different tumoral lesions and normal tissue samples that led to unveil a germline POLE mutation (p.Ser297Cys) coexisting with the PMS2 c.2174 + 1 G > A splicing mutation. This clinical scenario defines a "POLE-LYNCH" collision syndrome, which explains the ultra-mutator phenotype observed in the tumor lesions, and the presence of MMR deficiency-associated unusual signatures. The patient was successfully treated with immune checkpoint inhibitors but subsequently developed a high-grade urothelial carcinoma cured by surgery. We complement this analysis with a transcriptomic characterization of tumoral lesions with a panel targeting 770 genes related to the tumor microenvironment and immune evasion thus getting insight on cancer progression and response to immunotherapy.

Distribution and Progression of Inflammatory Chorioretinal Lesions Related to Multifocal Choroiditis and Their Correlations with Clinical Outcomes at 24 Months.

PURPOSE: To analyze distribution and progression of multifocal choroiditis (MFC) inflammatory lesions and their correlations with clinical outcomes at 24 months. METHODS: Distribution and progression of inflammatory lesions were evaluated in eyes with MFC using a semi-automatic approach based on fundus autofluorescence. Twenty-four-months clinical outcomes were correlated with baseline features. RESULTS: Twenty-five eyes from 20 patients were enrolled. Visual acuity (VA) significantly improved from baseline to 24 months. Chorioretinal lesions spared the fovea in most eyes. The area of inflammatory lesions at 24 months significantly increased. Final number and area of lesions were significantly influenced by baseline features. Inflammatory lesions enlarged over time regardless of MFC recurrences. New lesion and MFC relapses did not affect final outcomes. CONCLUSIONS: Final VA correlated with baseline VA. Scars resulting from MFC lesions enlarged overtime even when the disease was under control. New lesions and MFC relapses did not affect final outcomes.

Molecular Characterization of Prostate Cancers in the Precision Medicine Era.

Prostate cancer (PCa) therapy has been recently revolutionized by the approval of new therapeutic agents in the metastatic setting. However, the optimal therapeutic strategy in such patients should be individualized in the light of prognostic and predictive molecular factors, which have been recently studied: androgen receptor (AR) alterations, PTEN-PI3K-AKT pathway deregulation, homologous recombination deficiency (HRD), mismatch repair deficiency (MMRd), and tumor microenvironment (TME) modifications. In this review, we highlighted the clinical impact of prognostic and predictive molecular factors in PCa patients' outcomes, identifying biologically distinct subtypes. We further analyzed the relevant methods to detect these factors, both on tissue, i.e., immunohistochemistry (IHC) and molecular tests, and blood, i.e., analysis of circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA). Moreover, we discussed the main pros and cons of such techniques, depicting their present and future roles in PCa management, throughout the precision medicine era.

Intravitreal injection associated rhegmatogenous retinal detachment: outcomes of a European analysis.

PURPOSE: As the number of intravitreal injections (IVI) increases annually, this study aimed to assess the anatomical and functional outcomes following rhegmatogenous retinal detachment (RRD) surgery for IVI-associated RRD (IVARD). METHODS: All non-vitrectomized eyes developing IVARD since 2007 in two European vitreoretinal centers (Department of Ophthalmology, LMU Munich, Germany, and Eye Clinic Luigi Sacco, University of Milan, Milan, Italy) were included. Main outcomes were primary and secondary retinal attachment rate after surgery, rate of proliferative vitreoretinopathy (PVR), and final functional result. Ten years of incidence rates per injection were calculated for one center. RESULTS: Fifty-two eyes of 52 patients comprised the study. Primary anatomic success rate was 83% (n = 43) and secondary 96% (n = 50). PVR was observed in all uveitic eyes (n = 3), in eyes with postoperative cystoid macular edema (n = 2), and in 8 of 9 eyes that received the dexamethasone implant (DEX). Age, number of prior injections, duration of symptoms, or time between last IVI and RRD did not show any statistically significant differences with regard to presence of PVR or not. Mean BCVA improved in 28 cases, remained stable in 16 cases, and worsened in 8 cases. The RRD incidence rate was statistically significant higher for DEX and ocriplasmin compared with that for anti-VEGF agents. CONCLUSION: The anatomical result after one surgical intervention seems acceptable, but the final visual outcome remains rather poor, because of the underlying macular disease. In our population, injection with DEX is associated with higher IVARD rate, presence and development of PVR, and recurrent RRD in comparison with anti-VEGF agents.

Outer Retinal Layer Thickening Predicts the Onset of Exudative Neovascular Age-Related Macular Degeneration.

PURPOSE: To assess changes in outer retinal layer (ORL) thickness before the development of exudative macular neovascularization (MNV) in eyes with age-related macular degeneration. DESIGN: Retrospective observational case series. METHODS: Eyes with age-related macular degeneration that eventually developed exudative MNV followed with sequential optical coherence tomography for ≥2 years before the exudation occurred were enrolled. The ORL thickness was automatically calculated by the optical coherence tomography software for each sector of the early treatment diabetic retinopathy study map at each follow-up visit. The ORL thickness change from baseline to the day when the exudative MNV developed was compared between sectors that eventually developed exudative MNV and those that did not. RESULTS: Forty-seven eyes (47 patients) were included. At baseline (24 ± 3 months before exudative MNV), mean (standard deviation) ORL thickness of sectors that eventually developed exudative MNV was similar to that of sectors that did not (85.2 [8.2] µm vs 86.8 [5.7] µm, P = .08). ORL thickness significantly increased in sectors that developed exudative MNV compared with those that did not (+5.8 [10.4] µm vs -2.8 [3.6] µm, P < .01). The regression model based on these data predicted an increase in ORL thickness from baseline of +4.2% 55 days and +11.1% 30 days before exudative MNV was detected. The ORL thickness of areas that did not develop exudative MNV did not change. CONCLUSION: Thickening of the ORL begins in the area where exudative MNV will develop long before the exudation, accelerating significantly in the last 2 months. The occurrence of exudative MNV could be predicted by 2 months using this simple analysis.

Artificial Intelligence in Bulk and Single-Cell RNA-Sequencing Data to Foster Precision Oncology.

Artificial intelligence, or the discipline of developing computational algorithms able to perform tasks that requires human intelligence, offers the opportunity to improve our idea and delivery of precision medicine. Here, we provide an overview of artificial intelligence approaches for the analysis of large-scale RNA-sequencing datasets in cancer. We present the major solutions to disentangle inter- and intra-tumor heterogeneity of transcriptome profiles for an effective improvement of patient management. We outline the contributions of learning algorithms to the needs of cancer genomics, from identifying rare cancer subtypes to personalizing therapeutic treatments.

Optical coherence tomography features of the repair tissue following RPE tear and their correlation with visual outcomes.

To assess the optical coherence tomography (OCT) features of the repair tissue after retinal pigment epithelial (RPE) tear in neovascular age-related macular degeneration. Retrospective, observational study. Medical and imaging records of patients that developed tears after starting anti-VEGF treatment and with at least 12 months of follow-up were reviewed. OCT reflectivity of the RPE-subretinal hyperreflective tissue (SHT) complex was measured at 6, 12 and 18 months (when available). Reflectivity of the adjacent unaffected RPE-Bruch's membrane was taken as internal reference. Other variables: grade and rip occurrence (early/late); number of intravitreal injections; type of macular neovascularization; sub-macular hemorrhage (SMH) at onset. Forty-nine eyes (age: 76.1 ± 7.0 years; VA: 0.54 ± 0.27 LogMAR) were included. Thirty-eight eyes had OCT signs of healing during the follow-up, with 21 showing SMH at baseline. Final VA positively correlated with the number of injections and negatively correlated with the RPE-SHT reflectivity and the presence of SMH (p < 0.001). Reflectivity of the RPE-SHT complex was positively associated with time and SMH at baseline (p < 0.05). In our study, most eyes showed signs of tissue repair after RPE tear. The reflectivity of repair tissue, the SMH presence and the number of anti-VEGF injections appeared to be major predictors of visual outcomes.

Precision Medicine in Osteosarcoma: MATCH Trial and Beyond.

Osteosarcoma (OS) is a rare bone malignant tumour with a poor prognosis in the case of recurrence. So far, there is no agreement on the best systemic therapy for relapsed OS. The availability of next generation sequencing techniques has recently revolutionized clinical research. The sequencing of the tumour and its matched normal counterpart has the potential to reveal a wide landscape of genetic alterations with significant implications for clinical practice. The knowledge that the genomic profile of a patient's tumour can be precisely mapped and matched to a targeted therapy in real time has improved the development of precision medicine trials (PMTs). PMTs aiming at determining the effectiveness of targeted therapies could be advantageous for patients with a tumour refractory to standard therapies. Development of PMTs for relapsed OS is largely encouraging and is in its initial phase. Assessing OS features, such as its rarity, its age distribution, the technical issues related to the bone tissue origin, and its complex genomic landscape, represents a real challenge for PMTs development. In this light, a multidisciplinary approach is required to fully exploit the potential of precision medicine for OS patients.