There is interest in whether COVID-19 cases respond to environmental conditions. If an effect is present, seasonal changes in local environmental conditions could alter the global spatial pattern of COVID-19 and inform local public health responses. Using a comprehensive global dataset of daily COVID-19 cases and local environmental conditions, we find that increased daily ultraviolet (UV) radiation lowers the cumulative daily growth rate of COVID-19 cases over the subsequent 2.5 wk. Although statistically significant, the implied influence of UV seasonality is modest relative to social distancing policies. Temperature and specific humidity cumulative effects are not statistically significant, and total COVID-19 seasonality remains to be established because of uncertainty in the net effects from seasonally varying environmental variables. With nearly every country combating the 2019 novel coronavirus (COVID-19), there is a need to understand how local environmental conditions may modify transmission. To date, quantifying seasonality of the disease has been limited by scarce data and the difficulty of isolating climatological variables from other drivers of transmission in observational studies. We combine a spatially resolved dataset of confirmed COVID-19 cases, composed of 3,235 regions across 173 countries, with local environmental conditions and a statistical approach developed to quantify causal effects of environmental conditions in observational data settings. We find that ultraviolet (UV) radiation has a statistically significant effect on daily COVID-19 growth rates: a SD increase in UV lowers the daily growth rate of COVID-19 cases by  Coronaviridae, referred to as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), began spreading throughout China , span 1 January 2020 to 10 April 2020, and have nearly global coverage since March 2020 . For purposes of testing for heterogeneity in response, these case records are also combined with data on location-specific containment policies and testing regimes . Daily COVID-19 growth rates are assessed in relation to local population-weighted daily temperature, specific humidity, precipitation, and UV from a 0.We construct our outcome variable as the daily growth rate of confirmed cases, hereafter “daily COVID-19 growth rate,” calculated as the daily change in the logarithm of confirmed cases. Confirmed COVID-19 cases are used because data on recoveries and deaths are not consistently available globally. Growth rates are analyzed because they are a well-established measure for disease spread that reflects changes in transmission characteristics  addresses four key issues associated with prior observational analyses. First, a location’s prevailing social and economic characteristics, which likely influence COVID-19 transmission, may also be correlated with its average environmental conditions. For example, countries that are cooler on average tend to have higher income per capita . We address the concern that such trends may bias causal estimates through the inclusion of flexible location-specific temporal controls that remove low-frequency temporal variation in both COVID-19 and environmental conditions. We additionally employ global-scale, day-of-sample controls to account for any high-frequency common shocks to the evolution of the disease or its reporting across the globe. The resulting location-specific, high-frequency fluctuations in environmental conditions after removal of trends appear to exhibit quasi-random variation . For instance, solar radiation is correlated with relative humidity and precipitation through cloud formation and convection. Such associations confound causal estimates if key variables are omitted from the analysis . We examine the sensitivity of our conclusions to a range of alternative statistical model formulations that, among other things, vary the stringency of the spatiotemporal controls and additionally control for the local timing of COVID-19 outbreaks, testing regimes, and COVID-19 containment policies. The ability of our statistical approach to recover the effects of environmentally driven changes in transmission on the COVID-19 growth rate is confirmed by applying our statistical model to synthetic data simulated by a standard susceptible–exposed–infected–recovered (SEIR) model with an environmentally perturbed transmission parameter  and with strengthened containment efforts over time, although they may also reflect other factors.On average across our sample, confirmed COVID-19 cases grow at a rate of 13.2%/d (SD of 24.4%), equivalent to a doubling time of 5.2 d. Growth rates generally decreased over the first months of the outbreak, with the sample average growth rate falling from 15.7% during the first month of a location’s outbreak to 2.3% in subsequent months B. These SI Appendix, section A.2) demonstrates a statistically significant effect whereby increases in surface UV intensity lower subsequent COVID-19 growth rates. In our primary specification, we estimate that a 1 SI Appendix, Table S1). The effects associated with UV are consistently negative across lags and peak in magnitude after 9 to 11 d . This delay between UV exposure and changes in the COVID-19 growth rate is consistent with the reported time frame between exposure to the virus and its detection . A SD increase in temperature (2.84 °C) and specific humidity (0.13%) leads to small and uncertain estimated effects on the growth rate of 0.49 and 0.29 percentage points, respectively.Applying a panel-regression model to growth rates ; linear, country-specific time trends ; controls for future weather as a test of reverse causality ; controls for temporally and spatially varying policies, such as work-from-home policies, school closures, and event cancellations ; controls for the extent of COVID-19 testing availability ; semiparametric controls that allow for within-country differential seasonality, in addition to country-wide seasonal patterns ; removal of country-level data and addition of semiparametric controls that allow for country-by-day specific shocks ; analysis using a Poisson regression model  in place of ordinary least squares ; replacing specific humidity with relative humidity ; and changing the number of lags included in the estimating equation . We further show our estimates are insensitive to outliers using a procedure whereby we reestimate our cumulative effect after systematically dropping each of our 3,235 geospatial units . Finally, we estimate an alternative model that allows for nonlinearities between weather conditions and COVID-19 growth rates and find that the UV effect exhibits strong linearity . Whereas the significance and magnitude of the cumulative UV effect are stable across the different model specifications, the cumulative effects of temperature and humidity are insignificant across all model specifications and have inconsistent sign .The effect of UV radiation on the COVID-19 growth rate A and B iLeft), the estimated effect of UV is of the opposite sign to that hypothesized in prior literature and estimated in our primary specification (gold line in Right). Similarly, omission of temporal controls (brown line in Center) overestimates the negative effect of UV on COVID-19 growth rates, as time-trending unobservable factors, such as changes in reporting and the general progression of the disease, correlate with gradual changes in UV exposure. These results highlight the empirical importance of adequately removing the influence of key confounding factors that have to date limited the ability to determine whether and how environmental conditions constrain the evolution of COVID-19 , we find that the cumulative effect of 1 The cumulative lagged effect of weather conditions on COVID-19 growth rates reflects the average treatment effect over all geospatial units and over the course of the observed pandemic A. It canin place C.SI Appendix, Fig. S8), although cumulative effects of temperature and specific humidity are statistically indistinguishable from zero both with and without public health policies in place . This period, besides encompassing our entire data period, also covers the full range of seasonal UV exposure experienced in any location, as shown in SI Appendix, Fig. S11). These changes in COVID-19 growth correspond to lowering the average doubling time to 3.3 d in northern latitudes and raising it to 12.6 d in southern latitudes.The estimated effect of UV on the COVID-19 growth rate has seasonal implications . To illu° south) B and C. As a whole, the tropics display moderate seasonal changes driven by UV, with our simulations generating an increase of 0.57 . Because factors such as social distancing policies have a larger influence than seasonal variation in UV, and high susceptibility to date among the global population permits for rapid transmission, COVID-19 growth is unlikely to exhibit substantial seasonality, at least in the near term . We additionally address this concern statistically by accounting for location-specific trends in confirmed COVID-19 cases and by controlling for the availability of COVID-19 testing at the country level , although reporting issues could remain.Our study has a number of important limitations. First, as is true in any empirical study of disease, we can only observe cases that are confirmed. The fact that confirmed cases of COVID-19 are likely well below the actual number of infections  would noSI Appendix, section A.1 and Fig. S13G).Second, it is possible that the behavioral response to random day-to-day fluctuations in UV  differs from the behavioral response to expected seasonal changes. For example, an anomalously sunny day in March may elicit different human behavior than a day in July with the same UV exposure. It may be possible to estimate such state-dependent effects after the accrual of multiple seasons of data. There is also a potential concern that the slow response of the dynamic system of the disease would damp the amplitude of the response to high-frequency day-to-day environmental fluctuations. We find using stochastic simulations of the SEIR model, however, that simulated UV-induced variations in transmission are generally insensitive to the frequency of perturbations . We additionally combine case records with data on location-specific containment policies and testing regimes from refs.  product  by calcuom refs. , 16.UV radiation is represented as including wavelengths from 200 to 440 nm. Limitations associated with the representation of radiative transfer associated with ERA5 reanalysis prevent us from distinguishing between UVa (400 to 315 nm), UVb (315 to 280 nm), and UVc (280 to 100 nm) effects. Higher-energy UVc and UVb radiation may more rapidly deactivate SARS-CoV-2 than UVa but is also more readily absorbed in the atmosphere , 62. A mSI Appendix, Eq. S1). We include indicator variables  for each subnational or national administrative unit, for each day of the sample, and for each country by week in the sample. We calculate standard errors accounting for serial correlation across days within each administrative unit. Clustering at the country level, which further allows for spatial correlation of arbitrary form within administrative units from the same country, does not discernibly change the precision of our estimates. All P values are calculated using two-sided tests. We test for heterogeneity in the estimated effect of weather conditions on COVID-19 growth rates by interacting the lagged weather variables with binary variables indicating whether containment policies are in place and whether the observation is in the first month of the outbreak .We statistically estimate the effect of weather on the daily growth rate of confirmed COVID-19 cases using a longitudinal  regression model. Daily COVID-19 growth rates are estimated as a linear function of UV, temperature, specific humidity, and precipitation exposure over the preceding 17 d in a model allowing the effect of each environmental variable to differ across lag intervals of 3 d .Seasonal simulations  use the Throughout the analysis daily growth rates of confirmed COVID-19 cases are shown using units of percent. For example, a growth rate of 0.13 is approximately equivalent to a growth rate of 13%. Changes in growth rates are given in units of percentage points. For example, a reduction of the growth rate by 1 percentage point would change the growth rate from 13% to 12%.Supplementary File


In the introductory chapter, Muresan and Orna-Montesinos provide an overview of the multiple dimensions of academic literacy development, with a focus on its relevance for plurilingual scholars engaged in academic research writing and publishing processes. They situate the ethnographic and pedagogical studies presented in the subsequent chapters within a cognitive/socio-cultural theoretical framework, providing insights into higher education and academic literacy in glocal contexts. Literacy, as an overarching concept, encompasses multiple dimensions of reading and writing abilities. As a term it has been used to promote a view of writing as social, personal, cognitive and attitudinal practice . As we expect to illustrate throughout the volume, there is sufficient evidence across geographical contexts worldwide regarding the challenges scholars face in the process of acquiring academic literacy in an L1 and in other languages. Pedagogical interventions can, therefore, play a beneficial role, particularly for enhancing awareness of discourse and genre conventions, and for raising rhetorical consciousness through appropriate tasks and materials  that underlie academic enculturation, the latter conceptualised by Prior and Bilbro  as the pInspired by previous research perspectives on the complex uses of academic languages in changing times  contexts, the volume starts with a selection of chapters that provide empirically based reflections on and enquiry into linguistic policies, norms or guidelines regarding the use of both academic English and other academic languages. These contributions also explore the impact of co-existing, controversial academic norms and guidelines on research writing in different linguistic eco-systems.In Chap. 10.1007/978-3-030-62877-2_2, Hynninen analyses the textual trajectory of a successful research paper, co-authored by six multilingual scholars. Text history data allow her to explore how different actors are allocated literacy broker roles and how they negotiate the writing norms and conventions for their interventions in the process of academic text production. In Chap. 10.1007/978-3-030-62877-2_3, Giannoni offers a critical overview of the language requirements and opportunities for thesis submission in languages other than English at UK universities. The implications of vague linguistic policies for academic literacy development are discussed in the light of recent research on multilingualism in higher education. Chapter 10.1007/978-3-030-62877-2_4 starts with the provocative question, “What constitutes ‘good’ academic writing” for multilingual students in EMI programs? McCambridge critically discusses the interplay of different academic writing norms and underlines the importance of supporting the students’ academic literacy development in ways that would help them to understand and negotiate the meaning of the different kinds of literacy practices and norms they will encounter.This section includes studies on academic writing development that draw on ethnographic or ethno-methodological enquiry, with data collected from observation, interviews and questionnaire-based surveys, conducted with graduate, Master and doctoral students, in a variety of disciplines and a diversity of international academic contexts .Chapter 10.1007/978-3-030-62877-2_5 focuses on the development of literacy in professional and academic contexts using a protocol-assisted modelling (PAM) approach, implemented to help student writers enrolled in a technical communication program to develop audience awareness by constructing a more accurate mental model of how readers think and react to information. Dressen-Hammouda discusses how the PAM method, applied in a French academic setting, contributes to the students’ literacy development by helping them better adapt their attitudes, behaviours and abilities to often unstated and shifting professional expectations. The emergence of situated work-based literacy development is thus linked by the author to mastering professional genres as well as to building disciplinary or professional identity and epistemology.Chapter 10.1007/978-3-030-62877-2_6 explores the development of academic writing literacy in a group of Spanish doctoral students participating in a writing course. Through a thematic analysis of the difficulties and challenges faced by students in their writing processes in English as an additional language, Guillén-Galve and Vela-Tafalla critically discuss how the acquisition of operational skills appears to be based on the identification of sources and strategies of independent learning and of self-efficacy, complemented by literacy brokering practices.Chapter 10.1007/978-3-030-62877-2_7 reports on the implementation of an EMI program which seeks to develop students’ academic writing, at the University of Iceland. In order to explore the students’ perceptions, Prinz and Arnbjörnsdóttir have conducted pre- and post-course surveys, with a focus on the students’ ability to regulate academic writing and their writing self-efficiency beliefs. Strategy instruction in writing appeared to help students to transfer conceptual knowledge about academic writing to functional writing skills and, thus, to develop the operational strategies and skills needed to become autonomous writers.Chapter 10.1007/978-3-030-62877-2_8 builds on an ethnographic study of a group of novice Chemistry researchers (in the US academic context) participating in a series of writing workshops with a focus on science communication genres. McCarty debates the perception of a clear divide between disciplinary writing addressed to specialists and science communication (Sci comm), targeted at non-specialist audiences. These novice writers question the creation of the narrative of science as newsworthy and the incorporation of more nuanced information about the research process. The author concludes that learning to write these new genres to address non-specialist audiences in new contexts imposes a more flexible approach to writing, one in which textual innovation results in the expression of learners’ shifting identity as scientists.The chapters in this section take further the exploration of academic literacy development journeys by placing a special focus on authorial voice, manifestations of identity, or the researcher’s and participants’ self-reflection and self-representation in the process of knowledge co-construction. The truly international dimension of the studies reported on here is reflected not only by the range of academic contexts where the chapter authors are based , but also by the diversity of linguistic and cultural backgrounds of the participants in their research, whose original country contexts included, for instance, Bosnia-and-Herzegovina, Brazil, China, Korea, R. N. Macedonia, Malta, Norway, Portugal, Romania, the UK, Ukraine.Chapter 10.1007/978-3-030-62877-2_9 focuses on the development of authorial voice in multilingual international students writing their theses in an additional language. Seeking to provide both students and supervisors with clear and pertinent advice on how to develop and express authorial voice, Morton and Storch critically evaluate the treatment of voice in thesis writing guidebooks and online material produced by universities as well as by popular blogs. Their research revealed that the resources targeting students were simplistic, providing only limited advice on authorial voice, and therefore, they stress the role of supervisors in guiding doctoral students to acquire strategies that can contribute to the development of writers’ authorial voice across disciplines.self, revealed through personal opinions, authoritativeness and presence in the interviews conducted. The study focuses on the manifestation of identity as a linguistic phenomenon, that is, on the discoursal resources which convey the writers’ personal opinions, beliefs, and attitudes, and which are used to justify their actions during the thesis writing process. This scrutiny of linguistic choices sheds light into how the students’ identities and own versions of reality are constructed. It also foregrounds a complex, multiaspectual nature of their self-presentation as anxious, supported, independent or triumphant writers.In Chap. 10.1007/978-3-030-62877-2_10, Bekar and Yakhontova report on a qualitative study of the various aspects of student writers’ Chapter 10.1007/978-3-030-62877-2_11 provides an account of the author’s own experience as a facilitator in writing conference groups for graduate students writing their thesis. In her ethnographic study, Mochizuki examines knowledge and meaning co-creation between the participants and herself, as a researcher, aiming to understand the significance of the role of researcher reflexivity in academic literacy development. The findings presented show how her interaction with the participants has contributed to transforming their views and to the negotiation of their identities and goals. The chapter illustrates how direct observation and reflexivity allow the researcher to be actively involved not only in knowledge gathering but also in knowledge construction.The studies included here delve into multilingual scholars’ literacy development stages, exploring the challenges they face in constructing academic texts, the strategies they have developed and the resources they are resorting to.In Chap. 10.1007/978-3-030-62877-2_12, Habibie presents the findings of a case study that explored the strategies, pedagogies and mentorship practices of a multilingual early-career academic in writing for scholarly publication. The author framed his ethnographically oriented study resorting to Tardy’s  notion oIn Chap. 10.1007/978-3-030-62877-2_13, Villares explores the perceptions of Spanish multilingual scholars regarding academic literacy development. The main challenges referred to in interviews consisted in both internal factors  and external factors . In addition, the study reveals the scholars’ perceptions of linguistic disadvantage during the writing and manuscript submission process. The use of both individual learning-by-doing strategies and collaborative strategies highlights the crucial role of the social aspect of academic literacy development. The implementation of policy decisions which foster the enculturation process of novice researchers and the mastery of academic literacy are discussed as a response to insufficient training and institutional support services.Chapter 10.1007/978-3-030-62877-2_14 explores academics’ perspectives on the development of feedback and scholarly writing literacies. Using concept map-mediated interviews, Gravett provides insights into academics’ experiences on moving from the highly emotive nature of learning from critical feedback and rejection, to taking critical feedback forward in order to offer a considered response. Despite the benefits of the collaborative nature of the peer review process, the study exposes the perception of power inequalities illustrated by the pressures experienced by multilingual scholars writing in English and the concerns about the debilitating and frustrating nature of the review process which might lead to the lack of faith in reviewers’ credibility, motivation or expertise.Chapter 10.1007/978-3-030-62877-2_15 presents longitudinal interview data from two plurilingual early career researchers participating in a writing for publication tutoring programme. Janssen and Crites explain how the evolution of the participants’ writing practices is connected to the interaction with academic literacy brokers and to the development of their research networks. Their findings show how engagement in networks with different literacy brokers  or collaboration and face-to-face discussion with other members of their community of practice, including friends, colleagues, co-authors, play a crucial role in mediating the publication process.In Chap. 10.1007/978-3-030-62877-2_16, Tercero brings to the fore the challenges and changes experienced by academia in the spring of 2020, which caught both students and instructors in Higher Education fairly unprepared. On the basis of the findings of a small-scale exploratory study of instructor and student perceptions of their online writing courses, the author proposes a framework for designing and teaching an online academic writing course for L2 writers. As Tercero concludes, the COVID-19 pandemic might have fostered the expansion of online teaching and learning in the near future in unprecedented ways, which makes the design of quality online teaching and learning a crucial endeavour.coda chapter, Englander and Corcoran review recent research on multilingual scholars’ trajectories and experiences in writing academic texts to bring to the fore the need for addressing advanced stages of literacy development from multiple perspectives. After highlighting the conceptual complexity of literacy, with its multilayered and intersecting dimensions, the authors engage with several salient themes addressed in the contributions to this volume. They underline the “variety of ethnographic research methodologies” employed in the studies included in this collection, as well as the value of bringing together such a range of “glocalized perspectives from broad geolinguisitic locales”. Englander and Corcoran conclude by problematising possible implications of the current tendencies towards achieving homogeneity in academic publishing and invite for further reflections on the multiple facets of academic knowledge exchange.In this


The application of single homogeneous materials produced through the fused deposition modelling (FDM) technology restricts the production of high-level multi-material components. The fabrication of a sandwich-structured specimen with different material combinations using conventional thermoplastics such as poly (lactic acid) (PLA), acrylonitrile butadiene styrene (ABS) and high impact polystyrene (HIPS) through the filament-based extrusion process can demonstrate an improvement on its properties. This paper aims to assess among these materials, the best material sandwich-structured arrangement design, to enhance the mechanical properties of a part and to compare the results with the homogeneous materials selected. The samples were subjected to tensile testing to identify the tensile strength, elongation at break and Young’s modulus of each material combination. The experimental results demonstrate that applying the PLA-ABS-PLA sandwich arrangement leads to the best mechanical properties between these materials. This study enables users to consider sandwich structure designs as an alternative to manufacturing multi-material components using conventional and low-cost materials. Future work will consider the flexural tests to identify the maximum stresses and bending forces under pressure. Additive manufacturing (AM) began in 1987 when it was known as generative manufacturing or rapid prototyping ,3,4,5. AIn recent years, the fabrication of multi-material polymer-based products and sandwich structure methods applied to the FDM process have brought the attention of the industry and research community. The benefits of multi-material enable the performance of smart components by improving 3D printing quality through topology optimization of different process parameters ,15. ThusFernandez-Vicente et al.  evaluateIn the authors’ previous work, tensile tests for single material specimens made of PLA ,23 and AThe present paper investigates and tests the influence of sandwich structures applied to conventional 3D printing materials to improve the mechanical properties of 3D printed prototypes. The new sandwich structure here proposed are specimens embedding rectilinear cores pattern with a 100% infill density and applying multiple independent nozzle extruders for each material within the same carriage. This new scheme could provide a piece of knowledge for comparison on the effect of combining conventional materials  as a sandwich structure to achieve and enhance higher strength of polymeric parts, which can be used for various applications, e.g. unmanned aerial vehicles (UAV). The tensile experiments aimed to identify whether the use of conventional materials printed as a sandwich structure is suitable and beneficial to implement and able to improve their mechanical behaviour  when compared with a single material 3D printed part. Therefore, the contribution of this paper lies in finding the best final product performance of the selected materials.The first material selected for this study was PLA. It is widely used in FDM 3D printing  and provFirstly, specimens were printed as a single homogeneous material with fixed parameters to be compared with the multi-material specimens. Secondly, the combination of materials for the sandwich structure was performed with four-layer sections of each material. It contained symmetric 1.2 mm thick polymeric inner and outer cores  giving a total dimension of a 3.6 mm thick specimen in accordance with the dimensions of the standard test method for tensile properties of plastics (ASTM D638-Type 1 standard) . All spePLA-ABS-PLA and PLA-HIPS-PLAABS-PLA-ABS and ABS-HIPS-ABSHIPS-PLA-HIPS and HIPS-ABS-HIPSThe material configurations to fabricate the sandwich structure specimen are shown below. For the present study, a multi-material 3D printer was developed and customized to allow the use of multiple filaments. It was necessary to integrate a multi-nozzle head extruder to enable producing different materials. The extrusion unit consisted of four nozzles within the same carriage. The module is based on the Bowden extrusion system , where tThe printed parts were measured before tested using an advanced Mitutoyo caliper. The measurements were compared with the exact dimensions of the CAD model design . All speThe machine used to perform the tensile tests was the Instron 5966. It was configured with a minimum speed of 2 mm/min, a load cell of 10 kN and a gripper with a maximum load of 5 kN. The selection of the speed rate was in accordance with the loading rate used in other studies ,26,49,50The specimens were loaded along the longitudinal axis until failure. According to the standard and for the evaluation of dispersion, five specimens were printed for each material configuration. In, total, 45 specimens were printed to validate the results. The mechanical properties considered for the comparison were the tensile strength, Young’s modulus and the elongation, as shown in The mechanical results obtained from the tensile test show differences between the homogeneous materials and the different combinations of sandwich structures. PLA demonstrated the highest tensile strength (47.46 MPa) and Young’s modulus (1396.90 MPa) among all specimens but presented the lowest elongation at break (4.16 mm). HIPS exhibited plastic deformation and showed the lowest tensile strength (20.06 MPa) and Young’s modulus (933.33 MPa) but had a higher elongation at break (6.69 mm) when compared to the other two. Moreover, it was expected that the performance of combining different materials would have a positive impact on the mechanical properties in comparison to the single material ones. In general, these three types of specimens showed higher ultimate strength and elastic modulus when fabricated with the sandwich configuration compared to a single material. Among these specimens, the combination of PLA-ABS-PLA had the best tensile strength and Young’s modulus with means of 44.40 and 1364.57 MPa, respectively. This behaviour may be due that both polymers are considered rigid materials. Besides, this combination had a significant improvement in the elongation at break with a mean of 6.14 mm. Considering that PLA had the highest tensile strength but the lowest elongation followed by ABS as homogeneous materials, it was observed that combining these two materials as a sandwich structure where PLA was printed as the outer skins and ABS as the inner core of the specimen provided a significant performance combining their properties. Secondly, PLA-HIPS-PLA had slightly higher tensile strength and Young’s modulus (38.77 and 1351.27 MPa) compared to ABS-PLA-ABS with a tensile strength of 38.28 MPa and Young’s modulus of 1232.96 MPa. In addition, the highest elongation at break exhibited from these three specimens was 6.14 mm from the PLA-HIPS-PLA sandwich structure.The infill fibers of the skin layers deform rapidly and absorb the stress, causing to break the bonds within materials. The location of the ruptures of the sandwich-structured specimens was mostly generated near the jaws of the test equipment, and elsewhere for two cases (ABS-PLA-ABS and ABS-HIPS-ABS). In addition, it was determined that the weakest sections of the specimens in which the fractures occurred were due to the boundary interface within the transition between different regions of the specimen. In addition, the fractures occurred due to the boundary interface between materials. Despite this condition, using a sandwich structure arrangement for conventional materials can improve the mechanical properties, as demonstrated and described previously. The generated matrix of the corresponding results of the different sandwich structures is shown in This research presented a unique polymer-based sandwich structure to evaluate the tensile properties of different material combinations produced via the FDM process. The aim of the study was applied to the fabrication of rectilinear infill cores and outer skins of several materials via multiple nozzle extruders. Experimental findings in the 3D printing process showed that the best sandwich-structured arrangement was the combination of outer skins of PLA and ABS cores. The average values were 44.40 MPa for the tensile strength and 1364.25 MPa for Young’s modulus. In addition, the elongation at break (6.14 mm) for this configuration was higher when compared to a homogeneous material. Specimens with outer skins of HIPS and ABS cores showed the lowest performance compared to the rest sandwich specimens. The mechanical behaviour between the sandwich structures is different. The change of the material arrangement determines mainly the tensile strength, elongation at break and stiffness between 30% and 50%.Ultimately, this study demonstrated the capabilities and flexibilities of conventional 3D printing materials to be used to improve the efficiency of an AM product. Therefore, the use of the sandwich structure applied to conventional 3D printing materials gain applicability in generating functional parts that can withstand tensile loading for longer periods over single homogenous materials by integrating the properties of two different materials in the same part. It can also provide higher elongation at break, which can absorb energy and therefore give long-term use before failing. The advantages of multi-material and sandwich structures methods are suitable to implement and able to achieve the requirements of various applications using low-cost materials.


IntroductionTreatment options for metastatic renal cell carcinoma disease have been improved in recent years. However, there is still no optimal treatment sequence or combination for metastatic disease. We aimed to investigate whether patients differed in terms of disease outcomes regarding pre-nivolumab tyrosine kinase inhibitors (TKIs).Material and methodsThe analysis of patients was performed after all cohorts were sub-grouped into two groups according to pre-nivolumab TKIs as following the sunitinib arm and the pazopanib arm.ResultA total of 75 patients were included in this study. The median follow-up time was eight months for all cohorts. The objective response rate was statistically significantly higher in the pazopanib arm as compared to the sunitinib arm . Progression-free survival was significantly higher in pazopanib than sunitinib . Multivariate analysis revealed that pazopanib treatment was associated with better progression-free survival . While the median overall survival for patients who had received sunitinib was 11.0 months, it has not been reached the median in the pazopanib arm .DiscussionWe demonstrated significantly better progression-free survival and a higher objective response rate with nivolumab treatment in patients who had received pazopanib as compared with patients who received sunitinib in the pre-nivolumab period. Kidney and renal pelvis carcinoma are estimated to constitute approximately 4.2% of all new cancer cases and 2.4% of all cancer-related deaths in 2019 [Surgery is still the mainstay of the treatment for patients with localized disease . HoweverAlthough sunitinib and pazopanib have demonstrated similar efficacy in the first-line settings, they had differences in terms of side effects and tolerability. Furthermore, it is still not known whether they have any impact on the outcomes of following ICI treatments. In the literature, there are various studies giving indirect information about the influence of TKIs on the following treatments. However, there is still no suggestion about the selection of any of the TKIs in first-line settings.In this retrospective multicenter study, we aimed to investigate whether patients who had received sunitinib or pazopanib differed in terms of disease outcomes with nivolumab treatment in mRCC.Patient characteristicsWe retrospectively evaluated the data of 286 patients who were admitted to seven different oncology centers. Of those, 75 patients who received nivolumab due to progression on treatment with either sunitinib or pazopanib were included in this study. The analysis of patients was performed after all cohorts were sub-classified into two groups according to pre-nivolumab TKIs as following the sunitinib arm and the pazopanib arm.The inclusion criteria were as follows: i) aged at or over 18 years, ii) histopathologically proven clear cell renal cell carcinoma, iii) Eastern Cooperative Oncology Group (ECOG) performance score (PS) ≤2, iv) measurable metastatic disease according to Immune Response Evaluation Criteria in Solid Tumors (iRECIST) criteria, v) adequate liver, kidney, and bone marrow function. Patients with brain metastasis, non-clear cell histology, and those who had received Interferon-alfa for more than three months were excluded from the study.The following data were obtained from patients' oncologic follow-up files: age, gender, ECOG performance score, International Metastatic RCC Database Consortium (IMDC) risk score, presence of cytoreductive nephrectomy, duration of metastatic disease, number of metastatic sites, previous treatment and duration, and the results of the biochemical analysis performed before nivolumab treatment.All patients had received Interferon-alfa (IFN-ɑ) after the diagnosis of metastatic settings due to the restriction of health policies in our country. All patients received either sunitinib  or pazopanib  after IFN-ɑ treatment due to unacceptable toxicity. Patients who progressed on sunitinib or pazopanib were treated with nivolumab 3 mg/kg every two weeks. All patients underwent physical examination, hematological, biochemical, and hormonal (thyroid-stimulating hormone (TSH), cortisol) evaluation every two weeks. Tumor response was evaluated by computed tomography every 12 weeks according to iRECIST criteria.According to immune RECIST criteria, complete response (CR) included the disappearance of all target lesions and reduction in the short axis measurement of all pathologic lymph nodes to ≤10 mm, partial response (PR) was defined as a ≥30% decrease in tumor size, progressive disease (PD) was defined as a ≥20% increase in tumor size, and stable disease (SD) was considered for patients who met neither PR nor PD criteria. The objective response rate (ORR) was defined as the sum of complete and partial responses. The disease control rate (DCR) was defined as the sum of the complete response, partial response, and stable disease rates. Hyperprogression was defined as a two-fold increase of tumor burden at first evaluation for nivolumab treatment and the presence of symptoms related to progression. The detection of new lesions was not considered as progression, and comparisons were performed after new lesions were added to the total tumor burden measured before treatment .Univariate and multivariate analysisFor univariate and multivariate analysis, seven variables that could have an impact on progression-free survival were selected based on previous studies. Variables were categorized into two groups as follows: age (<65 years or ≥65 years), gender , ECOG PS (0-1 or 2), number of metastatic sites (single or multiple), bone metastasis (present or absent), liver metastasis (present or absent), and lung metastasis (present or absent). Variables with a p-value of <0.05 were included in the multivariate analysis.Statistical analysisStatistical analysis was performed by using Statistical Package for the Social Sciences Version 22.0 for Windows . Patients’ characteristics and response rates were compared by the Pearson chi-square test and Fisher’s exact test. The Kaplan-Meier test was used for survival analysis, and the survival outcomes were analyzed by the log-rank test. The Cox proportional-hazards model was applied for the multivariate analysis. All reported p-values were two-sided and p-values of less or equal than 0.05 were considered to as a significant result.The overall survival was defined as the time between the date of starting nivolumab to death or the last visit for living patients. Progression-free survival was defined as the time between the date of starting nivolumab treatment to disease progression or death, whichever occurred first.A total of 75 patients were included in this study. Of those, 41 patients were in the sunitinib arm and 34 patients were in the pazopanib arm. The median age was 59 years (range: 26-78) for all patients, and it was 59 years in both arms. The baseline characteristics of both groups were similar to each other except for the rate of bone and lung metastases. The ratio of patients with bone metastasis was higher in the pazopanib arm than in patients in the sunitinib arm . The baseline characteristics of both groups were summarized in Table The median duration from the diagnosis of metastatic disease to starting nivolumab treatment was 21.1 months for patients in the sunitinib arm, whereas it was 18.9 months for patients in the pazopanib arm. The median duration of interferon-alfa treatment was 1.6 months and 1.0 months for the sunitinib and pazopanib arms, respectively. While the median duration of TKI treatment was 8.7 months in the sunitinib arm, it was 9.8 months for pazopanib treatment. Objective response rates were 30% and 46% for sunitinib and pazopanib treatments in the pre-nivolumab period, respectively. Table The median follow-up time was 8.0 months for all cohorts. Complete response was observed in two patients 3%) and partial response in 30 patients (40%) in all patients. Complete response was observed in one patient with nivolumab treatment in both the sunitinib and pazopanib arms. However, the rate of partial response was statistically significantly higher in the pazopanib arm as compared to the sunitinib arm . Response rates are summarized in Table % and parMedian progression-free survival was 8.9 months  for all cohorts. Progression-free survival was significantly higher in the pazopanib arm than the sunitinib arm   . Survival analysis could not be performed because survival data were immature at the time of the final analysis.Hyperprogession was observed in three patients and all of those cases were in the sunitinib arm. Nivolumab was permanently discontinued in two patients due to prolonged grade 4 hematologic toxicity (neutropenia) and grade 4 neurological toxicity  in the pazopanib arm. In the sunitinib arm, nivolumab had to be discontinued in one patient due to grade 4 nephropathy (proteinuria).There are various studies demonstrating the effectiveness of sunitinib and pazopanib in first-line settings and nivolumab in second-line settings for mRCC. However, to the best of our knowledge, there is no study directly comparing the impact of TKIs on subsequent immunotherapy. This is the first study performing this evaluation in a head-to-head fashion. In this study, we evaluated the influence of pre-nivolumab TKIs on the following nivolumab treatments. After a median eight-months follow-up, our findings showed that patients who had received pazopanib before nivolumab achieved better progression-free survival as compared to patients who had received sunitinib in the pre-nivolumab period . There was no statistically significant difference in terms of baseline characteristics, except for bone and liver metastasis, between the sunitinib arm and the pazopanib arm. Additionally, patients who had received pazopanib as pre-nivolumab TKI achieved higher partial response rates compared to patients who had received sunitinib . On the other hand, we could not perform overall survival analysis due to it not reaching the median. Taken together, patients who received pazopanib as pre-nivolumab TKI experienced better progression-free survival with nivolumab treatment as compared to patients who had received sunitinib before nivolumab treatment.The first trial demonstrating a head-to-head comparison of sunitinib and pazopanib in mRCC as first-line treatment was the COMPARZ  study. The primary endpoint of this phase 3, prospective randomized study was to show the non-inferiority of pazopanib compared to sunitinib regarding progression-free survival. According to the result of this study, median progression-free survival was 9.5 months for sunitinib and 8.4 months for pazopanib. Although progression-free survival for the sunitinib arm was numerically longer, the hazard ratio for progression or death was 1.05 (95% CI: 0.90-1.22), which was lower than the predefined upper bound margin for non-inferiority . FurtherThe phase 3 Checkmate-025 (Study of Nivolumab (BMS-936558) vs. Everolimus in Pre-Treated Advanced or Metastatic Clear-cell Renal Cell Carcinoma) trial has obviously demonstrated the superiority of nivolumab over everolimus in patients with mRCC who progressed on one or two anti-angiogenic TKI. This study showed that overall survival for patients who received nivolumab was 25.0 months and for patients who received everolimus was 19.6 (p<0.001) . AdditioThe impact of prior anti-angiogenic treatment on subsequent treatments has been evaluated in previous studies indirectly. After patients were stratified according to prior treatments, the analysis of the Checkmate-025 trial showed that while median overall survival for patients who received sunitinib as pre-nivolumab TKI was 23.6 months, it has not reached the median for patients who received pazopanib as prior nivolumab treatment. Although there was no direct comparison between those two groups, the authors did not report any difference in overall survival regarding prior TKIs. Notably, the SUNPAZ  study had an appropriate study design for evaluating the influence of sunitinib and pazopanib on subsequent treatment . The SUNAlthough we do not know the exact mechanism of difference observed in those two groups, according to an in vitro study, sunitinib has been shown to reduce myeloid-derived suppressor cells, which in turn modulate the immune response . AdditioThe major limitations of our study are its retrospective nature, relatively low number of patients, and the absence of overall survival data.Taken together, to the best of our knowledge, our study is the first study reporting a direct comparison of the influence of prior TKIs (sunitinib vs pazopanib) on subsequent nivolumab treatment. We demonstrated significantly better progression-free survival with nivolumab in patients who had received pazopanib in the pre-nivolumab period compared with patients who had received sunitinib as prior treatment. Our study may guide clinicians for selection between these two TKIs in countries that require utilizing one-line TKI before immunotherapy. Therefore, we believe our results need to be confirmed by further prospective clinical trials with a larger number of patients.


Complementary datasets on heart-rate, whole-body oxygen consumption, proximal arm-muscle electrical activity and power output, were obtained in a mock-up scanner. The utility of the platform was tested by a preliminary study over 4 weeks of skill practice on the efficiency of execution of a dynamic arm-cranking task in healthy subjects.Evaluation of the effect of human upper-body training regimens may benefit from knowledge of local energy expenditure in arm muscles. To that end, we developed a novel arm-crank ergometry platform for use in a clinical magnetic resonance (MR) scanner with in vivo31P MR spectra from the human biceps brachii (BB) muscle during dynamic exercise in five healthy subjects. Changes in BB energy- and pH balance varied considerably between individuals. Surface electromyography and mechanical force recordings revealed that individuals employed different arm muscle recruitment strategies, using either predominantly elbow flexor muscles , elbow extensor muscles  or a combination of both (two subjects). The magnitude of observed changes in BB energy- and pH balance during ACT execution correlated closely with each strategy. Skill practice improved muscle coordination but did not alter individual strategies. Mechanical efficiency on group level seemed to increase as a result of practice, but the outcomes generated by the new platform showed the additional caution necessary for the interpretation that total energy cost was actually reduced at the same workload.The new platform successfully recorded the first ever in vivo31P MRS recordings from proximal arm muscles with whole-body calorimetry, surface electromyography and biomechanical measurements. This new methodology enables evaluation of cyclic motor performance and outcomes of upper-body training regimens in healthy novices. It may be equally useful for investigations of exercise physiology in lower-limb impaired athletes and wheelchair users as well as frail patients including patients with debilitating muscle disease and the elderly.The presented platform integrates dynamic  Optimal upper-extremity functioning is a prerequisite for healthy performance under different conditions, such as activities of daily living of persons in general and in particular for individuals with lower limb impairments, the work of many industrial laborers, or during different athletic disciplines. Upper-extremity cyclic exercise is more straining and less efficient than leg exercise, while shoulders and wrists are prone to overuse complaints and pain . Whole-bWhole body physiological outcomes in upper-body exercise e.g.,  are limiin vivo biochemical and physiological data gathering from individual arm muscles as well as ensembles of arm muscles all the way up to the whole body. An existing MR-compatible leg-crank ergometer .Here, we report on a novel investigative platform for experimental study of upper-extremity cyclic exercise in humans enabling rgometer  was herergometer  and implSix able-bodied naive participants were recruited for the study. The physical task of arm-cranking inside the 60 cm diameter bore of a clinical MRI scanner imposed physical constraints on body-height (maximum of 1.80 m) and upper-body width . The physical characteristics of the five subjects that completed the study are shown in m. Biceps brachii , m. Brachioradialis and m. Triceps  muscles, respectively, of the right arm as well as a mask for spirometry and a chest strap for heart rate (HR) monitoring  enabling dynamic recording of surface EMG (sEMG), breath-by-breath VO2 and VCO2 and HR data, respectively, in study subjects during ACT execution  was positioned over the BB muscle of the right arm of the study subjects. The coil was manually tuned and matched at the Larmor frequency of phosphorus-31 at 3 Tesla (51.73 MHz). Free induction decays (FIDs) were next acquired using a 90-degree adiabatic excitation pulse and characterized by sampling 2,048 points with a dwell time of 0.33 ms. Firstly, a measurement of the basal phosphorus metabolite levels in the biceps muscle was performed  13,333 ms). During exercise, continuous MR spectroscopic data gathering from the BB muscle was synchronized with the arm-cranking frequency as described elsewhere , phosphocreatine (PCr), and phosphomonoesters (PME) and quantified using the AMARES  algorithm in the jMRUI software package (version 3.0) using custom prior knowledge of starting values, line width, frequency and shape of the resonance peaks . Intramum. biceps brachii longus and brevis, triceps longus, triceps lateralis, and brachioradialis. Subjects were next asked to take place in the starting position of the handcycle trial and perform a total of six Maximum Voluntary Contractions (MVCs) on the handgrip equipped with the LCM300 load cell while the flywheel of the ergometer was locked. Each MVC lasted 5 s and the participants had 2 min of rest between each MVC. Three MVCs of elbow flexion were performed followed by three MVCs of elbow extension.Surface EMG (sEMG) recordings were collected from the participants’ right arm during pre-test 1 and post-test 1 using the Porti-5 system. The Porti-5 system measured EMG activity in mV at a sample frequency of 1,600 Hz, PortiLab2 software was used for data acquisition. The skin of the participants was shaved, scrubbed and cleaned with alcohol to improve EMG electrode conductivity. After evaporation, two EMG electrodes were patched upon the muscle belly of interest with approximately 0.02 m distance between the center of the electrodes . The following six muscles were, respectively, patched with electrodes and connected to the Porti system; Raw sEMG recordings were processed using custom written scripts in Matlab. sEMG signals were filtered using a fourth order dual pass Butterworth filter setting. A high pass filter with a cut-off frequency of 6 Hz was performed on the raw sEMG signal to remove low frequency noise. Subsequently, full wave rectification was performed by taking the absolute value of the signals. The rectified signal was low-pass filtered using a cut-off frequency of 10 Hz to create the linear envelope of the sEMG signal. sEMG signal amplitudes during ACT execution were determined for each muscle and normalized to maximal amplitude recorded during MVC of elbow flexion  or extension (triceps) yielding values in units of “percentage of maximal” (%). Maximal amplitude was calculated as the mean of three 500 ms sEMG recordings during MVC around the time point at which force on the load cell reached its peak (0.125 s before the peak till 0.375 s after the peak).An additional pole was constructed and instrumented with a non-MR compatible unidimensional force sensor , and a MT-9 accelerometer . Each device was connected to a Porti 5 system  to record its millivolt (mV) outputs with a sample frequency of 1,600 Hz, yielding data characterizing of both pushing and pulling forces and pole acceleration in X, Y, and Z direction, respectively. Prior to recordings during ACT execution, baseline mV output of the load cell during 10 s in the static was recorded and nulled. The post-practice datasets of two subjects (#2 and #4) recorded during ACT execution were corrupted due to faulty wiring to the Porti-5 system.Raw signals from the LCM300 load cell were processed using custom-written Matlab code. First, the signal was filtered using a fourth-order low-pass Butterworth filter at a cut-off frequency of 6 Hz to remove any movement noise and subsequently transformed to N using prior calibration with weights. A typical example of the measured force output is shown in The mobile Spirometer Cosmed K4B2  was used to record gas exchange dynamics during ACT execution. Heart rate data was collected using a strap and software provided by the same supplier. Data were collected during a 10 min rest period followed by the 4 min ACT and a 10 min long recovery period.2 (L/min) and VCO2 (L/min) recordings were used to calculate the internal energy expenditure   and mechanical efficiency (ME)) vs. proximal arm muscle outcomes  of ACT execution in individual subjects is presented in in vivo data gathering on energy metabolism and pH balance in proximal muscles of the arm performing a calibrated concentric exercise task. Two previous applications of in vivo31P MRS to the investigation of energy metabolism in proximal muscle of the human arm employed either a qualitative isometric task  over the biceps muscle  to record in vivo31P MR spectra from the biceps muscle. In light of our findings of inter-individual differences in pull- vs. push- handcycling strategies, future studies should also collect MRS data from the triceps muscle and preferably simultaneously from both muscles. In principle, use of a two-element 31P surface coil in combination with dual receive channels should render simultaneous MRS data collection from both upper-arm muscles feasible. Modern clinical 3T MRI scanners with multinuclear capability from major vendors typically support use of such advanced coil designs. Moreover, these scanners are typically also available in a 70 cm diameter bore size. This both greatly enhances room for in-magnet exercise as well as relaxes some of the constraint on physical dimensions of study subjects that we encountered.The present study employed a conventional single element 2 and VCO2 data are available including Douglas bag-based methodologies  for the publication of any potentially identifiable images or data included in this article.RV, SB, LW, and JJ contributed to the conception and design of the work, the acquisition, analysis, and interpretation of data and drafted the work. LM contributed to the conception and design of the work, interpretation of data and drafted the work. AS contributed to the design of the work, the acquisition, analysis, and interpretation of data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer MM declared a past co-authorship with one of the authors JJ to the handling editor.


Distinguishing low-grade phyllodes tumor from fibroadenoma is practically challenging due to their overlapping histologic features. However, the final interpretation is essential to surgeons, who base their management on the final pathology report. Patients who receive a diagnosis of fibroadenoma might not undergo any additional intervention while lumpectomy with wide margins is the standard of care for phyllodes tumor, which can have significant cosmetic consequences. We studied the clinical, immunophenotypic, and proteomics profiles of 31 histologically confirmed low-grade phyllodes tumor and 30 fibroadenomas. Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) and immunohistochemistry for Ki-67, p53, β-catenin, and E-cadherin were performed on all cases. After the mass spectra for all 31 cases of low-grade phyllodes tumor and 30 cases of fibroadenoma were collected, an average peak value for all cases was generated. There was no significant difference in the overall mass spectra pattern in any of the peaks identified. There was also overlap in the percentage of cells staining positive for Ki-67, p53, β-catenin, and E-cadherin. The two groups of patients showed no statistically significant difference in age, tumor size, or disease-free survival. Neither group developed malignant transformation, distant metastases, or disease-related mortality. We have demonstrated low-grade phyllodes tumor and fibroadenoma to show significant overlapping clinical and proteomics features. However, phyllodes tumor represents a heterogenous group of neoplasms that display highly variable prognosis2.Fibroepithelial lesions of the breast are a heterogeneous group of biphasic tumors that include the common benign fibroadenomas and the relatively rare phyllodes tumors . Current treatment guidelines rely on histologic diagnosis on either core or excisional biopsy and recommend wide excision without axillary staging to treat phyllodes tumors regardless of grade2 recommended a three-tier grading system , while the Armed Forces Institute of Pathology (AFIP) proposed a two-tier system 3 with slight variation in their cutoff criteria. AFIP defines low-grade phyllodes tumors as fibroepithelial neoplasms with well-defined borders, uniform leaf-like processes, rare to moderate mitoses (<3 mitoses per 10 high-power fields), mildly increased cellularity often with subepithelial accentuation, no to mild cytological atypia and no sarcomatous stromal overgrowth. The low-grade category proposed by the AFIP would include the majority of benign phyllodes tumors and a subset of borderline phyllodes tumors.Many grading systems for phyllodes tumor have been proposed, all based on the assessment of tumor borders, stromal cellularity and atypia, mitotic activity, and stromal overgrowth. The World Health Organization (WHO)4 showed that there is significant inter-observer variability, even among pathologists who specialize in breast pathology, when making diagnosis of either benign phyllodes tumor or fibroadenoma in a research setting. The issue is more significant in small core biopsies, which determines whether the fibroepithelial lesion should be observed (fibroadenoma) or excised with a wide margin (phyllodes tumor)5. The justification for the latter is on the basis of reported local recurrences of phyllodes tumors6. This diagnostic dilemma on core needle biopsies has been highlighted during recent attempts of incorporating digital pathology into routine practice7. To date, there are no clinically validated immunohistochemical assays to facilitate the differential diagnosis.Despite the above well-established diagnostic criteria, distinguishing low-grade phyllodes tumor from fibroadenoma is challenging due to their overlapping histologic features Fig. . Recent 8. Since the development of histology-directed MALDI-IMS technique8, there have been several feasibility studies exploring its utility in the facilitation of challenging histologic diagnoses11. One main advantage of histology-directed MALDI-IMS is the ability to interrogate the proteomics profile of individual tumor components. This approach has definite appeal in our case, as both phyllodes tumors and fibroadenomas are biphasic tumors, it would be of significant interest to compare the epithelial and stromal components between the two biphasic lesions independently.The application of matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) on formalin-fixed, paraffin-embedded tissue allows for the label-free, multiplex analysis of thousands of analytes across the tissue surface and elucidate both the localization and relative abundance of endogenous metabolites, lipids, peptides, and proteinsThe aim of this study was to perform a comprehensive evaluation of the clinical, histopathologic, immunophenotypic, and proteomics characteristics of low-grade phyllodes tumor and fibroadenoma to exploit similarities and differences of these two entities.A total of 31 patients with low-grade phyllodes tumor  cases with the diagnosis of low-grade phyllodes tumor had re-excision of the tumor bed performed, whereas none of the cases with the diagnosis of fibroadenoma undergone additional intervention. This is consistent with the current standard of care. The median follow-up time for low-grade tumor was 47.0 months , compared to the 61.5 months median follow-up time for fibroadenoma . Two of 31 patients (6.5%) with low-grade phyllodes tumor subsequently developed recurrence of disease, and upon a second excision procedure, there were no further recurrences. Although none of the 30 patients with fibroadenoma developed “recurrences,” it is important to note that one patient presented with new ipsilateral breast mass, one patient with contralateral breast mass, and one patient with bilateral breast masses. None of which were biopsied under the clinical impression that these were fibroadenomas. The disease-free Kaplan–Meier curve is presented to show no significant difference in Fig. There is significant overlap in the percentage of cells staining positive for Ki-67, p53, β-catenin, and E-cadherin between low-grade phyllodes tumor and fibroadenoma Figs.  and 5. KIn contrast, high-grade phyllodes tumor showed minimal overlap and statistically significantly different staining pattern for Ki-67, p53, and β-catenin Figs.  and 5 coThe results of the immunohistochemistry study are summarized in Table A total of 21 cases of low-grade phyllodes tumor and another 20 cases of fibroadenoma were randomly selected as the study set to generate an algorithm to distinguish low-grade phyllodes tumor and fibroadenoma. This algorithm was then tested on a validation set of remaining ten cases of low-grade phyllodes tumor and fibroadenoma. The initial mass spectra generated from the study set showed only few peaks with >1-fold difference and fewer peaks that showed statistical significance. There is greater proteomic variability between the stromal compared to the epithelial component in these two tumor types Fig. . However12. Within the study cohort, the local recurrence rate of low-grade phyllodes tumor was 6.5%, all of which were cured by re-excision of the tumor bed. Whether this represented incomplete excision of the initial surgery, or rather a recurrence of the tumor could not be further discerned in the retrospective data. We did confirm that the margin status was all negative on evaluation of initial resection. We also noted, however, that although there were no reported “recurrences” for fibroadenomas, new lesions were seen in the breast on mammography, and were often not biopsied due to minimal risk of progression. There were no malignant transformation or distal metastases in any of the low-grade phyllodes tumor or fibroadenoma cases. In the three largest studies to date, only 0.3% of low-grade phyllodes tumor eventually developed metastatic disease15.Distinguishing a fibroadenoma from a low-grade phyllodes tumor by pure morphology can be challenging, particularly on core needle biopsies. Our study shows that, even when strictly adhering to the diagnostic criteria, there is minimal difference in the clinical characteristic and overall outcome between low-grade phyllodes tumor and fibroadenoma and that perhaps separating the two entities is neither possible nor desirable. Low-grade phyllodes tumor are slightly larger in size and present in older patients, but neither age or size differences are statistically significant. We also demonstrated that low-grade phyllodes tumor have a low risk for recurrence and malignant potential on long term follow-up, which is similar to findings in other studies in the literature15, likely due to the limited number of patients with high-grade phyllodes tumor in our study. This highlights the importance of distinguishing high-grade phyllodes tumor from low-grade phyllodes tumor, which do show significant prognostic differences.We reviewed the clinical outcome for high-grade phyllodes tumor and 2 of the 8 (25.0%) patients with available follow-up information died of metastatic disease 18 and 19 months after surgery, respectively. This number is slightly above the 16.3% rate reported in the literature18. The difference between fibroadenomas and low-grade phyllodes tumor may be explained by a bias to designate as phyllodes tumor those lesions with increased stromal mitotic figures. Previous studies have shown p53 positivity to be increased in phyllodes tumors18. We also observed the lowest positivity in fibroadenomas , second low-grade phyllodes tumor , followed by high-grade phyllodes tumor . β-catenin nuclear staining has been used as a surrogate marker for Wnt signaling pathway, which have been proposed to play a role in the pathogenesis of phyllodes tumor19. β-catenin had highest nuclear positivity in low-grade phyllodes tumor , followed by fibroadenoma , with high-grade phyllodes tumor rarely expressing the marker . The loss of nuclear β-catenin expression in high-grade phyllodes tumor has been shown in previous studies and correlate with worse prognosis20. Given the overlap in immunohistochemistry results for Ki-67, p53, and β-catenin, it would seem unlikely that these markers would provide much additional information to help distinguish fibroadenoma and phyllodes tumor. There was one study showing E-cadherin in the stromal cells to correlate with recurrence and shorter tumor-specific survival21, however, we did not identify any staining for E-cadherin in the stromal component of any of our cases. This difference may be attributed to the alternative antibody clone used in our study.There are no established criteria for evaluation of immunohistochemistry stains to help classify or stratify fibroadenoma and phyllodes tumor. In our study, Ki-67 showed lowest proliferative index in fibroadenomas , followed by low-grade phyllodes tumor , with high-grade phyllodes tumor showing the highest proliferation index . This is similar to results from previous studies, which some also found high proliferation index to be associated with worse clinical outcomeAlthough immunohistochemistry studies have the advantage of examining protein expression in the context of histomorphology, the main caveat to this approach is that only selected proteins of interest can be studied at a time. The choice of proteins that can be studied is also restricted by the antibodies that are available. Conventional mass-spectrometry can be utilized to study multiple peptides, lipids, and other molecules, but lacks the capability of spatial recognition. MALDI-IMS provides a platform with spatial specificity to separately compare the stromal and epithelial components between fibroadenoma and low-grade phyllodes tumor. To our knowledge, our study is the first to look at the proteomics constitution of the stromal and epithelial component of low-grade phyllodes tumor and fibroadenoma separately.Our results showed no significant difference in proteomic profile of the stromal or the epithelial component between low-grade phyllodes tumor and fibroadenoma, as the algorithm generated from the mass spectra in the study set did not confidently distinguish the two entities in the validation set. Furthermore, the combined average of all the peaks generated for the mass spectra showed no significant difference between the two entities. Clinically, none of the low-grade phyllodes tumor or the fibroadenoma cases in our study developed distant metastasis on follow-up and by the fact that a total of five cases developed subsequent lesions in the breast, two cases were low-grade phyllodes tumor which were cured of disease on re-resection, and the other three were cases of fibroadenoma and the new lesions were not biopsied, likely a consequence of interpreting new lesions preceded by a diagnosis of phyllodes tumor as a recurrence and those by a diagnosis of fibroadenoma as multicentricity. It seems that a propensity for recurrence/multicentricity cannot be accurately outlined by known histologic criteria nor by the histology-directed mass spectral protein profiles. Given the limited number of cases included in our study and the low number of proteins analyzed, further investigation is warranted.MED12, an X-linked gene encoding mediator complex subunit 12, in fibroadenomas and phyllodes tumors28, in the stromal components26. Mutational analysis in several cohorts showed higher rate of MED12 exon 2 hotspot mutations in fibroadenomas and lower grade phyllodes tumors, particularly benign phyllodes tumors31, while genetic aberrations in TP53, RB1, NF1, PIK3CA, ERBB4, and EGFR were more commonly found in malignant phyllodes tumors33. Pareja et al.34 reported a higher frequency of MED12 mutations in phyllodes tumors with fibroadenoma-like areas, compared with those without fibroadenoma-like areas, suggesting the progression of MED12-mutant fibroadenomas to a subset of phyllodes tumors. Tan et al.31 used combined whole exome sequencing and targeted deep sequencing of fibroepithelial lesions and revealed similar genetic signatures between fibroadenomas and a subset of benign phyllodes tumors. For diagnostic purposes, there have been proposal of using an assay of five-gene transcript set  for classifying breast fibroepithelial lesions into fibroadenomas and phyllodes tumors35.In the last two decades, the utility of next generation sequencing has provided new insights in the classification and pathogenesis of breast fibroepithelial lesions. The most important and well-established finding has been the identification of hotspot mutations in exon 2 of 12 acknowledged that the definitive distinction between cellular fibroadenoma and benign phyllodes tumor may not be crucial and indeed not necessary. We further showed that conventional fibroadenoma and low-grade phyllodes tumor  are identical by proteomics analysis. Overall, the results from our study as well as those of focusing on genetic aspects support the interpretation these lesions are one and the same and a more conservative surgical approach is justified by its indolent nature, something that can be facilitated by avoiding the diagnosis of phyllodes tumor, at least for those lesions designated as benign phyllodes and those in which a distinction between fibroadenoma and phyllodes tumor is difficult or not possible. In any case of uncertainty, especially in core biopsies, rendering the diagnosis of “benign fibroepithelial tumor” is preferable. Separating out the high-grade  phyllodes tumor, on the other hand, has significant clinical implications. There are strict histologic criteria and immunohistochemistry studies that add supporting evidence to this distinction.Most recent consensusThere were several limitations to our study that need to be acknowledged. First, our study was retrospective in nature, and was limited by the inherent bias associated with such approach. Second, the number of cases in our study was relatively small. This is mainly due to the fact that phyllodes tumor are much less common in the area populated with Caucasians and African Americans. Third, we did not include specific information on laterality and location of the masses, which may provide additional information in predicting availability for intervention and/or tumor behavior. Lastly, we did not have enough cases of phyllodes tumor with recurrence and/or metastasis to perform MALDI-IMS, which would be of clinical relevance to differentiate from other cases. Additional prospective studies with larger cohorts, especially with large number of cases with recurrences and/or metastases, would be necessary to fully address the issues above.2 and AFIP3 grading criteria. Fibroadenoma variants, including juvenile fibroadenoma, cellular fibroadenoma, myxoid fibroadenoma, and complex fibroadenoma with florid changes that obscure the underlying fibroadenomatous nature were excluded due to potential challenges in accurate classification. Fibroadenomas with focal complex changes were not excluded. A total of 31 cases with confirmed diagnosis of low-grade phyllodes tumor and 30 consecutive cases of fibroadenoma were identified and retrieved from archives. Clinical data for all cases of low-grade phyllodes tumor and fibroadenoma were extracted from the electronic medical records system for analysis. We additionally analyzed all ten cases of high-grade phyllodes tumor identified with immunohistochemistry using Ki-67, p53, β-catenin, and E-cadherin.Our study was approved by the Washington University School of Medicine Institutional Review Board, including a waiver of consent given the retrospective nature of this study. The surgical pathology archives were searched for partial or total mastectomies with a diagnosis, including the word “phyllodes” from year 2006 to 2014. Archived histologic slides of these cases and of a matching number of fibroadenomas were retrieved and reviewed by three pathologists  blinded to the clinical outcome information. All tumors were subclassified using both the WHOOne representative formalin-fixed paraffin-embedded tissue block from each case  was selected to generate 4 μm thick unstained slides for immunohistochemical staining for the following antibodies: Ki-67 , p53 , β-catenin , and E-cadherin . The immunohistochemistry staining was performed on Ventana Benchmark automated immunostainer  according to standard protocols with appropriate positive and negative controls. The immunohistochemistry staining results were interpreted individually by two pathologists (C.Y. and L.Z.) blinded of the diagnosis. The percentage of cells positive was visually estimated and recorded and the average of the two results were used for analysis. Only nuclear staining for Ki-67, p53, and β-catenin in the stromal tumor cells were considered as positive. In contrast, cytoplasmic and membranous staining in the stromal tumor cells for E-cadherin was interpreted as positive. The percentage of staining was estimated relative to total number of stromal cells.36. Two pathologists (L.Z. and C.Y.) then annotated the scanned images with color-coded circular marks (300 μm in diameter). In each image of fibroadenoma or phyllodes tumor, 20 marks (red circles) were assigned for the epithelial component and 20 marks (purple circles) were assigned for the stromal components   was deposited over a series of 40 iterations, 40 passes, one drop each pass (roughly 170 pL/drop) at each designated coordinate to achieve a final concentration of ~29 ng/mm2. This was completed over a total of 2 h to allow drying time between iterations. Alpha-cyano-4-hydroxycinnamic acid matrix  was then applied over the digested spots over 72 iterations, 8 passes, 9 drops each pass. The samples were analyzed in profiling mode on a Bruker UltrafleXtreme TOF MS in reflectron positive mode. Data was sorted by patient and tissue type.Serial sections, 6 μm thick, were cut from the above selected blocks. One section per sample was mounted onto indium tin oxide coated slide for MALDI time-of-flight (TOF) analysis, whereas the consecutive section was mounted onto a regular glass slide as a reference histologic section. The slides were dried overnight at 37 °C and stored out of light until ready to use. The reference slides were stained with H&E and scanned with an automated slide scanner  at x20 magnification and uploaded to a web-based pathology interface for mass-spectrometry (PIMS)na) Fig. . In a su38 in the R environment. After data import, the spectra were baseline corrected to remove MALDI chemical noise using the SNIP algorithm39 with 20 iterations. After baseline correction, the spectral data was normalized by the total ion current to account for pixel-to-pixel signal artifacts. Following normalization, the spectral data was statistically re-aligned to a reference spectrum to remove inherent mass shifts from the TOF instrument that can greatly affect classification quality. The reference spectrum for realignment was closest spectrum to the 50th quantile of the TIC distribution of all data. Following mass axis alignment, peaks with signal-to-noise ratio above 3 were selected on the overall mean spectrum from all data. The peak intensity data was integrated for each spectrum resulting in a data matrix where observations (rows) are individual pixels labeled by de-identified patient serial number and pathologist annotation and variables (columns) are picked peak intensity data from the dataset from each spectrum.Acquired MALDI-IMS profiling data was processed using MALDIquantMachine learning on the picked peak MS data implemented the support vector machine algorithm with a linear kernel. The model and cost function (regularization) were tuned using 5-fold cross validation. The tuned cost function was found to be 0.5 in this instance. Analysis of significant different peaks between the two data classes was performed using area under the receiver operator characteristic curve.Relationships between the variables were examined by Wilcoxon-Mann–Whitney tests for comparison of means, Fisher-Exact tests for comparison of categorical variables, and log-rank tests for comparison of survival. All tests were two-sided.Further information on research design is available in the Supplementary Table 1.Reporting Summary Checklist