Risk and Prognostics of Second Primary Cancer After Prostate Radiation Therapy.

INTRODUCTION: As overall survival in prostate cancer increases due to advances in early detection and management, there is a growing need to understand the long-term morbidity associated with treatment, including secondary tumors. The significance of developing radiation-associated secondary cancers in an elderly population remains unknown. METHODS: Patients diagnosed with prostate cancer between 1975 and 2016 in one of 9 Surveillance, Epidemiology, and End Results registries were included in this study. Risk of second primary pelvic malignancies (SPPMs) were assessed with death as a competing risk using the Fine-Gray model. Time-varying Cox proportional hazard models were employed to analyze risk to overall mortality based on secondary tumor status. RESULTS: A total of 569,167 primary prostate cancers were included in analysis with an average follow-up of 89 months. Among all prostate cancer patients, 4956 SPPMs were identified. After controlling for differences in age, year of diagnosis, and surgery at time of prostate cancer treatment, radiation receipt was associated with a significantly higher incidence of SPPMs (1.1% vs 1.8% at 25 years). Among those who received radiation during initial prostate cancer treatment (n = 195,415), developing an SPPM is significantly associated with worse survival (adjusted hazard ratio = 1.76), especially among younger patients (under age 63, adjusted hazard ratio = 2.36). CONCLUSIONS: While developing a secondary malignancy carries a detrimental effect on overall survival, the absolute risk of developing such tumors is exceedingly low regardless of radiation treatment.

Cell-Cell Interactions Enhance Cartilage Zonal Development in 3D Gradient Hydrogels.

Cartilage tissue is characterized by zonal organization with gradual transitions of biochemical and mechanical cues from superficial to deep zones. We previously reported that 3D gradient hydrogels made of polyethylene glycol and chondroitin sulfate can induce zonal-specific responses of chondrocytes, resulting in zonal cartilage formation that mimics native tissues. While the role of cell-matrix interactions has been studied extensively, how cell-cell interactions across different zones influence cartilage zonal development remains unknown. The goal of this study is to harness gradient hydrogels as a tool to elucidate the role of cell-cell interactions in driving cartilage zonal development. When encapsulated in intact gradient hydrogels, chondrocytes exhibited strong zonal-specific responses that mimic native cartilage zonal organization. However, the separate culture of each zone of gradient hydrogels resulted in a significant decrease in cell proliferation and cartilage matrix deposition across all zones, while the trend of zonal dependence remains. Unexpectedly, mixing the coculture of all five zones of hydrogels in the same culture well largely abolished the zonal differences, with all zones behaving similarly to the softest zone. These results suggest that paracrine signal exchange among cells in different zones is essential in driving cartilage zonal development, and a spatial organization of zones is required for proper tissue zonal development. Intact, separate, or coculture groups resulted in distinct gene expression patterns in mechanosensing and cartilage-specific markers, suggesting that cell-cell interactions can also modulate mechanosensing. We further showed that 7 days of priming in intact gradient culture was sufficient to instruct the cells to complete the zonal development, and the separate or mixed coculture after 7 days of intact culture had minimal effects on cartilage formation. This study highlights the important role of cell-cell interactions in driving cartilage zonal development and validates gradient hydrogels as a useful tool to elucidate the role of cell-matrix and cell-cell interactions in driving zonal development during tissue morphogenesis and regeneration.

Association of hyperglycemia and molecular subclass on survival in IDH-wildtype glioblastoma.

Background: Hyperglycemia has been associated with worse survival in glioblastoma. Attempts to lower glucose yielded mixed responses which could be due to molecularly distinct GBM subclasses. Methods: Clinical, laboratory, and molecular data on 89 IDH-wt GBMs profiled by clinical next-generation sequencing and treated with Stupp protocol were reviewed. IDH-wt GBMs were sub-classified into RTK I (Proneural), RTK II (Classical) and Mesenchymal subtypes using whole-genome DNA methylation. Average glucose was calculated by time-weighting glucose measurements between diagnosis and last follow-up. Results: Patients were stratified into three groups using average glucose: tertile one (<100 mg/dL), tertile two (100-115 mg/dL), and tertile three (>115 mg/dL). Comparison across glucose tertiles revealed no differences in performance status (KPS), dexamethasone dose, MGMT methylation, or methylation subclass. Overall survival (OS) was not affected by methylation subclass (P = .9) but decreased with higher glucose (P = .015). Higher glucose tertiles were associated with poorer OS among RTK I (P = .08) and mesenchymal tumors (P = .05), but not RTK II (P = .99). After controlling for age, KPS, dexamethasone, and MGMT status, glucose remained significantly associated with OS (aHR = 5.2, P = .02). Methylation clustering did not identify unique signatures associated with high or low glucose levels. Metabolomic analysis of 23 tumors showed minimal variation across metabolites without differences between molecular subclasses. Conclusion: Higher average glucose values were associated with poorer OS in RTKI and Mesenchymal IDH-wt GBM, but not RTKII. There were no discernible epigenetic or metabolomic differences between tumors in different glucose environments, suggesting a potential survival benefit to lowering systemic glucose in selected molecular subtypes.

Risk of Second Primary Neoplasms of the Central Nervous System.

Purpose: Second primary (SP) neoplasms of the central nervous system (CNS) among cancer survivors are devastating but poorly understood processes. The absolute risk, or true incidence, of developing an SP CNS tumor among cancer survivors is not well characterized. Methods and Materials: Patients diagnosed with cancer between 1975 and 2016 were queried using the Surveillance, Epidemiology, and End Results Program. Cumulative incidence rates (CIRs) were estimated using competitive risk analysis. The effects of covariates were assessed using multivariate competitive risk regression. Results: More than 3.8 million patient records were extracted. The absolute risk of developing an SP CNS neoplasm at 25 years was highest among long-term survivors of CNS cancers (CIR, 6.6%). Cranial radiation increased the incidence of SP tumors in pediatric patients (25-year CIR, 5.7% vs 1.1%; P = .0012) but not adults (25-year CIR, 5.8% vs 5.0%; P = .66). Multivariate cumulative risk regression identified radiation among pediatric patients as the greatest risk for an increased CIR (subdistribution hazard ratio, 2.50; 95% CI, 1.86-3.38; P = 2e-9). Meningiomas (42.9% vs 24.1%; P = 2e-7) and glioblastomas (20.5% vs 14.5%; P = .046) represented a greater proportion of the SP CNS tumors in those who received cranial irradiation. The median age of an SP diagnosis was decreased among those who received prior radiation (41 years [interquartile range (IQR), 30-65 years] vs 49 years [IQR, 30-65 years]; P = 7e-5). Conclusions: The risk of developing a second primary CNS neoplasm is elevated in patients with a prior CNS cancer independent of treatment history. The association between cranial radiation therapy and risk for subsequent cancers may be limited to the pediatric population.

Radiation therapy modalities for keloid management: A critical review.

OBJECTIVE: To provide a critical overview of current radiation modalities for keloid management. BACKGROUND: Despite multimodal therapies, keloids that can develop following injury are poorly controlled. A number of studies have suggested that post-excisional radiation therapy can reduce rates of keloid recurrence. However, existing reports span multiple radiation modalities, including brachytherapy, electron beam radiation, and photon radiation. In this review, we describe the advantages and disadvantages of commonly used radiation techniques and highlight their efficacy in keloid management. RESULTS: Electron beam radiation and high-dose rate brachytherapy are the two most commonly used modalities for adjuvant radiotherapeutic management of keloids and can provide effective keloid control but may be suited for different kinds of keloid growth patterns. Increasing biologically equivalent dose (BED) likely improves rates of control, though the clinical significance of this finding remains to be elucidated. Though radiation treatments are associated with acute and chronic side effects, the risk of developing a secondary malignancy is minimal. CONCLUSIONS: While radiation therapy is a promising modality for treating keloids, more studies of a prospective, randomized nature are needed to standardize its utility.

Comprehensive profiling of myxopapillary ependymomas identifies a distinct molecular subtype with relapsing disease.

BACKGROUND: Myxopapillary ependymoma (MPE) is a heterogeneous disease regarding histopathology and outcome. The underlying molecular biology is poorly understood, and markers that reliably predict the patients' clinical course are unknown. METHODS: We assembled a cohort of 185 tumors classified as MPE based on DNA methylation. Methylation patterns, copy number profiles, and MGMT promoter methylation were analyzed for all tumors, 106 tumors were evaluated histomorphologically, and RNA sequencing was performed for 37 cases. Based on methylation profiling, we defined two subtypes MPE-A and MPE-B, and explored associations with epidemiological, clinical, pathological, and molecular characteristics of these tumors. RESULTS: MPE-A occurred at a median age of 27 years and were enriched with tumors demonstrating papillary morphology and MGMT promoter hypermethylation. Half of these tumors could not be totally resected, and 85% relapsed within 10 years. Copy number alterations were more common in MPE-A. RNA sequencing revealed an enrichment for extracellular matrix and immune system-related signatures in MPE-A. MPE-B occurred at a median age of 45 years and included many tumors with a histological diagnosis of WHO grade II and tanycytic morphology. Patients within this subtype had a significantly better outcome with a relapse rate of 33% in 10 years (P = 3.4e-06). CONCLUSIONS: We unraveled the morphological and clinical heterogeneity of MPE by identifying two molecularly distinct subtypes. These subtypes significantly differed in progression-free survival and will likely need different protocols for surveillance and treatment.

Gradient Hydrogels for Optimizing Niche Cues to Enhance Cell-Based Cartilage Regeneration.

Hydrogels have been widely used for cell delivery to enhance cell-based therapies for cartilage tissue regeneration. To better support cartilage deposition, it is imperative to determine hydrogel formulation with physical and biochemical cues that are optimized for different cell populations. Previous attempts to identify optimized hydrogels rely mostly on testing hydrogel formulations with discrete properties, which are time-consuming and require large amounts of cells and materials. Gradient hydrogels encompass a range of continuous changes in niche properties, therefore offering a promising solution for screening a wide range of cell-niche interactions using less materials and time. However, harnessing gradient hydrogels to assess how matrix stiffness modulates cartilage formation by different cell types in vivo have never been investigated before. The goal of this study is to fabricate gradient hydrogels for screening the effects of varying hydrogel stiffness on cartilage formation by mesenchymal stem cells (MSCs) and chondrocytes, respectively, the two most commonly used cell populations for cartilage regeneration. We fabricated stiffness gradient hydrogels with tunable dimensions that support homogeneous cell encapsulation. Using gradient hydrogels with tunable stiffness range, we found MSCs and chondrocytes exhibit opposite trend in cartilage deposition in response to stiffness changes in vitro. Specifically, MSCs require soft hydrogels with Young's modulus less than 5 kPa to support faster cartilage deposition, as shown by type II collagen and sulfated glycosaminoglycan staining. In contrast, chondrocytes produce cartilage more effectively in stiffer matrix (>20 kPa). We chose optimal ranges of stiffness for each cell population for further testing in vivo using a mouse subcutaneous model. Our results further validated that soft matrix (Young's modulus <5 kPa) is better in supporting MSC-based cartilage deposition in three-dimensional, whereas stiffer matrix (Young's modulus >20 kPa) is more desirable for supporting chondrocyte-based cartilage deposition. Our results show the importance of optimizing niche cues in a cell-type-specific manner and validate the potential of using gradient hydrogels for optimizing niche cues to support cartilage regeneration in vitro and in vivo. Impact statement The present study validates the utility of gradient hydrogels for determining optimal hydrogel stiffness for supporting cartilage regeneration using both chondrocytes and stem cells. We demonstrate that such gradient hydrogels can be used for fast optimizing matrix stiffness for specific cell type to support optimal cartilage regeneration. To our knowledge, this is the first demonstration of applying gradient hydrogels for assessing optimal niche cues that support tissue regeneration in vivo and may be used for assessing optimal niche cues for different cell types to regeneration of different tissues.

Full automation of spinal stereotactic radiosurgery and stereotactic body radiation therapy treatment planning using Varian Eclipse scripting.

The purpose of this feasibility study is to develop a fully automated procedure capable of generating treatment plans with multiple fractionation schemes to improve speed, robustness, and standardization of plan quality. A fully automated script was implemented for spinal stereotactic radiosurgery/stereotactic body radiation therapy (SRS/SBRT) plan generation using Eclipse v15.6 API. The script interface allows multiple dose/fractionation plan requests, planning target volume (PTV) expansions, as well as information regarding distance/overlap between spinal cord and targets to drive decision-making. For each requested plan, the script creates the course, plans, field arrangements, and automatically optimizes and calculates dose. The script was retrospectively applied to ten computed tomography (CT) scans of previous cervical, thoracic, and lumbar spine SBRT patients. Three plans were generated for each patient - simultaneous integrated boost (SIB) 1800/1600 cGy to gross tumor volume (GTV)/PTV in one fraction; SIB 2700/2100 cGy to GTV/PTV in three fractions; and 3000 cGy to PTV in five fractions. Plan complexity and deliverability patient-specific quality assurance (QA) was performed using ArcCHECK with an Exradin A16 chamber inserted. Dose objectives were met for all organs at risk (OARs) for each treatment plan. Median target coverage was GTV V100% = 87.3%, clinical target volume (CTV) V100% = 95.7% and PTV V100% = 88.0% for single fraction plans; GTV V100% = 95.6, CTV V100% = 99.6% and PTV V100% = 97.2% for three fraction plans; and GTV V100% = 99.6%, CTV V100% = 99.1% and PTV V100% = 97.2% for five fraction plans. All plans (n = 30) passed patient-specific QA (>90%) at 2%/2 mm global gamma. A16 chamber dose measured at isocenter agreed with planned dose within 3% for all cases. Automatic planning for spine SRS/SBRT through scripting increases efficiency, standardizes plan quality and approach, and provides a tool for target coverage comparison of different fractionation schemes without the need for additional resources.

Racial and socioeconomic disparities differentially affect overall and cause-specific survival in glioblastoma.

INTRODUCTION: The prognostic role of racial and socioeconomic factors in patients with glioblastoma is controversially debated. We aimed to evaluate how these factors may affect survival outcomes in an overall and cause-specific manner using large, national cancer registry cohort data in the temozolomide chemoradiation era. METHODS: The National Cancer Institute's Surveillance, Epidemiology, and End Results database was queried for patients diagnosed with glioblastoma between 2005 and 2016. Overall survival was assessed using Cox proportional hazard models using disease intrinsic and extrinsic factors. Cause-specific mortality was assessed using cumulative incidence curves and modeled using multivariate cumulative risk regression. RESULTS: A total of 28,952 patients met the prespecified inclusion criteria and were included in this analysis. The following factors were associated with all-cause mortality: age, calendar year of diagnosis, sex, treatment receipt, tumor size, tumor location, extent of resection, median household income, and race. Asian/Pacific Islanders and Hispanic Whites had lower mortality compared to Non-Hispanic Whites. Cause-specific mortality was associated with both racial and socioeconomic groups. After adjusting for treatment and tumor-related factors, Asian/Pacific and black patients had lower glioblastoma-specific mortality. However, lower median household income and black race were associated with significantly higher non-glioblastoma mortality. CONCLUSIONS: Despite the aggressive nature of glioblastoma, racial and socioeconomic factors influence glioblastoma-specific and non-glioblastoma associated mortality. Our study shows that patient race has an impact on glioblastoma-associated mortality independently of tumor and treatment related factors. Importantly, socioeconomic and racial differences largely contribute to non-glioblastoma mortality, including death from other cancers, cardio- and cerebrovascular events.

Stereotactic Radiation for Treating Primary and Metastatic Neoplasms of the Spinal Cord.

Stereotactic radiation treatment can be used to treat spinal cord neoplasms in patients with either unresectable lesions or residual disease after surgical resection. While treatment guidelines have been suggested for epidural lesions, the utility of stereotactic radiation for intradural and intramedullary malignancies is still debated. Prior reports have suggested that stereotactic radiation approaches can be used for effective tumor control and symptom management. Treatment-related toxicity has been documented in rare subsets of patients, though the incidences of injury are not directly correlated with higher radiation doses. Further studies are needed to assess the factors that influence the risk of radiation-induced myelopathy when treating spinal cord neoplasms with stereotactic radiation, which can include, but may not be limited to, maximum dose, dose-fractionation, irradiated volume, tumor location, histology and treatment history. This review will discuss evidence for current treatment approaches.

Stereotactic Radiosurgery for Resected Brain Metastases: Does the Surgical Corridor Need to be Targeted?

PURPOSE: Although consensus guidelines for postresection stereotactic radiosurgery (SRS) for brain metastases recommend the surgical corridor leading to the resection cavity be included in the SRS plan, no study has reported patterns of tumor recurrence based on inclusion or exclusion of the corridor as a target. We reviewed tumor control and toxicity outcomes of postresection SRS for deep brain metastases based on whether or not the surgical corridor was targeted. MATERIALS AND METHODS: We retrospectively reviewed patients who had resected brain metastases treated with SRS between 2007 and 2018 and included only "deep" tumors (defined as located ≥1.0 cm from the pial surface before resection). RESULTS: In 66 deep brain metastases in 64 patients, the surgical corridor was targeted in 43 (65%). There were no statistical differences in the cumulative incidences of progression at 12 months for targeting versus not targeting the corridor, respectively, for overall local failure 2% (95% confidence interval [CI], 0%-11%) versus 9% (95% CI, 1%-25%; P = .25), corridor failure 0% (95% CI, 0%-0%) versus 9% (95% CI, 1%-25%; P = .06), cavity failure 2% (95% CI, 0%-11%) versus 0% (95% CI, 0%-0%; P = .91), and adverse radiation effect 5% (95% CI, 1%-15%) versus 13% (95% CI, 3%-30%; P = .22). Leptomeningeal disease (7%; 95% CI, 2%-18%) versus 26% (95% CI, 10%-45%; P = .03) was higher in those without the corridor targeted. CONCLUSIONS: Omitting the surgical corridor in postoperative SRS for resected brain metastases was not associated with statistically significant differences in corridor or cavity recurrence or adverse radiation effect. As seen in recent prospective trials of postresection SRS, the dominant pattern of progression is within the resection cavity; omission of the corridor would yield a smaller SRS volume that could allow for dose escalation to potentially improve local cavity control.

Novel Therapies for Glioblastoma.

PURPOSE OF REVIEW: Glioblastoma (GBM) is the most common malignant primary brain tumor, and the available treatment options are limited. This article reviews the recent preclinical and clinical investigations that seek to expand the repertoire of effective medical and radiotherapy options for GBM. RECENT FINDINGS: Recent phase III trials evaluating checkpoint inhibition did not result in significant survival benefit. Select vaccine strategies have yielded promising results in early phase clinical studies and warrant further validation. Various targeted therapies are being explored but have yet to see breakthrough results. In addition, novel radiotherapy approaches are in development to maximize safe dose delivery. A multitude of preclinical and clinical studies in GBM explore promising immunotherapies, targeted agents, and novel radiation modalities. Recent phase III trial failures have once more highlighted the profound tumor heterogeneity and diverse resistance mechanisms of glioblastoma. This calls for the development of biomarker-driven and personalized treatment approaches.

Evaluating Surgical Resection Extent and Adjuvant Therapy in the Management of Gliosarcoma.

Introduction: Gliosarcomas are clinically aggressive tumors, histologically distinct from glioblastoma. Data regarding the impact of extent of resection and post-operative adjuvant therapy on gliosarcoma outcomes are limited. Methods: Patients with histologically confirmed gliosarcoma diagnosed between 1999 and 2019 were identified. Clinical, molecular, and radiographic data were assembled based on historical records. Comparisons of categorical variables used Pearson's Chi-square and Fisher's exact test while continuous values were compared using the Wilcoxon signed-rank test. Survival comparisons were assessed using Kaplan-Meier statistics and Cox regressions. Results: Seventy-one gliosarcoma patients were identified. Secondary gliosarcoma was not associated with worse survival when compared to recurrent primary gliosarcoma (median survival 9.8 [3.8 to 21.0] months vs. 7.6 [1.0 to 35.7], p = 0.7493). On multivariable analysis, receipt of temozolomide (HR = 0.02, 95% CI 0.001-0.21) and achievement of gross total resection (GTR; HR = 0.13, 95% CI 0.02-0.77) were independently prognostic for improved progression-free survival (PFS) while only receipt of temozolomide was independently associated with extended overall survival (OS) (HR = 0.03, 95% CI 0.001-0.89). In patients receiving surgical resection followed by radiotherapy and concomitant temozolomide, achievement of GTR was significantly associated with improved PFS (median 32.97 [7.1-79.6] months vs. 5.45 [1.8-26.3], p = 0.0092) and OS (median 56.73 months [7.8-104.5] vs. 14.83 [3.8 to 29.1], p = 0.0252). Conclusion: Multimodal therapy is associated with improved survival in gliosarcoma. Even in patients receiving aggressive post-operative multimodal management, total surgical removal of macroscopic disease remains important for optimal outcomes.

Evolving role of biomaterials in diagnostic and therapeutic radiation oncology.

Radiation therapy to treat cancer has evolved significantly since the discovery of x-rays. Yet, radiation therapy still has room for improvement in reducing side effects and improving control of cancer. Safer and more effective delivery of radiation has led us to novel techniques and use of biomaterials. Biomaterials in combination with radiation and chemotherapy have started to appear in pre-clinical explorations and clinical applications, with many more on the horizon. Biomaterials have revolutionized the field of diagnostic imaging, and now are being cultivated into the field of theranostics, combination therapy, and tissue protection. This review summarizes recent development of biomaterials in radiation therapy in several application areas.

Stereotactic Radiosurgery for Resected Brain Metastases: Single-Institutional Experience of Over 500 Cavities.

PURPOSE: Postoperative stereotactic radiosurgery (SRS) has less detrimental effect on cognition and quality of life compared with whole brain radiation therapy (WBRT) and is increasingly used for resected brain metastases (BMs). Postoperative SRS techniques are not standardized, and there is a concern for a different pattern of failure after postoperative SRS compared with WBRT. We aim to study the efficacy, toxicity, and failure pattern of postoperative SRS. METHODS AND MATERIALS: We retrospectively reviewed outcomes of patients with resected BMs treated with postoperative SRS between 2007 and 2018. Overall survival and cumulative incidences of local failure, overall distant intracranial failure (distant parenchymal failure, nodular leptomeningeal disease [nLMD], classical leptomeningeal disease [cLMD]), and adverse radiation effect were reported. Neurologic death was determined for patients with leptomeningeal disease (LMD). RESULTS: A total of 442 patients with 501 resected BMs were treated over 475 total SRS courses. Median clinical follow-up and overall survival after SRS were 10.1 months (interquartile range, 3.6-20.7 months) and 13.9 months (95% confidence interval [CI], 11.8-15.2 months), respectively. At 12 months, event rates were 7% (95% CI, 5%-10%) for local failure, 9% (95% CI, 7%-12%) for adverse radiation effect, 44% (95% CI, 40%-49%) for overall distant intracranial failure, 37% (95% CI, 33%-42%) for distant parenchymal failure, and 13% (95% CI, 10%-17%) for LMD. The overall incidence of LMD was 15.8% (53% cLMD, 46% nLMD). cLMD was associated with shorter survival than nLMD (2.0 vs 11.2 months, P < .01) and a higher proportion of neurologic death (67% vs 41%, P = .02). A total of 15% of patients ultimately received WBRT. CONCLUSIONS: We report the largest clinical experience of postoperative SRS for resected BMs, showing excellent local control and low toxicity. Intracranial failure was predominantly distant, with a rising incidence of LMD.

Biochemical and Mechanical Gradients Synergize To Enhance Cartilage Zonal Organization in 3D.

Articular cartilage is characterized by zonal organizations containing dual gradients of biochemical cues and mechanical cues. However, how biochemical gradient interacts with the mechanical gradient to drive the cartilage zonal development remains largely unknown. Here, we report the development of a dual-gradient hydrogel platform as a 3D niche to elucidate the relative contributions of biochemical and mechanical niche gradients in modulating zonal-specific chondrocyte responses and cartilage zonal organization. Chondroitin sulfate (CS), a major constituent of cartilage extracellular matrix, was chosen as the biochemical cue. Poly(ethylene glycol), a bioinert polymer, was used to create the stiffness gradient. Dual-gradient hydrogels upregulated cartilage marker expressions and increased chondrocyte proliferation and collagen deposition in a zonal-dependent manner. Hydrogels with CS gradient alone exhibited poor mechanical strength and degraded prematurely after 1 week of culture. While CS gradient alone did not support long-term culture, adding CS gradient to mechanical-gradient hydrogels substantially enhanced cell proliferation, glycosaminoglycan production, and collagen deposition compared to mechanical-gradient hydrogels alone. These results suggest that biochemical and mechanical gradient cues synergize to enhance cartilage zonal organization by chondrocytes in 3D. Together, our results validate the potential of dual-gradient hydrogels as a 3D cell niche for cartilage regeneration with zonal organization and may be used to recreate other tissue interfaces.